Fig. 2: Identification and validation of SARS-CoV-2 antigen-reactive T cell receptors. | Nature Communications

Fig. 2: Identification and validation of SARS-CoV-2 antigen-reactive T cell receptors.

From: Single-cell RNA sequencing reveals ex vivo signatures of SARS-CoV-2-reactive T cells through ‘reverse phenotyping’

Fig. 2

a Top: IFNG expression of CD8 clonotypes after antigenic or no stimulation. Only clonotypes with at least three cells in each condition and as defined by a unique αβ CDR3 sequence were included in this analysis. Each dot represents one cell and lines indicate the median. n = 56/66 (clone 81), 38/30 (clone 304), 25/30 (clone 244), 16/16 (clone 152), and 23/22 (clone 104) cells for stim/unstim condition, respectively. Exact cell numbers per clonotype can be found in Supplementary Data 11. Antigen-reactive clonotypes (defined through statistically significant upregulation of IFNG) are highlighted. p < 0.0001 for clones 81, 244, and 104; p = 0.0033 for clone 304. Bottom: Clonotype sizes in numbers of total cells analyzed. Clonotypes defined as “reactive” are highlighted in black. b As in a, but for CD4 clonotypes. n = 28/42 (clone 138), 17/26 (clone 19), 11/18 (clone 256), and 4/4 (clone 574) cells for stim/unstim condition, respectively. p < 0.0001 for clones 138, 19, 256, and 574 c Experimental setup; T cells from healthy donors were equipped with TCRs identified from COVID-19 patients by CRISPR/Cas9-mediated orthotopic TCR replacement (OTR); transgenic T cells were co-incubated with antigen-loaded patient PBMCs and reactivity was investigated by intracellular cytokine staining. d Flow-cytometric analysis of antigen-stimulated TCR-engineered T cells, 1 week after OTR; representative data are shown for CD4 TCR138 after stimulation with SARS-CoV-2 spike protein–peptide mix, no stimulation, and stimulation with irrelevant EBV antigen BZLF1 peptide mix (negative controls); mTRBC: murine constant region of the TCR beta chain incorporated into transgenic TCRs for detection; shown gates are pre-gated for CD3+ CD8− living lymphocytes. e Quantification of spike antigen-specific reactivity for selected clonotypes tested in (b) as well as two additional small clonotypes; for antigen-specific transcriptional shifts detected by initial scRNA seq for the respective clones see Fig. 1e, Supplementary Fig. 5a and Supplementary Fig. 7b. n = 1 technical replicate (no stim) and n = 2 technical replicates (stim). Statistical analysis by two-way ANOVA (**** each for the treatment effect and clonotype distribution) followed by Sidak’s multiple comparisons test *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (a, b). Data are shown for patient GT_3.

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