Humoral immune responses are initiated by the activation of B cells in the T cell-rich areas of secondary lymphoid organs that lead to the recruitment of germinal center (GC) precursors into secondary follicles, where they differentiate into memory cells. B cell activation requires cognate receptor/ligand interactions, among which, the ligation of B cetl- CD40 by the T celI-CD40 ligand (CD154) provides the essential stimuli for cell proliferation, survival, and induction of B cell memory. The present study focuses on the AT-hook protein AKNA that is expressed by germinal center B- lymphocytes, binds to A/T-rich regulatory elements of CD40 and CD154 promoters and regulates their transcription. In keeping with this notion, mouse AKNA (moAKNA) is also a nuclear protein that is expressed by B and T lymphocytes, exhibits the AT-hook DNA-binding motifs, upregulates CD154 and induces the expression of CD40. Collectively these findings suggest that AKNA plays a role in the regulation of Ag-dependent responses within GCs.
The specific aims are: 1. To assess in vitro the role of AKNA in gene transcription. Using the murine model, we shall determine moAKNA's role in the expression of CD40, CD154 and other gene targets. We shall study the mechanisms of moAKNA-mediated gene regulation and assess its DNA-binding requirements and specificity. 2. To examine whether moAKNA is expressed by dendritic cells (DC) and whether it plays any role in DC maturation and function, moAKNA expression will be analyzed following in vivo Fit3 ligand(FItL)-dependent DC mobilization, and sorting of distinct stage-specific DC subsets. We shall also examine whether AKNA expression peaks with the acquisition of the antigen presenting cell (APC) phenotype and the expression of CD40 and CD154.3. To assess in vivo the significance of AKNA's function. AKNA-deficient mice will be generated to elucidate its function in the immune response. We shall examine the development of secondary immune responses after immunization, examine the expression of costimulatory molecules, formation of germinal centers and antibody production. The histological analysis of lymphoid architecture, cell proliferation, phenotype, CD40 and CD154 expression, and determination of serum Ig will assess the presence or absence of functional mature B lymphocyte repertoires. DC maturation and their ability to stimulate T cells in an Ag and CD40/CD154-dependent manner will be examined. Intrathymic T cell differentiation, selection, and their capacity to home to the secondary follicles and provide cognate help will be tested. Immune functions of AKNA-deficient cells will be examined in vivo and in vitro for their capacity to respond to Ag stimuli, proliferate, produce antibodies and/or cytokines, respond to survival/death signaling, and develop immunological memory.
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