Tuning T helper cell differentiation by ITK

Abstract

CD4⁺ effector T cells effectuate T cell immune responses, producing cytokines to orchestrate the nature and type of immune responses. The non-receptor tyrosine kinase IL-2 inducible T cell kinase (ITK), a mediator of T cell Receptor signaling, plays a critical role in tuning the development of these effector cells. In this review we discussed the role that signals downstream of ITK, including the Ras/MAPK pathway, play in differentially controlling the differentiation of T_H17, Foxp3⁺ T regulatory (Treg) cells, and Type 1 regulatory T (Tr1) cells, supporting a model of ITK signals controlling a decision point in the effector T cell differentiation process.

Summary sentence:.

TCR signals traveling via ITK differentially regulates distinct T helper cell differentiation.

Introduction

Naïve CD4⁺ T cells are activated by interaction with antigen presenting cells carrying their cognate antigen, and in the presence of specific cytokine environments, can differentiate into a number of different effector T helper (T_H) cell lineages. These T helper effector cells produce critical cytokines that drive the immune response, including influencing the nature of the response that can lead to specific disease, or alternatively, immune suppression. Among these effector T_H cells, this includes cells that produce inflammatory cytokines including T_H1 cells (that express the transcription factor (TF) Tbet and secrete IFNγ), T_H2 cells (that express TF GATA3 and secrete cytokines such as IL-4, IL-5 and IL-13), T_H9 cells (that express TF PU.1 and secrete IL-9), and T_H17 cells (that express TF RAR–related Orphan Receptor gamma T (RORγt) and secrete IL-17) [1–3]. In addition, there are effector T_H cells that produce suppressive cytokines, including Foxp3⁺ T regulatory (Treg) cells (that can produce TGF-β and IL-10), and Foxp3⁻ Type 1 regulatory T (Tr1) cells (that also produce high levels of IL-10 (see Fig. 1A)) [4, 5]. The balance of generation of these cells, and their production of cytokines, can control whether the immune response will be inflammatory or suppressive, and can alter the course of an immune response.

Figure 1. TCR tuning of T_H differentiation.

A) Different T_H effector cell fates that can emerge from early T cell activation and differentiation, and the cytokines that they produce. Green arrows indicate enhanced differentiation in the absence of ITK. Gray arrows indicate reduced differentiation in the absence of ITK. B) Depiction of specific TCR signaling networks downstream of ITK that influence Tr1 cells, T_H17 cells and Treg cells as reported [31, 37, 43, 58, 63, 76–78]. Inhibition or absence of Itk (depicted by the red arrows) results in reduced Tr1 cells, T_H17 cells and enhanced Treg cells. Furthermore, inhibition or absence of Itk during T_H17 differentiation results in enhanced Treg cells. Cytokines depicted are those involved in the specific T_H cell fate differentiation.

Regardless of their subsequent fate, activation of CD4⁺ T cells via their antigen specific TCR by peptide/MHC complexes induce a series of downstream signaling networks that drive the production of cytokines, expression of cytokine receptors and critical TF that drive their differentiation, depending on the surrounding cytokine milieu [6, 7]. TCR triggering activates the Src family kinase Lck, which phosphorylates ITAMs located on the cytoplasmic tails of the TCR/CD3 complex proteins. The phosphorylated ITAMs recruit the tyrosine kinase ZAP70, which leads to the phosphorylation of adaptor proteins LAT and SLP-76 [8–11]. The lipid kinase PI3K, also activated by TCR triggered Lck activation, results in the production of Phosphatidylinositol (3,4,5)-trisphosphate (PIP3) lipids in the plasma membrane and recruits the Tec family tyrosine ITK to the plasma membrane [12], where it interacts with LAT and SLP-76, the latter via Tyrosine 145 (Y145) [7, 13–19]. An important substrate for the kinase activity of ITK is PLC-γ [20], which when activated, generates IP₃ leading to increased intracellular calcium [21–24], and DAG. Increases in intracellular calcium activates the TF NFAT [25], and DAG activates PKCθ pathways leading to the activation of Akt and NF-κB [26–29], as well as the RAS/MAPK pathways [25]. The network of these activated pathways eventually leads to T cell proliferation and differentiation. However, the role of these pathways and networks in T cell differentiation is not well understood. Furthermore, although TCR signaling is necessary, and a positive regulator in the differentiation of CD4⁺ naïve progenitors to effector T_H cells [30][31], its regulation of the differentiation of Foxp3⁺ Treg cells is more complex [31–34]. It should also be noted that these intracellular signaling networks triggered by the TCR intersect with the specific cytokine signals that regulate specific differentiation of T_H subsets.

As a critical regulator of intracellular signaling downstream of the TCR, ITK plays an important role in the differentiation of effector T_H cells, among other functions (for review of ITK functions, see [17, 19, 35]). While Itk has also been shown to be important in other T cell populations, including CD8⁺ T cells and intestinal ILC2 populations, [36–42], in this review, we focus on recent work indicating that the absence of ITK impairs differentiation into T_H17 and Tr1 cell subsets [31, 43], but enhances the development of Foxp3⁺ T regulatory cells (Tregs, see Fig. 1B) [31, 43]. Thus ITK plays an important role in regulating signaling networks downstream of the TCR that govern the differentiation of effector T_H cells.

ITK signaling negatively regulates differentiation of Foxp3⁺ Treg cells

Foxp3⁺ Treg cells are derived from the thymus (tTreg) and can also be induced in the periphery (iTreg). These Tregs are critical in maintaining tolerance and immunological homeostasis [44, 45]. Tregs suppress immune responses through the production of TGF-β and IL-10, depletion of IL-2, and/or the expression of coinhibitory receptors such as CTLA-4 and PD-1 [46–48]. In the periphery, naïve CD4⁺ T cells can differentiate into iTreg cells upon TCR activation in the presence of IL-2 and TGF-β [49]. Downstream of these cytokines, the STAT5 (IL-2) and Foxo3a (TGF-β) pathways induce Foxp3 expression, which drives Treg differentiation [50–52]. However, not all TCR signals have the same effect, as low antigen dose or low concentrations of anti-CD3 to stimulate cells leads to increased Treg differentiation both in vitro and in vivo, suggesting that strong TCR signals can inhibit Treg differentiation [32, 33]. Strong TCR signals activate the PI3K/mTOR pathway which suppresses Foxp3 expression by promoting nuclear export of Foxo3a and Foxo1 [34, 50]. Indeed, T cells carrying mutated TCR CD3/ζ chains that disrupt all six ITAMs, or a SLP-76 Y145F mutation which disrupts the interaction with ITK [53] and thus have attenuated TCR activity, show increased frequency of Treg cells. In support of this model, attenuating TCR signaling strength such as in the absence of ITK signaling, results in increased frequency of Treg cells in Itk^−/− mice compared to WT mice (see Fig. 1B) [31, 43]. In addition, naïve Itk^−/− CD4⁺ T cells and Treg cells are hyperresponsive to IL-2 in vitro and in vivo [31, 43]. IL-2 signals through both STAT5 and PI3K/mTOR pathways, however only STAT5 displays increased phosphorylation in the absence of ITK, due to enhanced degradation of PTEN [31]. PTEN is a negative regulator of PI3K, which is downregulated upon sufficient TCR signaling through ITK [31]. In addition, Foxp3 negatively regulates Itk expression adding another layer to how the Treg phenotype is maintained [49, 54]. This intersection between TCR signals via ITK, and the IL-2 and TGF-β signaling pathways results in a tuning function for Itk^−/− Treg differentiation (Fig. 1). We note that experiments probing the function of ITK using kinase dead mutants reveal subtle differences in interpreting ITK function. T cells from mice expressing a kinase dead ITK exhibit a phenotype that is largely similar to those lacking Itk [55, 56], with the exception that no difference was observed in proportion of splenic Tregs [57]. Given the similarities in the Itk deficiency and ITK specific inhibition with regards to enhancing Treg development [43], it is possible that precursor development in the presence of a kinase deficient ITK leads to differences in Treg development in vivo. Indeed, in human T cells, knockdown of ITK leads to a similar increase in Treg development [58]. While the related kinase Rlk/Txk also plays a role in the activation of T cells, its role is less clear given the lack of clear phenotype observed in Rlk/Txk deficient T cells [23]. While a kinase inhibitor that affects both ITK and Rlk/Txk inhibits the development of Tregs, it should be noted that such inhibitors may affect other kinases and pathways as well, complication clear interpretation of such results [58].

ITK positively regulates T_H17 differentiation

Initially considered a subset of either T_H1 or T_H2 cells, T_H17 cells are generally characterized as proinflammatory [59]. These cells express the master TF RORγT and produce T_H17 cytokines, IL-17A, IL-17F, IL-21 and IL-23 [59]. These cells have been shown to be involved in the immune response at mucosal surfaces, as well as in the development of autoimmune conditions such as rheumatoid arthritis [60, 61]. Differentiation of naïve CD4⁺ T cells by the TCR in the presence of IL-6 and TGF-β leads to T_H17 cells [59], although other cytokine combinations can also generate T_H17 cells e.g. IL-21 and TGF-β [62]. Although IL-6 and TGF-β ostensibly have opposing roles, both are critical for T_H17 cell differentiation characterized by expression of RORγt. IL-6 signaling activates JAK1/2 and STAT3, and the absence of IL-6 in mice results in impaired T_H17 responses, and enhanced generation of Treg cells [62]. TGF-β thus acts to regulate the balance of T_H17 cells and Treg cells.

Although the precise molecular pathway driving T_H17 differentiation following TCR activation is not fully understood, Itk deficient naive CD4⁺ T cells have defects in the differentiation of Th17 cells (see Fig. 1B) [63]. Similar findings have been reported for human Th17 cells [58]. In addition, humans with Itk deficiency have reduced proportions of T_H17 cells, and biochemical inhibition of ITK blocks Th17 differentiation in vitro [37]. This is likely due to the ability of ITK to trigger increases in intracellular calcium and calmodulin activation via its substrate PLCγ1 leading the NFAT activation [63, 64]. Other studies have reported that STIM1, and PKCθ signals are important for T_H17 differentiation as well [65].

Interestingly, differentiation of Itk deficient naïve CD4⁺ T cells under T_H17 conditions results in reduced T_H17 differentiation as described above, but also, instead, the development of Foxp3⁺ T cells (see Fig. 1B) [31]. This relationship between ITK, T_H17 and Treg cells is interesting given the shared use of TGF-β signals along with IL-6 (for T_H17) or IL-2 (for Treg cells). There are a number of shared pathways downstream of ITK that may regulate this relationship, including PKCθ (which can act downstream of ITK [29]) and inhibits the differentiation of iTreg cells and their function in vitro [66, 67], while acting as a positive mediator of T_H17 cells [65].

ITK positively regulates differentiation of Type 1 Regulatory (Tr1) T cells

Type 1 regulatory (Tr1) cells are Foxp3⁻ CD4⁺ cells that regulate immune responses via multiple mechanisms including, IL-10 production and coinhibitory receptors [5, 68, 69]. Their ability to promote immune tolerance has made Tr1 cells an attractive target for development of immunotherapies [70, 71]. Tr1 cells can differentiate from naïve CD4⁺ T cells upon TCR engagement in the presence of IL-27 [72], and although Tr1 cells have been reported to be able to express IFN-γ, production of IFN-γ or T-bet are not required for Tr1 cell development [72]. Additionally, T_H17 are able to transdifferentiate into Tr1 cells during the resolution of inflammation [73]. Tr1 differentiation is controlled by a network of transcription factors which include IRF4, Blimp-1, Ahr, c-Maf and others [74, 75]. We have recently shown that Tr1 differentiation requires ITK, and that expression of these transcription factors is impaired in the absence of ITK signaling [76]. Furthermore, downstream of ITK, activation of the HRas/MAPK pathway upregulates IRF4 expression, which is upstream of Blimp-1 and Ahr [75, 76]. Importantly, Ahr agonists and reintroduction of Blimp-1 into Itk deficient naïve T cells were unable to rescue Tr1 differentiation, highlighting the importance of the HRas/IRF4 pathway in the Tr1 differentiation program [76].

Perspective

(i). Importance of the field.

The differentiation of naive CD4⁺ T cells into effector cells is critical for subsequent production of cytokines that can orchestrate the nature and type of immune response. This has implications for the response to infection, as well as autoimmunity and immunotherapy, including cellular immunotherapy.

(ii). Summary of the current thinking.

ITK, as a critical mediator of the strength of signal delivered by the TCR, plays a key function in tuning the development of these effector cells. These TCR signals regulated by ITK seem to be able to tune the cytokine response of the differentiating naïve CD4⁺ T cells, such that effector T cells that have so far been shown to depend on ITK signals, T_H1, T_H2, T_H9, T_H17 and Tr1 cells, as well Foxp3⁺ Treg cells, may utilize different pathways and TFs downstream of ITK to achieve those goals. This differential usage of the signaling network downstream of ITK may intersect with the relevant differentiating cytokines to tune T_H differentiation. While a role for Rlk/Txk has not been evaluated in Treg, T_H17 and Tr1 cell differentiation, and it is possible that the absence of Rlk could affect the differentiation of these cells, our work suggests that ITK plays a dominant role on the differentiation of these cells.

(iii). Comment on future directions.

The exploration of the signaling networks that dictate the differentiation of T_H cells continues to be of significant interest given the potential ability to manipulate these pathways to control the nature of the immune response, but also to enhance immunotherapy. In addition, exploring the network of TFs that respond to these signals, as well as the changes in chromatin landscape that set up the transcriptional networks that are mobilized to drive this differentiation is of considerable interest. Temporal analysis of these systems promise to reveal the impact early TCR signals have on driving changes leading the specific T_H cell types.

Abbreviations used:

Ahr

Aryl Hydrocarbon Receptor

Blimp-1

B lymphocyte-induced Maturation Protein

c-Maf

cellular musculoaponeurotic fibrosarcoma oncogene homolog

DAG

Di-Acyl Glycerol

Treg

Foxp3⁺ T regulatory cells

IP₃

Inositol tri-Phosphate

IRF4

Interferon Regulatory Factor 4

iTreg

Inducible Treg

ITAMs

Immunoreceptor Tyrosine-based Activation Motif

ITK

IL-2 inducible T cell kinase

IL

Interleukin

LAT

Linker of Activated T cells

PIP3

Phosphatidylinositol (3,4,5)-trisphosphate

PI3K

Phosphatidyl Inositol 3-Phosphate

PLC-γ

Phospholipase C-gamma

RORγt

RAR–related Orphan Receptor gamma T

SLP-76

SH2 domain-containing leukocyte protein

STIM1

Stromal interaction molecule 1

TCR

T cell Receptor

TF

Transcription factor

T_H

T helper

Treg

Tr1

Type 1 regulatory T cell

tTreg

thymus derived Treg

ZAP70

Zeta chain Associated protein 70