Fig 2: Functional impact of mutations in key SARS-CoV-2 dominant epitopes(A–F) Recognition of wild-type (black) and mutant (red) peptide titrations by polyclonal epitope-specific T cell lines in IFN-? ELISpot assays. SFU, spot forming units. Shown are mean values from three or more replicates +/- standard deviation.(G–J) Ability of CD8+ T cell lines to kill autologous B cells loaded with wild-type (black) or mutant (red) peptides in carboxyfluorescein succinimidyl ester (CFSE) assays. The effector:target ratio denotes the proportion of CD8+ T cells:B cells in each assay.(K–N) Recognition of wild-type (black) and mutant (red) peptide titrations by a polyclonal CD8+ T cell line specific for the HLA*A01:01-restricted ORF1a epitope TTDPSFLGRY1637-1646, using intra-cellular cytokine staining for interferon-gamma (IFNg, K), tumor necrosis factor (TNFa, L), and the degranulation factor CD107a (M), and a killing assay (N). Similar findings were seen with a T cell line generated from another donor (Figure S4).

Fig 3: mem(sc)LTa2ß but not mem(sc)LTaß2 activate TNFR1 and TNFR2.A Scheme of single-chain encoded memLTaß variants and memTNF. B Flow cytometry analysis of Flp-In HEK293 transfectants stably expressing mem(sc)LTa2ß and mem(sc)LTaß2 and of a CHO transfectant stably expressing a non-cleavable mutant of memTNF. Empty vector (EV) transfected cells served as negative controls. C The indicated Flp-In HEK293 and CHO-?(1-12)TNF cells were analyzed with respect to binding of TNFR1ed-GpL, TNFR2ed-GpL, and LTßRed-GpL. One representative experiment of three independent experiments are shown. D HeLa, HeLa-TNFR2, and HeLa-TNFR2-TNFR1KO cells were cocultured 1:1 (50,000 cells each) with the indicated ligand transfectants. Cell culture supernatants were analyzed the next day for their IL8 content by ELISA. Shown are results from three different experiments. ***p < 0.001; **p < 0.01; n.s., not significant, repeated-measures ANOVA.

Fig 4: The soluble ectodomains of TNFR1, TNFR2, and LTßR bind to different cell-bound heteromeric LTaß species.A Scheme of the TNFRed–GpL fusion proteins used in (B). B HEK293T cells were transfected with empty vector (EV) or a mixture of LTa- and memLTß-encoding expression plasmids. Next day, binding of 500 ng/ml TNFR1ed–GpL, TNFR2ed–GpL, and LTßRed–GpL was analyzed in the presence and absence of 20 µg/ml TNF. Data shown are technical replicates of one representative experiment of three independent experiments.

Fig 5: Cytokine and Granzyme B production of AIM+ CD8 T cells of patients with MS treated with aCD20 and healthy controls after SARS-CoV-2 mRNA vaccination.a) Representative flow cytometry plots for quantifying IFN-?, TNF, IL-2 and Granzyme B expression in AIM+ CD8 T cells. Numbers represent the percentage of AIM+ non-naive CD8 T cells that co-express 41BB and the corresponding cytokine or Granzyme B. b) Tukey boxplots (median, Q1 and Q3 quartiles, left) and individual points (right) of the frequency of non-naive CD8 T cells that are 41BB+ and express the indicated cytokines and/or Granzyme B at timepoints T1 and T4 following vaccination. Lines connect individual donors sampled longitudinally. Grey indicates HCs (n = 8), orange indicates MS-aCD20 patients (n = 10). c) Tukey boxplots (median, Q1 and Q3 quartiles) of the percentage of 41BB+ non-naive CD8 T cells that express the indicated cytokines and/or Granzyme B at timepoint T4 following vaccination. Grey indicates HCs (n = 8), orange indicates MS-aCD20 patients (n = 10). Any function = at least one of IFN-?, TNF, IL-2, and Granzyme B. Any cytokine = at least one of IFN-?, TNF, and IL-2.