Regulatory mechanisms of immune function: Class II transactivator expression in T lymphocytes and the role of plexin -A1 in dendritic cells

The presentation of foreign antigen on major histocompatibility complex (MHC) class II molecules by antigen presenting cells (APCs) to CD4+ T cells results in activation of an immune response against the antigen. The class II transactivator (CIITA) is the master regulator of MHC class II genes and functions as a molecular scaffold to recruit transcription factors to the MHC promoter to enhance MHC class II expression. CIITA expression is controlled by four distinct promoters (PI, PII, PIII and PIV) in humans, each of which is expressed in varied cell types and under different stimuli. In our first study we find that PIII CIITA is expressed in activated, human T cells. Transient transfection of wildtype and mutant PIII constructs indicate that two sites, ARE-1 and ARE-2, are required for promoter activity. EMSA analysis shows that ARE-2 inducibly binds to two proteins: an ATF/CREB/CREM family member and an unidentified protein. In the second study, we utilize Affymetrix microarray to identify novel targets of CIITA. We find the neuronal receptor, plexin-A1 as being downregulated in CIITA-/- but not IA-beta -/- or wildtype DCs. Ablation of plexin-A1 expression by RNA interference in the 3B11 DC-like cell line indicates that in both an allogeneic mixed lymphocyte reaction and antigen presentation assay, T cell activation is inhibited in T cells cultured with the plexin-A1 deficient 3B11 cells. The impaired T cell activation is not due to the ability of peptide to bind MHC class II as there were no differences in peptide binding between plexin-A1 deficient and control cells. The final study investigated the function of plexin-A1 in DCs. Plexin-A1 localizes to the cell surface and is present in the lipid rafts of mature DCs. During DC-T cell interaction, it polarizes towards the site of DC-T cell interface. Removing plexin-A1 expression with RNA interference in DCs inhibited actin polarization toward the DC-T cell interface but did not affect the localization of immune synapse molecules. Plexin-A1 deficient DCs also have reduced levels of activated Rho but not Rac and Cdc42. This work suggests a prominent role for plexin-A1 in DCs which involves Rho activation and actin polarization.