Arthur A. Hurwitz

Augmentation of T Cell Levels and Responses Induced by Androgen Deprivation

Androgen has been implicated as a negative regulator of host immune function and a factor contributing to the gender dimorphism of autoimmunity. Conversely, androgen deprivation has been suggested to potentiate male host immunity. Studies have shown that removal of androgen in postpubertal male mice produces an increase in size and cellularity of primary and peripheral lymphoid organs, and enhances a variety of immune responses. Yet, few details are known about the effect of androgen removal on T cell-mediated immunity. In this study, we demonstrate two pronounced and independent alterations in T cell immunity that occur in response to androgen deprivation, provided by castration, in postpubertal male mice. First, we show that levels of T cells in peripheral lymphoid tissues of mice are increased by androgen deprivation. Second, T cells from these mice transiently proliferate more vigorously to TCR- and CD28-mediated costimulation as well as to Ag-specific activation. In addition, androgen deprivation accelerates normalization of host T and B cell levels following chemotherapy-induced lymphocyte depletion. Such alterations induced by androgen deprivation may have implications for enhancing immune responses to immunotherapy and for accelerating the recovery of the immune system following chemotherapy.

Manipulation of costimulatory signals to enhance antitumor T-cell responses

One of the major goals of tumor immunotherapy is the induction of tumor-specific T-cell responses that will be effective in eradicating disseminated tumors. Emerging information on the role of costimulatory molecules in T-cell activation offers several new strategies for enhancing antitumor responses, including the induction of expression of costimulatory molecules on tumor cells, enhancement of the presentation of transferred tumor antigen by host antigen-presenting cells, and ex vivo antigen priming of autologous antigen-presenting cells.

FOXO3 programs tumor-associated DCs to become tolerogenic in human and murine prostate cancer

The limited success of cancer immunotherapy is often attributed to the loss of antigen-specific T cell function in situ. However, the mechanism for this loss of function is unknown. In this study, we describe a population of tumor-associated DCs (TADCs) in both human and mouse prostate cancer that tolerizes and induces suppressive activity in tumor-specific T cells. In tumors from human prostate cancer patients and transgenic adenocarcinoma of the mouse prostate (TRAMP) mice, TADCs expressed elevated levels of FOXO3 and Foxo3, respectively, which correlated with expression of suppressive genes that negatively regulate T cell function. Silencing FOXO3 and Foxo3 with siRNAs abrogated the ability of human and mouse TADCs, respectively, to tolerize and induce suppressive activity by T cells. Silencing Foxo3 in mouse TADCs was also associated with diminished expression of tolerogenic mediators, such as indoleamine-2,3-dioxygenase, arginase, and TGF-β, and upregulated expression of costimulatory molecules and proinflammatory cytokines. Importantly, transfer of tumor-specific CD4+ Th cells into TRAMP mice abrogated TADC tolerogenicity, which was associated with reduced Foxo3 expression. These findings demonstrate that FOXO3 may play a critical role in mediating TADC-induced immune suppression. Moreover, our results identify what we believe to be a novel target for preventing CTL tolerance and enhancing immune responses to cancer by modulating the immunosuppressive activity of TADCs found in the tumor microenvironment.

MicroRNA-1 is a candidate tumor suppressor and prognostic marker in human prostate cancer

We previously reported that miR-1 is among the most consistently down-regulated miRs in primary human prostate tumors. In this follow-up study, we further corroborated this finding in an independent data set and made the novel observation that miR-1 expression is further reduced in distant metastasis and is a candidate predictor of disease recurrence. Moreover, we performed in vitro experiments to explore the tumor suppressor function of miR-1. Cell-based assays showed that miR-1 is epigenetically silenced in human prostate cancer. Overexpression of miR-1 in these cells led to growth inhibition and down-regulation of genes in pathways regulating cell cycle progression, mitosis, DNA replication/repair and actin dynamics. This observation was further corroborated with protein expression analysis and 3′-UTR-based reporter assays, indicating that genes in these pathways are either direct or indirect targets of miR-1. A gene set enrichment analysis revealed that the miR-1-mediated tumor suppressor effects are globally similar to those of histone deacetylase inhibitors. Lastly, we obtained preliminary evidence that miR-1 alters the cellular organization of F-actin and inhibits tumor cell invasion and filipodia formation. In conclusion, our findings indicate that miR-1 acts as a tumor suppressor in prostate cancer by influencing multiple cancer-related processes and by inhibiting cell proliferation and motility.

Engagement of OX40 Enhances Antigen-Specific CD4+ T Cell Mobilization/Memory Development and Humoral Immunity: Comparison of αOX-40 with αCTLA-4

Increasing the long-term survival of memory T cells after immunization is key to a successful vaccine. In the past, the generation of large numbers of memory T cells in vivo has been difficult because Ag-stimulated T cells are susceptible to activation-induced cell death. Previously, we reported that OX40 engagement resulted in a 60-fold increase in the number of Ag-specific CD4+ memory T cells that persisted 60 days postimmunization. In this report, we used the D011.10 adoptive transfer model to examine the kinetics of Ag-specific T cell entry into the peripheral blood, the optimal route of administration of Ag and αOX40, and the Ag-specific Ab response after immunization with soluble OVA and αOX40. Finally, we compared the adjuvant properties of αOX40 to those of αCTLA-4. Engagement of OX-40 in vivo was most effective when the Ag was administered s.c. Time course studies revealed that it was crucial for αOX40 to be delivered within 24–48 h after Ag exposure. Examination of anti-OVA Ab titers revealed a 10-fold increase in mice that received αOX40 compared with mice that received OVA alone. Both αOX40 and αCTLA-4 increased the percentage of OVA-specific CD4+ T cells early after immunization (day 4), but αOX40-treated mice had much higher percentages of OVA-specific memory CD4+ T cells from days 11 to 29. These studies demonstrate that OX40 engagement early after immunization with soluble Ag enhances long-term T cell and humoral immunity in a manner distinct from that provided by blocking CTLA-4.

Specific blockade of CTLA-4/B7 interactions results in exacerbated clinical and histologic disease in an actively-induced model of experimental allergic encephalomyelitis

In addition to an antigen-specific signal, T cell activation requires an antigen-independent costimulatory signal provided by interaction of CD28 with B7 (CD80 and CD86) on the APC. By blocking B7 interactions, previous studies demonstrated the requirement for costimulation in the induction of experimental allergic encephalomyelitis (EAE). Recent studies suggest that unlike CD28, CTLA-4 (a second B7 ligand) delivers an inhibitory signal. To address the regulatory role of CTLA-4 in EAE, we used an antibody directed against CTLA-4 administered at the time of disease induction. This resulted in a significantly more severe clinical course and more inflammatory and demyelinating lesions in the CNS of anti-CTLA-4-treated mice. These data suggest that CTLA-4-mediated inhibitory signals can regulate the clinical severity and histologic parameters of neuroautoimmune disease.

Tolerization of Tumor-Specific T Cells Despite Efficient Initial Priming in a Primary Murine Model of Prostate Cancer

In this report, we studied T cell responses to a prostate cancer Ag by adoptively transferring tumor Ag-specific T cells into prostate tumor-bearing mice. Our findings demonstrate that CD8+ T cells initially encountered tumor Ag in the lymph node and underwent an abortive proliferative response. Upon isolation from the tumor, the residual tumor-specific T cells were functionally tolerant of tumor Ag as measured by their inability to degranulate and secrete IFN-γ and granzyme B. We next sought to determine whether providing an ex vivo-matured, peptide-pulsed dendritic cell (DC) vaccine could overcome the tolerizing mechanisms of tumor-bearing transgenic adenocarcinoma of the mouse prostate model mice. We demonstrate that tumor Ag-specific T cells were protected from tolerance following provision of the DC vaccine. Concurrently, there was a reduction in prostate tumor size. However, even when activated DCs initially present tumor Ag, T cells persisting within the tolerogenic tumor environment gradually lost Ag reactivity. These results suggest that even though a productive antitumor response can be initiated by a DC vaccine, the tolerizing environment created by the tumor still exerts suppressive effects on the T cells. Furthermore, our results demonstrate that when trying to elicit an effective antitumor immune response, two obstacles must be considered: to maintain tumor Ag responsiveness, T cells must be efficiently primed to overcome tumor Ag presented in a tolerizing manner and protected from the suppressive mechanisms of the tumor microenvironment.

The TRAMP mouse as a model for prostate cancer.

The transgenic adenocarcinoma of the mouse prostate (TRAMP) model closely mirrors the pathogenesis of human prostate cancer. Male TRAMP mice uniformly and spontaneously develop autochthonous (orthotopic) prostate tumors following the onset of puberty. Prostate cancer occurs consequent to the expression of SV40 T antigen. The versatility of the TRAMP model has been extended by establishment of several TRAMP‐derived prostate tumor cells lines that can be injected into syngeneic male nontransgenic C57BL/6 hosts to induce ectopic prostate tumorigenesis. Subcutaneous tumor induction using the TRAMP‐C cell lines has provided the basis for two additional murine models, the first of which can be used for rapid screening of experimental therapies for the treatment of primary prostate tumors and the second for testing the effectiveness of adjunctive therapies targeting prostate cancer metastases. Detailed descriptions for the harvesting and microdissection of TRAMP prostates and tumors, and the evaluation and scoring of tumors are also provided.

Costimulatory wars: the tumor menace.

Advances in our understanding of T cell costimulatory molecules have provided a vast array of novel approaches to tumor immunotherapy. In the past year, combinatorial immunotherapy based on earlier studies of CTLA-4 blockade, the identification of novel B7-family members, the modulation of CD40 to reverse tolerance to tumor-associated antigens and the use of OX40 to enhance antitumor responses of CD4+ T cells have all contributed to the development of more-powerful immunomodulatory cancer therapies.

Cytotoxic T lymphocyte antigen-4 (CTLA-4) limits the expansion of encephalitogenic T cells in experimental autoimmune encephalomyelitis (EAE)-resistant BALB/c mice.

We and others previously reported that cytotoxic T lymphocyte antigen-4 (CTLA-4) regulates the severity of peptide-induced experimental autoimmune encephalomyelitis (EAE) in mouse strains that are inherently susceptible to the disease. In this report, we show that CTLA-4 engagement also controls disease susceptibility in BALB/c mice, a strain considered to be resistant to EAE induction. Although immunization of BALB/c mice with syngeneic spinal cord homogenate or an I-Ad-binding myelin peptide antigen failed to result in EAE, immunization with either antigen preparation in conjunction with anti-CTLA-4 resulted in both clinical and histological EAE. CTLA-4 blockade also resulted in a preferential increase in the frequency of antigen-specific T cells secreting IFN-γ. We conclude that CTLA-4 controls susceptibility in BALB/c mice by limiting the expansion of autoreactive T cells present in the periphery, suggesting a mechanism whereby CTLA-4 contributes to the maintenance of peripheral T cell tolerance to self antigens.