While infectious disease antigens, in general, stimulate a very robust T cell immune response that can easily be detected using a variety of assays, immunity to tumor antigens is generally low level. For this reason, to evaluate the immune response against cancer antigens, assays are needed that can detect a broad range of T cell responses i.e. from innate to induced immunity. We focused on evaluating the sensitivity of two commonly used methods for assessing tumor specific immunity; the proliferation of T cells after antigen exposure measured by the incorporation of tritiated thymidine and IFN-gamma ELISPOT. Compared to other methods of T cell assessment, such as flow cytometry or tetramer analysis, these assays are technically simple, easy to perform for all levels of lab personnel, require smaller numbers of cells, and reagents which are readily available and inexpensive.
Very few studies have attempted to determine the sensitivity and specificity of assays that measure cellular immune responses after vaccination. A major problem preventing such an analysis is the definition of a parameter that predicts that immunity has been successfully elicited. We used the development of humoral immunity to indicate that tumor antigen and foreign antigen vaccinations were effective in stimulating a detectable immune response. Humoral immunity has long been used as an accepted measure of successful immunization. Presence of antigen specific antibodies indicates exposure to antigen and as such is considered proof of immunity (via natural exposure or vaccination) to common infectious diseases such as measles and influenza [6, 7]. Vaccine development for infectious disease continues to rely on antibody presence as an indicator not only of exposure to antigen , but as a surrogate of protection against disease. Indeed, antigen specific humoral immunity is an indicator for the development of an antigen specific T cell response. In one study, T cell responses measured by IFN-gamma ELISPOT were significantly associated with antibody responses induced by vaccination against P. falciparum protein . A recent population-based analysis of immune responses to MMR vaccination found that an antigen specific antibody response was significantly associated with antigen specific T cell proliferation, and that lymphoproliferation was in turn associated with production of IFN-gamma as measured by cytokine ELISA . Thus, although not a perfect measure or "gold standard", humoral immunity can be used as a meaningful determinate of effective immunization by which the T cell assays could be assessed and compared.
Antibody responses to tumor associated antigen demonstrate exposure to tumor, and may be significantly associated with antigen specific cellular responses. However, the relationship between presence of antigen specific antibodies, presence of antigen specific T cells, and clinical response in cancer is not as well characterized as the relationship between antibody and T cell responses, and clinical response in infectious diseases such as malaria. Recently, the presence of tumor-infiltrating T cells has recently been associated with clinical response in various cancers, and antigen specific T cells have been shown necessary for tumor destruction [10–12]. Thus, direct assay of T cell responses, both in terms of measuring quantities and frequencies of T cells and in terms of characterizing T cell function, remains an essential component in elucidating the relationship between immune responses and clinical outcome, and in the immunologic monitoring of clinical trials.
Due to the large number of cells required to perform the proliferation and ELISPOT assays, not all of the samples contained sufficient quantity to perform both assays. It is interesting to note that although the ELISPOT assay for response to CMV tested only 12 samples, compared to the 18 samples tested for CMV responses by proliferation assay, both methods produced results which predicted positive humoral immunity to CMV. In contrast, all 27 samples were tested for HER-2/neu specific responses using both ELISPOT and proliferation assays, and only the proliferation assay produced results which were a significant predictor of antibody immunity. It is possible that the lack of concordance between tt specific results by ELISPOT and tt specific antibody immunity is due to the difference in samples size between T cell assays, as only 12 samples were of sufficient quantity to obtain results by ELISPOT, compared to the 18 available for testing by proliferation assay. Similarly, cell quantities limited the ELISPOT assay to measurement of only IFN-gamma. This may in part explain the ELISPOT's lack of sensitivity in determining low level responses. Although increasing the breadth of cytokine analysis to include measurement of other cytokine responses such as IL-4 or IL-5 may lead to greater sensitivity, the measurement of IFN-gamma alone is standard for most clinical trials.