Fig. 4. Negative selection sets apart TCR repertoires of CD4+Foxp3+ and Foxp3− thymocytes.
a. Phenotype and recovery of thymocytes from the indicated bone marrow chimeras 6 weeks after bone marrow transplantation. Numbers indicate proportions of gated cells, and bar graphs show the mean values +/− SD from two experiments (4 mice per group). Statistical significance of data was calculated using two sample t – test (OriginLab). b. Repertoire of TCRαCDR3 regions from CD4+Foxp3− and CD4+Foxp3+ thymocytes from indicated chimeras. Color coding of the particular TCRs is the same as in Fig. 3a and b, and Supplementary Fig. 4 shows CDR3 sequences of dominant TCRs. c. An overlay of all CDR3 sequences obtained from thymocytes from indicated chimeras. CDR3 regions represented at least at 0.01% are shown. d. The hierarchical diagrams depict similarity indices (MII) for TCR repertoires from CD4+Foxp3− and CD4+Foxp3+ thymocytes (based on HT sequencing, see Supplementary Table 1 for the number of TCR CDR3 sequences analyzed). Note: The clustering of CD4+Foxp3+ and CD4+Foxp3− repertoires in AbEp63KTCRmini→AbEpTCRα− chimeras was not due to their high similarity but to their low TCR diversity (see 4e), and the similarity between repertoires of CD4+Foxp3− subsets from AbEp63KTCRmini and AbEpKTCRmini→AbEp63KTCRα− chimeras is likely due to the negative selection by AbEp63K complex expressed on thymic medullary epithelial cells. e. Diversity (ENS) and coverage (CVG) of TCRα CDR3 regions on CD4+Foxp3+ thymocytes selected in the indicated chimeras. CD4+ subsets were separately isolated from three chimeras of each kind and their TCRs were sequenced.