Intermittent Tacrolimus Treatment Delays CD8+ Tumor-Infiltrating Lymphocyte Exhaustion and Enhances PD1 Blockade Therapy in Melanoma-Bearing Mice.

Abstract

Continuous antigen exposure initiates the developmental process of CD8+ T cell exhaustion, accompanied by a gradual increase in the expression of inhibitory receptors (IRs). Our previous study conceptualized T cell exhaustion as an over-activation status induced by chronic antigen stimuli. To intervene in the developmental process of CD8+ T cell exhaustion, we adjusted the over-activation status by intermittently blocking T cell activation signals with tacrolimus, a T cell activation inhibitor. Melanoma-bearing mice received intermittent tacrolimus (IT) and/or programmed cell death 1 (PD1) blockade antibody. Exhaustion phenotype of CD8+ tumor-infiltrating lymphocytes (CD8+TILs) was detected by flow cytometry. The expression levels of IRs (PD1 and CD223), as well as memory markers (Ly108 and CD127), were utilized to demarcate the extent of CD8+TILs exhaustion. Then, tyrosinase-related protein 2 peptide (Trp2) was used to detect tumor-specific CD8+ TILs. Furthermore, tumor growth rate and tumor weight were also evaluated. We found that IT and/or PD1 blockade enhanced the infiltration of functional CD8+TILs. The findings demonstrated that both IT therapy and PD1 blockade enhanced the antitumor immunity. Combining IT and PD1 blockade led to a greater reduction in tumor growth rate and tumor weight. IT therapy also improved the response of CD8+TILs to melanoma-specific Trp2peptide. A detailed analysis of the CD8+TILs showed that Ly108 and CD127 reduced dramatically as the expression of PD1 increased. In comparison to PD1 blockade, combined treatment (IT plus PD1 blockade) significantly increased the number of intermediate IRs-expressing CD8+TILs, while reducing the number of high IRs-expressing CD8+TILs. These results indicated that combined treatment enhanced the function and decelerated the increase in IRs expression of CD8+TILs, deferring the developmental process of CD8+TILs exhaustion.