CMV-specific T-cell monitoring is currently explored as a valuable tool to identify transplant recipients at risk for viral complications. As both CMV-specific CD4 and CD8 T cells are critically involved in efficient CMV control and currently used assays are optimised to either detect CD4 or CD8 T cells, the use of urea-modified CMV proteins (IE-1 and pp65) was analysed for their potential to simultaneously stimulate CD4 and CD8 T cells. CMV-specific T cells were quantified by IFN-Γ induction using an ELISPOT assay (CMV T-Track, Lophius Biosciences) or flow-cytometry and compared with a non-modified CMV-lysate. To assess validity in serologically defined immunocompromised patients, 40 healthy controls (43.3±13.7 yrs), 40 hemodialysis patients (65.9±13.3 yrs) and 40 renal transplant patients(54.7±14.3 yrs) were screened.
Median ELISPOT counts in CMV IgG positive individuals were low for IE-1 (3.8, IQR 10.5), medium for pp65 (65, IQR 179.5) and highest for CMV-lysate (120, IQR 160), and no significant difference was observed between patients and controls. IgG negative individuals had almost no spots regardless of the antigens used (0-0.5, IQR 0-0.5). Flow-cytometry revealed that median CMV-specific CD4 T-cell frequencies were substantially lower after stimulation with urea-modified pp65 (0.09%, IQR 0.30) compared to non-modified CMV-lysate (1.93%, IQR 3.57). In contrast, induction of CMV-specific CD8 T cells was stronger after stimulation with the urea-modified pp65 (0.21%, IQR 0.94) as compared to the CMV-lysate (0.14%, IQR 0.46). In line with ELISPOT results, median IE-1 specific CD4 and CD8 T-cell frequencies were low (0.02%, IQR 0.03 and 0.04%, IQR 0.17, respectively). Although significant for all three antigens (p<0.0001), correlation between ELISPOT and flow-cytometry was highest with pp65 (r=0.83) and CMV-lysate (r=0.86).
Conclusion: While the non-modified CMV-lysate is well-suited for detection of CD4 T cells, urea-modification of protein antigens represents a viable approach to improve concomitant detection of CMV-specific CD8 T cells. As compared to peptide stimulation, this approach does not require knowledge of antigenic epitopes or HLA type. Both ELISPOT and the flow-cytometry are feasible readouts and allow monitoring of CMV-specific cellular immunity in controls and immunocompromised patients.
Deml, L.: Employee, Lophius Biosciences is the Manufacturer of ELISPOT.
To cite this abstract in AMA style:Schub D, Schmidt T, Guckelmus C, Kirsch S, Deml L, Sester U, Sester M. Urea-Modified Protein Antigens to Simultaneously Assess CMV-Specific CD4 and CD8 T-Cell Responses Using a New CMV-ELISPOT Assay and Flow-Cytometry [abstract]. Am J Transplant. 2013; 13 (suppl 5). https://atcmeetingabstracts.com/abstract/urea-modified-protein-antigens-to-simultaneously-assess-cmv-specific-cd4-and-cd8-t-cell-responses-using-a-new-cmv-elispot-assay-and-flow-cytometry/. Accessed October 31, 2020.
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