Fig. 4: ENI reduces epitope spreading and T cell activation in the distant DLN and tumor. | Nature Communications

Fig. 4: ENI reduces epitope spreading and T cell activation in the distant DLN and tumor.

From: Elective nodal irradiation mitigates local and systemic immunity generated by combination radiation and immunotherapy in head and neck tumors

Fig. 4

A Schematic of experimental design. An adoptive transfer of DO11 CD4 T cells (1 × 105 cells) was given to the mice two days after the first dose of SBRT. Tissue was harvested three days after the last dose of SBRT. Created with BioRender.com. B Tumor growth curves of the mice depicted in (A) (tumor only, n = 9; ENI, n = 10). C Quantitation of the percentage of CD4+ T cells that were positive for the DO11 TCR (KJ+) in the blood (ENI, n = 6; tumor only, n = 5). D Quantification of Tbet+ antigen-specific CD4 T cells (DO11 TCR+ or KJ+) in the inguinal DLN (ENI, n = 6; tumor only, n = 6). E Quantitation of IFNg+IL-2+CD4 T cells in the inguinal lymph node (ENI, n = 6; tumor only, n = 6). Gating strategy for IFNg+IL-2+ cells are provided on the right. Representative flow plots of Tbet expression in IFNg+IL-2+ and IFNg-IL-2- cells are also shown. F Quantitation of the mean florescent intensity (MFI) of Granzyme B in CD8 T cells in the inguinal lymph node. Histogram of MFI is provided to the left (ENI, n = 6; tumor only, n = 6). G Quantification of cleaved caspase 3 expression on KJ+CD4 T cells and on DCs (CD11c+MHCII+) in the DLNs (ENI, n = 7; tumor only, n = 7). H Quantification of the number of CD3+ T cells in the flank tumor. Flow plot of CD3+ cells are provided to the right (ENI, n = 5; tumor only, n = 5). I Quantification of KJ+CD4 T cells in the flank tumor (ENI, n = 6; tumor only, n = 6). J t-SNE with FlowSOM population overlay of CD45+ cells (ENI, n = 6; tumor only, n = 6). CD8 T cells (brown) were defined as having CD3 and CD8 expression. Activation CD8 T cells (pink) were defined as having expression of CD3, CD8, IFNg and Granzyme B expression. CD4 T cells (blue) were defined as having CD3 and CD4 expression. For tumor growth at selected time points, treatment difference was determined by a Two-Way ANOVA test. For the flow cytometry analysis, a two-tailed student’s t test was used. Significance was determined if the p-value was <0.05*, <0.01**, and <0.001***. The error bars represent the standard error of the mean (± SEM). Source data are provided as a Source Data file. p-values are indicated for figures B. *0.015, C. *0.0260, D. *0.0313, E. *0.015, F. *0.011, G. **0.0077, ****<0.0001, H. *0.0391, and I. **0.0068.

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