Optimization of interferon gamma ELISPOT assay to detect human cytomegalovirus specific T-cell responses in solid organ transplants

Abstract

Assessing the CMV specific CMI in transplant subjects represents a promising strategy to determine the risk of infection on individual basis. In this study 61 adult CMV IgG seropositive solid organ transplant recipients were examined in order to improve the efficacy of CMI detection. For this purpose, pair-wise comparisons were conducted comparing positive control stimuli PWM and PMA/iono and CMV stimuli, pp65 peptide pool and whole CMV particle. Rosette pre-depletion of blood was also investigated for detecting CD4+ or CD8+ T-cell responses using the IFN-g ELISPOT assay. In the time-points 30-180 days after transplantation, PMA/iono produced statistically significant higher responses compared to PWM, probably because PMA/iono activation pathway is independent from the effect of immunosuppressive drugs. The data showed that 11% of transplant patients displayed very low or undetectable responses to pp65 peptide pool antigen while having sustained high responses to whole CMV particle. In addition, in all the subjects analyzed, CMI responses to CMV particle produced a statistically significant higher number of spots compared to pp65 peptide pool antigen. Rosette pre-depletion of whole blood proved to be effective in detecting CD4+ or CD8+ T-cell responses similarly to flow cytometry. Taken together, the following recommendations are suggested to optimize the CMV-ELISPOT for transplantation settings: (1) use PMA/iono as positive control; (2) whole virus particle should be used to avoid peptide-related false negative responses; (3) a rosette pre-depletion step may be useful to detect CD4+ or CD8+ T-cell responses.

Keywords: AZA; CMI; CMV; Cell mediated immunity; ConA; CsA; ELISPOT; HLA; Human cytomegalovirus; Interferon gamma releasing assay; MMF; PBMCs; PFU; PHA; PKC; PMA/iono; PWM; T-cell; Transplantation; azathioprine; cell-mediated immunity; concanavalin A; cyclosporine A; human cytomegalovirus; human leukocyte antigen; mycophenolic acid; peripheral blood mononuclear cells; phorbol myristate acetate and ionomycin; phytohemoagglutinin; plaque forming unit; pokeweed mitogen; protein kinase C.