A method using PCR amplification and primer extension with fluorescent oligonucleotides was developed to analyze T-celi repertoires. The sizes of the hypervariable CDR3-like regions of the murine T-celi antigen receptor .3 chains were measured for all possible Vp-Jp combinations. This analysis shows that ,B chains are distributed into at least 2000 groups, a value that provides a lower limit to their complexity. The CDR3 sizes appear to be dependent on the Jp and especially the Vp segment used and correlates with amino acid sequence motifs in the corresponding CDR1 region. This feature of T-cell receptors is discussed.The specific receptors of T lymphocytes (T-cell antigen receptors; TCRs) are membrane-bound heterodimers, two types of which, af3 and 'yS, have been identified so far (1-4). The gene segments encoding the a, /3, y, and 6 chains have been cloned in several species (surveyed in ref. 5). These segments are rearranged by mechanisms similar to those that operate in the immunoglobulin genes. Thus, the genes encoding the TCR a and l3 chains are produced by the combination of the Va,,, J.a, and C,. or Vp, J, D3, and Cp segments, respectively. Junctional diversity is generated by a variety of mechanisms (6), in which terminal deoxynucleotidyltransferase is known to play a role a few days after birth (reviewed in ref. 7). The three-dimensional structure ofTCRs is still unknown, but sequence homologies, the conservation of key amino acids, and modelization studies have suggested that TCRs have an antibody-like structure (2,(8)(9)(10). Accordingly, complementarity determining region (CDR) 1-, 2-, and 3-like regions have been defined in TCRs (5). The CDR3-like regions (encoded by the V-J or V-D-J junctions) are the only ones to display extensive diversity.The diversity of TCRs, although still unknown, is thought to be as large as that of antibodies (2, MATERIALS AND METHODSMice. BALB/c and C57BL/6 mice were bred in the local facilities of the Pasteur Institute. C57BL/1O.A, BALB.B, and BALB.K strains were from Harlan Olac (Bicester, U.K.).Oligonucleotides and Fluorescent Dye Labeling. Oligonucleotides were synthesized by using an Applied Biosystems DNA synthesizer. Fluorescent dye labeling (with Fam, Joe, Tamra, or Rox) was performed as recommended by the supplier (Applied Biosystems). Dye-labeled oligonucleotides were purified by ethanol precipitation to eliminate unreacted dye, followed by reverse-phase chromatography to remove the unlabeled oligonucleotides. The primer sequences are given in Table 1. RNA and cDNA Preparation. RNA was prepared with the guanidinium isothiocyanate procedure followed by CsCl ultracentrifugation (16).Single-strand cDNA synthesis was performed by using the Boehringer Mannheim cDNA synthesis kit. Briefly, 10 ,Ag of total RNA was incubated for 10 min at 70°C with (dT)15 (5 ,uM) and each dNTP at 1 mM. After cooling, Boehringer Mannheim buffer I and avian myeloblastosis virus reverse transcriptase were added together with 40 units of RNasin (Promega). The solution was th...

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The diversity of the T cell repertoire of mature T splenocytes is generated, in the thymus, by pairing of α and β variable domains of the αβ TCR and by the rearrangements of various gene segments encoding these domains. In the periphery, it results from competition between various T cell subpopulations including recent thymic migrants and long-lived T cells. Quantitative data on the actual size of the T cell repertoire are lacking. Using PCR methods and extensive sequencing, we have measured for the first time the size of the TCR-αβ repertoire of naive mouse T splenocytes. There are 5–8 × 105 different nucleotide sequences of BV chains in the whole spleen of young adult mice. We have also determined the size of the BV repertoire in a subpopulation of AV2+ T splenocytes, which allows us to provide a minimum estimate of the αβ repertoire. We find that the mouse spleen harbors about 2 × 106 clones of about 10 cells each. This figure, although orders of magnitude smaller than the maximum theoretical diversity (estimated up to 1015), is still large enough to maintain a high functional diversity.