Oct-1 DNA binding activity unresponsive to retinoblastoma protein expression prevents MHC class II induction in a non-small cell lung carcinoma cell line

Numerous human tumor lines fail to induce major histocompatibility (MHC) class II expression following interferon-gamma (IFN-gamma) treatment, a response that is considered to be a normal function for almost all human parenchymal and connective tissue cell-types. The effect of MHC class II non-inducibility on solid tumor growth is controversial, but an extensive body of literature indicates that tumor cell MHC class II expression can lead to an antitumor response or tumor tolerance, depending on a number of variables. Thus, understanding the molecular basis for MHC class II induction failures in solid tumor cells will likely lead to ideas for manipulating the antitumor immune response. To date, a handful of tumor associated molecular anomalies have accounted for all the known failures of MHC class II inducibility. In particular, lack of the retinoblastoma tumor suppressor protein (Rb) has been shown in both human and mouse cells to be strongly associated with failure to induce MHC class II. The basis for this relationship is traceable to, among other things, high level Oct-1 DNA binding activity in Rb-defective cells, which represses the prototypical human MHC class II gene, HLA-DRA. Ordinarily, re-establishment of Rb expression leads to elimination of, or substantially reduced Oct-1 DNA binding activity and to rescue of HLA-DRA inducibility. However, in the case of one non-small cell lung carcinoma line (NSCLC), Rb re-expression failed to rescue HLA-DRA inducibility despite successful re-establishment of Rb-function. We now report that this failure is traceable to the failure of Rb to rescue normal Oct-1 function. Furthermore, histone deacetylase inhibitor treatment allows a bypass of the Rb requirement and facilitates the MHC class II induction in this NSCLC line.     

Direct role of NF-kappaB activation in Toll-like receptor-triggered HLA-DRA expression

Microbial components, such as DNA containing immunostimulatory CpG motifs (CpG-DNA) and lipopolysaccharides (LPS), elicit the cell surface expression of MHC class II (MHC-II) through Toll-like receptor (TLR)/IL-1R. Here, we show that CpG-DNA and LPS induce expression of the HLA-DRA in the human B cell line, RPMI 8226. Ectopic expression of the dominant negative mutant of CIITA and RNA interference targeting the CIITA gene indicate that CIITA activation is not enough for the maximal MHC-II expression induced by CpG-DNA and LPS. Additionally, nuclear factor (NF)-kappaB activation is required for the CpG-DNA-activated and LPS-activated HLA-DRA expression, whereas IFN-gamma-induced MHC-II expression depends on CIITA rather than on NF-kappaB. Comprehensive mutant analyses, electrophoretic mobility shift assays and chromatin immunoprecipitation assays, reveal that the functional interaction of NF-kappaB with the promoter element is necessary for the TLR-mediated HLA-DRA induction by CpG-DNA and LPS. This novel mechanism provides the regulation of MHC-II gene expression with complexity and functional diversity.     

Description of a polymorphism in the regulatory region of the HLA-DRA gene.

It was previously reported that the cell membrane expression of HLA class II molecules is tissue specific and variable among individuals. This variation could be explained at the level of gene regulation. Due to the fact that a coordinate regulation of the HLA-DR genes seems to exist, we focused on the HLA-DRA monomorphic gene. In order to study the polymorphism of the HLA-DRA gene regulatory region, we used restriction fragment length polymorphism analysis of polymerase chain reaction (PCR)-amplified genomic DNA. The DdeI and PvuII digestions of the amplified DNA permitted the definition of the two alleles according to the absence (allele A) or the presence (allele B) of the two polymorphic restriction sites. The presence of these sites was confirmed by direct sequencing after PCR. Using homozygous typing cells, a close relationship between the HLA-DRB coding region and the HLA-DRA regulatory region polymorphisms was shown. Furthermore, a linkage between the HLA-DRA regulatory region and DRB3 gene coding region polymorphisms could be established. These results suggested a structural argument for different levels of HLA class II genes and, consequently, of cell-surface expression of class II antigens according to the allelic specificities of DRA, DRB1, and DRB3.     

Quantitative control of MHC class II expression by the transactivator CIITA

Activation of T lymphocytes is quantitatively controlled by the level of expression of MHC class II molecules. Both constitutive and inducible expression of MHC class II genes is regulated by the transactivator CIITA, which is itself tightly regulated. Since the level of MHC class II molecules expressed is a functionally essential parameter, it was of interest to explore whether MHC class II expression is quantitatively controlled by the level of the transactivator. This report shows that in a variety of experimental conditions one does indeed observe, in both mouse and man, a quantitative control of MHC class II expression by the level of CIITA. This relationship between the regulator gene, which behaves as a rate-limiting factor, and its target genes clarifies our understanding of the quantitative modulation of MHC class II expression, and thus of T lymphocyte activation.     

Immune complex-FcgammaR interaction modulates monocyte/macrophage molecules involved in inflammation and immune response

The interaction between receptors for the Fc portion of IgG (FcgammaRs) from monocytes/macrophages and immune complexes (IC) triggers regulatory and effector functions. Recently, we have demonstrated that IC exert a drastic inhibition of basal and IFN-gamma-induced expression of MHC class II on human monocytes. Taking into account that the regulation of MHC class II molecules is a crucial event in the immune response, in this report we extend our previous studies analysing the effect of STAT-1 phosphorylation in the down-regulatory process, the fate of the intracellular pool of MHC class II molecules and the effect of complement on MHC class II down-regulation induced by IC. We also studied the effect of IC on the expression of MHC class II (I-A(d)) in macrophages using a mouse model of chronic inflammation. We demonstrate that IC induce a depletion not only on surface expressed but also on intracellular MHC class II content and that IC-induced down-regulation of MHC class II is not mediated by the inhibition of STAT-1 phosphorylation. On the other hand, the effect of IC is not specific for the down-regulation of MHC class II, for it could be restricted to other molecules involved in inflammatory processes. Our experiments also show that the activation of the complement system could be a crucial step on the regulation of the effect of IC on MHC class II expression. In agreement with our in vitro experiments using human monocytes, IC treatment reduces the expression of MHC class II in a mouse model of chronic inflammation.     

Ligation of MHC class II molecules differentially upregulates TNF beta gene expression in B cell lines of different MHC class II haplotypes.

Although the production of selected cytokines by B cells is important for their regulation, little is known about MHC class II-induced cytokine expression in these cells. We designed the present studies to investigate MHC class II-mediated TNF-beta gene expression in 19 EBV-transformed homozygote B cell lines at similar stage of differentiation but presenting different MHC class II haplotypes. Our results demonstrate that in contrast to PMA, engagement of MHC class II with staphylococcal enterotoxin A (SEA), a natural ligand, or with anti-HLA-DR mAb L243, stimulates TNF-beta gene expression in some but not all B cell lines. The differential stimulation of TNF-beta gene expression via MHC class II was not due to the cells MHC class II expression level, nor to their capacity to bind the ligands as evidenced by SEA binding affinity studies. Together these results demonstrate that ligation of MHC class II molecules can stimulate TNF-beta gene expression in a B cell line-dependent manner. The differential cytokine gene expression might be due to an influence of MHC class II haplotype either by a linkage disequilibrium with TNF-beta gene or by a differential association with effector or cell surface molecules.