Characterization of Preexisting MAGE-A3-Specific CD4+ T Cells in Cancer Patients and Healthy Individuals and Their Activation by Protein Vaccination

Abstract

Vaccination with cancer/testis Ag MAGE-A3 in the form of recombinant protein often induces specific humoral and cellular immune responses. Although Ag-specific CD4+ T cells following vaccination are detectable by cytokine production after a single in vitro stimulation, their detection before vaccination is difficult because of low frequency. In this study, we have applied a sensitive method using CD154 (CD40L) staining to detect MAGE-A3-specific CD4+ T cells. MAGE-A3-specific T cell responses were analyzed in four healthy donors, two lung cancer patients with spontaneous serum Abs to MAGE-A3, and two baseline seronegative lung cancer patients throughout vaccination with MAGE-A3 protein. MAGE-A3-specific CD4+ T cells were detected in all individuals tested, at low frequency in healthy donors and seronegative cancer patients and higher frequency in patients seropositive for MAGE-A3. Polyclonal expansion of CD154-expressing CD4+ T cells after cell sorting generated a large number of MAGE-A3-specific CD4+ T cell lines from all individuals tested, enabling full characterization of peptide specificity, HLA-restriction, and avidity. Application of this method to cancer patients vaccinated with MAGE-A3 protein with or without adjuvant revealed that protein vaccination induced oligoclonal activation of MAGE-A3-specific CD4+ T cells. It appeared that MAGE-A3 protein vaccination in the presence of adjuvant selectively expanded high avidity CD4+ T cells, whereas high avidity T cells disappeared after multiple vaccinations with MAGE-A3 protein alone.

Effect of Montanide and Poly-ICLC Adjuvant on Human Self/Tumor Antigen-Specific CD4+ T Cells in Phase I Overlapping Long Peptide Vaccine Trial

“…These results indicated that OLP-alone vaccination selectively deleted or anergized high-avidity precursors. A similar observation was made in patients vaccinated with MAGE-A3 protein alone in comparison with patients vaccinated with MAGE-A3 in the adjuvant system AS02B, indicating that this phenomenon is not limited to peptide vaccination (18). Interestingly, patients who received MAGE-A3 protein alone did not respond to booster vaccinations with MAGE-A3þAS02B adjuvant, indicating that deletion or persistent anergy of high-avidity CD4 þ T cells led to long-term tolerance (27,28 (35).…”

“…To investigate the effects of Montanide and poly-ICLC in the differential immunogenicity of OLP, we addressed the quantitative and qualitative changes in vaccine-induced NY-ESO-1-specific CD4 þ T cells depending on vaccine compositions using an in-depth sensitive approach. As we reported previously, upregulation of CD154 (CD40-ligand) on CD4 þ T cells restimulated with tumor antigen peptides after a single in vitro peptide sensitization was used successfully to isolate tumor antigen-specific CD4 þ T cells (18). CD154 upregulation is sufficiently sensitive to allow the isolation of low-frequency tumor antigen-specific CD4 þ T-cell precursors even from healthy donors or from patients before vaccination.…”

“…We previously reported that tumor antigen-specific CD4 þ T cells with different functions such as IFN-g-producing Th1 and IL-4-producing Th2 are sensitively detected by the expression of CD154, also known as CD40-ligand, which is transiently expressed on antigen-specific CD4 þ T cells after antigenic stimulation (18,20). To assess NY-ESO-1-specific cellular immune response, CD4 þ T cells isolated from PBMCs of OLP-vaccinated patients were presensitized once with an assay NY-ESO-1 peptide pool (assay OLP) and after a 20-day culture period, CD154-expressing cells were enumerated after restimulation with peptide-pulsed or -unpulsed target cells by flow cytometry.…”

Efficacy of vaccination with recombinant vaccinia and fowlpox vectors expressing NY-ESO-1 antigen in ovarian cancer and melanoma patients

“…1A). Next, we established NY-ESO-1-specific CD4 + Tcell lines in a subset of ovarian cancer patients pre-and postvaccination, as previously described (9). We determined Th1, Th2, and Th17 differentiation based on IFN-γ, IL-4, IL-13, or IL-17 production upon stimulation with NY-ESO-1 pooled peptides.…”

Heat Shock Protein 90-Mediated Peptide-Selective Presentation of Cytosolic Tumor Antigen for Direct Recognition of Tumors by CD4+ T Cells

“…CD4 + T cells, and peptide-pulsed T celldepleted PBMCs were cocultured for 20 d in the presence of 10 U/ml IL-2 (Roche Diagnostics) and 20 ng/ml IL-7 (R&D Systems) to allow expansion of NY-ESO-1-specific CD4 + T cells. NY-ESO-1 peptide-specific T cell lines were established as described (15). CD154-expressing CD4 + T cells were isolated after restimulation with PHA-activated CD4 + T cells (T-APC) pulsed with a subpool (#9-#12 peptides) of NY-ESO-1 overlapping peptides using a FACSAria instrument and expanded with PHA (Remel).…”