Journal: Neoplasia (New York, N.Y.)

Article Title: Screening and identification of HLA-A2-restricted neoepitopes for immunotherapy in endocrine therapy-resistant breast cancer.

doi: 10.1016/j.neo.2025.101200

Figure Lengend Snippet: Fig. 2. Immunoreactivity of mutant peptides targeting T2A2 cells in vitro. CTLs were induced with autologous MUT peptide-pulsed DCs from peripheral blood lymphocytes of HLA-A2+ healthy donors. A-B. CTLs were collected and co- cultured with T2A2 cells loaded with MUT/WT peptide and then were detected for IFN-γ production and FasL expression. C. CTLs were co-cultured with T2A2 cells that were pulsed with MUT peptide or corresponding WT peptide at an effector/target (E/T) ratio of 12.5:1, 25:1, and 50:1, respectively. LDH cytotoxicity killing assay was used to detect the immunogenicity of mutated epitope peptides. The T2A2 cells loaded with WT peptide group was defined as the negative control group. Data were represented as means ± SD. Statistical significance was determined by unpaired Student’s t-test, *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Human breast cancer cell lines MDA-MB-231 (HLA-A*0201-positive) and MCF7 (HLA-A*0201-positive), along with the human lymphoblast cell line T2A2 (transporter associated with antigen processing deficient) were obtained from the American Type Culture Collection (ATCC, USA) and maintained under standard culture conditions in our laboratory.

Techniques: Mutagenesis, In Vitro, Cell Culture, Expressing, Immunopeptidomics, Negative Control

Journal: Neoplasia (New York, N.Y.)

Article Title: Screening and identification of HLA-A2-restricted neoepitopes for immunotherapy in endocrine therapy-resistant breast cancer.

doi: 10.1016/j.neo.2025.101200

Figure Lengend Snippet: Fig. 4. CTLs obtained by MUT peptide pool induction in HLA-A2.1/Kb transgenic mice are able to specifically distinguish antigenic epitopes loaded on T2A2 cells. Immunogenicity of the MUT peptide in HLA-A2.1/Kb transgenic mice. Splenocytes and lymph node cells from HLA-A2.1/Kb transgenic mice immunized with either a peptide pool (Peptide pool group) or normal saline (Vehicle group) in combination with CpG ODN 1826 (30 µg per mouse) were restimulated in vitro with MUT peptides for 5 days. Then CTLs were co-cultured with different target cells to detect the CTL response in (A-B) intracellular cytokine assay and (C) cytotoxicity assay. The T2A2 cells loaded with WT peptide pool and T2A2 cells loaded with MUT peptide pool were severed as stimulator cells and target cells. The T2A2 cells loaded with WT peptide pool group was defined as the negative control group. Data were represented as means ± SD. Statistical significance was determined by unpaired Student’s t-test, **P < 0.01, ***P < 0.001.

Article Snippet: Human breast cancer cell lines MDA-MB-231 (HLA-A*0201-positive) and MCF7 (HLA-A*0201-positive), along with the human lymphoblast cell line T2A2 (transporter associated with antigen processing deficient) were obtained from the American Type Culture Collection (ATCC, USA) and maintained under standard culture conditions in our laboratory.

Techniques: Transgenic Assay, Immunopeptidomics, Saline, In Vitro, Cell Culture, Cytokine Assay, Cytotoxicity Assay, Negative Control

Journal: Neoplasia (New York, N.Y.)

Article Title: Screening and identification of HLA-A2-restricted neoepitopes for immunotherapy in endocrine therapy-resistant breast cancer.

doi: 10.1016/j.neo.2025.101200

Figure Lengend Snippet: Fig. 5. CTLs obtained from MUT peptide pool induction in HLA-A2.1/Kb transgenic mice specifically recognize single mutant epitopes loaded on T2A2 cells. Splenocytes and lymph node cells from HLA-A2.1/Kb transgenic mice immunized with either a peptide pool (Peptide pool group) or normal saline (Vehicle group) in combination with CpG ODN 1826 (30 µg per mouse) were restimulated in vitro with MUT peptides for 5 days. Then CTLs were co-cultured with different target cells to detect the CTL response with cytotoxicity assay. The T2A2 cells loaded with single WT peptide and T2A2 cells loaded with single MUT peptide were severed as stimulator cells and target cells. The T2A2 cells loaded with single WT peptide group was defined as the negative control group. Data were represented as means ± SD. Statistical significance was determined by unpaired Student’s t-test, *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Human breast cancer cell lines MDA-MB-231 (HLA-A*0201-positive) and MCF7 (HLA-A*0201-positive), along with the human lymphoblast cell line T2A2 (transporter associated with antigen processing deficient) were obtained from the American Type Culture Collection (ATCC, USA) and maintained under standard culture conditions in our laboratory.

Techniques: Transgenic Assay, Mutagenesis, Saline, In Vitro, Cell Culture, Cytotoxicity Assay, Negative 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: 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, Binding Assay, Synthesized, Incubation, Staining, Flow Cytometry, Fluorescence, Enzyme-linked Immunosorbent Assay, Negative Control, 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, Labeling

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