Elena N. Klyushnenkova

CD4 and CD8 T-lymphocyte recognition of prostate specific antigen in granulomatous prostatitis.

In order to develop immunotherapies for prostate cancer, many groups are exploring vaccination strategies to induce an immune response against prostate specific antigen (PSA). To determine if T-cell recognition of PSA might be a feature of a naturally occurring human disease, we have studied patients with prostatitis, a poorly understood clinical syndrome of men in which there is evidence that an immune response directed against the prostate may be occurring. We wished to determine if a T-cell response to PSA might be occurring in these patients. We generated long-term T-cell lines from peripheral blood mononuclear cells (PBMC) of one patient with granulomatous prostatitis using purified PSA as an antigen. Several CD4+ and CD8+ TcR &agr;/&bgr;+ T-cell lines were selected for PSA reactivity as measured by at least a threefold increase in IFN-&ggr; secretion in response to PSA presented by irradiated autologous PBMC. CD4 and CD8 T-cell lines recognized PSA in the context of HLA-DR&bgr;1*1501 and HLA-B*0702, respectively. The specificity and HLA restriction of the lines was confirmed using EBV-B cell lines infected with a recombinant PSA-expressing vaccinia virus and also engineered to express PSA by retroviral transfection. HLA-matched targets infected by control vector as well as HLA-mismatched PSA-expressing targets did not induce the response. The data demonstrate that PSA-specific T cells are present in the PBMC of this patient with granulomatous prostatitis, who may be manifesting naturally the type of immune response directed at the prostate that is the goal of prostate cancer immunotherapy. However, the Class I-restricted epitope has not yet been demonstrated to be expressed on the surface of prostate cancer cells. To our knowledge, this is the first demonstration of HLA-DRB1*1501- or HLA-B*0702-restricted responses to PSA and extends the number of HLA molecules accommodating the use of PSA antigen as a candidate vaccine for prostate cancer immunotherapy.

A cytomegalovirus-based vaccine expressing a single tumor-specific CD8+ T cell epitope delays tumor growth in a murine model of prostate cancer

Cytomegalovirus (CMV) is a highly immunogenic virus that results in a persistent, life-long infection in the host typically with no ill effects. Certain unique features of CMV, including its capacity to actively replicate in the presence of strong host CMV-specific immunity, may give CMV an advantage compared with other virus-based vaccine delivery platforms. In the present study, we tested the utility of mouse CMV (mCMV)-based vaccines expressing human prostate-specific antigen (PSA) for prostate cancer immunotherapy in double-transgenic mice expressing PSA and HLA-DRB1*1501 (DR2bxPSA F1 mice). We assessed the capacity of 2 mCMV-based vectors to induce PSA-specific CD8 T-cell responses and affect the growth of PSA-expressing Transgenic Adenocarcinoma of the Mouse Prostate tumors (TRAMP-PSA). In the absence of tumor challenge, immunization with mCMV vectors expressing either a H2-Db-restricted epitope PSA65–73 (mCMV/PSA65–73) or the full-length gene for PSA (mCMV/PSAFL) induced comparable levels of CD8 T-cell responses that increased (inflated) with time. Upon challenge with TRAMP-PSA tumor cells, animals immunized with mCMV/PSA65–73 had delay of tumor growth and increased PSA-specific CD8 T-cell responses, whereas animals immunized with mCMV/PSAFL showed progressive tumor growth and no increase in number of splenic PSA65–73-specific T cells. The data show that a prototype CMV-based prostate cancer vaccine can induce an effective antitumor immune response in a “humanized” double-transgenic mouse model. The observation that mCMV/PSAFL is not effective against TRAMP-PSA is consistent with our previous findings that HLA-DRB1*1501-restricted immune responses to PSA are associated with suppression of effective CD8 T-cell responses to TRAMP-PSA tumors.

Challenges to Prostate Cancer Immunotherapy

Recent approval of Provenge (Sipuleucel-T, Dendreon Corporation, Seattle, WA) by the United States Food and Drug Administration (FDA) has stimulated a new wave of interest in prostate cancer immunotherapy. Sipuleucel-T is an autologous peripheral blood product (APC8015) comprised of partially purified antigen-presenting cells (including monocytes, dendtritic cells, B cells and T cells) loaded in vitro with a recombinant fusion protein PA2024, human prostatic acid phosphatase (hPAP) linked to granulocyte-macrophage colony stimulatory factor (GM-CSF) (Small et al., 2000). The conceptual development of this therapy was derived from the fundamental achievements in several different areas of life science in the past several decades. First, the discovery of dendritic cells (DCs) has led to the exploration of these cells as vaccines for cancer immunotherapy (Steinman & Cohn, 1973). Second, the pioneer studies in melanoma indicated that lineage-specific “self” differentiation antigens can be recognized by the immune system, and could be the targets for the immunotherapy (van der Bruggen et al., 2002). Finally, the technological advances in the blood products manufacturing as well as a satisfactory safety record of other cell-based therapies provided an opportunity for the smooth transition of the basic research ideas into the clinical science (ADIS R&D profile, 2006). However, the road to the approval of the first immunological therapy product was not straightforward. Significant difficulties and controversies surrounded all steps from the initial characterization of the immunogenic properties of hPAP to clinical trials and final regulatory approval of Provenge for the use in patients with asymptomatic metastatic castrate-resistant prostate cancer (mCRPC).

Abstract 5318: CD4+CD25+ regulatory T cells are involved in the downregulation of antitumor CD8 T-cell response and tumor progression in HLA-DR2b transgenic mouse model of prostate cancer

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DCnnWe have recently described a novel mouse model of prostate cancer in transgenic mice engineered to express Human Leukocyte Antigen (HLA)-DRB1*1501 (DR2b in old nomenclature) (J. Immunol. 2009, 182:1242-1246). Using these mice, we investigated the impact of HLA-DR2b on the growth of prostate-specific antigen (PSA)-expressing Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) tumor cells. We demonstrated that a “permissive” HLA class II allele could change the pattern of anti-tumor immune response resulting in failure of tumor rejection. The simple presence of HLA-DR2b was all that was required in our model for the development of strong humoral immune responses to the tumor antigen (PSA), suppression of CD8 T cell responses and enhanced growth of the tumor. In contrast, mice bearing class II allele that did not support CD4 T cell response to PSA (I-Ab), developed strong CD8 T cell response in the absence of antibody response, and rejected PSA-expressing tumors. One of the major mechanisms by which the presence of CD4 T cell epitope in PSA can negatively affect anti-tumor immunity may include CD4+CD25+ regulatory T cells (Treg) that can be selectively activated in an antigen-specific manner in DR2b+ mice. We found that the depletion of CD4+CD25+ Treg cells prior to tumor inoculation resulted in the significant enhancement of PSA-specific CD8 T cell response in DR2b+ mice. Moreover, we also found that depletion of CD25+ T cells prior to tumor inoculation significantly delayed TRAMP-PSA tumor growth (p=0.01, log rank test). These results indicated that low level of CD8 T cell-mediated IFN-γ response against TRAMP-PSA tumors in DR2b+ mice is an active process of immune suppression mediated by CD4+CD25+ Treg cells, and that the activation of Treg cells can be involved in tumor progression in our model. We also analyzed a composition of the tumor-infiltrating leukocytes (TIL) in DR2b+ and DR2b- mice bearing TRAMP-PSA tumors by flow cytometry. The analysis of infiltrating CD3+ T cells revealed significant strain-specific differences. In DR2b+ mice, CD4+ T cell were accumulated in larger number compared to DR2b- mice, and a significant proportion of CD4+ cells expressed CD25. These cells were also foxp3+. These observations are consistent with other data in our model, and confirm that 1) HLA-DR2b-dependent Treg cell activation may be involved in tumor progression in DR2b+ mice, and 2) lack of such activation in DR2b- mice can result in the efficient CD8 T cell response and tumor rejection.nnSupported by a grant from the US Department of Veterans Affairs and a pilot grant from the Baltimore Research and Education Foundation.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5318.

Identification of HLA-DRB1*1501-Restricted T Cell Epitopes from Prostate-Specific Antigen (PSA) Using DR2b Transgenic Mice

augment IFN-DC secretion of inhibitory cytokine IL-10, and down-regulate IL-12p70. Previously, we have shown that a low concentration of IFNa (100 IU/ml) induces IFN-DCs to secrete a large amount of IL-10 and very little, if any, IL-12p70 in response to CD40 ligand. With the increase of IFNa concentration (up to 100 times), however, the production of IL-10 declined, which was associated with the upregulation of IL-12p70. In this study, we explored the possibility that poor secretion of IL-12p70 was due to endogenously produced IL-10. We implemented serum-free tissue culture conditions to minimize the effect of serum-related factors. Human peripheral mononuclear blood cells from breast cancer patients were grown in macrophage serum-free medium with granulocyte-macrophage colonystimulating factor (GM-CSF, 1000 IU/ml) and increasing concentrations of IFNa (100 IU/ml to 10,000 IU/ml) for 5–7 days. The CD40 ligand–induced IL-10 and IL-12p70 production in the presence of IL-10 neutralizing antibody or control antibody was measured by ELISA. We found that CD40L-induced production of IL-12p70 by low dose IFN-DCs was enhanced after incubation with anti-IL-10 antibody but still remained well below the levels of high dose IFN-DCs incubated with isotype control. We conclude that the endogenously produced IL-10 is only partially responsible for poor secretion of IL-12p70 by low dose IFN-DCs and the additional DC maturation signals provided by high concentrations of IFNa are necessary for production of immunostimulatory molecules.