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EBV-associated gastric adenocarcinomas (EBVaGCs) often exhibit better clinical outcomes than EBV negative gastric cancers (GCs), which could be related to their consistent expression of foreign viral antigens. Antigen-presenting cells (APCs) present peptide antigens in the context of the class-II major histocompatibility complex (MHC-II). During inflammatory conditions, epithelial cells express MHC-II and function as accessory APCs. Utilizing RNA-seq data from nearly 400 GC patients, we determined the impact of EBV-status on expression of MHC-II components, genes involved in their regulation, and T-cell co-stimulation. Virtually all MHC-II genes were significantly upregulated in EBVaGCs compared to normal tissues, or other GC subtypes. Genes involved in antigen presentation were also significantly upregulated in EBVaGCs, as were the key MHC-II transcriptional regulators CIITA and RFX5. This was unexpected as the EBV encoded BZLF1 protein can repress CIITA transcription and is expressed in many EBVaGCs. Furthermore, MHC-II upregulation was strongly correlated with elevated intratumoral levels of interferon-gamma. In addition, expression of co-stimulatory molecules involved in T-cell activation and survival was also significantly increased in EBVaGCs. Thus, gastric adenocarcinoma cells may functionally contribute to the highly immunogenic tumor microenvironment observed in EBVaGCs via a previously unappreciated role in interferon-induced antigen presentation.

Major histocompatibility complex (MHC) II deficiency is a rare primary immunodeficiency disorder that is characterized by the deficiency of MHC class II molecules. The disease is caused by transcription factor mutations including class II transactivator (CIITA), regulatory factor X-5 (RFX5), RFX-associated protein (RFXAP), and RFXAP-containing ankyrin repeat (RFXANK), respectively. Mutations in the RFXANK gene account for >70% of all known patients worldwide. Herein, we reported a 10-month-old boy with MHC II deficiency caused by a novel mutation in the RFXANK gene (c.337 + 1G>C). The boy was admitted to the hospital due to pneumonia and diarrhea at 4 months of age. Genetic analysis revealed a novel homozygous mutation in the RFXANK gene, which derived from the c.337 + 1G>C heterozygous mutations in the RFXANK gene of his parents. The boy died 3 months after diagnosis. More than 200 cases have been reported, and a review of the literature revealed different mutation rates of 4 transcription factors in different countries or regions. This is the first case report of MHC II deficiency from East Asia. We also describe all gene mutations that cause MHC II deficiency and the epidemiology of MHC II deficiency with gene mutations in this paper.

Supplemental Digital Content is available in the text., Major histocompatibility complex (MHC) class II regulatory genes play a paramount role in immune response that can exert a predominant influence on clinical outcome of Epstein–Barr virus infection consistently assumed as the main pathogenetic factor for nasopharyngeal carcinoma. To elucidate the relationship between allelic variants of MHC class II regulatory genes and susceptibility to nasopharyngeal carcinoma, a total of 28 polymorphic loci at MHC class II regulatory genes, involving CIITA, CREB1, RFX family genes (RFX5, RFXAP, and RFXANK), and NFY family genes (NFYA, NFYB, and NFYC), were genotyped by multiplex SNaPshot minisequencing in 137 patients with nasopharyngeal carcinoma and 107 healthy controls from the southern Chinese population. Allelic analysis disclosed that rs7404873, rs6498121, rs6498126, and rs56074043 shared correlations with nasopharyngeal carcinoma (Ptrend < 0.05). Further, rs6498126 on CIITA was independently associated with the risk of developing nasopharyngeal carcinoma (CC vs. GG, odds ratio: 7.386, 95% confidence interval: 1.934–28.207, Ptrend < 0.01). Conversely, rs7404873 on CIITA and rs56074043 on NFYB manifested epistatic interaction to decreased susceptibility of nasopharyngeal carcinoma (rs7404873, TT vs. GG, odds ratio: 0.256, 95% confidence interval: 0.088–0.740, Ptrend < 0.05; rs56074043, AA vs. AG, odds ratio: 0.341, 95% confidence interval: 0.129–0.900, Ptrend < 0.05). Additionally, bioinformatics analysis revealed that the three variants were transcriptional regulatory in function and might impact the expression of nearby genes. The findings suggested genetic variants on MHC class II regulatory genes contributed to nasopharyngeal carcinoma susceptibility and might provide new insights for screening high-risk population.

The MHC II deficiency is a rare autosomal recessive primary immunodeficiency syndrome with increased susceptibility to respiratory and gastrointestinal infections, failure to thrive and early mortality. This syndrome is caused by mutations in transcription regulators of the MHC II gene and results in development of blind lymphocytes due to the lack of indicatory MHC II molecules. Despite homogeneity of clinical manifestations of patients with MHC II deficiency, the genetic defects underlying this disease are heterogeneous. Herein, we report an Iranian patient with MHC II deficiency harbouring a novel mutation in RFXANK and novel misleading clinical features. He had ataxic gait and dysarthria from 30 months of age. Epidemiology, clinical and immunological features, therapeutic options and prognosis of patients with MHC II are reviewed in this paper.

Macrophages are professional antigen-presenting cells relying on the expression of class II major histocompatibility complex (MHC II) genes. Interferon-γ (IFN-γ) activates MHC II transcription via the assembly of an enhanceosome centred on class II trans-activator (CIITA). In the present study, we investigated the role of the forkhead transcription factor FOXO3a in IFN- γ-induced MHC II transcription in macrophages. Knockdown of FOXO3a, but not FOXO1 or FOXO4, diminished IFN-γ-induced MHC II expression in RAW cells. On the contrary, over-expression of FOXO3a, but neither FOXO1 nor FOXO4, enhanced CIITA-mediated trans-activation of the MHC II promoter. IFN-γ treatment promoted the recruitment of FOXO3a to the MHC II promoter. Co-immunoprecipitation and RE-ChIP assays showed that FOXO3a was a component of the MHC II enhanceosome forming interactions with CIITA, RFX5, RFXB and RFXAP. FOXO3a contributed to MHC II transcription by altering histone modifications surrounding the MHC II promoter. Of interest, FOXO3a was recruited to the type IV CIITA promoter and directly activated CIITA transcription by interacting with signal transducer of activation and transcription 1 in response to IFN-γ stimulation. In conclusion, our data unveil a novel role for FOXO3a in the regulation of MHC II transcription in macrophages.

Major histocompatibility complex (MHC) class II deficiency, also called the bare lymphocyte syndrome, is a rare primary immunodeficiency disease characterized by a virtual absence of MHC class II expression associated with a variable reduction in MHC class I expression. The disease is genetically heterogeneous. It can result from loss-of-function mutations in any one of four genes – CIITA , RFX5 , RFXAP , and RFXANK – encoding regulatory factors required for transcription of class I and/or class II MHC genes. RFX5 , RFXAP , and RFXANK encode subunits of regulatory factor X, a DNA-binding transcription factor required for transcription of both class I and class II MHC genes. Class II transactivator (CIITA) encodes a transcriptional coactivator showing high specificity for MHC class II genes. CIITA is referred to as the ‘master regulator’ of MHC class II genes because its highly regulated pattern of expression dictates most qualitative and quantitative aspects of MHC class II expression. At MHC class I promoters, CIITA is replaced by a related coactivator, NLRC5. Deficient MHC expression leads to the inability to mount antigen-specific adaptive immune responses against infectious agents. Patients consequently suffer from a broad range of severe and recurrent infections, generally resulting in death at a young age, unless they are treated by hematopoietic stem cell transplantation.

The extraordinary homology between major histocompatibility complex class II (MHC II) proteins across species from human to bony fish suggests that transcription factors that regulate these proteins might be conserved as well. Deficiencies in four proteins that regulate MHC II genes in humans (RFX-B, RFX5, RFXAP, and CIITA) cause an inherited immunodeficiency disorder known as the bare lymphocyte syndrome (BLS). To understand the structure and mechanism of function of the BLS transcription factors, we analyzed the evolutionary history of RFX-B, the factor deficient in the majority of patients with BLS. Sequence comparison and analysis of the RFX-B proteins showed that RFX-B and a closely related protein, ANKRA2, are present in humans to bony fish and that specific domains are highly conserved. In addition to sequence conservation, functional conservation exists, as mouse and Xenopus RFX-B orthologues, but not the paralogous protein ANKRA2, were able to complement the MHC II deficiency in a BLS-patient-derived cell line deficient in RFX-B. The remarkable conservation of the RFX-B lineage attests to the conservation of the regulation mechanism for this gene system and its importance to precisely regulate MHC class II molecules in both the developing and active immune response.

Major histocompatibility complex (MHC) class II genes are regulated at the transcriptional level by coordinate action of a limited number of transcription factors that include regulatory factor X (RFX), class II transcriptional activator (CIITA), nuclear factor Y (NF-Y), and cyclic AMP-response element binding protein (CREB). Here, the MHC class-II-specific transcription factors and CREB were expressed in insect cells with recombinant baculoviruses, isolated, and characterized by biochemical and biophysical methods. Analytical ultracentrifugation (AUC) has demonstrated that RFX is a heterotrimer. A heterodimer of RFX5 and RFX-AP was also observed. A high-affinity interaction (K(d) = 25 nM) between RFX5 and RFX-AP was measured by isothermal titration calorimetry (ITC), while the interaction between RFX-AP and RFX-ANK is at least an order of magnitude weaker. The biophysical data show that the interaction between RFX-AP and RFX5 is a key event in the assembly of the heterotrimer. Fluorescence anisotropy was used to determine protein-nucleic acid binding affinities for the RFX subunits and complexes binding to duplex DNA. The RFX5 subunit was found to drive recognition of the promoter, while the auxiliary RFX-AP and RFX-ANK subunits were shown to contribute to the specificity of binding for the overall complex. AUC experiments demonstrate that in the absence of additional subunits, monomeric RFX5 binds to X-box DNA with a 1:1 stoichiometry. Interactions between CREB, CIITA, and RFX in the absence of DNA were demonstrated using bead-based immunoprecipitation assays, confirming that preassociation with DNA is not required for forming the macromolecular assemblies that drive MHC class II gene expression.

We analysed the regulation of human leucocyte antigen (HLA)-E, -F and -G genes, focusing on the SXY module, a promoter region that controls major histocompatibility complex (MHC) class II expression and participates in the expression of classical HLA class I molecules. It comprises the X1, X2 and Y boxes, bound by RFX, X2-BP/ATF/CREB and NFY factors, respectively. The complex recruits the master control factor CIITA. The SXY module is conserved in HLA-E and HLA-F gene promoters, whereas in the HLA-G promoter, the only conserved boxes are S and X1. Chromatin immunoprecipitation assays, performed on HLA-G positive and negative cell lines, demonstrated the in situ binding of RFX5 and CIITA to HLA-E and HLA-F, but not to HLA-G, promoters. In B cells from bare lymphocyte syndrome patients lacking RFX5 or CIITA, we observed lower steady-state levels of HLA-E and HLA-F transcripts but did not find any significant decrease in the cell-surface expression of HLA-E/classical HLA class I. In RFX5-deficient fibroblasts, the cell-surface expression of HLA molecules was decreased. RFX5 and CIITA are thus not involved in HLA-G expression and their importance for the surface expression of HLA-E/classical HLA class I molecules may vary depending on the cell type.

MHC class II deficiency provokes a severe immunodeficiency characterized by a lack of antigen-specific immune response. In the absence of bone marrow transplantation (the only curative treatment), patients affected by this genetic recessive disease die in early childhood. However, others and we have recently described cases of mild or asymptomatic immunodeficiencies with defects in either CIITA (class II transactivator) or RFX5, both proteins required for the transcription of HLA-D genes. We describe in this report the first case of moderate immunodeficiency resulting from a defect in RFXANK, another transcription factor essential for HLA-D expression. The patient did not display any detectable expression of MHC class II molecules on B lymphocytes, monocytes or activated T lymphocytes. Accordingly HLA-D transcription was altered in the corresponding B-lymphoblastoid cell line. The defect in RFXANK was observed both at the transcript and protein level. Indeed a homozygous IVS4+5G>A mutation was evidenced in RFXANK, and shown to hamper the splicing of intron 4. However, we had shown previously that a defect in intron 4 can lead to the skipping of exon 4, and that the resulting truncated protein retains the capacity to activate HLA-DR expression. Therefore, like the two cases of moderate immunodeficiencies described previously, we demonstrate that the RFXANK defect presented here is coherent with a residual activity of the mutant protein. We thus propose that the common feature displayed by mildly immunodeficient patients is the leakiness of the mutations, which might allow a local or temporal expression of MHC class II molecules.

The class II transactivator (CIITA) is a master regulator of major histocompatibility complex class II (MHC-II), Ii, and DM genes (8, 9, 10, 55, 56). CIITA was initially identified by complementation of an HLA-DR− mutant B-cell line, RJ2.2.5 (55), which was created by mutagenesis followed by negative immunoselection for MHC-II-defective cells (1). CIITA is important for the constitutive expression of MHC-II in B cells and dendritic cells and the cytokine induction of these genes in a variety of other cell types. Gamma interferon (IFN-γ) is a prime example of a cytokine which induces CIITA and subsequently MHC-II expression. CIITA is a transcriptional coactivator that does not bind DNA (55) but does interact with RFX (consisting of RFX5-RFXANK-RFXAP), CREB, and NF-Y (NF-YA/B/C) (65), all of which directly bind to the X and Y element of the MHC-II promoters. Specifically, CIITA interacts with both RFX5 and RFXANK and NF-YB and NF-YC but not with NF-YA. In addition, CIITA also interacts with the histone acetyltransferase (HAT) CREB-binding protein (CBP) (20, 30), which can acetylate histone at lysine residues to allow gene activation (13, 19, 31, 57, 59, 62). Thus, CIITA appears to be a focal point of interaction for both DNA-binding proteins specific for the MHC-II promoters and for HATs to allow chromatin opening. CIITA expression is strongly induced by IFN-γ (56). Two of its promoters, promoter III and promoter IV, are responsible for IFN-γ inducibility (39, 42, 43). Promoter III contains a proximal sequence that is responsible for the constitutive expression of CIITA in B cells and a distal sequence containing STAT1-responsive element which confers IFN-γ responsiveness. Promoter IV contains the major IFN-responsive sequence regulated by both STAT1 and IRF-1. An invaluable tool to study IFN-γ pathways has been the use of mutant cell lines. Mutant lines which are defective in the IFN-γ induction of MHC-II genes but not defective in the induction of other IFN-γ functions have been useful in elucidating this pathway (9, 37). The G3A cell line was derived from the commonly used IFN-γ-responsive parental cell line 2fTGH (10). IFN-γ treatment of G3A cells causes the normal induction of most responsive genes but causes a selective failure to induce MHC-II, Ii, and DM. This is caused by a defect in the IFN induction of CIITA and places CIITA downstream of the JAK/STAT pathway. In addition to its well-known function in inducing gene expression, IFN-γ also suppresses the expression of a number of genes. There are several examples of IFN-γ-suppressible promoters or genes, and these can be loosely grouped as the following: genes important for cell proliferation and cell differentiation, such as cyclin (51) and c-myc and N-myc (59); certain cytokine genes expressed by the TH2 subpopulation, such as interleukin 4 (IL-4) (15) and IL-10 (17); and genes coding for matrix proteins, such as the collagen (26, 29, 58) and proteoglycan (16, 50) genes. The mechanism by which IFN-γ causes gene suppression is not well known, and the present study shows that one mechanism involves the CIITA molecule. The rationale for this study stems from a previous report, where we and others detected interactions of CIITA with a number of DNA-binding proteins that bind the MHC-II promoters (65). This prompted us to determine if CIITA can selectively induce the expression of MHC-II but simultaneously sequester DNA-binding proteins from their other gene targets. The squelching of transcription factors has been proposed before (4, 21, 34, 43); however, there are few good physiologic examples of squelching. In this report, we determined that CIITA can squelch the expression of NF-Y-dependent promoters. However, this was not reversed by the addition of NF-Y, and the CIITA domain that is required for this effect does not match the CIITA–NF-Y interaction site. Instead, the region within CIITA that is required for squelching maps to a 36-amino-acid (aa) region which binds the CBP. Significantly, squelching was observed under biologic conditions where the effects of endogenous CIITA on endogenous collagen gene expression were examined. These experiments show that this small peptide domain is a potent inhibitor of CBP function and elucidate a possible mechanism by which IFN-γ suppresses gene expression.

Tight control of MHC expression is essential for the outcome of a successful pregnancy. The lack of MHC class II and class I mediated antigen presentation by fetal trophoblast cells is an important mechanism to evade maternal immune recognition. Interestingly, the deficient expression of MHC class II molecules (HLA-DR, -DQ and -DP) and of the classical MHC class I molecules HLA-A and HLA-B is also noted after IFN-gamma treatment in trophoblast-derived cell lines. Our studies show that in trophoblast cell lines the IFN-gamma induced transactivation of HLA-A and HLA-B promoters is repressed. Furthermore, it was found that trophoblast cells lacked IFN-gamma mediated induction of the class II transactivator (CIITA). This lack of CIITA expression in trophoblast cells is due to CIITA promoter hypermethylation. In addition to lack of CIITA expression, trophoblast cells also displayed a repressed expression of RFX5. Together, these observations reveal a silencing of multiple activation pathways that are critical to the transcriptional control of MHC class II and class I antigen presentation functions by trophoblast cells.

The major histocompatibility complex (MHC) class II transactivator CIITA plays a pivotal role in the control of the cellular immune response through the quantitative regulation of MHC class II expression. We have analyzed a region of CIITA with similarity to leucine-rich repeats (LRRs). CIITA LRR alanine mutations abolish both the transactivation capacity of full-length CIITA and the dominant-negative phenotype of CIITA mutants with N-terminal deletions. We demonstrate direct interaction of CIITA with the MHC class II promoter binding protein RFX5 and could also detect novel interactions with RFXANK, NF-YB, and -YC. However, none of these interactions is influenced by CIITA LRR mutagenesis. On the other hand, chromatin immunoprecipitation shows that in vivo binding of CIITA to the MHC class II promoter is dependent on LRR integrity. LRR mutations lead to an impaired nuclear localization of CIITA, indicating that a major function of the CIITA LRRs is in nucleocytoplasmic translocation. There is, however, evidence that the CIITA LRRs are also involved more directly in MHC class II gene transactivation. CIITA interacts with a novel protein of 33 kDa in a manner sensitive to LRR mutagenesis. CIITA is therefore imported into the nucleus by an LRR-dependent mechanism, where it activates transcription through multiple protein-protein interactions with the MHC class II promoter binding complex.

Major histocompatibility complex (MHC) class II molecules are the predominant presenters of exogenous antigens to T helper cells (reviewed in references 21, 77, and 99). These key molecules are critical for numerous aspects of immune function, including T-cell selection, tolerance induction, antibody production, T-cell-mediated immunity, and the inflammatory response. As principal mediators of transplant rejection, these molecules are often common targets for immune therapies to prevent the rejection of grafted tissues. Class II MHC is implicated as a contributing factor in a host of diseases ranging from rheumatoid arthritis and diabetes to Alzheimer's disease and multiple sclerosis. Constitutive expression of class II MHC is restricted to “professional” antigen-presenting cells but can be induced on various tissues by gamma interferon (IFN-γ). In humans, a congenital lack of both constitutive and inducible class II results in a profound and generally fatal immunodeficiency (type II bare lymphocyte syndrome [BLS]) (7, 29, 45, 61, 67, 75, 112) marked by a significant reduction of CD4+ T cells. Early molecular forays addressing BLS revealed that the genes encoding class II MHC were not defective. Instead, the defect lay in transcription factors controlling class II MHC gene expression. BLS thus became the first disease known to be caused by defective or absent transcription factors. The availability of patient-derived cell lines with class II MHC transcription defects provided a unique tool of nature to identify the requisite transcription factors. Transcriptional regulation of class II MHC expression is complex. Class II MHC and related promoters are characterized by the presence of conserved W (or S), X, and Y boxes (Fig. ​(Fig.1)1) (reviewed in references 10, 67, and 75). The X element is bipartite. The upstream X1 region is recognized by RFX, a trimeric complex of RFX family members including RFX5, RFXANK (RFX-B), and RFXAP (32, 74, 87, 117). The downstream X2 box is bound by X2BP (NF-X2), a complex comprising CREB, and an unidentified 120-kDa protein (83, 84). Another trimeric complex, NF-Y (CBF), which is highly conserved in eukaryotes, binds the Y box (69, 71, 146; reviewed in reference 70). A number of factors interacting with the W box have been described, including the RFX complex (26, 48, 120). The factors involved in X and Y box binding are ubiquitous and expressed constitutively yet fail to account for either constitutive or IFN-γ-inducible class II MHC expression. Somatic cell fusions using BLS patient-derived cells allowed the definition of complementation groups, with each group containing a defect in a single genetic locus. This type of analysis revealed a crucial locus, aIr-1, which in all likelihood encodes the class II transactivator (CIITA), which explained the lack of class II transcription in BLS complementation group A (1, 118). Group A cells express the requisite X and Y binding proteins but fail to transcribe class II. CIITA expression appears to be a nearly absolute requisite for expression of class II MHC, whether constitutive or inducible (17, 19, 23, 47, 85, 103, 114, 118, 119). A number of class II MHC-related genes including genes encoding HLA-DM (H-2M in mice) and invariant chain (Ii), with promoters similar to those for classical class II genes, are also regulated by CIITA (17, 18, 22, 23, 50, 137). CIITA can also upregulate expression of class I MHC genes and beta-2-microglobulin (β2m) through effects at site α in addition to X- and Y-like sequences in the promoters for these genes (40, 72, 104). These initial observations have led to the view that CIITA is a master, or global, regulator for expression of class II MHC and related genes. FIG. 1 Organization of W, X, Y, and other motifs in the promoters of class II MHC and related genes. Genes coding for class II MHC and related proteins contain well conserved W, X, and Y boxes, the presence of which correlates with transcriptional regulation ... Since the discovery of CIITA, numerous primary articles and several reviews on its role in regulating the class II MHC have been published. In this review, we will discuss the molecular structure of this novel protein, its mechanism of function, and its biologic and clinical relevance, which is broad.

The X2 box of MHC class II promoters is homologous to TRE/CRE elements and is required for expression of MHC class II genes. The X2 box-specific DNA binding activity, X2BP, was purified to homogeneity, sequenced, and identified as CREB. Transient transactivation experiments showed that CREB can cooperate with CIITA to enhance activation of transcription from MHC class II promoters in a dose-dependent manner. Binding of CREB to the class II promoter in vivo was demonstrated by a chromatin immunoprecipitation assay. Additionally, ICER, a dominant inhibitor of CREB function, was found to repress class II expression. These results demonstrate that CREB binds to the X2 box in vivo and cooperates with CIITA to direct MHC class II expression.

The class II transactivator (CIITA) is a master transcription regulator of gene products involved in the exogenous antigen presentation pathway, including major histocompatibility complex (MHC) class II, invariant chain, and DM. An extensive analysis of the putative functional domains of CIITA is undertaken here to explore the action of CIITA. Antibodies to CIITA protein were produced to verify that these mutant proteins are expressed. Both acidic and proline/serine/threonine-rich domains are essential for class II MHC promoter activation. In addition, three guanine nucleotide-binding motifs are essential for CIITA activity. Of these mutants, two exhibited strong transdominant-negative functions. These two mutants provide a plausible approach to manipulate MHC class II expression and immune responses.

MHC-encoded HLA-DMA and -DMB molecules are atypical MHC chains that play an essential role in antigen presentation by MHC class II molecules. They resemble both MHC class I and II molecules but are not expressed at the cell surface. From the study of MHC class II regulatory mutants, it was found recently that two novel transactivators, CIITA and RFX5, are essential for the control of MHC class II gene expression. We report here that CIITA and RFX5, although operating at different levels of transcriptional control, are also both essential regulators of HLA-DMA and -DMB genes. This is true for both the constitutive and the inducible mode of DM gene expression. Indeed, both CIITA and RFX5 cDNA can correct the HLA-DMA and -DMB gene expression defect in the respective regulatory mutants. The involvement of these two transcription factors accounts for the coordinate expression of MHC class II and HLA-DM, two sets of molecules that perform quite different functions in the overall process of antigen presentation.

Presenting processed antigens to CD4+ lymphocytes during the immune response involves major histocompatibility complex class II molecules. MHC class II genes transcription is regulated by four transcription factors: CIITA, RFXANK, RFX5 and RFXAP. Defects in these factors result in major histocompatibility complex class II expression deficiency, a primary combined immunodeficiency frequent in North Africa. Autosomal recessive mutations in the RFXANK gene have been reported as being the principal defect found in North African patients with this disorder. In this paper, we describe clinical, immunological and genetic features of 11 unrelated Algerian patients whose monocytes display a total absence of MHC class II molecules. They shared mainly the same clinical picture which included protracted diarrhoea and respiratory tract recurrent infections. Genetic analysis revealed that 9 of the 11 patients had the same RFXANK founder mutation, a 26 bp deletion (named I5E6-25_I5E6+1, also known as 752delG26). Immunological and genetic findings in our series may facilitate genetic counselling implementation for Algerian consanguineous families. Further studies need to be conducted to determine 752delG26 heterozygous mutation frequency in Algerian population.

Major histocompatibility complex (MHC) class II genes are expressed constitutively in only a few cell types, but they can be induced in the majority of them, in particular by interferon-gamma (IFN-gamma). The MHC class II transactivator gene CIITA is defective in a form of primary MHC class II deficiency. Here it is shown that CIITA expression is controlled and induced by IFN-gamma. A functional CIITA gene is necessary for class II induction, and transfection of CIITA is sufficient to activate expression of MHC class II genes in class II-negative cells in the absence of IFN-gamma. CIITA is therefore a general regulator of both inducible and constitutive MHC class II expression.

The class II transactivator (CIITA) has been shown to be required for major histocompatibility complex (MHC) class II gene expression in B cells and its deficiency is responsible for a hereditary MHC class II deficiency. Here we show that CIITA is also involved in the inducible expression of class II genes upon interferon gamma (IFN-gamma) treatment. The expression of CIITA is also inducible with IFN-gamma before the induction of MHC class II mRNA. In addition, CIITA mRNA expression does not require new protein synthesis, although new protein synthesis is necessary for the transcription of class II. This suggests that synthesis of new CIITA protein may be essential to induce class II gene expression. We also showed that the JAK1 protein tyrosine kinase activity is required to induce the expression of CIITA upon IFN-gamma stimulation. This finding indicates that CIITA is part of the signaling cascade from the IFN-gamma receptor to the activation of class II genes. In addition, the expression of CIITA is sufficient to activate class II genes in the absence of IFN-gamma stimulation suggesting that CIITA is the major regulatory factor for the inducible expression of class II genes. Together, these data suggest that CIITA is the IFN-inducible cycloheximide sensitive factor previously shown to be required for the induction of MHC class II gene expression.

This review focuses on recent progress made in MHC regulation. The better characterization of proteins that interact with MHC class I and II promoters and the isolation of genes encoding several of these transcription factors, such as H-2RIIBP/RXR beta, NK kappa B, I-kappa B, hXBP-1 and NF-Y, allow the functional analysis of these molecules in MHC gene regulation. The application of new techniques, such as genomic in vivo footprinting analysis, to the study of these promoters provides insights into the status of in vivo protein-DNA interaction over these promoters. New insights have also been gained in the understanding of MHC-associated genes.

Major histocompatibility complex class II plays a key role in the immune response, by presenting processed antigens to CD4+ lymphocytes. Major histocompatibility complex class II expression is controlled at the transcriptional level by at least four trans-acting genes: CIITA, RFXANK, RFX5 and RFXAP. Defects in these regulatory genes cause MHC class II immunodeficiency, which is frequent in North Africa. The aim of this study was to describe the immunological and molecular characteristics of ten unrelated Moroccan patients with MHC class II deficiency. Immunological examinations revealed a lack of expression of MHC class II molecules at the surface of peripheral blood mononuclear cells, low CD4+ T lymphocyte counts and variable serum immunoglobulin (IgG, IgM and IgA) levels. In addition, no MHC class II (HLA DR) expression was observed on lymphoblasts. The molecular analysis identified the same homozygous 752delG26 mutation in the RFXANK genes of all patients. This finding confirms the association between the high frequency of the combined immunodeficiency and the defect in MHC class II expression and provides strong evidence for a founder effect of the 752delG26 mutation in the North African population. These findings should facilitate the establishment of molecular diagnosis and improve genetic counselling for affected Moroccan families.

MHC class II (MHCII) genes are transactivated by the NOD-like receptor (NLR) family member CIITA, which is recruited to SXY enhancers of MHCII promoters via a DNA-binding “enhanceosome” complex. NLRC5, another NLR protein, was recently found to control transcription of MHC class I (MHCI) genes. However, detailed understanding of NLRC5’s target gene specificity and mechanism of action remained lacking. We performed ChIP-sequencing experiments to gain comprehensive information on NLRC5-regulated genes. In addition to classical MHCI genes, we exclusively identified novel targets encoding non-classical MHCI molecules having important functions in immunity and tolerance. ChIP-sequencing performed with Rfx5−/− cells, which lack the pivotal enhanceosome factor RFX5, demonstrated its strict requirement for NLRC5 recruitment. Accordingly, Rfx5-knockout mice phenocopy Nlrc5 deficiency with respect to defective MHCI expression. Analysis of B cell lines lacking RFX5, RFXAP, or RFXANK further corroborated the importance of the enhanceosome for MHCI expression. Although recruited by common DNA-binding factors, CIITA and NLRC5 exhibit non-redundant functions, shown here using double-deficient Nlrc5−/−CIIta−/− mice. These paradoxical findings were resolved by using a “de novo” motif-discovery approach showing that the SXY consensus sequence occupied by NLRC5 in vivo diverges significantly from that occupied by CIITA. These sequence differences were sufficient to determine preferential occupation and transactivation by NLRC5 or CIITA, respectively, and the S box was found to be the essential feature conferring NLRC5 specificity. These results broaden our knowledge on the transcriptional activities of NLRC5 and CIITA, revealing their dependence on shared enhanceosome factors but their recruitment to distinct enhancer motifs in vivo. Furthermore, we demonstrated selectivity of NLRC5 for genes encoding MHCI or related proteins, rendering it an attractive target for therapeutic intervention. NLRC5 and CIITA thus emerge as paradigms for a novel class of transcriptional regulators dedicated for transactivating extremely few, phylogenetically related genes., Author Summary Major histocompatibility complex class I (MHCI) molecules are central to immunity and immunological disorders, and constitute a major obstacle in organ transplantation. It is therefore vital to gain insight into the regulation of their expression. NLRC5 was recently found to regulate MHCI gene transcription. However, we lack a thorough understanding of its target gene specificity and mechanism of action. Our work addresses these questions, delineating the unique consensus sequence required for NLRC5 recruitment and pinpointing conserved features conferring its specificity. Furthermore, through genome-wide analyses, we confirm that NLRC5 regulates classical MHCI genes and identify novel target genes, all encoding non-classical MHCI molecules exerting an array of functions in immunity and tolerance. We thereby demonstrate that NLRC5 exclusively transactivates genes of the MHCI pathway, rendering it an attractive target for future therapeutic intervention. The most striking feature of NLRC5 is its restricted and highly focused transcriptional activity, which has been described so far only for one related factor, CIITA. NLRC5 and CIITA therefore emerge as prototypes for a novel kind of extremely specific transcriptional regulator.

Major histocompatibility class II (MHC-II) genes are coordinately regulated by conserved, upstream promoter elements that are bound cooperatively by cyclic AMP response element binding protein (CREB), regulatory factor X (RFX), and nuclear factor Y (NF-Y). These DNA-binding proteins serve as a scaffold for the transcriptional coactivator class II transactivator (CIITA). To determine how CREB interacts with RFX and CIITA, co-immunoprecipitations and reporter assays were performed using a variety of CREB mutants. These assays demonstrated that CREB interacted with CIITA and the RFX5 subunit of RFX through the C-terminal portion of CREB. This C-terminal portion of CREB was fully functional in MHC-II promoter reporter assays. Phosphorylation of CREB enhanced transcription from the reporter, but was not required for transcription. Phospho-CREB was found at the HLA-DRA promoter by chromatin immunoprecipitation, providing evidence for its role. Together, these data provide genetic and biochemical evidence of the specific associations between CREB and two elements of the MHC-II regulatory complex and of the role played by phosphorylated CREB at MHC-II promoters.

MHC class II (MHCII) deficiency or bare lymphocyte syndrome (BLS) is a severe immunodeficiency characterized by deficient T helper (Th)-cell-dependent immunity. The disease is caused by defects of the MHCII promoter complex resulting in low or absent MHCII expression. We demonstrate in a murine model of MHCII deficiency (RFX5- or CIITA-deficient mice) that residual MHCII expression by professional antigen-presenting cells (APCs) is sufficient to support activation of adoptively transferred Th cells. Furthermore, upon transplantation of WT thymic epithelium, we observed development of endogenous Th cells with restoration of Th-cell-dependent antibody responses and immunity to cytomegalovirus infection, thus opening the possibility of an alternative treatment regimen for BLS. Residual MHCII expression was further induced by the presence of Th cells and also other stimuli. Analysis of CIITA/RFX5 double-deficient animals revealed that this inducible MHCII expression is genetically independent of the known promoter complex and thus constitutes an alternative MHCII expression pathway. In these experiments, we also detected a novel repressive function of the RFX complex in the absence of CIITA.

Interferon γ (IFN-γ) plays an important role during inflammation by repressing collagen and activating major histocompatibility class II (MHC-II) expression. Activation of MHC-II by IFN-γ requires regulatory factor for X-box 5 (RFX5) complex as well as class II transactivator (CIITA). We have shown that the RFX family binds to the COL1A2 transcription start site (Sengupta, P. K., Fargo, J., and Smith, B. D. (2002) J. Biol. Chem. 277, 24926–24937), and the RFX5 complex represses COL1A2 gene expression during IFN-γ response (Xu, Y., Wang, L., Buttice, G., Sengupta, P. K., and Smith, B. D. (2003) J. Biol. Chem. 278, 49134–49144). In this report, we demonstrate that CIITA is a key mediator of COL1A2 repression by IFN-γ. IFN-γ up-regulates the expression of CIITA in a time-dependent manner in lung fibroblasts and promotes CIITA protein occupancy on COL1A2 transcription start site in vivo as judged by chromatin immunoprecipitation (ChIP) assays. There are coordinate decreases in the occupancy of RNA polymerase II on the collagen transcription start site with increasing CIITA occupancy during IFN-γ treatment. In addition, we are able to specifically knockdown the IFN-γ-stimulated expression of CIITA utilizing short hairpin interference RNA (shRNA) against CIITA. This leads to the alleviation of COL1A2 repression and MHC-II activation by IFN-γ. RFX5 recruits CIITA to the collagen site as evidenced by DNA affinity chromatography. The presence of RFX5 complex proteins enhances the collagen repression by CIITA reaching levels occurring during IFN-γ treatment. Co-expression of CIITA with deletion mutations and collagen promoter constructs demonstrates that CIITA represses collagen promoter mainly through its N-terminal region including the acidic domain and the proline/serine/threonine domain. Our data suggest that CIITA is a crucial member of a repressor complex responsible for mediating COL1A2 transcription repression by IFN-γ.

The MHC class II transactivator (CIITA) plays a central role in adaptive immune responses by controlling the expression of MHC class II genes. CIITA binds DNA-binding proteins and co-activator proteins to form an enhanceosome complex necessary for MHC class II gene expression. Here we demonstrate that CIITA interactions depend upon the phosphorylation status of CIITA. Hyper-phosphorylated CIITA interacts with co-activator p300, RFX5 and CIITA itself, which in turn results in induction of MHC class II promoter activity. Moreover, the C-terminal region of CIITA containing leucine-rich repeats (LRR) is a regulatory domain for CIITA self-association and LRR binding to CIITA is negatively regulated by phosphorylation. cAMP-dependent protein kinase (PKA) phosphorylates CIITA, and serine residues residing in a region between the proline/serine/threonine-rich domain and the GTP-binding domain are phosphorylated by PKA in vitro. The maximum transactivation potential of CIITA requires PKA phosphorylation as demonstrated by reduced transactivation activities of the mutant bearing substitutions of serine residues at the PKA site.

International audience; MHC class II (MHCII) determinants play a crucial role in the immune response by presenting antigenic peptides to T cells. Their expression is controlled from compact promoters at the transcriptional level. Pre-assembled regulatory factor X (RFX) and nuclear factor Y (NFY) complexes form a platform on DNA. The class II transactivator (CIITA) can then be recruited through multiple protein-protein interactions. In this report, we defined domains of CIITA that are responsible for its interactions with these DNA-bound factors. Furthermore, using DNA-affinity precipitation, we demonstrated that although CIITA binds at least five activators, RFX5, RFXAP, RFXANK/B, NFYB and NFYC, its assembly on the promoter requires the addition of nuclear extracts. We conclude that not only does the platform bind DNA via multiple, spatially constrained nteractions, but that it can recruit only modified and/or complexed CIITA to MHCII promoters.

The expression of MHC class II molecules is essential for all Ag-dependent immune functions and is regulated at the transcriptional level. Four trans-acting proteins control the coordinate expression of MHC class II molecules: class II trans-activator (CIITA), regulatory factor binding to the X box (RFX)-associated protein; RFX protein containing ankyrin repeats, and RFX5. In humans, defects in these genes result in MHC class II expression deficiency and cause combined immunodeficiency. Most patients with this deficiency suffer from severe recurrent infections that frequently lead to death during early childhood. We investigated three sisters, now ages 21, 22, and 24 years, in whom MHC-II deficiency was detected. Even though the eldest sibling was asymptomatic and the other two had only mild immunodeficiency, none of the three class II isotypes was expressed on T cell blasts, fibroblasts, EBV B cell lines, or epidermal dendritic cells. Residual HLA-II expression was detected in fresh PBMC. Somatic complementation identified the disease as CIITA deficiency. A homozygous T1524C (L469P) substitution was found in the coding region of the CIITA cDNA and was shown to be responsible for the defect in MHC-II expression. This missense mutation prevents the normal functioning of MHC-II but does not lead to the nuclear exclusion of the L469P CIITA. Transfection experiments demonstrated that the CIITA L469P mutant had residual MHC class II trans activation activity, which might explain the unusual clinical course of the patients studied. This study shows that an attenuated clinical phenotype or an asymptomatic clinical course can be observed in patients despite a profound defect in the expression of MHC class II genes. The frequency of the inherited MHC class II deficiency might thus be underestimated.

The bare lymphocyte syndrome (BLS) is a hereditary immunodeficiency resulting from the absence of major istocompatibility complex class II (MHCII) expression. Considering the central role of MHCII molecules in the development and activation of CD4+T cells, it is not surprising that the immune system of the patients is severely impaired. BLS is the prototype of a “disease of gene regulation.” The affected genes encode RFXANK, RFX5, RFXAP, and CIITA, four regulatory factors that are highly specific and essential for MHCII genes. The first three are subunits of RFX, a trimeric complex that binds to all MHCII promoters. CIITA is a non-DNA-binding coactivator that functions as the master control factor for MHCII expression. The study of RFX and CIITA has made major contributions to our comprehension of the molecular mechanisms controlling MHCII genes and has made this system into a textbook model for the regulation of gene expression.

The bare lymphocyte syndrome, a severe combined immunodeficiency due to loss of major histocompatibility complex (MHC) class II gene expression, is caused by inherited mutations in the genes encoding the heterotrimeric transcription factor RFX (RFX-B, RFX5, and RFXAP) and the class II transactivator CIITA. Mutagenesis of the RFX genes was performed, and the properties of the proteins were analyzed with regard to transactivation, DNA binding, and protein-protein interactions. The results identified specific domains within each of the three RFX subunits that were necessary for RFX complex formation, including the ankyrin repeats of RFX-B. DNA binding was dependent on RFX complex formation, and transactivation was dependent on a region of RFX5. RFX5 was found to interact with CIITA, and this interaction was dependent on a proline-rich domain within RFX5. Thus, these studies have defined the protein domains required for the functional regulation of MHC class II genes. Type II bare lymphocyte syndrome (BLS), an inherited severe combined immunodeficiency in humans, is caused by the inability to transcribe major histocompatibility complex (MHC) class II genes (9, 15, 32). MHC class II genes encode heterodimeric glycoproteins that present antigens to CD4 1 T cells to initiate the acquired arm of the immune response. They are also crucial for determining the repertoire of CD4 1 T cells during positive and negative selection in the thymus. Patients with BLS typically present in the first year of life with recurrent infections and have reduced levels of CD4 1 T cells (9, 11). Their humoral immune response is severely impaired as well, and most patients die before reaching puberty. Patient and experimentally derived cell lines were used to separate the BLS phenotype into four complementation groups: BLS groups A, B, C, and D (3, 46, 54). The genes responsible for each of these groups have been identified and found to encode proteins required for MHC class II gene transcription. MHC class II genes are expressed on the surface of B cells, dendritic cells, macrophages, thymic epithelia, and activated T cells. Additionally, non-antigen-presenting cells can be induced to express MHC class II by exposure to the cytokine gamma interferon (IFN-g) (8). Aberrant expression of MHC class II genes is associated with autoimmunity, tumor growth, and failure to mount an immune response. The three MHC class II isotypes, HLA-DR, HLA-DP, and HLA-DQ, contain conserved cis-acting elements in their promoters (the W, X1, X2, and Y boxes) that allow their coordinate regulation (reviewed in references 4 and 31). Homologous sequence elements are also found in the HLA-DM, invariant chain, and MHC class I genes. These elements allow the coordinate expression of the different isotypes in antigen-presenting cells and the induction of these genes by IFN-g. Regulatory factor X (RFX) and the X2 box-binding protein (X2BP), which was identified as the cyclic AMP response element-binding protein (CREB) (34), bind to the X1 and X2 boxes, respectively. The Y box, an inverted CAAT box, is bound by the heterotrimeric nuclear factor Y (NF-Y) (4). The W box has not been extensively studied, but it was suggested to bind the X1 box factor, RFX (22). While all of these promoter-bound factors are required for MHC class II expression, they are not sufficient. The class II transactivator, CIITA, is also required. CIITA does not bind DNA and is believed to interact with factors on the MHC class II promoter, as well as the general transcriptional machinery, to activate transcription through its acidic activation domain (44, 49, 55). CIITA expression correlates directly with MHC class II expression and is regulated by IFN-g (6, 7, 50). Thus, the presence of CIITA functions as a molecular switch for MHC class II gene regulation.

Scaffold molecules interact with multiple effectors to elicit specific signal transduction pathways. CIITA, a non-DNA-binding regulator of class II major histocompatibility complex (MHC) gene transcription, may serve as a transcriptional scaffold. Regulation of the class II MHC promoter by CIITA requires strict spatial-helical arrangements of the X and Y promoter elements. The X element binds RFX (RFX5/RFXANK-RFXB/RFXAP) and CREB, while Y binds NF-Y/CBF (NF-YA, NF-YB, and NF-YC). CIITA interacts with all three. In vivo analysis using both N-terminal and C-terminal deletion constructs identified critical domains of CIITA that are required for interaction with NF-YB, NF-YC, RFX5, RFXANK/RFXB, and CREB. We propose that binding of NF-Y/CBF, RFX, and CREB by CIITA results in a macromolecular complex which allows transcription factors to interact with the class II MHC promoter in a spatially and helically constrained fashion.

MHC class II deficiency or bare lymphocyte syndrome is a severe combined immunodeficiency caused by defects in MHC-specific regulatory factors. Fibroblasts derived from two recently identified bare lymphocyte syndrome patients, EBA and FZA, were found to contain novel mutations in the RFX-B gene. RFX-B encodes a component of the RFX transcription factor that functions in the assembly of multiple transcription factors on MHC class II promoters. Unlike RFX5- and RFXAP-deficient cells, transfection of exogenous class II transactivator (CIITA) into these RFX-B-deficient fibroblasts resulted in the induction of HLA-DR and HLA-DP and, to a lesser extent, HLA-DQ. Similarly, CIITA-mediated induction of MHC class I, β2-microglobulin, and invariant chain genes was also found in these RFX-B-deficient fibroblasts. Expression of wild-type RFX-B completely reverted the noted deficiencies in these cells. Transfection of CIITA into Ramia cells, a B cell line that does not produce a stable RFX-B mRNA, resulted in induction of an MHC class II reporter, suggesting that CIITA overexpression may partially override the RFX-B defect.

SUMMARY In order to develop an effective immunotherapy for hematological malignancies, we investigated the applicability of class II transactivator (CIITA), which had been demonstrated to regulate the expression of MHC class II (MHC-II) by assembling the transcription factors of MHC-II molecules, for immunotherapy by potentiating the antigenicity of tumour cells by inducing MHC expression. First, 32 hematopoietic cell lines were analysed for the expression of HLA-DR, CIITA, RFX5 or HLA-ABC. Fourteen cell lines were positive and 18 were negative for HLA-DR. All the 14 HLA-DR positive cell lines were demonstrated to express CIITA mRNA by RT-PCR. On the other hand, in all the 18 HLA-DR negative cell lines, the expression of CIITA was not demonstrated. RFX5, which is one of the transcription factors of MHC-II, was expressed ubiquitously in all 32 cell lines. Three cell lines out of 23 hematopoietic cell lines examined were negative for HLA-ABC, and all three of these cell lines were negative for both HLA-DR and CIITA expression. Furthermore, CIITA cDNA was transfected into K562 cells, which were negative for HLA-ABC, -DR and -DQ, but positive for HLA-DP. The transfection rendered HLA-DR negative to positive and increased the expression level of HLA-DP, but HLA-DQ remained negative. In addition to HLA-DR, HLA-ABC was also induced to express by the transfection of CIITA gene. The present study demonstrated that the expression of HLA-DR in hematopoietic cells is regulated in subordination to CIITA and the expression of HLA-DR (and HLA-ABC in K562) is induced by transfection with the CIITA gene. These findings revealed the applicability of CIITA in potentiating antitumour immunity of HLA-DR negative tumour cells for immunotherapy of hematological malignancies. Copyright # 1999 John Wiley & Sons, Ltd.

The bare lymphocyte syndrome (BLS) is characterized by the absence of MHC class II transcription and humoral- and cellular-mediated immune responses to foreign antigens. Three of the four BLS genetic complementation groups have defects in the activity of the MHC class II transcription factor RFX. We have purified the RFX complex and sequenced its three subunits. The sequence of the smallest subunit describes a novel gene, termed RFX-B. RFX-B complements the predominant BLS complementation group (group B) and was found to be mutant in cell lines from this BLS group. The protein has no known DNA-binding domain but does contain three ankyrin repeats that are likely to be important in protein-protein interactions.

Class II major histocompatibility (class II) genes are regulated in a B-cell-specific and gamma interferon-inducible fashion. The master switch for the expression of these genes is the class II transactivator (CIITA). In this report, we demonstrate that one of the functions of CIITA is to recruit the CREB binding protein (CBP) to class II promoters. Not only functional but also specific binding interactions between CIITA and CBP were demonstrated. Moreover, a dominant negative form of CBP decreased the activity of class II promoters and levels of class II determinants on the surface of cells. Finally, the inhibition of class II gene expression by the glucocorticoid hormone could be attributed to the squelching of CBP by the glucocorticoid receptor. We conclude that CBP, a histone acetyltransferase, plays an important role in the transcription of class II genes.

The class II transactivator (CIITA) is a key regulatory factor that controls expression of the major histocompatibility complex (MHC) class II genes that are essential components for antigen presentation and thus regulation of the immune response. We show here that the adenovirus E1A protein interferes with the action of CIITA and inhibits both B-cell-specific and gamma interferon (IFN-gamma)-induced expression of MHC class II promoters. Transfection studies provide evidence for the functional role of the CREB-binding protein (CBP) in IFN-gamma and CIITA-mediated MHC class II promoter activation. We demonstrate that the N-terminally located transcription activation domain of CIITA physically interacts with both the N-terminal and the E1A-binding (C/H3) regions of CBP. These results suggest the involvement of a multisubunit complex, which contains the gene-specific coactivator CIITA and the versatile coactivator CBP, in MHC class II gene regulation, which may be responsible for both high-level expression and modulation by different signaling pathways.

Patients with major histocompatibility complex class II (MHC-II) deficiency are known to carry mutations in either the RFX complex or the trans-activator CIITA. While the pivotal role of CIITA for MHC-II gene transcription is supported by the essential absence of MHC-II molecules in CIITA-deficient mice, we demonstrate here that RFX5−/− mice retain expression of MHC-II in thymic medulla, mature dendritic cells, and activated B cells. Nevertheless, RFX5−/− mice develop a severe immunodeficiency due to the lack of MHC-II in thymic cortex, failure of positive selection of CD4+ T cells, and absence of MHC-II on resting B cells and resident or IFNγ-activated macrophages. This differential requirement for CIITA and RFX5 in subsets of antigen-presenting cells may be specific for the mouse; it may, however, also exist in humans without having been noticed so far.

BACKGROUND: Major-histocompatibility-complex (MHC) class II deficiency is an autosomal recessive primary immunodeficiency disease in which MHC class II molecules are absent. It is a genetically heterogeneous disease of gene regulation resulting from defects in several transactivating genes that regulate the expression of MHC class II genes. The mutations responsible for MHC class II deficiency are classified according to complementation group (a group in which the phenotype remains uncorrected in pairwise fusions of cells). There are three known complementation groups (A, B, and C). METHODS: To elucidate the genetic defect in patients with MHC class II deficiency that was not classified genetically, we performed direct complementation assays with the three genes known to regulate the expression of MHC class II genes, CIITA, RFX5, and RFXAP, and the relevant mutations were identified in each patient. RESULTS: Mutations in the RFXAP gene were found in three patients from unrelated families, and the resulting defect was classified as belonging to a novel complementation group (D). Transfection with the wild-type RFXAP gene restored the expression of MHC class II molecules in the patients' cells. CONCLUSIONS: Mutations in a novel MHC class II transactivating factor, RFXAP, can cause MHC class II deficiency. These mutations abolish the expression of MHC class II genes and lead to the same clinical picture of immunodeficiency as in patients with mutations in the other two MHC class II regulatory genes.

CIITA activates the expression of multiple genes involved in antigen presentation and it is believed to be required for both constitutive and IFN gamma-inducible expression of these genes. To understand the role of CIITA in vivo, we have used gene targeting to generate mice that lack CIITA. CIITA-deficient (-/-) mice do not express conventional MHC class II molecules on the surface of splenic B cells and dendritic cells. In addition, macrophages resident in the peritoneal cavity do not express MHC class II molecules upon IFN gamma stimulation nor do somatic tissues of mice injected with IFN gamma, in contrast with wild-type mice. The levels of Ii and H-2M gene transcripts are substantially decreased but absent in CIITA (-/-) mice. The transcription of nonconventional MHC class II genes is, however not affected by CIITA deficiency. A subset of thymic epithelial cells express MHC class II molecules. Nonetheless, very few mature CD4 T cells are present in the periphery of CIITA (-/-) mice despite MHC class II expression in the thymus. Consequently, CIITA(-/-) mice are impaired in T-dependent antigen responses and MHC class II-mediated allogeneic responses.

CIITA (a major histocompatibility complex [MHC] class II transactivator) has been shown to be required for the expression of MHC class II genes in both B cells and interferon gamma-inducible cells. Here we demonstrate that CIITA not only activates MHC class II genes but also genes required for antigen presentation. Mutant HeLa cells, defective in the expression of classic MHC class II genes, invariant chain, and the newly described human histocompatibility leukocyte antigen-DM genes, were used to study the role of CIITA in the regulation of these genes. Upon transfection with CIITA cDNA, the mutant cells expressed all three genes, suggesting that CIITA is a global regulator for the expression of genes involved in antigen presentation.

Class II major histocompatibility complex (MHC) genes and the invariant (Ii) gene are inducible by interferon-gamma (IFN gamma) but not by interferon-alpha and interferon-beta. The promoter regions of these genes contain three regulatory elements that mediate constitutive and IFN gamma-induced expressions; however, none of the DNA-binding proteins that interact with these elements are regulated by IFN gamma. Recently, a gene coding for a transactivator (CIITA) of class II MHC genes that complements a HLA-DR-negative immunodeficiency has been isolated. Using one IFN gamma mutant cell line (G3A) that is selectively defective in HLA-DR and Ii induction, four lines of evidence are presented to show that CIITA mediates the IFN gamma induction of HLA-DR and Ii genes. Analysis of another mutant line, G1B, indicates that the lack of DRA and Ii gene induction by IFN gamma is correlated with the lack of RFX DNA binding activity, thus providing the link between RFX and an IFN gamma response.

Constitutive major histocompatibility complex (MHC) class II gene expression is tightly restricted to antigen presenting cells and is under developmental control. Cells of the B cell lineage acquire the capacity to express MHC class II genes early during ontogeny and lose this property during terminal differentiation into plasma cells. Cell fusion experiments have suggested that the extinction of MHC class II expression in plasma cells is due to a dominant repression, but the underlying mechanisms are not understood. CIITA was recently identified as an MHC class II transactivator that is essential for MHC class II expression in B lymphocytes. We show here that inactivation of MHC class II genes in plasmocytes is associated with silencing of the CIITA gene. Moreover, experimentally induced expression of CIITA in plasmocytes leads to reexpression of MHC class II molecules to the same level as that observed on B lymphocytes. We therefore conclude that the loss of MHC class II expression observed upon terminal differentiation of B lymphocytes into plasmocytes results from silencing of the transactivator gene CIITA.

Major histocompatibility complex class II plays a key role in the immune response, by presenting processed antigens to CD4+ lymphocytes. Major histocompatibility complex class II expression is controlled at the transcriptional level by at least four trans-acting genes: CIITA, RFXANK, RFX5 and RFXAP. Defects in these regulatory genes cause MHC class II immunodeficiency, which is frequent in North Africa. The aim of this study was to describe the immunological and molecular characteristics of ten unrelated Moroccan patients with MHC class II deficiency. Immunological examinations revealed a lack of expression of MHC class II molecules at the surface of peripheral blood mononuclear cells, low CD4+ T lymphocyte counts and variable serum immunoglobulin (IgG, IgM and IgA) levels. In addition, no MHC class II (HLA DR) expression was observed on lymphoblasts. The molecular analysis identified the same homozygous 752delG26 mutation in the RFXANK genes of all patients. This finding confirms the association between the high frequency of the combined immunodeficiency and the defect in MHC class II expression and provides strong evidence for a founder effect of the 752delG26 mutation in the North African population. These findings should facilitate the establishment of molecular diagnosis and improve genetic counselling for affected Moroccan families.

Purpose of the Study. Major histocompatibility complex (MHC) class II deficiency or the bare lymphocyte syndrome is an autosomal recessive congenital immunodeficiency which typically is associated with a severe combined immunodeficiency (SCID)-like picture. There are 4 gene defects that can cause this disorder: class II trans -activator (CIITA), RFX, RFX5, and RFX-associated protein defects. These are all transcription factors required for the coordinate transcriptional regulation of MHC class II genes. Approximately 70 patients with MHC class II deficiency have been described and …

CIITA , a gene that can complement a transcriptional mutation of the major histocompatibility complex (MHC) class II genes, was tested for its ability to function as a coactivator. CIITA cDNA clones isolated showed alternative RNA splicing, but only one splice site combination was able to restore class II MHC gene expression. DNA-mediated transfection experiments showed that CIITA directs its activity through the X ☐ element; the presence of CIITA leads to the formation of a higher order complex at the X ☐ region; and CIITA contains a potent activation domain. These findings support the hypothesis that CIITA directly interacts with the MHC class l1-specific transcription factors and is required for expression.

Type II major histocompatibility complex combined immune deficiency (type II MHC CID or bare lymphocyte syndrome) is a congenital Immunodeficiency disease characterized by absent MHC class II expression. Four distinct complementation groups have been identified. Recently, the defective gene in group II type II MHC CID has been isolated and termed CIITA. Here, we demonstrate that CIITA is an MHC class II gene-specific transcription activator. The transcription activation function is provided by the N-terminal acidic domain (amino acids 26–137), which is experimentally exchangeable with a heterologous viral transcription-activating domain. The specificity of CIITA for three major MHC class 11 genes, DR, DO and DP, is mediated by Its remaining C-terminal residues (amino acids 317–1130). The transactivation of multiple cis elements, especially S and X2, of the DRα proximal promoter in group II CID cells is CIITA dependent. Since CIITA over-expression in normal cells did not increase class II expression, we propose that initiation of CIITA expression serves as the on-off switch, while availability of downstream interactor(s) limits transcription.

The master transactivator CIITA is essential to the regulation of Major Histocompatibility Complex (MHC) class II genes and an effective immune response. CIITA is known to modulate a small number of non-MHC genes involved in antigen presentation such as CD74 and B2M but its broader genome-wide function and relationship with underlying genetic diversity has not been resolved. We report the first genome-wide ChIP-seq map for CIITA and complement this by mapping inter-individual variation in CIITA expression as a quantitative trait. We analyse CIITA recruitment for pathophysiologically relevant primary human B cells and monocytes, resting and treated with interferon-gamma, in the context of the epigenomic regulatory landscape and DNA-binding proteins associated with the CIITA enhanceosome including RFX, CREB1/ATF1 and NFY. We confirm recruitment to proximal promoter sequences in MHC class II genes and more distally involving the canonical CIITA enhanceosome. Overall, we map 843 CIITA binding intervals involving 442 genes and find 95% of intervals are located outside the MHC and 60% not associated with RFX5 binding. Binding intervals are enriched for genes involved in immune function and infectious disease with novel loci including major histone gene clusters. We resolve differentially expressed genes associated in trans with a CIITA intronic sequence variant, integrate with CIITA recruitment and show how this is mediated by allele-specific recruitment of NF-kB. Our results indicate a broader role for CIITA beyond the MHC involving immune-related genes. We provide new insights into allele-specific regulation of CIITA informative for understanding gene function and disease.