ROCK2 Inhibition Suppresses Cytotoxic T Lymphocyte-Mediated Platelet Destruction in Primary Immune Thrombocytopenia Running title: ROCK2 inhibition reduces CTL platelet injury

ROCK2 Inhibition Suppresses Cytotoxic T Lymphocyte-Mediated Platelet Destruction in Primary Immune Thrombocytopenia Running title: ROCK2 inhibition reduces CTL platelet injury | 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 ROCK2 Inhibition Suppresses Cytotoxic T Lymphocyte-Mediated Platelet Destruction in Primary Immune Thrombocytopenia Running title: ROCK2 inhibition reduces CTL platelet injury Juanjuan Song, Bingjie Ding, Qian Liu, Ao Xie, Mengjuan Li, Xuewen Song, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8913555/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 13 You are reading this latest preprint version Abstract Cytotoxic T lymphocyte (CTL)-mediated platelet destruction represents an important pathogenic mechanism in ITP patients. Rho-associated coiled-coil kinase 2 (ROCK2) is an emerging regulator of immune balance, but its role in pathogenic CTL activation in ITP remains undefined. Here, we demonstrated that selective ROCK2 inhibition with KD025 potently suppressed CTL-mediated platelet destruction. In vitro , KD025 treatment of CTLs from ITP patients suppressed key effector functions, reducing degranulation as measured by CD107a expression, diminishing the secretion of cytotoxic molecules such as granzyme B and perforin, and decreasing CTL-platelet conjugate formation, resulting in reduced platelet apoptosis and activation. RNA-sequencing revealed downregulation of cytotoxic and glycolytic programs, with enrichment of JAK-STAT signaling. Mechanistically, KD025 reversed the pathogenic metabolic shift in ITP CTLs by lowering glycolytic flux and restoring mitochondrial respiration, accompanied by decreased STAT3 phosphorylation. IL-6-mediated STAT3 activation largely reversed these effects, indicating a ROCK2-STAT3-dependent mechanism. In vivo , both daily KD025 administration to an active ITP mouse model and transplantation of KD025-pretreated CD8 + T cells into irradiated Rag1 −/− mice alleviated CTL-mediated platelet apoptosis and increased platelet counts. Collectively, these findings identified a ROCK2-STAT3 regulatory axis integrating transcriptional and metabolic control of CTL pathogenicity and support ROCK2 inhibition as a promising therapeutic strategy for ITP. primary immune thrombocytopenia cytotoxic T lymphocyte ROCK2 KD025 STAT3 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Primary immune thrombocytopenia (ITP) is an acquired autoimmune disorder characterized by accelerated platelet destruction and impaired platelet production, leading to an increased risk of bleeding. 1 , 2 Traditionally, autoantibody-mediated platelet clearance via Fcγ receptor (FcγR)-bearing macrophages has been considered the central pathogenic mechanism. 3 However, accumulating evidence highlights a complementary and clinically important antibody-independent pathway: cytotoxic T lymphocyte (CTL)-mediated platelet destruction. 4 , 5 CTLs can kill platelets directly through granzyme/perforin-mediated lysis and death receptor signaling pathways. 6 , 7 The formation of CTL-platelet conjugates and the ensuing polarized secretion of cytotoxic granules represent key mechanistic step in CTL-driven platelet lysis. 8 Despite the increasing availability of new therapeutic agents, a substantial proportion of patients remain refractory to treatments, underscoring the urgent need for novel strategies that target alternative pathogenic mechanisms, particularly CTL-mediated platelet destruction. 9 Rho-associated coiled-coil kinase 2 (ROCK2) is a serine/threonine kinase with dual functions in orchestrating cellular processes. 10 , 11 While initially recognized for its canonical function in orchestrating actin cytoskeletal dynamics, which is crucial for cell adhesion and migration, 12,13 a growing body of work has revealed its non-canonical role as a direct modulator of key signaling pathway. 14 , 15 This includes its ability to interact with and regulate transcription factors such as STAT, thereby controlling T cell differentiation and metabolism. 16 This dual functionality makes ROCK2 a particularly attractive therapeutic target in T-cell-driven autoimmune diseases. The selective ROCK2 inhibitor KD025, approved for the treatment of chronic graft-versus-host disease (GVHD), exerts robust immunomodulatory effects by dampening inflammatory signaling pathways and facilitating the restoration of immune homeostasis. 17 , 18 However, its direct effects on CTL activity—particularly cytotoxic molecule expression, degranulation capacity, and CTL-platelet conjugate formation—remain poorly characterized in ITP. Elucidating these mechanisms is crucial, as modulation of CTL-mediated cytotoxic pathways may provide therapeutic benefit for patients with refractory ITP who respond inadequately to conventional immunosuppressive therapy. In this study, we examined how ROCK2 inhibition with KD025 affected the effector function and metabolic profile of CTLs from ITP patients, focusing on cytotoxicity, degranulation, and CTL-platelet conjugate formation. By integrating functional, transcriptional, and metabolic analyses, we delineated a ROCK2-STAT3-dependent immunometabolic program that drove pathogenic CTL activity in ITP, thereby supporting ROCK2 inhibition as a potential therapeutic strategy for CTL-mediated autoimmunity. Materials and Methods Patients and healthy controls Adult patients with ITP were prospectively enrolled at Qilu Hospital, Shandong University, and Henan Cancer Hospital, Zhengzhou University. Active ITP was defined according to the 2019 ASH guidelines. 19 Peripheral blood samples were obtained before initiation of ITP-specific therapy. An initial cohort of 35 patients with active ITP was screened. CTL induced platelet apoptosis was quantified as described in the Supplementary Methods , and patients with values above the upper limit of the healthy volunteer range were classified as CTL-mediated ITP. 20 Seventeen patients fulfilled this criterion and were included in all subsequent immunologic and mechanistic analyses. At enrollment, these 17 patients (11 females and 6 males; age range, 19–68 years; median age, 45 years), exhibited platelet counts ranging from 1 × 10⁹/L to 29 × 10⁹/L (median platelet count, 15 × 10⁹/L). Six ITP patients in remission with normal platelet counts and of treatment were enrolled as a comparator group. Baseline demographic and hematologic characteristics were provided in Supplementary Table 1 . Thirty-two age- and sex-matched healthy volunteers (18 females and 14 males; age range, 26–72 years; median age, 48 years) were recruited as healthy controls (HCs), with platelet counts ranging from 157 × 10 9 to 294 × 10 9 /L (median, 254 × 10 9 /L). All participants gave written informed consent, and the study was approved by the ethics committees of the participating centers in accordance with the Declaration of Helsinki. Active ITP mouse model An active ITP mouse model was established as previously described. 21 Briefly, C57BL/6 Cd61 -knockout ( Cd61 −/− ) mice were immunized weekly with platelets isolated from syngeneic wild-type (WT) C57BL/6 male mice (8–12 weeks old). Splenocytes from immunized mice were harvested, and 5 × 10⁴ cells per mouse were intravenously transferred into sublethally irradiated severe combined immunodeficient ( Rag1 −/− ) mice to construct the active ITP murine model. The strains and backgrounds of the mice are detailed in the Online Supplementary Methods. At the time of splenocyte transfer, recipient mice were randomized to receive the following treatments: 2% DMSO control (intraperitoneal injection, once every 2 days and oral gavage, every day; Sigma-Aldrich), KD025 (100 mg/kg/day, oral gavage; MedChemExpress), IL‑6 (2 mg/kg, intraperitoneal injection, once every 2 days; PeproTech), and the combination of IL-6 and KD025. Platelet counts were determined weekly. On day 28, mice were anesthetized and euthanized, and splenocytes were collected for flow cytometric (FCM) analysis. To determine whether the therapeutic benefit of KD025 was mediated through direct modulation of CD8⁺ T cells, CD8⁺ T cells were purified from anti-CD61-sensitized splenocytes and cultured in vitro with KD025 (1 µM) or 0.1% DMSO for 72 hours. These pretreated CD8⁺ T cells were then recombined with CD8⁺ T-cells-depleted splenocytes and adoptively transferred into irradiated Rag1 −/− mice to induce ITP. All animal experiments were approved by the Animal Care and Use Committee of Qilu Hospital and conducted in accordance with institutional guidelines of Shandong University. Detailed descriptions of PBMC and CD8⁺ T-cell isolation and culture, FCM, qRT-PCR, ELISA, Seahorse metabolic flux analysis, and immunofluorescence staining are provided in the Supplementary Methods. Results ROCK2 expression was elevated in ITP patients and mice Using an ELISA-based assay, we observed that ROCK kinase activity was significantly higher in PBMCs from patients with active ITP than from HCs, and was markedly reduced in ITP patients in remission (Fig. 1 A). Consistently, qRT-PCR analysis confirmed upregulation of ROCK2 mRNA expression in PBMCs from ITP patients compared to HCs (Fig. 1 B). Moreover, immunofluorescence staining of spleen sections revealed stronger ROCK2 signals in CD8 + T cells from ITP patients than from HCs who underwent traumatic splenectomy (Fig. 1 C). To further evaluate the role of ROCK2 in the pathogenesis of ITP, we next assessed ROCK2 expression in spleen tissues from a murine model of active ITP (Fig. 1 D). In line with human data, qRT-PCR analysis showed a significant increase in Rock2 mRNA levels in the spleens of ITP mice compared with WT mice (Fig. 1 E). Furthermore, immunofluorescence analysis demonstrated enhanced ROCK2 expression within CD8⁺ T cells in the spleens of ITP mice relative to WT mice (Fig. 1 F). KD025 suppressed CD8 T-cell proliferation, degranulation, cytotoxicity, and CTL cell-platelet interactions in ITP patients The optimal concentration of KD025, a selective ROCK2 inhibitor, was determined using a range of gradient doses. Early apoptotic PBMCs were identified as Annexin V⁺/PI⁻ cells by FCM. To determine the working concentration of KD025, PBMCs from ITP patients were cultured with graded doses, and early apoptosis (Annexin V⁺/PI⁻) was assessed by FCM. KD025 at 5 and 10 µM significantly increased PBMC apoptosis, whereas 0.5 and 1 µM had no pro-apoptotic effect ( Supplemental Fig. 1A ). Therefore, 1 µM KD025 was selected for subsequent in vitro experiments. To explore the functional effects of KD025 on CTLs, 5(6)-carboxyfluorescein diacetate N-succinimidyl ester (CFSE)-labeled CD8⁺ T cells stimulated with anti-CD3/CD28 antibodies were cultured in the presence or absence of KD025. As shown in Supplemental Fig. 1B , the division index in the KD025-treated group was significantly lower than that in the DMSO control group, indicating that KD025 suppresses CTL proliferation. Next, CTL cytotoxicity was assessed by analyzing platelet apoptosis after 72 hours of co-culture, as determined by mitochondrial membrane depolarization using JC‑1 staining (loss of JC-1 aggregate signal and increase in JC‑1 monomer signal). Platelets cocultured with CTLs exhibited higher levels of apoptosis than platelets cultured alone. In contrast, the addition of KD025 to CTL-platelet cocultures significantly reduced platelet apoptosis compared with coculture with CTLs alone (Fig. 2 A). Additionally, platelet CD62P expression, as one of the marker for platelet activation, was markedly increased after co-culture with CTLs, and this increase was significantly attenuated when KD025 was present during the co-culture (Fig. 2 B), suggesting that KD025 could reduce CTL-mediated platelet activation. CD107a is normally expressed on granule membranes of CTLs and becomes exposed on the cell surface following degranulation, thereby serving as a standard marker of CTL degranulation 8 . Membranous CD107a expression on CD8 + T cells was significantly elevated after co-culture with platelets compared with CTLs cultured alone, and was significantly reduced when KD025 was present during the co‑culture (Fig. 2 C). To assess whether ROCK2 inhibition affects the ability of CTLs to physically engage with their targets, CD8 + T cells from ITP patients were co-cultured with platelets from HCs for 4 hours. KD025 treatment substantially reduced CTL-platelet aggregate formation compared to DMSO controls, as quantified by CD8 + CD61 + aggregates (Fig. 2 D). Consistently, confocal microscopy further confirmed reduced CTL-platelet aggregate formation in KD025-treated co-culture compared with the DMSO controls (Fig. 2 D). These observations suggested that ROCK2 activity was involved in the stable adhesion between CTLs and platelets, a critical prerequisite for targeted cytotoxicity. We next assessed cytotoxic granule secretion by CTLs. Levels of granzyme B (GZMB) and perforin (PRF1) in culture supernatants were significantly decreased after KD025 treatment, whereas granzyme A (GZMA) remained unchanged (Fig. 2 E). At the transcriptional level, KD025-treated CTLs exhibited significantly lower mRNA expression of GZMA , GZMB , PRF1 , TBX21 and Eomesodermin ( EOMES ; Fig. 2 F). Thus, KD025 attenuated the cytotoxic machinery of CTLs at both protein and transcriptional levels and suppressed proliferation, degranulation, and CTL-platelet interactions in ITP. RNA sequencing revealed substantial transcriptional reprogramming between DMSO- and KD025-treated CD8⁺ T cells. The volcano plot demonstrated that 397 genes were upregulated and 624 were downregulated after KD025 treatment, while 15,322 genes remained unchanged (Fig. 3 A). Multiple cytotoxic effector molecules, including GZMA , GZMB , GZMK , GZMH , FASLG , and IFNG , were consistently reduced by KD025 (Fig. 3 B). RNA-sequencing further showed that multiple glycolysis-related genes, including HK2, PFKM, PKM, ENO1, LDHA, and PFKFB3, were downregulated after KD025 treatment (Fig. 3 C). KEGG pathway and GO enrichment analyses of differentially expressed genes (DEGs) indicated that KD025 predominantly downregulated cell-cycle- and proliferation-related programs, and modulated cytokine-driven signaling, including JAK-STAT pathways, while GSEA revealed decreased enrichment of MYC targets and mTORC1 signaling (Fig. 3 D - F). Together, these results indicated that KD025 induced coordinated transcriptional changes that reshaped CTL effector function. KD025 attenuated CD61-driven activation and cytotoxicity of CD8⁺ T cells in Cd61 −/− mice To investigate the in vivo impact of KD025 on platelet antigen-specific CD8⁺ T-cell responses, Cd61 −/− mice were intravenous transfused weekly with platelets from WT mice and concurrently administered KD025 (100 mg/kg) or a 2% DMSO daily for 4 consecutive weeks. At the end of treatment, splenocytes were harvested, CD8 + T cells were isolated, and then cocultured with WT platelets for 4 hours (Fig. 4 A). Consistent with the in vitro findings, CD8⁺ T cells from KD025-treated Cd61 −/− mice induced significantly less platelet apoptosis (Fig. 4 B) and activation (Fig. 4 C) than those from DMSO-treated controls. Functionally, KD025 also restrained CD8⁺ T-cell degranulation, as evidenced by reduced surface CD107a expression (Fig. 4 D). In parallel, KD025-treated mice displayed diminished CTL-platelet aggregates, reflected by a lower proportion of CD8 + CD61 + population after coculture (Fig. 4 E). These results suggested that KD025 mitigated CD61-induced activation and effector responses of CD8⁺ T cells in Cd61 −/− mice. Metabolic reprogramming in CD8 + T cells from ITP patients and its regulation by ROCK2 inhibition Metabolic reprogramming is a hallmark of CD8 + T-cell activation and proliferation, characterized by enhanced glycolysis, amino acid turnover, and protein synthesis, concomitant with decreased aerobic glucose oxidation. 7 To evaluate metabolic activity in CD8 + T cells, we performed a Seahorse glycolysis and mitochondrial stress assay, measuring the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) as indicators of glycolytic flux and mitochondrial respiration, respectively. Following glucose injection, CTLs from ITP patients displayed a markedly greater increase in ECAR compared to HCs, indicating hyperactivated glycolytic metabolism. Quantitative analysis confirmed that basal glycolysis, glycolytic capacity, and glycolytic reserve were all significantly elevated in ITP-derived CTLs (Fig. 5 A). In contrast, basal OCR prior to oligomycin injection was significantly lower in CTLs from the ITP patients than from HCs. Consistently, basal respiration, maximal respiratory, ATP-linked respiration and spare respiratory capacity were all markedly reduced in ITP CTLs (Fig. 5 B), indicating impaired mitochondrial oxidative phosphorylation (OXPHOS). At the transcriptional level, the expression of key glycolytic genes—including glucose transporter 1 ( GLUT1 ), hexokinase 2 ( HK2 ), pyruvate kinase M2 ( PKM2 ), and lactate dehydrogenase ( LDHA )—was significantly upregulated in CD8⁺ T cells from ITP patients compared with HCs (Fig. 5 C), further supporting a glycolytic metabolic bias. To delineate the underlying mechanisms by which ROCK2 inhibition regulates CD8 + T cells, we next evaluated the bioenergetic status of cultured CD8 + T cells from ITP patients. KD025 treatment significantly decreased ECAR, including reductions in basal glycolysis, glycolytic capacity, and glycolytic reserve (Fig. 5 D). By contrast, OCR was markedly increased, accompanied by enhanced basal respiration, maximal respiratory, ATP-linked respiration and spare respiratory capacity (Fig. 5 E). Consistent with these functional metabolic changes, qRT-PCR analysis revealed that KD025 treatment downregulated glycolysis-related genes, including GLUT1 , HK2 , PKM2 , and LDHA , in CD8 + T cells from ITP patients (Fig. 5 F). ROCK2 inhibition suppressed STAT3 phosphorylation to attenuate CTL-mediated platelet destruction Having established that ROCK2 inhibition profoundly suppresses CTL effector functions, we next sought to identify the upstream signaling mechanism. Pathway enrichment analysis of our RNA sequencing data revealed the JAK-STAT signaling pathway as a top hit. Given the pivotal role of STAT3 in controlling T-cell cytotoxicity, proliferation, and metabolism 22 – 24 , we specifically hypothesized that STAT3 is the critical signaling node downstream of ROCK2 that orchestrates CTL pathogenicity. To test this, we evaluated whether ROCK2 inhibition could restrain STAT3 activation in vitro . PBMCs were treated with KD025 or DMSO, and p-STAT3 expression in CD8 + T cells were quantified by FCM. KD025 treatment significantly reduced p-STAT3 MFI compared to DMSO controls (Fig. 6 A). Consistent with this in vitro effect, in an active ITP mouse model, oral administration of KD025 substantially reduced p-STAT3 expression in splenic CD8⁺ T cells compared relative to DMSO-treated mice (Fig. 6 B). Together, these data demonstrated that ROCK2 activity was required for robust STAT3 phosphorylation in CTLs both in vitro and in vivo . To functionally confirm that the suppression of STAT3 signaling was the key mechanism responsible for KD025's effects, we designed a comprehensive rescue experiment. We reasoned that if the STAT3 pathway was the critical mediator, then reactivating it downstream of the ROCK2 block should restore the full spectrum of CTL effector functions. Indeed, co-treatment with IL-6, a potent STAT3 activator, not only enhanced CTL proliferation (Fig. 6 C), but also restored the overall cytotoxic capacity of CTLs suppressed by KD025. To determine if this restoration translated to the ultimate pathological outcome, we assessed direct CTL-mediated platelet damage. In these assays, the protective effect of KD025 on platelet apoptosis was significantly reversed by the addition of IL-6 (Fig. 6 D). This hierarchical response was mirrored across a range of parallel functional readouts. Specifically, platelet activation (CD62P), CTL degranulation (CD107a), and CTL-platelet aggregate formation were all maximal in the IL-6 group, minimal in the KD025 group, and displayed an intermediate phenotype with the combination treatment. (Fig. 6 E - G). Taken together, this comprehensive array of rescue data provided definitive evidence that the ROCK2-STAT3 signaling axis was a central molecular pathway fueling CTL activation and cytotoxicity toward platelets in ITP. IL‑6/STAT3 signaling counteracted ROCK2 inhibition-mediated metabolic reprogramming in CD8⁺ T cells Given that ROCK2 is a critical upstream regulator of STAT3 activation, we next examined whether IL‑6-STAT3 signaling could interfere with KD025‑induced metabolic reprogramming in CD8⁺ T cells from ITP patients. In the glycolysis stress test (Fig. 7 A), KD025 treatment markedly reduced ECAR over time, with glycolysis, glycolytic capacity, and glycolytic reserve all significantly decreased compared with DMSO (Fig. 7 B), indicating a broad suppression of glycolytic function. IL-6 co-stimulation largely restored ECAR in KD025-treated cells, significantly increasing glycolysis, glycolytic capacity, and glycolytic reserve relative to KD025 alone, thereby re-establishing a glycolytic phenotype. Conversely, in the mitochondrial stress test (Fig. 7 C), KD025 significantly enhanced OCR, with increased basal respiration, maximal respiration, ATP production and spare respiratory capacity compared with DMSO (Fig. 7 D), consistent with a shift toward oxidative phosphorylation. IL-6 partially reversed these effects: all 4 parameters were significantly reduced in the IL-6 plus KD025 group versus KD025 alone, trending back toward the IL-6-driven glycolytic profile. These results indicated that ROCK2 inhibition ameliorated abnormal CTL immunometabolism in ITP patients through STAT3 suppression. KD025 ameliorated thrombocytopenia in active ITP mice To determine whether the in vitro findings of KD025 could be recapitulated in vivo , an active murine model of ITP was established (Fig. 8 A). Following irradiation and adoptive transfer of anti-CD61-immunized splenocytes into recombination-activating gene 1 ( Rag1 −/− ) mice, platelet counts gradually declined, reaching a nadir by day 14. KD025 treatment significantly ameliorated thrombocytopenia, with platelet counts remaining markedly higher than control on day 21 and day 28. In contrast, IL-6 administration induced the most profound platelet suppression on day 28. Notably, co-administration of IL-6 with KD025 abolished the therapeutic benefit of KD025, as platelet counts in the IL-6 plus KD025 group failed to increase and were markedly lower than those in the KD025 group, indicating that IL-6 abolished the therapeutic benefit of KD025 (Fig. 8 B). To further verify whether the platelet-protective effect of KD025 was maintained at the effector-cell level, splenic CD8⁺ T cells were purified from each treatment group and co-cultured with WT platelets for 4 hours. CD8⁺ T cells from KD025-treated group induced significantly less platelet apoptosis than those from DMSO control group, confirming an attenuation of platelet-destructive activity. In contrast, IL-6 treatment significantly increased platelet apoptosis, and IL-6 plus KD025 conditioning completely abrogated the protective effect of KD025, yielding apoptosis levels comparable to those observed in the DMSO control group (Fig. 8 C and Supplemental Fig. 2A ). Platelet CD62P expression showed a consistent pattern, with KD025 markedly reducing platelet activation compared with control, whereas IL-6 co-treatment significantly weakened this inhibitory effect of KD025 on platelet activation (Fig. 8 C and Supplemental Fig. 2B ). Consistent with these findings, KD025 directly dampened CD8⁺ T-cell cytotoxic function in vivo . Compared to DMSO controls, KD025 significantly reduced the MFI of CD107a on CTLs (Fig. 8 C and Supplemental Fig. 2C ) and decreased the frequency of CLT-platelet aggregates (Fig. 8 C and Supplemental Fig. 2D ). Importantly, concurrent IL-6 administration fully reversed these inhibitory effects, restoring CD107a expression and CTL-platelet conjugate formation to levels comparable to those in controls (Fig. 8 C). To directly determine whether the therapeutic benefit of KD025 was mediated through intrinsic reprogramming of CD8⁺ T cells, CD8⁺ T cells were purified from anti-CD61-sensitized splenocytes and pretreated in vitro with KD025 or 0.1% DMSO for 72 hours. These pretreated CD8⁺ T cells were then recombined with CD8⁺ T-cells-depleted splenocytes and adoptively transferred into irradiated Rag1 −/− mice (Fig. 8 D). Mice receiving KD025-treated CD8⁺ T cells exhibited significantly higher platelet counts on day 21 (Fig. 8 E) and markedly reduced platelet apoptosis (Fig. 8 F) compared with mice receiving DMSO‑treated CD8⁺ T cells. These results suggested that KD025‑treated CD8⁺ T cells exhibited diminished platelet‑destructive activity in vivo . Discussion ITP has long been regarded as an autoantibody-mediated disorder, while accumulating evidence supports an important contribution of CTL-mediated platelet destruction. 25 – 27 Effector CD8⁺ T cells induce platelet apoptosis via exocytosis of cytotoxic granules, forming stable conjugates with platelets and delivering polarized granule secretion that triggers caspase activation and programmed platelet death. 28 , 29 In parallel, T-cell receptor (TCR) engagement triggers a metabolic switch from mitochondrial OXPHOS toward aerobic glycolysis to support clonal expansion and acquisition of effector function. 30 Our study extended these concepts to ITP by demonstrating that CD8⁺ T cells from patients underwent pathological immunometabolic reprogramming, characterized by enhanced glycolysis, reduced oxidative phosphorylation, and upregulation of glycolysis-related genes, consistent with a glycolysis-addicted, bioenergetically inflexible state that sustained pathogenic CTL activation. In this context, we showed that KD025, a selective ROCK2 inhibitor, restored metabolic balance by suppressing glycolytic flux and enhancing OXPHOS, thereby attenuating CTL-mediated platelet destruction. The therapeutic efficacy of KD025 in ITP appeared to be multifaceted, integrating effects on both the physical interaction and the intrinsic cytotoxic programming of CTLs. The observed reduction in CTL-platelet conjugate formation was consistent with the canonical function of ROCK2 as a master regulator of the actin cytoskeleton, which was essential for stable immune synapse assembly and cell adhesion. 31 , 32 While this modulation of cell mechanics likely represented an important layer of regulation, our data revealed a more profound impact on the core pathogenic identity of CTLs. We found that KD025 could dismantle the STAT3-OXPHOS signaling axis, a central metabolic and transcriptional engine for T-cell effector function. This aligned with the emerging paradigm of ROCK2 acting as a non-canonical signal transducer that directly influenced transcription factor activity in the nucleus. 16 , 33 Therefore, we proposed that KD025's potent suppressive effect stemmed from a powerful synergy: it not only disrupted the physical engagement of CTLs with platelets but, more critically, it de-energized and transcriptionally rewired the CTLs, stripping them of their pathogenic capabilities. STAT3 phosphorylation is a central regulator of CD8⁺ T-cell biology. 23 , 34 It promotes survival and clonal expansion, upregulates perforin and granzyme B, and augments production of inflammatory cytokines such as IFN-γ and TNF-α. 24,35 Activating STAT3 mutations in CD8⁺ T cells correlate with heightened cytotoxicity in rheumatoid arthritis 36 , and STAT3 also contributes to the shift from OXPHOS to glycolysis during CD8⁺ T-cell activation by enhancing glycolytic enzymes and glucose transporters. 36 , 37 ROCK2 has recently emerged as an upstream node that couples cellular metabolism, inflammation, and cytoskeletal dynamics. 38 , 39 Meanwhile, ROCK2 signaling can drive glycolysis-dependent pathogenic programs in multiple tissues and disease settings, 40–42 and pharmacologic ROCK2 inhibition with KD025 has shown therapeutic efficacy in immune-mediated and fibrotic conditions. 33 , 43 , 44 These observations led us to hypothesize that aberrant ROCK2 activity might sustain a STAT3-dependent, glycolysis-biased program in CTLs from ITP patients. In line with this, we observed increased ROCK kinase activity and elevated ROCK2 mRNA expression in PBMCs from ITP patients, as well as in spleens from active ITP mice. Previous studies have reported that ROCK2 could phosphorylate and activate downstream transcription factor STAT3, thereby promoting pathogenic Th17 polarization and impairing regulatory T-cell function in autoimmune settings. 18 , 41 Consistent with a ROCK2-STAT3 signaling axis in CTLs, we found that ROCK2 inhibition with KD025 significantly reduced STAT3 phosphorylation in vitro and in vivo . This suppression of STAT3 signaling coincided with normalization of CTL metabolism and attenuation of their cytotoxic program, suggesting that aberrant ROCK2-STAT3 activity sustains both the effector and metabolic phenotypes of pathogenic CTLs in ITP. Functionally, ROCK2 inhibition with KD025 curtailed multiple steps in the CTL-mediated platelet destruction cascade. In human ITP CTLs, KD025 reduced proliferation, degranulation (CD107a exposure), and secretion of granzyme B and perforin, and it diminished CTL-platelet conjugate formation. As a result, platelets co-cultured with KD025-treated CTLs exhibited lower apoptosis and decreased CD62P expression, indicating reduced activation and damage. Similar inhibitory effects were observed in Cd61 −/− mice immunized with WT platelets, where KD025 treatment reduced platelet apoptosis, CD62P upregulation, CTL degranulation, and CTL-platelet aggregates. These findings supported the notion that ROCK2 inhibition attenuated antigen-specific CTL responses against platelet antigens. A key mechanistic insight from our study was that KD025 corrected abnormal CTL immunometabolism in ITP via STAT3 suppression. In CD8⁺ T cells from cytotoxic ITP patients, KD025 inhibited glycolysis and enhanced mitochondrial respiration, accompanied by downregulation of glycolytic genes. Conversely, IL-6, a canonical STAT3 activator, restored glycolytic activity and reversed KD025-mediated increases in OXPHOS. Functionally, IL-6 also rescued CTL proliferation, degranulation, cytotoxicity, and CTL-platelet aggregate formation that had been suppressed by KD025. Thus, the ROCK2-STAT3 axis appeared to act as a metabolic and transcriptional hub: ROCK2 inhibition down-modulated STAT3 to shift CTLs from a glycolysis-dominated, highly cytotoxic state toward a more quiescent, OXPHOS-supported phenotype, whereas IL-6-driven STAT3 activation counteracted this reprogramming and reinstated pathogenic CTL function. Our in vivo data provided proof-of-concept that targeting this axis could ameliorate disease. In an active ITP model that recapitulates both humoral and cellular anti-platelet immunity, KD025 treatment significantly increased platelet counts and reduced CTL-mediated platelet destruction. Phenotypically, CD8⁺ T cells from KD025-treated mice displayed less degranulation and fewer interactions with platelets. These findings indicated that ROCK2 inhibition could attenuate ongoing platelet damage in a complex autoimmune milieu. However, because transferred splenocytes contained heterogeneous lymphocyte populations, including CD4⁺ T cells and B cells, we could not fully exclude KD025 effects on other immune subsets that contributed to disease modulation. To more directly interrogate CD8⁺ T cells, we performed adoptive transfer experiments using ex vivo KD025-pretreated CTLs recombined with CD8-depleted splenocytes. Mice receiving KD025-conditioned CD8⁺ T cells exhibited higher platelet counts and lower platelet apoptosis on day 21 than mice receiving DMSO-treated CD8⁺ T cells, although this benefit diminished by day 28 as thrombocytopenia naturally improved in both groups. These results indicated that intrinsic reprogramming of CD8⁺ T cells by KD025 was sufficient to transiently mitigate platelet destruction even in the presence of humoral autoimmunity. Our study had several limitations. The number of cytotoxic ITP patients undergoing detailed metabolic profiling was modest, and larger cohorts were needed to validate ROCK2-STAT3-driven metabolic signatures as biomarkers. We focused mainly on CD8⁺ T cells in the study. However, the effects of ROCK2-STAT3 signaling on CD4⁺ T cells, B cells, and myeloid cells in ITP were also worth further evaluation. Potential off-target effects of KD025 were not systematically assessed, and murine ITP models only partially recapitulated the chronic and heterogeneous nature of human disease. In conclusion, we identified the ROCK2-STAT3 axis as a central immunometabolic checkpoint in ITP. ROCK2 inhibition with KD025 downregulated STAT3 phosphorylation, shifted CD8⁺ T-cell metabolism from pathological glycolysis toward OXPHOS, and attenuated CTL-mediated platelet destruction in vitro and in vivo . These findings supported ROCK2 inhibition as a strategy to restore immune homeostasis and reduce platelet destruction, particularly in CTL-driven disease. Declarations Data availability statement For original data, please contact [email protected] . Ethics statement Our study was approved by the ethics committees of Qilu Hospital of Shandong University according to the Helsinki Principles. Patient consent statement Written informed consent was obtained from all participants included in the study. Authorship contributions Contribution: J.S., A.X., Q.L., and B.D. performed the experiments, M.L., X.S., and F.W. analyzed the results and created the figures, J.H., L.L., and J.S. designed the research and wrote the paper; X.L., and H.Z. funded the research and edited the manuscript. Competing Interests The authors declare no competing financial interests. Funding information This work was supported by the National Key Research and Development Program of China (2024YFC2510500), National Natural Science Foundation of China (No. 82570171 and No. 82170123), Young Taishan Scholar Foundation of Shandong Province (Grant/Award No. tsqn202312325), Natural Science Foundation of Henan Province (No. 252300421374) and Henan Province Young and Middle-aged Health Science and Technology Innovation Leading Talent Training Program (No. YXKC2022008). Author Contribution Contribution: J.S., A.X., Q.L., and B.D. performed the experiments, M.L., X.S., and F.W. analyzed the results and created the figures, J.H., L.L., and J.S. designed the research and wrote the paper; X.L., and H.Z. funded the research and edited the manuscript. References Kuen DS, Park M, Ryu H, et al. Critical regulation of follicular helper T cell differentiation and function by Gα(13) signaling. 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of Medicine, Shandong University","correspondingAuthor":true,"prefix":"","firstName":"Xinguang","middleName":"","lastName":"Liu","suffix":""},{"id":597766856,"identity":"5f2661bb-2a1a-47bf-9e08-b219becf2c90","order_by":10,"name":"Hu Zhou","email":"","orcid":"","institution":"Affiliated Cancer Hospital of Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Hu","middleName":"","lastName":"Zhou","suffix":""}],"badges":[],"createdAt":"2026-02-19 03:38:48","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8913555/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8913555/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104404540,"identity":"4e23d839-ba99-46ef-94f6-2f9aca6065f2","added_by":"auto","created_at":"2026-03-11 12:20:29","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3172643,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eROCK2 expression was elevated in active ITP patients and in an active ITP mouse model.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) ROCK kinase activity in PBMCs from patients with active ITP, ITP patients in remission, and HCs. \u003cem\u003eP \u003c/em\u003evalues: active ITP \u003cem\u003evs\u003c/em\u003e. ITP in remission, \u003cem\u003eP\u003c/em\u003e = 0.014; active ITP \u003cem\u003evs\u003c/em\u003e. HCs, \u003cem\u003eP\u003c/em\u003e = 0.003. (\u003cstrong\u003eB\u003c/strong\u003e) \u003cem\u003eROCK2\u003c/em\u003e mRNA expression in PBMCs from patients with active ITP and HCs, assessed by qRT‑PCR (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001). (\u003cstrong\u003eC\u003c/strong\u003e) Representative immunofluorescence staining of ROCK2 (green) and CD8 (red) in spleen sections from active ITP patients and HCs. Nuclei were counterstained with DAPI (blue). Quantitative analysis of the proportion of ROCK2⁺CD8⁺ cells among total CD8⁺ cells was performed using ImageJ software (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001). Images were acquired at × 200 magnification. (\u003cstrong\u003eD\u003c/strong\u003e) Schematic of the active ITP mouse model: irradiated \u003cem\u003eRag1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice were infused with 5 × 10\u003csup\u003e4\u003c/sup\u003e splenocytes from \u003cem\u003eCd61\u003c/em\u003e‑knockout (\u003cem\u003eCd61\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e) mice immunized with wild‑type (WT) C57BL/6 platelets. (\u003cstrong\u003eE\u003c/strong\u003e) \u003cem\u003eRock2\u003c/em\u003e mRNA expression in spleen tissue from active ITP mice and WT controls (\u003cem\u003eP\u003c/em\u003e = 0.047). (\u003cstrong\u003eF\u003c/strong\u003e) Immunofluorescence staining of ROCK2 (green) and CD8 (red) in spleen sections from active ITP and WT mice. Nuclei were counterstained with DAPI (blue). Quantitative analysis of the proportion of ROCK2⁺CD8⁺ cells among total CD8⁺ cells was performed using ImageJ software (\u003cem\u003eP\u003c/em\u003e = 0.005). Images were acquired at × 200 magnification. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8913555/v1/3c7add784a9cb514e0cf4c70.png"},{"id":104170777,"identity":"61c2a3d4-8611-494a-b31e-3699a312faa8","added_by":"auto","created_at":"2026-03-08 14:50:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1945000,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKD025 attenuated proliferation, cytotoxic function, and platelet interactions of CTLs from ITP patients \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) Representative flow cytometry plots of mitochondrial membrane potential in platelets, assessed by JC‑1 staining under the indicated culture conditions. The proportion of JC‑1 red (J‑aggregates) versus green (monomers) fluorescence reflects ΔΨm, with a decrease in the red/green ratio indicating loss of mitochondrial membrane potential during platelet injury. Quantification of platelet apoptosis: CD8⁺ CTLs from ITP patients markedly increased platelet apoptosis compared with platelets alone (\u003cem\u003eP \u0026lt;\u003c/em\u003e0.001), whereas KD025 significantly reduced CTL‑induced platelet apoptosis (\u003cem\u003eP \u003c/em\u003e= 0.031). (\u003cstrong\u003eB\u003c/strong\u003e) Representative histogram of CD62P expression on platelets under the indicated treatment conditions. Platelet CD62P expression increased significantly after co-cultured with CD8⁺ T cells from ITP patients, and KD025 significantly decreased CD8⁺ T-cell-induced upregulation of CD62P compared to DMSO (\u003cem\u003eP \u003c/em\u003e= 0.030). (\u003cstrong\u003eC\u003c/strong\u003e) Representative histogram of CD107a expression on CD8⁺ T cells under the indicated treatment conditions. CD107a was higher on CD8⁺ T‑cell-platelet cocultures than CD8⁺ T cells cultured alone (\u003cem\u003eP =\u003c/em\u003e 0.002), and KD025 significantly lowered CD107a levels on CD8⁺ T‑cell-platelet cocultures versus DMSO (\u003cem\u003eP \u003c/em\u003e= 0.040). (\u003cstrong\u003eD\u003c/strong\u003e) Representative dot plots of CD8⁺ T‑cell-platelet aggregates (CD8⁺CD61⁺). KD025 significantly reduced the frequency of CD8⁺ T‑cell-platelet aggregates compared with DMSO (\u003cem\u003eP \u003c/em\u003e= 0.008). Confocal imaging of CD8⁺ T-cell (red) and platelet (green) interactions under DMSO or KD025 treatment. Nuclei were counterstained with DAPI (blue). Images were acquired at × 500 magnification. (\u003cstrong\u003eE\u003c/strong\u003e) Concentrations of granzyme A, granzyme B, and perforin in culture supernatants of CD8⁺ T cells pretreated with KD025 or DMSO and then co‑cultured with platelets (all \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.050). (\u003cstrong\u003eF\u003c/strong\u003e) mRNA expression of cytotoxic and transcriptional regulators in CD8⁺ T cells treated with KD025 versus DMSO, showing reduced expression of \u003cem\u003eGZMA\u003c/em\u003e, \u003cem\u003eGZMB\u003c/em\u003e, \u003cem\u003ePRF1\u003c/em\u003e, \u003cem\u003eTBX21\u003c/em\u003e, and \u003cem\u003eEOMES \u003c/em\u003e(all \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.050). *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05\u003cstrong\u003e, \u003c/strong\u003e**\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01\u003cstrong\u003e,\u003c/strong\u003e and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003cstrong\u003e \u003c/strong\u003eΔΨm, mitochondrial membrane potential; DEGs, differentially expressed genes.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8913555/v1/93a427f581bba6db9d11af81.png"},{"id":104403883,"identity":"68cdc463-198a-422f-982e-0b7d6a4ebde1","added_by":"auto","created_at":"2026-03-11 12:19:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1431302,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptome sequencing of CD8⁺ T cells from ITP patients cultured \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e with DMSO or KD025. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Volcano plot of DEGs in CD8⁺ T cells treated with KD025 versus DMSO, showing 397 upregulated genes, 624 downregulated genes, and 15322 genes without significant change. (\u003cstrong\u003eB\u003c/strong\u003e) Heatmap of selected cytotoxicity‑related genes, demonstrating reduced expression of \u003cem\u003eGZMA\u003c/em\u003e, \u003cem\u003eGZMB\u003c/em\u003e, \u003cem\u003eGZMK\u003c/em\u003e, \u003cem\u003eGZMH\u003c/em\u003e, \u003cem\u003eFASLG\u003c/em\u003eand \u003cem\u003eIFNG\u003c/em\u003e after KD025 treatment. (\u003cstrong\u003eC\u003c/strong\u003e) RNA‑sequencing analysis of glycolysis‑related genes in CD8⁺ T cells from ITP patients treated with KD025 versus DMSO, showing reduced expression of \u003cem\u003eHK2\u003c/em\u003e, \u003cem\u003ePFKM\u003c/em\u003e, \u003cem\u003ePKM\u003c/em\u003e, \u003cem\u003eENO1\u003c/em\u003e, \u003cem\u003eLDHA\u003c/em\u003e, and \u003cem\u003ePFKFB3\u003c/em\u003e after KD025 treatment. (\u003cstrong\u003eD\u003c/strong\u003e) KEGG pathway enrichment analysis of DEGs, highlighting significant enrichment of pathways involved in DNA replication and repair and cytokine‑mediated signaling, including the JAK-STAT pathway. (\u003cstrong\u003eE\u003c/strong\u003e) GO enrichment analysis indicated that attenuated cell-cycle associated processes, including DNA replication, chromosomal segregation, and mitotic phase transition. (\u003cstrong\u003eF\u003c/strong\u003e) GSEA enrichment analysis showed decreased enrichment of MYC targets and mTORC1 signaling, alongside increased activation of immune- and stress-response pathways such as allograft rejection, IL2-STAT5 signaling, TNFα-NFκB signaling, and the unfolded protein response.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8913555/v1/4562b053e35d2c653fdec1b4.png"},{"id":104404144,"identity":"7cc234b3-c483-4256-90a1-9e8b91e5585d","added_by":"auto","created_at":"2026-03-11 12:19:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2209543,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKD025 inhibited CD8\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e T-cell activation by platelet antigen in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCd61\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003e-/-\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cstrong\u003e mice. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Experimental scheme: \u003cem\u003eCd61\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice received weekly transfusions of platelets from WT C57BL/6 mice and daily oral gavage of KD025 (100 mg/kg) or DMSO (2%) for 4 weeks; WT mice served as controls. Splenic CD8⁺ T cells were then magnetically sorted and co‑cultured with platelets for 4 h \u003cem\u003ein vitro\u003c/em\u003e. (\u003cstrong\u003eB\u003c/strong\u003e) Representative FCM plot of platelet apoptosis (loss of JC‑1 aggregate signal and increase in JC‑1 monomer signal) in co‑cultures with CTLs from the indicated groups. Quantification of platelet apoptosis showed that KD025 significantly reduced CTL‑mediated platelet apoptosis in \u003cem\u003eCd61\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003emice compared to DMSO‑treated \u003cem\u003eCd61\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003econtrols (\u003cem\u003eP \u003c/em\u003e= 0.006). (\u003cstrong\u003eC\u003c/strong\u003e) MFI of CD62P on platelets from the indicated co‑culture conditions. Quantification of CD62P MFI on platelet, showing reduced degranulation in KD025‑treated \u003cem\u003eCd61\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice compared to DMSO‑treated \u003cem\u003eCd61\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice (\u003cem\u003eP\u003c/em\u003e = 0.010). (\u003cstrong\u003eD\u003c/strong\u003e) Representative histogram of CD107a expression on CD8⁺ T cells from WT, \u003cem\u003eCd61\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e-DMSO, and \u003cem\u003eCd61\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e-KD025 mice. Quantification of CD107a MFI on CD8⁺ T cells, showing reduced degranulation in KD025‑treated \u003cem\u003eCd61\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice compared to DMSO‑treated \u003cem\u003eCd61\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003emice (\u003cem\u003eP\u003c/em\u003e = 0.020). (\u003cstrong\u003eE\u003c/strong\u003e) Representative dot plots of CD8⁺ T‑cell-platelet aggregates (CD8⁺CD61⁺ events) in the indicated groups. KD025 significantly reduced the frequency of CD8⁺ T‑cell-platelet aggregates in \u003cem\u003eCd61\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice compared to DMSO‑treated \u003cem\u003eCd61\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice (\u003cem\u003eP\u003c/em\u003e = 0.003). *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05\u003cstrong\u003e, \u003c/strong\u003e**\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01\u003cstrong\u003e,\u003c/strong\u003e and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8913555/v1/6299677801566774f45d2e27.png"},{"id":104779344,"identity":"d1c3f831-3591-400a-b6b3-77a3bae14336","added_by":"auto","created_at":"2026-03-17 07:39:06","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":346848,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMetabolic reprogramming in CD8\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e T cells from ITP patients and its regulation by ROCK2 inhibition. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Representative glycolytic stress assay of CD8\u003csup\u003e+\u003c/sup\u003e T cells from ITP patients and HCs, showing ECAR changes after sequential injection of glucose (10 mM), oligomycin (1 μM), and 2‑deoxyglucose (2‑DG, 50 mM). Comparison of glycolysis, glycolytic capacity, and glycolytic reserve in CD8⁺ T cells from ITP patients and HCs (glycolysis: \u003cem\u003eP\u003c/em\u003e = 0.008; glycolytic capacity: \u003cem\u003eP\u003c/em\u003e = 0.023; glycolytic reserve: \u003cem\u003eP\u003c/em\u003e = 0.009). (\u003cstrong\u003eB\u003c/strong\u003e) Representative mitochondrial stress test (OCR) of CD8⁺ T cells from ITP patients and HCs after oligomycin (10 μM), FCCP (1.5 μM), and rotenone/antimycin A (1 μM). Quantification of mitochondrial respiration parameters in CD8⁺ T cells from ITP patients and HCs, including basal respiration (\u003cem\u003eP\u003c/em\u003e = 0.002), maximal respiration (\u003cem\u003eP\u003c/em\u003e = 0.001), ATP‑linked respiration (\u003cem\u003eP\u003c/em\u003e = 0.003), and spare respiratory capacity (\u003cem\u003eP\u003c/em\u003e = 0.001). (\u003cstrong\u003eC\u003c/strong\u003e) qRT-PCR analysis of glycolysis‑related genes in CD8⁺ T cells, showing increased mRNA expression of \u003cem\u003eGLUT1\u003c/em\u003e, \u003cem\u003eHK2\u003c/em\u003e, \u003cem\u003ePKM2\u003c/em\u003e, and \u003cem\u003eLDHA\u003c/em\u003e in ITP patients compared to HCs (\u003cem\u003eGLUT1\u003c/em\u003e: \u003cem\u003eP\u003c/em\u003e = 0.032; \u003cem\u003eHK2\u003c/em\u003e: \u003cem\u003eP\u003c/em\u003e = 0.001; \u003cem\u003ePKM2\u003c/em\u003e: \u003cem\u003eP\u003c/em\u003e = 0.009; \u003cem\u003eLDHA\u003c/em\u003e: \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001) (\u003cstrong\u003eD\u003c/strong\u003e) Representative glycolytic stress test of CD8⁺ T cells from ITP patients treated with KD025 or DMSO, after sequential addition of glucose (10 mM), oligomycin (1 μM), and 2‑DG (50 mM). Comparison of glycolysis, glycolytic capacity, and glycolytic reserve in CD8⁺ T cells from ITP patients treated with KD025 or DMSO (glycolysis: \u003cem\u003eP\u003c/em\u003e = 0.001; glycolytic capacity: \u003cem\u003eP\u003c/em\u003e = 0.002; glycolytic reserve: \u003cem\u003eP\u003c/em\u003e = 0.001). (\u003cstrong\u003eE\u003c/strong\u003e) Representative mitochondrial stress test (OCR) of CD8⁺ T cells from ITP patients treated with KD025 or DMSO, after oligomycin (10 μM), FCCP (1.5 μM), and rotenone/antimycin A (1 μM). Quantification of mitochondrial respiration parameters in KD025‑ and DMSO‑treated CD8⁺ T cells, including basal respiration (\u003cem\u003eP\u003c/em\u003e = 0.046), maximal respiration (\u003cem\u003eP\u003c/em\u003e = 0.007), ATP‑linked respiration (\u003cem\u003eP\u003c/em\u003e = 0.043), and spare respiratory capacity (\u003cem\u003eP\u003c/em\u003e = 0.011). (\u003cstrong\u003eF\u003c/strong\u003e) qRT-PCR analysis of glycolysis‑related genes in CD8⁺ T cells from ITP patients treated with KD025 or DMSO, showing down‑regulation of key glycolytic enzymes (\u003cem\u003eGLUT1\u003c/em\u003e: \u003cem\u003eP\u003c/em\u003e = 0.050; HK2: \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; \u003cem\u003ePKM2\u003c/em\u003e: \u003cem\u003eP\u003c/em\u003e = 0.017; \u003cem\u003eLDHA\u003c/em\u003e: \u003cem\u003eP\u003c/em\u003e = 0.026). *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-8913555/v1/88683ad39e85214323318430.png"},{"id":104403369,"identity":"62e607a4-f9f7-41d8-901a-e6bd652044cb","added_by":"auto","created_at":"2026-03-11 12:18:11","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2525057,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eROCK2 inhibition suppressed STAT3 phosphorylation to attenuates CTL-mediated platelet destruction. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) MFI of p‑STAT3 in CD8⁺ T cells treated with KD025 or DMSO in PBMCs (\u003cem\u003eP \u003c/em\u003e= 0.010). (\u003cstrong\u003eB\u003c/strong\u003e) MFI of p‑STAT3 in CD8⁺ T cells from spleens of KD025‑treated mice compared to DMSO controls (\u003cem\u003eP\u003c/em\u003e = 0.002). (\u003cstrong\u003eC\u003c/strong\u003e) Representative CFSE histograms of CD8⁺ T cells from ITP patients treated with DMSO, IL‑6, KD025, or KD025 plus IL‑6. Division index of CD8⁺ T‑cell proliferation from ITP patients in the presence of DMSO, IL‑6, KD025, or KD025 plus IL‑6. Compared with DMSO, IL‑6 increased proliferation (\u003cem\u003eP\u003c/em\u003e = 0.001) and KD025 reduced proliferation (\u003cem\u003eP\u003c/em\u003e = 0.001). KD025 plus IL‑6 significantly increased the division index compared with KD025 alone (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001). (\u003cstrong\u003eD\u003c/strong\u003e) Representative dot plots showing platelet apoptosis in the presence of platelet, platelet + CTL, platelet + IL-6-CTL, platelet + KD025-CTL, platelet + (IL-6 + KD025)-CTL groups \u003cem\u003ein vitro. \u003c/em\u003eKD025 pretreatment markedly suppressed CTL‑mediated platelet apoptosis (\u003cem\u003eP \u0026lt;\u003c/em\u003e 0.001), whereas IL‑6 attenuated the inhibitory effect of KD025 on CTL‑induced platelet apoptosis (\u003cem\u003eP \u0026lt;\u003c/em\u003e 0.001) (\u003cstrong\u003eE\u003c/strong\u003e) MFI of CD62P on platelets after co‑culture with CD8⁺ T cells pretreated with DMSO, IL‑6, KD025, or KD025 plus IL‑6 \u003cem\u003ein vitro\u003c/em\u003e. (\u003cstrong\u003eF\u003c/strong\u003e) MFI of CD107a on CD8⁺ T cells pretreated with DMSO, IL‑6, KD025, or KD025 plus IL‑6. (\u003cstrong\u003eG\u003c/strong\u003e) Levels of CD8⁺ T cell-platelet aggregates in the DMSO, IL‑6, KD025, and KD025 plus IL‑6 groups. KD025 reduced CD8⁺ T cell-platelet aggregates compared to DMSO (\u003cem\u003eP\u003c/em\u003e = 0.031), whereas IL‑6 counteracted this inhibitory effect (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001). *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05; **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01; ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-8913555/v1/68dc7dcbb51d0a7dbab3cfa3.png"},{"id":104403677,"identity":"222db665-d903-4278-a1b9-eddc7dd378e8","added_by":"auto","created_at":"2026-03-11 12:18:49","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1405668,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eROCK2 inhibition reshaped CD8⁺ T‑cell metabolism via STAT3-dependent regulation. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Representative glycolytic stress test of CD8⁺ T cells from ITP patients sequentially treated with glucose (10 mM), oligomycin (1 μM), and 2‑DG (50 mM). (\u003cstrong\u003eB\u003c/strong\u003e) Quantification of glycolysis, glycolytic capacity, and glycolytic reserve in CD8⁺ T cells from ITP patients pretreated with DMSO, IL‑6, KD025, or KD025 plus IL‑6. (\u003cstrong\u003eC\u003c/strong\u003e) Representative mitochondrial stress test of CD8⁺ T cells from ITP patients sequentially treated with oligomycin (10 μM), FCCP (1.5 μM), and rotenone/antimycin A (1 μM). (\u003cstrong\u003eD\u003c/strong\u003e) Quantification of basal respiration, maximal respiration, ATP production and spare respiratory capacity in CD8⁺ T cells from ITP patients pretreated with DMSO, IL‑6, KD025, or KD025 plus IL‑6. KD025 significantly reduced glycolysis and enhanced mitochondrial oxidative metabolism, whereas IL‑6 partially attenuated these KD025‑induced metabolic effects (all \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05). * \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05; ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003e P \u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-8913555/v1/5f71d722cb3df9c356c4f68f.png"},{"id":104404803,"identity":"fe3c10de-1b85-45e3-9ce8-593826d6e2e5","added_by":"auto","created_at":"2026-03-11 12:21:07","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1486681,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eROCK2 inhibition mitigates CD8⁺ T cell-mediated thrombocytopenia and mitochondrial dysfunction \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Schematic of the active ITP model: anti-CD61-immunized splenocytes were adoptively transferred into irradiated \u003cem\u003eRag1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice, followed by treatment with 2% DMSO, KD025, IL‑6, or IL‑6 plus KD025. (\u003cstrong\u003eB\u003c/strong\u003e) Platelet counts in \u003cem\u003eRag1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice over 28 days after splenocyte transfer and treatment as indicated. KD025 markedly alleviated thrombocytopenia in this active ITP model (day 21: KD025 \u003cem\u003evs.\u003c/em\u003e control: \u003cem\u003eP = \u003c/em\u003e0.030; day 28: KD025 \u003cem\u003evs.\u003c/em\u003e control: \u003cem\u003eP = \u003c/em\u003e0.021), whereas IL‑6 attenuated the platelet‑raising effect of KD025 on day 28 (IL-6 \u003cem\u003evs.\u003c/em\u003e KD025: \u003cem\u003eP = \u003c/em\u003e0.023). (\u003cstrong\u003eC\u003c/strong\u003e) The CTL‑mediated platelet apoptosis in control group, IL-6 group, KD025 group, and IL‑6 plus KD025 group (all \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001). The platelet CD62P expression in control, IL-6, KD025, and IL‑6 plus KD025 groups (all \u003cem\u003eP\u003c/em\u003e = 0.001). KD025 group significantly decreased CD107a MFI on splenic CD8⁺ T cells (\u003cem\u003eP\u003c/em\u003e = 0.002), whereas IL‑6 group reversed this effect and restored CD107a expression to levels comparable to control group (\u003cem\u003eP\u003c/em\u003e = 0.043). KD025 group reduced the frequency of splenic CTL-platelet aggregates (\u003cem\u003eP\u003c/em\u003e = 0.001), and concurrent IL‑6 administration abrogated this reduction (\u003cem\u003eP\u003c/em\u003e = 0.006). (\u003cstrong\u003eD\u003c/strong\u003e) Schematic of the CD8⁺ T cell transfer experiment: anti‑CD61-sensitized CD8⁺ T cells were pretreated\u003cem\u003e in vitro\u003c/em\u003e with KD025 or DMSO, then mixed with CD8⁺ T-cell-depleted splenocytes, and adoptively transferred into irradiated \u003cem\u003eRag1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice. Mice receiving CD8⁺ T-cell-depleted splenocytes alone were used as the control group. (\u003cstrong\u003eE\u003c/strong\u003e) Mice receiving KD025‑treated CD8⁺ T cells showed significantly higher platelet counts than mice receiving DMSO‑treated CD8⁺ T cells on day 21 (\u003cem\u003eP\u003c/em\u003e = 0.010). (\u003cstrong\u003eF\u003c/strong\u003e) KD025‑treated CD8⁺ T cells group induced markedly less platelet apoptosis \u003cem\u003ein vivo \u003c/em\u003ethan DMSO‑treated CD8⁺ T cells group (\u003cem\u003eP\u003c/em\u003e = 0.011). * \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05; ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003e P \u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-8913555/v1/300d8596f74d1814bb9844dc.png"},{"id":104785488,"identity":"dccfe432-65da-4cd0-a625-d917b173ce75","added_by":"auto","created_at":"2026-03-17 08:11:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":15711603,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8913555/v1/1569a2c9-8a36-43d2-af49-ec06434e5962.pdf"},{"id":104170768,"identity":"6e124c50-279c-4654-bf8e-6631a5d3b658","added_by":"auto","created_at":"2026-03-08 14:50:15","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":917499,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8913555/v1/734c61294e973f91b9c3ada9.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"ROCK2 Inhibition Suppresses Cytotoxic T Lymphocyte-Mediated Platelet Destruction in Primary Immune Thrombocytopenia Running title: ROCK2 inhibition reduces CTL platelet injury","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePrimary immune thrombocytopenia (ITP) is an acquired autoimmune disorder characterized by accelerated platelet destruction and impaired platelet production, leading to an increased risk of bleeding.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e Traditionally, autoantibody-mediated platelet clearance via Fcγ receptor (FcγR)-bearing macrophages has been considered the central pathogenic mechanism.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e However, accumulating evidence highlights a complementary and clinically important antibody-independent pathway: cytotoxic T lymphocyte (CTL)-mediated platelet destruction.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e CTLs can kill platelets directly through granzyme/perforin-mediated lysis and death receptor signaling pathways.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e The formation of CTL-platelet conjugates and the ensuing polarized secretion of cytotoxic granules represent key mechanistic step in CTL-driven platelet lysis.\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e Despite the increasing availability of new therapeutic agents, a substantial proportion of patients remain refractory to treatments, underscoring the urgent need for novel strategies that target alternative pathogenic mechanisms, particularly CTL-mediated platelet destruction.\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eRho-associated coiled-coil kinase 2 (ROCK2) is a serine/threonine kinase with dual functions in orchestrating cellular processes.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e While initially recognized for its canonical function in orchestrating actin cytoskeletal dynamics, which is crucial for cell adhesion and migration,\u003csup\u003e12,13\u003c/sup\u003e a growing body of work has revealed its non-canonical role as a direct modulator of key signaling pathway.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e This includes its ability to interact with and regulate transcription factors such as STAT, thereby controlling T cell differentiation and metabolism.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e This dual functionality makes ROCK2 a particularly attractive therapeutic target in T-cell-driven autoimmune diseases. The selective ROCK2 inhibitor KD025, approved for the treatment of chronic graft-versus-host disease (GVHD), exerts robust immunomodulatory effects by dampening inflammatory signaling pathways and facilitating the restoration of immune homeostasis.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e However, its direct effects on CTL activity\u0026mdash;particularly cytotoxic molecule expression, degranulation capacity, and CTL-platelet conjugate formation\u0026mdash;remain poorly characterized in ITP. Elucidating these mechanisms is crucial, as modulation of CTL-mediated cytotoxic pathways may provide therapeutic benefit for patients with refractory ITP who respond inadequately to conventional immunosuppressive therapy.\u003c/p\u003e \u003cp\u003eIn this study, we examined how ROCK2 inhibition with KD025 affected the effector function and metabolic profile of CTLs from ITP patients, focusing on cytotoxicity, degranulation, and CTL-platelet conjugate formation. By integrating functional, transcriptional, and metabolic analyses, we delineated a ROCK2-STAT3-dependent immunometabolic program that drove pathogenic CTL activity in ITP, thereby supporting ROCK2 inhibition as a potential therapeutic strategy for CTL-mediated autoimmunity.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatients and healthy controls\u003c/h2\u003e \u003cp\u003eAdult patients with ITP were prospectively enrolled at Qilu Hospital, Shandong University, and Henan Cancer Hospital, Zhengzhou University. Active ITP was defined according to the 2019 ASH guidelines.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e Peripheral blood samples were obtained before initiation of ITP-specific therapy. An initial cohort of 35 patients with active ITP was screened. CTL induced platelet apoptosis was quantified as described in the \u003cb\u003eSupplementary Methods\u003c/b\u003e, and patients with values above the upper limit of the healthy volunteer range were classified as CTL-mediated ITP.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e Seventeen patients fulfilled this criterion and were included in all subsequent immunologic and mechanistic analyses. At enrollment, these 17 patients (11 females and 6 males; age range, 19\u0026ndash;68 years; median age, 45 years), exhibited platelet counts ranging from 1 \u0026times; 10⁹/L to 29 \u0026times; 10⁹/L (median platelet count, 15 \u0026times; 10⁹/L). Six ITP patients in remission with normal platelet counts and of treatment were enrolled as a comparator group. Baseline demographic and hematologic characteristics were provided in \u003cb\u003eSupplementary Table\u0026nbsp;1\u003c/b\u003e. Thirty-two age- and sex-matched healthy volunteers (18 females and 14 males; age range, 26\u0026ndash;72 years; median age, 48 years) were recruited as healthy controls (HCs), with platelet counts ranging from 157 \u0026times; 10\u003csup\u003e9\u003c/sup\u003e to 294 \u0026times; 10\u003csup\u003e9\u003c/sup\u003e/L (median, 254 \u0026times; 10\u003csup\u003e9\u003c/sup\u003e/L). All participants gave written informed consent, and the study was approved by the ethics committees of the participating centers in accordance with the Declaration of Helsinki.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eActive ITP mouse model\u003c/h3\u003e\n\u003cp\u003eAn active ITP mouse model was established as previously described.\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e Briefly, C57BL/6 \u003cem\u003eCd61\u003c/em\u003e-knockout (\u003cem\u003eCd61\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e) mice were immunized weekly with platelets isolated from syngeneic wild-type (WT) C57BL/6 male mice (8\u0026ndash;12 weeks old). Splenocytes from immunized mice were harvested, and 5 \u0026times; 10⁴ cells per mouse were intravenously transferred into sublethally irradiated severe combined immunodeficient (\u003cem\u003eRag1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e) mice to construct the active ITP murine model. The strains and backgrounds of the mice are detailed in the Online Supplementary Methods.\u003c/p\u003e \u003cp\u003eAt the time of splenocyte transfer, recipient mice were randomized to receive the following treatments: 2% DMSO control (intraperitoneal injection, once every 2 days and oral gavage, every day; Sigma-Aldrich), KD025 (100 mg/kg/day, oral gavage; MedChemExpress), IL‑6 (2 mg/kg, intraperitoneal injection, once every 2 days; PeproTech), and the combination of IL-6 and KD025. Platelet counts were determined weekly. On day 28, mice were anesthetized and euthanized, and splenocytes were collected for flow cytometric (FCM) analysis.\u003c/p\u003e \u003cp\u003eTo determine whether the therapeutic benefit of KD025 was mediated through direct modulation of CD8⁺ T cells, CD8⁺ T cells were purified from anti-CD61-sensitized splenocytes and cultured \u003cem\u003ein vitro\u003c/em\u003e with KD025 (1 \u0026micro;M) or 0.1% DMSO for 72 hours. These pretreated CD8⁺ T cells were then recombined with CD8⁺ T-cells-depleted splenocytes and adoptively transferred into irradiated \u003cem\u003eRag1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice to induce ITP.\u003c/p\u003e \u003cp\u003e All animal experiments were approved by the Animal Care and Use Committee of Qilu Hospital and conducted in accordance with institutional guidelines of Shandong University.\u003c/p\u003e \u003cp\u003eDetailed descriptions of PBMC and CD8⁺ T-cell isolation and culture, FCM, qRT-PCR, ELISA, Seahorse metabolic flux analysis, and immunofluorescence staining are provided in the \u003cb\u003eSupplementary Methods.\u003c/b\u003e\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eROCK2 expression was elevated in ITP patients and mice\u003c/h2\u003e \u003cp\u003eUsing an ELISA-based assay, we observed that ROCK kinase activity was significantly higher in PBMCs from patients with active ITP than from HCs, and was markedly reduced in ITP patients in remission (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Consistently, qRT-PCR analysis confirmed upregulation of \u003cem\u003eROCK2\u003c/em\u003e mRNA expression in PBMCs from ITP patients compared to HCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Moreover, immunofluorescence staining of spleen sections revealed stronger ROCK2 signals in CD8\u003csup\u003e+\u003c/sup\u003e T cells from ITP patients than from HCs who underwent traumatic splenectomy (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further evaluate the role of ROCK2 in the pathogenesis of ITP, we next assessed ROCK2 expression in spleen tissues from a murine model of active ITP (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). In line with human data, qRT-PCR analysis showed a significant increase in \u003cem\u003eRock2\u003c/em\u003e mRNA levels in the spleens of ITP mice compared with WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Furthermore, immunofluorescence analysis demonstrated enhanced ROCK2 expression within CD8⁺ T cells in the spleens of ITP mice relative to WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eKD025 suppressed CD8 T-cell proliferation, degranulation, cytotoxicity, and CTL cell-platelet interactions in ITP patients\u003c/h3\u003e\n\u003cp\u003eThe optimal concentration of KD025, a selective ROCK2 inhibitor, was determined using a range of gradient doses. Early apoptotic PBMCs were identified as Annexin V⁺/PI⁻ cells by FCM. To determine the working concentration of KD025, PBMCs from ITP patients were cultured with graded doses, and early apoptosis (Annexin V⁺/PI⁻) was assessed by FCM. KD025 at 5 and 10 \u0026micro;M significantly increased PBMC apoptosis, whereas 0.5 and 1 \u0026micro;M had no pro-apoptotic effect (\u003cb\u003eSupplemental Fig.\u0026nbsp;1A\u003c/b\u003e). Therefore, 1 \u0026micro;M KD025 was selected for subsequent \u003cem\u003ein vitro\u003c/em\u003e experiments.\u003c/p\u003e \u003cp\u003eTo explore the functional effects of KD025 on CTLs, 5(6)-carboxyfluorescein diacetate N-succinimidyl ester (CFSE)-labeled CD8⁺ T cells stimulated with anti-CD3/CD28 antibodies were cultured in the presence or absence of KD025. As shown in \u003cb\u003eSupplemental Fig.\u0026nbsp;1B\u003c/b\u003e, the division index in the KD025-treated group was significantly lower than that in the DMSO control group, indicating that KD025 suppresses CTL proliferation.\u003c/p\u003e \u003cp\u003eNext, CTL cytotoxicity was assessed by analyzing platelet apoptosis after 72 hours of co-culture, as determined by mitochondrial membrane depolarization using JC‑1 staining (loss of JC-1 aggregate signal and increase in JC‑1 monomer signal). Platelets cocultured with CTLs exhibited higher levels of apoptosis than platelets cultured alone. In contrast, the addition of KD025 to CTL-platelet cocultures significantly reduced platelet apoptosis compared with coculture with CTLs alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Additionally, platelet CD62P expression, as one of the marker for platelet activation, was markedly increased after co-culture with CTLs, and this increase was significantly attenuated when KD025 was present during the co-culture (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), suggesting that KD025 could reduce CTL-mediated platelet activation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCD107a is normally expressed on granule membranes of CTLs and becomes exposed on the cell surface following degranulation, thereby serving as a standard marker of CTL degranulation\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Membranous CD107a expression on CD8\u003csup\u003e+\u003c/sup\u003e T cells was significantly elevated after co-culture with platelets compared with CTLs cultured alone, and was significantly reduced when KD025 was present during the co‑culture (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eTo assess whether ROCK2 inhibition affects the ability of CTLs to physically engage with their targets, CD8\u003csup\u003e+\u003c/sup\u003e T cells from ITP patients were co-cultured with platelets from HCs for 4 hours. KD025 treatment substantially reduced CTL-platelet aggregate formation compared to DMSO controls, as quantified by CD8\u003csup\u003e+\u003c/sup\u003eCD61\u003csup\u003e+\u003c/sup\u003e aggregates (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Consistently, confocal microscopy further confirmed reduced CTL-platelet aggregate formation in KD025-treated co-culture compared with the DMSO controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). These observations suggested that ROCK2 activity was involved in the stable adhesion between CTLs and platelets, a critical prerequisite for targeted cytotoxicity.\u003c/p\u003e \u003cp\u003eWe next assessed cytotoxic granule secretion by CTLs. Levels of granzyme B (GZMB) and perforin (PRF1) in culture supernatants were significantly decreased after KD025 treatment, whereas granzyme A (GZMA) remained unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). At the transcriptional level, KD025-treated CTLs exhibited significantly lower mRNA expression of \u003cem\u003eGZMA\u003c/em\u003e, \u003cem\u003eGZMB\u003c/em\u003e, \u003cem\u003ePRF1\u003c/em\u003e, \u003cem\u003eTBX21\u003c/em\u003e and Eomesodermin (\u003cem\u003eEOMES\u003c/em\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). Thus, KD025 attenuated the cytotoxic machinery of CTLs at both protein and transcriptional levels and suppressed proliferation, degranulation, and CTL-platelet interactions in ITP.\u003c/p\u003e \u003cp\u003eRNA sequencing revealed substantial transcriptional reprogramming between DMSO- and KD025-treated CD8⁺ T cells. The volcano plot demonstrated that 397 genes were upregulated and 624 were downregulated after KD025 treatment, while 15,322 genes remained unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Multiple cytotoxic effector molecules, including \u003cem\u003eGZMA\u003c/em\u003e, \u003cem\u003eGZMB\u003c/em\u003e, \u003cem\u003eGZMK\u003c/em\u003e, \u003cem\u003eGZMH\u003c/em\u003e, \u003cem\u003eFASLG\u003c/em\u003e, and \u003cem\u003eIFNG\u003c/em\u003e, were consistently reduced by KD025 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). RNA-sequencing further showed that multiple glycolysis-related genes, including HK2, PFKM, PKM, ENO1, LDHA, and PFKFB3, were downregulated after KD025 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). KEGG pathway and GO enrichment analyses of differentially expressed genes (DEGs) indicated that KD025 predominantly downregulated cell-cycle- and proliferation-related programs, and modulated cytokine-driven signaling, including JAK-STAT pathways, while GSEA revealed decreased enrichment of MYC targets and mTORC1 signaling (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD - F). Together, these results indicated that KD025 induced coordinated transcriptional changes that reshaped CTL effector function.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eKD025 attenuated CD61-driven activation and cytotoxicity of CD8⁺ T cells in\u003c/b\u003e \u003cb\u003eCd61\u003c/b\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e \u003cb\u003emice\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate the \u003cem\u003ein vivo\u003c/em\u003e impact of KD025 on platelet antigen-specific CD8⁺ T-cell responses, \u003cem\u003eCd61\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice were intravenous transfused weekly with platelets from WT mice and concurrently administered KD025 (100 mg/kg) or a 2% DMSO daily for 4 consecutive weeks. At the end of treatment, splenocytes were harvested, CD8\u003csup\u003e+\u003c/sup\u003e T cells were isolated, and then cocultured with WT platelets for 4 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Consistent with the \u003cem\u003ein vitro\u003c/em\u003e findings, CD8⁺ T cells from KD025-treated \u003cem\u003eCd61\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice induced significantly less platelet apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) and activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC) than those from DMSO-treated controls.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFunctionally, KD025 also restrained CD8⁺ T-cell degranulation, as evidenced by reduced surface CD107a expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). In parallel, KD025-treated mice displayed diminished CTL-platelet aggregates, reflected by a lower proportion of CD8\u003csup\u003e+\u003c/sup\u003eCD61\u003csup\u003e+\u003c/sup\u003e population after coculture (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). These results suggested that KD025 mitigated CD61-induced activation and effector responses of CD8⁺ T cells in \u003cem\u003eCd61\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMetabolic reprogramming in CD8\u003csup\u003e+\u003c/sup\u003e T cells from ITP patients and its regulation by ROCK2 inhibition\u003c/h2\u003e \u003cp\u003eMetabolic reprogramming is a hallmark of CD8\u003csup\u003e+\u003c/sup\u003e T-cell activation and proliferation, characterized by enhanced glycolysis, amino acid turnover, and protein synthesis, concomitant with decreased aerobic glucose oxidation.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e To evaluate metabolic activity in CD8\u003csup\u003e+\u003c/sup\u003e T cells, we performed a Seahorse glycolysis and mitochondrial stress assay, measuring the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) as indicators of glycolytic flux and mitochondrial respiration, respectively. Following glucose injection, CTLs from ITP patients displayed a markedly greater increase in ECAR compared to HCs, indicating hyperactivated glycolytic metabolism. Quantitative analysis confirmed that basal glycolysis, glycolytic capacity, and glycolytic reserve were all significantly elevated in ITP-derived CTLs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn contrast, basal OCR prior to oligomycin injection was significantly lower in CTLs from the ITP patients than from HCs. Consistently, basal respiration, maximal respiratory, ATP-linked respiration and spare respiratory capacity were all markedly reduced in ITP CTLs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), indicating impaired mitochondrial oxidative phosphorylation (OXPHOS). At the transcriptional level, the expression of key glycolytic genes\u0026mdash;including glucose transporter 1 (\u003cem\u003eGLUT1\u003c/em\u003e), hexokinase 2 (\u003cem\u003eHK2\u003c/em\u003e), pyruvate kinase M2 (\u003cem\u003ePKM2\u003c/em\u003e), and lactate dehydrogenase (\u003cem\u003eLDHA\u003c/em\u003e)\u0026mdash;was significantly upregulated in CD8⁺ T cells from ITP patients compared with HCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), further supporting a glycolytic metabolic bias.\u003c/p\u003e \u003cp\u003eTo delineate the underlying mechanisms by which ROCK2 inhibition regulates CD8\u003csup\u003e+\u003c/sup\u003e T cells, we next evaluated the bioenergetic status of cultured CD8\u003csup\u003e+\u003c/sup\u003e T cells from ITP patients. KD025 treatment significantly decreased ECAR, including reductions in basal glycolysis, glycolytic capacity, and glycolytic reserve (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). By contrast, OCR was markedly increased, accompanied by enhanced basal respiration, maximal respiratory, ATP-linked respiration and spare respiratory capacity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Consistent with these functional metabolic changes, qRT-PCR analysis revealed that KD025 treatment downregulated glycolysis-related genes, including \u003cem\u003eGLUT1\u003c/em\u003e, \u003cem\u003eHK2\u003c/em\u003e, \u003cem\u003ePKM2\u003c/em\u003e, and \u003cem\u003eLDHA\u003c/em\u003e, in CD8\u003csup\u003e+\u003c/sup\u003e T cells from ITP patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eROCK2 inhibition suppressed STAT3 phosphorylation to attenuate CTL-mediated platelet destruction\u003c/h3\u003e\n\u003cp\u003eHaving established that ROCK2 inhibition profoundly suppresses CTL effector functions, we next sought to identify the upstream signaling mechanism. Pathway enrichment analysis of our RNA sequencing data revealed the JAK-STAT signaling pathway as a top hit. Given the pivotal role of STAT3 in controlling T-cell cytotoxicity, proliferation, and metabolism\u003csup\u003e\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, we specifically hypothesized that STAT3 is the critical signaling node downstream of ROCK2 that orchestrates CTL pathogenicity. To test this, we evaluated whether ROCK2 inhibition could restrain STAT3 activation \u003cem\u003ein vitro\u003c/em\u003e. PBMCs were treated with KD025 or DMSO, and p-STAT3 expression in CD8\u003csup\u003e+\u003c/sup\u003e T cells were quantified by FCM. KD025 treatment significantly reduced p-STAT3 MFI compared to DMSO controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Consistent with this \u003cem\u003ein vitro\u003c/em\u003e effect, in an active ITP mouse model, oral administration of KD025 substantially reduced p-STAT3 expression in splenic CD8⁺ T cells compared relative to DMSO-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Together, these data demonstrated that ROCK2 activity was required for robust STAT3 phosphorylation in CTLs both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. To functionally confirm that the suppression of STAT3 signaling was the key mechanism responsible for KD025's effects, we designed a comprehensive rescue experiment. We reasoned that if the STAT3 pathway was the critical mediator, then reactivating it downstream of the ROCK2 block should restore the full spectrum of CTL effector functions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIndeed, co-treatment with IL-6, a potent STAT3 activator, not only enhanced CTL proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC), but also restored the overall cytotoxic capacity of CTLs suppressed by KD025. To determine if this restoration translated to the ultimate pathological outcome, we assessed direct CTL-mediated platelet damage. In these assays, the protective effect of KD025 on platelet apoptosis was significantly reversed by the addition of IL-6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). This hierarchical response was mirrored across a range of parallel functional readouts. Specifically, platelet activation (CD62P), CTL degranulation (CD107a), and CTL-platelet aggregate formation were all maximal in the IL-6 group, minimal in the KD025 group, and displayed an intermediate phenotype with the combination treatment. (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE - G).\u003c/p\u003e \u003cp\u003eTaken together, this comprehensive array of rescue data provided definitive evidence that the ROCK2-STAT3 signaling axis was a central molecular pathway fueling CTL activation and cytotoxicity toward platelets in ITP.\u003c/p\u003e\n\u003ch3\u003eIL‑6/STAT3 signaling counteracted ROCK2 inhibition-mediated metabolic reprogramming in CD8⁺ T cells\u003c/h3\u003e\n\u003cp\u003eGiven that ROCK2 is a critical upstream regulator of STAT3 activation, we next examined whether IL‑6-STAT3 signaling could interfere with KD025‑induced metabolic reprogramming in CD8⁺ T cells from ITP patients. In the glycolysis stress test (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA), KD025 treatment markedly reduced ECAR over time, with glycolysis, glycolytic capacity, and glycolytic reserve all significantly decreased compared with DMSO (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB), indicating a broad suppression of glycolytic function. IL-6 co-stimulation largely restored ECAR in KD025-treated cells, significantly increasing glycolysis, glycolytic capacity, and glycolytic reserve relative to KD025 alone, thereby re-establishing a glycolytic phenotype.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eConversely, in the mitochondrial stress test (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC), KD025 significantly enhanced OCR, with increased basal respiration, maximal respiration, ATP production and spare respiratory capacity compared with DMSO (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD), consistent with a shift toward oxidative phosphorylation. IL-6 partially reversed these effects: all 4 parameters were significantly reduced in the IL-6 plus KD025 group versus KD025 alone, trending back toward the IL-6-driven glycolytic profile. These results indicated that ROCK2 inhibition ameliorated abnormal CTL immunometabolism in ITP patients through STAT3 suppression.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eKD025 ameliorated thrombocytopenia in active ITP mice\u003c/h2\u003e \u003cp\u003eTo determine whether the \u003cem\u003ein vitro\u003c/em\u003e findings of KD025 could be recapitulated \u003cem\u003ein vivo\u003c/em\u003e, an active murine model of ITP was established (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). Following irradiation and adoptive transfer of anti-CD61-immunized splenocytes into recombination-activating gene 1 (\u003cem\u003eRag1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e) mice, platelet counts gradually declined, reaching a nadir by day 14. KD025 treatment significantly ameliorated thrombocytopenia, with platelet counts remaining markedly higher than control on day 21 and day 28. In contrast, IL-6 administration induced the most profound platelet suppression on day 28. Notably, co-administration of IL-6 with KD025 abolished the therapeutic benefit of KD025, as platelet counts in the IL-6 plus KD025 group failed to increase and were markedly lower than those in the KD025 group, indicating that IL-6 abolished the therapeutic benefit of KD025 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further verify whether the platelet-protective effect of KD025 was maintained at the effector-cell level, splenic CD8⁺ T cells were purified from each treatment group and co-cultured with WT platelets for 4 hours. CD8⁺ T cells from KD025-treated group induced significantly less platelet apoptosis than those from DMSO control group, confirming an attenuation of platelet-destructive activity. In contrast, IL-6 treatment significantly increased platelet apoptosis, and IL-6 plus KD025 conditioning completely abrogated the protective effect of KD025, yielding apoptosis levels comparable to those observed in the DMSO control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC and \u003cb\u003eSupplemental Fig.\u0026nbsp;2A\u003c/b\u003e). Platelet CD62P expression showed a consistent pattern, with KD025 markedly reducing platelet activation compared with control, whereas IL-6 co-treatment significantly weakened this inhibitory effect of KD025 on platelet activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC and \u003cb\u003eSupplemental Fig.\u0026nbsp;2B\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eConsistent with these findings, KD025 directly dampened CD8⁺ T-cell cytotoxic function \u003cem\u003ein vivo\u003c/em\u003e. Compared to DMSO controls, KD025 significantly reduced the MFI of CD107a on CTLs (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC and \u003cb\u003eSupplemental Fig.\u0026nbsp;2C\u003c/b\u003e) and decreased the frequency of CLT-platelet aggregates (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC and \u003cb\u003eSupplemental Fig.\u0026nbsp;2D\u003c/b\u003e). Importantly, concurrent IL-6 administration fully reversed these inhibitory effects, restoring CD107a expression and CTL-platelet conjugate formation to levels comparable to those in controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eTo directly determine whether the therapeutic benefit of KD025 was mediated through intrinsic reprogramming of CD8⁺ T cells, CD8⁺ T cells were purified from anti-CD61-sensitized splenocytes and pretreated \u003cem\u003ein vitro\u003c/em\u003e with KD025 or 0.1% DMSO for 72 hours. These pretreated CD8⁺ T cells were then recombined with CD8⁺ T-cells-depleted splenocytes and adoptively transferred into irradiated \u003cem\u003eRag1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD). Mice receiving KD025-treated CD8⁺ T cells exhibited significantly higher platelet counts on day 21 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE) and markedly reduced platelet apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eF) compared with mice receiving DMSO‑treated CD8⁺ T cells. These results suggested that KD025‑treated CD8⁺ T cells exhibited diminished platelet‑destructive activity \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eITP has long been regarded as an autoantibody-mediated disorder, while accumulating evidence supports an important contribution of CTL-mediated platelet destruction.\u003csup\u003e\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e Effector CD8⁺ T cells induce platelet apoptosis via exocytosis of cytotoxic granules, forming stable conjugates with platelets and delivering polarized granule secretion that triggers caspase activation and programmed platelet death.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e In parallel, T-cell receptor (TCR) engagement triggers a metabolic switch from mitochondrial OXPHOS toward aerobic glycolysis to support clonal expansion and acquisition of effector function.\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e Our study extended these concepts to ITP by demonstrating that CD8⁺ T cells from patients underwent pathological immunometabolic reprogramming, characterized by enhanced glycolysis, reduced oxidative phosphorylation, and upregulation of glycolysis-related genes, consistent with a glycolysis-addicted, bioenergetically inflexible state that sustained pathogenic CTL activation. In this context, we showed that KD025, a selective ROCK2 inhibitor, restored metabolic balance by suppressing glycolytic flux and enhancing OXPHOS, thereby attenuating CTL-mediated platelet destruction.\u003c/p\u003e \u003cp\u003eThe therapeutic efficacy of KD025 in ITP appeared to be multifaceted, integrating effects on both the physical interaction and the intrinsic cytotoxic programming of CTLs. The observed reduction in CTL-platelet conjugate formation was consistent with the canonical function of ROCK2 as a master regulator of the actin cytoskeleton, which was essential for stable immune synapse assembly and cell adhesion.\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e While this modulation of cell mechanics likely represented an important layer of regulation, our data revealed a more profound impact on the core pathogenic identity of CTLs. We found that KD025 could dismantle the STAT3-OXPHOS signaling axis, a central metabolic and transcriptional engine for T-cell effector function. This aligned with the emerging paradigm of ROCK2 acting as a non-canonical signal transducer that directly influenced transcription factor activity in the nucleus.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e Therefore, we proposed that KD025's potent suppressive effect stemmed from a powerful synergy: it not only disrupted the physical engagement of CTLs with platelets but, more critically, it de-energized and transcriptionally rewired the CTLs, stripping them of their pathogenic capabilities.\u003c/p\u003e \u003cp\u003eSTAT3 phosphorylation is a central regulator of CD8⁺ T-cell biology.\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e It promotes survival and clonal expansion, upregulates perforin and granzyme B, and augments production of inflammatory cytokines such as IFN-γ and TNF-α.\u003csup\u003e24,35\u003c/sup\u003e Activating STAT3 mutations in CD8⁺ T cells correlate with heightened cytotoxicity in rheumatoid arthritis\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, and STAT3 also contributes to the shift from OXPHOS to glycolysis during CD8⁺ T-cell activation by enhancing glycolytic enzymes and glucose transporters.\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e ROCK2 has recently emerged as an upstream node that couples cellular metabolism, inflammation, and cytoskeletal dynamics.\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e Meanwhile, ROCK2 signaling can drive glycolysis-dependent pathogenic programs in multiple tissues and disease settings,\u003csup\u003e40\u0026ndash;42\u003c/sup\u003e and pharmacologic ROCK2 inhibition with KD025 has shown therapeutic efficacy in immune-mediated and fibrotic conditions.\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e These observations led us to hypothesize that aberrant ROCK2 activity might sustain a STAT3-dependent, glycolysis-biased program in CTLs from ITP patients.\u003c/p\u003e \u003cp\u003eIn line with this, we observed increased ROCK kinase activity and elevated \u003cem\u003eROCK2\u003c/em\u003e mRNA expression in PBMCs from ITP patients, as well as in spleens from active ITP mice. Previous studies have reported that ROCK2 could phosphorylate and activate downstream transcription factor STAT3, thereby promoting pathogenic Th17 polarization and impairing regulatory T-cell function in autoimmune settings.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e Consistent with a ROCK2-STAT3 signaling axis in CTLs, we found that ROCK2 inhibition with KD025 significantly reduced STAT3 phosphorylation \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. This suppression of STAT3 signaling coincided with normalization of CTL metabolism and attenuation of their cytotoxic program, suggesting that aberrant ROCK2-STAT3 activity sustains both the effector and metabolic phenotypes of pathogenic CTLs in ITP.\u003c/p\u003e \u003cp\u003eFunctionally, ROCK2 inhibition with KD025 curtailed multiple steps in the CTL-mediated platelet destruction cascade. In human ITP CTLs, KD025 reduced proliferation, degranulation (CD107a exposure), and secretion of granzyme B and perforin, and it diminished CTL-platelet conjugate formation. As a result, platelets co-cultured with KD025-treated CTLs exhibited lower apoptosis and decreased CD62P expression, indicating reduced activation and damage. Similar inhibitory effects were observed in \u003cem\u003eCd61\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice immunized with WT platelets, where KD025 treatment reduced platelet apoptosis, CD62P upregulation, CTL degranulation, and CTL-platelet aggregates. These findings supported the notion that ROCK2 inhibition attenuated antigen-specific CTL responses against platelet antigens.\u003c/p\u003e \u003cp\u003eA key mechanistic insight from our study was that KD025 corrected abnormal CTL immunometabolism in ITP via STAT3 suppression. In CD8⁺ T cells from cytotoxic ITP patients, KD025 inhibited glycolysis and enhanced mitochondrial respiration, accompanied by downregulation of glycolytic genes. Conversely, IL-6, a canonical STAT3 activator, restored glycolytic activity and reversed KD025-mediated increases in OXPHOS. Functionally, IL-6 also rescued CTL proliferation, degranulation, cytotoxicity, and CTL-platelet aggregate formation that had been suppressed by KD025. Thus, the ROCK2-STAT3 axis appeared to act as a metabolic and transcriptional hub: ROCK2 inhibition down-modulated STAT3 to shift CTLs from a glycolysis-dominated, highly cytotoxic state toward a more quiescent, OXPHOS-supported phenotype, whereas IL-6-driven STAT3 activation counteracted this reprogramming and reinstated pathogenic CTL function.\u003c/p\u003e \u003cp\u003eOur \u003cem\u003ein vivo\u003c/em\u003e data provided proof-of-concept that targeting this axis could ameliorate disease. In an active ITP model that recapitulates both humoral and cellular anti-platelet immunity, KD025 treatment significantly increased platelet counts and reduced CTL-mediated platelet destruction. Phenotypically, CD8⁺ T cells from KD025-treated mice displayed less degranulation and fewer interactions with platelets. These findings indicated that ROCK2 inhibition could attenuate ongoing platelet damage in a complex autoimmune milieu. However, because transferred splenocytes contained heterogeneous lymphocyte populations, including CD4⁺ T cells and B cells, we could not fully exclude KD025 effects on other immune subsets that contributed to disease modulation.\u003c/p\u003e \u003cp\u003eTo more directly interrogate CD8⁺ T cells, we performed adoptive transfer experiments using \u003cem\u003eex vivo\u003c/em\u003e KD025-pretreated CTLs recombined with CD8-depleted splenocytes. Mice receiving KD025-conditioned CD8⁺ T cells exhibited higher platelet counts and lower platelet apoptosis on day 21 than mice receiving DMSO-treated CD8⁺ T cells, although this benefit diminished by day 28 as thrombocytopenia naturally improved in both groups. These results indicated that intrinsic reprogramming of CD8⁺ T cells by KD025 was sufficient to transiently mitigate platelet destruction even in the presence of humoral autoimmunity.\u003c/p\u003e \u003cp\u003eOur study had several limitations. The number of cytotoxic ITP patients undergoing detailed metabolic profiling was modest, and larger cohorts were needed to validate ROCK2-STAT3-driven metabolic signatures as biomarkers. We focused mainly on CD8⁺ T cells in the study. However, the effects of ROCK2-STAT3 signaling on CD4⁺ T cells, B cells, and myeloid cells in ITP were also worth further evaluation. Potential off-target effects of KD025 were not systematically assessed, and murine ITP models only partially recapitulated the chronic and heterogeneous nature of human disease.\u003c/p\u003e \u003cp\u003eIn conclusion, we identified the ROCK2-STAT3 axis as a central immunometabolic checkpoint in ITP. ROCK2 inhibition with KD025 downregulated STAT3 phosphorylation, shifted CD8⁺ T-cell metabolism from pathological glycolysis toward OXPHOS, and attenuated CTL-mediated platelet destruction \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. These findings supported ROCK2 inhibition as a strategy to restore immune homeostasis and reduce platelet destruction, particularly in CTL-driven disease.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eData availability statement\u003c/h2\u003e \u003cp\u003eFor original data, please contact [email protected].\u003c/p\u003e \u003c/div\u003e\u003cp\u003e \u003ch2\u003eEthics statement\u003c/h2\u003e \u003cp\u003e Our study was approved by the ethics committees of Qilu Hospital of Shandong University according to the Helsinki Principles.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003ePatient consent statement\u003c/strong\u003e \u003cp\u003eWritten informed consent was obtained from all participants included in the study.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eAuthorship contributions\u003c/strong\u003e \u003cp\u003eContribution: J.S., A.X., Q.L., and B.D. performed the experiments, M.L., X.S., and F.W. analyzed the results and created the figures, J.H., L.L., and J.S. designed the research and wrote the paper; X.L., and H.Z. funded the research and edited the manuscript.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting Interests\u003c/h2\u003e \u003cp\u003eThe authors declare no competing financial interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding information\u003c/h2\u003e \u003cp\u003eThis work was supported by the National Key Research and Development Program of China (2024YFC2510500), National Natural Science Foundation of China (No. 82570171 and No. 82170123), Young Taishan Scholar Foundation of Shandong Province (Grant/Award No. tsqn202312325), Natural Science Foundation of Henan Province (No. 252300421374) and Henan Province Young and Middle-aged Health Science and Technology Innovation Leading Talent Training Program (No. YXKC2022008).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eContribution: J.S., A.X., Q.L., and B.D. performed the experiments, M.L., X.S., and F.W. analyzed the results and created the figures, J.H., L.L., and J.S. designed the research and wrote the paper; X.L., and H.Z. funded the research and edited the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKuen DS, Park M, Ryu H, et al. 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ROCK2 signaling is required to induce a subset of T follicular helper cells through opposing effects on STATs in autoimmune settings. Sci Signal. 2016;9(437):ra73.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZanin-Zhorov A, Blazar BR. ROCK2, a critical regulator of immune modulation and fibrosis has emerged as a therapeutic target in chronic graft-versus-host disease. Clin Immunol. 2021;230:108823.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-and-bioscience","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cbio","sideBox":"Learn more about [Cell \u0026 Bioscience](http://cellandbioscience.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/cbio/default.aspx","title":"Cell \u0026 Bioscience","twitterHandle":"@OACellBiology","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"primary immune thrombocytopenia, cytotoxic T lymphocyte, ROCK2, KD025, STAT3","lastPublishedDoi":"10.21203/rs.3.rs-8913555/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8913555/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCytotoxic T lymphocyte (CTL)-mediated platelet destruction represents an important pathogenic mechanism in ITP patients. Rho-associated coiled-coil kinase 2 (ROCK2) is an emerging regulator of immune balance, but its role in pathogenic CTL activation in ITP remains undefined. Here, we demonstrated that selective ROCK2 inhibition with KD025 potently suppressed CTL-mediated platelet destruction. \u003cem\u003eIn vitro\u003c/em\u003e, KD025 treatment of CTLs from ITP patients suppressed key effector functions, reducing degranulation as measured by CD107a expression, diminishing the secretion of cytotoxic molecules such as granzyme B and perforin, and decreasing CTL-platelet conjugate formation, resulting in reduced platelet apoptosis and activation. RNA-sequencing revealed downregulation of cytotoxic and glycolytic programs, with enrichment of JAK-STAT signaling. Mechanistically, KD025 reversed the pathogenic metabolic shift in ITP CTLs by lowering glycolytic flux and restoring mitochondrial respiration, accompanied by decreased STAT3 phosphorylation. IL-6-mediated STAT3 activation largely reversed these effects, indicating a ROCK2-STAT3-dependent mechanism. \u003cem\u003eIn vivo\u003c/em\u003e, both daily KD025 administration to an active ITP mouse model and transplantation of KD025-pretreated CD8\u003csup\u003e+\u003c/sup\u003e T cells into irradiated \u003cem\u003eRag1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice alleviated CTL-mediated platelet apoptosis and increased platelet counts. Collectively, these findings identified a ROCK2-STAT3 regulatory axis integrating transcriptional and metabolic control of CTL pathogenicity and support ROCK2 inhibition as a promising therapeutic strategy for ITP.\u003c/p\u003e","manuscriptTitle":"ROCK2 Inhibition Suppresses Cytotoxic T Lymphocyte-Mediated Platelet Destruction in Primary Immune Thrombocytopenia Running title: ROCK2 inhibition reduces CTL platelet injury","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-08 14:50:10","doi":"10.21203/rs.3.rs-8913555/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-21T00:20:44+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-18T10:31:18+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-18T08:02:01+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-17T13:13:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"293037750815240189231249760021674225010","date":"2026-03-13T12:53:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"234290565669844052087801956068768095256","date":"2026-03-13T10:11:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"202812910011906413669710990392675136887","date":"2026-03-13T09:57:02+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-09T05:59:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"54932291520871773558185092664815367018","date":"2026-02-26T20:34:46+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-26T11:41:56+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-21T02:13:39+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-19T08:32:50+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell \u0026 Bioscience","date":"2026-02-19T03:22:07+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-and-bioscience","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cbio","sideBox":"Learn more about [Cell \u0026 Bioscience](http://cellandbioscience.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/cbio/default.aspx","title":"Cell \u0026 Bioscience","twitterHandle":"@OACellBiology","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"101f0401-12f1-4a5f-981b-3e55c387418f","owner":[],"postedDate":"March 8th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-16T15:53:26+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-08 14:50:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8913555","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8913555","identity":"rs-8913555","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}