IL-21 counteracts the regulatory T cell-mediated suppression of human CD4+ T lymphocytes

High expression of IL-21 and/or IL-21R has been described in T cell-mediated inflammatory diseases characterized by defects of counterregulatory mechanisms. CD4(+)CD25(+) regulatory T cells (Treg) are a T cell subset involved in the control of the immune responses. A diminished ability of these cells to inhibit T cell activation has been documented in immune-inflammatory diseases, raising the possibility that inflammatory stimuli can block the regulatory properties of Treg. We therefore examined whether IL-21 controls CD4(+)CD25(+) T cell function. We demonstrate in this study that IL-21 markedly enhances the proliferation of human CD4(+)CD25(-) T cells and counteracts the suppressive activities of CD4(+)CD25(+) T cells on CD4(+)CD25(-) T cells without affecting the percentage of Foxp3(+) cells or survival of Treg. Additionally, CD4(+)CD25(+) T cells induced in the presence of IL-21 maintain the ability to suppress alloresponses. Notably, IL-21 enhances the growth of CD8(+)CD25(-) T cells but does not revert the CD4(+)CD25(+) T cell-mediated suppression of this cell type, indicating that IL-21 makes CD4(+) T cells resistant to suppression rather than inhibiting CD4(+)CD25(+) T cell activity. Finally, we show that IL-2, IL-7, and IL-15, but not IL-21, reverse the anergic phenotype of CD4(+)CD25(+) T cells. Data indicate that IL-21 renders human CD4(+)CD25(-) T cells resistant to Treg-mediated suppression and suggest a novel mechanism by which IL-21 could augment T cell-activated responses in human immune-inflammatory diseases.     

Testosterone acts directly on CD4+ T lymphocytes to increase IL-10 production

Males are less susceptible than females to experimental autoimmune encephalomyelitis and many other autoimmune diseases. Gender differences in cytokine production have been observed in splenocytes of experimental autoimmune encephalomyelitis mice stimulated with myelin proteins and may underlie gender differences in susceptibility. As these differences should not be limited to responses specific for myelin proteins, gender differences in cytokine production upon stimulation with Ab to CD3 were examined, and the mechanisms were delineated. Splenocytes from male mice stimulated with Ab to CD3 produced more IL-10 and IL-4 and less IL-12 than those from female mice. Furthermore, splenocytes from dihydrotestosterone (DHT)-treated female mice produced more IL-10 and less IL-12 than those from placebo-treated female mice, whereas there was no difference in IL-4. IL-12 knockout mice were then used to determine whether changes in IL-10 production were mediated directly by testosterone vs indirectly by changes in IL-12. The results of these experiments favored the first hypothesis, because DHT treatment of female IL-12 knockout mice increased IL-10 production. To begin to delineate the mechanism by which DHT may be acting, the cellular source of IL-10 was determined. At both the RNA and protein levels, IL-10 was produced primarily by CD4+ T lymphocytes. CD4+ T lymphocytes were then shown to express the androgen receptor, raising the possibility that testosterone acts directly on CD4+ T lymphocytes to increase IL-10 production. In vitro experiments demonstrated increased IL-10 production following treatment of CD4+ T lymphocytes with DHT. Thus, testosterone can act directly via androgen receptors on CD4+ T lymphocytes to increase IL-10 gene expression.     

Suppressor of cytokine signaling 1 regulates IL-15 receptor signaling in CD8+CD44high memory T lymphocytes

T lymphocyte survival, proliferation, and death in the periphery are dependent on several cytokines. Many of these cytokines induce the expression of suppressor of cytokine signaling-1 (SOCS1), a feedback inhibitor of JAK kinases. However, it is unclear whether the cytokines that regulate T lymphocyte homeostasis are critically regulated by SOCS1 in vivo. Using SOCS1(-/-)IFN-gamma(-/-) mice we show that SOCS1 deficiency causes a lymphoproliferative disorder characterized by decreased CD4/CD8 ratio due to chronic accumulation of CD8+CD44(high) memory phenotype T cells. SOCS1-deficient CD8+ T cells express elevated levels of IL-2Rbeta, show increased proliferative response to IL-15 and IL-2 in vitro, and undergo increased bystander proliferation and vigorous homeostatic expansion in vivo. Sorted CD8+CD44(high) T cells from SOCS1(-/-)IFN-gamma(-/-) mice respond 5 times more strongly than control cells, indicating that SOCS1 is a critical regulator of IL-15R signaling. Consistent with this idea, IL-15 stimulates sustained STAT5 phosphorylation in SOCS1-deficient CD8+ T cells. IL-15 strongly induces TNF-alpha production in SOCS1-deficient CD8+ T cells, indicating that SOCS1 is also a critical regulator of CD8+ T cell activation by IL-15. However, IL-15 and IL-2 induce comparable levels of Bcl-2 and Bcl-x(L) in SOCS1-deficient and SOCS1-sufficient CD8+ T cells, suggesting that cytokine receptor signals required for inducing proliferation and cell survival signals are not identical. These results show that SOCS1 differentially regulates common gamma-chain cytokine signaling in CD8+ T cells and suggest that CD8+ T cell homeostasis is maintained by distinct mechanisms that control cytokine-mediated survival and proliferation signals.     

Stimulation via the CD3 and CD28 molecules induces responsiveness to IL-4 in CD4+CD29+CD45R- memory T lymphocytes

Although both IL-2 and IL-4 can promote the growth of activated T cells, IL-4 appears to selectively promote the growth of those helper/inducer and cytolytic T cells which have been activated via their CD3/TCR complex. The present study examines the participation of CD28 and certain other T cell-surface molecules in inducing T cell responsiveness to IL-4. Purified small high density T cells were cultured in the absence of accessory cells with various soluble anti-human T cell mAb with or without soluble anti-CD3 mAb and their responsiveness to IL-4 was studied. None of the soluble anti-T cell mAb alone was able to induce T cell proliferation in response to IL-4. A combination of soluble anti-CD3 with anti-CD28 mAb but not with mAb directed at the CD2, CD5, CD7, CD11a/CD18, or class I MHC molecules induced T cell proliferation in response to IL-4. Anti-CD2 and anti-CD5 mAb enhanced and anti-CD18 mAb inhibited this anti-CD3 + anti-CD28 mAb-induced T cell response to IL-4. In addition, anti-CD2 in combination with anti-CD3 and anti-CD28 mAb induced modest levels of T cell proliferation even in the absence of exogenous cytokines. IL-1, IL-6, and TNF were each unable to replace either anti-CD3 or anti-CD28 mAb in the induction of T cell responsiveness to IL-4, but both IL-1 and TNF enhanced this response. The anti-CD3 + anti-CD28 mAb-induced response to IL-4 was exhibited only by cells within the CD4+CD29+CD45R- memory T subpopulation, and not by CD8+ or CD4+CD45R+ naive T cells. When individually cross-linked with goat anti-mouse IgG antibody immobilized on plastic surface, only anti-CD3 and anti-CD28 mAb were able to induce T cell proliferation. These results indicate that the CD3 and CD28 molecules play a crucial role in inducing T cell responsiveness to IL-4 and that the CD2, CD5, and CD11a/CD18 molecules influence this process.     

Production of IL-10 by CD4+ T lymphocytes correlates with down-regulation of Th1 cytokine synthesis in helminth infection.

After the onset of parasite egg deposition, mice infected with the helminth Schistosoma mansoni mount strong Th2 cytokine responses in the absence of significant Th1 cytokine synthesis. To examine the basis of this immunoregulatory state, spleen or lymph node cells from schistosome-infected mice were stimulated with parasite-specific Ag and the supernatants tested for their capacity to suppress IFN-gamma synthesis by a Th1 cell line. Strong inhibition was observed that was neutralized by a mAb against IL-10, a cytokine previously shown to down-regulate Th1 cytokine synthesis. By means of ELISA measurements the production of IL-10 in schistosome infection was confirmed and shown to depend on CD4+ T cells. IL-10 synthesis stimulated by either mitogen or Ag was observed only at those stages of infection when Th2 response induction and Th1 cytokine down-regulation also occurred and was not detected in mice vaccinated with attenuated parasites. Moreover, the addition of the neutralizing anti-IL-10 mAb to Ag-stimulated spleen cell cultures from infected mice caused a dramatic augmentation in IFN-gamma synthesis. These findings suggest that IL-10 is responsible for the down-regulation of Th1 responses observed in schistosome infections, a phenomenon that may enable the parasite to escape potentially harmful cell-mediated responses.     

Uncoupling of IL-2 signaling from cell cycle progression in naive CD4+ T cells by regulatory CD4+CD25+ T lymphocytes

Prior reports have shown that CD4(+)CD25(+) regulatory T cells suppress naive T cell responses by inhibiting IL-2 production. In this report, using an Ag-specific TCR transgenic system, we show that naive T cells stimulated with cognate Ag in the presence of preactivated CD4(+)CD25(+) T cells also become refractory to the mitogenic effects of IL-2. T cells stimulated in the presence of regulatory T cells up-regulated high affinity IL-2R, but failed to produce IL-2, express cyclins or c-Myc, or exit G(0)-G(1). Exogenous IL-2 failed to break the mitotic block, demonstrating that the IL-2 production failure was not wholly responsible for the proliferation defect. This IL-2 unresponsiveness did not require the continuous presence of CD4(+)CD25(+) regulatory T cells. The majority of responder T cells reisolated after coculture with regulatory cells failed to proliferate in response to IL-2, but were not anergic and proliferated in response to Ag. The mitotic block was also dissociated from the antiapoptotic effects of IL-2, because IL-2 still promoted the survival of T cells that had been cocultured with CD4(+)CD25(+) T cells. IL-2-induced STAT5 phosphorylation in the cocultured responder cells was intact, implying that the effects of the regulatory cells were downstream of receptor activation. Our results therefore show that T cell activation in the presence of CD4(+)CD25(+) regulatory T cells can induce an alternative stimulation program characterized by up-regulation of high affinity IL-2R, but a failure to produce IL-2, and uncoupling of the mitogenic and antiapoptotic effects of IL-2.     

IL-10 is produced by subsets of human CD4+ T cell clones and peripheral blood T cells.

Murine IL-10 has been reported originally to be produced by the Th2 subset of CD4+ T cell clones. In this study, we demonstrate that human IL-10 is produced by Th0, Th1-, and Th2-like CD4+ T cell clones after both Ag-specific and polyclonal activation. In purified peripheral blood T cells, low, but significant, levels of IL-10 were found to be produced by the CD4+CD45RA+ population, whereas CD4+CD45RA- "memory" cells secreted 5- to 20-fold higher levels of IL-10. In addition, IL-10 was produced by activated CD8+ peripheral blood T cells. Optimal induction of IL-10 was observed after activation by specific Ag and by the combination of anti-CD3 mAb and the phorbol ester tetradecanoyl phorbol acetate, whereas the combination of calcium ionophore A23187 and 12-O-tetradecanoylphorbol-13-acetate acetate was a poor inducer of IL-10 production. Kinetic studies indicated that IL-10 was produced relatively late as compared with other cytokines. Maximal IL-10 mRNA expression in CD4+ T cell clones and purified peripheral blood T cells was obtained after 24 h, whereas maximal IL-10 protein synthesis occurred between 24 h and 48 h after activation. No differences were observed in the kinetics of IL-10 production among Th0, Th1-, and Th2-like subsets of CD4+ T cell clones. The results indicate a regulatory role for IL-10 in later phases of the immune response.     

Dysfunction of simian immunodeficiency virus/simian human immunodeficiency virus-induced IL-2 expression by central memory CD4+ T lymphocytes

Production of IL-2 and IFN-gamma by CD4+ T lymphocytes is important for the maintenance of a functional immune system in infected individuals. In the present study, we assessed the cytokine production profiles of functionally distinct subsets of CD4+ T lymphocytes in rhesus monkeys infected with pathogenic or attenuated SIV/simian human immunodeficiency virus (SHIV) isolates, and these responses were compared with those in vaccinated monkeys that were protected from immunodeficiency following pathogenic SHIV challenge. We observed that preserved central memory CD4+ T lymphocyte production of SIV/SHIV-induced IL-2 was associated with disease protection following primate lentivirus infection. Persisting clinical protection in vaccinated and challenged monkeys is thus correlated with a preserved capacity of the peripheral blood central memory CD4+ T cells to express this important immunomodulatory cytokine.     

CD25+ CD4+ T cells regulate the expansion of peripheral CD4 T cells through the production of IL-10

The mechanisms by which the immune system achieves constant T cell numbers throughout life, thereby controlling autoaggressive cell expansions, are to date not completely understood. Here, we show that the CD25(+) subpopulation of naturally activated (CD45RB(low)) CD4 T cells, but not CD25(-) CD45RB(low) CD4 T cells, inhibits the accumulation of cotransferred CD45RB(high) CD4 T cells in lymphocyte-deficient mice. However, both CD25(+) and CD25(-) CD45RB(low) CD4 T cell subpopulations contain regulatory cells, since they can prevent naive CD4 T cell-induced wasting disease. In the absence of a correlation between disease and the number of recovered CD4(+) cells, we conclude that expansion control and disease prevention are largely independent processes. CD25(+) CD45RB(low) CD4 T cells from IL-10-deficient mice do not protect from disease. They accumulate to a higher cell number and cannot prevent the expansion of CD45RB(high) CD4 T cells upon transfer compared with their wild-type counterparts. Although CD25(+) CD45RB(low) CD4 T cells are capable of expanding when transferred in vivo, they reach a homeostatic equilibrium at lower cell numbers than CD25(-) CD45RB(low) or CD45RB(high) CD4 T cells. We conclude that CD25(+) CD45RB(low) CD4 T cells from nonmanipulated mice control the number of peripheral CD4 T cells through a mechanism involving the production of IL-10 by regulatory T cells.     

Expression and regulation of IL-22 in the IL-17-producing CD4＋ T lymphocytes

IL-22 is a novel cytokine in the IL-10 family that functions to promote innate immunity of tissues against infection. Although CD4＋ helper T lymphocytes （TH） were found as a source of IL-22, the regulation of this cytokine has been poorly understood. Here, we show that IL-22 is expressed at both mRNA and protein levels by a novel subset of TH cells that also makes IL-17. IL-22 and IL-17 were found to be coordinately regulated by TGFI3 and IL-6 during TH differentiation by real-time PCR as well as ELISA analysis. However, IL-22 does not regulate TH differentiation; exogenous IL-22 or an IL-22 antagonist had no effect on TH differentiation. These data demonstrate a novel cytokine expressed by IL-17-producing T cells, and suggest interaction and synergy of IL-22 and IL-l 7 signaling pathways in tissue inflammation and autoimmune diseases.     

CD8+ T cell-mediated airway hyperresponsiveness and inflammation is dependent on CD4+IL-4+ T cells

CD4+ T cells, particularly Th2 cells, play a pivotal role in allergic airway inflammation. However, the requirements for interactions between CD4+ and CD8+ T cells in airway allergic inflammation have not been delineated. Sensitized and challenged OT-1 mice in which CD8+ T cells expressing the transgene for the OVA(257-264) peptide (SIINFEKL) failed to develop airway hyperresponsiveness (AHR), airway eosinophilia, Th2 cytokine elevation, or goblet cell metaplasia. OT-1 mice that received naive CD4+IL-4+ T cells but not CD4+IL-4- T cells before sensitization developed all of these responses to the same degree as wild-type mice. Moreover, recipients of CD4+IL-4+ T cells developed significant increases in the number of CD8+IL-13+ T cells in the lung, whereas sensitized OT-1 mice that received primed CD4+ T cells just before challenge failed to develop these responses. Sensitized CD8-deficient mice that received CD8+ T cells from OT-1 mice that received naive CD4+ T cells before sensitization increased AHR and eosinophil numbers in bronchoalveolar lavage fluid when challenged with allergen. In contrast, sensitized CD8-deficient mice receiving CD8+ T cells from OT-1 mice without CD4+ T cells developed reduced AHR and eosinophil numbers in bronchoalveolar lavage fluid when challenged. These data suggest that interactions between CD4+ and CD8+ T cells, in part through IL-4 during the sensitization phase, are essential to the development of CD8+IL-13+ T cell-dependent AHR and airway allergic inflammation.     

A protective role for human IL-10-expressing CD4+ T cells in colitis

IL-10 is an immunoregulatory cytokine expressed by numerous cell types. Studies in mice confirm that different IL-10-expressing cell subsets contribute differentially to disease phenotypes. However, little is known about the relationship between cell- or tissue-specific IL-10 expression and disease susceptibility in humans. In this study, we used the previously described human (h)IL10BAC transgenic model to examine the role of hIL-10 in maintaining intestinal homeostasis. Genomically controlled hIL-10 expression rescued Il10(-/-) mice from Helicobacter-induced colitis and was associated with control of proinflammatory cytokine expression and Th17 cell accumulation in gut tissues. Resistance to colitis was associated with an accumulation of hIL-10-expressing CD4(+)Foxp3(+) regulatory T cells specifically within the lamina propria but not other secondary lymphoid tissues. Cotransfer of CD4(+)CD45RB(lo) cells from Il10(-/-)/hIL10BAC mice rescued Rag1(-/-) mice from colitis, further suggesting that CD4(+) T cells represent a protective source of hIL-10 in the colon. In concordance with an enhanced capacity to express IL-10, CD4(+)CD44(+) T cells isolated from the lamina propria exhibited lower levels of the repressive histone mark H3K27Me3 and higher levels of the permissive histone mark acetylated histone H3 in both the human and mouse IL10 locus compared with the spleen. These results provide experimental evidence verifying the importance of T cell-derived hIL-10 expression in controlling inflammation within the colonic mucosa. We also provide molecular evidence suggesting the tissue microenvironment influences IL-10 expression patterns and chromatin structure in the human (and mouse) IL10 locus.     

IL-10 signaling in CD4+ T cells is critical for the pathogenesis of collagen-induced arthritis

Introduction

IL-10 is a very important anti-inflammatory cytokine. However, the role of this cytokine in T cells in the pathogenesis of collagen-induced arthritis is unclear. The purpose of this study was to define the role of IL-10 signaling in T cells in the pathogenesis of collagen-induced arthritis.

Methods

IL-10 receptor dominant-negative transgenic (Tg) and control mice were immunized with bovine type II collagen to induce arthritis. The severity of arthritis was monitored and examined histologically. T-cell activation and cytokine production were analyzed using flow cytometry. T-cell proliferation was examined by [³H]thymidine incorporation. Antigen-specific antibodies in serum were measured by ELISA. Foxp3 expression in CD4⁺ regulatory T cells (Tregs) was determined by intracellular staining or Foxp3-RFP reporter mice. The suppressive function of Foxp3⁺CD4⁺ Tregs was determined in vitro by performing a T-cell proliferation assay. The level of IL-17 mRNA in joints was measured by real-time PCR. A two-tailed nonparametric paired test (Wilcoxon signed-rank test) was used to calculate the arthritis and histological scores. Student's paired or unpaired t-test was used for all other statistical analyses (InStat version 2.03 software; GraphPad Software, San Diego, CA, USA).

Results

Blocking IL-10 signaling in T cells rendered mice, especially female mice, highly susceptible to collagen-induced arthritis. T-cell activation and proliferation were enhanced and produced more IFN-γ. The suppressive function of CD4⁺Foxp3⁺ regulatory T cells was significantly impaired in Tg mice because of the reduced ability of Tregs from Tg mice to maintain their levels of Foxp3. This was further confirmed by transferring Foxp3-RFP cells from Tg or wild-type (Wt) mice into a congenic Wt host. The higher level of IL-17 mRNA was detected in inflammatory joints of Tg mice, probably due to the recruitment of IL-17⁺γδ T cells into the arthritic joints.

Conclusion

IL-10 signaling in T cells is critical for dampening the pathogenesis of collagen-induced arthritis by maintaining the function of Tregs and the recruitment of IL-17⁺γδ T cells.     

Human cellular immune response to the saliva of Phlebotomus papatasi is mediated by IL-10-producing CD8+ T cells and Th1-polarized CD4+ lymphocytes

Background

The saliva of sand flies strongly enhances the infectivity of Leishmania in mice. Additionally, pre-exposure to saliva can protect mice from disease progression probably through the induction of a cellular immune response.

Methodology/Principal Findings

We analysed the cellular immune response against the saliva of Phlebotomus papatasi in humans and defined the phenotypic characteristics and cytokine production pattern of specific lymphocytes by flow cytometry. Additionally, proliferation and IFN-γ production of activated cells were analysed in magnetically separated CD4+ and CD8+ T cells. A proliferative response of peripheral blood mononuclear cells against the saliva of Phlebotomus papatasi was demonstrated in nearly 30% of naturally exposed individuals. Salivary extracts did not induce any secretion of IFN-γ but triggered the production of IL-10 primarily by CD8+ lymphocytes. In magnetically separated lymphocytes, the saliva induced the proliferation of both CD4+ and CD8+ T cells which was further enhanced after IL-10 blockage. Interestingly, when activated CD4+ lymphocytes were separated from CD8+ cells, they produced high amounts of IFN-γ.

Conclusion

Herein, we demonstrated that the overall effect of Phlebotomus papatasi saliva was dominated by the activation of IL-10-producing CD8+ cells suggesting a possible detrimental effect of pre-exposure to saliva on human leishmaniasis outcome. However, the activation of Th1 lymphocytes by the saliva provides the rationale to better define the nature of the salivary antigens that could be used for vaccine development.     

Characterization of Foxp3+CD4+CD25+ and IL-10-secreting CD4+CD25+ T cells during cure of colitis

CD4+CD25+ regulatory T cells can prevent and resolve intestinal inflammation in the murine T cell transfer model of colitis. Using Foxp3 as a marker of regulatory T cell activity, we now provide a comprehensive analysis of the in vivo distribution of Foxp3+CD4+CD25+ cells in wild-type mice, and during cure of experimental colitis. In both cases, Foxp3+CD4+CD25+ cells were found to accumulate in the colon and secondary lymphoid organs. Importantly, Foxp3+ cells were present at increased density in colon samples from patients with ulcerative colitis or Crohn's disease, suggesting similarities in the behavior of murine and human regulatory cells under inflammatory conditions. Cure of murine colitis was dependent on the presence of IL-10, and IL-10-producing CD4+CD25+ T cells were enriched within the colon during cure of colitis and also under steady state conditions. Our data indicate that although CD4+CD25+ T cells expressing Foxp3 are present within both lymphoid organs and the colon, subsets of IL-10-producing CD4+CD25+ T cells are present mainly within the intestinal lamina propria suggesting compartmentalization of the regulatory T cell response at effector sites.     

Preservation of functional virus-specific memory CD8+ T lymphocytes in vaccinated, simian human immunodeficiency virus-infected rhesus monkeys

Functional impairment of virus-specific memory CD8(+) T lymphocytes has been associated with clinical disease progression following HIV, SIV, and simian human immunodeficiency virus infection. These lymphocytes have a reduced capacity to produce antiviral cytokines and mediators involved in the lysis of virally infected cells. In the present study, we used polychromatic flow cytometry to assess the frequency and functional capacity of central memory (CD28(+)CD95(+)) and effector memory (CD28(-)CD95(+)) subpopulations of Gag-specific CD8(+) T cells in SIV/simian human immunodeficiency virus-infected rhesus monkeys. The aim of this study was to determine whether Ag-specific, memory CD8(+) T cell function could be preserved in infected monkeys that had been immunized before infection with a vaccine regimen consisting of a plasmid DNA prime followed by a recombinant viral vector boost. We observed that vaccination was associated with the preservation of Gag-specific central memory CD8(+) T cells that were functionally capable of producing IFN-gamma, and effector memory CD8(+) T cells that were capable of producing granzyme B following viral Ag exposure.