Microbial infection-induced expansion of effector T cells overcomes the suppressive effects of regulatory T cells via an IL-2 deprivation mechanism

Foxp3(+) regulatory T (Treg) cells are a critical cell population that suppresses T cell activation in response to microbial and viral pathogens. We identify a cell-intrinsic mechanism by which effector CD4(+) T cells overcome the suppressive effects of Treg cells in the context of three distinct infections: Toxoplasma gondii, Listeria monocytogenes, and vaccinia virus. The acute responses to the parasitic, bacterial, and viral pathogens resulted in a transient reduction in frequency and absolute number of Treg cells. The infection-induced partial loss of Treg cells was essential for the initiation of potent Th1 responses and host protection against the pathogens. The observed disappearance of Treg cells was a result of insufficiency in IL-2 caused by the expansion of pathogen-specific CD4(+) T cells with a limited capacity of IL-2 production. Exogenous IL-2 treatment during the parasitic, bacterial, and viral infections completely prevented the loss of Treg cells, but restoration of Treg cells resulted in a greatly enhanced susceptibility to the pathogens. These results demonstrate that the transient reduction in Treg cells induced by pathogens via IL-2 deprivation is essential for optimal T cell responses and host resistance to microbial and viral pathogens.     

IL-2, regulatory T cells, and tolerance

IL-2 is a potent T cell growth factor that for many years was assumed to amplify lymphocyte responses in vivo. Accordingly, IL-2 has been used clinically to enhance T cell immunity in patients with AIDS or cancer, and blocking Abs to the IL-2R are used to inhibit T cell responses against transplanted tissues. It was later shown in mice that, unexpectedly, disruption of the IL-2 pathway results in lymphoid hyperplasia and autoimmunity rather than immune deficiency, indicating that the major physiological function of IL-2 is to limit rather than enhance T cell responses. This apparent paradox has recently been resolved with the discovery that IL-2 is critical for the development and peripheral expansion of CD4(+)CD25(+) regulatory T cells, which promote self-tolerance by suppressing T cell responses in vivo. Our new understanding of IL-2 biology prompts a re-evaluation of how best to clinically manipulate this important immunoregulatory pathway.     

Involvement of IL-2 in homeostasis of regulatory T cells: the IL-2 cycle

A large body of evidence on the activity of regulatory T (Treg) cells was gathered during the last decade, and a similar number of reviews and opinion papers attempted to integrate the experimental findings. The abundant literature clearly delineates an exciting area of research but also underlines some major controversies. A linear cause-result interpretation of experimental maneuvers often ignores the fact that the activity of Treg cells is orchestrated with the effector T (Teff) cells within an intricate network of physiological immune homeostasis. Every modulation of the activity of the effector (cytotoxic) immune system revolves to affect the activity of regulatory (suppressive) cells through elaborate feedback loops of negative and positive regulation. The lack of IL-2 production by innate Treg cells makes this cytokine a prime coupler of the effector and suppressive mechanisms. Here we attempt to integrate evidence that delineates the involvement of IL-2 in primary and secondary feedback loops that regulate the activity of suppressive cells within the elaborate network of physiological immune homeostasis.     

IL-2 unresponsiveness in anergic CD4+ T cells is due to defective signaling through the common gamma-chain of the IL-2 receptor

Repeated administration of the superantigen staphylococcal enterotoxin A to mice transduces a state of anergy in the CD4+ T cell compartment, characterized by inhibition of IL-2 production and clonal expansion in vivo. In contrast to what has been reported on anergic T cell clones in vitro, culture of in vivo anergized CD4+ T cells in the presence of exogenous IL-2 did not overcome the block in responsiveness. In this study, we demonstrate that CD4+ T cells from mice anergized with staphylococcal enterotoxin A also exhibit a reduced proliferative capacity in response to IL-7 and IL-15, cytokines that share a common gamma-chain with the IL-2R. Flow-cytometric analysis revealed only modest changes in the expression of the different IL-2R chains. In a number of experiments, our results also provide evidence that excludes a major role of the IL-2R alpha-chain in this system. According to these results, the inability of anergic cells to respond to IL-2 is not mainly due to a down-regulation of the high affinity IL-2R, but to a perturbation in intracellular signaling. Our study confirmed that the activation and tyrosine phosphorylation of Janus-associated kinase 3 and STAT5 were considerably weaker after anergy induction. Moreover, anergic CD4+ T cells showed significantly reduced DNA-binding ability to STAT5-specific elements. Taken together, we suggest that the observed IL-2 unresponsiveness in anergic CD4+ T cells could be due to a defect in signaling through the common gamma-chain of the IL-2R.     

8-Mercaptoguanosine overcomes unresponsiveness of human neonatal B cells to polysaccharide antigens

We have shown previously that pneumococcal polysaccharides behave as human T cell-independent type 2 Ag. When cultured in vitro with type 4 pneumococcal polysaccharides (PS4), human neonatal B cells do not or only marginally respond. Limiting dilution analysis of neonatal B cells polyclonally activated by a combination of phorbol esters, calcium ionophore, and T cells and T cell factors, however, showed that Ag-reactive B cells are present in cord blood. The frequency of anti-PS4 reactive B cells in cord blood is comparable with that of adult peripheral blood. In order to obtain more insight into the activation requirements of these PS4-reactive neonatal B cells, 8-mercaptoguanosine (8MGuo) was added to in vitro cultures. Addition of 0.5 to 1.0 mM 8MGuo resulted in a 3- to 10-fold amplification of the anti-PS4 response. the effect of 8 MGuo was most prominent when added 3 days after initiation of the culture. Based on kinetic studies, we propose that in vitro activated cells are target for 8MGuo. These data further indicate that neonatal unresponsiveness to polysaccharide Ag is not due to the physical absence of Ag-reactive B cells but more likely to be the consequence of different activation requirements as compared with adult B cells.     

Cytokine expanded myeloid precursors function as regulatory antigen-presenting cells and promote tolerance through IL-10-producing regulatory T cells

The initiation of graft-vs-host disease (GVHD) after stem cell transplantation is dependent on direct Ag presentation by host APCs, whereas the effect of donor APC populations is unclear. We studied the role of indirect Ag presentation in allogenic T cell responses by adding populations of cytokine-expanded donor APC to hemopoietic grafts that would otherwise induce lethal GVHD. Progenipoietin-1 (a synthetic G-CSF/Flt-3 ligand molecule) and G-CSF expanded myeloid dendritic cells (DC), plasmacytoid DC, and a novel granulocyte-monocyte precursor population (GM) that differentiate into class II+,CD80/CD86+,CD40- APC during GVHD. Whereas addition of plasmacytoid and myeloid donor DC augmented GVHD, GM cells promoted transplant tolerance by MHC class II-restricted generation of IL-10-secreting, Ag-specific regulatory T cells. Importantly, although GM cells abrogated GVHD, graft-vs-leukemia effects were preserved. Thus, a population of cytokine-expanded GM precursors function as regulatory APCs, suggesting that G-CSF derivatives may have application in disorders characterized by a loss of self-tolerance.     

Distinct effects of IL-18 on the engraftment and function of human effector CD8 T cells and regulatory T cells

IL-18 has pleotropic effects on the activation of T cells during antigen presentation. We investigated the effects of human IL-18 on the engraftment and function of human T cell subsets in xenograft mouse models. IL-18 enhanced the engraftment of human CD8(+) effector T cells and promoted the development of xenogeneic graft versus host disease (GVHD). In marked contrast, IL-18 had reciprocal effects on the engraftment of CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) in the xenografted mice. Adoptive transfer experiments indicated that IL-18 prevented the suppressive effects of Tregs on the development of xenogeneic GVHD. The IL-18 results were robust as they were observed in two different mouse strains. In addition, the effects of IL-18 were systemic as IL-18 promoted engraftment and persistence of human effector T cells and decreased Tregs in peripheral blood, peritoneal cavity, spleen and liver. In vitro experiments indicated that the expression of the IL-18Ralpha was induced on both CD4 and CD8 effector T cells and Tregs, and that the duration of expression was less sustained on Tregs. These preclinical data suggest that human IL-18 may have use as an adjuvant for immune reconstitution after cytotoxic therapies, and to augment adoptive immunotherapy, donor leukocyte infusions, and vaccine strategies.     

Anti-CD3 antibody induces unresponsiveness to IL-2 in Th1 clones but not in Th2 clones

Culture of murine T cells with immobilized (platebound) anti-CD3 antibody results in autocrine growth factor secretion in both Th1 (IL-2 producing) and Th2 (IL-4 producing) cells. Using a panel of murine T cell clones, we demonstrate that the IL-2-induced proliferation of Th1 clones is dramatically inhibited by immobilized anti-CD3 antibody, whereas that of Th2 clones is not. This unresponsiveness of Th1 clones to IL-2 is not due to decreases in IL-2R expression. Supernatants from Th1 or Th2 cell cultures fail to alter the effects of anti-CD3 on the two types of clones, suggesting that unresponsiveness induced in Th1 clones or the lack thereof in Th2 clones is not mediated by a stable cytokine(s). Accessory cells enhance the proliferation of Th1 cells exposed to low concentrations of anti-CD3, but the unresponsiveness induced by high concentrations of anti-CD3 is not prevented by accessory cells. Finally, soluble anti-CD4 antibody prevents the induction of the unresponsive state even at high concentrations of anti-CD3. These experiments demonstrate that two subsets of cloned CD4+ T cells differ in their responses to anti-CD3, and that CD4 molecules may play a critical role in regulating the outcome of receptor-mediated stimulation.     

Natural but not inducible regulatory T cells require TNF-alpha signaling for in vivo function

TNF-α has a multifunctional role in autoimmune diseases as reflected in the variable responses of different human diseases to anti-TNF-α therapy. Recent studies have suggested that TNF-α modulates autoimmunity partially via effects on regulatory T cells (Tregs) and that these effects are mediated through the type II TNFR (TNFR2). We have investigated the requirement for TNFR2-expression on murine natural Tregs (nTregs) and induced Tregs (iTregs) in mediating suppression of colitis. Surprisingly, we find that TNFR2-expression is required for both spleen- and thymus-derived nTreg-mediated suppression, but is not required for iTreg-mediated suppression. Abnormal TNFR2(-/-) nTreg function was not associated with an in vivo decrease in accumulation, stability, or expression of markers known to be relevant in Treg function. Because iTregs are generated in the presence of TGF-β, we investigated whether activation in the presence of TGF-β could overcome the functional defect in TNFR2(-/-) nTregs. Although preactivation alone did not restore suppressive function of nTregs, preactivation in the presence of TGF-β did. These results identify potentially critical differences in activation requirements for nTregs versus iTregs. Furthermore, our findings are consistent with reports suggesting that nTregs are activated in sites of inflammation while iTregs are activated in lymph nodes. Finally, by demonstrating that nTregs require TNF-α for optimal function whereas iTregs do not, our results suggest that the enigma of variable responses of different human diseases to anti-TNF-α therapy may relate to whether nTregs or iTregs have the predominant regulatory role in a given disease.     

Recombinant murine IL-3 fails to stimulate T or B lymphopoiesis in vivo, but enhances immune responses to T cell-dependent antigens

We have explored the in vivo effect of IL-3 on the lymphopoiesis and humoral responses of mice bearing osmotic minipumps loaded with murine rIL-3 for 1 to 4 wk. A marked splenomegaly due to the accumulation of hemopoietic precursors was seen, but no increase was found in the lymphoid organs in the total number of cells belonging to the T or B lymphocyte lineage, i.e., of L3T4+ or Lyt-2+, or of allospecific cytotoxic T lymphocyte precursor for the T lineage, or of sIg+ or B220+ cells, or of B colony-forming cells for the B lineage; total activity of natural killer and lymphokine-activated killer cells was decreased. In contrast to the splenomegaly, a marked diminution in the number of thymocytes was observed, suggesting that rIL-3 in large amounts does suppress the T lymphopoiesis, perhaps as the result of the selective stimulation of early progenitor cells toward the hemopoietic pathway. rIL-3 perfusion during immunization increased the IgM and IgG responses to a T cell-dependent antigen, human IgG, and prevented tolerance induction by the deaggregated human IgG, although in the same conditions it did not modify the response to a T cell-independent antigen. Our results suggest that in vivo IL-3 does not act directly on lymphocytes or their precursors, but may potentiate the humoral immune response to T cell-dependent antigens, presumably by acting on accessory cells.     

Cutting edge: mechanisms of IL-2-dependent maintenance of functional regulatory T cells

IL-2 controls the survival of regulatory T cells (Tregs), but it is unclear whether IL-2 also directly affects Treg suppressive capacity in vivo. We have found that eliminating Bim-dependent apoptosis in IL-2- and CD25-deficient mice restored Treg numbers but failed to cure their lethal autoimmune disease, demonstrating that IL-2-dependent survival and suppressive activity can be uncoupled in Tregs. Treatment with IL-2-anti-IL-2-Ab complexes enhanced the numbers and suppressive capacity of IL-2-deprived Tregs with striking increases in CD25, CTLA-4, and CD39/CD73 expression. Although cytokine treatment induced these suppressive mechanisms in both IL-2(-/-) and IL-2(-/-)Bim(-/-) mice, it only reversed autoimmune disease in the latter. Our results suggest that successful IL-2 therapy of established autoimmune diseases will require a threshold quantity of Tregs present at the start of treatment and show that the suppressive capacity of Tregs critically depends on IL-2 even when Treg survival is independent of this cytokine.     

Preferential migration of T regulatory cells induced by IL-16

As a natural ligand for CD4, IL-16 has been shown to preferentially induce migration in Th1 cells, and, in long-term cultures with IL-2, IL-16 facilitates the expansion of CD4(+)CD25(+) cells. In addition, IL-16 has an immunomodulatory role in asthmatic inflammation, as exogenous administration significantly reduces inflammation and airway hyperreactivity. The mechanism for this, however, is not clear. Based on its functional characteristics and potential immunomodulatory role, we investigated the ability of IL-16 to recruit and influence the development of T regulatory (Treg) cells. We now demonstrate that IL-16 preferentially induces migration in a CD25(+)CTLA-4(+) human T cell subset and that responding cells produce IFNgamma and TGFbeta but not IL-10. These cells are relatively unresponsive to antigenic stimulation and can suppress proliferation and IL-5, but not IFNgamma, production by autologous T cells. We further demonstrate that IL-16-recruited cells are enriched for Forkhead box P3 (Foxp3). In addition, we find that IL-16 stimulation may facilitate de novo induction of Foxp3(+) Treg cells, because the stimulation of FoxP3-negative T cells for 48 h results in the expression of FoxP3 mRNA and protein. These data indicate that at sites of inflammation IL-16 may contribute to selective Treg cell expansion through the preferential induction of a migratory response from existing Treg cells, as well as by the induction of de novo generation of FoxP3(+) cells. These findings offer a potential mechanism for the immunosuppressive effects of IL-16 seen in Th2-mediated inflammation.     

IL-9 production by regulatory T cells recruits mast cells that are essential for regulatory T cell-induced immune suppression

Both mast cells (MCs) and regulatory T cells (Tregs) have gained attention as immunosuppressive cell populations. To investigate a possible interaction, we used the Th1- and Th17-dependent model of nephrotoxic serum nephritis (NTS), in which both MCs and Tregs have been shown to play a protective role. Transfer of wild-type (wt) Tregs into wt recipients almost completely prevents development of NTS and leads to a profound increase of MCs in the renal draining lymph nodes (LNs). By contrast, transfer of wt Tregs into animals deficient in MCs, which are characterized by an exaggerated susceptibility to NTS, no longer exhibited protective effects. Blocking the pleiotropic cytokine IL-9, known to be involved in MC recruitment and proliferation, by means of a mAb in mice receiving Tregs abrogated protection from NTS. Moreover, transfer of IL-9-deficient Tregs also failed to protect from NTS. In the absence of Treg-derived IL-9, MCs fail to accumulate in the LNs, despite the fact that IL-9 deficiency does not alter the general suppressive activity of Tregs. In summary, to our knowledge, we provide the first direct in vivo evidence that the nephroprotective, anti-inflammatory effects of Tregs critically depend on IL-9-mediated attraction of MCs into kidney-draining LNs.     

Antigen-induced IL-10+ regulatory T cells are independent of CD25+ regulatory cells for their growth, differentiation, and function

Recent studies have emphasized the importance of T cells with regulatory/suppressor properties in controlling autoimmune diseases. A number of different types of regulatory T cells have been described with the best characterized being the CD25(+) population. In addition, it has been shown that regulatory T cells can be induced by specific Ag administration. In this study, we investigate the relationship between peptide-induced, CD4(+) regulatory T cells and naturally occurring CD4(+)CD25(+) cells derived from the Tg4 TCR-transgenic mouse. Peptide-induced cells were FoxP3(-) and responded to Ag by secreting IL-10, whereas CD25(+) cells failed to secrete this cytokine. Both cell types were able to suppress the proliferation of naive lymphocytes in vitro although with distinct activation sensitivities. Depletion of CD25(+) cells did not affect the suppressive properties of peptide-induced regulators. Furthermore, peptide-induced regulatory/suppressor T cells could be generated in RAG(-/-), TCR-transgenic mice that do not spontaneously generate CD25(+) regulatory cells. These results demonstrate that these natural and induced regulatory cells fall into distinct subsets.     

Myelin-specific regulatory T cells accumulate in the CNS but fail to control autoimmune inflammation

Treatment with ex vivo-generated regulatory T cells (T-reg) has been regarded as a potentially attractive therapeutic approach for autoimmune diseases. However, the dynamics and function of T-reg in autoimmunity are not well understood. Thus, we developed Foxp3gfp knock-in (Foxp3gfp.KI) mice and myelin oligodendrocyte glycoprotein (MOG)(35-55)/IA(b) (MHC class II) tetramers to track autoantigen-specific effector T cells (T-eff) and T-reg in vivo during experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. MOG tetramer-reactive, Foxp3(+) T-reg expanded in the peripheral lymphoid compartment and readily accumulated in the central nervous system (CNS), but did not prevent the onset of disease. Foxp3(+) T cells isolated from the CNS were effective in suppressing naive MOG-specific T cells, but failed to control CNS-derived encephalitogenic T-eff that secreted interleukin (IL)-6 and tumor necrosis factor (TNF). Our data suggest that in order for CD4(+)Foxp3(+) T-reg to effectively control autoimmune reactions in the target organ, it may also be necessary to control tissue inflammation.     

Suppression of murine allergic airway disease by IL-2:anti-IL-2 monoclonal antibody-induced regulatory T cells

Regulatory T cells (Treg) play a decisive role in many diseases including asthma and allergen-induced lung inflammation. However, little progress has been made developing new therapeutic strategies for pulmonary disorders. In the current study we demonstrate that cytokine:antibody complexes of IL-2 and anti-IL-2 mAb reduce the severity of allergen-induced inflammation in the lung by expanding Tregs in vivo. Unlike rIL-2 or anti-IL-2 mAb treatment alone, IL-2:anti-IL-2 complexes dampened airway inflammation and eosinophilia while suppressing IL-5 and eotaxin-1 production. Mucus production, airway hyperresponsiveness to methacholine, and parenchymal tissue inflammation were also dramatically reduced following IL-2:anti-IL-2 treatment. The suppression in allergic airway disease was associated with a marked expansion of Tregs (IL-10(+)CD4(+)CD25(+) and Foxp3(+)CD4(+)CD25(+)) in the tissues, with a corresponding decrease in effector T cell responses. The ability of IL-2:anti-IL-2 complexes to suppress airway inflammation was dependent on Treg-derived IL-10, as IL-10(+/+), but not IL-10(-/-) Tregs, were capable of mediating the suppression. Furthermore, a therapeutic protocol using a model of established airway allergy highlighted the ability of IL-2:anti-IL-2 complexes to expand Tregs and prevent successive airway inflammation and airway hyperresponsiveness. This study suggests that endogenous Treg therapy may be a useful tool to combat the rising incidence of allergic airway disease.     

IL-10 is required for regulatory T cells to mediate tolerance to alloantigens in vivo

We present evidence that donor-reactive CD4(+) T cells present in mice tolerant to donor alloantigens are phenotypically and functionally heterogeneous. CD4(+) T cells contained within the CD45RB(high) fraction remained capable of mediating graft rejection when transferred to donor alloantigen-grafted T cell-depleted mice. In contrast, the CD45RB(low) CD4(+) and CD25(+)CD4(+) populations failed to induce rejection, but rather, were able to inhibit rejection initiated by naive CD45RB(high) CD4(+) T cells. Analysis of the mechanism of immunoregulation transferred by CD45RB(low) CD4(+) T cells in vivo revealed that it was donor Ag specific and could be inhibited by neutralizing Abs reactive with IL-10, but not IL-4. CD45RB(low) CD4(+) T cells from tolerant mice were also immune suppressive in vitro, as coculture of these cells with naive CD45RB(high) CD4(+) T cells inhibited proliferation and Th1 cytokine production in response to donor alloantigens presented via the indirect pathway. These results demonstrate that alloantigen-specific regulatory T cells contained within the CD45RB(low) CD4(+) T cell population are responsible for the maintenance of tolerance to donor alloantigens in vivo and require IL-10 for functional activity.     

IL-4 inhibits IL-2 synthesis and IL-2-induced up-regulation of IL-2R alpha but not IL-2R beta chain in CD4+ human T cells.

There is growing evidence to suggest a regulatory role of IL-4 in the immune system affecting both proliferation and lymphokine production. In the present work we have analyzed the effect of IL-4 on IL-2 and IFN-gamma synthesis by stimulating CD4+ human T cells (+10% accessory cells) with Con A in the presence of several doses (1 to 100 U/ml) of human rIL-4. The results showed an impaired IL-2 and IFN-gamma synthesis in the presence of IL-4. This inhibition was dose dependent and was evident only when IL-4 was added in the first 2 h of culture. Moreover, the external addition of IL-2 did not revert the inhibitory effect of IL-4 on IL-2 and IFN-gamma synthesis induced by Con A. We have also analyzed the effect of IL-4 on the expression of both alpha- and beta-chains of the IL-2R. Although the expression of IL-2R alpha mRNA was not modified after 6 h in culture in the presence of IL-4, a decrease was observed at 24 and 48 h. The addition of rIL-2 showed that the inhibition in IL-2R alpha expression could be explained by an impairment in the up-regulatory signal transmitted through the IL-2R. In addition to this, IL-4 did not modify the IL-2R beta mRNA expression at 6 and 24 h although a decreased expression was observed at 48 h which could be explained by the defective IL-2 production. The differential effect of IL-4 on the up-regulatory effect of IL-2 in the expression of IL-2R alpha and IL-2R beta suggest the existence of different regulatory mechanisms acting on the expression of both chains.