Decoding the molecular composition of individual Ngn3 + endocrine progenitors (EPs) during pancreatic morphogenesis could provide insight into the mechanisms regulating hormonal cell fate. Here, we identify population markers and extensive cellular diversity including four EP subtypes reflecting EP maturation using high-resolution single-cell RNA-sequencing of the e14.5 and e16.5 mouse pancreas. While e14.5 and e16.5 EPs are constantly born and share select genes, these EPs are overall transcriptionally distinct concomitant with changes in the underlying epithelium. As a consequence, e16.5 EPs are not the same as e14.5 EPs: e16.5 EPs have a higher propensity to form beta cells. Analysis of e14.5 and e16.5 EP chromatin states reveals temporal shifts, with enrichment of beta cell motifs in accessible regions at later stages. Finally, we provide transcriptional maps outlining the route progenitors take as they make cell fate decisions, which can be applied to advance the in vitro generation of beta cells.

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The ability to express and study a single T cell receptor (TCR) in vivo is an important aspect of both basic and translational immunological research. Traditionally, this was achieved by using TCR transgenic mice. In the past decade, a more efficient approach for single TCR expression was developed. This relatively rapid and accessible method utilizes retrovirus‐mediated stem cell–based gene transfer and is commonly referred to as the TCR retrogenic approach. In this approach, hematopoietic bone marrow precursors are transduced with retroviral vector carrying both alpha and beta chains of a T cell receptor. After successful transduction, bone marrow is injected into recipient mice, in which T cell development is driven by expression of the vector‐encoded TCR. This article details the materials and methods required to generate TCR retrogenic mice. It is divided into three sections and provides detailed methods for generation of stable retroviral producer cell lines, isolation and optimal transduction of hematopoietic bone marrow cells, and subsequent analysis of TCR retrogenic T cells. A detailed example of such analysis is provided. The current protocol is a culmination of many years of optimization and is the most efficient approach to date. Bone marrow transduction and transfer into recipient mice can now be achieved in a short period of four days. The protocol can be followed in most laboratories with standard biomedical equipment, and is supported by a troubleshooting guide that covers potential pitfalls and unexpected results. © 2019 by John Wiley & Sons, Inc.

Accumulating evidence supports a critical role for post-translationally modified (PTM) islet neo-antigens in type 1 diabetes. However, our understanding regarding thymic development and peripheral activation of PTM autoantigen reactive T cells is still limited. Using HLA-DR4 humanized mice, we observed that deamidation of GAD65115-127 generates a more immunogenic epitope that recruits T cells with promiscuous recognition of both the deamidated and native epitopes, and reduced frequency of regulatory T cells. Using humanized HLA/TCR mice we observed that TCRs reactive to the native or deamidated GAD65115-127 led to efficient development of CD4+ effector T cells; however, regulatory T cell development was reduced in mice expressing the PTM reactive TCR, which was partially restored with exogenous PTM peptide. Upon priming, both the native specific and the deamidated specific T cells accumulated in pancreatic islets, suggesting that both specificities can recognize endogenous GAD65 and contribute to anti-beta cell responses. Collectively, our observations in polyclonal and single TCR systems suggest that while effector T cell responses can exhibit cross-reactivity between native and deamidated GAD65 epitopes, regulatory T cell development is reduced in response to the deamidated epitope, pointing to Treg development as a key mechanism for loss of tolerance to PTM antigenic targets.

Thymic presentation of self-antigens is critical for establishing a functional yet self-tolerant T cell population. Hybrid peptides formed through transpeptidation within pancreatic beta cell lysosomes have been proposed as a new class of autoantigens in Type 1 Diabetes (T1D). While the production of hybrid peptides in the thymus has not been explored, due to the nature of their generation, it is thought to be highly unlikely. Therefore, hybrid peptide-reactive thymocytes may preferentially escape thymic selection and contribute significantly to T1D progression. Using an antibody-peptide conjugation system, we targeted the 2.5HIP hybrid peptide towards thymic resident Langerin+ dendritic cells to enhance thymic presentation during the early neonatal period. Our results indicated that anti-Langerin-2.5HIP delivery can enhance T cell central tolerance toward cognate thymocytes in NOD.BDC2.5 mice. Strikingly, a single dose treatment with anti-Langerin-2.5HIP during neonatal period delayed diabetes onset in NOD mice, indicating the potential of antibody-mediated delivery of autoimmune neo-antigens during early stages of life as a therapeutic option in the prevention of autoimmune diseases.

Foxp3+ regulatory T cells (Tregs) are capable suppressors of aberrant self-reactivity. However, TCR affinity and specificities that support Treg function, and how these compare to autoimmune T cells remain unresolved. In this study, we used antigen agnostic and epitope-focused analyses to compare TCR repertoires of regulatory and effector T cells that spontaneously infiltrate pancreatic islets of non-obese diabetic mice. We show that effector and regulatory T cell-derived TCRs possess similar wide-ranging reactivity for self-antigen. Treg-derived TCRs varied in their capacity to confer optimal protective function, and Treg suppressive capacity was in part determined by effector TCR affinity. Interestingly, when expressing the same TCR, Tregs showed higher Nur77-GFP expression than Teffs, suggesting Treg-intrinsic ability to compete for antigen. Our findings provide a new insight into TCR-dependent and independent mechanisms that regulate Treg function and indicate a TCR-intrinsic insufficiency in tissue-specific Tregs that may contribute to the pathogenesis of type 1 diabetes.

<a>Accumulating evidence supports a critical role for post-translationally modified (PTM) islet neoantigens in type 1 diabetes. However, our understanding regarding thymic development and peripheral activation of PTM autoantigen reactive T cells is still limited. Using HLA-DR4 humanized mice, we observed that deamidation of GAD65_115-127generates a more immunogenic epitope that recruits T cells with promiscuous recognition of both the deamidated and native epitopes, and reduced frequency of regulatory T cells. Using humanized HLA/TCR mice we observed that TCRs reactive to the native or deamidated GAD65_115-127led to efficient development of CD4+ effector T cells; however, regulatory T cell development was reduced in mice expressing the PTM reactive TCR, which was partially restored with exogenous PTM peptide. Upon priming, both the native specific and the deamidated specific T cells accumulated in pancreatic islets, suggesting that both specificities can recognize endogenous GAD65 and contribute to anti-beta-cell responses. Collectively, our observations in polyclonal and single TCR systems suggest that while effector T cell responses can exhibit cross-reactivity between native and deamidated GAD65 epitopes, regulatory T cell development is reduced in response to the deamidated epitope, pointing to Treg development as a key mechanism for loss of tolerance to PTM antigenic targets. </a>

T cells specific for post-translationally modified (PTM) islet antigens have been implicated as critical players in the development of type 1 diabetes. T cells reactive to the deamidated version of glutamic acid decarboxylase 65 (GAD65) 115-127 epitope accumulate in peripheral blood of patients as they progress to disease. However, knowledge regarding the development, activation, differentiation and islet recruitment of PTM reactive T cells is limited. Using retroviral gene delivery, we have developed a humanized TCR/HLA mouse model to assess the in vivo function of human TCRs reactive to either the wild type (GADWT) or the deamidated (GAD120E) GAD65115-127 epitope. Both TCRs supported efficient thymic selection; however, fewer regulatory T cells developed in mice expressing the PTM reactive TCR, which was partially rescued by the thymic delivery of GAD120E peptide. Immunization of immune sufficient polyclonal DR4.NOD mice with GAD120E/CFA elicited a stronger response compared to GADWT/CFA, confirming that tolerance to the wild type autoantigen is more robust. Interestingly, while islet infiltration in GAD120E single TCR mice was dependent on priming with the cognate PTM peptide, GADWT-TCR T cells could be induced to infiltrate islets after priming with either GADWT or GAD120E peptide, despite the vastly different functional affinity (EC50, GADWT: 0.047μM vs. GAD120E: ≥1.4μM). These findings suggest that the development and expansion of GAD65 PTM reactive CD4+ T cells are less restricted in peripheral lymphoid organs, but their recruitment to the tissue site is dependent on the availability of PTM antigen. Disclosure Y. Jing: None. Y. Kong: None. J. McGinty: None. G. Blahnik-Fagan: None. M. Bettini: None. E.A. James: None. M. Bettini: None. Funding National Institutes of Health (R01AI125301)