PD-1 blockade-induced CD4 + T cell dysregulation triggers dopaminergic neurodegeneration in Parkinson’s disease

PD-1 blockade-induced CD4 + T cell dysregulation triggers dopaminergic neurodegeneration in Parkinson’s disease | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article PD-1 blockade-induced CD4 + T cell dysregulation triggers dopaminergic neurodegeneration in Parkinson’s disease Luyan Gu, Xiaoli Si, Yi Fan, Jie Liang, Mengjie Shi, Chao Chen, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9090665/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 11 You are reading this latest preprint version Abstract With the widespread use of PD-1 inhibitors in the treatment of cancer patients, emerging clinical evidence has linked them to the onset of parkinsonism, raising critical questions about the role of PD-1 in Parkinson’s disease (PD) pathogenesis—a connection previously unexplored. Here, we found that PD-1 blockade aggravated, while its activation mitigated, dopaminergic neuronal damage and motor deficits in PD mouse models. Immunofluorescence and flow cytometry analyses revealed that PD-1 blockade promoted blood-brain barrier (BBB) permeabilization and drove neuroinflammation by enhancing cerebral infiltration of CD4 + T cell and a Th1-biased differentiation. Notably, these effects were reversed by PD-1 activation both in vivo and in vitro. We further identify the AKT/GSK3β signaling pathway in CD4 + T cells as the central mediator of this immunomodulatory effect. Clinically relevance demonstrated by dysregulated PD-1 and PD-L1 expression in peripheral CD4 + T cells from PD patients, with notable variation across clinical subtypes. Together, these findings demonstrated that PD-1 blockade-induced CD4 + T cell dysregulation exacerbates neuroinflammation and neurodegeneration in PD, providing a novel perspective on immune pathogenesis in PD and presenting a significant, mechanistic advance with translational implications. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Parkinson's disease (PD) is the most common neurodegenerative movement disorder, affecting 1–2% of people aged over 65 years 1 . Its pathological hallmarks include the progressive loss of dopaminergic (DA) neurons and the accumulation of alpha-synuclein (α-Syn) in the substantia nigra compacta (SNc) 2 . While traditional view holds PD as a neurodegenerative disorder, increasing evidence has highlighted the key role of immune dysregulation in its onset and progression 3,4 . Notably, with the widespread application of immune checkpoint inhibitors (ICIs) in cancer patients, several reports have found ICIs triggered parkinsonism as immune-related adverse events 5–7 . These clinical findings suggest that immune dysfunction induced by ICIs may be involved in the pathogenesis of PD, but its mechanism remains unclear. Anti-programmed cell death protein-1 (anti-PD-1), the most widely used ICIs, exerts its anti-tumor efficacy by inducing systemic immune activation, primarily through enhancing T-cell responses 8,9 . Accumulating evidence suggests an important role of PD-1 in the central nervous system (CNS) 10 . PD-1 suppresses the cerebral immune response via CNS-resident immune cells and infiltrating peripheral T cells, while PD-1 blockade exacerbates neuroinflammation by activating these immune cells. Preclinical study reported that dual inhibition of PD-1 and Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) induces DA neuron loss within the SNc and enhances cerebral CD4 + T cell infiltration in lung tumor mice model 11 . Moreover, Cheng et al. found that PD-1 deficiency exacerbates DA neuron damage and motor deficits in mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a neurotoxin that selectively targets dopaminergic neurons to model PD 12 . These findings highlight the damaging effect of PD-1 blockade on DA neurons, however, it is currently unclear how it triggers immune cell dysfunction and infiltration into the CNS, and further drives DA neuronal degeneration in PD. PD-1 is mainly expressed on activated T cells and maintains immune homeostasis by regulating their differentiation and function. Notably, CD4⁺ T cell infiltration near DA neuron has been observed from postmortem PD patients and mouse model, which may drive DA neuron degeneration 13,14 . Accumulating evidence further indicates an imbalance among CD4 + T cell subsets in PD patinets 15 . Specifically, Th1 and Th17 cells exacerbate DA neuron injury by producing IFN-γ and IL-17, whereas Th2 cells and regulatory T cells (Tregs) mitigate inflammation and injury via anti-inflammatory cytokines such as IL-4 and IL-10 16,17 . Moreover, PD-1 is involved in regulating CD4 + T cell differentiation, as evidenced in cancer and immune-mediated inflammatory diseases like asthma and Crohn's disease 18,19 . In vivo, PD-1 deficiency drives a cytokine shift toward Th1/Th17 profiles and away from Th2 responses. Correspondingly, in vitro studies corroborate that PD-1 ligation suppresses Th1 and Th17 polarization while enhancing Th2 and Treg response 20,21 . Therefore, it remains to be elucidated whether manipulating PD-1 exerts damaging or protective effects on DA neurons through CD4⁺ T cell homeostasis. Here, we investigated the role and mechanism of PD-1 in PD pathogenesis at multiple levels. We found that PD-1 blockade exacerbates DA neuron damage and motor deficits which also impairing BBB integrity. This permeabilization drove neuroinflammation by enhancing cerebral CD4 + T cell infiltration and a Th1-biased differentiation, which was mediated by activated AKT/GSK3β pathway in CD4 + T cells. Strikingly, PD-1 activation reversed the entire pathological cascade in vivo and in vitro. Extending the clinical relevance of these findings, dysregulated PD-1 and PD-L1 expression was observed in peripheral CD4 + T cells from PD patients, with notable variation across clinical subtypes. Our findings demonstrated that PD-1 blockade-induced CD4 + T cell dysregulation exacerbates neuroimmune inflammation and neurodegeneration in PD, whereas PD-1 activation exerts a protective effect, providing a novel perspective on immune pathogenesis in PD and presenting a significant, mechanistic advance with translational implications. Results PD-1 blockade exacerbates dopaminergic neuronal damage and motor dysfunction in PD mice, whereas PD-1 activation exertes a protective effect Use of PD-1 inhibitors in cancer patients has led to Parkinsonism (e.g., bradykinesia, tremor) that is alleviated upon supplementation with dopaminergic drugs 7,22 . To further investigate the impact of PD-1 manipulation on PD pathology and behavior, mice were treated intraperitoneally with either an anti-PD-1 antibody (αPD-1, 0.25 mg × 2 doses, 3-day interval) to block PD-1, or a recombinant PD-L1 Fc fusion protein (0.25 mg × 2 doses, 3-day interval) to activate PD-1 signaling. Four weeks post-injection, a 5-day subacute MPTP model was established. Behavioral function and dopaminergic neuropathology were then evaluated before sacrifice (Fig. 1 A). Compared to controls, MPTP-induced PD mice exhibited significant hypolocomotion in both open-field and rotarod tests. Although not statistically significant, a downward trend was observed in αPD-1-treated PD mice. Notably, PD-L1-Fc treatment surprisingly rescued motor deficits in PD mice (Fig. 1 B, C). Tyrosine hydroxylase (TH) is a rate-limiting enzyme responsible for dopamine biosynthesis, and its deficiency reflects the loss of dopaminergic neurons and the progression of PD 23 . Western blotting analysis of striatal TH expression showed that compared with PD mice, TH depletion was exacerbated in αPD-1-treated PD mice, while PD-L1 Fc treatment markedly attenuated TH loss (Fig. 1 D, E). Immunofluorescence detection of striatal TH intensity corroborated these findings, supporting the neuroregulatory effects of PD-1 immunotherapy. Furthermore, unbiased stereological counting of TH-positive neurons in the SNc demonstrated that αPD-1 blockade exacerbated neuronal loss, reducing TH-positive neurons by approximately 55%, while PD-L1 Fc resulted in a ~ 24% recovery in TH-positive neuron number (Fig. 1 F, G). These results demonstrate that PD-1 blockade exacerbates motor deficits and DA neuronal damage in PD mice, whereas PD-1 activation ameliorates both behavioral impairment and neuronal pathology. PD-1 blockade promotes blood-brain barrier permeability, while PD-1 activation restores barrier function in PD mice. Anti-PD-1 can uniquely compromise BBB integrity in brain metastases, thereby enhancing the infiltration of peripheral immune cells into the brain and exerting anti-tumor effects 24 . To investigate whether PD-1 influence BBB integrity during dopaminergic neuronal damage in PD, MPTP-induced PD mice were pre-conditioned with αPD-1 or PD-L1 Fc and BBB permeability was assessed. Western blot was performed to measure the levels of tight junction proteins (e.g., ZO-1, claudin-5 and occludin) in the striatum and substantia nigra. These proteins act as structural barriers in the BBB. We found that the tight junction proteins expression level decreased in MPTP mice striatum and SN, which was aggravated following PD-1 blockade. In contrast, PD-L1 Fc treatment rescued its expression by different degrees (Fig. 2 A-D). Immunofluorescence analysis revealed lower structural overlap between ZO-1/claudin-5 with blood vessels (labeled with CD31) in αPD-1-treated PD mice than MPTP alone, whereas PD-L1 Fc treatment partially restored this colocalization (Fig. 2 E, F). To complement the protein-level analysis, we also assessed the transcriptional levels of tight junction genes across all experimental groups by RT-qPCR. The results showed that PD-1 blockade significantly reduced the expression of tight junction genes in the SN. In contrast, PD-1 activation promoted the recovery of their expression levels (Fig. 2 G). Taken together, these results explicitly indicate that PD-1 blockade aggravates BBB permeability, whereas PD-1 activation rescues the barrier function. PD-1 blockade enhances cerebral CD4 + T cells infiltration and Th1/Treg differentiation, while PD-1 activation mitigates these effects The intracranial efficacy of PD-1 blockade is primarily mediated by activated peripheral T cells that infiltrate the brain parenchyma 25 . Given that PD-1 blockade promotes BBB permeabilization, peripheral immune cells may infiltrate the brain and exert immune responses. To investigate alterations in the brain microenvironment following PD-1 blockade, we assessed brain-infiltrating immune cell populations, including CD4 + T cells, CD8 + T cells, microglia and related subsets. Using flow cytometric analysis of the brain, we found that PD-1 blockade significantly increased the infiltration of CD4⁺ T cells into the brains of PD mice, while PD-1 activation reduced CD4⁺ T cells recruitment. In contrast, CD8⁺ T cell infiltration remained unchanged across experimental groups (Fig. 3 A, B). Alterations in brain CD4 + T cell populations were corroborated by both CD4 expression levels in the striatum (Fig. 3 C, D) and immunofluorescence quantification of CD4⁺ T cells in the SNc (Fig. 3 E, F). The functional regulation of CD4 + T cells primarily depends on alterations in distinct subset proportions or activity. To further investigate the impact of PD-1 immunotherapy on cerebral CD4 + T cell subsets in PD, we performed intracellular cytokine staining on CD4 + T cells and analyzed changes in Th1, Th2, Th17, and Tregs populations using flow cytometry. Compared to controls, MPTP group exhibited a bias toward Th1 cell differentiation along with a reduction in Tregs, leading to a disruption of the Th1/Treg balance. Notably, this Th1/Treg differentiation shift was further aggravated following PD-1 blockade but was partially restored by PD-L1 Fc stimulation (Fig. 3 G, H). In addition to investigating the effects of PD-1/PD-L1 signaling on CD4⁺ T cell-mediated adaptive immunity, we also assessed the innate immune response mediated by microglia. Consistent with previous reports, PD mice exhibited significantly elevated expression of the microglial marker Iba1 along with an increase in microglial cell number, suggesting exacerbated neuroinflammation. However, no changes in microglial activation or abundance were observed in PD mice following PD-1 immunotherapy (Figure S1 A, B). These findings indicate that the PD-1 primarily modulates adaptive immunity through CD4⁺ T cell regulation. Collectively, PD-1 blockade exacerbates the pathology by enhancing both the infiltration and pro-inflammatory differentiation of CD4⁺ T cells in the brains of PD mice, while PD-1 activation attenuates these effects. PD-1 signaling modulates Th1 and Tregs differentiation in PD mice To elucidate the regulatory role of PD-1 signaling in Th1 and Treg cell differentiation, we further analyzed the expression of PD-1 and its ligand PD-L1 in PD mice and assessed its correlation with Th1 and Treg frequencies. Western blotting showed MPTP group demonstrated a declining trend of PD-1 expression in the striatum compared to controls. PD-1 blockade further reduced PD-1 levels relative to MPTP group, while PD-1 activation modestly elevated PD-1 expression. No significant alterations in PD-L1 expression were detected among the groups (Fig. 4 A, B). Flow cytometry analysis of PD-1 and PD-L1 expression on CD4 + T cells similarly demonstrated that PD-1 blockade reduced PD-1 expression levels while PD-1 activation counteracted this reduction. Notably, PD-L1 expression on CD4 + T cells exhibited a strikingly parallel trend across the groups (Fig. 4 C, D). Notably, PD-L1 expression on CD4⁺ T cells exhibited a similar trend, showing reduction after αPD-1 blockade and elevation following PD-L1 Fc stimulation. Furthermore, we performed correlation analyses between PD-1/PD-L1 expression on CD4 + T cells and cytokine production (IFN-γ, IL-4, IL-17a, and IL-10) defining Th1, Th2, Th17, and Treg frequencies. Both PD-1 and PD-L1 expression negatively correlated with Th1 frequencies but positively correlated with Treg frequencies. No significant correlation was detected with either Th2 or Th17 subsets (Fig. 4 E). These results indicate that PD-1 signaling intensity participates in the regulation of Th1 and Treg differentiation in PD mice. PD-1 blockade promotes Th1-biased differentiation while decreasing Tregs, whereas PD-1 activation restores Th1/Treg homeostasis in vitro Next, we aimed to validate the regulatory role of PD-1 on CD4⁺ T cell immune homeostasis in vitro. To establish optimal MPP⁺ concentrations for inducing changes in PD-1/PD-L1 expression and Th1/Treg polarization in CD4⁺ T cells, we isolated primary CD4⁺ T cells from C57BL/6 mouse spleens via Magnetic-Activated Cell Sorting (MACS). The cells were then stimulated with graded MPP + concentrations and analyzed by flow cytometry (Fig. 5 A). MACS yielded CD4 + T cells with 92.8% purity, satisfying the requirements for subsequent in vitro stimulation assays (Fig. 5 B). Primary CD4 + T cells were stimulated with αCD3 + αCD28 and exposed to MPP + at concentrations of 0.25 mM, 0.5 mM, or 1.0 mM for 72h, followed by surface staining and intracellular staining. Flow cytometric analysis revealed that increasing MPP⁺ concentrations elevated IFN-γ expression while reducing Foxp3 expression in CD4⁺ T cells, indicating Th1 polarization and Treg suppression concurrent with PD-1 signaling downregulation. At 1.0 mM MPP + stimulation, significant difference in PD-1/PD-L1 expression and Th1/Treg differentiation bias were observed, establishing this concentration as the reference standard for subsequent in vitro modeling (Figure S2A, B). To investigate whether PD-1 signaling modulates Th1/Treg differentiation under MPP + stimulation, CD4 + T cells were co-stimulated with αCD3/CD28 and MPP + , followed by treatment with either PD-1 blockade or activation to regulate the pathway. After 72 h, cells were harvested for flow cytometric analysis, and culture supernatants were assessed for IFN-γ and IL-10 levels by ELISA. We observed that stimulation with αCD3/CD28 antibodies significantly increased the proportion of Th1 cells, which was further elevated following MPP⁺ stimulation. PD-1 activation reduced Th1 polarization, and this suppression was attenuated upon PD-1 blockade. Moreover, PD-1 activation rescued the loss of Tregs induced by MPP⁺, while PD-1 blockade further reduced the Treg population (Fig. 5 C, D). These findings were corroborated by ELISA, which showed corresponding changes in IFN-γ and IL-10 secretion (Fig. 5 E). Consistent with the in vivo results, these findings reveal that PD-1 blockade exacerbates the Th1/Treg differentiation imbalance under MPP⁺ stimulation in vitro, whereas PD-1 stimulation alleviates this effect. PD-1 blockade leads to dysregulated CD4⁺ T cell differentiation via AKT/GSK3β pathway. To further elucidate the mechanism underlying CD4⁺ T cell differentiation imbalance, we isolated CD4⁺ T cells from the brains of mice treated with PD-1 blockade or activation and performed transcriptomic analysis (Fig. 6 A). Gene Ontology (GO) enrichment analysis revealed that PD-1 signaling is implicated in biological processes (BP) such as monatomic ion transmembrane transport, cell differentiation, and monatomic ion transport (Fig. 6 B). In terms of cellular components (CC), the analysis encompassed cell projection, the extracellular matrix, and the cell surface (Figure. S3A). For molecular function (MF), significant terms included calcium ion binding and signal receptor activity (Figure. S3B). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis further identified neuroactive ligand-receptor interaction, calcium signaling pathway, and PI3K-AKT signaling pathway as the top three pathways modulated by PD-1 immunotherapy in PD (Fig. 6 C). Previous studies have suggested that glycogen synthase kinase 3β (GSK3β), a downstream effector of AKT, is involved in the CD4 + T cell differentiation and function, including Th1 and Treg lineages 26 , and PD-1 signaling can regulate immune cell differentiation through the AKT/GSK3β pathway 27 . Integrating our sequencing data with published evidence, we interrogated the role of AKT/GSK3β pathway in PD-1-mediated CD4 + T cell differentiation. Subsequently, magnetically isolated splenic CD4⁺ T cells were treated with αPD-1 or PD-L1 Fc, then co-stimulated with MPP + in αCD3/CD28-coated plates for 72 hours. Western blot analysis revealed that MPP⁺ stimulation alone reduced phosphorylation levels of AKT (Ser473) and GSK3β (Ser9), whereas PD-L1 Fc co-stimulation increased p-AKT and p-GSK3β expression. Conversely, PD-1 blockade again reduced phosphorylation of both AKT and GSK3β (Fig. 6 D, E). Total AKT and GSK3β protein expression remained comparable across experimental groups. To further verify the role of the AKT/GSK3β pathway in PD-1 regulation of CD4 + T cell differentiation, the cells were pretreated with specific inhibitors of AKT (MK2206) and GSK3β (Tideglusib/NP-12) to block the signaling. Western blot analysis revealed that inhibition of AKT with MK2206 abolished PD-L1 Fc-induced augmentation of phosphorylation in both AKT and GSK3β (Fig. 6 F, G), while GSK3β blockade with NP-12 further increased PD-L1 Fc-mediated phosphorylation of GSK3β (Fig. 6 H). These findings suggest that PD-1 blockade exacerbates CD4⁺ T cell differentiation dysregulation by activating the AKT/GSK3β pathway. Conversely, PD-1 stimulation suppresses this pathway and restores t CD4⁺ T cell homeostasis. Dysregulated PD-1 and PD-L1 expression in peripheral blood in PD patients, with distinct levels across clinical subtypes. Previous studies have revealed distinct peripheral immune profiles in PD patients stratified by age at onset 30,31 . To comprehensively characterize the expression and distribution of PD-1 and PD-L1 on T cells in PD patients across different age-onset subtypes, mass cytometry and CyTOF analysis were used on peripheral blood samples from early-onset PD (EOPD) and late-onset PD (LOPD) cohorts (Fig. 7 A). The baseline demographic and clinical characteristics of PD patients and healthy controls are shown in Table S1 . Compared to healthy controls, PD patients overall showed no significant differences in the expression of PD-1 or PD-L1 (Fig. 7 B, C). Intriguingly, reduced PD-1 and PD-L1 levels were observed in EOPD patients, whereas their expression was elevated in LOPD patients (Fig. 7 D, E). Moreover, a similar pattern was observed for PD-1 and PD-L1 expression specifically on CD4 + T cells across age-onset subgroups (Fig. 7 F, G). Further phenotyping of CD4 + T cell subsets revealed that PD-1 expression was significantly reduced on Th1, Th2, and Treg in EOPD patients, but increased on Treg and Tfh in LOPD patients (Fig. 7 H-K). Similarly, PD-L1 expression is reduced on Th2 and Treg in EOPD patients, while it is increased on Treg in LOPD (Fig. 7 L-O). No differential expression of PD-1 or PD-L1 was found on CD8 + T cells among PD subtypes (Fig. 7 P, Q). These findings suggest that PD-1/PD-L1 expression profiles differ among PD patients with different age onset, primarily on CD4 + T cells and their subsets, reflecting the distinct peripheral immune homeostasis. Discussion In this study, we demonstrate that PD-1 blockade exacerbates, while PD-1 activation mitigates, dopaminergic neuronal damage and motor deficits in a PD mouse model. Mechanistically, PD-1 blockade promotes blood-brain barrier permeability and drives neuroinflammation by enhancing cerebral CD4 + T cell infiltration and a Th1-biased differentiation, a process reversible upon PD-1 activation both in vivo and in vitro. We further identify the AKT/GSK3β signaling pathway in CD4 + T cells as the key mediator of this immunomodulatory effect. Clinically, dysregulated PD-1 and PD-L1 expression was also observed in peripheral CD4 + T cells from PD patients and varied across clinical subtypes. Immune checkpoint inhibitors (ICIs) have shown remarkable efficacy in various types of tumors and become the most commonly used cancer immunotherapy. With the widespread clinical application, there have been increasing reports of immune-related adverse events (irAEs) occurring in patients receiving ICIs 28 . Movement disorders (MD) are rare neurological irAEs, accounting for 3%–5% of cases, and primarily manifest as tremor, rigidity, and parkinsonism 29 . As the most frequently administrated ICIs, anti-PD-1-induced MD (including parkinsonism) account for approximately half of such cases 7 . Preclinical studies have also observed damage to dopaminergic neurons in tumor-bearing mice treated with PD-1 inhibitors 11 . Furthermore, PD-1 deficiency aggravates motor dysfunction and the DA neuronal damage in MPTP Model of PD 12 . Consistent with previous studies, we found that PD-1 blockade exacerbates motor deficits and DA neuronal damage in MPTP-induced PD mice. Notably, we also administered PD-1 activation therapy and discovered that activating this pathway mitigates motor dysfunction and reduce the loss of dopaminergic neurons. This is opposite to the therapeutic effect of PD-1 in tumors, possibly due to disease-specific variations in the immune microenvironment 30,31 . Unlike the immunosuppressive environment in cancer, PD is characterized by immune activation, accompanied by the release of pro-inflammatory cytokines and lymphocyte infiltration 32 . PD-1 blockade would further lead to overactivation of both brain-resident immune cells and peripheral T cells, thereby contributing to the DA neurons degeneration. We observed that PD-1 blockade promotes BBB permeability in the substantia nigra of PD mice, leading to increased infiltration of CD4 + T cells, whereas PD-1 activation restores BBB function and reduces the level of CD4 + T cells in the substantia nigra. Anti-PD-1 can hardly cross the BBB into the brain, though its intracranial efficacy is mediated largely by activated peripheral T cells that infiltrate the brain parenchyma 25,33 . Recent studies showed that PD-1 blockade promotes BBB permeability, which is mediated by activated CD8⁺ T cells or microglia 24,34 . The BBB function is also regulated by CD4⁺ T cells, through the release of pro-inflammatory cytokines and the recruitment of other immune cells. Under pathological conditions, CD4⁺ T cells accumulate around the BBB, where they release inflammatory factors and induce endothelial cell apoptosis, thereby disrupting the tight junction structure of the BBB and increasing its permeability 35 . Increased BBB permeabilization conversely facilitates the infiltration of peripheral CD4⁺ T cells into the brain, triggering an imbalance in the CNS immune microenvironment and leading to neuronal death. Early pioneering study found that CD4⁺ T cells had invaded the brain in both postmortem human PD specimens and in MPTP mouse model of PD 13 . Further studies found that mice lacking CD4 + T cells attenuated MPTP-induced DA neuronal death, but not mice lacking CD8 + T cells. CD4 + T cells directly participate in the process of DA neuron degeneration by recognizing α-syn antigens, disrupting immune balance, and driving chronic neuroinflammation 36–38 . Thus, we speculate that the pathological impact of PD-1 blockade on DA neuron mediated by increased cerebral CD4⁺ T cell infiltration via impairing BBB in the substantia nigra. Another significant finding is that PD-1 therapy modulates CD4⁺ T cell subset differentiation. Specifically, PD‑1 blockade promotes inflammatory differentiation (enhanced Th1 polarization and diminished Tregs), while PD‑1 activation induces an anti‑inflammatory phenotype (reduced Th1 and expanded Tregs). Most studies have demonstrated elevated frequencies of Th1 cells in the peripheral blood of PD patients as well as in the brains of PD mouse models, which lead to a Th1 bias 39,40 . Adoptive transfer of Th1 cells also significantly enhance MPTP-induced neurotoxicity. Activated Th1 cells significantly increase the production of cytokines such as IFN-γ and TNF-α, which exacerbate neuroinflammation and affect neuronal survival 41 . Additionally, these cytokines may increase BBB permeability, allowing peripheral immune cells and inflammatory mediators into the brain, which further disrupts the neuronal microenvironment 42 . On the other hands, the reduction of Tregs is also observed in animal models of PD. MPTP-treated mice exhibits fewer Tregs, and the adoptive transfer of Tregs reduces the brain’s inflammatory response and alleviated neuronal death by suppressing glial activation 16,43,44 . Another study also revealed that the imbalance between Th1 and Tregs plays a detrimental role in PD mice 45 . In our study, dysregulation of CD4⁺ T cell subset differentiation in PD was exacerbated after PD-1 therapy. Previous studies found that PD-1-deficient mice displayed elevated Th1/Th17 and reduced Th2 cytokine responses. Correspondingly, PD-1 ligation limited cytokine production in polarized Th1 and Th17 cells in vitro but slightly enhanced it in Th2 cells 20,46 . However, we did not find significant changes in Th2 and Th17 cells following PD-1 immunotherapy, which may be resulted from the heterogeneity and stage-dependent effects of the disease. Mechanistically, we identified the AKT/GSK3β signaling axis as a key intracellular pathway downstream of PD-1 that regulates CD4⁺ T cell differentiation. Transcriptomic profiling of brain-infiltrating CD4⁺ T cells pointed to alterations in the PI3K-AKT pathway after PD-1 immunotherapy. Subsequent in vitro experiments validated that PD-1 signaling activation augmented phosphorylation of both AKT (Ser473) and its downstream target GSK3β (Ser9), an inhibition signal for GSK3β, in CD4⁺ T cells under MPP⁺ stimulation. This PD-1-induced signaling cascade is both necessary and sufficient to suppress Th1 differentiation and promote Tregs generation, as evidenced by pharmacologic inhibition of the AKT/GSK3β pathway. These findings are consistent with established role of AKT/GSK3β activity in regulating T cells lineage commitment 47,48 . In T cells, active GSK3β generally promotes pro-inflammatory subsets while inhibiting Tregs development. Additionally, GSK3β can modulate T cell activity in breast cancer through its interaction with PD-L1, whereas PD-1 regulates GSK3β activity and tau phosphorylation in AD mouse models 49,50 . Song et al. demonstrated Th1 cytokine IFN-γ could affect the expression of PD-1 in T lymphocytes through the AKT/GSK3β signaling pathway 51 . Hu et al also showed that PD-1 regulates macrophage polarization through the Akt/GSK3β/β-catenin pathway 27 . Our findings establish a critical link between surface checkpoint signaling and intracellular pathways in CD4⁺ T cells, demonstrating that PD-1 regulates CD4 + T cell differentiation in PD via the AKT/GSK3β axis. Our study demonstrated that peripheral immune modulation via the PD-1 pathway can significantly influence the central immune environment and disease progression in PD. The central question in neuroimmunology is how peripheral immune signals communicate with the brain 52 . There is complex immune cross-talk between the periphery and the central nervous system. For example, BBB disruption during neuroinflammation enables peripheral T cells to infiltrate the brain directly 53 . In addition, immune factors released by peripherally activated T cells can diffuse into the brain or signal through endothelial cells to recruit and educate resident immune cells 54,55 . Recent study has also shown that peripheral inflammatory signals can reach the brain via the vagus nerve pathway 56 . In this study, we found PD-1 blockade disrupted the BBB, suggesting that the infiltration of peripheral CD4⁺ T cells into the brain and the diffusion of peripheral cytokines may occur concurrently. Therefore, peripheral immune signature likely reflects a parallel and possibly causative process occurring within the brain. The PD-1-low CD4⁺ T cells in the periphery may represent a pathogenic subset with heightened activation capacity and a propensity to infiltrate the brain, where they exacerbate neuroinflammation and drive the loss of DA neurons. However, this study also has several limitations. First, the MPTP-induced mouse model, while classic, does not fully replicate the slow progression and α-synucleinopathy of human PD. Future research should employ models like α-synuclein pre-formed fibrils (PFF). Second, although we linked PD-1 signaling to the AKT/GSK3β pathway in CD4⁺ T cells, the precise molecular intermediates remain unclear. Third, the impact of AKT/GSK3β on the differentiation of CD4⁺ T cells has not been well studied in vivo. Additionally, while the correlation between PD-1/PD-L1 expression and clinical symptoms was observed in PD patients, the small sample size limited the statistical power. Future larger and longitudinal cohort studies are warranted to fully evaluate the clinical translational potential of the PD-1/PD-L1 axis as a therapeutic target and biomarker for PD. In summary, we demonstrate the role and mechanism of PD-1 signaling in regulating neuroinflammation and neurodegeneration in PD by both blocking and stimulating PD-1. Specifically, the immune homeostasis of CD4⁺ T cell was identified as a key mediator of this process. Our findings offer a novel perspective on immune pathogenesis in PD and present a significant, mechanistic advance with translational implications. Materials and Methods Animals and drugs C57BL/6 mice (8 weeks old; 22–25 g) were used for the experiments. Animals were housed in a centralized location with a 12-h light/dark cycle and the same sleeping condition. For the regulation of the PD-1/PD-L1 pathway, we utilized PD-1-specific blocking antibody (αPD-1; rat IgG2a isotype; clone RPM1-14; BIOXCELL) to blockade PD-1/PD-L1 pathway, and recombinant mouse PD-L1 (PD-L1-Fc; Cat. No. CJ89; Novoprotein) to binding to PD-1 receptor activates PD-1/PD-L1 pathway. αPD-1 or PD-L1 were administrated two injections, 0.25 mg at a 3-day interval, to modulate the PD-1/PD-L1 immune pathway in mice. Four weeks after the last injection, MPTP mouse model was carried out in reference to a protocol of our previous studies. Briefly, MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, Sigma) was intraperitoneally injected at dose of 30 mg/kg/day for consecutive 5 days, and injection of same amount of 0.9% saline was given as control. All mice were euthanized 7 days after the last injection of MPTP or saline. Motor behavior was detected before sacrifice, and flow cytometry, immunofluorescence and western blot experiment were analyzed using postmortem brain tissue. Behavioral analyses Motor behavior detection, including rotarod test and open-field test, were performed 7 days after the last MPTP or saline injection. For the rotarod test, mice were trained to run on an accelerated rotating rod (LE8205; Panlab, Barcelona, Spain) for three consecutive days, prior to the final test. After training, mice were placed on the rotating rod with the rotation speed gradually increased from 4 to 40 rpms within a 5 min period. Te duration that mice remained on the rod until their first slip to the base (also called latency to falling) was recorded. Measurements were averaged over three trials, with at least 1 h of rest. To assess general locomotor activity and exploratory behavior, mice were subjected to open-field test. The mice were transported to a testing room 30 min prior to testing for acclimatization and placed in the center of a nontransparent Plexiglas arena under bright lighting for 5 min subsequently. The total distance moved in 5 min was recorded using SMART video tracking software (Smart 3.0; Panlab, Cornellà de Llobregat, Spain). Western blotting Samples were lysed using radioimmunoprecipitation assay (RIPA) buffer and proteinase inhibitor (Thermo Fisher Scientific), and protein concentrations were determined using a bicinchoninic acid protein kit (Thermo Fisher Scientific). Protein was separated using 10–12% sodium dodecyl-sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. After blocking with 5% skim milk or 5% bovine serum albumin (BSA) in 0.1% Tween-20/Tris-buffered saline (TBS-T) for 1 h at RT, the membranes were incubated overnight with the following primary antibodies at 4℃. Then the blots were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature. Immunoblots (IB) were detected using a chemiluminescence reagent (Thermo Fisher Scientific) and analyzed using ImageJ. In this study, the membranes were incubated with primary antibodies against TH (1:1,000, AB152, Sigma), CD4 (1:1,000, ab183685, Abcam), PD-1(1:1000, 66220-1-Ig, Proteintech), PD-L1(1:1000, 66248-1-Ig, Proteintech), ZO-1(1:1000, A0659), Occludin(1:1000, A2601), Claudin-5(1:1000, A10207) (Abclonal), AKT (1:1,000, #4691), GSK-3β (1:1,000, #12456), phospho-AKT (1:500, #4060), phospho-GSK-3β Ser9 (1:500, #9323) (Cell Signaling Technology) and GAPDH (1:2000, Proteintech); secondary antibodies against goat anti-rabbit (1:5,000) or goat anti-mouse (1:5,000) (Proteintech). Immunofluorescence Mice under complete anesthetic state were perfused with phosphate-buffered saline, and then fixed with 4% paraformaldehyde. The brains were dissected, fixed in 4% PFA solution for 6 h at 4°C, and placed into 30% sucrose solution for dehydrating. Then, the brains were embedded in OCT compound and sliced at thickness of 30 µm. After blocking and permeabilization, brain slices containing substantia nigra and striatum were incubated in the presence of primary antibodies at 4°C overnight. The next day, the brain slices were incubated with secondary antibodies for 1 h at room temperature. After DAPI counter stain, the sections were observed using a confocal microscope (TCS SP8, Leica) and a VS120 virtual slide microscope (DM6B, Leica), and interested images were captured for demonstration representatively. Photomicrographs were analyzed by a blinded investigator using ImageJ or Fiji software. Antibodies and working dilutions were used in immunohistochemical staining. These primary and secondary antibodies were purchased from Sigma, abcam and Thermo. Primary antibodies included rabbit anti-TH (1:500, AB152; Sigma), CD4 (1:200, ab183685, Abcam), ZO-1 (1:200, A0659, Abclonal), Claudin-5 (1:200, A10207, Abclonal) and CD31 (1:200, R&D, AF3682). Second antibodies included Alexa Fluo488-labeled donkey anti-rabbit IgG (1:500, Thermo). Quantitative reverse transcription PCR (RT-qPCR) Total RNA was extracted with RNAiso Plus (Takara 9108) according to the manufacturer's instructions. 5× Prime Script RT master mix (Takara, RR036A) was used for reverse transcription. All primers for RT-qPCR were designed with Primer-BLAST( https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi ) according to the sequences and are listed in Table S2 . All primers used spanned an exon–exon junction. The 20 µL reaction volume contained the following components: 10 µL of TB GreenPremix Ex Taq II (Takara, RR420A), 0.8 µL each of forward and reverse primers, 0.4µL ROX Reference Dye, 2 µL of cDNA, and 6 µL of nuclease-free water. The qRT-PCR analyses were carried out using a two-step method with the StepOnePlus Real-Time PCR System (Applied Biosystems), and the cycling parameters were as follows: 95 ℃ for 30 s, 40 cycles of denaturation (95 ℃, 5 s), and annealing (60 ℃, 30 s). All reactions were performed in duplicate or in triplicate with GAPDH as the internal reference, and the relative mRNA expression data were calculated using the 2 ΔΔ Ct method. Mononuclear cell isolation and flow cytometry Mononuclear cells were isolated from hemibrains of mouse that received PD-1/PD-L1 immunotherapy and MPTP according to previously published methods 57 . Briefly, brain was minced and homogenized using a tissue dissociation tube, followed by centrifugation at 800×g for 20 minutes under minimal acceleration and deceleration in 30%/70% Percoll to remove myelin debris. Erythrocytes were lysed using erythrocyte lysis buffer (420301, BioLegend), yielding a single-cell suspension. Single-cell suspension was first incubated with an anti-CD16/CD32 antibody to block Fc receptors and reduce nonspecific binding. Cell viability was assessed using fixable viability dye (Zombie AquaTM Fixable Viability Kit, 423101, BioLegend), following the manufacturer's protocol. Then surface staining was performed by adding fluorescent-conjugated antibodies at a 1:200 dilution, and incubating the samples in the dark at 4°C for 30 minutes. The following antibodies were used: CD45 (BV605, 103139, BioLegend), CD11b (PE/Cyanine7, 101215, BioLegend), CD3 (FITC, 100203, BioLegend), CD4 (PerCP/Cyanine5.5, 100539, BioLegend), CD8a (APC/Fire, 100765, BioLegend), CD25 (BV650, 102037, BioLegend), PD-1 (BV421, 135221, BioLegend) and anti-mouse PD-L1 (PE, 153606, BioLegend) For intracellular staining of transcription factors, cells were fixed with Transcription Factor Buffer Set (BD Pharmingen) according to manufacturer’s instructions. For intracellular cytokine staining, isolated mononuclear cells were stimulated with phorbol myristate acetate (PMA) (50 ng/ml, InvivoGen) and ionomycin (1 ug/ml, InvivoGen) in the presence of monensin (BioLegend) for 4 h at 37 C/5% CO2. The following antibodies were used: IFN-γ (BV785, 505838, BioLegend), IL-4 (PE/Cyanine7, 504117, BioLegend), IL-17A (APC, 506915, BioLegend), IL-10 (PE, 505007, BioLegend), and Foxp3 (PE, 12-5773-82, Invitrogen). Samples were acquired using a flow cytometer (Beckman Coulter, USA), and data were analyzed using FlowJo software. CD4 + T cells isolation and culture In order to explore the mechanism of the effect of PD-1/PD-L1 on CD4 + T cell differentiation in vitro, splenic CD4 + T cells were sorted from C57BL/6 mice using CD4 + T Cell Isolation Kit for mice (130-104-454; Miltenyi Biotec, Auburn, CA, USA). Sorted T cells were cultured in RPMI 1640 medium, supplemented with 10% FBS and 1% pen/strep (all from Gibco, Shanghai, China) for further study. According to the different groups, CD4 + T cells were cultured under αCD3/CD28 (5µg/mL), MPP+ (1.0mmol), αPD-1(5µg/mL) and PD-L1 Fc (5µg/mL) stimulation for 3 days. Subsequently, cells were collected for flow cytometry analysis and western blot assay, and the supernatant was used to detect cytokines by ELISA. Enzyme-linked immunosorbent assay (ELISA) The concentration of cytokine in the culture supernatants was determined with mouse IFN Gamma/IFNG ELISA Kit (EK0375, BOSTER) and mouse IL-10 ELISA Kit (EK0417, BOSTER). The steps are carried out according to the manufacturers’ instruction. Absorbance was measured at 450 nm using a plate reader (BioTek), and 540nm was measured for correction. Cell transcriptome sequencing Single-cell suspensions were extracted from the brain tissues of PD-1/PD-L1 immunotherapy PD mice. Following Fc receptor blocking, fixable viability dye-based viability staining (Zombie AquaTM Fixable Viability Kit, 423101, BioLegend), and surface staining for CD45 (BV605, 103139, BioLegend), CD3 (FITC, 100203, BioLegend) and CD4 (APC, 100515, BioLegend), fluorescence-activated cell sorting (FACS) was performed to isolate CD4 + T cells for Switching Mechanism at the 5' end of RNA Template Sequencing (SMART-Seq2). SMARTer cDNA synthesis starts with picogram amounts of total RNA or single cell/several cells. A modified oligo(dT) primer (the SMART CDS Primer) primes the first-strand synthesis reaction. When SMARTScribeTM Reverse Transcriptase reaches the 5' end of the mRNA, the enzyme's terminal transferase activity adds a few additional nucleotides to the 3' end of the cDNA. The carefully-designed SMARTer Oligonucleotide base-pairs with the non-template nucleotide stretch, creating an extended template to enable SMARTScribe RT continue replicating to the end of the oligonucleotide. The resulting full-length, single-stranded (ss) cDNA contains the complete 5' end of the mRNA, as well as sequences that are complementary to the SMARTer Oligonucleotide. Amplify sscDNA by LD PCR and get enough dscDNA for library construction. cDNA was fragmented by dsDNA Fragmentase (NEB, M0348S) by incubate at 37℃ for 30min. Library construction begins with fragmented cDNA. Blunt-end DNA fragments are generated using a combination of fill-in reactions and exonuclease activity, and size selection is performed with provided sample purification beads. An A-base is then added to the blunt ends of each strand, indexed Y adapters are ligated to the fragments, and the ligated products are amplified with PCR. And then we performed the paired-end sequencing on an Illumina NovaseqTM 6000 at the (LC Sceiences, USA) following the vendor's recommended protocol. Human samples Our previous studies have found that the peripheral immune status of PD patients at different ages of onset is different 58,59 . Thus, peripheral blood samples were collected from 10 early-onset PD patients (EOPD; ≤50 years), 12 late-onset PD patients (LOPD; >50 years of age) and age-matched healthy volunteers from the neurology department at the Second Affiliated Hospital of Zhejiang University. PD patients had a diagnosis of PD according to Movement Disorder Society (MDS) criteria 60 . The study has excluded subjects suffering from immune-related diseases or using immune-related drugs. Clinical characteristics including the age of onset, duration of disease, Hoehn and Yahr (H-Y) stage, UPDRS, MMSE and MoCA scores were collected. All of the study participants provided informed consent. Mass cytometry and CyTOF analysis The blood samples were transferred to a 50 ml centrifuge tube, 10 ml of Ficoll separation solution (GE Healthcare) was added, and the centrifuge tube was placed in a centrifuge with a plate rotor (Avanti J-15R, Beckman), centrifuged at 400 x g for 15 min, and the white layer was transferred to a new 50 ml centrifuge tube to obtain a PBMC initial extract. PBMC suspension obtained after cell lysis using 1 ml ammonium-chloride–potassium (ACK). Cells were washed once with PBS and incubated in Fc receptor blocking solution before staining with surface antibodies cocktail for 30 min on ice. After fixation in intercalation solution (Maxpar Fix and Perm Buffer containing 250 nM 191/193 Ir, Fluidigm) overnight, cells were washed once with the FACS buffer and then stained with intracellular antibodies cocktail for 30 min on ice. Cells were washed and resuspended with deionized water, adding to 20% EQ beads (Fluidigm), acquired on a mass cytometer (Helios, Fluidigm) 61 . CyTOF analysis was performed according to previously reported methods 62 . Data of each sample were debarcoded from raw data with unique mass-tagged barcodes using a doublet-filtering scheme.The beads normalization method was used to normalize FCS files generated by different batches. Data were manually gated using the FlowJo software to exclude debris, dead cells, and doublets, leaving live, single immune cells. Cells were partitioned based on marker expression level using the X-shift clustering algorithm. Cell types of each cluster were annotated according to the marker expression pattern on a heatmap of cluster versus marker. Then, T-distributed stochastic neighbor embedding (t-SNE), a dimensionality reduction algorithm, was used to visualize high-dimensional data in two dimensions and to show the distribution of each cluster and differences between groups. Statistical analysis Statistical analysis was performed using GraphPad Prism 8.2.1 (Graph Pad Software, San Diego, CA, USA) and SPSS 24.0 (IBM Corp., Armonk, NY, USA). The Shapiro-Wilk test was used to test the normality of the distribution of the variables. Student’s t-test or two-way analyses of variance (ANOVA) was used to compare means between and within groups, followed by Tukey’s post hoc test. Data are expressed as mean ± SEM for at least n = 3. Statistical significances are indicated as *p < 0.05; **p < 0.01; ***p < 0.001 Declarations Ethics statement All experiments involving human and animals were conducted according to the ethical policies and procedures approved by the Medical Ethics Committees of the Second Affiliated Hospital of Zhejiang University, China (Approval no. 2020 − 596 and 2022 − 0865). Clinical trial number Not applicable. Competing interests The authors declare no competing interests. Author Contribution J.P. conceived the study; J.P., L.G.and X.S. designed the experiments. L.G., X.S. and Y.F. performed the experiments; J.L., M.S., C.C., Y.W. and W.H. collected the data. X.T., Z.L. and N.X. analyzed the data. L.G. and X.S. prepared the figures. J.P. and B.Z. supervised the project. L.G.and X.S. wrote the original manuscript, which was critically reviewed and edited by Y.J., J.T., B.Z., and J.P. Acknowledgement This work was supported by the National Natural Science Foundation of China [No.82571419] and the Key Research and Development Program of Zhejiang Pcovince [No. 2025C02113]. Data Availability All data are available within this article and its Supplementary information. References Su, D. et al. Projections for prevalence of Parkinson's disease and its driving factors in 195 countries and territories to 2050: modelling study of Global Burden of Disease Study 2021. BMJ (Clinical research ed.) 388 , e080952 (2025). Tanner, C. M. & Ostrem, J. L. Parkinson's Disease. The New England journal of medicine 391 , 442–452 (2024). Tansey, M. G. et al. Inflammation and immune dysfunction in Parkinson disease. Nature reviews. Immunology 22 , 657–673 (2022). Roodveldt, C. et al. The immune system in Parkinson's disease: what we know so far. Brain : a journal of neurology 147 , 3306–3324 (2024). Özistanbullu, D., Kaufmann, R. & Meissner, M. Immune checkpoint inhibitor triggered parkinsonism in a patient with metastatic melanoma. Journal der Deutschen Dermatologischen Gesellschaft = Journal of the German Society of Dermatology : JDDG 18 , 884–885 (2020). Prasertpan, T., Teeyapun, N., Bhidayasiri, R. & Sringean, J. Immune Checkpoint Inhibitor-Associated Striatal Encephalitis Presenting with Subacute Progressive Parkinsonism. Movement disorders clinical practice 10 , S7-s11 (2023). Dinoto, A. et al. Autoimmune Movement Disorders Complicating Treatment with Immune Checkpoint Inhibitors. Movement disorders clinical practice 11 , 543–549 (2024). Chamoto, K., Yaguchi, T., Tajima, M. & Honjo, T. Insights from a 30-year journey: function, regulation and therapeutic modulation of PD1. Nature reviews. Immunology 23 , 682–695 (2023). Weiss, S. A. et al. Epigenetic tuning of PD-1 expression improves exhausted T cell function and viral control. Nature immunology 25 , 1871–1883 (2024). Zhao, J., Roberts, A., Wang, Z., Savage, J. & Ji, R. R. Emerging Role of PD-1 in the Central Nervous System and Brain Diseases. Neuroscience bulletin 37 , 1188–1202 (2021). Dong, L., Zheng, Y. M., Luo, X. G. & He, Z. Y. High Inflammatory Tendency Induced by Malignant Stimulation Through Imbalance of CD28 and CTLA-4/PD-1 Contributes to Dopamine Neuron Injury. Journal of inflammation research 14 , 2471–2482 (2021). Cheng, Y. Y., Chen, B. Y., Bian, G. L., Ding, Y. X. & Chen, L. W. Programmed Death-1 Deficiency Aggravates Motor Dysfunction in MPTP Model of Parkinson's Disease by Inducing Microglial Activation and Neuroinflammation in Mice. Molecular neurobiology 59 , 2642–2655 (2022). Brochard, V. et al. Infiltration of CD4 + lymphocytes into the brain contributes to neurodegeneration in a mouse model of Parkinson disease. The Journal of clinical investigation 119 , 182–192 (2009). Zirra, A. & Huxford, B. CD4(+) T Cells Contribute to Neurodegeneration in Lewy Body Dementia. Movement disorders : official journal of the Movement Disorder Society 37 , 268 (2022). Sun, X., Gu, R. & Bai, J. Differentiation and regulation of CD4(+) T cell subsets in Parkinson's disease. Cellular and molecular life sciences : CMLS 81 , 352 (2024). Reynolds, A. D. et al. Regulatory T cells attenuate Th17 cell-mediated nigrostriatal dopaminergic neurodegeneration in a model of Parkinson's disease. Journal of immunology (Baltimore, Md. : 1950) 184 , 2261–2271 (2010). Tahmasebinia, F. & Pourgholaminejad, A. The role of Th17 cells in auto-inflammatory neurological disorders. Progress in neuro-psychopharmacology & biological psychiatry 79 , 408–416 (2017). Snell, L. M. et al. Dynamic CD4(+) T cell heterogeneity defines subset-specific suppression and PD-L1-blockade-driven functional restoration in chronic infection. Nature immunology 22 , 1524–1537 (2021). Lin, X. et al. Regulatory mechanisms of PD-1/PD-L1 in cancers. Molecular cancer 23 , 108 (2024). McAlees, J. W. et al. Differential control of CD4(+) T-cell subsets by the PD-1/PD-L1 axis in a mouse model of allergic asthma. European journal of immunology 45 , 1019–1029 (2015). Ma, F., Zhao, M., Song, Z. & Wang, Z. T-bet interferes with PD-1/PD-L1-mediated suppression of CD4(+) T cell inflammation and survival in Crohn's disease. Clinical and experimental pharmacology & physiology 46 , 798–805 (2019). Zimmer, L. et al. Neurological, respiratory, musculoskeletal, cardiac and ocular side-effects of anti-PD-1 therapy. European journal of cancer (Oxford, England : 1990) 60 , 210–225 (2016). Haavik, J. & Toska, K. Tyrosine hydroxylase and Parkinson's disease. Molecular neurobiology 16 , 285–309 (1998). Deo, A. et al. PD1 blockade-induced DKK1 expression by CD8 + T cells promotes blood-brain barrier permeabilization. Cancer discovery (2026). Portnow, J. et al. Systemic Anti-PD-1 Immunotherapy Results in PD-1 Blockade on T Cells in the Cerebrospinal Fluid. JAMA oncology 6 , 1947–1951 (2020). Xia, Y. et al. Glycogen synthase kinase 3β inhibition promotes human iTreg differentiation and suppressive function. Immunologic research 62 , 60–70 (2015). Hu, X. H. et al. A newly intervention strategy in preeclampsia: Targeting PD-1/Tim-3 signaling pathways to modulate the polarization of decidual macrophages. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 36 , e22073 (2022). Fletcher, K. & Johnson, D. B. Chronic immune-related adverse events arising from immune checkpoint inhibitors: an update. Journal for immunotherapy of cancer 12 (2024). Fonseca, E. et al. Neurological adverse events related to immune-checkpoint inhibitors in Spain: a retrospective cohort study. The Lancet. Neurology 22 , 1150–1159 (2023). Filippou, P. S. & Outeiro, T. F. Cancer and Parkinson's Disease: Common Targets, Emerging Hopes. Movement disorders : official journal of the Movement Disorder Society 36 , 340–346 (2021). Surguchov, A. & Surguchev, A. A. Association between Parkinson's Disease and Cancer: New Findings and Possible Mediators. International journal of molecular sciences 25 (2024). Harms, A. S., Ferreira, S. A. & Romero-Ramos, M. Periphery and brain, innate and adaptive immunity in Parkinson's disease. Acta neuropathologica 141 , 527–545 (2021). van Bussel, M. T. J., Beijnen, J. H. & Brandsma, D. Intracranial antitumor responses of nivolumab and ipilimumab: a pharmacodynamic and pharmacokinetic perspective, a scoping systematic review. BMC cancer 19 , 519 (2019). Vinnakota, J. M. et al. Anti-PD-1 cancer immunotherapy induces central nervous system immune-related adverse events by microglia activation. Science translational medicine 16 , eadj9672 (2024). Shi, S. X. et al. CD4(+) T cells aggravate hemorrhagic brain injury. Science advances 9 , eabq0712 (2023). Liu, Z. et al. Dopamine receptor D2 on CD4(+) T cells is protective against neuroinflammation and neurodegeneration in a mouse model of Parkinson's disease. Brain, behavior, and immunity 98 , 110–121 (2021). Williams, G. P. et al. CD4 T cells mediate brain inflammation and neurodegeneration in a mouse model of Parkinson's disease. Brain : a journal of neurology 144 , 2047–2059 (2021). Karikari, A. A. et al. Neurodegeneration by α-synuclein-specific T cells in AAV-A53T-α-synuclein Parkinson's disease mice. Brain, behavior, and immunity 101 , 194–210 (2022). Kustrimovic, N. et al. Parkinson's disease patients have a complex phenotypic and functional Th1 bias: cross-sectional studies of CD4 + Th1/Th2/T17 and Treg in drug-naïve and drug-treated patients. Journal of neuroinflammation 15 , 205 (2018). Mamula, D., Khosousi, S., He, Y., Lazarevic, V. & Svenningsson, P. Impaired migratory phenotype of CD4(+) T cells in Parkinson's disease. NPJ Parkinson's disease 8 , 171 (2022). Bai, X. B. et al. Oral pathogens exacerbate Parkinson's disease by promoting Th1 cell infiltration in mice. Microbiome 11 , 254 (2023). Maes, M., Almulla, A. F., You, Z. & Zhang, Y. Neuroimmune, metabolic and oxidative stress pathways in major depressive disorder. Nature reviews. Neurology 21 , 473–489 (2025). Rostami, J. et al. Astrocytes have the capacity to act as antigen-presenting cells in the Parkinson's disease brain. Journal of neuroinflammation 17 , 119 (2020). Reynolds, A. D., Stone, D. K., Mosley, R. L. & Gendelman, H. E. Nitrated {alpha}-synuclein-induced alterations in microglial immunity are regulated by CD4 + T cell subsets. Journal of immunology (Baltimore, Md. : 1950) 182 , 4137–4149 (2009). Li, W., Luo, Y., Xu, H., Ma, Q. & Yao, Q. Imbalance between T helper 1 and regulatory T cells plays a detrimental role in experimental Parkinson's disease in mice. The Journal of international medical research 49 , 300060521998471 (2021). Lee, J. et al. The Multifaceted Role of Th1, Th9, and Th17 Cells in Immune Checkpoint Inhibition Therapy. Frontiers in immunology 12 , 625667 (2021). Ji, S. et al. Mesenchymal stem cell transplantation inhibits abnormal activation of Akt/GSK3β signaling pathway in T cells from systemic lupus erythematosus mice. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology 29 , 705–712 (2012). Hong, L. J., Chen, A. J., Li, F. Z., Chen, K. J. & Fang, S. The HSP90 Inhibitor, 17-AAG, Influences the Activation and Proliferation of T Lymphocytes via AKT/GSK3β Signaling in MRL/lpr Mice. Drug design, development and therapy 14 , 4605–4612 (2020). Li, C. W. et al. Glycosylation and stabilization of programmed death ligand-1 suppresses T-cell activity. Nature communications 7 , 12632 (2016). Zou, Y. et al. Programmed Cell Death Protein 1 Blockade Reduces Glycogen Synthase Kinase 3β Activity and Tau Hyperphosphorylation in Alzheimer's Disease Mouse Models. Frontiers in cell and developmental biology 9 , 769229 (2021). Song, M. et al. IFN-γ decreases PD-1 in T lymphocytes from convalescent COVID-19 patients via the AKT/GSK3β signaling pathway. Scientific reports 14 , 5038 (2024). Zhang, S. et al. Peripheral and central neuroimmune mechanisms in Alzheimer's disease pathogenesis. Molecular neurodegeneration 20 , 22 (2025). Sweeney, M. D., Sagare, A. P. & Zlokovic, B. V. Blood-brain barrier breakdown in Alzheimer disease and other neurodegenerative disorders. Nature reviews. Neurology 14 , 133–150 (2018). Xu, Y. et al. The reciprocal interactions between microglia and T cells in Parkinson's disease: a double-edged sword. Journal of neuroinflammation 20 , 33 (2023). Kedia, S. et al. T cell-mediated microglial activation triggers myelin pathology in a mouse model of amyloidosis. Nature neuroscience 27 , 1468–1474 (2024). Jin, H., Li, M., Jeong, E., Castro-Martinez, F. & Zuker, C. S. A body-brain circuit that regulates body inflammatory responses. Nature 630 , 695–703 (2024). Williams, G. P. et al. Targeting of the class II transactivator attenuates inflammation and neurodegeneration in an alpha-synuclein model of Parkinson's disease. Journal of neuroinflammation 15 , 244 (2018). Tian, J. et al. Specific immune status in Parkinson's disease at different ages of onset. NPJ Parkinson's disease 8 , 5 (2022). Jiang, S. S. et al. Cytokine and chemokine map of peripheral specific immune cell subsets in Parkinson's disease. NPJ Parkinson's disease 9 , 117 (2023). Postuma, R. B. et al. MDS clinical diagnostic criteria for Parkinson's disease. Movement disorders : official journal of the Movement Disorder Society 30 , 1591–1601 (2015). Abrecht, C. et al. Simplified mass cytometry protocol for in-plate staining, barcoding, and cryopreservation of human PBMC samples in clinical trials. STAR protocols 3 , 101362 (2022). Esaulova, E. et al. Single-cell RNA-seq analysis of human CSF microglia and myeloid cells in neuroinflammation. Neurology(R) neuroimmunology & neuroinflammation 7 (2020). Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterials.docx GraphicalAbstract.tif Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 02 Apr, 2026 Reviews received at journal 02 Apr, 2026 Reviews received at journal 02 Apr, 2026 Reviews received at journal 31 Mar, 2026 Reviewers agreed at journal 19 Mar, 2026 Reviewers agreed at journal 19 Mar, 2026 Reviewers agreed at journal 17 Mar, 2026 Reviewers invited by journal 17 Mar, 2026 Editor assigned by journal 17 Mar, 2026 Submission checks completed at journal 16 Mar, 2026 First submitted to journal 11 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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University","correspondingAuthor":false,"prefix":"","firstName":"Naijia","middleName":"","lastName":"Xue","suffix":""},{"id":608391484,"identity":"f16cb8c2-5e9f-4b23-9f75-fcf947888a2f","order_by":11,"name":"Wenhao Huang","email":"","orcid":"","institution":"Second Affiliated Hospital of Zhejiang University","correspondingAuthor":false,"prefix":"","firstName":"Wenhao","middleName":"","lastName":"Huang","suffix":""},{"id":608391485,"identity":"1e44d6e0-bf7b-4ede-999f-e0de0651b600","order_by":12,"name":"Jun Tian","email":"","orcid":"","institution":"Second Affiliated Hospital of Zhejiang University","correspondingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Tian","suffix":""},{"id":608391486,"identity":"119229f6-a653-4c57-9ee1-ff888871d436","order_by":13,"name":"Baorong Zhang","email":"","orcid":"","institution":"Second Affiliated Hospital of Zhejiang University","correspondingAuthor":false,"prefix":"","firstName":"Baorong","middleName":"","lastName":"Zhang","suffix":""},{"id":608391487,"identity":"581a930e-4dd6-426d-b03a-4db77c5e3e76","order_by":14,"name":"Jiali Pu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0klEQVRIie3RsQqDMBCA4RPBLhZXJ32FSMe+jE6ZBCfniGCXQlehQ5/BzTEl0C7BuUMHfQPHdig0J3RtMhaafxCF+7gEAWy2H8ypweEpQATA1adnRgDJxpxgOJux5dWEuDt35FMv6KmSBOZSQHBkuoN5hGdS5BWTxGkHAeGd6wgo0oi8BkncdSOAhKmOrGYk1EPyMiP+siX1kTiGpFB3oUkLl+K8H6gf3jQkOVy76dlv47gV3fgot1HQ6gjDVfgI+fKD/O/zqhg+JGDaYZvNZvvT3nTASOPlTZrmAAAAAElFTkSuQmCC","orcid":"","institution":"Second Affiliated Hospital of Zhejiang University","correspondingAuthor":true,"prefix":"","firstName":"Jiali","middleName":"","lastName":"Pu","suffix":""}],"badges":[],"createdAt":"2026-03-11 06:25:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9090665/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9090665/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104999030,"identity":"d41dfac4-eca6-45d3-b7c0-cde3561874ed","added_by":"auto","created_at":"2026-03-19 16:32:35","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1321477,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePD-1 blockade exacerbated dopaminergic neuronal damage and motor dysfunction in PD mice, whereas PD-1 activation exerted a protective effect. (A) \u003c/strong\u003eTime schedules of the experimental design. Black arrows indicate time points of drug treatment. Time points of behavioral testing are also indicated. \u003cstrong\u003e(B)\u003c/strong\u003e Representative images of the open-field and rotarod test in control, MPTP, anti-PD-1-treated and PD-L1 Fc-treated group. \u003cstrong\u003e(C)\u003c/strong\u003eQuantification of the total traveled distance in the open-field test (n=9–10) and the latency to falling in the rotarod test (n=9-10) in the different treatment groups. \u003cstrong\u003e(D)\u003c/strong\u003e Immunoblot images showing TH levels in the striatum of control, MPTP, anti-PD-1-treated and PD-L1 Fc-treated mice. \u003cstrong\u003ee\u003c/strong\u003eRepresentative immunoblot bands of TH. \u003cstrong\u003e(E)\u003c/strong\u003e and densitometric quantification of TH (n=6). \u003cstrong\u003e(F)\u003c/strong\u003e Representative immunofluorescence staining of TH-positive neurons in the striatum and SN compacta (SNc) in the different treatment groups. White dotted circles indicate the SNc regions. Scale bar: 200 µm. \u003cstrong\u003e(G)\u003c/strong\u003e Quantification of the TH intensity in the striatum and TH-positive cell numbers in SNc in the different treatment groups (n=5). Data are represented as mean ± SEM. *p \u0026lt; 0.05; **p \u0026lt; 0.01; ***p \u0026lt; 0.001. One-way ANOVA with LSD post-hoc test was performed.\u003c/p\u003e","description":"","filename":"Figures21.png","url":"https://assets-eu.researchsquare.com/files/rs-9090665/v1/801906e0b77c01a0d555c8b4.png"},{"id":105035778,"identity":"afca7956-749f-4f44-bece-a03f417c90c0","added_by":"auto","created_at":"2026-03-20 07:26:37","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1512923,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePD-1 blockade compromised blood-brain barrier integrity, while PD-1 activation restored barrier function in PD mice. (A-B) \u003c/strong\u003eWestern blot showed the expressions of ZO-1, occludin, and claudin-5 of striatum and substantia nigra (SN) in control, MPTP, anti-PD-1-treated and PD-L1 Fc-treated group. \u003cstrong\u003e(C)\u003c/strong\u003e Quantification of ZO-1, occludin, and claudin-5 levels of striatum in the different treatment groups (n=6). \u003cstrong\u003e(D)\u003c/strong\u003e Quantification of the indicated proteins levels of SN in the different treatment groups (n=6). \u003cstrong\u003e(E)\u003c/strong\u003e Immunofluorescence staining of ZO-1 and CD31 expression in blood vessels in SNc. Scale bar: 20 µm. \u003cstrong\u003e(F)\u003c/strong\u003e Immunofluorescence staining of claudin-5 and CD31 expression in blood vessels in SN. Scale bar: 20 µm. \u003cstrong\u003e(G)\u003c/strong\u003e RT-qPCR were performed to determine ZO-1, occludin, claudin-5, and CD31 expression levels of mice SN in the different treatment groups (n=3). Data are represented as mean ± SEM. *p \u0026lt; 0.05; **p \u0026lt; 0.01; ***p \u0026lt; 0.001. One-way ANOVA with LSD post-hoc test was performed.\u003c/p\u003e","description":"","filename":"Figures22.png","url":"https://assets-eu.researchsquare.com/files/rs-9090665/v1/81d9298aa0984a507ecbbc37.png"},{"id":105035726,"identity":"609dce3b-972f-49a3-bf75-bfe59a4e8f09","added_by":"auto","created_at":"2026-03-20 07:26:32","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":827601,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePD-1 blockade enhanced cerebral CD4\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+ \u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eT cells infiltration and Th1/Treg differentiation, while PD-1 activation reversed these effects. (A) \u003c/strong\u003eRepresentative flow cytometry plots of CD4\u003csup\u003e\u003cstrong\u003e+ \u003c/strong\u003e\u003c/sup\u003eT and CD8\u003csup\u003e\u003cstrong\u003e+ \u003c/strong\u003e\u003c/sup\u003eT cells in PD mouse brain after anti-PD-1 or PD-L1 Fc treatment. \u003cstrong\u003e(B)\u003c/strong\u003e Quantify the population frequency of CD4\u003csup\u003e\u003cstrong\u003e+ \u003c/strong\u003e\u003c/sup\u003eT and CD8\u003csup\u003e\u003cstrong\u003e+ \u003c/strong\u003e\u003c/sup\u003eT cells in the different treatment groups (n=5). \u003cstrong\u003e(C)\u003c/strong\u003e Western blot showed the expressions of CD4 of striatum in control, MPTP, anti-PD-1-treated and PD-L1 Fc-treated group. \u003cstrong\u003e(D)\u003c/strong\u003e Quantification of CD4 levels of striatum in the different treatment groups (n=6). \u003cstrong\u003e(E)\u003c/strong\u003e Representative immunofluorescence staining of CD4-positive cells in the SNc in four groups. Scale bar: 50 µm. \u003cstrong\u003e(F)\u003c/strong\u003e Quantification of CD4-positive cells in the SNc after anti-PD-1 or PD-L1 Fc treatment (n=5). \u003cstrong\u003e(G)\u003c/strong\u003e Representative flow plots of the CD4\u003csup\u003e\u003cstrong\u003e+ \u003c/strong\u003e\u003c/sup\u003eT cell subsets, including Th1, Th2, Th17, and Tregs, identified by cytokines IFN-γ, IL-4, IL-17a, and IL-10 in the different treatment groups. \u003cstrong\u003e(H)\u003c/strong\u003e Quantification of population frequency of CD4\u003csup\u003e+ \u003c/sup\u003eT cell subsets in control, MPTP, anti-PD-1-treated and PD-L1 Fc-treated group (n=5). Data are represented as mean ± SEM. *p \u0026lt; 0.05; **p \u0026lt; 0.01; ***p \u0026lt; 0.001. One-way ANOVA with LSD post-hoc test was performed.\u003c/p\u003e","description":"","filename":"Figures23.png","url":"https://assets-eu.researchsquare.com/files/rs-9090665/v1/b3f93872c3b213d4282cdf61.png"},{"id":104999031,"identity":"28517ad8-6b03-414b-84d7-262ecd7a4162","added_by":"auto","created_at":"2026-03-19 16:32:35","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":658064,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePD-1 signaling modulates Th1 and Tregs differentiation in MPTP induced PD mice.\u003c/strong\u003e \u003cstrong\u003e(A) \u003c/strong\u003eRepresentative immunoblot bands of PD-1 and PD-L1 in control, MPTP, anti-PD-1-treated and PD-L1 Fc-treated group. \u003cstrong\u003e(B)\u003c/strong\u003e Quantification of PD-1 and PD-L1 levels of striatum in the different treatment groups (n=6). \u003cstrong\u003e(C)\u003c/strong\u003e Representative flow cytometry plots of PD-1 and PD-L1 on cerebral CD4\u003csup\u003e\u003cstrong\u003e+ \u003c/strong\u003e\u003c/sup\u003eT cells after anti-PD-1 or PD-L1 Fc treatment. \u003cstrong\u003e(D)\u003c/strong\u003e Quantification of population frequency of PD-1 and PD-L1 on CD4\u003csup\u003e\u003cstrong\u003e+ \u003c/strong\u003e\u003c/sup\u003eT cells in the different treatment groups (n=5). \u003cstrong\u003e(E)\u003c/strong\u003e Correlation between Th1/2/17/Reg cytokines IFN-γ, IL-4, IL-17a, and IL-10 and PD-1/PD-L1 flow cytometry results. Data are represented as mean ± SEM. *p \u0026lt; 0.05; **p \u0026lt; 0.01; ***p \u0026lt; 0.001. One-way ANOVA with LSD post-hoc test was performed.\u003c/p\u003e","description":"","filename":"Figures24.png","url":"https://assets-eu.researchsquare.com/files/rs-9090665/v1/636d19c27b2f8b425bd231f7.png"},{"id":104999034,"identity":"cc7e1c6c-cd0f-416e-8609-efdb99ec851e","added_by":"auto","created_at":"2026-03-19 16:32:35","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":909571,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePD-1 blockade potentiates Th1 commitment while attenuating Treg differentiation, whereas PD-1 activation restores Th1/Treg homeostasis in vitro. (A)\u003c/strong\u003e Experimental workflow from obtaining single cell suspension from spleen of C57BL/6 mice to the magnetic sorting of CD4\u003csup\u003e+ \u003c/sup\u003eT cells. \u003cstrong\u003e(B)\u003c/strong\u003e The sorting efficiency of magnetically isolated of CD4\u003csup\u003e+ \u003c/sup\u003eT cells. \u003cstrong\u003e(C)\u003c/strong\u003e Representative flow cytometry plots of Th1 and Tregs after anti-PD-1 or PD-L1 Fc treatment. \u003cstrong\u003e(D)\u003c/strong\u003e Quantification of population frequency of Th1 and Tregs in the different treatment groups (n=3). \u003cstrong\u003e(E)\u003c/strong\u003e enzyme-linked immunosorbent assay (ELISA) showed Th1/Treg cytokines IFN-γ and IL-10 levels in culture supernatants in the different treatment groups (n=3). Data are represented as mean ± SEM. *p \u0026lt; 0.05; **p \u0026lt; 0.01; ***p \u0026lt; 0.001. One-way ANOVA with LSD post-hoc test was performed.\u003c/p\u003e","description":"","filename":"Figures25.png","url":"https://assets-eu.researchsquare.com/files/rs-9090665/v1/62bbd7e5e99ecf8c0bcfdc70.png"},{"id":104999035,"identity":"66c9d9f3-683a-4286-b66a-aae49d8018e9","added_by":"auto","created_at":"2026-03-19 16:32:35","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":880193,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePD-1 blockade leads to dysregulated CD4⁺ T cell differentiation via AKT/GSK3β pathway.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Experimental workflow flow cytometry sorting brain CD4\u003csup\u003e+ \u003c/sup\u003eT cells from MPTP-induced PD mice that received PD-1/PD-L1 immunotherapy for Smart-seq. \u003cstrong\u003e(B)\u003c/strong\u003e Gene Ontology (GO) enrichment analysis revealed that PD-1/PD-L1 signaling is implicated in following biological processes (BP), including monatomic ion transmembrane transport, cell differentiation, and monatomic ion transport. \u003cstrong\u003e(C) \u003c/strong\u003eKyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis furtherly identified neuroactive ligand-receptor interaction; calcium signaling pathway, and PI3K-AKT signaling pathway as the top three molecular pathways after PD-1/PD-L1 immunotherapy in PD. \u003cstrong\u003e(D)\u003c/strong\u003e Protein levels of p-AKT, AKT, p-GSK3β, and GSK3β in CD4\u003csup\u003e+ \u003c/sup\u003eT cells after PD-1 intervention under MPP+stimulation in vitro. \u003cstrong\u003e(E)\u003c/strong\u003e Quantification of p-AKT, AKT, p-GSK3β, and GSK3β in CD4\u003csup\u003e+ \u003c/sup\u003eT cells in the different treatment groups (n=3). \u003cstrong\u003e(F, G)\u003c/strong\u003e The protein expression and quantification of p-AKT and p-GSK3β in CD4\u003csup\u003e+ \u003c/sup\u003eT cells pretreated with MK2206 (an inhibitor of AKT) and PD-L1 Fc (n=3). \u003cstrong\u003e(H)\u003c/strong\u003e The protein expression and quantification of p-GSK3β in CD4\u003csup\u003e+ \u003c/sup\u003eT cells pretreated with NP-12 (an inhibitor of GSK3β) and PD-L1 Fc (n=3). Data are represented as mean ± SEM. *p \u0026lt; 0.05; **p \u0026lt; 0.01; ***p \u0026lt; 0.001. One-way ANOVA with LSD post-hoc test was performed.\u003c/p\u003e","description":"","filename":"Figures26.png","url":"https://assets-eu.researchsquare.com/files/rs-9090665/v1/b0cb55e033871510ac3f793c.png"},{"id":104999040,"identity":"774f80fb-56d0-4cf6-86ac-e357cf061905","added_by":"auto","created_at":"2026-03-19 16:32:35","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1253963,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDysregulated PD-1 and PD-L1 expression in peripheral blood in PD patients, with distinct levels across clinical subtypes. (A)\u003c/strong\u003e Experimental and analytical workflow from obtaining PBMCs to the CyTOF analysis. \u003cstrong\u003e(B, C)\u003c/strong\u003e Overall expression levels of PD-1 and PD-L1 in PD and healthy controls. \u003cstrong\u003e(D, E)\u003c/strong\u003e Overall expression levels of PD-1 and PD-L1 expression in EOPD patients, LOPD patients and age-matched controls. \u003cstrong\u003e(F, G)\u003c/strong\u003e PD-1 and PD-L1 on CD4\u003csup\u003e+ \u003c/sup\u003eT cells in PD patients at different ages of onset. \u003cstrong\u003e(H-K) \u003c/strong\u003ePD-1 expression on CD4\u003csup\u003e+ \u003c/sup\u003eT cell subsets, including Th1, Th2, Treg and Tfh in different groups. \u003cstrong\u003e(L-O)\u003c/strong\u003e PD-L1 expression on Th1, Th2, Treg and Tfh in EOPD patients, LOPD patients and age-matched controls. \u003cstrong\u003e(P, Q)\u003c/strong\u003e PD-1 and PD-L1 on CD8\u003csup\u003e+ \u003c/sup\u003eT cells in PD patients at different ages of onset. One-way ANOVA with LSD post-hoc test was used to compare EOPD patients, LOPD patients, and age-matched controls; Data are represented as mean ± SEM. *p \u0026lt; 0.05; **p \u0026lt; 0.01; ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figures27.png","url":"https://assets-eu.researchsquare.com/files/rs-9090665/v1/315cb9c8120108c793f88c7f.png"},{"id":104999039,"identity":"cdc06df3-327f-4ffa-bb47-8ce7c2758b7d","added_by":"auto","created_at":"2026-03-19 16:32:35","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":542979,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePD-1 and PD-L1 expression profiles on CD4\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+ \u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eT cells significantly associate with clinical symptoms. (A, B)\u003c/strong\u003e Pearson correlation between CD4\u003csup\u003e+ \u003c/sup\u003eT frequency and UPDRS III score (r=0.513, p=0.042) and UPDRS score (r=0.525, p=0.037). \u003cstrong\u003e(C, D)\u003c/strong\u003e Pearson correlation between CD4\u003csup\u003e+ \u003c/sup\u003eNaive frequency and age (r=−0.500, p=0.049) as well as age onset (r=-0.535, p=0.033). \u003cstrong\u003e(E, F) \u003c/strong\u003ePearson correlation between CD4\u003csup\u003e+ \u003c/sup\u003eTreg frequency and age (r=0.549, p=0.028) as well as age onset (r=0.580, p=0.019). \u003cstrong\u003e(G-I) \u003c/strong\u003ePearson correlation between PD-1 expression and UPDRS III score (r=-0.501, p=0.048), MMSE score (r=0.505, p=0.046) and MoCA score (r=0.575, p=0.020).\u003cstrong\u003e (J, K)\u003c/strong\u003e Pearson correlation between PD-1\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e T and disease progression (r=-0.490, p=0.049), and between PD-L1\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e T and H-Y score (r=-0.502, p=0.047). *p \u0026lt; 0.05; **p \u0026lt; 0.01; ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figures28.png","url":"https://assets-eu.researchsquare.com/files/rs-9090665/v1/53297dfd6be5926be6c641cd.png"},{"id":105036838,"identity":"a70e5730-82cf-46ee-9366-cc51b5d7ff76","added_by":"auto","created_at":"2026-03-20 07:36:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8628632,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9090665/v1/2411b4a2-7f97-478f-8aaa-cfb8fb883836.pdf"},{"id":104999037,"identity":"416e719d-4ae3-4c00-8f91-27cfa274279a","added_by":"auto","created_at":"2026-03-19 16:32:35","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":6376163,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-9090665/v1/7b9d9e3461486ac292065dd3.docx"},{"id":104999033,"identity":"26fac017-ea09-4aa5-b62d-7b4974435b29","added_by":"auto","created_at":"2026-03-19 16:32:35","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":467453,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.tif","url":"https://assets-eu.researchsquare.com/files/rs-9090665/v1/8bda007b99c5c12661a7bfb2.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"PD-1 blockade-induced CD4 + T cell dysregulation triggers dopaminergic neurodegeneration in Parkinson’s disease","fulltext":[{"header":"Introduction","content":"\u003cp\u003eParkinson's disease (PD) is the most common neurodegenerative movement disorder, affecting 1\u0026ndash;2% of people aged over 65 years\u003csup\u003e1\u003c/sup\u003e. Its pathological hallmarks include the progressive loss of dopaminergic (DA) neurons and the accumulation of alpha-synuclein (α-Syn) in the substantia nigra compacta (SNc)\u003csup\u003e2\u003c/sup\u003e. While traditional view holds PD as a neurodegenerative disorder, increasing evidence has highlighted the key role of immune dysregulation in its onset and progression\u003csup\u003e3,4\u003c/sup\u003e. Notably, with the widespread application of immune checkpoint inhibitors (ICIs) in cancer patients, several reports have found ICIs triggered parkinsonism as immune-related adverse events\u003csup\u003e5\u0026ndash;7\u003c/sup\u003e. These clinical findings suggest that immune dysfunction induced by ICIs may be involved in the pathogenesis of PD, but its mechanism remains unclear.\u003c/p\u003e \u003cp\u003eAnti-programmed cell death protein-1 (anti-PD-1), the most widely used ICIs, exerts its anti-tumor efficacy by inducing systemic immune activation, primarily through enhancing T-cell responses\u003csup\u003e8,9\u003c/sup\u003e. Accumulating evidence suggests an important role of PD-1 in the central nervous system (CNS)\u003csup\u003e10\u003c/sup\u003e. PD-1 suppresses the cerebral immune response via CNS-resident immune cells and infiltrating peripheral T cells, while PD-1 blockade exacerbates neuroinflammation by activating these immune cells. Preclinical study reported that dual inhibition of PD-1 and Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) induces DA neuron loss within the SNc and enhances cerebral CD4\u003csup\u003e+\u003c/sup\u003e T cell infiltration in lung tumor mice model\u003csup\u003e11\u003c/sup\u003e. Moreover, Cheng et al. found that PD-1 deficiency exacerbates DA neuron damage and motor deficits in mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a neurotoxin that selectively targets dopaminergic neurons to model PD\u003csup\u003e12\u003c/sup\u003e. These findings highlight the damaging effect of PD-1 blockade on DA neurons, however, it is currently unclear how it triggers immune cell dysfunction and infiltration into the CNS, and further drives DA neuronal degeneration in PD.\u003c/p\u003e \u003cp\u003ePD-1 is mainly expressed on activated T cells and maintains immune homeostasis by regulating their differentiation and function. Notably, CD4⁺ T cell infiltration near DA neuron has been observed from postmortem PD patients and mouse model, which may drive DA neuron degeneration\u003csup\u003e13,14\u003c/sup\u003e. Accumulating evidence further indicates an imbalance among CD4\u003csup\u003e+\u003c/sup\u003e T cell subsets in PD patinets\u003csup\u003e15\u003c/sup\u003e. Specifically, Th1 and Th17 cells exacerbate DA neuron injury by producing IFN-γ and IL-17, whereas Th2 cells and regulatory T cells (Tregs) mitigate inflammation and injury via anti-inflammatory cytokines such as IL-4 and IL-10\u003csup\u003e16,17\u003c/sup\u003e. Moreover, PD-1 is involved in regulating CD4\u003csup\u003e+\u003c/sup\u003e T cell differentiation, as evidenced in cancer and immune-mediated inflammatory diseases like asthma and Crohn's disease\u003csup\u003e18,19\u003c/sup\u003e. In vivo, PD-1 deficiency drives a cytokine shift toward Th1/Th17 profiles and away from Th2 responses. Correspondingly, in vitro studies corroborate that PD-1 ligation suppresses Th1 and Th17 polarization while enhancing Th2 and Treg response\u003csup\u003e20,21\u003c/sup\u003e. Therefore, it remains to be elucidated whether manipulating PD-1 exerts damaging or protective effects on DA neurons through CD4⁺ T cell homeostasis.\u003c/p\u003e \u003cp\u003eHere, we investigated the role and mechanism of PD-1 in PD pathogenesis at multiple levels. We found that PD-1 blockade exacerbates DA neuron damage and motor deficits which also impairing BBB integrity. This permeabilization drove neuroinflammation by enhancing cerebral CD4\u003csup\u003e+\u003c/sup\u003e T cell infiltration and a Th1-biased differentiation, which was mediated by activated AKT/GSK3β pathway in CD4\u003csup\u003e+\u003c/sup\u003e T cells. Strikingly, PD-1 activation reversed the entire pathological cascade in vivo and in vitro. Extending the clinical relevance of these findings, dysregulated PD-1 and PD-L1 expression was observed in peripheral CD4\u003csup\u003e+\u003c/sup\u003e T cells from PD patients, with notable variation across clinical subtypes. Our findings demonstrated that PD-1 blockade-induced CD4\u003csup\u003e+\u003c/sup\u003e T cell dysregulation exacerbates neuroimmune inflammation and neurodegeneration in PD, whereas PD-1 activation exerts a protective effect, providing a novel perspective on immune pathogenesis in PD and presenting a significant, mechanistic advance with translational implications.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003ePD-1 blockade exacerbates dopaminergic neuronal damage and motor dysfunction in PD mice, whereas PD-1 activation exertes a protective effect\u003c/b\u003e \u003c/p\u003e \u003cp\u003eUse of PD-1 inhibitors in cancer patients has led to Parkinsonism (e.g., bradykinesia, tremor) that is alleviated upon supplementation with dopaminergic drugs\u003csup\u003e7,22\u003c/sup\u003e. To further investigate the impact of PD-1 manipulation on PD pathology and behavior, mice were treated intraperitoneally with either an anti-PD-1 antibody (αPD-1, 0.25 mg \u0026times; 2 doses, 3-day interval) to block PD-1, or a recombinant PD-L1 Fc fusion protein (0.25 mg \u0026times; 2 doses, 3-day interval) to activate PD-1 signaling. Four weeks post-injection, a 5-day subacute MPTP model was established. Behavioral function and dopaminergic neuropathology were then evaluated before sacrifice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Compared to controls, MPTP-induced PD mice exhibited significant hypolocomotion in both open-field and rotarod tests. Although not statistically significant, a downward trend was observed in αPD-1-treated PD mice. Notably, PD-L1-Fc treatment surprisingly rescued motor deficits in PD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTyrosine hydroxylase (TH) is a rate-limiting enzyme responsible for dopamine biosynthesis, and its deficiency reflects the loss of dopaminergic neurons and the progression of PD\u003csup\u003e23\u003c/sup\u003e. Western blotting analysis of striatal TH expression showed that compared with PD mice, TH depletion was exacerbated in αPD-1-treated PD mice, while PD-L1 Fc treatment markedly attenuated TH loss (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, E). Immunofluorescence detection of striatal TH intensity corroborated these findings, supporting the neuroregulatory effects of PD-1 immunotherapy. Furthermore, unbiased stereological counting of TH-positive neurons in the SNc demonstrated that αPD-1 blockade exacerbated neuronal loss, reducing TH-positive neurons by approximately 55%, while PD-L1 Fc resulted in a\u0026thinsp;~\u0026thinsp;24% recovery in TH-positive neuron number (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, G). These results demonstrate that PD-1 blockade exacerbates motor deficits and DA neuronal damage in PD mice, whereas PD-1 activation ameliorates both behavioral impairment and neuronal pathology.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePD-1 blockade promotes blood-brain barrier permeability, while PD-1 activation restores barrier function in PD mice.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAnti-PD-1 can uniquely compromise BBB integrity in brain metastases, thereby enhancing the infiltration of peripheral immune cells into the brain and exerting anti-tumor effects\u003csup\u003e24\u003c/sup\u003e. To investigate whether PD-1 influence BBB integrity during dopaminergic neuronal damage in PD, MPTP-induced PD mice were pre-conditioned with αPD-1 or PD-L1 Fc and BBB permeability was assessed. Western blot was performed to measure the levels of tight junction proteins (e.g., ZO-1, claudin-5 and occludin) in the striatum and substantia nigra. These proteins act as structural barriers in the BBB. We found that the tight junction proteins expression level decreased in MPTP mice striatum and SN, which was aggravated following PD-1 blockade. In contrast, PD-L1 Fc treatment rescued its expression by different degrees (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-D). Immunofluorescence analysis revealed lower structural overlap between ZO-1/claudin-5 with blood vessels (labeled with CD31) in αPD-1-treated PD mice than MPTP alone, whereas PD-L1 Fc treatment partially restored this colocalization (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, F). To complement the protein-level analysis, we also assessed the transcriptional levels of tight junction genes across all experimental groups by RT-qPCR. The results showed that PD-1 blockade significantly reduced the expression of tight junction genes in the SN. In contrast, PD-1 activation promoted the recovery of their expression levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). Taken together, these results explicitly indicate that PD-1 blockade aggravates BBB permeability, whereas PD-1 activation rescues the barrier function.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePD-1 blockade enhances cerebral CD4\u003c/b\u003e \u003csup\u003e \u003cb\u003e+\u003c/b\u003e \u003c/sup\u003e \u003cb\u003eT cells infiltration and Th1/Treg differentiation, while PD-1 activation mitigates these effects\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe intracranial efficacy of PD-1 blockade is primarily mediated by activated peripheral T cells that infiltrate the brain parenchyma\u003csup\u003e25\u003c/sup\u003e. Given that PD-1 blockade promotes BBB permeabilization, peripheral immune cells may infiltrate the brain and exert immune responses. To investigate alterations in the brain microenvironment following PD-1 blockade, we assessed brain-infiltrating immune cell populations, including CD4\u003csup\u003e+\u003c/sup\u003e T cells, CD8\u003csup\u003e+\u003c/sup\u003e T cells, microglia and related subsets. Using flow cytometric analysis of the brain, we found that PD-1 blockade significantly increased the infiltration of CD4⁺ T cells into the brains of PD mice, while PD-1 activation reduced CD4⁺ T cells recruitment. In contrast, CD8⁺ T cell infiltration remained unchanged across experimental groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B). Alterations in brain CD4\u003csup\u003e+\u003c/sup\u003e T cell populations were corroborated by both CD4 expression levels in the striatum (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, D) and immunofluorescence quantification of CD4⁺ T cells in the SNc (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, F). The functional regulation of CD4\u003csup\u003e+\u003c/sup\u003e T cells primarily depends on alterations in distinct subset proportions or activity. To further investigate the impact of PD-1 immunotherapy on cerebral CD4\u003csup\u003e+\u003c/sup\u003e T cell subsets in PD, we performed intracellular cytokine staining on CD4\u003csup\u003e+\u003c/sup\u003e T cells and analyzed changes in Th1, Th2, Th17, and Tregs populations using flow cytometry. Compared to controls, MPTP group exhibited a bias toward Th1 cell differentiation along with a reduction in Tregs, leading to a disruption of the Th1/Treg balance. Notably, this Th1/Treg differentiation shift was further aggravated following PD-1 blockade but was partially restored by PD-L1 Fc stimulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG, H).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn addition to investigating the effects of PD-1/PD-L1 signaling on CD4⁺ T cell-mediated adaptive immunity, we also assessed the innate immune response mediated by microglia. Consistent with previous reports, PD mice exhibited significantly elevated expression of the microglial marker Iba1 along with an increase in microglial cell number, suggesting exacerbated neuroinflammation. However, no changes in microglial activation or abundance were observed in PD mice following PD-1 immunotherapy (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA, B). These findings indicate that the PD-1 primarily modulates adaptive immunity through CD4⁺ T cell regulation. Collectively, PD-1 blockade exacerbates the pathology by enhancing both the infiltration and pro-inflammatory differentiation of CD4⁺ T cells in the brains of PD mice, while PD-1 activation attenuates these effects.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePD-1 signaling modulates Th1 and Tregs differentiation in PD mice\u003c/h2\u003e \u003cp\u003eTo elucidate the regulatory role of PD-1 signaling in Th1 and Treg cell differentiation, we further analyzed the expression of PD-1 and its ligand PD-L1 in PD mice and assessed its correlation with Th1 and Treg frequencies. Western blotting showed MPTP group demonstrated a declining trend of PD-1 expression in the striatum compared to controls. PD-1 blockade further reduced PD-1 levels relative to MPTP group, while PD-1 activation modestly elevated PD-1 expression. No significant alterations in PD-L1 expression were detected among the groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B). Flow cytometry analysis of PD-1 and PD-L1 expression on CD4\u003csup\u003e+\u003c/sup\u003e T cells similarly demonstrated that PD-1 blockade reduced PD-1 expression levels while PD-1 activation counteracted this reduction. Notably, PD-L1 expression on CD4\u003csup\u003e+\u003c/sup\u003e T cells exhibited a strikingly parallel trend across the groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, D). Notably, PD-L1 expression on CD4⁺ T cells exhibited a similar trend, showing reduction after αPD-1 blockade and elevation following PD-L1 Fc stimulation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurthermore, we performed correlation analyses between PD-1/PD-L1 expression on CD4\u003csup\u003e+\u003c/sup\u003e T cells and cytokine production (IFN-γ, IL-4, IL-17a, and IL-10) defining Th1, Th2, Th17, and Treg frequencies. Both PD-1 and PD-L1 expression negatively correlated with Th1 frequencies but positively correlated with Treg frequencies. No significant correlation was detected with either Th2 or Th17 subsets (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). These results indicate that PD-1 signaling intensity participates in the regulation of Th1 and Treg differentiation in PD mice.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePD-1 blockade promotes Th1-biased differentiation while decreasing Tregs, whereas PD-1 activation restores Th1/Treg homeostasis in vitro\u003c/b\u003e \u003c/p\u003e \u003cp\u003eNext, we aimed to validate the regulatory role of PD-1 on CD4⁺ T cell immune homeostasis in vitro. To establish optimal MPP⁺ concentrations for inducing changes in PD-1/PD-L1 expression and Th1/Treg polarization in CD4⁺ T cells, we isolated primary CD4⁺ T cells from C57BL/6 mouse spleens via Magnetic-Activated Cell Sorting (MACS). The cells were then stimulated with graded MPP\u003csup\u003e+\u003c/sup\u003e concentrations and analyzed by flow cytometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). MACS yielded CD4\u003csup\u003e+\u003c/sup\u003e T cells with 92.8% purity, satisfying the requirements for subsequent in vitro stimulation assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePrimary CD4\u003csup\u003e+\u003c/sup\u003e T cells were stimulated with αCD3\u0026thinsp;+\u0026thinsp;αCD28 and exposed to MPP\u003csup\u003e+\u003c/sup\u003e at concentrations of 0.25 mM, 0.5 mM, or 1.0 mM for 72h, followed by surface staining and intracellular staining. Flow cytometric analysis revealed that increasing MPP⁺ concentrations elevated IFN-γ expression while reducing Foxp3 expression in CD4⁺ T cells, indicating Th1 polarization and Treg suppression concurrent with PD-1 signaling downregulation. At 1.0 mM MPP\u003csup\u003e+\u003c/sup\u003e stimulation, significant difference in PD-1/PD-L1 expression and Th1/Treg differentiation bias were observed, establishing this concentration as the reference standard for subsequent in vitro modeling (Figure S2A, B).\u003c/p\u003e \u003cp\u003eTo investigate whether PD-1 signaling modulates Th1/Treg differentiation under MPP\u003csup\u003e+\u003c/sup\u003e stimulation, CD4\u003csup\u003e+\u003c/sup\u003e T cells were co-stimulated with αCD3/CD28 and MPP\u003csup\u003e+\u003c/sup\u003e, followed by treatment with either PD-1 blockade or activation to regulate the pathway. After 72 h, cells were harvested for flow cytometric analysis, and culture supernatants were assessed for IFN-γ and IL-10 levels by ELISA. We observed that stimulation with αCD3/CD28 antibodies significantly increased the proportion of Th1 cells, which was further elevated following MPP⁺ stimulation. PD-1 activation reduced Th1 polarization, and this suppression was attenuated upon PD-1 blockade. Moreover, PD-1 activation rescued the loss of Tregs induced by MPP⁺, while PD-1 blockade further reduced the Treg population (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, D). These findings were corroborated by ELISA, which showed corresponding changes in IFN-γ and IL-10 secretion (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Consistent with the in vivo results, these findings reveal that PD-1 blockade exacerbates the Th1/Treg differentiation imbalance under MPP⁺ stimulation in vitro, whereas PD-1 stimulation alleviates this effect.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePD-1 blockade leads to dysregulated CD4⁺ T cell differentiation via AKT/GSK3β pathway.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo further elucidate the mechanism underlying CD4⁺ T cell differentiation imbalance, we isolated CD4⁺ T cells from the brains of mice treated with PD-1 blockade or activation and performed transcriptomic analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Gene Ontology (GO) enrichment analysis revealed that PD-1 signaling is implicated in biological processes (BP) such as monatomic ion transmembrane transport, cell differentiation, and monatomic ion transport (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). In terms of cellular components (CC), the analysis encompassed cell projection, the extracellular matrix, and the cell surface (Figure. S3A). For molecular function (MF), significant terms included calcium ion binding and signal receptor activity (Figure. S3B). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis further identified neuroactive ligand-receptor interaction, calcium signaling pathway, and PI3K-AKT signaling pathway as the top three pathways modulated by PD-1 immunotherapy in PD (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePrevious studies have suggested that glycogen synthase kinase 3β (GSK3β), a downstream effector of AKT, is involved in the CD4\u003csup\u003e+\u003c/sup\u003e T cell differentiation and function, including Th1 and Treg lineages\u003csup\u003e26\u003c/sup\u003e, and PD-1 signaling can regulate immune cell differentiation through the AKT/GSK3β pathway\u003csup\u003e27\u003c/sup\u003e. Integrating our sequencing data with published evidence, we interrogated the role of AKT/GSK3β pathway in PD-1-mediated CD4\u003csup\u003e+\u003c/sup\u003e T cell differentiation. Subsequently, magnetically isolated splenic CD4⁺ T cells were treated with αPD-1 or PD-L1 Fc, then co-stimulated with MPP\u0026thinsp;+\u0026thinsp;in αCD3/CD28-coated plates for 72 hours. Western blot analysis revealed that MPP⁺ stimulation alone reduced phosphorylation levels of AKT (Ser473) and GSK3β (Ser9), whereas PD-L1 Fc co-stimulation increased p-AKT and p-GSK3β expression. Conversely, PD-1 blockade again reduced phosphorylation of both AKT and GSK3β (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD, E). Total AKT and GSK3β protein expression remained comparable across experimental groups.\u003c/p\u003e \u003cp\u003eTo further verify the role of the AKT/GSK3β pathway in PD-1 regulation of CD4\u003csup\u003e+\u003c/sup\u003e T cell differentiation, the cells were pretreated with specific inhibitors of AKT (MK2206) and GSK3β (Tideglusib/NP-12) to block the signaling. Western blot analysis revealed that inhibition of AKT with MK2206 abolished PD-L1 Fc-induced augmentation of phosphorylation in both AKT and GSK3β (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF, G), while GSK3β blockade with NP-12 further increased PD-L1 Fc-mediated phosphorylation of GSK3β (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH). These findings suggest that PD-1 blockade exacerbates CD4⁺ T cell differentiation dysregulation by activating the AKT/GSK3β pathway. Conversely, PD-1 stimulation suppresses this pathway and restores t CD4⁺ T cell homeostasis.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDysregulated PD-1 and PD-L1 expression in peripheral blood in PD patients, with distinct levels across clinical subtypes.\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePrevious studies have revealed distinct peripheral immune profiles in PD patients stratified by age at onset\u003csup\u003e30,31\u003c/sup\u003e. To comprehensively characterize the expression and distribution of PD-1 and PD-L1 on T cells in PD patients across different age-onset subtypes, mass cytometry and CyTOF analysis were used on peripheral blood samples from early-onset PD (EOPD) and late-onset PD (LOPD) cohorts (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). The baseline demographic and clinical characteristics of PD patients and healthy controls are shown in \u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCompared to healthy controls, PD patients overall showed no significant differences in the expression of PD-1 or PD-L1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, C). Intriguingly, reduced PD-1 and PD-L1 levels were observed in EOPD patients, whereas their expression was elevated in LOPD patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD, E). Moreover, a similar pattern was observed for PD-1 and PD-L1 expression specifically on CD4\u003csup\u003e+\u003c/sup\u003e T cells across age-onset subgroups (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF, G). Further phenotyping of CD4\u003csup\u003e+\u003c/sup\u003e T cell subsets revealed that PD-1 expression was significantly reduced on Th1, Th2, and Treg in EOPD patients, but increased on Treg and Tfh in LOPD patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH-K). Similarly, PD-L1 expression is reduced on Th2 and Treg in EOPD patients, while it is increased on Treg in LOPD (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eL-O). No differential expression of PD-1 or PD-L1 was found on CD8\u003csup\u003e+\u003c/sup\u003e T cells among PD subtypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eP, Q). These findings suggest that PD-1/PD-L1 expression profiles differ among PD patients with different age onset, primarily on CD4\u003csup\u003e+\u003c/sup\u003e T cells and their subsets, reflecting the distinct peripheral immune homeostasis.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we demonstrate that PD-1 blockade exacerbates, while PD-1 activation mitigates, dopaminergic neuronal damage and motor deficits in a PD mouse model. Mechanistically, PD-1 blockade promotes blood-brain barrier permeability and drives neuroinflammation by enhancing cerebral CD4\u003csup\u003e+\u003c/sup\u003e T cell infiltration and a Th1-biased differentiation, a process reversible upon PD-1 activation both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro.\u003c/em\u003e We further identify the AKT/GSK3β signaling pathway in CD4\u003csup\u003e+\u003c/sup\u003e T cells as the key mediator of this immunomodulatory effect. Clinically, dysregulated PD-1 and PD-L1 expression was also observed in peripheral CD4\u003csup\u003e+\u003c/sup\u003e T cells from PD patients and varied across clinical subtypes.\u003c/p\u003e \u003cp\u003eImmune checkpoint inhibitors (ICIs) have shown remarkable efficacy in various types of tumors and become the most commonly used cancer immunotherapy. With the widespread clinical application, there have been increasing reports of immune-related adverse events (irAEs) occurring in patients receiving ICIs\u003csup\u003e28\u003c/sup\u003e. Movement disorders (MD) are rare neurological irAEs, accounting for 3%\u0026ndash;5% of cases, and primarily manifest as tremor, rigidity, and parkinsonism\u003csup\u003e29\u003c/sup\u003e. As the most frequently administrated ICIs, anti-PD-1-induced MD (including parkinsonism) account for approximately half of such cases\u003csup\u003e7\u003c/sup\u003e. Preclinical studies have also observed damage to dopaminergic neurons in tumor-bearing mice treated with PD-1 inhibitors\u003csup\u003e11\u003c/sup\u003e. Furthermore, PD-1 deficiency aggravates motor dysfunction and the DA neuronal damage in MPTP Model of PD\u003csup\u003e12\u003c/sup\u003e. Consistent with previous studies, we found that PD-1 blockade exacerbates motor deficits and DA neuronal damage in MPTP-induced PD mice. Notably, we also administered PD-1 activation therapy and discovered that activating this pathway mitigates motor dysfunction and reduce the loss of dopaminergic neurons. This is opposite to the therapeutic effect of PD-1 in tumors, possibly due to disease-specific variations in the immune microenvironment\u003csup\u003e30,31\u003c/sup\u003e. Unlike the immunosuppressive environment in cancer, PD is characterized by immune activation, accompanied by the release of pro-inflammatory cytokines and lymphocyte infiltration\u003csup\u003e32\u003c/sup\u003e. PD-1 blockade would further lead to overactivation of both brain-resident immune cells and peripheral T cells, thereby contributing to the DA neurons degeneration.\u003c/p\u003e \u003cp\u003eWe observed that PD-1 blockade promotes BBB permeability in the substantia nigra of PD mice, leading to increased infiltration of CD4\u003csup\u003e+\u003c/sup\u003e T cells, whereas PD-1 activation restores BBB function and reduces the level of CD4\u003csup\u003e+\u003c/sup\u003e T cells in the substantia nigra. Anti-PD-1 can hardly cross the BBB into the brain, though its intracranial efficacy is mediated largely by activated peripheral T cells that infiltrate the brain parenchyma\u003csup\u003e25,33\u003c/sup\u003e. Recent studies showed that PD-1 blockade promotes BBB permeability, which is mediated by activated CD8⁺ T cells or microglia\u003csup\u003e24,34\u003c/sup\u003e. The BBB function is also regulated by CD4⁺ T cells, through the release of pro-inflammatory cytokines and the recruitment of other immune cells. Under pathological conditions, CD4⁺ T cells accumulate around the BBB, where they release inflammatory factors and induce endothelial cell apoptosis, thereby disrupting the tight junction structure of the BBB and increasing its permeability\u003csup\u003e35\u003c/sup\u003e. Increased BBB permeabilization conversely facilitates the infiltration of peripheral CD4⁺ T cells into the brain, triggering an imbalance in the CNS immune microenvironment and leading to neuronal death. Early pioneering study found that CD4⁺ T cells had invaded the brain in both postmortem human PD specimens and in MPTP mouse model of PD\u003csup\u003e13\u003c/sup\u003e. Further studies found that mice lacking CD4\u003csup\u003e+\u003c/sup\u003e T cells attenuated MPTP-induced DA neuronal death, but not mice lacking CD8\u003csup\u003e+\u003c/sup\u003e T cells. CD4\u003csup\u003e+\u003c/sup\u003e T cells directly participate in the process of DA neuron degeneration by recognizing α-syn antigens, disrupting immune balance, and driving chronic neuroinflammation\u003csup\u003e36\u0026ndash;38\u003c/sup\u003e. Thus, we speculate that the pathological impact of PD-1 blockade on DA neuron mediated by increased cerebral CD4⁺ T cell infiltration via impairing BBB in the substantia nigra.\u003c/p\u003e \u003cp\u003eAnother significant finding is that PD-1 therapy modulates CD4⁺ T cell subset differentiation. Specifically, PD‑1 blockade promotes inflammatory differentiation (enhanced Th1 polarization and diminished Tregs), while PD‑1 activation induces an anti‑inflammatory phenotype (reduced Th1 and expanded Tregs). Most studies have demonstrated elevated frequencies of Th1 cells in the peripheral blood of PD patients as well as in the brains of PD mouse models, which lead to a Th1 bias\u003csup\u003e39,40\u003c/sup\u003e. Adoptive transfer of Th1 cells also significantly enhance MPTP-induced neurotoxicity. Activated Th1 cells significantly increase the production of cytokines such as IFN-γ and TNF-α, which exacerbate neuroinflammation and affect neuronal survival\u003csup\u003e41\u003c/sup\u003e. Additionally, these cytokines may increase BBB permeability, allowing peripheral immune cells and inflammatory mediators into the brain, which further disrupts the neuronal microenvironment\u003csup\u003e42\u003c/sup\u003e. On the other hands, the reduction of Tregs is also observed in animal models of PD. MPTP-treated mice exhibits fewer Tregs, and the adoptive transfer of Tregs reduces the brain\u0026rsquo;s inflammatory response and alleviated neuronal death by suppressing glial activation\u003csup\u003e16,43,44\u003c/sup\u003e. Another study also revealed that the imbalance between Th1 and Tregs plays a detrimental role in PD mice\u003csup\u003e45\u003c/sup\u003e. In our study, dysregulation of CD4⁺ T cell subset differentiation in PD was exacerbated after PD-1 therapy. Previous studies found that PD-1-deficient mice displayed elevated Th1/Th17 and reduced Th2 cytokine responses. Correspondingly, PD-1 ligation limited cytokine production in polarized Th1 and Th17 cells in vitro but slightly enhanced it in Th2 cells\u003csup\u003e20,46\u003c/sup\u003e. However, we did not find significant changes in Th2 and Th17 cells following PD-1 immunotherapy, which may be resulted from the heterogeneity and stage-dependent effects of the disease.\u003c/p\u003e \u003cp\u003eMechanistically, we identified the AKT/GSK3β signaling axis as a key intracellular pathway downstream of PD-1 that regulates CD4⁺ T cell differentiation. Transcriptomic profiling of brain-infiltrating CD4⁺ T cells pointed to alterations in the PI3K-AKT pathway after PD-1 immunotherapy. Subsequent in vitro experiments validated that PD-1 signaling activation augmented phosphorylation of both AKT (Ser473) and its downstream target GSK3β (Ser9), an inhibition signal for GSK3β, in CD4⁺ T cells under MPP⁺ stimulation. This PD-1-induced signaling cascade is both necessary and sufficient to suppress Th1 differentiation and promote Tregs generation, as evidenced by pharmacologic inhibition of the AKT/GSK3β pathway. These findings are consistent with established role of AKT/GSK3β activity in regulating T cells lineage commitment\u003csup\u003e47,48\u003c/sup\u003e. In T cells, active GSK3β generally promotes pro-inflammatory subsets while inhibiting Tregs development. Additionally, GSK3β can modulate T cell activity in breast cancer through its interaction with PD-L1, whereas PD-1 regulates GSK3β activity and tau phosphorylation in AD mouse models\u003csup\u003e49,50\u003c/sup\u003e. Song et al. demonstrated Th1 cytokine IFN-γ could affect the expression of PD-1 in T lymphocytes through the AKT/GSK3β signaling pathway\u003csup\u003e51\u003c/sup\u003e. Hu et al also showed that PD-1 regulates macrophage polarization through the Akt/GSK3β/β-catenin pathway\u003csup\u003e27\u003c/sup\u003e. Our findings establish a critical link between surface checkpoint signaling and intracellular pathways in CD4⁺ T cells, demonstrating that PD-1 regulates CD4\u003csup\u003e+\u003c/sup\u003e T cell differentiation in PD via the AKT/GSK3β axis.\u003c/p\u003e \u003cp\u003eOur study demonstrated that peripheral immune modulation via the PD-1 pathway can significantly influence the central immune environment and disease progression in PD. The central question in neuroimmunology is how peripheral immune signals communicate with the brain\u003csup\u003e52\u003c/sup\u003e. There is complex immune cross-talk between the periphery and the central nervous system. For example, BBB disruption during neuroinflammation enables peripheral T cells to infiltrate the brain directly\u003csup\u003e53\u003c/sup\u003e. In addition, immune factors released by peripherally activated T cells can diffuse into the brain or signal through endothelial cells to recruit and educate resident immune cells\u003csup\u003e54,55\u003c/sup\u003e. Recent study has also shown that peripheral inflammatory signals can reach the brain via the vagus nerve pathway\u003csup\u003e56\u003c/sup\u003e. In this study, we found PD-1 blockade disrupted the BBB, suggesting that the infiltration of peripheral CD4⁺ T cells into the brain and the diffusion of peripheral cytokines may occur concurrently. Therefore, peripheral immune signature likely reflects a parallel and possibly causative process occurring within the brain. The PD-1-low CD4⁺ T cells in the periphery may represent a pathogenic subset with heightened activation capacity and a propensity to infiltrate the brain, where they exacerbate neuroinflammation and drive the loss of DA neurons.\u003c/p\u003e \u003cp\u003eHowever, this study also has several limitations. First, the MPTP-induced mouse model, while classic, does not fully replicate the slow progression and α-synucleinopathy of human PD. Future research should employ models like α-synuclein pre-formed fibrils (PFF). Second, although we linked PD-1 signaling to the AKT/GSK3β pathway in CD4⁺ T cells, the precise molecular intermediates remain unclear. Third, the impact of AKT/GSK3β on the differentiation of CD4⁺ T cells has not been well studied in vivo. Additionally, while the correlation between PD-1/PD-L1 expression and clinical symptoms was observed in PD patients, the small sample size limited the statistical power. Future larger and longitudinal cohort studies are warranted to fully evaluate the clinical translational potential of the PD-1/PD-L1 axis as a therapeutic target and biomarker for PD.\u003c/p\u003e \u003cp\u003eIn summary, we demonstrate the role and mechanism of PD-1 signaling in regulating neuroinflammation and neurodegeneration in PD by both blocking and stimulating PD-1. Specifically, the immune homeostasis of CD4⁺ T cell was identified as a key mediator of this process. Our findings offer a novel perspective on immune pathogenesis in PD and present a significant, mechanistic advance with translational implications.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eAnimals and drugs\u003c/h2\u003e \u003cp\u003eC57BL/6 mice (8 weeks old; 22\u0026ndash;25 g) were used for the experiments. Animals were housed in a centralized location with a 12-h light/dark cycle and the same sleeping condition.\u003c/p\u003e \u003cp\u003eFor the regulation of the PD-1/PD-L1 pathway, we utilized PD-1-specific blocking antibody (αPD-1; rat IgG2a isotype; clone RPM1-14; BIOXCELL) to blockade PD-1/PD-L1 pathway, and recombinant mouse PD-L1 (PD-L1-Fc; Cat. No. CJ89; Novoprotein) to binding to PD-1 receptor activates PD-1/PD-L1 pathway. αPD-1 or PD-L1 were administrated two injections, 0.25 mg at a 3-day interval, to modulate the PD-1/PD-L1 immune pathway in mice.\u003c/p\u003e \u003cp\u003eFour weeks after the last injection, MPTP mouse model was carried out in reference to a protocol of our previous studies. Briefly, MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, Sigma) was intraperitoneally injected at dose of 30 mg/kg/day for consecutive 5 days, and injection of same amount of 0.9% saline was given as control. All mice were euthanized 7 days after the last injection of MPTP or saline. Motor behavior was detected before sacrifice, and flow cytometry, immunofluorescence and western blot experiment were analyzed using postmortem brain tissue.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eBehavioral analyses\u003c/h2\u003e \u003cp\u003eMotor behavior detection, including rotarod test and open-field test, were performed 7 days after the last MPTP or saline injection.\u003c/p\u003e \u003cp\u003eFor the rotarod test, mice were trained to run on an accelerated rotating rod (LE8205; Panlab, Barcelona, Spain) for three consecutive days, prior to the final test. After training, mice were placed on the rotating rod with the rotation speed gradually increased from 4 to 40 rpms within a 5 min period. Te duration that mice remained on the rod until their first slip to the base (also called latency to falling) was recorded. Measurements were averaged over three trials, with at least 1 h of rest.\u003c/p\u003e \u003cp\u003eTo assess general locomotor activity and exploratory behavior, mice were subjected to open-field test. The mice were transported to a testing room 30 min prior to testing for acclimatization and placed in the center of a nontransparent Plexiglas arena under bright lighting for 5 min subsequently. The total distance moved in 5 min was recorded using SMART video tracking software (Smart 3.0; Panlab, Cornell\u0026agrave; de Llobregat, Spain).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eWestern blotting\u003c/h3\u003e\n\u003cp\u003eSamples were lysed using radioimmunoprecipitation assay (RIPA) buffer and proteinase inhibitor (Thermo Fisher Scientific), and protein concentrations were determined using a bicinchoninic acid protein kit (Thermo Fisher Scientific). Protein was separated using 10\u0026ndash;12% sodium dodecyl-sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. After blocking with 5% skim milk or 5% bovine serum albumin (BSA) in 0.1% Tween-20/Tris-buffered saline (TBS-T) for 1 h at RT, the membranes were incubated overnight with the following primary antibodies at 4℃. Then the blots were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature. Immunoblots (IB) were detected using a chemiluminescence reagent (Thermo Fisher Scientific) and analyzed using ImageJ.\u003c/p\u003e \u003cp\u003eIn this study, the membranes were incubated with primary antibodies against TH (1:1,000, AB152, Sigma), CD4 (1:1,000, ab183685, Abcam), PD-1(1:1000, 66220-1-Ig, Proteintech), PD-L1(1:1000, 66248-1-Ig, Proteintech), ZO-1(1:1000, A0659), Occludin(1:1000, A2601), Claudin-5(1:1000, A10207) (Abclonal), AKT (1:1,000, #4691), GSK-3β (1:1,000, #12456), phospho-AKT (1:500, #4060), phospho-GSK-3β Ser9 (1:500, #9323) (Cell Signaling Technology) and GAPDH (1:2000, Proteintech); secondary antibodies against goat anti-rabbit (1:5,000) or goat anti-mouse (1:5,000) (Proteintech).\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence\u003c/h3\u003e\n\u003cp\u003eMice under complete anesthetic state were perfused with phosphate-buffered saline, and then fixed with 4% paraformaldehyde. The brains were dissected, fixed in 4% PFA solution for 6 h at 4\u0026deg;C, and placed into 30% sucrose solution for dehydrating. Then, the brains were embedded in OCT compound and sliced at thickness of 30 \u0026micro;m. After blocking and permeabilization, brain slices containing substantia nigra and striatum were incubated in the presence of primary antibodies at 4\u0026deg;C overnight. The next day, the brain slices were incubated with secondary antibodies for 1 h at room temperature. After DAPI counter stain, the sections were observed using a confocal microscope (TCS SP8, Leica) and a VS120 virtual slide microscope (DM6B, Leica), and interested images were captured for demonstration representatively. Photomicrographs were analyzed by a blinded investigator using ImageJ or Fiji software.\u003c/p\u003e \u003cp\u003eAntibodies and working dilutions were used in immunohistochemical staining. These primary and secondary antibodies were purchased from Sigma, abcam and Thermo. Primary antibodies included rabbit anti-TH (1:500, AB152; Sigma), CD4 (1:200, ab183685, Abcam), ZO-1 (1:200, A0659, Abclonal), Claudin-5 (1:200, A10207, Abclonal) and CD31 (1:200, R\u0026amp;D, AF3682). Second antibodies included Alexa Fluo488-labeled donkey anti-rabbit IgG (1:500, Thermo).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative reverse transcription PCR (RT-qPCR)\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted with RNAiso Plus (Takara 9108) according to the manufacturer's instructions. 5\u0026times; Prime Script RT master mix (Takara, RR036A) was used for reverse transcription. All primers for RT-qPCR were designed with Primer-BLAST(\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) according to the sequences and are listed in \u003cb\u003eTable S2\u003c/b\u003e. All primers used spanned an exon\u0026ndash;exon junction. The 20 \u0026micro;L reaction volume contained the following components: 10 \u0026micro;L of TB GreenPremix Ex Taq II (Takara, RR420A), 0.8 \u0026micro;L each of forward and reverse primers, 0.4\u0026micro;L ROX Reference Dye, 2 \u0026micro;L of cDNA, and 6 \u0026micro;L of nuclease-free water. The qRT-PCR analyses were carried out using a two-step method with the StepOnePlus Real-Time PCR System (Applied Biosystems), and the cycling parameters were as follows: 95 ℃ for 30 s, 40 cycles of denaturation (95 ℃, 5 s), and annealing (60 ℃, 30 s). All reactions were performed in duplicate or in triplicate with GAPDH as the internal reference, and the relative mRNA expression data were calculated using the 2 \u003csup\u003eΔΔ\u003c/sup\u003eCt method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eMononuclear cell isolation and flow cytometry\u003c/h2\u003e \u003cp\u003eMononuclear cells were isolated from hemibrains of mouse that received PD-1/PD-L1 immunotherapy and MPTP according to previously published methods\u003csup\u003e57\u003c/sup\u003e. Briefly, brain was minced and homogenized using a tissue dissociation tube, followed by centrifugation at 800\u0026times;g for 20 minutes under minimal acceleration and deceleration in 30%/70% Percoll to remove myelin debris. Erythrocytes were lysed using erythrocyte lysis buffer (420301, BioLegend), yielding a single-cell suspension.\u003c/p\u003e \u003cp\u003eSingle-cell suspension was first incubated with an anti-CD16/CD32 antibody to block Fc receptors and reduce nonspecific binding. Cell viability was assessed using fixable viability dye (Zombie AquaTM Fixable Viability Kit, 423101, BioLegend), following the manufacturer's protocol. Then surface staining was performed by adding fluorescent-conjugated antibodies at a 1:200 dilution, and incubating the samples in the dark at 4\u0026deg;C for 30 minutes. The following antibodies were used: CD45 (BV605, 103139, BioLegend), CD11b (PE/Cyanine7, 101215, BioLegend), CD3 (FITC, 100203, BioLegend), CD4 (PerCP/Cyanine5.5, 100539, BioLegend), CD8a (APC/Fire, 100765, BioLegend), CD25 (BV650, 102037, BioLegend), PD-1 (BV421, 135221, BioLegend) and anti-mouse PD-L1 (PE, 153606, BioLegend)\u003c/p\u003e \u003cp\u003eFor intracellular staining of transcription factors, cells were fixed with Transcription Factor Buffer Set (BD Pharmingen) according to manufacturer\u0026rsquo;s instructions. For intracellular cytokine staining, isolated mononuclear cells were stimulated with phorbol myristate acetate (PMA) (50 ng/ml, InvivoGen) and ionomycin (1 ug/ml, InvivoGen) in the presence of monensin (BioLegend) for 4 h at 37 C/5% CO2. The following antibodies were used: IFN-γ (BV785, 505838, BioLegend), IL-4 (PE/Cyanine7, 504117, BioLegend), IL-17A (APC, 506915, BioLegend), IL-10 (PE, 505007, BioLegend), and Foxp3 (PE, 12-5773-82, Invitrogen). Samples were acquired using a flow cytometer (Beckman Coulter, USA), and data were analyzed using FlowJo software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eCD4\u003csup\u003e+\u003c/sup\u003e T cells isolation and culture\u003c/h2\u003e \u003cp\u003eIn order to explore the mechanism of the effect of PD-1/PD-L1 on CD4\u0026thinsp;+\u0026thinsp;T cell differentiation in vitro, splenic CD4\u003csup\u003e+\u003c/sup\u003e T cells were sorted from C57BL/6 mice using CD4\u0026thinsp;+\u0026thinsp;T Cell Isolation Kit for mice (130-104-454; Miltenyi Biotec, Auburn, CA, USA). Sorted T cells were cultured in RPMI 1640 medium, supplemented with 10% FBS and 1% pen/strep (all from Gibco, Shanghai, China) for further study. According to the different groups, CD4\u0026thinsp;+\u0026thinsp;T cells were cultured under αCD3/CD28 (5\u0026micro;g/mL), MPP+ (1.0mmol), αPD-1(5\u0026micro;g/mL) and PD-L1 Fc (5\u0026micro;g/mL) stimulation for 3 days. Subsequently, cells were collected for flow cytometry analysis and western blot assay, and the supernatant was used to detect cytokines by ELISA.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eEnzyme-linked immunosorbent assay (ELISA)\u003c/h2\u003e \u003cp\u003eThe concentration of cytokine in the culture supernatants was determined with mouse IFN Gamma/IFNG ELISA Kit (EK0375, BOSTER) and mouse IL-10 ELISA Kit (EK0417, BOSTER). The steps are carried out according to the manufacturers\u0026rsquo; instruction. Absorbance was measured at 450 nm using a plate reader (BioTek), and 540nm was measured for correction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eCell transcriptome sequencing\u003c/h2\u003e \u003cp\u003eSingle-cell suspensions were extracted from the brain tissues of PD-1/PD-L1 immunotherapy PD mice. Following Fc receptor blocking, fixable viability dye-based viability staining (Zombie AquaTM Fixable Viability Kit, 423101, BioLegend), and surface staining for CD45 (BV605, 103139, BioLegend), CD3 (FITC, 100203, BioLegend) and CD4 (APC, 100515, BioLegend), fluorescence-activated cell sorting (FACS) was performed to isolate CD4\u003csup\u003e+\u003c/sup\u003e T cells for Switching Mechanism at the 5' end of RNA Template Sequencing (SMART-Seq2).\u003c/p\u003e \u003cp\u003eSMARTer cDNA synthesis starts with picogram amounts of total RNA or single cell/several cells. A modified oligo(dT) primer (the SMART CDS Primer) primes the first-strand synthesis reaction. When SMARTScribeTM Reverse Transcriptase reaches the 5' end of the mRNA, the enzyme's terminal transferase activity adds a few additional nucleotides to the 3' end of the cDNA. The carefully-designed SMARTer Oligonucleotide base-pairs with the non-template nucleotide stretch, creating an extended template to enable SMARTScribe RT continue replicating to the end of the oligonucleotide. The resulting full-length, single-stranded (ss) cDNA contains the complete 5' end of the mRNA, as well as sequences that are complementary to the SMARTer Oligonucleotide. Amplify sscDNA by LD PCR and get enough dscDNA for library construction.\u003c/p\u003e \u003cp\u003ecDNA was fragmented by dsDNA Fragmentase (NEB, M0348S) by incubate at 37℃ for 30min. Library construction begins with fragmented cDNA. Blunt-end DNA fragments are generated using a combination of fill-in reactions and exonuclease activity, and size selection is performed with provided sample purification beads. An A-base is then added to the blunt ends of each strand, indexed Y adapters are ligated to the fragments, and the ligated products are amplified with PCR. And then we performed the paired-end sequencing on an Illumina NovaseqTM 6000 at the (LC Sceiences, USA) following the vendor's recommended protocol.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eHuman samples\u003c/h2\u003e \u003cp\u003eOur previous studies have found that the peripheral immune status of PD patients at different ages of onset is different\u003csup\u003e58,59\u003c/sup\u003e. Thus, peripheral blood samples were collected from 10 early-onset PD patients (EOPD; \u0026le;50 years), 12 late-onset PD patients (LOPD; \u0026gt;50 years of age) and age-matched healthy volunteers from the neurology department at the Second Affiliated Hospital of Zhejiang University. PD patients had a diagnosis of PD according to Movement Disorder Society (MDS) criteria\u003csup\u003e60\u003c/sup\u003e. The study has excluded subjects suffering from immune-related diseases or using immune-related drugs. Clinical characteristics including the age of onset, duration of disease, Hoehn and Yahr (H-Y) stage, UPDRS, MMSE and MoCA scores were collected. All of the study participants provided informed consent.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eMass cytometry and CyTOF analysis\u003c/h2\u003e \u003cp\u003eThe blood samples were transferred to a 50 ml centrifuge tube, 10 ml of Ficoll separation solution (GE Healthcare) was added, and the centrifuge tube was placed in a centrifuge with a plate rotor (Avanti J-15R, Beckman), centrifuged at 400 x g for 15 min, and the white layer was transferred to a new 50 ml centrifuge tube to obtain a PBMC initial extract. PBMC suspension obtained after cell lysis using 1 ml ammonium-chloride\u0026ndash;potassium (ACK). Cells were washed once with PBS and incubated in Fc receptor blocking solution before staining with surface antibodies cocktail for 30 min on ice. After fixation in intercalation solution (Maxpar Fix and Perm Buffer containing 250 nM 191/193 Ir, Fluidigm) overnight, cells were washed once with the FACS buffer and then stained with intracellular antibodies cocktail for 30 min on ice. Cells were washed and resuspended with deionized water, adding to 20% EQ beads (Fluidigm), acquired on a mass cytometer (Helios, Fluidigm)\u003csup\u003e61\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCyTOF analysis was performed according to previously reported methods\u003csup\u003e62\u003c/sup\u003e. Data of each sample were debarcoded from raw data with unique mass-tagged barcodes using a doublet-filtering scheme.The beads normalization method was used to normalize FCS files generated by different batches. Data were manually gated using the FlowJo software to exclude debris, dead cells, and doublets, leaving live, single immune cells. Cells were partitioned based on marker expression level using the X-shift clustering algorithm. Cell types of each cluster were annotated according to the marker expression pattern on a heatmap of cluster versus marker. Then, T-distributed stochastic neighbor embedding (t-SNE), a dimensionality reduction algorithm, was used to visualize high-dimensional data in two dimensions and to show the distribution of each cluster and differences between groups.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed using GraphPad Prism 8.2.1 (Graph Pad Software, San Diego, CA, USA) and SPSS 24.0 (IBM Corp., Armonk, NY, USA). The Shapiro-Wilk test was used to test the normality of the distribution of the variables. Student\u0026rsquo;s t-test or two-way analyses of variance (ANOVA) was used to compare means between and within groups, followed by Tukey\u0026rsquo;s post hoc test. Data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM for at least n\u0026thinsp;=\u0026thinsp;3. Statistical significances are indicated as *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":" \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eEthics statement\u003c/h2\u003e \u003cp\u003eAll experiments involving human and animals were conducted according to the ethical policies and procedures approved by the Medical Ethics Committees of the Second Affiliated Hospital of Zhejiang University, China (Approval no. 2020\u0026thinsp;\u0026minus;\u0026thinsp;596 and 2022\u0026thinsp;\u0026minus;\u0026thinsp;0865).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eClinical trial number\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/div\u003e\u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJ.P. conceived the study; J.P., L.G.and X.S. designed the experiments. L.G., X.S. and Y.F. performed the experiments; J.L., M.S., C.C., Y.W. and W.H. collected the data. X.T., Z.L. and N.X. analyzed the data. L.G. and X.S. prepared the figures. J.P. and B.Z. supervised the project. L.G.and X.S. wrote the original manuscript, which was critically reviewed and edited by Y.J., J.T., B.Z., and J.P.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e \u003cp\u003eThis work was supported by the National Natural Science Foundation of China [No.82571419] and the Key Research and Development Program of Zhejiang Pcovince [No. 2025C02113].\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data are available within this article and its Supplementary information.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSu, D. \u003cem\u003eet al.\u003c/em\u003e Projections for prevalence of Parkinson's disease and its driving factors in 195 countries and territories to 2050: modelling study of Global Burden of Disease Study 2021. \u003cem\u003eBMJ (Clinical research ed.)\u003c/em\u003e \u003cb\u003e388\u003c/b\u003e, e080952 (2025).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTanner, C. M. \u0026amp; Ostrem, J. L. Parkinson's Disease. \u003cem\u003eThe New England journal of medicine\u003c/em\u003e \u003cb\u003e391\u003c/b\u003e, 442\u0026ndash;452 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTansey, M. G. \u003cem\u003eet al.\u003c/em\u003e Inflammation and immune dysfunction in Parkinson disease. \u003cem\u003eNature reviews. Immunology\u003c/em\u003e \u003cb\u003e22\u003c/b\u003e, 657\u0026ndash;673 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRoodveldt, C. \u003cem\u003eet al.\u003c/em\u003e The immune system in Parkinson's disease: what we know so far. \u003cem\u003eBrain : a journal of neurology\u003c/em\u003e \u003cb\u003e147\u003c/b\u003e, 3306\u0026ndash;3324 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u0026Ouml;zistanbullu, D., Kaufmann, R. \u0026amp; Meissner, M. Immune checkpoint inhibitor triggered parkinsonism in a patient with metastatic melanoma. \u003cem\u003eJournal der Deutschen Dermatologischen Gesellschaft\u0026thinsp;=\u0026thinsp;Journal of the German Society of Dermatology : JDDG\u003c/em\u003e \u003cb\u003e18\u003c/b\u003e, 884\u0026ndash;885 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePrasertpan, T., Teeyapun, N., Bhidayasiri, R. \u0026amp; Sringean, J. Immune Checkpoint Inhibitor-Associated Striatal Encephalitis Presenting with Subacute Progressive Parkinsonism. \u003cem\u003eMovement disorders clinical practice\u003c/em\u003e \u003cb\u003e10\u003c/b\u003e, S7-s11 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDinoto, A. \u003cem\u003eet al.\u003c/em\u003e Autoimmune Movement Disorders Complicating Treatment with Immune Checkpoint Inhibitors. \u003cem\u003eMovement disorders clinical practice\u003c/em\u003e \u003cb\u003e11\u003c/b\u003e, 543\u0026ndash;549 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChamoto, K., Yaguchi, T., Tajima, M. \u0026amp; Honjo, T. Insights from a 30-year journey: function, regulation and therapeutic modulation of PD1. \u003cem\u003eNature reviews. Immunology\u003c/em\u003e \u003cb\u003e23\u003c/b\u003e, 682\u0026ndash;695 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWeiss, S. A. \u003cem\u003eet al.\u003c/em\u003e Epigenetic tuning of PD-1 expression improves exhausted T cell function and viral control. \u003cem\u003eNature immunology\u003c/em\u003e \u003cb\u003e25\u003c/b\u003e, 1871\u0026ndash;1883 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao, J., Roberts, A., Wang, Z., Savage, J. \u0026amp; Ji, R. R. Emerging Role of PD-1 in the Central Nervous System and Brain Diseases. \u003cem\u003eNeuroscience bulletin\u003c/em\u003e \u003cb\u003e37\u003c/b\u003e, 1188\u0026ndash;1202 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDong, L., Zheng, Y. M., Luo, X. G. \u0026amp; He, Z. Y. High Inflammatory Tendency Induced by Malignant Stimulation Through Imbalance of CD28 and CTLA-4/PD-1 Contributes to Dopamine Neuron Injury. \u003cem\u003eJournal of inflammation research\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e, 2471\u0026ndash;2482 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCheng, Y. Y., Chen, B. Y., Bian, G. L., Ding, Y. X. \u0026amp; Chen, L. W. Programmed Death-1 Deficiency Aggravates Motor Dysfunction in MPTP Model of Parkinson's Disease by Inducing Microglial Activation and Neuroinflammation in Mice. \u003cem\u003eMolecular neurobiology\u003c/em\u003e \u003cb\u003e59\u003c/b\u003e, 2642\u0026ndash;2655 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrochard, V. \u003cem\u003eet al.\u003c/em\u003e Infiltration of CD4\u0026thinsp;+\u0026thinsp;lymphocytes into the brain contributes to neurodegeneration in a mouse model of Parkinson disease. \u003cem\u003eThe Journal of clinical investigation\u003c/em\u003e \u003cb\u003e119\u003c/b\u003e, 182\u0026ndash;192 (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZirra, A. \u0026amp; Huxford, B. CD4(+) T Cells Contribute to Neurodegeneration in Lewy Body Dementia. \u003cem\u003eMovement disorders : official journal of the Movement Disorder Society\u003c/em\u003e \u003cb\u003e37\u003c/b\u003e, 268 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun, X., Gu, R. \u0026amp; Bai, J. Differentiation and regulation of CD4(+) T cell subsets in Parkinson's disease. \u003cem\u003eCellular and molecular life sciences : CMLS\u003c/em\u003e \u003cb\u003e81\u003c/b\u003e, 352 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReynolds, A. D. \u003cem\u003eet al.\u003c/em\u003e Regulatory T cells attenuate Th17 cell-mediated nigrostriatal dopaminergic neurodegeneration in a model of Parkinson's disease. \u003cem\u003eJournal of immunology (Baltimore, Md. : 1950)\u003c/em\u003e \u003cb\u003e184\u003c/b\u003e, 2261\u0026ndash;2271 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTahmasebinia, F. \u0026amp; Pourgholaminejad, A. The role of Th17 cells in auto-inflammatory neurological disorders. \u003cem\u003eProgress in neuro-psychopharmacology \u0026amp; biological psychiatry\u003c/em\u003e \u003cb\u003e79\u003c/b\u003e, 408\u0026ndash;416 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSnell, L. M. \u003cem\u003eet al.\u003c/em\u003e Dynamic CD4(+) T cell heterogeneity defines subset-specific suppression and PD-L1-blockade-driven functional restoration in chronic infection. \u003cem\u003eNature immunology\u003c/em\u003e \u003cb\u003e22\u003c/b\u003e, 1524\u0026ndash;1537 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin, X. \u003cem\u003eet al.\u003c/em\u003e Regulatory mechanisms of PD-1/PD-L1 in cancers. \u003cem\u003eMolecular cancer\u003c/em\u003e \u003cb\u003e23\u003c/b\u003e, 108 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcAlees, J. W. \u003cem\u003eet al.\u003c/em\u003e Differential control of CD4(+) T-cell subsets by the PD-1/PD-L1 axis in a mouse model of allergic asthma. \u003cem\u003eEuropean journal of immunology\u003c/em\u003e \u003cb\u003e45\u003c/b\u003e, 1019\u0026ndash;1029 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMa, F., Zhao, M., Song, Z. \u0026amp; Wang, Z. T-bet interferes with PD-1/PD-L1-mediated suppression of CD4(+) T cell inflammation and survival in Crohn's disease. \u003cem\u003eClinical and experimental pharmacology \u0026amp; physiology\u003c/em\u003e \u003cb\u003e46\u003c/b\u003e, 798\u0026ndash;805 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZimmer, L. \u003cem\u003eet al.\u003c/em\u003e Neurological, respiratory, musculoskeletal, cardiac and ocular side-effects of anti-PD-1 therapy. \u003cem\u003eEuropean journal of cancer (Oxford, England : 1990)\u003c/em\u003e \u003cb\u003e60\u003c/b\u003e, 210\u0026ndash;225 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHaavik, J. \u0026amp; Toska, K. Tyrosine hydroxylase and Parkinson's disease. \u003cem\u003eMolecular neurobiology\u003c/em\u003e \u003cb\u003e16\u003c/b\u003e, 285\u0026ndash;309 (1998).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDeo, A. \u003cem\u003eet al.\u003c/em\u003e PD1 blockade-induced DKK1 expression by CD8\u0026thinsp;+\u0026thinsp;T cells promotes blood-brain barrier permeabilization. \u003cem\u003eCancer discovery\u003c/em\u003e (2026).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePortnow, J. \u003cem\u003eet al.\u003c/em\u003e Systemic Anti-PD-1 Immunotherapy Results in PD-1 Blockade on T Cells in the Cerebrospinal Fluid. \u003cem\u003eJAMA oncology\u003c/em\u003e \u003cb\u003e6\u003c/b\u003e, 1947\u0026ndash;1951 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXia, Y. \u003cem\u003eet al.\u003c/em\u003e Glycogen synthase kinase 3β inhibition promotes human iTreg differentiation and suppressive function. \u003cem\u003eImmunologic research\u003c/em\u003e \u003cb\u003e62\u003c/b\u003e, 60\u0026ndash;70 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHu, X. H. \u003cem\u003eet al.\u003c/em\u003e A newly intervention strategy in preeclampsia: Targeting PD-1/Tim-3 signaling pathways to modulate the polarization of decidual macrophages. \u003cem\u003eFASEB journal : official publication of the Federation of American Societies for Experimental Biology\u003c/em\u003e \u003cb\u003e36\u003c/b\u003e, e22073 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFletcher, K. \u0026amp; Johnson, D. B. Chronic immune-related adverse events arising from immune checkpoint inhibitors: an update. \u003cem\u003eJournal for immunotherapy of cancer\u003c/em\u003e \u003cb\u003e12\u003c/b\u003e (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFonseca, E. \u003cem\u003eet al.\u003c/em\u003e Neurological adverse events related to immune-checkpoint inhibitors in Spain: a retrospective cohort study. \u003cem\u003eThe Lancet. Neurology\u003c/em\u003e \u003cb\u003e22\u003c/b\u003e, 1150\u0026ndash;1159 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFilippou, P. S. \u0026amp; Outeiro, T. F. Cancer and Parkinson's Disease: Common Targets, Emerging Hopes. \u003cem\u003eMovement disorders : official journal of the Movement Disorder Society\u003c/em\u003e \u003cb\u003e36\u003c/b\u003e, 340\u0026ndash;346 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSurguchov, A. \u0026amp; Surguchev, A. A. Association between Parkinson's Disease and Cancer: New Findings and Possible Mediators. \u003cem\u003eInternational journal of molecular sciences\u003c/em\u003e \u003cb\u003e25\u003c/b\u003e (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHarms, A. S., Ferreira, S. A. \u0026amp; Romero-Ramos, M. Periphery and brain, innate and adaptive immunity in Parkinson's disease. \u003cem\u003eActa neuropathologica\u003c/em\u003e \u003cb\u003e141\u003c/b\u003e, 527\u0026ndash;545 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003evan Bussel, M. T. J., Beijnen, J. H. \u0026amp; Brandsma, D. Intracranial antitumor responses of nivolumab and ipilimumab: a pharmacodynamic and pharmacokinetic perspective, a scoping systematic review. \u003cem\u003eBMC cancer\u003c/em\u003e \u003cb\u003e19\u003c/b\u003e, 519 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVinnakota, J. M. \u003cem\u003eet al.\u003c/em\u003e Anti-PD-1 cancer immunotherapy induces central nervous system immune-related adverse events by microglia activation. \u003cem\u003eScience translational medicine\u003c/em\u003e \u003cb\u003e16\u003c/b\u003e, eadj9672 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShi, S. X. \u003cem\u003eet al.\u003c/em\u003e CD4(+) T cells aggravate hemorrhagic brain injury. \u003cem\u003eScience advances\u003c/em\u003e \u003cb\u003e9\u003c/b\u003e, eabq0712 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu, Z. \u003cem\u003eet al.\u003c/em\u003e Dopamine receptor D2 on CD4(+) T cells is protective against neuroinflammation and neurodegeneration in a mouse model of Parkinson's disease. \u003cem\u003eBrain, behavior, and immunity\u003c/em\u003e \u003cb\u003e98\u003c/b\u003e, 110\u0026ndash;121 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWilliams, G. P. \u003cem\u003eet al.\u003c/em\u003e CD4 T cells mediate brain inflammation and neurodegeneration in a mouse model of Parkinson's disease. \u003cem\u003eBrain : a journal of neurology\u003c/em\u003e \u003cb\u003e144\u003c/b\u003e, 2047\u0026ndash;2059 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKarikari, A. A. \u003cem\u003eet al.\u003c/em\u003e Neurodegeneration by α-synuclein-specific T cells in AAV-A53T-α-synuclein Parkinson's disease mice. \u003cem\u003eBrain, behavior, and immunity\u003c/em\u003e \u003cb\u003e101\u003c/b\u003e, 194\u0026ndash;210 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKustrimovic, N. \u003cem\u003eet al.\u003c/em\u003e Parkinson's disease patients have a complex phenotypic and functional Th1 bias: cross-sectional studies of CD4\u0026thinsp;+\u0026thinsp;Th1/Th2/T17 and Treg in drug-na\u0026iuml;ve and drug-treated patients. \u003cem\u003eJournal of neuroinflammation\u003c/em\u003e \u003cb\u003e15\u003c/b\u003e, 205 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMamula, D., Khosousi, S., He, Y., Lazarevic, V. \u0026amp; Svenningsson, P. Impaired migratory phenotype of CD4(+) T cells in Parkinson's disease. \u003cem\u003eNPJ Parkinson's disease\u003c/em\u003e \u003cb\u003e8\u003c/b\u003e, 171 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBai, X. B. \u003cem\u003eet al.\u003c/em\u003e Oral pathogens exacerbate Parkinson's disease by promoting Th1 cell infiltration in mice. \u003cem\u003eMicrobiome\u003c/em\u003e \u003cb\u003e11\u003c/b\u003e, 254 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMaes, M., Almulla, A. F., You, Z. \u0026amp; Zhang, Y. Neuroimmune, metabolic and oxidative stress pathways in major depressive disorder. \u003cem\u003eNature reviews. Neurology\u003c/em\u003e \u003cb\u003e21\u003c/b\u003e, 473\u0026ndash;489 (2025).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRostami, J. \u003cem\u003eet al.\u003c/em\u003e Astrocytes have the capacity to act as antigen-presenting cells in the Parkinson's disease brain. \u003cem\u003eJournal of neuroinflammation\u003c/em\u003e \u003cb\u003e17\u003c/b\u003e, 119 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReynolds, A. D., Stone, D. K., Mosley, R. L. \u0026amp; Gendelman, H. E. Nitrated {alpha}-synuclein-induced alterations in microglial immunity are regulated by CD4\u0026thinsp;+\u0026thinsp;T cell subsets. \u003cem\u003eJournal of immunology (Baltimore, Md. : 1950)\u003c/em\u003e \u003cb\u003e182\u003c/b\u003e, 4137\u0026ndash;4149 (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi, W., Luo, Y., Xu, H., Ma, Q. \u0026amp; Yao, Q. Imbalance between T helper 1 and regulatory T cells plays a detrimental role in experimental Parkinson's disease in mice. \u003cem\u003eThe Journal of international medical research\u003c/em\u003e \u003cb\u003e49\u003c/b\u003e, 300060521998471 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee, J. \u003cem\u003eet al.\u003c/em\u003e The Multifaceted Role of Th1, Th9, and Th17 Cells in Immune Checkpoint Inhibition Therapy. \u003cem\u003eFrontiers in immunology\u003c/em\u003e \u003cb\u003e12\u003c/b\u003e, 625667 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJi, S. \u003cem\u003eet al.\u003c/em\u003e Mesenchymal stem cell transplantation inhibits abnormal activation of Akt/GSK3β signaling pathway in T cells from systemic lupus erythematosus mice. \u003cem\u003eCellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology\u003c/em\u003e \u003cb\u003e29\u003c/b\u003e, 705\u0026ndash;712 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHong, L. J., Chen, A. J., Li, F. Z., Chen, K. J. \u0026amp; Fang, S. The HSP90 Inhibitor, 17-AAG, Influences the Activation and Proliferation of T Lymphocytes via AKT/GSK3β Signaling in MRL/lpr Mice. \u003cem\u003eDrug design, development and therapy\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e, 4605\u0026ndash;4612 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi, C. W. \u003cem\u003eet al.\u003c/em\u003e Glycosylation and stabilization of programmed death ligand-1 suppresses T-cell activity. \u003cem\u003eNature communications\u003c/em\u003e \u003cb\u003e7\u003c/b\u003e, 12632 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZou, Y. \u003cem\u003eet al.\u003c/em\u003e Programmed Cell Death Protein 1 Blockade Reduces Glycogen Synthase Kinase 3β Activity and Tau Hyperphosphorylation in Alzheimer's Disease Mouse Models. \u003cem\u003eFrontiers in cell and developmental biology\u003c/em\u003e \u003cb\u003e9\u003c/b\u003e, 769229 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSong, M. \u003cem\u003eet al.\u003c/em\u003e IFN-γ decreases PD-1 in T lymphocytes from convalescent COVID-19 patients via the AKT/GSK3β signaling pathway. \u003cem\u003eScientific reports\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e, 5038 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, S. \u003cem\u003eet al.\u003c/em\u003e Peripheral and central neuroimmune mechanisms in Alzheimer's disease pathogenesis. \u003cem\u003eMolecular neurodegeneration\u003c/em\u003e \u003cb\u003e20\u003c/b\u003e, 22 (2025).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSweeney, M. D., Sagare, A. P. \u0026amp; Zlokovic, B. V. Blood-brain barrier breakdown in Alzheimer disease and other neurodegenerative disorders. \u003cem\u003eNature reviews. Neurology\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e, 133\u0026ndash;150 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu, Y. \u003cem\u003eet al.\u003c/em\u003e The reciprocal interactions between microglia and T cells in Parkinson's disease: a double-edged sword. \u003cem\u003eJournal of neuroinflammation\u003c/em\u003e \u003cb\u003e20\u003c/b\u003e, 33 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKedia, S. \u003cem\u003eet al.\u003c/em\u003e T cell-mediated microglial activation triggers myelin pathology in a mouse model of amyloidosis. \u003cem\u003eNature neuroscience\u003c/em\u003e \u003cb\u003e27\u003c/b\u003e, 1468\u0026ndash;1474 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJin, H., Li, M., Jeong, E., Castro-Martinez, F. \u0026amp; Zuker, C. S. A body-brain circuit that regulates body inflammatory responses. \u003cem\u003eNature\u003c/em\u003e \u003cb\u003e630\u003c/b\u003e, 695\u0026ndash;703 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWilliams, G. P. \u003cem\u003eet al.\u003c/em\u003e Targeting of the class II transactivator attenuates inflammation and neurodegeneration in an alpha-synuclein model of Parkinson's disease. \u003cem\u003eJournal of neuroinflammation\u003c/em\u003e \u003cb\u003e15\u003c/b\u003e, 244 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTian, J. \u003cem\u003eet al.\u003c/em\u003e Specific immune status in Parkinson's disease at different ages of onset. \u003cem\u003eNPJ Parkinson's disease\u003c/em\u003e \u003cb\u003e8\u003c/b\u003e, 5 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiang, S. S. \u003cem\u003eet al.\u003c/em\u003e Cytokine and chemokine map of peripheral specific immune cell subsets in Parkinson's disease. \u003cem\u003eNPJ Parkinson's disease\u003c/em\u003e \u003cb\u003e9\u003c/b\u003e, 117 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePostuma, R. B. \u003cem\u003eet al.\u003c/em\u003e MDS clinical diagnostic criteria for Parkinson's disease. \u003cem\u003eMovement disorders : official journal of the Movement Disorder Society\u003c/em\u003e \u003cb\u003e30\u003c/b\u003e, 1591\u0026ndash;1601 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbrecht, C. \u003cem\u003eet al.\u003c/em\u003e Simplified mass cytometry protocol for in-plate staining, barcoding, and cryopreservation of human PBMC samples in clinical trials. \u003cem\u003eSTAR protocols\u003c/em\u003e \u003cb\u003e3\u003c/b\u003e, 101362 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEsaulova, E. \u003cem\u003eet al.\u003c/em\u003e Single-cell RNA-seq analysis of human CSF microglia and myeloid cells in neuroinflammation. \u003cem\u003eNeurology(R) neuroimmunology \u0026amp; neuroinflammation\u003c/em\u003e \u003cb\u003e7\u003c/b\u003e (2020).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-neuroinflammation","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jneu","sideBox":"Learn more about [Journal of Neuroinflammation](http://jneuroinflammation.biomedcentral.com)","snPcode":"12974","submissionUrl":"https://submission.nature.com/new-submission/12974/3","title":"Journal of Neuroinflammation","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-9090665/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9090665/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWith the widespread use of PD-1 inhibitors in the treatment of cancer patients, emerging clinical evidence has linked them to the onset of parkinsonism, raising critical questions about the role of PD-1 in Parkinson\u0026rsquo;s disease (PD) pathogenesis\u0026mdash;a connection previously unexplored. Here, we found that PD-1 blockade aggravated, while its activation mitigated, dopaminergic neuronal damage and motor deficits in PD mouse models. Immunofluorescence and flow cytometry analyses revealed that PD-1 blockade promoted blood-brain barrier (BBB) permeabilization and drove neuroinflammation by enhancing cerebral infiltration of CD4\u003csup\u003e+\u003c/sup\u003e T cell and a Th1-biased differentiation. Notably, these effects were reversed by PD-1 activation both in vivo and in vitro. We further identify the AKT/GSK3β signaling pathway in CD4\u003csup\u003e+\u003c/sup\u003e T cells as the central mediator of this immunomodulatory effect. Clinically relevance demonstrated by dysregulated PD-1 and PD-L1 expression in peripheral CD4\u003csup\u003e+\u003c/sup\u003e T cells from PD patients, with notable variation across clinical subtypes. Together, these findings demonstrated that PD-1 blockade-induced CD4\u003csup\u003e+\u003c/sup\u003e T cell dysregulation exacerbates neuroinflammation and neurodegeneration in PD, providing a novel perspective on immune pathogenesis in PD and presenting a significant, mechanistic advance with translational implications.\u003c/p\u003e","manuscriptTitle":"PD-1 blockade-induced CD4 + T cell dysregulation triggers dopaminergic neurodegeneration in Parkinson’s disease","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-19 16:32:30","doi":"10.21203/rs.3.rs-9090665/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-02T18:10:56+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-02T15:12:41+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-02T11:13:40+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-31T22:58:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"299855748567674194116859092090751511870","date":"2026-03-19T22:12:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"179799486615681866573824373093493791074","date":"2026-03-19T13:17:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"40251958223452693743506470753030529799","date":"2026-03-17T13:47:52+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-17T13:07:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-17T05:27:11+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-17T02:25:15+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Neuroinflammation","date":"2026-03-11T06:18:16+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-neuroinflammation","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jneu","sideBox":"Learn more about [Journal of Neuroinflammation](http://jneuroinflammation.biomedcentral.com)","snPcode":"12974","submissionUrl":"https://submission.nature.com/new-submission/12974/3","title":"Journal of Neuroinflammation","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e3a3f224-973b-4396-9f17-9a506136c3b1","owner":[],"postedDate":"March 19th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-05-18T17:38:44+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-19 16:32:30","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9090665","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9090665","identity":"rs-9090665","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}