Abul K. Abbas Department of Pathology University of California San Francisco M-590, 505 Parnassus Avenue San Francisco, CA 94143 USA Tel.: +1 415 514 0681 Fax: +1 415 502 4563 e-mail: firstname.lastname@example.org
Summary: Interleukin-2 (IL-2) has multiple, sometimes opposing, functions during an inflammatory response. It is a potent inducer of T-cell proliferation and T-helper 1 (Th1) and Th2 effector T-cell differentiation and provides T cells with a long-lasting competitive advantage resulting in the optimal survival and function of memory cells. In a regulatory role, IL-2 is important for the development, survival, and function of regulatory T cells, it enhances Fas-mediated activation-induced cell death, and it inhibits the development of inflammatory Th17 cells. Thus, in its dual and contrasting functions, IL-2 contributes to both the induction and the termination of inflammatory immune responses.
Our view of interleukin-2 (IL-2)'s function and its multi-faceted effects on the activation and regulation of immune responses has changed over time. Initially, IL-2 was seen as the canonical T-cell growth factor, inducing clonal expansion of T cells following antigen stimulation. It acts primarily as an autocrine growth factor, but can also act in a paracrine fashion on nearby cells. In addition to its effects on CD4+ and CD8+ T cells, IL-2 also stimulates natural killer (NK) cells to proliferate and induces cytolytic activity when present at high levels and stimulates B cells to divide and produce antibody. The phenotype of IL-2- and CD25-deficient mice has led to an appreciation of the importance of IL-2 in controlling immune responses. It is thought that IL-2 acts primarily through the development and maintenance of regulatory T cells (Tregs) but also perhaps by suppression of T-helper 17 (Th17) effector cell differentiation and promotion of Fas-mediated cell death. Thus, IL-2 is critical to both the induction and the resolution of inflammatory immune responses.
IL-2, a 15 kDa α-helical cytokine, is produced almost exclusively by activated T cells and promotes proliferation of lymphocytes, macrophages, and NK cells (1, 2). IL-2 is important for the differentiation of CD4+ T cells into Th1 and Th2 effector subsets, while inhibiting Th17 differentiation (3–5). Furthermore, we and others have shown that IL-2 is required for the development of memory T cells (6, 7). The IL-2 receptor (IL-2R) consists of three membrane-bound subunits: α (CD25), β (CD122), and common γ (γc) (CD132) chains. In T cells, IL-2 binding to the IL-2R activates the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway, as well as mitogen-activated protein kinase and phosphoinositide 3-kinase signaling, resulting in the transcription of proinflammatory cytokine, survival, and cell cycle genes. Through these pathways, IL-2 upregulates expression of CD25 and IL-2Rβ, modulates genes involved in cell cycle regulation, and promotes T-cell survival and differentiation into effector and memory cells (6, 8–10). Based on these early data, IL-2 was thought to be a lymphocyte-activating and immune-stimulatory factor. The surprising finding that absence of IL-2 or CD25 results in inflammatory autoimmune disease, rather than immunodeficiency, led to the initial suggestion that lymphoproliferation occurs in these mice because IL-2 is required for activation-induced cell death (11, 12). However, subsequent studies have shown that disease is primarily the result of decreased thymic and peripheral Tregs and a failure to control inflammatory responses (13).
IL-2 in the generation of effector and memory T cells
Requirements for IL-2 in CD4+ T-cell responses
Despite the current focus on the regulatory role of IL-2 in autoimmune disease and the assumption that this may be its physiologically predominant function, several recent studies have furthered our understanding of the immunostimulatory actions of IL-2. The picture that is emerging from these studies is that IL-2 does not significantly contribute to the initial cycling of antigen-stimulated T cells but is necessary for the survival of activated cells and the successful generation of effector and memory responses (Fig. 1). Experiments using in vitro-stimulated T-cell receptor (TCR)-transgenic T cells demonstrated that in the absence of IL-2, signals generated by TCR stimulation were sufficient to initiate cell cycling and upregulation of classic activation markers such as CD25 and CD44 (12, 14). Early in the response, IL-2-deficient ovalbumin-specific DO11 CD4+ T cells undergo a similar number of cell divisions as wildtype cells after antigen challenge in vitro and in vivo (6, 15, 16). During the second phase of the primary immune response, activated T cells differentiate into cytokine-producing effector cells. There is ample evidence that the numbers of fully differentiated Th1 and Th2 cells are diminished when T cells are unable to receive IL-2 signals during priming (6, 12, 14, 17). However, when T-cell responses are initiated in an environment that is completely devoid of IL-2, and as a consequence not controlled by Tregs, the requirement for IL-2 in effector cell differentiation is reduced. IL-2- and CD25-deficient animals mount strong Th1- and Th2-dependent immune responses that contribute to the development of autoimmune disease (18). These data support the intriguing possibility that IL-2 is only required for proper effector cell differentiation in situations where regulation needs to be overcome. Thus, the role of IL-2 in the development of a primary immune response may be limited, leading to the following question: what is the purpose of early IL-2 production by antigen-activated T cells? Work by Long et al. (19) hints that after viral infection the first targets of IL-2 are Tregs, not the responding effector population. Such a paracrine action of IL-2 on Tregs could represent a mechanism by which Tregs negatively regulate anti-viral responses (20, 21).
In addition to its role in Th1 and Th2 generation, current data suggest that IL-2 may be particularly important for programming CD4+ and CD8+ T-cell memory responses. We initially showed that in vitro-primed IL-2-deficient CD4+ T cells did not develop into long-lived memory cells after adoptive transfer in vivo (6). However, addition of recombinant IL-2 during the 4-day period of priming restored the subsequent development of memory cells, demonstrating that transient IL-2 signals during priming had long-lasting effects on memory responses. For CD8+ cells, Williams et al. (7) showed that IL-2Rα-deficient cells expanded and produced interferon γ (IFNγ) similar to wildtype cells during the primary response to lymphocytic choriomeningitis virus infection but failed to mount an effective memory response. Delivery of IL-2 signals to IL-2Rα-deficient T cells during the primary response, using IL-2/anti-IL-2 antibody complexes (22), enabled these cells to proliferate and produce IFNγ during a secondary response. However, restoring IL-2 signals only during the secondary response failed to reverse the defect in the memory response. Together, these data suggest that IL-2 imprints a program for memory cell differentiation during the primary response of CD4+ and CD8+ T cells.
Mechanism of action of IL-2 in memory T-cell generation
The obvious next goal in unraveling the role of IL-2 in programming the memory response is to identify the IL-2 target genes executing this ‘memory’ program. One candidate gene is IL-7Rα. IL-7 is a constitutively produced cytokine that shares one receptor component, γc, with IL-2 while achieving specificity through usage of IL-7Rα. IL-7 is important for T-cell thymic development and for the survival of naive cells. However, the highest expression of IL-7Rα is observed in CD4+ and CD8+ memory cells (16, 23). Several studies using IL-7Rα-deficient T cells and IL-7-deficient hosts have now established that IL-7 is indispensable for the survival of both CD4+ and CD8+ memory cells (23–26). It has also become clear that upon activation, T cells lose IL-7Rα expression (16, 23, 27). Re-expression of IL-7Rα on primed CD8+ T cells correlates with preferential entry into the memory cell lineage (27). The kinetics of IL-7Rα downregulation and re-expression inversely coincides with the acquisition of CD25 expression after priming and loss of this receptor at the end of the primary response (16). This observation suggested that IL-2 signals were responsible for inducing IL-7Rα re-expression leading to memory cell differentiation. We found that IL-2- and CD25-deficient T cells primed in vitro and in vivo indeed failed to re-express the IL-7Rα at the end of the response and this correlated with poor memory formation (16) (Fig. 1). Administration of exogenous IL-2 to primed IL-2-deficient T cells restored IL-7Rα re-expression and memory formation. Forced expression of IL-7Rα in IL-2-deficient cells by retroviral transduction was sufficient to regain the capacity to become long-lived memory cells (16). Thus, we propose that IL-7Rα is one IL-2-regulated gene participating in memory programming.
Systemic inflammatory disease caused by IL-2/IL-2R deficiency
Phenotype of disease
Although perhaps no longer surprising, it was an unexpected discovery that elimination of IL-2 or IL-2R leads to systemic autoimmunity (28–31). IL-2-, CD25-, and IL-2Rβ-deficient mice develop a multi-organ inflammatory disorder, with elevated Th1-dependent serum immunoglobulin G (IgG), antibodies against red blood cells (RBCs) and DNA, and activated CD4+ T cells. On the BALB/c background, mice rapidly die by 5 weeks of age due to complications from autoimmune hemolytic anemia (AIHA) (32). The further development of other autoimmune manifestations appears to be prevented by this early death. In contrast, on the C57BL/6 background, approximately 50% of mice die within 9 weeks from AIHA; the remainder develop colitis and AIHA and die between 12 and 25 weeks of age (28). The primary defect in IL-2- and CD25-deficient mice is thought to be a Treg deficiency that leads to a breakdown of self-tolerance and homeostasis, promoting uncontrolled activation and proliferation of CD4+ T cells. Although no analogous human example of complete IL-2-deficiency exists, in many human diseases [including systemic lupus erythematosus, multiple sclerosis, Crohn's disease, and diabetes (33–37)], autoimmunity is attributed to decreased Treg numbers or suppressive function. Genome-wide association studies and studies defining single nucleotide polymorphisms have shown that polymorphisms in CD25 are associated with multiple sclerosis, Graves' disease, and autoimmune thyroid disease (38–40).
Role of IL-2 in the generation, maintenance, and function of Tregs
Tregs comprise 5–15% of the peripheral CD4+ T-cell population and are characterized by expression of CD25 and the transcription factor forkhead box protein 3 (Foxp3). Tregs are generated in the thymus and are thought to be specific for self-antigens. They can also be generated in the periphery in response to foreign or self-antigens. Functionally, Tregs are known to suppress effector T-cell proliferation and cytokine production and perhaps the activities of dendritic cells (41).
The generation and maintenance of Tregs are not well understood, nor is the mechanism by which they suppress self-reactive lymphocytes. What is known suggests that cytokines alter Treg activities. For example, transforming growth factor β (TGFβ) induces the differentiation of Tregs, and Treg suppressor activities are inhibited when TGFβ is blocked (42, 43). The link between IL-2 and Treg development and function is also unclear. It is known that IL-2 is not necessary to activate the Treg-specific transcription factor Foxp3 in the thymus (44), but likely does alter Foxp3 levels in peripheral Tregs through STAT5-binding sites in the Foxp3 promoter (45). Foxp3 is required, however, for the generation of Tregs (46). Debate regarding the requirement for IL-2 in Treg development is well underway and still in flux.
There is conflicting evidence for a requirement for IL-2 in thymic generation of CD25+ Tregs (13, 44, 45, 47). More recently, it has been shown that IL-2 is critical for the generation and survival of peripheral Tregs (48). Treatment of mice with anti-CD25 or anti-IL-2 antibodies leads to depletion of Tregs and development of systemic autoimmunity (49, 50). Autoimmunity in IL-2-deficient mice can be prevented by transfer of IL-2-treated IL-2-deficient splenocytes, and IL-2Rβ-knockout mice can be protected by adoptive transfer of normal CD4+CD25+ T cells (51, 52).
The initial reports on IL-2- and IL-2R-deficient mice interpreted the elimination of IL-2 signaling as preventing Treg development (13). However, these studies had only CD25 as an available marker for Treg differentiation. It later became clear that IL-2 upregulates expression of CD25 (53), and thus in the absence of IL-2, CD25 expression on T cells is low. Utilizing Foxp3 to identify Tregs, it is now apparent that IL-2- and CD25-deficient mice have near-normal percentages and numbers of thymic Tregs, and the percentage of Tregs in the periphery is reduced by only about 50%. However, the total number of peripheral Foxp3+ Tregs in IL-2- and CD25-deficient mice is similar to that found in wildtype littermates (Fig. 2). This disparity between Treg number and percentage is likely due to the massive lymphoproliferation in these mice. Either peripheral maintenance of Tregs is impaired in the absence of IL-2 signaling, or Tregs cannot keep pace with the rapid expansion of effector T cells during this lymphoproliferative disorder. This could be due to a defect in Treg survival or function in the absence of IL-2 signaling or due to an inherent advantage provided to effector T cells in this setting. Using a soluble systemic antigen system, we have demonstrated that peripheral generation of Tregs does not occur in the absence of IL-2 (48). Therefore, we can speculate that the remaining peripheral Tregs in the IL-2- and CD25-deficient mice are recent thymic emigrants to the periphery due to continuous generation of thymic Tregs.
Tregs constitutively express CD28, and costimulation through CD28 is required for the induction of Foxp3 (54). Elimination of CD28 costimulation decreases the number of thymic and peripheral Tregs, as is found in CD28- and CD80/CD86-deficient mice (55). CD28 signals within Tregs upregulate CD25 expression, and it has been suggested that this occurs through direct effects on IL-2 production (56). CD28 is important for IL-2 production by activated CD4+ T cells (57), and IL-2 production is impaired when CD28 costimulation is missing (56). Thus, the decrease in Treg numbers in CD28-deficient mice was initially attributed to a diminution of IL-2 production. However, although IL-2-deficient mice have a modest decrease in thymic Treg numbers, CD28-deficient mice have a 50% reduction in thymic Tregs (58). Thus, the overall weakening of thymic Treg generation in CD28-deficient mice has some IL-2-independent cause(s). In fact, our data suggest that CD28 signals have a larger impact than IL-2 signals on the generation of thymic Tregs (Fig. 2). Tai et al. (54) also concluded, when evaluating the role of CD28 and IL-2 in the development of thymic Tregs, that CD28 is more critical. Our evaluation of IL-2/CD28-deficient mice indicates that IL-2 and CD28 have independent and additive effects on thymic Treg generation. The additive effect of CD28 and IL-2 absence is also observed in the number of peripheral Tregs. Less than 1% of peripheral CD4+ T cells in IL-2/CD28-deficient mice are Foxp3+ Tregs (58). There are 10 times as many peripheral Tregs in the lymph node (LN) of IL-2- than IL-2/CD28-deficient mice, while the absolute number of Tregs in CD28-deficient LN is only 3.5 times that in the IL-2/CD28-deficient LN (Fig. 2). Our data from IL-2/CD28-deficient mice suggest that there are both IL-2-dependent and CD28-dependent/IL-2-independent requirements for the maintenance of peripheral Tregs. It is likely that cell-intrinsic IL-2 and CD28 signals in Tregs and IL-2-dependent signals derived from effector T cells are both important in the peripheral generation or maintenance of Tregs. Because thymic development of Tregs is fairly normal in the absence of IL-2, what, if any, defects are present in the Tregs of IL-2-deficient mice? IL-2 signals may be more important for the maintenance of Foxp3 expression than for the induction of Foxp3, because Foxp3 expression is increased in wildtype peripheral Tregs that are stimulated with IL-2 and Foxp3 levels decline if IL-2 is blocked with antibody (44). In contrast, Foxp3 levels are unchanged in the peripheral Tregs of IL-2-deficient mice. There are also conflicting data regarding the impact of IL-2 on the suppressive function of Tregs. In some studies, pretreatment of Tregs with IL-2 enhances suppression in co-culture assays (59, 60), while other data indicate that the suppressor activity of IL-2-deficient Tregs is unimpaired (44). Administering IL-2 prevents development of diabetes (61), and blocking IL-2 triggers autoimmune gastritis (49). Such treatments rapidly change the number and phenotype of Tregs, and IL-2 stimulation boosts metabolic pathways and TGFβ1 and IL-10 production in Tregs (44, 59). Some of these apparently opposing conclusions regarding the role of IL-2 in the function of Tregs may be reconciled, if, in the absence of IL-2, other cytokines partially restore Treg development or functional activities. To fully elucidate the role of IL-2 in tolerance, it will be essential to determine the separate contributions of IL-2 to Treg development, survival, proliferation, and suppressive functions. It will also be important to assess the consequences of IL-2's effects on the level and stability of Foxp3 expression and to identify other cytokines that may support Treg differentiation and maintenance.
Role of other growth factors in the absence of IL-2 signaling
Because IL-2 is considered the canonical growth factor necessary for effector T-cell differentiation, we wondered what factors promote autoimmunity and lymphoproliferation in the absence of IL-2. In other words, which factors within the cytokine milieu promote the expansion and activation of self-reactive lymphocytes in the absence of IL-2 and peripheral Tregs, or when Treg controls are overcome (due to decreased numbers or function)? We reasoned that other γc chain-dependent cytokines may substitute for IL-2 when IL-2 is limiting or absent. The growth-promoting activities of IL-7 on immature and mature lymphocytes made this cytokine a likely candidate. It is a major cytokine involved in homeostasis, and transgenic overexpression of IL-7 results in severe lymphoproliferative and autoimmune disease (62, 63). We have shown that IL-7Rα expression is elevated in a portion of CD4+ T cells from IL-2-deficient mice, suggesting that CD4+ T cells may be more responsive to IL-7 in the absence of IL-2 (58). This increased expression of IL-7Rα on IL-2-deficient CD4+ cells may provide a survival or a proliferative advantage as has been suggested for memory CD4+ T cells (16). When IL-2 is absent, IL-7:IL-7R signals can induce spontaneous activation or survival of self-reactive lymphocytes. Elimination of IL-7 signaling delays the onset of autoimmunity in IL-2-deficient mice (58). Of note, when IL-7 signaling was blocked in IL-2-deficient mice, anti-RBC antibody levels were dramatically reduced, while total serum IgG1 and IgG2a levels remained elevated. These data suggest that elimination of IL-7:IL-7R signaling delays or prevents the self-reactive response preferentially. Work using CD8+ T cells supports the idea that IL-7 promotes the expansion of self-reactive lymphocytes (64, 65). IL-7 compensates, at least in part, for the absence of IL-2 in the activation of self-reactive lymphocytes (58). We suggest that IL-7 signaling compensates for the loss of IL-2 during T-cell expansion by promoting survival or activation of T cells. It is conceivable that the significance of IL-7 in providing a growth or a survival advantage to activated T cells might only be relevant in situations of low IL-2 or in the absence of functional Tregs.
Effector cytokines in IL-2-deficient mice
Dysregulation of helper cell differentiation and subsequent imbalance in Th1/Th2/Th17 populations factor into the onset of several autoimmune diseases (66–70). Previous work concluded that systemic inflammatory disease is caused by uncontrolled expansion of Th1 effector cells producing the proinflammatory cytokines IFNγ and TNF. Many autoantibody-mediated diseases are driven by helper T-cell responses, especially those of the Th1 subset. However, the recent identification of the Th17 effector subset has prompted the re-evaluation of the cause of disease in inflammatory disease models. IL-17 is now believed to be the major mediator of tissue inflammation in several autoimmune inflammatory diseases (reviewed in 71). Dysregulated IL-17 plays a role in the induction of collagen-induced arthritis (72), experimental autoimmune encephalitis (73), and trinitrobenzene sulfonic acid-induced colitis (74). Also, IL-17 expression is elevated in the colon and serum of patients with active Crohn's disease and ulcerative colitis (75). In humans, there is evidence of a role for Th1 cytokines in lupus nephritis and thyroiditis, Th17 cytokines in psoriasis, rheumatoid arthritis, and multiple sclerosis, and Th2 cytokines in scleroderma. Although the inflammatory environment and cytokine burst that occur during autoimmunity may indirectly contribute to the expansion of IL-17A-producing cells in mice lacking IL-2, it is now clear that IL-2 acts directly to inhibit Th17 differentiation via STAT5 signaling (4). IL-2 inhibits Th17 development but enhances Treg differentiation both in vitro and in vivo (5). Therefore, IL-2 acts to block inflammation by preventing the generation and therefore the expansion of the highly inflammatory Th17 cells, while increasing the number of suppressive Tregs (Fig. 3). However, it cannot be excluded that in the absence of IL-17A, Th17, or Th17-like cells producing IL-17F, IL-21, or IL-22 may still be present. We hypothesize that different cytokines play distinct roles in the multiple manifestations of autoimmunity. T cells from BALB/c IL-2-deficient mice produce large amounts of IFNγ and slightly elevated levels of IL-17 (76). It is clear that IFNγ is responsible for the early autoimmune lethality in these mice, as elimination of IFNγ significantly delayed the development of autoimmunity. In the absence of IFNγ, IL-17 is further elevated and intestinal inflammation becomes the major pathologic manifestation. It has been demonstrated that IFNγ inhibits Th17 differentiation in vitro (77). The increase in IL-17-producing CD4+ T cells in the IL-2/IFNγ-deficient mice supports this notion. In contrast, IL-17 is not required for the autoantibody-mediated autoimmunity that occurs in the IL-2-deficient mice (76). We suggest that in the absence of IL-2 and Treg control, Th1 effectors drive the early antibody-mediated autoimmunity and Th17 cells mobilize tissue-mediated inflammation. This idea is further supported by studies showing that Th17 cells are potent inducers of tissue inflammation.
Role of costimulation in inflammation and autoimmunity
We have also examined the importance of costimulatory pathways in various aspects of the autoimmune disorder in IL-2-deficient mice. To this end, we crossed IL-2-deficient mice with mice lacking two molecules involved in the activation of lymphocytes: CD28 and CD40L. Elimination of CD28 reduced the activation of autoreactive T cells and lymphoproliferation as well as production of autoantibodies, whereas elimination of CD40L reduced autoantibody production without affecting T-cell expansion and accumulation (58). Elimination of CD28 or CD40L from IL-2-deficient mice drastically increased the survival of the mice. Thus, the CD28 T-cell costimulatory pathway and the CD40L-dependent B-cell activation pathway are each necessary for development of the autoimmunity, but CD28 appears to have a larger impact on the early inflammatory disease. In contrast, Boone et al. (78) demonstrated that CD28 is not necessary for the ulcerative colitis-like disease that develops in the C57BL/6 IL-2-deficient mice. Similar to the elimination of CD28, cytotoxic T-lymphocyte antigen-4 transgenic overexpression in T cells prevents the early autoimmunity but not the later development of ulcerative colitis in C57BL/6 IL-2-deficient mice (79). Together, the studies from our laboratory and others have demonstrated that the early autoimmunity and later intestinal inflammation that develop in the absence of IL-2 evolve by divergent mechanisms and that these disease manifestations can be separated.
Conclusions: mechanisms of inflammatory disease in the absence of IL-2
These studies reveal some of the pathways leading to inflammatory autoimmune disease. We will continue to build from this empirical foundation to elucidate novel triggers for inflammation and autoimmunity that refine our understanding of the mechanisms of these diseases. In our evaluation of the role of IL-2 in inflammation and autoimmunity, we have demonstrated the importance of several effector cytokines, growth factors, and costimulatory receptors in the development of inflammation and autoimmunity. Blocking many of these profoundly inhibits the generation of antigen-specific lymphocyte responses and the onset of disease. In addition, we have shown that autoreactive cells do indeed utilize many of the same signals that conventional cells require for activation and survival, including CD28 for activation, CD40L for B-cell help, IL-7 likely for survival, and IFNγ for effector functions. Elimination of any of these pathways delays the onset of autoimmune disease presumably by reducing the survival or the activation state of the self-reactive lymphocytes.
Under normal conditions, self-reactive lymphocytes are suppressed by Tregs, resulting in unresponsiveness to self-antigens (80–83). Disruption of these control mechanisms results in the survival and pathogenic activation of self-reactive lymphocytes. It remains unclear how self-reactive lymphocytes may be spontaneously activated in the absence of overt infection or other stimuli, leading to inflammation and autoimmunity. The role of IL-2 in the generation or the survival of Tregs and the ability of IL-2 to inhibit Th17 responses provide simple explanations for the Treg deficiency and Th17 expansion in IL-2-deficient mice. It is more puzzling that Th1 (and, in some studies, Th2) responses are increased in IL-2-deficient mice, given that IL-2 is known to be necessary for the generation of Th1 and Th2 cells in normal cell populations. We postulate that in the absence of Tregs, controls on T-cell activation are reduced, so that the normal requirements for generation of effector and memory cells are no longer critical. We postulate that the triggers for spontaneous activation of self-reactive lymphocytes are specific costimulatory and cytokine signals. Our recent work suggests that both B- and T-cell activation (through CD28 and CD40:CD40L signaling) and cytokine signaling (IL-7:IL-7Rα, IFNγ, and IL-17) are important in the development of inflammation and autoimmune disease in the absence of IL-2 (58, 76). We suggest that the signals that normally maintain homeostasis instead promote autoimmunity and inflammation in the absence of IL-2, mainly because the absence of Tregs reduces the threshold for activation.
Dual roles of IL-2 in a systemic inflammatory disease and its control
A well-balanced regulation of effector and Treg numbers is essential during an immune response to eradicate infection and prevent long-term destructive inflammation and to develop tolerance rather than autoimmunity to self-antigens. To define the mechanisms of tolerance versus chronic inflammation, we have utilized the DO11 TCR-transgenic mouse in combination with the soluble ovalbumin transgenic (sOVA-Tg) mouse. The sOVA-Tg mouse expresses chicken ovalbumin as a soluble protein under control of the metallothionine promoter. Transfer of DO11 CD4+ T cells into sOVA-Tg/recombination-activating gene (Rag)-deficient mice results in tissue inflammation and a wasting disease that resembles graft versus host disease (84). We have shown that IL-17 is responsible for the tissue inflammation in this model, while Th1 responses are protective. Following transfer of naive CD4+ T cells, there is a rapid Th1 response, followed by a later, Th17-dominant response. Recovery from disease is associated with peripheral Foxp3+ Treg generation. In the absence of IL-2, acute disease is reduced, and a chronic, inflammatory disorder develops instead. A reduced percentage of IFNγ-producing cells in the early response (although the absolute number is the same as in wildtype mice) likely explains the delay in disease, but infiltration by IL-17-producing cells into the skin is elevated during late disease. We attribute the late chronic inflammatory disease in the absence of IL-2 to the lack of peripheral Treg generation. Thus, IL-2 production by T cells activates and expands peripheral Tregs, which in turn may suppress the antigen-specific effector T-cell response. Together with accumulated data from the IL-2- and CD25-deficient mice, these results indicate that IL-2 signals are important for the generation of Tregs in the periphery to protect from autoimmunity and uncontrolled effector responses.
Conclusions: biological and clinical implications
Inflammation is a complex immune response to an array of cytokines, chemokines, and signaling pathways. Unraveling the multitude of responses contributed by various immune cells will require both basic in vitro studies and the application of in vivo models. IL-2 is a good example of the step-wise clarification that occurs when combining in vitro and in vivo experiments to reveal the functions of a cytokine in tolerance, inflammation, and autoimmunity. The early in vitro studies demonstrated a critical role for IL-2 in growth and differentiation of T cells. Later work has added many additional functions for IL-2. The IL-2- and CD25-deficient mice showed that IL-2 is also important in the regulation of immune responses through suppression by Tregs and promotion of apoptosis through Fas signaling. Future studies will continue to add layers to our knowledge about the function of IL-2 in immune responses. We are now left with numerous questions about the dominant role of IL-2 during an immune response. Are the effects of IL-2 on effector cell differentiation and proliferation or on the generation and maintenance of Tregs more critical to maintaining homeostasis? Does this depend on the stage of the immune response, the local concentration of IL-2, or the kinetics of IL-2 production? Among the many cell types that express IL-2R, is IL-2 signaling more critical in some cells than in others? We know that IL-2 signaling confers a long-term competitive advantage to T cells during activation but that strong IL-7 signals can also provide this ‘competitiveness.’ We expect that this might also be true for other cell types. Finally, and probably most important, can we control the amount, kinetics, or targets of IL-2 in such a way as to selectively target different effector and regulatory populations and better utilize it in therapy? High-dose IL-2 administration is currently being used to boost immunity in human immunodeficiency virus-infected patients (85) and to treat various cancers such as metastatic renal carcinoma (reviewed in 86). IL-2R blockade has shown effectiveness in reducing autoimmunity and transplant rejection (87, 88). However, high-dose IL-2 therapy has recently been shown to increase the frequency of Foxp3+ Tregs in patients with metastatic melanoma or renal cell carcinoma. Thus, there is a clear need to predict the effects of boosting or inhibiting IL-2 signaling during disease. It will be important to define the dominant effect of IL-2 therapy, whether it is on Th1 and Th2 effector T cells, Tregs, Fas-induced cell death, or Th17 inflammatory T cells. Combined in vitro and in vivo studies will continue to resolve our view of IL-2's multi-faceted effects on the activation and control of immune responses and lead to improved prediction of treatment outcome.