CD8+ T-cell responses rapidly select for antigen-negative tumor cells in the prostate.

Stimulation of patients' immune systems for the treatment of solid tumors is an emerging therapeutic paradigm. The use of enriched autologous T cells for adoptive cell therapy or vaccination with antigen-loaded dendritic cells have shown clinical efficacy in melanoma and prostate cancer, respectively. However, the long-term effects of immune responses on selection and outgrowth of antigen-negative tumor cells in specific tumor types must be determined to understand and achieve long-term therapeutic effects. In this study, we have investigated the expression of a tumor-specific antigen in situ after treatment with tumor-specific CD8(+) T cells in an autochthonous mouse model of prostate cancer. After T-cell treatment, aggregates of dead antigen-positive tumor cells were concentrated in the lumen of the prostate gland and were eventually eliminated from the prostate tissue. Despite the elimination of antigen-positive tumor cells, prostate tumor continued to grow in T-cell-treated mice. Interestingly, the remaining tumor cells were antigen negative and downregulated MHC class I expression. These results show that CD8(+) T cells are effective in eliminating antigen-bearing prostate tumor cells but they also can select for the outgrowth of antigen-negative tumor cells. These findings provide insights into the requirements for an effective cancer immunotherapy within the prostate that not only induces potent immune responses but also avoids selection and outgrowth of antigen-negative tumor cells.

Differential requirement for CD70 and CD80/CD86 in dendritic cell-mediated activation of tumor-tolerized CD8 T cells.

A major obstacle to efficacious T cell-based cancer immunotherapy is the tolerizing-tumor microenvironment that rapidly inactivates tumor-infiltrating lymphocytes. In an autochthonous model of prostate cancer, we have previously shown that intratumoral injection of Ag-loaded dendritic cells (DCs) delays T cell tolerance induction as well as refunctionalizes already tolerized T cells in the tumor tissue. In this study, we have defined molecular interactions that mediate the effects of DCs. We show that pretreating Ag-loaded DCs with anti-CD70 Ab abolishes the ability of DCs to delay tumor-mediated T cell tolerance induction, whereas interfering with 4-1BBL, CD80, CD86, or both CD80 and CD86 had no significant effect. In contrast, CD80(-/-) or CD80(-/-)CD86(-/-) DCs failed to reactivate already tolerized T cells in the tumor tissue, whereas interfering with CD70 and 4-1BBL had no effect. Furthermore, despite a high level of programmed death 1 expression by tumor-infiltrating T cells and programmed death ligand 1 expression in the prostate, disrupting programmed death 1/programmed death ligand 1 interaction did not enhance T cell function in this model. These findings reveal dynamic requirements for costimulatory signals to overcome tumor-induced tolerance and have significant implications for developing more effective cancer immunotherapies.

Phenotypic and functional delineation of murine CX(3)CR1 monocyte-derived cells in ovarian cancer.

Ovarian tumor progression is marked by the peritoneal accumulation of leukocytes. Among these leukocytes, an immunosuppressive CD11b(+)CD11c(+) population has been identified in both human and ovarian tumors. The use of transplantable models of murine ovarian tumors has demonstrated that this population promotes ovarian tumor growth, whereas elimination of this population has been shown to inhibit ovarian tumor progression. Despite the demonstrated importance of these cells to ovarian tumor progression, the mechanisms by which these cells are recruited to the peritoneal tumor are largely unknown. Therefore, this study analyzes the mechanisms these cells use to migrate to the peritoneum with the goal of therapeutically blocking their recruitment and subsequent immunosuppressive activity. Recent studies have identified that CX(3)CR1, Gr-1, and CCR2 delineate phenotypic and functional murine monocyte subsets. Here, we report that CX(3)CR1(lo)Gr-1(hi) cells dominate the population of peritoneal CD11b(+) leukocytes early in murine tumor development; however, the CX(3)CR1(hi) population of cells present in the peritoneum dramatically increases in both total numbers and percentage during tumor progression. Functional analyses reveal that both of these CX(3)CR1 subsets are immunosuppressive to naive CD8(+) and CD4(+) T-cell responses. Importantly, we demonstrate that CCR2 is a critical functional facilitator of leukocyte recruitment to the ovarian tumor microenvironment, and its genetic deletion results in a reduced tumor burden compared with wild-type mice. These results demonstrate that subsets of immunosuppressive leukocytes are recruited to the ovarian tumor environment through the CCR2 pathway, which offers a viable therapeutic target to inhibit their migration to the tumor site.

Murine ovarian cancer vascular leukocytes require arginase-1 activity for T cell suppression.

The predominant leukocyte population present in both human and murine peritoneal ovarian tumors is the Vascular Leukocyte (VLC). VLCs are recruited en masse to the ovarian tumor microenvironment whereupon they promote tumor progression. Importantly, the presence of VLCs is requisite for peritoneal ovarian cancer progression: selective elimination of VLCs inhibits tumor burden and ascites accumulation. Despite the critical importance of VLCs to ovarian tumors, their derivation and the mechanisms by which they facilitate tumor progression are not well understood. Here we demonstrate in vivo that the murine ID8 ovarian tumor model can usurp the host peritoneal macrophage pathway to elicit and recruit VLCs. Moreover, we demonstrate that VLCs express CD11b and Gr-1, a characteristic phenotype shared amongst heterogeneous populations of leukocytes referred to as myeloid-derived suppressor cells (MDSCs). In accord with their MDSC phenotype, both murine and human VLCs express arginase-1 (ARG1). Importantly, we demonstrate that the VLCs suppress both CD8(+) and CD4(+) T cells responses and that this immunosuppression is ARG1-dependent, since blockade of VLC ARG1 activity with nor-NOHA reversed the immunosuppression. These data further characterize the tumor-associated leukocytes in ovarian cancer and provide insights into the mechanisms by which they promote tumor growth.

Calreticulin requires an ancillary adjuvant for the induction of efficient cytotoxic T cell responses.

Molecular chaperones stimulate the immune system to induce both protective immune responses and therapeutic tumor rejection. However, the underlying basis for this immunogenic activity is not well understood. A variety of chaperones, including calreticulin, hsp70 and grp94, function as vehicles to efficiently traffic associated peptides into professional antigen presenting cells. Importantly, these chaperones have also been proposed to function as adjuvants by stimulating the dendritic cell activation and co-stimulatory responses required to elicit peptide-specific CD8(+) T cell cytolytic activity. The efficacy of chaperone-mediated tumor rejection has been attributed to the ability of chaperones to function in both of these capacities. However, purified calreticulin has not previously been assessed for its ability to elicit DC maturation and, moreover, recent data indicates that it is not efficient at inducing Nf-kappaB activity which often accompanies or stimulates DC maturation. Here we use two complementary methods to produce endotoxin-free calreticulin and demonstrate that it does not measurably mature or activate dendritic cells both in vitro and in vivo. Additionally, a calreticulin/peptide complex required the addition of an exogenous adjuvant to elicit in vivo cytotoxic CD8(+) T cell responses. These data are discussed with respect to current models for chaperone-derived immune responses and in regard to rational vaccine design.

Scavenger receptor-A-targeted leukocyte depletion inhibits peritoneal ovarian tumor progression.

Immunosuppressive leukocytes are emerging as a critical factor in facilitating tumor progression. These leukocytes are converted by the tumor microenvironment to become tolerogenic, facilitate metastasis, and to aid in neovascularization. The predominant variety of suppressive leukocytes found in human and murine ovarian cancer are called vascular leukocytes (VLC), due to sharing functions and cell surface markers of both dendritic cells and endothelial cells. Using the ID8 murine model of ovarian cancer, the aim of this study was to test the efficacy of VLC elimination as an ovarian tumor therapy. We show that carrageenan-mediated depletion of peritoneal tumor-associated leukocytes inhibits ovarian tumor progression. We then identified scavenger receptor-A (SR-A) as a cell surface receptor that is robustly and specifically expressed within human and murine ovarian tumor ascites upon VLCs. Administration of anti-SR-A immunotoxin to mice challenged with peritoneal ID8 tumors eliminated tumor-associated VLCs and, importantly, substantially inhibited peritoneal tumor burden and ascites accumulation. Moreover, the toxin required targeting to SR-A because mice that received untargeted toxin did not exhibit inhibition of tumor progression. We conclude that SR-A constitutes a novel and specific target for efficacious immunotherapeutic treatment of peritoneal ovarian cancer.