Detection of Epstein–Barr virus‐specific memory CD4⁺T cells using a peptide‐based cultured enzyme‐linked immunospot assay

Abstract

SummaryApproaches to evaluate T-cell responses to Epstein-Barr virus (EBV) include enzyme-linked immunospot (ELISPOT), which quantifies cells capable of immediate interferon-c secretion upon antigen stimulation. However, evaluation of expandable EBV-specific memory T cells in an ELISPOT format has not been described previously. We quantified EBVspecific T-cell precursors with high proliferative capacity by using a peptide-based cultured interferon-c ELISPOT assay. Standard and cultured ELISPOT responses to overlapping peptide pools (15-mers overlapping by 11 amino acids) covering the lytic (BZLF1 and BMRF1) and latent (EBNA1, EBNA3a, EBNA3b, EBNA3c, LMP1 and LMP2) EBV proteins were evaluated in 20 healthy subjects with remote EBV infection and, for comparison, in four solid organ transplant recipients. Cultured ELISPOT responses to both lytic and latent EBV antigens were significantly higher than standard ELISPOT responses. The distribution of EBV-specific T-cell responses detected in healthy virus carriers showed more consistent cultured ELISPOT responses compared with standard ELISPOT responses. T-cell responses quantified by cultured ELISPOT were mainly mediated by CD4+ T cells and a marked pattern of immunodominance to latentphase antigens (EBNA1 > EBNA3 family antigens > LMP2 > LMP1) was shown. Both the magnitude and distribution of EBV-specific T-cell responses were altered in solid organ transplant recipients; in particular, cultured ELISPOT responses were almost undetectable in a lungtransplanted patient with EBV-associated diseases. Analysis of T-cell responses to EBV by ELISPOT assays might provide new insights into the pathogenesis of EBV-related diseases and serve as new tools in the monitoring of EBV infection in immunocompromised patients.

Clinical immune‐monitoring strategies for predicting infection risk in solid organ transplantation

“…In contrast, the pathogen-specific immune-monitoring strategies rely on antigen-specific assays that estimate the magnitude and functionality of adaptive immune responses generated by T cells or B cells against a given pathogen, usually by measuring the production of Th 1 effector cytokines (that is, interferon (IFN)-γ or tumor necrosis factor-α) upon stimulation with a known antigen. Although progress has been made in the assessment of different virus-specific cell-mediated immune responses, including Epstein–Barr virus (EBV) 9 or BK polyomavirus (BKPyV), 10 we will mainly focus on current developments in cytomegalovirus (CMV)-specific T-cell monitoring, in view of its relative state of maturity and wide potential implications for the management of the SOT population.…”

“…126, 127, 128 Therefore, the monitoring of EBV-DNA replication in peripheral blood is routinely used to identify those patients at risk of developing this complication, particularly in the EBV-seronegative pediatric population. 129 Previous studies have demonstrated the feasibility of measuring the EBV-specific T-cell-mediated response by MHC-tetramer staining, 130 ICS 131 and ELISpot assay, 9, 132 although with limited clinical application at this time. One potential role of these approaches would be to monitor the effect of newer therapeutic interventions in patients with uncontrolled EBV replication (that is, adoptive cellular immunotherapy with donor EBV-specific cytotoxic T cells after in vitro expansion).…”

Memory T cells specific for HBV enumerated by a peptide-based cultured enzyme-linked immunospot assay in healthy HBV-vaccinated subjects

“…Additionally, we found a low variation across replicate wells and the mean inter-assay CV was below 50%, which is reasonably accepted for a biological assay. 25,26 Specifically, we reported that SEB data had a lower variability than HBV data. It is mainly due to the fact that net spots/ million PBMC were generally higher in response to SEB than to HBV pool stimulation.…”

Zika Virus Infection in Pregnancy: Advanced Diagnostic Approaches in Dengue-Naive and Dengue-Experienced Pregnant Women and Possible Implication for Cross-Reactivity and Cross-Protection

“…Values lower than 0.36 were specific for ZIKV infection; (ii) ELISpot assay for DENV using peptide pools, 15-18 amino acids in length with an 11-12 amino acid overlap, as antigens. Pool of NS3 peptide from Dengue 1, Dengue 2, Dengue 3, and Dengue 4 were used as stimuli, while human IFN-γ release was determined by ELISpot kits (Diaclone, Besancon, France) as described [22,23]. The mean number of spots from duplicates was adjusted to 1 × 10 6 PBMC.…”