William H.D. Hallett

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.

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.

Sensitization of human breast cancer cells to natural killer cell‐mediated cytotoxicity by proteasome inhibition

The proteasome inhibitor, bortezomib, has direct anti‐tumour effects and has been demonstrated to sensitize tumour cells to tumour necrosis factor‐related apoptosis‐inducing ligand‐mediated apoptosis. Natural killer (NK) cells are effective mediators of anti‐tumour responses, both through cytotoxic granule killing and apoptosis‐inducing pathways. We therefore investigated if bortezomib sensitized human breast cancer cells to killing by the human NK cell line, NK‐92. Bortezomib was unable to sensitize MDA‐231 breast cancer cells to NK cell‐mediated killing in short‐term in vitro assays. However, bortezomib did cause these cells to up‐regulate apoptosis‐related mRNA as well as death receptors on the cell surface. In a long‐term in vitro tumour outgrowth assay that allows NK cells to use their full repertoire of killing pathways, bortezomib sensitized three breast cancer cell lines to NK cell‐mediated killing, which led to greater anti‐tumour effects than either treatment alone. We then used a xenogeneic mouse model in which CB‐17 SCID mice were injected with human breast cancer cells. This model displayed the effectiveness of NK‐92 cells, but the addition of bortezomib did not increase the survival further or reduce the number of lung metastases in tumour‐bearing mice. However, while bortezomib was highly cytotoxic to NK‐92 cells in vitro, bortezomib treatment in vivo did not decrease NK‐92 function, suggesting that through alternative dosing or timing of bortezomib, greater efficacy may occur from combined therapy. These data demonstrate that combined treatment of human breast cancer with bortezomib and NK cells has the potential to generate superior anti‐tumour responses than either therapy alone.

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.

5-Azacytidine- and Hoechst-induced aneuploidy in Indian muntjac

Hoechst 33258 (bis-benzimidazole) and 5-azacytidine (5-AC) cause decondensation of the pericentric heterochromatin in mouse and aberrations in the sequence of centromere separation apparently by different mechanisms. We treated the male Indian muntjac cells (2n=7), which do not undergo decondensation of the pericentric heterochromatin, to study if these chemicals would result in induction of aneuploidy limited to the Y(2) chromosome. This paper reports that both agents result in aneuploidy primarily limited to one chromosome, the Y(2). It is likely that other chromosomes are not tolerated in aneuploid condition because every chromosome carries some household genes including those essential for mitotic progression. The loss/gain of the Y(2) chromosome is tolerated because it is the smallest chromosome and is almost entirely composed of constitutive heterochromatin. Since Indian muntjac has only three pairs of large chromosomes comprising its basic genome, which can be clearly viewed under high dry objective, these cells are very suitable for the preliminary analysis of aneuploidy-inducing ability of various chemicals.

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.

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.

Exploiting arginase to prevent GVHD

In this issue of Blood , Highfill and colleagues introduce a novel procedure to generate arginase-producing MDSCs that can prevent GVHD while maintaining antileukemic responses. An alternative non-cell–based approach using pegylated arginase-1 is also introduced. Allogeneic hematopoietic stem