Introduction

Pulmonary hemorrhage (PH) is a life-threatening manifestation of systemic autoimmunity, with reported mortality rates ranging from 40% to 90%. PH occurs in autoimmune disorders such as Goodpasture syndrome, systemic lupus erythematosus (SLE), and antiphospholipid syndrome, in which immune-mediated injury to the alveolar-capillary interface triggers rapid and often fatal pulmonary bleeding^1–3. Current standard of care relies on high-dose corticosteroids together with systemic immunosuppression, including cyclophosphamide and rituximab, and in some cases antifibrinolytic agents to stabilize clot formation^3,4. Despite these aggressive interventions, outcomes remain poor, highlighting a major unmet need to define the immune mechanisms that drive organ-specific injury versus those that promote tissue protection and repair^5–8.

Interleukin-2-inducible T cell kinase (ITK), a member of the Tec family of tyrosine kinases, has emerged as a key checkpoint in shaping T cell responses. We previously demonstrated that targeting ITK signaling uncouples graft-versus-host disease (GVHD) from graft-versus-leukemia (GVL) activity, supporting the concept that ITK differentially regulates pathogenic and protective T cell functions⁹. We have also shown that ITK deficiency gives rise to highly suppressive regulatory T cells (Tregs) with enhanced functional capacity¹⁰. However, whether ITK-dependent Treg reprogramming can be harnessed to preserve the alveolar-capillary barrier during acute autoimmune lung injury remains unknown.

Defining the mechanisms by which ITK deficiency protects against pulmonary hemorrhage is a central objective of this study. ITK functions as a critical pro-inflammatory amplifier in autoimmunity by strengthening T cell receptor (TCR) signaling, promoting the differentiation of pathogenic T helper 17 (Th17) and T follicular helper (Tfh) cells, and constraining the development or function of regulatory T (Treg) cells^11,12. Through these effects, ITK shifts the immune balance away from tolerance and toward sustained inflammation and tissue injury. Increased ITK activation in CD4⁺ T cells have been implicated in the pathogenesis of several autoimmune diseases, including rheumatoid arthritis (RA) and multiple sclerosis (MS), positioning ITK as an important molecular checkpoint and a potential therapeutic target for restoring immune homeostasis^13,14. Despite these established roles, its contribution to pristane-induced pulmonary hemorrhage remains unknown. Here, using ITK-deficient (ITK^-/-) mice^9,15, we show that loss of ITK drives marked remodeling of the T cell compartment. Specifically, both CD8⁺ and CD4⁺ T cells from ITK^-/- mice exhibited significant expansion of central memory (CM) and effector memory (EM) subsets compared with wild-type (WT) controls. These populations were characterized by increased expression of CD44 and CD122, together with the T-box transcription factors Eomesodermin (Eomes) and T-bet, consistent with a shift toward a memory-like and potentially protective immune state.

To define the functional contribution of ITK to autoimmune lung injury, we challenged WT and ITK^-/- mice with pristane. ITK^-/- mice were fully protected from pristane-induced pulmonary hemorrhage, showing a striking absence of alveolar bleeding compared with WT controls. This protective effect extended beyond the lung, as ITK^-/- mice also exhibited significantly reduced pathology in multiple organs, indicating that loss of ITK broadly attenuates systemic autoimmune injury. This protection was accompanied by a marked reduction in the infiltration of proinflammatory immune cells into the lung and other affected tissues. Consistent with diminished inflammatory cell accumulation, ITK^-/- mice also displayed significantly lower levels of proinflammatory cytokines than WT mice. A key cellular feature associated with this phenotype was the expansion of regulatory T cell populations. Specifically, ITK-deficient mice exhibited significantly increased frequencies of both canonical (CD4⁺CD25⁺Foxp3⁺) and non-canonical (CD4⁺CD25⁻Foxp3⁺) Tregs, consistent with our previous findings^10,16. Together, these data suggest that loss of ITK signaling establishes a dominant regulatory immune environment that preserves tissue integrity during autoimmune injury.

To determine whether the protection observed in ITK^-/- mice was intrinsic to the regulatory T cell compartment, we performed adoptive transfer experiments¹⁷. Tregs isolated from ITK-deficient donors and transferred into WT recipients at the time of pristane challenge conferred robust protection against PH. This transfer significantly reduced pulmonary inflammation, suppressed systemic proinflammatory cytokine production, and increased anti-inflammatory mediator expression. These findings demonstrate that ITK-deficient Tregs possess enhanced suppressive capacity and are sufficient to override pathogenic inflammation in a WT host environment. To define the molecular basis of this heightened regulatory state, we performed unbiased RNA sequencing (RNA-seq). Transcriptomic profiling revealed that ITK-deficient Tregs were significantly enriched for programs associated with immune homeostasis, cytokine regulation, metabolism, and tissue repair. Collectively, these findings identify a previously unrecognized role for ITK signaling in regulating alveolar-capillary barrier integrity. More broadly, our work establishes the ITK axis as a promising therapeutic target for pulmonary hemorrhage and other severe inflammatory lung diseases characterized by dysregulated cytokine responses.

Results

ITK Deficiency Promotes a Predominantly Memory-like T Cell Phenotype

Interleukin-2-inducible T cell kinase (ITK) is a Tec-family tyrosine kinase required for optimal T cell receptor (TCR) signaling and is therefore critical for T cell development, activation, and effector function¹⁸. To determine how ITK deficiency reshapes the T cell compartment prior to autoimmune challenge, we analyzed splenic T cell subsets in WT and ITK^-/- mice on a C57BL/6 background^9,19. Because the differentiation state of αβ T cells strongly influences effector potential and disease outcome^20–23, we used flow cytometry to profile freshly isolated splenic CD3⁺ T cells. T cells were first divided into CD4⁺ and CD8⁺ populations and then classified as naïve (CD44^lo CD62L^hi), central memory (CM; CD44^hi CD62L^hi), or effector memory (EM; CD44^hiCD62L^lo) subsets^10,24. ITK^-/- mice displayed a significantly increased proportion of CM CD8⁺ T cells compared with WT controls (Fig. 1A-D). Within the CD4⁺ compartment, this shift was even more pronounced, with ITK^-/- mice showing significant expansion of both EM and CM subsets, accompanied by a corresponding reduction in the naïve T cell population (Fig. 1E-H). Together, these findings indicate that ITK deficiency skews the T cell compartment toward a memory-like state, establishing a distinct immune baseline that may favor protective and regulatory responses over pathogenic inflammation.

Figure 1.

ITK Deficiency Enhances Expression of Memory-Associated Activating Markers and Transcriptional Regulators

We have previously shown that T cells with attenuated T cell receptor (TCR) signaling can acquire an activated phenotype without driving pathogenic alloimmunity^{9,10,19,25,26}. Consistent with this, flow cytometric analysis revealed significantly increased CD44 expression on CD3⁺CD8⁺ T cells from ITK^-/- mice compared with WT controls (Fig. 2A-B). We next examined transcriptional regulators associated with memory differentiation. The T-box transcription factors T-bet and Eomes cooperatively promote memory formation in part through induction of CD122 (IL-2Rβ), a key component of the IL-2 and IL-15 receptor complexes^27,28. Consistent with an enhanced memory-like state, CD8⁺ T cells from ITK^-/- mice expressed significantly higher levels of T-bet, Eomes, and CD122 than WT controls (Fig. 2C-H). Notably, a similar pattern was observed in the CD4⁺ T cell compartment, where T cells from ITK^-/- mice also exhibited increased expression of CD44, Eomes, CD122, and T-bet (Supplementary Fig. 1A-H). Together, these data indicate that ITK deficiency reprograms both CD8⁺ and CD4⁺ T cell lineages toward a memory-like phenotype. Importantly, these findings suggest that attenuated TCR signaling does not produce a loss-of-function state, but instead preserves—and in some respects enhances—features associated with T cell fitness and cytokine responsiveness.

Figure 2.

ITK Deficiency Confers Complete Protection Against Pristane-Induced PH and Systemic Pathology

We and others have previously shown that targeting ITK signaling attenuates pathogenic immune responses across multiple models of alloimmunity^{9,10,19,25,26}. To investigate the role of ITK in autoimmune lung injury, we used the pristane-induced pulmonary hemorrhage (PH) model, which recapitulates key features of immune-mediated capillary damage observed in clinical conditions such as systemic lupus erythematosus (SLE) and Goodpasture syndrome^1–3. WT and ITK^-/- mice were challenged with a single intraperitoneal injection of pristane (0.5 mL) and monitored for 14 days, as previously established in our studies and others^17,20,29,30 (Fig. 3A). Although neither group exhibited significant weight loss during this acute phase, gross and histologic analyses at day 14 revealed striking differences in disease severity. Lungs from WT mice showed severe diffuse pulmonary hemorrhage, with extensive erythrocyte accumulation and marked tissue injury on histologic examination. In contrast, ITK^-/- mice were fully protected from PH, preserving normal lung architecture and clear alveolar spaces (Fig. 3B-E). Because progression of PH is driven in part by recruitment of pathogenic innate immune cells^31–33, we next quantified inflammatory monocyte infiltration. WT mice displayed a marked influx of CD11b⁺Ly6C⁺ inflammatory monocytes into the lung, whereas this population was significantly reduced in ITK^-/- mice (Fig. 3F-G). Beyond the lung, ITK deficiency also mitigated systemic manifestations of pristane-induced injury^34,35. WT mice developed pronounced splenomegaly associated with megakaryocyte infiltration, whereas spleens from ITK^-/- mice remained normal in size and histologic appearance (Fig. 3H-I; Supplementary Fig. 2). Similarly, WT mice exhibited substantial small intestinal injury, including epithelial hyperplasia and focal acute inflammation, both of which were markedly attenuated in ITK^-/- mice (Fig. 3J; Supplementary Figs. 3-4). Together, these findings establish that ITK deficiency confers multi-organ protection against the systemic inflammatory injury triggered by pristane. Having established this protective phenotype, we next asked whether loss of ITK also restrains systemic proinflammatory cytokine production.

Figure 3.

ITK Deficiency Attenuates Proteinuria and Shifts the Systemic Cytokine Balance Toward Resolution

Proteinuria is a clinical hallmark of pulmonary-renal syndrome and frequently accompanies PH in systemic autoimmune disease, serving as an important indicator of glomerular involvement³⁶. To determine whether the protection observed in ITK^-/- mice extended to the kidney, we quantified urinary protein levels at baseline (day 0) and at the study endpoint (day 14). Whereas WT mice developed a significant increase in proteinuria following pristane challenge, ITK^-/- mice showed a markedly attenuated response, indicating that loss of ITK signaling protects against PH-associated renal dysfunction (Fig. 4A-C). Histologic analysis at this acute 14-day time point did not reveal overt structural kidney injury, although WT mice displayed focal inflammatory cell infiltration that was absent in ITK^-/- mice (Supplementary Fig. 4). These findings suggest that although glomerular injury remains at an early stage in this acute model, the ITK axis already regulates the inflammatory processes that precede overt renal damage.

Figure 4.

Because PH is associated with a robust systemic inflammatory response, we next quantified circulating cytokines to determine how the ITK axis influences the balance between pathogenic and reparative immunity. We measured key mediators implicated in PH pathogenesis, including IFN-γ and TNF-α, which promote procoagulant activity and tissue factor expression³⁷, as well as IL-6 and IL-17, which drive neutrophil recruitment and vascular permeability^38–42. Serum analysis by ELISA showed that ITK^-/- mice had significantly lower levels of all four proinflammatory cytokines than WT controls (Fig. 4D-G). Conversely, because anti-inflammatory cytokines can restrain pathogenic immune responses^43,44, we asked whether ITK deficiency also promotes a pro-resolving cytokine environment. Indeed, ITK^-/- mice produced significantly higher levels of IL-10 and IL-13 during PH (Fig. 4H-I). Together, these findings indicate that ITK^-/- does not simply impose broad immunosuppression, but instead redirects the systemic immune response toward regulation and resolution, further supporting ITK as a promising therapeutic target in PH and related autoimmune disease.

ITK Deficiency Expands Canonical and Non-canonical Treg Populations During PH

Regulatory T cells (Tregs) are key mediators of immune suppression and tissue repair, acting in part through anti-inflammatory cytokines such as IL-10 to restrain neutrophils and inflammatory monocytes^45,46. In addition to their suppressive function, Tregs contribute directly to restoration of the alveolar-capillary barrier following inflammatory injury^45,47. We have previously shown that ITK deficiency increases the frequency of both canonical (CD4⁺CD25⁺Foxp3⁺) and non-canonical (CD4⁺CD25⁻Foxp3⁺) Tregs^15,48. To determine whether these subsets expand during autoimmune lung injury, we analyzed splenic Tregs from WT and ITK^-/- mice following pristane challenge. ITK^-/- mice exhibited significantly higher absolute numbers and frequencies of both canonical and non-canonical Treg subsets than WT controls (Fig. 5A-B; Supplementary Fig. 6A-B). These findings suggest that ITK signaling normally constrains Treg expansion during systemic inflammation and that its absence establishes a broader regulatory reservoir capable of counteracting PH pathogenesis. Furthermore, the expansion of non-canonical CD25⁻Foxp3⁺ Tregs—a population often considered less stable or transient in other inflammatory contexts—suggests that ITK deficiency creates a uniquely permissive environment for robust, multi-lineage regulatory responses.

Figure 5.

To determine whether the expanded Treg populations in ITK^-/- mice possess enhanced therapeutic activity in vivo, we performed adoptive transfer experiments in WT recipients with established disease. WT mice were challenged with pristane and monitored for 10 days to allow initiation of systemic inflammation and lung injury. On day 10, mice were assigned to three groups: an untreated control group, a group receiving FACS-purified WT Tregs (CD3⁺CD4⁺CD25⁺Foxp3⁺), and a group receiving ITK^-/- Tregs (1 x 10⁶) cells per mouse; Fig. 5C-D). By day 21 after pristane injection, untreated WT mice developed severe gross pulmonary pathology and a significant increase in proteinuria relative to baseline (day 0) (Fig. 5E). Recipients of WT Tregs showed partial protection, with a moderate reduction in proteinuria compared with untreated controls, although levels remained significantly elevated above baseline (Fig. 5F). In contrast, WT mice treated with ITK^-/- Tregs were fully protected from PH, with proteinuria returning to baseline levels comparable to those observed before challenge (Fig. 5G). Together, these findings demonstrate that ITK-deficient Tregs possess enhanced suppressive and tissue-protective capacity in vivo and support modulation of the ITK axis as a promising therapeutic strategy for PH and severe autoimmune inflammatory injury.

Consistent with the reduced cytokine levels observed in ITK^-/- mice (Fig. 4), we next asked whether adoptive transfer of ITK-deficient Tregs could similarly reshape the systemic cytokine milieu in WT recipients. Serum ELISA analysis confirmed that pristane-challenged WT mice developed marked increases in IFN-γ, TNF-α, IL-6, and IL-17 (Fig. 5H-K). Although transfer of WT Tregs produced a modest reduction in these proinflammatory cytokines, recipients of ITK^-/- Tregs exhibited significantly greater suppression of this inflammatory response. Notably, WT mice treated with ITK^-/- Tregs also showed a significant increase in serum IL-10, a key mediator of immune regulation and resolution, exceeding levels observed in both untreated and WT Treg-treated groups (Fig. 5L). These findings indicate that ITK^-/- Tregs not only suppress pathogenic Th1- and Th17-associated inflammatory responses more effectively than WT Tregs, but also actively promote an anti-inflammatory systemic environment. Collectively, these results show that ITK^-/- Tregs confer enhanced protection against PH by shifting the cytokine balance from a proinflammatory toward a pro-resolving state.

ITK Deficiency Reprograms the Treg Transcriptome Toward a Metabolically and Functionally Enhanced State

Having established that ITK-deficient Tregs confer enhanced protection against PH, we next sought to define the global transcriptomic programs underlying this phenotype. We performed RNA sequencing (RNA-seq) on FACS-purified canonical (CD3⁺CD4⁺CD25⁺Foxp3⁺) Tregs from WT and ITK^-/- mice, as well as non-canonical (CD3⁺CD4⁺CD25⁻Foxp3⁺) Tregs from ITK^-/- mice. Notably, the non-canonical subset was selectively expanded in ITK^-/- mice, precluding direct comparison with an equivalent WT population. Hierarchical clustering of normalized gene expression profiles revealed distinct transcriptomic segregation, with samples partitioning into two principal modules: Module 1, consisting of genes downregulated in ITK^-/- subsets, and Module 2, comprising genes upregulated in ITK^-/- Tregs (Fig. 6A-B). To identify the biological programs associated with the enhanced function of ITK-deficient Tregs, we performed Gene Set Enrichment Analysis (GSEA) and hallmark pathway profiling. Dot plot and ridge plot analyses of normalized enrichment scores (NES) revealed significant enrichment of multiple pathways in both canonical and non-canonical ITK^-/- Tregs, including MYC targets, mTORC1 signaling, oxidative phosphorylation (OXPHOS), and glycolysis (Fig. 6C-H). Additional enrichment was observed in pathways related to DNA repair, E2F targets, and immune homeostasis, together with differential regulation of IFN-γ and NF-κB signaling. GSEA enrichment plots further supported the conclusion that loss of ITK signaling promotes a metabolically active and functionally enhanced Treg state, characterized by increased cell cycle- and metabolism-associated programs (Supplementary Fig. 7A-K). Together, these data indicate that ITK deficiency does not simply expand Treg numbers, but instead reprograms the Treg transcriptome toward a state consistent with greater metabolic fitness and regulatory function. These findings provide a mechanistic framework for the potent tissue-protective activity of ITK-deficient Tregs in PH.

Figure 6.

Discussion

Pulmonary hemorrhage (PH) remains a life-threatening clinical emergency in systemic autoimmune disease. With mortality rates frequently exceeding 50%, there is a pressing need to move beyond broad immunosuppression toward therapeutic strategies that promote immune resolution and tissue protection^49–53. In this study, we identify ITK as a key molecular checkpoint that regulates the balance between destructive pulmonary inflammation and reparative immunity. Our findings demonstrate that ITK deficiency confers marked protection against PH and is associated with broad remodeling of the T cell compartment toward a dominant regulatory state with features of enhanced metabolic fitness.

ITK Signaling has long been viewed as a signaling rheostat, whereby its absence or inhibition attenuates T cell receptor (TCR) signaling in ways that favor the development of memory CD8⁺ T cells and regulatory T cell lineages over pathogenic Th17 and Tfh populations^{14,15,54–57}. Although inherited ITK deficiency in humans is associated with impaired IFN-γ immunity and defective viral control^58,59, our findings suggest that in the setting of acute autoimmune injury, reduced ITK signaling does not result in immune failure, but instead supports a more protective regulatory response. In particular, we show that ITK-deficient Tregs are not only expanded in number, but are also sufficient to rescue established PH in wild-type recipients. This regulatory reprogramming may help explain why ITK-deficient T cells, which we previously showed retain anti-tumor activity without inducing graft-versus-host disease (GVHD)⁹, are also capable of protecting the alveolar-capillary barrier during autoimmune injury

Our study further shows that ITK deficiency reshapes the T cell compartment, with expansion of effector memory (EM) and central memory (CM) populations in both the CD4⁺ and CD8⁺ lineages. This memory-like shift is accompanied by increased expression of activation-associated markers and the T-box transcription factors Eomes and T-bet, which are known to support effector competence and durable memory differentiation^27,60. Interestingly, although T-bet is typically induced downstream of TCR, IL-12, and mTOR signaling and can promote Eomes expression through IL-2/STAT5-dependent pathways^27,61–64. our findings suggest that attenuation of ITK signaling creates a distinct signaling context that favors acquisition of an Eomes-rich, memory-like phenotype. While the role of ITK in T cell development is well established^65–67, our results extend its significance to systemic autoimmunity and inflammatory lung injury. We propose that this preconfigured memory-like state may contribute to protection during acute injury by enabling more controlled and effective immune responses, thereby limiting the unchecked innate inflammation that characterizes pristane-induced PH.

Utilizing the pristane-induced murine model, we show that ITK deficiency confers marked protection against PH, as evidenced by preservation of lung architecture and a striking reduction in pulmonary hemorrhage. Acute PH is associated with robust recruitment of proinflammatory innate immune cells that disrupt the alveolar-capillary barrier^68,69. In this context, loss of ITK signaling significantly reduced the accumulation of CD11b⁺Ly6C⁺ inflammatory monocytes in the lung and was also associated with reduced inflammatory pathology in the spleen and gastrointestinal tract. These findings suggest that ITK contributes broadly to the inflammatory cascade that drives both pulmonary and systemic tissue injury in PH. This tissue protection was paralleled by marked suppression of systemic inflammatory cytokines. ITK deficiency significantly reduced circulating levels of IFN-γ, IL-6, TNF-α, and IL-17, mediators known to promote vascular permeability, inflammatory amplification, and tissue injury during severe autoimmune inflammation^15,70. By limiting this proinflammatory milieu, modulation of the ITK axis may provide dual therapeutic benefit: preserving alveolar-capillary barrier integrity while reducing the systemic inflammatory injury that contributes to poor outcomes in PH.

Beyond suppressing proinflammatory mediators, ITK deficiency also promoted a pro-resolving cytokine environment, including increased IL-10 and IL-13, and in some settings IL-4^71,72. This shift is particularly relevant in PH, where early containment of inflammatory injury is likely critical for preserving alveolar-capillary barrier integrity. These mediators are known to act not only on immune cells but also directly on the lung epithelium and endothelium. This observation suggests a dual-action mechanism in which ITK-deficient Tregs suppress inflammatory infiltration while simultaneously signaling to the alveolar-capillary unit to enhance barrier integrity and promote tissue resilience. A central finding of our study is that adoptive transfer of ITK-deficient Tregs was sufficient to rescue established PH in WT recipients^10,73. Because the therapeutic efficacy of Tregs is often limited by impaired stability or function under inflammatory stress^74,75, these findings suggest that loss of ITK signaling supports a more durable regulatory program in vivo.

Our transcriptomic analysis provides a mechanistic framework for this phenotype. RNA sequencing revealed that ITK deficiency reprograms the Treg transcriptional landscape, with enrichment of pathways linked to oxidative phosphorylation (OXPHOS), mTORC1, STAT5 signaling, and tissue repair. Together with the observed changes in glycolytic and cell cycle-associated programs, these data suggest that ITK-deficient Tregs acquire a state of enhanced metabolic fitness that may support their persistence and suppressive activity in inflamed tissue. Thus, loss of ITK signaling appears to generate not simply more Tregs, but Tregs with molecular features consistent with heightened regulatory capacity.

Collectively, these findings establish ITK as a promising therapeutic target in PH. Modulating the ITK axis may offer a strategy to suppress systemic inflammation while promoting tissue-protective immune regulation and restoration of alveolar-capillary barrier integrity. More broadly, our study provides a mechanistic rationale for developing ITK-directed approaches in severe inflammatory lung disease. While our findings in the pristane-induced model are striking, further studies are required to determine whether pharmacological ITK inhibition can fully phenocopy the genetic deficiency observed here and to evaluate the long-term stability of these reprogrammed Tregs in chronic models of systemic autoimmunity.

Materials and Methods

Mice

Both male and female mice were included as a biological variable in all studies, as previously described^25,76. ITK^-/- mice were generously provided by Dr. Avery August (Cornell University)^9,15,77. C57BL/6 mice were obtained from Charles River Laboratories or The Jackson Laboratory. All experiments were performed using mice 8–12 weeks of age, with age- and sex-matched controls included throughout.

Reagents, cell lines, flow cytometry

Spleen or lung tissues were processed into single-cell suspensions. For surface staining, cells were incubated with Fc-receptor blocking antibody (anti-CD16/32) followed by fluorochrome-conjugated monoclonal antibodies at a 1:100 dilution (unless otherwise specified) for 30 minutes at 4°C. Antibodies were purchased from BioLegend, eBioscience, or BD Pharmingen and included: anti-mouse CD3 (BV605, 100237), CD4 (PE, 100408), CD8 (PE/Cy7, 100722), CD44 (Pacific Blue, 156006), CD122 (APC, 105912), CD62L (APC/Cy7, 304814), CD25 (FITC, 101908), CD11b (FITC, 101205), and Ly-6C (APC, 128015). For transcription factor and intracellular cytokine staining, cells were fixed and permeabilized using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience) according to the manufacturer’s protocol. Intracellular antibodies included: Eomes (PE/Cy7, 25-4877-42), T-bet (BV421, 644816), TCF-1 (PE, 564217), Foxp3 (Pacific Blue, 126410), TNF-α (FITC, 502906), and IFN-ψ (APC, 505810). We also used Ly-6C (APC, 128015), CD62L (APC, 161218), CD4 (BV510, 100559), CD25 (FITC, 101907), CD25 (PB, 102021), CD122 (APC, 123213), CD11b (Percp 5.5 450112-82), CD8 (APC Cy7 140421), Eomes (PE, 12-4875-80), and T-bet (FITC, 64481), Dead cells were excluded using a Live/Dead fixable viability dye (BioLegend). Data were acquired on a BD LSRFortessa (BD Biosciences) and analyzed using FlowJo software (Tree Star), with singlets identified by forward and side-scatter properties as previously described^15,48.

Consumable

All general laboratory consumables and plasticware were sourced from EIMMUNA Medical Supply.

Proteinuria Assays

To assess renal involvement, urine was collected from mice at baseline (Day 0) and at specified intervals (Day 14 or Day 21) following pristane administration. Total urinary protein concentrations were quantified using the Pierce BCA Protein Assay Kit (Thermo Scientific, Cat# 23225) according to the manufacturer’s instructions. Briefly, urine samples were diluted 1:10 in PBS, and 25 µL of the diluted sample was combined with 200 µL of the BCA working reagent (50:1 ratio of Reagents A:B) in a 96-well microplate. The plate was briefly centrifuged (30 s at 1,250 rpm) to ensure uniform mixing and incubated at 37°C for 30 minutes. Absorbance was measured at 562 nm using a BioTek FLx800 plate reader. Protein concentrations (μg/mL) were determined using Gen5 software by interpolation from a bovine serum albumin (BSA) standard curve. Data were normalized to baseline values to account for physiological variation¹⁷.

Serum Cytokine Profiling

Systemic cytokine concentrations were assessed at baseline (Day 0) and at the study endpoints (Day 14 or Day 21). Whole blood was collected via cardiac puncture, and serum was isolated by centrifugation. A comprehensive panel of cytokines—including IFN-ψ, TNF-α IL-5, IL-12, IL-6, IL-10, IL-9, IL-17A, IL-17F, IL-22, and IL-13—was quantified using the LEGENDplex™ Mouse Th Cytokine Panel (13-plex) (BioLegend, Cat# 741011) according to the manufacturer’s instructions^9,19. Briefly, serum samples were incubated with capture beads, followed by detection antibodies and streptavidin-phycoerythrin (SA-PE). Data were acquired on a BD LSRFortessa (BD Biosciences). Absolute cytokine concentrations (pg/mL) were calculated using the LEGENDplex Data Analysis Software (BioLegend) by interpolation from a 5-parameter logistic (5PL) standard curve. All samples were analyzed in duplicate to ensure technical reproducibility.

Histopathological Evaluation

Following euthanasia at Day 14 or Day 21 post-pristane challenge, systemic organs—including the lungs, liver, spleen, kidneys, and small intestine—were harvested and fixed in 10% neutral-buffered formalin for 24–48 hours. Tissues were processed for paraffin embedding, sectioned at 5μm, and stained with hematoxylin and eosin (H&E) by the Histology Core Facility at SUNY Upstate Medical University. Histopathological evaluation, specifically focusing on the severity of PH, alveolar-capillary disruption, and systemic inflammatory infiltration, was performed by a board-certified pathologist (L.C.) who was blinded to the experimental groups. Tissue injury was graded according to previously established semi-quantitative criteria^17,78,79. Representative images were captured using a light microscope, and histopathology scores were analyzed using the Mann-Whitney U test for non-parametric comparisons

Isolation and Purification of Regulatory T Cells

To facilitate live isolation of regulatory T cells, ITK^-/- and WT mice were bred onto the C57BL/6 background and crossed with the Foxp3^tm1Flv/J reporter strain (kindly provided by Dr. Avery August, Cornell University). In this X-linked knock-in model, monomeric red fluorescent protein (mRFP) faithfully reports endogenous Foxp3 expression. Foxp3-RFP expression was confirmed in both WT and ITK^-/- cohorts prior to experimentation. Single-cell suspensions were prepared from spleens, and CD4⁺ lymphocytes were pre-enriched using anti-CD4 magnetic microbeads and MACS column-based separation (Miltenyi Biotec, Cat# 130-114-043). For high-purity isolation, pre-enriched cells were stained with antibodies against CD3 and CD25. Canonical Tregs were identified and purified as CD3⁺CD4⁺CD25⁺Foxp3(RFP)⁺ cells, while non-canonical Tregs were identified as CD3⁺CD4⁺CD25⁻Foxp3(RFP)⁺ cells. Fluorescence-activated cell sorting (FACS) was performed on a BD FACSAria IIIu (BD Biosciences), with sorted Treg purity routinely exceeding 98%. Purified cells were used immediately for RNA sequencing or adoptive transfer. Unless otherwise noted, all cell culture reagents were obtained from Sigma-Aldrich or Invitrogen, consistent with previously established protocols^10,17,48.

Statistics

Statistical analyses were performed using GraphPad Prism 10. Unless otherwise specified in the figure legends, data are presented as mean ± SD. For comparisons between two groups, an unpaired two-tailed Student’s t-test was used. For comparisons involving multiple groups, one-way or two-way ANOVA was performed, followed by Tukey’s post hoc test for multiple comparisons.

Non-parametric data, such as histopathology scores, were analyzed using the Mann-Whitney U test. A p value of ≤ 0.05 was considered statistically significant (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001). For in vivo studies, cohorts of n = 10–22 age- and sex-matched mice were used per group. All experiments were independently repeated at least twice to ensure reproducibility. Sample sizes were determined based on power calculations to achieve a minimum power of 0.8, and the experimental design was consistent with previously established protocols^{9,10,15–17,19,24–26,48,80–91}.

RNA Sequencing and Transcriptomic Analysis

For transcriptomic profiling, canonical and non-canonical Tregs were FACS-purified from the spleens of WT (n = 3) and ITK-/- (n = 3) mice as described above. Total RNA was extracted, and library preparation and high-throughput sequencing were performed by the Molecular Analysis Core Facility at SUNY Upstate Medical University. Raw sequencing data were processed in R (v4.5.1) using RStudio (v2025.05.1) and Bioconductor packages. Transcript abundance was quantified via pseudoalignment with Kallisto (v0.46.2)⁹². Transcript abundance estimates were imported with tximport⁹³, summarized to the gene level, and converted to scaled TPM values. Genes with low expression were filtered prior to model fitting. Differentially expressed genes (DEGs) were identified using DESeq2⁹⁴, with significance defined using a Benjamini-Hochberg⁹⁵ adjusted FDR ≤ 0.05 and a |log₂ fold change| ≥ 0.5. Variance-stabilized expression data were visualized using PCA and hierarchical clustering heatmaps generated with pheatmap⁹⁶.Functional annotation of identified up- and downregulated genes was performed by Gene Ontology (GO) enrichment analysis using gprofiler2 (gost)⁹⁷. Gene Set Enrichment Analysis (GSEA) was conducted using clusterProfiler against the MSigDB Hallmark gene set, with the DESeq2 Wald statistic as the ranked input list. Normalized enrichment scores (NES) and ridge plots were used to identify metabolic and signaling pathways unique to ITK-deficient Treg subsets. All RNA-seq data have been deposited in the NCBI Gene Expression Omnibus (GEO) under accession number GSE185327.

Ethics Statement and Animal Welfare

All animal housing and experimental procedures were performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The study protocol was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of SUNY Upstate Medical University (Protocol #443). All mice were maintained in a pathogen-free facility under a 12-hour light/dark cycle with ad libitum access to food and water. Every effort was made to minimize animal suffering during the pristane challenge and subsequent procedures.

Data availability

NCBI Gene Expression Omnibus (GEO) under accession number GSE185327.

Acknowledgements

The authors thank all members of the Karimi Laboratory for their helpful discussions and technical support throughout this study. We also thank the Molecular Analysis Core and the Histology Core Facility at SUNY Upstate Medical University for their expertise and assistance with high-throughput sequencing and pathological processing.

Additional information

Author contributions

M.S.H., H.X., A.M., and R.T.: Investigation, methodology, and data acquisition. L.C. and L.S.: Formal analysis and histological evaluation M.K.: Conceptualization, supervision, project administration, funding acquisition, resources, data curation, and writing—original draft preparation, review, and editing.

Funding Support

This research was supported by the National Institute on Aging (NIA) of the National Institutes of Health under Award Number 1R21AG098389 (to M.K.). This work was also supported by the Upstate Medical University Cancer Center (PTA 1184369 to M.K.), the Carol M. Baldwin Breast Cancer Research Fund (PTA 1192750 to M.K.), and the Paige’s Butterfly Run Fund (Grant #33875 to M.K.). This work is subject to the NIH Public Access Policy. Through the acceptance of federal funding, the NIH has been granted the right to make this work publicly available in PubMed Central.

Funding

HHS | NIH | NIH Office of the Director (OD) (1R21AG098389)

• Mobin Karimi

Upstate Cancer Center (UCC) (PTA 118436)

• Mobin Karimi

Baldwin Breast Cancer Research (1192750)

• Mobin Karimi