Figure 3.

Stimulated MYC-deficient T cells are blocked in an activated state

(A and B) Ex vivo stimulation assays. T lymphocytes from control and Myc^del spleens were labeled with CTV, stimulated with anti-CD3/CD28 beads for 24 and 72 hr and analyzed by flow cytometry. CTV staining at 72 H (A) and CD25/CD69 expression at 24H and 72H (B) of eYFP⁺ cells from Control and Myc^del spleens.

(C–G) In vivo stimulation assays. (C) Overview of the procedure used for monitoring OT-II T lymphocytes proliferation in vivo. T lymphocytes from Control OT-II or Myc^del OT-II spleens were labeled with CTV and injected in recipient C57BL/6 mice, which were then immunized with Ovalbumin and finally euthanized 5 days later for lymph nodes analysis (D–G). (D) CD4⁺ cells from mouse injected with control OT-II or Myc^delOT-II cells were analyzed for OT-II expression, then eYFP expression was assessed in OT-II⁺ cells (right histograms). (E-G) All Myc-deficient CD4⁺OT-II⁺YFP⁺ cells (113 cells) were concatenated with 120 control CD4⁺OT-II⁺YFP⁺ T cells and then analyzed using the UMAP tool from FlowJo software. The resulting UMAP graph is colored according to the origin of the cells (E) and to CTV labeling (F). (G) Expression of CD25 and CD69 in clusters 1 and 2 that were defined in the UMAP (panel E).