Breaking immune tolerance by targeting CD25+ regulatory T cells is essential for the anti-tumor effect of the CTLA-4 blockade in an HLA-DR transgenic mouse model of prostate cancer.

INTRODUCTION: Recent studies suggest that the cancer immunotherapy based on the blockade of the CTLA-4-mediated inhibitory pathway is efficacious only in select populations, predominantly for immunogenic tumors or when delivered in combination with modalities that can break immunologic tolerance to tumor antigens. METHODS: We studied the effect of CD25+ cell depletion and CTLA-4 blockade on the growth of Transgenic Mouse Adenocarcinoma of Prostate (TRAMP)-PSA tumor cells in DR2bxPSA F1 mice. In these mice, immunological tolerance to PSA was established in a context of the HLA-DRB1*1501(DR2b) allele. RESULTS: In our model, single administration of anti-CD25 antibody prior to tumor inoculation significantly increased IFN-γ production in response to the CD8 T cell epitope PSA65-73 , and delayed TRAMP-PSA tumor growth compared to mice treated with isotype control antibodies. In contrast, the anti-tumor effect of the anti-CTLA-4 antibody as a monotherapy was marginal. The combinatory treatment with anti-CD25/anti-CTLA-4 antibodies significantly enhanced anti-tumor immunity and caused more profound delay in tumor growth compared to each treatment alone. The proportion of tumor-free animals was higher in the group that received combination treatment (21%) compared to other groups (2-7%). The enhanced anti-tumor immunity in response to the CD25 depletion or CTLA-4 blockade was only seen in the immunogenic TRAMP-PSA tumor model, whereas the effect was completely absent in mice bearing poorly immunogenic TRAMP-C1 tumors. DISCUSSION: Our data suggest that breaking immunological tolerance to "self" antigens is essential for the therapeutic effect of CTLA-4 blockade. Such combinatory treatment may be a promising approach 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-D(b)-restricted epitope PSA(65-73) (mCMV/PSA(65-73)) or the full-length gene for PSA (mCMV/PSA(FL)) induced comparable levels of CD8 T-cell responses that increased (inflated) with time. Upon challenge with TRAMP-PSA tumor cells, animals immunized with mCMV/PSA(65-73) had delay of tumor growth and increased PSA-specific CD8 T-cell responses, whereas animals immunized with mCMV/PSA(FL) showed progressive tumor growth and no increase in number of splenic PSA(65-73)-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/PSA(FL) 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.

Immunological and pathogenic properties of poliovirus variants selected for resistance to antiviral drug V-073.

BACKGROUND: The National Research Council has recommended development of polio antiviral drugs to assist in management of outbreaks and to mitigate adverse consequences of vaccination. V-073 is a small molecule poliovirus capsid inhibitor that is being developed for these purposes. Antiviral use raises the potential of treatment-emergent resistance. Understanding virological consequences of resistance is important. METHODS: Six independent laboratory-derived V-073-resistant poliovirus variants were characterized for their ability to be neutralized by conventional vaccine-induced immune sera, to elicit serum neutralizing antibodies upon CD-1 mouse immunization, and to replicate in and to cause paralysis of TgPVR21 mice. RESULTS: V-073-resistant variants were effectively neutralized by oral poliovirus vaccine and inactivated poliovirus vaccine human immune sera. All variants elicited virus neutralizing antibody titres in CD-1 mice that were comparable to drug-susceptible parental and Sabin vaccine strain viruses. Infection efficiency of TgPVR21 mice by variants was comparable to (1 of 6 variants) or considerably lower than (5 of 6 variants) parental viruses. Drug-resistant variants replicated to levels comparable to (1 of 6 variants) or substantially less than (5 of 6 variants) their drug-susceptible parental viruses and were on average 1.4 log(10) (range 0.3 to >2.8 log10) less neurovirulent. CONCLUSIONS: Laboratory-derived V-073-resistant variants exhibit clear attenuation of pathogenic properties while maintaining immunological features of drug-susceptible viruses.

T-cell recognition of prostatic peptides in men with chronic prostatitis/chronic pelvic pain syndrome.

PURPOSE: A potential etiology of chronic prostatitis/chronic pelvic pain syndrome is autoimmunity. We determined whether T cells from men with chronic prostatitis/chronic pelvic pain syndrome would recognize peptides derived from the normal self-prostatic proteins prostate specific antigen and prostatic acid phosphatase. MATERIALS AND METHODS: CD4 T cells purified from peripheral blood of 31 patients with chronic prostatitis/chronic pelvic pain syndrome and from the buffy coat preparation of 27 normal male blood donors were stimulated in vitro with a panel of immunogenic peptides from prostate specific antigen and prostatic acid phosphatase, and assayed for reactivity with the peptides by interferon-gamma enzyme-linked immunosorbent spot assay. Intermediate resolution HLA typing was done by polymerase chain reaction. Peptides were also tested by binding assay against different class II alleles. RESULTS: Peptide PAP(173-192) was recognized more frequently by CD4 T cells from patients with chronic prostatitis/chronic pelvic pain syndrome than from healthy donors. The recognition of prostate specific antigen peptides was not statistically different when comparing cases to normal male blood donors individually. Peptide reactivity was more common in patients than in normal male blood donors for any prostate specific antigen peptide or any tested peptide. All peptides showed high promiscuity on binding assays. There was no association of cases with any specific HLA class II phenotype at intermediate resolution. CONCLUSIONS: CD4 T cells from patients with chronic prostatitis/chronic pelvic pain syndrome have a higher rate of recognizing the self-prostatic proteins prostatic acid phosphatase and prostate specific antigen compared to those from normal male blood donors. Data provide further evidence to support the role of autoimmunity in some men with chronic prostatitis/chronic pelvic pain syndrome.

Vaccination with agonist peptide PSA: 154-163 (155L) derived from prostate specific antigen induced CD8 T-cell response to the native peptide PSA: 154-163 but failed to induce the reactivity against tumor targets expressing PSA: a phase 2 study in patients with recurrent prostate cancer.

We conducted a clinical trial of peptide prostate specific antigen (PSA): 154-163 (155L) vaccination in human leukocyte antigen (HLA)-A2 patients with detectable and rising serum PSA after radical prostatectomy for prostate cancer (Clinicaltrials.gov identifier NCT00109811). The trial was a single dose-level, phase 2 pilot trial of 1 mg of PSA: 154-163 (155L) emulsified with adjuvant (Montanide ISA-51). The primary endpoint was the determination of immunogenicity of the vaccine; secondary outcomes were determination of toxicity and effect on serum PSA. The vaccine was given subcutaneously 7 times on weeks 0, 2, 4, 6, 10, 14, and 18. Peptide-specific CD8 T-cell responses in the peripheral blood mononuclear cells (PBMC) of patients were measured by interferon (IFN)-gamma enzyme-linked immunosorbent spot assay. CD8 T-cell cultures were also established by in vitro stimulation with the peptide presented by autologous dendritic cells. Five patients were enrolled and completed all vaccinations. No IFN-gamma response to PSA: 154-163 (155L) was detected in unfractioned PBMC in any patient either before or after vaccination. Three of 5 patients demonstrated strong IFN-gamma responses to PSA: 154-163 (155L) and native PSA: 154-163 peptides in CD8 T-cell cultures derived from postvaccination PBMC. However, peptide-specific T cells failed to recognize HLA-A2 positive targets expressing endogenous PSA. There were no significant changes in serum PSA level in any subject. No serious adverse events were observed. PSA: 154-163 (155L) is not an effective immunogen when given with Montanide ISA-51. The PSA: 154-163 peptide is poorly processed from endogenous PSA and therefore represents a cryptic epitope of PSA in HLA-A2 antigen-presenting cells.

Cutting edge: permissive MHC class II allele changes the pattern of antitumor immune response resulting in failure of tumor rejection.

We studied the growth of transgenic adenocarcinoma of mouse prostate (TRAMP)-C1 tumor cells expressing human prostate-specific Ag (PSA) in HLA-DRB1*1501 (DR2b) transgenic mice. TRAMP-PSA tumors were frequently rejected by HLA-DR2b(-) mice but had increased incidence in HLA-DR2b(+) littermates. The levels of PSA-specific CD8 T cell responses were significantly higher in the HLA-DR2b(-) mice that rejected TRAMP-PSA tumors compared with HLA-DR2b(+) tumor-bearing littermates. In contrast, Ab responses to PSA were strong in HLA-DR2b(+) mice bearing TRAMP-PSA tumors and were virtually undetectable in HLA-DR2b(-) littermates. The analysis of CD4 T cell responses to PSA revealed the presence of several CD4 T cell epitopes in HLA-DR2b(+) mice but failed to identify strong I-A(b)-restricted epitopes in HLA-DR2b(-) mice. Our data demonstrate that the expression of a permissive HLA class II allele can change the pattern of the immune response to a tumor Ag, resulting in the failure of tumor rejection.

The binding sites for the very low density lipoprotein receptor and low-density lipoprotein receptor-related protein are shared within coagulation factor VIII.

Coagulation factor VIII (FVIII) is a ligand for two members of the low-density lipoprotein receptor family, low-density lipoprotein receptor-related protein (LRP) and low-density lipoprotein receptor, which cooperate in regulating clearance of FVIII from the circulation. This study was aimed to explore the mechanism of interaction of FVIII with very low density lipoprotein receptor (VLDLR), another member of the family, and map receptor-binding sites. Binding of plasma-derived FVIII and its fragments to recombinant soluble ectodomain of VLDLR (sVLDLR) was studied in solid-phase and surface plasmon resonance assays. Full-length FVIII and its light chain bound to sVLDLR with similar affinities (KD = 114 +/- 14 and 95 +/- 11 nmol/l, respectively); in contrast, exposure of high-affinity VLDLR-binding site within the heavy chain (KD = 30 +/- 2 nmol/l) required proteolytic cleavage by thrombin. The VLDLR-binding sites within heavy and light chains were mapped to the A2 domain residues 484-509 and the A3-C1 fragment, based on the inhibitory effects of anti-A2 monoclonal antibody 413 and anti-A3-C1 antibody fragment scFv KM33, respectively, previously shown to inhibit FVIII/LRP interaction. Soluble ligand-binding fragment of VLDLR inhibited activation of factor X by the intrinsic Xase in purified system. In cell culture, a higher Xase activity was associated with wild-type human embryonic kidney cells compared with transfected cells that express VLDLR on the cell surface. We conclude that the binding sites for VLDLR and LRP within FVIII overlap and the A2 site becomes exposed upon physiological activation of FVIII. A functional role of FVIII/VLDLR interaction may be related to regulation of intrinsic Xase activity.

Identification of HLA-DRB1*1501-restricted T-cell epitopes from human prostatic acid phosphatase.

BACKGROUND: The crucial role of CD4 T-cells in anti-tumor immune response is widely recognized, yet the identification of HLA class II-restricted epitopes derived from tumor antigens has lagged behind compared to class I epitopes. This is particularly true for prostate cancer. Based on the hypothesis that successful cancer immunotherapy will likely resemble autoimmunity, we searched for the CD4 T-cell epitopes derived from prostatic proteins that are restricted by human leukocyte antigen (HLA)-DRB1*1501, an allele associated with granulomatous prostatitis (GP), a disease that may have an autoimmune etiology. One of the antigens implicated in the development of autoimmunity in the prostate is prostatic acid phosphatase (PAP), which is also considered a promising target for prostate cancer immunotherapy. METHODS: We immunized transgenic (tg) mice engineered to express HLA-DRB1*1501 with human PAP. A library of overlapping 20-mer peptides spanning the entire human PAP sequence was screened in vitro for T-cell recognition by proliferative and interferon (IFN)-gamma enzyme-linked immunosorbent spot (ELISPOT) assays. RESULTS: We identified two 20-mer peptides, PAP (133-152), and PAP (173-192), that were immunogenic and naturally processed from whole PAP in HLA-DRB1*1501 tg mice. These peptides were also capable of stimulating CD4 T lymphocytes from HLA-DRB1*1501-positive patients with GP and normal donors. CONCLUSIONS: These peptides can be used for the design of a new generation of peptide-based vaccines against prostate cancer. The study can also be helpful in understanding the role of autoimmunity in the development of some forms of chronic prostatitis.

Intrinsic pathway of blood coagulation contributes to thrombogenicity of atherosclerotic plaque.

Thrombosis is the major mechanism underlying acute complications of atherosclerosis. Although thrombogenicity of atherosclerotic plaques has been ascribed to activation of the extrinsic pathway of blood coagulation, in the present study we investigated contribution of the intrinsic factor VIII (fVIII)-dependent pathway. We found that in vitro exposure of human macrophages and smooth muscle cells (SMCs) to atherogenic oxidized low-density lipoprotein (oxLDL) enhances their ability to support activity of 2 major complexes of the intrinsic pathway, Xase and prothrombinase, leading to a 20- and 10-fold increase in thrombin formation, respectively. In contrast, human aortic endothelial cells were less responsive to oxLDL. The increase in the intrinsic procoagulant activity was related to formation of additional fVIII binding sites due to enhanced translocation of phosphatidylserine to the outer surface of oxLDL-treated cells and a 5-fold higher affinity of interaction between components of the Xase complex, activated factors VIII and IX. Processes occurring at early apoptotic stages, including changes in the cell membrane induced by free radicals, may be related to activation of the intrinsic pathway as suggested by effects of inhibitors of early apoptosis on thrombin formation. Immunohistochemical studies on human atherectomy specimens revealed the presence of fVIII in the vicinity of macrophages and SMCs in atheromatous regions with massive deposits of oxLDL, supporting the possible involvement of the intrinsic pathway in thrombus formation in vivo. Our data predict that the intrinsic pathway significantly enhances thrombogenicity of atherosclerotic lesions after removal of the endothelial layer and exposure of SMCs and macrophages to blood flow.