Journal: bioRxiv

Article Title: SARS-CoV-2 variant B.1.1.7 caused HLA-A2 + CD8 + T cell epitope mutations for impaired cellular immune response

doi: 10.1101/2021.03.28.437363

Figure Lengend Snippet: A: The schematic of mutation sites of the SARS-CoV-2 variant B.1.1.7. B: List of the predicted epitopes for following experiments. The mutated amino acids were highlighted as red in varian B.1.1.7t. a The antigenic vaule threshold was > 0.4000 C - D: Comparison of ancestral and mutant epitope binding affinity to HLA-A2 in T2A2 cells. ancestral and mutated epitopes were synthesized and incubated with T2A2 cells. The binding of the peptide on T2A2 was measured with anti-HLA-A2 staining with flow cytometry. Binding capacity was presented as mean fluorescence intensity (MFI) of HLA-A2 staining. C was the representative plot of D. Each symbol represents an independent experiment. Ancestral: Wuhan strain epitope; Mutant: varian B.1.1.7t epitope. E: Evaluation of epitope binding to HLA-A2 with ELISA assay. Peptide exchanged assay was performed with coated UV-sensitive peptide/MHC complex and given peptides. The binding capability was measured with pMHC ELISA assay. Data shown are mean plus standard error of the mean (SEM). Threshold for pMHC formation positivity was set as above the average OD value of the negative-control cohort. Blank: no peptides; Neg ctrl: negative control, Zika virus peptide GLQRLGYVL; Pos ctrl: positive control, influenza A M1 peptide GILGFVFTL.

Article Snippet: 0.5×10 6 CD8 + T cells isolated from health donors were co-cultured with 0.5 × 10 6 peptide-loaded T2A2 cells stained with 5 μmol/L CFSE (TargetMol), and stimulated with 1 μg/mL anti-human CD28 antibodies (BioLegend) and 50 IU/mL IL-2 (SL PHARM, Recombinant Human Interleukin-2( 125 Ala) Injection).

Techniques: Mutagenesis, Variant Assay, Comparison, Binding Assay, Synthesized, Incubation, Staining, Flow Cytometry, Fluorescence, Enzyme-linked Immunosorbent Assay, Negative Control, Virus, Positive Control

Journal: bioRxiv

Article Title: SARS-CoV-2 variant B.1.1.7 caused HLA-A2 + CD8 + T cell epitope mutations for impaired cellular immune response

doi: 10.1101/2021.03.28.437363

Figure Lengend Snippet: Mitomycin pretreated T2A2 cells were loaded with mixed peptides from ancestral or mutant, and incubated with CD8 + T cell from health donors at 1:1 ratio, respectively. Epitope specific CD8 + T cells were generated after 7 day stimulation. A - B: The CD69 expression level of activated CD8 + T cell was evaluated with flow cytometry after 16 hour stimulation. A was the representative plot of B. n=4 per group. Day 0 ctrl: staining before stimulation; T2A2 ctrl: T2A2 without peptide loading; Pos ctrl: T2A2 loaded with influenza A M1 peptide GILGFVFTL. C - E: Epitope specific CD8 + T cell measurement before (C) and after (D) 7 day stimulation. The cells were stained with corresponding ancestral or mutated tetramer, and compared before and after stimulation (E). Four and five repeats were performed for decreased and nonchanged comparison, respectively. Please also see Supplementary Figure 1 A-C. F-I: Cross-detection of epitope specific CD8 + T cells with tetramers based on ancestral and corresponding mutant peptides. F-G: ancestral or mutant epitopes stimulated CD8 + T cells were stained with ancestral peptide-based tetramer. H-I: mutant or ancestral epitopes stimulated CD8 + T cells were stained with mutant peptide-based tetramer. n=3 per group. Symbols in G and I represented individual person. The p values were calculated by paired-samples T test. ** p < 0.01. Neg ctrl: T2A2 without peptide loading; Pos ctrl: T2A2 loaded with influenza A M1 peptide GILGFVFTL. J - M: Epitope specific CD8 + T cell mediated cytotoxicity was evaluated after 7 day culture (J&K). The remained CFSE labeled T2A2 cells were calculated as survived target cells. J was the representative plot of K. Apoptosis of T2A2 cells at day 7 after culture. The proportion of CFSE + AnnexinV + cells was calculated as indicator for epitope stimulated T cell mediated T2A2 apoptosis (L&M). L was the representative plot of M. n=4 per group. N - O: The expression of IFN-γ after epitope stimulation for 7 days. IFN-γ was measured with intracellular stained flow cytometry. N was the representative plot of O. n=4 per group. P - Q: The expression of Granzyme B after epitope stimulation for 7 days. Granzyme B was measured with intracellular stained flow cytometry. P was the representative plot of Q. n=4 per group. Day 0 ctrl: staining before stimulation; T2A2 ctrl: T2A2 without peptide loading; Pos ctrl: T2A2 loaded with influenza A M1 peptide GILGFVFTL. The p values were calculated by paired-samples T test, *** p < 0.001.

Article Snippet: 0.5×10 6 CD8 + T cells isolated from health donors were co-cultured with 0.5 × 10 6 peptide-loaded T2A2 cells stained with 5 μmol/L CFSE (TargetMol), and stimulated with 1 μg/mL anti-human CD28 antibodies (BioLegend) and 50 IU/mL IL-2 (SL PHARM, Recombinant Human Interleukin-2( 125 Ala) Injection).

Techniques: Mutagenesis, Incubation, Generated, Expressing, Flow Cytometry, Staining, Comparison, Labeling