Table 1.

Functions and methods of T cell detection assays.

Assay (References)	Function/method	Notes
ELISpot20–24	Quantify the frequency of isolated T cells secreting cytokines (i.e., functional T cells) in response to stimulation with SARS-CoV-2-specific peptide pools. Chromogenic detection of cytokine “spots” correlates with individual-activated T cells. T cell subsets can be identified by their cytokine signature (e.g., Th1 cells secrete IFN-γ, IL-2, and TNF-α)	• In vitro stimulation of PBMCs using SARS-CoV-2 peptide pools revealed pre-existing memory CD4 T cells cross-reactive to SARS-CoV-2 and common cold HCoVs • SARS-CoV-2 peptide pools mapped T cell responses to individual SARS-CoV-2 epitopes, which were predominantly S-specific
Whole-blood cytokine release19,20	Detect secreted cytokines from natural SARS-CoV-2 infection- and COVID-19 vaccine-induced T cells in peptide-stimulated whole blood	• Facilitated rapid quantification of secreted IFN-γ and IL-2 in whole blood stimulated with S-specific peptide pools from BNT162b2-vaccinated individuals, and from convalescent asymptomatic and symptomatic COVID-19 patients • IL-2 demonstrated better sensitivity than IFN-γ in detecting S-specific T cell responses 2–3 months post-vaccination and 12 months post-infection in convalescent COVID-19 patients
ICS23,25–27	Quantify frequency of T cells secreting cytokines in response to stimulation with SARS-CoV-2-specific peptide pools. Immunostaining of cytokines enables quantification by FACS	• Levels of IFN-γ-secretion of SARS-CoV-2-specific memory CD4 and CD8 T cells were greater in recovered COVID-19 patients than in their close contacts (i.e., exposed to SARS-CoV-2, but lacking detectable infection) • Polyfunctionality (the ability to secrete multiple cytokines, such as IFN-γ, IL-2, or TNF-a) was observed in the SARS-CoV-2-specific CD4 (25%–40%) and CD8 (30%–50%) T cells of recovered patients 2 months post-symptom onset, and was maintained for ~9 months post-symptom onset
AIM10,23,24	Detect natural SARS-CoV-2 infection- and COVID-19 vaccine-induced T cells by measuring upregulation of TCRs upon stimulation with antigen-specific peptide libraries followed by FACS	• AIM assays using SARS-CoV-2 peptide pools (S and non-S) and markers for CD4 (e.g., CD137+ OX40+) or CD8 (e.g., CD137+ CD69+) T cells identified SARS-CoV-2-specific CD4 and CD8 T cells in recovered patients • Memory CD4 T cells of individuals unexposed to SARS-CoV-2 are cross-reactive to homologous SARS-CoV-2 and HCoV peptide pools
MHC Multimer Staining23,28,29	Detect T cells expressing TCRs capable of binding specific complexes of SARS-CoV-2 epitopes and multimers of MHC/HLA class I or II molecules by FACS	• SARS-CoV-2-specific MHC-I multimer staining detected subsets of stem cell-like memory T cells in recovered individuals, peaking ~4 months post-symptom onset
Single-cell immune profiling: scRNA-seq and scTCR-seq 29	Elucidate gene expression profiles of FACS-sorted, SARS-CoV-2-specific T cells by next-generation sequencing and analysis of the single-cell transcriptome (scRNA-seq) or TCR sequence (scTCR-seq)	• A combination of MHC multimer staining, scRNA-seq, and scTCR-seq using pools of 18 DNA-barcoded MHC-I multimers revealed similar levels of S-specific T cell responses in individuals after natural SARS-CoV-2 infection and mRNA COVID-19 vaccination, though the target antigens differ. These T cell responses were boosted in recovered, vaccinated individuals

AIM: activation-induced marker; COVID-19: coronavirus disease 2019; ELISpot: enzyme-linked immune absorbent spot; FACS: fluorescence-activated cell sorting; HCoV: human coronavirus; HLA: human leukocyte antigen; IFN: interferon; IL: interleukin; ICS: intracellular cytokine staining; MHC: major histocompatibility complex; PMBC: peripheral blood mononuclear cell; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; scRNA-seq: single-cell RNA sequencing; scTCR-seq: single-cell T cell receptor sequencing; S: spike; TCR: T cell receptor; Th1: T helper cell type 1; TNF: tumor necrosis factor.