# A novel mass cytometry protocol optimized for immunophenotyping of low-frequency antigen-specific T cells

## TL;DR

This paper introduces a new mass cytometry method to better study rare T-cell responses to antigens, useful for vaccine and therapeutic development.

## Contribution

A dual-barcoding system combined with a centrifugation-free washing method for improved immunophenotyping of low-frequency T cells.

## Key findings

- The dual-barcoding approach reduces cell loss and improves debarcoding efficiency in multiplexed samples.
- The protocol reveals heterogeneous T-cell phenotypes in SARS-CoV-2–vaccinated and infected patients.
- The method enables analysis of antigen-specific T-cell responses from minute populations in the same donor sample.

## Abstract

Understanding antigen-specific T-cell responses, for example, following virus infections or allergen exposure, is of high relevance for the development of vaccines and therapeutics. We aimed on optimizing immunophenotyping of T cells after antigen stimulation by improving staining procedures for flow and mass cytometry. Our method can be used for primary cells of both mouse and human origin for the detection of low-frequency T-cell response using a dual-barcoding system for individual samples and conditions. First, live-cell barcoding was performed using anti-CD45 antibodies prior to an in vitro T-cell stimulation assay. Second, to discriminate between stimulation conditions and prevent cell loss, sample barcoding was combined with a commercial barcoding solution. This dual-barcoding approach is cell sparing and, therefore, particularly relevant for samples with low cell numbers. To further reduce cell loss and to increase debarcoding efficiency of multiplexed samples, we combined our dual-barcoding approach with a new centrifugation-free washing system by laminar flow (Curiox™). Finally, to demonstrate the benefits of our established protocol, we assayed virus-specific T-cell response in SARS-CoV-2–vaccinated and SARS-CoV-2–infected patients and compared with healthy non-exposed individuals by a high-parameter CyTOF analysis. We could reveal a heterogeneity of phenotypes among responding CD4, CD8, and gd-T cells following antigen-specific stimulations. Our protocol allows to assay antigen-specific responses of minute populations of T cells to virus-derived peptides, allergens, or other antigens from the same donor sample, in order to investigate qualitative and quantitative differences.