Analysis of the T cell response to tumor and viral peptide antigens by an IFNγ-ELISPOT assay

Abstract

We have established a sensitive ELISPOT assay measuring interferon gamma (IFN gamma) release on a single-cell basis to detect influenza peptide-specific CD8+ T cells in uncultured peripheral blood mononuclear cells (PBMC). Using this method, we studied the T cell response to HLA-A1 and HLA-A2.1 binding peptide epitopes derived from the MAGE-1 and MAGE-3 proteins, from the melanoma-associated antigens tyrosinase, Melan-A/MART-1 and gp100, and from influenza proteins in stage IV melanoma patients and healthy controls. In 18 of 24 HLA-A2-positive donors (75%), but only in 9 of 25 HLA-A2-positive melanoma patients (36%) T cells reactive with the influenza matrix peptide were demonstrated (p = 0.007). T cells responding to one or several of the melanoma-associated peptides were detected in 5 of 25 HLA-A2-positive patients with metastatic melanoma. Four of these 5 patients had been treated with interleukin-2- and IFN alpha-containing therapy. Two of the 24 healthy donors had T cells reactive with the MART-1 27-35 peptide. No reactivity with the HLA-A1-binding peptides from MAGE-1 or MAGE-3 was detected in any of the HLA-A1-positive healthy controls or melanoma patients. These results show that the IFN gamma-ELISPOT assay is suitable to determine quantitatively T cells reactive with melanoma-associated and influenza peptide epitopes in uncultured PBMC. The failure to detect T cells responding to influenza in many melanoma patients with progressive disease may indicate an impairment of their T cell function.

IFN‐γ stimulates CpG‐induced IL‐10 production in B cells via p38 and JNK signalling pathways

“…In these conditions, endogenous production of IL-10 hampers an optimal immune response [75][76][77][78]. Upon vaccination, IFN-γ production is often used as a readout for Th1 responses, cell-mediated cytotoxicity and NK cell activity [75,79,80]. However, our results show that the presence of IFN-γ may also have an inhibitory effect on the immune response through stimulation of IL-10, a mechanism that may explain the absence of an adequate vaccine response in some patients [78].…”

Dendritic cells enhance detection of antigen‐specific cellular immune responses by lymphocytes from rhesus macaques immunized with an HIV envelope peptide cocktail vaccine

“…Further, the tetramer assay does not differentiate between functional and non-functional T cells. The other two functional assays, CFC and ELISPOT, provide quantitative information on the frequencies of MHC-restricted T lymphocytes in terms of antigen-specific cytokine production which could be detected to single cell level [11,13,22,23,31]. While the CFC assay relies on determining intracellular cytokine production that requires expensive and often less frequently accessible instrumentation (flowcytometer), the ELISPOT assay is performed by using the cytokine-specific antibodies in a simple microtiter well format following the colorimetric detection principle of enzyme-linked immunosorbent assay (ELISA).…”

“…In the ELISPOT assay [9,11,12,13,14,22,23,31], PBMC are stimulated with specific antigen(s) and the cytokines produced by individual cells is trapped on the filters in the microtiter wells that were pre-coated with a specific anti-cytokine antibody. Thus, in this protocol the responder T cells present among the PBMC rely on other cells like the monocyte/macrophage population for antigen presentation.…”

Protective CD8 Memory T Cell Responses to Mouse Melanoma Are Generated in the Absence of CD4 T Cell Help

“…IFN-γ ELISPOT (MabTech) was performed as previously described [2], [49]. Briefly, CD8 + T cells from pooled spleens or inguinal lymph nodes were purified using anti-CD8 MACS magnetic beads (Miltenyi Biotec).…”