TGF-beta-mediated suppression by CD4+CD25+ T cells is facilitated by CTLA-4 signaling

CD4+CD25+ T cells play a pivotal role in immunological homeostasis by their capacity to exert immunosuppressive activity. However, the mechanism by which these cells function is still a subject for debate. We previously reported that surface (membrane) TGF-beta produced by CD4+CD25+ T cells was an effector molecule mediating suppressor function. We now support this finding by imaging surface TGF-beta on Foxp3+CD4+CD25+ T cells in confocal fluorescence microscopy. Then, using a TGF-beta-sensitive mink lung epithelial cell (luciferase) reporter system, we show that surface TGF-beta can be activated to signal upon cell-cell contact. Moreover, if such TGF-beta signaling is blocked in an in vitro assay of CD4+CD25+ T cell suppression by a specific inhibitor of TGF-betaRI, suppressor function is also blocked. Finally, we address the role of CTLA-4 in CD4+CD25+ T cell suppression, showing first that whereas anti-CTLA-4 does not block in vitro suppressor function, it does complement the blocking activity of anti-TGF-beta. We then show with confocal fluorescence microscopy that incubation of CD4+CD25+ T cells with anti-CTLA-4- and rB7-1/Fc-coated beads results in accumulation of TGF-beta at the cell-bead contact site. This suggests that CTLA-4 signaling facilitates TGF-beta-mediated suppression by intensifying the TGF-beta signal at the point of suppressor cell-target cell interaction.     

Cutting edge: the phosphoinositide 3-kinase p110 delta is critical for the function of CD4+CD25+Foxp3+ regulatory T cells

CD4+CD25+Foxp3+ regulatory T cells (Tregs) contribute to the maintenance of peripheral tolerance by inhibiting the expansion and function of conventional T cells. Treg development and homeostasis are regulated by the Ag receptor, costimulatory receptors such as CD28 and CTLA-4, and cytokines such as IL-2, IL-10, and TGF-beta. Here we show that the proportions of Tregs in the spleen and lymph nodes of mice with inactive p110delta PI3K (p110deltaD910A/D910A) are reduced despite enhanced Treg selection in the thymus. p110deltaD910A/D910A CD4+CD25+Foxp3+ Tregs showed attenuated suppressor function in vitro and failed to secrete IL-10. In adoptive transfer experiments, p110deltaD910A/D910A T cells failed to protect against experimental colitis. The identification of p110delta as an intracellular signaling protein that regulates the activity of CD4+CD25+Foxp3+ Tregs may facilitate the further elucidation of the molecular mechanisms responsible for Treg-mediated suppression.     

TGF-beta requires CTLA-4 early after T cell activation to induce FoxP3 and generate adaptive CD4+CD25+ regulatory cells

Although positive CD28 costimulation is needed for the generation of natural CD4+CD25+ regulatory T cells, we report that negative CTLA-4 costimulation is necessary for generating phenotypically and functionally similar adaptive CD4+CD25+ suppressor cells. TGF-beta could not induce CD4+CD25- cells from CTLA-4(-/-) mice to express normal levels of FoxP3 or to develop suppressor activity. Moreover, blockade of CTLA-4 following activation of wild-type CD4+ cells abolished the ability of TGF-beta to induce FoxP3-expressing mouse suppressor cells. TGF-beta accelerated expression of CTLA-4, and time course studies suggested that CTLA-4 ligation of CD80 shortly after T cell activation enables TGF-beta to induce CD4+CD25- cells to express FoxP3 and develop suppressor activity. TGF-beta also enhanced CD4+ cell expression of CD80. Thus, CTLA-4 has an essential role in the generation of acquired CD4+CD25+ suppressor cells in addition to its other inhibitory effects. Although natural CD4+CD25+ cells develop normally in CTLA-4(-/-) mice, the lack of TGF-beta-induced, peripheral CD4+CD25+ suppressor cells in these mice may contribute to their rapid demise.     

CD18 is required for optimal development and function of CD4+CD25+ T regulatory cells

CD4+CD25+ T regulatory (Treg) cells inhibit immunopathology and autoimmune disease in vivo. CD4+CD25+ Treg cells' capacity to inhibit conventional T cells in vitro is dependent upon cell-cell contact; however, the cell surface molecules mediating this cell:cell contact have not yet been identified. LFA-1 (CD11a/CD18) is an adhesion molecule that plays an established role in T cell-mediated cell contact and in T cell activation. Although expressed at high levels on murine CD4+CD25+ Treg cells, the role of LFA-1 in these cells has not been defined previously. We hypothesized that LFA-1 may play a role in murine CD4+CD25+ Treg function. To evaluate this, we analyzed LFA-1-deficient (CD18-/-) CD4+CD25+ T cells. We show that CD18-/- mice demonstrate a propensity to autoimmunity. Absence of CD18 led to diminished CD4+CD25+ T cell numbers and affected both thymic and peripheral development of these cells. LFA-1-deficient CD4+CD25+ T cells were deficient in mediating suppression in vitro and in mediating protection from colitis induced by the transfer of CD4+CD25- T cells into lymphopenic hosts. Therefore, we define a crucial role for CD18 in optimal CD4+CD25+ Treg development and function.     

Follicular lymphoma intratumoral CD4+CD25+GITR+ regulatory T cells potently suppress CD3/CD28-costimulated autologous and allogeneic CD8+CD25- and CD4+CD25- T cells

Regulatory T cells (T(R)) play a critical role in the inhibition of self-reactive immune responses and as such have been implicated in the suppression of tumor-reactive effector T cells. In this study, we demonstrate that follicular lymphoma (FL)-infiltrating CD8+ and CD4+ T cells are hyporesponsive to CD3/CD28 costimulation. We further identify a population of FL-infiltrating CD4+CD25+GITR+ T(R) that are significantly overrepresented within FL nodes (FLN) compared with that seen in normal (nonmalignant, nonlymphoid hyperplastic) or reactive (nonmalignant, lymphoid hyperplastic) nodes. These T(R) actively suppress both the proliferation of autologous nodal CD8+CD25- and CD4+CD25- T cells, as well as cytokine production (IFN-gamma, TNF-alpha and IL-2), after CD3/CD28 costimulation. Removal of these cells in vitro by CD25+ magnetic bead depletion restores both the proliferation and cytokine production of the remaining T cells, demonstrating that FLN T cell hyporesponsiveness is reversible. In addition to suppressing autologous nodal T cells, these T(R) are also capable of suppressing the proliferation of allogeneic CD8+CD25- and CD4+CD25- T cells from normal lymph nodes as well as normal donor PBL, regardless of very robust stimulation of the target cells with plate-bound anti-CD3 and anti-CD28 Abs. The allogeneic suppression is not reciprocal, as equivalent numbers of CD25+FOXP3+ cells derived from either normal lymph nodes or PBL are not capable of suppressing allogeneic CD8+CD25- and CD4+CD25- T cells, suggesting that FLN T(R) are more suppressive than those derived from nonmalignant sources. Lastly, we demonstrate that inhibition of TGF-beta signaling partially restores FLN T cell proliferation suggesting a mechanistic role for TGF-beta in FLN T(R)-mediated suppression.     

Naturally arising CD4+CD25+ regulatory T cells suppress the expansion of colitogenic CD4+CD44highCD62L- effector memory T cells

Naturally arising CD4+CD25+ regulatory T (T(R)) cells have been shown to prevent and cure murine T cell-mediated colitis. However, their exact mechanism of controlling colitogenic memory CD4+ T cells in in vivo systems excluding the initial process of naive T cell activation and differentiation has not been examined to date. Using the colitogenic effector memory (T(EM)) CD4+ cell-mediated colitis model induced by adoptive transfer of colitogenic CD4+CD44(high)CD62L(-) lamina propria (LP) T cells obtained from colitic CD4+CD45RB(high) T cell-transferred mice, we have shown in the present study that CD4+CD25+ T(R) cells are able not only to suppress the development of colitis, Th1 cytokine production, and the expansion of colitogenic LP CD4+ T(EM) cells but also to expand these cells by themselves extensively in vivo. An in vitro coculture assay revealed that CD4+CD25+ T(R) cells proliferated in the presence of IL-2-producing colitogenic LP CD4+ T(EM) cells at the early time point (48 h after culture), followed by the acquisition of suppressive activity at the late time point (96 h after culture). Collectively, these data suggest the distinct timing of the IL-2-dependent expansion of CD4+CD25+ T(R) cells and the their suppressive activity on colitogenic LP CD4+ T(EM) cells.     

Regulation of murine inflammatory bowel disease by CD25+ and CD25- CD4+ glucocorticoid-induced TNF receptor family-related gene+ regulatory T cells

CD4(+)CD25(+) regulatory T cells in normal animals are engaged in the maintenance of immunological self-tolerance and prevention of autoimmune disease. However, accumulating evidence suggests that a fraction of the peripheral CD4(+)CD25(-) T cell population also possesses regulatory activity in vivo. Recently, it has been shown glucocorticoid-induced TNFR family-related gene (GITR) is predominantly expressed on CD4(+)CD25(+) regulatory T cells. In this study, we show evidence that CD4(+)GITR(+) T cells, regardless of the CD25 expression, regulate the mucosal immune responses and intestinal inflammation. SCID mice restored with the CD4(+)GITR(-) T cell population developed wasting disease and severe chronic colitis. Cotransfer of CD4(+)GITR(+) population prevented the development of CD4(+)CD45RB(high) T cell-transferred colitis. Administration of anti-GITR mAb-induced chronic colitis in mice restored both CD45RB(high) and CD45RB(low) CD4(+) T cells. Interestingly, both CD4(+)CD25(+) and CD4(+)CD25(-) GITR(+) T cells prevented wasting disease and colitis. Furthermore, in vitro studies revealed that CD4(+)CD25(-)GITR(+) T cells as well as CD4(+)CD25(+)GITR(+) T cells expressed CTLA-4 intracellularly, showed anergic, suppressed T cell proliferation, and produced IL-10 and TGF-beta. These data suggest that GITR can be used as a specific marker for regulatory T cells controlling mucosal inflammation and also as a target for treatment of inflammatory bowel disease.     

CD4+CD25+ regulatory T cells suppress differentiation and functions of Th1 and Th2 cells,Leishmania major infection,and colitis in mice

Regulatory T cells play a major role in modulating the immune response. However, most information on these cells centers on autoimmunity, and there is also considerable controversy on the functional characteristics of these cells. Here we provide direct in vitro and in vivo evidence that CD4+CD25+ regulatory T cells inhibit the differentiation and functions of both Th1 and Th2 cells. Importantly, CD4+CD25+ T cells suppressed the disease development of Leishmania major infection in SCID mice reconstituted with naive CD4+CD25- T cells. Furthermore, CD4+CD25+ T cells inhibited the development of colitis induced by both Th1 and Th2 cells in SCID mice. Our results therefore document that CD4+CD25+ regulatory T cells suppress both Th1 and Th2 cells and that these regulatory T cells have a profound therapeutic potential against diseases induced by both Th1 and Th2 cells in vivo.     

Protection from type 1 diabetes by invariant NK T cells requires the activity of CD4+CD25+ regulatory T cells

Invariant NK T (iNKT) cells regulate immune responses, express NK cell markers and an invariant TCR, and recognize lipid Ags in a CD1d-restricted manner. Previously, we reported that activation of iNKT cells by alpha-galactosylceramide (alpha-GalCer) protects against type 1 diabetes (T1D) in NOD mice via an IL-4-dependent mechanism. To further investigate how iNKT cells protect from T1D, we analyzed whether iNKT cells require the presence of another subset(s) of regulatory T cells (Treg), such as CD4+ CD25+ Treg, for this protection. We found that CD4+ CD25+ T cells from NOD.CD1d(-/-) mice deficient in iNKT cell function similarly in vitro to CD4+ CD25+ T cells from wild-type NOD mice and suppress the proliferation of NOD T responder cells upon alpha-GalCer stimulation. Cotransfer of NOD diabetogenic T cells with CD4+ CD25+ Tregs from NOD mice pretreated with alpha-GalCer demonstrated that activated iNKT cells do not influence the ability of T(regs) to inhibit the transfer of T1D. In contrast, protection from T1D mediated by transfer of activated iNKT cells requires the activity of CD4+ CD25+ T cells, because splenocytes pretreated with alpha-GalCer and then inactivated by anti-CD25 of CD25+ cells did not protect from T1D. Similarly, mice inactivated of CD4+ CD25+ T cells before alpha-GalCer treatment were also not protected from T1D. Our data suggest that CD4+ CD25+ T cells retain their function during iNKT cell activation, and that the activity of CD4+ CD25+ Tregs is required for iNKT cells to transfer protection from T1D.     

TGF-beta: the missing link in CD4+CD25+ regulatory T cell-mediated immunosuppression

A unique population of CD4(+) T lymphocytes that constitutively express CD25 has been recognized as anergic/suppressor cells. While the immunosuppressive activity of these CD4(+)CD25(+) cells has been validated and implicated in tolerance, autoimmunity, transplantation, cancer and infectious diseases, the mechanism(s) by which they function still remains controversial. Although the involvement of TGF-beta was initially discounted, emerging evidence now links this cytokine with CD4(+)CD25(+) T cell-mediated suppression of antigen-activated T cells. In this perspective, we summarize recently published studies, as well as our own data, which shed light on how cell membrane-bound TGF-beta can deliver a regulatory signal to target cells via a contact-dependent process. Moreover, suppressor T cell function is a complex process, tightly regulated by multiple factors, including IL-2, cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and glucocorticoid induced TNF receptor (GITR). Collectively, multiple previously unconnected puzzle pieces are beginning to be linked into a more coherent, albeit incomplete picture of CD4(+)CD25(+) T cell-mediated suppression.     

In vitro induction of CD25+ CD4+ regulatory T cells by the neuropeptide alpha-melanocyte stimulating hormone (alpha-MSH)

Recently, we have found that the neuropeptide alpha-melanocyte stimulating hormone (alpha-MSH) not only suppresses IFN-gamma production, but also induces TGF-beta1 production by activated effector T cells. These alpha-MSH- treated effector T cells function as regulatory T cells in that they suppress IFN-gamma production and hypersensitivity mediated by other effector T cells. Experimental autoimmune uveoretinitis (EAU) was suppressed in its severity and incidence in mice that were injected with primed T cells activated in vitro by APC and antigen in the presence of alpha-MSH. Moreover, it appeared that alpha-MSH had converted a population of effector T cells polarized to mediate hypersensitivity into a population of T cells that now mediated immunoregulation. To characterize these alpha-MSH- treated T cells, primed T cells were TCR-stimulated in the presence of alpha-MSH in vitro and their lymphokine profile was examined. Such effector T cells displayed enhanced levels of TGF-beta1 production and no IFN-gamma or IL-10, with IL-4 levels remaining unchanged in comparison with inactivated T cells. In addition, if soluble TGF-beta receptor II was added to cocultures of alpha-MSH-treated T cells and activated Th1 cells, the alpha-MSH-treated T cells could not suppress IFN-gamma production by the Th1 cells. These results suggest that alpha-MSH induces T cells with a regulatory lymphokine pattern, and that through their production of TGF-beta1 these cells suppress other effector T cells. Examination of the alpha-MSH-treated T cells showed that alpha-MSH did not alter the phosphorylation of CD3 molecules following TCR engagement. Primed T cells express the melanocortin 5 receptor (MC5r), a receptor that is linked to an intracellular signalling pathway shared by other cytokine receptors. Blocking the receptor with antibody prevented alpha-MSH from suppressing IFN-gamma production by the activated regulatory T cells, suggesting that alpha-MSH immunoregulation is through the MC5r on primed T cells. Surface staining and cell sorting of the alpha-MSH- treated primed T cells showed that the regulatory T cells are CD25+ CD4+ T cells. From these results we find that alpha-MSH can mediate the induction of CD25+ CD4+ regulatory T cells. These regulatory T cells require specific antigen for activation, but through non-specific TGF-beta1-mediated mechanisms they can suppress other effector T cells.     

Cutting edge: TGF-beta signaling is required for the in vivo expansion and immunosuppressive capacity of regulatory CD4+CD25+ T cells

Data regarding the role of TGF-beta for the in vivo function of regulatory CD4(+)CD25(+) T cells (Treg) are controversial. A transgenic mouse model with impaired TGF-beta signaling specifically in T cells was used to assess the role of endogenous TGF-beta for the in vivo function of CD4(+)CD25(+) Treg in a murine model of colitis induced by dextran sulfate. Transfer of wild-type, but not transgenic CD4(+)CD25(+) Treg was found to suppress colitis in wild-type mice. In addition, by transferring CFSE-labeled CD4(+)CD25(+) Treg we could demonstrate that endogenous TGF-beta promotes the expansion of CD4(+)CD25(+) Treg in vivo. Transgenic mice themselves developed reduced numbers of peripheral CD4(+)CD25(+) Treg and were more susceptible to the induction of colitis, which could be prevented by the transfer of wild-type Treg. These data indicate that TGF-beta signaling in CD4(+)CD25(+) Treg is required for their in vivo expansion and suppressive capacity.     

Suppression of CD4+ T lymphocyte effector functions by CD4+CD25+ cells in vivo

CD4+CD25+ regulatory T cells have been extensively studied during the last decade, but how these cells exert their regulatory function on pathogenic effector T cells remains to be elucidated. Naive CD4+ T cells transferred into T cell-deficient mice strongly expand and rapidly induce inflammatory bowel disease (IBD). Onset of this inflammatory disorder depends on IFN-gamma production by expanding CD4+ T cells. Coinjection of CD4+CD25+ regulatory T cells protects recipient mice from IBD. In this study, we show that CD4+CD25+ regulatory T cells do not affect the initial activation/proliferation of injected naive T cells as well as their differentiation into Th1 effectors. Moreover, naive T cells injected together with CD4+CD25+ regulatory T cells into lymphopenic hosts are still able to respond to stimuli in vitro when regulatory T cells are removed. In these conditions, they produce as much IFN-gamma as before injection or when injected alone. Finally, when purified, they are able to induce IBD upon reinjection into lymphopenic hosts. Thus, prevention of IBD by CD4+CD25+ regulatory T cells is not due to deletion of pathogenic T cells, induction of a non reactive state (anergy) among pathogenic effector T cells, or preferential induction of Th2 effectors rather than Th1 effectors; rather, it results from suppression of T lymphocyte effector functions, leading to regulated responses to self.     

pConsensus peptide induces tolerogenic CD8+ T cells in lupus-prone (NZB x NZW)F1 mice by differentially regulating Foxp3 and PD1 molecules

Systemic lupus erythematosus is an autoimmune disease caused primarily by autoantibodies (including IgG anti-DNA) and immune complexes that cause tissue damage. After tolerization with an artificial peptide (pConsensus, pCons) based on murine anti-DNA IgG sequences containing MHC class I and class II T cell determinants, lupus-prone (NZB x NZW)F(1) female (BWF(1)) mice develop regulatory CD4+CD25+ T cells and inhibitory CD8+ T cells, both of which suppress anti-DNA Ig production and immune glomerulonephritis. In the present work, we show that splenocytes from BWF(1) mice treated with pCons had significant expansion of primarily CD8+ T cells. CD4+ T cells and B cells were each directly suppressed by CD8+ T cells from tolerized mice in a contact-independent manner. Both pCons-induced CD8+CD28+ and CD8+CD28- T cells suppressed production of anti-DNA in vitro. Silencing with small interfering RNA of Foxp3 abrogated the suppression mediated by both CD8+ T cell subsets. Additionally, CD8+ T cells from tolerized mice were weakly cytotoxic against syngeneic B cells from old anti-DNA-producing mice, but not from young mice. Importantly, pCons treatment had dual effects on CD8+ suppressor T cells from tolerized mice, increasing the intracellular expression of Foxp3 while decreasing the surface expression of PD1 molecules. Blocking PD1/PDL1 interactions in the CD8+ T cells from tolerized mice reduced their expression of Foxp3 and their ability to suppress CD4+CD25- proliferation. In contrast, blocking PD1/PDL1 in naive T cells increased Foxp3 expression. Our data suggest that tolerization with pCons activates different subsets of inhibitory/cytotoxic CD8+ T cells whose targets are both CD4+CD25- effector T cells and B cells.     

Protection against autoimmunity in nonlymphopenic hosts by CD4+ CD25+ regulatory T cells is antigen-specific and requires IL-10 and TGF-beta

CD4+ CD25+ regulatory T cells (T(Reg)) play a critical role in the control of autoimmunity. However, little is known about how T(Reg) suppress self-reactive T cells in vivo, thus limiting the development of T(Reg)-based therapy for treating autoimmune diseases. This is in large part due to the dependency on a state of lymphopenia to demonstrate T(Reg)-mediated suppression in vivo and the unknown Ag specificity of T(Reg) in most experimental models. Using a nonlymphopenic model of autoimmune pneumonitis and T(Reg) with known Ag specificity, in this study we demonstrated that these T(Reg) can actively suppress activation of self-reactive T cells and protect mice from fatal autoimmune pneumonitis. The protection required T(Reg) with the same Ag specificity as the self-reactive T cells and depended on IL-10 and TGF-beta. These results suggest that suppression of autoimmunity by T(Reg) in vivo consists of multiple layers of regulation and advocate for a strategy involving Ag-specific T(Reg) for treating organ-specific autoimmunity, because they do not cause generalized immune suppression.     

CD4+CD25+ regulatory T cells inhibit the antigen-dependent expansion of self-reactive T cells in vivo

A deficiency of CD4+CD25+ regulatory T cells (CD25+ Tregs) in lymphopenic mice can result in the onset of autoimmune gastritis. The gastric H/K ATPase alpha (H/Kalpha) and beta (H/Kbeta) subunits are the immunodominant autoantigens recognized by effector CD4+ T cells in autoimmune gastritis. The mechanism by which CD25+ Tregs suppress autoimmune gastritis in lymphopenic mice is poorly understood. To investigate the antigenic requirements for the genesis and survival of gastritis-protecting CD25+ Tregs, we analyzed mice deficient in H/Kbeta and H/Kalpha, as well as a transgenic mouse line (H/Kbeta-tsA58 Tg line 224) that lacks differentiated gastric epithelial cells. By adoptive transfer of purified T cell populations to athymic mice, we show that the CD25+ Treg population from mice deficient in either one or both of H/Kalpha and H/Kbeta, or from the H/Kbeta-tsA58 Tg line 224 mice, is equally effective in suppressing the ability of polyclonal populations of effector CD4+ T cells to induce autoimmune gastritis. Furthermore, CD25+ Tregs, from either wild-type or H/Kalpha-deficient mice, dramatically reduced the expansion of pathogenic H/Kalpha-specific TCR transgenic T cells and the induction of autoimmune gastritis in athymic recipient mice. Proliferation of H/Kalpha-specific T cells in lymphopenic hosts occurs predominantly in the paragastric lymph node and was dependent on the presence of the cognate H/Kalpha Ag. Collectively, these studies demonstrate that the gastritis-protecting CD25+ Tregs do not depend on the major gastric Ags for their thymic development or their survival in the periphery, and that CD25+ Tregs inhibit the Ag-specific expansion of pathogenic T cells in vivo.     

Generation ex vivo of TGF-beta-producing regulatory T cells from CD4+CD25- precursors

Previously we reported that TGF-beta has an important role in the generation and expansion of human "professional" CD4(+)CD25(+) regulatory T cells in the periphery that have a cytokine-independent mechanism of action. In this study we used low-dose staphylococcal enterotoxin to induce T cell-dependent Ab production. We report that TGF-beta induces activated CD4(+)CD25(-) T cells to become Th3 suppressor cells. While stimulating CD4(+) cells with TGF-beta modestly increased expression of CD25 and intracellular CTLA-4 in primary cultures, upon secondary stimulation without TGF-beta the total number and those expressing these markers dramatically increased. This expansion was due to both increased proliferation and protection of these cells from activation-induced apoptosis. Moreover, adding as few as 1% of these TGF-beta-primed CD4(+) T cells to fresh CD4(+) cells and B cells markedly suppressed IgG production. The inhibitory effect was mediated by TGF-beta and was also partially contact dependent. Increased TGF-beta production was associated with a decreased production of IFN-gamma and IL-10. Depletion studies revealed that the precursors of these TGF-beta-producing CD4(+) suppressor cells were CD25 negative. These studies provide evidence that CD4(+)CD25(+) regulatory cells in human blood consist of at least two subsets that have TGF-beta-dependent and independent mechanisms of action. TGF-beta has an essential role in the generation of both of these T suppressor cell subsets from peripheral T cells. The ability to induce CD4(+) and CD8(+) cells to become regulatory cells ex vivo has the potential to be useful in the treatment of autoimmune diseases and to prevent transplant rejection.     

IL-10-producing CD4+CD25+ regulatory T cells play a critical role in granulocyte-macrophage colony-stimulating factor-induced suppression of experimental autoimmune thyroiditis

Our earlier study showed that GM-CSF has the potential not only to prevent, but also to suppress, experimental autoimmune thyroiditis (EAT). GM-CSF-induced EAT suppression in mice was accompanied by an increase in the frequency of CD4(+)CD25(+) regulatory T cells that could suppress mouse thyroglobulin (mTg)-specific T cell responses in vitro, but the underlying mechanism of this suppression was not elucidated. In this study we show that GM-CSF can induce dendritic cells (DCs) with a semimature phenotype, an important characteristic of DCs, which are known to play a critical role in the induction and maintenance of regulatory T cells. Adoptive transfer of CD4(+)CD25(+) T cells from GM-CSF-treated and mTg-primed donors into untreated, but mTg-primed, recipients resulted in decreased mTg-specific T cell responses. Furthermore, lymphocytes obtained from these donors and recipients after adoptive transfer produced significantly higher levels of IL-10 compared with mTg-primed, untreated, control mice. Administration of anti-IL-10R Ab into GM-CSF-treated mice abrogated GM-CSF-induced suppression of EAT, as indicated by increased mTg-specific T cell responses, thyroid lymphocyte infiltration, and follicular destruction. Interestingly, in vivo blockade of IL-10R did not affect GM-CSF-induced expansion of CD4(+)CD25(+) T cells. However, IL-10-induced immunosuppression was due to its direct effects on mTg-specific effector T cells. Taken together, these results indicated that IL-10, produced by CD4(+)CD25(+) T cells that were probably induced by semimature DCs, is essential for disease suppression in GM-CSF-treated mice.     

CD4+CD25+ T regulatory cells suppress NK cell-mediated immunotherapy of cancer

CD4+CD25+ regulatory T cells (Treg) that suppress T cell-mediated immune responses may also regulate other arms of an effective immune response. In particular, in this study we show that Treg directly inhibit NKG2D-mediated NK cell cytotoxicity in vitro and in vivo, effectively suppressing NK cell-mediated tumor rejection. In vitro, Treg were shown to inhibit NKG2D-mediated cytolysis largely by a TGF-beta-dependent mechanism and independently of IL-10. Adoptively transferred Treg suppressed NK cell antimetastatic function in RAG-1-deficient mice. Depletion of Treg before NK cell activation via NKG2D and the activating IL-12 cytokine, dramatically enhanced NK cell-mediated suppression of tumor growth and metastases. Our data illustrate at least one mechanism by which Treg can suppress NK cell antitumor activity and highlight the effectiveness of combining Treg inhibition with subsequent NK cell activation to promote strong innate antitumor immunity.