Restored Thymic Output after Androgen Blockade Participates in Antitumor Immunity

Abstract

The thymus is a hormone-sensitive organ, which involutes with age in response to production of sex steroids. Thymic involution leads to a decrease in the generation of recent thymic emigrants (RTEs), resulting in a reduced response to immune challenges such as cancer. Interestingly, the standard of care for prostate cancer patients is androgen deprivation therapy (ADT), which leads to thymic regeneration and an increase in thymic output. It remains unknown whether these newly produced T cells can contribute to the antitumor immune response. This study defines the kinetics of thymic regeneration in response to ADT in mice, determining that thymic epithelial cell proliferation is critical for the increase in RTE output. Using a mouse model to track RTE in vivo, we demonstrate that these newly generated RTEs can traffic to tumors, where they become activated and produce effector cytokines at levels similar to more mature T cells. Collectively, these data suggest that RTEs produced from ADT-induced thymic regeneration could be harnessed for the antitumor immune response.

Figure 1.. ADT promotes thymic regeneration through proliferation of TEC but not thymocytes.

12-week old male mice were orchiectomized, and thymi were harvested at 2, 5 and 12 weeks later. A) Thymic weight, B) total thymocyte count and C) number of thymic epithelial cells (TEC) at time of harvest. D) Representative flow plots gated on TECs (CD45−Epcam+) and showing EpCAM and Ki67 expression, and E) quantification of percent Ki67+ TEC at 0, 2, 5 and 12 weeks post orchiectomy. F) Numbers and G) percent Ki67+ of DN, DP, CD4 SP and CD8 SP in the thymus at the indicated times. H) 12 week old male mice were orchiectomized or chemically castrated using degarelix, and thymi were harvested 2 or 4 weeks later. Graphs show thymic weight and total thymocyte counts after the indicated treatments. 3 animals per group. Data representative of 3 experiments. One-way ANOVA with Tukey multiple comparison, * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001.

Figure 2.. RTE traffic to the tumor and display an activated phenotype.

A) Experimental design. B) Representative cytogram (gated on live, TCRβ+) showing CD45.1+ (host) and CD45.2+ (adoptively transferred cells) staining in the dLN and tumor. C-D) Representative histograms, gated on live, TCRβ+, CD45.1+ (host T cells) or CD45.2+ (RTE), showing Rag2-GFP, PD1, CD44 and CD69 expression in host CD8 T cells and adoptively transfered RTE CD8 T cells in dLN (C) and tumor (D). E) Quantification of percent PD1+, CD44hi and CD69+ RTE and host T cells in the tumor. 3 animals per group. Data representative of 3 experiments. Unpaired two-tailed Student t test, * P<0.05, ** P<0.01.

Figure 3.. Tumor-specific RTE contribute to the anti-tumor immune response.

A) Experimental design. B) Representative flow plots (gated on live, TCRβ+) showing Rag2-GFP and Thy1.1 staining in the dLN and tumor of animals that received adoptive transfer of RTE OTI or non-RTE OTI. C) Cell number of recovered OTI cells in the dLN and tumor. D) Representative histograms (gated on live, TCRβ+, CD8+, Thy1.1+ or Thy1.1-) showing Rag2-GFP, PD1 and CD44 expression in the OTI cells and host endogenous CD8 T cells. E) Quantification of PD1 and CD44 MFI in OTI the dLN and tumor. F) Percent Ki67+ OTI cells. G-I) dLN cells and tumor infiltrating lymphocytes (TIL) were stimulated in vitro with SIINFEKL peptide and stained for intracellular cytokines. G) Representative flow plots (gated on live, TCRβ+, CD8, Thy1.1+) showing IFNγ and TNFα expression adoptively transferred RTE and non-RTE in the dLN and tumor. H-I) Quantification of percent IFNγ+ and TNFα+ among adoptively transferred RTE and non-RTE in the dLN (H) and tumor (I). 3 animals per group. Data are representative of 2 experiments. Unpaired two-tailed Student t test, * P<0.05.

Figure 4.. Orchiectomy leads to increased numbers of functional CD8+ RTE in the tumor.

A) Experimental design. B) Representative flow plots (gated on live, TCRβ+, CD8+) showing CD44 and tdTomato expression in the dLN and tumor of intact and orchiectomized animals. Quantification of percent and numbers of tdTomato+ CD8 T cells in the C) dLN and D) tumor (numbers are per gram of tumor). E) Representative flow plots gated on live, TCRβ+, CD8, tdTomato+ or - showing IFNγ and TNFα expression. Intact TdTomato+ cells are not shown, but look identical to intact TdTomato- cells. F, G) Quantification of percent IFNγ+ and IFNγ+TNFα+ in the dLN (F) and tumor (G). H, I) Number of CD8+ RTE expressing IFNγ (H) and TNFα (I) per gram of tumor. 3 animals per group. Data representative of 3 repeat experiments. Unpaired two-tailed Student t test, * P<0.05, ** P<0.01, *** P<0.001.