Effect of bryostatin-1 on in vitro exhausted CD8+ T cells

Bryostatin-1, a pharmacological agent from marine organisms, has been studied for HIV and cancer therapies due to its modulation of protein kinase C. In our previous study (Zhao M et al. Pharmacological research. 2019;139:524-34.), we found that bryostatin-1 downregulated inhibitory receptor PD-1 on activated CD8+ T cells from people with HIV. Since HIV-specific CD8+ T cells become functionally exhausted during chronic HIV infection and PD-1 expression is known to be related to the exhaustion state of these cells, we hypothesized that bryostatin-1 may modulate CD8+ T cell exhaustion. To test this, we generated in vitro exhausted OT-I CD8+ T cells as previously described (Zhao M et al PLoS Pathog. 16(6): e1008555) by repeatedly stimulating cells with OVA(257-264) peptide. Then we treated both exhausted CD8+ T cells (repeat peptide stimulated cells) and non-exhausted CD8+ T cells (single peptide stimulated cells) with bryostatin-1 for 3 days. We found that bryostatin-1 decreased exhaustion-associated markers and improved the functionality of exhausted CD8+ T cells. To investigate the underlying mechanism of how bryostatin-1 exerts its effects on exhausted CD8+ T cells, we performed RNA-sequencing (RNA-seq) on in vitro exhausted OT- I CD8+ T cells which were treated with bryostatin-1 or DMSO, with single peptide stimulated cells as non-exhausted controls. Taken together, our study demonstrate bryostatin-1's effect on exhausted CD8+ T cells and bryostatin-1's potential in enhancing T cell immunity against chronic infection or cancer. OT-I CD8+ T cells are first exhausted in vitro for 5 days, then treated with either DMSO or bryostatin-1 for 3 days before RNA isolation and sequencing. Non-exhausted OT-I CD8+ T cells (stimulated only once with peptide) were treated with either DMSO or bryostatin-1 are controls.