Enumeration and Characterization of Human Memory T Cells by Enzyme-Linked Immunospot Assays

Abstract

The enzyme-linked immunospot (ELISPOT) assay has advanced into a useful and widely applicable tool for the evaluation of T-cell responses in both humans and animal models of diseases and/or vaccine candidates. Using synthetic peptides (either individually or as overlapping peptide mixtures) or whole antigens, total lymphocyte or isolated T-cell subset responses can be assessed either after short-term stimulation (standard ELISPOT) or after their expansion during a 10-day culture (cultured ELISPOT). Both assays detect different antigen-specific immune responses allowing the analysis of effector memory T cells and central memory T cells. This paper describes the principle of ELISPOT assays and discusses their application in the evaluation of immune correlates of clinical interest with a focus on the vaccine field.

Individual-level factors associated with variation in mycobacterial-specific immune response: Gender and previous BCG vaccination status

“…For example, HIV positive and latently infected individuals were excluded. Thirdly, in the outlined laboratory procedure [17e20] a 16-h ELISPOT assay was chosen, which may have potentially missed central memory CD4þ T-cells as they require a longer period of antigen re-stimulation to generate IFNg [35]. As such, our responses may underestimate the true "memory" cell presence, specifically at the later time point of 24 weeks.…”

Coxiella burnetii epitope-specific T-cell responses in chronic Q fever patients

“…T-cell responses were first analyzed by cultured IFNγ ELISpot, which both enhances detection of low frequency responses and preferentially measures central memory T-cells (13). Similar to previous observations for convalescent C. burnetii -exposed subjects, 9/14 chronic Q fever patients (64%) showed responses to 3-14 HLA class II peptides per donor (Figure 1A), while responses to HLA class I peptides were rare, with only three subjects showing responses to one or two peptides each (Figure 1B).…”

“…For a subset of chronic and convalescent individuals we next analyzed by direct ELISpot whether HLA class II C. burnetii -specific peptide responses would also be more readily detected in chronic patients. This assay preferentially measures effector memory responses (13). The proportion of individuals with direct ELISpot responses in both cohorts was comparable, with 5/11 responding convalescent individuals and 7/13 chronic subjects (Figure 4).…”

“…Two different ELISpot assays were employed to facilitate detection of central memory T-cell responses (cultured ELISpot) and effector memory T-cell responses (standard or direct ELISpot) (13). ELISpot was conducted using freshly isolated peripheral blood mononuclear cells (PBMCs) from lithium-heparin anti-coagulated blood, using Leukosep tubes prefilled with Ficoll (Greiner BioOne) according to the manufacturer’s recommendations.…”

Approaches for monitoring of non virus-specific and virus-specific T-cell response in solid organ transplantation and their clinical applications

“…The limitations are that the assay detects only one cytokine at a time and no information is provided on the phenotype or other characteristics of antigen-specific cells. Modified assays have been developed, such as dual/three color ELISPOT assays which allow simultaneous detection of different cytokines [62][63][64][65][66][67] or cultured ELISPOT assays which involve expansion of T cells in response to specific antigen during a 10-day culture before the ELISPOT is performed [68].…”