Monitoring of CD8؉ T-cell responses in cancer patients

Novel diagnostic tools are needed to diagnose latent infection and to provide biologically meaningful surrogate markers to define cellular immune responses against Mycobacterium tuberculosis (MTB). Interferon gamma‐based assays have recently been developed in addition to the more than 100‐year‐old tuberculin skin test (TST) for the immune diagnosis of MTB in blood. The advent of soluble MHC/peptide tetramer molecules allows to objectively enumerate antigen‐specific T cells. We identified novel MHC class II‐restricted MTB epitopes and used HLA‐DR4 tetrameric complexes to visualize ex vivo CD4+ T cells directed against the antigens Ag85B and the 19‐kDa lipoprotein, shared between MTB and other Mycobacterium species, and CD4+ T cells which recognize the MTB‐associated ESAT‐6 antigen. MTB‐reactive CD4+ T cells reside predominantly in the CD45RA+ CD28+ and CD45− CD28+ T‐cell subset and recognize naturally processed and presented MTB epitopes. HLA‐DR4‐restricted, Ag85B or ESAT‐6‐specific CD4+ T cells show similar dynamics over time in peripheral blood mononuclear cells (PBMC) when compared with CD8+ T cells directed against the corresponding HLA‐A2‐presented MTB epitopes in patients with pulmonary MTB infection and subsequent successful therapy. This was not found to be true for T‐cell responses directed against the 19‐kDa lipoprotein. The dissection of the cellular immune response in M. tuberculosis infection will enable novel strategies for monitoring MTB vaccine candidates and to gauge CD4+ T cells directed against MTB.

SUMMARYIn response to antigenic stimulation, naive MHC-class I restricted and antigen-specific + CD45 + CD28 -T cells define antigen/peptide-specific and MHCrestricted responses. These data were confirmed in PBL from patients with tuberculosis using HLA-A2 tetramer-complexes loaded with a peptide from the M. tuberculosis Ag85b antigen by flow cytometry. The sorting of this T cell subset enables to determine the fine specificity of CD8 + effector T cells without the need for in vitro manipulation.

Purpose: Cellular and humoral immune responses directed against autologous tumor cells in patients with cervical cancer may be directed against proteins provided by human papillomavirus (HPV) associated products or, alternatively, by yet undefined targets as well. The goal of our study was to evaluate the local cellular immune response in cervical cancer. Methods: From a fresh tumor sample of a patient with cervical carcinoma, tumor-infiltrating lymphocytes (TIL) were generated, expanded and characterized by immunohistochemistry, flow cytometry, cytokine release assays and DNA fragment analysis. Results: We established a MHC class II-restricted CD4+ T-cell line from a patient with cervical cancer which recognizes autologous (HPV35+, HPV59+) tumor cells and the HLA-DR4-matched cervical cancer cell line Me180 (HPV68+) as determined by TNFa secretion. Expression of different HPV-E7 genes in autologous B-cells revealed that this T-cell line defines a DR4-presented T-cell epitope which is shared among the E7 gene of HPV59 and HPV68. Conclusion: Tumor-HPV-specific and MHC-class II-restricted CD4+ T-cells are present within the tumor lesion and can be successfully expanded in the presence of IL-2 and IL-7. MHC class II presented peptides may be implemented to augment T-cell responses directed against autologous tumor cells.