The efficacy of cancer immunotherapy can be improved by treatment with full-length tumor antigen and by combining several antigens. This approach allows the induction of a broad immune response irrespective of the patient's HLA type which at the same time challenges immune monitoring. Also, the number of available lymphocytes is most often limited and minimal in vitro restimulations of the lymphocytes should maintain information about the actual in vivo situation. To overcome these hurdles, we developed a method to measure the CD8(+) and CD4(+) T-cell responses directly ex vivo. Skin biopsies taken from dendritic cell (DC)-induced DTH reactions from melanoma patients participating in a DC-clinical trial served as lymphocyte source. Antigen-specificity of skin infiltrating lymphocytes was investigated by coculture with antigen-presenting autologous B cells and assessed for CD137 upregulation and enhanced cytokine secretion. Using this approach we could detect treatment-specific CD8(+) T-cells without restimulation in vitro. Upregulation of the activation marker CD137 correlated with the upregulation of the lytic marker CD107a. CD137 upregulation by treatment-specific CD4(+) lymphocytes however was more pronounced after antigen-specific in vitro restimulation. Both CD8(+) and CD4(+) lymphocytes could be further expanded using the same B cells as for screening allowing characterization of the recognized antigenic region. In addition, this technique can be extended to detect a broader array of T-cell functions and to monitor a large cohort of patients. We believe that this approach of direct ex vivo monitoring, irrespective of the patient's HLA-type or the recognized peptide, and using a limited number of lymphocytes is a valuable tool in the immune monitoring of current cellular immunotherapies.
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