Novel use of T cell Actinobacillus actinomycetemcomitans Actinobacillus actinomycetemcomitans (Aa) a gram-negative bacterium, is strongly associated with Localized Juvenile periodontitis (LJP) which affects approximately 0.5% of the U.S. children. LJP is successfully managed by antibiotic therapy and surgery, but extensive damage to the periodontium has often occurred before detection or treatment. Thus an immune based therapy may provide a better method of controlling LJP. In animal studies, T cells have been shown to be important in protecting the host against Aa, but little is currently known about the specific Aa proteins that elicit a T cell response. To address this question, we have generated 24 T cell hybridomas using lymph nodes from mice orally inoculated with Aa. To determine the sizes of the Aa proteins recognized by these hybridomas, """"""""T cell Westerns"""""""" were employed. Interestingly, 6 of the 11 T cell hybridomas tested to date showed reactivity to an Aa protein of relatively high molecular weight. Since leukotoxin, the primary vi rulence factor produced by this bacterium, is one of only a few proteins in that size range, we suspected that it might be the relevant antigen. Thus, these hybridomas were tested for reactivity to protein extracts prepared from IP-2 and from AAM68 (an isogenic leukotoxin knockout strain that we generated); 5/6 T cell hybridomas were indeed specific for leukotoxin. Thus, we have defined the first Aa antigen that elicits a strong, potentially protective, T cell response. In addition, in order to eliminate sister clones, the sequences of the T cell receptor B chain junctional regions were sequenced. The response to Aa appears quite heterogeneous and many difference VB gene segments are represented; this suggests that Aa has no super-antigenic activity in mice. T cell hybridomas have never before been used in the study of Aa, they provide an important new tool useful in dissecting the immune response and disease etiology seen with this important periodontal pathogen. This work was supported by the National Institutes of Health, grants DE00152 and DE10731.
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