IL-2/IL-2Rβγ signaling in pruriceptors drives neuroimmune mechanisms of nivolumab- induced persistent itch

IL-2/IL-2Rβγ signaling in pruriceptors drives neuroimmune mechanisms of nivolumab- induced persistent itch | 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 IL-2/IL-2Rβγ signaling in pruriceptors drives neuroimmune mechanisms of nivolumab- induced persistent itch Lixuan Li, Huijuan Zhao, Jing Guo, Huaizhi Li, Aiping Li, Xiyuan Ba, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7654982/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Dec, 2025 Read the published version in Journal of Neuroinflammation → Version 1 posted 11 You are reading this latest preprint version Abstract Background Immune checkpoint inhibitor (ICI) therapy frequently induces pruritus as a cutaneous immune-related adverse event, affecting 13–25% of patients treated with anti–PD-1 antibodies. Unlike allergy-associated itch, ICI-induced pruritus often responds poorly to antihistamines, indicating a distinct mechanism. This study aimed to investigate the mechanisms by which repeated PD-1 blockade induces persistent itch and to identify molecular pathways linking immune activation with pruriceptor sensitization. Methods We established a mouse model of pruritus by repeated administration of Nivolumab subcutaneously. Behavioral assays were conducted to evaluate itch-like behaviors (scratching). The expression and distribution of IL-2 receptor subunits in dorsal root ganglia (DRG) were assessed using qPCR, RNAscope, and Western blotting. Electrophysiological recordings, fluorescent antibody labeling, immunostaining, and pharmacological interventions were employed to explore the cellular and molecular mechanisms. Results A single Nivolumab injection induced transient scratching, whereas three consecutive injections triggered persistent itch lasting about one week beyond drug withdrawal. Persistent itch was accompanied by dermal CD4⁺ T-cell infiltration and elevated serum IL-2. Neutralization of IL-2 abolished persistent but not transient itch. In DRG, repeated Nivolumab selectively upregulated IL-2 receptor β and γ subunits, localized predominantly to MrgprA3⁺ pruriceptors. These neurons exhibited enhanced excitability, c-Fos induction, and direct Nivolumab binding. Mechanistically, PD-1 blockade suppressed SHP-1 phosphorylation, promoted JNK and STAT5 activation, and drove IL-2Rβ/γ upregulation. JNK inhibition prevented IL-2R induction and alleviated persistent itch without affecting acute responses. Conclusion Our findings demonstrate that repeated Nivolumab administration upregulates IL-2Rβ/γ in MrgprA3⁺ neurons via SHP-1–JNK–STAT5 signaling, together with elevated systemic IL-2, establishing a neuroimmune loop that drives ICI-induced pruritus. PD-1 blockade Nivolumab itch Interleukin-2 (IL-2) IL-2 receptor Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Background Immune checkpoint inhibitors (ICIs) targeting PD-1 (e.g., Nivolumab) have revolutionized cancer therapy, markedly improving survival across multiple malignancies. However, their clinical success is tempered by immune-related adverse events (irAEs). Among the most common irAEs are cutaneous toxicities, notably chronic pruritus (itch)[ 1 ]. Recent clinical series and meta-analyses suggest that anti–PD–1 therapy frequently provokes pruritus in 13–25% of patients[ 1 , 2 ]. Notably, ICI-related pruritus is mechanistically distinct from classical allergic or atopic itch, as it often arises without a visible skin rash and is notoriously resistant to antihistamine therapy[ 3 ]. These observations indicate that checkpoint inhibitor–induced pruritus involves non-histaminergic mechanisms. Recent preclinical studies have shown that the intradermal administration of PD-1/PD-L1 antibodies induces acute itch through non-histaminergic pathways involving spinal microglial activation[ 4 ]. However, since ICI-induced pruritus frequently persists throughout treatment and may even continue after withdrawal[ 1 , 2 ], the mechanisms underlying PD-1 antibody–induced chronic itch remain unclear. Previous studies have further demonstrated that PD-1 is functionally and widely expressed on primary sensory neurons in the dorsal root ganglia (DRG), where it regulates neuronal excitability and pain transmission[ 5 – 7 ]. Indeed, a distinct subset of primary sensory neurons in the dorsal root ganglia (DRG) is dedicated to transmitting itch signals, often referred to as pruriceptors. Dysfunction of these neurons is closely associated with the development of chronic itch. Common molecular markers for itch-selective neurons include MrgprA3, Nppb, and the interleukin-31 receptor complex, as well as histamine receptors[ 8 – 10 ]. Among these, MrgprA3⁺ neurons have been established as a key pruriceptive lineage that mediates non-histaminergic itch responses[ 8 ]. Transcriptomic profiling further revealed that MrgprA3⁺ neurons are highly enriched in SHP-1 (Ptpn6), a phosphatase that serves as a critical downstream effector of PD-1 signaling[ 7 , 11 , 12 ]. Within the field of pain research, PD-1 antibody treatment has been demonstrated to modulate nociceptor ion channel activity via SHP-1[ 5 , 7 , 13 ], thereby implicating MrgprA3⁺ neurons as likely contributors to PD-1 antibody–induced chronic pruritus. Given the critical immunoregulatory role of PD-1, PD-1 antibody–induced chronic itch may represent a dual effect, driven not only by neuronal mechanisms but also by the activation of immune cells. PD-1 blockade unleashes T-cell activity, particularly in CD4⁺ helper T cells, leading to robust cytokine production[ 14 ]. Among these, interleukin-2 (IL-2) is a pivotal cytokine induced during T-cell activation[ 15 ]. Although IL-2 is classically considered a T-cell growth factor, accumulating evidence indicates that it can also modulate the nervous system. For example, early studies demonstrated that IL-2 receptors are constitutively expressed in dorsal root ganglion (DRG) neurons and that IL-2 application can alter nociceptive signaling[ 16 ]. Moreover, it is reported that IL-2 can directly induce itch, as demonstrated by a clinical study where a single intradermal injection of recombinant human IL-2 elicited low-intensity local itch[ 17 ]. In line with this, IL-2 is also considered one of the cytokines involved in chronic pruritus[ 18 ]. Importantly, IL-2 receptor (IL-2R) expression is not fixed and can be upregulated in sensory neurons during inflammation or dermatitis[ 8 , 16 , 19 ]. Together, these findings suggest that IL-2/IL-2R signaling may serve as a critical messenger linking immune hyperactivation to neuronal sensitization, thereby driving chronic itch during PD-1 antibody therapy. In this study, we demonstrate that repeated administration of Nivolumab in mice induces persistent itch by directly engaging MrgprA3⁺ pruriceptors and driving IL-2 receptor β/γ upregulation through SHP-1 inhibition and subsequent activation of the JNK–STAT5 pathway. This cascade renders sensory neurons hypersensitive to IL-2 released from activated CD4⁺ T cells, thereby establishing a neuroimmune feedforward loop that perpetuates chronic pruritus. Experimental Section Animals Adult C57BL/6 mice (8–12 weeks) were used for most experiments, purchased from the Medical Experimental Animal Center of Guangdong Province, China. For neuron labeling, we used Trpv1 Cre , and Ai9 mice (JAX #017769 and #007909) to generate TRPV1-reporting mice. Animal experiments were conducted in accordance with the guidelines set by the International Association for the Study of Pain and approved by the Animal Care and Use Committee of Shenzhen University (A202300939). All animals were group-housed in cages with 2 ~ 5 mice under a 12-hour light/dark cycle at a stable temperature (22 ± 1℃) with access to food and water ad libitum. Both male and female mice were included in each group in a sex-matched manner. In all experiments, animals were randomly assigned to each group. The sample size was determined based on our previous studies[ 20 ]. All investigators were blinded to both treatment assignment and outcome assessments. Reagents Nivolumab (Opdivo®) is a human IgG4 monoclonal antibody against PD-1; control human IgG4 was obtained from BioXCell. For single-injection experiments, mice received a single subcutaneous injection (50 µL) of either Nivolumab (10–300 µg) or an equal dose of control IgG4. For repeated-injection experiments, mice were given three subcutaneous injections of Nivolumab (30 µg per dose) or IgG4 at the same site on days 1, 2, and 3. For IL-2 neutralization, anti–mouse IL-2 antibody (JES6-1A12, BioXCell, # BE0043) was administered intraperitoneally (i.p., 20–25 mg/kg), starting 3 days before the first Nivolumab injection and repeated every 3 days for a total of four doses. Control mice received PBS i.p. on the same schedule. For JNK inhibition, the cell-permeant peptide D-JNKI-1 (MCE, #HY-P0069; 10 µg in 5 µL saline) or vehicle (saline) was administered via intrathecal (i.t.) injection. D-JNKI-1 was administered 2 days before the first Nivolumab injection and then every 2 days for a total of five injections. Itch Behavior Assay Mice were habituated in individual observation chambers for 3 days before the experiments and additionally acclimated for 30–60 min before each injection. For transient itch assessment, mice were recorded for 30 min immediately after injection. For persistent itch evaluation, scratching behavior was recorded in 30 min sessions on baseline day 0 and on days 1, 2, 3, 4, 7, 10, and 14 following the initiation of injections. Scratching was defined as a mouse using its hind paw to scratch the neck region, and bouts were summed over each 30 min observation period for individual mice. Real-time quantitative PCR (qPCR) for mRNAs Total RNA was isolated from DRG using Trizol reagent (Invitrogen, Carlsbad, CA, USA). Four DRGs were pooled together for each RNA extraction. Briefly, total RNA (1 µg) was reverse-transcribed by oligo (dT) primer using a HiScript II 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China). The qPCR was performed using an ABI Step-One Plus system (Applied Biosystems, CA, USA) with TB Green Premix Ex Taq II (Takara, Dalian, China) and the primers listed in Table S1 . The qPCR cycling conditions were 95°C for 3 min, followed by 40 cycles of amplification at 95°C for 10 s and 60°C for 30 s. Gapdh was used as a control to normalize differences for mRNA detection. Melt curves were performed after the cycles to ensure the absence of nonspecific products. Quantification was performed by normalizing the Ct (cycle threshold) values to those of Gapdh Ct (mRNA) and analyzed using the 2-ΔΔCT method. Immunofluorescence and RNAscope Mice of both sexes (8–12 weeks old) were deeply anesthetized with isoflurane, followed by transcardial perfusion with PBS and then 4% paraformaldehyde (PFA) containing 1.5% picric acid. For skin immunostaining, tissue at the injection site was shaved, fixed, and cryoprotected before being cryosectioned (12 µm). Sections were incubated overnight at 4°C with anti-CD4 antibody (rabbit mAb, 1:200, Abcam, Cat# ab183685), followed by Alexa Fluor 594–conjugated goat anti-rabbit secondary antibody (1:500, Invitrogen). Nuclei were counterstained with DAPI. Images were captured using a fluorescence microscope at 20× magnification. CD4⁺ T cells were quantified in the dermis as the mean number of red cells per high-power field (0.25 mm²) across 4–5 randomly selected fields per mouse. For DRG immunostaining, cervical DRGs (C5–C8) were dissected, post-fixed for 2 h at room temperature, cryoprotected sequentially in 20% and 30% sucrose, and sectioned at 12 µm thickness. Sections were blocked and incubated overnight at 4°C with anti–c-Fos (rabbit mAb, 1:500, Cell Signaling, clone 9F6, Cat# 2250) and anti-MrgprA3 (goat polyclonal, 1:200, Santa Cruz Biotechnology, Cat# sc-23348), followed by Alexa Fluor 488– and 594–conjugated secondary antibodies (1:500; Invitrogen). For anti-Nivolumab staining, cultured cortical neurons were incubated with Nivolumab (300 ng/mL), post-fixed in 4% PFA for 15 min, and permeabilized with PBS containing 0.3% Triton X-100 for 1 h. Neurons were incubated overnight at 4°C with primary antibodies against PD-1 (rabbit, 1:300, Sigma, Cat# PRS4065) and NeuN (mouse, 1:250, Millipore, Cat# MAB377), followed by Cy3- or Cy5-conjugated secondary antibodies (1:500, Jackson ImmunoResearch) and/or anti-IgG4-FITC (1:200, Abcam, Cat# ab99281). Nuclei were counterstained with DAPI in mounting medium (Sigma, Cat# SLCC8848). Images were acquired with an FV3000 confocal laser scanning microscope (Olympus). In situ hybridization was performed using the RNAscope Multiplex Fluorescent V2 kit (Advanced Cell Diagnostics) according to the manufacturer’s instructions. Commercial probes included Il2rb (Mm-Il2rb, Cat# 502761-C2), Il2rg (Mm-Il2rg, Cat# 445011-C1), and MrgprA3 (Mm-MrgprA3, Cat# 548161-C3). Fluorescent puncta representing target RNA expression were quantified using QuPath software. To visualize neurons in some experiments, sections were counterstained with Nissl (1:200, Invitrogen, Cat# N21483) for 2 h. A neuron was considered positive if three or more fluorescent puncta were detected within the soma. Cell counts were performed in ImageJ using the cell counter plugin, with investigators blinded to experimental conditions. Images were obtained using an FV3000 confocal microscope (Olympus). Each experimental group consisted of five mice. ELISA Blood samples were collected by submandibular venipuncture at designated time points. Serum was isolated and stored at − 80°C until analysis. Interleukin-2 (IL-2) concentrations were quantified using a high-sensitivity IL-2 Quantikine ELISA kit (Sigma-Aldrich, # RAB0287) following the manufacturer’s instructions. Absorbance was measured with a microplate reader, and cytokine concentrations were interpolated from a standard curve. Whole-cell patch clamp recordings in cultured DRG neurons The mouse DRGs were removed aseptically and incubated with collagenase (1.25mg/ml, Roche)/dispase-II (2.4 units/ml, Roche) at 37°C for 90 min, then digested with 0.25% trypsin for 8 min at 37°C, followed by 0.25% trypsin inhibitor. Cells were mechanically dissociated with a flame polished Pasteur pipette in the presence of 0.05% DNase I (Sigma). DRG cells were plated on glass coverslips and grown in a neurobasal defined medium (with 2% B27 supplement, Invitrogen) with 5 mM AraC and 5% carbon dioxide at 36.5°C. DRG neurons were grown for at least 6 hours before use. Whole-cell patch clamp recordings were performed at room temperature using an Axopatch-700B amplifier (Axon Instruments) with a Digidata 1440B (Axon Instruments). The patch pipettes were pulled from borosilicate capillaries (World Precision Instruments, Inc.) using a P-97 Flaming/Brown micropipette puller (Sutter Instrument Co.). The pipette resistance was 4–6 MΩ. The internal pipette solution contains (in mM): 126 K-gluconate,10 NaCl, 1 MgCl 2 , 10 EGTA, 10 HEPES, and 2 Na-ATP (adjusted to pH 7.4 with KOH, Osmolarity 295–300 mOsm), and the extracellular solution contains (in mM): 140 NaCl, 5 KCl, 2 MgCl 2 , 2 CaCl 2 , 10 HEPES, 10 glucose, adjusted to pH 7.4 with KOH. Data were filtered at 2 kHz and digitized at 10 kHz using a data acquisition interface (1440A, Molecular Devices). The liquid junction potential was corrected. The pClamp 10 software (Axon Instruments) was used for signal acquisition and analysis. The TRPV1-positive neurons were chosen for recording in the experiment. The action potentials (APs) evoked by a series of ramp current stimulation (time: 1 sec; current intensity: 100 pA, 200 pA, 300 pA) were recorded. Western blot analysis After isoflurane-induced anesthesia, animals were transcardially perfused with 0.9% NaCl, and the Cervical DRGs (C5–C8) were dissected. The tissues were homogenized in a RIPA lysis buffer containing protease and phosphatase inhibitors (Roche, Basel, Switzerland). Protein concentrations were determined by BCA Protein Assay (Thermo Scientific, Waltham, MA, USA). Protein samples (30 µg) were separated on SDS–PAGE gel and transferred to the PVDF membrane. The membranes were blocked with 5% milk and incubated overnight at 4°C with primary antibodies against: phospho-STAT5 (Tyr694, Cell Signaling #9359, 1:1000), total STAT5 (#94205, 1:1000), phospho-JNK (Thr183/Tyr185, Cell Signaling #4668, 1:500), total JNK (#9252, 1:1000), phospho-SHP-1 (Tyr564, Abcam ab131498, 1:500), total SHP-1 (Cell Signaling #3759, 1:1000), IL-2Rβ (CD122, R&D AF225, 1:500), IL-2Rγ (CD132, Santa Cruz sc-668, 1:200), and β-actin or GAPDH (loading controls, 1:5000). After washing, membranes were incubated with appropriate HRP-conjugated secondary antibodies (1:5000, Jackson Labs) for 1 h. Bands were visualized with ECL substrate (Thermo) on a ChemiDoc imager (Bio-Rad). Specific bands were evaluated by predicted molecular size, and the intensity of selected bands was analyzed by ImageJ software. Phospho-protein signals were normalized to the corresponding total protein, and receptor subunit signals were normalized to GAPDH. Each blot included 3–4 samples per group. Single-cell RNA sequencing analysis Publicly available DRG neurons scRNA-seq data were obtained from the study by Xu et al. [ 21 ]. The raw data were downloaded from the Gene Expression Omnibus (GEO) under accession number GSE167910. We reanalyzed this data using the Seurat package (version 4.0) in R (version 4.1.2), by focusing on Ptpn6 . Statistical Analysis All sample sizes and experimental designs were based on previously published data from our lab and similar experiments in the field[ 20 ]. All data were mean ± SEM, and all statistical tests were conducted using two-tailed hypothesis testing. If only two groups were applied, the Student’s t-test was used. For the single injection of Nivolumab at different doses, one-way ANOVA was used. If the differences were significant, post hoc Bonferroni’s test was applied to compare the differences between the two groups. For behavioral tests, qPCR data, electrophysiological data, and Western blotting data, two-way repeated-measure ANOVA was used. If the differences were significant, post hoc Bonferroni’s test was applied to compare values at different points. All statistical analyses were performed using GraphPad Prism 8 (GraphPad Software, Inc.). The value of P < 0.05 was considered statistically significant. Results Nivolumab provokes acute transient itch with a single dose, but repeated dosing leads to persistent pruritus. As shown in Fig. 1 A, we employed subcutaneous injection to establish the model. Administration of Nivolumab at doses of 30 µg, 100 µg, and 300 µg robustly elicited scratching behavior compared with IgG4 controls (Fig. 1 B). Based on these findings, we selected a dose of 100 µg for subsequent single-injection experiments. Time-course analysis revealed that a single subcutaneous injection of Nivolumab induced scratching behavior that peaked at 15–20 minutes and subsided within ~ 30 minutes (Fig. 1 C). Strikingly, however, daily injections of Nivolumab for three consecutive days produced a persistent itch phenotype. Quantification of scratching bouts within 30-minute observation periods showed that scratching increased during the treatment phase and persisted for nearly one week after the final injection (Fig. 1 D–F). Repeated Nivolumab administration triggers dermal CD4 + T-cell infiltration and systemic IL-2 elevation, whereas a single injection has no such effect. Given reports that ICI-induced skin toxicities are associated with T-cell infiltrates (particularly helper T cells) and elevated IL-2 levels in patients, we examined immune changes in our model. In mice receiving only one Nivolumab injection, there was no significant increase in CD4 + T cell numbers in the skin at the injection site compared to controls, and serum IL-2 concentrations remained at baseline levels (Fig. 2 A–D). In contrast, mice subjected to three daily Nivolumab injections showed a marked accumulation of CD4 + T cells in the dermis (Fig. 2 E,F) alongside a significant rise in circulating IL-2 in the circulation (Fig. 2 G). Neutralization of IL-2 selectively abolishes persistent itch after repeated Nivolumab but does not affect the transient itch from a single dose. To test whether IL-2 is required for Nivolumab-induced persistent itch, we administered an IL-2–neutralizing antibody (JES6-1A12, i.p., 20 mg/kg) or vehicle (PBS) control[ 22 ]. In the transient itch model, JES6-1A12 was given intraperitoneally once, both 3 days before and on the day of subcutaneous injection of IgG4 or Nivolumab. Blocking IL-2 had no effect on the acute scratching response to a single Nivolumab injection. Mice in the PBS + Nivolumab(×1) group exhibited significantly increased scratching compared with the PBS + IgG4(×1) group, and mice in the JES6-1A12 + Nivolumab(×1) group also showed significantly increased scratching compared with the JES6-1A12 + IgG4(×1) group. However, no statistical differences were observed between the PBS + IgG4(×1) and JES6-1A12 + IgG4(×1) groups, or between the PBS + Nivolumab(×1) and JES6-1A12 + Nivolumab(×1) groups. (Fig. 3 A,B). By contrast, IL-2 neutralization profoundly influenced the persistent itch phenotype in the repeated dosing paradigm. JES6-1A12 or PBS was administered every three days starting 3 days before the first subcutaneous injection of IgG4 or Nivolumab and continued until day 14 (Fig. 3 C). In a four-group comparison (PBS + IgG4×3, PBS + Nivolumab×3, JES6-1A12 + IgG4×3, JES6-1A12 + Nivolumab×3), all Nivolumab-treated mice exhibited increased scratching during the 3-day treatment period regardless of IL-2 neutralization, indicating that IL-2 is not essential for acute itch during ongoing Nivolumab exposure (Fig. 3 D). Although scratching behavior in the JES6-1A12 + Nivolumab (×3) group differed from that in the JES6-1A12 + IgG4 (×3) group, JES6-1A12 + Nivolumab(×3) mice displayed markedly reduced overall scratching compared with PBS + Nivolumab(×3), with significant differences at days 4, 7, and 10 after the initial injection (Fig. 3 D). Moreover, PBS + Nivolumab(×3) mice continued to exhibit high scratching counts for many days after treatment relative to PBS + IgG4(×3). Repeated Nivolumab upregulates IL-2 receptor β and γ subunits in dorsal root ganglia (DRG) neurons, particularly in itch-selective MrgprA3 + pruriceptors. The elevation of IL-2 prompted us to investigate the expression of IL-2 receptor (IL-2R) in sensory neurons after repeated Nivolumab treatments. Quantitative PCR analysis of cervical DRG showed that three consecutive Nivolumab injections significantly upregulated Il2rb (encoding IL-2Rβ) and Il2g (encoding IL-2Rγ) transcripts compared with IgG4 controls at days 4 and 7 after the initial injection, with expression returning to control levels by day 10 (Fig. 4 B,D). In contrast, Il2ra (encoding IL-2Rα) expression remained unchanged following Nivolumab treatment at days 1, 4, 7, and 10 after the initial injection (Fig. 4 B), indicating that the intermediate-affinity IL-2Rβ/γ (heterodimer) is specifically upregulated in neurons without induction of the α subunit. Notably, on day 1 after the initial injection, the expression levels of Il2ra , Il2rb , and Il2g in the DRG were unchanged compared with the control group (Fig. 4 C,D). To determine the cellular localization of Il2rb and Il2g , we performed RNAscope in situ hybridization on DRG sections. We found that Il2rb and Il2g were predominantly expressed in neurons (identified by Nissl stain) (Fig. 5 B,D,E,F), and their expression was greatly enhanced in the Nivolumab multi-dose group. Quantification showed a significant rise in the percentage of positive neurons for Il2rb and Il2g after repeated Nivolumab at day 7 after initial injection, compared to IgG4 group. But there was no difference from IgG4 controls at day 1 after initial injection (Fig. 5 B-I). Western blotting of DRG protein similarly confirmed that IL-2Rβ and IL-2Rγ protein levels were elevated in Nivolumab-treated mice (multi-dose) compared to controls, at day 7 after initial injection (Fig. 5 K-M). We next examined whether Il2rb and Il2rg induction was restricted to specific subsets of sensory neurons. Remarkably, our experiments showed that Il2rb and Il2rg were predominantly localized in MrgprA3 ⁺ pruriceptive neurons. Using fluorescent probes for MrgprA3 together with Il2rb and Il2rg , we found that more than 80% of MrgprA3 ⁺ neurons co-expressed both Il2rb and Il2rg , and conversely, near 50% of Il2rb / Il2rg double-positive neurons were MrgprA3⁺ (Fig. 6 B,C). Nivolumab directly binds to MrgprA3 + neurons and heightens their excitability. We next examined whether Nivolumab can directly interact with sensory neurons. In primary cultured DRG neuros, we found that fluorescently labeled Nivolumab bound to the surface of MrgprA3 + neurons (Fig. 7 B,C). No binding was observed on MrgprA3-labeling neurons treated with control IgG4 (Fig. 7 B,C). This direct neuron-antibody interaction implies a direct activity of Nivolumab on sensory neurons. We then asked if such binding has functional consequences for neuronal activity. In vivo, the repetitive Nivolumab treatment caused robust activation of MrgprA3 + neurons, as evidenced by a significant increase in c-Fos expression in these neurons in Nivolumab-treated mice compared to IgG controls (Fig. 8 A,B) at day 7 after initial injection. To directly assess changes in neuronal excitability, we performed whole-cell patch-clamp recordings on small-diameter nociceptive neurons from TRPV1 cre ::Ai9 mice, which largely overlap with the pruriceptive population[ 23 , 24 ]. The TRPV1 cre ::Ai9 mice were also treated with IgG4 or Nivolumab for three injections, and the DRGs were removed to culture at day 7 after initial injection. We found that TRPV1 + Neurons exhibited enhanced excitability compared to vehicle-treated control neurons. (Fig. 8 D-F). PD-1 blockade suppresses SHP-1 and activates JNK–STAT5 signaling, while JNK inhibition blocks IL-2R upregulation and Nivolumab-induced persistent itch. We investigated the intracellular signaling in sensory ganglia on days 1 and 7 after the initial injection. Using Western blot analysis of DRG lysates, we found that mice receiving the three Nivolumab treatments had a dramatic reduction in phosphorylated SHP-1 (p-SHP-1) levels compared to IgG4-treated mice (Fig. 9 B). Meanwhile, phosphorylation of c-Jun N-terminal kinase (p-JNK) was significantly increased in Nivolumab-treated DRG relative to controls (Fig. 9 C), as was phosphorylation of the transcription factor STAT5 (p-STAT5; Fig. 9 D). To assess whether JNK activation is functionally required for nivolumab-induced itch, a selective JNK phosphorylation inhibitor (D-JNKI-1, 10 µg in 5 µL) was administered intrathecally every two days, beginning 2 days before the first subcutaneous injection of IgG4 or nivolumab and continuing through day 14. Four groups were divided: PBS + IgG4(×3), PBS + Nivolumab(×3), D-JNKI-1 + IgG4(×3), and D-JNKI-1 + Nivolumab(×3). As shown in Fig. 9 F, mice in the PBS + Nivolumab(×3) group significantly increased scratching behavior compared with the PBS + IgG4(×3) group. Although mice in the D-JNKI-1 + Nivolumab(×3) group showed increased scratching compared with the D-JNKI-1 + IgG4(×1) group, D-JNKI-1 + Nivolumab(×3) displayed markedly reduced overall scratching compared with PBS + Nivolumab(×3), with significant differences at days 4, 7, and 10 after the initial injection (Fig. 9 F). Furtherly, in JNK-inhibited mice, DRG extracts showed no significant increase in p-STAT5 after repeated Nivolumab at days 1 and 7 post injection, in contrast to the D-JNKI-1 + IgG4 group (Fig. 9 H). Moreover, the upregulation of IL-2Rβ and IL-2Rγ in DRG was blocked by the JNK inhibitor, comparable to controls (Fig. 9 I). Discussion In this study, we uncovered the mechanism by which PD-1 blockade induces persistent pruritus. Repeated administration of Nivolumab induced a persistent itch state that extended beyond drug withdrawal, reflecting a complex neuroimmune mechanism rather than a simple extension of transient scratching. This chronic phase was characterized by pronounced immune activation, with increased dermal infiltration of CD4⁺ T cells and elevated systemic IL-2, which acted as a key mediator of sustained pruritus. In parallel, DRG exhibited selective upregulation of IL-2Rβ and IL-2Rγ, particularly within MrgprA3⁺ pruriceptors, where Nivolumab directly engaged the neuronal membrane and enhanced excitability. Mechanistically, PD-1 blockade diminished SHP-1 activity in DRG, shifting intracellular signaling toward JNK and STAT5 activation, a cascade that promoted IL-2 receptor expression and maintained neuronal hypersensitivity. Significantly, pharmacological inhibition of JNK disrupted this pathway, preventing receptor upregulation and alleviating chronic itch. Collectively, these findings identify the SHP-1–JNK–STAT5–IL-2R axis in pruriceptive neurons as a cellular mechanism by which PD-1 blockade drives persistent itch. Clinically, anti–PD-1 therapies such as Nivolumab often elicit pruritus that is not a fleeting side effect but a prolonged condition. Patients have been reported to continue experiencing itch for weeks or even months after discontinuing PD-1 inhibitors[ 3 , 25 ]. For example, pruritus from anti–PD-1 antibody has been noted to persist for numerous months beyond therapy cessation[ 25 ]. In this study, we observed that repeated Nivolumab administrations elicited persistent itch-like behaviors in mice, which continued for nearly one week even after drug withdrawal. This phenomenon, to some extent, mirrors clinical observation. By contrast, a single administration of Nivolumab induced only transient scratching behavior, consistent with the findings reported by Tian, who further demonstrated that the activation of spinal microglia mediates acute itch in this setting[ 4 ]. However, the sustained scratching observed after three consecutive injections was markedly intensified and long-lasting, suggesting that the mechanisms underlying Nivolumab-induced persistent itch are more complex and might not be explained solely by microglial activation. Indeed, it has been well-documented that perception and chronic itch are often involved in cytokines and neuroimmune signaling [ 19 , 26 – 28 ]. Given this, we examined the expression of immune cells and cytokines in our mouse model. We found that repeated administrations of Nivolumab induced a pronounced infiltration of CD4⁺ T cells into the subcutaneous tissue, accompanied by a significant increase in IL-2 levels. This finding is consistent with prior studies, which have shown that PD-1 blockade enhances T-cell activation and IL-2 secretion[ 29 – 31 ]. To substantiate the role of IL-2 in mediating the persistent itch induced by repeated Nivolumab administration, we employed an IL-2 neutralizing antibody. Strikingly, blockade of IL-2 signaling markedly attenuated Nivolumab-induced persistent itch, thereby confirming that elevated IL-2 is a key driver of this sustained pruritic response. Consistent with our animal findings, clinical studies in humans have shown that IL-2 immunotherapy frequently induces severe pruritus and rash, and intradermal administration of IL-2 in healthy volunteers produces a prolonged, low-grade itch[ 17 , 32 – 34 ]. Moreover, it is reported that gabapentin, which is commonly used for neuropathic pain, can safely and effectively relieve IL-2–related pruritus, suggesting that IL-2–induced itching may involve a neuropathic mechanism[ 35 ]. Accordingly, we examined the expression of IL-2 receptor subunits in DRG neurons following Nivolumab treatments. qPCR analysis showed that Il2rb and Il2rg , but not Il2ra , transcripts were significantly upregulated after repeated Nivolumab administrations, a finding further validated at the protein level by RNAscope and Western blot. In contrast, a single Nivolumab injection failed to alter Il2rb or Il2rg expression. Given that IL-2Rβ and IL-2Rγ heterodimerize to form a βγ complex that binds IL-2 with intermediate affinity and is fully competent to transduce IL-2 signaling, these results suggest that repeated PD-1 blockade selectively enhances the IL-2Rβ/γ axis, thereby establishing a permissive neuronal environment for sustained IL-2 signaling and persistent itch. To further explore the cellular mechanisms underlying Nivolumab-induced itch, we analyzed single-cell datasets of DRG neurons. We found that Ptpn6 , the gene encoding SHP-1, a key downstream effector of PD-1, is highly enriched in MrgprA3⁺ neurons. This observation is consistent with the findings reported by Han and colleagues[ 8 ]. Building on this, we employed RNAscope to examine whether Il2rb and Il2rg mRNA are also localized in MrgprA3⁺ neurons following model induction. We found that Il2rb, Il2rg and MrgprA3 showed a high level of co-expression. Furthermore, using primary cultures of sensory neurons, we demonstrated that Nivolumab directly binds to MrgprA3⁺ neurons. Because it has been reported that Nivolumab could increase the excitability of DRG neurons via modulating ion channels[ 7 ], this might explain the reason why Nivolumab induced transient scratching behavior under its application. In vivo, c-Fos expression was markedly increased in MrgprA3⁺ neurons after model induction, indicating robust neuronal activation. Although electrophysiological recordings were performed in TRPV1-reporter mice, excitability was significantly enhanced following Nivolumab treatment. Given that MrgprA3 is well known to mediate itch, and TRPV1 and MrgprA3⁺ neurons are highly co-expressed[ 23 , 24 ], these findings strongly suggest that Nivolumab promotes persistent itch through direct activation of pruriceptive MrgprA3⁺ neurons. Together with our observation that repeated PD-1 blockade upregulates IL-2Rβ/γ in MrgprA3⁺ neurons, this highlights a neuro–immune crosstalk mechanism in which Nivolumab binding and enhanced IL-2 signaling converge to drive neuronal hyperexcitability and sustained itch transmission. Finally, we investigated the molecular mechanisms by which repeated Nivolumab injections induce increased expression of Il2rb and Il2rg in neurons. Our findings revealed that repeated Nivolumab (anti–PD-1) administration reduced SHP-1 phosphorylation while concomitantly increasing JNK and STAT5 activation in the DRG. Previous studies have shown that PD-1 engagement normally recruits and activates the phosphatase SHP-1, and PD-1 blockade (e.g. with Nivolumab) diminishes SHP-1 activity[ 5 , 7 ], thereby releasing the inhibition of downstream MAPK signaling, including the JNK pathway[ 36 ]. Importantly, JNK activation has been demonstrated to enhance STAT5 phosphorylation and transcriptional activity. In particular, JNK (a MAP kinase) can phosphorylate STAT5 on key serine residues, which augments STAT5’s nuclear translocation and transcriptional potency[ 37 ]. Moreover, phosphorylated STAT5 directly promotes the transcription of IL-2 receptor subunits. For example, STAT5 is known to bind to and upregulate Il2rb (encoding the IL-2Rβ chain) in response to IL-2 signaling[ 38 ], and IL-2 stimulation (via STAT5) also contributes to the expression of the common γ-chain Il2rg [ 38 ]. Taken together, these results support a model in which PD-1/SHP-1 inactivation unleashes the JNK–STAT5 signaling axis, thereby driving the upregulation of IL-2Rβ/γ in pruriceptive DRG neurons, amplifying IL-2 signaling, and ultimately sustaining chronic itch. Conclusion Our study establishes IL-2/IL-2Rβγ signaling in pruriceptive neurons as a driver of checkpoint inhibitor–related chronic itch. Beyond providing mechanistic insight, these findings underscore the need to consider neuro–immune pathways when addressing immune-related adverse events. Targeted modulation of IL-2 signaling may thus represent a promising strategy to improve PD-1–based cancer immunotherapy. Abbreviations DRG : Dorsal root ganglion ICI : Immune checkpoint inhibitor IgG4 : Immunoglobulin G4 (isotype control antibody) IL-2 : Interleukin-2 IL-2R : Interleukin-2 receptor i.p. : Intraperitoneal injection i.t. : Intrathecal injection JNK : c-Jun N-terminal kinase PD-1 : Programmed cell death protein-1 qPCR : Quantitative polymerase chain reaction SHP-1 : Src homology region 2 domain-containing phosphatase-1 STAT5 : Signal transducer and activator of transcription 5 TRPV1 : Transient receptor potential vanilloid 1 Declarations Ethics approval and consent to participate All animal experiments were approved by the Animal Care and Use Committee of Shenzhen University (A202300939) and conducted in accordance with the guidelines of the International Association for the Study of Pain. Consent to participate is not applicable. Consent for publication Not applicable Availability of data and materials The datasets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request. Publicly available single-cell RNA sequencing data used in this study were obtained from the Gene Expression Omnibus (GEO) under accession number GSE167910. Competing interests The authors declare that they have no competing interests. Funding The present study was supported by the National Natural Science Foundation of China (82171221 and 82471240), Guangdong S&T Program (2023B0303010002), Funding by Science and Technology Projects in Guangzhou (Grant2023A03J1020), the Shenzhen Science and Technology Program (JCYJ20220818103206013), and Science and Technology Project of Shenzhen Nanshan District Health System (NSZD2025007, NSZD2023017). Authors' contributions L.L., H.Z., and J.G. performed the majority of the experiments, data acquisition, and statistical analyses. H.L. and A.L. carried out animal experiments, behavioral assays, and molecular analyses. X.B., F.W., N.L., and X.L. assisted with immunostaining, RNAscope, electrophysiology, and data interpretation. X.W., Y.L., and C.J. conceived and supervised the study, designed experiments, and interpreted results. L.L. and C.J. drafted the manuscript. All authors reviewed and approved the final version of the manuscript. Acknowledgements Not applicable. References Geisler, A.N., et al., Immune checkpoint inhibitor-related dermatologic adverse events. J Am Acad Dermatol, 2020. 83 (5): p. 1255-1268. Salinas, N., et al., Causes of Pruritus in Patients Treated With Immune Checkpoint Inhibitors for Melanomas or Skin Carcinomas. Front Med (Lausanne), 2021. 8 : p. 632683. Nikolaou, V., A. Tsimpidakis, and A. Stratigos, Cutaneous Adverse Reactions of Immunotherapy in Patients with Advanced Melanoma. Cancers (Basel), 2023. 15 (7). 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Additional Declarations No competing interests reported. Supplementary Files FigS1.tif Figure S1. Single-cell RNA-seq analysis reveals Ptpn6 enrichment in MrgprA3 ⁺ DRG neurons. t-SNE plots showing Ptpn6 expression is highly enriched in MrgprA3 ⁺ neuron clusters. Fig5KGAPDHSupplementaryFigureUncroppedblots.tif Fig5KIL2RbSupplementaryFigureUncroppedblots.tif Fig5LGAPDHSupplementaryFigureUncroppedblots.tif Fig5LIL2RgSupplementaryFigureUncroppedblots.tif Fig9BpSHP1SupplementaryFigureUncroppedblots.tif Fig9BSHP1SupplementaryFigureUncroppedblots.tif Fig9CJNKSupplementaryFigureUncroppedblots.tif Fig9CpJNKSupplementaryFigureUncroppedblots.tif Fig9DpSTAT5SupplementaryFigureUncroppedblots.tif Fig9DSTAT5SupplementaryFigureUncroppedblots.tif Fig9HpSTAT5SupplementaryFigureUncroppedblots.tif Fig9HSTAT5SupplementaryFigureUncroppedblots.tif Fig9IGAPDHSupplementaryFigureUncroppedblots.tif Fig9IIL2RbSupplementaryFigureUncroppedblots.tif Fig9IIL2RgSupplementaryFigureUncroppedblots.tif table1.docx Cite Share Download PDF Status: Published Journal Publication published 05 Dec, 2025 Read the published version in Journal of Neuroinflammation → Version 1 posted Editorial decision: Revision requested 21 Oct, 2025 Reviews received at journal 16 Oct, 2025 Reviews received at journal 14 Oct, 2025 Reviewers agreed at journal 30 Sep, 2025 Reviewers agreed at journal 30 Sep, 2025 Reviews received at journal 27 Sep, 2025 Reviewers agreed at journal 26 Sep, 2025 Reviewers invited by journal 24 Sep, 2025 Editor assigned by journal 22 Sep, 2025 Submission checks completed at journal 21 Sep, 2025 First submitted to journal 19 Sep, 2025 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. 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(B) Quantification of scratching bouts within 30 min after a single injection of Nivolumab at different doses (10–300 µg). \u0026nbsp;*p \u0026lt; 0.05, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001 vs. IgG4. One-way ANOVA. N = 6 mice/group. (C) Time course of scratching behavior in the mice with a single injection of IgG4 or Nivolumab (100 μg). ****p \u0026lt; 0.0001, Two-way ANOVA. N = 8 mice/group. (D) Schematic of repeated dosing paradigm (3 consecutive daily injections). (E) Behavioral assessment schedule for monitoring itch. (F) Time course of scratching behavior in the mice with repeated injections of IgG4 or Nivolumab (30 µg × 3). ### p \u0026lt; 0.001, #### p \u0026lt; 0.0001; ****p \u0026lt; 0.0001. Two-way ANOVA. N = 8 mice/group. # indicates comparisons between groups at the same point. * indicates overall comparisons between two groups across the row and column factors. The blue-shaded area indicates the period of drug administration.\u003c/p\u003e","description":"","filename":"Fig1new.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7654982/v1/d5a924d400ff2ff92acf2509.jpg"},{"id":93011758,"identity":"a23303c5-5d38-4530-9406-16b7d9fbe1d6","added_by":"auto","created_at":"2025-10-08 07:20:17","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3523515,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRepeated Nivolumab increases dermal CD4⁺ T-cell infiltration and systemic IL-2.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic diagram of experimental design. (B) Representative images of dermal CD4⁺ T cells after a single injection of IgG4 or Nivolumab (day 1). (C) Quantification of dermal CD4⁺ T cells. ns (not significant). Unpaired t-test. N = 5 mice/group. (D) Serum IL-2 concentration after a single injection of IgG4 or Nivolumab (day 1). ns, Unpaired t-test. N = 5 mice/group. (E) Representative images of dermal CD4⁺ T cells after repeated injections of IgG4 or Nivolumab (30 µg × 3, day 4). (F) Quantification of dermal CD4⁺ T cells. ***p \u0026lt; 0.001. Unpaired t-test. N = 5 mice/group. (G) Serum IL-2 concentration after repeated injections of IgG4 or Nivolumab (day 4). **p \u0026lt; 0.01. Unpaired t-test. N = 5 mice/group. The area between the yellow dashed lines represents the epidermal layer.\u003c/p\u003e","description":"","filename":"Fig2new.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7654982/v1/2ca48a6c42d11d51169be80e.jpg"},{"id":93011761,"identity":"b34f4348-9656-4c91-8c09-3c1a70d40ae0","added_by":"auto","created_at":"2025-10-08 07:20:17","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1979876,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIL-2 neutralization abolishes persistent but not transient itch.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic of experimental design for a single injection of IgG4 or Nivolumab with PBS or IL-2 neutralizing antibody (JES6-1A12). (B) Quantification of scratching bouts within 30 min after a single injection of IgG4 or Nivolumab in four groups: PBS + IgG4, PBS + Nivolumab, JES6-1A12 + IgG4, and JES6-1A12 + Nivolumab. *p \u0026lt; 0.05. Two-way ANOVA. N = 8 mice/group. (C) Schematic of repeated injections of IgG4 or Nivolumab with IL-2 neutralization (PBS as control) administered every 3 days. (D) Quantification of scratching bouts within 30 min at each indicated day in four groups: PBS + IgG4 ×3, PBS + Nivolumab ×3, JES6-1A12 + IgG4 ×3, and JES6-1A12 + Nivolumab ×3. #p \u0026lt; 0.05, ****p \u0026lt; 0.0001. Two-way ANOVA. N = 6 mice/group. # indicates comparisons between PBS + Nivolumab ×3 and JES6-1A12 + Nivolumab ×3.\u003c/p\u003e","description":"","filename":"Fig3.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7654982/v1/ad79f30eacab277f144c51e4.jpg"},{"id":93011742,"identity":"94b53fe3-5822-42bd-89f0-d4e012850c3f","added_by":"auto","created_at":"2025-10-08 07:20:16","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2293471,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRepeated Nivolumab upregulates \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eIl2rb\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eIl2rg\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mRNA in DRG.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic of experimental design. (B-D) Relative \u003cem\u003eIl2ra, Il2rb\u003c/em\u003e, and \u003cem\u003eIl2rg\u003c/em\u003emRNA expression at different time points after repeated Nivolumab injections. *p \u0026lt; 0.05, **p \u0026lt; 0.01. Two-way ANOVA. N = 6 mice/group.\u003c/p\u003e","description":"","filename":"Fig4.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7654982/v1/fcf12818b8c087b1375ecc7c.jpg"},{"id":93014087,"identity":"d8ec8363-2e87-4d5e-b614-fa52a24fd148","added_by":"auto","created_at":"2025-10-08 07:36:18","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3710837,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLocalization and protein validation of IL-2Rβ/γ upregulation in DRG neurons.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic of experimental design. (B) Representative RNAscope images of \u003cem\u003eIl2rb\u003c/em\u003emRNA in DRG neurons after a single injection of IgG4 or Nivolumab. (C) Quantification of \u003cem\u003eIl2rb\u003c/em\u003e⁺ neurons after a single injection of IgG4 or Nivolumab. ns. \u0026nbsp;Unpaired t-test. N = 5 mice/group. (D) Representative RNAscope images of \u003cem\u003eIl2rb\u003c/em\u003e mRNA in DRG neurons after repeated injections of IgG4 or Nivolumab. (E) Quantification of \u003cem\u003eIl2rb\u003c/em\u003e⁺ neurons after repeated injections of IgG4 or Nivolumab. *p \u0026lt; 0.05. \u0026nbsp;Unpaired t-test. N = 5 mice/group. (F) Representative RNAscope images of \u003cem\u003eIl2rg\u003c/em\u003e mRNA in DRG neurons after a single injection of IgG4 or Nivolumab. (G) Quantification of \u003cem\u003eIl2rg\u003c/em\u003e⁺ neurons after a single injection of IgG4 or Nivolumab. ns. \u0026nbsp;Unpaired t-test. N = 5 mice/group. (H) Representative RNAscope images of \u003cem\u003eIl2rg\u003c/em\u003e mRNA in DRG neurons after repeated injections of IgG4 or Nivolumab. (I) Quantification of \u003cem\u003eIl2rg\u003c/em\u003e⁺ neurons after repeated injections of IgG4 or Nivolumab. *p \u0026lt; 0.05. \u0026nbsp;Unpaired t-test. N = 5 mice/group. (J) Schematic of experimental design. (K–M) Western blot analysis and quantification of IL-2Rβ and IL-2Rγ protein expression in DRG after repeated injections. **p \u0026lt; 0.01. Two-way ANOVA. N = 5 mice/group.\u003c/p\u003e","description":"","filename":"Fig5.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7654982/v1/f9cdfa09907d574cd429c5ca.jpg"},{"id":93011744,"identity":"a19b49f1-d40b-48c3-a121-bef77b2d68f3","added_by":"auto","created_at":"2025-10-08 07:20:16","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2324578,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIL-2Rβ/γ are highly co-expressed in MrgprA3⁺ pruriceptors.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic of experimental design. (B) Representative RNAscope images showing co-localization of \u003cem\u003eIl2rb\u003c/em\u003e and \u003cem\u003eIl2rg\u003c/em\u003e with \u003cem\u003eMrgprA3\u003c/em\u003e mRNA in DRG neurons after repeated injections of IgG4 or Nivolumab. (C) Quantification of the percentage of \u003cem\u003eMrgprA3\u003c/em\u003e⁺ neurons co-expressing \u003cem\u003eIl2rb/g\u003c/em\u003e, and the percentage of \u003cem\u003eIl2rb/g\u003c/em\u003e⁺ neurons that are \u003cem\u003eMrgprA3\u003c/em\u003e⁺.\u003c/p\u003e","description":"","filename":"Fig6.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7654982/v1/2ab03ee9e25de9ee6f72bb7e.jpg"},{"id":93011752,"identity":"80fe4978-10b1-482b-8fe6-05d0abfc3a03","added_by":"auto","created_at":"2025-10-08 07:20:17","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2874338,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNivolumab binds directly to MrgprA3⁺ sensory neurons in vitro.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic of experimental design. (B–C) Representative immunofluorescence images showing labeled Nivolumab binding to the surface of MrgprA3⁺ neurons, but not control IgG4. Scale bar, 50 µm. N = 4 independent experiments.\u003c/p\u003e","description":"","filename":"Fig7.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7654982/v1/42640448a0103737d16e13f5.jpg"},{"id":93011747,"identity":"faff1fdb-249d-4085-b370-1b6bffed2544","added_by":"auto","created_at":"2025-10-08 07:20:17","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":3574654,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRepeated Nivolumab activates MrgprA3⁺ neurons and enhances excitability.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic of experimental design. (B) Representative immunofluorescence images of c-Fos expression in MrgprA3⁺ neurons after repeated injections of IgG4 or Nivolumab. (C) Quantification of c-Fos⁺/MrgprA3⁺ neurons. ****p \u0026lt; 0.0001. Unpaired t-test. N = 5 mice/group. (D) Under the microscope, a TRPV1⁺ DRG neuron was recorded by a microelectrode. (E) Representative traces of action potential recorded from TRPV1⁺ DRG neurons from the mice that were injected with IgG4 or Nivolumab three times. (F) Quantification of action potential firing at different current injections (100–300 pA). ****p \u0026lt; 0.0001. Two-way ANOVA. N = 10 neurons from 5 mice/group.\u003c/p\u003e","description":"","filename":"Fig8.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7654982/v1/7ef4290a816b5a337956d962.jpg"},{"id":93013680,"identity":"159db9c3-8bb1-49f3-8323-2114d2a721b3","added_by":"auto","created_at":"2025-10-08 07:28:17","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":4481588,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePD-1 blockade suppresses SHP-1 and activates JNK–STAT5 signaling to drive IL-2Rβ/γ upregulation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic of experimental design. (B–D) Western blot and quantification showing the levels of p-SHP-1, p-JNK and p-STAT5 in DRG at different indicated day after repeated injections. ****p \u0026lt; 0.0001. Two-way ANOVA. N = 5 mice/group. (E) Schematic of experimental design for timeline of JNK inhibitor (D-JNKI-1) administration. (F) Quantification of scratching bouts within 30 min at each indicated day in four groups: PBS + IgG4 ×3, PBS + Nivolumab ×3, D-JNKI-1 + IgG4 ×3, and D-JNKI-1 + Nivolumab ×3. #p \u0026lt; 0.05, # #p \u0026lt; 0.01, # # # p \u0026lt; 0.001, ****p \u0026lt; 0.0001. Two-way ANOVA. N = 8 mice/group. # indicates comparisons between PBS + Nivolumab ×3 and D-JNKI-1 + Nivolumab ×3. (G) Schematic of experimental design. (H–I) Western blot and quantification showing the levels of p-STAT5 (H) and IL-2Rβand IL-2Rγ(I) in DRG at different indicated day after repeated injections under the effects of D-JNKI-1. ns. Two-way ANOVA. N = 5 mice/group.\u003c/p\u003e","description":"","filename":"Fig9new.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7654982/v1/62572f215b883b78f10ee268.jpg"},{"id":97724744,"identity":"973e8b79-54e5-44dd-8140-029debc4d6b8","added_by":"auto","created_at":"2025-12-08 16:13:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":28318236,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7654982/v1/79f19fd3-b9c2-456b-ab3f-5689c41e94f8.pdf"},{"id":93013679,"identity":"744574fd-fe00-4c8a-b93b-4c81c9b11ea1","added_by":"auto","created_at":"2025-10-08 07:28:16","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2835792,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S1. Single-cell RNA-seq analysis reveals \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePtpn6\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e enrichment in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMrgprA3\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e⁺ DRG neurons.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003et-SNE plots showing \u003cem\u003ePtpn6\u003c/em\u003e expression is highly enriched in \u003cem\u003eMrgprA3\u003c/em\u003e⁺ neuron clusters.\u003c/p\u003e","description":"","filename":"FigS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-7654982/v1/ace4aaea722613e703d92e8b.tif"},{"id":93011748,"identity":"b5f0e212-d7f5-4585-946c-68ce7bec4711","added_by":"auto","created_at":"2025-10-08 07:20:17","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":547804,"visible":true,"origin":"","legend":"","description":"","filename":"Fig5KGAPDHSupplementaryFigureUncroppedblots.tif","url":"https://assets-eu.researchsquare.com/files/rs-7654982/v1/6c4c617fa28242210df99127.tif"},{"id":93011766,"identity":"75ac4513-31cc-4bb5-b919-1db9dc1a9636","added_by":"auto","created_at":"2025-10-08 07:20:17","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":659656,"visible":true,"origin":"","legend":"","description":"","filename":"Fig5KIL2RbSupplementaryFigureUncroppedblots.tif","url":"https://assets-eu.researchsquare.com/files/rs-7654982/v1/f18f42f7b0fb26568f3f836b.tif"},{"id":93011779,"identity":"010e23a1-0d35-4e88-9d3f-33fceb3767c8","added_by":"auto","created_at":"2025-10-08 07:20:18","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":591812,"visible":true,"origin":"","legend":"","description":"","filename":"Fig5LGAPDHSupplementaryFigureUncroppedblots.tif","url":"https://assets-eu.researchsquare.com/files/rs-7654982/v1/cfe7dd497f21f73a5b046057.tif"},{"id":93011749,"identity":"f9b5159e-1644-4c5d-a19a-7f48caf489f3","added_by":"auto","created_at":"2025-10-08 07:20:17","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":542808,"visible":true,"origin":"","legend":"","description":"","filename":"Fig5LIL2RgSupplementaryFigureUncroppedblots.tif","url":"https://assets-eu.researchsquare.com/files/rs-7654982/v1/c5c47d73861a7c844081491b.tif"},{"id":93011760,"identity":"a42eab2d-6319-4eaa-9002-8c34fbde3baf","added_by":"auto","created_at":"2025-10-08 07:20:17","extension":"tif","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":13517632,"visible":true,"origin":"","legend":"","description":"","filename":"Fig9BpSHP1SupplementaryFigureUncroppedblots.tif","url":"https://assets-eu.researchsquare.com/files/rs-7654982/v1/6119679b23da7e02155353b5.tif"},{"id":93014086,"identity":"b1ba55c7-c60a-456a-8db8-c18ed55558c6","added_by":"auto","created_at":"2025-10-08 07:36:17","extension":"tif","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":14655588,"visible":true,"origin":"","legend":"","description":"","filename":"Fig9BSHP1SupplementaryFigureUncroppedblots.tif","url":"https://assets-eu.researchsquare.com/files/rs-7654982/v1/3b802e87f75f1edb21243766.tif"},{"id":93011817,"identity":"8d094177-5608-445d-9946-32f704e3377a","added_by":"auto","created_at":"2025-10-08 07:20:19","extension":"tif","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":13517660,"visible":true,"origin":"","legend":"","description":"","filename":"Fig9CJNKSupplementaryFigureUncroppedblots.tif","url":"https://assets-eu.researchsquare.com/files/rs-7654982/v1/44a65acba9de6d128ebe0ec6.tif"},{"id":93013701,"identity":"b7f400e3-2c1f-40d9-871e-ab0f2a8b7bcd","added_by":"auto","created_at":"2025-10-08 07:28:18","extension":"tif","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":16686892,"visible":true,"origin":"","legend":"","description":"","filename":"Fig9CpJNKSupplementaryFigureUncroppedblots.tif","url":"https://assets-eu.researchsquare.com/files/rs-7654982/v1/f2b5a5fa39fabecd85a23b6d.tif"},{"id":93014084,"identity":"71853d07-f6ed-4949-88b5-4005ca9f80ea","added_by":"auto","created_at":"2025-10-08 07:36:17","extension":"tif","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":28191184,"visible":true,"origin":"","legend":"","description":"","filename":"Fig9DpSTAT5SupplementaryFigureUncroppedblots.tif","url":"https://assets-eu.researchsquare.com/files/rs-7654982/v1/655f007d35a71745a3096af8.tif"},{"id":93013700,"identity":"877e3205-240e-4ad2-bf3d-944fac950cbb","added_by":"auto","created_at":"2025-10-08 07:28:18","extension":"tif","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":12520232,"visible":true,"origin":"","legend":"","description":"","filename":"Fig9DSTAT5SupplementaryFigureUncroppedblots.tif","url":"https://assets-eu.researchsquare.com/files/rs-7654982/v1/a37738a5a319cd0a6041deb1.tif"},{"id":93013704,"identity":"2e526710-c0d6-476e-a78b-ca848390eb28","added_by":"auto","created_at":"2025-10-08 07:28:19","extension":"tif","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":16687980,"visible":true,"origin":"","legend":"","description":"","filename":"Fig9HpSTAT5SupplementaryFigureUncroppedblots.tif","url":"https://assets-eu.researchsquare.com/files/rs-7654982/v1/2f212a24386d59c003ae5992.tif"},{"id":93011756,"identity":"05e1ff4c-3aaf-49fb-af95-93da8abe5449","added_by":"auto","created_at":"2025-10-08 07:20:17","extension":"tif","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":16686516,"visible":true,"origin":"","legend":"","description":"","filename":"Fig9HSTAT5SupplementaryFigureUncroppedblots.tif","url":"https://assets-eu.researchsquare.com/files/rs-7654982/v1/d285f86104f6427875ce2b23.tif"},{"id":93011753,"identity":"805066ad-c262-4de4-9d51-758e36442ebd","added_by":"auto","created_at":"2025-10-08 07:20:17","extension":"tif","order_by":14,"title":"","display":"","copyAsset":false,"role":"supplement","size":1538276,"visible":true,"origin":"","legend":"","description":"","filename":"Fig9IGAPDHSupplementaryFigureUncroppedblots.tif","url":"https://assets-eu.researchsquare.com/files/rs-7654982/v1/d45f741ee03cc71bb56b4930.tif"},{"id":93013689,"identity":"70ba66ed-2b16-4a8d-bf4f-e29ae3c07b38","added_by":"auto","created_at":"2025-10-08 07:28:17","extension":"tif","order_by":15,"title":"","display":"","copyAsset":false,"role":"supplement","size":1538432,"visible":true,"origin":"","legend":"","description":"","filename":"Fig9IIL2RbSupplementaryFigureUncroppedblots.tif","url":"https://assets-eu.researchsquare.com/files/rs-7654982/v1/a7f98dd11e10c363f5704251.tif"},{"id":93011810,"identity":"6c39af54-2333-44dc-991d-886a09e7c6b4","added_by":"auto","created_at":"2025-10-08 07:20:19","extension":"tif","order_by":16,"title":"","display":"","copyAsset":false,"role":"supplement","size":1537308,"visible":true,"origin":"","legend":"","description":"","filename":"Fig9IIL2RgSupplementaryFigureUncroppedblots.tif","url":"https://assets-eu.researchsquare.com/files/rs-7654982/v1/3bbda88c4f78ba18bb5d7f3b.tif"},{"id":93014824,"identity":"8c4e86a2-c575-40c9-ae13-5d9f775d5363","added_by":"auto","created_at":"2025-10-08 07:44:18","extension":"docx","order_by":17,"title":"","display":"","copyAsset":false,"role":"supplement","size":16830,"visible":true,"origin":"","legend":"","description":"","filename":"table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7654982/v1/a8224e8c4ca6c18b668ddad2.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"IL-2/IL-2Rβγ signaling in pruriceptors drives neuroimmune mechanisms of nivolumab- induced persistent itch","fulltext":[{"header":"Background","content":"\u003cp\u003eImmune checkpoint inhibitors (ICIs) targeting PD-1 (e.g., Nivolumab) have revolutionized cancer therapy, markedly improving survival across multiple malignancies. However, their clinical success is tempered by immune-related adverse events (irAEs). Among the most common irAEs are cutaneous toxicities, notably chronic pruritus (itch)[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Recent clinical series and meta-analyses suggest that anti\u0026ndash;PD\u0026ndash;1 therapy frequently provokes pruritus in 13\u0026ndash;25% of patients[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Notably, ICI-related pruritus is mechanistically distinct from classical allergic or atopic itch, as it often arises without a visible skin rash and is notoriously resistant to antihistamine therapy[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. These observations indicate that checkpoint inhibitor\u0026ndash;induced pruritus involves non-histaminergic mechanisms. Recent preclinical studies have shown that the intradermal administration of PD-1/PD-L1 antibodies induces acute itch through non-histaminergic pathways involving spinal microglial activation[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. However, since ICI-induced pruritus frequently persists throughout treatment and may even continue after withdrawal[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], the mechanisms underlying PD-1 antibody\u0026ndash;induced chronic itch remain unclear.\u003c/p\u003e\u003cp\u003ePrevious studies have further demonstrated that PD-1 is functionally and widely expressed on primary sensory neurons in the dorsal root ganglia (DRG), where it regulates neuronal excitability and pain transmission[\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Indeed, a distinct subset of primary sensory neurons in the dorsal root ganglia (DRG) is dedicated to transmitting itch signals, often referred to as pruriceptors. Dysfunction of these neurons is closely associated with the development of chronic itch. Common molecular markers for itch-selective neurons include MrgprA3, Nppb, and the interleukin-31 receptor complex, as well as histamine receptors[\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Among these, MrgprA3⁺ neurons have been established as a key pruriceptive lineage that mediates non-histaminergic itch responses[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Transcriptomic profiling further revealed that MrgprA3⁺ neurons are highly enriched in SHP-1 (Ptpn6), a phosphatase that serves as a critical downstream effector of PD-1 signaling[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Within the field of pain research, PD-1 antibody treatment has been demonstrated to modulate nociceptor ion channel activity via SHP-1[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], thereby implicating MrgprA3⁺ neurons as likely contributors to PD-1 antibody\u0026ndash;induced chronic pruritus.\u003c/p\u003e\u003cp\u003eGiven the critical immunoregulatory role of PD-1, PD-1 antibody\u0026ndash;induced chronic itch may represent a dual effect, driven not only by neuronal mechanisms but also by the activation of immune cells. PD-1 blockade unleashes T-cell activity, particularly in CD4⁺ helper T cells, leading to robust cytokine production[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Among these, interleukin-2 (IL-2) is a pivotal cytokine induced during T-cell activation[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Although IL-2 is classically considered a T-cell growth factor, accumulating evidence indicates that it can also modulate the nervous system. For example, early studies demonstrated that IL-2 receptors are constitutively expressed in dorsal root ganglion (DRG) neurons and that IL-2 application can alter nociceptive signaling[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Moreover, it is reported that IL-2 can directly induce itch, as demonstrated by a clinical study where a single intradermal injection of recombinant human IL-2 elicited low-intensity local itch[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In line with this, IL-2 is also considered one of the cytokines involved in chronic pruritus[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Importantly, IL-2 receptor (IL-2R) expression is not fixed and can be upregulated in sensory neurons during inflammation or dermatitis[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Together, these findings suggest that IL-2/IL-2R signaling may serve as a critical messenger linking immune hyperactivation to neuronal sensitization, thereby driving chronic itch during PD-1 antibody therapy.\u003c/p\u003e\u003cp\u003eIn this study, we demonstrate that repeated administration of Nivolumab in mice induces persistent itch by directly engaging MrgprA3⁺ pruriceptors and driving IL-2 receptor β/γ upregulation through SHP-1 inhibition and subsequent activation of the JNK\u0026ndash;STAT5 pathway. This cascade renders sensory neurons hypersensitive to IL-2 released from activated CD4⁺ T cells, thereby establishing a neuroimmune feedforward loop that perpetuates chronic pruritus.\u003c/p\u003e"},{"header":"Experimental Section","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eAnimals\u003c/h2\u003e\u003cp\u003eAdult C57BL/6 mice (8\u0026ndash;12 weeks) were used for most experiments, purchased from the Medical Experimental Animal Center of Guangdong Province, China. For neuron labeling, we used Trpv1\u003csup\u003eCre\u003c/sup\u003e, and Ai9 mice (JAX #017769 and #007909) to generate TRPV1-reporting mice. Animal experiments were conducted in accordance with the guidelines set by the International Association for the Study of Pain and approved by the Animal Care and Use Committee of Shenzhen University (A202300939). All animals were group-housed in cages with 2\u0026thinsp;~\u0026thinsp;5 mice under a 12-hour light/dark cycle at a stable temperature (22\u0026thinsp;\u0026plusmn;\u0026thinsp;1℃) with access to food and water ad libitum. Both male and female mice were included in each group in a sex-matched manner. In all experiments, animals were randomly assigned to each group. The sample size was determined based on our previous studies[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. All investigators were blinded to both treatment assignment and outcome assessments.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eReagents\u003c/h3\u003e\n\u003cp\u003eNivolumab (Opdivo\u0026reg;) is a human IgG4 monoclonal antibody against PD-1; control human IgG4 was obtained from BioXCell. For single-injection experiments, mice received a single subcutaneous injection (50 \u0026micro;L) of either Nivolumab (10\u0026ndash;300 \u0026micro;g) or an equal dose of control IgG4. For repeated-injection experiments, mice were given three subcutaneous injections of Nivolumab (30 \u0026micro;g per dose) or IgG4 at the same site on days 1, 2, and 3.\u003c/p\u003e\u003cp\u003eFor IL-2 neutralization, anti\u0026ndash;mouse IL-2 antibody (JES6-1A12, BioXCell, # BE0043) was administered intraperitoneally (i.p., 20\u0026ndash;25 mg/kg), starting 3 days before the first Nivolumab injection and repeated every 3 days for a total of four doses. Control mice received PBS i.p. on the same schedule. For JNK inhibition, the cell-permeant peptide D-JNKI-1 (MCE, #HY-P0069; 10 \u0026micro;g in 5 \u0026micro;L saline) or vehicle (saline) was administered via intrathecal (i.t.) injection. D-JNKI-1 was administered 2 days before the first Nivolumab injection and then every 2 days for a total of five injections.\u003c/p\u003e\n\u003ch3\u003eItch Behavior Assay\u003c/h3\u003e\n\u003cp\u003eMice were habituated in individual observation chambers for 3 days before the experiments and additionally acclimated for 30\u0026ndash;60 min before each injection. For transient itch assessment, mice were recorded for 30 min immediately after injection. For persistent itch evaluation, scratching behavior was recorded in 30 min sessions on baseline day 0 and on days 1, 2, 3, 4, 7, 10, and 14 following the initiation of injections. Scratching was defined as a mouse using its hind paw to scratch the neck region, and bouts were summed over each 30 min observation period for individual mice.\u003c/p\u003e\n\u003ch3\u003eReal-time quantitative PCR (qPCR) for mRNAs\u003c/h3\u003e\n\u003cp\u003eTotal RNA was isolated from DRG using Trizol reagent (Invitrogen, Carlsbad, CA, USA). Four DRGs were pooled together for each RNA extraction. Briefly, total RNA (1 \u0026micro;g) was reverse-transcribed by oligo (dT) primer using a HiScript II 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China). The qPCR was performed using an ABI Step-One Plus system (Applied Biosystems, CA, USA) with TB Green Premix Ex Taq II (Takara, Dalian, China) and the primers listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. The qPCR cycling conditions were 95\u0026deg;C for 3 min, followed by 40 cycles of amplification at 95\u0026deg;C for 10 s and 60\u0026deg;C for 30 s. Gapdh was used as a control to normalize differences for mRNA detection. Melt curves were performed after the cycles to ensure the absence of nonspecific products. Quantification was performed by normalizing the Ct (cycle threshold) values to those of Gapdh Ct (mRNA) and analyzed using the 2-ΔΔCT method.\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence and RNAscope\u003c/h3\u003e\n\u003cp\u003eMice of both sexes (8\u0026ndash;12 weeks old) were deeply anesthetized with isoflurane, followed by transcardial perfusion with PBS and then 4% paraformaldehyde (PFA) containing 1.5% picric acid. For skin immunostaining, tissue at the injection site was shaved, fixed, and cryoprotected before being cryosectioned (12 \u0026micro;m). Sections were incubated overnight at 4\u0026deg;C with anti-CD4 antibody (rabbit mAb, 1:200, Abcam, Cat# ab183685), followed by Alexa Fluor 594\u0026ndash;conjugated goat anti-rabbit secondary antibody (1:500, Invitrogen). Nuclei were counterstained with DAPI. Images were captured using a fluorescence microscope at 20\u0026times; magnification. CD4⁺ T cells were quantified in the dermis as the mean number of red cells per high-power field (0.25 mm\u0026sup2;) across 4\u0026ndash;5 randomly selected fields per mouse.\u003c/p\u003e\u003cp\u003eFor DRG immunostaining, cervical DRGs (C5\u0026ndash;C8) were dissected, post-fixed for 2 h at room temperature, cryoprotected sequentially in 20% and 30% sucrose, and sectioned at 12 \u0026micro;m thickness. Sections were blocked and incubated overnight at 4\u0026deg;C with anti\u0026ndash;c-Fos (rabbit mAb, 1:500, Cell Signaling, clone 9F6, Cat# 2250) and anti-MrgprA3 (goat polyclonal, 1:200, Santa Cruz Biotechnology, Cat# sc-23348), followed by Alexa Fluor 488\u0026ndash; and 594\u0026ndash;conjugated secondary antibodies (1:500; Invitrogen).\u003c/p\u003e\u003cp\u003eFor anti-Nivolumab staining, cultured cortical neurons were incubated with Nivolumab (300 ng/mL), post-fixed in 4% PFA for 15 min, and permeabilized with PBS containing 0.3% Triton X-100 for 1 h. Neurons were incubated overnight at 4\u0026deg;C with primary antibodies against PD-1 (rabbit, 1:300, Sigma, Cat# PRS4065) and NeuN (mouse, 1:250, Millipore, Cat# MAB377), followed by Cy3- or Cy5-conjugated secondary antibodies (1:500, Jackson ImmunoResearch) and/or anti-IgG4-FITC (1:200, Abcam, Cat# ab99281). Nuclei were counterstained with DAPI in mounting medium (Sigma, Cat# SLCC8848). Images were acquired with an FV3000 confocal laser scanning microscope (Olympus).\u003c/p\u003e\u003cp\u003eIn situ hybridization was performed using the RNAscope Multiplex Fluorescent V2 kit (Advanced Cell Diagnostics) according to the manufacturer\u0026rsquo;s instructions. Commercial probes included Il2rb (Mm-Il2rb, Cat# 502761-C2), Il2rg (Mm-Il2rg, Cat# 445011-C1), and MrgprA3 (Mm-MrgprA3, Cat# 548161-C3). Fluorescent puncta representing target RNA expression were quantified using QuPath software. To visualize neurons in some experiments, sections were counterstained with Nissl (1:200, Invitrogen, Cat# N21483) for 2 h. A neuron was considered positive if three or more fluorescent puncta were detected within the soma. Cell counts were performed in ImageJ using the cell counter plugin, with investigators blinded to experimental conditions. Images were obtained using an FV3000 confocal microscope (Olympus). Each experimental group consisted of five mice.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eELISA\u003c/h2\u003e\u003cp\u003eBlood samples were collected by submandibular venipuncture at designated time points. Serum was isolated and stored at \u0026minus;\u0026thinsp;80\u0026deg;C until analysis. Interleukin-2 (IL-2) concentrations were quantified using a high-sensitivity IL-2 Quantikine ELISA kit (Sigma-Aldrich, # RAB0287) following the manufacturer\u0026rsquo;s instructions. Absorbance was measured with a microplate reader, and cytokine concentrations were interpolated from a standard curve.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eWhole-cell patch clamp recordings in cultured DRG neurons\u003c/h3\u003e\n\u003cp\u003e The mouse DRGs were removed aseptically and incubated with collagenase (1.25mg/ml, Roche)/dispase-II (2.4 units/ml, Roche) at 37\u0026deg;C for 90 min, then digested with 0.25% trypsin for 8 min at 37\u0026deg;C, followed by 0.25% trypsin inhibitor. Cells were mechanically dissociated with a flame polished Pasteur pipette in the presence of 0.05% DNase I (Sigma). DRG cells were plated on glass coverslips and grown in a neurobasal defined medium (with 2% B27 supplement, Invitrogen) with 5 mM AraC and 5% carbon dioxide at 36.5\u0026deg;C. DRG neurons were grown for at least 6 hours before use. Whole-cell patch clamp recordings were performed at room temperature using an Axopatch-700B amplifier (Axon Instruments) with a Digidata 1440B (Axon Instruments). The patch pipettes were pulled from borosilicate capillaries (World Precision Instruments, Inc.) using a P-97 Flaming/Brown micropipette puller (Sutter Instrument Co.). The pipette resistance was 4\u0026ndash;6 MΩ. The internal pipette solution contains (in mM): 126 K-gluconate,10 NaCl, 1 MgCl\u003csub\u003e2\u003c/sub\u003e, 10 EGTA, 10 HEPES, and 2 Na-ATP (adjusted to pH 7.4 with KOH, Osmolarity 295\u0026ndash;300 mOsm), and the extracellular solution contains (in mM): 140 NaCl, 5 KCl, 2 MgCl\u003csub\u003e2\u003c/sub\u003e, 2 CaCl\u003csub\u003e2\u003c/sub\u003e, 10 HEPES, 10 glucose, adjusted to pH 7.4 with KOH. Data were filtered at 2 kHz and digitized at 10 kHz using a data acquisition interface (1440A, Molecular Devices). The liquid junction potential was corrected. The pClamp 10 software (Axon Instruments) was used for signal acquisition and analysis. The TRPV1-positive neurons were chosen for recording in the experiment. The action potentials (APs) evoked by a series of ramp current stimulation (time: 1 sec; current intensity: 100 pA, 200 pA, 300 pA) were recorded.\u003c/p\u003e\n\u003ch3\u003eWestern blot analysis\u003c/h3\u003e\n\u003cp\u003eAfter isoflurane-induced anesthesia, animals were transcardially perfused with 0.9% NaCl, and the Cervical DRGs (C5\u0026ndash;C8) were dissected. The tissues were homogenized in a RIPA lysis buffer containing protease and phosphatase inhibitors (Roche, Basel, Switzerland). Protein concentrations were determined by BCA Protein Assay (Thermo Scientific, Waltham, MA, USA). Protein samples (30 \u0026micro;g) were separated on SDS\u0026ndash;PAGE gel and transferred to the PVDF membrane. The membranes were blocked with 5% milk and incubated overnight at 4\u0026deg;C with primary antibodies against: phospho-STAT5 (Tyr694, Cell Signaling #9359, 1:1000), total STAT5 (#94205, 1:1000), phospho-JNK (Thr183/Tyr185, Cell Signaling #4668, 1:500), total JNK (#9252, 1:1000), phospho-SHP-1 (Tyr564, Abcam ab131498, 1:500), total SHP-1 (Cell Signaling #3759, 1:1000), IL-2Rβ (CD122, R\u0026amp;D AF225, 1:500), IL-2Rγ (CD132, Santa Cruz sc-668, 1:200), and β-actin or GAPDH (loading controls, 1:5000). After washing, membranes were incubated with appropriate HRP-conjugated secondary antibodies (1:5000, Jackson Labs) for 1 h. Bands were visualized with ECL substrate (Thermo) on a ChemiDoc imager (Bio-Rad). Specific bands were evaluated by predicted molecular size, and the intensity of selected bands was analyzed by ImageJ software. Phospho-protein signals were normalized to the corresponding total protein, and receptor subunit signals were normalized to GAPDH. Each blot included 3\u0026ndash;4 samples per group.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eSingle-cell RNA sequencing analysis\u003c/h2\u003e\u003cp\u003ePublicly available DRG neurons scRNA-seq data were obtained from the study by Xu et al. [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The raw data were downloaded from the Gene Expression Omnibus (GEO) under accession number GSE167910. We reanalyzed this data using the Seurat package (version 4.0) in R (version 4.1.2), by focusing on \u003cem\u003ePtpn6\u003c/em\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eAll sample sizes and experimental designs were based on previously published data from our lab and similar experiments in the field[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. All data were mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM, and all statistical tests were conducted using two-tailed hypothesis testing. If only two groups were applied, the Student\u0026rsquo;s t-test was used. For the single injection of Nivolumab at different doses, one-way ANOVA was used. If the differences were significant, post hoc Bonferroni\u0026rsquo;s test was applied to compare the differences between the two groups. For behavioral tests, qPCR data, electrophysiological data, and Western blotting data, two-way repeated-measure ANOVA was used. If the differences were significant, post hoc Bonferroni\u0026rsquo;s test was applied to compare values at different points. All statistical analyses were performed using GraphPad Prism 8 (GraphPad Software, Inc.). The value of P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eNivolumab provokes acute transient itch with a single dose, but repeated dosing leads to persistent pruritus.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, we employed subcutaneous injection to establish the model. Administration of Nivolumab at doses of 30 \u0026micro;g, 100 \u0026micro;g, and 300 \u0026micro;g robustly elicited scratching behavior compared with IgG4 controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Based on these findings, we selected a dose of 100 \u0026micro;g for subsequent single-injection experiments. Time-course analysis revealed that a single subcutaneous injection of Nivolumab induced scratching behavior that peaked at 15\u0026ndash;20 minutes and subsided within ~\u0026thinsp;30 minutes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Strikingly, however, daily injections of Nivolumab for three consecutive days produced a persistent itch phenotype. Quantification of scratching bouts within 30-minute observation periods showed that scratching increased during the treatment phase and persisted for nearly one week after the final injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD\u0026ndash;F).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eRepeated Nivolumab administration triggers dermal CD4\u003c/b\u003e\u003csup\u003e\u003cb\u003e+\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eT-cell infiltration and systemic IL-2 elevation, whereas a single injection has no such effect.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eGiven reports that ICI-induced skin toxicities are associated with T-cell infiltrates (particularly helper T cells) and elevated IL-2 levels in patients, we examined immune changes in our model. In mice receiving only one Nivolumab injection, there was no significant increase in CD4\u003csup\u003e+\u003c/sup\u003e T cell numbers in the skin at the injection site compared to controls, and serum IL-2 concentrations remained at baseline levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u0026ndash;D). In contrast, mice subjected to three daily Nivolumab injections showed a marked accumulation of CD4\u003csup\u003e+\u003c/sup\u003e T cells in the dermis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE,F) alongside a significant rise in circulating IL-2 in the circulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eNeutralization of IL-2 selectively abolishes persistent itch after repeated Nivolumab but does not affect the transient itch from a single dose.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo test whether IL-2 is required for Nivolumab-induced persistent itch, we administered an IL-2\u0026ndash;neutralizing antibody (JES6-1A12, i.p., 20 mg/kg) or vehicle (PBS) control[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In the transient itch model, JES6-1A12 was given intraperitoneally once, both 3 days before and on the day of subcutaneous injection of IgG4 or Nivolumab. Blocking IL-2 had no effect on the acute scratching response to a single Nivolumab injection. Mice in the PBS\u0026thinsp;+\u0026thinsp;Nivolumab(\u0026times;1) group exhibited significantly increased scratching compared with the PBS\u0026thinsp;+\u0026thinsp;IgG4(\u0026times;1) group, and mice in the JES6-1A12\u0026thinsp;+\u0026thinsp;Nivolumab(\u0026times;1) group also showed significantly increased scratching compared with the JES6-1A12\u0026thinsp;+\u0026thinsp;IgG4(\u0026times;1) group. However, no statistical differences were observed between the PBS\u0026thinsp;+\u0026thinsp;IgG4(\u0026times;1) and JES6-1A12\u0026thinsp;+\u0026thinsp;IgG4(\u0026times;1) groups, or between the PBS\u0026thinsp;+\u0026thinsp;Nivolumab(\u0026times;1) and JES6-1A12\u0026thinsp;+\u0026thinsp;Nivolumab(\u0026times;1) groups. (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA,B).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eBy contrast, IL-2 neutralization profoundly influenced the persistent itch phenotype in the repeated dosing paradigm. JES6-1A12 or PBS was administered every three days starting 3 days before the first subcutaneous injection of IgG4 or Nivolumab and continued until day 14 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). In a four-group comparison (PBS\u0026thinsp;+\u0026thinsp;IgG4\u0026times;3, PBS\u0026thinsp;+\u0026thinsp;Nivolumab\u0026times;3, JES6-1A12\u0026thinsp;+\u0026thinsp;IgG4\u0026times;3, JES6-1A12\u0026thinsp;+\u0026thinsp;Nivolumab\u0026times;3), all Nivolumab-treated mice exhibited increased scratching during the 3-day treatment period regardless of IL-2 neutralization, indicating that IL-2 is not essential for acute itch during ongoing Nivolumab exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Although scratching behavior in the JES6-1A12\u0026thinsp;+\u0026thinsp;Nivolumab (\u0026times;3) group differed from that in the JES6-1A12\u0026thinsp;+\u0026thinsp;IgG4 (\u0026times;3) group, JES6-1A12\u0026thinsp;+\u0026thinsp;Nivolumab(\u0026times;3) mice displayed markedly reduced overall scratching compared with PBS\u0026thinsp;+\u0026thinsp;Nivolumab(\u0026times;3), with significant differences at days 4, 7, and 10 after the initial injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Moreover, PBS\u0026thinsp;+\u0026thinsp;Nivolumab(\u0026times;3) mice continued to exhibit high scratching counts for many days after treatment relative to PBS\u0026thinsp;+\u0026thinsp;IgG4(\u0026times;3).\u003c/p\u003e\u003cp\u003e\u003cb\u003eRepeated Nivolumab upregulates IL-2 receptor β and γ subunits in dorsal root ganglia (DRG) neurons, particularly in itch-selective MrgprA3\u003c/b\u003e\u003csup\u003e\u003cb\u003e+\u003c/b\u003e\u003c/sup\u003e \u003cb\u003epruriceptors.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe elevation of IL-2 prompted us to investigate the expression of IL-2 receptor (IL-2R) in sensory neurons after repeated Nivolumab treatments. Quantitative PCR analysis of cervical DRG showed that three consecutive Nivolumab injections significantly upregulated \u003cem\u003eIl2rb\u003c/em\u003e (encoding IL-2Rβ) and \u003cem\u003eIl2g\u003c/em\u003e (encoding IL-2Rγ) transcripts compared with IgG4 controls at days 4 and 7 after the initial injection, with expression returning to control levels by day 10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB,D). In contrast, \u003cem\u003eIl2ra\u003c/em\u003e (encoding IL-2Rα) expression remained unchanged following Nivolumab treatment at days 1, 4, 7, and 10 after the initial injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), indicating that the intermediate-affinity IL-2Rβ/γ (heterodimer) is specifically upregulated in neurons without induction of the α subunit. Notably, on day 1 after the initial injection, the expression levels of \u003cem\u003eIl2ra\u003c/em\u003e, \u003cem\u003eIl2rb\u003c/em\u003e, and \u003cem\u003eIl2g\u003c/em\u003e in the DRG were unchanged compared with the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC,D).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo determine the cellular localization of \u003cem\u003eIl2rb\u003c/em\u003e and \u003cem\u003eIl2g\u003c/em\u003e, we performed RNAscope in situ hybridization on DRG sections. We found that \u003cem\u003eIl2rb\u003c/em\u003e and \u003cem\u003eIl2g\u003c/em\u003e were predominantly expressed in neurons (identified by Nissl stain) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB,D,E,F), and their expression was greatly enhanced in the Nivolumab multi-dose group. Quantification showed a significant rise in the percentage of positive neurons for \u003cem\u003eIl2rb\u003c/em\u003e and \u003cem\u003eIl2g\u003c/em\u003e after repeated Nivolumab at day 7 after initial injection, compared to IgG4 group. But there was no difference from IgG4 controls at day 1 after initial injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB-I). Western blotting of DRG protein similarly confirmed that IL-2Rβ and IL-2Rγ protein levels were elevated in Nivolumab-treated mice (multi-dose) compared to controls, at day 7 after initial injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eK-M).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe next examined whether \u003cem\u003eIl2rb\u003c/em\u003e and \u003cem\u003eIl2rg\u003c/em\u003e induction was restricted to specific subsets of sensory neurons. Remarkably, our experiments showed that \u003cem\u003eIl2rb\u003c/em\u003e and \u003cem\u003eIl2rg\u003c/em\u003e were predominantly localized in \u003cem\u003eMrgprA3\u003c/em\u003e⁺ pruriceptive neurons. Using fluorescent probes for \u003cem\u003eMrgprA3\u003c/em\u003e together with \u003cem\u003eIl2rb\u003c/em\u003e and \u003cem\u003eIl2rg\u003c/em\u003e, we found that more than 80% of \u003cem\u003eMrgprA3\u003c/em\u003e⁺ neurons co-expressed both \u003cem\u003eIl2rb\u003c/em\u003e and \u003cem\u003eIl2rg\u003c/em\u003e, and conversely, near 50% of \u003cem\u003eIl2rb\u003c/em\u003e/\u003cem\u003eIl2rg\u003c/em\u003e double-positive neurons were MrgprA3⁺ (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB,C).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eNivolumab directly binds to MrgprA3\u003c/b\u003e\u003csup\u003e\u003cb\u003e+\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eneurons and heightens their excitability.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe next examined whether Nivolumab can directly interact with sensory neurons. In primary cultured DRG neuros, we found that fluorescently labeled Nivolumab bound to the surface of MrgprA3\u003csup\u003e+\u003c/sup\u003e neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB,C). No binding was observed on MrgprA3-labeling neurons treated with control IgG4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB,C). This direct neuron-antibody interaction implies a direct activity of Nivolumab on sensory neurons. We then asked if such binding has functional consequences for neuronal activity. In vivo, the repetitive Nivolumab treatment caused robust activation of MrgprA3\u003csup\u003e+\u003c/sup\u003e neurons, as evidenced by a significant increase in c-Fos expression in these neurons in Nivolumab-treated mice compared to IgG controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA,B) at day 7 after initial injection. To directly assess changes in neuronal excitability, we performed whole-cell patch-clamp recordings on small-diameter nociceptive neurons from TRPV1\u003csup\u003ecre\u003c/sup\u003e::Ai9 mice, which largely overlap with the pruriceptive population[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The TRPV1\u003csup\u003ecre\u003c/sup\u003e::Ai9 mice were also treated with IgG4 or Nivolumab for three injections, and the DRGs were removed to culture at day 7 after initial injection. We found that TRPV1\u003csup\u003e+\u003c/sup\u003e Neurons exhibited enhanced excitability compared to vehicle-treated control neurons. (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD-F).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003ePD-1 blockade suppresses SHP-1 and activates JNK\u0026ndash;STAT5 signaling, while JNK inhibition blocks IL-2R upregulation and Nivolumab-induced persistent itch.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe investigated the intracellular signaling in sensory ganglia on days 1 and 7 after the initial injection. Using Western blot analysis of DRG lysates, we found that mice receiving the three Nivolumab treatments had a dramatic reduction in phosphorylated SHP-1 (p-SHP-1) levels compared to IgG4-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB). Meanwhile, phosphorylation of c-Jun N-terminal kinase (p-JNK) was significantly increased in Nivolumab-treated DRG relative to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eC), as was phosphorylation of the transcription factor STAT5 (p-STAT5; Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo assess whether JNK activation is functionally required for nivolumab-induced itch, a selective JNK phosphorylation inhibitor (D-JNKI-1, 10 \u0026micro;g in 5 \u0026micro;L) was administered intrathecally every two days, beginning 2 days before the first subcutaneous injection of IgG4 or nivolumab and continuing through day 14. Four groups were divided: PBS\u0026thinsp;+\u0026thinsp;IgG4(\u0026times;3), PBS\u0026thinsp;+\u0026thinsp;Nivolumab(\u0026times;3), D-JNKI-1\u0026thinsp;+\u0026thinsp;IgG4(\u0026times;3), and D-JNKI-1\u0026thinsp;+\u0026thinsp;Nivolumab(\u0026times;3). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eF, mice in the PBS\u0026thinsp;+\u0026thinsp;Nivolumab(\u0026times;3) group significantly increased scratching behavior compared with the PBS\u0026thinsp;+\u0026thinsp;IgG4(\u0026times;3) group. Although mice in the D-JNKI-1\u0026thinsp;+\u0026thinsp;Nivolumab(\u0026times;3) group showed increased scratching compared with the D-JNKI-1\u0026thinsp;+\u0026thinsp;IgG4(\u0026times;1) group, D-JNKI-1\u0026thinsp;+\u0026thinsp;Nivolumab(\u0026times;3) displayed markedly reduced overall scratching compared with PBS\u0026thinsp;+\u0026thinsp;Nivolumab(\u0026times;3), with significant differences at days 4, 7, and 10 after the initial injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eF).\u003c/p\u003e\u003cp\u003eFurtherly, in JNK-inhibited mice, DRG extracts showed no significant increase in p-STAT5 after repeated Nivolumab at days 1 and 7 post injection, in contrast to the D-JNKI-1\u0026thinsp;+\u0026thinsp;IgG4 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eH). Moreover, the upregulation of IL-2Rβ and IL-2Rγ in DRG was blocked by the JNK inhibitor, comparable to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eI).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we uncovered the mechanism by which PD-1 blockade induces persistent pruritus. Repeated administration of Nivolumab induced a persistent itch state that extended beyond drug withdrawal, reflecting a complex neuroimmune mechanism rather than a simple extension of transient scratching. This chronic phase was characterized by pronounced immune activation, with increased dermal infiltration of CD4⁺ T cells and elevated systemic IL-2, which acted as a key mediator of sustained pruritus. In parallel, DRG exhibited selective upregulation of IL-2Rβ and IL-2Rγ, particularly within MrgprA3⁺ pruriceptors, where Nivolumab directly engaged the neuronal membrane and enhanced excitability. Mechanistically, PD-1 blockade diminished SHP-1 activity in DRG, shifting intracellular signaling toward JNK and STAT5 activation, a cascade that promoted IL-2 receptor expression and maintained neuronal hypersensitivity. Significantly, pharmacological inhibition of JNK disrupted this pathway, preventing receptor upregulation and alleviating chronic itch. Collectively, these findings identify the SHP-1\u0026ndash;JNK\u0026ndash;STAT5\u0026ndash;IL-2R axis in pruriceptive neurons as a cellular mechanism by which PD-1 blockade drives persistent itch.\u003c/p\u003e\u003cp\u003eClinically, anti\u0026ndash;PD-1 therapies such as Nivolumab often elicit pruritus that is not a fleeting side effect but a prolonged condition. Patients have been reported to continue experiencing itch for weeks or even months after discontinuing PD-1 inhibitors[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. For example, pruritus from anti\u0026ndash;PD-1 antibody has been noted to persist for numerous months beyond therapy cessation[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In this study, we observed that repeated Nivolumab administrations elicited persistent itch-like behaviors in mice, which continued for nearly one week even after drug withdrawal. This phenomenon, to some extent, mirrors clinical observation. By contrast, a single administration of Nivolumab induced only transient scratching behavior, consistent with the findings reported by Tian, who further demonstrated that the activation of spinal microglia mediates acute itch in this setting[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. However, the sustained scratching observed after three consecutive injections was markedly intensified and long-lasting, suggesting that the mechanisms underlying Nivolumab-induced persistent itch are more complex and might not be explained solely by microglial activation.\u003c/p\u003e\u003cp\u003eIndeed, it has been well-documented that perception and chronic itch are often involved in cytokines and neuroimmune signaling [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Given this, we examined the expression of immune cells and cytokines in our mouse model. We found that repeated administrations of Nivolumab induced a pronounced infiltration of CD4⁺ T cells into the subcutaneous tissue, accompanied by a significant increase in IL-2 levels. This finding is consistent with prior studies, which have shown that PD-1 blockade enhances T-cell activation and IL-2 secretion[\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. To substantiate the role of IL-2 in mediating the persistent itch induced by repeated Nivolumab administration, we employed an IL-2 neutralizing antibody. Strikingly, blockade of IL-2 signaling markedly attenuated Nivolumab-induced persistent itch, thereby confirming that elevated IL-2 is a key driver of this sustained pruritic response. Consistent with our animal findings, clinical studies in humans have shown that IL-2 immunotherapy frequently induces severe pruritus and rash, and intradermal administration of IL-2 in healthy volunteers produces a prolonged, low-grade itch[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Moreover, it is reported that gabapentin, which is commonly used for neuropathic pain, can safely and effectively relieve IL-2\u0026ndash;related pruritus, suggesting that IL-2\u0026ndash;induced itching may involve a neuropathic mechanism[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Accordingly, we examined the expression of IL-2 receptor subunits in DRG neurons following Nivolumab treatments. qPCR analysis showed that \u003cem\u003eIl2rb\u003c/em\u003e and \u003cem\u003eIl2rg\u003c/em\u003e, but not \u003cem\u003eIl2ra\u003c/em\u003e, transcripts were significantly upregulated after repeated Nivolumab administrations, a finding further validated at the protein level by RNAscope and Western blot. In contrast, a single Nivolumab injection failed to alter \u003cem\u003eIl2rb\u003c/em\u003e or \u003cem\u003eIl2rg\u003c/em\u003e expression. Given that IL-2Rβ and IL-2Rγ heterodimerize to form a βγ complex that binds IL-2 with intermediate affinity and is fully competent to transduce IL-2 signaling, these results suggest that repeated PD-1 blockade selectively enhances the IL-2Rβ/γ axis, thereby establishing a permissive neuronal environment for sustained IL-2 signaling and persistent itch.\u003c/p\u003e\u003cp\u003eTo further explore the cellular mechanisms underlying Nivolumab-induced itch, we analyzed single-cell datasets of DRG neurons. We found that \u003cem\u003ePtpn6\u003c/em\u003e, the gene encoding SHP-1, a key downstream effector of PD-1, is highly enriched in MrgprA3⁺ neurons. This observation is consistent with the findings reported by Han and colleagues[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Building on this, we employed RNAscope to examine whether \u003cem\u003eIl2rb and Il2rg\u003c/em\u003e mRNA are also localized in MrgprA3⁺ neurons following model induction. We found that \u003cem\u003eIl2rb, Il2rg\u003c/em\u003e and \u003cem\u003eMrgprA3\u003c/em\u003e showed a high level of co-expression. Furthermore, using primary cultures of sensory neurons, we demonstrated that Nivolumab directly binds to MrgprA3⁺ neurons. Because it has been reported that Nivolumab could increase the excitability of DRG neurons via modulating ion channels[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], this might explain the reason why Nivolumab induced transient scratching behavior under its application. In vivo, c-Fos expression was markedly increased in MrgprA3⁺ neurons after model induction, indicating robust neuronal activation. Although electrophysiological recordings were performed in TRPV1-reporter mice, excitability was significantly enhanced following Nivolumab treatment. Given that MrgprA3 is well known to mediate itch, and TRPV1 and MrgprA3⁺ neurons are highly co-expressed[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], these findings strongly suggest that Nivolumab promotes persistent itch through direct activation of pruriceptive MrgprA3⁺ neurons. Together with our observation that repeated PD-1 blockade upregulates IL-2Rβ/γ in MrgprA3⁺ neurons, this highlights a neuro\u0026ndash;immune crosstalk mechanism in which Nivolumab binding and enhanced IL-2 signaling converge to drive neuronal hyperexcitability and sustained itch transmission.\u003c/p\u003e\u003cp\u003eFinally, we investigated the molecular mechanisms by which repeated Nivolumab injections induce increased expression of Il2rb and Il2rg in neurons. Our findings revealed that repeated Nivolumab (anti\u0026ndash;PD-1) administration reduced SHP-1 phosphorylation while concomitantly increasing JNK and STAT5 activation in the DRG. Previous studies have shown that PD-1 engagement normally recruits and activates the phosphatase SHP-1, and PD-1 blockade (e.g. with Nivolumab) diminishes SHP-1 activity[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], thereby releasing the inhibition of downstream MAPK signaling, including the JNK pathway[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Importantly, JNK activation has been demonstrated to enhance STAT5 phosphorylation and transcriptional activity. In particular, JNK (a MAP kinase) can phosphorylate STAT5 on key serine residues, which augments STAT5\u0026rsquo;s nuclear translocation and transcriptional potency[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Moreover, phosphorylated STAT5 directly promotes the transcription of IL-2 receptor subunits. For example, STAT5 is known to bind to and upregulate \u003cem\u003eIl2rb\u003c/em\u003e (encoding the IL-2Rβ chain) in response to IL-2 signaling[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], and IL-2 stimulation (via STAT5) also contributes to the expression of the common γ-chain \u003cem\u003eIl2rg\u003c/em\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Taken together, these results support a model in which PD-1/SHP-1 inactivation unleashes the JNK\u0026ndash;STAT5 signaling axis, thereby driving the upregulation of IL-2Rβ/γ in pruriceptive DRG neurons, amplifying IL-2 signaling, and ultimately sustaining chronic itch.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur study establishes IL-2/IL-2Rβγ signaling in pruriceptive neurons as a driver of checkpoint inhibitor\u0026ndash;related chronic itch. Beyond providing mechanistic insight, these findings underscore the need to consider neuro\u0026ndash;immune pathways when addressing immune-related adverse events. Targeted modulation of IL-2 signaling may thus represent a promising strategy to improve PD-1\u0026ndash;based cancer immunotherapy.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cstrong\u003eDRG\u003c/strong\u003e: Dorsal root ganglion\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eICI\u003c/strong\u003e: Immune checkpoint inhibitor\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIgG4\u003c/strong\u003e: Immunoglobulin G4 (isotype control antibody)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIL-2\u003c/strong\u003e: Interleukin-2\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIL-2R\u003c/strong\u003e: Interleukin-2 receptor\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ei.p.\u003c/strong\u003e: Intraperitoneal injection\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ei.t.\u003c/strong\u003e: Intrathecal injection\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJNK\u003c/strong\u003e: c-Jun N-terminal kinase\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePD-1\u003c/strong\u003e: Programmed cell death protein-1\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eqPCR\u003c/strong\u003e: Quantitative polymerase chain reaction\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSHP-1\u003c/strong\u003e: Src homology region 2 domain-containing phosphatase-1\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSTAT5\u003c/strong\u003e: Signal transducer and activator of transcription 5\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTRPV1\u003c/strong\u003e: Transient receptor potential vanilloid 1\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were approved by the Animal Care and Use Committee of Shenzhen University (A202300939) and conducted in accordance with the guidelines of the International Association for the Study of Pain. Consent to participate is not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request. Publicly available single-cell RNA sequencing data used in this study were obtained from the Gene Expression Omnibus (GEO) under accession number GSE167910.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe present study was supported by the National Natural Science Foundation of China (82171221 and 82471240), Guangdong S\u0026amp;T Program (2023B0303010002), Funding by Science and Technology Projects in Guangzhou (Grant2023A03J1020), the Shenzhen Science and Technology Program (JCYJ20220818103206013), and Science and Technology Project of Shenzhen Nanshan District Health System (NSZD2025007, NSZD2023017).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eL.L., H.Z., and J.G. performed the majority of the experiments, data acquisition, and statistical analyses. H.L. and A.L. carried out animal experiments, behavioral assays, and molecular analyses. X.B., F.W., N.L., and X.L. assisted with immunostaining, RNAscope, electrophysiology, and data interpretation. X.W., Y.L., and C.J. conceived and supervised the study, designed experiments, and interpreted results. L.L. and C.J. drafted the manuscript. All authors reviewed and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGeisler, A.N., et al., \u003cem\u003eImmune checkpoint inhibitor-related dermatologic adverse events.\u003c/em\u003e J Am Acad Dermatol, 2020. \u003cstrong\u003e83\u003c/strong\u003e(5): p. 1255-1268.\u003c/li\u003e\n\u003cli\u003eSalinas, N., et al., \u003cem\u003eCauses of Pruritus in Patients Treated With Immune Checkpoint Inhibitors for Melanomas or Skin Carcinomas.\u003c/em\u003e Front Med (Lausanne), 2021. \u003cstrong\u003e8\u003c/strong\u003e: p. 632683.\u003c/li\u003e\n\u003cli\u003eNikolaou, V., A. Tsimpidakis, and A. 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\u003cstrong\u003e391-392\u003c/strong\u003e: p. 104758.\u003c/li\u003e\n\u003cli\u003eMaurer, B., et al., \u003cem\u003eSTAT5A and STAT5B-Twins with Different Personalities in Hematopoiesis and Leukemia.\u003c/em\u003e Cancers (Basel), 2019. \u003cstrong\u003e11\u003c/strong\u003e(11).\u003c/li\u003e\n\u003cli\u003eLin, J.X., et al., \u003cem\u003eTyrosine phosphorylation of both STAT5A and STAT5B is necessary for maximal IL-2 signaling and T cell proliferation.\u003c/em\u003e Nat Commun, 2024. \u003cstrong\u003e15\u003c/strong\u003e(1): p. 7372.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"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":"PD-1 blockade, Nivolumab, itch, Interleukin-2 (IL-2), IL-2 receptor","lastPublishedDoi":"10.21203/rs.3.rs-7654982/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7654982/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eBackground\u003c/b\u003e\u003c/p\u003e\u003cp\u003eImmune checkpoint inhibitor (ICI) therapy frequently induces pruritus as a cutaneous immune-related adverse event, affecting 13\u0026ndash;25% of patients treated with anti\u0026ndash;PD-1 antibodies. Unlike allergy-associated itch, ICI-induced pruritus often responds poorly to antihistamines, indicating a distinct mechanism. This study aimed to investigate the mechanisms by which repeated PD-1 blockade induces persistent itch and to identify molecular pathways linking immune activation with pruriceptor sensitization.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe established a mouse model of pruritus by repeated administration of Nivolumab subcutaneously. Behavioral assays were conducted to evaluate itch-like behaviors (scratching). The expression and distribution of IL-2 receptor subunits in dorsal root ganglia (DRG) were assessed using qPCR, RNAscope, and Western blotting. Electrophysiological recordings, fluorescent antibody labeling, immunostaining, and pharmacological interventions were employed to explore the cellular and molecular mechanisms.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA single Nivolumab injection induced transient scratching, whereas three consecutive injections triggered persistent itch lasting about one week beyond drug withdrawal. Persistent itch was accompanied by dermal CD4⁺ T-cell infiltration and elevated serum IL-2. Neutralization of IL-2 abolished persistent but not transient itch. In DRG, repeated Nivolumab selectively upregulated IL-2 receptor β and γ subunits, localized predominantly to MrgprA3⁺ pruriceptors. These neurons exhibited enhanced excitability, c-Fos induction, and direct Nivolumab binding. Mechanistically, PD-1 blockade suppressed SHP-1 phosphorylation, promoted JNK and STAT5 activation, and drove IL-2Rβ/γ upregulation. JNK inhibition prevented IL-2R induction and alleviated persistent itch without affecting acute responses.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusion\u003c/b\u003e\u003c/p\u003e\u003cp\u003eOur findings demonstrate that repeated Nivolumab administration upregulates IL-2Rβ/γ in MrgprA3⁺ neurons via SHP-1\u0026ndash;JNK\u0026ndash;STAT5 signaling, together with elevated systemic IL-2, establishing a neuroimmune loop that drives ICI-induced pruritus.\u003c/p\u003e","manuscriptTitle":"IL-2/IL-2Rβγ signaling in pruriceptors drives neuroimmune mechanisms of nivolumab- induced persistent itch","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-08 07:20:10","doi":"10.21203/rs.3.rs-7654982/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-21T11:21:46+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-16T07:29:59+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-15T00:49:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"94978915506511875755658836440387435326","date":"2025-10-01T00:09:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"275886043972832211921878745640216246345","date":"2025-09-30T12:23:20+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-27T23:51:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"77584380696208458381374270747611833457","date":"2025-09-26T17:15:20+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-24T14:02:41+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-22T05:18:44+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-22T02:01:15+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Neuroinflammation","date":"2025-09-19T06:00:29+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":"f154a870-1949-40cc-ad2e-0a1dad68e903","owner":[],"postedDate":"October 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-08T16:10:39+00:00","versionOfRecord":{"articleIdentity":"rs-7654982","link":"https://doi.org/10.1186/s12974-025-03626-w","journal":{"identity":"journal-of-neuroinflammation","isVorOnly":false,"title":"Journal of Neuroinflammation"},"publishedOn":"2025-12-05 15:58:28","publishedOnDateReadable":"December 5th, 2025"},"versionCreatedAt":"2025-10-08 07:20:10","video":"","vorDoi":"10.1186/s12974-025-03626-w","vorDoiUrl":"https://doi.org/10.1186/s12974-025-03626-w","workflowStages":[]},"version":"v1","identity":"rs-7654982","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7654982","identity":"rs-7654982","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}