Detection of CD4 T-cell responses to a tumor vaccine by cytokine flow cytometry

Cytokine flow cytometry (CFC) is a simple and powerful method for measuring antigen-specific T-cell responses by detection of intracellular cytokine staining. We applied this method to the detection of CD4 T-cell responses to tumor vaccines. Patients with multiple myeloma were immunized against their autologous tumor immunoglobulin idiotype, using antigen-pulsed dendritic cell vaccination. Blood samples were drawn before and after vaccination, and CFC and proliferation assays were performed. For CFC, whole blood was incubated overnight with antigen in the presence of costimulatory antibodies to CD28 and CD49d. The blood was then treated with EDTA, erythrocytes were lysed, and leukocytes were fixed, permeabilized, and stained for intracellular cytokines [tumor necrosis factor-alpha (TNF-alpha) or IFN-gamma], CD4, and CD69. Cells were analyzed by flow cytometry and cytokine-producing CD69+ cells enumerated as a percentage of CD4 cells. Of nine patients analyzed, three demonstrated detectable CFC responses to tumor immunoglobulin and/or keyhole limpet hemocyanin (KLH) after vaccination. One of these patients responded only to KLH, whereas the other two responded to both tumor immunoglobulin and KLH. Most responses were detected with both TNF-alpha and IFN-gamma, but one patient's KLH response was detected only with TNF-alpha. There was a positive, but not strong, correlation of cytokine responses with proliferative responses to KLH. Although further follow-up and correlation with clinical outcome is needed, CFC may represent a simple yet detailed assessment of T-cell frequencies and subsets responding to cancer vaccines.