Supplementary Figure 5: Detailed analysis of selection and activation responses in thymocytes with transgenic expression of a TCR.

(a) Identification of post-selection (TCR^hiCD69⁺ thymocytes) from total viable LLO56 and LLO118 thymocytes, representative of at least three experiments. (b) Frequencies of NK (CD3^-NK1.1⁺), NKT (CD3⁺NK1.1⁺), and γδ T cells (GL3⁺) among DN thymocytes. Data are representative of analyses of three mice from two experiments. (c) Gradual emergence of LLO56 and LLO118 responses during the DP to CD4SP thymocyte transition (top plots, labeled 1 through 5; DN thymocytes gated out for clarity). PMA + ionomycin-induced IL-2 responses (bottom left, numbers indicate % IL-2⁺ cells) and ERK phosphorylation after 3 minute PMA stimulation (bottom right, red and blue numbers indicate LLO56 and LLO118 pERK MFI, respectively) are presented. Data are representative of at least three experiments. (d) PMA + ionomycin-induced intracellular IL-2 responses of CD4⁺ T cells transgenically expressing the AND TCR on H2^b or H2^k MHC haplotype backgrounds representative of three experiments. (e) Analysis of cell survival-associated markers among LLO56 and LLO118 thymocyte subsets and mature CD4⁺ T cells, representative of two or three experiments. Bar graphs depict means ± SEM, with statistical analyses done using unpaired two-tailed Student's t tests. *P < 0.0001.

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