Mice deficient for Tec (Tec^−/− mice) were described previously (32). OT-II TCR transgenic mice were kindly provided by Dr. Stoiber-Sakaguchi (Medical University of Vienna). C57BL/6 Rag1^−/− , C57BL/6 Rag2^−/− , and Rosa26^floxSTOP^flox eYFP mice were purchased from The Jackson Laboratories. IL-17A^CRE mice were kindly provided by B. Stockinger (21).

The IL-17 fate mapping mice resulted from the breeding of WT R26^YFP or Tec^−/− R26^YFP mice with WT IL-17A^CRE or Tec^−/− IL-17A^CRE mice, respectively. All mice were bred and maintained in the preclinical research facility of the Medical University of Vienna, and animal experiments were done according to protocols approved by the Federal Ministry for Education, Science and Art (GZ:BMWF-66.009/0105-WF/II/3b/2014, BMBWF-66.009/0039-V/3b/2019). Animal experiments were performed under national laws in agreement with guidelines of the Federation of European Laboratory Animal Science Associations (FELASA), which correspond to the National Center for the Replacement, Refinement, and Reduction of Animals in Research (ARRIVE) guidelines.

The following antibodies and reagents were used in our study: from BD Biosciences (Franklin Lakes, NJ, USA): IL-17A (TC11-18H10.1), IL-10 (JES5-16E3), pSTAT1 (4a), pSTAT3 (4/P-STAT3), pSTAT4 (38/p-Stat4), and pSTAT5 (47); from Thermo Fisher (Waltham, MA, USA): CD4 (clone RM4-5), CD8a (53-6,7), CD45.1 (A20), CD45.2 (104), Foxp3 (FJK-16s), IL-22 (IL22JOP), and fixable viability dye eFluor™ 506; and from BioLegend (San Diego, CA, USA): CD16/CD32 (2.4G2), CD44 (IM7), CD19 (6D5), CD45.2 (104), CD62L (MEL-14), CD45RB (MB4B4), IFN-γ (XMG1.2), IL-23R (12B2B64), TCR-β (H57-597), and IL-6Rα (D7715A7). The clone number is indicated in brackets.

CD4⁺ T cells were isolated from spleen and lymph nodes of 8-week-old mice as previously described (28). Sorted naive (CD44^lowCD62L⁺) CD4⁺ T cells were stimulated (day 0) with plate-bound anti-CD3ϵ (1 µg/ml) and anti-CD28 (3 µg/ml) on 96-well plates (2 × 10⁵ cells/well) in 200 µl T-cell medium (RPMI GlutaMAX-I supplemented with 10% FCS, antibiotics, and 2-ME; all from Invitrogen, Waltham, MA, USA) under various cytokine conditions as indicated in the figure legend. For Th17 conditions, IL-6 (20 ng/ml, or as indicated in the figure legend) and TGF-β1 (1 ng/ml, or as indicated in the figure legend) were used. Cells were cultured for 72 h or as indicated in the figure legend. Where indicated, IL-1β (20 ng/ml, BioLegend) and IL-23 (20 ng/ml, R&D, Minneapolis, MN, USA) were added to the differentiation medium. Cells were analyzed after 60 h by flow cytometry on a LSRII Fortessa (BD Biosciences).

T cells were cultured under various conditions as described above. For intracellular cytokine detection, cells were stimulated in 96-well plates for 3 h with PMA (50 ng/ml) and ionomycin (500 ng/ml) (Sigma-Aldrich, St. Louis, MO, USA) in the presence of GolgiStop (BD Biosciences). After harvesting, T cells were surface stained with appropriate antibodies. Subsequently, cells were fixed for 10 min with Cytofix Fixation Buffer (BD Biosciences), permeabilized 15 minutes with Perm/Wash Buffer (BD Biosciences), and stained for intracellular proteins according to the manufacturer’s protocol. Intracellular phosphorylated STAT levels were assessed using BD Phosflow protocol.

Cells were harvested, and total RNA was isolated with Qiagen RNA isolation kit according the manufacturer’s instructions. RNA was reversely transcribed using the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA). Quantitative real-time PCR (qRTPCR) analysis was performed with the SuperScript III qPCR MasterMix (Invitrogen) on the CFX 96 Real-Time PCR detection system (Bio-Rad). The list of primers used can be found in the Supplementary Table .

Mouse IL-17A and IFN-γ ELISA MAX Deluxe sets (BioLegend) and Mouse IL-21 DuoSet Elisa (R&D) were used to measure IL-17A, IFN-γ, and IL-21 from the culture supernatant of Th17 cultures from CD4⁺ T cells isolated from WT or Tec-deficient mice.

T cells (0.5 × 10⁶) were lysed in (50 µl) Carin lysis buffer (20 mM Tris-HCl [pH 8.0], 138 mM NaCl, 10 mM EDTA, 100 mM NaF, 1% Nonidet P-40, 10% glycerol, 2 mM Na orthovanadate) supplemented with complete protease inhibitors (Roche, Basel, Switzerland). Proteins were separated on 10% SDS-polyacrylamide gels and blotted onto PVDF membranes according to standard protocols. The following primary antibodies were used: anti-actin (AC-74; Sigma-Aldrich, St. Louis, MO, USA) and rabbit anti-Tec. Secondary antibody was a peroxidase-conjugated AffiniPure goat anti-rabbit IgG (H+L) (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Signals were detected using ECL (SuperSignal West Dura Extended Duration Substrate from ThermoScientific).

Recipient mice (CD45.1⁺) received 5 × 10⁵ CD45.2⁺ Tec ^−/− or Tec ^+/+ OTII IL-17A^cre R26^YFP CD4⁺ T cells intravenously 1 day before immunization with 100 μg OVA(323–339) in CFA subcutaneously in the flank. Draining inguinal lymph nodes were isolated, and phenotype of OTII⁺ cells was assessed by flow cytometry, gating on live CD4⁺CD45.2⁺ cells.

CD4⁺CD45RB^hiCD25⁻CD45.2⁺ T cells were purified from CD4⁺ T cells isolated from spleens and LNs of Tec^−/− and control mice, and intraperitoneally injected into Rag1 ^−/− CD45.1⁺ mice or Rag2^−/− CD45.2⁺ mice (4 × 10⁵ cells per mouse) as indicated in the figures. After 3 or 7 weeks, as indicated in the figures, mice were sacrificed and intestines removed and placed in ice-cold PBS. The colon length was measured, and the tissue was kept for histology or used for cell isolation. After removal of residual mesenteric fat tissue, the small intestine or colon was opened longitudinally. The intestine was then thoroughly washed in ice-cold PBS and cut into 1.5 cm pieces. The pieces were washed twice with 10 mM Hepes, 10% fetal bovin serum in Hanks’ balanced salt solution (HBSS) to remove excess mucus and incubated twice in 5 ml of 5 mM EDTA in HBSS for 15–20 min at 37°C with shaking. After each incubation, the epithelial cell layer, containing the intraepithelial lymphocytes (IELs), was removed by intensive vortexing and passing through a 100-μm cell strainer, and new EDTA solution was added. After the second EDTA incubation, the pieces were washed in HBSS, cut in small pieces using scissors, and placed in 5 ml digestion solution containing 4% fetal calf serum and 0.5 mg/ml each of collagenase D (Roche) and DNase I (Sigma). For isolation of lymphocytes from small intestine or colon 3 weeks after transfer, cells were not incubated with EDTA for IEL isolation and processed directly for digestion. Digestion was performed by incubating the pieces at 37°C for 20 min with shaking. After the initial 20 min, the solution was vortexed intensely and passed through a 40-μm cell strainer. The pieces were collected and placed into fresh digestion solution, and the procedure was repeated a total of three times. Supernatants from all three digestions (or from the EDTA treatment for IEL isolation) from a single small intestine were combined, washed once in cold FACS buffer, resuspended in 10 ml of the 40% fraction of a 40:80 Percoll gradient, and overlaid on 5 ml of the 80% fraction in a 15-ml Falcon tube. Percoll gradient separation was performed by centrifugation for 20 min at 2,500 rpm at room temperature. Lamina propria lymphocytes (LPLs) were collected at the interphase of the Percoll gradient, washed once, and resuspended in FACS buffer or T-cell medium. The cells were used immediately for experiments.

Colon was fixed in 4% buffered formalin and embedded in paraffin. Blocks were cut at 2 µm thickness and stained with hematoxylin and eosin (HE). A pathologist blinded to treatment groups evaluated slides in a semiquantitative manner. Extent and severity of the inflammatory cell infiltrate (1-5), epithelial changes (1-5), and lesions of the mucosal architecture (1-5) were graded according to a scoring scheme for colonic inflammation mediated by disturbed immune cell homeostasis (33). Furthermore, crypt length from surface of the epithelium to the lamina muscularis mucosae was measured with an Olympus BX53 microscope and attached Olympus DP26 camera in three locations of each colon perpendicular to the serosal surface. The same microscope and camera were used for acquisition of representative images. White balance and assembly of images were done using Adobe Photoshop 2021. The total histopathological score was calculated by adding the scores for each of the parameters mentioned above as described in (34).

RNA-sequencing data were downloaded as reads per kilobase of transcript, per million mapped reads (RPKM) from NCBI GEO datasets (GSE16452, GSE163894, GSE168288, (35)), and Tec expression was normalized to Hprt.

All data are expressed as mean with SEM. Statistical analysis was performed by using a nonpaired Student’s t-test or by one-way ANOVA corrected with Tukey post-hoc test as indicated in the figure legend. For multiple comparison testing, two-way ANOVA using a Sidak’s multiple comparisons test was applied as indicated in the figure legend. The p-values were defined as follows: ^* p < 0.05; ^** p < 0.01; ^*** p < 0.001; n.s., not significant.

Tec kinases were shown to have a T helper-cell subtype-specific expression pattern (28, 36). We revisited Tec expression profiles among T helper subsets by reanalyzing published RNA-sequencing data (35) (see Result section for complete datasets used) ( Figure 1A ). Tec was expressed at low levels in naïve and Th1 cells, exhibiting an intermediate expression level in Treg cells and highest expression in Th2 and Th17 cells ( Figure 1A ). To investigate how Tec expression is regulated in Th17 cells, we isolated naïve CD4⁺ T cells from WT mice and cultured them with TGF-β1 and/or IL-6 ( Figure 1B ). Different concentrations of either TGF-β1 or IL-6 were used in order to test a dose dependency. The cells were then further processed for assessment of Tec mRNA levels. Addition of TGF-β1 alone but not IL-6 led to a mild induction of Tec in a dose-dependent manner. However, the combination of TGF-β1 and IL-6 resulted in the highest induction of Tec, indicating a synergistic activity of TGF-β1 and IL-6 signaling pathways in inducing Tec expression. Other Th17 driving cytokines like IL-1 or IL-23 did not further enhance Tec induction ( Figure 1C ). We confirmed the mRNA expression data on a protein level ( Figure 1D ). Naïve CD4⁺ T cells cultured under Th17 driving conditions from Tec^−/− mice were used as negative control. An analysis of the Tec locus on the NCBI database indicates several transcript variants (1 and 2, 3, and 4) resulting in 3 Tec protein isoforms (named a, b, and c, respectively) ( Figure 1E ). In Th17 cells, we detected isoform a, the dominant form in immune cells (37), as the major isoform expressed ( Figure 1F ). Taken together, our data show that in Th17 cells, Tec isoform a is predominantly induced via synergistic activity of IL-6 and TGF-β1 signaling.

As Tec is highly induced under Th17 priming conditions, we investigated how TGF-β1 or IL-6 signaling impacts on the frequency of IL-17A-positive cells and IL-17A secretion during activation of WT and Tec^−/− naïve CD4⁺ T cells ( Figure 2 ; Supplementary Figure S1 ). Therefore, we activated naïve CD4⁺ T cells from WT or Tec^−/− mice with plate-bound CD3 and CD28 in the presence of a fixed concentration of IL-6 (20 ng/ml) and increasing concentrations of TGF-β1 ( Supplementary Figure S1 ). As expected, Th17 differentiation of both WT and Tec^−/− naïve CD4⁺ T cells increased with higher TGF-β1 concentrations until reaching a plateau and even decreasing at higher concentrations. The sensing of various TGF-β1 concentrations by naïve Tec^−/− CD4⁺ cells was comparable with the sensing of naïve WT CD4⁺ T cells. We also activated naïve CD4⁺ T cells from WT or Tec^−/− mice in the presence of a fixed concentration of TGF-β1 (1 ng/ml) and increasing concentrations of IL-6 ( Figures 2A–C ). Again, Th17 differentiation of both WT and Tec^−/− naïve CD4⁺ T cells increased with higher IL-6 concentrations. Notably, Tec^−/− Th17 cells exhibited higher percentages of IL-17A-positive cells and higher IL-17A secretion at IL-6 concentrations below 20 ng/ml (further defined as low IL-6 concentrations) compared with WT Th17 cells implying a stronger IL-6 sensing in the absence of Tec ( Figures 2A–C ). In agreement with the IL-17A secretion data, STAT3 phosphorylation was enhanced in cells cultured for 48 or 72 h with TGF-β1 and low IL-6 concentration in the absence of Tec, equaling out with WT levels at higher IL-6 concentration ( Figure 2D ). Of note, STAT5 phosphorylation in contrast to STAT1 and STAT4 phosphorylation was enhanced in Tec-deficient cells cultured for 48 h with low IL-6 concentration, indicating modulation of other STAT molecules by Tec ( Supplementary Figure S2 ). To assess whether Tec directly regulates IL-6R signaling independently of T-cell receptor activation, WT and Tec^−/− naïve CD4⁺ T cells were activated with anti-CD3 and anti-CD28 in the presence of TGF-β1 and IL-6 for 48 h, rested in PBS with 2% FCS, and activated for 15 min with 5 or 20 ng/ml IL-6 ( Supplementary Figure S3A ). A kinetic study of Tec expression revealed that Tec protein is clearly detectable at 48 h ( Supplementary Figure S3B ). STAT3 phosphorylation was enhanced in the absence of Tec with 5 ng/ml IL-6 but not 20 ng/ml IL-6, showing that Tec directly regulates IL-6R signaling at low IL-6 concentration ( Supplementary Figure S3C ). Furthermore, the effects of Tec were specific for the Th17 lineage as Foxp3 and IFN-γ expression in Tec^−/− Th17 cells generated with increasing IL-6 concentrations was similar to WT Th17 cells ( Supplementary Figures S4A, B ). IL-21 secretion which is also dependent on STAT3 activity (38) was increased at 2.5 and 5 ng/ml IL-6 in Tec-deficient conditions ( Supplementary Figure S4B ). As a result of a stronger IL-6 signaling, the expression of Il23r was enhanced in Tec^−/− Th17 cells at low IL-6 concentration as compared with their WT counterparts ( Figure 2E ). Of note, the IL-6 receptor levels were similar between WT and Tec^−/− naïve CD4⁺ T cells, excluding a stronger IL-6 response due to higher receptor expression as a consequence of T-cell developmental alterations ( Supplementary Figure S5 ). Taken together, our data indicate that Tec dampens Th17 differentiation at low IL-6 concentrations via inhibition of STAT3 phosphorylation, and thereby restrains IL-21 secretion and Il23r expression.

To study whether the enhanced in vitro Th17 differentiation in the absence of Tec can also be observed in vivo, we took advantage of an adoptive T-cell transfer model which allows to monitor Th17 differentiation in vivo in an antigen-specific way (39) ( Figure 3A ). Th17 cells were shown to either retain IL-17A expression and induce expression of cytokines of other lineages or even switch to another T helper-type-like Th1, Tr1 or Tfh, a feature also described as “plasticity” (11, 21–23, 40, 41). We combined therefore this adoptive transfer model with the IL-17A fate mapping mouse (21) to ensure that we also monitor Th17 cells that switched to another T helper lineage and stopped producing IL-17A, and to further evaluate Th17 in vivo plasticity. We transferred a low number of naïve WT or Tec^−/− OT-II IL-17A^cre R26^YFP CD45.2⁺CD4⁺ T cells (which are specific for ovalbumin, and allowed IL-17A fate mapping) into CD45.1⁺C57BL/6-recipient mice which were subcutaneously immunized 24 h later with ovalbumin in complete Freund’s adjuvant (CFA). Six days later, draining lymph nodes were investigated for IL-17A⁺ cells and eYFP⁺ CD45.2⁺CD4⁺ T cells (for a gating strategy, see Supplementary Figure S6 ). Transferred Tec^−/− CD4⁺ T cells displayed enhanced Th17 differentiation compared with their WT counterpart, as revealed by the expression of IL-17A after PMA/ionomycin ex vivo restimulation of draining lymph node cells ( Figures 3B–D ). It was shown that during in vitro and in vivo Th17 differentiation, eYFP-positive cells correlate partially with IL-17A expression (21). This difference (IL-17A⁺YFP^– cells) was explained by a threshold Cre protein expression level required to faithfully mark Th17 cells which have acquired full effector functions. Therefore eYFP⁺ CD4⁺ T cells represent a subset of more mature effector Th17 cells (21). We observed more eYFP⁺ CD45.2⁺CD4⁺ T cells from Tec^−/− mice compared with those from WT mice, indicating that Tec deficiency results in an enhanced fraction of mature effector Th17 cells ( Figures 3B–D ).

Independent of the genotype, immunization led to a robust induction of Th17 and Th1 cells, but only to a marginal IL-10 production by CD4⁺ T cells, indicating an ongoing inflammatory immune reaction which is still not resolving ( Supplementary Figures S7A–C ) (23). To understand if, in addition to an enhanced Th17 effector differentiation, Tec^−/− CD4⁺ T cells show an altered plasticity and/or Th17 subset distribution in vivo, we analyzed the proportion of IL-17 and IFN-γ single- and double-positive CD4⁺ T cells gated on eYFP⁺ cells among transferred CD45.2⁺CD4⁺ T cells in the draining lymph nodes of CD45.1⁺ immunized recipient animals ( Figure 4 ; Supplementary Figure S8 for the gating strategy). This allows the analysis of Th17 cells which produce only IL-17A, IFN-γ⁺Th17 cells which secrete both IL-17A and IFN-γ and Th1/exTh17 cells which secrete only IFN-γ. Upon transfer, Tec^−/− CD4⁺ T cells differentiated more towards IFN-γ⁺Th17 cells ( Figure 4 ), suggesting a higher degree of pathogenicity in the absence of Tec, as these cells were described to have a higher pathogenic potential (12). Taken together, our results indicate that Tec regulates the differentiation of Th17 cells and in particular IFN-γ⁺Th17 cells during immunization.

Th1 and Th17 cells and especially IFN-γ⁺Th17 cells and Th1/exTh17 cells were shown to play an important role in inflammatory bowel disease (IBD) (42, 43). To understand whether in the absence of Tec an enhanced mucosal Th17 cell differentiation occurs during gut-related inflammatory conditions and disease, and even contributes to the disease severity, we took advantage of the T-cell-driven colitis model (44, 45). In this model, naïve CD4⁺ T cells are adoptively transferred into Rag2^−/− mice deficient for T and B cells ( Figure 5A ). These transferred naïve CD4⁺ T cells develop into Th1 and Th17 effector cells by exposure to gut microbiota antigens and cause severe colitis which recapitulates major aspects of human IBD (46). We therefore transferred naïve WT or Tec^−/− naïve CD4⁺ T cells from IL-17A fate mapping mice into Rag2^−/− mice and analyzed the small intestine and colon for appearance of preconversion (Th17) and postconversion (exTh17) effector cells 3 weeks after transfer by monitoring eYFP⁺ cells ( Figure 5A ; Supplementary Figure S9A ), a time frame which corresponds to the onset of colitis disease (47), as exemplified by similar colon lengths between control and treated groups ( Supplementary Figure S9B ). Despite similar disease severity, we observed an increase in eYFP⁺ cells in small intestine and colon of mice transferred with Tec^−/− naïve CD4⁺ T cells compared with those that received WT cells ( Figure 5B ; Supplementary Figure S9C ). Therefore, more Tec-deficient naïve CD4⁺ T cells underwent Th17 differentiation before manifestation of strong disease phenotypes.

Th17 cells have a high degree of functional heterogeneity and plasticity (41), in particular in the gut (48, 49), where pathogenic IFN-γ producing Th17 cells and anti-inflammatory IL-10 producing Th17 cells were described. Therefore, we analyzed the production of IL-17A versus IFN-γ and IL-17A versus IL-10 by eYFP⁺ CD4⁺ T cells ( Figure 5C ; Supplementary Figures S9D , 10 ) to identify IFN-γ⁺ Th17, Th1/exTh17, IL-10⁺Th17, and Tr1/exTh17 cells. Tec^−/− eYFP⁺ cells from small intestine or colon of diseased mice showed a high degree of plasticity of Th17 cells towards Th1/exTh17 cells, as Tec^−/− eYFP⁺ had a higher frequency of eYFP⁺ IFN-γ⁺IL-17A^– cells in comparison with WT cells ( Figures 5C, D ; Supplementary Figures S9D, E ). In addition, these Tec^−/− Th1/exTh17 cells showed enhanced IFN-γ production, as MFI of these cells was significantly higher compared with WT Th1/exTh17 cells in small intestine ( Figure 5E ) and as a tendency in colon ( Supplementary Figure S9F ). However, IL-10⁺ cell frequencies were not increased among YFP⁺ intestinal cells in the absence of Tec ( Supplementary Figures S10B, C ). Taken together, these data indicate that Tec negatively regulates Th17 cells in the gut during onset of disease, restraining their plasticity towards Th1/exTh17 cells.

One of the most important molecular factors regulating Th17 plasticity is IL-23 (50), which was shown to promote expression of IFN-γ in Th17 cells and their transdifferentiation towards Th1 cells (51). The IL-23R has to be upregulated on Th17 cells during their differentiation in response to IL-6 (16). Thus, mechanisms that enhance IL-23R levels on Th17 cells also promote their plasticity. We therefore monitored the expression of the IL-23R on Th17, IFN-γ⁺Th17, and Th1/exTh17 cells from small intestine or colon of diseased mice. We observed a significant higher frequency of IL-23R-positive cells among Tec^−/− Th17, IFN-γ⁺Th17, and Th1/exTh17 cells from small intestine ( Figures 5F, G , upper panel) correlating with higher IL-23R expression levels ( Figure 5G , lower panel). The frequency of IL-23R-positive cells was enhanced among Tec^−/− IFN-γ⁺Th17 cells from colon origin ( Supplementary Figures S9G, H , upper panel) correlating with a tendency towards higher receptor levels ( Supplementary Figure S9H , lower panel). Taken together, our data show enhanced IL-23R-positive cells with higher receptor levels among eYFP⁺ cells in the intestine in the absence of Tec during onset of colitis.

To understand whether the altered Th17 differentiation in the absence of Tec also affects the disease severity in the T-cell-driven colitis model, a histopathological analysis of the colon from Rag1^−/− mice 7 weeks after transfer of WT or Tec^−/− naïve CD4⁺ T cell was performed ( Figure 6A ). At this time point, disease severity is pronounced (44). In general, colitis was significantly induced irrespective whether WT or Tec^−/− naïve CD4⁺ T cell were transferred as revealed by transmural infiltration with immune cells, epithelial cell hyperplasia, loss of goblet cells, crypt abscesses, and focal erosions ( Figure 6B ). A score was assigned by assessing all these parameters and revealed a moderate but significantly stronger inflammation in colons of animals that received Tec^−/− naïve CD4⁺ T cells in comparison with those receiving WT naïve CD4⁺ T cells ( Figure 6C ). In agreement, colon length was significantly shorter in mice that received Tec^−/− naïve CD4⁺ T cells, further indicating that a stronger colon inflammation was induced by Tec^−/− naïve CD4⁺ T cells ( Figures 6D, E ). These data together support an increased pathogenicity of Th17 subsets generated during colitis in the absence of Tec, either because of increased frequency of Th17 cells or because of the occurrence of more pathogenic-type Th17 cells, or both.

To assess whether Th17 cell differentiation and plasticity is also altered during an inflammatory stage of colitis where disease is pronounced, we analyzed intraepithelial lymphocytes (IEL) and lamina propria lymphocyte (LPL) subsets 7 weeks after transfer. We observed more eYFP⁺ cells among small intestine associated IEL and LPL of diseased Tec^−/− mice compared with diseased WT mice ( Figure 7A ; Supplementary Figure S11 ). In addition. Th1/exTh17 LPLs were significantly increased in frequency and number in the absence of Tec ( Figures 7B–D ; Supplementary Figure S12A for a gating strategy). IL-10 production was not changed between eYFP⁺ WT and Tec^−/− LPLs ( Supplementary Figures S12B–D ). Th1 cells (eYFP⁻) that did not originate from Th17 cells were strongly induced in both experimental groups, however no difference was observed in the frequency between WT and Tec^−/− Th1 cells ( Supplementary Figures S12E, F ). Taken together, Tec-deficient naïve CD4⁺ T cells differentiated markedly more towards effector Th17 cells with enhanced plasticity towards Th1/exTh17 cells during overt colitis with pronounced inflammatory phenotype.