Analysis of Antigen‐Specific T and B Cells for Monitoring Immune Protection Against SARS‐CoV‐2

Abstract

Immunological memory is the basis of protection against most pathogens. Long‐living memory T and B cells able to respond to specific stimuli, as well as persistent antibodies in plasma and in other body fluids, are crucial for determining the efficacy of vaccination and for protecting from a second infection by a previously encountered pathogen. Antigen‐specific cells are represented at a very low frequency in the blood, and indeed, they can be considered “rare events” present in the memory T‐cell pool. Therefore, such events should be analyzed with careful attention. In the last 20 years, different methods, mostly based upon flow cytometry, have been developed to identify such rare antigen‐specific cells, and the COVID‐19 pandemic has given a dramatic impetus to characterize the immune response against the virus. In this regard, we know that the identification, enumeration, and characterization of SARS‐CoV‐2‐specific T and B cells following infection and/or vaccination require i) the use of specific peptides and adequate co‐stimuli, ii) the use of appropriate inhibitors to avoid nonspecific activation, iii) the setting of appropriate timing for stimulation, and iv) the choice of adequate markers and reagents to identify antigen‐specific cells. Optimization of these procedures allows not only determination of the magnitude of SARS‐CoV‐2‐specific responses but also a comparison of the effects of different combinations of vaccines or determination of the response provided by so‐called “hybrid immunity,” resulting from a combination of natural immunity and vaccine‐generated immunity. Here, we present two methods that are largely used to monitor the response magnitude and phenotype of SARS‐CoV‐2‐specific T and B cells by polychromatic flow cytometry, along with some tips that can be useful for the quantification of these rare events. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Identification of antigen‐specific T cells Basic Protocol 2: Identification of antigen‐specific B cells

Detailed characterization of SARS-CoV-2-specific T and B cells after infection or heterologous vaccination

“…To investigate the percentage of Ag-specific T cells, we used T-cell receptor (TCR)-dependent activation-induced marker (AIM) assays to identify and quantify SARS-CoV-2-specific CD4 + T cells ( 31 – 34 ). We stimulated peripheral blood mononuclear cells (PBMCs) from nine REC patients and 11 MIX and 12 RNA donors overnight with 15-mer peptides with 11-amino acid overlap, covering the complete sequence of Wuhan SARS-CoV-2 spike glycoprotein (see Methods for details).…”

“…PBMCs were cultured in a 96-well plate in the presence of 15-mer peptides with 11-amino acid overlap, covering the complete sequence of Wuhan SARS-CoV-2 spike glycoprotein (PepTivator SARS-CoV-2 Prot_S complete, Miltenyi Biotec, Bergisch Gladbach, Germany) together with 1 μg/ml of anti-CD28 (Miltenyi Biotec, Germany). PBMCs were stimulated for 18 h at 37°C in a 5% CO 2 atmosphere in complete culture medium (RPMI 1640 supplemented with 10% FBS and 1% each of L-glutamine, sodium pyruvate, nonessential amino acids, antibiotics, 0.1 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and 55 μM β-mercaptoethanol) ( 33 , 34 ). For each stimulated sample, an unstimulated one was prepared as a negative control.…”

Metabolic pathways engaged by antigen-specific T and B cells after SARS-CoV-2 vaccination in multiple sclerosis patients on different immunomodulatory drugs reveal immunosenescence and predict vaccine efficacy.

“…Then, we identi ed Ag + T cells, de ned as cells expressing CD137 and CD69 after 18 hours of in vitro stimulation with SARS-CoV-2 peptides (Supplementary Fig. 1) 9,35 . As shown in Fig.…”

“…For this reason we broadly interrogated SARS-CoV-2 antigen-speci c T and B cells 6 months after the third dose of mRNA vaccine in a cohort of 106 MS patients treated with different DMT such as cladribine, DMF, FTY, IFN-β, natalizumab, teri unomide or aCD20. By using 21-parameter ow cytometry, we have investigated the phenotype and function of antigen-speci c T and B cells, while the metabolic pro le of such cells was studied by 45-parameter mass cytometry that allows to pro le the metabolic regulome at the single cell level (scMEP) 9,35,36 . We nd that almost all patients develop a detectable and functional SARS-CoV-2 immune response.…”

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“…This type of protection is therefore only to a minor extent influenced by a mismatch between the vaccine variant and the circulating variant. Furthermore, the duration of T cell protection is apparently higher than that provided by antibodies, due to both the generation of long living memory cells and the capacity of virus-specific T lymphocytes to exert different functions at the same moment [4,5].…”