mice were maintained in the joslin diabetes center barrier facility ( protocol 9919 , 9920 approved by the joslin diabetes center 's international animal care and use committee ) . lymphocytes from thymi , lns , and plns were first sorted in bulk as bdc2.5 clonotype - expressing , v2cd4 , and either cd25 or cd25 , before re - sorting as individual cells into wells of 96-well pcr plates containing the rt reaction mix containing primer 5-cctcttcttgcgaaactcaaattcatctacg-3 for foxp3specific priming ; lymphocytes from the pancreas were sorted directly into plates . the plates were incubated for 90 min at 37c , and then heat inactivated for 10 min at 70c . nested pcr amplification was performed and contamination was monitored in the replicates for foxp3 or v2 transcripts , as previously described ( 11 , 27 ) . v2 amplifications were prepared for automated sequencing using shrimp alkaline phosphatase ( ge healthcare ) and exonuclease i ( new england biolabs ) , as previously detailed ( 11 ) . products were subjected to automated sequencing ( dana - farber / harvard cancer center high - throughput sequencing core ) . raw sequencing files were filtered for sequence quality , and processed in an automated fashion . data for each cell were tabulated in a database ( access ; microsoft ) , together with the cell 's origin , surface phenotype , and foxp3 rt - pcr result . s1 depicts the gross characteristics of the t reg and t conv cell thymocyte populations . table s1 shows the complete listing of tcr cdr3 sequences , along with their occurrences in each population listed at the top of the chart . table s2 outlines the v2 nomenclature was applied throughout the text , including cross - references to genbank accession nos . mice were maintained in the joslin diabetes center barrier facility ( protocol 9919 , 9920 approved by the joslin diabetes center 's international animal care and use committee ) . lymphocytes from thymi , lns , and plns were first sorted in bulk as bdc2.5 clonotype - expressing , v2cd4 , and either cd25 or cd25 , before re - sorting as individual cells into wells of 96-well pcr plates containing the rt reaction mix containing primer 5-cctcttcttgcgaaactcaaattcatctacg-3 for foxp3specific priming ; lymphocytes from the pancreas were sorted directly into plates . the plates were incubated for 90 min at 37c , and then heat inactivated for 10 min at 70c . nested pcr amplification was performed and contamination was monitored in the replicates for foxp3 or v2 transcripts , as previously described ( 11 , 27 ) . v2 amplifications were prepared for automated sequencing using shrimp alkaline phosphatase ( ge healthcare ) and exonuclease i ( new england biolabs ) , as previously detailed ( 11 ) . products were subjected to automated sequencing ( dana - farber / harvard cancer center high - throughput sequencing core ) . raw sequencing files were filtered for sequence quality , and processed in an automated fashion . data for each cell were tabulated in a database ( access ; microsoft ) , together with the cell 's origin , surface phenotype , and foxp3 rt - pcr result . s1 depicts the gross characteristics of the t reg and t conv cell thymocyte populations . table s1 shows the complete listing of tcr cdr3 sequences , along with their occurrences in each population listed at the top of the chart . table s2 outlines the v2 nomenclature was applied throughout the text , including cross - references to genbank accession nos .
foxp3-expressing regulatory t ( t reg ) cells derive primarily from selection in the thymus . 
 yet conversion of mature conventional cd4 + t ( t conv ) cell lymphocytes can be achieved in several conditions , such as transforming growth factor  treatment , homeostatic expansion , or chronic exposure to low - dose antigen . 
 such conversion might provide a means to generate peripheral tolerance by  converting  potentially damaging t cells that react to self - antigens . 
 we tested this hypothesis in mice transgenic for the bdc2.5 t cell receptor ( tcr ) , which is representative of a diabetogenic specificity that is naturally present in nod mice and reactive against a pancreatic self - antigen . in the thymus , before any exposure to antigen , clonotype - positive t reg and t conv cells express a second tcr chain derived from endogenous loci . 
 high - throughput single - cell sequencing of secondary tcrs of the v2 family showed their joining cdr3 regions to be very different in t reg and t conv cell thymocytes . 
 these specific cdr3 motifs , thus , provided a 
 tag  with which to test the actual impact of t conv to t reg cell conversion in response to peripheral self - antigen ; should the autoreactive clonotypic tcr induce t conv to t reg cell conversion upon encounter of cognate antigen in the pancreas or draining lymph node , one would expect to detect tag cdr3 motifs from t conv cells in the t reg cell populations . 
 sequencing large numbers of peripheral bdc+v2 + cells showed that little to no conversion occurs in response to this pancreatic autoantigen .