Establishment of CD8+ T Cell Thymic Central Tolerance to Tissue-Restricted Antigen Requires PD-1.

Highly self-reactive T cells are censored from the repertoire by both central and peripheral tolerance mechanisms upon receipt of high-affinity TCR signals. Clonal deletion is considered a major driver of central tolerance; however, other mechanisms such as induction of regulatory T cells and functional impairment have been described. An understanding of the interplay between these different central tolerance mechanisms is still lacking. We previously showed that impaired clonal deletion to a model tissue-restricted Ag did not compromise tolerance. In this study, we determined that murine T cells that failed clonal deletion were rendered functionally impaired in the thymus. Programmed cell death protein 1 (PD-1) was induced in the thymus and was required to establish cell-intrinsic tolerance to tissue-restricted Ag in CD8+ thymocytes independently of clonal deletion. In bone marrow chimeras, tolerance was not observed in PD-L1-deficient recipients, but tolerance was largely maintained following adoptive transfer of tolerant thymocytes or T cells to PD-L1-deficient recipients. However, CRISPR-mediated ablation of PD-1 in tolerant T cells resulted in broken tolerance, suggesting different PD-1 signaling requirements for establishing versus maintaining tolerance. Finally, we showed that chronic exposure to high-affinity Ag supported the long-term maintenance of tolerance. Taken together, our study identifies a critical role for PD-1 in establishing central tolerance in autoreactive T cells that escape clonal deletion. It also sheds light on potential mechanisms of action of anti-PD-1 pathway immune checkpoint blockade and the development of immune-related adverse events.

γδ Thymocyte Maturation and Emigration in Adult Mice.

Several unique waves of γδ T cells are generated solely in the fetal/neonatal thymus, whereas additional γδ T cell subsets are generated in adults. One intriguing feature of γδ T cell development is the coordination of differentiation and acquisition of effector function within the fetal thymus; however, it is less clear whether this paradigm holds true in adult animals. In this study, we investigated the relationship between maturation and thymic export of adult-derived γδ thymocytes in mice. In the Rag2pGFP model, immature (CD24+) γδ thymocytes expressed high levels of GFP whereas only a minority of mature (CD24-) γδ thymocytes were GFP+ Similarly, most peripheral GFP+ γδ T cells were immature. Analysis of γδ recent thymic emigrants (RTEs) indicated that most γδ T cell RTEs were CD24+ and GFP+, and adoptive transfer experiments demonstrated that immature γδ thymocytes can mature outside the thymus. Mature γδ T cells largely did not recirculate to the thymus from the periphery; rather, a population of mature γδ thymocytes that produced IFN-γ or IL-17 remained resident in the thymus for at least 60 d. These data support the existence of two populations of γδ T cell RTEs in adult mice: a majority subset that is immature and matures in the periphery after thymic emigration, and a minority subset that completes maturation within the thymus prior to emigration. Additionally, we identified a heterogeneous population of resident γδ thymocytes of unknown functional importance. Collectively, these data shed light on the generation of the γδ T cell compartment in adult mice.

Deciphering the Immunomodulatory Capacity of Oncolytic Vaccinia Virus to Enhance the Immune Response to Breast Cancer.

Vaccinia virus (VACV) is a double-stranded DNA virus that devotes a large portion of its 200 kbp genome to suppressing and manipulating the immune response of its host. Here, we investigated how targeted removal of immunomodulatory genes from the VACV genome impacted immune cells in the tumor microenvironment with the intention of improving the therapeutic efficacy of VACV in breast cancer. We performed a head-to-head comparison of six mutant oncolytic VACVs, each harboring deletions in genes that modulate different cellular pathways, such as nucleotide metabolism, apoptosis, inflammation, and chemokine and interferon signaling. We found that even minor changes to the VACV genome can impact the immune cell compartment in the tumor microenvironment. Viral genome modifications had the capacity to alter lymphocytic and myeloid cell compositions in tumors and spleens, PD-1 expression, and the percentages of virus-targeted and tumor-targeted CD8+ T cells. We observed that while some gene deletions improved responses in the nonimmunogenic 4T1 tumor model, very little therapeutic improvement was seen in the immunogenic HER2/neu TuBo model with the various genome modifications. We observed that the most promising candidate genes for deletion were those that interfere with interferon signaling. Collectively, this research helped focus attention on the pathways that modulate the immune response in the context of VACV oncolytic virotherapy. They also suggest that the greatest benefits to be obtained with these treatments may not always be seen in "hot tumors."