RAS P21 Protein Activator 3 finetunes Type I immunity by limiting HCK-mediated STAT4 phosphorylation

Abstract

Type I immunity, mediated by IFN-γ, is essential for combating intracellular pathogens but also drives inflammatory diseases. How the immune system balances protective type I responses with pathological inflammation is elusive. While the IL-12-STAT4 pathway that promotes IFN-γ production is well-established, the intrinsic negative regulators remain unclear. We establish that RASA3 serves as a permissive checkpoint for type I immune responses. T cell-specific ablation of RASA3 amplified Th1/Tc1 immunity and IFN-γ expression, enhancing the clearance of Listeria monocytogenes but exacerbating allergic contact dermatitis. We further identify the hematopoietic cell kinase (HCK) as a direct kinase for STAT4, which binds to and phosphorylates it at the Tyr693 residue to elicit IFN-γ production. Mechanistically, RASA3 represses the translation of HCK via constraining ribosomal protein RPL36A expression. Additionally, the RASA3-HCK axis is conserved in human Th1 cells. Thus, we define a critical role of the RASA3-HCK-STAT4 axis in fine-tuning type I immunity and offer promising targets for intervening in Th1/Tc1-driven pathologies.

IFN-γ-mediated type I immunity is important for clearing intracellular pathogens yet promotes inflammatory diseases. However, T cell-intrinsic negative regulators controlling IFN-γ expression remain poorly characterized. The authors here identify a role of the RASA3-HCK-STAT4 axis in tempering type I immunity.

Introduction

The type I immune response presents a major health dilemma: its deficiency increases susceptibility to infections like Listeria monocytogenes infection in immunocompromised individuals, while its overactivity contributes to the rising prevalence of inflammatory disorders, such as allergic contact dermatitis^1–4. The IL-12-induced production of IFN-γ is a cornerstone of this response, polarizing CD4⁺ Th1 and CD8⁺ Tc1 cells to clear pathogens. However, its dysregulation can lead to inflammatory diseases^5–7. Understanding the mechanisms that keep Th1 cells in check can provide insight into targets for inflammatory diseases and anti-bacterial immunity. Although cytokine milieu and dendritic cells play a crucial role in directing Th1 cells^8,9, the intrinsic molecular mechanisms that restrain type I immunity within T cells remain poorly characterized.

Upon IL-12 binding, the receptor-associated kinases JAK2 and TYK2 are activated and phosphorylate the IL-12 receptor, then recruit STAT4 for its phosphorylation^10–12. Following dimerization, phosphorylated STAT4 translocates to the nucleus, where it cooperates with the transcription factor T-bet to regulate the expression of type I immunity-related genes such as IFN-γ and TNF-α^13–15. Signal transducer and activator of transcription (STAT) proteins are often regulated by a variety of important posttranslational modifications (PTMs), including phosphorylation, acetylation, and ubiquitination, which dynamically control their activation, stability, and subcellular localization^3,16–18. Of note, the phosphorylation of STAT4 is the key event in IFN-γ-related T cell immune responses, and its dysregulation leads to various diseases^19–21. Although JAK2, TYK2, and p38/MAPK are the primary kinases that phosphorylate STAT4^11,19, the pharmacological inhibition of these targets is often associated with severe adverse effects, owing to their pleiotropic functions in broader cellular physiology^22,23. Despite the critical importance of STAT4 phosphorylation in type I immunity, kinases capable of this modification beyond the established JAK2/TYK2 and p38 pathways remain poorly defined.

Using murine models of Listeria monocytogenes infection and DNFB-induced contact hypersensitivity (CHS)-both driven by Th1/Tc1-mediated immunity^24,25, we demonstrate that RAS P21 Protein Activator 3 (RASA3) is a critical negative regulator of type I immunity and IFN-γ production. RASA3 is progressively decreased following Th1 differentiation both in vitro and in vivo. Its deficiency results in increased Th1/Tc1 cell generation, causing exacerbated contact hypersensitivity and reduced bacterial burden. Mechanistically, loss of RASA3 unleashes ribosomal protein L36a (RPL36A)-mediated hematopoietic cell kinase (HCK) translation, which phosphorylates STAT4 to drive IFN-γ production. This study illuminates the negative regulation of type I immunity by uncovering the RASA3-RPL36A-HCK axis, a critical pathway controlling STAT4 phosphorylation.

Results

RASA3 negatively regulated type I immunity and IFN-γ generation

To explore the endogenous factors that negatively regulate type I T cell immunity, we differentiated Th1 cells over a time course and harvested RNA and protein at each time point for RNA-seq and LC-MS analysis, respectively. We then characterized the overlapping hits from RNA-seq, LC-MS, and public datasets as genes that are downregulated during Th1 cell differentiation (Supplementary Fig. 1A and Fig. 1A). By integrating the enriched genes from all datasets, we uncovered 2 molecules RASA3 and RASGRP2 (Fig. 1A). Among which, RASA3, a GTPase activating protein of the GAP1 family, has been implicated in T cell migration, differentiation, platelet function and erythropoiesis^26–30, while its role in type I immunity remains poorly understood. Notably, both protein and mRNA levels of RASA3 progressively decreased during Th1 cell polarization (Fig. 1B, C). The strong inverse correlation between Rasa3 and Ifng in Th1 cells in vitro and in T cells from Listeria monocytogenes-OVA (Lm-OVA)-infected mice (Fig. 1D–F) further suggests RASA3 may be involved in the negative regulation of Th1 cells and subsequent IFN-γ expression. Additionally, we observed a significant reduction of RASA3 expression in the skin of patients with allergic contact dermatitis, a type I immune response-mediated inflammatory skin disease^31,32, compared to healthy individuals (Fig. 1G). Of interest, RASA3 expression level in Th1 cells is regulated by TCR signals but not by IL-12 stimulation (Supplementary Fig. 1C). TCR stimulation decreased RASA3 levels in T cells, and this reduction was enhanced by anti-CD28 co-stimulation (Supplementary Fig. 1C). Therefore, RASA3 may play a role in negative regulation of type I immunity.

Fig. 1. RASA3 is decreased in Th1 cells and negatively regulates Th1 cell differentiation.

A Numbers of overlapping genes from top 2 hits of each screen. Genes are listed by log₂FoldChange of protein intensity. B Comparison of Rasa3 mRNA expression in CD4⁺ T cells activated under indicated conditions for different time points. (n = 6 samples from 3 independent experiments; means ± s.d.; ***p < 0.001. One-way ANOVA with Tukey’s test. C Immunoblotting to detect RASA3 protein expression in CD4⁺ T cells activated under indicated conditions for different time points. Results are representative of 3 independent experiments. D Ifng and Rasa3 mRNA expression in activated CD4⁺ T cells under indicated conditions as in (B) and  Supplementary Fig. 1B. Colors depict different time points of CD4⁺ T cells activation. (Spearman’s rank correlation test, 95% confidence intervals −0.9763 to −0.8758. Pearson’s correlation analysis). E Comparison of Ifng and Rasa3 mRNA expression in splenic CD4⁺ T cells separated from mice infected with Lm-OVA at different time points. (n = 6 samples from 3 independent experiments; means ± s.d.; *p < 0.05, **p < 0.01, ***p < 0.001. One-way ANOVA with Tukey’s test). F Ifng and Rasa3 mRNA expression in splenic CD4⁺ T cells separated from mice infected with Lm-OVA, as in (E). Colores depict different time points after Lm-OVA infection. (Spearman’s rank correlation test, 95% confidence intervals −0.9454 to −0.6496. Pearson’s correlation analysis). G Immunohistochemical staining for RASA3 in healthy control and allergic contact dermatitis (ACD) human skin. Scale bar: 200μm. The arrow indicates the stained RASA3 protein in the skin. H IFN-γ produced by CD4⁺ T cells from Cd4Cre; Rasa3^flox/+ (WT) and Cd4Cre; Rasa3^flox/flox (KO) mouse, activated in the presence of IL-12 for 3 days, analyzed by FACS and ELISA. (n = 8 samples from 5 independent experiments for the percentage of IFN-γ; n = 6 samples from 3 independent experiments for the secretion of IFN-γ; means ± s.d.; ***p < 0.001. Two-sided t-test). I Flow-cytometry of IFN-γ produced by human CD4⁺ T cells after knocking down Rasa3 by lentiviral-based shRNAs, activated in the presence of IL-12 for 3 days. (n = 6 samples from 3 independent experiments; means ± s.d.; ***p < 0.001. One-way ANOVA with Tukey’s test). J qRT-PCR analysis to detect mRNA levels expressed by CD4⁺ T cells of indicated genotypes, activated under Th1 cell-polarizing conditions for 3 days. (n = 6 samples from 3 independent experiments; means ± s.d.; *p < 0.05, **p < 0.01, ***p < 0.001. Per two-sided t-test). K Th1-related genes by Rasa3-deficient versus Rasa3-sufficient CD4⁺ T cells, 3 days after activation under Th1 cell-polarizing conditions, assayed by bulk RNA-seq. (Results are averages of 4 independent experiments). L qRT-PCR analysis to detect mRNA levels expressed by CD8⁺ T cells of indicated genotypes, activated under Tc1 cell-polarizing conditions for 3 days. (n = 6 samples from 3 independent experiments; means ± s.d.; **p < 0.01, ***p < 0.001. Per two-sided t-test and two-sided unpaired nonparametric Mann-Whitney test). M Tc1-related genes by Rasa3-deficient versus Rasa3-sufficient CD8⁺ T cells, 3 days after activation under Tc1 cell polarizing conditions, assayed by bulk RNA-seq. (Results are averages of 3 independent experiments). Source data are provided as a Source Data file.

To investigate RASA3 function in T cells, we generated Rasa3^flox/flox mice by flanking the third exon with loxP sites (Supplementary Fig. 1D) and crossed them with Cd4Cre mice to enable T cell-specific deletion of RASA3, confirmed by quantitative PCR and immunoblotting (Supplementary Fig. 1E, F). The percentage of IFN-γ⁺CD4⁺ and IFN-γ⁺TNF-α⁺CD8⁺ T cells in lymph nodes and spleen remained unperturbed in the absence of RASA3 in homeostatic conditions (Supplementary Fig. 1G, H). Nonetheless, we found that type I immunity was significantly increased in draining lymph nodes of Cd4Cre; Rasa3^flox/flox mice (also named as RASA3 KO) after ovalbumin (OVA) immunization, compared to WT controls (Cd4Cre; Rasa3^flox/wt mice) (Supplementary Fig. 1I, J). Similarly, following IL-12 stimulation in vitro, Rasa3-deficient CD4⁺ T cells produced higher percentages of IFN-γ⁺ cells, and Rasa3-deficient CD8⁺ T cells produced higher percentages of IFN-γ⁺TNF-α⁺ cells than their Rasa3-sufficient counterparts (Fig. 1H and Supplementary Fig. 1K). Moreover, in human CD4⁺ T cells, short hairpin RNA (shRNA)-mediated Rasa3 knockdown also led to increased IFN-γ expression under Th1 polarization conditions (Fig. 1I). Consistently, compared to Rasa3-sufficient CD4⁺ T cells, Rasa3-deficient CD4⁺ T cells expressed higher mRNA amounts of Th1 signature genes as determined by qRT-PCR and bulk RNA-seq analysis (Fig. 1J, K). In addition, RASA3 also impaired the expression of Tc1 signature genes in CD8⁺ T cells (Fig. 1L, M). These data therefore suggest that RASA3 is critical for negatively regulating Th1 and Tc1 cell differentiation.

The deficiency of RASA3 led to increased IFN-γ expression during DNFB-elicited CHS disease and Lm-OVA infection

Type I immunity is essential for both the pathogenesis of contact hypersensitivity (CHS), a murine model of allergic contact dermatitis, and protective immunity against Lm-OVA infection^24,25. To address whether RASA3 is required for Th1/Tc1 cell generation and immune-pathology in vivo, we topically applied 2,4-dinitrofluorobenzene (DNFB) to induce skin CHS mouse model^33,34 as elucidated in Fig. 2A. RASA3 deficiency in Cd4Cre; Rasa3^flox/flox mice resulted in more severe skin disease, as shown by increased ear swelling and tissue immunopathology compared to Cd4Cre; Rasa3^flox/+ mice (Fig. 2B, C). Consistently, mice with RASA3 deletion revealed increased percentage of proinflammatory IFN-γ⁺CD4⁺, IFN-γ⁺TNF-α⁺CD8⁺ T in the submaxillary lymph nodes (smLN), axillary lymph nodes (aLN), inguinal lymph nodes (iLN), blood, and skin lesions (Fig. 2D, E). However, the fraction of immune suppressive Foxp3⁺ Treg cells was slightly increased in the lymph nodes of Cd4Cre; Rasa3^flox/flox mice while IL-4⁺CD4⁺ T cells remained unaltered (Supplementary Fig. 2A, B), indicating that RASA3 deficiency triggers a profound type I immune response during dermatitis.

Fig. 2. RASA3 inhibits type I immunity during DNFB-elicited CHS and Lm-OVA infection.

A CHS responses were induced, as shown in the scheme. B Twelve hours after sensitization with DNFB of the ear of Cd4Cre; Rasa3^flox/+ (WT) and Cd4Cre; Rasa3^flox/flox (KO) mice, ear swelling was determined by vernier caliper (n = 6 samples from 3 independent experiments; means ± s.d.; ***p < 0.001. Per two-sided t-test). C Representative histological sections are shown in (B). (Hematoxylin and eosin staining; scale bar = 400 µm). D–E IFN-γ-producing CD4⁺ (D) and IFN-γ/TNF-α-producing CD8⁺ (E) T cells in the submaxillary lymph nodes (smLN), axillary lymph nodes (aLN), inguinal lymph nodes (iLN), blood, and skin lesion of sensitized mice of indicated genotypes, assessed by flow cytometry, 12 hours after 2nd DNFB challenge. (n = 6 samples from 3 independent experiments; means ± s.d.; *p < 0.05, **p < 0.01, ***p < 0.001. Per two-sided t-test and two-sided unpaired nonparametric Mann-Whitney test). F Lm-OVA CFUs per gram of spleen and liver of Cd4Cre; Rasa3^flox/+ (WT) and Cd4Cre; Rasa3^flox/flox (KO) mice infected 4 days with 2 × 10⁵ Lm-OVA i.v. (n = 6 samples from 3 independent experiments; means ± s.d.; *p < 0.05, **p < 0.01. Per two-sided t-test and two-sided unpaired nonparametric Mann-Whitney test). G–H Flow-cytometry of IFN-γ⁺CD4⁺ (G) and IFN-γ⁺TNF-α⁺CD8⁺ (H) T cells in the LNs and SP of Lm-OVA-infected mice of indicated genotypes at day 7 post infection. (n = 6 samples from 3 independent experiments; means ± s.d.; *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant. Per two-sided t-test. I qRT-PCR analysis to detect mRNA levels of Th1-related genes expressed by splenic CD4⁺ T cells from Cd4Cre; Rasa3^flox/+ (WT) and Cd4Cre; Rasa3^flox/flox (KO) mice infected 7 days with Lm-OVA. (n = 6 samples from 4 independent experiments; means ± s.d.; *p < 0.05, ***p < 0.001; ns, not significant. Per two-sided t-test). Source data are provided as a Source Data file.

To investigate the role of RASA3 in regulating IFN-γ expression during pathogen infection, Cd4Cre; Rasa3^flox/flox and Cd4Cre; Rasa3^flox/+ mice were challenged with Lm-OVA. Notably, the numbers of viable bacteria recovered from the spleen and liver of Cd4Cre; Rasa3^flox/flox mice was up to 10 times lower than that from Cd4Cre; Rasa3^flox/+ mice (Fig. 2F). Consistently, IFN-γ expression levels were increased in CD4⁺ and CD8⁺ T cells from spleen of Cd4Cre; Rasa3^flox/flox mice compared to Cd4Cre; Rasa3^flox/+ mice (Fig. 2G, H), suggest enhanced bacteria clearance and elevated IFN-γ production in Rasa3-deficient mice. In the meantime, Foxp3⁺ Treg cell population remained unaffected (Supplementary Fig. 2C). Furthermore, Rasa3-deficient CD4⁺ T cells isolated from spleens of Lm-OVA-infected mice exhibited higher Th1-related genes expression, while regulatory gene Il10 remained unaltered (Fig. 2I). The higher percentage of IFN-γ-producing T cells in Cd4Cre; Rasa3^flox/flox mice was not due to hyper-proliferation as Ki67 level was comparable between Rasa3-sufficient and Rasa3-deficient T cells (Supplementary Fig. 2D). We further determined the role of RASA3 in regulating IFN-γ expression in protective memory T cell responses to secondary bacterial infection^35,36. We observed that RASA3 was dispensable for memory T cell generation (Supplementary Fig. 2E, F). However, RASA3 deficiency enhances the recall response to Lm-OVA by promoting IFN-γ production in antigen-specific T cells (Supplementary Fig. 2G, H). Our findings position RASA3 as a key regulatory checkpoint that fine-tunes Th1 and Tc1-driven type I immunity, preventing excessive inflammation while preserving host defense in various disease settings.

RASA3 dominantly suppressed Th1 cell generation via restraining STAT4 phosphorylation

To determine if RASA3 acts dominantly or recessively to limit Th1 generation, we co-cultured Rasa3-deficient and -sufficient T cells during Th1 differentiation. Rasa3-deficient CD4⁺ T cells maintained elevated IFN-γ expression even when co-cultured with Rasa3-sufficient cells, indicating a cell-intrinsic role for RASA3 in restraining Th1 differentiation (Fig. 3A). Additionally, Rasa3-deficient CD4⁺ T cells exhibited an increased G1-S phase transition and higher CFSE incorporation, indicating slightly increased cell proliferation in T cells lacking RASA3 (Fig. 3B and Supplementary Fig. 3A). In addition, the cell viability of Th1 cells was unaltered in the absence of RASA3 (Fig. 3C, D). We thus wondered if RASA3 directly modulated key regulators of IFN-γ expression. Firstly, the mRNA and protein levels of T-bet, a key transcription factor for Th1 cells and IFN-γ expression, were comparable between Rasa3-deficient and sufficient Th1 cells (Supplementary Fig. 3B and Fig. 3E). Consistent with our in vitro data, T-bet expression was largely intact in CD4⁺ T cells from Cd4Cre; Rasa3^flox/flox mice during both CHS and Lm-OVA infection models (Supplementary Fig. 3C, D). Secondly, IL-12 binding to the IL-12 receptor complex initiates JAK2 and TYK2 phosphorylation, which subsequently phosphorylate STAT4 at Tyr693, the critical event in eliciting IFN-γ expression^11,12,37. Nonetheless, RASA3 did not affect Il12r level and the IL-12-related pathway in CD4⁺ T cells (Fig. 3F, G). Notably, Rasa3-deficient CD4⁺ T cells exhibited elevated STAT4 Tyr693 phosphorylation compared to Rasa3-sufficient controls, both in vitro under Th1-polarizing conditions and in CD4⁺ T cells in vivo (Fig. 3H, I). However, RASA3 deficiency did not affect the phosphorylation of key IL-12 signaling components (p38, TYK2, JAK2), indicating that RASA3’s effect on STAT4 occurs independently of the initial activation of these kinases (Supplementary Fig. 3E and Fig. 3J, K). In summary, RASA3 functions to restrain IFN-γ production by specifically repressing STAT4 phosphorylation.

Fig. 3. Rasa3-deficient CD4⁺ T cells dominantly promote IFN-γ generation via increasing STAT4 phosphorylation level.

A Flow cytometry of IFN-γ-producing CD4⁺ T cells isolated from Cd4Cre; Rasa3^flox/+ (WT, CD45.1⁺) and Cd4Cre; Rasa3^flox/flox (KO, CD45.2⁺) mice, 3 days after activation either separately or mixed at the ratio of 1:1 under Th1 cell-polarizing conditions. (n = 6 samples from 3 independent experiments; means ± s.d.; ***p < 0.001. Per two-sided t-test). B Quantification of cell-incorporated BrdU and total DNA content (7-AAD). (n = 6 samples from 4 independent experiments; means ± s.d.; **p < 0.01, ***p < 0.001. Per two-sided t-test). C Cell viability of IFN-γ-producing CD4⁺ T cells of indicated genotypes activated in the presence of IL-12 for 3 days, monitored by Annexin V staining and flow cytometry. (n = 7 samples from 3 independent experiments; means ± s.d.; ns, not significant. Per two-sided t-test). D Gene set enrichment analysis (GSEA) plot showing apoptotic signaling pathway within the CD4⁺ T cells activated under indicated conditions. FDR, false discovery rate; NES, normalized enrichment score. E Flow-cytometry of T-bet-producing CD4⁺ T cells of indicated genotypes, 3 days after activation under Th1 cell-polarizing conditions. (n = 6 samples from 3 independent experiments; means ± s.d.; ns, not significant. Per two-sided t-test). F qRT-PCR analysis to detect mRNA levels of Il12r expressed by CD4⁺ T cells of indicated genotypes, activated under Th1 cell-polarizing conditions at indicated time points. (n = 6 samples from 3 independent experiments; means ± s.d.; ns, not significant. Per two-sided t-test). G Gene set enrichment analysis (GSEA) plot showing the IL-12 pathway within the CD4⁺ T cells activated under indicated conditions. FDR, false discovery rate; NES, normalized enrichment score. H Immunoblotting of p-STAT4 and STAT4 in the Rasa3-sufficient and -deficient CD4⁺ T cells activated at different time points under Th1 cell-polarizing conditions. Results are representative of 3 independent experiments. I Immunoblotting of p-STAT4 and STAT4 in the splenic CD4⁺ T cells from Cd4Cre; Rasa3^flox/+ (WT) and Cd4Cre; Rasa3^flox/flox (KO) mice infected 7 days with Lm-OVA. Results are representative of 3 independent experiments. J Immunoblotting to detect p-TYK2 and TYK2 protein expression in CD4⁺ T cells activated under indicated conditions at different time points. Results are representative of 3 independent experiments. K Immunoblotting to detect p-JAK2 and JAK2 protein expression in CD4⁺ T cells activated under indicated conditions at different time points. Results are representative of 3 independent experiments. Source data are provided as a Source Data file.

Hematopoietic cell kinase (HCK) promoted type I immunity by enhancing STAT4 phosphorylation

Given that RASA3 is not a kinase, we hypothesized that it controls STAT4 phosphorylation, likely by regulating an intermediary kinase. By analyzing the proteins upregulated in Rasa3-deficient CD4⁺ T cells compared to Rasa3-sufficient CD4⁺ T cells through LC-MS, we identified that two kinases, HCK and KAPCA, were the most upregulated kinases in Rasa3-deficienct Th1 cells (Fig. 4A). Then, CD4⁺ T cells were activated under a Th1-polarized condition and treated with iHCK-37 or dasatinib, the inhibitors for HCK, and KAPCA inhibitor BLU0588. We found that inhibition of HCK, but not KAPCA, suppressed IFN-γ expression in both Rasa3-sufficient and -deficient T cells (Fig. 4B and Supplementary Fig. 4A, B).

Fig. 4. RASA3 restricts Th1 differentiation by limiting HCK expression and STAT4 phosphorylation.

A The log2 protein intensity in Rasa3-sufficient vs -deficient CD4⁺ T cells; cells were activated under Th1 polarization conditions for 3 days. The proteins marked in red squares are related to phosphokinases, and the protein names marked in red are phosphokinases that rank among the top 20 proteins. B Flow-cytometry to assess the effect of HCK inhibition (using 5 nM dasatinib) on IFN-γ production by CD4⁺ T cells from Cd4Cre; Rasa3^flox/+ (WT) and Cd4Cre; Rasa3^flox/flox (KO) mouse, activated under Th1 cell-polarizing conditions for 3 days. (n = 6 samples from 3 independent experiments; means ± s.d.; **p < 0.01, ***p < 0.001. Two-way ANOVA with Tukey’s test). C Ifng and Hck mRNA expression in activated CD4⁺ T cells under indicated conditions as in Supplementary Fig. 4D. Colors depict different time points of CD4⁺ T cell activation. (Spearman’s rank correlation test, 95% confidence intervals 0.8589 to 0.9729. Pearson’s correlation analysis). D Immunoblotting to detect HCK protein expression in Rasa3-sufficient and -deficient CD4⁺ T cells activated under Th1 cell-polarizing conditions at indicated time points. Results are representative of 3 independent experiments. E Immunoblotting of HCK in the splenic CD4⁺ T cells of Cd4Cre; Rasa3^flox/+ and Cd4Cre; Rasa3^flox/flox mice infected 7 days with Lm-OVA. Results are representative of 3 independent experiments. F Flow-cytometry of IFN-γ produced by CD4⁺ T cells of indicated genotypes after knocking down Hck by lentiviral-based shRNAs. (n = 6 samples from 3 independent experiments; means ± s.d.; **p < 0.01, ***p < 0.001; ns, not significant. Two-way ANOVA with Tukey’s test). G Treated CD4⁺ T cells of indicated genotypes with dasatinib under Th1 cell-polarizing conditions for 3 days, and the level of p-STAT4 and STAT4 was detected by Western blot analysis. Results are representative of 3 independent experiments. H Immunoblotting to assess HCK knockdown efficiency and the level of p-STAT4 and STAT4 in Rasa3-sufficient and -deficient CD4⁺ T cells as in (F). Results are representative of 3 independent experiments. I Flow cytometry of IFN-γ⁺CD4⁺ T cells in the SP of Lm-OVA-infected mice for 7 days of indicated genotypes, dasatinib treated at day 4, 5 and 6 after Lm-OVA infection. (n = 7 samples from 4 independent experiments; means ± s.d.; **p < 0.01, ***p < 0.001; ns, not significant. Two-way ANOVA with Tukey’s test). J Lm-OVA CFUs per gram of SP and liver of Cd4Cre; Rasa3^flox/+ and Cd4Cre; Rasa3^flox/flox mice infected 4 days with 2 × 10⁵Lm-OVA i.v. after dasatinib treatment. (n = 7 samples from 4 independent experiments for Spleen; n = 6 samples from 3 independent experiments for Liver; means ± s.d.; *p < 0.05, **p < 0.01. One-way ANOVA with Tukey’s test). K CHS responses were induced and treated with dasatinib, as shown in the scheme. L Dasatinib treatment at day3.5, 4.5, and 5, twelve hours after sensitization with DNFB on the ear of Cd4Cre; Rasa3^flox/+ and Cd4Cre; Rasa3^flox/flox mice, ear swelling was determined by vernier caliper (n = 6 samples from 3 independent experiments; means ± s.d.; ***p < 0.001; ns, not significant. Two-way ANOVA with Tukey’s test). M Representative histological sections are shown in (L). (Hematoxylin and eosin staining; scale bar = 400 µm). N Proportion of IFN-γ-producing CD4⁺ T cells in the blood of sensitized mice of indicated genotypes after treatment with dasatinib, assessed by flow cytometry. (n = 6 samples from 3 independent experiments; means ± s.d.; **p < 0.01, ***p < 0.001; ns, not significant. Two-way ANOVA with Tukey’s test). Source data are provided as a Source Data file.

Hematopoietic cell kinase (HCK) is a member of the SRC family kinases expressed in hematopoietic cells and implicated in distinct molecular signaling pathways governing phagocytosis, cell adhesion, cell death and migration^38–42. However, its role in type I immunity remains unclear. Dasatinib most potently inhibited HCK phosphorylation and only slightly affected other SRC family kinases such as FYN, LYN and LCK (Supplementary Fig. 4C). We then demonstrated that HCK expression was progressively increased during Th1 cell differentiation and was positively correlated with IFN-γ levels (Supplementary Fig. 4D, E and Fig. 4C). Furthermore, the upregulation of HCK protein in Rasa3-deficient Th1 cells was validated by immunoblotting both in vitro and in vivo (Fig. 4D, E), suggesting an essential role of HCK in Th1 cell differentiation. Additionally, short hairpin RNA (shRNA)-mediated HCK knockdown normalized the elevated IFN-γ expression in Rasa3-deficient cells, while ectopic expression of HCK in CD4⁺ T cells was sufficient to increase IFN-γ production (Fig. 4F and Supplementary Fig. 4F). To determine if HCK is involved in human Th1 immunity, we assessed the effect of HCK inhibition on IFN-γ expression. HCK inhibitor corrected IFN-γ levels in T cells with lentiviral-mediated Rasa3 knockdown (Supplementary Fig. 4G). The critical role of HCK activity in regulating IFN-γ expression was further validated by reduced STAT4 phosphorylation levels in T cells treated with HCK inhibitors but not KAPCA inhibitor, as determined by phospho-STAT4 (Tyr693) immunoblotting (Supplementary Fig. 4H, I and Fig. 4G). Moreover, shRNA-mediated knockdown of HCK impaired STAT4 phosphorylation, whereas HCK overexpression promoted it (Fig. 4H and Supplementary Fig. 4J). Thus, RASA3 suppresses HCK protein levels and subsequent STAT4 phosphorylation.

Intrigued by the above findings, we sought to address the role of HCK in type I immunity in vivo. During Lm-OVA infection, inhibition of HCK by dasatinib, a commercial drug for leukemia^43,44, repressed Th1 and Tc1 cell generation of Cd4Cre; Rasa3^flox/flox mice compared to control mice (Fig. 4I and Supplementary Fig. 4K), leading to increased Lm-OVA bacterial burden in the spleen and liver (Fig. 4J). Of interest, dasatinib administration, on the other hand, was able to decrease the ear swelling and histopathology of Cd4Cre; Rasa3^flox/flox mice with DNFB-induced CHS skin disease (Fig. 4K–M). Immunological analysis revealed that the percentage of IFN-γ⁺CD4⁺ and IFN-γ⁺TNF-α⁺CD8⁺ T cell subsets were decreased in mice with dasatinib injection (Fig. 4N and Supplementary Fig. 4L). Additionally, we also demonstrated that another HCK-specific inhibitor iHCK-37, alleviated CHS disease and type I immune responses (Supplementary Fig. 4M–P). In summary, RASA3 deficiency promotes HCK expression, which in turn enhances STAT4-phosphorylation and ultimately increases IFN-γ-production.

HCK directly phosphorylated and activated STAT4

Then we are prompted to determine how HCK regulates STAT4 phosphorylation in Th1 cells. Interestingly, HCK binds to STAT4 during Th1 cell generation (Fig. 5A), detected by endogenous co-immunoprecipitation assays and further validated in HEK 293 T cells (Supplementary Fig. 5A). In addition, the introduce of HCK protein in HEK 293 T cells resulted in STAT4 phosphorylation at phospho-STAT4 (Tyr693) and STAT4 dimer formation which is the critical event for STAT4 activation^12,19,45 (Fig. 5B, C), indicating that HCK binds to and phosphorylates STAT4. In vitro GST pulldown assay using purified GST-tagged STAT4 and Flag-HCK protein further confirmed a direct interaction between STAT4 protein and HCK (Supplementary Fig. 5B and Fig. 5D). To determine whether HCK was able to phosphorylate STAT4, we performed an in vitro kinase assay using ATPγS as a phosphate donor to trace HCK protein kinase activities by protein thiophosphorylation^46,47. Incubation of GST-STAT4 and Flag-tagged HCK with ATPγS resulted in intense thiophosphorylation of GST-STAT4 (Fig. 5E), suggesting HCK could directly phosphorylate STAT4. Notably, HCK was found to phosphorylate STAT4 at Tyr693, the site that is critical for STAT4 dimerization, nuclear translocation, and transcriptional activity^11,48, as the thiophosphorylation was abolished when using a Tyr693Phe (Y693F) mutant of GST-STAT4 during in vitro kinase assay (Supplementary Fig. 5B and 5F).

Fig. 5. HCK is the kinase that directly binds to and phosphorylates STAT4 at Tyr 693 residue.

A The interactions between HCK and STAT4, detected by co-immuno-precipitation in Rasa3-sufficient CD4⁺ T cells activated for 3 days under Th1 cell-polarizing conditions. Results are representative of 3 independent experiments. B HEK 293 T cells were transfected with plasmids encoding Flag-STAT4 and HA-HCK as indicated. The protein levels of p-STAT4, STAT4, and HCK were determined by immunoblotting. Results are representative of 3 independent experiments. C HEK 293 T cells were transfected with plasmids encoding Flag-STAT4 and HA-HCK as indicated. Native-PAGE was used to detect the dimer and unimer of STAT4 by immunoblotting. Results are representative of 3 independent experiments. D GST pull-down assay was performed with purified GST-STAT4 and Flag-HCK. Results are representative of 3 independent experiments. E In vitro phosphorylation assays of recombinant GST and GST-STAT4 by HCK kinase. Results are representative of 3 independent experiments. F In vitro phosphorylation assays of recombinant GST-STAT4 and GST-STAT4-Y693F by HCK kinase. Results are representative of 3 independent experiments. G HEK 293 T cells were transfected with plasmids encoding Flag-STAT4, HA-HCK, HA-HCK-∆SH1, HA-HCK-∆SH2 and HA-HCK-∆SH3 as indicated, and the level of p-STAT4, STAT4 and HCK was detected by Western blot analysis. Results are representative of 3 independent experiments. H HEK 293 T cells were transfected with plasmids encoding Flag-STAT4, HA-HCK, HA-HCK-Y499F and HA-HCK-Y388F as indicated, and the level of p-STAT4, STAT4 and HCK was detected by Western blot analysis. Results are representative of 3 independent experiments. I The interactions between HCK and STAT4, detected by co-immunoprecipitation in Rasa3-sufficient and -deficient CD4⁺ T cells activated for 3 days under Th1 cell-polarizing conditions. Results are representative of 3 independent experiments. J HEK 293 T cells were transfected with plasmids encoding Flag-STAT4, HA-HCK, HA-HCK-Y499F and MYC-RASA3 as indicated, and the levels of p-STAT4, STAT4, HCK and RASA3 were detected by Western blot analysis. Results are representative of 3 independent experiments. Source data are provided as a Source Data file.

HCK protein comprises a myristoylated N-terminal domain, followed by three conserved SRC Homology (SH) domains (SH3, SH2 and SH1) and a C-terminal tail^42,49,50 (Supplementary Fig. 5C). Notably, truncated SH1 domain of HCK, but not SH2 and SH3 domain, deprived HCK function in activating STAT4, as evidenced by reduced STAT4 phosphorylation and dimerization, suggesting SH1 domain is required for HCK role in STAT4 activation (Fig. 5G and Supplementary Fig. 5D). Of interest, the catalytic activity of HCK is tightly regulated by key positive Y388 and regulatory Y499 residues^51,52. Despite the Y388F and Y499F mutations having no effect on the HCK-STAT4 interaction, the inactivated HCK Y388F mutation abrogated STAT4 phosphorylation and dimerization. Conversely, the constitutively activated HCK Y499F mutant form resulted in robust STAT4 activation (Fig. 5H and Supplementary Fig. 5E, F). Thus, HCK directly binds to and phosphorylates STAT4, leading to its dimerization.

In addition, the interaction between HCK and STAT4 appears to be modulated by RASA3. Immunoprecipitation analysis revealed that RASA3 impaired the STAT4-HCK complex in both Th1 and HEK 293 T cells (Fig. 5I and Supplementary Fig. 5G). Consistent with the phenotype observed in RASA3-deficient T cells, we found that RASA3 overexpression reduced HCK expression and impaired the phosphorylation and dimerization of STAT4 (Fig. 5J and Supplementary Fig. 5H). Furthermore, RASA3 retained its ability to inhibit HCK even when HCK was constitutively activated by the Y499F mutation (Fig. 5J). Taken together, our data support a model in which RASA3 deletion promotes HCK expression and its subsequent association with STAT4, enabling the direct phosphorylation of STAT4 by HCK.

RASA3 inhibited Hck mRNA translation through repression of RPL36A

Intrigued by the above findings, we sought to elucidate the mechanism by which RASA3 regulates HCK protein expression. The mRNA level of HCK was comparable between Rasa3-sufficient and Rasa3-deficient CD4⁺ T cells during Th1 differentiation (Fig. 6A), indicating that RASA3 regulates HCK expression at the post-transcriptional level. The mRNA and protein stability of HCK remained unaltered in Rasa3-deficient Th1 cells (Fig. 6B, C), prompting us to address whether RASA3 controls HCK translation. We used sucrose gradient centrifugation to resolve polysome fractions from Rasa3-sufficient and Rasa3-deficient Th1 cells. Although RASA3 depletion did not alter the overall polysome profile (Fig. 6D), the distribution of Hck mRNA in polysome fractions was more abundant in Rasa3-deficient Th1 cells (Fig. 6E), and the rate of HCK de novo synthesis increased (Fig. 6F), indicating impaired HCK translation in the presence of RASA3.

Fig. 6. RASA3 controls HCK translation in T cells by RPL36A.

A Comparison of Hck mRNA expression in CD4⁺ T cells from Cd4Cre; Rasa3^flox/+ (WT) and Cd4Cre; Rasa3^flox/flox (KO) mouse, activated under Th1 cell-polarizing conditions at indicated time points by qRT-PCR analysis. (n = 6 samples from 3 independent experiments; means ± s.d.; ns, not significant. Per two-sided t-test). B Immunoblotting of HCK in Rasa3-sufficient and -deficient CD4⁺ T cells activated for 3 days under Th1 cell-polarizing conditions, after treating with translation inhibitor cycloheximide (CHX) for the indicated time to determine HCK protein half-life. Protein gray values were analyzed. (n = 6 samples from 6 independent experiments; means ± s.d.; ns, not significant. Per two-sided t-test). C qRT-PCR analysis of the mRNA level of Hck in CD4⁺ T cells of indicated genotypes, activated under Th1 cell -polarizing conditions for 3 days, after treating with Actinomycin D for the indicated time to determine Hck mRNA half-life. (n = 6 samples from 4 independent experiments; means ± s.d.; ns, not significant. Per two-sided t-test). D Sucrose gradient-based polysome profiling of Rasa3-sufficient and -deficient CD4⁺ T cells activated for 3 days under Th1 cell-polarizing conditions. E Hck mRNA in each ribosomal fraction was quantified by qRT-PCR and plotted as the fractional distribution, as in (D). (n = 6 samples from 4 independent experiments; means ± s.d.; **p < 0.01, ***p < 0.001. Per two-sided t-test). F HCK protein synthesis rates in Rasa3-sufficient and -deficient CD4⁺ T cells activated for 3 days under Th1 cell-polarizing conditions, as measured by puromycin labeling. Results are representative of 3 independent experiments. G Numbers of overlapping genes of screen. H Comparison of Rpl36a mRNA expression in CD4⁺ T cells of indicated genotypes, activated under Th1 cell-polarizing conditions at indicated time points by qRT-PCR analysis. (n = 6 samples from 3 independent experiments; means ± s.d.; ***p < 0.001. Per two-sided t-test). I Flow-cytometry of IFN-γ produced by CD4⁺ T cells of indicated genotypes after knocking down Rpl36a by lentiviral-based shRNAs. (n = 6 samples from 3 independent experiments; means ± s.d.; ***p < 0.001. Two-way ANOVA with Tukey’s test). J Immunoblotting to assess the level of HCK, p-STAT4 and STAT4 in Rasa3-sufficient and -deficient CD4⁺ T cells after knocking down Rpl36a by lentiviral-based shRNAs. Results are representative of 3 independent experiments. K Sucrose gradient-based polysome profiling of CD4⁺ T cells of indicated genotypes after knocking down Rpl36a by lentiviral-based shRNAs, activated under Th1 cell-polarizing conditions for 3 days. L Hck mRNA in each ribosomal fraction was quantified by qRT-PCR and plotted as the fractional distribution, as in (K). (n = 6 samples from 4 independent experiments; means ± s.d.; ***p < 0.001. Per two-sided t-test). M RPL36A-RIP followed by qRT-PCR to determine the binding of RPL36A to the indicated regions of Hck mRNA using Th1 cells, activated in the presence of IL-12 for 3 days. The qRT-PCR primers correspond to those three regions. (n = 5 samples from 3 independent experiments; means ± s.d.; ***p < 0.001; ns, not significant. One-way ANOVA with Tukey’s test). N The firefly luciferase activity in 293 T cells transfected with the HCK-3′UTR or HCK-5′UTR reporter plasmids was measured and normalized to Renilla luciferase activity. (n  =  3 samples from 3 independent experiments; means ± s.d.; **p < 0.01; ns, not significant. Per two-sided t-test). Source data are provided as a Source Data file.

Interestingly, by intersecting the LC-MS dataset with the cytosolic ribosomes gene set, we identified several ribosomal proteins, such as RPL36A, RPL28 and RPL35 (Fig. 6G). shRNA-mediated Rpl28 and Rpl35 knockdown in Rasa3-deficient CD4⁺ T cells did not reduce IFN-γ expression during Th1 cell differentiation (Supplementary Fig. 6A). We then focused on RPL36A, a structural component of the 60S ribosomal subunit, is known to regulate the translation of mRNAs^53,54. In addition to increased RPL36A expression in Rasa3-deficient Th1 cells (Fig. 6H), RPL36A was also progressively upregulated during Th1 polarization and positively correlated with IFN-γ expression (Supplementary Fig. 6B, C), suggesting a role of RPL36A in Th1 cell generation. Indeed, shRNA-mediated Rpl36a knockdown in Rasa3-deficient CD4⁺ T cells reduced IFN-γ expression during Th1 cell differentiation (Supplementary Fig. 6D and Fig. 6I), and more importantly suppressed the protein level of HCK and STAT4 phosphorylation (Fig. 6J). The contribution of RPL36A in Hck mRNA translation was further investigated by polysome profiling assay. The distribution of Hck mRNA in polysome fractions was decreased in Th1 cells that were transduced with lenti-based RPL36A shRNA (Fig. 6K, L). Of note, RPL36A favored to bind to the 3′UTR of Hck mRNA as determined by RNA immunoprecipitation (RIP) (Fig. 6M). Luciferase reporter assay in HEK 293 T cells further revealed that RPL36A enhanced HCK translation through the 3′UTR of Hck mRNA (Fig. 6N). Collectively, our data suggest that RASA3 curbs HCK translation by reducing RPL36A expression, which in turn suppresses STAT4 phosphorylation and IFN-γ expression (Supplementary Fig. 6E).

Discussion

IFN-γ-expressing Th1 and Tc1 cells play crucial roles in both protective immunity and inflammatory disorders^1,55–58. While their polarization and pathogenicity have been extensively studied, the intrinsic mechanisms that restrict IFN-γ expression remain poorly understood. Our study identifies RASA3 in T cells as a self-control mechanism that negatively regulates type I immunity by restraining the RPL36A-HCK-STAT4 signaling axis. Targeting the RASA3-HCK pathway may therefore offer a strategy to modulate type I immunity in inflammatory diseases and antibacterial defense.

We found that RASA3 is progressively repressed following Th1 stimulation, consistent with previous reports of its rapid downregulation upon TCR activation^27,59. This downregulation appears to permit the activation of naïve T cells and their differentiation into effector T cells. Interestingly, RASA3 expression varies among T cell subsets; it is high in Treg cells, memory CD8⁺ T cells and pathogenic Th17 cells but relatively low in Tfh cells, suggesting its function is highly context-dependent^28,60–62. Furthermore, RASA3 has been reported to suppress LFA-1 activation, thereby inhibiting accumulation of T cells in the lung parenchyma^27,63,64. This raises the possibility that the elevated skin inflammation observed upon T cell-specific RASA3 deletion could also be due to an accumulation of effector Th1 cells in skin lesions, an open question needs to be addressed in the future.

IL-12 induces the tyrosine phosphorylation of JAK2 and TYK2, which in turn phosphorylate the IL-12 receptor, leading to the recruitment of STAT4 and phosphorylation of STAT4 at Tyr693 by activated JAK2 and TYK2^12,16,19,20. Nonetheless, the development of drugs targeting STATs is challenging due to their lack of enzymatic activity. JAK inhibitors such as Ruxolitinib and Baricitinib have been approved for treating rheumatoid arthritis and atopic dermatitis^65–67. However, their escalating clinical application has been accompanied by serious adverse effects, which are partly attributable to the broad role of the JAK-STAT pathway in orchestrating the immune system, cell proliferation, and hematopoiesis^22,68. Our study identifies a role for the SRC kinase HCK, demonstrating that it can directly bind to and phosphorylate STAT4, thereby driving IFN-γ expression. Furthermore, HCK inhibition alleviated contact hypersensitivity but impaired antibacterial immunity, highlighting its critical role in type I responses. Mechanistically, we established that RASA3 suppresses STAT4 activation independently of the canonical JAK2/TYK2 pathway by instead repressing HCK expression. Therefore, our findings not only reveal a non-canonical pathway for STAT4 activation but also identify the RASA3-HCK axis as a potential therapeutic target for modulating type I immunity.

The transcription factors STAT4 and T-bet are established as central, non-redundant regulators of Th1 differentiation and IFN-γ production^69–72. However, the precise hierarchy and cooperative dynamics between these two factors have remained a subject of debate, with two principal models proposed. The first is a linear pathway, wherein STAT4 activation initiates a positive feedback loop involving IFN-γ, STAT1, and T-bet to amplify and stabilize the Th1 phenotype^73–75. The second is a parallel pathway, which posits that STAT4 can drive a significant portion of IFN-γ expression independently of T-bet^70,76–78. We demonstrate that RASA3 deficiency specifically enhances STAT4 phosphorylation and subsequent IFN-γ production without altering T-bet expression. This uncoupling of STAT4 activation from T-bet regulation provides direct experimental support for the parallel pathway model. Nonetheless, a mutual reinforcement loop between STAT4 and T-bet likely exists to fully potentiate Th1 cell differentiation. Therefore, we propose that RASA3 acts as a critical gatekeeper to restrain this T-bet-independent, STAT4-driven arm of type I immunity.

Methods

Human study

Lesional skin samples were collected from a patient with allergic contact dermatitis, and normal skin samples were gathered from the peripheries of removed nevi at the Department of Dermatology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University. Skin samples were applied for IHC. Experiments involving human samples were approved by the research ethics board of Sun Yat-sen Memorial Hospital (SYSKY-2025-1014-01). Informed consent of donors was obtained.

Mice

Cd4Cre; Rasa3^flox/flox mice were used on a C57BL/6 background. Rasa3^flox/flox mice were generated using the CRISPR-Cas9 technology with loxP sites flanking exon 3 of Rasa3 (Shanghai Model Organisms). Cd4Cre mice were obtained from the Jackson Laboratory (strain no. 022071). Mice used for experiments were 8-12-weeks age and sex- (both males and females) and age- matched. Littermates were used unless stated otherwise. All mice were bred and maintained under specific pathogen-free conditions (19–23 °C and 45-65% humidity) on a 12-hour dark-light cycle at the Medical Research Institute of Wuhan University. All animal experiments were performed in accordance with the protocols approved by the Animal Care and Use Committee of the Medical Research Institute, Wuhan University.

According to the requirements of the Ethics Committee of the Medical Research Institute of Wuhan University, the mouse experiments will be terminated under any of the following conditions: mice were dying, unable to move, unresponsive to gentle stimulation, exhibited breathing difficulties, diarrhea, urinary incontinence, had a 20% weight loss, or were unable to eat or drink. This research strictly complied with these regulations.

Listeria monocytogenes-OVA infection and drug administration

Listeria monocytogenes-OVA (Lm-OVA) (provided by H. Zhang, Wuhan University, 10403S-OVA strain) was stored as glycerol stocks at −80 °C and cultured in brain heart infusion (BHI, Cat: 237500, BD) broth containing 100 μg/mL streptomycin (Cat: S8290, Solarbio) and 50 μg/mL erythromycin (Cat: E8100, Solarbio) overnight at 37 °C with shaking to reach mid-log growth phase. 8-week-old mice were intravenously injected via the tail vein with 2 × 10⁵ CFUs of Listeria monocytogenes-OVA in PBS. Bacterial load was assessed on day 4 after infection, and the T cell responses were analyzed on day 7. The second infection was performed 50 days after the primary infection, with analysis conducted on day 4 post-infection.

For application of dasatinib, mice were intraperitoneally injected with dasatinib (2.5 mg/kg, Cat: S1021, Selleck) starting on day 4, 5, and 6 after Listeria monocytogenes-OVA infection.

After Listeria monocytogenes-OVA infection, infected mice were sacrificed and perfused with ice-cold phosphate-buffered saline containing 20 U/ml heparin (Cat: A417626, Sangon Biotech). The liver was separated and minced with scissors, then ground with a syringe through a 70 μm cell strainer. Cells were suspended with 40% Percoll (Cat: BS909-100ml, Biosharp); 80% Percoll was added at the bottom of the centrifuge tube with a syringe. Lymphocytes were then isolated by centrifugation at 1244 × g for 15 minutes and cells in the middle layer were subjected to subsequent analysis.

For antigen-specific T cell detection, T cells were stimulated for 4 hours with 5 μg/mL OVA257-264 (Cat: HY-P1489, MCE) or 5 μg/mL OVA329-339 (Cat: HY-P0286, MCE) in the presence of 5 μg/mL brefeldin A (BFA, Cat: 420601, Biolegend) at 37 °C in 5% CO₂-containing humidified incubators.

DNFB-induced CHS model and drug administration

Mice were challenged with 50 μL of 0.5% DNFB (v/v) (Cat: D1529, Sigma-Aldrich) dissolved in acetone (Cat: 40064460, HUSHI) /olive oil (Cat: IO9000, Solarbio) (4:1, v/v) on pre-shaved abdominal skin on day 0. On day 5, the ear was rechallenged with 20 μL 0.5% DNFB and the dorsal skin with 50 μL 0.5% DNFB (elicitation phase).

For application of dasatinib, mice were intraperitoneally injected with dasatinib (5 mg/kg, Cat: S1021, Selleck) starting on day 3.5, 4.5, and 5 after DNFB challenge. The ear thickness was measured at pre-challenge and at 12 hours post-rechallenge using a vernier caliper.

For application of iHCK-37, mice were intraperitoneally injected with iHCK-37 (10 mg/kg, Cat: HY-139147, MCE) starting on day 3.5, 4.5, and 5 after DNFB challenge. The ear thickness was measured at pre-challenge and at 12 hours post-rechallenge using a vernier caliper.

For the isolation of skin T cells, the skin was excised from the mouse dorsum, and the hair was removed. The inner side of the skin was gently scored with a scalpel (without cutting through) to facilitate enzyme penetration, and any remaining mucus was carefully removed. The skin was then placed inner-side down into a well of a culture plate. A digestion solution containing collagenase IV (2.5 mg/mL, Cat: A004186, Sangon Biotech) in serum‑free medium was prepared. The solution was slowly added along the edges of the skin until the tissue floated freely. Digestion was carried out at 37 °C for 4 hours. After digestion, the tissue was gently ground through a 70 µm cell strainer, centrifuged, and washed twice with serum‑free medium. The resulting cell suspension was filtered again before proceeding to subsequent experiments.

OVA immunization

2 mg/mL OVA (Cat: A5503, Sigma) in PBS was emulsified with an equal volume of 2 mg/mL Complete Freund’s Adjuvant (CFA, Cat: F5881, Sigma). 100 μL of the emulsion was injected subcutaneously into the inner sides of both thighs of each mouse, and the T cell responses were analyzed on day 7 after immunization.

Flow-cytometry

Lymphocytes were isolated from age-matched and sex-matched mice according to the experimental protocol. Fluorescence-conjugated anti-CD4 (RM4-5, Cat: 100534, Biolegend), anti-CD4 (RM4-5, Cat: 100509, Biolegend), anti-CD4 (RM4-5, Cat:100515, Biolegend), anti-CD4 (RM4-5, Cat:100539, Biolegend), anti-CD4 (RPA-T4, Cat: 300514, Biolegend), anti-CD8 (53-6.7, Cat: 100721, Biolegend), anti-CD8 (53-6.7, Cat: 100728, Biolegend), anti-IFN-γ (XMG1.2, Cat: 505809, Biolegend), anti-IFN-γ (B27, Cat: 506538, Biolegend), anti-T-bet (4B10, Cat: 644812, Biolegend), anti-FoxP3 (FJK-16s, Cat: 17-5773-82, Invitrogen), anti-IL-4 (11B11, Cat: 504104, Biolegend), anti-CD44 (IM-7, Cat: 103021, Biolegend), anti-CD62L (MEL-14, Cat: 104412, Biolegend), anti-TNF-α (MP6-XT22, Cat: 561063, BD), anti-CD45.1 (A20, Cat: 110706, Biolegend), anti-BrdU-FITC (3D4, Cat: 364103, Biolegend), 7-amino-actinomycin D (Cat: 420404, Biolegend), Annexin V (Cat:640907, Biolegend) and anti-Ki-67 (11F6, Cat:151209, Biolegend) were used. Cells with fluorescence-conjugated antibodies were incubated for 15 min at 4 °C in the dark for surface staining. For intracellular staining, cells were stimulated for 4 hours with 50 ng/mL of PMA (phorbol 12-myristate 13-acetate, Cat: P8139, Sigma) and 1 µM ionomycin (Cat: HY-13434A, MCE) in the presence of brefeldin A, then fixed and permeabilized for 20 min at 4 °C with the Fixation/Permeabilization Solution Kit (Cat: 73158, BioLegend) and stained with fluorescence-conjugated antibodies overnight at 4 °C. Annexin V was diluted to 1:200, anti-BrdU-FITC was diluted to 1:20, 7-amino-actinomycin D was diluted to 1:30, and other antibodies were diluted at 1:400 for staining. The detailed information of antibodies for flow cytometry is provided in Supplementary Table 1 and the gating strategies for flow cytometry in this study are presented in Supplementary Fig. 7.

Stained cells were analyzed on a CytoFLEX LX Flow Cytometer (Cat: C11185, Beckman Coulter) or CytoFLEX Flow Cytometer (Cat: B53000, Beckman Coulter). All FACS data were analyzed with FlowJo v.10 (Tree Star).

T cell activation, differentiation and proliferation in vitro

Primary mouse CD4⁺ or CD8⁺ T cells were isolated from spleen and lymph nodes by mouse CD4 (Cat: 130-117-043, Miltenyi Biotec) or CD8 magnetic beads (Cat: 130-117-044, Miltenyi Biotec) per manufacturer’s protocols. 1 × 10⁶ CD4⁺ or CD8⁺ T cells were activated in 24 well-plate pre-coated with 5 μg/mL anti-CD3 (Cat: BE0001-1, BioXCell) and 2 μg/mL anti-CD28(Cat: BE0015-1, BioXCell). For Th1 cell differentiation, 40 ng/mL IL-12 (Cat: 577004, Biolegend), 10 μg/mL anti-IL-4 (Cat: BE0045, BioXCell) were added to the culture. For Tc1 cell differentiation, 10 ng/mL IL-12 (Cat: 577004, BioLegend), 10 μg/mL anti-IL-4 (Cat: BE0045, BioXCell) were added to the culture.

For application of iHCK-37, dasatinib and BLU0588 (Cat: HY-153967, MCE) during T cell differentiation in vitro, 5 nM dasatinib, 5 nM BLU0588 and 5 μM iHCK-37 were added on day 0.

To assess proliferation, isolated CD4⁺ T cells were labeled with 10 μM carboxyfluorescein diacetate succinimidyl ester (CFSE, Cat: AS-89000, AnaSpec) for 7 minutes at room temperature and then washed with 10 mL RPMI-1640 culture medium (Cat: C11875500BT, Gibco). Labelled T cells were assessed 72 hours post activation based on CFSE dilution by flow cytometry.

Human T cells

Human T cells were obtained from Milecell Biotechnologies (Cat: PB04-N-0C, Lot: P424060906C). Purity of CD4⁺ cells was routinely tested as >98%. 1 × 10⁵ human CD4⁺ T cells were activated in 96 well-plate pre-coated with 10 μg/mL anti-CD3 (Cat: 300301, Biolegend) and 10 μg/mL anti-CD28 (Cat: 302901, Biolegend). For human Th1 cell differentiation, 10 ng/mL rhIL-12 (Cat: CI58, Novoprotein) was added to the culture.

For lentiviral transduction in vitro, human CD4⁺ T cells were cultured under Th1 differentiation conditions on day 0 and spin-inoculated with the indicated retroviruses at 1500 g for 1.5 hours with 10 mM HEPES (Cat: 15630106, Gibco) and 8 μg/mL polybrene at 30 °C on day 1. Cells were harvested and analyzed by flow cytometry on day 3.

Quantitative RT-PCR (qRT-PCR) and RNA-seq analysis

For qRT-PCR analysis, total RNA from T cells was extracted by TRIzol reagent (Cat: 15596018CN, Invitrogen) and reverse-transcribed into cDNA using TRUEscript RT Master Mix Kit (Cat: PC5802, Aidlab Biotechnology) according to the manufacturer’s instructions. Quantitative PCR (qPCR) was then performed on the CFX384 Touch Real-Time PCR Detection System (Bio-Rad Laboratories). The relative mRNA amounts of genes of interest to endogenous control β-actin were presented using arbitrary units and analyzed with classic ΔΔCT method; the primers were provided as Supplementary Table 2.

For the RNA-seq analysis, total RNA was extracted from T cells using the Direct-zol RNA MicroPrep kit (Cat: R2060, Zymo Research). Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first-strand cDNA was synthesized using random hexamer primers followed by the second-strand cDNA synthesis. The library was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification. Indexed samples were sequenced using the 150 bp paired-end mode via the NovoSeq 6000 system (Illumina) according to the manufacturer’s protocol. The raw reads were processed through fastp software. Clean data were obtained by removing reads containing adapters, reads containing poly-N, and low-quality reads from raw data. At the same time, Q20, Q30, and GC content of the clean data were calculated. All the downstream analyses were based on the clean data of high quality. The quantification results from “HTSeq (Version 2.0.3)” were then analyzed with the Bioconductor package DESeq2, which fits a negative binomial distribution to estimate technical and biological variability. Differential expression genes (DEGs) were identified, and a gene was judged to be differentially expressed when the p-value was less than 0.05. Visualization of DEGs was performed with the heatmap library in R (Version 4.3.3). Gene Set Enrichment Analysis (GSEA) was conducted through two complementary approaches: Signal-to-noise ratio-based GSEA using Broad Institute GSEA (v4.3.3).

Mass-Spectrometry (MS)

For mass spectrometry analysis, naïve CD4⁺ T cells were activated in the presence of IL-12 and eluted with the same volume of buffer (8 M Urea, 100 mM Tris-HCl, pH = 8.5). Protein samples were then reduced and alkylated with Tris (2-carboxyethyl) phosphine hydrochloride (TCEP, Cat: C4706-10G, SignalChem) and 2-Chloroacetamide (CAA, Cat: C0267-100G, SignalChem). Trypsin (Cat: T575-31N-10, SignalChem) was added overnight at 37 °C below 2 M urea buffer (Cat: 51456-500 G, SignalChem). The SDB-RPS desalting column was used for peptide purification. Peptides were loaded on a C18 Trap column (Thermo), and analyzed on timsTOF Pro (Bruker Daltonics) coupled with an UltiMate 3000 RSLCnano system (Thermo). Mobile phase A (0.1% formic acid in water) and mobile phase B (0.1% formic acid in ACN) were used to establish the separation gradient. The MS was operated in DIA PASEF mode. The MS and MS/MS spectra were acquired from 100 to 1700 m/z and the ion mobility was scanned from 0.6 to 1.6 Vs/cm2. The collision energy was ramped linearly as a function of the mobility from 59 eV at 1/K0 = 1.6 Vs/cm2 to 20 eV at 1/K0 = 0.6 Vs/cm2. DIA raw data were analyzed with DIA-NN (DIA-NN 1.9.2) in library-free mode. Spectra files were searched against the Mouse protein sequence database (2024-09-10, 17217 entries) downloaded from Uniprot. Precursor ion generation options were enabled for in silico-predicted spectral library generation; Trypsin/P with a maximum 2 missed cleavages. The missing values were imputed from a random Gaussian-shaped distribution around the detection limit of the mass spectrometer, which applied a downshift of 1.8 times the standard deviations and a width of 0.25 times the standard deviations. Valid value filtering was performed with at least 50% of valid values in at least one group. Significantly differentially expressed proteins (DEPs) were determined by the P value from Student’s t-test and fold change thresholds.

Immuno-blotting and Immuno-precipitation (IP)

T cells were lysed with IP-lysis-buffer (50 mM pH 7.5 Tris-HCl (Cat: A501492-0500, Sangon Biotech), 150 mM NaCl (Cat: A501218-0001, Sangon Biotech), 1% NP40 (Cat: N8030, Solarbio), 2 mM EDTA (Cat: abs9133, Absin), 1% Protease Inhibitor Cocktail (Cat: C0001, TargetMoI) and 1% Phosphatase Inhibitor Cocktail (Cat: C0004, TargetMoI)). The lysates were resolved by SDS-PAGE, transferred to a polyvinylidene fluoride membrane (Cat: IPVH00010, Millipore) and blocked with 5% skim milk at room temperature for 1 hour and analyzed by immuno-blotting with the following antibodies: anti-RASA3 (Cat: H00022821-M01, Novus Biologicals), anti-p-STAT4 (Cat: 5267S, Cell Signaling Technology), anti-STAT4 (Cat: 2653S, Cell Signaling Technology), anti-HCK (Cat: A14537, Abclonal), anti-p-HCK (Cat: ab61055, Abcam), anti-p-TYK2 (Cat: F0956, Selleck), anti-TYK2 (Cat: A2128, Abclonal), anti-JAK2 (Cat: F0231, Selleck), anti-p-JAK2 (Cat: 3771S, Cell Signaling Technology), anti-P38 (Cat: 9212S, Cell Signaling Technology), anti-p-P38 (Cat: 9211S, Cell Signaling Technology), anti- thiophosphate ester (Cat: ab92570, Abcam) anti-HA (Cat: AE105, Abclonal), anti-GST (Cat: AE077, Abclonal), anti-Flag (Cat: AE092, Abclonal), anti-Puromycin (Cat: A23031, Abclonal), anti-FYN (Cat: A9165, Abclonal), anti-p-FYN (Cat: AP0510, Abclonal), anti-LYN (Cat: A2093, Abclonal), anti-p-LYN (Cat: AP1050, Abclonal), anti-LCK (Cat: A23988, Abclonal), anti-p-LCK (Cat: AP0182, Abclonal), anti-Puromycin (Cat: A23031, Abclonal), anti-MYC (Cat: 60003-2-Ig, Proteintech) and anti-β-Actin (Cat: AC026, Abclonal), at 4 °C overnight. The blot was subsequently incubated with horseradish peroxidase-linked goat anti-mouse (Cat: AS003, Abclonal) and goat anti-rabbit (Cat: AS014, Abclonal) secondary antibodies at room temperature for 1 hour and the signal was detected using a ChemiDoc MP Imaging System (Bio-Rad). For Native-PAGE, the lysates were resolved by PAGE and did not contain SDS and β-ME.

For IP, cells were lysed with IP-lysis-buffer. Cell lysates were incubated with 50 μL agarose beads (Cat: 17061801, Cytiva) and antibodies. After 3 hours incubation at 4 degrees, beads were washed 3 times with IP-lysis-buffer containing 0.5 M NaCl. Associated protein was eluted by 2 × Laemmli sample buffer (Cat: 1610737, Bio-Rad) added with 5% β-Mercaptoethanol (Cat: M828395, Macklin) and incubated at 95 °C for 5 min. Eluted proteins were separated by SDS-PAGE gel and analyzed by immuno-blotting.

For the de novo synthesis of HCK, Th1 cells are differentiated in vitro for three days. The cells are collected and incubated with 5 µg/mL puromycin (Cat: HY-K1057, MCE) for 10 minutes to label newly synthesized proteins. The cells are then centrifuged, and the supernatant is removed. Subsequently, the cell pellet is washed twice with ice‑cold PBS and is ready for HCK immunoprecipitation (IP).

Anti-HCK, anti-p-HCK, anti-FYN, anti-p-FYN, anti-LCK, anti-p-LCK, anti-LYN, anti-p-LYN and anti-RASA3 was diluted at 1:500; anti-β-Actin, anti-HA, anti-Flag, anti-MYC, anti-GST and anti-puromycin was diluted at 1:5000; other antibodies were diluted at 1:1000. Detailed information regarding the antibodies used for Western blot analysis is listed in Supplementary Table 3 and the statistics of all Western blots in the manuscript are provided in Supplementary Fig. 8.

Cell cycle assay

Naïve CD4⁺ T cells were activated under Th1 polarizing conditions for 2 days and incubated with 10 μM 5-bromo-20-deoxyuridine (BrdU, Cat: 423401, Biolegend) for 24 hours. T cells were then collected and fixed overnight at 4 °C, permealized and incubated with DNase I (Cat: E1009-A, Zymo Research) for 45 min at 37 °C, and FITC-conjugated anti-IFN-γ and PB-conjugated anti-CD4 antibody were added for 2 hours at room temperature. PE-conjugated anti-BrdU antibody (dilution: 1:20) was added for 20 min at 4 °C. Total DNA was stained with 7-amino-actinomycin D (7-AAD), followed by flow cytometric analysis.

Protein and mRNA stability assay

For the protein stability assay, CD4⁺ T cells were cultured under Th1 differentiation conditions for 3 days and treated with 100 μg/mL cycloheximide (CHX, Cat: HY-12320, MCE). Cells were harvested, and lysates were analyzed by Western blot.

For the mRNA stability assay, CD4⁺ T cells were cultured under Th1 differentiation conditions for 3 days and treated with 5 μg/mL actinomycin D (Act D, Cat: HY-17559, MCE). Cells were harvested for RNA isolation and cDNA synthesis for subsequent RT-PCR.

Purification of Flag or GST fusion proteins

For GST fusion proteins, recombinant GST-STAT4 and GST-STAT4-Y693F were produced in BL21(DE3) (Cat: B528414, Sangon Biotech) super competent cells. Single clones expressing the plasmid of each individual GST fusion protein were cultured until the OD₆₀₀ reached 0.5–0.6. The proteins were induced by 0.75 mM IPTG (Isopropyl β-D-thiogalactoside, Cat: S6826, Selleck) at 16 °C at 160 rpm for 16 hours. The bacterial pellet was lysed with lysis buffer (PBS containing 1% Triton-X 100 (Cat: T8200, Solarbio) and 0.2 mM PMSF (Cat: S3025, Selleck)) and ultrasound for 15-20 minutes (30% power, 5 s on, 5 s off). The supernatant was incubated with 50 μL glutathione-coated agarose beads (Cat: SA008010, Smart-Lifesciences) 4 hours at 4 °C in a rotator. Then, the beads were washed 2 times with 5 mL lysis buffer and incubated with 1 mL eluent (PBS containing 50 mM Tris-HCl, pH = 8.0 and 10 mM glutathione (Cat: 143313, Biofroxx)) for 5 minutes at 4°C.

For Flag fusion proteins, recombinant Flag-HCK was produced in HEK 293 T cells. The pEF-Flag-HCK construct was transfected into HEK 293 T cells as described in the methods. Cells were lysed with lysis-buffer (50 mM HEPES, 300 mM NaCl, 1 mM MgCl₂ (Cat: 20059028, HUSHI), 1 mM EGTA (Cat: A360509, Sangon Biotech), 0.5% Triton-X 100, 1 mM DTT (Cat: B645939, Sangon Biotech), 0.1 mM ATP (Cat: 60605ES03, YEASEN), 1% protease inhibitor cocktail) and then incubated with 50 μL flag-coated agarose beads (Cat: M7001, Mabnus) for 4 h at 4°C in a rotational manner. Beads were washed 4 times with lysis buffer and 2 times with wash buffer C (50 mM HEPES, 150 mM NaCl, 1 mM MgCl₂, 1 mM EGTA, 0.01% Triton-X 100, 1 mM DTT, 0.1 mM ATP) and then incubated with 200 μL eluent (TBS containing a final concentration of 400 μg/mL flag peptides (Cat: B23111, Selleck)).

GST pull-down assay

50 μL glutathione-agarose beads were washed twice with 500 μL GST pull-down lysis buffer (20 mM Tris-HCl, pH = 8.0; 150 mM NaCl; 0.5% NP-40; 1 mM EDTA; 1% protease inhibitor cocktail), resuspended with 500 μL GST pull-down lysis buffer, and then purified fusion proteins Flag-HCK and GST-STAT4 were added. After rotating incubation overnight at 4 °C, the beads were washed 4 times with GST pull-down lysis buffer and analyzed by Western blot.

In vitro kinase assay

For in vitro HCK assays, GST-STAT4, GST-STAT4-Y693F, and Flag HCK were purified. GST fusion protein was incubated with Flag-HCK in kinase assay buffer containing 500 μM ATPγS (Cat: ab138911, Abcam) in a final volume of 30 μL for 1 hour at 30 °C. Then the 0.5 mM p-Nitrobenzyl mesylate (Cat: ab138910, Abcam) was added and incubated for 1 hour at 30 °C. The protein samples were prepared and detected with anti-thiophosphate-ester antibody (Cat: ab92570, Abcam).

RNA immunoprecipitation

For RNA immunoprecipitation (RIP), Th1 cells that have been differentiated in vitro for three days are cross‑linked with 1% paraformaldehyde (Cat: 252549, Sigma) for 10 minutes. The cross‑linking reaction was quenched by adding one‑tenth volume of 2.66 M glycine (Cat: 56-40-6, VWR). After centrifugation, the cells were collected and lysed in 1 mL of IP‑lysis buffer supplemented with RNase (Cat: N8080119, Thermo) and protease inhibitors. The lysis was performed for 20 minutes, followed by sonication using a cycle of 30 seconds on and 30 seconds off, repeated for 13 cycles. A 10% aliquot of the lysate was saved as the input control. The sonicated lysate was then incubated overnight at 4 °C with either anti-RPL36A (Cat: 15185-1-AP, Proteintech) antibody or control IgG (Cat: 2729S, Cell Signaling Technology) antibody, both pre‑bound to washed agarose beads. After incubation, the beads were washed twice with IP‑lysis buffer. RNA was subsequently extracted from the beads with TRIzol reagent and analyzed by qRT‑PCR. The mRNA level of Hck was analyzed. (fold enrichment=2(-∆∆^{CT(normalized RPL36A RIP)}); ∆∆^{CT(normalized RPL36A RIP)} = ∆^{CT (normalized RPL36A RIP)}-∆^{CT(normalized IgG)}; ∆^{CT(normalized RIP)}=(average^CT(RIP)-(average^CT(Input)-log2(input dilution factor)))).

Dual luciferase reporter assay

The 3’‑UTR and 5’‑UTR of HCK mRNA were constructed into the pGL4‑SV40 reporter plasmid. The pEF‑Flag‑RPL36A plasmid, the reporter plasmid, and the TK‑Renilla control plasmid were co‑transfected into HEK 293 T cells using the polyethylenimine (PEI, Cat: MX2203, MKBio) transfection reagent. The relative luciferase activity in the protein samples was measured using a dual‑luciferase reporter assay (Cat: DL101-01, Vazyme) according to the manufacturer’s instructions.

Ectopic gene expression and shRNA-mediated gene knockdown

For ectopic gene expression, the murine full-length cDNA encoding RASA3, STAT4, and HCK were cloned into pCMV-MYC and pEF-Flag vectors. For short hairpin RNA (shRNA)-mediated gene knockdown, the primers were provided as Supplementary Table 4.

Retroviruses and lentiviruses were prepared by transfecting HEK 293 T cells. For retrovirus production, the murine full-length cDNA encoding HCK was cloned into a retroviral expression vector (RVKM). When HEK 293 T cells in a 10 cm dish grow to 80%, 7.5 μg retrovirus vector and 2.5 μg pCL-Eco packaging vector were mixed evenly and transfected at a ratio of 3:1 with PEI according to the manufacturer’s instructions. For lentivirus production, the lentiviral shRNA vector, psPAX2, and pMD2.G were transfected at a 3:2:1 ratio using BES reagent according to the manufacturer’s instructions. After 48 hours, viruses were collected, filtered through 0.45μm syringe filter, and stocked in −80 °C in the freezer.

For retroviral or lentiviral transduction in vitro, isolated CD4⁺ T cells were cultured under Th1 differentiation conditions on day0 and spin-inoculated with indicated retroviruses at 1500 g for 1.5 hours with 10 mM HEPES and 8 μg/mL polybrene at 30 °C on day 1. Cells were harvested and analyzed by flow cytometry on day 3. All cells were cultured at 37 °C in 5% CO₂-containing humidified incubators.

Ribosome separation experiment

Th1 cells were treated with 100 μg/mL cycloheximide for 10 minutes at 37 °C before harvesting. Cells were washed 3 times with PBS containing 100 μg/ml CHX and then resuspended in 600 μL cell lysis buffer (20 mM Tris-HCl pH 7.5, 100 mM NaCl, 5 mM MgCl₂, 0.5% NP-40, supplemented with 100 μg/ml CHX, 1% Protease Inhibitor Cocktail, and 40U/ml RNase inhibitor (Cat: EO0381, Thermo)). 500 μL of protein solution was loaded onto 10–50% sucrose gradients and centrifuged at 190,000 × g for 90 minutes at 4 °C using a Beckman SW-41Ti rotor. Subsequently, the RNA from each fraction collected by the density gradient fractionation system (Biocomp) was extracted and analyzed by qRT-PCR.

Statistical analysis

Two-tailed/sided Student’s t-test, one-way ANOVA test, and two-way ANOVA test were used. Nonparametric t-test and Dunn’s test were used for samples that failed to pass the normal distribution test. In the figures, *, **, and *** were used to indicate p < 0.05, p < 0.01, and p < 0.001, respectively; ns means not significant. All results shown are mean ± SD unless stated otherwise.

Supplementary information

Supplementary Information ^{(8.1MB, pdf)}

Transparent Peer Review file ^{(2.1MB, pdf)}

Source data

Source Data ^{(6.8MB, xlsx)}

Author contributions

J.Y.S. contributed to the design and implementation of the cellular, molecular, biochemical, and animal experiments and the writing of the manuscript; X.Y.L. contributed to the flow cytometry and animal experiments. J.S. contributed to the bioinformatic analysis; Y.H.Z., X.C., and J.J.Z. contributed to protein purification and edited the manuscript; C.H. and J.F.Z. contributed to kinase activity experiments; H.X. designed the experiment and contributed to the patient’s samples; B.W. conceived the project, designed the experiments, analyzed the data, and wrote the manuscript.

Peer review

Peer review information

Nature Communications thanks Hiromitsu Hara and the other anonymous reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

Funding

B.W. discloses support for the research of this work from National Natural Science Foundation of China [2470939, 32170886], Fundamental Research Funds for the Central Universities [2042022dx0003, 2042022rc0032], Natural Science Foundation of Wuhan [2024040701010031], and National Key R&D Program of China [2021YFC2701602]. H.X. discloses support for the research of this work from the Natural Science Foundation of Guangdong Province of China [2023A1515030139].

Data availability

The RNA-seq data generated in this study have been deposited in the Gene Expression Omnibus repository at the National Center for Biotechnology Information under accession code GSE313543 and GSE337937. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (https://proteomecentral.proteomexchange.org) via the iProX partner repository^79,80 under accession code PXD071978 (http://www.iprox.org/page/project.html?id=IPX0014657000). Source data are provided with this paper.

Competing interests

The authors declare no competing interests.

Supplementary information

The online version contains supplementary material available at https://doi.org/10.1038/s41467-026-76201-3.