Examining T cells at vaccine sites of tumor-bearing hosts provides insights to dysfunctional T-cell immunity.

When tumor vaccines are administered as cancer immunotherapy, cellular interactions at the vaccine site are crucial to the generation of antitumor immunity. Examining interactions at the vaccine site could provide important insights to the success or failure of vaccination. Our laboratory previously showed that while administration of a cell-based vaccine to tumor-free mice leads to productive antineuroblastoma immunity, vaccination of tumor-bearing mice does not. The goal of this study was to examine immune effectors at the vaccine site to identify mechanisms responsible for the generation of ineffective antitumor immunity in tumor-bearing mice. The results of this study show that vaccine sites of tumor-bearing mice contained significantly fewer T cells than vaccine sites of tumor-free mice. Similar migration and proliferation of T cells was observed in the vaccine sites of tumor-bearing and tumor-free mice, but T cells in the sites of tumor-bearing mice were more apoptotic. T cells at the vaccine sites of both tumor-free and tumor-bearing mice had an effector-memory phenotype and expressed activation markers. Despite the activated phenotype, T cells from tumor-bearing mice elicited defective antitumor immune responses. Although T cells from vaccine sites of tumor-bearing mice were capable of producing inflammatory cytokines, the T cells from tumor-bearing mice produced lower levels of cytokines compared with T cells from the tumor-free mice. Remarkably, this defect seems to be systemic, affecting distal T cells in tumor-bearing mice. This study demonstrates that the defective vaccine-induced immune response to neuroblastoma in tumor-bearing hosts originates as a result of tumor burden, resulting in poor antitumor immunity.

Immunosuppressive effects of multiple myeloma are overcome by PD-L1 blockade.

Multiple myeloma is an incurable plasma cell malignancy. Patients who fail conventional therapy are frequently treated with hematopoietic stem cell transplantation (HSCT), which results in reduced tumor burden, but the patients subsequently relapse from sites of chemotherapy-resistant disease. Using the 5T33 murine model of myeloma and a previously successful immunotherapy regimen consisting of autologous (syngeneic) HSCT and cell-based vaccine administration, we were unable to improve survival of myeloma-bearing mice. The 5T33 tumor line, similar to malignant plasma cells from myeloma patients, expresses high levels of programmed death receptor ligand-1 (PD-L1), which binds to the inhibitory receptor, PD-1. We observed that T cells from myeloma-bearing mice express high levels of PD-1, which has also been observed in patients with multiple myeloma. These PD-1(+) T cells were exhausted and produced IL-10. Based on these observations, we combined HSCT with whole-cell vaccination and PD-L1 blockade. Inhibition of the PD-1/PD-L1 pathway with HSCT and whole-cell vaccination increased the survival of myeloma-bearing mice from 0% to 40%. These data demonstrate a role for PD-L1 in suppressing immune responses to myeloma and suggest that blockade of this pathway may enhance immunotherapy for this disease.

Depletion of CD25⁺ T cells from hematopoietic stem cell grafts increases posttransplantation vaccine-induced immunity to neuroblastoma.

A multifaceted immunotherapeutic strategy that includes hematopoietic stem cell (HSC) transplantation, T-cell adoptive transfer, and tumor vaccination can effectively eliminate established neuroblastoma tumors in mice. In vivo depletion of CD4⁺ T cells in HSC transplantation recipients results in increased antitumor immunity when adoptively transferred T cells are presensitized, but development of T-cell memory is severely compromised. Because increased percentages of regulatory T (Treg) cells are seen in HSC transplantation recipients, here we hypothesized that the inhibitory effect of CD4⁺ T cells is primarily because of the presence of expanded Treg cells. Remarkably, adoptive transfer of presensitized CD25-depleted T cells increased tumor vaccine efficacy. The enhanced antitumor effect achieved by ex vivo depletion of CD25⁺ Treg cells was similar to that achieved by in vivo depletion of all CD4⁺ T cells. Depletion of CD25⁺ Treg cells resulted in elevated frequencies of tumor-reactive CD8 and CD4⁺ T cells and increased CD8-to-Treg cell ratios inside tumor masses. All mice given presensitized CD25-depleted T cells survived a tumor rechallenge, indicating the development of long-term CD8⁺ T-cell memory to tumor antigens. These observations should aid in the future design of immunotherapeutic approaches that promote the generation of both acute and long-term antitumor immunity.

Early expression of stem cell-associated genes within the CD8 compartment after treatment with a tumor vaccine.

Using a mouse neuroblastoma cell line, we have demonstrated that vaccination of tumor-free mice with a cell-based vaccine leads to productive immunity and resistance to tumor challenge, while vaccination of tumor-bearing mice does not. The T cell immunity induced by this vaccine, as measured by in vitro assays, is amplified by the depletion of Treg. Our goal is to understand this barrier to the development of protective cellular immunity. mRNA microarray analyses of CD8(+) T cells from naïve or tumor-bearing mice undergoing vaccination were carried out with or without administering anti-CD25 antibody. Gene-expression pathway analysis revealed the presence of CD8(+) T cells expressing stem cell-associated genes early after induction of productive anti-tumor immunity in tumor-free mice, prior to any phenotypic changes, but not in tumor-bearing mice. These data demonstrate that early after the induction of productive immune response, cells within the CD8(+) T cell compartment adopt a stem cell-related genetic phenotype that correlates with increased anti-tumor function.

Combination therapy using IL-2 and anti-CD25 results in augmented natural killer cell-mediated antitumor responses.

Interleukin (IL)-2 has been extensively examined to promote clinical T and natural killer (NK) cell responses. Regulatory T cells (Tregs) have been shown to regulate many aspects of the immune system, including NK cell-mediated responses. We have demonstrated that in vivo administration of IL-2 led to activation and expansion of both NK cells and immunosuppressive Tregs. Therefore, we attempted to augment NK cell antitumor effects by concurrently depleting Tregs using anti-CD25. Increased NK cell activation by IL-2 was found to be correlated with an increase in classical, short-term NK cell in vitro killing assays regardless of the depletion of Tregs. But when splenocytes of the treated mice were used in long-term tumor outgrowth experiments, we observed that prior depletion of Tregs from IL-2 administration led to improved antitumor effects compared with either treatment alone. Importantly, these in vitro data are correlated with subsequent in vivo survival of leukemia-bearing mice, in which co-treatment of IL-2 with anti-CD25 led to significantly improved survival compared with mice treated with either IL-2 alone or with Treg depletion. Prior depletion of NK1.1(+) cells, but not of CD8(+) cells, completely abrogated all antitumor effects mediated by IL-2 and anti-CD25 combination therapy. These findings demonstrate that superior NK cell-mediated antileukemic effects can be achieved with IL-2 administration and concurrent depletion of CD25(+) cells.

Sensitization of tumor cells to NK cell-mediated killing by proteasome inhibition.

Bortezomib is a proteasome inhibitor that has direct antitumor effects. We and others have previously demonstrated that bortezomib could also sensitize tumor cells to killing via the death ligand, TRAIL. NK cells represent a potent antitumor effector cell. Therefore, we investigated whether bortezomib could sensitize tumor cells to NK cell-mediated killing. Preincubation of tumor cells with bortezomib had no effect on short-term NK cell killing or purified granule killing assays. Using a 24-h lysis assay, increases in tumor killing was only observed using perforin-deficient NK cells, and this increased killing was found to be dependent on both TRAIL and FasL, correlating with an increase in tumor Fas and DR5 expression. Long-term tumor outgrowth assays allowed for the detection of this increased tumor killing by activated NK cells following bortezomib treatment of the tumor. In a tumor purging assay, in which tumor:bone marrow cell mixtures were placed into lethally irradiated mice, only treatment of these mixtures with a combination of NK cells with bortezomib resulted in significant tumor-free survival of the recipients. These results demonstrate that bortezomib treatment can sensitize tumor cells to cellular effector pathways. These results suggest that the combination of proteasome inhibition with immune therapy may result in increased antitumor efficacy.

Positive and negative regulation of Natural Killer cells: therapeutic implications.

Natural Killer (NK) cells can mediate numerous anti-tumor and anti-viral effector functions as well as play important immunoregulatory roles in various disease states. Promoting the ability of NK cells to respond in an immunotherapeutic setting has often been sought by the addition of NK cell-stimulating factors. However, such therapies are often found to be insufficient, which may in part be due to the presence of inhibitory influences on the NK cell. NK cells can respond to a plethora of cytokines which are generated by numerous cell types and these interactions can markedly affect NK cell survival and activity. NK cells also possess multiple activating and inhibiting receptors which can alter their function. Whether the NK cell will become activated or not can depend on a complex balance of activating and inhibitory signals received by the cell and modulation of these signals may shift the balance on NK activation. This review discusses the various activating and inhibitory stimuli which can act on NK cells, and suggests that future NK cell-based therapies consider not only activating stimuli but also removal of possible inhibitory elements which could prevent optimal NK cell function and/or survival.

Natural killer cells: biology and clinical use in cancer therapy.

Natural killer (NK) cells have the ability to mediate both bone marrow rejection and promote engraftment, as well as the ability to elicit potent anti-tumor effects. However the clinical results for these processes are still elusive. Greater understanding of NK cell biology, from activating and inhibitory receptor functions to the role of NK cells in allogeneic transplantation, needs to be appreciated in order to draw out the clinical potential of NK cells. Mechanisms of bone marrow cell (BMC) rejection are known to be dependent on inhibitory receptors specific for major histocompatibility complex (MHC) molecules and on activating receptors that have many potential ligands. The modulation of activating and inhibitory receptors may hold the key to clinical success involving NK cells. Pre-clinical studies in mice have shown that different combinations of activating and inhibitory receptors on NK cells can reduce graft-versus-host disease (GVHD), promote engraftment, and provide superior graft-versus-tumor (GVT) responses. Recent clinical data have shown that the use of KIR-ligand incompatibility produces tremendous graft-versus-leukemia effect in patients with acute myeloid leukemia at high risk of relapse. This review will attempt to be a synthesis of current knowledge concerning NK cells, their involvement in BMT, and their use as an immunotherapy for cancer and other hematologic malignancies.