Supplementary Figure 3: Additional characterization of the expansion potential of naive CD5^hi and CD5^lo CD8⁺ T cell populations.

(A and B) Detection of CD5^lo/hi B8R/K^b-specific (A) and bulk LM-specific cells (B) by IFN-γ. Bulk splenocytes were stimulated with 1 μM B8R_20-27 peptide (A) or PMA/ionomycin (B) for 5 hrs in the presence of BFA and then stained for intracellular IFN-γ. (C-E) The preferential expansion of naive CD5^hi CD8 T cells is not unique to B8R/K^b specific cells or to LM infection. (C) shows the ratio of donor CD5^hi: CD5^lo OVA/K^b-specific cells on day 7 after primary LM-B8R infection or 5 days after re-challenge of memory recipients with virulent LM-B8R. Red symbols indicate mice where OVA/K^b-specific responses were not detected within the CD5^lo donor population and the ratio was arbitrarily set to 100. (D and E) Ratio of donor CD5^hi:CD5^lo gp33/D^b (D) or bulk pathogen-specific cells (E) on days 7 and 30+ after primary acute infection with LCMV Armstrong. (F) In vitro stimulation of CFSE-labeled polyclonal CD5^lo/hi cells with plate-bound αCD3/αCD28 (2.5ug/mL) for 6 days. (G) CD5^hi donor cells still have an advantage over CD5^lo cells in RAG^-/- recipients. Donor cells were transferred into RAG^-/- recipients, infected the next day with LM-B8R, and the splenic responses were analyzed 7 days later. (H) CD5^lo/hi cells were sorted on equally low CD44 expression and adoptively transferred into congenic wild type recipients. Mice were infected with LM-B8R and responses in peripheral blood were analyzed 7 days later. Red symbol indicates mice were the B8R/K^b-specific response was not detected within the CD5^lo donor population and the ratio was arbitrarily set to 100. (I) CD5 expression levels on WT and CD25^-/- naïve CD8+ T cells derived from bone marrow chimeras (described in Fig. 3g). Data in (a,b) are compiled from 1 to 4 experiments (n=4-11), (c) compiled from 2-4 experiments (n=6-11), (d) compiled from 2 experiments (n=4-7) (f) is representative of 2 experiments (g,h) from 1 experiment (n=4) (i) is representative of 3 chimera sets (n=6).