Very low-dose tolerance with nucleosomal peptides controls lupus and induces potent regulatory T cell subsets

We induced very low-dose tolerance by injecting lupus prone (SWR x NZB)F1 (SNF1) mice with 1 mug nucleosomal histone peptide autoepitopes s.c. every 2 wk. The subnanomolar peptide therapy diminished autoantibody levels and prolonged life span by delaying nephritis, especially by reducing inflammatory cell reaction and infiltration in kidneys. H4(71-94) was the most effective autoepitope. Low-dose tolerance therapy induced CD8+, as well as CD4+ CD25+ regulatory T (Treg) cell subsets containing autoantigen-specific cells. These adaptive Treg cells suppressed IFN-gamma responses of pathogenic lupus T cells to nucleosomal epitopes at up to a 1:100 ratio and reduced autoantibody production up to 90-100% by inhibiting nucleosome-stimulated T cell help to nuclear autoantigen-specific B cells. Both CD4+ CD25+ and CD8+ Treg cells produced and required TGF-beta1 for immunosuppression, and were effective in suppressing lupus autoimmunity upon adoptive transfer in vivo. The CD4+ CD25+ T cells were partially cell contact dependent, but CD8+ T cells were contact independent. Thus, low-dose tolerance with highly conserved histone autoepitopes repairs a regulatory defect in systemic lupus erythematosus by generating long-lasting, TGF-beta-producing Treg cells, without causing allergic/anaphylactic reactions or generalized immunosuppression.     

Antigen-specific therapy of murine lupus nephritis using nucleosomal peptides: tolerance spreading impairs pathogenic function of autoimmune T and B cells

In the (SWR x NZB)F1 mouse model of lupus, we previously localized the critical autoepitopes for nephritogenic autoantibody-inducing Th cells in the core histones of nucleosomes at aa positions 10-33 of H2B and 16-39 and 71-94 of H4. A brief therapy with the peptides administered i.v. to 3-mo-old prenephritic (SWR x NZB)F1 mice that were already producing pathogenic autoantibodies markedly delayed the onset of severe lupus nephritis. Strikingly, chronic therapy with the peptides injected into 18-mo-old (SWR x NZB)F1 mice with established glomerulonephritis prolonged survival and even halted the progression of renal disease. Remarkably, tolerization with any one of the nucleosomal peptides impaired autoimmune T cell help, inhibiting the production of multiple pathogenic autoantibodies. However, cytokine production or proliferative responses to the peptides were not grossly changed by the therapy. Moreover, suppressor T cells were not detected in the treated mice. Most interestingly, the best therapeutic effect was obtained with nucleosomal peptide H416-39, which had a tolerogenic effect not only on autoimmune Th cells, but autoimmune B cells as well, because this peptide contained both T and B cell autoepitopes. These studies show that the pathogenic T and B cells of lupus, despite intrinsic defects in activation thresholds, are still susceptible to autoantigen-specific tolerogens.     

Human CD4+CD25high regulatory T cells modulate myeloid but not plasmacytoid dendritic cells activation

Human CD4(+)CD25(+) regulatory T cells (Treg) play an essential role in the prevention of autoimmune diseases. However, the mechanisms of immune suppression and the spectrum of cells they target in vivo remain incompletely defined. In particular, although Treg directly suppress conventional T cells in vitro, they have been shown to inhibit the Ag-presenting functions of macrophage- and monocyte-derived dendritic cells (DC). We have now studied the maturation of human blood-derived myeloid DC and plasmacytoid DC activated with TLR ligands in the presence of Treg. Preactivated Treg suppressed strongly TLR-triggered myeloid DC maturation, as judged by the blocking of costimulatory molecule up-regulation and the inhibition of proinflammatory cytokines secretion that resulted in poor Ag presentation capacity. Although IL-10 played a prominent role in inhibiting cytokines secretion, suppression of phenotypic maturation required cell-cell contact and was independent of TGF-beta and CTLA-4. In contrast, the acquisition of maturation markers and production of cytokines by plasmacytoid DC triggered with TLR ligands were insensitive to regulatory T cells. Therefore, human Treg may enlist myeloid, but not plasmacytoid DC for the initiation and the amplification of tolerance in vivo by restraining their maturation after TLR stimulation.     

A defect in deletion of nucleosome-specific autoimmune T cells in lupus-prone thymus: role of thymic dendritic cells

To study central tolerance to the major product of ongoing apoptosis in the thymus, we made new lines of transgenic (Tg) mice expressing TCR of a pathogenic autoantibody-inducing Th cell that was specific for nucleosomes and its histone peptide H4(71-94). In the lupus-prone (SWR x NZB)F1 (SNF1) thymus, introduction of the lupus TCR transgene caused no deletion, but marked down-regulation of the Tg TCR and up-regulation of endogenous TCRs. Paradoxically, autoimmune disease was suppressed in the alphabetaTCR Tg SNF1 mice with induction of highly potent regulatory T cells in the periphery. By contrast, in the MHC-matched, normal (SWR x B10. D2)F1 (SBF1), or in the normal SWR backgrounds, marked deletion of transgenic thymocytes occurred. Thymic lymphoid cells of the normal or lupus-prone mice were equally susceptible to deletion by anti-CD3 Ab or irradiation. However, in the steady state, spontaneous presentation of naturally processed peptides related to the nucleosomal autoepitope was markedly greater by thymic dendritic cells (DC) from normal mice than that from lupus mice. Unmanipulated thymic DC of SNF1 mice expressed lesser amounts of MHC class II and costimulatory molecules than their normal counterparts. These results indicate that apoptotic nucleosomal autoepitopes are naturally processed and presented to developing thymocytes, and a relative deficiency in the natural display of nucleosomal autoepitopes by thymic DC occurs in lupus-prone SNF1 mice.     

CD8+ CD205+ splenic dendritic cells are specialized to induce Foxp3+ regulatory T cells

Foxp3(+)CD25(+)CD4(+) regulatory T cells (Treg) mediate immunological self-tolerance and suppress immune responses. A subset of dendritic cells (DCs) in the intestine is specialized to induce Treg in a TGF-beta- and retinoic acid-dependent manner to allow for oral tolerance. In this study we compare two major DC subsets from mouse spleen. We find that CD8(+) DEC-205/CD205(+) DCs, but not the major fraction of CD8(-) DC inhibitory receptor-2 (DCIR2)(+) DCs, induce functional Foxp3(+) Treg from Foxp3(-) precursors in the presence of low doses of Ag but without added TGF-beta. CD8(+)CD205(+) DCs preferentially express TGF-beta, and the induction of Treg by these DCs in vitro is blocked by neutralizing Ab to TGF-beta. In contrast, CD8(-)DCIR2(+) DCs better induce Foxp3(+) Treg when exogenous TGF-beta is supplied. In vivo, CD8(+)CD205(+) DCs likewise preferentially induce Treg from adoptively transferred, Ag-specific DO11.10 RAG(-/-) Foxp3(-)CD4(+) T cells, whereas the CD8(-)DCIR2(+) DCs better stimulate natural Foxp3(+) Treg. These results indicate that a subset of DCs in spleen, a systemic lymphoid organ, is specialized to differentiate peripheral Foxp3(+) Treg, in part through the endogenous formation of TGF-beta. Targeting of Ag to these DCs might be useful for inducing Ag-specific Foxp3(+) Treg for treatment of autoimmune diseases, transplant rejection, and allergy.     

CD8+ T cell-mediated suppression of autoimmunity in a murine lupus model of peptide-induced immune tolerance depends on Foxp3 expression

Systemic lupus erythematosus is an autoimmune disease caused by autoantibodies, including IgG anti-DNA. New Zealand Black/New Zealand White F(1) female mice, a model of spontaneous polygenic systemic lupus erythematosus, tolerized with an artificial peptide (pConsensus) based on anti-DNA IgG sequences containing MHC class I and class II T cell determinants, develop regulatory CD4+CD25+ T cells and CD8+ inhibitory T cells (CD8+ Ti), both of which suppress autoantibody production. CD8+ Ti inhibit primarily via secretion of TGF-beta. In the present study, we show that the inhibitory function of CD8+ T cells from tolerized mice is sustained for up to 8 wk and at all times depends on expression of Foxp3. Both CD28-positive and CD28-negative CD8+ T cells contain inhibitory cells, but the expression of mRNA for Foxp3 and for TGF-beta is higher and lasts longer in the CD28- subset. In vitro addition of TGF-beta (in the presence of IL-2) induces Foxp3 expression in a dose-response manner. Gene inhibition or blockade with small interfering RNA of Foxp3 abrogates the ability of the CD8+ Ti to inhibit anti-DNA production and the proliferation of CD4+ Th cells. Moreover, a significant correlation between expression of Foxp3 and ability of CD8+ Ti to secrete TGF-beta is observed. Therefore, CD8+ Ti in this system of tolerance are similar to CD4+CD25+ regulatory T cells in their dependence on expression of Foxp3, and there may be a bidirectional Foxp3/TGF-beta autocrine loop that determines the ability of the CD8+ T cells to control autoimmunity.     

Identification of a minimal T cell epitope recognized by antinucleosome Th cells in the C-terminal region of histone H4

Autoreactive T cells responding to systemic autoantigens have been characterized in patients and mice with autoimmune diseases and in healthy individuals. Using peptides covering the whole sequence of histone H4, we characterized several epitopes recognized by lymph node Th cells from nonsystemic lupus erythematosus-prone mice immunized with the same peptides, the H4 protein, or nucleosomes. Multiple T epitopes were identified after immunizing H-2d BALB/c mice with H4 peptides. They spanned residues 28-42, 30-47, 66-83, 72-89, and 85-102. Within the region 85-102, a minimal CD4+ T epitope containing residues 88-99 was characterized. Although Abs to peptide 88-99 recognized H4, this peptide does not contain a dominant B cell epitope recognized by anti-H4 Abs raised in BALB/c mice or Abs from NZB/NZW H-2d/z lupus mice. Th cells primed in vivo with H4 responded to H4, but not to peptide 88-99. However, this peptide was able to stimulate the proliferation and IL-2 secretion of Th cells generated after immunization with nucleosomes. H488-99 thus represents a cryptic epitope with regard to H4 and a supradominant epitope presented by nucleosome, a supramolecular complex that plays a key role in lupus. This study shows that in the normal repertoire of naive BALB/c mice, autoreactive Th cells specific for histones are not deleted. The reactivity of these Th cells seems to be relatively restricted and resembles that of Th clones generated from SNF1 ((SWR x NZB)F1; I-Ad/q) lupus mice described earlier.     

Mechanisms of nasal tolerance induction in experimental autoimmune myasthenia gravis: identification of regulatory cells

Autoantigen administration via nasal mucosal tissue can induce systemic tolerance more effectively than oral administration in a number of experimental autoimmune diseases, including Ab-mediated experimental autoimmune myasthenia gravis, a murine model of myasthenia gravis. The mechanisms underlying nasal tolerance induction are not clear. In this study, we show that nasal administration of acetylcholine receptor (AChR) in C57BL/6 mice, before immunizations with AChR in adjuvant, results in delayed onset and reduced muscle weakness compared with control mice. The delayed onset and reduced muscle weakness were associated with decreased AChR-specific lymphocyte proliferation and decreased levels of anti-AChR Abs of the IgG2a and IgG2b isotypes in serum. The clinical and immunological changes in the AChR-pretreated C57BL/6 wild-type (wt) mice were comparable with those observed in AChR-pretreated CD8-/- mice, indicating that CD8+ T cells were not required for the generation of nasal tolerance. AChR-pretreated wt and CD8-/- mice showed augmented TGF-beta and reduced IFN-gamma responses, whereas levels of IL-4 were unaltered. Splenocytes from AChR-pretreated wt and CD8-/- mice, but not from CD4-/- mice, suppressed AChR-specific lymphocyte proliferation. This suppression could be blocked by Abs against TGF-beta. Thus, our results demonstrate that the suppression induced in the present model is independent of CD8+ T cells and suggest the involvement of Ag-specific CD4+ Th3 cells producing TGF-beta.     

Pertussis toxin by inducing IL-6 promotes the generation of IL-17-producing CD4 cells

Compelling evidence has now demonstrated that IL-17-producing CD4 cells (Th17) are a major contributor to autoimmune pathogenesis, whereas CD4+CD25+ T regulatory cells (Treg) play a major role in suppression of autoimmunity. Differentiation of proinflammatory Th17 and immunosuppressive Treg from naive CD4 cells is reciprocally related and contingent upon the cytokine environment. We and others have reported that in vivo administration of pertussis toxin (PTx) reduces the number and function of mouse Treg. In this study, we have shown that supernatants from PTx-treated mouse splenic cells, which contained IL-6 and other proinflammatory cytokines, but not PTx itself, overcame the inhibition of proliferation seen in cocultures of Treg and CD4+CD25- T effector cells. This stimulatory effect could be mimicked by individual inflammatory cytokines such as IL-1beta, IL-6, and TNF-alpha. The combination of these cytokines synergistically stimulated the proliferation of CD4+CD25- T effector cells despite the presence of Treg with a concomitant reduction in the percentage of FoxP3+ cells and generation of IL-17-expressing cells. PTx generated Th17 cells, while inhibiting the differentiation of FoxP+ cells, from naive CD4 cells when cocultured with bone marrow-derived dendritic cells from wild-type mice, but not from IL-6-/- mice. In vivo treatment with PTx induced IL-17-secreting cells in wild-type mice, but not in IL-6-/- mice. Thus, in addition to inhibiting the development of Treg, the immunoadjuvant activity of PTx can be attributable to the generation of IL-6-dependent IL-17-producing CD4 cells.     

TGF-beta expression by allogeneic bone marrow stromal cells ameliorates diabetes in NOD mice through modulating the distribution of CD4+ T cell subsets

BMSCs could promote the regeneration of islet beta-cell, but the status of BMSCs under diabetes is still unknown. Our study verified the effect of allogeneic BMSCs (ICR) transferred into NOD mice on blood glucose and CD4+ T cells subsets function. In vivo experiment, BMSCs could decrease blood glucose, weaken lymphocytes proliferation. In vitro experiment, the distribution of CD4+ T cell subsets was changed after co-culture with BMSCs, resulting in a greater frequency of Treg cells and reduced representation of Th17 cells. After TGF-beta blockade, CD4+ T cells differentiated along a route favoring development of Th17, but not Treg cells. Thus, NOD can be treated by BMSCs which changes the distribution of CD4+ T cells, increases the number of Treg cells, and inhibits the differentiation of Th17 cells. And the positive effects of allogeneic BMSCs in the treatment of NOD mice depend on the regulation of TGF-beta secreted by BMSCs.     

Th3 cells in peripheral tolerance. I. Induction of Foxp3-positive regulatory T cells by Th3 cells derived from TGF-beta T cell-transgenic mice

TGF-beta has been shown to be critical in the generation of CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs). Because Th3 cells produce large amounts of TGF-beta, we asked whether induction of Th3 cells in the periphery was a mechanism by which CD4(+)CD25(+) Tregs were induced in the peripheral immune compartment. To address this issue, we generated a TGF-beta1-transgenic (Tg) mouse in which TGF-beta is linked to the IL-2 promoter and T cells transiently overexpress TGF-beta upon TCR stimulation but produce little or no IL-2, IL-4, IL-10, IL-13, or IFN-gamma. Naive TGF-beta-Tg mice are phenotypically normal with comparable numbers of lymphocytes and thymic-derived Tregs. We found that repeated antigenic stimulation of pathogenic myelin oligodendrocyte glycoprotein (MOG)-specific CD4(+)CD25(-) T cells from TGF-beta Tg mice crossed to MOG TCR-Tg mice induced Foxp3 expression in both CD25(+) and CD25(-) populations. Both CD25 subsets were anergic and had potent suppressive properties in vitro and in vivo. Furthermore, adoptive transfer of these induced regulatory CD25(+/-) T cells suppressed experimental autoimmune encephalomyelitis when administrated before disease induction or during ongoing experimental autoimmune encephalomyelitis. The suppressive effect of TGF-beta on T cell responses was due to the induction of Tregs and not to the direct inhibition of cell proliferation. The differentiation of Th3 cells in vitro was TGF-beta dependent as anti-TGF-beta abrogated their development. Thus, Ag-specific TGF-beta-producing Th3 cells play a crucial role in inducing and maintaining peripheral tolerance by driving the differentiation of Ag-specific Foxp3(+) regulatory cells in the periphery.     

Enhanced regulatory T cell activity is an element of the host response to injury

CD4+CD25+ regulatory T cells (Tregs) play a critical role in suppressing the development of autoimmune disease, in controlling potentially harmful inflammatory responses, and in maintaining immune homeostasis. Because severe injury triggers both excessive inflammation and suppressed adaptive immunity, we wished to test whether injury could influence Treg activity. Using a mouse burn injury model, we demonstrate that injury significantly enhances Treg function. This increase in Treg activity is apparent at 7 days after injury and is restricted to lymph node CD4+CD25+ T cells draining the injury site. Moreover, we show that this injury-induced increase in Treg activity is cell-contact dependent and is mediated in part by increased cell surface TGF-beta1 expression. To test the in vivo significance of these findings, mice were depleted of CD4+CD25+ T cells before sham or burn injury and then were immunized to follow the development of T cell-dependent Ag-specific immune reactivity. We observed that injured mice, which normally demonstrate suppressed Th1-type immunity, showed normal Th1 responses when depleted of CD4+CD25+ T cells. Taken together, these observations suggest that injury can induce or amplify CD4+CD25+ Treg function and that CD4+CD25+ T cells contribute to the development of postinjury immune suppression.     

Reprogrammed FoxP3+ T regulatory cells become IL-17+ antigen-specific autoimmune effectors in vitro and in vivo

Lymphocyte differentiation from naive CD4(+) T cells into mature Th1, Th2, Th17, or T regulatory cell (Treg) phenotypes has been considered end stage in character. In this study, we demonstrate that dendritic cells (DCs) activated with a novel immune modulator B7-DC XAb (DC(XAb)) can reprogram Tregs into T effector cells. Down-regulation of FoxP3 expression after either in vitro or in vivo Treg-DC(XAb) interaction is Ag-specific, IL-6-dependent, and results in the functional reprogramming of the mature T cell phenotype. The reprogrammed Tregs cease to express IL-10 and TGFbeta, fail to suppress T cell responses, and gain the ability to produce IFN-gamma, IL-17, and TNF-alpha. The ability of IL-6(+) DC(XAb) and the inability of IL-6(-/-) DC(XAb) vaccines to protect animals from lethal melanoma suggest that exogenously modulated DC can reprogram host Tregs. In support of this hypothesis and as a test for Ag specificity, transfer of DC(XAb) into RIP-OVA mice causes a break in immune tolerance, inducing diabetes. Conversely, adoptive transfer of reprogrammed Tregs but not similarly treated CD25(-) T cells into naive RIP-OVA mice is also sufficient to cause autoimmune diabetes. Yet, treatment of normal mice with B7-DC XAb fails to elicit generalized autoimmunity. The finding that mature Tregs can be reprogrammed into competent effector cells provides new insights into the plasticity of T cell lineage, underscores the importance of DC-T cell interaction in balancing immunity with tolerance, points to Tregs as a reservoir of autoimmune effectors, and defines a new approach for breaking tolerance to self Ags as a strategy for cancer immunotherapy.     

Neuron-mediated generation of regulatory T cells from encephalitogenic T cells suppresses EAE

Neurons have been neglected as cells with a major immune-regulatory function because they do not express major histocompatibility complex class II. Our data show that neurons are highly immune regulatory, having a crucial role in governing T-cell response and central nervous system (CNS) inflammation. Neurons induce the proliferation of activated CD4+ T cells through B7-CD28 and transforming growth factor (TGF)-beta1-TGF-beta receptor signaling pathways, resulting in amplification of T-cell receptor signaling through phosphorylated ZAP-70, interleukin (IL)-2 and IL-9. The interaction between neurons and T cells results in the conversion of encephalitogenic T cells to CD25+ TGF-beta1+ CTLA-4+ FoxP3+ T regulatory (Treg) cells that suppress encephalitogenic T cells and inhibit experimental autoimmune encephalomyelitis. Suppression is dependent on cytotoxic T lymphocyte antigen (CTLA)-4 but not TGF-beta1. Autocrine action of TGF-beta1, however, is important for the proliferative arrest of Treg cells. Blocking the B7 and TGF-beta pathways prevents the CNS-specific generation of Treg cells. These findings show that generation of neuron-dependent Treg cells in the CNS is instrumental in regulating CNS inflammation.     

Expression of a soluble TGF-beta receptor by tumor cells enhances dendritic cell/tumor fusion vaccine efficacy

Dendritic cell (DC)-based antitumor immunotherapy is a promising cancer therapy. We have previously shown that tumor-derived TGF-beta limits the efficacy of the DC/tumor fusion vaccine in mice. In the current study we investigated the effect of neutralizing tumor-derived TGF-beta on the efficacy of the DC/tumor fusion vaccine. An adenovirus encoding human TGF-beta receptor type II fused to the Fc region of human IgM (Adv-TGF-beta-R) or a control adenovirus encoding LacZ (Adv-LacZ) was used to express a soluble form of the neutralizing TGF-beta receptor (TGF-beta-R). Murine breast carcinoma cells, 4T1, but not bone marrow-derived DCs, were successfully transfected with Adv-TGF-beta-R (4T1+Adv-TGF-beta-R) using a multiplicity of infection of 300. Immunization with irradiated 4T1+Adv-TGF-beta-R tumor cells conferred enhanced antitumor immunity compared with immunization with irradiated 4T1+Adv-LacZ tumor cells. The DC/4T1+Adv-TGF-beta-R fusion vaccine offered enhanced protective and therapeutic efficacy compared with the DC/4T1-Adv-LacZ fusion vaccine. Because TGF-beta is known to induce regulatory T cells (Tregs), we further showed that the DC/4T1+Adv-TGF-beta-R fusion vaccine induced fewer CD4(+)CD25(+)Foxp3(+) Tregs than the DC/4T1+Adv-LacZ fusion vaccine in vitro and in vivo. The suppressive role of splenic CD4(+)CD25(+) Tregs isolated from mice immunized with DC/4T1+Adv-LacZ was demonstrated using a CTL killing assay. Similar enhanced therapeutic efficacy was observed in murine renal cell carcinoma, RenCa, which expresses a high level of TGF-beta. We conclude that the blockade of tumor-derived TGF-beta reduces Treg induction by the DC/tumor fusion vaccine and enhances antitumor immunity. This may be an effective strategy to enhance human DC-based antitumor vaccines.     

In vitro and in vivo down-regulation of regulatory T cell activity with a peptide inhibitor of TGF-beta1

Down-regulation of CD4+CD25+ regulatory T (Treg) cell function might be beneficial to enhance the immunogenicity of viral and tumor vaccines or to induce breakdown of immunotolerance. Although the mechanism of suppression used by Treg cells remains controversial, it has been postulated that TGF-beta1 mediates their immunosuppressive activity. In this study, we show that P17, a short synthetic peptide that inhibits TGF-beta1 and TGF-beta2 developed in our laboratory, is able to inhibit Treg activity in vitro and in vivo. In vitro studies demonstrate that P17 inhibits murine and human Treg-induced unresponsiveness of effector T cells to anti-CD3 stimulation, in an MLR or to a specific Ag. Moreover, administration of P17 to mice immunized with peptide vaccines containing tumor or viral Ags enhanced anti-vaccine immune responses and improved protective immunogenicity against tumor growth or viral infection or replication. When CD4+ T cells purified from OT-II transgenic mice were transferred into C57BL/6 mice bearing s.c. EG.7-OVA tumors, administration of P17 improved their proliferation, reduced the number of CD4+Foxp3+ T cells, and inhibited tumor growth. Also, P17 prevented development of immunotolerance induced by oral administration of OVA by genetically modified Lactococcus lactis in DO11.10 transgenic mice sensitized by s.c. injection of OVA. These findings demonstrate that peptide inhibitors of TGF-beta may be a valuable tool to enhance vaccination efficacy and to break tolerance against pathogens or tumor Ags.     

Dendritic cell type determines the mechanism of bystander suppression by adaptive T regulatory cells specific for the minor antigen HA-1

One hallmark of acquired tolerance is bystander suppression, a process whereby Ag-specific (adaptive) T regulatory cells (TR) inhibit the T effector cell response both to specific Ag and to a colocalized third-party Ag. Using peripheral blood T cells from recipients of HLA-identical kidney transplants as responders in the trans vivo-delayed type hypersensitivity assay, we found that dendritic cells (DC), but not monocyte APCs, could mediate bystander suppression of EBV-specific recall response. When HA-1(H) peptide was added to mixtures of plasmacytoid DC (pDC) and T cells, bystander suppression of the response to a colocalized recall Ag occurred primarily via indolamine-2,3-dioxygenase (IDO) production. Similarly, addition of HA-1(H) peptide to cocultures of T cells and pDC, but not myeloid DC (mDC), induced IDO activity in vitro. When mDC presented HA-1(H) peptide to Ag-specific CD8+ TR, cytokine release (TGF-beta, IL-10, or both) was the primary mode of bystander suppression. Bystander suppression via mDC was reversed not only by Ab to TGF-beta and its receptor on T cells, but also by Ab to thrombospondin-1. EBV addition did not induce IDO or thrombospondin-1 in T-DC cocultures, suggesting that these DC products are not induced by T effector cells, but only by TR cells. These results shed light upon the mechanism of bystander suppression by donor Ag-specific TR in patients with organ transplant tolerance and underscores the distinct and critical roles of mDC and pDCs in this phenomenon.     

Dicer insufficiency and microRNA-155 overexpression in lupus regulatory T cells: an apparent paradox in the setting of an inflammatory milieu

Systemic lupus erythematosus is a chronic autoimmune disease characterized by loss of tolerance to self-Ags and activation of autoreactive T cells. Regulatory T (Treg) cells play a critical role in controlling the activation of autoreactive T cells. In this study, we investigated mechanisms of potential Treg cell defects in systemic lupus erythematosus using MRL-Fas(lpr/lpr) (MRL/lpr) and MRL-Fas(+/+) mouse models. We found a significant increase in CD4(+)CD25(+)Foxp3(+) Treg cells, albeit with an altered phenotype (CD62L(-)CD69(+)) and with a reduced suppressive capacity, in the lymphoid organs of MRL strains compared with non-autoimmune C3H/HeOuj mice. A search for mechanisms underlying the altered Treg cell phenotype in MRL/lpr mice led us to find a profound reduction in Dicer expression and an altered microRNA (miRNA, miR) profile in MRL/lpr Treg cells. Despite having a reduced level of Dicer, MRL/lpr Treg cells exhibited a significant overexpression of several miRNAs, including let-7a, let-7f, miR-16, miR-23a, miR-23b, miR-27a, and miR-155. Using computational approaches, we identified one of the upregulated miRNAs, miR-155, that can target CD62L and may thus confer the altered Treg cell phenotype in MRL/lpr mice. In fact, the induced overexpression of miR-155 in otherwise normal (C3H/HeOuj) Treg cells reduced their CD62L expression, which mimics the altered Treg cell phenotype in MRL/lpr mice. These data suggest a role of Dicer and miR-155 in regulating Treg cell phenotype. Furthermore, simultaneous appearance of Dicer insufficiency and miR-155 overexpression in diseased mice suggests a Dicer-independent alternative mechanism of miRNA regulation under inflammatory conditions.     

Targeted CTLA-4 engagement induces CD4+CD25+CTLA-4high T regulatory cells with target (allo)antigen specificity

CTLA-4 (CD152) is actively involved in down-regulating T cell activation and maintaining lymphocyte homeostasis. Our earlier studies showed that targeted engagement of CTLA-4 can down-modulate T cell response and suppress allo- and autoimmune responses. In this study, we report that targeted CTLA-4 engagement can induce immune tolerance to a specific target through selective induction of an Ag-specific CD4(+)CD25(+)CTLA-4(high) regulatory T cell (Treg cell) population. Allogeneic cells coated with anti-CTLA-4 Ab induced immune hyporesponsiveness through suppression of proinflammatory cytokines IFN-gamma and IL-2, and up-regulation of the regulatory cytokines IL-10, TGF-beta1, and IL-4, presumably through the engagement of CTLA-4 on activated T cells. Although rechallenge with alloantigen failed to break the unresponsiveness, a transient recovery from tolerance was observed in the presence of high concentrations of exogenous IL-2, saturating concentrations of neutralizing anti-TGF-beta1 and anti-IL-10 Abs, and blocking anti-CTLA-4 Ab, and upon depletion of CD4(+)CD25(+) Treg cells. The CD4(+)CD25(+)CTLA-4(high) Treg cells from tolerant mice suppressed the effector function of CD25(-) T cells from Ag-primed mice. Adoptive transfer of these Treg cells into Ag-primed mice resulted in a significantly reduced alloantigen-specific response. Further characterization demonstrated that the Treg cells with memory phenotype (CD62L(-)) were more potent in suppressing the alloantigen-specific T cell response. These results strongly support that the targeted engagement of CTLA-4 has therapeutic potential for the prevention of transplant rejection.