In most cases, early islet infiltrates appear to have oligoclonality superimposed on a background of relatively diverse TCR usage. have used this method to analyze the TCR repertoire of CD4+ T cell infiltrates at the earliest stages of insulitis in NOD mice. Two motifs in the TCR -chain have been identified in these early islet infiltrates. reconstitution of representative TCRs with these -chain motifs demonstrates their islet-cell specificity. Furthermore, these TCRs do not appear to recognize GAD65 or insulin. Our results suggest that autoimmune diabetes in NOD may be initiated by a limited number of autoantigens distinct from GAD65 or insulin. Materials and Methods Single-Cell PCR Protocol. Pancreata were harvested from NOD mice and digested with collagenase P according to the manufacturer’s protocol (Roche Molecular Biochemicals) in which the digestion buffer was supplemented with 1 mM PMSF/100 M leupeptin/1 M pepstatin A. Ficoll gradient purification of islets and preparation of a single-cell suspension from islets were performed as described (10). Single CD4+ T cells were sorted from this mixture by standard fluorescence activated cell sorter (FACS) methods into 5 l of sorting buffer (6% Triton X-100/0.3 mg/ml BSA/1.5 mM spermidine/1.5 mM dNTPs (each)/2.08 Frentizole mM CH3HgOH/16.67 g/ml oligo dT). cDNA synthesis was preceded Frentizole by the addition of 4.35 l of DTT buffer [51.72 mM DTT/0.25 U RNAsin (Promega)]. Reverse transcription (RT)-PCR was performed according to the manufacturer’s protocol (SuperScript II, United States Biochemical) in a final volume of 15 l. Resulting cDNA (2.5 l) was used for each PCR reaction. PCR amplification of TCR – and -chains from the cDNA of individual T cells was performed as described in polymerase. PCR products were subcloned into pGEM-T (Promega) and sequenced. Sequences of the oligonucleotides and Frentizole the method for their design are given in Tables 1 and 2, which are published as supporting information on the PNAS web site, www.pnas.org. Nomenclature used in Tables 1 and 2 is taken from Arden (28). Construction of TCR Expression Vectors. Cassettes encoding TCR and variable chain sequences with the appropriate CDR3 were generated by PCR from genomic DNA templates. Restriction sites for subcloning, leader sequences, and unique V(D)J junctional sequences were contributed by primer oligonucleotides. TCR -chain cassettes were subcloned into a previously described Frentizole TCR -shuttle vector, which retains a portion of the genomic TCR -gene structure (29). TCR -chain cassettes were combined with the full TCR constant region sequences to generate a full-length cDNA, which then was subcloned into the mammalian expression vector pBJ1-neo (30). Maps and sequences of these constructs are available upon request. Reconstitution of TCRs in D10.G4.1. Linearized TCR – and -chain constructs were transfected into the mouse T cell clone D10.G4.1 as described (31). Briefly, D10 cells were harvested 14 to 28 days after stimulation with Mouse monoclonal to NANOG antigen-presenting cells and conalbumin (Sigma), washed and resuspended in PBS + 10 mM MgCl2 at 3.1 106 cells per ml. For each TCR, 750 l of this cell suspension was mixed with 4 g of linearized TCR -chain construct and 20 g of linearized TCR -chain construct, incubated on ice for 10 min, pulsed (300 V, 960 F) in a Bio-Rad GenePulser electroporation cuvette, placed back on ice for 10 min, diluted with 1 ml of serum-free RPMI medium 1640 and incubated at room temperature for 10 min; finally, the suspension was diluted into 10 ml RPMI medium 1640 supplemented with 10% (vol/vol) FCS/1 mM glutamine/-ME/sodium pyruvate/penicillin/streptomycin. Transfections were plated in 96-well tissue-culture plates at 100 l per well with 2 105 irradiated (3,500 rad) H-2k splenocytes and 200 g/ml conalbumin (Sigma). After 2 days at 37C, an additional 100 l of media was added to.