New insights into the regulation of T cells by γ_c family cytokines

Preface

Common cytokine-receptor γ-chain (γ_c) family cytokines have critical roles in the development, proliferation, survival and differentiation of multiple cell lineages of both the innate and adaptive immune system. In this review, we focus on our current understanding of the distinct and overlapping effects of IL-2, IL-7, IL-9, IL-15, and IL-21, as well as the IL-7-related cytokine TSLP (thymic stromal lymphopoietin), on the survival and proliferation of conventional αβ T cells, γδ T cells, and regulatory T cells. This knowledge potentially allows for the therapeutic manipulation of immune responses for the treatment of cancer, autoimmunity, allergic diseases and immunodeficiency, as well as for vaccine development.

Introduction

Cytokines are hormones of the immune system that have important functions related to cellular proliferation, differentiation and survival. Type I cytokines have four α-helical bundle structures and include many interleukins, as well as some growth and haematopoietic factors. One important family of type I cytokines is the common cytokine-receptor γ-chain (γ_c) family, which consists of interleukin-2 (IL-2), IL-4, IL-7, IL-9, IL-15 and IL-21 (FIG. 1), and is so named because the receptors for these cytokines share γ_c^1,2.

Figure 1. Receptors for γ_c family cytokines and TSLP.

Shown are the receptors for IL-2, IL-4, IL-7, IL-9, IL-15, IL-21, and TSLP. IL-2 and IL-15 are the only two of these cytokines to have three receptor chains. These two cytokines share IL-2Rβ, whereas IL-7 and TSLP share TSLPR, and of the cytokines shown, only TSLP does not share γ_c. There are three classes of IL-2 receptors, binding IL-2 with low affinity (IL-2Rα alone), intermediate affinity (IL-2Rβ + γ_c), and high affinity (IL-2Rα + IL-2Rβ + γ_c); only the high affinity IL-2 receptor is shown in the figure. Each γ_c family cytokine activates JAK1 and JAK3¹, whereas TSLP was reported to not activate any JAK kinase²⁵,²⁶. The major STAT proteins activated by these cytokines are shown. STAT5 refers to both STAT5A and STAT5B.

γ_c was first discovered as a component of the receptor for IL-2³, the prototypic member of this family. The IL-2 receptor (IL-2R) consists of three chains, which together form the high-affinity IL-2R (FIG. 1), but which in other combinations bind IL-2 with low affinity (IL-2Rα alone) or intermediate affinity (IL-2Rβ and γ_c). The structures for some of the receptors for γ_c family members are known, providing an insight into how different cytokines can each interact with γ_c⁴.

The gene encoding γ_c (IL2RG) is mutated in humans with X-linked severe combined immunodeficiency (X-SCID)⁵, who lack T cells and NK cells, which indicates that γ_c is crucial for the development of these cells. However, the finding that the immune defects in patients with X-SCID are much more severe than those of humans or mice lacking IL-2, in which the development of T and NK cells is intact, originally led to the hypothesis and subsequent confirmation that γ_c was shared by multiple cytokines¹.

IL-2 functions as a T cell growth factor, can augment NK cell cytolytic activity, and promotes immunoglobulin production by B cells⁶. In addition, it contributes to the development of regulatory T (Treg) cells and therefore peripheral T cell tolerance⁷, as well as regulating the expansion and apoptosis of activated T cells^8,9. IL-4 is required for the development and function of T helper 2 (Th2) cells and is therefore regarded as the classical Th2-type cytokine. IL-4 also has an important role in allergy and immunoglobulin class switching¹⁰. IL-7 has a central role in the development of T cells in both humans and mice. Indeed, defective IL-7-induced signaling is responsible for the defective T cell development that is observed in patients with XSCID⁵, as well as in patients with SCID caused by mutations in a signaling molecule downstream of γ_c, Janus kinase 3 (JAK3),^11,12 or by mutations in IL7R¹³ (Table 1). Interestingly, IL-7 is also required for the development of B cells in mice but it is not necessary for their development in humans as evidenced by normal B cell development in patients with XSCID (IL2RG deficiency), JAK3-deficient-SCID, and IL7R-deficient SCID¹. Nevertheless, in an in vitro model, IL-7 can promote the development of human B cells from adult bone marrow hematopoietic stem cells (HSCs), although not from cord blood HSCs¹⁴. IL-7 is well known for its potent role as a survival factor^15,16. IL-9 is produced by a subset of activated CD4⁺ T cells^17,18 and induces the activation of epithelial cells, B cells, eosinophils, and mast cells¹⁹ (FIG. 1). Although IL-9 has been shown to function as a T cell growth factor late during an immune response²⁰, its role in T cell biology remains unclear. IL-15 is essential for the development of NK cells, and it is the defective signaling by IL-15 that results in the failure of NK-cell development in both X-SCID and JAK3-deficient SCID¹. IL-15 also has an essential role in CD8⁺ T cell homeostasis¹⁶. IL-21 is the most recently described member of this family², and it has broad actions that include promoting terminal B-cell differentiation to plasma cells, cooperating with IL-7 or IL-15 to drive the expansion of CD8⁺ T cell populations, and acting as a pro-apoptotic factor for NK cells and incompletely activated B cells². IL-21 has also been shown to be an essential mediator of the development of type 1 diabetes^21,22 and systemic lupus erythematosus²³ in animal models, and to have potent anti-tumor actions².

Table 1. The basis of defects in Severe Combined Immunodeficiency.

Mutations in IL2RG or JAK3 genes cause defects in T and NK cell development, with functional abnormalities of B cell lineage in human and lack of B cells in mice. IL7R mutations greatly diminish T cell numbers, with normal or even increased number of B and NK cells. IL2RB mutations result in defective NK cell development but do not affect T and B cell development.

Form of SCID	Lineage abnormalities	Causes of defects	References
X-linked SCID	TB+NK−. T and NK cells absent; B cells present but non-functional.	The disease results from mutations in the IL2RG gene. Decreased T cell development is due to defective IL-7- induced signaling. Lack of NK cell development is the result of defective IL-15-induced signaling. Functional B cell abnormalities are due to a lack of T-cell help and defects in IL-4 and IL- 21-induced signaling, as indicated by the pan-hypogammaglobulinemia found in Il4/Il21r double KO mice, which is associated with germinal center abnormalities in these mice57	1,5
JAK3- deficient SCID	TB+NK−. T and NK cells absent; B cells present but non-functional.	The disease results from mutations in the JAK3 gene. Abnormalities are due to same reasons as in XSCID.	11,12
IL7R- deficient SCID	TB+NK+−. T cells absent. In humans B cells are present but in Il7r- deficient mice, B cells are absent.	Defective IL-7-induced signaling with a possible partial contribution from TSLP. Humans with IL7-deficiency have not been reported. Mice with Il7r deficiency have a somewhat more severe T cell phenotype than mice with Il7 deficiency.	13,42,43
IL2RB- deficient SCID	T+B+NK−. NK cells absent.	Defective IL-15-induced signaling, based on Il15, Il15ra, and Il2rb deficient mice, IL2RB-deficient SCID, and in vitro studies.	1,146

γ_c family cytokines all signal through the JAK–STAT (signal transducer and activator of transcription) pathway. Interestingly, IL-2, IL-7, IL-9 and IL-15 mainly activate STAT5A and STAT5B (together referred to as STAT5 proteins), whereas IL-4 generally activates STAT6 and IL-21 mainly activates STAT3²⁴ (FIG. 1). The activation of different STAT proteins may help to explain the different actions induced by these cytokines.

As partially indicated above, some of the γ_c family cytokines broadly contribute to lymphocyte homeostasis, which will be a main focus of this review. We also discuss another cytokine that is not a member of the γ_c family but has overlapping functions with IL-7, known as thymic stromal lymphopoietin (TSLP)²⁵. Whereas the IL-7 receptor contains IL-7Rα (also known as CD127) and γ_c, the TSLP receptor consists of IL-7Rα and TSLPR, which is closely related to γ_c^26,27 (FIG. 1).

Direct effects of γ_c family cytokines on T cells

Regulation of naïve and memory αβ T cell homeostasis

γ_c-deficient mice show a low thymic output of T cells and lymphopaenia, and those T cells that do develop have impaired survival²⁸. IL-7 seems to be the most important γ_c family cytokine for regulating the homeostasis of naïve and memory T cells^29–32 (FIG. 2). In contrast to other γ_c cytokines, the levels of which increase after immune cell activation, IL-7 is constitutively produced by stromal and epithelial cells in the bone marrow and thymus and by fibroblastic reticular cells in the T-cell zones of secondary lymphoid organs^16,33. The availability of IL-7 is regulated both by its production and by its consumption by CD4⁺ T cells^16,34. Thus, diminished numbers of CD4⁺ T cells in humans are associated with increased levels of IL-7³⁴.

Figure 2. Direct and indirect actions of γ_c cytokines and TSLP on T cell expansion, homeostasis, and differentiation.

γ_c cytokines can directly influence the survival, activation, proliferation, and differentiation of T cells (see top half of the figure), as well as indirectly affecting these processes via actions on DCs, macrophages, and Treg cells (bottom half of the figure). Although IL-15 is a critical factor for memory CD8⁺ T cell homeostasis, it also is responsible for the recovery of naïve CD8⁺ T cells in lymphopenic conditions. In the absence of IL-7, IL-15 has major effects on the homeostasis of memory CD4⁺ T cells. Both IL-7 and TSLP are important for the survival of naïve T cells, with IL-7 playing the greater role. The action of TSLP on memory T cells has not been evaluated. IL-2, IL-4 and IL-21 are produced by activated T cells and play essential role in T cell differentiation. In addition, IL-2 and IL-15 can augment expansion of T cells upon antigen-stimulation.

A distinctive feature of IL-7 compared with other γ_c cytokines relates to the expression of its receptor. Whereas most of the cognate chains for γ_c cytokines are up-regulated after T-cell receptor (TCR) activation, IL-7Rα is expressed by naïve and memory resting T cells but is down-regulated after TCR activation^29,35 (Table 2). This indicates that IL-7 primarily mediates its functions on naïve and memory T cells rather than on activated T cells (see below). IL-2, IL-7 and other pro-survival cytokines can transiently reduce the expression of IL-7Rα^35–37, which could decrease IL-7 consumption, thus increasing the availability of IL-7 for other cells that express high levels of IL-7Rα and are poised to receive survival signals in vivo³⁷. Maintaining IL-7Rα expression is dependent, at least in part, on the transcription factor GABP³⁸, and GABP and Gfi-1, respectively, control the up- and down-regulation of IL-7Rα expression³⁹.

Table 2. Expression of receptors for γ_c family cytokines and TSLP.

The expression of various cytokine receptors in different stages of the T cell immune response. γ_c is constitutively expressed by T cells. IL-2Rα is rapidly up-regulated in the early stage of T cell activation, thus augmenting the number of high-affinity IL-2Rs. The levels of expression of IL-2Rβ, IL-4Rα, IL-15Rα, IL-21R, and TSLPR are increased after T cell activation, and IL-2Rβ expression is sustained at a high level on CD8⁺ T memory cells.

type of T cells receptor chain	Naive T cells	Effector T cells	Memory T cells
γc (CD132)	++	++	++
IL-2Rα (CD25)	−	+++	−
IL-2Rβ (CD122)	+	+++	+++
IL-4Rα (CD124)	−	+++	Nd
IL-7Rα (CD127)	++	−*	+++
IL-15Rα	+	+++	+++
IL-21R	+	+++	Nd
TSLPR**	+	++	Nd

^*Only a few effector T cells express IL-7Rα.

^**The level of TSLPR expression on T cells is shown relative to expression on dendritic cells. Levels of IL-4Rα, IL-21R, and TSLPR on memory T cells have not been evaluated (nd, not done).

IL-7 uses at least two different mechanisms for supporting T cell homeostasis. First, it promotes survival by activating the pro-survival phosphoinositol 3-kinase/AKT signaling pathway and by increasing the expression of survival factors such as BCL2 and myeloid cell leukemia sequence 1 (MCL-1) whereas it inhibits the expression of pro-apoptotic factors BAX and BAD^15,16. Second, it induces the proliferation of naïve and memory T cells in lymphopaenic conditions and of memory T cells but not of naïve T cells under normal physiological conditions^29,30,40,41 (FIG. 2).

Although Il7ra^−/− and Il7^−/− mice each have markedly decreased numbers of T cells, the absence of IL-7 can be partially compensated for by increasing TSLP, which can result in the partial restoration of normal T and B cell numbers^42,43. These observations suggested that TSLP might have a role in T cell homeostasis. Indeed, administration of recombinant TSLP to Il2rg^−/− mice, which cannot respond to IL-7 or other γ_c family cytokines, results in a partial increase in CD4⁺ and CD8⁺ T cell numbers. Consistent with this, restoration of CD4⁺ and CD8⁺ T cells following irradiation is less efficient in Tslpr^–/– mice than in irradiated wild-type mice⁴². Moreover, TSLP promotes the survival of CD8⁺ T cells in both normal and lymphopaenic conditions⁴⁴. Interestingly, whereas IL-7 induces proliferation (as well as survival) of naïve CD8⁺ T cells in lymphopaenic mice, TSLP does not affect the proliferation of these cells⁴⁴. A possible explanation for this observation is that IL-7, but not TSLP, can activate JAK3 and is a more potent activator of STAT5^25,26.

IL-15 is another important homeostatic cytokine, which preferentially has activity for memory CD8⁺ T cells^30,45–47 (FIG. 2) that express high levels of IL-2Rβ (CD122), particularly the IL-2Rβ^hi Ly49⁺ subset of CD8 memory cells⁴⁸. Although IL-15 does not play an essential role in the homeostastic expansion of memory CD4⁺ T cells, in the absence of IL-7 IL-15 was reported to affect the homeostasis of these cells⁴⁹. Furthermore, depleting CD8⁺ T cells and NK cells, which are main consumers of IL-15, results in a greater availability of IL-15 and increases the homeostatic proliferation of memory CD4⁺ T cells⁴⁹. Although IL-15 is not vital for the development of naïve T cells, Il15^−/− mice have reduced numbers of naïve CD8⁺ T cells and exhibit diminished proliferative rates, likely explaining a slower recovery of adoptively transferred naïve CD8⁺ T cells in these mice than in wild-type mice^40,47,50. Memory CD8⁺ T cells are dependent on IL-15; interestingly, however, although these cells express high levels of IL-15Rα⁴⁶ (Table 2), they proliferate better to the administration of an IL-15–IL-15Rα complex than to purified IL-15 alone.^51,52. This term refers to a process whereby IL-15 that is bound to IL-15Rα is presented to cells expressing the other IL-15R subunits, IL-2Rβ and γ_c⁵³. Indeed, under physiological conditions, trans-presentation of IL-15 by IL-15Rα on the surface of other non-T cells is required⁵⁴, underscoring the importance of this mode of signaling for IL-15.

IL-7 and IL-15 can also function cooperatively with IL-21 to expand CD8⁺ T cells in vitro⁵⁵. Although IL-21 alone induces the survival of naïve but not memory mouse CD8⁺ T cells, in the presence of IL-15 the rate of apoptosis in both populations of CD8⁺ T cells is markedly reduced and cell proliferation is strongly augmented⁵⁵. Similarly, the combination of IL-15 and IL-21 increases antigen-independent proliferation of human naïve CD8⁺ T cells in vitro⁵⁶, increases the number of CD8⁺ T cells producing IL-2 and IFNγ and enhances the cytotoxic activity of these cells following TCR activation^55,56. Although Il21r^−/− mice have normal numbers of peripheral CD8⁺ T cells⁵⁷, over-expression of IL-21 increases memory CD8⁺ T cell numbers⁵⁸; whether this is an effect of IL-21 or the result of synergistic actions of IL-21 with constitutively produced IL-7 or IL-15 is unclear. These data overall underscore the wide range of actions of various γ_c family cytokines, particularly IL-7 and IL-15, in naïve and memory T cell homeostasis.

Proliferation and survival of effector T cells

IL-2 is perhaps the earliest cytokine secreted by T cells following TCR stimulation⁵⁹ and is important in the initiation of Th2 cell differentiation^60,61. It is well known that IL-2 can induce the proliferation and survival of TCR-activated human and mouse T cells^6,62 and is required for sustained expansion of T cell populations⁸. Although administration of supra-physiological levels of IL-2 to LCMV-infected mice during the expansion phase of the anti-viral T cell response unexpectedly decreases the numbers of antigen-specific CD4⁺ T cells⁶³, this might reflect the ability of IL-2 to induce apoptosis of T cells that have been recently activated through their TCR⁹. Conversely, IL-2 treatment during the contraction phase of the T cell response results in increased survival and accumulation of T cells^8,63,64. Overall, the role of IL-2 is multi-faceted due to its complex effects on driving T cell proliferation, promoting the expansion of Treg cells (see later) and mediating activation-induced cell death.

The role of IL-7 and IL-15 in the expansion of effector and memory T cell populations has been widely studied. Immediately after TCR activation, most T cells down-regulate IL-7Rα and up-regulate IL-2Rα, IL-15Rα, and IL-2Rβ expression (Table 2). It was thus predicted that IL-7 is not required for the function of activated T cells²⁹, and IL-2 and/or IL-15, the production of which is also increased by activated DCs^65–67, may increase the proliferative rate and/or diminish the contraction of effector T cells^64,68,69. Although a selective population of primed T cells re-expresses IL-7Rα⁷⁰, IL-7 signaling is not essential for the formation of functional memory CD4⁺ and CD8⁺ T cells^71–74, which could reflect potentially redundant functions of TSLP and IL-7 or instead important actions of other cytokines. Several findings support a possible role for TSLP in the expansion of effector and memory T cell populations: TSLPR expression is increased after TCR activation^44,75 (Table 2), TSLP increases the proliferation of TCR-activated CD4⁺ T cells in vitro^42,75, and TSLP production is increased during the acute phase of the immune response to pathogens and allergens⁷⁶.

Regulation of γδ T cell homeostasis

γδ T cells are a population of T cells that arise from the same precursors as αβ T cells in the thymus. They migrate to the periphery, mostly to epithelial tissues, and have broad immunological actions, which include their production of cytokines and chemokines, cytolytic activity in response to pathogens, regulation of the viability and proliferation of keratinocytes, induction of macrophage and DC responses, and presentation of antigen to T cells^77,78. The expression of γ_c and IL-7Rα are both essential for normal γδ T cell development⁷⁸. Under lymphopaenic conditions, γδ T cells undergo MHC-independent homeostatic proliferation that requires IL-7 or IL-15^79,80. Although αβ and γδ T cells express comparable levels of IL-7Rα and IL-2Rβ⁷⁹, partial depletion of αβ T cells, NK cells, or γδ T cells themselves significantly enhances the homeostatic proliferation of γδ T cells, revealing that these cells compete for limited quantities of IL-7 and IL-15^79–81. Thus, the maintenance of γδ T cells as well as αβ T cells is regulated by IL-7 and IL-15.

The maintenance of Treg cells

Treg cells are a subset of CD4⁺ T cells that were defined in part by their constitutive expression of the IL-2 receptor α chain (CD25) and the Treg-cell specific transcription factor forkhead box P3 (FOXP3), which controls the development and function of these cells⁷. Although IL-2 induces the proliferation and clonal expansion of conventional T cells^6,62, IL-2 also mediates immune tolerance at least in part through its regulation of Treg cells. IL-2 deficiency is characterized by a reduction in the number and function of Treg cells, and accordingly, leads to lymphoproliferation and autoimmunity⁷. However, the lack of IL-2, IL-2Rα, or IL-2Rβ (CD122) does not alter FOXP3 expression nor result in a complete loss of Treg cells^82–85. By contrast, γ_c-deficient (Il2rg^−/−) mice and Jak3^−/− mice, in addition to having very low numbers of T cells, are devoid of FOXP3⁺ Treg cells^83,86.

STAT5 appears to be vital for Treg cells development. STAT5 activation is sufficient to increase the number of CD4⁺CD25⁺ Treg cells even in the absence of IL-2 production⁸⁷ or when there is defective IL-2-induced signaling⁸⁸, whereas, conversely, deletion of the Stat5a and Stat5b genes is characterized by a dramatic reduction in the number of Treg cells in the thymus and in the periphery⁸⁶. Correspondingly, a patient with a missense mutation in the human STAT5B gene exhibited diminished numbers of CD4⁺CD25^hi T cells and these cells had a dramatic reduction in FOXP3 expression levels and impaired suppressive activity⁸⁹. These findings confirm earlier observations indicating that STAT5A and STAT5B are critical factors downstream of IL-2R^90–93 and suggest that other γ_c family cytokines, such as IL-7 and IL-15, which also activate STAT5 proteins, might contribute to Treg cell development and maintenance.

Although deficiency of IL-7 or IL-15 (which both activate STAT5 proteins) does not alter the number of FOXP3⁺ Treg cells^88,94, the absence of IL-7- or IL-15-induced signaling in combination with disrupted IL-2R signaling results in a greater reduction in Treg cells than is observed in mice lacking either IL-2 or IL-2Rα alone^88,95. Interestingly, mouse Treg cells express low levels of IL-7Rα^94,95, but in contrast to other subsets of T cells that can re-express IL-7Rα after culture in vitro, peripheral Treg cells do not upregulate IL-7Rα expression after culture in vitro⁹⁴. Nevertheless, the low level of IL-7Rα that is expressed seems to be functional, as IL-7, even in the absence of IL-2-induced signaling, can mediate the survival, although not the proliferation, of mouse Treg cells⁹⁵. Similarly, most human CD4⁺CD25⁺FOXP3⁺ Treg cells have lower levels of IL-7Rα expression than do CD4⁺ CD25⁺FOXP3⁻ T cells^96,97. Il7ra^−/− mice have a marked reduction in the number of Treg cells in lymphoid tissues and decreased suppressive activity, as compared with Il7^−/− mice⁹⁴. These differences can be explained by the ability of TSLP (which signals through a receptor that contains IL-7Rα) to also mediate the induction of Treg cells⁹⁴. Thus, IL-2, IL-7, IL-15, and TSLP all contribute to Treg cell development and function.

Although IL-2, IL-4, IL-7, IL-15, and IL-21 can promote the survival of Treg cells and rescue them from apoptosis in vitro, only IL-2 induces their proliferation and clonal expansion⁹⁸. Consistent with this, peripheral homeostasis of Treg cells in vivo is more dependent on IL-2 than on other γ_c cytokines⁷, and neutralizing IL-2 not only reduces the number of mouse Treg cells in the thymus but also prevents their clonal expansion in lymph nodes⁹⁹. Correspondingly, IL-2 therapy during immune reconstitution after chemotherapy markedly increases the size of the Treg-cell compartment¹⁰⁰.

Indirect effects of γ_c cytokines on T cells

T cell survival and proliferation through DCs

It is well known that γ_c family cytokines have pleiotropic effects on the immune system and that they can stimulate various populations of cells in addition to T cells, which in turn affect T cell homeostasis. For example, DCs are key players in the activation of the adaptive immune response, and γ_c family and related cytokines that are induced by pathogens may activate (e.g. IL-15 or TSLP) or inhibit (e.g. IL-7 or IL-21) the function of DCs (FIG. 2).

DCs constitutively express IL-2Rβ and γ_c^66,101,102 and up-regulate the expression of IL-15Rα in response to type I and II interferons (IFNs) and inducers of NF-κB activation, such as ligands for Toll-like receptors (TLRs)^65,66. Similar signals promote IL-15 production by DCs and epithelial cells^65,66. Therefore, IL-15 has paracrine and autocrine actions on DCs that result in increased survival of mature DCs, up-regulation of co-stimulatory molecules and enhanced presentation of antigen by DCs to CD4⁺ and CD8⁺ T cells^65–67,103. DCs from aged mice produce less IL-15 that those from young mice, and the functional defects of DCs from aged mice can be reversed by IL-15 treatment¹⁰⁴. Thus, reduction of IL-15 production might be a factor that contributes to reduced immunity to pathogens in the elderly. Moreover, infection of humans with hepatitis C virus reduces IFNα-mediated IL-15 production by DCs and therefore decreases the maturation of functional DCs, which has been suggested as a possible mechanism for the poor T-cell immunity against this virus¹⁰⁵.

Both IL-4 and TSLP are involved in Th2-cell responses and have essential roles in allergic diseases^10,25,76; however, their actions on DCs differ. IL-4 is a survival factor for DCs and in combination with GM-CSF promotes the differentiation of DCs from mouse bone marrow progenitor cells and from human monocytes in vitro^148,149. DCs pre-cultured in the presence of IL-4 express a relatively low level of MHC and co-stimulatory molecules, which is indicative of an immature phenotype, and these cells poorly respond to IFNα¹⁰⁶. Unlike IL-4, TSLP is not needed for DC differentiation, but as shown by in vitro experiments, it promotes their activation and upregulation of expression of MHC class II and co-stimulatory molecules, including CD80, CD86 and OX40L²⁵. TSLP-activated human DCs support naïve CD4⁺ T cell homeostasis and induce robust expansion and differentiation of human CD4⁺ T cells into inflammatory Th2 cells²⁵. In mice, TSLP plays a role in intestine immunity, by inhibiting production of IL-12 by DCs induced by LPS and thereby reducing the number of IFNγ⁺ CD4⁺ T cells¹⁰⁷. Interestingly, IL-7, which shares IL-7Rα with TSLP, maintains the immature phenotype of DCs and can down-regulate MHC class II expression by mature mouse DCs, correlating with decreased homeostatic proliferation of CD4⁺ T cells¹⁰⁸. IL-21 is not needed for DC differentiation, but pre-treatment of DCs with IL-21 inhibits their maturation in response to TLR stimuli, thus suppressing DC functions, such as antigen presentation and cytokine and chemokine secretion^109,110. Because IL-21 is produced by CD4⁺ T cells following antigen stimulation² and IL-21-primed DCs have inhibitory effects on T cell responses, the production of IL-21 by T cells may activate a DC-mediated negative loop.

Conventional T cells homeostasis through Treg cells

As discussed above, γ_c cytokines have a key role in the development and maintenance of Treg cells. In turn, these cells inhibit the expansion of auto-reactive T cells, preventing autoimmunity and suppressing the response of conventional T cells to foreign and self-antigens. Several mechanisms have been proposed to explain how Treg cells mediate this suppression (FIG. 3), including their inhibiting of responder T cells by producing suppressive cytokines such as TGFβ, IL-10, and IL-35; inactivating antigen-presenting cells (APCs) through the expression of the inhibitory molecules cytotoxic T-lymphocyte antigen 4 (CTLA4) and lymphocyte-activation gene 3 (LAG3); killing target cells through cytolytic activity; and consuming pro-survival γ_c cytokines, thus resulting in apoptosis of conventional T cells in vitro^98,111. Although it is not yet clear whether this latter cytokine-deprivation mechanism occurs in vivo, Treg cells also induce cytokine-dependent apoptosis of conventional CD4⁺ T cells in mice⁹⁸. Note that the mechanisms listed above are not mutually exclusive, so that more than one mechanism might be used.

Figure 3. Mechanisms of T cell regulation by Treg cells.

Treg cells exhibit several mechanisms to suppress activation and expansion of conventional T cells. Treg cells modulate functions of APCs by inhibiting their maturation and blocking of MHC molecules and co-stimulatory molecules (CD80 and CD86) on the surface of APCs and thus attenuating interactions between APCs and T cells. Treg cells might have cytolytic effects on target T cells as well as on APCs. Treg cells suppress activation and proliferation of T cells by their secretion of inhibitory cytokines, such as TGFβ, IL-10, and IL-35 and by consumption of γ_c cytokines. Deprivation of γ_c cytokines induces expression of pro-apoptotic proteins and elevates apoptotic rate of conventional T cells.

γ_c family cytokines and T-cell differentiation

Naive T cells can differentiate during a primary antigen response into several distinct polarized subsets, such as Th1, Th2, Th17, and T follicular helper (T_FH) cells. These subsets produce discrete sets of cytokines and chemokines to collectively allow responses to different classes of pathogens. Four γ_c family cytokines are among the main cytokines that are produced by these polarized cells: IL-2 by Th1 cells, IL-4 and IL-9 by Th2 cells and IL-21 by Th17 and T_FH cells as well as Th1 and Th2 cells. These cytokines act on other target cells to direct immune responses, but IL-2, IL-4 and IL-21 also have important roles in the early development of CD4⁺ T cell subsets.

Th1 cell differentiation

Th1 cell differentiation depends primarily on APC-derived IL-12, which then leads to IFNγ production and increased IL-12Rβ2 expression¹¹². Although IL-2 is the earliest detected cytokine produced by naive CD4⁺ T cells following TCR stimulation⁵⁹, and this cytokine is a major product of Th1 cells¹¹³, it is not clear whether IL-2 signaling contributes to early commitment to the Th1 cell lineage. IL-21 was reported to be a Th2-type cytokine that had inhibitory effects on Th1 cells¹¹⁴, but IL-21 does not affect the expression of Th1 cell-associated transcription factor T-bet or of IL-12Rβ2 by mouse CD4⁺ T cells¹¹⁴. Instead, IL-21 can inhibit IFNγ production by naive CD4⁺ T cells that are undergoing Th1 differentiation by the repressing the expression of the T-box transcription factor Eomesodermin¹¹⁵. It is unclear whether this inhibition of IFNγ by IL-21 has a role in modulating Th1 cell responses in vivo as opposed to promoting Th17 cell differentiation (see below). Interestingly, in human peripheral blood T cells stimulated via the TCR, IL-21 actually induces IFNγ, T-bet and IL-12Rβ2 expression¹¹⁶, which suggests that IL-21 under certain circumstances might promote Th1 cell differentiation.

Th2 cell differentiation

In vitro studies have demonstrated that IL-2 and IL-4 are both required for the efficient induction of Th2 cells. IL-2 is produced early following the activation of naive CD4⁺ T cells and activates STAT5A and STAT5B to promote increased transcription of the Il4ra gene, thus leading to the enhanced cell surface receptor expression of IL-4Rα and subsequent increased responsiveness to IL-4⁶¹. IL-2 also induces STAT5 binding to consensus binding sites located within DNase-I hypersensitive sites in the Il4 locus, thus promoting increased accessibility of this locus to the formation of transcriptional complexes⁶⁰. A genome-wide in vivo analysis revealed that IL-2 via its actions on STAT5 activates not only the Il4ra locus but also the entire Th2 cytokine locus, which includes the Sept8, Kif3a, Il4, Il13, Rad50, Il15, and Irf1 genes. Interestingly, this analysis showed that STAT5A and STAT5B bind first at the Il4ra locus and then at the Th2 locus in vivo, consistent with observation that IL-4 is produced by Th2 cells after they express IL-4Rα⁶¹. Thus, IL-2-induced signaling during Th2 cell differentiation results in both increased production of IL-4 and increased responsiveness to IL-4, leading to stabilization of this lineage. Other cytokines that can activate STAT5 proteins including IL-7 and IL-15 were also shown to induce IL-4Rα expression, indicating that multiple STAT5 activators may be able to prime T cells for Th2 cell differentiation⁶¹. Although IL-21 also can promote STAT5 activation, IL-21-induced signaling does not affect the efficiency of Th2 cell differentiation in vitro⁵⁷, which is consistent with its primarily activating STAT3 rather than STAT5. Il21r-deficient mice exhibit reduced responses to Th2 cell-inducing pathogens; however, it is possible that this results from diminished effects of IL-21 on macrophage activation¹¹⁷ rather than IL-21 having direct Th2-related effects.

Th17 cell differentiation

Th17 cell differentiation depends in part on TGFβ, an immunosuppressive cytokine that also has a role in Treg cell differentiation. The presence of either IL-6 or IL-21 during priming with TGFβ event subverts cell differentiation from the FOXP3-directed Treg cell pathway to the Th17 cell pathway through the induction of the orphan nuclear receptor RORγt^118–120. Th17 versus Treg cell differentiation is therefore determined by the presence of IL-6 or IL-21.

IL-2 can promote the development of Treg cells, and it inhibits the differentiation of naïve CD4⁺ T cells into Th17 cells^121,122. Accordingly, administration of IL-2 to tumor-bearing mice can diminish the number of IL-17-producing cells while increasing the number of Treg cells¹²³. Correspondingly, Il2^−/− mice exhibit a decrease in the number of Treg cells⁸⁴ and an increase in the production of IL-17¹²¹, suggesting that IL-17 producing cells may contribute to the autoimmune disease that develops in Il2^−/− mice⁷. However, Il17^−/−Il2^−/− double knockout mice develop systemic autoimmune hemolytic anemia to the same extent as Il2^−/− mice, suggesting that IL-17 producing cells are not absolutely essential for this disease process and that other potentially redundant cytokines contribute as well¹²⁴. Although IL-2 inhibits Th17 cell differentiation, it can provide proliferative signals to human Th17 cells, as indicated by the IL-2-induced expansion of these Th17 cells from normal donors and from patients with uveitis or scleritis in vitro¹²⁵. Interestingly, the inhibitory effects of IL-2 on the Th17 cell lineage is prevented by IL-1, suggesting that the local cytokine profile controls the IL-17⁺ T cell pool¹²⁶.

The role of IL-21 in the differentiation of Th17 cells is controversial. In vitro experiments have shown that IL-21 is critical for upregulating IL-23R expression by Th17 cells¹²⁰. Although IL-23, which is produced by APCs, is an important factor in the differentiation and expansion of Th17 cells and therefore in inflammatory diseases, IL-23R is not expressed by naive CD4⁺ T cells. IL-21 therefore, promotes the expansion of Th17 cell populations by increasing their responsiveness to IL-23. Although Th17 cell differentiation is reduced in the absence of IL-21 signaling in vitro^118–120, the role of IL-21 in Th17 cell development in vivo and in Th17 cell-mediated autoimmune disease is less clear. Specifically, Th17 cell development in the lamina propria of the small intestine can occur in the absence of IL-21-induced signaling¹²⁷. Moreover, although one study reported that the development of experimental autoimmune encephalomyelitis (EAE) was significantly reduced in IL-21-deficient mice¹¹⁹, two other studies found no difference in the development of EAE in either IL-21- or IL-21R-deficient mice^128,129. Thus, although IL-21 can promote the differentiation of Th17 cells, its actions can apparently be subserved by other cytokines, as well, at least in certain circumstances.

T_FH cell differentiation

T_FH cells are a distinct subset of CD4⁺ T cells that provide help to B cells within germinal centers during the generation of T cell dependent antibody responses. T_FH cells are characterized by the expression of high levels of CXC-chemokine receptor 5 (CXCR5) and the co-stimulatory molecules ICOS and CD40L. T_FH cells produce high levels of IL-21¹³⁰, which can act on B cells in germinal centers and as an autocrine factor for T_FH cells. Il21^−/− mice exhibit defective germinal center formation as well as reduced levels of T_FH cells¹³¹. Unlike Th17 cells, which can also produce high levels of IL-21², T_FH cells develop independently of RORγt and do not produce IL-17¹³². Although the differentiation of T_FH cells during a normal T cell-dependent antibody response requires IL-21 production, the excessive differentiation of T_FH cells that accompanies systemic autoimmunity in sanroque mice is independent of IL-21, suggesting that there are alternative mechanisms for the maintenance and/or expansion of T_FH cells within germinal centers in some systemic autoimmune diseases¹³³.

CD8⁺ T cell differentiation

CD8⁺ T cells also undergo differentiation into polarized Tc1, Tc2, and Tc17 cell populations, which parallel the CD4⁺ Th1, Th2, and Th17 cell populations. One distinction is that naive CD8⁺ T cells produce only minimal levels of IL-2 and no IL-21, so that the source of these cytokines during an immune response must be from either activated CD4⁺ T cells or other cells, such as NKT cells (which can produce IL-21¹³⁴). In addition to a role for γ_c cytokines in the expansion of CD8⁺ T cell populations, IL-2 and IL-21 have distinct effects on CD8⁺ T cell differentiation when they are present during TCR priming. The presence of IL-21 during priming leads to the generation of CD28^hiCD8⁺ T cells that can produce IL-2, thus potentially overcoming the requirement for IL-2 from helper CD4⁺ T cells¹³⁵. Moreover, whereas priming of tumor-specific CD8⁺ T cells in vitro in the presence of IL-2 can potently promote their proliferation and enhance their cytolytic activity, priming in the presence of IL-21 was shown to inhibit these processes¹³⁶. However, when these two populations of cells primed under different conditions were transferred into tumor-bearing mice, the IL-21-primed CD8⁺ T cells displayed superior anti-tumor immunity and greater secondary clonal expansion and persistence than did the IL-2-primed CD8⁺ T cells. These differences that persisted in vivo in the absence of further cytokine stimulation were associated with distinctive and persistent gene expression profiles in either IL-2- or IL-21-primed CD8⁺ T cells¹³⁶, suggesting the possibility of these cytokines induced epigenetic changes at the time of priming. Thus, distinct γ_c cytokines exhibit different factors for CD8⁺ T cell differentiation, with particularly dramatic diversity for IL-2 and IL-21 in priming for anti-tumor effects.

Therapeutic implications

As is evident from the information presented above, γ_c family cytokines and TSLP have vital roles in regulating numerous immune cell activities, which have been harnessed to modulate immune responses for therapeutic purposes (Table 3). IL-2 is already used in the clinic to expand and maintain CD4⁺ T cell numbers in patients with HIV infection^137,138 and as an anti-cancer agent, and this is showing efficacy in treating some patients with melanoma and renal cell carcinoma¹³⁹. The related cytokine IL-15, the receptor of which consists of IL-2Rβ, γ_c and IL-15Rα (FIG. 1), holds promise as an adjuvant for vaccines¹³⁹. IL-15 preferentially induces the proliferation of CD8⁺ T cells rather than Treg cells and therefore, is not expected to induce enhanced tolerance in a similar fashion to IL-2 and has stronger effects on NK cell and CTL activity¹³⁹. IL-7 and TSLP are other potential agents that might be using for expanding the number of T cells in individuals with inherited or acquired immunodeficiency (Table 3). Indeed, the treatment of SIV-infected primates with IL-7 was shown to increase the number of circulating naïve and memory T cells¹⁴⁰ and similarly, the administration of IL-7 to humans induces a selective increase in the number of CD4⁺ and CD8⁺ T cells but does not affect Treg cell numbers^32,141.

Table 3. Effect of diminished versus augmented signaling by γ_c family cytokines and TSLP.

Aberrant regulation of γ_c cytokine signals associated with the development of immunodeficiency, autoimmunity, allergic diseases, inflammation, and cancer. Different strategies of manipulating these signals might be used in immunotherapy.

	Disruption of signaling (knock out or blocking/neutralization of receptors or cytokines)		Enhancing of signaling (overexpression or administration of cytokines)
	Positive effect	Negative effect	Positive effect	Negative effect
IL-2	Immunosupression in organ allografts and leukemia/lymphomas (daclizumab)*	Lymphoproliferative disorders and autoimmunity associated with loss of Treg cells	Anti-cancer and immunodeficiency (HIV) treatment, increased NK cells, and NK and LAK activity	Capillary leak syndrome
IL-4	Diminish asthma symptoms, resistance to leishmania	Defect in Ig class switch, no IgE production, failure to protection against worms	None	Allergy, atopic dermatits, airway inflammation, pro- fibrotic agent
IL-7	None	Severe combined immunodeficiency disease (SCID), defect in T cell homeostasis	Immunodeficiency treatment	Lymphomas, dermatitis, chronic colitis
IL-9	Diminish asthma symptoms, impaired goblet cell hyperplasia	None	None	Airway inflammation
IL-15	None	Diminish innate immunity associated with loss of NK cells, defective memory CD8+ T cell homeostasis	Adjuvant with viral vaccine, anti-cancer and immunodeficiency treatment	Lymphomas
IL-21	Prevent autoimmune diseases (EAE, SLE, diabetes)	Defect in B cell maturation, with reduced IgG1 and IgG3 production, pan- hypogammaglobulinemia, defect in Th17 differentiation	Anti-cancer agent	Aberrant CD8+ T cell homeostasis, autoimmunity
TSLP	Prevent development of allergic lung inflammation	Possible relationship to Crohn’s disease	Increase lymphoid cellularity	Atopic dermatitis, airway inflammation, inflammatory arthritis

^*Daclizumab (Zenapax, anti-Tac) is a humanized anti-IL-2Rα blocking antibody that prevents IL-2 binding to the high affinity IL-2 receptor complex that is expressed on leukemia/lymphomas cells and allograft activated CTLs¹⁴⁷.

IL-21 also may have substantial clinical potential (Table 3); its potent anti-tumor effects have been described in animal models with large established tumors, and it is now in Phase II clinical trials for the treatment of humans with cancer^2,142. By contrast, blocking IL-21 may prove valuable in treating autoimmune diseases. In this regard, diabetes does not develop in the non-obese diabetic (NOD) mouse model of type 1 diabetes when the animals are crossed to the Il21r^−/− background²¹, and similarly, manifestations of systemic lupus erythematosus no longer develops when BXSB-Yaa mice are crossed to the Il21r^−/− background²³. These studies underscore the potential role of IL-21 in autoimmunity and suggest that interfering with the action of IL-21 might have therapeutic potential for several autoimmune disorders. Finally, the IL-7-related cytokine TSLP seems to have a role in the development of atopic dermatitis and asthma^25,76 and thus perhaps other allergic diseases as well. Blocking of TSLP with a soluble TSLPR-specific antibody has already been shown to protect against the development of pulmonary allergic inflammation in a mouse model^143–145. These studies collectively underscore a range of therapeutic roles for γ_c cytokines and TSLP.

Concluding remarks and future directions

The γ_c family cytokines have central roles in the regulation of a range of immunological processes. The sharing of γ_c could represent a mechanism for inducing overlapping actions but it also could be a basis for the ability of this family of cytokines to compete with each other for the recruitment of γ_c. Additionally, these cytokines can affect signaling by other γ_c cytokines changing expression of their receptors, creating a system of intricate cross-regulation (for example, IL-2 increases the expression of its own receptor and IL-4Rα but represses the expression of IL-7Rα). The actions of these cytokines have clear clinical relevance, and increasing or decreasing their effects have implications for the treatment of cancer, autoimmunity, allergy and immunodeficiency. Future efforts will be directed not only towards further elucidation of the basic biology of these cytokines, including aspects of gene regulation and signaling, but also towards achieving therapeutic benefits in a range of pathological states.