Despite accumulating evidence that regulatory T cells play a crucial role in preventing autoimmunity, the processes underlying their generation during immune repertoire formation are unknown. We show here that interactions with a single self-peptide can induce thymocytes that bear an autoreactive T cell receptor (TCR) to undergo selection to become CD4+CD25+ regulatory T cells. Selection of CD4+CD25+ thymocytes appears to require a TCR with high affinity for a self peptide because thymocytes that bear TCRs with low affinity do not undergo selection into this pathway. Our findings indicate that specificity for self-peptides directs the selection of CD4+CD25+ regulatory thymocytes by a process that is distinct from positive selection and deletion.

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CD4+25+ T cells are a unique population of immunoregulatory T cells which are critical for the prevention of autoimmunity. To address the thymic selection of these cells we have used two models of attenuated thymic deletion. In K14-Aβ b mice, major histocompatibility complex (MHC) class II I-Ab expression is limited to thymic cortical epithelium and deletion by hematopoietic antigen-presenting cells does not occur. In H2-DMα–deficient mice, MHC class II molecules contain a limited array of self-peptides resulting in inefficient clonal deletion. We find that CD4+25+ T cells are present in the thymus and periphery of K14-Aβ b and H2-DMα–deficient mice and, like their wild-type counterparts, suppress the proliferation of cocultured CD4+25− effector T cells. In contrast, CD4+25+ T cells from MHC class II–deficient mice do not suppress responder CD4+ T cells in vitro or in vivo. Thus, development of regulatory CD4+25+ T cells is dependent on MHC class II-positive thymic cortical epithelium. Furthermore, analysis of the specificities of CD4+25+ T cells in K14-Aβ b and H2-DMα–deficient mice suggests that a subset of CD4+25+ T cells is subject to negative selection on hematopoietic antigen-presenting cells.

SummaryWe have examined at the molecular level the CDR3 and adjacent regions in peripheral blood B lymphocytes of normal individuals. A total of 111 sequences (12-28 sequences from six individuals) were obtained after cloning of the polymerase chain reaction-amplified segments into plasmids or phage . The average length of the VDJ joining was 109 nucleotides, with a range from 79 to 151 . Approximately 75% of the sequences were in frame when translated into amino acids . Among the JH segments, J H4 was found most frequently (in 52 .5% of the sequences), and JH 1 and JH 2 segments the least frequently (-I% of the clones) . A polymorphic JH6 gene with a one-codnn deletion accompanied by a base change was present in two of six patients. Preferential breakpoints were found for JH 2, J.3, J 4, and Jx5, although the breakpoints of JH6 were distributed more heterogenously.In -90% of the cases, significant homology of the D regions with published D sequences was found . Preferential usage of a particular coding frame was observed in in-frame sequences utilizing DA, D21/9, and DMl segments. However, in general, all coding frames of germline D genes were used to generate CDR3s . Eight sequences that have a DN1-like D sequence with two base changes at the same positions were identified, suggesting the likely existence of a new germ line D gene belonging to the DN families. Using probes specific for a particular CDR3, the frequency of a specific B cell clone in the peripheral blood of normal individuals was estimated to be at most as high as 1/20,000 .T he most variable region of the immunoglobulin heavy chain is the third complementarity determining region (CDR3) (1, 2) . This region spans the junction between the variable (V ) diversity (D), and junctional 0,,) segments in the rearranged IgH genes (1, 2) . The hypervariability of this region is due to the combinatorial assortment of the many V , D, and JH segments that are utilized to generate a particular CDR3, to the imprecise joining mechanisms that include deletion of bases from the potential coding regions of each segment to be joined (3), and the addition of new bases that can be enzymatically added at the point of joining (N regions) (4) . Finally, somatic mutations of the rearranged region can contribute to the production of higher affinity antibodies (5) .The nucleotide sequences of all the human D genes, estimated to be -30 in number (6), have not been fully defined, and questions remain about the relative usage of different D genes used in VDJjoinings during development and in adult individuals . Furthermore, the characteristics of the extent of base excision and addition, including the identification of preferred sequence boundaries for the V., D, and J regions, have not been well delineated. The relative frequency of inframe translation products reflecting productive rearrangements, and the possibility of specific translation frames being preferred for particular D gene families have not been determined on a large sample size.To address these questions, we ha...