regulation of mhc class ii gene expression by the class ii transactivator

© 2005 Nature Publishing Group

*Department of Pathology and Immunology, University of Geneva Medical School, Centre Médical Universitaire, 1 Rue Michel-Servet, CH-1211, Geneva, Switzerland.‡Immunobiology Laboratory, Cancer Research UK London Research Institute, 44 Lincoln’s Inn Fields, London WC2A 3PX, UK.Correspondence to W.R.e-mail: [email protected]:10.1038/nri1708

In the past decade, there has been an explosion in our knowledge of the molecular mechanisms that regulate transcription of the genes encoding MHC class II molecules in health and disease. Of prime importance has been an unusually detailed genetic dissection of this system. This was achieved by the study of a severe immunodeficiency disease that is known as bare lymphocyte syndrome (BLS)1,2 BOX 1 and by the generation of several informative gene-knockout mice3–7. This genetic dissection, combined with a wealth of information that has been derived from biochemical and cell-culture-based studies, has placed the regulation of MHC class II genes among the best understood transcription-control systems in mammals. It has, in many respects, also turned out to be one of the most atypical systems. As discussed here, the task of regulating MHC class II expression, which is exquisitely controlled at a cell-type-specific level and precisely fine tuned, is shouldered almost entirely by the class II transactivator (CIITA), a highly regulated non-DNA-binding co-activator that has a remarkable degree of specificity for MHC class II genes2,8–10. It is the only known system in which a complex pattern of gene expression is imparted by a single dedicated co-activator rather than by combinatorial control

exerted by several DNA-binding transcription factors. In this Review, we summarise our current knowledge of the role of CIITA as the master regulator of MHC class II genes. We concentrate on novel insights from recent gene-targeting experiments in mice that have allowed us to integrate a profusion of results from numerous in vitro studies into a single coherent model of how the differential usage of three independent promoters of the gene encoding CIITA determines the tightly regulated pattern of cell-type-specific and inducible MHC class II expression in vivo.

Expression of MHC class II moleculesMHC class II molecules are cell-surface glycoproteins that are of central importance to the adaptive immune system because they present peptides — derived mainly from extracellular proteins — to the antigen receptor of CD4+ T cells. MHC-class-II-mediated peptide presentation is essential for the following: the positive and negative selection processes that shape the specificity of the T-cell-receptor repertoire of the CD4+ T-cell population during its development in the thymus; the homeostasis of the mature CD4+ T-cell population in the periphery; and the initiation, ampli-fication and regulation of protective immune responses

REGULATION OF MHC CLASS II GENE EXPRESSION BY THE CLASS II TRANSACTIVATORWalter Reith*, Salomé LeibundGut-Landmann*‡ and Jean-Marc Waldburger*

Abstract | MHC class II molecules are pivotal for the adaptive immune system, because they guide the development and activation of CD4+ T helper cells. Fulfilling these functions requires that the genes encoding MHC class II molecules are transcribed according to a strict cell-type-specific and quantitatively modulated pattern. This complex gene-expression profile is controlled almost exclusively by a single master regulatory factor, which is known as the class II transactivator. As we discuss here, differential activation of the three independent promoters that drive expression of the gene encoding the class II transactivator ultimately determines the exquisitely regulated pattern of MHC class II gene expression.

NATURE REVIEWS | IMMUNOLOGY VOLUME 5 | OCTOBER 2005 | 793

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ASTROCYTE A star-shaped glial cell of the central-nervous system that forms a structural and functional interface between non-nervous tissues and neurons.

TROPHOBLAST A cell of the outer layer of the mammalian blastocyst that gives rise to the embryonic portion of the placenta.

ENHANCER A composite regulatory region that is composed of several distinct sequence elements that are bound by sequence-specific transcription factors that act positively or negatively on the transcription of an adjacent gene.

REGULATORYFACTORX FAMILY (RFX family). A family of evolutionarily related DNA-binding proteins that has diverse functions in eukaryotic organisms, ranging from yeast to man. There are five family members (RFX1, RFX2, RFX3, RFX4 and RFX5) in mammals.

to pathogens and tumours. Moreover, it is pivotal for the maintenance of self-tolerance, as well as for the breakdown of tolerance in autoimmune diseases.

To ensure tight control of these functions, MHC class II genes are themselves regulated in a precise cell-type-specific manner2. Their expression is largely restricted to thymic epithelial cells (TECs) and to cells that are specialized for the capture and presentation of extracellular antigens. The latter — which are col-lectively referred to as antigen-presenting cells (APCs) — include B cells, cells of the monocyte–macrophage lineage and dendritic cells (DCs). In humans, acti-vated T cells also express MHC class II molecules. Within APC lineages, MHC class II expression is fine tuned as a function of various parameters, including developmental stage, activation status and exposure to extracellular stimuli. For example, MHC class II expression increases in B cells that are stimulated with interleukin-4 (IL-4) and lipopolysaccharide, and in macrophages that are activated with interferon-γ (IFN-γ). Conversely, the differentiation of B cells into plasma cells, as well as the maturation of DCs, is con-comitant with a shut down of de novo MHC class II synthesis.

Cells other than TECs and APCs — such as fibro-blasts, ASTROCYTES, endothelial cells and epithelial cells — do not express MHC class II molecules unless they are exposed to specific stimuli, particularly IFN-γ, that are produced during infection, inflammation or trauma. With the notable exception of TROPHOBLASTS, cells other than TECs and APCs activate expression

of their MHC class II genes in response to stimula-tion with IFN-γ2. This IFN-γ-induced expression of MHC class II molecules can be further modulated by various secondary stimuli, including transforming growth factor-β (TGF-β), IFN-β, tumour-necrosis factor, IL-1, IL-10, infection with various pathogens, and certain drugs.

Regulation of MHC class II expressionMHC class II expression is controlled mainly at the level of transcription, by a highly conserved regula-tory module that is situated 150–300 base pairs (bp) upstream of the transcription-initiation site in all MHC class II genes2,8,9 (FIG. 1). This MHC-class-II-specific regulatory module, known as the SXY module, consists of four sequences — the S, X, X2 and Y boxes — that are present in a tightly constrained order, orientation and spacing (FIG. 1). Characteristic SXY modules are found in the promoters of the genes encoding the α-chain and β-chain of all MHC class II molecules in all vertebrate species that have been examined, includ-ing the genes encoding the three human MHC class II isotypes (HLA-DP, HLA-DQ and HLA-DR) and the two mouse MHC class II isotypes (H2-A and H2-E). Typical SXY modules are also found in the promoters of the genes encoding invariant chain (Ii) and the non-classical MHC class II molecules HLA-DM (known as H2-M in mice) and HLA-DO (known as H2-O in mice), which are accessory proteins that are required for intracellular trafficking and peptide loading of MHC class II molecules. More recently, additional SXY modules that function as ENHANCERS and control MHC class II genes from a distance have been identi-fied at distal positions throughout the MHC class II locus and in the first intron of the gene encoding Ii11,12. Sequences that resemble SXY modules also contribute to the function of MHC class I promoters2,8,9.

Four key trans-acting factors that regulate the trans-cription of MHC class II genes by interacting with the SXY module were identified by studying B-cell lines with regulatory defects in MHC class II expres-sion13,14 and by isolating the genes that are mutated in individuals with BLS, a hereditary immunodeficiency disease that results from an almost complete absence of MHC class II expression1,2 BOX 1. BLS is a geneti-cally heterogeneous disease that is caused by mutations in the genes encoding the regulatory factors CIITA, REGULATORY FACTOR X 5 (RFX5), RFX-associated protein (RFXAP) and RFX-associated ankyrin-containing protein (RFXANK)15–19. All four are dedicated to the transcription of MHC class II genes and related genes. They are, to a large extent, essential for this function, because only weak, residual MHC class II expression is retained in patients with BLS and in mice that lack RFX5 or CIITA1–5.

RFX5, RFXAP and RFXANK form the hetero-trimeric RFX complex, which specifically binds the X box of the SXY module16–20. RFX nucleates the assem-bly of a higher-order nucleoprotein complex through cooperative-binding interactions with the X2-box-specific cyclic-AMP-responsive-element-binding

Box 1 | Bare lymphocyte syndrome

Bare lymphocyte syndrome (BLS) — also known as MHC class II deficiency — is a rare autosomal recessive immunodeficiency1,2. In this disease, failure to express MHC class II molecules at the surface of all cells that would usually express them — including thymic epithelial cells, B cells, macrophages, dendritic cells and interferon-γ-stimulated cells — leads to the reduced positive selection of CD4+ T cells in the thymus and to the inability of mature CD4+ T cells to respond to antigens in the periphery. A combined deficiency in cellular and antibody-mediated immune responses ensues. As a result, patients suffer from severe and recurrent bacterial, viral, fungal and protozoan infections, mainly of the gastrointestinal, pulmonary, respiratory and urinary tracts. These infections lead to chronic diarrhoea, malabsorption, growth retardation, and death in early childhood. Bone-marrow transplantation remains the only curative therapy.

The genetic lesions that are responsible for BLS do not lie in the MHC class II genes themselves but in the genes encoding trans-acting factors that are required for their transcription. Patients have been assigned to four complementation groups (A, B, C and D), using cell-fusion experiments. The regulatory genes that are mutated in these groups encode the class II transactivator (CIITA), RFXANK (regulatory factor X (RFX)-associated ankyrin-containing protein), RFX5 and RFXAP (RFX associated protein), respectively2,15–19. Despite this genetic heterogeneity, BLS is phenotypically homogeneous in the sense that phenotypes that are restricted to a particular complementation group have not been identified1,2. Moreover, all clinical and immunological abnormalities can be accounted for by the absence of MHC class II expression, and no obvious perturbations of other biological systems have been documented. The same is true for mouse models of BLS that are generated by disruption of C2ta (the gene encoding CIITA) or Rfx5 REFS 35. Taken together, these observations imply that CIITA and the three RFX factors are, to a large extent, dedicated to the regulation of MHC class II genes.

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MHC class Imolecule

MHC class II molecule(HLA-DR, HLA-DQor HLA-DP)

CBP, p300, PCAF,BRG1, CARM1, TFIID,TFIIB and P-TEFb

Nuclear membrane

Highly regulatedpattern ofexpression

Plasma membrane

? RFX CREB

HLA-DO

HLA-DM

Ii

?

NFYTranscription

S X X2 Y

CIITA

CIITA

? RFX CREB NFY

S X X2 Y

?

RFX

CREBNFY

Gene encodingMHC class II molecule,HLA-DM, HLA-DO,Ii or MHC class I molecule

S X X2 Y

MHC class IIenhanceosome

Ubiquitouslyexpressedfactors

RFXANK

RFX

5

RFX

AP

protein (CREB), the Y-box-specific nuclear transcrip-tion factor Y (NFY) and a factor that binds the S box21–23 (FIG. 1). Although the S box can be bound by RFX in in vitro-binding assays, its function in enhancing the activity of MHC class II promoters is mediated by an as-yet-unidentified factor23. The multiprotein complex that is assembled at the SXY module — which is known as the MHC class II enhanceosome — is a platform to which CIITA is recruited by multiple synergistic protein–protein interactions2,8,9,24. The enhanceosome and CIITA then cooperate to activate transcription of MHC class II genes2,8,9.

The MHC class II transactivatorBecause several comprehensive reviews have addressed the structure and mode of action of CIITA2,8,9,25, we limit ourselves to summarizing the most salient points. Important features in the primary sequence of CIITA include an amino (N)-terminal region that is rich in acidic amino acids, three segments that are rich in proline, serine and threonine, a centrally placed GTP-binding domain, and a carboxy (C)-terminal region that consists of leucine-rich repeats (LRRs) BOX 2. The association of a central nucleotide-binding domain with a C-terminal LRR region is characteris-tic of the nucleotide-binding oligomerization domain (NOD) family of proteins, which is also known as the CATERPILLER family (caspase-recruitment domain (CARD), transcription enhancer, R (purine)-binding, pyrin, lots of LRRs family)26,27 BOX 2. The N-terminal moiety of CIITA contains transcription-activation domains that are thought to mediate interactions with effector proteins that are implicated in promoting transcription, including components of the GENERAL

TRANSCRIPTION MACHINERY, factors that are involved in CHROMATIN REMODELLING and other co-activators. The C-terminal two-thirds of the protein is implicated in self-association, localization to the nucleus and recruitment to the enhanceosome2,8,9.

CIITA functions as a non-DNA-binding co-activator that coordinates multiple events that are required for the activation of transcription (FIG. 1). It has been implicated in promoting transcription by various mechanisms: first, recruiting components such as trans-cription factor IID (TFIID) and TFIIB of the general transcription-initiation machinery28,29; second, induc-ing phosphorylation of RNA polymerase II REF. 30; third, interacting with the positive transcription elonga-tion factor b (P-TEFb)31; fourth, recruiting co-activators that alter chromatin accessibility by inducing histone acetylation or methylation25; and last, recruiting the chromatin-remodelling factor BRAHMARELATED GENE 1 (BRG1)32. CIITA has also been reported to have an endogenous histone-acetylase activity33. The relative importance of these effector functions and the precise order in which they are enlisted during the activation of MHC class II genes remain to be established.

The function of CIITA can be modulated by post-translational modifications. For example, phosphory-lation of certain residues in CIITA can increase its oligomerization, its interactions with other key

Figure 1 | Regulation of the transcription of MHC class II genes. The SXY module that is present in all classical MHC class II genes — as well as in the genes encoding invariant chain (Ii), HLA-DM, HLA-DO and MHC class I molecules — is bound cooperatively by four factors: the heterotrimeric X-box-binding factor regulatory factor X (RFX), which is composed of RFX5, RFX-associated protein (RFXAP) and RFX-associated ankyrin-containing protein (RFXANK); the X2-box-binding factor cyclic-AMP-responsive-element-binding protein (CREB); the Y-box-binding factor nuclear transcription factor Y (NFY); and an as-yet-unidentified S-box-binding factor. This multiprotein complex — which is known as the MHC class II enhanceosome — is a ‘landing pad’ for the class II transactivator (CIITA), which is a non-DNA-binding co-activator that is recruited by multiple protein–protein interactions with several components of the enhanceosome. CIITA coordinates the recruitment of additional factors that are involved in CHROMATIN MODIFICATION and remodelling: these are CREB-binding protein (CBP), p300, p300/CBP-associated factor (PCAF), brahma-related gene 1 (BRG1) and co-activator-associated arginine methyltransferase 1 (CARM1). CIITA also coordinates the recruitment of factors that are involved in transcription initiation (that is, transcription factor IID (TFIID) and TFIIB) and in transcription elongation (that is, positive transcription elongation factor b, P-TEFb).


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CARD

EBD

NOD1 protein NOD LRRs

CARDCARD NODNOD2 protein LRRs

PYD NODCryopyrin LRRs

CARD NODCIITA (isoform I) AD LRRs

NODPlant R proteins(95 in Arabidopsis thaliana) LRRsTIR

( (

CHROMATIN MODIFICATION Alterations that are induced in chromatin by enzymes that modify the extent of acetylation, methylation or other covalent modifications of histones.

GENERAL TRANSCRIPTION MACHINERY Factors that are required for the initiation of transcription of all genes that are transcribed by RNA polymerase II. They include RNA polymerase II itself and numerous general transcription factors that assemble at the core promoter (that is, the DNA sequence that surrounds the transcription-initiation site) of these genes.

CHROMATIN REMODELLING Alterations that are induced in chromatin through the displacement of nucleosomes by ATP-dependent multiprotein complexes.

factors and its ability to transactivate MHC class II promoters34,35. Conversely, phosphorylation at other sites downregulates CIITA function36. In addition, ubiq-uitylation of CIITA increases its ability to transactivate MHC class II genes37.

In contrast to the components of the enhance osome — which are produced widely in most cell types irre-spective of their MHC class II expression status — the synthesis of CIITA is tightly controlled. Indeed, it is the highly regulated pattern of transcription of the gene encoding CIITA (which is known as CIITA in humans and C2ta in mice and was originally mapped to the AIR1 locus (activator of immune-response gene Ir1 locus14)) that ultimately dictates where, when and to what level MHC class II genes are expressed2,8–10. This is exemplified by the following key observations. First, MHC class II expression by TECs and APCs strictly depends on activation of the CIITA or C2ta gene2,3,5–7,38. Second, IFN-γ-induced MHC class II expression by other cell types is mediated by induc-tion of expression of the CIITA or C2ta gene7,39–42. Third, the abrogation of MHC class II expression by trophoblasts, plasma cells, mature DCs and vari-ous types of tumour cell is a direct consequence of silencing of the CIITA or C2ta gene43–50. Last, stimuli that downregulate IFN-γ-induced MHC class II

expression — including TGF-β, IL-1, IL-4, IL-10, vari-ous pathogens and certain drugs — generally achieve this by interfering with induction of expression of the CIITA or C2ta gene31,51–66.

The classical and non-classical MHC class II genes and the Ii gene are the best-documented targets of CIITA. Although several other potential targets have been proposed, the influence of CIITA on their expression is minor, is not observed in an in vivo setting, is a result of indirect mechanisms that are distinct from those operating at MHC class II genes or is a subject of controversy10 BOX 3. CIITA is there-fore remarkably specific for MHC class II expression. Because of its restricted target-gene specificity and decisive regulatory role, CIITA is known as the mas-ter control factor or master regulator of MHC class II genes and related genes.

Regulation of the gene encoding CIITAThe role of CIITA as the master regulator of MHC class II expression has motivated a considerable amount of interest in the molecular mechanisms that control its expression. CIITA expression is regulated mainly at the level of transcription, although it can be further modulated by changes in mRNA and protein stability 67,68. Transcription of CIITA is driven by a large regulatory region that contains four distinct promot-ers, which are known as pI, pII, pIII and pIV REF. 42 (FIG. 2). Three of these promoters — pI, pIII and pIV — are strongly conserved in the mouse C2ta gene. A mouse equivalent of pII has not been identified, and the function of pII is not known; therefore, pII is not discussed further here.

Promoters pI, pIII and pIV precede alternative first exons that are spliced to the shared downstream exons, and this gives rise to three types of CIITA or C2ta mRNA, which differ at their 5′ ends (FIG. 2). All three mRNAs have a translation-initiation codon in the common second exon. However, the alternative 5′ exons that follow pI and pIII contain additional upstream initiation codons and therefore encode spe-cific N-terminal extensions of 101 and 24 amino acids, respectively. As a consequence, three CIITA isoforms that differ at their N termini are produced (FIG. 2). All three restore MHC class II expression when produced in CIITA-deficient B cells, and they all activate MHC class II genes to equivalent levels when expressed in cells of non-haematopoietic origin. Whether these isoforms have different functions is therefore not clear. The isoform that is derived from pI is, however, slightly more efficient than the others at activating MHC class II promoters (REF. 69, and S.L.L. and W.R., unpub-lished observations). This increased efficiency has been attributed to the N-terminal sequence that is encoded by the alternative first exon that follows pI, which con-tains a weak homology to CARDs69. This CARD-like sequence strengthens the similarity between CIITA and other NOD-family proteins, several of which contain an N-terminal CARD(s)26,27 BOX 2.

Cell-type-specific, cytokine-induced and devel-opmentally modulated MHC class II expression is

Box 2 | CIITA is a member of a family of structurally related proteins

The class II transactivator (CIITA) is a member of a family of cytosolic proteins that is known by several names, including the nucleotide-binding oligomerization domain (NOD)-protein family and the CATERPILLER (caspase-recruitment domain (CARD), transcription enhancer, R (purine)-binding, pyrin, lots of leucine-rich repeats (LRRs))-protein family26,27. Family members have a conserved tripartite structure that consists of a variable amino (N)-terminal effector-binding domain (EBD), a centrally located NOD and a carboxy-terminal region that contains a variable number of LRRs. The family is evolutionarily ancient, because this tripartite-domain arrangement is conserved in certain disease-resistance proteins (R proteins) that mediate immune responses in plants. In most mammalian family members, the N-terminal EBD is either a CARD or pyrin domain (PYD). The EBD in many R proteins is a Toll/interleukin-1 receptor (TIR) domain. CIITA is the only member that has an N-terminal transcription-activation domain (AD) and that has a documented role as a transcription factor. The N-terminal extension that is specific to the CIITA isoform derived from use of promoter I (pI) of the CIITA gene has weak CARD homology.

NOD proteins have diverse roles in inflammation, apoptosis and immunoregulation. NOD1 protein and NOD2 protein function as intracellular receptors for specific motifs in bacterial peptidoglycan. Mutations that affect the genes encoding several important family members are associated with immunological or inflammatory disorders: the CIITA gene in bare lymphocyte syndrome BOX 1; the NOD2 gene in Crohn’s disease and Blau syndrome; and the cold autoinflammatory syndrome 1 (CIAS1) gene (which encodes cryopyrin) in familial cold urticaria syndrome, Muckle–Wells syndrome and neonatal-onset multisystem inflammatory disease.


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BRAHMARELATED GENE 1 (BRG1). An ATPase subunit that is present in chromatin-remodelling complexes that are known as SWI–SNF (switching-defective–sucrose non-fermenting) complexes.

MICROGLIA Small glial cells that are distributed throughout the grey and white matter in the central-nervous system. They are monocyte-derived cells that invade neural tissue before birth and can differentiate into macrophages.

determined by transcriptional control of the CIITA or C2ta gene through the differential usage of pI, pIII and pIV. Initial experiments with established cell lines and several primary-cell populations indicated that pI is used mainly in DCs, that pIII is activated specifically in B cells and that pIV is induced by IFN-γ41,42. Subsequent in vitro studies, however, chal-lenged this relatively simple picture. For example, all three promoters were reported to be inducible by IFN-γ, and both pI and pIII were found to be active in DCs. In addition, pIII was shown to be used in cer-tain tumours of non-haematopoietic origin47,66,70–74. Therefore, to define the true specificity of these pro-moters, mice carrying targeted deletions of the regu-latory region of the C2ta gene were generated. The mice lack pIV carry a deletion that excises pIV and its associated first exon but leaves pI and pIII intact7. The mice that lack pIII and pIV (denoted p(III+IV)-deficient mice) carry a deletion that excises pIII, pIV and their associated first exons such that only pI remains intact6. The analysis of MHC class II expression in several cell types from these mice has led to an unambiguous and accurate definition of the functions of the three C2ta promoters in vivo. The ‘division of labour’ between these promoters is remarkably strict and is much more precise in vivo than was anticipated from earlier in vitro studies. Moreover, the promoters function independently of one another without any significant crosstalk.

Lessons from pIV-deficient mice. In vitro studies initially led to the suggestion that pIV is activated by IFN-γ in cells of the macrophage lineage, as well as numerous non-haematopoietic cells, including astrocytes, fibroblasts, endothelial cells and epithelial cells40–42,52,72. A key role for pIV in IFN-γ-mediated MHC class II gene induction was confirmed by the phenotype of pIV-deficient mice, which show selective abrogation of IFN-γ-induced MHC class II expression by a wide variety of cells of non-haematopoietic origin, including glandular, respiratory and intestinal epithe-lial cells, endothelial cells, astrocytes and fibroblasts7. In the absence of pIV, pI and pIII are not sufficient to support IFN-γ-induced MHC class II expression in these cell types, despite the fact that both promoters have been reported to be activated by IFN-γ in certain freshly isolated cells and in cell lines6,7,66,72–74. So, pIV is essential for driving IFN-γ-induced CIITA expression in cells of non-haematopoietic origin (FIG. 3).

In contrast to non-haematopoietic cells, IFN-γ-stimulated macrophages from pIV-deficient mice have normal levels of MHC class II expression7. This is true for peritoneal macrophages, as well as tissue-resident macrophages, such as MICROGLIA in the central-nervous system. Therefore, although transcription from pIV is activated in macrophages by IFN-γ 66,73–76, this does not markedly contribute to IFN-γ-induced MHC class II expression by these cells.

Another key role of pIV was uncovered by the find-ing that pIV-deficient mice have a marked reduction in CD4+ T-cell numbers7,38. This results from a severe defect in the positive selection of CD4+ T cells in the thymus, which is normally driven by MHC class II+ epithelial cells in the thymic cortex (cTECs)77. Positive selection of CD4+ T cells is abolished in pIV-deficient mice, because MHC class II expression by cTECs is eliminated7,38. CD4+ T-cell numbers are as low as those in mice that are deficient in MHC class II molecules, indicating that the defect in MHC class II expression by cTECs in pIV-deficient mice is complete. Owing to the absence of CD4+ T cells, protective immune responses are severely hampered7,38, and pIV-deficient mice succumb to infection with pathogens that are commonly found in non-barrier housing facili-ties (J.-M.W. and W.R., unpublished observations). Nevertheless, weak T-cell-dependent immune responses are observed, perhaps as a consequence of the posi-tive selection of some CD4+ T cells by MHC class II+ bone-marrow-derived APCs78.

Induction of pIV by IFN-γ. A conserved 300 bp pro-moter-proximal region is sufficient for the activation of pIV by IFN-γ. This region contains three regulatory elements — an IFN-γ-activated site (GAS), an E box and an IFN-regulatory factor (IRF) element (IRF-E) — all of which are required for the induction of pIV REFS 4042,72,75 (FIG. 4). The GAS and E box are bound cooperatively by signal transducer and activator of tran-scription 1 (STAT1) and upstream transcription factor 1 (USF1)41. The IRF-E is co-occupied by IRF1 REFS 40,41 and IRF2 REFS 79,80. Binding of STAT1 is induced by

Box 3 | Target-gene specificity of CIITA

By far the most well-established target genes of the class II transactivator (CIITA) remain those encoding MHC class II molecules and invariant chain, HLA-DM and HLA-DO proteins that are required for MHC-class-II-mediated antigen presentation2,8–10. CIITA also has a more limited role in the activation of MHC class I genes132,133. This high specificity of CIITA was recently challenged by reports indicating that it can influence — albeit, in most cases, relatively modestly — the expression of a surprising variety of genes that are involved in numerous distinct functions within and outside the immune system10. The plexin-A1 gene was found to be stimulated by CIITA in mouse dendritic cells134. DNA-microarray experiments identified more than 40 genes that were proposed to be upregulated by CIITA in human B cells and in interferon-γ-stimulated cells135. Genes that were proposed to be repressed by CIITA in specific cell types include those encoding interleukin-4 (IL-4), IL-10, CD95 ligand (also known as FAS ligand), cathepsin E, collagen type I α2, cyclin D1 and 16 additional proteins of diverse function135–139. Repression of the genes encoding IL-4, CD95 ligand, collagen type I α2, cyclin D1 and cathepsin E was proposed to be mediated by sequestration of the co-activator CBP (cyclic-AMP-responsive-element-binding protein (CREB)-binding protein) by CIITA136–139. The mechanisms that mediate activation or repression of the other candidate target genes have not been defined; however, they are likely to be distinct from those operating at MHC class II genes, because none of these genes contains the characteristic SXY module to which CIITA is recruited at MHC class II promoters. For most of the potential new target genes, the biological importance of their regulation by CIITA has not been evaluated in vivo. It remains to be determined whether their altered expression leads to a clear-cut phenotype in CIITA-deficient mice or patients with BLS. Furthermore, conflicting results have been reported for certain target genes, such as Il4 REFS 97,139,140. Consequently, until further studies have been carried out, CIITA should not be regarded as a pleiotropic factor having widespread functions that extend beyond its role in the control of MHC class II expression.


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pI pII pIII pIVExons 2–19

AUG Type IV mRNA

Type III mRNA

Type I mRNA

AUGAUG

121 kDa

121 kDa

121 kDa

CARD DE

Transcription activation:binding to TFIID, TFIIB,P-TEFb, CBP and PCAF

NLS1 NLS2 GTP NLS3 LRRsPST

132 kDa

124 kDa24 aa

AUGAUG

101 aa

( )

Recruitment to enhanceosome

Self-associationSelf-association Self-association

CIITA

FIBROSARCOMA A malignant tumour that is derived from connective-tissue fibroblasts.

REAGGREGATE THYMIC ORGAN CULTURE Three-dimensional thymus-lobe structures that are formed in cell culture by the reaggregation of cells that are present in mixtures of purified thymocytes and thymic stromal-cell subsets.

the classical IFN-γ-mediated signal-transduction path-way. On binding of IFN-γ to its receptor, activation of the receptor-associated protein tyrosine kinases Janus kinase 1 (JAK1) and JAK2 leads to the phosphory lation of STAT1, which then dimerizes, translocates to the nucleus and activates its target promoters, including pIV. IRF1 is encoded by a gene that is also controlled by STAT1, so its expression is also induced by IFN-γ. The dependence on IRF1 explains the delayed kinetics of IFN-γ-induced CIITA expression relative to the rapid induction of expression of genes that are controlled only by STAT1 REFS 39,81. The relative importance of STAT1 and IRF1 for the activation of pIV varies as a function of cell type40,41,72,76,82. For example, the GAS and the IRF-E are equally crucial in certain melanoma and FIBROSARCOMA cell lines41,72, whereas the IRF-E is more important than the GAS in astrocytes40,76.

Activation of pIV by IFN-γ depends on remodelling of the local chromatin structure. This requires BRG1, which is the ATPase subunit of some nucleosome-remodelling complexes83. Moreover, binding of STAT1 to pIV increases chromatin accessibility by inducing histone acetylation81.

Several cytokines suppress IFN-γ-induced expression of CIITA. For example, TGF-β inhibits IFN-γ-induced activation of pIV REFS 51,5355,72. The inhibitory effect of TGF-β is mediated by SMAD3 (mothers against decapentaplegic homologue 3)55. Several interleukins also inhibit the induction of CIITA expression in human astrocytes (IL-1) and mouse microglia (IL-4 or IL-10), but the molecular mechanisms that are implicated remain undefined52,53.

Trophoblasts are unique in that they cannot acti-vate MHC class II genes in response to IFN-γ. The absence of MHC class II molecules at the cell surface of trophoblasts is thought to have a crucial role in pre-venting rejection of the fetus by the maternal immune system. The inability of trophoblasts to express MHC class II genes is a consequence of epigenetic silencing of the gene encoding CIITA43,44. Most studies have indicated that DNA methylation is responsible for silencing43,44,50, but alternative mechanisms have also been proposed84,85.

Regulation of pIV in TECs. TECs retain MHC class II expression and the ability to drive positive selection of CD4+ T cells when cultured in three-dimensional REAGGREGATE THYMIC ORGAN CULTURES86. By contrast, they dedifferentiate and lose expression of MHC class II molecules when they are maintained in monolayer cultures87,88. This indicates that pIV activity depends on signals that are provided by the thymic micro-environment. However, the nature of these signals and the transcription factors on which they converge remain elusive38,89. Although CIITA and MHC class II expression can be induced by IFN-γ in ex vivo TEC lines and thymic stromal cell lines90,91, the mecha-nism operating in vivo is independent of the IFN-γ-mediated signalling pathway (FIG. 4). Indeed, positive selection of CD4+ T cells is normal in mice that lack essential components of the IFN-γ-mediated signal-ling pathway — including IFN-γ, the IFN-γ receptor, STAT1 and IRF1 REFS 92,93 — indicating that MHC class II expression by TECs is not perturbed in these mice. Moreover, a defect in CD4+ T-cell selection that results from the absence of MHC class II expression in the thymus has not been described in any other mouse that lacks a specific cytokine, cytokine receptor, STAT or IRF38,89. Other signalling pathways that are involved in the development and function of the thymus (such as the WNT-, fibroblast-growth-factor-, TGF-β and sonic-hedgehog-homologue-mediated pathways)94 have also not been implicated in driving MHC class II expression by TECs. Elucidation of the mechanism that mediates pIV activation in TECs therefore remains an important challenge.

Lessons from p(III+IV)-deficient mice. Several lines of evidence initially indicated that pIII is active in B cells. The first CIITA cDNA clone to be isolated was derived from a B-cell line and corresponded to a type III CIITA mRNA15. Moreover, transcripts that are derived from pIII are the most abundant of the three transcript types in B-cell lines, and pIII has B-cell specificity in

Figure 2 | Modular structure of the regulatory region of the gene encoding CIITA. The gene encoding the class II transactivator (CIITA) is controlled by three independent promoters (pI, pIII and pIV) that precede alternative first exons that are spliced to the shared downstream exons (exons 2–19). A fourth promoter (pII) is present in the human gene but is not conserved in the mouse gene. The three types of mRNA encoding CIITA (type I, III and IV) are derived from pI, pIII and pIV, and they encode protein isoforms of 132, 124 and 121 kDa, respectively. These isoforms differ only at their amino (N) termini. The region shared by all three isoforms contains an acidic domain (DE), three segments that are rich in proline, serine and threonine (PST), a centrally placed GTP-binding domain (GTP), a series of leucine-rich repeats (LRRs) and at least three nuclear-localization signals (NLS1, NLS2 and NLS3). The N-terminal one-third of the protein functions as a transcription-activation domain, whereas the central and carboxy-terminal regions are implicated in self-association and recruitment to the MHC class II enhanceosome. Effector proteins that have been shown to interact with the transcription-activation domain include cyclic-AMP-responsive-element-binding protein (CREB)-binding protein (CBP), p300/CBP-associated factor (PCAF), transcription factor IID (TFIID), TFIIB and positive transcription elongation factor b (P-TEFb).The type-I-specific N-terminal extension contains a sequence that shows a weak caspase-recruitment domain (CARD) homology. aa, amino acids.


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Normal CIITA or C2ta promoter specificities

CIITA orC2TA

Dysregulated expression in disease

Some melanomas,myelomas and gliomas Many tumours Various pathogens Statins

Exons 2–19pI pIII pIV

?

Cells of myeloidoriginConventional DCsMacrophages(+/– IFN-γ)

Cells of lymphoidoriginB cellsT cells (human)pDCs

Cells of non-haematopoieticoriginTECsIFN-γ-stimulated cells

TCELLDEPENDENT ANTIGEN CD4+ T-cell help is required for the production of high-affinity antibodies specific for this type of antigen.

PLASMACYTOID DC (pDC). A unique type of dendritic cell (DC). These cells are also known as interferon (IFN)-producing cells because they are the main source of type I IFNs (that is, IFN-α and IFN-β) during viral infections.

GLIOBLASTOMA A malignant tumour that is derived from glial cells.

reporter-gene assays42,95. That pIII is indeed crucial for B cells in vivo was shown by the finding that p(III+IV)-deficient mice lack CIITA and MHC class II expression in all B-cell subsets, including B2 cells in the spleen, lymph nodes, thymus, peritoneum and blood, B1 cells in the peritoneum and blood, and marginal-zone B cells in the spleen6. The functional consequence of the loss of expression of MHC class II molecules by B cells is exemplified by the failure of p(III+IV)-deficient mice to produce antibodies that are specific for TCELLDEPENDENT ANTIGENS6.

PLASMACYTOID DCs (pDCs) are a unique and highly specialized type of DC. They have also been referred to as IFN-producing cells, because their most remarkable characteristic is that they are the main source of type I IFNs during viral infection96. Surprisingly, pDCs from p(III+IV)-deficient mice are MHC class II– and lack CIITA6. This sets pDCs apart from all other DC sub-sets, which do not show altered CIITA or MHC class II expression in p(III+IV)-deficient mice. Defective MHC class II expression by pDCs results from the deletion of pIII rather than pIV, because CIITA and MHC class II expression are normal in pDCs from pIV-deficient mice6. The dependence of pDCs on pIII is consistent with the finding that most C2ta transcripts synthesized by wild-type mouse pDCs6 and human pDCs (S.L.L. and W.R., unpublished observations) are derived from pIII.

There are several aspects, associated with their function and origin, in which pDCs differ from conventional (that is, myeloid) DCs. Two distinctive

features are directly relevant to their dependence on pIII for driving MHC class II expression. First, compared with other DCs, pDCs have low antigen-presentation and T-cell-stimulatory activity96. This is, at least in part, a consequence of the reduced abundance of cell-surface MHC class II molecules, which is likely to result from their use of pIII rather than pI (which is used in conventional DCs). A second difference is associated with the origin of pDCs. Several lines of evidence indicate that, unlike conventional DCs, pDCs could be more closely related to lymphoid cells than myeloid cells96. Because pIII is highly specific to B cells, as well as to activated T cells in humans (dis-cussed later), the use of pIII in pDCs could stem from their heritage.

Differences in pIII function between humans and mice. IFN-γ can induce pIII of the CIITA gene in human fibrosarcoma and GLIOBLASTOMA cells72. This induction is mediated by a STAT1-dependent enhancer that is situated 5 kilobases (kb) upstream of the transcription-initiation site72 (FIG. 4). Surprisingly, this IFN-γ responsiveness of pIII is not observed in rodents: pIII is not induced by IFN-γ in primary rat astrocytes76 or mouse macrophages7,74. Moreover, pIII is not sufficient to drive IFN-γ-induced CIITA expres-sion in non-haematopoietic cells of pIV-deficient mice7. There is therefore a species-specific difference in the induction of pIII by IFN-γ, probably because the IFN-γ-responsive enhancer that is found upstream of human pIII is not present in the mouse gene.

Figure 3 | Regulation of CIITA expression in health and disease. The three regulatory modules (pI, pIII and pIV) of the class II transactivator gene (CIITA in humans and C2ta in mice) have distinct functions. The promoter pI is a myeloid-cell-specific promoter that is sufficient to drive CIITA expression by conventional dendritic cells (DCs) and interferon-γ (IFN-γ)-activated macrophages. The promoter pIII is a lymphoid-cell-specific promoter that is essential for CIITA expression by B cells and activated human T cells. It is also required for CIITA expression by plasmacytoid DCs (pDCs). The promoter pIV is essential for driving CIITA expression by thymic epithelial cells (TECs) and for mediating induction by IFN-γ in cells of non-haematopoietic origin, such as endothelial cells, epithelial cells, fibroblasts and astrocytes. Various pathogens have developed ways of interfering with the expression of CIITA, often by attenuating the activation of pIV in response to IFN-γ. Statins have been proposed to interfere with the activation of pIV by IFN-γ, but this mechanism is controversial. Many tumours tend to lose MHC class II expression as a result of epigenetic silencing of pIII or pIV. Conversely, certain melanomas, myelomas and gliomas acquire abnormal expression of MHC class II as a result of the constitutive activation of pIII or pIV.


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IRF1

USF1

USF1 IRF2

IRF1

IRF2

IRF1

–126 bp

Transcription+1 bp

Plasma membrane

Nuclear membrane

PP

P

STA

T1

JAK1 JAK2

IFN-γa

IFN-γreceptor

GAS E box IRF-E–110 bp –66 bp –55 bp

pIV

PP

PP

Transcription

Plasma membrane

Nuclear membrane

b

–5 kb

+1bp

–322 bp

GAS E box ARE1

–183 bp –65 bpETS–ISRE

E47 PU.1

ARE2

IRF4 CREB

Site B

OCT?

Site A

NF1?

IFN-γIFN-γreceptor

AML2or AML3pIII

PP

STAT1

TECs: unknown activation pathway

BLIMP1

It is well established that activated human T cells are MHC class II+, whereas activated mouse T cells have low or undetectable levels of MHC class II molecules97. This results from a difference in the function of pIII, which is activated strongly in human T cells98,99 but not in mouse T cells97. The molecular basis of this differential activation of pIII is not known.

Regulation of pIII. The molecular mechanisms that control the B-cell-specific activity of pIII have been studied in human cell lines (FIG. 4). A 320 bp promoter-proximal regulatory region is sufficient to confer B-cell specificity42,95. This region contains five sequences that are shown by genomic footprinting to be occupied in vivo100. At least two of these sequences — activation response element 1 (ARE1) and ARE2 — are key regu-latory elements in B-cell lines100,101. CREB1 and activat-ing transcription factor 1 (ATF1) have been proposed to bind ARE2 REF. 101. However, these factors are expressed widely, by many cell types, and are therefore unlikely to confer B-cell specificity. More recently, an E-box motif, as well as an ETS–IRSE motif (that is, a composite ETS-binding site and IFN-stimulated response element), has been described upstream of the ARE motifs102. The E-box-binding factor E47, the ETS-family member PU.1 (also known as SPI1) and IRF4 were found to bind these elements in vivo and to function synergistically to promote B-cell-specific activation of pIII REF. 102. All three factors have previously been shown to control the expression of genes that are required for the development or func-tion of B cells. Notably, synergy between E47, PU.1 and IRF4 has been implicated in the activation of several B-cell-specific genes. Interestingly, a polymorphism that is located adjacent to the ISRE was recently found to be associated with reduced expression of CIITA and increased susceptibility to autoimmune disease103.

The pattern of transcription-factor occupation that is observed at pIII in vivo differs slightly between activated human T cells and B cells98. In addition, the factors that bind the ARE1 and ETS–ISRE motifs in activated T cells differ from those that bind these motifs in B cells98,99. These differences are likely to reflect that constitutive expression in B cells and induced expression in T cells are mediated by distinct mechanisms.

MHC class II expression is lost during terminal dif-ferentiation of B cells into plasma cells, because the CIITA or C2ta gene is switched off 45,46. This repres-sion has been proposed to be mediated by binding of B-lymphocyte-induced maturation protein 1 (BLIMP1) to a site in pIII REFS 104,105. BLIMP1 is a transcriptional repressor that participates in plasma-cell differentiation by repressing B-cell gene-expression programmes. The BLIMP1-binding site in pIII overlaps with the ISRE, indicating that it might repress pIII by interfering with activation of pIII by IRF4 REF. 102 (FIG. 4).

Function of pI. Transcripts encoding CIITA are derived mainly from pI in most DC subsets, including mouse splenic DCs, mouse bone-marrow-derived DCs and

Figure 4 | Molecular regulation of pIII and pIV. a | Promoter IV (pIV) is activated in response to the classical interferon-γ (IFN-γ)-mediated signalling pathway. Binding of IFN-γ to its receptor induces the activation of Janus kinase 1 (JAK1) and JAK2, which leads to the phosphorylation, dimerization and nuclear translocation of signal transducer and activator of transcription 1 (STAT1). STAT1 binds cooperatively with upstream transcription factor 1 (USF1) to an IFN-γ-activated site (GAS)–E-box motif. STAT1 also activates expression of the gene encoding IFN-regulatory factor 1 (IRF1), which then binds (together with IRF2) its cognate IRF element (IRF-E) in pIV. The mechanisms that activate pIV in thymic epithelial cells (TECs) in response to signals from the thymic microenvironment are unknown, but these signals must be distinct from the IFN-γ-mediated induction pathway. b | Several regulatory elements have been defined in pIII, and factors that can recognize these elements have been identified in B and T cells by in vitro- and in vivo-binding studies. A member of the cyclic-AMP-responsive-element-binding protein (CREB) and activating transcription factor (ATF) family binds activation response element 2 (ARE2) in B and T cells. ARE1 is bound by acute myeloid leukaemia 2 (AML2) in B cells and by AML2 and AML3 in T cells. The E-box motif and the ETS–IRSE motif (that is, a composite ETS-binding site and IFN-stimulated response element) are bound by E47, and PU.1 and IRF4, respectively, in B cells. Silencing of pIII in plasma cells is thought to be mediated by binding of B-lymphocyte-induced maturation protein 1 (BLIMP1) to a site that coincides with the ISRE. Site A and site B are potential binding sites for octamer-binding transcription factors (OCTs) and nuclear factor 1 (NF1). The human gene contains an additional distal enhancer that is activated by STAT1 in response to the classical IFN-γ-mediated induction pathway. This distal IFN-γ-inducible enhancer is not conserved in the mouse gene. bp, base pairs; kb, kilobases.


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human monocyte-derived DCs7,42,47,73. This DC specificity was confirmed by the analysis of p(III+IV)-deficient mice, in which only pI remains functional6. With the exception of pDCs, MHC class II expression is normal in all DC subsets in p(III+IV)-deficient mice, including bone-marrow-derived DCs, epidermal Langerhans cells and conventional DCs in the spleen, thymus and lymph nodes6. So, pI is sufficient to sustain CIITA and MHC class II expression in all conventional DC subsets.

As observed for pIV-deficient mice, macrophages from the p(III+IV)-deficient mice express normal lev-els of basal and IFN-γ-induced MHC class II expres-sion, indicating that pI is sufficient for maintaining CIITA expression by these cells6,7. This was a surpris-ing finding in view of earlier reports showing that IFN-γ induces pIV in macrophage cell lines, perito neal macrophages and tissue-resident macrophages, such as microglia66,73–76. To reconcile these findings, we6 and others74 compared the induction of transcription from pI and pIV in macrophages that were stimulated with IFN-γ. The abundance of mRNAs that were derived from each promoter was found to increase rapidly. However, the levels of mRNA that were derived from pIV increased only transiently, whereas mRNA derived from pI reached a level that was tenfold higher (S.L.L. and W.R., unpublished observations), and its expression was sustained for long periods6,74. It is therefore the robust activation of pI that is crucial for maintaining CIITA and MHC class II expression in IFN-γ-stimulated macrophages. This observation, together with the finding that pI is the key promoter in all conventional DC subsets, led us to redefine pI as a myeloid-cell-specific promoter.

Regulation of pI. The molecular mechanisms that underlie the activation of transcription from pI in DCs remain largely unknown. A 400 bp promoter-proximal region is sufficient to confer specificity for cells of myeloid origin (S.L.L. and W.R., unpublished observations). This region contains several sequence motifs that are potential binding sites for known transcription factors, and footprints in or near some of these motifs have been observed in vivo in human monocyte-derived DCs47. However, these observations have not yet been reinforced by functional studies. Even less is known about the mechanisms that drive IFN-γ-mediated induction of pI in macrophages. In contrast to pIV, IFN-γ-responsive sequences have not been identified near pI, raising the possibility that pI is not controlled directly by the factors that mediate the classical IFN-γ-mediated signalling pathway. A likely alternative is that enhanced pI function is an indirect consequence of IFN-γ-induced macrophage activation, which could lead to an increase in the concentration or activity of transcription factors that are required for the expression of pI.

The activity of pI is markedly decreased in DCs when their maturation is induced by inflamma tory or pathogenic stimuli47. Changes in the synthesis, pep-tide loading and cellular localization of MHC class II

molecules are key aspects of DC maturation. The density of cell-surface MHC class II molecules is increased as a result of changes in the intracellular localization and an increase in the stability of pre-existing MHC class II proteins. By contrast, de novo MHC class II synthesis is shut down. This is a con-sequence of transcriptional inactivation of the CIITA or C2ta gene by a global repression mechanism that involves histone deacetylation of a large domain that spans the entire regulatory region of the gene47.

Expression of CIITA in diseasePathogens use various strategies to avoid protective immune responses that are mounted by the host. To inhibit the expression of MHC class II molecules and the establishment of CD4+ T-cell-dependent immune responses, many pathogens have developed ways of interfering with the function or expression of CIITA TABLE 1. The transcriptional transactivator (Tat) protein of HIV interferes with the function of CIITA by competing for binding to cyclin T1, a component of the transcription elongation factor P-TEFb31,56. Varicella-zoster virus57, human cytomegalovirus58,59, human parainfluenza virus type 3 REF. 60, Chlamydia trachomatis61, Toxoplasma gondii62, Mycobacterium tuberculosis63 and Mycobacterium bovis64 interfere at various known or unknown steps of the pathway that mediates the induction of CIITA expression by IFN-γ. For example, M. tuberculosis produces a 19 kDa lipo-protein that inhibits IFN-γ-induced expression of IRF1 and CIITA63.

In contrast to pathogens that target CIITA to evade the immune response, another emerging concept is that CIITA might interfere with the activity of certain pathogen-derived products TABLE 1. For example, CIITA can inhibit the replication of HIV and human T-cell leukaemia virus type 2 (HTLV-2) by block-ing the function of the viral transactivators Tat and transcriptional activator X 2 (Tax-2), respectively106,107.

The loss of MHC expression contributes to the abil-ity of cancers to escape host immune surveillance108. Because malignant cells are eliminated mainly by cyto-toxic CD8+ T cells, the loss of MHC class I expression is observed most frequently. However, because effi-cient rejection requires CD4+ T-cell help during both the priming and the effector phases of anti tumour responses109, silencing of MHC class II expression is also commonly observed. This is particularly rel-evant for cancers of haematopoietic origin48,110,111. For example, the loss of MHC class II expression is a negative prognostic marker in diffuse large B-cell lymphoma112. Similarly, the MHC class II expression level in certain mouse B-cell lymphomas correlates with increased immunogenicity and reduced tumori-genicity113. There is no clear relationship between MHC class II expression and prognosis for malignan-cies of non-haematopoietic origin114. Nevertheless, non-haematopoietic tumour cells also frequently lose their ability to activate MHC class II genes in response to IFN-γ. The main cause for the loss of constitutive or inducible MHC class II expression by tumour cells


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GLOMERULONEPHRITIS An inflammation of the kidney glomeruli that can result in destruction of the glomeruli and renal failure.

STATINSA family of inhibitors of hydroxymethyl glutaryl-coenzyme A reductase (HMG-CoA reductase), an enzyme that catalyses the conversion of HMG-CoA to l-mevalonate. These molecules are mainly used as cholesterol-lowering drugs, but they also have immunoregulatory and anti-inflammatory properties. l-Mevalonate and its metabolites are implicated in cholesterol synthesis and other intracellular pathways.

of haematopoietic or non-haematopoietic origin is epigenetic silencing of the gene encoding CIITA48–50. The mechanism that is implicated most frequently is DNA methylation at pIII or pIV.

By contrast, several tumours acquire abnormal MHC class II expression. Myeloma cells — the malignant counterpart of plasma cells — tend to reac-tivate MHC class II genes as a result of constitutive activation of transcription from pIV REF. 115. MHC class II+ myelomas might escape immune surveillance in vivo by various mechanisms, including induction of T-cell anergy through a lack of co-stimulatory molecule expression116. Certain melanomas and gliomas acquire MHC class II molecules as a result of constitutive activation of pIII REFS 70,117 or both pIII and pIV REF. 71. The existence of these highly malignant MHC class II+ tumours indicates that the loss of MHC class II expression is not always essential for immune escape.

The induction of MHC class II expression by non-haematopoietic cells is closely associated with patho-logical immune responses118. It is typically observed at the surface of the parenchyma and endothelia of various organs during allograft rejection, as well as in many autoimmune and inflammatory disorders. Although it has not been shown in most situations, this aberrant MHC class II expression is thought to

contribute to disease pathology. A good example is provided by a mouse model of GLOMERULONEPHRITIS in which disease development strictly depends on MHC class II expression by renal parenchymal cells119. This is probably mediated by pIV, which is induced by most inflammatory stimuli in the kidney82. Given its key role in driving CIITA expression in non-haematopoietic cells, pIV is likely to be implicated in most pathologi-cal situations that involve upregulation of MHC class II expression by non-bone-marrow-derived cells.

Therapeutic modulation of CIITA expressionDetailed knowledge of the regulation of CIITA expression paves the way for the development of novel therapeutic strategies aimed at the modulation of MHC-class-II-mediated antigen presentation. Inhibition of CIITA expression might be an advantage in situations in which upregulation of MHC class II expression is detrimental, such as after transplanta-tion or in autoimmune disease. Conversely, increasing CIITA expression could be beneficial for promoting tumour immunogenicity or bolstering protective immune responses to certain pathogens, particularly those that downregulate MHC class II expression.

STATINS (which are hydroxymethylglutaryl-coenzyme A (HMG-CoA)-reductase inhibitors) have recently received considerable attention because they

Table 1 | CIITA and host–pathogen interactions

Pathogen Effect observed References

Inhibition of CIITA and MHC class II expression

HIV HIV Tat protein interferes with the function of CIITA by competing with CIITA for binding to cyclin T1, a subunit of the transcription elongation factor P-TEFb

31,56

Varicella-zoster virus Inhibits IFN-γ-induced CIITA expression by downregulating the expression of STAT1 and JAK2

57

Human cytomegalovirus Inhibits IFN-γ-induced CIITA expression at a step downstream of STAT1 phosphorylation and nuclear translocationInhibits IFN-γ-induced CIITA expression by enhancing proteasome mediated degradation of JAK1

58

59

Human parainfluenza virus type 3

Inhibits IFN-γ-induced MHC class II expression directly, by interfering with the induction of CIITA expression at a step downstream of STAT1 activation, and indirectly, by inducing type I IFN expression, which targets a step downstream of CIITA

60

Chlamydia trachomatis Inhibits IFN-γ-induced CIITA expression by inducing the degradation of USF1

61

Toxoplasma gondii Inhibits IFN-γ-induced CIITA expression by an unknown mechanism 62

Mycobacterium tuberculosis

Produces a 19 kDa lipoprotein that inhibits IFN-γ-induced IRF1 and CIITA expression

63

Mycobacterium bovis BCG

Inhibits IFN-γ-induced CIITA expression by a mechanism that depends on NRAMP1

64

Inhibition of viral products by CIITA

HIV CIITA inhibits viral replication by blocking the function of the viral transactivator Tat

106

HTLV-2 CIITA inhibits viral replication by blocking the function of the viral transactivator Tax-2

107

BCG, bacillus Calmette–Guérin; CIITA, class II transactivator; HTLV-2, human T-cell leukaemia virus type 2; IFN, interferon; IRF1, IFN-regulatory factor 1; JAK, Janus kinase; NRAMP1, natural resistance-associated macrophage protein 1; P-TEFb, positive transcription elongation factor b; STAT1, signal transducer and activator of transcription 1; Tat, transcriptional transactivator; Tax-2, transcriptional activator X 2; USF1, upstream transcription factor 1.


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EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS (EAE). An experimental model of multiple sclerosis that is induced by immunization of susceptible animals with myelin-derived antigens, such as myelin basic protein, proteolipid protein or myelin oligodendrocyte glycoprotein.

COLLAGENINDUCED ARTHRITIS (CIA). An experimental model of rheumatoid arthritis that is induced by immunization of susceptible animals with collagen type II.

DNAALKYLATING AGENT An anticancer drug that is cytotoxic to rapidly proliferating cells because it leads to the alkylation of bases in DNA.

have anti-inflammatory properties that might be beneficial for the treatment of autoimmune diseases. They were found to protect against EXPERIMENTAL AUTO

IMMUNE ENCEPHALOMYELITIS66. A protective effect was also documented for COLLAGENINDUCED ARTHRITIS120, although discordant results were reported by oth-ers121. Furthermore, a recent clinical trial in patients with rheumatoid arthritis showed a weak anti-inflammatory trend in treated individuals122. The favourable outcome for individuals with auto immune disorders was proposed to result, among other effects, from an inhibition of MHC class II expression. However, the mechanism that has been proposed to be responsible for this inhibition is controversial; the initial hypothesis that statins attenuate IFN-γ-induced CIITA expression65,66 was recently challenged by evidence indicating that another mechanism — namely, an impairment of cell-surface MHC class II expression levels — is likely to be responsible123.

The idea that MHC class II expression by tumour cells can increase their immunogenicity has fostered hopes that it might be possible to boost antitumour immune responses using tumour cells that have been rendered MHC class II+ using CIITA expression vec-tors. Although this was not successful in a model of lung carcinoma124, this strategy increased tumour immunogenicity and rejection, and elicited tumour-antigen-specific immune memory, in a mouse model of mammary adenocarcinoma125. Promising results were also obtained by combining overexpression of CIITA with downregulation of Ii expression126. Such MHC class II+Ii– tumour cells have been shown to be both prophylactic and therapeutic in studies of primary and metastatic cancers in mice127. Downregulation of Ii expression was proposed to increase the potency of antitumour vaccines by favouring the presenta-tion of endogenous antigens over exogenous anti-gens. However, the precise pathway that is involved in the presentation of endogenous antigens by MHC class II+Ii– tumour cells remains unclear128. The efficacy of such approaches can be increased by combining them with adjuvant therapies, such as radiotherapy and administration of cytokines, to strengthen the antitumour response129.

Another promising anticancer strategy relies on therapeutic monoclonal antibodies specific for MHC class II molecules. Although encouraging preclinical results have been obtained using this approach130, clinical trials (mainly for the treatment of B-cell

malignancies) have not lived up to expectations so far131. However, the efficacy of this new treatment might benefit from an improved understanding of the regulation of MHC class II expression in cancer cells. One could, for example, envisage coupling MHC-class-II-specific antibodies with drugs, such as DNAALKYLATING AGENTS, that prevent silencing of the CIITA gene, and this could prevent methylation-dependent silencing of pIII in lymphomas49.

Concluding remarksWe have acquired a remarkably detailed understanding of how CIITA expression is regulated in vivo by mul-tiple promoters that have different cell-type-specific and inducible activities. However, several important gaps in our knowledge remain. What are the signal-transduction pathways and transcription factors that activate pIV in TECs? Is pIII activated through the same pathways or distinct pathways in B cells and pDCs? What is responsible for the activation of pI in DCs? How is pI turned on in IFN-γ-activated macrophages? What mechanisms trigger silencing of the gene encoding CIITA when B cells differentiate into plasma cells and when DCs mature? And last, which features of the CIITA or C2ta gene confer its high propensity to undergo epigenetic silencing in tumour cells? Answering many of these questions will be particularly challenging, because we are hampered by the absence of suitable TEC, DC and pDC lines that accurately reproduce the pattern of MHC class II gene regulation that is shown by the corresponding primary cells in vivo. Consequently, studying the regulation of CIITA expression in these cells will require time-consuming in vivo approaches or sophisticated in vitro experiments with small numbers of primary cells. Nevertheless, we can expect active progress in these areas, because research into the molecular mechanisms that regulate MHC class II expression is driven by the combined interests of both fundamental biologists and clinicians who are studying the function of the adaptive immune system in health and its dysregulation in dis-ease. We anticipate that this research will set the stage for the development of novel therapeutic interventions based on the modulation of MHC class II expression, including strategies that are aimed at boosting protec-tive immune responses to pathogens and tumours or at attenuating unwanted immune responses during transplantation, and autoimmune and inflammatory diseases.

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3. Chang, C. H., Guerder, S., Hong, S. C., van Ewijk, W. & Flavell, R. A. Mice lacking the MHC class II transactivator (CIITA) show tissue-specific impairment of MHC class II expression. Immunity 4, 167–178 (1996).This paper, together with reference 5, describes the generation of C2ta-knockout mice, which have a global loss of MHC class II expression, thereby reproducing the phenotype of patients with BLS.

4. Clausen, B. E. et al. Residual MHC class II expression on mature dendritic cells and activated B cells in RFX5-deficient mice. Immunity 8, 143–155 (1998).

5. Itoh-Lindstrom, Y. et al. Reduced IL-4-, lipopolysaccharide-, and IFN-γ-induced MHC class II expression in mice lacking class II transactivator due to targeted deletion of the GTP-binding domain. J. Immunol. 163, 2425–2431 (1999).

6. LeibundGut-Landmann, S., Waldburger, J. M., Reis e Sousa, C., Acha-Orbea, H. & Reith, W. MHC class II expression is differentially regulated in plasmacytoid and conventional dendritic cells. Nature Immunol. 5, 899–908 (2004).This paper describes the generation of mice that lack pIII and pIV of the C2ta gene and shows that pDCs

differ from all other DC subsets with respect to C2ta promoter usage. The results define pI as a myeloid-cell-specific promoter and pIII as a lymphoid-cell-specific promoter.

7. Waldburger, J. M., Suter, T., Fontana, A., Acha-Orbea, H. & Reith, W. Selective abrogation of major histocompatibility complex class II expression on extrahematopoietic cells in mice lacking promoter IV of the class II transactivator gene. J. Exp. Med. 194, 393–406 (2001).This paper describes the generation of mice that lack pIV of the C2ta gene. It shows that this promoter is essential for driving IFN-γ-induced CIITA and MHC class II expression by non-haematopoietic cells but is dispensable for this function in macrophages.


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8. Ting, J. P. & Trowsdale, J. Genetic control of MHC class II expression. Cell 109, S21–S33 (2002).

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11. Krawczyk, M. et al. Long distance control of MHC class II expression by multiple distal enhancers regulated by regulatory factor X complex and CIITA. J. Immunol. 173, 6200–6210 (2004).

12. Masternak, K., Peyraud, N., Krawczyk, M., Barras, E. & Reith, W. Chromatin remodeling and extragenic transcription at the MHC class II locus control region. Nature Immunol. 4, 132–137 (2003).References 11 and 12 show that CIITA is recruited not only to MHC class II promoters but also to multiple distant enhancers that are scattered throughout the MHC class II locus. One of these is situated in a locus control region that is found upstream of the HLA-DRA gene.

13. Accolla, R. S. Human B cell variants immunoselected against a single Ia antigen subset have lost expression in several Ia antigen subsets. J. Exp. Med. 157, 1053–1058 (1983).

14. Accolla, R. S. et al. aIr-1, a newly found locus on mouse chromosome 16 encoding a trans-acting activator factor for MHC class II gene expression. J. Exp. Med. 164, 369–374 (1986).

15. Steimle, V., Otten, L. A., Zufferey, M. & Mach, B. Complementation cloning of an MHC class II transactivator mutated in hereditary MHC class II deficiency. Cell 75, 135–146 (1993).This seminal work describes isolation of the gene encoding CIITA and shows that it is mutated in certain in vitro-generated MHC class II– cell lines and in patients with BLS who are classified in complementation group A.

16. Steimle, V. et al. A novel DNA binding regulatory factor is mutated in primary MHC class II deficiency (bare lymphocyte syndrome). Genes Dev. 9, 1021–1032 (1995).

17. Masternak, K. et al. A gene encoding a novel RFX-associated transactivator is mutated in the majority of MHC class II deficiency patients. Nature Genet. 20, 273–277 (1998).

18. Durand, B. et al. RFXAP, a novel subunit of the RFX DNA binding complex is mutated in MHC class II deficiency. EMBO J. 16, 1045–1055 (1997).

19. Nagarajan, U. M. et al. RFX-B is the gene responsible for the most common cause of the bare lymphocyte syndrome, an MHC class II immunodeficiency. Immunity 10, 153–162 (1999).References 16–19 describe isolation of the genes encoding the RFXANK, RFX5 and RFXAP subunits of the RFX complex. They also show that these genes are mutated in patients with BLS who are classified in complementation group B, C and D, respectively.

20. Reith, W. et al. Congenital immunodeficiency with a regulatory defect in MHC class II gene expression lacks a specific HLA-DR promoter binding protein, RF-X. Cell 53, 897–906 (1988).

21. Moreno, C. S., Beresford, G. W., Louis-Plence, P., Morris, A. C. & Boss, J. M. CREB regulates MHC class II expression in a CIITA-dependent manner. Immunity 10, 143–151 (1999).

22. Mantovani, R. The molecular biology of the CCAAT-binding factor NF-Y. Gene 239, 15–27 (1999).

23. Muhlethaler-Mottet, A. et al. The S box of major histocompatibility complex class II promoters is a key determinant for recruitment of the transcriptional co-activator CIITA. J. Biol. Chem. 279, 40529–40535 (2004).

24. Masternak, K. et al. CIITA is a transcriptional coactivator that is recruited to MHC class II promoters by multiple synergistic interactions with an enhanceosome complex. Genes Dev. 14, 1156–1166 (2000).This paper was the first to show that CIITA is physically recruited to the promoters of its target genes in vivo and in vitro through multiple protein–protein interactions with an enhanceosome complex formed by DNA-bound transcription factors.

25. Zika, E. & Ting, J. P. Epigenetic control of MHC class II: interplay between CIITA and histone-modifying enzymes. Curr. Opin. Immunol. 17, 58–64 (2005).

26. Ting, J. P. & Davis, B. K. CATERPILLER: a novel gene family important in immunity, cell death, and diseases. Annu. Rev. Immunol. 23, 387–414 (2005).

27. Inohara, N., Chamaillard, M., McDonald, C. & Nunez, G. NOD-LRR proteins: role in host–microbial interactions and inflammatory disease. Annu. Rev. Biochem. 74, 355–383 (2005).

28. Fontes, J. D., Jiang, B. & Peterlin, B. M. The class II trans-activator CIITA interacts with the TBP-associated factor TAFII32. Nucleic Acids Res. 25, 2522–2528 (1997).

29. Mahanta, S. K., Scholl, T., Yang, F. C. & Strominger, J. L. Transactivation by CIITA, the type II bare lymphocyte syndrome-associated factor, requires participation of multiple regions of the TATA box binding protein. Proc. Natl Acad. Sci. USA 94, 6324–6329 (1997).

30. Spilianakis, C. et al. CIITA regulates transcription onset via Ser5-phosphorylation of RNA Pol II. EMBO J. 22, 5125–5136 (2003).

31. Kanazawa, S., Okamoto, T. & Peterlin, B. M. Tat competes with CIITA for the binding to P-TEFb and blocks the expression of MHC class II genes in HIV infection. Immunity 12, 61–70 (2000).

32. Mudhasani, R. & Fontes, J. D. The class II transactivator requires brahma-related gene 1 to activate transcription of major histocompatibility complex class II genes. Mol. Cell. Biol. 22, 5019–5026 (2002).

33. Raval, A. et al. Transcriptional coactivator, CIITA, is an acetyltransferase that bypasses a promoter requirement for TAFII250. Mol. Cell 7, 105–115 (2001).

34. Tosi, G., Jabrane-Ferrat, N. & Peterlin, B. M. Phosphorylation of CIITA directs its oligomerization, accumulation and increased activity on MHCII promoters. EMBO J. 21, 5467–5476 (2002).

35. Sisk, T. J., Nickerson, K., Kwok, R. P. & Chang, C. H. Phosphorylation of class II transactivator regulates its interaction ability and transactivation function. Int. Immunol. 15, 1195–1205 (2003).

36. Greer, S. F. et al. Serine residues 286, 288, and 293 within the CIITA: a mechanism for down-regulating CIITA activity through phosphorylation. J. Immunol. 173, 376–383 (2004).

37. Greer, S. F., Zika, E., Conti, B., Zhu, X. S. & Ting, J. P. Enhancement of CIITA transcriptional function by ubiquitin. Nature Immunol. 4, 1074–1082 (2003).

38. Waldburger, J. M. et al. Promoter IV of the class II transactivator gene is essential for positive selection of CD4+ T cells. Blood 101, 3550–3559 (2003).This paper shows that pIV of the C2ta gene is essential for driving positive selection of CD4+ T cells, because it is required for the induction of CIITA and MHC class II expression in TECs.

39. Steimle, V., Siegrist, C.-A., Mottet, A., Lisowska-Grospierre, B. & Mach, B. Regulation of MHC class II expression by interferon-γ mediated by the transactivator gene CIITA. Science 265, 106–109 (1994).This seminal paper was the first to show that IFN-γ-induced MHC class II expression is mediated by the induction of CIITA expression.

40. Dong, Y., Rohn, W. M. & Benveniste, E. N. IFN-γ regulation of the type IV class II transactivator promoter in astrocytes. J. Immunol. 162, 4731–4739 (1999).

41. Muhlethaler-Mottet, A., Di Berardino, W., Otten, L. A. & Mach, B. Activation of the MHC class II transactivator CIITA by interferon-γ requires cooperative interaction between Stat1 and USF-1. Immunity 8, 157–166 (1998).This paper was the first detailed description of the molecular mechanisms that mediate the IFN-γ induction of pIV of CIITA.

42. Muhlethaler-Mottet, A., Otten, L. A., Steimle, V. & Mach, B. Expression of MHC class II molecules in different cellular and functional compartments is controlled by differential usage of multiple promoters of the transactivator CIITA. EMBO J. 16, 2851–2860 (1997).This key paper was the first to describe how multiple promoters with different functions drive transcription of the gene encoding CIITA.

43. van den Elsen, P. J. et al. Lack of CIITA expression is central to the absence of antigen presentation functions of trophoblast cells and is caused by methylation of the IFN-γ inducible promoter (PIV) of CIITA. Hum. Immunol. 61, 850–862 (2000).

44. Morris, A. C., Spangler, W. E. & Boss, J. M. Methylation of class II trans-activator promoter IV: a novel mechanism of MHC class II gene control. J. Immunol. 164, 4143–4149 (2000).References 43 and 44 report that the gene encoding CIITA is irreversibly silenced in trophoblasts by DNA methylation of pIV.

45. Silacci, P., Mottet, A., Steimle, V., Reith, W. & Mach, B. Developmental extinction of major histocompatibility complex class II gene expression in plasmocytes is mediated by silencing of the transactivator gene CIITA. J. Exp. Med. 180, 1329–1336 (1994).

46. Sartoris, S., Tosi, G., De Lerma, B., Cestari, T. & Accolla, R. S. Active suppression of the class II transactivator-encoding AIR-1 locus is responsible for the lack of major histocompatibility complex class II gene expression observed during differentiation from B cells to plasma cells. Eur. J. Immunol. 26, 2456–2460 (1996).

47. Landmann, S. et al. Maturation of dendritic cells is accompanied by rapid transcriptional silencing of class II transactivator (CIITA) expression. J. Exp. Med. 194, 379–392 (2001).

48. Holling, T. M., Schooten, E., Langerak, A. W. & van den Elsen, P. J. Regulation of MHC class II expression in human T-cell malignancies. Blood 103, 1438–1444 (2004).

49. Murphy, S. P., Holtz, R., Lewandowski, N., Tomasi, T. B. & Fuji, H. DNA alkylating agents alleviate silencing of class II transactivator gene expression in L1210 lymphoma cells. J. Immunol. 169, 3085–3093 (2002).

50. van der Stoep, N., Biesta, P., Quinten, E. & van den Elsen, P. J. Lack of IFN-γ-mediated induction of the class II transactivator (CIITA) through promoter methylation is predominantly found in developmental tumor cell lines. Int. J. Cancer 97, 501–507 (2002).

51. Lee, Y. J. et al. TGF-β suppresses IFN-γ induction of class II MHC gene expression by inhibiting class II transactivator messenger RNA expression. J. Immunol. 158, 2065–2075 (1997).

52. Rohn, W., Tang, L. P., Dong, Y. & Benveniste, E. N. IL-1β inhibits IFN-γ-induced class II MHC expression by suppressing transcription of the class II transactivator gene. J. Immunol. 162, 886–896 (1999).

53. O’Keefe, G. M., Nguyen, V. T. & Benveniste, E. N. Class II transactivator and class II MHC gene expression in microglia: modulation by the cytokines TGF-β, IL-4, IL-13 and IL-10. Eur. J. Immunol. 29, 1275–1285 (1999).

54. Navarrete, S. A., Kehlen, A., Schutte, W., Langner, J. & Riemann, D. Regulation by transforming growth factor-β1 of class II mRNA and protein expression in fibroblast-like synoviocytes from patients with rheumatoid arthritis. Int. Immunol. 10, 601–607 (1998).

55. Dong, Y., Tang, L., Letterio, J. J. & Benveniste, E. N. The Smad3 protein is involved in TGF-β inhibition of class II transactivator and class II MHC expression. J. Immunol. 167, 311–319 (2001).

56. Okamoto, H., Asamitsu, K., Nishimura, H., Kamatani, N. & Okamoto, T. Reciprocal modulation of transcriptional activities between HIV-1 Tat and MHC class II transactivator CIITA. Biochem. Biophys. Res. Commun. 279, 494–499 (2000).

57. Abendroth, A. et al. Modulation of major histocompatibility class II protein expression by varicella-zoster virus. J. Virol. 74, 1900–1907 (2000).

58. Le Roy, E., Muhlethaler-Mottet, A., Davrinche, C., Mach, B. & Davignon, J. L. Escape of human cytomegalovirus from HLA-DR-restricted CD4+ T-cell response is mediated by repression of γ interferon-induced class II transactivator expression. J. Virol. 73, 6582–6589 (1999).

59. Miller, D. M. et al. Human cytomegalovirus inhibits major histocompatibility complex class II expression by disruption of the Jak/Stat pathway. J. Exp. Med. 187, 675–683 (1998).

60. Gao, J. et al. Human parainfluenza virus type 3 inhibits γ interferon-induced major histocompatibility complex class II expression directly and by inducing α/β interferon. J. Virol. 75, 1124–1131 (2001).

61. Zhong, G., Fan, T. & Liu, L. Chlamydia inhibits interferon γ-inducible major histocompatibility complex class II expression by degradation of upstream stimulatory factor 1. J. Exp. Med. 189, 1931–1938 (1999).

62. Luder, C. G. et al. Toxoplasma gondii inhibits MHC class II expression in neural antigen-presenting cells by down-regulating the class II transactivator CIITA. J. Neuroimmunol. 134, 12–24 (2003).

63. Pai, R. K., Convery, M., Hamilton, T. A., Boom, W. H. & Harding, C. V. Inhibition of IFN-γ-induced class II transactivator expression by a 19-kDa lipoprotein from Mycobacterium tuberculosis: a potential mechanism for immune evasion. J. Immunol. 171, 175–184 (2003).

64. Wojciechowski, W., DeSanctis, J., Skamene, E. & Radzioch, D. Attenuation of MHC class II expression in macrophages infected with Mycobacterium bovis bacillus Calmette–Guerin involves class II transactivator and depends on the Nramp1 gene. J. Immunol. 163, 2688–2696 (1999).

65. Kwak, B., Mulhaupt, F., Myit, S. & Mach, F. Statins as a newly recognized type of immunomodulator. Nature Med. 6, 1399–1402 (2000).

66. Youssef, S. et al. The HMG-CoA reductase inhibitor, atorvastatin, promotes a TH2 bias and reverses paralysis in central nervous system autoimmune disease. Nature 420, 78–84 (2002).


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67. De Lerma, B. A., Procopio, F. A., Mortara, L., Tosi, G. & Accolla, R. S. The MHC class II transactivator (CIITA) mRNA stability is critical for the HLA class II gene expression in myelomonocytic cells. Eur. J. Immunol. 35, 603–611 (2005).

68. Schnappauf, F. et al. N-terminal destruction signals lead to rapid degradation of the major histocompatibility complex class II transactivator CIITA. Eur. J. Immunol. 33, 2337–2347 (2003).

69. Nickerson, K. et al. Dendritic cell-specific MHC class II transactivator contains a caspase recruitment domain that confers potent transactivation activity. J. Biol. Chem. 276, 19089–19093 (2001).

70. Deffrennes, V. et al. Constitutive expression of MHC class II genes in melanoma cell lines results from the transcription of class II transactivator abnormally initiated from its B cell-specific promoter. J. Immunol. 167, 98–106 (2001).

71. Goodwin, B. L. et al. Varying functions of specific major histocompatibility class II transactivator promoter III and IV elements in melanoma cell lines. Cell Growth Differ. 12, 327–335 (2001).

72. Piskurich, J. F., Linhoff, M. W., Wang, Y. & Ting, J. P. Two distinct γ interferon-inducible promoters of the major histocompatibility complex class II transactivator gene are differentially regulated by STAT1, interferon regulatory factor 1, and transforming growth factor β. Mol. Cell. Biol. 19, 431–440 (1999).This paper shows that pIII of the human CIITA gene can be induced by IFN-γ through a STAT1-dependent enhancer that is situated 5 kb upstream.

73. Suter, T. et al. Dendritic cells and differential usage of the MHC class II transactivator promoters in the central nervous system in experimental autoimmune encephalitis. Eur. J. Immunol. 30, 794–802 (2000).

74. Pai, R. K., Askew, D., Boom, W. H. & Harding, C. V. Regulation of class II MHC expression in APCs: roles of types I, III, and IV class II transactivator. J. Immunol. 169, 1326–1333 (2002).Together with reference 6, this detailed kinetic study of CIITA expression shows that transcription from pI is sustained in IFN-γ-stimulated macrophages, whereas pIV is induced only transiently.

75. O’Keefe, G. M., Nguyen, V. T., Ping Tang, L. L. & Benveniste, E. N. IFN-γ regulation of class II transactivator promoter IV in macrophages and microglia: involvement of the suppressors of cytokine signaling-1 protein. J. Immunol. 166, 2260–2269 (2001).

76. Nikcevich, K. M., Piskurich, J. F., Hellendall, R. P., Wang, Y. & Ting, J. P. Differential selectivity of CIITA promoter activation by IFN-γ and IRF-1 in astrocytes and macrophages: CIITA promoter activation is not affected by TNF-α. J. Neuroimmunol. 99, 195–204 (1999).

77. Starr, T. K., Jameson, S. C. & Hogquist, K. A. Positive and negative selection of T cells. Annu. Rev. Immunol. 21, 139–176 (2003).

78. Martinic, M. M. et al. Efficient T cell repertoire selection in tetraparental chimeric mice independent of thymic epithelial MHC. Proc. Natl Acad. Sci. USA 100, 1861–1866 (2003).

79. Xi, H. et al. Co-occupancy of the interferon regulatory element of the class II transactivator (CIITA) type IV promoter by interferon regulatory factors 1 and 2. Oncogene 18, 5889–5903 (1999).

80. Xi, H., Goodwin, B., Shepherd, A. T. & Blanck, G. Impaired class II transactivator expression in mice lacking interferon regulatory factor-2. Oncogene 20, 4219–4227 (2001).

81. Morris, A. C., Beresford, G. W., Mooney, M. R. & Boss, J. M. Kinetics of a γ interferon response: expression and assembly of CIITA promoter IV and inhibition by methylation. Mol. Cell. Biol. 22, 4781–4791 (2002).This paper reports a detailed time course of the events that lead to the activation of pIV by IFN-γ.

82. Takeuchi, O. et al. Differential usage of class II transactivator promoters PI and PIV during inflammation and injury in kidney. J. Am. Soc. Nephrol. 14, 2823–2832 (2003).

83. Pattenden, S. G., Klose, R., Karaskov, E. & Bremner, R. Interferon-γ-induced chromatin remodeling at the CIITA locus is BRG1 dependent. EMBO J. 21, 1978–1986 (2002).

84. Holtz, R., Choi, J. C., Petroff, M. G., Piskurich, J. F. & Murphy, S. P. Class II transactivator (CIITA) promoter methylation does not correlate with silencing of CIITA transcription in trophoblasts. Biol. Reprod. 69, 915–924 (2003).

85. Murphy, S. P., Choi, J. C. & Holtz, R. Regulation of major histocompatibility complex class II gene expression in trophoblast cells. Reprod. Biol. Endocrinol. 2, 52 (2004).

86. Anderson, G., Jenkinson, E. J., Moore, N. C. & Owen, J. J. MHC class II-positive epithelium and mesenchyme cells are both required for T-cell development in the thymus. Nature 362, 70–73 (1993).

87. Anderson, G. & Jenkinson, E. J. Lymphostromal interactions in thymic development and function. Nature Rev. Immunol. 1, 31–40 (2001).

88. Anderson, G., Hare, K. J., Platt, N. & Jenkinson, E. J. Discrimination between maintenance- and differentiation-inducing signals during initial and intermediate stages of positive selection. Eur. J. Immunol. 27, 1838–1842 (1997).

89. Honey, K. & Rudensky, A. The pIV-otal class II transactivator promoter regulates major histocompatibility complex class II expression in the thymus. J. Exp. Med. 194, F15–F18 (2001).

90. Rigaud, G. et al. Induction of CIITA and modification of in vivo HLA-DR promoter occupancy in normal thymic epithelial cells treated with IFN-γ: similarities and distinctions with respect to HLA-DR-constitutive B cells. J. Immunol. 156, 4254–4258 (1996).

91. Hare, K. J., Jenkinson, E. J. & Anderson, G. In vitro models of T cell development. Semin. Immunol. 11, 3–12 (1999).

92. Dalton, D. K. et al. Multiple defects of immune cell function in mice with disrupted interferon-γ genes. Science 259, 1739–1742 (1993).

93. Huang, S. et al. Immune response in mice that lack the interferon-γ receptor. Science 259, 1742–1745 (1993).

94. Gill, J. et al. Thymic generation and regeneration. Immunol. Rev. 195, 28–50 (2003).

95. Lennon, A. M. et al. Isolation of a B-cell-specific promoter for the human class II transactivator. Immunogenetics 45, 266–273 (1997).

96. Colonna, M., Trinchieri, G. & Liu, Y. J. Plasmacytoid dendritic cells in immunity. Nature Immunol. 5, 1219–1226 (2004).

97. Otten, L. A. et al. Deregulated MHC class II transactivator expression leads to a strong TH2 bias in CD4+ T lymphocytes. J. Immunol. 170, 1150–1157 (2003).

98. Holling, T. M., van der Stoep, N., Quinten, E. & van den Elsen, P. J. Activated human T cells accomplish MHC class II expression through T cell-specific occupation of class II transactivator promoter III. J. Immunol. 168, 763–770 (2002).

99. Wong, A. W. et al. Regulation and specificity of MHC2TA promoter usage in human primary T lymphocytes and cell line. J. Immunol. 169, 3112–3119 (2002).References 98 and 99 establish that pIII of the CIITA gene drives CIITA and MHC class II expression by human T cells.

100. Ghosh, N. et al. A novel element and a TEF-2-like element activate the major histocompatibility complex class II transactivator in B-lymphocytes. J. Biol. Chem. 274, 32342–32350 (1999).

101. van der Stoep, N., Quinten, E. & van den Elsen, P. J. Transcriptional regulation of the MHC class II trans-activator (CIITA) promoter III: identification of a novel regulatory region in the 5′-untranslated region and an important role for cAMP-responsive element binding protein 1 and activating transcription factor-1 in CIITA-promoter III transcriptional activation in B lymphocytes. J. Immunol. 169, 5061–5071 (2002).

102. van der Stoep, N., Quinten, E., Marcondes, R. M. & van den Elsen, P. J. E47, IRF-4, and PU.1 synergize to induce B-cell-specific activation of the class II transactivator promoter III (CIITA-PIII). Blood 104, 2849–2857 (2004).This paper shows that the B-cell-specific activity of pIII of the CIITA gene can be attributed to binding of the transcription factors E47, IRF4 and PU.1.

103. Swanberg, M. et al. MHC2TA is associated with differential MHC molecule expression and susceptibility to rheumatoid arthritis, multiple sclerosis and myocardial infarction. Nature Genet. 37, 486–494 (2005).

104. Piskurich, J. F. et al. BLIMP-I mediates extinction of major histocompatibility class II transactivator expression in plasma cells. Nature Immunol. 1, 526–532 (2000).

105. Ghosh, N., Gyory, I., Wright, G., Wood, J. & Wright, K. L. Positive regulatory domain I binding factor 1 silences class II transactivator expression in multiple myeloma cells. J. Biol. Chem. 276, 15264–15268 (2001).

106. Accolla, R. S., Mazza, S., De Lerma, B. A., De, M. A. & Tosi, G. The HLA class II transcriptional activator blocks the function of HIV-1 Tat and inhibits viral replication. Eur. J. Immunol. 32, 2783–2791 (2002).

107. Casoli, C. et al. The MHC class II transcriptional activator (CIITA) inhibits HTLV-2 viral replication by blocking the function of the viral transactivator Tax-2. Blood 103, 995–1001 (2004).

108. Garcia-Lora, A., Algarra, I., Collado, A. & Garrido, F. Tumour immunology, vaccination and escape strategies. Eur. J. Immunogenet. 30, 177–183 (2003).

109. Toes, R. E., Schoenberger, S. P., van der Voort, E. I., Offringa, R. & Melief, C. J. CD40–CD40Ligand interactions and their role in cytotoxic T lymphocyte priming and anti-tumor immunity. Semin. Immunol. 10, 443–448 (1998).

110. Liu, A. et al. Regulation of the expression of MHC class I and II by class II transactivator (CIITA) in hematopoietic cells. Hematol. Oncol. 17, 149–160 (1999).

111. Momburg, F., Herrmann, B., Moldenhauer, G. & Moller, P. B-cell lymphomas of high-grade malignancy frequently lack HLA-DR, -DP and -DQ antigens and associated invariant chain. Int. J. Cancer 40, 598–603 (1987).

112. Rimsza, L. M. et al. Loss of MHC class II gene and protein expression in diffuse large B-cell lymphoma is related to decreased tumor immunosurveillance and poor patient survival regardless of other prognostic factors: a follow-up study from the Leukemia and Lymphoma Molecular Profiling Project. Blood 103, 4251–4258 (2004).

113. Fuji, H. & Iribe, H. Clonal variation in tumorigenicity of L1210 lymphoma cells: nontumorigenic variants with an enhanced expression of tumor-associated antigen and Ia antigens. Cancer Res. 46, 5541–5547 (1986).

114. Altomonte, M., Fonsatti, E., Visintin, A. & Maio, M. Targeted therapy of solid malignancies via HLA class II antigens: a new biotherapeutic approach? Oncogene 22, 6564–6569 (2003).

115. Morimoto, Y. et al. Inactivation of class II transactivator by DNA methylation and histone deacetylation associated with absence of HLA-DR induction by interferon-γ in haematopoietic tumour cells. Br. J. Cancer 90, 844–852 (2004).

116. Cook, G. & Campbell, J. D. Immune regulation in multiple myeloma: the host–tumour conflict. Blood Rev. 13, 151–162 (1999).

117. Takamura, Y. et al. Regulation of MHC class II expression in glioma cells by class II transactivator (CIITA). Glia 45, 392–405 (2004).

118. Guardiola, J. & Maffei, A. Control of MHC class II gene expression in autoimmune, infectious, and neoplastic diseases. Crit. Rev. Immunol. 13, 247–268 (1993).

119. Li, S., Kurts, C., Kontgen, F., Holdsworth, S. R. & Tipping, P. G. Major histocompatibility complex class II expression by intrinsic renal cells is required for crescentic glomerulonephritis. J. Exp. Med. 188, 597–602 (1998).This paper provides convincing evidence that MHC class II expression in renal tissues is required for the development of experimental glomerulonephritis in mice.

120. Leung, B. P. et al. A novel anti-inflammatory role for simvastatin in inflammatory arthritis. J. Immunol. 170, 1524–1530 (2003).

121. Palmer, G. et al. Assessment of the efficacy of different statins in murine collagen-induced arthritis. Arthritis Rheum. 50, 4051–4059 (2004).

122. McCarey, D. W. et al. Trial of Atorvastatin in Rheumatoid Arthritis (TARA): double-blind, randomised placebo-controlled trial. Lancet 363, 2015–2021 (2004).

123. Kuipers, H. F. & van den Elsen, P. J. Statins and control of MHC2TA gene transcription. Nature Med. 11, 365–366 (2005).

124. Martin, B. K., Frelinger, J. G. & Ting, J. P. Combination gene therapy with CD86 and the MHC class II transactivator in the control of lung tumor growth. J. Immunol. 162, 6663–6670 (1999).

125. Meazza, R., Comes, A., Orengo, A. M., Ferrini, S. & Accolla, R. S. Tumor rejection by gene transfer of the MHC class II transactivator in murine mammary adenocarcinoma cells. Eur. J. Immunol. 33, 1183–1192 (2003).

126. Hillman, G. G. et al. Generating MHC class II+/Ii– phenotype after adenoviral delivery of both an expressible gene for MHC class II inducer and an antisense Ii-RNA construct in tumor cells. Gene Ther. 10, 1512–1518 (2003).

127. Clements, V. K., Baskar, S., Armstrong, T. D. & Ostrand-Rosenberg, S. Invariant chain alters the malignant phenotype of MHC class II+ tumor cells. J. Immunol. 149, 2391–2396 (1992).

128. Dissanayake, S. K., Tuera, N. & Ostrand-Rosenberg, S. Presentation of endogenously synthesized MHC class II-restricted epitopes by MHC class II cancer vaccines is independent of transporter associated with Ag processing and the proteasome. J. Immunol. 174, 1811–1819 (2005).


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129. Wang, Y. et al. Curative antitumor immune response is optimal with tumor irradiation followed by genetic induction of major histocompatibility complex class I and class II molecules and suppression of Ii protein. Hum. Gene Ther. 16, 187–199 (2005).This paper describes a mouse model of prostate cancer in which 50% of the mice were cured by combining radiotherapy with gene therapy using CIITA, IFN-γγ and IL-2 expression vectors, together with an antisense construct directed towards Ii mRNA. Moreover, 100% of cured animals were resistant to rechallenge with tumour cells.

130. Nagy, Z. A. et al. Fully human, HLA-DR-specific monoclonal antibodies efficiently induce programmed death of malignant lymphoid cells. Nature Med. 8, 801–807 (2002).

131. Dechant, M., Bruenke, J. & Valerius, T. HLA class II antibodies in the treatment of hematologic malignancies. Semin. Oncol. 30, 465–475 (2003).

132. Martin, B. K. et al. Induction of MHC class I expression by the MHC class II transactivator CIITA. Immunity 6, 591–600 (1997).

133. Gobin, S. J., Peijnenburg, A., Keijsers, V. & van den Elsen, P. J. Site α is crucial for two routes of IFN γ-induced MHC class I transactivation: the ISRE-mediated route and a novel pathway involving CIITA. Immunity 6, 601–611 (1997).

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AcknowledgementsWe are grateful to B. Mach, in whose laboratory this subject was first initiated. We are also grateful to all current and past members of our laboratories, particularly B. Durand, M. Krawczyk, K. Masternak, A. Muhlethaler-Mottet, L. Otten and V. Steimle, who

made pivotal contributions to the field. We thank H. Acha-Orbea, A. Fontana and C. Reis e Sousa for important collaborations. Work in our laboratory was supported by the Swiss National Science Foundation, the Roche Research Foundation (Switzerland), the Gabriella Giorgi-Cavaglieri Foundation (Switzerland), the Ernst and Lucie Schmidheiny Foundation (Switzerland), the Swiss Multiple Sclerosis Society and the National Center of Competence in Research on Neural Plasticity and Repair (Switzerland).

Competing interests statementThe authors declare no competing financial interests.

Online links

DATABASESThe following terms in this article are linked online to:Entrez Gene:http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=geneCIITA | CREB | E47 | IFN-γ | IRF1 | IRF2 | IRF4 | NFY | P-TEFb | PU.1 | RFX5 | RFXANK | RFXAP | TFIIB | TFIID | USF1 OMIM: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIMbare lymphocyte syndrome

FURTHER INFORMATIONWalter Reith’s homepage: http://pathology.unige.ch/patim/group-reith.htmlAccess to this interactive links box is free online.


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regulation of mhc class ii gene expression by the class ii transactivator

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