Abstract 3613: CD28 signaling controls maintenance and differentiation of PD1+CD8 T cells during chronic antigen stimulation
Authors: Humblin Etienne , Van der Heide Verena , Filipescu Dan , Lu Ashley , Soares-Schanoski Alessandra , Selvan Myvizhi , Horta Laila , Gumus Zeynep , Bernstein Emily , Chipuk Jerry , Homann Dirk , Kamphorst Alice O. 
Published: June 2022

Abstract
During chronic infections and cancer, CD8 T cells that recognize pathogens/tumors can persist in a dysfunctional state characterized by high expression of the inhibitory receptor Programmed Cell Death (PD)-1. PD-1+ CD8 T cells are heterogeneous: T cell factor (TCF)-1+PD-1+ progenitor exhausted cells (Tpex) are capable of self-renewal and give rise to the more differentiated subpopulation of TCF-1negPD-1+ CD8 T cells that can retain some effector-like function. Tpex are essential for the maintenance of the antigen-specific T cell pool during chronic stimulation, and in animal models, only these cells respond to PD-1 blockade. In cancer patients, the presence of Tpex cells in tumors has been associated with response to PD-1 targeted therapy. However, it has been reported that the presence of these cells in the tumor microenvironment requires a specific niche, and the molecular mechanisms supporting maintenance of Tpex remains to be investigated. Tpex have high CD28 expression, and we have shown that during PD-1 targeted therapies, CD28 costimulation is required for the reinvigoration of CD8 T cell responses. In this study, we sought to understand the role of sustained CD28 signaling for self-renewal and differentiation of Tpex cells. In mice chronically infected with lymphocytic choriomeningitis virus (LCMV), we abrogated CD28 signaling by blockade of anti-B7-1/B7-2, and observed a sharp reduction of virus-specific CD8 T cells (both TCF-1+ and TCF-1neg subsets). By contrast, B7-2 single blockade resulted in reduction of differentiated TCF-1neg virus-specific cells, yet the Tpex subset was not affected. We hypothesized that stronger CD28 signaling might be required for differentiation of Tpex cells into TCF-1neg, whereas weaker CD28 signaling would be sufficient for Tpex self-renewal. To address this question, we performed conditional deletion of Cd28 on PD-1+ CD8 T cells in mice with established chronic infection. Deletion of both Cd28 alleles resulted in reduction of TCF-1+ and TCF-1neg subsets. In contrast, when CD28 signaling was reduced (deletion of one allele), TCF-1neg PD-1+ CD8 T cells were decreased but the number of Tpex cells was not affected. We performed RNA sequencing and metabolic assays to better understand the role of CD28 signaling on PD-1+ CD8 T cells. These data revealed that sustained CD28 signaling during persistent antigen stimulation is required to maintain mitochondrial fitness. Our work support the novel hypothesis that CD28 signaling strength modulates cell fate decision of Tpex cells through metabolic regulation. Our data also suggest that antigen presenting cells capable of providing ligands to CD28 are required for the long-term maintenance of antigen-specific T cells during chronic stimulation. These findings provide key insights about PD-1+ CD8 T cells, with important implications for checkpoint therapy in cancer, including longevity of responses.
Citation Format: Etienne Humblin, Verena Van der Heide, Dan Filipescu, Ashley Lu, Alessandra Soares-Schanoski, Myvizhi Selvan, Laila Horta, Zeynep Gumus, Emily Bernstein, Jerry Chipuk, Dirk Homann, Alice O. Kamphorst. CD28 signaling controls maintenance and differentiation of PD1+CD8 T cells during chronic antigen stimulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3613.