Isoforms of the class II transactivator protein

The class II transactivator (CIITA) controls both the constitutive and IFN-gamma inducible expression of HLA-D genes. In addition, through the squelching of another transactivator CREB-binding protein, CIITA was more recently shown to have a wider cellular function, including cell cycle control or cellular response to IFN-gamma and IL-4. However, due to its low expression level, its analysis mainly relies on the study of recombinant overexpressed forms of the protein. We report here the analysis of native CIITA in various cell types. We first show the precise timing of CIITA protein expression in a fibroblast cell line in response to IFN-gamma. This expression is observed 2 h after the cytokine addition with a peak of expression ranging from 16 to 24 h. We next show the existence of two major isoforms of the CIITA protein differentially expressed in fibroblast, B lymphocyte or melanoma cell lines. We present the first demonstration that these isoforms originate from alternative translation initiation codons. We finally show that CIITA isoforms translocate to the nucleus with an apparently similar efficiency. Our data therefore demonstrate the existence of CIITA isoforms whose respective ratio depends on the cell type examined. However, we present evidence for a modulation of this ratio in a melanoma cell line with an abnormal constitutive expression of MHC class II molecules.     

Phosphorylation of Ser-3 of cofilin regulates its essential function on actin

Background: Cofilin is a low-molecular weight actin-modulating protein, and is structurally and functionally conserved in eucaryotes from yeast to mammals. The functions of cofilin appear to be regulated by phosphorylation and dephosphorylation. Results: A proteolytic study of phosphorylated porcine cofilin and expression of a mutated cofilin in cultured cells revealed that Ser-3 is the unique phosphorylation site. Phosphorylated cofilin was found not to bind to either F-or G-actin while unphosphorylated cofilin binds to both. S3D-cofilin, in which Ser-3 was replaced with Asp, did not bind in vitro to actin while S3A-cofilin did. The transient overexpression of wild-type or S3A-cofilin in cultured cells caused disruption of pre-existing actin structures and induced cytoplasmic actin bundles. Heat shock-induced nuclear or NaCl buffer-induced cytoplasmic actin/cofilin rods contained the expressed cofilin. In contrast, the overexpression of S3D-cofilin did not alter the actin structures. Induced actin rods did not contain S3D-cofilin. S3D-porcine cofilin did not complement the lethality associated with Δcof1 mutations in Saccharomyces cerevisiae while wild-type and S3A-cofilin did. Furthermore, we found that S2A/S4D- and S2D/S4D-yeast cofilin mutants were not viable. Conclusion: We conclude that the function of cofilin is negatively regulated in vivo by phosphorylation of Ser-3 and that cells require the functions of unphosphorylated cofilin for viability.     

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.     

Revisiting the specificity of the MHC class II transactivator CIITA in vivo

CIITA is a master regulatory factor for the expression of MHC class II (MHC-II) and accessory genes involved in Ag presentation. It has recently been suggested that CIITA also regulates numerous other genes having diverse functions within and outside the immune system. To determine whether these genes are indeed relevant targets of CIITA in vivo, we studied their expression in CIITA-transgenic and CIITA-deficient mice. In contrast to the decisive control of MHC-II and related genes by CIITA, nine putative non-MHC target genes (Eif3s2, Kpna6, Tap1, Yars, Col1a2, Ctse, Ptprr, Tnfsf6 and Plxna1) were found to be CIITA independent in all cell types examined. Two other target genes, encoding IL-4 and IFN-gamma, were indeed found to be up- and down-regulated, respectively, in CIITA-transgenic CD4(+) T cells. However, there was no correlation between MHC-II expression and this Th2 bias at the level of individual transgenic T cells, indicating an indirect control by CIITA. These results show that MHC-II-restricted Ag presentation, and its indirect influences on T cells, remains the only pathway under direct control by CIITA in vivo. They also imply that precisely regulated MHC-II expression is essential for maintaining a proper Th1-Th2 balance.     

Brucella abortus regulates bovine macrophage-T-cell interaction by major histocompatibility complex class II and interleukin-1 expression

T-cell activation is dependent on nominal antigen associated with major histocompatibility complex (MHC) class II molecules and interleukin-1 (IL-1), both provided by antigen-presenting cells. We have studied the effects of Brucella abortus and recombinant bovine gamma interferon (IFN-gamma) on bovine macrophage expression of MHC class II and IL-1 molecules and subsequent T-cell proliferation in response to B. abortus. When peripheral blood mononuclear cells were cocultured with B. abortus and IFN-gamma, increasing amounts of IFN-gamma, from 1 to 100 U/ml, down regulated T-cell proliferation. Expression of MHC class II molecules on macrophages was increased independently by IFN-gamma or B. abortus treatment. Thus, class II molecule expression was not the cause of down regulation of T-cell proliferation as observed in other systems. However, B. abortus-IFN-gamma-treated macrophages obtained from overnight cultures had minimal membrane IL-1 compared with macrophages treated with B. abortus alone. Loss of membrane IL-1 required IFN-gamma and the o-polysaccharide of the lipopolysaccharide. IFN-gamma at 1 U/ml in combination with B. abortus produced a 32% decrease in T-cell response, while IFN-gamma at 100 U/ml added to B. abortus-treated cultures produced an 82% reduction in T-cell response. Membrane IL-1 levels were not altered when recombinant bovine IFN-alpha or the rough strain 45/20 of B. abortus, which lacks the o-polysaccharide, was used. Secreted IL-1 levels were unaffected by IFN-gamma and B. abortus treatment. The addition of recombinant bovine IL-1 beta (0.001 to 0.1 ng/ml) to B. abortus- and IFN-gamma-treated cultures failed to provide a signal necessary for T-cell proliferation. These data suggest that membrane IL-1 has a key role in T-cell activation in response to B. abortus. When the o-polysaccharide of B. abortus lipopolysaccharide is combined with IFN-gamma at an inappropriate time during an immune response, T-cell proliferation is prevented and cannot be restored by the addition of exogenous IL-1.     

Structural and functional characteristics of a dominant-negative isoform of porcine MHC class II transactivator.

The MHC class II transactivator, CIITA, is critical for MHC class II gene expression in all species studied to date. We isolated an interferon (IFN)-gamma-inducible isoform of porcine CIITA (pCIITA') encoding a protein of 566 amino acids (aa) with significant homology to human CIITA (hCIITA). Analysis indicated that pCIITA' lacks the entire GTP-binding domain that is important for nuclear translocation and activation of target genes by hCIITA. In pCIITA' this region is replaced by a 14-aa motif with homology to several signalling peptide sequences. Expression of pCIITA' in porcine (ST-IOWA) and human (HeLa) cell lines resulted in suppression of IFN-gamma-stimulated MHC class II gene expression, at the protein and mRNA levels. We also identified two IFN-gamma-inducible variants of hCIITA, hCIITAlo and hCIITA' from Hela cells, both exhibiting dominant-negative suppression of MHC class II gene expression. Interestingly, hCIITA' encodes a predicted protein of 546 aa with a strikingly similar organization to pCIITA' including the 14-aa GTP-binding domain-replacement motif in which 10 out of 14 amino acids are identical to the pig sequence. Expression of hCIITA' and hCIITAlo sequences in Hela cells suppressed IFN-gamma-induced MHC class II gene expression. hCIITAlo, a predicted 303-aa protein with deleted GTP-binding and carboxy-terminal domain, displayed a more subtle suppression of IFN-gamma-induced MHC class II expression. These in vitro data indicate that there may be a role in vivo for isoforms of CIITA that can suppress full-length CIITA-mediated MHC class II gene expression. Both humans and now, potentially, pigs are candidate donors for organ and tissue allografts and xenografts, respectively. Regulation of MHC class II gene expression by manipulation of CIITA isoform expression in humans and pigs may provide a useful strategy for attenuation of T-cell-mediated cellular rejection.     

Inactivating mutations of class II transactivator (CIITA) gene in gastric and colorectal cancers

Expression of MHC class II, which is important against cancer immunity, depends on the transcactivator CIITA. These data suggest a possibility that CIITA gene might be inactivated in cancers. In this study, we studied inactivating mutation status of CIITA gene in gastric (GC) and colorectal (CRC) cancers by analyzing the C7 repeat in the CIITA (exon 11) gene. We found frameshift mutations in 3 GCs and 6 CRCs in cancers with high microsatellite instability (MSI-H) (9/113), but not in those with microsatellite-stable (MSS) (0/90) (P = 0.004). They were all deletion or duplication of one base in the C7 repeat that would result in truncation of amino acid synthesis. Immune therapy is now a major option in cancer therapy and our results on the genetic alterations of MHC II-related CTIITA in MSH-H GC and CRC might provide useful information for the treatment of MSI-H cancers.     

Quantitative analysis of peptide-MHC class II interaction

The tremendous progress in the field of basic immunology and immunochemistry made in the last decade has significantly advanced our understanding of antigen processing and presentation by MHC class I and II proteins. In this review different techniques to study peptide interaction with MHC class II molecules are summarized and their impact on the elucidation of quantitative parameters, like affinities or kinetic data, is discussed. A recently introduced method based on synthetic combinatorial peptide libraries allows to quantify the binding contribution of each amino acid residue in a class II ligand and is presented in more detail. As this knowledge is fundamental for current investigations in modern medicine, e.g. for novel immune system based therapy concepts, further aspects like the design of new high affinity MHC class II ligands and the prediction of peptide antigens are discussed.