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, 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.

ITK Deficiency Confers Complete 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 innate immune cells. Representative flow cytometry plots (G) and quantification (H) of inflammatory monocytes, gated as CD11b⁺Ly6C⁺ within live singlets. (I and J) Evaluation of splenic pathology. Representative gross images (I) and H&E-stained sections (J) of spleens from WT and ITK-/- mice after pristane injection. (K) 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 ≤ 0.0001; **P ≤ 0.01.

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–L) 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 two-way ANOVA with Tukey’s post hoc correction for time-course data. **P < 0.01; ***P < 0.001; ****P < 0.0001.

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

(A and B) Characterization of the regulatory T cell reservoir. Representative flow cytometry plots (A) and quantification (B) of splenic CD4⁺Foxp3⁺ Tregs in WT and ITK-/- mice, illustrating expansion of both canonical (CD25⁺) and non-canonical (CD25⁻) populations in the absence of ITK signaling. (C and D) Experimental schematic for therapeutic rescue. WT recipient mice were challenged with pristane. At day 10 post-injection, a time point at which pulmonary injury was already established, mice were either left untreated or received an adoptive transfer of $1\times 10^6$ FACS-purified CD3⁺CD4⁺CD25⁺Foxp3⁺ Tregs from either WT or ITK-/- donor mice. All cohorts were euthanized at day 21 for systemic analysis. (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) for untreated mice (E), WT Treg-treated mice (F), and ITK-/- Treg-treated mice (G). Transfer of ITK-deficient Tregs resulted in near-complete resolution of gross hemorrhage and significant attenuation of proteinuria. (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 two-way ANOVA with Tukey’s post hoc correction. NS, p > 0.05; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****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 of differentially expressed genes. Plots identify top DEGs between ITK-/- and WT Tregs. Genes meeting the thresholds of FDR ≤ 0.05 and |log₂FC| ≥ 0.5 are highlighted, with 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, green indicates the non-canonical group. (G) Tables showing GO enrichment analysis of the DEG of 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 Tregs mice. On the table, p-values were color-coded as light blue for insignificant findings to orange with highest significance. (n = 3 mice per group). (H and I) Gene Set Enrichment Analysis (GSEA) of regulatory pathways in canonical (H) and noncanonical (I) samples. Ridge plots showing 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.

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 ≤ 0.0001; *P ≤ 0.05.

ITK Deficiency Preserves Splenic Architecture and Limits Inflammatory Infiltration.

(A and B) Histopathological evaluation of the spleen during the systemic inflammatory phase. Representative H&E-stained sections of spleens from WT (A) and ITK-/- (B) mice at Day 14 post-pristane challenge. Note that WT sections exhibit significant architectural disruption and the accumulation of proinflammatory immune cells. Black arrows indicate clusters of infiltrating inflammatory cells and regions of follicular expansion/disruption in the WT, which are markedly attenuated in the ITK-/-cohorts.

ITK Deficiency Protects Against Systemic Gastrointestinal Injury.

(A and B) Histopathological evaluation of the small intestine. Representative H&E-stained sections of the small intestine (SI) from WT (A) and ITK-/- (B) 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 Attenuates Hepatic Inflammatory Infiltration.

(A and B) Histopathological evaluation of the liver. Representative H&E-stained sections of liver tissue from WT (A) and ITK-/- (B) mice at Day 14 post-pristane challenge. Black arrows indicate focal regions of infiltrating proinflammatory immune cells and periportal inflammation in WT mice, which are markedly reduced in ITK-/- cohorts. Notably, while inflammatory infiltration is attenuated in ITK-/- mice, no significant differences in hepatocellular hyperplasia were observed between genotypes at this time point. Results are representative of 3 independent experiments.

ITK Deficiency Attenuates Renal Inflammatory Infiltration.

(A and B) Histopathological evaluation of the kidneys during the systemic inflammatory phase. Representative H&E-stained sections of kidney tissue from WT (A) and ITK-/- (B) mice at Day 14 post-pristane challenge. In WT mice, black arrows highlight focal clusters of infiltrating proinflammatory immune cells within the renal interstitium. While ITK-/- mice show a reduction in these inflammatory clusters, the overall glomerular and tubular architecture remains largely preserved in both genotypes at this time point. Notably, while immune cell infiltration is localized, no significant differences in gross renal structural damage or tubular hyperplasia were observed between groups. 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.

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.