expression vectors encoding hla a2 kb  (Addgene inc)

Journal: bioRxiv

Article Title: Mapping kinase-dependent tumor immune adaptation with multiplexed single-cell CRISPR screens

doi: 10.64898/2026.01.08.698516

Figure Lengend Snippet: (a) Experimental schematic of CRISPRi/a perturbations in NY-ESO-1/HLA-A*02:01-engineered GBM cells co-cultured with T cells. (b) UMAP embeddings of cancer cells under CRISPRi (left) or CRISPRa (right) showing transcriptomic trajectories across E:T ratios. (c) Module-level changes in MHC-I, MHC-II, IFN-γ, and NF-κB pathway programs under CRISPRi versus CRISPRa. Colors indicate effect size, quantified as the β coefficient for the genotype term (perturbation) in a regression model evaluated under the no–T cell condition. (d) Schematic for MrVI analysis, and (e) Forest plots summarizing perturbation effects on T cell-induced program across E:T ratios from MrVI analysis. Color and circle size denote p values, while arrow length and thickness represent distance differences. The statistical tests were performed with a one-sided Mann-Whitney U test (Bonferroni-corrected, α < 0.05).

Article Snippet: A plasmid encoding HLA-A*02:01 was obtained from (Addgene, Plasmid #108213).

Techniques: Cell Culture, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Comprehensive Analysis of CD4 + T Cell Response Cross-Reactive to SARS-CoV-2 Antigens at the Single Allele Level of HLA Class II

doi: 10.3389/fimmu.2021.774491

Figure Lengend Snippet: CD4 + T cell responses to SARS-CoV-2 antigens according to HLA class II loci and alleles. CD4 + T cells were stimulated with an allotype-matched aAPCs pulsed with a mixture of peptide pools of spike, nucleocapsid, and membrane protein. The responses by HLA-DR, -DQ, and -DP loci (A) , and HLA-DR alleles (B) , HLA-DQ alleles (C) , and HLA-DP alleles (D) in 42 donors were compared. Each dot presents the magnitude of response by a locus or an allele in an individual. Error bars indicate the mean ± standard error of the mean (SEM), and the number of donors with the allele are shown in parentheses. The stacked bar graph presents a proportion of the classified response in donors out of the donors with the allele. Statistical analysis was performed using a one-way ANOVA. *P < 0.05, ***P < 0.001. (E) Correlation of allele frequency with the proportion of positive response by HLA-DR, -DQ, and -DP alleles that are in at least three individuals. Statistical analysis was performed using Pearson’s correlation analysis. A line of best fit (solid lines) and the 95% confidence bands (dotted lines) were analyzed using linear regression analysis.

Article Snippet: As previously described , 5 × 10 6 293TN producer cells (System Biosciences) were transfected (Lipofectamine 2000; Invitrogen) with 1.3 pmol psPAX2 (RRID: Addgene_12260), 0.72 pmol pMD.2G (RRID: Addgene_12259), and 1.64 pmol single HLA class II allele-encoding pCDH, and the supernatant was harvested after 2 days.

Techniques:

Journal: Frontiers in Immunology

Article Title: Comprehensive Analysis of CD4 + T Cell Response Cross-Reactive to SARS-CoV-2 Antigens at the Single Allele Level of HLA Class II

doi: 10.3389/fimmu.2021.774491

Figure Lengend Snippet: CD4 + T cell responses restricted by single HLA class II allotype within individuals. CD4 + T cells were stimulated with an allotype-matched aAPCs pulsed with a mixture of peptide pools of spike, nucleocapsid, and membrane protein. (A) CD4 + T cell responses (vertical) by two allotypes of HLA-DR, -DQ, and -DP loci (depth) in each individual (horizontal). The magnitude of response above 50 SFCs/5 × 10 5 are colored and the alleles corresponding to the response are shown in the same color. (B) A schematic diagram of the analysis of dominant response in individuals. (C) The order of highest response by an allotype within an individual is presented as the dominant response in an individual. Each dot represents a response by an allotype. Data are shown as mean ± standard deviation (SD) of the order-specific response. Statistical analysis was performed using one-way ANOVA. ****P < 0.0001. (D) The probability of intra-individual dominance presents (the number of donors with the corresponding order from the responses by the allotype)/(the number of donors with the allotype). The donors who did not respond by any allotype were excepted. The order and color of the allele follow (B) and (A) , respectively.

Article Snippet: As previously described , 5 × 10 6 293TN producer cells (System Biosciences) were transfected (Lipofectamine 2000; Invitrogen) with 1.3 pmol psPAX2 (RRID: Addgene_12260), 0.72 pmol pMD.2G (RRID: Addgene_12259), and 1.64 pmol single HLA class II allele-encoding pCDH, and the supernatant was harvested after 2 days.

Techniques: Standard Deviation

Journal: Frontiers in Immunology

Article Title: Comprehensive Analysis of CD4 + T Cell Response Cross-Reactive to SARS-CoV-2 Antigens at the Single Allele Level of HLA Class II

doi: 10.3389/fimmu.2021.774491

Figure Lengend Snippet: CD4 + T cell responses by each HLA class II allotype to each SARS-CoV-2 antigen in each individual. (A, B) CD4 + T cell responses by an allotype were measured individually with spike (S), nucleocapsid (N), membrane (M), or CMV pp65, and with a mixture (MIX) of peptide pools of S, N, and M protein. High responses are highlighted at the blue end of the spectrum. (C) The order of immunogenicity indicates the order of highest response to S, N, or M among the responses by an allele that showed the strong response in an individual. Each dot represents a response by an allotype to a peptide pool of a protein. Data are shown as mean ± SD. Statistical analysis was performed using one-way ANOVA. *P < 0.05.

Article Snippet: As previously described , 5 × 10 6 293TN producer cells (System Biosciences) were transfected (Lipofectamine 2000; Invitrogen) with 1.3 pmol psPAX2 (RRID: Addgene_12260), 0.72 pmol pMD.2G (RRID: Addgene_12259), and 1.64 pmol single HLA class II allele-encoding pCDH, and the supernatant was harvested after 2 days.

Techniques:

Journal: Cancer immunology research

Article Title: Direct Detection and Quantification of Neo-antigens

doi: 10.1158/2326-6066.CIR-19-0107

Figure Lengend Snippet: Quantification of neo-antigens through MANA-SRM.

Article Snippet: Plasmids encoding K-RAS (wild-type, G12C, G12D, G13D, Q61H, Q61L, and Q61R), IDH2 (wild-type and R140Q mutant), and HLA class I alleles (A1, A2, A3, B7) followed by a T2A sequence and the GFP gene were synthesized and cloned into pcDNA3.1 by GeneArt (ThermoFisher Scientific).

Techniques: Transfection, Plasmid Preparation, Control, Binding Assay, Knock-In