Tumor necrosis factor receptor superfamily member 25 (TNFRSF25) agonists in islet transplantation: Endogenous in vivo regulatory T cell expansion promotes prolonged allograft survival.

Regulatory T cells (Tregs) modulate alloimmune responses and may facilitate minimization or withdrawal of immunosuppression posttransplant. Current approaches, however, rely on complex ex vivo Treg expansion protocols. Herein, we explore endogenous in vivo Treg expansion through antibody-mediated agonistic stimulation of the tumor necrosis factor receptor superfamily member 25 (TNFRSF25) pathway and its potential to prolong graft survival in a mouse model of islet allotransplantation. C57BL/6 male mice were treated with a single dose of TNFRSF25 agonistic antibodies (4C12 or mPTX-35) or IgG control. Diabetes was induced using streptozotocin. Four days later, flow cytometry was completed to corroborate Treg expansion, and 500 islets (CBA/J male mice) were transplanted. Glycemia was assessed thrice weekly until rejection/endpoint. Early intra-graft Treg infiltration was assessed 36 h posttransplant. TNFRSF25 antibodies enabled pronounced Treg expansion and treated mice had significantly prolonged graft survival compared with controls (p < .001). Additionally, the degree of Treg expansion significantly correlated with graft survival (p < .001). Immunohistochemistry demonstrated marked Treg infiltration in long-term surviving grafts; intra-graft Treg infiltration occurred early posttransplant. In conclusion, a single dose of TNFRSF25 antibodies enabled in vivo Treg expansion, which promotes prolonged graft survival. TNFRSF25-mediated in vivo Treg expansion could contribute to achieving lasting immunological tolerance in organ transplantation.

Prevention of Type 1 Diabetes with Acetalated Dextran Microparticles Containing Rapamycin and Pancreatic Peptide P31.

Type 1 diabetes (T1D) is a common autoimmune disease with no cure. T1D subjects are dependent on daily exogenous insulin administration, due to the loss of functional insulin-producing ß cells. Needed are immunotherapies that prevent and/or treat T1D. One approach of immunotherapy is to administer an autoantigen to selectively tolerize diabetogenic effector T cells without global immunosuppression. To date, however, strategies of antigen-specific immunotherapy are largely ineffective in the clinic. Using an antigen-specific approach, a biodegradable polymeric delivery vehicle, acetalated dextran microparticles (Ace-DEX MPs), is applied and T1D development is prevented through coadministration of the immunosuppressant rapamycin and the diabetogenic peptide P31 (Rapa/P31/MPs), via alterations of both innate and adaptive immunity. Ex vivo, adoptively transferred CD4+ T cells exhibit reduced proliferation and an increased ratio of FoxP3+ to IFNγ+ T cells. In vitro analysis indicates dendritic cells exhibit a less mature phenotype following coculture with Rapa/P31/MPs, which results in reduced CD4+ T cell proliferation and proinflammatory cytokine production (IFNγ and IL-2), but promotes PD-1 expression. Together these results demonstrate Ace-DEX MP-based antigen-specific therapy effectively tolerizes diabetogenic CD4+ T cells to prevent T1D, thereby demonstrating one of the first successful attempts of T1D prevention using a single-formulation particulate delivery platform.

Thymic Dendritic Cell Subsets Display Distinct Efficiencies and Mechanisms of Intercellular MHC Transfer.

Thymic dendritic cells (DC) delete self-antigen-specific thymocytes, and drive development of Foxp3-expressing immunoregulatory T cells. Unlike medullary thymic epithelial cells, which express and present peripheral self-antigen, DC must acquire self-antigen to mediate thymic negative selection. One such mechanism entails the transfer of surface MHC-self peptide complexes from medullary thymic epithelial cells to thymic DC. Despite the importance of thymic DC cross-dressing in negative selection, the factors that regulate the process and the capacity of different thymic DC subsets to acquire MHC and stimulate thymocytes are poorly understood. In this study intercellular MHC transfer by thymic DC subsets was investigated using an MHC-mismatch-based in vitro system. Thymic conventional DC (cDC) subsets signal regulatory protein α (SIRPα+) and CD8α+ readily acquired MHC class I and II from thymic epithelial cells but plasmacytoid DC were less efficient. Intercellular MHC transfer was donor-cell specific; thymic DC readily acquired MHC from TEC plus thymic or splenic DC, whereas thymic or splenic B cells were poor donors. Furthermore DC origin influenced cross-dressing; thymic versus splenic DC exhibited an increased capacity to capture TEC-derived MHC, which correlated with direct expression of EpCAM by DC. Despite similar capacities to acquire MHC-peptide complexes, thymic CD8α+ cDC elicited increased T cell stimulation relative to SIRPα+ cDC. DC cross-dressing was cell-contact dependent and unaffected by lipid raft disruption of donor TEC. Furthermore, blocking PI3K signaling reduced MHC acquisition by thymic CD8α+ cDC and plasmacytoid DC but not SIRPα+ cDC. These findings demonstrate that multiple parameters influence the efficiency of and distinct mechanisms drive intercellular MHC transfer by thymic DC subsets.

Thymic development of autoreactive T cells in NOD mice is regulated in an age-dependent manner.

Inefficient thymic negative selection of self-specific T cells is associated with several autoimmune diseases, including type 1 diabetes. The factors that influence the efficacy of thymic negative selection, as well as the kinetics of thymic output of autoreactive T cells remain ill-defined. We investigated thymic production of ß cell-specific T cells using a thymus-transplantation model. Thymi from different aged NOD mice, representing distinct stages of type 1 diabetes, were implanted into NOD.scid recipients, and the diabetogenicity of the resulting T cell pool was examined. Strikingly, the development of diabetes-inducing ß cell-specific CD4(+) and CD8(+) T cells was regulated in an age-dependent manner. NOD.scid recipients of newborn NOD thymi developed diabetes. However, recipients of thymi from 7- and 10-d-old NOD donor mice remained diabetes-free and exhibited a progressive decline in islet infiltration and ß cell-specific CD4(+) and CD8(+) T cells. A similar temporal decrease in autoimmune infiltration was detected in some, but not all, tissues of recipient mice implanted with thymi from NOD mice lacking expression of the autoimmune regulator transcription factor, which develop multiorgan T cell-mediated autoimmunity. In contrast, recipients of 10 d or older thymi lacked diabetogenic T cells but developed severe colitis marked by increased effector T cells reactive to intestinal microbiota. These results demonstrate that thymic development of autoreactive T cells is limited to a narrow time window and occurs in a reciprocal manner compared with colonic microbiota-responsive T cells in NOD mice.

MerTK regulates thymic selection of autoreactive T cells.

T cell-mediated autoimmune diseases such as type 1 diabetes (T1D) are believed to be the result in part of inefficient negative selection of self-specific thymocytes. However, the events regulating thymic negative selection are not fully understood. In the current study, we demonstrate that nonobese diabetic (NOD) mice lacking expression of the Mer tyrosine kinase (MerTK) have reduced inflammation of the pancreatic islets and fail to develop diabetes. Furthermore, NOD mice deficient in MerTK expression (Mer(-/-)) exhibit a reduced frequency of beta cell-specific T cells independent of immunoregulatory effectors. The establishment of bone marrow chimeric mice demonstrated that the block in beta cell autoimmunity required hematopoietic-derived cells lacking MerTK expression. Notably, fetal thymic organ cultures and self-peptide administration showed increased thymic negative selection in Mer(-/-) mice. Finally, thymic dendritic cells (DC) prepared from Mer(-/-) mice exhibited an increased capacity to induce thymocyte apoptosis in a peptide-specific manner in vitro. These findings provide evidence for a unique mechanism involving MerTK-mediated regulation of thymocyte negative selection and thymic DC, and suggest a role for MerTK in contributing to beta cell autoimmunity.

Cutting edge: Dendritic cells prime a high avidity CTL response independent of the level of presented antigen.

CTL that possess a high functional avidity are known to be optimal for the clearance of pathogens in vivo. We have shown that the amount of peptide encountered by a CD8+ CTL determines its functional avidity. Notably, in these studies nonprofessional APC were used. However, it is mature dendritic cells (DC) that are predominantly responsible for the activation of naive T cells in vivo. Whether DC also direct dose dependent-differences in avidity is unknown. In this work we examined the ability of mature DC presenting a high vs low level of peptide to generate CTL of distinct avidities. In contrast to what was observed with nonprofessional APC, CTL generated by stimulation with mature DC were of high avidity regardless of the amount of peptide presented. This DC property may promote generation of highly effective CTL that retain plasticity, which would allow the tuning of avidity in the periphery to promote optimal pathogen recognition and clearance.