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The Bare Lymphocyte Syndrome: Molecular Clues to the Transcriptional Regulation of Major Histocompatibility Complex Class II Genes

1999; Elsevier BV; Volume: 65; Issue: 2 Linguagem: Inglês

10.1086/302519

1537-6605

Angela M. DeSandro, Uma M. Nagarajan, Jeremy M. Boss,

Cytokine Signaling Pathways and Interactions

Major histocompatibility complex (MHC) class II proteins are cell-surface heterodimeric glycoproteins that function in the initiation of acquired immune responses. In antigen-presenting cells, antigenic peptides are loaded onto MHC class II molecules in a process that depends on the class II invariant chain and another accessory protein, human leukocyte antigen (HLA-DM). This process is completed in a specialized, post-Golgi intracellular compartment, the MHC class II compartment (MIIC; Peters et al. Peters et al., 1991Peters PJ Neefjes JJ Oorschot V Ploegh HL Geuze HJ Segregation of MHC class II molecules from MHC class I molecules in the Golgi complex for transport to lysosomal compartments.Nature. 1991; 349: 669-676Crossref PubMed Scopus (548) Google Scholar), where peptides with the highest affinity for the MHC class II proteins are selected for presentation (Denzin and Cresswell Denzin and Cresswell, 1995Denzin LK Cresswell P HLA-DM induces CLIP dissociation from MHC class II α β dimers and facilitates peptide loading.Cell. 1995; 82: 155-165Abstract Full Text PDF PubMed Scopus (618) Google Scholar). Subsequently, the MHC-peptide complex is transported to the surface where it can interact with antigen-specific CD4+ T-helper cells, initiating an immune response. MHC class II molecules are expressed in a limited subset of immune cells, including B cells, macrophages, activated T cells, thymic epithelial cells, and dendritic cells. MHC class II genes may also be induced in many other cell types by the cytokine interferon-γ (IFN-γ). Induction by IFN-γ may allow presentation of antigens to the immune system by nonimmune cells. Because of this intimate involvement in immune responses, aberrant expression of MHC class II genes could potentially lead to or sustain autoimmune disorders, tumor growth, or failure to mount an immune response. Lack of expression of MHC class II genes results in a severe combined immunodeficiency, called bare lymphocyte syndrome (BLS). Patients with BLS are 5% of all cases of severe combined immunodeficiency (Elhasid and Etzioni Elhasid and Etzioni, 1996Elhasid R Etzioni A Major histocompatibility complex class II deficiency: a clinical review.Blood Rev. 1996; 10: 242-248Abstract Full Text PDF PubMed Scopus (41) Google Scholar). Cell lines derived from these patients and a collection of lab-generated cell lines that share the same characteristics as BLS-derived cells have provided unique tools for elucidating the regulation of MHC class II genes. This review will focus on the molecular basis for BLS and on how understanding the genetics of this system has provided the current models of MHC class II gene regulation. MHC class II genes are located on the short arm of chromosome 6. The MHC class II locus encodes separate α and β chains for each of the three isotypes: HLA-DR, HLA-DQ, and HLA-DP. Driving the expression of these isotypes—and the expression of other gene products that are involved in antigen presentation—is a conserved upstream cis-acting regulatory region that coordinately regulates their expression. This regulatory region consists of four major elements: the W, X1, X2, and Y boxes. Maximal expression in B lymphocytes and during IFN-γ induction requires the presence of all these elements (reviewed by Boss Boss, 1997Boss JM Regulation of transcription of MHC class II genes.Curr Opin Immunol. 1997; 9: 107-113Crossref PubMed Scopus (174) Google Scholar). The W box is the least fully characterized of the four elements, but the DNA-binding factor regulatory-factor X (RFX; discussed extensively below) has been suggested to interact with this region. RFX and the X2-box–binding protein (X2BP), recently identified as the cAMP response-element–binding protein (CREB), bind to the X1 and X2 boxes, respectively (Boss Boss, 1997Boss JM Regulation of transcription of MHC class II genes.Curr Opin Immunol. 1997; 9: 107-113Crossref PubMed Scopus (174) Google Scholar; Moreno et al. Moreno et al., 1999Moreno CS Beresford G Louis-Plence P Morris AC Boss JM CREB regulates MHC class II expression in a CIITA-dependent manner.Immunity. 1999; 10: 143-151Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). The Y box, an inverted CCAAT box, is bound by the heterotrimeric factor nuclear-factor y (NF-Y). These three DNA-binding factors are required, but are not alone sufficient, for MHC class II expression. A fourth factor, the class II transactivator (CIITA), which does not appear to bind DNA (Steimle et al. Steimle et al., 1993Steimle V Otten LA Zufferey M Mach B Complementation cloning of an MHC class II transactivator mutated in hereditary MHC class II deficiency (or bare lymphocyte syndrome).Cell. 1993; 75: 135-146Abstract Full Text PDF PubMed Scopus (730) Google Scholar), is also required. CIITA is proposed to interact with the DNA-bound factors RFX, CREB, and NF-Y at the class II promoter and to use its acidic activation domain to activate transcription. BLS is a rare autosomal recessive disease characterized by severe combined immunodeficiency. More than 40 cases have been diagnosed since its description (Griscelli et al. Griscelli et al., 1989Griscelli C Lisowska-Grospierre B Mach B Combined immunodeficiency with defective expression in MHC class II genes.Immunodefic Rev. 1989; 1: 135-153PubMed Google Scholar). Although some patients show very low levels of MHC class II cell-surface expression, most patients do not express MHC class II proteins or mRNA in their cells. Patients can also vary in the levels of expression of MHC class I proteins, which are expressed on virtually all cell types. Patients with BLS usually present in the first year of life with infections in the respiratory system and the gastrointestinal tract. In addition, viral infections are extremely dangerous, and, as a result of intestinal infections, affected children exhibit protracted diarrhea, severe malabsorption, and failure to thrive. The humoral immune response in these patients is severely impaired, varying from panhypogammaglobulinemia to reduction in one or two of the Ig isotypes (Klein et al. Klein et al., 1993Klein C Lisowska-Grospierre B LeDeist F Fischer A Griscelli C Major histocompatibility complex class II deficiency: Clinical manifestations, immunologic features, and outcome.J Pediatr. 1993; 123: 921-928Abstract Full Text PDF PubMed Scopus (155) Google Scholar). The prognosis for these patients is very poor, and most do not reach puberty (Elhasid and Etzioni Elhasid and Etzioni, 1996Elhasid R Etzioni A Major histocompatibility complex class II deficiency: a clinical review.Blood Rev. 1996; 10: 242-248Abstract Full Text PDF PubMed Scopus (41) Google Scholar). The current treatment for BLS includes regular intravenous immunoglobulin support and prophylactic antibiotics (Klein et al. Klein et al., 1993Klein C Lisowska-Grospierre B LeDeist F Fischer A Griscelli C Major histocompatibility complex class II deficiency: Clinical manifestations, immunologic features, and outcome.J Pediatr. 1993; 123: 921-928Abstract Full Text PDF PubMed Scopus (155) Google Scholar; Elhasid and Etzioni Elhasid and Etzioni, 1996Elhasid R Etzioni A Major histocompatibility complex class II deficiency: a clinical review.Blood Rev. 1996; 10: 242-248Abstract Full Text PDF PubMed Scopus (41) Google Scholar). The only effective long-term treatment is a bone-marrow transplant. The prognosis for post-transplant BLS patients depends on the age of the patient, with younger patients responding more favorably. Infection as a result of the immune suppression after transplant causes many of the complications associated with recovery (Elhasid and Etzioni Elhasid and Etzioni, 1996Elhasid R Etzioni A Major histocompatibility complex class II deficiency: a clinical review.Blood Rev. 1996; 10: 242-248Abstract Full Text PDF PubMed Scopus (41) Google Scholar). Haplotyping of family members of patients with BLS shows that the MHC locus segregates independently of the BLS phenotype (de Preval et al. de Preval et al., 1985de Preval C Lisowska-Grospierre B Loche M Griscelli C Mach B A trans-acting class II regulatory gene unlinked to the MHC controls expression of HLA class II genes.Nature. 1985; 318: 291-293Crossref PubMed Scopus (133) Google Scholar), suggesting, as proposed by Gladstone and Pious (Gladstone and Pious, 1980Gladstone P Pious D Identification of a trans-acting function regulation HLA-DR expression in a DR-negative B cell variant.Somat Cell Genet. 1980; 6: 285-298Crossref PubMed Scopus (59) Google Scholar), that one or more transacting factors that regulate expression of MHC class II genes are mutated in this syndrome. To determine how many genes are involved in BLS, somatic cell-fusion experiments were performed between the different patient cell lines and in vitro–generated cell lines. Four complementation groups—A, B, C, and D—have been defined (Benichou and Strominger Benichou and Strominger, 1991Benichou B Strominger JL Class II-antigen-negative patient and mutant B-cell lines represent at least three, and probably four, distinct genetic defects defined by complementation analysis.Proc Natl Acad Sci USA. 1991; 88: 4285-14288Crossref PubMed Scopus (120) Google Scholar; Seidl et al. Seidl et al., 1992Seidl C Saraiya C Osterweil Z Fu YP Lee JS Genetic complexity of regulatory mutants defective for HLA class II expression.J Immunol. 1992; 148: 1576-1584PubMed Google Scholar), demonstrating that, although patients with BLS share a similar phenotype, the genetic basis of the disease is heterogeneous (table 1).Table 1Mutations in BLS GenesBLS Group and Gene (Location)Cell and Patient LinesGenotypeMutation(s)Result of MutationMHC LevelsA: CIITA (16p)BLS-2HG→A at s.d.72 bp in-frame Δ of exon encoding a.a. 940–963 that contains nuclear localization signalNDBCHCHG→T; G→A at s.d.GAA (Glu381)→TAA (stop);84 bp in-frame Δ (a.a 1079–1106)NDRJ2.2.5aExperimentally derived mutant cell lines.CH1,811-bp Δ in mRNA completely deletedFrameshift; complete nullNDB: RFX-B or RFX-ANK (19p12)Bequit (Ab), Nacera (Nh), and Ramia (RA)bUnrelated patients.H26-bp Δ in s.a. of exon 6FrameshiftNDBLS-1H58-bp Δ in s.d. of exon 6FrameshiftNDEBAHG→T in exon 5GAG (Glu102)→TAG (stop)LowFZAHT→C in exon 8CTG (Leu195)→CCG195 (Pro)LowC: RFX5 (1q21.1-q21.3)RoHC1032 →T1032CGA (Arg294)→TGA (stop)LowSJOCHG→A in the s.a. of exon 5; other allele is not definedUse of a cryptic s.a. at +5 results in 5-bp Δ and frameshift; not expressedNDTHF, EVFcSiblings or cousins.HG→A at + 5 s.d. of exon 2Use of a cryptic s.d. at −10 results in 10-bp Δ and frameshiftNDOSEHG→A in the s.a. of exon 4Use of a cryptic s.a. at −4 results in 4-bp Δ and frameshiftNDSSIHC1122→TCAG (Gln321)→TAG (stop)NDD: RFXAP (13q)ABI, AkObUnrelated patients.HC279→T279CAG (Gln55)→TAG (stop)LowDA, ZM, SSbUnrelated patients.HΔG484FrameshiftLow6.1.6aExperimentally derived mutant cell lines.CHG insertion at 418 bp; G insertion at 508 bpFrameshift; frameshiftLowShA, ShGcSiblings or cousins.H7-bp insertion at 151 as a result of duplication of 144–150 bpFrameshiftLowNote.—See text for references. Abbrieviations: H = homozygous; CH = compound heterozygous; s.a. = splice acceptor; s.d.= splice donor; Δ = deletion; and ND = not detectable.a Experimentally derived mutant cell lines.b Unrelated patients.c Siblings or cousins. Open table in a new tab Note.— See text for references. Abbrieviations: H = homozygous; CH = compound heterozygous; s.a. = splice acceptor; s.d.= splice donor; Δ = deletion; and ND = not detectable. This complementation analysis provided a powerful system with which to identify the defective genes in two of the complementation groups. Further clues about the different molecular defects in the various complementation groups came from in vitro and in vivo studies of protein-DNA interactions in the MHC class II gene promoters. In a variety of DNA-binding assays, X2BP/CREB and NF-Y binding is seen, both in wild-type cells and in all complementation groups of BLS cells. However, two patterns emerged with regard to RFX binding. RFX activity was present in BLS group A cells but not in cells from groups B–D, suggesting that RFX activity may be encoded in three or more distinct genes. Furthermore, in vivo footprint analysis reveals two patterns that correlate with RFX activity (Kara and Glimcher Kara and Glimcher, 1991Kara CJ Glimcher LH In vivo footprinting of MHC class II genes: bare promoters in the bare lymphocyte syndrome.Science. 1991; 252: 709-712Crossref PubMed Scopus (148) Google Scholar, Kara and Glimcher, 1993Kara CJ Glimcher LH Developmental and cytokine-mediated regulation of MHC class II gene promoter occupancy in vivo.J Immunol. 1993; 150: 4934-4942PubMed Google Scholar), corresponding to the X1-, X2-, and Y-box regions of the MHC class II promoters. Both wild-type and BLS group A cells contain fully occupied promoters, but the other BLS groups show no binding to X1, X2, or Y elements. Defects in CIITA are common to all cells in complementation group A—the first of the BLS groups to be characterized at the molecular level. MHC2TA, the gene that encodes CIITA, maps to 16p13. CIITA was cloned by cDNA complementation in an in vitro–generated cell line (Steimle et al. Steimle et al., 1993Steimle V Otten LA Zufferey M Mach B Complementation cloning of an MHC class II transactivator mutated in hereditary MHC class II deficiency (or bare lymphocyte syndrome).Cell. 1993; 75: 135-146Abstract Full Text PDF PubMed Scopus (730) Google Scholar). The N-terminal portion of the 1,130 amino acid CIITA protein carries a transcriptional-activation domain (Riley et al. Riley et al., 1995Riley JL Westerheide SD Price JA Brown JA Boss JM Activation of class II MHC genes requires both the X box region and the class II transactivator (CIITA).Immunity. 1995; 2: 533-543Abstract Full Text PDF PubMed Scopus (175) Google Scholar; Zhou and Glimcher Zhou and Glimcher, 1995Zhou H Glimcher LH Human MHC class II gene transcription directed by the carboxyl terminus of CIITA, one of the defective genes in type II MHC combined immune deficiency.Immunity. 1995; 2: 545-553Abstract Full Text PDF PubMed Scopus (145) Google Scholar). The finding that CIITA does not bind to DNA (Steimle et al. Steimle et al., 1993Steimle V Otten LA Zufferey M Mach B Complementation cloning of an MHC class II transactivator mutated in hereditary MHC class II deficiency (or bare lymphocyte syndrome).Cell. 1993; 75: 135-146Abstract Full Text PDF PubMed Scopus (730) Google Scholar) is consistent with the in vivo footprinting results showing a fully occupied promoter region in BLS group A cells and suggests that CIITA interacts with the DNA-bound W-, X-, and Y-box factors. Indeed, CIITA appears to act as a master regulator, because it is developmentally regulated in B cells (Chang et al. Chang et al., 1992Chang C-H Fodor WL Flavell RA Reactivation of a major histocompatibility complex class II gene in mouse plasmacytoma cells and mouse T cells.J Exp Med. 1992; 176: 1465-1469Crossref PubMed Scopus (22) Google Scholar) and because it is induced by IFN-γ (Steimle et al. Steimle et al., 1994Steimle V Siegrist C-A Mottet A Lisowska-Grospierre B Mach B Regulation of MHC class II expression by interferon-gamma mediated by the transactivator gene CIITA.Science. 1994; 265: 106-108Crossref PubMed Scopus (649) Google Scholar) in nonlymphoid cells prior to their induction of MHC class II genes. The cloning of MHC2TA has allowed for mutation analysis of cells in complementation group A, whether from cultured mutant cell lines or from the two known patients in this group, patient BCH and patient BLS-2. Mutations in the BLS group A–like cell line, RJ2.2.5, comprise a complete loss of one allele (Steimle et al. Steimle et al., 1993Steimle V Otten LA Zufferey M Mach B Complementation cloning of an MHC class II transactivator mutated in hereditary MHC class II deficiency (or bare lymphocyte syndrome).Cell. 1993; 75: 135-146Abstract Full Text PDF PubMed Scopus (730) Google Scholar) and an internal deletion of 1,811 bp in the second allele (Brown et al. Brown et al., 1995Brown JA He X-F Westerheide SD Boss JM Characterization of the expressed CIITA allele in the class II MHC transcriptional mutant RJ2.2.5.Immunogenetics. 1995; 43: 88-91Crossref Scopus (33) Google Scholar). Patient BCH is a compound heterozygote with a G→T transversion that results in a nonsense mutation and a severely truncated protein in one allele (Bontron et al. Bontron et al., 1997Bontron S Steimle V Ucla C Eibl MM Mach B Two novel mutations in the MHC class II transactivator CIITA in a second patient from MHC class II deficiency complementation group A.Hum Genet. 1997; 99: 541-546Crossref PubMed Scopus (48) Google Scholar). The second allele contains a G→A transition in a splice-donor sequence leading to an 84 bp in-frame deletion of an exon. Both mutations completely inactivate the CIITA protein. Patient BLS-2 is homozygous for a G→A transition in a splice-donor sequence that results in a 72 bp in-frame deletion (Steimle et al. Steimle et al., 1993Steimle V Otten LA Zufferey M Mach B Complementation cloning of an MHC class II transactivator mutated in hereditary MHC class II deficiency (or bare lymphocyte syndrome).Cell. 1993; 75: 135-146Abstract Full Text PDF PubMed Scopus (730) Google Scholar). This deletion removes a 5 amino acid nuclear-localization sequence, rendering CIITA unable to translocate to the nucleus (Cressman et al. Cressman et al., 1999Cressman DE Chin KC Taxman DJ Ting JP A defect in the nuclear translocation of CIITA causes a form of type II bare lymphocyte syndrome.Immunity. 1999; 10: 163-171Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Two CIITA knockout mice have been generated (Chang et al. Chang et al., 1996Chang C-H Guerder S Hong S-C Van Ewijk W Flavell RA Mice lacking the MHC class II transactivator (CIITA) show tissue-specific impairment of MHC class II expression.Immunity. 1996; 4: 167-178Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar; Williams et al. Williams et al., 1998Williams GS Malin M Vremec D Chang C-H Boyd R Benoist C Mathis D Mice lacking the transcription factor CIITA-a second look.Int Immunol. 1998; 10: 1957-1967Crossref PubMed Scopus (81) Google Scholar). Both mutant mouse strains display a phenotype similar to that of patients with BLS in that they lack MHC class II molecules on their antigen-presenting cells and have a reduced number of peripheral CD4+ T cells. Interestingly, both mutant strains have residual MHC class II expression on a subset of thymic epithelial cells and on a subset of dendritic cells. Mice of both groups show a reduction, but not a complete loss, in their expression of the invariant chain and of H-2M (the mouse orthologue of HLA-DM; Chang et al. Chang et al., 1996Chang C-H Guerder S Hong S-C Van Ewijk W Flavell RA Mice lacking the MHC class II transactivator (CIITA) show tissue-specific impairment of MHC class II expression.Immunity. 1996; 4: 167-178Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar; Williams et al. Williams et al., 1998Williams GS Malin M Vremec D Chang C-H Boyd R Benoist C Mathis D Mice lacking the transcription factor CIITA-a second look.Int Immunol. 1998; 10: 1957-1967Crossref PubMed Scopus (81) Google Scholar). A reduction in global MHC class I expression that is observed in some human patients was not detected in either mouse. Unexpectedly, cells from both CIITA-deficient mouse strains up-regulate MHC class I when treated with IFN-γ. However, the level of IFN-γ–induced MHC class I was definitively lower in the C2ta−/− mouse, described by Williams et al. (Williams et al., 1998Williams GS Malin M Vremec D Chang C-H Boyd R Benoist C Mathis D Mice lacking the transcription factor CIITA-a second look.Int Immunol. 1998; 10: 1957-1967Crossref PubMed Scopus (81) Google Scholar), than in wild-type mice. Thus, although these knockout mice do not provide a perfect model for BLS, they do follow some of the variation that has been documented in patients with BLS. Importantly, these studies suggest CIITA-independent mechanisms of MHC class II regulation that require investigation. Patient-derived cell lines lacking RFX binding to MHC class II promoters are represented in the complementation groups B, C, and D. Co-immunoprecipitation experiments with the RFX5 subunit of RFX identified two associated proteins, with apparent molecular weights of 41 and 33 kDa (Moreno et al. Moreno et al., 1997Moreno CS Rogers EM Brown JA Boss JM RFX, a bare lymphocyte syndrome transcription factor, is a multimeric phosphoprotein complex.J Immunol. 1997; 158: 5841-5848PubMed Google Scholar). The co-immunoprecipitation of p41 requires the presence of p33 and vice versa, suggesting that RFX5 and the two associated subunits form a tight complex. The study by Moreno et al. (Moreno et al., 1997Moreno CS Rogers EM Brown JA Boss JM RFX, a bare lymphocyte syndrome transcription factor, is a multimeric phosphoprotein complex.J Immunol. 1997; 158: 5841-5848PubMed Google Scholar) also predicted that BLS groups B, C, and D would be affected in the genes encoding the three subunits of RFX. The largest of the four BLS complementation groups is group B, with >24 patients (Fondaneche et al. Fondaneche et al., 1998Fondaneche M Villard J Wiszniewski W Jouanguy E Etzioni A Le Deist F Peijnenburg A et al.Genetic and molecular definition of complementation group D in MHC class II deficiency.Hum Mol Gen. 1998; 7: 879-885Crossref PubMed Scopus (25) Google Scholar). The defective gene in this group was cloned by isolating the RFX complex, sequencing the 33-kDa protein, and isolating the cDNA on the basis of the protein sequence. The gene, which maps to 19p12, was termed “RFX-B” (Nagarajan et al. Nagarajan et al., 1999Nagarajan UM Louis-Plence P DeSandro A Nilsen R Bushey A Boss JM RFX-B is the gene responsible for the most common cause of the bare lymphocyte syndrome, a MHC class II immunodeficiency.Immunity. 1999; 10: 153-162Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar) or “RFX-ANK” (Masternak et al. Masternak et al., 1998Masternak K Barras E Zufferey M Conrad B Corthals G Aebersold R Sanchez J-C et al.A gene encoding a novel RFX-associated transactivator is mutated in the majority of MHC class II deficiency patients.Nat Genet. 1998; 20: 273-277Crossref PubMed Scopus (237) Google Scholar), the latter name indicating that the gene protein contains three ankyrin repeats, domains that typically mediate protein-protein interactions. These regions could be involved in RFX-complex formation and/or interaction with other DNA-bound factors, such as X2BP/CREB or NF-Y. BLS group B patients display the greatest diversity of disease alleles. BLS group B–derived cell lines Bequit, Nacera, and Ramia carry a common homozygous deletion of 26 bp that removes the splice-acceptor region of exon 6, resulting in the deletion of exon 6, and a frameshift that results in a truncated protein (Nagarajan et al. Nagarajan et al., 1999Nagarajan UM Louis-Plence P DeSandro A Nilsen R Bushey A Boss JM RFX-B is the gene responsible for the most common cause of the bare lymphocyte syndrome, a MHC class II immunodeficiency.Immunity. 1999; 10: 153-162Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar; Masternak et al. Masternak et al., 1998Masternak K Barras E Zufferey M Conrad B Corthals G Aebersold R Sanchez J-C et al.A gene encoding a novel RFX-associated transactivator is mutated in the majority of MHC class II deficiency patients.Nat Genet. 1998; 20: 273-277Crossref PubMed Scopus (237) Google Scholar) (table 1). These three patients are unrelated but come from the same geographical area of North Africa, suggesting a common ancestor for this allele. Patient BLS-1 has a 58-bp deletion in the splice-donor region of exon 6, resulting in a frameshift (Masternak et al. Masternak et al., 1998Masternak K Barras E Zufferey M Conrad B Corthals G Aebersold R Sanchez J-C et al.A gene encoding a novel RFX-associated transactivator is mutated in the majority of MHC class II deficiency patients.Nat Genet. 1998; 20: 273-277Crossref PubMed Scopus (237) Google Scholar). Patient EBA has a nonsense mutation in exon 5, resulting in a truncated protein (U. Nagarajan and J. Boss, unpublished data). Patient FZA has a homozygous point mutation, changing a conserved leucine in the third ankyrin repeat to a proline in exon 8 (U. Nagarajan and J. Boss, unpublished data). Cell lines from patient EBA and patient FZA can be induced to express low levels of MHC class II genes, which may be due to an alternatively spliced product or to some residual activity of the variant protein. Complementation group C mutations are in the gene encoding RFX5, which maps to 1q21. RFX5 was the first of the RFX subunits to be identified genetically. RFX5 was cloned by complementation of the MHC class II–negative cell line of patient SJO (Steimle et al. Steimle et al., 1995Steimle V Durand B Emmanuele B Zufferey M Hadam MR Mach B Reith W A novel DNA-binding regulatory factor is mutated in primary MHC class II deficiency (bare lymphocyte syndrome).Genes Dev. 1995; 9: 1021-1032Crossref PubMed Scopus (302) Google Scholar). At 66 kDa and 616 aa, RFX5 is the largest of the RFX subunits and is the only subunit to contain a known DNA-binding motif. Patient Ro and patient SSI both carry nonsense mutations, at position 1032 and 1122, respectively (Steimle et al. Steimle et al., 1995Steimle V Durand B Emmanuele B Zufferey M Hadam MR Mach B Reith W A novel DNA-binding regulatory factor is mutated in primary MHC class II deficiency (bare lymphocyte syndrome).Genes Dev. 1995; 9: 1021-1032Crossref PubMed Scopus (302) Google Scholar; Peijnenburg et al. Peijnenburg et al., 1999Peijnenburg A van Eggermond MCJA van den Berg R Sanal O Vossen JMJJ van den Elsen PJ Molecular analysis of an MHC class II deficiency patient reveals a novel mutation in the RFX5 gene.Immunogenetics. 1999; 49: 338-345Crossref PubMed Scopus (20) Google Scholar). The remaining patients all carry G→A transitions at splice junctions that result in the use of cryptic splice sites and premature truncations; these splice junctions occur in exon 5 in SJO cells (Steimle et al. Steimle et al., 1995Steimle V Durand B Emmanuele B Zufferey M Hadam MR Mach B Reith W A novel DNA-binding regulatory factor is mutated in primary MHC class II deficiency (bare lymphocyte syndrome).Genes Dev. 1995; 9: 1021-1032Crossref PubMed Scopus (302) Google Scholar), in exon 2 in patient THF and patient EVF cells (Villard et al. Villard et al., 1997bVillard J Reith W Barras E Gos A Morris MA Antonarakis Sã E van den Elsen PJ et al.Analysis of mutations and chromosomal localisation of the gene encoding RFX5, a novel transcription factor affected in major histocompatibility complex class II deficiency.Hum Mutat. 1997; 10: 430-435Crossref PubMed Scopus (25) Google Scholar), and in exon 4 in patient OSE cells (Peijnenburg et al. Peijnenburg et al., 1999Peijnenburg A van Eggermond MCJA van den Berg R Sanal O Vossen JMJJ van den Elsen PJ Molecular analysis of an MHC class II deficiency patient reveals a novel mutation in the RFX5 gene.Immunogenetics. 1999; 49: 338-345Crossref PubMed Scopus (20) Google Scholar). Clausen et al. (Clausen et al., 1998Clausen BE Waldburger JM Schwenk F Barras E Mach B Rajewsky K Forster I et al.Residual MHC class II expression on mature dendritic cells and activated B cells in RFX5-deficient mice.Immunity. 1998; 8: 143-155Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) have generated an Rfx5 knockout mouse and have determined that the phenotype recapitulates many aspects of human BLS. Rfx5−/− mice have a severe immunodeficiency because of absence of CD4+ T cells and a lack of MHC class II molecules on resting B cells and macrophages. As observed in C2ta−/− mice, MHC class II expression is present on dendritic cells, and invariant chain and H2-M were reduced but not absent. In contrast to the C2ta−/− mice, however, strong MHC class II expression was detected in thymic medullary epithelial cells and activated B cells. The finding of RFX5-independent MHC class II expression is intriguing and suggests a novel mechanism of MHC class II gene control. The gene mutated in complementation group D is RFXAP, which maps to 13q14. This gene was cloned by isolation of the RFX complex and sequencing of the 41-kDa protein. With its cloning, the question of whether a fourth complementation group existed was resolved. The question arose from the fact that cell line 6.1.6—the BLS group D experimental cell line most studied (Gladstone and Pious Gladstone and Pious, 1978Gladstone P Pious D Stable variants affecting B cell alloantigens in human lymphoid cells.Nature. 1978; 271: 459-461Crossref PubMed Scopus (82) Google Scholar)—as well as the patient cell lines, showed slight MHC class II expression. However, this expression did not lead to fully functional MHC class II proteins, since the patients exhibited BLS symptoms. Patient ABI (Villard et al. Villard et al., 1997aVillard J Lisowska-Grospierre B van den Elsen P Fischer A Reith W Mach B Mutation of RFXAP, a regulator of MHC class II genes, in primary MHC class II deficiency.N Engl J Med. 1997; 337: 748-753Crossref PubMed Scopus (57) Google Scholar) and patient AkO (Fondaneche et al. Fondaneche et al., 1998Fondaneche M Villard J Wiszniewski W Jouanguy E Etzioni A Le Deist F Peijnenburg A et al.Genetic and molecular definition of complementation group D in MHC class II deficiency.Hum Mol Gen. 1998; 7: 879-885Crossref PubMed Scopus (25) Google Scholar) carry, in homozygous form, a C→T transition that results in a premature stop codon and truncated protein. Patient ZM, patient SS (Fondaneche et al. Fondaneche et al., 1998Fondaneche M Villard J Wiszniewski W Jouanguy E Etzioni A Le Deist F Peijnenburg A et al.Genetic and molecular definition of complementation group D in MHC class II deficiency.Hum Mol Gen. 1998; 7: 879-885Crossref PubMed Scopus (25) Google Scholar), and patient DA (Durand et al. Durand et al., 1997Durand B Sperisen P Emery P Barras E Zufferey M Mach B Reith W RFXAP, a novel subunit of the RFX DNA binding complex, is mutated in MHC class II deficiency.EMBO J. 1997; 16: 1045-1055Crossref PubMed Scopus (201) Google Scholar) have a deletion of a G at position 484 that causes a frameshift and premature stop codon. Patient ShA and pateint ShG, who are cousins, have a 7-bp insertion (duplication) at bp 151 that also results in a frameshift and premature stop codon (Fondaneche et al. Fondaneche et al., 1998Fondaneche M Villard J Wiszniewski W Jouanguy E Etzioni A Le Deist F Peijnenburg A et al.Genetic and molecular definition of complementation group D in MHC class II deficiency.Hum Mol Gen. 1998; 7: 879-885Crossref PubMed Scopus (25) Google Scholar). Although both RFX-B and RFX5 make sequence-specific contacts with the X1 box, RFXAP binds at many positions (Westerheide and Boss Westerheide and Boss, 1999Westerheide SD Boss JM Site-specific crosslinking mapping of RFX and X2BP transcription factor subunits to the major histocompatibility complex class II transcriptional enhancer.Nucleic Acids Res. 1999; 27: 1635-1641Crossref PubMed Scopus (30) Google Scholar). The role that RFXAP plays in the RFX complex remains to be determined, but its multiple contacts with the X1 box may indicate that it helps to stabilize the binding of the RFX complex to specific DNA sequences. Figure 1 illustrates the current model of MHC class II gene regulation and the role of the BLS proteins. The RFX subunits X2BP/CREB and NF-Y are expressed in a ubiquitous manner, although their levels may vary between cell types. If present in high enough concentrations, these factors form an extremely stable complex on the X- and Y-box–regulatory region, both in vivo and in vitro. This complex is inactive in the absence of CIITA, a factor that is constitutively expressed in B lymphocytes and other antigen-presenting cells, and that is inducible by IFN-γ in most other cell types (Steimle et al. Steimle et al., 1994Steimle V Siegrist C-A Mottet A Lisowska-Grospierre B Mach B Regulation of MHC class II expression by interferon-gamma mediated by the transactivator gene CIITA.Science. 1994; 265: 106-108Crossref PubMed Scopus (649) Google Scholar). The N-terminal–activation domain of CIITA most likely interacts with the X- and Y-box factors to activate transcription (Riley et al. Riley et al., 1995Riley JL Westerheide SD Price JA Brown JA Boss JM Activation of class II MHC genes requires both the X box region and the class II transactivator (CIITA).Immunity. 1995; 2: 533-543Abstract Full Text PDF PubMed Scopus (175) Google Scholar; Zhou and Glimcher Zhou and Glimcher, 1995Zhou H Glimcher LH Human MHC class II gene transcription directed by the carboxyl terminus of CIITA, one of the defective genes in type II MHC combined immune deficiency.Immunity. 1995; 2: 545-553Abstract Full Text PDF PubMed Scopus (145) Google Scholar). CIITA may also stabilize the binding of the RFX-CREB-NF–Y complex to DNA in cells in which the concentration of the latter factors is limiting (Moreno et al. Moreno et al., 1997Moreno CS Rogers EM Brown JA Boss JM RFX, a bare lymphocyte syndrome transcription factor, is a multimeric phosphoprotein complex.J Immunol. 1997; 158: 5841-5848PubMed Google Scholar; Wright et al. Wright et al., 1998Wright KL Chin K-C Linhoff M Skinner C Li G Boss JM Stark GR et al.CIITA stimulation of transcription factor binding to major histocompatibility complex class II and associated promoters in vivo.Proc Natl Acad Sci USA. 1998; 95: 6267-6272Crossref PubMed Scopus (62) Google Scholar). Kretsovali et al. (Kretsovali et al., 1998Kretsovali A Agaloti T Spilianakis C Tzortzakaki E Merika M Papamatheakis J Involvement of CREB binding protein in expression of major histocompatibility complex class II genes via interaction with the class II transactivator.Mol Cell Biol. 1998; 18: 6777-6783Crossref PubMed Scopus (153) Google Scholar) and Fontes et al. (Fontes et al., 1999Fontes JD Kanazawa S Jean D Peterlin BM Interactions between the class II transactivator and CREB binding protein increase transcription of major histocompatibility complex class II genes.Mol Cell Biol. 1999; 19: 941-947Crossref PubMed Scopus (136) Google Scholar) showed that the histone acetyltransferase CREB-binding protein can bind to CIITA, perhaps allowing the assembled class II transcription factor–complex to open chromatin for more efficient transcription and factor binding. Both CIITA and RFX have been found to be involved in the IFN-γ induction of MHC class I genes (Gobin et al. Gobin et al., 1997Gobin SJP Peijnenburg A Keijsers V van den Elsen PJ Site α is crucial for two routes of IFNλ-induced MHC class I transactivation: The ISRE-mediate route and a novel pathway involving CIITA.Immunity. 1997; 6: 601-611Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, Gobin et al., 1998Gobin SJP Peijneburg A van Eggermond M van Zutphen M van den Berg R van den Elsen P The RFX complex is crucial for the constitutive and CIITA-mediated transactivation of MHC class I and beta 2-microglobulin genes.Immunity. 1998; 9: 531-541Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar; Martin et al. Martin et al., 1997Martin BK Chin K-C Olsen JC Skinner CA Dey A Ozato K Ting JP-Y Induction of MHC class I expression by the MHC class II transactivator CIITA.Immunity. 1997; 6: 591-600Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar), suggesting that the control of antigen presentation is an ancient system that may have existed prior to the divergence of MHC class I and class II genes. The analysis of BLS has provided genetic proof that the RFX complex and CIITA are integral components of MHC class II expression. The cooperative binding of RFX with X2BP/CREB and NF-Y has substantiated their roles in the assembly and regulation of the class II promoter. Thus, this unfortunate disorder has provided a unique set of tools to unravel an important aspect of the control of the immune response: control of antigen processing and presentation. Future work will no doubt focus on how the regulatory complex assembles, additional components that may be required for activation, possible treatments for patients with BLS, and pharmaceuticals that can control the assembly and activation of these factors for use in immune-based therapies.