Figure 2. Several Tumor dLN Myeloid Populations Stimulate Naive OTI Proliferation In Vitro.
(A) Representative flow cytometric plot showing expression of the Venus transgene within the myeloid populations in the tumor dLN.
(B) Time projection of representative multiphoton microscopy image of tumor dLN explants of B78ChOVA-bearing Xcr1-Venus mice 2 days after transfer of 2 × 106 GFP+ OTI T cells. XCR1+ cell time projections are represented in yellow, OTI T cells in blue, and mCherry in red. Two regions of interest (ROI) are shown.
(C) Time projection of the XCR1+ DC and interacting OTI T cells in ROI 1 and ROI 2.
(D) Representative OTI T cell dwell time on XCR1+ population determined by mCherry fluorescence within the dLN. Data are plotted as median ± interquartile range and statistics were performed using the Mann-Whitney U test. ****p < 0.0001.
(E) Schematic of T cell activation assay with sorted populations from the dLN of B78ChOVA tumor-bearing mice.
(F) Representative flow cytometric histograms of the induction of Nur77EGFP transgene and the early activation marker CD69 by OTI T cells cultured for 12 hr on APCs sorted directly from the tumor dLN of mice. CD103+ DC is green, migratory CD11b+ DC orange, resident CD11b+ DC purple, CD8α+ DC blue, and macrophage red. Data are representative of three independent experiments.
(G) Representative flow cytometric plots of naive OTI T cell proliferation, as measured by dye dilution of eFluor 670 plotted against Nur77EGFP, at 72 hr following co-culture with tumor dLN APC populations. Gray, BM-derived DC + OTI cognate peptide (SL8). Data are representative of four independent experiments.
(H) Proportion of OTI T cells at 72 hr that have divided following co-culture with tumor dLN APC populations. Plots show mean ± SEM. Data are representative of four independent experiments.
(I) Proportion of pmel T cells at 72 hr that have divided following co-culture with tumor dLN APC populations ± addition of gp100 peptide. Plots show mean ± SEM. Data are representative of two independent experiments.
See also Movie S1.