Medullary thymic epithelial cells and CD8α+ dendritic cells coordinately regulate central tolerance but CD8α+ cells are dispensable for thymic regulatory T cell production.

In the thymus, antigen presenting cells (APCs) namely, medullary thymic epithelial cells (mTECs) and thymic dendritic cells (tDCs) regulate T cell tolerance through elimination of autoreactive T cells and production of thymic T regulatory (tTreg) cells. How the different APCs in the thymus share the burden of tolerazing the emerging T cell repertoire remains unclear. For example, while mutations that inhibit mTEC development or function associate with peripheral autoimmunity, the role of tDCs in organ-specific autoimmunity and tTreg cell production remains controversial. In this report we used mice depleted of mTECs and/or CD8α+ DCs, to examine the contributions of these cell populations in thymic tolerance. We found that while mice depleted of CD8α+ DCs or mTECs were normal or developed liver inflammation respectively, combined depletion of mTECs and CD8α+ DCs resulted in overt peripheral autoimmunity. The autoimmune manifestations in mice depleted of both mTECs and CD8α+ cDCs associated with increased percentages of CD4+ and CD8+ T cells in the thymus. In contrast, while mTEC depletion resulted in reduced percentages of tTreg cells, no additional effect was observed when CD8α+ DCs were also depleted. These results reveal that: 1) mTECs and CD8α+ DCs cooperatively safeguard against peripheral autoimmunity through thymic T cell deletion; 2) CD8α+ DCs are dispensable for tTreg cell production, whereas mTECs play a non-redundant role in this process; 3) mTECs and CD8α+ DCs make unique contributions to tolerance induction that cannot be compensated for by other thymic APCs such as migratory SIRPα+ or plasmacytoid DCs.

Medullary thymic epithelial cells and central tolerance in autoimmune hepatitis development: novel perspective from a new mouse model.

Autoimmune hepatitis (AIH) is an immune-mediated disorder that affects the liver parenchyma. Diagnosis usually occurs at the later stages of the disease, complicating efforts towards understanding the causes of disease development. While animal models are useful for studying the etiology of autoimmune disorders, most of the existing animal models of AIH do not recapitulate the chronic course of the human condition. In addition, approaches to mimic AIH-associated liver inflammation have instead led to liver tolerance, consistent with the high tolerogenic capacity of the liver. Recently, we described a new mouse model that exhibited spontaneous and chronic liver inflammation that recapitulated the known histopathological and immunological parameters of AIH. The approach involved liver-extrinsic genetic engineering that interfered with the induction of T-cell tolerance in the thymus, the very process thought to inhibit AIH induction by liver-specific expression of exogenous antigens. The mutation led to depletion of specialized thymic epithelial cells that present self-antigens and eliminate autoreactive T-cells before they exit the thymus. Based on our findings, which are summarized below, we believe that this mouse model represents a relevant experimental tool towards elucidating the cellular and molecular aspects of AIH development and developing novel therapeutic strategies for treating this disease.

Medullary thymic epithelial cell depletion leads to autoimmune hepatitis.

TRAF6, an E3 ubiquitin protein ligase, plays a critical role in T cell tolerance by regulating medullary thymic epithelial cell (mTEC) development. mTECs regulate T cell tolerance by ectopically expressing self-antigens and eliminating autoreactive T cells in the thymus. Here we show that mice with mTEC depletion due to conditional deletion of Traf6 expression in murine thymic epithelial cells (Traf6ΔTEC mice) showed a surprisingly narrow spectrum of autoimmunity affecting the liver. The liver inflammation in Traf6ΔTEC mice exhibited all the histological and immunological characteristics of human autoimmune hepatitis (AIH). The role of T cells in AIH establishment was supported by intrahepatic T cell population changes and AIH development after transfer of liver T cells into immunodeficient mice. Despite a 50% reduction in natural Treg thymic output, peripheral tolerance in Traf6ΔTEC mice was normal, whereas compensatory T regulatory mechanisms were evident in the liver of these animals. These data indicate that mTECs exert a cell-autonomous role in central T cell tolerance and organ-specific autoimmunity, but play a redundant role in peripheral tolerance. These findings also demonstrate that Traf6ΔTEC mice are a relevant model with which to study the pathophysiology of AIH, as well as autoantigen-specific T cell responses and regulatory mechanisms underlying this disease.

Lymph-migrating, tissue-derived dendritic cells are minor constituents within steady-state lymph nodes.

Observations that dendritic cells (DCs) constitutively enter afferent lymphatic vessels in many organs and that DCs in some tissues, such as the lung, turnover rapidly in the steady state have led to the concept that a major fraction of lymph node DCs are derived from migratory DCs that enter the lymph node through upstream afferent lymphatic vessels. We used the lysozyme M-Cre reporter mouse strain to assess the relationship of lymph node and nonlymphoid organ DCs. Our findings challenge the idea that a substantial proportion of lymph node DCs derive from the upstream tissue during homeostasis. Instead, our analysis suggests that nonlymphoid organ DCs comprise a major population of DCs within lymph nodes only after introduction of an inflammatory stimulus.

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.

Dendritic cells are specialized accessory cells along with TGF- for the differentiation of Foxp3+ CD4+ regulatory T cells from peripheral Foxp3 precursors.

Foxp3(+)CD25(+)CD4(+) regulatory T cells are produced in the thymus (natural T regs) but can also differentiate from peripheral Foxp3(-)CD4(+) precursors (induced or adaptive T regs). We assessed antigen presenting cell (APC) requirements for the latter differentiation. With added transforming growth factor (TGF)-beta, both immature and mature populations of dendritic cells (DCs) induced antigen-specific Foxp3(+) T regs from Foxp3(-) precursors. Using endogenous TGF-beta, DCs from gut-associated mesenteric lymph nodes were capable of differentiating Foxp3(+)T regs. Spleen DCs were 100-fold more potent than DC-depleted APCs for the induction of T regs and required 10-fold lower doses of peptide antigen. Interleukin-2 (IL-2) was essential, but could be provided endogenously by T cells stimulated by DCs, but not other APCs. The required IL-2 was induced by DCs that expressed CD80/CD86 costimulatory molecules. The DC-induced Foxp3(+)T regs divided up to 6 times in 6 days and were comprised of CD62L and CD103 positive and negative forms. The induced Foxp3(+)T regs exerted suppression in vitro and blocked tumor immunity in vivo. These results indicate that DCs are specialized to differentiate functional peripheral Foxp3(+)T regs and help set the stage to use DCs to actively suppress the immune response in an antigen-specific manner.

Glycolipid alpha-C-galactosylceramide is a distinct inducer of dendritic cell function during innate and adaptive immune responses of mice.

alpha-Galactosylceramide (alpha-GalCer) is the prototype compound for studying the presentation of glycolipids on CD1d molecules to natural killer T (NKT) lymphocytes. A single i.v. dose of glycolipid triggers a cascade of events involving the production of several cytokines over the course of a day, a short-lived activation of NKT and natural killer (NK) cells, and a more prolonged adaptive T cell immune response if certain antigens are given together with alpha-GalCer. We find that a recently described analogue, alpha-C-galactosylceramide (alpha-C-GalCer), more potently induces these innate and adaptive immune responses in mice. alpha-C-GalCer acts as a more effective trigger for IL-12 and IFN-gamma production, although it minimally elicits IL-4 and TNF-alpha release into the serum. Also, alpha-C-GalCer better mobilizes NKT and natural killer cells to resist B16 melanoma. To help understand these effects, we find that alpha-C-GalCer binds more stably to dendritic cells than alpha-GalCer and that dendritic cells loaded with alpha-C-GalCer induce larger and more long lasting NKT cell responses in vivo. When glycolipid is targeted to dendritic cells in spleen together with antigens in dying cells, such as irradiated tumor cells, alpha-C-GalCer is active as an adjuvant for T cell-mediated immunity at lower doses, just 20 ng per mouse, where it is also able to up-regulate the required CD40L costimulatory molecule on NKT cells. Therefore, alpha-C-GalCer represents a glycolipid that binds more stably to dendritic cells and acts as a more effective link between innate and adaptive immunity in vivo.

The linkage of innate to adaptive immunity via maturing dendritic cells in vivo requires CD40 ligation in addition to antigen presentation and CD80/86 costimulation.

Dendritic cell (DC) maturation is an innate response that leads to adaptive immunity to coadministered proteins. To begin to identify underlying mechanisms in intact lymphoid tissues, we studied alpha-galactosylceramide. This glycolipid activates innate Valpha14(+) natural killer T cell (NKT) lymphocytes, which drive DC maturation and T cell responses to ovalbumin antigen. Hours after giving glycolipid i.v., tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma were released primarily by DCs. These cytokines induced rapid surface remodeling of DCs, including increased CD80/86 costimulatory molecules. Surprisingly, DCs from CD40(-/-) and CD40L(-/-) mice did not elicit CD4(+) and CD8(+) T cell immunity, even though the DCs exhibited presented ovalbumin on major histocompatibility complex class I and II products and expressed high levels of CD80/86. Likewise, an injection of TNF-alpha up-regulated CD80/86 on DCs, but CD40 was required for immunity. CD40 was needed for DC interleukin (IL)-12 production, but IL-12p40(-/-) mice generated normal ovalbumin-specific responses. Therefore, the link between innate and adaptive immunity via splenic DCs and innate NKT cells has several components under distinct controls: antigen presentation in the steady state, increases in costimulatory molecules dependent on inflammatory cytokines, and a distinct CD40/CD40L signal that functions together with antigen presentation ("signal one") and costimulation ("signal two") to generate functioning CD4(+) T helper cell 1 and CD8(+) cytolytic T lymphocytes.