Fig 2: YLQPRTFLL-specific T cell clonotypes from vaccinated donors do not activate with P272L-YLQLRTFLL peptide(A) Wuhan-YLQPRTFLL tetramer enriched T cell lines from 6 of the 7 vaccinees in Figure 5. HLA A*02:01-restricted SLYNTVATL peptide from HIV used as an irrelevant tetramer (Cole et al., 2017). Percentage of CD3+ cells is shown.(B) The lines were re-sorted with Wuhan-YLQPRTFLL tetramer for TCR analysis. Pie charts display the proportion (chart segments) and frequency (numbers in center) of public (blue) and private (grey) TCRs. Public or private status based on data from convalescent patient studies. Variable (V) (arc on the right) and joining (J) (arc on the left) gene rearrangements are annotated with dominant and public chains with CDR3s of interest. CDR3 sequences are listed in Figure S5A.(C) CD107a upregulation of the T cell lines with Wuhan-YLQPRTFLL or P272L-YLQLRTFLL peptides. Peptides (10-6 M) were pulsed on to T2 antigen-presenting cells prior to assay. CD3/CD28 Dynabeads used as a positive control. Error bars depict SD of duplicates. Flow cytometry data shown in Figure S5B.See also Figures S3 and S5.

Fig 3: TCRs and peptide reactivity of Wuhan-YLQPRTFLL tetramer enriched T cell lines from six vaccinated donors, related to Figures 5 and 6(A) TCR usage and CDR3 sequences of Wuhan-YLQPRTFLL specific T cells from vaccinated donors. TCRs that appear in multiple individuals highlighted as public, according to the key.(B) CD107a upregulation of the T cell lines with Wuhan and P272L-YLQLRTFLL peptides. Peptides (10-6 M) were pulsed on to T2 antigen-presenting cells prior to assay. CD3/CD28 Dynabeads used as a positive control. Performed in duplicate. Gating of positive cells shown for vaccinee 201a.

Fig 4: Differential gene expression analysis between CARpos and CARneg/vectorpos cellsVolcano plots of DEGs (Wilcoxon rank-sum test) between CARpos T cells and CARneg/vectorpos T cells in the (A) CD3-LV-, (B) CD4-LV-, (C) CD8-LV-, and (D) VSV-LV-treated samples from two healthy donors (D#1 & D#2). The CAR values were excluded from the graphs for better visualization. Negative fold changes indicate genes upregulated in CARpos T cells. Positive fold changes indicate genes upregulated in CARneg/vectorpos T cells. ns, non-significant; FDR, false discovery rate.

Fig 5: ERK2 inhibition promotes T cell transduction with LVs and AAVs(A) Percentages of CAR expression 72 h after transduction with CD4-LV (left) (one donor, D#7) or (CD8-LV) (one donor, D#8) in the presence of different doses of ERK2 inhibitors. 6,000 particles per cell were used.(B) Fold change differences in CAR T cell percentages 72 h after transduction using different LVs (three donors, D#4, D#5, and D#6, mean of two technical replicates) with or without 5 μΜ ulixertinib or 20 μM VX-11e. 2,000 particles per cell for CD3-LV, CD4-LV, and CD8-LV and 500 particles per cell for VSV-LV were used.(C) Representative flow cytometry plots of T cells 72 h after transduction with different LVs, with or without 20 μM VX-11e. Depending on the vector type, CAR expression was measured on total CD3 T cells (CD3-LV and VSV-LV), on CD4 T cells (CD4-LV), or on CD8 T cells (CD8-LV). V, VX-11e; U, ulixertinib; R, ravoxertinib; S, selumetinib. See also Figure S14A.(D) Fold change differences in the percentage of GFP-positive cells 72 h after transduction with sc-AAV6 (top) (five donors, D#4, D#5, D#6, D#12, and D#13, mean of two technical replicates) or ss-AAV6 (bottom) (three donors, D#4, D#5, and D#6, mean of two technical replicates) in the absence (Ctrl) or presence (V) of Vx-11e 20 μM.(E) Representative flow cytometry plots of GFP expression in T cells with sc-AAV6 (top) or ss-AAV6 (bottom) in the absence (Ctrl) or presence (V) of 20 μM Vx-11e. Ctrl, control; V, Vx-11e.