NSF Award
0211785
The TCR beta chain constant (TCRBC) regions of both channel catfish and bicolor damselfish are encoded by two substantially different genes in contrast to the situation in mammals where these two genes are extremely similar. The objective of this project is to test the hypothesis that these major differences in fish TCRBC are important for immune functions. Determination of V region usage and expression of the two TCRBC in catfish will indicate whether the two genes use the same V region repertoire and whether cells expressing these genes are compartmentalized within host tissues. Development of monoclonal antibodies directed toward the two TCRBC gene products as well as antibodies to catfish CD3 epsilon and zeta family members (zeta and Fc-gamma) will be used in immunoprecipitation and Western blot analyses to differentiate members of the TCR receptor complex. Cloning of catfish lymphoid cells lines expressing different TCRBC region genes will allow in vitro comparisons of antigen-binding and receptor signaling processes using uniform cell populations. To identify the membrane associated accessory molecules used by cells bearing the different TCR receptors and to determine the initial signaling events following ligation of the TCR, two types of experiments will be done. First, the composition of immune synapses will be defined following receptor ligation using antibody reactive with TCR complex members or cholera toxin. Secondly, the generation of intracellular signals including tyrosine phosphorylation and calcium mobilization will be measured. If differences in accessory molecules or signaling capacities are observed, hybrid genes formed from C region exons of both TCRB will be used to distinguish which portion of the C region influences the observed differences. Thus, these experiments will determine whether divergent TCRBC segments have the ability to recruit or activate different signaling peptides. Regardless of the outcome, these experiments will define the TCR complex used in catfish. The phylogenetic significance of two very divergent TCRBC genes is unknown, but is potentially important for understanding the evolution of immune reactivity in all vertebrates. This project will determine whether the C regions of the two TCRB proteins have different regulatory functions or merely serve as scaffolds for assembly of TCR alpha/CD3/zeta and for holding the variable region distal to the membrane. <br/><br/>Two substantially different T cell receptors are used by lymphocytes from certain fish species whereas those used by mammalian cells are essentially identical. The goal of this research is to understand the impact of these fish T cell receptor differences on immune and host defense functions. This project will determine whether or not the different T cell receptors are capable of using the same antigen-binding segments, and thus directing immune function toward similar pathogens. The research will also determine whether the two different T cell receptors use the same mechanisms to communicate with the inside of the cell, thus influencing the type or magnitude of ensuing response. At present, the significance of these fish T cell receptor differences is not known, but may be important for understanding changes in host defense capabilities during evolution.
