Blythe D. Sather

Altering the distribution of Foxp3+ regulatory T cells results in tissue-specific inflammatory disease

CD4+Foxp3+ regulatory T cells (T reg) are essential for maintaining self-tolerance, but their functional mechanisms and sites of action in vivo are poorly defined. We examined the homing receptor expression and tissue distribution of T reg cells in the steady state and determined whether altering their distribution by removal of a single chemokine receptor impairs their ability to maintain tissue-specific peripheral tolerance. We found that T reg cells are distributed throughout all nonlymphoid tissues tested, and are particularly prevalent in the skin, where they express a unique CCR4+CD103hi phenotype. T reg cell expression of CCR4 and CD103 is induced by antigen-driven activation within subcutaneous lymph nodes, and accumulation of T reg cells in the skin and lung airways is impaired in the absence of CCR4 expression. Mice with a complete loss of CCR4 in the T reg cell compartment develop lymphocytic infiltration and severe inflammatory disease in the skin and lungs, accompanied by peripheral lymphadenopathy and increased differentiation of skin-tropic CD4+Foxp3+ T cells. Thus, selectively altering T reg cell distribution in vivo leads to the development of tissue-specific inflammatory disease.

Interleukin-15 rescues tolerant CD8 + T cells for use in adoptive immunotherapy of established tumors

CD8+ T cells can mediate eradication of established tumors, and strategies to amplify tumor-reactive T-cell numbers by immunization or ex vivo expansion followed by adoptive transfer are currently being explored in individuals with cancer. Generating effective CD8+ T cell–mediated responses to tumors is often impeded by T-cell tolerance to relevant tumor antigens, as most of these antigens are also expressed in normal tissues. We examined whether such tolerant T cells could be rescued and functionally restored for use in therapy of established tumors. We used a transgenic T-cell receptor (TCR) mouse model in which peripheral CD8+ T cells specific for a candidate tumor antigen also expressed in liver are tolerant, failing to proliferate or secrete interleukin (IL)-2 in response to antigen. Molecular and cellular analysis showed that these tolerant T cells expressed the IL-15 receptor α chain, and could be induced to proliferate in vitro in response to exogenous IL-15. Such proliferation abrogated tolerance and the rescued cells became effective in treating leukemia. Therefore, high-affinity CD8+ T cells are not necessarily deleted by encounter with self-antigen in the periphery, and can potentially be rescued and expanded for use in tumor immunotherapy.

Trypanosoma cruzi trans-sialidase initiates a program independent of the transcription factors RORγt and Ahr that leads to IL-17 production by activated B cells

We identified B cells as a major source for rapid, innate-like interleukin 17 (IL-17) production in vivo in response to Trypanosoma cruzi infection. IL-17+ B cells exhibited a plasmablast phenotype, outnumbered TH17 cells and were required for optimal response to this pathogen. Using both murine and human primary B cells, we demonstrate that exposure to parasite-derived trans-sialidase in vitro was sufficient to trigger modification of the cell surface mucin, CD45, leading to Btk-dependent signaling and IL-17A or IL-17F production via an ROR-γt and AHR-independent transcriptional program. Our combined data suggest that generation of IL-17+ B cells may be an unappreciated feature of innate immune responses required for pathogen control or IL-17-mediated autoimmunity.Here we identified B cells as a major source of rapid, innate-like production of interleukin 17 (IL-17) in vivo in response to infection with Trypanosoma cruzi. IL-17+ B cells had a plasmablast phenotype, outnumbered cells of the TH17 subset of helper T cells and were required for an optimal response to this pathogen. With both mouse and human primary B cells, we found that exposure to parasite-derived trans-sialidase in vitro was sufficient to trigger modification of the cell-surface mucin CD45, which led to signaling dependent on the kinase Btk and production of IL-17A or IL-17F via a transcriptional program independent of the transcription factors RORγt and Ahr. Our combined data suggest that the generation of IL-17+ B cells may be a previously unappreciated feature of innate immune responses required for pathogen control or IL-17-mediated autoimmunity.

CD8+ T Cell Tolerance to a Tumor-associated Antigen Is Maintained at the Level of Expansion Rather than Effector Function

CD8+ T cell tolerance to self-proteins prevents autoimmunity but represents an obstacle to generating T cell responses to tumor-associated antigens. We have made a T cell receptor (TCR) transgenic mouse specific for a tumor antigen and crossed TCR-TG mice to transgenic mice expressing the tumor antigen in hepatocytes (gag-TG). TCRxgag mice showed no signs of autoimmunity despite persistence of high avidity transgenic CD8+ T cells in the periphery. Peripheral CD8+ T cells expressed phenotypic markers consistent with antigen encounter in vivo and had upregulated the antiapoptotic molecule Bcl-2. TCRxgag cells failed to proliferate in response to antigen but demonstrated cytolytic activity and the ability to produce interferon γ. This split tolerance was accompanied by inhibition of Ca2+ flux, ERK1/2, and Jun kinasephosphorylation, and a block in both interleukin 2 production and response to exogenous interleukin 2. The data suggest that proliferation and expression of specific effector functions characteristic of reactive cells are not necessarily linked in CD8+ T cell tolerance.

Age-Dependent T Cell Tolerance and Autoimmunity to Myelin Basic Protein

Experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis, is induced by activating a subset of myelin basic protein (MBP)-specific T cells that have escaped tolerance induction. Here, we define the tolerance mechanisms that eliminate the majority of MBP-specific T cells from the periphery. We show that MBP-specific T cells undergo central tolerance mediated by bone marrow-derived antigen-presenting cells presenting exogenously derived MBP epitopes. The efficiency of tolerance is age dependent, reflecting the developmentally regulated expression of MBP. Dependence of tolerance on the amount of MBP expressed in vivo results in an age window of susceptibility to EAE in mice that peaks during puberty. These results suggest that factors regulating expression of self-antigens in vivo can influence susceptibility to autoimmunity.

Coupling endonucleases with DNA end-processing enzymes to drive gene disruption

Targeted DNA double-strand breaks introduced by rare-cleaving designer endonucleases can be harnessed for gene disruption applications by engaging mutagenic nonhomologous end-joining DNA repair pathways. However, endonuclease-mediated DNA breaks are often subject to precise repair, which limits the efficiency of targeted genome editing. To address this issue, we coupled designer endonucleases to DNA end–processing enzymes to drive mutagenic break resolution, achieving up to 25-fold enhancements in gene disruption rates.

B cell–specific lentiviral gene therapy leads to sustained B-cell functional recovery in a murine model of X-linked agammaglobulinemia

The immunodeficiency disorder, X-linked agammaglobulinemia (XLA), results from mutations in the gene encoding Bruton tyrosine kinase (Btk). Btk is required for pre-B cell clonal expansion and B-cell antigen receptor signaling. XLA patients lack mature B cells and immunoglobulin and experience recurrent bacterial infections only partially mitigated by life-long antibody replacement therapy. In pursuit of definitive therapy for XLA, we tested ex vivo gene therapy using a lentiviral vector (LV) containing the immunoglobulin enhancer (Emu) and Igbeta (B29) minimal promoter to drive B lineage-specific human Btk expression in Btk/Tec(-/-) mice, a strain that reproduces the features of human XLA. After transplantation of EmuB29-Btk-LV-transduced stem cells, treated mice showed significant, albeit incomplete, rescue of mature B cells in the bone marrow, peripheral blood, spleen, and peritoneal cavity, and improved responses to T-independent and T-dependent antigens. LV-treated B cells exhibited enhanced B-cell antigen receptor signaling and an in vivo selective advantage in the peripheral versus central B-cell compartment. Secondary transplantation showed sustained Btk expression, viral integration, and partial functional responses, consistent with long-term stem cell marking; and serial transplantation revealed no evidence for cellular or systemic toxicity. These findings strongly support pursuit of B lineage-targeted LV gene therapy in human XLA.

Regulatory T Cells Maintain Long-Term Tolerance to Myelin Basic Protein by Inducing a Novel, Dynamic State of T Cell Tolerance

The pathogenesis of multiple sclerosis involves a breakdown in T cell tolerance to myelin proteins like myelin basic protein (MBP). Most MBP-specific T cells are eliminated by central tolerance in adult mice, however, the developmentally regulated expression of MBP allows MBP-specific thymocytes in young mice to escape negative selection. It is not known how these T cells that encounter MBP for the first time in the periphery are regulated. We show that naive MBP-specific T cells transferred into T cell-deficient mice induce severe autoimmunity. Regulatory T cells prevent disease, however, suppression of the newly transferred MBP-specific T cells is abrogated by activating APCs in vivo. Without APC activation, MBP-specific T cells persist in the periphery of protected mice but do not become anergic, raising the question of how long-term tolerance can be maintained if APCs presenting endogenous MBP become activated. Our results demonstrate that regulatory T cells induce naive MBP-specific T cells responding to nonactivated APCs to differentiate into a unique, tolerized state with the ability to produce IL-10 and TGF-β1 in response to activated, but not nonactivated, APCs presenting MBP. This tolerant response depends on continuous activity of regulatory T cells because, in their absence, these uniquely tolerized MBP-specific T cells can again induce autoimmunity.

Cellular Requirements for Diabetes Induction in DO11.10xRIPmOVA Mice

Type 1 diabetes (T1D) results from the immune-mediated destruction of the insulin-producing β-islet cells in the pancreas. The genetic and environmental mechanisms promoting the development of this disease remain poorly understood. We have explored the cellular requirements for T1D development in DO11.10xRIPmOVA (DORmO) mice, which carry a TCR transgene specific for an MHC class II-restricted epitope from OVA and express membrane-bound OVA in the pancreas under the control of the rat insulin promoter. We found that DORmO.RAG2−/− mice do not develop insulitis and are completely protected from diabetes, demonstrating that endogenous lymphocyte receptor rearrangement is required for disease development. Diabetes in DORmO mice is preceded by the development of OVA-specific autoantibodies and is delayed in B cell-deficient DORmO.JhD−/− mice, demonstrating that B cells contribute to disease progression. In addition, transfer of CD8+ T cells from diabetic animals into DORmO.RAG2−/− mice promoted insulitis by OVA-specific CD4+ T cells. Finally, although diabetes develops in DORmO mice in the presence of a significant population of Foxp3+ OVA-specific regulatory T cells, boosting regulatory T cell numbers by injecting IL-2 immune complexes dampens autoantibody production and prevents development of insulitis and overt diabetes. These results help define the events leading to diabetes in DORmO mice and provide new insights into the cellular interactions required for disease development in an Ag-specific model of T1D.

572. Successful Generation of CAR+PD-1-Primary T Cells Using Cas9-Mediated Genome Editing

Cancer immunotherapy is an exciting area of cancer treatment bolstered by recent success of anti-immune checkpoint antibodies in the clinic. Engineered T cells that are programmed to attack tumors via chimeric antigen receptors (CARs) have also shown promise in early clinical trials. It is speculated, however, that the PD-1/PD-L1 axis dampens the effectiveness of CAR T therapy in certain cancer types. In order to improve the function of transplanted CAR T cells in these contexts, we have deleted PDCD1 using the genome editing system, CRISPR/Cas9. Delivery of the Cas9 protein and a PDCD1 specific gRNA to primary T cells resulted in the deletion of PD1 expression in over 90% of cells. Additionally, we were able to achieve similar editing efficiencies in the context of a lentivirus CAR infection and successfully generated CAR+/PD-1 knockout primary T cells with a very high level (>60%) of duel CAR expression and PD-1 editing. Utilizing a variety of assays, we have determined that the CAR+/PD-1− primary T cells are viable, proliferate normally and can kill target expressing cells both in the presence and absence of PD-L1 on the target cells. In conclusion, we have successfully generated CAR+/PD-1− primary T cells using Cas9-mediated editing that can be directed to kill PD-L1 expressing target cells.