Although Mls-1a has been described nearly 30 years ago as a single gene trait that induces strong T cell proliferation in H-2 matched combinations, the nature of this determinant has remained an enigma. Recently, attention has been newly focused on Mls, because of its profound effect on the expressed TCR Vbeta repertoire and its similarity to bacterial toxins, the superantigens. The goal of this revised grant renewal is the biochemical and molecular characterization of Mls-1a. This ambitious proposal is based on our preliminary results which, we believe, represent major breakthroughs in two areas of Mls research; namely, i) the identification of an experimental approach leading to the production of Mls-specific mAb, and ii) the demonstration that Mls-1a is tightly linked to the endogenous provirus Mtv-7. This latter observation is exciting, because Mtv-9 is already known to be tightly associated with a B cell specific co-tolerogen whose product is recognized by Vbeta5.2+ T cells in the context of MHC class II molecules. We propose the following projects: 1) We will stabilize our existing Mls-specific IgM mAb, and prepare new IgG2a mAb of the same specificity by in vitro priming and boosting of the B cells. Alternatively, we will use the IgM mAb to partially purify Mls for immunization of rabbits. We will analyze the tissue distribution of Mls and the control of its expression by cytokines. In addition, we will test the biochemical nature of Mls. 2) We will clone the endogenous provirus Mtv-7 to test for Mls-1a expression. If we obtain positive results, we will identify the proviral gene encoding Mls-1a and test whether the endogenous MMTV proviruses constitute a gene family encoding related immunological functions. 3) We will analyze the chromosomal segregation and map the gene encoding the """"""""Mls-1a-like"""""""" stimulatory activity found in MA/MyJ mice. 4) If Mtv-7 does not encode Mls-1a, we will search for Mls-1a in the flanking regions of this endogenous provirus.
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