­­Delineating the role of co-stimulatory signals on human virus-specific memory CD8+ T cell activation and expansion using highly selective T cell receptor-targeted biologics

To delineate the minimum signals required to activate and expand antigen-specific memory CD8+ T cells, we recapitulated the discrete activation signals delivered by antigen-presenting cells using antigen-specific T-cell receptor-specific biologics termed Immuno-STAT (IST), which can specifically deliver a T cell receptor (TCR) signal alone or paired with a defined co-stimulatory signal to antigen-specific CD8+ T cells. Using a reductionist approach that provides defined TCR and co-stimulatory signaling, we demonstrated that different IST could engage TCR alone or in combination with either CD28 or 4-1BB to initiate distinct transcriptional programs in memory CMV-specific CD8+ T cells. As compared to activation by TCR signaling alone, the combination of TCR and CD28 or 4-1BB co-stimulatory signals significantly altered the transcriptome and drove expression of genes related to growth and survival. Nevertheless, TCR signaling in the presence of IL-2 (5 U/ml) was sufficient for robust recall activation and expansion of functional cytomegalovirus CMV(NLV)-specific and HIV(SL9)-specific memory CD8+ T cells. This response contrasts with naïve CD8+ T cells, which required CD28 co-stimulation in addition to TCR stimulation for antigen-specific activation and expansion even in the presence of high levels of IL-2 (100 U/ml). These results have important implications for antigen-specific immunotherapy by indicating that biologics that deliver TCR signals alone in the presence of IL-2 may be sufficient for immunotherapeutic strategies designed to expand memory CD8+ T cells to eliminate cancerous or infected cells. In contrast, strategies that aim to stimulate naïve T cell responses may benefit from the codelivery of TCR and CD28 signals. Overall design: To understand the transcriptional response of CMV-specific memory CD8 T cells to TCR-stimulation with or without CD28 or 4-1BB co-stimulation, human PBMC were stimulated with a pMHC-based TCR agonist linked to either a CD28 or 4-1BB agonist. After stimulation, CMV-specific CD8 T cells were collected using FACS and RNA was isolated. Replicates were samples of PBMC from the same donor that underwent treatment, cell sorting, RNA isolation, and sequencing independently. Samples that received no treatment or retreatment were used as controls.