Comparison of Cytomegalovirus-Specific Immune Cell Response to Proteins versus Peptides Using an IFN-γ ELISpot Assay after Hematopoietic Stem Cell Transplantation.

Eva Wagner-Drouet ,Mareike Verbeek,Martin Schreder,Ludwig Deml,Anne Rascle,Martina Koch,Sascha Barabas,Ralf Wagner,Monika Lindemann,Daniel Teschner,Traudel Schmidt,Inken Hilgendorf,Guido Kobbe,Markus Ditschkowski,Johannes Gärtner,Daniel Wolff,Christine Wolschke,Stefan Klein,Stephan Mielke,Kerstin Schäfer‐Eckart

doi:10.3390/diagnostics11020312

Abstract

Cytomegalovirus (CMV) infection is a major cause of morbidity and mortality following hematopoietic stem cell transplantation (HSCT). Measuring CMV-specific cellular immunity may improve the risk stratification and management of patients. IFN-γ ELISpot assays, based on the stimulation of peripheral blood mononuclear cells with CMV pp65 and IE-1 proteins or peptides, have been validated in clinical settings. However, it remains unclear to which extend the T-cell response to synthetic peptides reflect that mediated by full-length proteins processed by antigen-presenting cells. We compared the stimulating ability of pp65 and IE-1 proteins and corresponding overlapping peptides in 16 HSCT recipients using a standardized IFN-γ ELISpot assay. Paired qualitative test results showed an overall 74.4% concordance. Discordant results were mainly due to low-response tests, with one exception. One patient with early CMV reactivation and graft-versus-host disease, sustained CMV DNAemia and high CD8+ counts showed successive negative protein-based ELISpot results but a high and sustained response to IE-1 peptides. Our results suggest that the response to exogenous proteins, which involves their uptake and processing by antigen-presenting cells, more closely reflects the physiological response to CMV infection, while the response to exogenous peptides may lead to artificial in vitro T-cell responses, especially in strongly immunosuppressed patients.

Highlights

Cytomegalovirus (CMV) infection is a serious cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT) [1]
February 2014) and all subjects gave written informed consent in accordance with the Declaration of Helsinki, as previously reported [4]. This investigation aimed to compare the response of isolated peripheral blood mononuclear cells (PBMC) to cytomegalovirus (CMV) antigens used as either proteins or peptides in an IFN-γ enzyme-linked immunospot (ELISpot) assay
Six out the 13 discordant included (pp65) and 120 (IE-1) test results showed a similar pattern of low spot counts near the limit of quantitation (LoQ) (SFC [SRM2]/200,000 PBMC ranging from 0.00 to 23.2; Table S1, patients no. 2, 3, 8, 11 and 14), not truly discordant either

Summary

Introduction

Cytomegalovirus (CMV) infection is a serious cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT) [1]. Two standardized CMV-specific IFN-γ ELISpot assays, based on the in vitro stimulation of peripheral blood mononuclear cells (PBMC) with pp and IE-1 peptides (T-SPOT®.CMV) or T-activated® proteins (T-Track® CMV), recently demonstrated their suitability to measure CMV-specific cellular immunity in clinical settings [4,5,8,9,13,14,15,16,17,18]. Peptide-loaded MHC class I and II molecules are transported to the plasma membrane and presented to CD8+ and CD4+ T cells, respectively [19,20]. Exogenous peptides are taken up by APC and intracellularly loaded to de novo-synthesized MHC molecules before being transported to the cell surface and presented to T cells. Direct loading of peptides onto MHC class II molecules at the cell surface is unlikely to occur, due to the high stability of peptide-bound MHC class II complexes [19,21,22]

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