ITK deficiency promotes a predominantly memory-like T cell phenotype.

(A–D) Analysis of the CD8⁺ T cell compartment in WT and Itk-/- mice. Freshly isolated splenocytes were gated on CD3⁺ lymphocytes and further subdivided into CD8⁺ and CD4⁺ T cell populations. (A) Representative flow cytometry plots showing CD44 and CD62L expression within CD8⁺ T cells. (B–D) Quantification of effector memory (EM; CD44⁺CD62L⁻), central memory (CM; CD44⁺CD62L⁺), and naïve (CD44⁻CD62L⁺) CD8⁺ T cell subsets. (E– H) Analysis of the CD4⁺ T cell compartment. (E) Representative flow cytometry plots showing CD44 and CD62L expression within CD4⁺ T cells. (F–H) Quantification of EM, CM, and naïve CD4⁺ T cell frequencies. Data are presented as mean ± SEM (n = 10–15 mice per group). Results are representative of 6 independent experiments. Statistical significance was determined by an unpaired two-tailed Student’s t-test. ****p ≤ 0.0001; ns, not significant (p > 0.05).

ITK deficiency enhances expression of memory-associated activating markers and transcriptional regulators.

(A and B) Surface expression of CD44 on splenic T cells. Representative flow cytometry plots (A) and quantification of frequencies (B) in WT and Itk-/- mice. (C and D) Expression of the T-box transcription factor Eomes. Representative flow cytometry plots (C) and quantification of total Eomes⁺ T cells (D). (E and F) Surface expression of CD122 (IL-2Rβ). Representative flow cytometry plots (E) and quantification of CD122 frequencies on T cells (F) from WT and Itk-/- mice. (G and H) Intracellular expression of the transcription factor T-bet. Representative flow cytometry plots (G) and quantification of T-bet frequencies within the indicated population (H). Data are presented as mean ± SEM (n = 15–20 mice per group, as indicated in the panels). Results are representative of at least 3 independent experiments. Statistical significance was determined by an unpaired two-tailed Student’s t-test. *p ≤ 0.05; ****p ≤ 0.0001.

TK deficiency confers protection against pristane-induced PH and systemic pathology.

(A) Experimental schematic: WT and Itk-/- mice received a single i.p. injection of 0.5 mL pristane to induce pulmonary hemorrhage (PH) and were euthanized on day 14 for tissue analysis. (B–E) Assessment of lung pathology. Representative gross morphology (B, D) and H&E-stained lung sections (C, E) from WT and Itk-/- mice after pristane injection. WT mice exhibit severe pulmonary hemorrhage and loss of architectural integrity, whereas lungs from Itk-/- mice remain largely free of blood and retain preserved alveolar structures. (F) Quantification of blinded histopathologic scoring. Lung injury was graded by a board-certified pathologist blinded to genotype and treatment. (G and H) Analysis of lung-infiltrating proinflammatory monocytes. Representative flow cytometry plots (G) and quantification (H) of inflammatory monocytes, gated as CD11b⁺Ly6C⁺ within live singlets. (I) Representative flow cytometry plots and quantification of neutrophils (CD11b⁺Ly6G⁺) within live singlets from WT and Itk-/- mice after pristane injection. (J and K) Evaluation of splenic pathology. Representative gross images (J) and H&E-stained sections (K) of spleens from WT and Itk-/- mice after pristane injection. (L) Histopathological analysis of systemic inflammation. Representative H&E-stained sections of the small intestine from WT and Itk-/- mice. Data are presented as mean ± SEM (n = 10 mice per group). Results are representative of 3 independent experiments. Statistical significance was determined by an unpaired two-tailed Student’s t-test, except for panel F, which was analyzed using the Mann-Whitney U test. p ≤ 0.01; ** P ≤ 0.01, ****p ≤ 0.0001.

ITK deficiency attenuates proteinuria and shifts the systemic cytokine balance toward resolution.

(A) Experimental schematic: WT and Itk-/- mice were challenged with pristane. Urine and blood were collected at baseline (day 0) and at the day 14 endpoint to assess systemic pathology. (B and C) Assessment of renal function by proteinuria. Total urinary protein was quantified by BCA assay in WT (B) and Itk-/- mice (C). Itk-/- mice show significant protection against the glomerular leakage observed in WT controls following pristane injection. (D–I) Multiplex serum cytokine profiling. Serum levels of proinflammatory mediators (IFN-γ, TNF-α, IL- 17, and IL-6) and pro-resolving cytokines (IL-13 and IL-10) were quantified by bead-based immunoassay. Itk-/- mice exhibited marked suppression of proinflammatory cytokines together with preservation of regulatory cytokines. Data are presented as mean ± SEM (n = 15–25 mice per group). Results are representative of 3 independent experiments. Statistical significance was determined by a two-tailed Student’s t-test for single comparisons or a two-way ANOVA with Tukey’s post hoc correction for time-course data. p < 0.01; ***p < 0.001; ****p < 0.0001, ** P ≤ 0.01

Adoptively transferred Itk-/- Tregs exhibit enhanced AREG expression and more effectively ameliorate PH.

(A) Representative flow cytometry plots and quantitative analysis of splenic CD4⁺Foxp3⁺ Tregs in WT and Itk-/- mice. (B–D) Experimental schematic for Treg adoptive transfer. (B) WT mice (CD45.1⁺) received a single i.p. injection of 0.5 mL pristane. (C) On day 10, CD4⁺Foxp3⁺ Tregs were FACS-sorted from WT or Itk-/- donor mice (CD45.2⁺), with post-sort purity confirmed as CD25⁺Foxp3⁺. These cells were injected intravenously (i.v.) into the pristane-treated CD45.1⁺ recipients as a therapeutic intervention. (D) On day 21, CD45.1⁺ host mice were euthanized for examination of the lungs and draining lymph nodes (dLNs). (E) Representative flow cytometry plots and quantitative analysis of lung CD4⁺Foxp3⁺ Tregs in WT and Itk-/- mice at baseline (without pristane injection). (F and G) Representative flow cytometry plots and quantitative analysis of total donor CD45.2⁺ cells (F) and donor-derived Tregs (G) engrafted in the lungs of CD45.1⁺ recipient mice. (H and I) Representative flow cytometry plots and quantitative analysis of total donor CD45.2⁺ cells (H) and donor-derived Tregs (I) in the dLNs of CD45.1⁺ recipient mice. (J) Immunofluorescence microscopy of lung sections confirming the presence of donor Tregs in inflamed lung tissue. Sections were stained for CD4, the donor congenic marker (CD45.2), and Foxp3, with merged images demonstrating colocalization; nuclei were counterstained with DAPI. (K) CD25⁺Foxp3⁺ Tregs were FACS-sorted from lung tissue and analyzed by immunoblot for amphiregulin (AREG), with β-actin as a loading control. Data are presented as mean ± SEM; sample sizes (n = 15–25 mice per group) are indicated in the panels. Each experiment was repeated 3 times. Statistical significance was determined using a two-tailed Student’s t-test, one-way ANOVA, or two-way ANOVA as appropriate; p < 0.01, ***p < 0.001, * P ≤ 0.05 ****p < 0.0001, ** P ≤ 0.01.

Adoptively transferred Itk-/- Tregs protect mice from PH, reduce proinflammatory cytokine levels, and increase anti- inflammatory cytokine levels.

(A–D) Experimental schematic for the therapeutic adoptive transfer model. (A) Baseline serum and urine were collected from WT host mice (CD45.1⁺), followed by a single i.p. injection of 0.5 mL pristane. (B and C) On day 10, CD4⁺Foxp3⁺ Tregs were FACS-sorted from WT or Itk-/- donor mice (CD45.2⁺), confirmed positive for CD25 and Foxp3, and injected i.v. into the pristane-treated CD45.1⁺ recipients. A control group of pristane-challenged CD45.1⁺ mice was left untreated. (D) On day 21, host mice were euthanized and analyzed for urinary protein and gross anatomical damage to the lungs. (E–G) Evaluation of therapeutic efficacy in lung and kidney injury. Representative gross lung images and longitudinal quantification of urinary protein (day 0 versus day 21) are shown for (E) untreated pristane-challenged mice, (F) mice treated with WT Tregs, and (G) mice treated with Itk-/- Tregs. (H–L) Suppression of systemic inflammatory responses. Serum levels of proinflammatory cytokines (IFN-γ, TNF-α, IL-6, and IL-17) and the pro-resolving mediator IL-10 were quantified at baseline and day 21. Itk-/- Tregs promoted a high-IL-10 regulatory environment while effectively suppressing the proinflammatory cytokine response. Data are presented as mean ± SEM (n = 15–25 mice per group). Results are representative of 3 independent experiments. Statistical significance was determined by a two-tailed Student’s t- test or a two-way ANOVA with Tukey’s post hoc correction. ns, p > 0.05; *p ≤ 0.05; p ≤ 0.01; ****p ≤ 0.001; ** P ≤ 0.01, *****p ≤ 0.0001.

ITK deficiency reprograms the Treg transcriptome toward a metabolically and functionally altered state.

(A) Global transcriptomic relationships. Principal component analysis (PCA) of transcriptomes from WT and Itk-/- canonical (CD25⁺) and non- canonical (CD25⁻) Tregs. All replicates are shown (n = 3). (B) Table showing the number of up- or downregulated differentially expressed genes (DEGs) between groups. (C and D) Transcriptomic landscape of ITK-deficient Tregs. Volcano plots identify the top DEGs between Itk-/- and WT Tregs. Genes meeting the thresholds of FDR ≤ 0.05 and |log₂FC| ≥ 0.5 are highlighted, with a positive log₂FC indicating enrichment in Itk-/- canonical (C) and non-canonical (D) Tregs. (E and F) Hierarchical clustering heatmaps showing row-scaled normalized expression of all significant DEGs in canonical CD4⁺CD25⁺Foxp3⁺ Tregs from WT and Itk-/- mice (n = 3 per group). A grey bar indicates WT, orange indicates the canonical group, and green indicates the non-canonical group. (G) Tables showing GO enrichment analysis of the DEGs from Itk-/- canonical (CD25⁺) and non-canonical (CD25⁻) Tregs. Using the online tool gProfiler and the ordered g:GOSt query, we assessed which biological processes (BP) were linked to the genes in the different modules from Itk- /- and WT mice. In the table, p-values are color-coded from light blue (not significant) to orange (highest significance) (n = 3 mice per group). (H and I) Gene Set Enrichment Analysis (GSEA) of regulatory pathways in canonical (H) and non-canonical (I) samples. Ridge plots show the distribution of ranked DESeq2 Wald statistic values for selected Hallmark pathways, including oxidative phosphorylation (OXPHOS), mTORC1 signaling, and MYC targets, which together reflect enhanced metabolic fitness in ITK-deficient regulatory subsets.

Gene-Level Analyses Reveal Distinct Transcriptional Programs in Canonical and Non-Canonical Itk⁻/⁻ Tregs.

(A, B) Leading edge genes of the Hallmark pathways shown in Figure 7, for Itk−/− canonical and Itk−/− non-canonical Tregs against WT canonical Tregs. Within each pathway the leading-edge genes, that is the core enrichment set returned by GSEA, were ranked by the absolute DESeq2 Wald statistic and the top fifteen are shown. Color is variance stabilized, batch corrected expression, z-scored across all nine samples, so the two panels are directly comparable. Asterisks mark genes that are also individually differentially expressed (FDR ≤ 0.05, |log2FC| ≥ 0.5). Row headings give the normalized enrichment score and the group in which each pathway is higher. (C, D, E) Genes annotated to the most significant Gene Ontology biological process terms for the differentially expressed genes in each comparison, tested against a background of the 16,723 expressed genes. Up to eight terms are shown per comparison and up to twelve genes per term, ranked by absolute fold change; color is the apeglm shrunken log2 fold change. No biological process term reached significance among the genes downregulated in canonical Itk−/− Tregs against this background.

Gating strategies for effector and central memory phenotypes.

Freshly isolated splenocytes from WT (A– G) and Itk-/- (H–N) mice were analyzed by flow cytometry. (A, H) Lymphocyte populations were initially gated based on side scatter area (SSC-A) versus forward scatter area (FSC-A). (B, I) Lymphocytes were then gated for single cells using forward scatter height (FSC-H) versus forward scatter width (FSC-W) to discriminate doublets. (C, J) Additional singlet gating was performed using side scatter height (SSC-H) versus side scatter width (SSC-W) to exclude any remaining doublets. (D, K) CD3⁺ T lymphocytes were identified from the single-cell population based on SSC-A versus CD3 expression. (E, L) The CD3⁺ compartment was further subdivided into CD8⁺ and CD4⁺ T cell populations. (F, M) CD8⁺ and (G, N) CD4⁺ T cells were then analyzed to identify and quantify effector memory (EM; CD44⁺CD62L⁻), central memory (CM; CD44⁺CD62L⁺), and naïve (CD44⁻CD62L⁺) subsets, as depicted in the contour plots.

ITK deficiency promotes an activated, memory-like program in CD4⁺ T cells.

(A and B) Surface expression of CD44 on splenic CD4⁺ T cells. Representative flow cytometry plots (A) and quantification of CD44⁺ frequencies (B) in WT and Itk-/- mice. (C and D) Expression of the T-box transcription factor Eomes. Representative flow cytometry plots (C) and quantification of total Eomes⁺ CD4⁺ T cells (D) within the CD3⁺CD4⁺ gate. (E and F) Surface expression of CD122 (IL-2Rβ). Representative flow cytometry plots (E) and quantification of CD122 frequencies on CD4⁺ T cells (F) from WT and Itk-/- mice. (G and H) Intracellular expression of the transcription factor T-bet. Representative flow cytometry plots (G) and quantification of T-bet frequencies within the CD3⁺CD4⁺ population (H). Data are presented as mean ± SEM (n = 15–20 mice per group). Results are representative of at least 3 independent experiments. Statistical significance was determined by an unpaired two-tailed Student’s t-test ***p < 0.001, * P ≤ 0.05 ****p < 0.0001, ** P ≤ 0.01.

ITK deficiency protects mice from pristane-induced PH and systemic pathology.

(A and B) Representative gross morphology of lungs and H&E-stained lung sections from WT and Itk-/- mice without pristane injection. (C and D) Representative gross morphology and H&E-stained lung sections from WT and Itk-/- mice 14 days after pristane injection

ITK deficiency prolongs survival following pristane-induced PH.

WT and Itk-/- mice received a single i.p. injection of 0.5 mL pristane to induce pulmonary hemorrhage (PH). Survival was monitored for 50 days post-injection.

ITK deficiency protects mice from pristane-induced PH and systemic pathology.

(A–C) Representative gross morphology of lungs and H&E-stained lung sections from either WT or Itk-/- mice without pristane injection. (D–F) Representative gross images and H&E-stained sections of spleens from WT and Itk-/- mice after pristane injection

ITK deficiency protects mice from pristane-induced small intestine pathology.

(A and B) Representative gross morphology and H&E-stained sections of the small intestine from either WT or Itk-/- mice without pristane injection. (C and D) Histopathological evaluation of the small intestine. Representative H&E-stained sections of the small intestine from WT (C) and Itk-/- (D) mice at day 14 post-pristane administration. In WT mice, systemic inflammatory injury is associated with evident mucosal damage, characterized by villus architecture disruption and increased inflammatory cell infiltration (black arrows). In contrast, Itk-/- mice exhibited significant protection against this systemic injury, with preservation of mucosal integrity. Results are representative of 3 independent experiments.

ITK deficiency promotes the expansion of non-canonical Tregs.

(A and B) Quantitative analysis of Treg subsets at baseline. Representative flow cytometry plots (A) and statistical quantification (B) of freshly isolated splenic regulatory T cells from WT and Itk-/- mice. Tregs were identified as CD3⁺CD4⁺Foxp3⁺ cells. ITK deficiency led to a significant increase in the frequency and absolute number of non-canonical (CD25⁻) Tregs compared with WT controls, while maintaining the canonical (CD25⁺) population. This expansion contributes to the broader regulatory reservoir observed in Itk-/- mice. Data are presented as mean ± SEM (n = 15–20 mice per group). Results are representative of 3 independent experiments. Statistical significance was determined by an unpaired two-tailed Student’s t-test. ****p ≤ 0.0001.

Adoptive transfer of Itk-/- Tregs prolongs survival following pristane-induced PH.

WT host mice (CD45.1⁺) received a single i.p. injection of 0.5 mL pristane. Mice were divided into three groups: left untreated, or adoptively transferred with Tregs isolated from either WT or Itk-/- donor mice (CD45.2⁺). Survival was monitored for over 40 days post-injection.

ITK Deficiency Reprograms Regulatory T Cells through Enhanced Metabolic and Signaling Circuitry.

(A–J) Enrichment of metabolic and survival pathways. Summary of MSigDB Hallmark pathway enrichment in canonical and non- canonical ITK-/- Tregs relative to WT controls. GSEA enrichment score plots show running enrichment scores for selected Hallmark gene sets. Representative GSEA plots are shown for high-impact pathways, including cell cycle progression, mTORC1 signaling, PI3K/Akt/mTOR signaling, IL-2/STAT5 signaling, MYC targets, and oxidative phosphorylation. Notably, ITK-/- Tregs exhibit coordinated upregulation of metabolic, survival, and stress-adaptation pathways, including interferon response and UV response/DNA repair signatures, consistent with enhanced resilience in inflammatory environments. Data are derived from transcriptomic analysis of n = 3 independent biological replicates per group.