Thomas Woelfel

Quantitation of antigen-reactive T cells in peripheral blood by IFNγ-ELISPOT assay and chromium-release assay: a four-centre comparative trial

The ELISPOT assay is increasingly being used for the monitoring of the induction of antigen-reactive T cells in cancer vaccination trials. In order to evaluate the reliability of T cell frequency analysis with the ELISPOT assay, a comparative study was performed in four European laboratories. Six samples from healthy subjects were analyzed for the frequency of influenza-reactive CD8+ T cells in peripheral blood mononuclear cells (PBMC) by IFNgamma-ELISPOT assay. In addition, one laboratory determined cytotoxic T cell precursor (CTL) frequencies in these samples by limiting dilution chromium-release assay (LDA), and three laboratories performed a variant of the LDA, the multiple microculture assay (MMA). Consistent frequencies of influenza peptide-reactive T cells were obtained with the ELISPOT assay in all four laboratories. The numbers detected by ELISPOT assay correlated closely with those determined by LDA. In contrast, the frequencies obtained with the MMA differed considerably and showed little correlation with the other two assays. This study shows that it is possible to use the ELISPOT assay to determine with reliability antigen-reactive T cells in a multicenter setting. We suggest that this assay may be suitable for monitoring cancer vaccine trials.

Rapid T cell–based identification of human tumor tissue antigens by automated two-dimensional protein fractionation

Identifying the antigens that have the potential to trigger endogenous antitumor responses in an individual cancer patient is likely to enhance the efficacy of cancer immunotherapy, but current methodologies do not efficiently identify such antigens. This study describes what we believe to be a new method of comprehensively identifying candidate tissue antigens that spontaneously cause T cell responses in disease situations. We used the newly developed automated, two-dimensional chromatography system PF2D to fractionate the proteome of human tumor tissues and tested protein fractions for recognition by preexisting tumor-specific CD4+ Th cells and CTLs. Applying this method using mice transgenic for a TCR that recognizes an OVA peptide presented by MHC class I, we demonstrated efficient separation, processing, and cross-presentation to CD8+ T cells by DCs of OVA expressed by the OVA-transfected mouse lymphoma RMA-OVA. Applying this method to human tumor tissues, we identified MUC1 and EGFR as tumor-associated antigens selectively recognized by T cells in patients with head and neck cancer. Finally, in an exemplary patient with a malignant brain tumor, we detected CD4+ and CD8+ T cell responses against two novel antigens, transthyretin and calgranulin B/S100A9, which were expressed in tumor and endothelial cells. The immunogenicity of these antigens was confirmed in 4 of 10 other brain tumor patients. This fast and inexpensive method therefore appears suitable for identifying candidate T cell antigens in various disease situations, such as autoimmune and malignant diseases, without being restricted to expression by a certain cell type or HLA allele.

Short Peptide Vaccine Induces CD4+ T Helper Cells in Patients with Different Solid Cancers

The possibility that short peptide vaccines induce antitumor CD4+ T-cell responses has been widely ignored. Peripheral blood from vaccinated patients revealed that short peptides often activate specific T helper cells, facilitating a strong combined CD4+ and CD8+ T-cell response. Previous cancer vaccination trials often aimed to activate CD8+ cytotoxic T-cell (CTL) responses with short (8–10mer) peptides and targeted CD4+ helper T cells (TH) with HLA class II–binding longer peptides (12–16 mer) that were derived from tumor antigens. Accordingly, a study of immunomonitoring focused on the detection of CTL responses to the short, and TH responses to the long, peptides. The possible induction of concurrent TH responses to short peptides was widely neglected. In a recent phase I vaccination trial, 53 patients with different solid cancers were vaccinated with EMD640744, a cocktail of five survivin-derived short (9- or 10-mer) peptides in Montanide ISA 51VG. We monitored 49 patients and found strong CD8+ T-cell responses in 63% of the patients. In addition, we unexpectedly found CD4+ TH cell responses against at least two of the five short peptides in 61% (23/38) of the patients analyzed. The two peptides were recognized by HLA-DP4– and HLA-DR–restricted TH1 cells. Some short peptide–reactive (sp)CD4 T cells showed high functional avidity. Here, we show that a short peptide vaccine is able to activate a specific CD4+ T-cell repertoire in many patients, facilitating a strong combined CD4+/CD8+ T-cell response. Cancer Immunol Res; 4(1); 18–25. ©2015 AACR.

Parvovirus H1-induced tumor cell death enhances human immune response via crosspresentation of dendritic cells

Address: 1I. Medical Department, University of Mainz, Langenbeckstr.1, D-55101 Mainz, Germany, 2Deutsches Krebsforschungszentrum, Applied Tumor Virology, Dept. F0100, and Institut National de la Sante et de la Recherche Medicale Unite 375, Im Neuenheimer Feld 242, D-69120 Heidelberg, Germany, 3III. Medical Department, University of Mainz, Langenbeckstr.1, D-55101 Mainz, Germany and 4Med. Department Mitte, Klinikum Dortmund GmbH, Beurhausstr. 10, 44137 Dortmund, Germany