Positive selection of T-cell precursors is the process by which a diverse T-cell repertoire is established. Positive selection begins at the CD4 1 CD8 1 double positive (DP) stage of development and involves at least two steps. First, DP thymocytes down-regulate CD8 to become transitional single positive (TSP) CD4 1 thymocytes. Then, cells are selected to become either mature single positive CD4 1 or mature single positive CD8 1 thymocytes. We sought to define the function of Gads during the two steps of positive selection by analyzing a Gads-deficient mouse line. In Gads 1/1 mice, most TSP CD4 1 thymocytes are TCR hi Bcl-2 hi CD69 1 , suggesting that essential steps in positive selection occurred in the DP stage. Despite that Gads À/À mice could readily generate TSP CD4 1 thymocytes, many Gads À/À TSP CD4 1 cells were TCR lo Bcl-2 lo CD69 À , suggesting that Gads À/À cells proceeded to the TSP CD4 1 stage prior to being positively selected. These data suggest that positive selection is not a prerequisite for the differentiation of DP thymocytes into TSP CD4 1 thymocytes. We propose a model in which positive selection and differentiation into the TSP CD4 1 stage are separable events and Gads is only required for positive selection.
Summary
During their development, B lymphocytes undergo V(D)J recombination events and selection processes that, if successfully completed, produce mature B cells expressing a non‐self‐reactive B‐cell receptor (BCR). Primary V(D)J rearrangements yield self‐reactive B cells at high frequency, triggering attempts to remove, silence, or reprogramme them through deletion, anergy induction, or secondary V(D)J recombination (receptor editing), respectively. In principle, expressing a catalytically inactive V(D)J recombinase during a developmental stage in which V(D)J rearrangement is initiated may impair this process. To test this idea, we generated transgenic mice expressing a RAG1 active site mutant (dnRAG1 mice); RAG1 transcript was elevated in splenic, but not bone marrow, B cells in dnRAG1 mice relative to wild‐type mice. The dnRAG1 mice accumulate splenic B cells with a B1‐like phenotype that exhibit defects in B‐cell activation, and are clonally diverse, yet repertoire restricted with a bias toward Jκ1 gene segment usage. The dnRAG1 mice show evidence of impaired B‐cell development at the immature‐to‐mature transition, immunoglobulin deficiency, and poorer immune responses to thymus‐independent antigens. Interestingly, dnRAG1 mice expressing the anti‐dsDNA 3H9H56R heavy chain fail to accumulate splenic B1‐like cells, yet retain peritoneal B1 cells. Instead, these mice show an expanded marginal zone compartment, but no difference is detected in the frequency of heavy chain gene replacement. Taken together, these data suggest a model in which dnRAG1 expression impairs secondary V(D)J recombination. As a result, selection and/or differentiation processes are altered in a way that promotes expansion of B1‐like B cells in the spleen.
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