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Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with poor prognosis and overall survival. In 2018 it is estimated that there will be 44,333 deaths making PDAC the 3rd cause of cancer related deaths. A distal pancreatomy for PDAC in the body/tail of the pancreas usually includes a splenectomy, with current practice to discard the spleen post-surgery. Spleens can provide a large number of lymphocytes, including CD8+ T-cells with potential to be utilized for adoptive cellular therapy. Our studies have focused on spleen from this patient population, which we posit (and show herein) provides a large number of CD8+PD1+ cells for expansion and the first clinical strategy to expand activated, tumor specific T-cells for subsequent infusion with therapeutic intent. In our studies we used Milteny magnetic bead isolation of CD8+PD1+ T-cells from the spleens of PDAC patients with disease in the distal pancreas. The purity of the cells was 98% with a yield of 79% providing 9x108 CD8+PD1+ cells from an idealized 160 gm spleen. CD8 expansion was undertaken with various growth factor cocktails incorporating combinations of three interleukins (IL); IL-7, IL-15, and IL-21 at varying concentrations. IL-7 supports T-cell survival and proliferation, IL-15 is a potent T-cell growth factor and promotes naïve and memory T-cell survival, and IL-21 limits T-cell exhaustion in response to chronic stimulation. The optimized cocktail incorporated IL-7, IL-15, and IL-21 at 40 ng/mL, which demonstrated maximum T-cell expansion, based on cellularity and cell viability. The predominant T-cell memory phenotype of the CD8+PD1+ cells following expansion was transitional memory (TTM) T-cells that increased from 35.6 ± 4.3 to 52 ± 3%. This memory T-cell phenotype has a high proliferative potential while retaining effector function. Following six days of culture with the three interleukins we observed a 10 fold increase in CD8+PD1+ cells such that 9x109 cells are obtained from the 160 gm spleen. These cells, based on ELISPOT analysis with autologous tumor lysate pulsed dendritic cells retained tumor specificity. In conclusion, CD8+PD1+ cells can be isolated from resected spleen, expanded with retention of tumor specificity and potentially used for adoptive cellular therapy. Citation Format: Kathryn Cole, Quan P. Ly, Jesse L. Cox, Michael A. Hollingsworth, James C. Padussis, Jason M. Foster, Luciano M. Vargas, James E. Talmadge. Novel expansion of CD8+PD1+ spleen cells for therapeutic intent in pancreatic adenocarcinoma cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1420.

The spleen is a rich resource of large numbers of lymphocytes, with potential for use in adoptive cellular therapy (ACT). This is of particular relevance to patients with a distal pancreatic ductal adenocarcinoma (PDAC) who frequently undergo a pancreatectomy that required a splenectomy to clear the nodal basin. In this study we compared the phenotypes of eight paired sets of peripheral blood (PB) and spleen cell populations to assess the frequency of myeloid derived suppressor cells (MDSC) and lymphocyte subpopulations. Because a positive clinical response to ACT requires a low frequency of T-regs and MDSC’s, both systemically and in the tumor microenvironment these cells were one of the foci for our studies. A high frequency of these cells negatively correlate with survival and positively with tumor burden. Indeed, in this study we observed a significant decrease in the frequency of granulocytic, monocytic and immature MDSC’s in the spleen vs PB (4.8±1.6 vs. 54.4±6.1; 0.2±0.1 vs. 0.9±0.4; and 0.6±0.1 vs. 2.3±0.8 percent respectively) but not T-regulatory (T-reg) cells (0.8+0.5 vs. 0.1+0.04 percent) supporting the potential superiority of spleen cells relative to PB as a cellular source for ACT. In addition to a significantly higher number of spleen cells as compared to the PB for expansion/infusion, spleens have a higher frequency of T-cells as compared to the PB including CD3+ (35+5 vs. 16+4%), CD4+ (19+2 vs. 10+2%) and CD8+ (10+1 vs. 5+2%) T-cells. The frequency of transitional memory (Ttm) T-cells, which have a high proliferative potential were also significantly increased in the spleen vs. PB (37.5±6 vs. 26±3.0%) as was the frequency of PD1+CD8+ T-cells (8+2 vs 1+0.5%), which have been shown to have tumor specific cytotoxicity in cancer patients. In summary the increased frequency of PD-1+CD8+ cells, a Ttm memory phenotype and low levels of an exhaustion phenotype (TIM3 and Lag3 expression) in spleen cells vs the PB is supportive of the potential utility of these cells for expansion and therapeutic utility for these cells. Citation Format: Kathryn Cole, Quan Ly, Jesse L. Cox, Michael A. Hollingsworth, James C. Padussis, Jason M. Foster, Luciano M. Vargas, James E. Talmadge. Cellular phenotypes of spleen cells in cancer patients targeted for adoptive cellular therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4049.