Establishing an in vitro system to validate antigen specificity of TCRs from Calr KO tumors. (A) Venn diagrams showing the number of CDR3 TCR clonotypes in Calr KO and control tumor, analyzed by single-cell TCR-seq. Results are pooled from five mice for each group. (B) Top 20 most abundant TCR CDR3 clonotypes from single-cell TCR-seq of sgNTC and sgCalr tumors. Clonal size is represented as the percentage of each clonotype relative to the total number of TCRs. (C) Proportion of CD8 T cell subsets from single-cell RNA-seq in sgNTC and sgCalr tumors. (D) Venn diagram depicting the number of TCR patterns in sgNTC and sgCalr tumors. Single-cell TCR-seq data were analyzed and clustered using the GLIPH2 algorithm, with TCR patterns defined as the cluster types identified in the GLIPH2 output. (E) Enrichment of TCR patterns identified by GLIPH2 in sgNTC and sgCalr tumors. Fold change represents the frequency of each TCR pattern in sgCalr tumors relative to sgNTC tumors. P values were calculated using the Poisson test. (F) Representative flow cytometry plots showing the expression of mouse CD8 and TCRβ in Jurkat cells transduced with OT-I TCR. (G) Coculture of OT1-Jurkat cells with LLC cells that were either untreated or pulsed with SIINFEKL peptide. The percentage of human CD69+ cells within the TCRβ+ population was measured by flow cytometry. Results are presented as the mean ± SD and representative of two independent experiments. ****P < 0.0001 by an unpaired t test. (H) Representative flow cytometry plots showing the expression of mouse CD8 and TCRβ in engineered Jurkat cell lines expressing TCR-1, TCR-2, TCR-3, and TCR-5. (I) Level of SIINFEKL-bound H2Kb of control or Pdia3 KO LLC tumors after SIINFEKL pulsing, measured by flow cytometry using the antibody 25-D1.16. Tumor cells were pretreated with 20 ng/ml IFN-γ for 16 h, and then pulsed with 100 ng/ml SIINFEKL peptide for 4 h. Results are presented as the mean ± SD and representative of two independent experiments. ***P < 0.001; ****P < 0.0001 by an unpaired t test. (J and K) Number of CD45+ and CD8+ T cells infiltrating LLC tumors after vehicle or LOC14 treatment, measured by flow cytometry. Data are presented as the mean ± SEM and evaluated by an unpaired t test. ns, not significant. (L and M) Frequency of granzyme B+ CD8 T cells and granzyme B+ NK cells in total CD8 or NK cells from vehicle or LOC14-treated LLC tumors, respectively. Data are presented as the mean ± SEM and evaluated by an unpaired t test. *P < 0.05.
Figure S3.

Establishing an in vitro system to validate antigen specificity of TCRs from Calr KO tumors. (A) Venn diagrams showing the number of CDR3 TCR clonotypes in Calr KO and control tumor, analyzed by single-cell TCR-seq. Results are pooled from five mice for each group. (B) Top 20 most abundant TCR CDR3 clonotypes from single-cell TCR-seq of sgNTC and sgCalr tumors. Clonal size is represented as the percentage of each clonotype relative to the total number of TCRs. (C) Proportion of CD8 T cell subsets from single-cell RNA-seq in sgNTC and sgCalr tumors. (D) Venn diagram depicting the number of TCR patterns in sgNTC and sgCalr tumors. Single-cell TCR-seq data were analyzed and clustered using the GLIPH2 algorithm, with TCR patterns defined as the cluster types identified in the GLIPH2 output. (E) Enrichment of TCR patterns identified by GLIPH2 in sgNTC and sgCalr tumors. Fold change represents the frequency of each TCR pattern in sgCalr tumors relative to sgNTC tumors. P values were calculated using the Poisson test. (F) Representative flow cytometry plots showing the expression of mouse CD8 and TCRβ in Jurkat cells transduced with OT-I TCR. (G) Coculture of OT1-Jurkat cells with LLC cells that were either untreated or pulsed with SIINFEKL peptide. The percentage of human CD69+ cells within the TCRβ+ population was measured by flow cytometry. Results are presented as the mean ± SD and representative of two independent experiments. ****P < 0.0001 by an unpaired t test. (H) Representative flow cytometry plots showing the expression of mouse CD8 and TCRβ in engineered Jurkat cell lines expressing TCR-1, TCR-2, TCR-3, and TCR-5. (I) Level of SIINFEKL-bound H2Kb of control or Pdia3 KO LLC tumors after SIINFEKL pulsing, measured by flow cytometry using the antibody 25-D1.16. Tumor cells were pretreated with 20 ng/ml IFN-γ for 16 h, and then pulsed with 100 ng/ml SIINFEKL peptide for 4 h. Results are presented as the mean ± SD and representative of two independent experiments. ***P < 0.001; ****P < 0.0001 by an unpaired t test. (J and K) Number of CD45+ and CD8+ T cells infiltrating LLC tumors after vehicle or LOC14 treatment, measured by flow cytometry. Data are presented as the mean ± SEM and evaluated by an unpaired t test. ns, not significant. (L and M) Frequency of granzyme B+ CD8 T cells and granzyme B+ NK cells in total CD8 or NK cells from vehicle or LOC14-treated LLC tumors, respectively. Data are presented as the mean ± SEM and evaluated by an unpaired t test. *P < 0.05.

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