Characterisation of immune infiltrates in malignant and benign prostate tissues

1. Characterisation of immune infiltrates in malignant and benign prostate tissues No. 122 D.T.S. Woon1,2, G. Whitty1, M. Saxena1,  D.M. Bolton1,2 , I. D. Davis1,3 1Ludwig Institute for Cancer Research Uro-oncology Laboratory 2Urology, Austin Health, Heidelberg, VIC, Australia 3Joint Ludwig-Austin Oncology Unit, Austin Health • Introduction • The role of immunity is not well defined for prostate cancer (PCa) • Regulatory T cells (Treg) are a subpopulation of T cells that can suppress the immune system • Treg maintain immune homeostasis by preventing overactivity of immune response by actively suppressing activation of the immune system • Hypothesis: Local immune modulation is relevant to prostate cancer biology. • Relatively high numbers of Treg and a low CD8/Treg ratio would tend to be present in patients with PCatissue. • A neutral pattern comparable to peripheral blood ratio would be expected in BPH tissue and normal prostate tissue • Results • A total of 33 patients were recruited for this project (table 1). • 24 of these patients had PCa. When comparing PCa with BPH and cystoprostatectomy group: • Percentage of activated CD8 cells in all three tissue groups is >20x higher than matching PBMC (Fig 3) • Percentage of Tregn in all three prostate tissue types is >2x higher than matching PBMC (Fig 4) • CD8/Treg ratio of all three tissue types are not very different (Fig 5). • CD8/Treg ratio of PBMC of all three patient groups were very similar • In seven patients, where we collected both PCa involved and non-involved samples from the same specimens. We found: • Percentage of Treg is higher in PCa tissue and is statistically significant. (Fig 6) • CD8:Treg Ratio in PCa involved tissue is lower than non-involved tissue (not statistically significant) (Fig 7) • No difference in the % of activated CD8 • Aim • To characterise and compare T cell responses in blood and tumour from patients with PCa, Benign Prostatic Hyperplasia (BPH) and no prostate pathology. • Method • Fresh prostate tissue from patients and their matching peripheral blood were collected. • Non-cancerous tissues were obtained from men with BPH undergoing radical prostatectomy, or with bladder cancer undergoing cystoprostatectomy. In each case the absence of PCa was confirmed by histology. • Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll gradient centrifugation. • Tissues were digested with collagenase/hyaluronidase overnight then incubated with trypsin to obtain a single cell population. • The tissue infiltrating lymphocytes (TILs) and the matching PBMCs were then subjected to detailed flow cytometric analysis to assess the following: • Proportion of Treg (Fig. 1) • CD45, CD3, CD4, CD25, and FoxP3+ • Activation status of T cells (Fig. 2) • CD45, CD8, CD69+ Table 1: Patients recruited Fig. 5: CD8:Treg ratio of tissue compared with PBMC Fig. 3: Percentage of activated CD8+ cells in tissue compared with PBMC Fig. 6: Percentage of Treg of PCa involved vs non-involved tissue Fig. 1:Treg Flow-cytometry gating strategy for tissue Fig. 7: CD8:Treg ratio of PCa involved vs non-involved tissue Fig. 4: Percentage of Treg in tissue compared with PBMC 1. FSC vs SCC plot: All cells in tissue except debris were gated 3. Dump vs CD45 plot: CD45+ cells were gated 2. Dump vsLivedeadAmCyan plot: Only live cells were gated • Conclusions • There is a higher proportion of Treg in all tissue types compared with PBMC. • PCa shows a trend consistent with a pattern of immunosuppression (higher % Treg, lower CD8:Treg ratio) when compared with non-involved tissue. • In all tissue types, the percentage of activated CD8 cells is >20 times that of PBMC • A high level of CD8+ T cell activation in the setting of high numbers and proportions of Treg is unexpected. This could be due to: • Our local immunosuppression hypothesis is incorrect; OR • the activated CD8+ cells are impaired and may not be acting as cytotoxic T cells and producing “anti-tumour” cytokines (IFNγ and TNFα), and instead producing other cytokines to favor cancer growth (IL-4, IL-10, TGF-β1) • Future direction: Using real time quantitative RT-PCR to assess production of relevant cytokines in all tissue types • Th0: IL10 TGF-β1; Th1: IFNγ, TNFα; Th2: IL4, IL5, IL13 6 CD25 vs FoxP3 plot: Treg (CD25 & FoxP3) positive cells were gated. 4. CD4 vs CD3 plot: CD4 and CD3 positive cells were gated 5. CD25 vs FoxP3 (isotype) plot: CD25 positive cells were gated Fig. 2: Activated CD8+ cells Flow cytometry gating strategy for Tissue 2. Dump vsSytox blue plot: Only live cells were gated 1. FSC vs SCC plot: All cells in tissue except cell debris were gated 3. Dump vs CD45 plot: CD45+ cells were gated • Acknowledgements • The authors would like to thank the patients who donated tissue and blood. • DTSW would like to acknowledge the RACS and Sporting Chance Australia for scholarship support • This study is sponsored by the Ludwig Institute for Cancer Research. • IDD is an NHMRC Practitioner Fellow. 4. Dump vs CD8 plot: CD8+ cells were gated 6 CD69 vs CD8 plot: Activated CD8+ cells were gated 5. CD69 Isotypevs CD8 plot: Isotype control against CD69 Poster presentation sponsor