Supplementary Figure 1: Deletion of VHL, splenic composition, resting phenotype and TCR responsiveness for the Vhl^fl/lf dLck model

(a) Deletion of VHL in gDNA from sorted CD44^lo and CD44^hi Vhl^fl/fl dLck (VHL-KO) CD8⁺ T cells relative to WT cells as determined by qPCR, n=2, error bars indicate range. (b) Absolute number of splenic B220⁺ cells and TCRβ⁺ cells for wild-type and VHL-KO mice; WT n=6, VHL-KO n=3. * P = 0.014 (Student's unpaired t-test). (c) Representative CD44 and CD62L phenotype of polyclonal CD8⁺CD4⁻ T cells and the absolute number and relative splenic percentage of indicated subsets; WT n=6, VHL-KO n=3, * P = 0.011, ** P = 0.0011, *** P = 0.033, **** P = 0.023 and ***** P = 0.006 (Student's unpaired t-test). (d) CD127 and KLRG1 phenotype of uninfected splenic polyclonal CD8⁺CD4⁻ T cells, number and %; n=3, derived from different mice than c; * P = 0.011 (Student's unpaired t-test). (e) CD44 and CD62L phenotype of splenic P14 (CD8α+CD4-Vα2⁺) T cells from VHL-sufficient and VHL-deficient P14 TCR transgenic mice. (f,g) Equivalent TCR sensitivity/early activation of VHL-sufficient and VHL-deficient P14 T cells to a range of gp33 peptide in vitro and to LCMV clone 13 in vivo. (f) P14 VHL-sufficient or VHL-deficient splenocytes were mixed at a 1:1 ratio and the indicated amount of gp33 peptide was added to duplicate wells. Upregulation of CD69 on VHL-sufficient and VHL-deficient cells was assessed by flow cytometry using congenic markers to distinguish each population after 18 hrs of stimulation; representative of two independent experiments. (g) Experimental set up as in f, but the mixture of P14 CD8⁺ T cells was transferred into B6 mice followed by infection with LCMV clone 13. Splenocytes were analyzed 36 hrs after infection. Grey-filled histogram indicates the fluorescence of naïve CD8⁺ T cells from uninfected mice; n=3, representative of two independent experiments. Error bars indicate s.e.m.