Darren J. Lee

Both MC5r and A2Ar Are Required for Protective Regulatory Immunity in the Spleen of Post–Experimental Autoimmune Uveitis in Mice

The ocular microenvironment uses a poorly defined melanocortin 5 receptor (MC5r)-dependent pathway to recover immune tolerance following intraocular inflammation. This dependency is seen in experimental autoimmune uveoretinitis (EAU), a mouse model of endogenous human autoimmune uveitis, with the emergence of autoantigen-specific regulatory immunity in the spleen that protects the mice from recurrence of EAU. In this study, we found that the MC5r-dependent regulatory immunity increased CD11b+F4/80+Ly-6ClowLy-6G+CD39+CD73+ APCs in the spleen of post-EAU mice. These MC5r-dependent APCs require adenosine 2A receptor expression on T cells to activate EAU-suppressing CD25+CD4+Foxp3+ regulatory T cells. Therefore, in the recovery from autoimmune disease, the ocular microenvironment induces tolerance through a melanocortin-mediated expansion of Ly-6G+ regulatory APCs in the spleen that use the adenosinergic pathway to promote activation of autoantigen-specific regulatory T cells.

Following EAU Recovery There Is an Associated MC5r-Dependent APC Induction of Regulatory Immunity in the Spleen

PURPOSE IRBPp-specific regulatory immunity is found in the spleens of mice recovered from experimental autoimmune uveoretinitis (EAU). Induction of this regulatory immunity is dependent on the expression of the melanocortin 5 receptor (MC5r). Therefore, the authors investigated whether dependence on the expression of MC5r was with the T cells or with the APCs mediating protective regulatory immunity in the EAU-recovered mouse spleen. METHODS Wild-type and MC5r-/- mice were immunized to induce EAU. The IRBPp-stimulated T-cell response in spleens of wild-type and MC5r-/- mice were compared for surface markers and cytokine production. Spleen APC were isolated and used to stimulate cytokine production and regulatory activity in IRBP-specific T cells from wild-type or MC5r-/- mice assayed in culture by ELISA, by flow cytometry, and in vivo by adoptive transfer into EAU mice. RESULTS IRBPp-specific CD25+CD4+ T cells from spleens of EAU-recovered wild-type mice express a Treg cell phenotype of FoxP3 and TGF-β compared with the effector T-cell phenotype of IFN-γ and IL-17 production in EAU-recovered MC5r-/- mice. APCs from the spleens of wild-type mice recovering from EAU promoted regulatory T-cell activation in IRBP-specific effector T cells from the spleens of EAU-recovering MC5r-/- mice. Spleen APCs from EAU-recovering wild-type, but not MC5r-/-, mice induced TGF-β expression by primed IRBP-specific effector T cells. CONCLUSIONS Dependence on MC5r expression is with an APC that promotes or selectively activates IRBP-specific FoxP3+ TGF-β+ CD25+CD4+ Treg cells in the spleens of EAU-recovered mice.

Recovery from experimental autoimmune uveitis promotes induction of antiuveitic inducible Tregs

The recovery of EAU, a mouse model of endogenous human autoimmune uveitis, is marked with the emergence of autoantigen‐specific regulatory immunity in the spleen that protects the mice from recurrence of EAU. This regulatory immunity is mediated by a melanocortin‐driven suppressor APC that presents autoantigen and uses adenosine to activate an antigen‐specific CD4+ Tregs through the A2Ar. These cells are highly effective in suppressing uveitis, and they appear to be inducible Tregs. In this study, we determined whether they are inducible or natural Tregs and identified the dependent mechanism for the function of these post‐EAU Tregs. The post‐EAU spleen CD25+CD4+ T cells were sorted for NRP‐1 expression and transferred to recipient mice immunized for EAU. The sorted NRP‐1−, but not the NRP‐1+, Tregs suppressed EAU. These NRP‐1− Tregs coexpress PD‐1 and PD‐L1. Treatment of naive APCs with α‐MSH promoted a regulatory APC that induced CD25+ CD4+ Tregs in a CD73‐dependent manner. These Tregs were PD‐L1+ PD‐1+ NRP‐1− FOXP3+ HELIOS− and suppressed EAU when transferred to recipient mice. In contrast, PD‐1− T cells did not suppress EAU, indicating that PD‐1 is necessary for the suppressive activity of iTregs. Moreover, these Tregs did not suppress effector T cells when the PD/‐1/PD‐L1 pathway was blocked. These results demonstrate that post‐EAU Tregs are inducible Tregs, which use a PD‐1/PD‐L1 mechanism to suppress disease.

The Alpha-Melanocyte Stimulating Hormone Induces Conversion of Effector T Cells into Treg Cells

The neuropeptide alpha-melanocyte stimulating hormone (α-MSH) has an important role in modulating immunity and homeostasis. The production of IFN-γ by effector T cells is suppressed by α-MSH, while TGF-β production is promoted in the same cells. Such α-MSH-treated T cells have immune regulatory activity and suppress hypersensitivity, autoimmune diseases, and graft rejection. Previous characterizations of the α-MSH-induced Treg cells showed that the cells are CD4+ T cells expressing the same levels of CD25 as effector T cells. Therefore, we further analyzed the α-MSH-induced Treg cells for expression of effector and regulatory T-cell markers. Also, we examined the potential for α-MSH-induced Treg cells to be from the effector T-cell population. We found that the α-MSH-induced Treg cells are CD25+  CD4+ T cells that share similar surface markers as effector T cells, except that they express on their surface LAP. Also, the α-MSH treatment augments FoxP3 message in the effector T cells, and α-MSH induction of regulatory activity was limited to the effector CD25+ T-cell population. Therefore, α-MSH converts effector T cells into Treg cells, which suppress immunity targeting specific antigens and tissues.

MC5r and A2Ar Deficiencies During Experimental Autoimmune Uveitis Identifies Distinct T cell Polarization Programs and a Biphasic Regulatory Response

Autoantigen-specific regulatory immunity emerges in the spleen of mice recovering from experimental autoimmune uveitis (EAU), a murine model for human autoimmune uveoretinitis. This regulatory immunity provides induced tolerance to ocular autoantigen, and requires melanocortin 5 receptor (MC5r) expression on antigen presenting cells with adenosine 2 A receptor (A2Ar) expression on T cells. During EAU it is not well understood what roles MC5r and A2Ar have on promoting regulatory immunity. Cytokine profile analysis during EAU revealed MC5r and A2Ar each mediate distinct T cell responses, and are responsible for a functional regulatory immune response in the spleen. A2Ar stimulation at EAU onset did not augment this regulatory response, nor bypass the MC5r requirement to induce regulatory immunity. The importance of this pathway in human autoimmune uveitis was assayed. PBMC from uveitis patients were assayed for MC5r expression on monocytes and A2Ar on T cells, and comparison between uveitis patients and healthy controls had no significant difference. The importance for MC5r and A2Ar expression in EAU to promote the induction of protective regulatory immunity, and the expression of MC5r and A2Ar on human immune cells, suggests that it may be possible to utilize the melanocortin-adenosinergic pathways to induce protective immunity in uveitic patients.