Carmen Visus

Tumor-Derived Microvesicles Promote Regulatory T Cell Expansion and Induce Apoptosis in Tumor-Reactive Activated CD8+ T Lymphocytes

Sera of patients with cancer contain membraneous microvesicles (MV) able to induce apoptosis of activated T cells by activating the Fas/Fas ligand pathway. However, the cellular origin of MV found in cancer patients’ sera varies as do their molecular and cellular profiles. To distinguish tumor-derived MV in cancer patients’ sera, we used MAGE 3/6+ present in tumors and MV. Molecular profiles of MAGE 3/6+ MV were compared in Western blots or by flow cytometry with those of MV secreted by dendritic cells or activated T cells. These profiles were found to be distinct for each cell type. Only tumor-derived MV were MAGE 3/6+ and were variably enriched in 42-kDa Fas ligand and MHC class I but not class II molecules. Effects of MV on signaling via the TCR and IL-2R and proliferation or apoptosis of activated primary T cells and T cell subsets were also assessed. Functions of activated CD8+ and CD4+ T lymphocytes were differentially modulated by tumor-derived MV. These MV inhibited signaling and proliferation of activated CD8+ but not CD4+ T cells and induced apoptosis of CD8+ T cells, including tumor-reactive, tetramer+CD8+ T cells as detected by flow cytometry for caspase activation and annexin V binding or by DNA fragmentation. Tumor-derived but not dendritic cell-derived MV induced the in vitro expansion of CD4+CD25+FOXP3+ T regulatory cells and enhanced their suppressor activity. The data suggest that tumor-derived MV induce immune suppression by promoting T regulatory cell expansion and the demise of antitumor CD8+ effector T cells, thus contributing to tumor escape.

Identification of Human Aldehyde Dehydrogenase 1 Family Member A1 as a Novel CD8+ T-Cell–Defined Tumor Antigen in Squamous Cell Carcinoma of the Head and Neck

Few epitopes are available for vaccination therapy of patients with squamous cell carcinoma of the head and neck (SCCHN). Using a tumor-specific CTL, aldehyde dehydrogenase 1 family member A1 (ALDH1A1) was identified as a novel tumor antigen in SCCHN. Mass spectral analysis of peptides in tumor-derived lysates was used to determine that the CTL line recognized the HLA-A*0201 (HLA-A2) binding ALDH1A1(88-96) peptide. Expression of ALDH1A1 in established SCCHN cell lines, normal mucosa, and primary keratinocytes was studied by quantitative reverse transcription-PCR and immunostaining. Protein expression was further defined by immunoblot analysis, whereas ALDH1A1 activity was measured using ALDEFLUOR. ALDH1A1(88-96) peptide was identified as an HLA-A2-restricted, naturally presented, CD8(+) T-cell-defined tumor peptide. ALDH1A1(88-96) peptide-specific CD8(+) T cells recognized only HLA-A2(+) SCCHN cell lines, which overexpressed ALDH1A1, as well as targets transfected with ALDH1A1 cDNA. Target recognition was blocked by anti-HLA class I and anti-HLA-A2 antibodies. SCCHN cell lines overexpressing ALDH1 had high enzymatic activity. ALDH1A1 protein was expressed in 12 of 17 SCCHN, and 30 of 40 dysplastic mucosa samples, but not in normal mucosa. ALDH1A1 expression levels in target cells correlated with their recognition by ALDH1A1(88-96) peptide-specific CD8(+) T cells. Our findings identify ALDH1A1, a metabolic antigen, as a potential target for vaccination therapy in the cohort of SCCHN subjects with tumors overexpressing this protein. A smaller cohort of subjects with SCCHN, whose tumors express little to no ALDH1A1, and thus are deficient in conversion of retinal to retinoic acid, could benefit from chemoprevention therapy.

Targeting ALDHbright Human Carcinoma–Initiating Cells with ALDH1A1-Specific CD8+ T Cells

Purpose: Cancer-initiating cells (CIC) are considered to represent the subpopulation of tumor cells that is resistant to conventional cancer treatments, highly tumorigenic in immunodeficient mice, and responsible for tumor recurrence and metastasis. Based on an elevated aldehyde dehydrogenase (ALDH) activity attributable to ALDH1/3 isoforms, ALDHbright cells have been identified and isolated from tumors and shown to have characteristics of CIC. The ALDH1A1 isoform was previously identified as a tumor antigen recognized by CD8+ T cells. This study examines the ability of ALDH1A1-specific CD8+ T cells to eliminate ALDHbright cells and control tumor growth and metastases. Experimental Design: ALDHbright cells were isolated by flow cytometry using ALDEFLUOR from HLA-A2+ human head and neck, breast, and pancreas carcinoma cell lines and tested for their tumorigenicity in immunodeficient mice. ALDH1A1-specific CD8+ T cells were generated in vitro and tested for their ability to eliminate CICs in vitro and in vivo by adoptive transfer to immunodeficient mice bearing human tumor xenografts. Results: ALDHbright cells isolated by flow cytometry from HLA-A2+ breast, head and neck, and pancreas carcinoma cell lines at low numbers (500 cells) were tumorigenic in immunodeficient mice. ALDHbright cells present in these cell lines, xenografts, or surgically removed lesions were recognized by ALDH1A1-specific CD8+ T cells in vitro. Adoptive therapy with ALDH1A1-specific CD8+ T cells eliminated ALDHbright cells, inhibited tumor growth and metastases, or prolonged survival of xenograft-bearing immunodeficient mice. Conclusions: The results of this translational study strongly support the potential of ALDH1A1-based immunotherapy to selectively target CICs in human cancer. Clin Cancer Res; 17(19); 6174–84. ©2011 AACR.

Function but not phenotype of melanoma peptide-specific CD8(+) T cells correlate with survival in a multiepitope peptide vaccine trial (ECOG 1696).

ECOG 1696 was a Phase II multi‐center trial testing vaccination with melanoma peptides, gp100, MART‐1 and tyrosinase delivered alone, with GM‐CSF, IFN‐α2b or both cytokines to HLA‐A2+ patients with metastatic melanoma. Here, the frequency of circulating CD8+tetramer+ (tet+) T cells and maturation stages of responding T cells were serially monitored and compared with baseline values in a subset of patients (n = 37) from this trial. Multiparameter flow cytometry was used to measure the frequency of CD8+ T cells specific for gp100, MART‐1, tyrosinase and influenza (FLU) peptides. Expression of CD45RA/CCR7 on CD8+tet+ T cells and CD25, CD27, CD28 on all circulating T cells was determined. Vaccine‐induced changes in the CD8+tet+ T cell frequency and phenotype were compared with results of IFN‐γ ELISPOT assays and with clinical responses. The frequency of CD8+tet+ T cells in the circulation was increased for the melanoma peptides (p < 0.03–0.0001) but not for FLU (p < 0.9). Only gp100‐ and MART‐1‐specific T cells differentiated to CD45RA+CCR7‐ effector/memory T cells. In contrast to the IFN‐γ ELISPOT frequency, previously correlated with overall survival (Kirkwood et al., Clin Cancer Res 2009;15:1443‐51), neither the frequency nor differentiation stage of CD8+tet+ T cells correlated with clinical responses. Delivery of GM‐CSF and/or IFN‐α2b had no effects on the frequency or differentiation of CD8+tet+, CD8+ or CD4+ T cells. Phenotypic analyses of CD8+tet+ T cells did not correlate with clinical responses to the vaccine, indicating that functional assessments of peptide‐specific T cells are preferable for monitoring of anti‐tumor vaccines.

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 ∼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.

The Wild-Type Sequence (wt) p53 25–35 Peptide Induces HLA-DR7 and HLA-DR11-Restricted CD4 + Th Cells Capable of Enhancing the Ex Vivo Expansion and Function of Anti-wt p53 264–272 Peptide CD8 + T Cells

Tumor peptide-based vaccines are more effective when they include tumor-specific Th cell-defined as well as CTL-defined peptides. Presently, two overlapping wild-type sequences (wt) p53 helper peptides, p53108–122 and p53110–124, have been identified as HLA-DR1- and/or HLA-DR4-restricted epitopes. These HLA-DR alleles are expressed by ∼35% of subjects with cancer. To identify Th cell-defined wt p53 peptides suitable for use on the remaining subject population, a dendritic cell (DC)-based coculture system was developed. CD4+ T cells isolated from PBMC obtained from HLA-DR4− normal donors were stimulated ex vivo with autologous DC transfected with wt p53 or mutant p53 cDNA. Reactivity of T cells was tested in ELISPOT IFN-γ assays against DC pulsed individually with a panel of algorithm-predicted, multiple HLA-DR-binding wt p53 peptides. The wt p5325–35 peptide was identified as capable of inducing and being recognized by CD4+ T cells in association, at a minimum, with HLA-DR7 and -DR11 molecules, each of which is expressed by ∼15% of the population. In addition, the presence of anti-p5325–35 CD4+ Th cells was shown to enhance the in vitro generation/expansion of HLA-A2-restricted, anti-wt p53264–272 CD8+ T cells, which from one donor were initially “nonresponsive” to the wt p53264–272 peptide. The wt p5325–35 peptide has attributes of a naturally presented Th cell-defined peptide, which could be incorporated into antitumor vaccines applicable to a broader population of subjects for whom a wt p53 helper peptide is presently unavailable, as well as used for monitoring anti-p53 Th cell activity in cancer subjects receiving p53-based immunotherapy.

Alterations in the T-Cell Receptor Variable β 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 β chain (Vβ)–restricted T cells were more sensitive to apoptosis than non–TCR Vβ-restricted T cells. Experimental Design: Flow cytometry analysis with anti-TCR Vβ antibodies was used to define expansions and contractions of Vβ-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 Vβ 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 Vβ-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 Vβ-restricted T cells. Conclusions: TCR Vβ 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 Vβ profile of antigen-responsive T-cell populations in patients with cancer.

Spontaneous apoptosis of tumor-specific tetramer+ CD8+ T lymphocytes in the peripheral circulation of patients with head and neck cancer.

In cancer, tumor escape from the host immune system includes apoptosis of circulating CD3+CD8+ effector T lymphocytes. Here, we compare sensitivity to apoptosis of virus‐ with tumor‐specific circulating CD8+ T cells in patients with head and neck cancer.

17β hydroxysteroid dehydrogenase type 12 (HSD17B12) is a marker of poor prognosis in ovarian carcinoma

OBJECTIVE 17β-hydroxysteroid dehydrogenase isoform 12 (HSD17B12) overexpression is associated with poor clinical outcome in invasive ductal carcinoma of the breast. Here, we evaluated HSD17B12 overexpression and its activity in ovarian carcinoma (OvCa) to determine its role in the growth and progression of this tumor. METHODS Immunohistochemical analysis of HSD17B12 expression was performed in 100 tissue samples of untreated OvCa and was correlated with clinicopathologic characteristics and patient outcome. In A2780 OvCa cell line expressing HSD17B12, siRNA knockdown of the enzyme was performed, and its effects on tumor cell growth and Annexin V binding were determined. RESULTS HSD17B12 expression was detected in all tumor samples, but the staining intensity was variable. Normal ovarian epithelium was negative. Patients with tumor showing weak/moderate expression of HSD17B12 had a better overall survival than those with strongly positive tumors (p<0.001). The time to first recurrence was longer for patients with tumors with heterogeneous staining relative to patients with tumors that were uniformly positive (p<0.001). Upon silencing of HSD17B12 in tumor cells, their growth was inhibited (p<0.005) and apoptosis was increased (p<0.05). Arachidonic acid but not estradiol reversed the growth inhibition mediated by HSD17B12 knockdown. CONCLUSION HSD17B12 overexpression is shown to be a marker of poor survival in patients with OvCa. Expression in the tumor and function of this enzyme facilitates OvCa progression.

Inhibition of Poly(ADP-Ribose) Polymerase (PARP) Induces Apoptosis in Lung Cancer Cell Lines

We have tested PJ34, a potent inhibitor of poly(ADP-ribose) polymerase (PARP), against various lung cancer cell lines (Calu-6, A549, and H460) and normal human bronchial epithelial cells (HBECs). While using WST1 dye assay, lung cancer cells exhibited LD50 values of approximately 30 μM PJ34 (72-hr assay). Molecular data showed that the effect of PJ34-induced apoptosis on lung cancer cells occurs via a caspase-dependent pathway. The present study has clearly shown that (a) PARP inhibitor can independently kill tumor cells, (b) caspase-3 has modest influence on PARP-inhibitor-mediated cancer-specific toxicity, and (c) a pan-caspase inhibitor decreases the apoptotic effect of PJ34.