Immune responses to p53 in patients with cancer: enrichment in tetramer+ p53 peptide-specific T cells and regulatory T cells at tumor sites

Abstract

Objective: A majority of human cancers, including head and neck cancer (HNC), "overexpress" p53. Although T cells specific for wild-type (wt) sequence p53 peptides are detectable in the peripheral blood of patients with HNC, it is unknown whether such T cells accumulate in tumor-involved tissues. Also, the localization of "regulatory" T cells (Treg) to tumor sites in HNC has not been investigated to date.

Methods: Tumor infiltrating lymphocytes (TIL), tumor-involved or non-involved lymph node lymphocytes (LNL) and peripheral blood mononuclear cells (PBMC) were obtained from 24 HLA-A2.1+ patients with HNC. Using tetramers and four-color flow cytometry, the frequency of Treg and CD3+CD8+ T cells specific for wt p53 epitopes as well as their functional attributes were determined.

Results: The CD3+CD8+ tetramer+ cell frequency was significantly higher (P<0.001) in TIL than autologous PBMC as was the percentage of CD4+CD25+ T cells (P<0.003). TIL were enriched in FOXp3+, GITR+ and CTLA-4+ Treg. CD8+ TIL had low Zeta expression and produced little IFN-gamma after ex vivo stimulation relative to autologous PBMC or PBMC from NC.

Conclusions: Anti-wt p53 epitope-specific T cells and Treg preferentially localize to tumor sites in patients with HNC. However, despite enrichment in tumor peptide-specific T cells, the effector cell population (CD3+CD8+) in TIL or PBMC was unresponsive to activation in the tumor microenvironment enriched in Treg.

Fig. 1

Specificity of tetramer binding to T cells expressing TCR for wt p53 epitopes. In a, T-cell lines were generated using wt p53 peptides presented on DC obtained from PBMC of HLA-A2+ patients with SCCHN. These T-cell lines were simultaneously stained with two different p53 tetramers and were shown to react only with the tetramers specific for the wt p53 peptides used for priming. In b, the generated wt p53 peptide-specific CTL lines were stained with the relevant and irrelevant (e.g., wt p53_65-73) tetramers. Only the relevant wt p53 peptide tetramers bound to CTL lines generated in cultures with wt p53_149-157 or wt p53_264-272 peptides. An irrelevant tetramer (wt p53_65-73) did not bind to these CTL. The percentages of positive cells are indicated below or next to every dot plot

Fig. 2

The log frequencies of CD3+CD8+tetramer+ cells in PBMC and TIL of an HLA-A2+ patient with SCCHN (#1). The frequencies of wt p53_149-157 tetramer+ T cells as well as p53_264-272 tetramer+ T cells are significantly higher in TIL than in PBMC (P<0.001) for both

Fig. 3

Increased percentages of CD4+CD25+ T cells in PBMC, TIL and one tumor non-involved LN in representative patients with SCCHN. The gate was set on CD3+CD4+ cells. Patient #1 shows the highest increase in CD4+CD25+ T cells in TIL among the patients studied. The tumor-non-involved LN has the same percentage of CD4+CD25+ T cells as PBMC. Patient #2 is the only one with a decreased percent of CD4+CD25+ cells in TIL relative to PBMC

Fig. 4

Low percentages of IFN-γ expressing CD3+CD8+ T cells in TIL or PBMC of patients with SCCHN compared to PBMC of normal donors. The cells were tested for IFN-γ expression by flow cytometry following 24 h activation with anti-CD3 Ab as described in Materials and methods. The gate was set on CD3+CD8+ T cells. The data are means ± SD