ß-cell-specific IL-35 therapy suppresses ongoing autoimmune diabetes in NOD mice.

IL-35 is a recently identified cytokine exhibiting potent immunosuppressive properties. The therapeutic potential and effects of IL-35 on pathogenic T effector cells (Teff) and Foxp3+ Treg, however, are ill defined. We tested the capacity of IL-35 to suppress ongoing autoimmunity in NOD mice. For this purpose, an adeno-associated virus vector in which IL-35 transgene expression is selectively targeted to ß cells via an insulin promoter (AAV8mIP-IL35) was used. AAV8mIP-IL35 vaccination of NOD mice at a late preclinical stage of type 1 diabetes (T1D) suppressed ß-cell autoimmunity and prevented diabetes onset. Numbers of islet-resident conventional CD4+ and CD8+ T cells, and DCs were reduced within 4 weeks of AAV8mIP-IL35 treatment. The diminished islet T-cell pool correlated with suppressed proliferation, and a decreased frequency of IFN-γ-expressing Teff. Ectopic IL-35 also reduced islet Foxp3+ Treg numbers and proliferation, and protection was independent of induction/expansion of adaptive islet immunoregulatory T cells. These findings demonstrate that IL-35-mediated suppression is sufficiently robust to block established ß-cell autoimmunity, and support the use of IL-35 to treat T1D and other T-cell-mediated autoimmune diseases.

Gene Therapy with an Adeno-Associated Viral Vector Expressing Human Interleukin-2 Alters Immune System Homeostasis in Humanized Mice.

Recombinant adeno-associated viruses (rAAVs) serve as vectors for in vivo gene delivery in both mice and humans, and have broad applicability for the treatment of genetic diseases. Clinical trials with AAV vectors have demonstrated promise and safety in several human diseases. However, the in vivo validation of novel AAV constructs expressing products that act specifically on human cells and tissues is limited by a paucity of effective translatable models. Humanized mice that are engrafted with human cells, tissues, and immune systems offer strong potential to test the biological effectiveness of AAV vectors on human cells and tissues. Using the BLT (bone marrow, liver, thymus) humanized NOD-scid Il2rgnull (NSG) mouse model, which enables efficient development of HLA-restricted effector and regulatory T cells (Tregs), we have evaluated the delivery and function of human interleukin (IL)-2 by an AAV vector. Humanized mice treated with an AAV vector expressing human IL-2 showed a significant and sustained increase in the number of functional human FOXP3+CD4+ Tregs. The expression of human IL-2 did not significantly change the levels or activation status of conventional T-cell subsets. Numbers of activated human natural killer cells were also increased significantly in humanized mice treated with the IL-2 vector. These data recapitulate observations in clinical trials of IL-2 therapy and collectively show that humanized mouse models offer a translational platform for testing the efficacy of AAV vectors targeting human immune cells.

Anti-coreceptor therapy drives selective T cell egress by suppressing inflammation-dependent chemotactic cues.

There continues to be a need for immunotherapies to treat type 1 diabetes in the clinic. We previously reported that nondepleting anti-CD4 and -CD8 Ab treatment effectively reverses diabetes in new-onset NOD mice. A key feature of the induction of remission is the egress of the majority of islet-resident T cells. How this occurs is undefined. Herein, the effects of coreceptor therapy on islet T cell retention were investigated. Bivalent Ab binding to CD4 and CD8 blocked TCR signaling and T cell cytokine production, while indirectly downregulating islet chemokine expression. These processes were required for T cell retention, as ectopic IFN-γ or CXCL10 inhibited Ab-mediated T cell purging. Importantly, treatment of humanized mice with nondepleting anti-human CD4 and CD8 Ab similarly reduced tissue-infiltrating human CD4+ and CD8+ T cells. These findings demonstrate that Ab binding of CD4 and CD8 interrupts a feed-forward circuit by suppressing T cell-produced cytokines needed for expression of chemotactic cues, leading to rapid T cell egress from the islets. Coreceptor therapy therefore offers a robust approach to suppress T cell-mediated pathology by purging T cells in an inflammation-dependent manner.

Isolation and transplantation of different aged murine thymic grafts.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and selection of T cells specific for self and foreign antigens. The approach entails implantation of thymic grafts derived from various aged mice into immunodeficient scid recipients. Over a relatively short period of time the recipients are fully reconstituted with T cells derived from the implanted thymus graft. Only thymocytes seeding the thymus at the time of isolation undergo selection and develop into mature T cells. As such, changes in the nature and specificity of the engrafted T cells as a function of age-dependent thymic events can be assessed. Although technical expertise is required for successful thymic transplantation, this method provides a unique strategy to study in vivo a wide range of pathologies that are due to or a result of aberrant thymic function and/or homeostasis.