Figure 6.

LAG3 expression on CD8⁺ T cells is linked to antigen dose in vitro and in vivo. (A) B16 melanoma cells were transfected with either a suboptimal (2 µg) or optimal (5 µg) amount of a plasmid expressing chicken ovalbumin (psOVA). One day later, the cells were assayed for levels of OVA mRNA by qPCR (left). Culture supernatant from the transfected cells was used to stimulate OT1 splenocytes in vitro. Levels of LAG3 on the CD3⁺CD8⁺ splenocytes stimulated with the different supernatants was assayed 24 h later by flow cytometry (right). (B) OT1 splenocytes were stimulated with different amounts of SIINFEKL peptide and assayed for LAG3 expression 24 h later as above (representative data from three independent experiments). (C) 10⁶ OT1 splenocytes were adoptively transferred into wild type C57BL6 mice (n = 5 per group), and then immunized with either 10 µg or 50 µg of psOVA. Three days later, LAG3 levels on SIINFEKL-specific tetramer⁺CD3⁺CD8⁺ CD44⁺ cells were assayed by flow cytometry. Given the absence of CD44⁺ tetramer⁺ cells in animals given vector control, LAG3 expression on cells from control animals is shown for all SIINFEKL tetramer positive CD8⁺ T cells. For all panels, * denotes a p-value < 0.05, two-sided t-test.