(B) Immunofluorescence microscopy of murine spleens after SD treatment showed increased expression of PD-1 and PD-L1 on splenocytes after TMZ exposure compared with the baseline. LAG-3 and TIM-3 in lymphocytes which was not seen with MD temozolomide. When temozolomide treatment was combined with programmed cell death 1 (PD-1) antibody therapy, the MD temozolomide/PD-1 antibody group demonstrated a decrease in exhaustion markers in tumor infiltrating lymphocytes that was not observed in the SD temozolomide/PD-1 antibody group. Also, the survival advantage of PD-1 antibody therapy in a murine syngeneic intracranial glioma model was abrogated by adding SD temozolomide to treatment. However, when MD temozolomide was added to PD-1 inhibition, it preserved the survival benefit that was seen by PD-1 antibody therapy alone. Conclusion The peripheral and intratumoral immune microenvironments are distinctively affected by dose modulation of temozolomide. 0.05, FDR 0.05).26 Immune checkpoints and co-inhibitor molecule expression of 2 groups were plotted to a volcano plot by R packages ggplot227 and ggrepel (https://github.com/slowkow/ggrepel). Statistical Analysis MannCWhitney 0.05. Statistical analysis was performed using GraphPad Prism 7.03 software. Results Impact of Temozolomide Dosing on Peripheral Immune Cell Phenotype Temozolomide is well known to result in lymphodepletion that can be leveraged for enhanced antigen-specific T-cell recovery when combined with cellular-based immunotherapy.17C19,28 The effects of TMZ-induced lymphodepletion on response to immune checkpoint inhibition are still unknown. Since our group has demonstrated that dosing of TMZ results in different effects on host antigen-specific T cells,17 we tested 2 different doses of TMZ. MD and SD TMZ were DBPR108 delivered to mice, and peripheral blood was collected to test different markers and absolute counts using flow cytometry. The absolute lymphocyte counts decreased at different timepoints posttreatment. The SD TMZ group had a mean CD4 DBPR108 T-cell count of 311.37 compared with 658.43 in the MD TMZ group at 1 week (Fig. 1A). Similarly, the mean absolute CD8 T-cell count in the SD TMZ group at 1 week was 150.64 compared with 247.61 in the MD TMZ group. As expected, higher doses of TMZ resulted in a ITGB2 more significant lymphopenia in both CD4 and CD8 T cells. Open in a separate window Fig. 1 Peripheral blood T-cell counts and PD-1 and PD-L1 expression on T cells after exposure to TMZ. (A) Peripheral blood was collected after animals were treated with SD or MD TMZ for T-cell count using flow cytometry. In the SD group, the mean number of CD4 T-cell count decreased 1 week (2.11-fold), 2 weeks (1.42-fold), and 6 weeks (1.7-fold) compared with the MD group. The mean number of CD8 T-cell count in the SD group decreased at 1 week (1.64-fold), 2 DBPR108 weeks (1.48-fold), and 6 weeks (1.73-fold) compared with the MD group ( 0.05). In both the SD and MD TMZ groups, lymphopenia was observed in the CD4 and CD8 populations compared with baseline ( 0.05). (B) Immunofluorescence microscopy of murine spleens after SD treatment showed increased expression of PD-1 and PD-L1 on splenocytes after TMZ exposure compared with the baseline. (C) PD-1 and PD-L1 expression on peripheral blood T cells was evaluated after TMZ treatment using flow cytometry. MD TMZ resulted in a 3.51-fold increase ( 0.001) of PD-1+/CD8 T cells at 2 weeks without a significant change in PD-1+/CD4 T cells. SD TMZ did not have a significant increase in PD-1+ CD4 or CD8 T cells. MD TMZ resulted in a 21-fold increase in PD-L1+/CD8 T cells and a 27.3-fold increase in PD-L1+/CD4 T cells ( 0.0001) after 2 weeks. SD TMZ resulted in a 9-fold increase in week 1 of PD-L1+/CD8 T cells, and a 25-fold increase in week 2 of PD-L1+/CD8 T cells ( 0.0001). There was a 29-fold increase in PD-L1+/CD4 T cells in week 2 ( 0.0001) without a significant change in week 1. = 5 per group; baseline = tumor bearing animals without any treatment. PD-1 inhibition efficacy has been linked to the expression of PD-1 and PD ligand 1 (PD-L1) on both T cells and tumor cells.29C31 In these experiments, we tested the expression of these markers on murine splenocytes using immunofluorescence microscopy. Evaluation of nonCtumor bearing murine spleens after SD TMZ treatment showed a qualitative upregulation of both PD-1 and PD-L1 (Fig. 1B). This finding was further investigated on circulating T cells in tumor-bearing mice with MD and SD TMZ treatment. GL261.