P53(110-124)-specific human CD4+ T-helper cells enhance in vitro generation and antitumor function of tumor-reactive CD8+ T cells

Current evidence suggests that the optimal vaccines for cancer should incorporate tumor-specific cytotoxic as well as helper epitopes. Wild-type sequence (wt) p53 peptides are attractive candidates for broadly applicable cancer vaccines, which could combine multiple tumor epitopes defined by CD8(+) CTLs, as well as CD4(+) T-helper cells. To test this possibility, we generated anti-p53 CD4(+) T cells from peripheral blood obtained from an HLA-DRB1*0401(+) donor by in vitro stimulation with dendritic cells and recombinant human p53 protein. We identified the wt p53(110-124) peptide as a naturally presented epitope. In a series of ex vivo experiments, performed in an autologous human system, we then demonstrated the ability of anti-wt p53(110-124) CD4(+) T cells to enhance the generation and antitumor functions of CD8(+) effector cells. The results demonstrate the crucial role of T helper-defined epitopes in shaping the immune response to multiepitope cancer vaccines targeting p53. This model of tumor-specific CD8(+) and CD4(+) T-cell interactions suggests that future vaccination strategies targeting tumor cells should incorporate helper and cytotoxic T cell-defined epitopes.     

Immunological characterization of missense mutations occurring within cytotoxic T cell-defined p53 epitopes in HLA-A*0201+ squamous cell carcinomas of the head and neck

Previous analyses of p53 in 40 HLA-A*0201(HLA-A2)(+) squamous cell carcinomas of the head and neck (SCCHN) indicated that 6/13 p53 missense mutations that were detected, S149C, T150R, V157F, Y220C, Y220H and E271K, occurred within HLA-A2-restricted cytotoxic T lymphocyte (CTL)-defined p53 epitopes. Of the 6, the p53 S149C, Y220C and Y220H peptides were immunogenic. Anti-p53 mutant S149C and Y220H effector cells cross-reacted against the parental wild type sequence (wt) p53 peptides, whereas anti-p53 Y220C effector cells were specific for the mutant peptide, p53 Y220C cDNA-transfected HLA-A2(+) SaOS cells, and an HLA-A2(+) SCCHN cell line naturally expressing the mutation. These results indicate that the p53 Y220C mutation can be processed and presented for CD8(+) T cell recognition. Furthermore, using an autologous PBMC/tumor system, anti-p53 Y220C peptide-effector cells recognizing the autologous tumor could also be generated. Our analysis of p53 in 10 additional HLA-A2(+) SCCHN tumors detected the p53 Y220C in 2/10 tumors raising the overall frequency of the p53 Y220C mutation to 6/50 (12%) HLA-A2(+) SCCHN tumors. In contrast, independent of their HLA class I genotypes, the p53 Y220C mutation frequency for all human tumors analyzed to date is approximately 1.5%. This unexpectedly high frequency of the p53 Y220C mutation in HLA-A2(+) SCCHN suggests that vaccines targeting this mutation would not only be expected to induce robust anti-tumor immune responses in HLA-A2(+) subjects, but also be more widely applicable than previously envisioned for any given p53 missense mutation.     

Glioblastoma patients exhibit circulating tumor-specific CD8+ T cells.

PURPOSE: There is growing interest in developing cellular immune therapies for glioblastoma multiforme, but little is known about tumor-specific T-cell responses. A glioblastoma multiforme-specific T-cell assay was developed using monocyte-derived dendritic cells to present tumor antigens from the established glioblastoma multiforme cell line U118. EXPERIMENTAL DESIGN: Peripheral blood mononuclear cells (PBMC) and tumor cells were obtained from nine patients with newly diagnosed brain tumors: five glioblastoma multiforme, two oligodendroglioma, one ependymoma, and one astrocytoma. PBMCs were incubated overnight with autologous tumor cells or autologous dendritic cells loaded with a U118 cell lysate, and responses were detected by IFN-gamma ELISPOT and cytokine flow cytometry assays. RESULTS: PBMCs from all glioblastoma multiforme patients exhibited IFN-gamma responses to autologous tumor but not to HLA-mismatched U118 cells. Glioblastoma multiforme-specific IFN-gamma responses were primarily mediated by CD8+ T cells and represented approximately 2% of total CD8+ T cells. Additionally, all glioblastoma multiforme patients responded to autologous dendritic cells loaded with U118 lysate but not with low-grade astrocytoma cell lysates. PBMCs from four patients with other brain tumor types and one normal donor failed to respond to U118 lysate-loaded autologous dendritic cells. These data indicate that the IFN-gamma responses to U118 lysate-loaded autologous dendritic cells are glioblastoma multiforme specific. Moreover, PBMCs stimulated 1 to 2 weeks with U118 lysate-loaded dendritic cells exhibited MHC class I-restricted cytotoxicity against autologous tumor cells. CONCLUSIONS: Glioblastoma multiforme patients exhibit circulating tumor-specific CD8+ T cells that recognize shared tumor antigens from the glioblastoma multiforme cell line U118. These data show that glioblastoma multiformes are immunogenic and support the development of immunotherapy trials.     

A wild-type sequence p53 peptide presented by HLA-A24 induces cytotoxic T lymphocytes that recognize squamous cell carcinomas of the head and neck.

Evidence has accumulated indicating that HLA-A2-restricted CTLs specific for human wild-type sequence p53 epitopes lyse tumor cells expressing mutant p53. To explore the possibility that wild-type sequence p53 peptides could also be used in vaccines for patients expressing HLA-A24 antigen, another frequent HLA class I allele, we investigated the induction of HLA-A24-restricted p53-specific CTLs from the peripheral blood lymphocytes of normal donors. Of six p53-derived peptides possessing an HLA-A24 binding motif, the p53 peptide 125-134 (p53(125-134)) was found to have a high binding capacity and induced peptide-specific CTLs from peripheral blood mononuclear cells, using peptide-pulsed autologous dendritic cells and subsequent cultivation with cytokines interleukin 2 and interleukin 7. Bulk CTL populations lysed peptide-pulsed HLA-A24+ targets as well as HLA-A24+ squamous cell carcinoma of the head and neck (SCCHN) cell lines. However, IFN-gamma pretreatment of HLA-A24+ SCCHN cell lines was necessary for lysis, suggesting that a ligand density higher than that normally expressed by tumor cells is required for these CTLs to mediate lysis. Moreover, a cloned CTL, designated TH#99, isolated from the bulk population by limiting dilution, lysed HLA-A24+ SCCHN targets more efficiently than the bulk CTL population. Lysis was inhibited by anti-HLA class I monoclonal antibody but not by anti-HLA-DR monoclonal antibody. These results indicate that HLA-A24-restricted CTLs recognizing the wild-type sequence p53(125-134) can be generated using autologous dendritic cells from precursors present in peripheral blood lymphocytes obtained from normal HLA-A24+ donors. This finding suggests that vaccine strategies targeting wild-type sequence p53 epitopes can be extended to a wider range of cancer patients.     

Induction and clonal expansion of tumor-specific cytotoxic T lymphocytes from renal cell carcinoma patients after stimulation with autologous dendritic cells loaded with tumor cells

Melanoma and renal cell carcinoma (RCC) are considered to be the most immunogenic tumors in humans. To generate conditions to induce primary T-cell responses against RCC and to allow further expansion of tumor-specific cytotoxic T lymphocytes (CTL) for adoptive transfer, peripheral blood mononuclear cells from RCC patients were stimulated with primary autologous tumor cells or monocyte-derived dendritic cells (DC) loaded with either tumor lysate (TU-LY) or apoptotic tumor cells (TU-AP). Whereas repetitive stimulation (4x) with tumor cells alone induced a predominant population of CD3(-) natural killer cells, 4 weeks of stimulation with tumor-loaded DC favored induction and expansion of CD4+ T cells (>80%). However, 2 weekly stimulation cycles with tumor-loaded DC followed by restimulation with autologous irradiated tumor cells alone were optimal for induction of tumor-specific CTL responses in vitro. Using these culture conditions a marked increase of CD4+ T cells was observed during the first 2 weeks of stimulation with tumor-loaded DC. Subsequent restimulation with autologous tumor cells alone gave rise to 500-fold expansion of CD8+ T cells. These CD8+ T cells were shown to exhibit strong major histocompatibility complex class I-restricted cytotoxic activity against the autologous tumor. Comparison of TU-LY and TU-AP as a source of tumor antigen for loading DC did not show any difference in stimulating tumor-specific CTL. Length pattern analysis of the complementary determining region 3 (CDR3) of the T-cell receptor Vbeta chain revealed expansion of oligoclonal CTL populations with outgrowth of 1 or 2 clones after prolonged stimulation with autologous tumor cells. Our study demonstrated an efficient method for generating tumor-specific CTL in vitro that may be used to identify tumor cell antigens or that can be expanded for adoptive T-cell transfer in tumor immunotherapy.     

Antitumor activity of human papillomavirus type 16 E7-specific T cells against virally infected squamous cell carcinoma of the head and neck

Human papillomavirus (HPV)-associated squamous cell carcinoma of the head and neck (SCCHN) seems to be a suitable target for cancer vaccination. HPV-encoded oncogenic proteins, such as E7, are promising tumor-specific antigens and are obligatory for tumor growth. Because few immunologic studies have analyzed the endogenous HPV-specific immune response in this subset of SCCHN patients, we studied T-cell frequencies against HPV-16 E7(11-20) or E7(86-93) in tumor-bearing, human leukocyte antigen (HLA)-A*0201+ SCCHN patients, whose tumors were either HPV-16+ or HPV-16-. In HPV-16+ SCCHN patients, frequencies of T cells against either peptide were significantly elevated (P < 0.005) compared with HPV-16- patients or healthy volunteers. Tetramer+ T cells showed evidence of terminally differentiated phenotype (CD45RA+CCR7-) and an elevated level of CD107a staining for degranulation. Despite detectable expression of the restricting HLA class I allele, HLA-A*0201-E7(11-20)- or HLA-A*0201-E7(86-93)-specific CTL obtained by in vitro stimulation of healthy donor peripheral blood mononuclear cells only recognize a naturally HPV-16-transformed, HLA-A*0201+ SCCHN cell line after pretreatment with IFN-gamma. This cell line had little or no expression of LMP2, TAP1, and tapasin, critical components of the HLA class I antigen-processing machinery, which were up-regulated by IFN-gamma treatment. Immunohistochemistry of HPV-16+ SCCHN tumors showed that these antigen-processing machinery components are down-regulated in tumors in vivo compared with adjacent normal squamous epithelium. Thus, immunity to HPV-16 E7 is associated with the presence of HPV-16 infection and presentation of E7-derived peptides on SCCHN cells, which show evidence of immune escape. These findings support further development of E7-specific immunotherapy and strategies for up-regulation of antigen-processing machinery components in HPV-associated SCCHN.     

A CD4+, HLA-DR7-restricted T-helper lymphocyte clone recognizes an antigen shared by human malignant melanoma and glioma

CD4(+) Th cells that are restricted by MHC class II molecules play an important role in the induction of antitumor immune responses. We have established a stable CD4(+) Th cell clone (Th35-1A) from the PBMCs of a patient with primary cutaneous melanoma. The Th cell clone is noncytolytic and proliferates specifically in the presence of irradiated autologous melanoma cells or autologous EBV-transformed B cells pulsed with melanoma tumor cell lysates. Th35-1A produces IFN-gamma (a Th1-type cytokine) after autologous tumor cell stimulation, and its proliferative reactivity is HLA class II-restricted. Th cells showed helper activity for PWM responses of PBMCs. Using a panel of HLA class II-matched and unmatched EBV-B cells as APCs and allogeneic melanoma tumor cell lysate as stimulant, DR7 was delineated as the HLA class II restriction element used by the Th cell clone. In agreement with these results, transfection of an allogeneic melanoma cell line with HLA-DR7 isolated from autologous EBV-B cells rendered the cell line stimulatory for Th35-1A cells. Specificity studies using autologous EBV-B cells (EBV-B35) pulsed with a panel of allogeneic tumor cell lysates of various tissue origins indicated that the Th cell clone recognizes an antigen shared by melanoma and glioma cells. The availability of the Th cell clone may lead to the development of new therapies against melanoma, using adoptive Th cell transfer and/or active immunization with a shared Th cell antigen.     

Superior antitumor in vitro responses of allogeneic matched sibling compared with autologous patient CD8+ T cells

Allogeneic cell therapy as a means to break immunotolerance to solid tumors is increasingly used for cancer treatment. To investigate cellular alloimmune responses in a human tumor model, primary cultures were established from renal cell carcinoma (RCC) tissues of 56 patients. In three patients with stable RCC line and human leukocyte antigen (HLA)-identical sibling donor available, allogeneic and autologous RCC reactivities were compared using mixed lymphocyte/tumor cell cultures (MLTC). Responding lymphocytes were exclusively CD8(+) T cells, whereas CD4(+) T cells or natural killer cells were never observed. Sibling MLTC populations showed higher proliferative and cytolytic antitumor responses compared with their autologous counterparts. The allo-MLTC responders originated from the CD8(+) CD62L(high)(+) peripheral blood subpopulation containing naive precursor and central memory T cells. Limiting dilution cloning failed to establish CTL clones from autologous MLTCs or tumor-infiltrating lymphocytes. In contrast, a broad panel of RCC-reactive CTL clones was expanded from each allogeneic MLTC. These sibling CTL clones either recognized exclusively the original RCC tumor line or cross-reacted with nonmalignant kidney cells of patient origin. A minority of CTL clones also recognized patient-derived hematopoietic cells or other allogeneic tumor targets. The MHC-restricting alleles for RCC-reactive sibling CTL clones included HLA-A2, HLA-A3, HLA-A11, HLA-A24, and HLA-B7. In one sibling donor-RCC pair, strongly proliferative CD3(+)CD16(+)CD57(+) CTL clones with non-HLA-restricted antitumor reactivity were established. Our results show superior tumor-reactive CD8 responses of matched allogeneic compared with autologous T cells. These data encourage the generation of antitumor T-cell products from HLA-identical siblings and their potential use in adoptive immunotherapy of metastatic RCC patients.     

Effect of human papillomavirus-16 infection on CD8+ T-cell recognition of a wild-type sequence p53264-272 peptide in patients with squamous cell carcinoma of the head and neck.

PURPOSE: Wild-type sequence (wt) p53 peptides are attractive candidates for broadly applicable cancer vaccines, currently considered primarily for patients whose tumors overexpress p53. Circumstances exist, however, where increased p53 degradation may result in appreciable presentation of p53-derived peptides, despite low p53 expression. Squamous cell carcinoma of the head and neck is associated with oncogenic human papillomavirus (HPV) subtypes, which inactivate p53 through proteasomal degradation. The criterion of p53 overexpression would exclude these individuals from wt p53-based immunotherapy. EXPERIMENTAL DESIGN: We tested the correlation of HPV infection with enhanced antigenicity of the p53 protein and postulated that removal of HPV-16(+) tumors with enhanced p53(264)-(272) peptide presentation might lead to a drop in T cells specific for this peptide in vivo. Circulating frequencies of T cells specific for the HLA A*0201:p53(264)-(272) complex were measured ex vivo using dimeric HLA:peptide complexes in 15 head and neck cancer patients before and 6 months after tumor excision. RESULTS: CD8+ T-cell recognition of HLA A*0201 restricted wt p53(264)-(272) peptide presented by HPV-16(-) squamous cell carcinoma of the head and neck lines was enhanced by HPV-16 E6 expression, sometimes exceeding that of a naturally transformed, HPV-16(+) wt p53 expressing squamous cell carcinoma of the head and neck cell line. In patients with HPV-16(-) tumors, the frequency of wt p53(264-272)-specific T cells remained largely unchanged after tumor removal. However, a significant decline in frequency of anti-p53(264-272) T cells was observed postoperatively in HPV-16(+) patients (P < 0.005). CONCLUSIONS: Recognition of HPV-associated squamous cell carcinoma of the head and neck appears associated with levels of wt p53-specific T cells and inversely with p53 expression. p53 peptides may be useful tumor antigens for squamous cell carcinoma of the head and neck immunotherapy in addition to viral gene products.     

Clinical significance of decreased zeta chain expression in peripheral blood lymphocytes of patients with head and neck cancer.

Patients with squamous cell carcinoma of the head and neck (SCCHN) frequently have impaired immune responses. Alterations in T-cell receptor-associated signaling molecules in tumor-infiltrating as well as circulating lymphocytes have been reported in these patients. Using quantitative flow cytometry analysis, we have demonstrated that expression of the zeta chain is significantly decreased relative to normal controls in both CD8+ and CD4+ T cells as well as CD3- CD56+ CD16+ natural killer cells in the peripheral blood of patients with SCCHN who, as a result of previous therapies, have no evident disease. Patients with a more aggressive type of SCCHN and those who experienced a recurrence or had a second primary cancer within the last 2 years of the study had the lowest zeta chain expression. In addition, SCCHN patients showed a significantly greater spontaneous ex vivo apoptosis, as measured by a terminal deoxynucleotide transferase-mediated dUTP nick end labeling assay, in PBMCs, compared to normal controls. The observed decreased expression of zeta in T and natural killer cells coincided but did not directly correlate with significantly increased spontaneous apoptosis of lymphocytes obtained from treated patients with no evident disease. The results suggest that in patients with SCCHN, zeta chain defects and lymphocyte apoptosis are manifestations of long-lasting negative effects of tumor on the immune system.     

Elimination of CD4+ T cells may overcome suppression of anti-HER2 immune responses in tumor-bearing hosts

In this study, we analyzed specific anti-tumor immune responses in tumor-bearing hosts by measuring HER2-specific CD8+ T cell responses. No measurable HER2-derived peptide (HER2p63)-specific CD8+ T cells were present in the spleens of mice in the early to late phase of tumor-bearing. Vaccination with HER2 protein and cholesteryl group-bearing pullulan (CHP-HER2 complex) induced HER2-specific CD8+ T cells, but their numbers continuously declined as tumors continued growing. Removal of CD4+ T cells by anti-CD4 monoclonal antibody in the early tumor-bearing stage resulted in tumor regression. The combination of CHP-HER2 complex vaccination and depletion of CD4+ T cells enhanced and restored HER2-specific CD8+ T cells in the late stage of tumor-bearing, and also suppressed tumor growth. These results indicate the importance of manipulation of CD4+ T cells in developing effective immunotherapies as cancer vaccines.     

Eradication of disseminated lymphomas with CpG-DNA activated T helper type 1 cells from nontransgenic mice

Various evidence suggests that adoptive transfer of polyclonal, tumor-specific, IFN-gamma-producing CD4+ T cells [T helper type 1 (Th1) cells] should be highly efficient for tumor immune therapy. However, this approach could not be tested because very few MHC class II-restricted tumor peptides have been defined. Here we show that stimulation of freshly isolated T helper cells with syngeneic tumor cells and antigen-presenting cells in the presence of immunostimulatory CpG DNA allows the generation of large numbers of strongly polarized, tumor-specific Th1 cells within 3 weeks of culture, even when T helper cells were derived from tumor-bearing mice. A single injection of 0.5 x 10(6) A20-specific Th1 cells even eradicated disseminated A20 lymphomas and provided lifelong protection without inducing autoimmune disease. The therapy was largely independent of CD8+ cells but required IFN-gamma and CD40-CD40L interactions, suggesting that tumor-specific Th1 cells eradicate established tumors by activating proinflammatory macrophages.     

Inhibition of cytolytic T lymphocyte proliferation by autologous CD4+/CD25+ regulatory T cells in a colorectal carcinoma patient is mediated by transforming growth factor-beta

Cancer patients often develop CTLs that lyse autologous tumor cells in culture. However, tumors can progress in vivo despite the presence of CTLs. Various mechanisms have been reported to down-modulate CTL functions. In this study, the role of CD4+/CD25+ regulatory T cells in CTL induction and proliferation of established CTLs was investigated in a patient with CRC. CD4+ cytotoxic and regulatory T-cell lines were derived from the peripheral blood mononuclear cells of the same patient in mixed-lymphocyte tumor culture. The cytotoxic T-cell line and a clonal derivative specifically lysed the autologous tumor cells but not the B lymphocytes. Only HLA-A1-matched allogeneic CRC cells were lysed by the CTL clone. The clone produced IFN-gamma and TNF-alpha. The regulatory CD4+/CD25+ T-cell line was tumor cell-dependent in its growth but did not lyse autologous tumor cells. This T-cell line suppressed pokeweed mitogen responses of allogeneic lymphocytes, proliferative activity of the established, autologous CTLs, and induction of CTLs in autologous, freshly isolated peripheral blood mononuclear cells. The immunosuppressive effect of the CD4+/CD25+ regulatory T cells was mediated by transforming growth factor-beta and did not require cell-to-cell contact. Thus, although CRC patients can develop specific CTLs against their tumors, the development of regulatory T cells may allow the escape of tumor cells from immune surveillance by the CTLs in vivo.     

Generation of anti-p53 cytotoxic T lymphocytes from human peripheral blood using autologous dendritic cells.

CTLs recognizing the HLA-A2.1-restricted, wild-type sequence p53 epitopes p53(149-157) and p53(264-272) were generated from CD8-enriched populations of nonadherent peripheral blood lymphocytes (PBLs) obtained from healthy donors. The PBLs were restimulated in vitro with peptide-pulsed granulocyte macrophage colony-stimulating factor- and interleukin (IL)-4-induced autologous dendritic cells in the presence of IL-6 and IL-12 and subsequently cultivated with IL-1alpha, IL-2, IL-4, IL-6, and IL-7. Bulk anti-p53(264-272) CTL populations were generated from PBLs obtained from two of five donors. Both CTL populations were cytotoxic against peptide-pulsed HLA-A2+ target cells, but not against untreated target cells. A CD8+ anti-p53 CTL clone designated p264#2 was isolated from one of the bulk populations. It was found to have an intermediate affinity of approximately 10(-9) M for the epitope and to mediate cytotoxicity against several human tumor cell lines, including the squamous cell carcinoma of the head and neck cell line SCC-9, which is known to present the wild-type sequence p53(264-272) epitope. In addition, CTLs reactive against p53(149-157)-pulsed targets as well as a HLA-A2+ tumor cell line were cloned from a bulk population of antitumor CTLs obtained from one of the five normal PBLs restimulated with this epitope. The results indicate that CTLs recognizing wild-type sequence epitopes can be generated from precursors present in PBLs obtained from some normal individuals using autologous dendritic cells as antigen-presenting cells and suggest that vaccine strategies targeting these epitopes can lead to antitumor CTL generation, thereby emphasizing the therapeutic potential of p53-based cancer vaccines.     

Ex vivo generation and expansion of anti-tumor cytotoxic T-cell lines derived from patients or their HLA-identical sibling

Successful ex-vivo priming and long-term maintenance of anti-tumor cytotoxic T-cell (CTL) lines are preliminary conditions for their use in approaches of adoptive immunotherapy for patients with cancer. We describe the results of a novel procedure for generating in vitro anti-tumor CTL using CD8-enriched peripheral blood mononuclear cells (PBMC) and dendritic cells (DC), pulsed with irradiated tumor cells (TC) as source of tumor antigen. Eight patients were enrolled in our study: 4 sarcoma, 2 renal cell carcinoma, 1 ovarian carcinoma and 1 breast carcinoma. Ten anti-tumor CTL-lines cytotoxic towards patient TC were generated. Five CTL-lines were obtained using both DC and PBMC from the patients (autologous setting). For 5 CTL-lines, DC derived from an HLA-identical sibling were employed (allogeneic setting): patients or siblings PBMC were used to generate CTL-lines in 2 and 3 cases, respectively,. After tumor-specific rounds of stimulation, followed by antigen-independent cycle of expansion, CTL-lines obtained in both autologous and allogeneic setting showed an expansion of the absolute number of cultured cells. In 6 of 10 CTL-lines, the majority of effector cells (>70%) were CD3+/CD8+, while in the remaining 4, 40-70% of effector cells were CD3+/CD4+. Both CD8+ and CD4+ T cells displayed anti-tumor cytotoxic activity. Spectratyping analysis of the TCR-Vbeta subfamilies revealed a preferential expansion of oligoclonal populations in 18 of 24Vbeta subfamily. Altogether these results demonstrate that our experimental approach is suitable for efficiently generating and expanding anti-solid tumor CTL to be used for adoptive immunotherapy.     

Targeted Tumor Cell Death Induced by Autologous Tumor-Specific T Lymphocyte Recognition of Wild-Type p53-Derived Peptides

Summary Autologous tumor-specific T lymphocyte (ATTL) lines were derived from the peripheral blood mononuclear cells (PBMC) of a healthy volunteer with human leukocyte antigen (HLA) -A*0201. These lines were achieved using interleukins -1β, -2, -4, and -6 and the p53-based peptide from the 264–272 sequence of the wild-type p53 protein with a strong affinity against HLA-A*0201. ;The frequencies of CD3+, CD4+, and CD8+ lymphocytes were 94–96%, 30–34%, and 69–74%, respectively. ATTLs killed most of the T2 cells pulsed with p53-derived peptide, but not against the T2 cells non-pulsed or pulsed with an irrelevant peptide. ATTLs also killed TKB-14 cells, which have been derived from human glioblastoma multiforme, and exhibited HLA-A*0201 molecule and immunohistochemical accumulation of p53 protein. These cytotoxic activities were inhibited by anti-CD3, anti-CD8, and anti-class I antibodies. These findings suggested that these ATTL lines might include CTL populations, which could recognize p53-derived peptide on HLA-A*0201 and the p53-based peptide may play as an antigen on HLA-A*0201. When tumor antigens would be more analyzed in the future, ATTL could be induced without the primary-cultured cells from tumor tissue and could be applied for cancer therapy.     

Definition of an immunogenic region within the ovarian tumor antigen stratum corneum chymotryptic enzyme.

PURPOSE: The serine protease stratum corneum chymotryptic enzyme (SCCE) is overexpressed by ovarian tumor cells, but is not expressed by normal tissues, suggesting that SCCE may be an attractive target for immunotherapy. In this study, we tested the hypothesis that dendritic cells loaded with SCCE peptides will induce ovarian tumor antigen-specific CD8+ CTL responses and antigen-specific CD4+ helper T cell responses. EXPERIMENTAL DESIGN: Computer algorithms were used to identify candidate HLA-A2.1-restricted CD8+ CTL epitopes and HLA-DR-binding CD4+ helper T cell epitopes within SCCE. CD8+ CTL stimulated with peptide-loaded dendritic cells were tested against targets expressing endogenous SCCE, including HLA-A2.1-matched ovarian tumor cells. Dendritic cells were also loaded with an extended SCCE peptide, SCCE 110-139, which encompassed a defined CD8+ CTL epitope and multiple candidate CD4+ T helper cell epitopes. RESULTS: CD8+ CTL specific for SCCE 123-131 lysed autologous macrophages infected with an SCCE-expressing recombinant adenovirus, and also lysed HLA-A2.1-matched, SCCE-expressing ovarian tumor cells. Dendritic cells loaded with SCCE 5-13 peptide stimulated an HLA-A2.1-restricted CD8+ CTL response, but with a reduced level of lysis against ovarian tumor cells. Dendritic cells loaded with SCCE 110-139 induced antigen-specific CD4+ T cell and CD8+ T cell responses. Although SCCE 110-139-loaded dendritic cells processed and presented the 123-131 epitope, the dominant CD8+ CTL response was directed against alternative epitopes within SCCE 110-139. CONCLUSIONS: The 110-139 region of SCCE incorporates multiple CD8+ CTL and CD4+ helper T cell epitopes, and represents an attractive target antigen for immunotherapy of ovarian cancer.     

Alterations in the T-cell receptor variable beta gene-restricted profile of CD8+ T lymphocytes in the peripheral circulation of patients with squamous cell carcinoma of the head and neck.

PURPOSE: Apoptosis of activated CD8(+) T cells is often seen in tumor-infiltrating lymphocytes and circulating peripheral blood mononuclear cells (PBMC) in patients with squamous cell carcinoma of the head and neck (SCCHN). We investigated whether T-cell receptor (TCR) variable beta chain (Vbeta)-restricted T cells were more sensitive to apoptosis than non-TCR Vbeta-restricted T cells. EXPERIMENTAL DESIGN: Flow cytometry analysis with anti-TCR Vbeta antibodies was used to define expansions and contractions of Vbeta-restricted T cells in patients with SCCHN relative to normal donors. This staining was combined with Annexin V binding to indicate early T-cell apoptosis. RESULTS: The TCR Vbeta profiles of CD3(+) T cells in tumor-infiltrating lymphocytes and PBMCs of patients with SCCHN were altered relative to controls, with one to five expansions and numerous contractions of TCR Vbeta-restricted T cells detected. These types of alterations were significantly greater in CD8(+) than CD4(+) T cells. Enhanced Annexin V binding to CD8(+) T cells was evident in PBMCs obtained from all patients, with 3 of 13 showing preferential targeting for apoptosis of TCR Vbeta-restricted T cells. CONCLUSIONS: TCR Vbeta profiles of CD8(+) T cells were altered in patients with SCCHN relative to normal controls. This may reflect increased apoptosis of expanded or contracted CD8(+) T cells, which define the TCR Vbeta profile of antigen-responsive T-cell populations in patients with cancer.     

Characteristics of CD4+CD25+ regulatory T cells in the peripheral circulation of patients with head and neck cancer

Patients with squamous cell carcinoma of the head and neck (SCCHN) have depressed antitumour immunity. The presence of CD4+CD25+ (Treg) cells in these patients might be, in part, responsible for downregulation of antitumour immune responses. To evaluate the frequency and characteristics of Treg in the peripheral circulation of patients with SCCHN, we used multicolour flow cytometry. Expression of CCR7, CD62L, zeta chain and Annexin V binding to Treg and non-Treg CD4+ lymphocyte populations were evaluated. Treg were confirmed to be Foxp3+ and GITR+. The Treg frequency was significantly elevated in patients with active disease and those with no evidence of disease (NED) following curative therapies. Both Treg and non-Treg CD4+ T cells in patients were significantly enriched in CCR7- and CD62L- cell subsets. Although Treg in patients contained a higher proportion of double negative (CCR7-CD62L-) cells, the majority of Tregs were CCR7-CD62L+. The proportion of Annexin V+CD4+ T cells was higher in patients (P<0.00005) than normal controls (NC), and Treg were significantly more sensitive to apoptosis than non-Treg in patients and NC. Expression of zeta was reduced in all subsets of CD4+ T cells obtained from patients vs NC. The data suggest that Treg in patients with SCCHN largely contain T cells with the 'effector' phenotype, which bind Annexin V and have low zeta expression, consistent with their activation state and a rapid turnover in the peripheral circulation.