Journal: Science Advances

Article Title: Unconventional CD8 + T cell surveillance of cytomegalovirus via Qa-1/HLA-E–restricted epitope recognition

doi: 10.1126/sciadv.aea8707

Figure Lengend Snippet: ( A ) Schematic of the bioinformatics workflow used to generate the MCMV peptide library listed in table S1. ( B ) P815 cells were pulsed with peptides (non–Qa-1 binder M45 and known binders Qdm, FL9, and M-SL9), and Qa-1 staining was performed. Histograms are representative of eight independent experiments (gating strategy available in fig. S1). ( C ) Normalized Qa-1 antibody binding on the surface of P815 cells following incubation with each indicated peptide or dimethyl sulfoxide (DMSO) control. Peptides derived from the same MCMV protein are denoted by the first letter(s) of the peptide sequence in parentheses following the viral protein name. Data are pooled from eight independent experiments, n = 3 to 8 per group. Data are presented as the means ± standard error of the mean (SEM). Statistical significance was determined using a one-way analysis of variance (ANOVA), and significant differences are * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. ( D ) Peptide sequence schematic highlighting anchor positions between known Qa-1 binders and MCMV peptides. Solid lines indicate the same or extremely similar amino acid (AA) usage, while dotted lines indicate somewhat similar AA usage, based on biochemical properties. The strongest m59 peptide, m59(RL), is indicated as the two m59 peptides differed by one AA.

Article Snippet: P815 [American Type Culture Collection (ATCC) no. TIB-64, RRID:CVCL_2154] and K562 (ATCC no. CCL-243, RRID:CVCL_0004) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/high glucose with l -glutamine (4 mM), glucose (4500 mg/liter), and sodium pyruvate (Thermo Fisher Scientific no. SH30243.01).

Techniques: Staining, Binding Assay, Incubation, Control, Derivative Assay, Sequencing

Journal: Science Advances

Article Title: Unconventional CD8 + T cell surveillance of cytomegalovirus via Qa-1/HLA-E–restricted epitope recognition

doi: 10.1126/sciadv.aea8707

Figure Lengend Snippet: ( A ) Normalized Qa-1 antibody binding on the surface of P815 cells or ( B ) HLA-E expression on the surface of K562-HLA-E cells following incubation with each indicated peptide or DMSO control. Data are pooled from (A) 11 or (B) 5 independent experiments, n = 3 to 11 per condition. Data are presented as the means ± SEM. Statistical significance was determined using a one-way ANOVA, and significant differences are denoted by ** P < 0.01 or **** P < 0.0001. ( C ) AlphaFold3 was used to model Qa-1-peptide complexes with HLA-E binding peptides that bound to Qa-1 in (A). ( D ) AlphaFold3 was used to model HLA-E-peptide complexes with Qa-1 binding peptides that bound to HLA-E in (B). ( E ) AlphaFold3 was used to model m139 in the binding groove of HLA-E:01*03. pLDDT score calculations are presented for each peptide, and the peptide residues are colored blue when the score was >90 (very high confidence), cyan when the score was 70 to 90 (confident prediction), yellow when the score was 50 to 70 (low confidence, indicating possible disorder or flexibility), or orange when the score was <50 (very low confidence).

Article Snippet: P815 [American Type Culture Collection (ATCC) no. TIB-64, RRID:CVCL_2154] and K562 (ATCC no. CCL-243, RRID:CVCL_0004) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/high glucose with l -glutamine (4 mM), glucose (4500 mg/liter), and sodium pyruvate (Thermo Fisher Scientific no. SH30243.01).

Techniques: Binding Assay, Expressing, Incubation, Control

Journal: The Journal of Clinical Investigation

Article Title: Mitochondrial complex II orchestrates divergent effects in CD4 + and CD8 + T cells

doi: 10.1172/JCI194134

Figure Lengend Snippet: ( A ) B6 and BALB/c mice were transplanted with WT or SDHA-KO T cells. Survival rate and clinical GVHD score are shown (syngeneic n = 8/group, allogeneic n = 10/group). ( B – D ) BALB/c mice received HCT with P815 cells (MHC class I + class II – ). ( B ) BALB/c mice received syngeneic or allogeneic T cells from WT or SDHA-KO mice and were infused concurrently with 1.0 × 10 3 luciferase + P815 cells. ( C ) Tumor-related mortality (syngeneic n = 5, allogeneic n = 11/group) and clinical GVHD score ( n = 5/group). ( D ) Representative bioluminescence images and tumor burden on day 14 after HCT ( n = 3–10/group). ( E ) BALB/c mice received HCT (WT or SDHA-KO B6 → BALB/c). Representative flow cytometry images and Annexin V + 7-AAD + cells in donor-derived (H-2Kb + ) CD4 + and CD8 + T cells day 7 after HCT ( n = 6/group). ( F ) Representative flow cytometry measuring proliferation of donor-derived (H-2Kb+CD45.2 + ) CD4 + and CD8 + T cells day 7 after HCT ( n = 3–6/group). ( G ) Representative flow cytometry images and granzyme A and perforin levels in donor-derived (H-2Kb + CD45.2 + ) CD8 + T cells day 7 after HCT ( n = 3–10/group). ( H ) Representative flow cytometry images and granzyme A, granzyme B, and perforin levels in donor-derived (H-2Kb + CD45.2 + ) CD4 + T cells day 7 after HCT ( n = 3–10/group). ( I ) WT and SDHA-KO mice were inoculated with B16-F10 melanoma cells on day 0 and injected with anti–PD-1 or isotype control antibody on days 7, 10, 13, and 16. Tumor growth was measured ( n = 6–9/group). Two-tailed Mann-Whitney U test for GVHD score and Annexin V + 7-AAD + , Mantel-Cox log rank test for survival, 1-way ANOVA with Tukey’s post hoc test ( D and F – H ), and 2-way ANOVA with Tukey’s post hoc test ( I ) were used (mean ± SEM). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: P815 leukemia cells (catalog TIB-64) were purchased from ATCC and were transduced with luciferase ( ).

Techniques: Luciferase, Flow Cytometry, Derivative Assay, Injection, Control, Two Tailed Test, MANN-WHITNEY