Sensitivity and specificity of tritiated thymidine incorporation and ELISPOT assays in identifying antigen specific T cell immune responses

<p>Abstract</p> <p>Background</p> <p>Standardization of cell-based immunologic monitoring is becoming increasingly important as methods for measuring cellular immunity become more complex. We assessed the ability of two commonly used cell-based assays, tritiated thymidine incorporation (proliferation) and IFN-gamma ELISPOT, to predict T cell responses to HER-2/neu, tetanus toxoid (tt), and cytomegalovirus (CMV) antigens. These antigens were determined to be low (HER-2/neu), moderate (tt), and robustly (CMV) immunogenic proteins. Samples from 27 Stage II, III, and IV HER-2/neu positive breast cancer patients, vaccinated against the HER-2/neu protein and tt, were analyzed by tritiated thymidine incorporation and IFN-gamma ELISPOT for T cell response.</p> <p>Results</p> <p>Linear regression analysis indicates that both 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 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 robust 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.</p> <p>Conclusion</p> <p>These data underscore the importance of taking into consideration the performance characteristics of assays used to measure T cell immunity. This consideration is particularly necessary when determining which method to utilize for assessing responses to immunotherapeutic manipulations in cancer patients.</p

Sipuleucel-T Immune Parameters Correlate with Survival: an Analysis of the Randomized Phase 3 Clinical Trials in Men with Castration-Resistant Prostate Cancer

Purpose: Sipuleucel-T, the first FDA-approved autologous cellular immunotherapy for treatment of advanced prostate cancer, is manufactured by activating peripheral blood mononuclear cells, including antigen presenting cells (APCs), with a fusion protein containing prostatic acid phosphatase. Analysis of data from three phase 3 trials was performed to immunologically characterize this therapy during the course of the three doses, and to relate the immunological responses to overall survival (OS). Methods: Sipuleucel-T product characteristics [APC numbers, APC activation (CD54 upregulation), and total nucleated cell (TNC) numbers] were assessed in three randomized, controlled phase 3 studies (N = 737). Antigen-specific cellular and humoral responses were assessed in a subset of subjects. The relationships between these parameters and OS were assessed. Results: APC activation occurred in the first dose preparation [6.2-fold, (4.65, 7.70); median (25th, 75th percentile)] and increased in the second [10.6-fold (7.83, 13.65)] and third [10.5-fold (7.89, 13.65)] dose preparations. Cytokines and chemokines associated with activated APCs were produced during the manufacture of each dose; T-cell activation-associated cytokines were detected in the second and third dose preparations. Antigen-specific T cells were detectable after administration of the first sipuleucel-T dose. Cumulative APC activation, APC number, and TNC number correlated with OS (P < 0.05). Antigen-specific immune responses were observed in 78.8 % of monitored subjects and their presence correlated with OS (P = 0.003). Conclusion: Sipuleucel-T broadly engages the immune system by activating APCs ex vivo and inducing long-lived immune responses in vivo. These data indicate antigen-specific immune activation as a mechanism by which sipuleucel-T prolongs OS

Sensitivity and specificity of tritiated thymidine incorporation and ELISPOT assays in identifying antigen specific T cell immune responses-0

<p><b>Copyright information:</b></p><p>Taken from "Sensitivity and specificity of tritiated thymidine incorporation and ELISPOT assays in identifying antigen specific T cell immune responses"</p><p>http://www.biomedcentral.com/1471-2172/8/21</p><p>BMC Immunology 2007;8():21-21.</p><p>Published online 12 Sep 2007</p><p>PMCID:PMC2034595.</p><p></p>xis. Antigen specific IgG antibody responses are plotted on the log-scaled vertical axis. The diagonal line indicates line of regression

Sensitivity and specificity of tritiated thymidine incorporation and ELISPOT assays in identifying antigen specific T cell immune responses-5

<p><b>Copyright information:</b></p><p>Taken from "Sensitivity and specificity of tritiated thymidine incorporation and ELISPOT assays in identifying antigen specific T cell immune responses"</p><p>http://www.biomedcentral.com/1471-2172/8/21</p><p>BMC Immunology 2007;8():21-21.</p><p>Published online 12 Sep 2007</p><p>PMCID:PMC2034595.</p><p></p>ed boxes). Reporting value for the proliferation assay is SI, and reporting value for the ELISPOT assay is antigen-specific precursor/10PBMC. Antibody immunity for the same group of subjects is shown for comparison, with a reporting value of ug/ml antigen specific IgG

Sensitivity and specificity of tritiated thymidine incorporation and ELISPOT assays in identifying antigen specific T cell immune responses-3

<p><b>Copyright information:</b></p><p>Taken from "Sensitivity and specificity of tritiated thymidine incorporation and ELISPOT assays in identifying antigen specific T cell immune responses"</p><p>http://www.biomedcentral.com/1471-2172/8/21</p><p>BMC Immunology 2007;8():21-21.</p><p>Published online 12 Sep 2007</p><p>PMCID:PMC2034595.</p><p></p> (B) tt specific cellular immunity (n = 12), and (C) CMV specific cellular immunity (n = 12). Samples with antigen specific circulating antibodies were considered to have positive immunity

Sensitivity and specificity of tritiated thymidine incorporation and ELISPOT assays in identifying antigen specific T cell immune responses-4

<p><b>Copyright information:</b></p><p>Taken from "Sensitivity and specificity of tritiated thymidine incorporation and ELISPOT assays in identifying antigen specific T cell immune responses"</p><p>http://www.biomedcentral.com/1471-2172/8/21</p><p>BMC Immunology 2007;8():21-21.</p><p>Published online 12 Sep 2007</p><p>PMCID:PMC2034595.</p><p></p>xis. Antigen specific IgG antibody responses are plotted on the log-scaled vertical axis. The diagonal line indicates line of regression