IL-2–Inducible T Cell Kinase Tunes T Regulatory Cell Development and Is Required for Suppressive Function

The Tec family nonreceptor tyrosine kinase IL-2–inducible T cell kinase (ITK) is a key signaling mediator downstream of TCR (for reviews, see Ref. 1). ITK regulates T cell differentiation, including positive selection of thymocytes, development of naive T cells and memory phenotype T cells (2–5), invariant NKT cells (6, 7), and γδ T cell populations (8–11). Conventional CD4⁺ T cell subset differentiation is tightly regulated by ITK as well. Itk^−/− naive CD4⁺ T cells exhibit defects in the differentiation to Th2, leading to attenuation of Th2-mediated allergic asthma (12, 13), as well as Th17 effector cells (14). Conventional Foxp3-expressing regulatory T cells (Tregs) are essential in preventing autoimmunity in both human and mouse (15, 16), and share similar developmental requirements for the presence of TGF-β with Th17 cells (17). Foxp3 can directly target genes involved in T cell activation and function, among which is ITK (18). Direct suppression of ITK transcription by Foxp3 may contribute to attenuate effector cytokine production in response to TCR stimulation and maintain Treg fate (18). It has recently been shown that Itk^−/− naive CD4⁺ T cells preferentially develop into inducible Tregs even under Th17 differentiating conditions in vitro (19). However, it is unclear whether ITK plays any role during natural Treg (nTreg) development in vivo.

All mice were on a C57BL/6 background. Wild-type (WT), Mhc2^−/− (B6.129S2-H2^dlAb1-Ea/J), CD45.1 (B6.SJL-Ptprc^a Pepc^b/BoyJ), and Thy1a (B6.PL-Thy1^a/CyJ) mice were from The Jackson Laboratory (Bar Harbor, ME). Rag1^−/− mice were from Taconic (Hudson, NY). ITK_Tg/Itk^−/− (Tg(hCD2-Itk)Itk^−/−) mice express ITK driven by CD2 promoter on an Itk^−/− background, as previously described (2). ITKas/Itk^−/− (Tg(hCD2-Itkas)Itk^−/−) mice are similar to ITK_Tg/Itk^−/−, but with an altered ATP binding pocket allowing inhibition by 1-(tert-butyl)-3-(3-methylbenzyl)-[¹H]-pyrazolo[3,4-d]pyrimidin-4-amine (MB-PP1) (F434G/∆429, A.K. Kannan et al., submitted for publication). Itk^−/−Mhc2^−/− mice were derived by crossing Itk^−/− and Mhc2^−/− mice. All experiments were approved by the Office of Research Protection’s Institutional Animal Care and Use Committee at Cornell University.

Naive CD4⁺ T cells were purified using the mouse CD4⁺CD62L⁺ T Cell Isolation Kit II (Miltenyi Biotec), then cultured with mitomycin C (5 μg/ml; Sigma-Aldrich) treated APCs (T cell–depleted WT splenocytes) at a 1:5 ratio in the presence of 1 μg/ml anti-CD3ε, 3 μg/ml anti-CD28 (BD Biosciences), 25 ng/ml IL-2 (PeproTech), 5 ng/ml TGF-β (PeproTech), 10 μg/ml anti–IFN-γ, and anti–IL-12 (BioLegend). Seventy-two hours later, CD4⁺ T cells were analyzed for Foxp3 expression. MB-PP1 (Millipore) was used at 2 μM.

Bone marrow chimeras were generated as previously described (2). Briefly, recipients were lethally irradiated, and 1 × 10⁷ bone marrow cells were injected through the retro-orbital vein. Chimeras were used 5–8 wk post transplantation. Congenic markers Thy1a and CD45.1 were used to distinguish the origin of the cells.

WT and Itk^−/− mice were given rat IgG2a isotype control or anti-mouse ICOS ligand (ICOSL) (100 μg per mouse; HK5.3, Bio X Cell, West Lebanon, NH) by retro-orbital injection every 3 d, and analyzed 3 d after the fifth injection. Protein carrier–free mouse rIL-2 (eBioscience) and anti–IL-2 (JES6-1A12; eBioscience) were mixed and incubated in 37°C for 30 min, prior to retro-orbital injection into WT and Itk^−/− mice (1 μg mouse rIL-2 + 5 μg anti–IL-2 per mouse) every 24 h, and mice were analyzed 24 h after the third injection.

Naive CD4⁺ T cells were isolated from CD45.1 mice. Tregs were isolated from CD45.2⁺ mice (CD45.2⁺CD25^hi Treg), using the CD4⁺CD25⁺ Regulatory T Cell Isolation Kit (Miltenyi Biotec). A total of 2 × 10⁵ CD45.1⁺ naive CD4⁺ and 5 × 10⁴ CD45.2⁺ Tregs were injected via retro-orbital injection into Rag^−/− recipients. Mice were weighed at the same time of day every week.

All fluorochrome-conjugated Abs used are listed in “fluorochrome-target” format as follows: FITC–IL-17A, PE-Foxp3, allophycocyanin–IFN-γ, allophycocyanin-Cy7-CD4, PerCP-Cy5.5-CD25, PE-Cy5-ICOS, PerCP–eFluor 710–TNF-α, and PE-Cy7-Thy1a were from eBioscience (San Diego, CA); BDV500-CD44, FITC-CD25, FITC-CD45.1, FITC-Thy1a, PE-CF594-CD4, PE-CF594-CD8α, allophycocyanin-ICOS, Alexa Fluor 700–CD45.2, PE-Cy7-CD4, and allophycocyanin-Cy7-TCRβ were from BD Biosciences (San Diego, CA); Alexa Fluor 610–CD4 and PE–Texas Red–CD8α were from Invitrogen (Carlsbad, CA); Brilliant Violet 421–neuropilin-1 (NRP1), Alexa Fluor 700–CD45.1, and PE-Cy7-CD62L were from BioLegend (San Diego, CA); the Foxp3 Staining Buffer Kit was from eBioscience. To detect cytokines, cells were stimulated with PMA/ionomycin and analyzed as previously described (20).

Analyses were performed using GraphPad Prism version 5.00.

To examine the function of ITK in differentiation of Foxp3-expressing Tregs in vivo, we compared Treg abundance in murine models that re-express ITK in a T cell–specific manner (via the CD2 promoter) on an Itk^−/− background: ITKas/Itk^−/− expressing ITK at ∼50% of WT level (A.K. Kannan et al., submitted for publication) and ITK_Tg/Itk^−/− expressing ITK at ∼35% of WT level (2, 3). We found that the percentage of CD25⁺Foxp3⁺ CD4⁺ T cells in the thymus and spleen inversely correlated with ITK expression in a dose-dependent manner, although the absolute numbers of Treg varied owing to reduced numbers of CD4⁺ T cells in the absence of ITK (Fig. 1A, 1B). ITK expressed at ∼50% of WT levels (as in the ITKas/Itk^−/− mice) is sufficient to rescue both percentage and number of Tregs in the spleen, whereas ∼35% expression levels (as in the ITK_Tg/Itk^−/−) did not (Fig. 1B). We further found that in vitro, Itk^−/− naive CD4⁺ T cells gave rise to a higher proportion of Foxp3-expressing CD4⁺ T cells under Treg-inducing conditions (induced Treg, Fig. 1C). Expression of ITK at ∼50% of WT levels in the ITKas/Itk^−/− naive CD4⁺ T cells fully reversed the proportion of Foxp3⁺ Tregs to the WT level (Fig. 1C). The ITKas/Itk^−/− model allows the use of the compound MB-PP1 to specifically inhibit the kinase activity of ITK (A.K. Kannan et al., submitted for publication), which enhanced the proportion of Foxp3⁺ Tregs to the level observed with Itk^−/− T cells (Fig. 1C). These data suggest that the ability of ITK to regulate Treg differentiation is dose dependent and dependent on its kinase activity.

In the absence of ITK, γδ T cells (9) and innate memory CD4⁺ T cells (2) are preferentially selected during T cell development in a bone marrow–intrinsic manner. To investigate whether Tregs share this property, we generated mixed bone marrow chimeras and found that although Thy1a⁺ WT and CD45.1⁺ WT bone marrow gave rise to similar proportions of CD25⁺Foxp3⁺ CD4⁺ T cells, CD45.1⁺ Itk^−/− bone marrow gave rise to a significantly increased proportion of CD25⁺Foxp3⁺ CD4⁺ T cells relative to Thy1a⁺ WT bone marrow in the same recipients (Fig. 2). This trend, along with the results of the ITK transgenic mice, is consistent in both the thymus and the spleen (Fig. 2), indicating that ITK signals suppress Treg development in a T cell–intrinsic manner.

Two key signaling pathways influence Treg development: the common γ-chain cytokine-mediated signals (notably, IL-2) and the TCR-mediated signals (Ref. 21; see review in Ref. 22). Foxp3 expression in thymic progenitors is proapoptotic and requires subsequent IL-2–induced survival signals, such as Bcl-2 expression, for the survival of differentiating Tregs (23). In the absence of ITK, Foxp3⁺ CD4SP thymocytes express significantly lower levels of Foxp3 and Fas, suggestive of an attenuated proapoptotic program; however, IL-2R and Bcl-2 expression is downregulated, suggesting a lack of contribution by IL-2 signals to the increased frequency of Foxp3⁺ cells in Itk^−/− thymus (Fig. 3A). In the periphery, mature Tregs can be divided into two fundamental subsets: CD44^loCD62L^hi central memory Tregs (cTregs) that are dependent on paracrine IL-2 for maintenance, which develop into CD44^hiCD62L^lo effector memory Tregs (eTregs) that are insensitive to IL-2 but rely on continued signaling through costimulatory receptor ICOS for maintenance (24). The lack of ITK leads to significantly increased frequency of the eTreg subset (Fig. 3B). Despite the slightly lower/similar IL-2R and Bcl-2 expression (Fig. 3A), Itk^−/− splenic Tregs include a higher proportion of the ICOS^hi subset, and both cTregs and eTregs had significantly higher ICOS expression (Fig. 3C). Of note, the majority of Tregs in both WT and Itk^−/− spleens are of thymic origin, which express high levels of NRP1 (Fig. 3D). When ICOS signaling was disrupted by blocking ICOSL, both WT and the Itk^−/− Treg population underwent similar reductions (Fig. 3E); however, because there was a higher proportion of eTregs in the Itk^−/− mice, the eTreg/cTreg ratio was reduced to WT levels in these mice (Fig. 3E). By contrast, Itk^−/− splenic Tregs underwent significantly higher fold expansion in vivo in response to IL-2/anti–IL-2 complexes compared with WT Tregs (Fig. 3F). ICOS⁺ Tregs have been shown to be more sensitive to IL-2 (25), and so our results suggest that the altered homeostasis of Foxp3-expressing CD4⁺ T cells and the proportion of eTregs in the absence of ITK may be the result of increased response to IL-2 signals, with the ICOS⁺ Tregs being more sensitive than the ICOS⁻ Tregs.

Thymus-derived Foxp3-expressing CD4⁺ T cells are the predominant Treg population regulating self-tolerance and in pathogenic conditions (22, 26), and the TCRs on these Tregs exhibit higher affinity to self Ags than those on conventional CD4⁺ T cells (27). Reduction of TCR signals through attenuating MHC2 expression on medullary thymic epithelial cells dampens T cell deletion and increases the abundance of Tregs (28). Selective ablation of MHC2 expression from hematopoietic and endothelial progenitor cells leads to reduced Treg development, although not the complete absence of these cells (29). We found that in the complete absence of MHC2, the Treg population is severely reduced, and the presence or absence of ITK does not affect the proportion of Foxp3⁺ CD4⁺ cells (Fig. 4A). Using alternative bone marrow chimeras, we discovered that the Treg population is significantly reduced by the lack of either donor or recipient MHC2 (30), indicative of a cooperative relationship for MHC2 expression in these two compartments for proper Treg selection in WT mice (Fig. 4B). However, the absence of ITK allowed for better Treg development whether selected via bone marrow (donor) or via thymic (recipient) MHC2, as thymic MHC2 selection of Tregs was fully restored to WT levels by the removal of ITK (Fig. 4B). These data suggest that ITK influences Treg differentiation by tuning TCR signals, with preferential suppression of Treg development driven by thymic MHC2 selection.

Although ITK suppresses Treg development, it is unclear whether ITK is required for functional suppression of Tregs in inflammatory disease. To investigate this, we used Rag^−/− mice reconstituted with naive CD4⁺ T cells to induce Th1-mediated colitis (31). Cotransfer of CD4⁺CD25⁺ Tregs has been shown to effectively suppress naive T cell–derived effector function and prevent weight loss associated with the development of colitis (32). Whereas WT CD4⁺CD25⁺ Tregs prevented weight loss (Fig. 5A), enlargement of the spleen (Fig. 5B, upper), and pathogenesis in the colon (Fig. 5B, lower) as a result of colitis, Itk^−/− CD4⁺CD25⁺ Tregs failed to prevent these events and even slightly enhanced pathogenesis (Fig. 5A, 5B). WT and Itk^−/− Tregs remained similar before and after the cell transfer, composed of mainly NRP1⁺ (33) nTregs, suggesting that the absence of ITK did not affect the phenotype of the transferred Tregs after transfer (Fig. 5C). WT Tregs inhibited effector CD4⁺ T cell (Teff cell) expansion, and Treg numbers remained higher, leading to significantly better Treg/Teff ratios than with Itk^−/− Tregs (Fig. 5D). In the presence of WT Tregs, naive cell–derived Teff cells exhibited an attenuated Th1 program, whereas Itk^−/− Tregs were incapable in altering the pathogenic CD4⁺ effector phenotype (IFN-γ⁺, IFN-γ⁺IL-17A⁺, or IFN-γ⁺TNF-α⁺, Fig. 5E). These data suggest an indispensable role for ITK in functional suppression of Th1-mediated colitis by Tregs.

ITK has been shown to be a critical mediator of TCR signals, regulating T cell development, and differentiation and cytokine production of Teff cells such as Th2 and Th17 cells. In this work we show that ITK also negatively tunes development of nTregs and IL-2–induced expansion of these cells. Furthermore, we show that ITK is indispensable for the ability of nTregs in functional suppression of naive CD4⁺ T cell–induced colitis in Rag^−/− recipients. We conclude that ITK regulates the development and function of Tregs.

Tregs and Th17 cells share TGF-β signals for differentiation, and ITK positively regulates Th17 differentiation (14). Gomez-Rodriguez et al. (19) recently reported that the absence of ITK results in preferential differentiation of inducible Tregs even under Th17 differentiation conditions in vitro. These authors suggested that ITK regulates the sensitivity of IL-2 signaling to STAT5, although IL-2–induced mammalian target of rapamycin was reduced in the absence of ITK. Our data showing that Itk^−/− nTregs undergo significantly higher expansion in response to IL-2 in vivo would support these findings in the nTreg population and would argue that ITK signals suppress development of both inducible Tregs (iTregs) in vitro (19) and nTregs in vivo. However, our data suggest some contradictory roles in that although ITK is apparently dispensable for iTreg suppressive function (19), we find that ITK is required for effective nTreg functional suppression in naive CD4⁺ T cell–induced colitis.

TCR, IL-2, and likely ICOS mediate essential signals for differentiation and/or maintenance of Tregs, and we find that ICOS⁺ effector Tregs are the major proportion of nTregs in Itk^−/− mice compared with central memory Tregs. Although ICOSL has been suggested to have the capacity to drive expansion of ICOS⁺ Tregs (24), these Treg populations have also been shown to be more sensitive to IL-2 signaling (25). Our experiments blocking ICOS signaling versus enhancing IL-2 signals suggest that WT and Itk^−/− Tregs are similarly sensitive to ICOS signals (i.e., similar fold reductions when signals are blocked); however, Itk^−/− Tregs undergo higher fold expansion in response to IL-2. We therefore suggest that the increased proportion of ICOS⁺ Tregs in the Itk^−/− mice may be secondary to the enhanced sensitivity of these Tregs to IL-2 in the absence of ITK. Indeed, our previous work has shown that TCR signals negatively tune IL-4–induced CD8⁺ memory phenotype T cells (20), and the recent report by Gomez-Rodriguez et al. (19) reveals similar negative tuning of TCR signals in IL-2/TGF-β–induced iTreg development. Thus, although Itk^−/− T cells have a well-described defect in production of IL-2 (34), Itk^−/− Tregs may be able to respond better owing to enhanced sensitivity to this cytokine. Similar increases in the proportion of Tregs have been observed in other murine models carrying mutants that affect the TCR proximal signalosome, such as the Slp-76 Y145F mutant, which disrupts the activation of ITK (35), and a CD3ζ mutant that is defective in ITAM phosphorylation sites (36). We do note that in these cases, the development of conventional naive CD4⁺ T cells is stunted, which may contribute to the increased proportion of Tregs in these mice. However, it should also be noted that although compared with WT mice, the number of conventional naive CD4⁺ T cells is significantly reduced in the absence of ITK, the number of nTregs is not. This finding suggests that development of conventional naive CD4⁺ T cells and nTregs is differentially regulated by ITK signals. Furthermore, we also observed significantly better expansion of Itk^−/− Tregs in response to IL-2 in vivo, supporting our conclusions.

The increased proportion of nTregs in the absence of ITK is in contrast to the idea that increased TCR signals select nTregs in the thymus, in that ITK can regulate the strength of the TCR (37). However, it is possible that during development of nTregs in the thymus, attenuation of strong TCR signals, as would be expected in the absence of ITK, would result in enhanced selection of Tregs. ITK is one of the genes suggested to be downregulated by expression of Foxp3 in Tregs, indicating that attenuation of TCR signals plays a role in the development of Tregs (18). Indeed, Sauer et al. (38) has suggested that attenuation of TCR signals leads to enhanced development of Tregs by regulating lysine 4 methylation of histone H3 near the Foxp3 promoter. This proposal would reconcile these two ideas of strong TCR-induced selection of Tregs and the increased proportion of Tregs that develop in the absence of ITK and other mutants that affect the TCR proximal signalosome.

Our analysis of the function of nTregs also suggests that ITK plays a role in the effective suppression of effector T cells. This observation is in contrast to what has been reported by Gomez-Rodriguez et al. (19), who suggested that ITK is apparently dispensable for suppressive function of iTregs. It is possible that nTregs and iTregs have different requirements for suppressive function with regard to TCR signals. However, we cannot rule out that ITK signals play some role in the imprinting of function on nTregs early in their development that is not required in iTregs. Nevertheless, taken together with previously published reports, our work suggests that modulating ITK signaling can alter the development of Tregs, in addition to other T cell subsets.

We thank Amie Wood for animal care, Gabriel Balmus for assistance with mouse irradiation, Dr. Judith Appleton for supporting Lu Huang’s participation in this work, and Chong T. Luo at Memorial Sloan-Kettering Cancer Center and Dr. Margaret Bynoe at Cornell University for helpful discussions.

The authors have no financial conflicts of interest.