We used the development of humoral immunity to indicate that tumor antigen and foreign antigen vaccinations were effective in stimulating a detectable immune response. stimulation index (SI) (p = 0.011) and IFN-gamma secreting precursor frequency (p < 0.001) are significant indicators of antigen specific immunity. ROC curves plotted to assess the performance of tritiated thymidine incorporation and the ELISPOT assay indicate that SI is usually a significant indicator of low T cell response to the HER-2/neu vaccine (p = 0.05), and of moderate and robust responses to tt (p = 0.01) and CMV (p = 0.016), respectively. IFN-gamma precursor frequency is a significant indicator of a strong T cell response to CMV (p = 0.03), but not of moderate tt (p = 0.09), or low HER-2/neu (p = 0.09) T cell responses. Conclusion These data underscore the importance of taking into consideration the performance characteristics of assays used to measure T cell immunity. This concern is particularly necessary when determining which method to utilize for assessing responses to immunotherapeutic manipulations in cancer patients. Background A multitude of assays have been developed to measure T cell responses to a variety of antigens [1]. There is little agreement as to which method is the most superior for detecting immune responses. Antigen specific T cell proliferation as measured by tritiated thymidine incorporation and IFN-gamma ELISPOT are two of the most commonly used methods to measure T cell immunity. We questioned if there was a difference in the ability of these assays to measure a broad range of T cell responses. In this study we utilized T cells from cancer patients that had been immunized with both a HER-2/neu protein based vaccine as well as a tetanus toxoid vaccine. Both vaccines were successful in generating antigen specific antibody immunity, an indicator of immunization. Evaluating three immunogenic proteins, HER-2/neu, tt, and CMV in these patients as representative of low, intermediate, and strong responses we determined that this proliferation assay was a better discriminator of low level immune responses than ELISPOT. These data spotlight the need for immunologic monitoring core laboratories to define the performance characteristics of the methods chosen to assess the development of a T cell immune response. Results Antibody immunity to both foreign and tumor antigens is an indicator of a concomitant T cell response A standard measure of successful immunization is the development of antigen specific humoral immunity [2]. Furthermore, the presence of humoral immunity may serve as a marker for the presence of antigen specific CD4+ T cells [3,4]. Breast malignancy patients were immunized against the HER-2/neu ICD protein and tt, and humoral immunity to these antigens measured by ELISA. Antibody responses to CMV, a viral antigen presumed to stimulate a robust immune reaction, were also measured by ELISA [5]. Successful immunization against HER-2/neu, as indicated by the presence of HER-2/neu specific antibodies, was achieved in 89% (24 of 27) of patients. Successful immunization against tt, as indicated by the presence of tt specific antibodies, MK-0517 (Fosaprepitant) was achieved in 100% of patients. Forty-four percent of the patients were CMV seropositive. Linear regression analysis of all results was performed to determine the correlation of antibody immunity to a detectable T cell response. Antibody response predicted T cell response when T cell response was assessed by either proliferation assay (Fig. ?(Fig.1A)1A) or ELISPOT assay (Fig. ?(Fig.1B).1B). Humoral immunity significantly (p = 0.011) predicted CD4+ T cell proliferation with an R2 of 0.428, and humoral MK-0517 (Fosaprepitant) immunity significantly (p < 0.001) predicted MK-0517 (Fosaprepitant) IFN-gamma secretion with an R2 of 0.531. Thus, humoral immunity and T cell immunity correlated in both foreign and tumor antigen systems. Open in a separate window Physique 1 Antibody immunity to both foreign and tumor antigens is an indicator of a concomitant T cell response. Results from the (A) proliferation assay and (B) ELISPOT assay for antigens HER-2/neu, tt and CMV are plotted around the log-scaled horizontal axis. Antigen specific IgG antibody responses are plotted around the log-scaled vertical axis. The diagonal line indicates line of regression. The magnitude of the T cell response differed significantly between antigens (Fig. ?(Fig.2).2). The range of T cell immunity to HER-2/neu was low to moderate with a mean SI of 5.06 (1.6C15.8) and a mean precursor frequency of 4.61 precursors/106 PBMC (0C46). MK-0517 (Fosaprepitant) Responses to tt were moderate to strong with a mean SI of 23.4 (0.9C76.2) and mean precursor frequency of 146 precursors/106 PBMC (0C1153). Responses to CMV were robust with a mean SI of 47.1 (0.7C296) and a mean Rabbit Polyclonal to Syntaxin 1A (phospho-Ser14) precursor frequency of 329 precursors/106 PBMC (0C1037). Mean antibody responses to HER-2/neu, tt and CMV were 3.3 (0C9.3) ug/ml, 47 (8.9C70.7) ug/ml and 131 (0C1000) ug/ml, respectively. We questioned the sensitivity of these two assays, CD4+ proliferation and IFN-gamma ELISPOT, in detecting this wide range of responses..