In vitro activation of hTERT-specific T cell responses in lung cancer patients following chemotherapy

Abstract

Objective: The aim of this study was to examine chemotherapy concomitant in vitro activation of human telomerase reverse transcriptase (hTERT)-specific T cell responses in peripheral blood mononuclear cell (PBMC) samples of patients with advanced non-small cell lung cancer (NSCLC).

Methods: PBMCs depleted of regulatory T cells were stimulated by peptide loaded dendritic cells (DC) matured either by application of cytokines (cDC) or a Toll-like receptor 7/8-agonist combined with a soluble CD40-ligand (ligDC). The hTERT peptide-specific T cell responses were assessed using flow cytometry for intracellular interferon-γ (IFN-γ).

Results: After cDC activation, T cells producing IFN-γ in response to hTERT were found in PBMC samples of 4 patients. In 2 of these patients the hTERT-specific T cell responses were further increased after ligDC application. However, PBMC of 3 other patients showed little or no induction of hTERT-specific T cell responses as a result of the methods applied during this study.

Conclusions: These results indicate, that concomitant to chemotherapy hTERT-specific T cell responses can be activated in PBMC of NSCLC patients in vitro. This activation can be further increased by ligDC though the number of responding patients is still limited.

Keywords: Lung cancer; T cells; dendritic cells; telomerase.

Figure 1

Expression of costimulatory molecules in DCs of patient D: after culture of adherent PBMCs in GM-CSF and IL-4 cells showed typical DC morphology. These immature DCs were either activated by the standard cytokine cocktail (cDC) or by the CD7/8-agonist combined with the sCD40L (ligDC). The phenotype of these cells was further analyzed by flow cytometry and based on forward-side-scatter dot plots the large granular cells were gated as DCs (A). Within this gate of PBMC-derived large granular cells with typical DC morphology we next analyzed the surface-antigen expression. In comparison to standard cytokine maturation (B) DCs express lower levels of costimulatory molecules and maturation markers when matured with TLR7/8-agonists and sCD40-ligands (C). Grey filled traces represent DCs before maturation. DCs after maturation are shown in black filled traces.

Figure 2

Depletion of CD4⁺CD25⁺ regulatory T cells in patient G: before depletion of CD4⁺CD25⁺ T cells 3.5% regulatory T cells were present in non-adherent PBMC of patient G (A). After depletion of CD4⁺CD25⁺ T cells no T_reg were detectable (B).

Figure 3

Direct ex vivo FACS analysis of intracellular IFN-γ release by peripheral blood T-cells in responses to SEA/SEB. A, Representative data from patient B; B, Detailed results from all 7 patients after SEA (patient A-E) and SEB (patient F-G) stimulation in NSCLC patients.

Figure 4

Activation of T cell responses to hTERT in T_reg depleted PBMC of NSCLC patients after weekly stimulations with peptide loaded cDC. Figure shows the CD8/IFN-γ profile of CD3 gated lymphocytes after incubation of T cells with hTERT-peptide loaded autologous target cells/DC (dahed bars). Responses against non-peptide loaded targets/controls/DC serve as negative controls (black bars).

Figure 5

Increased activation of hTERT-specific T cells after stimulation of Treg depleted PBMC with peptide-loaded ligDC. Flow cytometric analysis of peripheral blood CD8⁺ T cells specifically producing IFN-γ in response to hTERT-peptide loaded targets. Target cells with no peptide were used as controls. The T cells were either stimulated by cytokine (cDC) or by sCD40-and TLR7/8-ligand (ligDC) matured, hTERT-peptide loaded DC. IFN-γ, interferon gamma. A, Representative data from patient A. Frequencies of CD8⁺ T cells that produced IFN-γ are shown as pecentages of total numbers of CD8⁺ T cells; B, In patient A and B the frequency of T cells that produced IFN-γ in response to hTERT-peptides was higher in T cells that had been stimulated with hTERT-peptide loaded ligDC than in T cells that had been stimulated with cDC.

Figure 6

Very low (patient C and E) and no hTERT-specific activation signal, respectively (patient D, F, G). The T cells were stimulated weekly by hTERT-peptide loaded ligDC. Figure shows the flow cytometric analysis of peripheral blood CD8⁺ T cells specifically producing IFN-γ in response to hTERT-peptides loaded targets. Target cells with no peptide were used as controls. IFN-γ, interferon gamma.