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Keywords:

  • development;
  • ERK;
  • Id3;
  • Interleukin-4;
  • PLC;
  • PLZF;
  • SLP-76;
  • signaling;
  • T-bet;
  • T cell receptor

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Itk and i NKT cell development
  5. Role of Itk in i NKT cell function
  6. Signaling by Itk in i NKT cells
  7. Itk and NKT γδ T-cell development
  8. Role of Itk in NKT-cell-like function of Vγ1.1/Vδ6.3 γδ T cells
  9. One signal, many outcomes
  10. Acknowledgements
  11. References

The Tec family tyrosine kinase interleukin-2 inducible T-cell kinase (Itk) is predominantly expressed in T cells and has been shown to be critical for the development, function and differentiation of conventional αβ T cells. However, less is known about its role in nonconventional T cells such as NKT and γδ T cells. In this minireview, we discuss evidence for a role for Itk in the development of invariant NKT αβ cells, as well as a smaller population NKT-like γδ T cells. We discuss how these cells take what could be the same signaling pathway regulated by Itk, and interpret it to give different outcomes with regards to development and function.


Abbreviations
DN

double negative

ERK

extracellular signal-regulated kinase

Id3

inhibitor of DNA binding 3

IFN

interferon

IL

interleukin

i NKT

invariant natural killer T cells

Itk

interlukin-2 inducible T-cell kinase

MAPK

mitogen-activated protein kinase

NFAT

nuclear factor for activated T cells

NFκB

nuclear factor kappa-light chain enhancer of activated B cells

NK

natural killer cells

PLC

phospholipase C

PLZF

promyelocytic leukemia zinc finger protein

SAP

signaling lymphocyte activating molecule-associated protein

SLP-76

Src homology 2-domain containing leukocyte protein of 76 kDa

TCR

T-cell receptor

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Itk and i NKT cell development
  5. Role of Itk in i NKT cell function
  6. Signaling by Itk in i NKT cells
  7. Itk and NKT γδ T-cell development
  8. Role of Itk in NKT-cell-like function of Vγ1.1/Vδ6.3 γδ T cells
  9. One signal, many outcomes
  10. Acknowledgements
  11. References

Interleukin-2-inducible T-cell kinase (Itk) is a member of the Tec family of nonreceptor protein tyrosine kinases which includes Rlk and Tec, and is important for effective signaling through the T-cell receptor (TCR) [1,2]. There are additional Tec family kinases that signal from other receptors and have essential functions in other cell types, and these are reviewed in the accompanying minireviews [3]. In the absence of Itk, there are severe defects in activation of key signaling components including phospholipase C (PLC)γ, which results in reduced influx of Ca2+, and defective activation of extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK), with resultant reduction in the activation of the transcription factors nuclear factor for activated T cells (NFAT), nuclear factor kappa-light chain enhancer of activated B cells (NFκB) and activator protein-1 [4]. A number of studies have examined the role of Itk in T-cell development. In the absence of Itk, there is a partial block in the development of αβ T cells and a reduced ratio of CD4 to CD8 single positive thymocytes in both the thymus and periphery [5]. In addition, the absence of Itk was also found to affect positive and negative selection of thymocytes using TCR transgenic mouse models, suggesting that Itk regulates the strength of the signal emanating from the TCR during T-cell selection [5–7]. Furthermore, combined deletion of Itk and Rlk leads to a further reduction in the TCR signal strength, resulting in the conversion of negative to positive selection and a rescue of T-cell numbers in T cell receptor transgenic mice [6].

More recently, Itk-deficient mice were reported to have reduced development of naïve or conventional CD4+ and CD8+ T cells, and normal or increased development of CD4+ and CD8+ T cells, which have an activated memory-cell-like phenotype ([8–12], reviewed in [13] and [14]). These cells have increased expression of CD44 and CD122, have preformed message for interferon (IFN)-γ, and are able to rapidly produce cytokines upon stimulation [8–12]. These cells, also referred to as innate memory phenotype T cells or nonconventional T cells, may develop via an independent pathway, dependent on expression of major histocompatability complex molecules on bone-marrow-derived cells. These nonconventional or innate memory phenotype T cells share characteristics with invariant natural killer T cells (iNKT) and γδ T cells, including the ability to rapidly produce cytokines, as well as alternative modes of development. The data that are accumulating suggest that the role of Itk in the development and function of iNKT cells and γδ T cells seems to be quite different from conventional αβ T cells. This minireview focuses on this aspect of Itk, its role in the development and function of iNKT and NKT-like γδ T cells.

Itk and i NKT cell development

  1. Top of page
  2. Abstract
  3. Introduction
  4. Itk and i NKT cell development
  5. Role of Itk in i NKT cell function
  6. Signaling by Itk in i NKT cells
  7. Itk and NKT γδ T-cell development
  8. Role of Itk in NKT-cell-like function of Vγ1.1/Vδ6.3 γδ T cells
  9. One signal, many outcomes
  10. Acknowledgements
  11. References

NKT cells are a subset of innate T cells characterized by their expression of the NK1.1 marker along with the αβ TCR. Although these cells carry an antigen-specific TCR, they are characterized by their shared functions with natural killer (NK) cells. Thus NKT cells can directly kill target cells in an antigen-nonspecific fashion, but can also respond to stimulation via their TCR in an antigen-specific fashion. Like NK cells, they have the ability to rapidly produce large amounts of cytokines upon stimulation by ligands that interact with either their NK receptors or their TCRs. These cells share portions of their developmental program with conventional T cells. iNKT are a subset of NKT cells that largely express an invariant αβ TCR. Both iNKT and conventional T cells develop in the thymus from T-cell progenitors derived from bone marrow, and progress through the CD4+ CD8+ double-positive thymocytes stage. However, iNKT cells diverge during positive selection and, in sharp contrast to conventional T cells, are selected to express a restricted αβ TCR repertoire characterized by a semiinvariant TCR chain formed through VDJ recombination. Although the process is stochastic, a majority of NKT cells carry a TCR composed of Vα14–Jα18 segments, combined with either Vβ8.2 or Vβ7. These cells recognize glycolipids, prototypically α-galactosyl ceramide (although a number of other ligands have been identified), in the context of the nonclassical major histocompatibility complex molecule CD1d [15]. Because of current technical difficulties in the isolation and analysis of other NKT cell subsets and their comparatively lower numbers, iNKT cells represent the most widely studied NKT cell lineage.

Unlike conventional T cells, iNKT cells are selected by CD1d expression on immature double-positive thymocytes [15,16]. Efficient selection of iNKT cells also depends on TCR signaling in response to cognate antigen in the context of CD1d. Indeed, a number of signaling molecules that lie downstream of the TCR can affect the development of iNKT cells (for review see [15,17]). iNKT cells pass through at least four stages of maturation based on their surface phenotype and expression of cytokines (Fig. 1) (reviewed in [17]). The earliest characterized iNKT cell progenitor is CD24+/NK1.1/CD44 (stage 0), and these progenitors can respond to interleukin (IL)-7. These cells then downregulate the expression of CD24 as they progress through to stage 1 (CD24/NK1.1/CD44). As the cells progress to stage 2 they upregulate CD44 (CD24/NK1.1/CD44+). At stages 1 and 2, these cells undergo extensive proliferation thus expanding the positively selected iNKT cell pool. Stage 3 marks the final maturation that can occur in either the thymus or the periphery. At this stage, most cells are CD44hi/NK1.1+ and can secrete large amounts IFN-γ and IL-4 [17]. These fully mature iNKT express high levels of the IL-15 receptor CD122 and their homeostasis is regulated by IL-15. The final maturation step, most clearly defined by the upregulation of NK1.1, is an important checkpoint to ensure normal numbers and frequency of iNKT cells in the periphery. This maturation step is also clinically relevant, as it has been implicated in ontogeny of autoimmunity induction in nonobese diabetic (NOD) mice [18,19].

image

Figure 1.  Involvement of Itk in the development of i NKT and NKT-like γδ T cells. During T-cell development in the thymus, γδ T cells separate from αβ T cells during the CD4CD8 DN thymocyte stage, although the exact separation point is unclear. Itk and SLP-76 regulate the development of NKT like Vγ1.1+/Vδ6.2/3+γδ T cells, likely due to its ability to mediate TCR signal strength, regulating MAPK signaling, thus affecting the expression of PLZF and Id3. i NKT cells arise from CD4+CD8+ double-positive thymocyte precursors through positive selection and develop through a series of developmental stages that ultimately become mature i NKT cells. Itk and Txk are involved in the final maturation of i NKT cells, which may be through regulating of the same pathway of TCR signal strength affecting the expression of transcription factors T-bet and PLZF, which is interpreted differently by developing i NKT cells. Dashed arrows indicate hypothesized or indirect interactions.

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The Tec family kinases that are expressed in conventional T cells are also expressed in iNKT cells. Itk is the most abundantly expressed, followed by Txk/Rlk (referred to as Txk) then Tec. All are upregulated in the mature NK1.1+ fraction when compared with NK1.1 cells in the thymus, although the expression levels are similar in the fractions in the periphery [20]. In the absence of Itk, there are reduced numbers of iNKT cells in the thymus and periphery [21]. More detailed analysis of the development of Itk-null i NKT cells revealed that they upregulate CD44, but fail to upregulate CD69, CD122 and NK1.1, thus failing to progress to stage 3. The absence of Txk along with Itk results in a more severe block at the stage 2/stage 3 transition point, suggesting that Txk may play some compensatory role in this developmental pathway [20]. In the absence of Itk, the splenic iNKT-cell population is increased in the CD44lo/NK1.1/CD69 population [20,22], which is exaggerated in the Itk/Txk double-knockout mice [20].

In the absence of Itk, there are also increased levels of apoptosis in peripheral iNKT cells [20]. This is suggested to correlate with decreased expression of the IL-15 receptor beta chain, CD122, which affects the IL-15 responsiveness of these cells in the periphery. CD122 expression is regulated in part by the transcriptional factor T-bet, the absence of which also results in a block on iNKT cell development [23,24]. Indeed, T-bet expression is reduced in Itk-deficient iNKT cells, and Itk may regulate the expression of T-bet in these cells, thus regulating iNKT cell development [20].

Role of Itk in i NKT cell function

  1. Top of page
  2. Abstract
  3. Introduction
  4. Itk and i NKT cell development
  5. Role of Itk in i NKT cell function
  6. Signaling by Itk in i NKT cells
  7. Itk and NKT γδ T-cell development
  8. Role of Itk in NKT-cell-like function of Vγ1.1/Vδ6.3 γδ T cells
  9. One signal, many outcomes
  10. Acknowledgements
  11. References

Analysis of the remaining Itk-null i NKT cells for cytokine production revealed that although these cells possess preformed mRNA for IL-4, IL-5, IL-13 and IFN-γ, they lack the capacity to translate and secrete these cytokines upon antigenic stimulation both in vitro and in vivo [20,22,25]. By contrast, bypassing the TCR with the addition of 4β-phorbol 12-myristate 13-acetate and the calcium ionophore ionomycin can rescue cytokine secretion, indicating that TCR signals are defective for cytokine secretion in these cells in the absence of Itk. Thus although iNKT cell development is reduced in the absence of Itk, cells that can make it through this pathway are functionally able to make and secrete cytokines if full TCR signals are applied. Thus the absence of Itk does not affect the capacity of these cells to generate preformed cytokine mRNA and become poised for cytokine secretion [20,22].

In conventional T cells, particularly Th1 cells, IFN-γ is predominantly regulated by T-bet, whereas IL-4 is regulated by the transcription factor GATA-3 [26]. However, in the absence of Itk, iNKT cells that do develop express IFN-γ mRNA, despite the reduced expression of T-bet (although GATA-3 expression is normal) [20,22]. These findings suggest that IFN-γ expression in iNKT cells may have less dependence on T-bet. Recently, the transcription factor promyelocytic leukemia zinc finger protein (PLZF), has been suggested to be a major regulator of i NKT cell development and function, with PLZF primarily expressed in iNKT cells and other nonconventional T cells [27]. PLZF belongs to the BTB-zinc finger family of transcription factors and in the absence of PLZF, the numbers of mature iNKT cells is greatly reduced [28]. The homeostasis of these cells is also affected as a majority of iNKT cells in PLZF-null mice accumulate to lymph nodes, whereas the majority of iNKT cells in wild-type mice are found in the liver [28]. These PLZF-null iNKT cells also lack preformed mRNA for cytokines and are defective in cytokine production following TCR stimulation [28]. Whether Itk regulates expression and/or function of PLZF is unclear at this time, although there are some interesting findings along these lines as discussed below.

Signaling by Itk in i NKT cells

  1. Top of page
  2. Abstract
  3. Introduction
  4. Itk and i NKT cell development
  5. Role of Itk in i NKT cell function
  6. Signaling by Itk in i NKT cells
  7. Itk and NKT γδ T-cell development
  8. Role of Itk in NKT-cell-like function of Vγ1.1/Vδ6.3 γδ T cells
  9. One signal, many outcomes
  10. Acknowledgements
  11. References

Itk plays a critical role in the increase in intracellular calcium in T cells, in part by interacting with Src homology 2-domain containing leukocyte protein of 76 kDa (SLP-76) and regulating tyrosine phosphorylation and activation of PLC-γ1 (Fig. 2) [1,2]. SLP-76 is also critical for the development of iNKT cells, particularly the Itk binding site Y145 [29]. Thus the absence of the Itk signaling pathway results in reduced NFAT activation and expression of NFAT-regulated genes [30]. NFATc1/NFAT2 is selectively upregulated after TCR stimulation in CD4+iNKT cells and this can lead to a substantial increase in IL-4 production by these cells [31]. This NFAT activation may be due to activation of the calcium calcineurin–NFAT–Erg2 axis [32]. Although, further experiments are needed, the well-documented regulation of the Ca2+ response, NFATc1 activation by Itk following TCR ligation in conventional T cells could be conserved in iNKT cells [1]. If conserved, this Itk-regulated signaling pathway leading to NFAT activation could be severely compromised, resulting in the observed deficiencies. Indeed, in conventional T cells, Itk is important for the induction of the transcription factor Egr2 (as well as Egr1 and -3), which lies downstream of NFAT [33]. Egr2 is uniquely critical for the development of iNKT cells, with the block observed at a similar stage to that observed in the absence of Itk in mice lacking this factor [20,32]. i NKT cell development and function in NFAT-deficient mice have not yet been analyzed, although the calcium pathway and calcineurin is critical for the development of these cells [32,34].

image

Figure 2.  Signaling pathway leading to i NKT and NKT-like γδ T cells regulated by Itk. Depiction of the signaling pathway used by the TCR and modulated by Itk that results in the development of i NKT αβ and γδ T cells. Note that in the case of the TCR, these pathways seem to be shared (negative regulation of PLZF, positive regulation of ERK), but lead to different developmental outcomes. Other pathways depicted such as PKC-θ, CARMA–MALT–Bcl10 and NFκB that are critical for the development of i NKT cells are depicted for comparison. Dashed lines indicate proposed but indirect interactions.

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As discussed above, Itk-deficient iNKT cells express low levels of T-bet, and Itk may regulate the expression of this critical transcription factor. Indeed, it has been suggested that Itk regulates T-bet levels in iNKT cells in the thymus, and that thymic egress favors those cells that express T-bet and CD122 [20]. In addition, iNKT cell development is dependent on signaling lymphocyte-activating molecule (SLAM), SLAM-associated protein (SAP), Src family kinase Fyn, PKCθ, Bcl-10 and NFκB [17]. NFκB and PKCθ are dispensable for selection of conventional T cells but critical for iNKT cell development [17]. Itk has been shown to modulate the localization of PKCθ, as well as the activation of NFκB, and it is likely that the signaling pathway that Itk regulates is conserved in both conventional and iNKT cells [35,36].

Recent reports suggest that there may be some functional interaction between Itk and PLZF. PLZF is selectively upregulated in the CD4+CD44hi memory phenotype T cells that are found in the absence of Itk, although none of the CD8+ subsets expressed PLZF. These CD4+CD44hi cells have features of innate memory phenotype cells discussed above [27]. In addition, the absence of PLZF leads to a block iNKT cell development at the initial stages of maturation [28], and both mice deficient in Itk and those that express a transgene for PLZF have a developmental block in stage 2 of iNKT cell maturation, all of which results in a severe reduction in the number and frequency of mature and functional iNKT cells. These seemingly opposing results might be explained by the tight regulation of PLZF expression during iNKT cell maturation. Cells in stage 1 express high levels of PLZF, and expression is decreased as the cells progress to stage 2, with levels are greatly reduced during final maturation to stage 3 [28]. Thus, the phenotype of PLZF-null mice could be due to the reliance of iNKT cells on this transcriptional regulator during the early stages of their development. The constitutive expression of PLZF in PLZF transgenic mice may lead to defective regulation of iNKT cell development and function.

The findings reported to date favor a view that Itk regulates TCR signals which, dependent on the T-cell type, will have differential outcomes. Itk signals are important for the development of naïve or conventional T cells, and are less important or not important for the development of nonconventional of innate memory phenotype T cells. By contrast, Itk is critical for the development of the iNKT-cell population. It remains to be seen if the molecular signals regulated by Itk are conserved in all of these T cell types. We next discuss another type of T cell whose development is dependent on Itk below, a small subset of γδ T cell that have properties of NKT cells and express the CD4 and NK1.1 markers.

Itk and NKT γδ T-cell development

  1. Top of page
  2. Abstract
  3. Introduction
  4. Itk and i NKT cell development
  5. Role of Itk in i NKT cell function
  6. Signaling by Itk in i NKT cells
  7. Itk and NKT γδ T-cell development
  8. Role of Itk in NKT-cell-like function of Vγ1.1/Vδ6.3 γδ T cells
  9. One signal, many outcomes
  10. Acknowledgements
  11. References

Compared with αβ T cells, the γδ T-cell population is minor, comprising ∼ 5–10% of the total T cells in the blood and lymphoid organs. Although the cell numbers are low in the periphery, γδ T cells are more abundant in the skin and reproductive tract (as reviewed previously [37]). In this section, we discuss the role of Itk in the development of peripheral γδ T cells, although Itk also plays a role in the development of skin γδ T cells [38].

The γδ T-cell population contains many distinct subsets which reside in different tissues, including the secondary lymphoid organs and the epithelial layers of tissue such as the skin, intestinal epithelium and lung. The different subsets of γδ T cells express distinct γδ TCRs and develop at different times in the thymus. Skin γδ T cells, also called skin-resident intraepithelial T lymphocytes, uniquely express Vγ3/Vδ1, arise from fetal thymic precursor at around day 13, and become mature and migrate to the skin before birth in mice [39]. Vγ4+γδ T cells are generated later than Vγ3+γδ T cells in the fetal thymus and migrate to epithelial layers of reproductive tract, lung and tongue [39,40]. By contrast, γδ T cells in the secondary lymphoid organs are only produced in the adult thymus, and they predominantly express Vγ2 and Vγ1.1 along with diverse Vδ chains [41–45]. Populations of γδ T cells that can uniquely secrete specific cytokines, including IL-17 or IL-4 have been described previously [45,46]. The IL-4 secreting population has been described as having properties of NKT cells [47].

An increasing number of studies suggest that TCR signaling strength determines T-cell lineage commitment, with stronger signals favoring γδ T-cell development, whereas weaker signals favor αβ T-cell development [48–50]. Although Itk may modulate TCR signal strength, and one might expect that reduced signal strength received by Itk-null developing T cells would lead to reduced γδ T-cell development, previous analysis of these cells in Itk-null mice suggested that this population is not affected [5]. However, we and others have recently reported that in the absence of Itk, the percentage and numbers of γδ T cells in the adult thymus and secondary lymphoid organs is dramatically increased [51,52]. Further analysis showed that this increase is mainly due to the accumulation of a Vγ1.1/Vδ6.3 subset of γδ T cells, which express high levels of CD4 and NK1.1 [51,52]. This Vγ1.1/Vδ6.3 subset of γδ T cells are the same γδ T-cell population previously shown to secrete IL-4 and exhibit properties of NKT cells [47,53]. It has been suggested that these NKT cell-like Vγ1.1/Vδ6.3 γδ T cells may receive stronger TCR signals than other γδ T-cell subsets, which in wild-type animals could lead to negative selection during development [53]. Because Itk may act as an amplifier in the TCR signaling, Itk deficiency may affect the SLP-76 signaling complex and dampen the TCR-mediated Ca2+ influx and activation of PLCγ1, weakening downstream signals, such as ERK/MAPK, NFAT and activator protein-1 [54]. Thus the Itk deficiency may decrease TCR signal strength and allow some Vγ1.1/Vδ6.3 γδ T cells to survive negative selection.

SLP-76 is an adaptor protein that interacts with, and is important for the activation of Itk and other signaling proteins during TCR signaling [55–57]. It is therefore of considerable interest that transgenic mice expressing two SLP-76 mutants including one carrying a mutant of the Itk binding site (Y145F, Y112-128F) also exhibit significantly increased numbers of Vγ1.1/Vδ6.3 γδ T cells [29,58]. Thymocytes expressing these SLP-76 mutants have defects in TCR mediated PLC-γ1 activation, Ca2+ influx and Erk activation, demonstrating that TCR signal strength is weakened in these T cells [29]. These data suggest that Itk regulates the development of Vγ1.1/Vδ6.3 γδ T cells through altered TCR signaling strength via SLP-76.

As discussed above, SAP is important in iNKT cell development. SAP deficiency in these SLP-76 transgenic mice results in normalization (i.e. reduced numbers compared to the SLP-76 mutants) of the altered numbers of Vγ1.1/Vδ6.3 γδ T cells, suggesting that SAP is also involved in the developmental pathway of these Vγ1.1/Vδ6.3 γδ T cells [58]. Because SLP-76 and Itk interact during TCR signaling (via Y145), it is possible that SAP also modulates the pathway regulated by Itk in the development of Vγ1.1/Vδ6.3 γδ T cells. Inhibitor of DNA binding 3 (Id3) is an E-protein inhibitor that is downstream of MAPK signaling pathway. Similar to Itk-null mice, mice lacking Id3 have alterations in γδ T-cell development, and also show increased numbers of Vγ1.1/Vδ6.3 γδ T cells [58–61]. PLZF, shown to regulate iNKT cell development, has also been shown to regulate the development and function of this Vγ1.1/Vδ6.2/3 γδ T-cell population [58]. Itk may thus modulate TCR signals that regulate expression of PLZF and Id3, thus affecting development of this unique γδ T-cell population.

Because the microenvironments in the fetal thymus and adult thymus are different, the production of distinct subsets of γδ T cells during different stages of ontogeny suggests that they have distinct developmental mechanisms. We have also found that that mice lacking Itk have significantly reduced numbers of another unique population of γδ T cells that carry the Vγ3/Vδ1 γδ TCR and home to the skin, skin-resident intraepithelial T lymphocytes, suggesting that Itk is important for their development [38,62]. Further analysis indicates that Itk regulates the migration and homing, but not maturation and homeostasis, of these γδ skin-resident intraepithelial T lymphocytes. Thus Itk plays distinct roles in the development of different γδ T-cell subsets.

Role of Itk in NKT-cell-like function of Vγ1.1/Vδ6.3 γδ T cells

  1. Top of page
  2. Abstract
  3. Introduction
  4. Itk and i NKT cell development
  5. Role of Itk in i NKT cell function
  6. Signaling by Itk in i NKT cells
  7. Itk and NKT γδ T-cell development
  8. Role of Itk in NKT-cell-like function of Vγ1.1/Vδ6.3 γδ T cells
  9. One signal, many outcomes
  10. Acknowledgements
  11. References

It has long been observed that naïve Itk-null mice have high levels of serum IgE despite the observed defects in Th2 cytokine secretion from conventional and iNKT cells. IgE production is highly dependent on IL-4, and the CD4+ NKT-like Vγ1.1/Vδ6.3 γδ T cells can rapidly secrete IL-4 in vitro and in vivo [53,58,63]. Several published studies suggest that IL-4-secreting γδ T cells contribute to helping B cells class switch to produce IgE. Mice lacking αβ T cells have normal B-cell phenotypes, germinal center formation and production of antibodies, particularly IgG1 and IgE, which was suggested to be due to the IL-4 production by γδ T cells [64,65]. Human γδ T cells can also induce class switching in B cells to produce IgE [66]. In an allergic asthma model, mice lacking γδ T cells had decreased production of IgE, which were rescued by adding IL-4, suggesting that γδ T cells are important for IL-4 production and help the production of IgE and IgG1 [67].

The CD4+ NKT-like γδ T cells observed in the Itk-null mice largely carry the Vγ1.1/Vδ6.2/3 TCR, and stimulation of these cells purified from either wild-type or Itk-null mice via the TCR induces large amounts of IL-4 [51,52]. The finding that these IL-4 producing CD4+ NKT-cell-like γδ T cells accumulate in Itk-null mice suggests that these cells may be responsible for this paradoxical finding. Indeed, removing γδ T cells from the Itk-null mice results in significantly reduced serum IgE [51,52], and transfer of these cells along with wild-type B cells induced class switch and IgE production in RAG-null mice [52]. These Itk-null NKT-like γδ T cells express CD40L and OX40, costimulatory molecules that provide B-cell help, in response to anti-TCR δ stimulation [51]. Interestingly, LAT mutant mice (Y175/195/235F), which have no αβ T cells but accumulate high numbers of γδ T cells in peripheral lymphoid organs, have high levels of serum IgE and IgG1, suggesting that LAT may also play a role in the development of similar if not the same population of NKT-like γδ T cells that secrete IL-4 and can induce B-cell class switch [68].

One signal, many outcomes

  1. Top of page
  2. Abstract
  3. Introduction
  4. Itk and i NKT cell development
  5. Role of Itk in i NKT cell function
  6. Signaling by Itk in i NKT cells
  7. Itk and NKT γδ T-cell development
  8. Role of Itk in NKT-cell-like function of Vγ1.1/Vδ6.3 γδ T cells
  9. One signal, many outcomes
  10. Acknowledgements
  11. References

The combination of studies on conventional αβ T-cell development, iNKT cell and γδ T-cell development, including NKT-like γδ T cells, suggests that signaling pathways regulated by Itk may be interpreted quite differently dependent on the cell type. Itk is critical for effective development of conventional αβ T cells, the major T-cell population that participates in the immune response [1,13]. Similarly, signals regulated by Itk are important for effective development of iNKT cells, but interestingly not their primed state with preformed cytokine message, although Itk is required for cytokine secretion. By contrast, Itk seems to play a negative regulatory role in the development of NKT-like γδ T cells (carrying the Vγ1.1/Vδ6.2/3 TCR), and does not affect their ability to secrete IL-4, but does affect the ability of other γδ T-cell populations to secrete IFN-γ.

What can we surmise from these findings about the signals regulated by Itk downstream of the TCR during the development of these various subsets of T cells? Based on the studies to date, it is clear that the calcium pathway and SLP-76 are critical mediators of Itk. SLP-76, and particularly the Itk-binding site within SLP-76, is critical for the development of iNKT cells and plays a role in restraining the development of NKT-like Vγ1.1/Vδ6.2/3 γδ T cells, perhaps due to negative selection. Similarly, the Ras/Erk/MAPK pathway, also downstream of Itk, was previously described as not being critical for the development of iNKT or γδ T cells [69]. However, given the more recent studies, a re-examination of the role of Ras in the development of these cells seems to be warranted. With regards to transcriptional targets of the Itk pathway, although the spotlight has been on NFAT, other factors are coming into focus, in particular, Egr family members (Egr1, -2 and -3), Id3 and PLZF. However, these pathways have different effects on iNKT cells versus NKT-like Vγ1.1/Vδ6.2/3 γδ T cells. In iNKT cells, the pathway is required, whereas in NKT-like Vγ1.1/Vδ6.2/3 γδ T cells, the pathway restrains. Given that both cell types can secrete IL-4, it is likely that the production of this cytokine, and the T-cell types that can produce it, need to be tightly controlled. Like iNKT cells, NKT-like Vγ1.1/Vδ6.2/3 γδ T cells seem to have a conserved ligand. Although yet to be identified, this ligand (or related ligands), may be involved in negative selection of these cells, likely via the Itk–SLP-76–ERK/MAPK module. Manipulating these pathways may result in differential manipulation of these cells and thus the immune response. Future experiments determining whether the same signaling pathway is used differentially by these different T-cell populations will be very informative. Nevertheless, these findings have important implications for the potential use of Itk inhibitors in various inflammatory diseases [2].

We have recently reported that iNKT cell development can be partially rescued by a kinase deleted mutant of Itk, suggesting that kinase activity is only partially required for the development of these cells. This partial rescue correlated with rescued expression of CD122 and T-bet, and suppression of Eomesodermin [70].

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Itk and i NKT cell development
  5. Role of Itk in i NKT cell function
  6. Signaling by Itk in i NKT cells
  7. Itk and NKT γδ T-cell development
  8. Role of Itk in NKT-cell-like function of Vγ1.1/Vδ6.3 γδ T cells
  9. One signal, many outcomes
  10. Acknowledgements
  11. References