Abstract While most patients with metastatic prostatic adenocarcinoma respond initially, nearly all develop resistance to multiple lines of androgen deprivation therapies. This is related to overexpression of the androgen receptor (AR), which is frequently driven by AR amplification. In some patients, increased AR renders tumor cells sensitive to high dose androgens which paradoxically inhibits their growth. Bipolar Androgen Therapy (BAT) was introduced in which patients with castration resistant prostate cancer (CRPC) are treated intermittently with high dose testosterone. This produces biochemical and objective responses, and may re-sensitize prostate cancer to subsequent second generation AR inhibitors. This study utilizes samples from a recently performed clinical trial for patients with CRPC that included sequential biopsies of soft tissue metastases before BAT and after 3 cycles of BAT and before patients were treated with nivolumab.Tumor cells were present in FFPE blocks for both the pretreatment and C4D1 time points for 24 of the patients. These FFPE samples were stained using a recently validated iterative multiplex IHC assay containing 6 antibodies including: CD3 (T cells), CD4 (Helper T Cells), CD8 (Cytotoxic T Cells), PD1 (T Cell Checkpoint), FOXP3 (T-Reg cells), and Keratin 8 (epithelial cell marker). Biopsy slides were scanned after each round of staining and whole slide scans were imported into the HALO image analysis where they underwent color deconvolution, image registration and fusion. Regions of tumor were demarcated manually, and computerized automated image analysis was used to determine cell densities for 8 cell phenotypes (total CD3+, CD3+CD4+, CD3+CD8+, CD3+CD4+Foxp3+, CD3+CD4+PD1+, CD3+CD8+PD1+, CD3+CD4+PD1-, CD3+CD8+PD1-). There was a higher density of CD3+CD8+PD1+ cells in the pretreatment biopsies of men who responded (R) (to BAT) as compared with those who did not respond (NR)(p=0.015 Wilcoxon rank-sum test; mean 92.7 [R] vs 13.9 [NR]). The difference in R vs. NR was less and not significant in the CD3+CD8+PD1- population. There was a trend towards a higher density of CD3+CD4+PD1+ cells in the pretreatment samples in responders, but less so in the CD3+CD4+PD1- population. In the C4D1 biopsies, only CD3+ total cells were significantly higher in the R vs. NR (P=0.02), but the other phenotypes were not. We conclude that increased density of CD3+C8+PD1+, but not CD3+CD8+PD1- cells was present in pre-treatment biopsies in responders as compared with non-responders to BAT. These results indicate that men who are likely to respond to high dose testosterone treatment harbor tumors that are immunologically distinct prior to treatment than those men unlikely to respond. Additional spatial analysis and comparisons with RNAseq data will be presented. Citation Format: Carolina Gomes Alexandre, Jessica Hicks, Tracy Jones, John T Isaacs, Kiara Bowers, Alyza Skist, Laura Sena, Jennifer Meyers, Emmanuel Antonarakis, David Sanin, Hanfei Qi, Samuel Denmeade, Mark Markowski, Srinivasan Yegnasubramanian, Angelo De Marzo. Molecular pathology studies reveal PD1+ CD8 T cell density correlates with response to supraphysiological testosterone treatment in pre-treatment biopsies and MYC mRNA and protein correlate with response after treatment. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4345.