2a) consistent with prior reports illustrating that this antibody actively reduces Treg frequency [26]. data have important implications for T cell immunotherapy in PDAC and demonstrate a novel role for CTLA-4/CD80 interactions in regulating T cell exclusion. In addition, our findings suggest distinct mechanisms govern CD4+ and CD8+ T cell infiltration in PDAC. (KPC) mice and (CiMist1) control mice. The increase in Tregs in KCiMist1 mice was even more marked after induction of chronic pancreatitis using cerulein (Supplementary Fig. 1). In this model, cerulein-induced chronic inflammation drives carcinogenesis and the development of invasive PDAC [12]. While an increased frequency of Tregs was also observed after cerulein treatment in CiMist1 mice which lack expression of the mutation in the pancreas, the Treg frequency was increased >2 fold in KCiMist1 mice, at a time point when the histopathology of the pancreas shows evidence of pancreatic intraepithelial neoplasia (PanIN) [20]. Thus, this finding suggests a role for malignant cells in directing Treg Troglitazone recruitment to pancreatic tumors (Supplementary Fig. 1). Using the (KPC) mouse model of invasive PDAC, we found by flow cytometry a similar result of increased frequency of Foxp3+ Tregs detected in the pancreas without an overt change in the frequency of CD4+ or CD8+ cells among total CD3+ T cells (Fig. 1c and Supplementary Fig. 2). However, since developing PDAC tumors commonly invade or metastasize to peritumoral lymph nodes, we Troglitazone next used microscopy to determine the location of Foxp3+ Tregs that were detected in pancreatic tissue in KPC mice. We found in malignant tissue that the presence of Foxp3+ cells was most pronounced around pancreatic intraepithelial neoplasia (PanIN) which are precursor lesions to the development of invasive PDAC (Fig. 1d, e). However, the majority of Tregs were detected in peritumoral lymph nodes rather than within the tumor bed. The frequency of Tregs detected in peritumoral lymph nodes was similar to non-tumor draining control lymph nodes. Thus, our findings are consistent with Treg recruitment to tumor tissue beginning early during tumorigenesis but with the majority of Tregs remaining confined to lymphatic structures that surround malignant lesions. Treg depletion and CTLA-4 blockade stimulate CD4 T cell infiltration in PDAC Tregs are well-recognized for their capacity to dampen T cell immune responses and in doing so, they can be key proponents of immune escape. Tregs can express high levels of the IL-2 receptor, CD25, which is important for Tregs to maintain an immunosuppressive phenotype [21]. In addition, CTLA-4 is constitutively expressed by Tregs and its expression is critical for Tregs to regulate conventional T cell activation [22] and to modulate suppressive properties of antigen presenting cells [23]. CTLA-4 binds to B7 ligands including CD80 and CD86 molecules that are expressed on antigen presenting cells and has a higher affinity for these ligands than the Troglitazone activating co-receptor CD28 which is essential for activation of conventional na?ve T cells [24]. In patients with metastatic breast cancer, CD25 antibodies produce a prolonged decrease in peripheral blood Tregs [21] and in patients with metastatic melanoma, CTLA-4 antibodies induce T cell infiltration into tumors [25]. Therefore, we examined the impact of targeting Tregs, using anti-CD25 and anti-CTLA-4 antibodies, on T cell infiltration into PDAC tumors in KPC mice. We found that treatment with anti-CD25 versus control reduced the frequency of Foxp3+CD4+ cells in the blood by >75% (Fig. 2a) consistent with prior reports illustrating that this antibody actively reduces Treg frequency [26]. Although CD25 can also be transiently expressed on conventional T cells upon activation, we found that only a small subset of CD8+ T cells (approximately 5%) expressed CD25 in the peripheral blood (Supplementary Fig. 3a). In addition, the presence of a PDAC tumor did not alter CD25 expression on CD8+ T cells compared to healthy control mice (Supplementary Fig. 3A). Further, anti-CD25 treatment did not produce any significant changes in CD8+ T frequency in the peripheral blood or peri-tumoral lymph nodes (Supplementary Fig. 3b, c). In contrast to selective Treg depletion seen with anti-CD25 antibodies, treatment with anti-CTLA-4 did not affect the number of Foxp3+CD4+ cells detected in the Troglitazone Rabbit polyclonal to p53 peripheral blood (Fig. 2b). Open in a separate window Fig. 2 Antibodies targeting CD25 and CTLA-4 induce CD4+ T cell infiltration into spontaneous PDAC tumorsMice (and is more rapidly upregulated on malignant cells than major histocompatibility molecules which are necessary for antigen.