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Class II transactivator: is it a master switch for MHC class II gene expression?
Microbes and Infection, 1, 1999, 879−885
Class II transactivator: is it a master switch for MHC class II gene expression? Cheong-Hee Chang*, Stacey Roys, Tania Gourley Department of Microbiology and Immunology, The University of Michigan Medical School, Ann Arbor, MI 48109-0620, USA
1. Introduction Major histocompatibility complex (MHC) class II molecules are heterodimeric cell surface glycoproteins whose expression is critical for the development of CD4 T cells and the ability of vertebrates to mount an immune response. These molecules are expressed on antigenpresenting cells (APCs) such as B cells, macrophages, and dendritic cells which take up, process, and present antigens to CD4 T cells. The expression of MHC class II on B cells is required for the collaboration between B and T cells which in turn is necessary for an efficient antibody response. These molecules are also expressed on epithelial cells of the thymus, where thymocytes go through positive and negative selection to generate the mature T-cell repertoire. The proper expression of MHC class II molecules is therefore crucial to regulate immune responses. Specifically, the lack of MHC class II expression can cause immunodeficiency whereas aberrant expression might result in autoimmune responses (reviewed in [33]). The expression of MHC class II genes is regulated primarily at the transcriptional level. The molecular mechanisms responsible for the regulation of MHC class II gene expression have been extensively studied (reviewed in [33]). Many cis-regulatory elements have been identified and DNA binding proteins have been characterized that could potentially be involved in the regulation of MHC class II gene expression. The conserved promoter elements, S, X, and Y boxes are present in all class II genes of both human and mouse and these elements are necessary and sufficient to confer both constitutive and inducible expression in transient transfection experiments. Multiple DNA binding proteins which recognize these cis elements, particularly X and Y boxes, have been characterized and cloned [21, 37, 45]. RFX and X2BP, ubiquitously expressed proteins, bind to the X box and X2 box of MHC class II promoter, respectively. The Y box is recognized by another ubiquitously expressed protein NFY [22, 46]. It has been observed that X2BP and NF-Y bind cooperatively with RFX, suggesting that they form a higher order complex [37]. MHC class II genes are inducible
* Correspondence and reprints Microbes and Infection 1999, 879-885
upon IFN-γ treatment and the proximal promoter elements including S, X, and Y boxes are sufficient to mediate the induction [33]. However, the proximal promoter does not contain any conventional IFN-γ responsive element. Rather, IFN-γ induction is mediated indirectly by a transcription factor, class II transactivator (CIITA), the expression of which is IFN-γ-inducible. Bare lymphocyte syndrome (BLS) is a hereditary severe combined immunodeficiency disease. It is characterized by the lack of HLA class II gene expression and a reduced number of mature CD4 T cells in the periphery of BLS patients (reviewed in [33]). There is genetic heterogeneity among BLS patients, and studies demonstrated the existence of at least four distinct complementation groups (group A-D) suggesting that there are four different regulatory genes that are essential for MHC class II gene expression [3, 49]. Cells derived from patients in group A and the in vitro-generated mutant Raji cell line RJ2.2.5 show intact MHC class II structural genes and the normal profile of DNA binding proteins to the MHC class II promoter elements [25]. These results suggest the requirement of other factor(s) necessary to activate class II genes. It has been shown that CIITA can complement MHC class II expression in these cells and that the defect in BLS-2 cells and RJ2.2.5 is caused by a mutation in the CIITA gene [54]. A deficiency in the binding of RFX to the X box of MHC class II promoters is observed in the complementation groups B, C, and D. Successful complementation of the cell line SJO which belongs to the complementation group C led to the identification of RFX5, a new family member of RFX [53]. RFX-associated protein RFXAP, a subunit of RFX, is responsible for the defect of complementation group D [14]. RFXAP interacts with RFX5 but it does not contain the characteristic RFX DNA binding motif. The defect in complementation group B has not yet been identified but it is believed to be a deficiency in an unidentified subunit of RFX.
2. CIITA CIITA was initially identified as a critical transcription factor which is required for both constitutive and IFN-γinducible expression of MHC class II genes [5, 54, 55]. CIITA also activates other genes involved in antigen pre879
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sentation such as invariant chain and H2-M genes [4, 9, 26]. In addition, introduction of the CIITA gene driven by a constitutive promoter is sufficient to activate MHC class II genes in plasmacytoma cells or mouse T cells where MHC class II genes are not normally expressed, suggesting that CIITA is a potent transcriptional activator regulating APC function [7, 51]. However, expression of CIITA in mouse T cells or melanoma cells, which are not professional APCs, is not sufficient to present protein antigens although they can present peptide antigens. Therefore, an additional molecule(s) which is not under the control of CIITA is required to either uptake or process protein antigens. The expression of the CIITA gene is controlled at least by four promoters (PI to PIV), leading to multiple CIITA transcripts with different first exons [39]. PI and PIII direct dendritic cell and B-cell-specific expression respectively, whereas PIV, residing downstream of PIII, mediates IFN-γinducible expression. Like many other IFN-γ-inducible genes CIITA cannot be induced in either JAK1 [5] or STAT-1-deficient cells [29, 36]. It has been shown that STAT-1 binding to the GAS site of the PIV and the subsequent interaction of STAT-1 and USF-1 dictates IFN-γinducible expression [38]. The study by Piskurich et al., however, demonstrated that IFN-γ-inducible expression of CIITA requires 4 kb upstream DNA in addition to sequences between nucleotides -545 to -113 relative to the PIII transcriptional start site [44]. Thus, it is not clear whether the CIITA gene has two independent promoters which respond to IFN-γ induction. It has also been shown that TGF-β inhibits IFN-γ-mediated induction of CIITA expression through the same promoter indicating the promoter is a target for TGF-β. Although CIITA is a potent transcriptional activator, it is not known to bind directly to DNA and its mode of action is not yet completely understood. It has been shown that acidic, proline/serine/threonine-rich, and GTP-binding regions of CIITA are critical for its function in MHC class II gene transactivation [8, 47, 63]. Although the role of the carboxyl-terminus of CIITA is not well characterized, the carboxyl-terminal 41 amino acids of CIITA are indispensable for the activation of MHC class II gene expression [10]. CIITA may function as a transcriptional coactivator interacting with the transcription factors bound to the MHC class II promoter. Studies have shown that the interaction between CIITA and multiple regions of the TATA box binding protein (TBP) and the TBP-associated factor (TAF) is critical to CIITA-mediated transactivation [17, 34]. Furthermore, CIITA interacts with the B-cellspecific transcription factor BOB [16] and RFX which is required for MHC class II gene expression [48]. CIITA is also required for the assembly of transcription factors specifically on the MHC class II and associated genes although this effect is limited in IFN-γ-inducible cell types [60]. It is not surprising, therefore, that CIITA might interact with many different proteins; the nature of the interaction between CIITA and specific DNA binding proteins or coactivators dictates the outcome. Recently, it has been demonstrated that CIITA interacts with the cAMP responsive element binding (CREB) binding protein (CBP) [28]. The acidic domain of CIITA has 880
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been shown to interact with two regions of CBP, 770 N-terminal amino acids of CBP, and the CH3 domain of CBP [28]. The CH3 domain of CBP/p300 interacts with E1A which is known to downregulate MHC class II expression [1, 20, 24]. Thus, E1A competes with CIITA for CBP, resulting in downregulation of MHC class II expression. CBP and p300 are the members of histone acetyltransferases (HATs) and a novel class of transcriptional coactivators that are suspected of being fundamentally important in various signal-modulating transcriptional events. HAT and histone deacetyltransferases have been clearly implicated in the mechanism of transcriptional activation and repression of many genes [50]. p300/CBP can also recruit other coactivators such as SRC-1, p/CIP, and P/CAF [50]. The ability of p300/CBP to interact with multiple, signal-dependent transcription factors has led to the proposal that these coactivators function as signal integrators by coordinating complex signal transduction events at the transcriptional level. Depending on the context, specific transcription factors can either cooperate or interfere with each other. This intriguing observation is consistent with the study of Wright et al., who showed that CIITA is required for the assembly of transcription factors on the MHC class II promoter in IFN-γ-inducible cell types [60]. Thus, it is tempting to speculate that upon IFN-γ signaling p300/CBP integrates signals with transcription factors and with chromatin. This can be carried out by their intrinsic acetyltransferase activities and their ability to associate with acetyltransferase, such as P/CAF. Upon acetylation of histones, the integrity of chromatin is altered and this in turn facilitates the assembly of general transcription factors, such as TATA-binding protein and TAFs, which can associate with p300/CBP into a preinitiation complex. CIITA therefore may play a critical role in assembly of a transcriptional unit by bringing necessary transcription factors, RFX5, BOB, TAFs, and CBP, to the MHC class II promoter. Since the CIITA gene itself has to be transcribed by IFN-γ induction in IFN-γ-inducible cells, the promoter cannot be assembled in the absence of CIITA or IFN-γ signaling. In contrast, in B cells with constitutive transcription of MHC class II, the promoter is already assembled as an active configuration. Thus a loss of CIITA causes only a loss of transcription but not a loss of promoter assembly. Retinoblastoma tumor suppresser protein (RB) can rescue IFN-γ induction of MHC class II gene transcription in noninducible breast carcinoma cells [32]. Furthermore, RB is required for MHC class II inducibility in nonsmall cell lung carcinoma line [31]. Another study revealed that RB facilitates promoter occupancy in the absence of IFN-γ, indicating that RB modifies the chromatin structure independently of transcription [41]. This notion is further supported by the fact that RB interacts with BRG-1 and hBRM which contain bromodomains like that of p300/CBP. This interaction is thought to potentiate the activity of certain sequence-specific DNA binding transcription factors, by disrupting nucleosome structure [13, 42, 52]. Taken together, these observations suggest that the MHC class II promoter, like many other promoters, depends on chromatin remodeling, such as histone acetylation which can increase the accessibility of transcriptional Microbes and Infection 1999, 879-885
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regulatory proteins to chromatin templates. CIITA is a critical mediator of this process to activate MHC class II gene expression both in B cells and IFN-γ-inducible cells.
3. CIITA-independent expression of MHC class II genes The significance of CIITA function in vivo was further confirmed by CIITA–/– mice [6, 59]. Two lines of CIITA–/– mice were generated independently using different strategies; the conventional gene targeting [6] and the cre-loxP recombination system [59]. Both lines show a similar phenotype. CIITA–/– mice do not express conventional MHC class II molecules on the surface of splenic B cells and dendritic cells. In addition, macrophages resident in the peritoneal cavity do not express MHC class II molecules upon IFN-γ stimulation nor do somatic tissues of mice injected with IFN-γ. The levels of Ii and H-2M gene transcripts are substantially decreased but not absent in CIITA–/– mice. The transcription of nonconventional MHC class II genes is, however, not affected by CIITA deficiency. Furthermore, in contrast to recent results on human cell lines [19, 35], CIITA is not critically involved in the IFNγ-induced upregulation of MHC class I genes. Interestingly, a substantial number of cells can express MHC class II molecules in CIITA–/– mice, and MHC classII-positive cells are present in both lines of CIITA–/– mice. When splenic B cells in suspension from CIITA–/– mice were stained with anti-MHC class II antibody, they failed to display class II molecules although MHC class I molecules were present at normal levels. Furthermore, activation of B cells in vitro with lipopolysaccharide (LPS) and IL-4 did not induce MHC class II expression [6]. However, when frozen sections of lymph nodes were examined for the expression of MHC class II molecules, staining was clearly present in the lymph nodes from mutant mice. These MHC class II molecules are properly paired for transport since the staining was detected with the conformation-specific mAb Y3P. Staining was restricted to the paracortex, and the vast majority of B cells in the follicles were negative, in agreement with the flow cytometry analysis. Their location suggests that the class IIpositive cells are dendritic cells. However, most dendritic cells in the corresponding T-cell areas of spleen (the peri-arteriolar lymphoid sheath) expressed very few MHC class II molecules in the same CIITA–/– mice. Thus, dendritic cells (DCs) residing in the spleen and subcutaneous lymph nodes appear to represent distinct subpopulations, differing at the molecular level in their ability to express MHC class II genes independently of CIITA. It is known that there are two distinct ontogenic origins for DCs in mouse: lymphoid and myeloid DCs which induce tolerance and immunity, respectively (reviewed in [15]). Lymphoid DCs (LDCs) originate from a lymphoid precursor present in the thymus and LDCs are located in the thymic medulla and the T-cell zones of the spleen and lymph nodes [2, 61]. In contrast, the precursor of myeloid DCs (MDCs) is shared with that of macrophages. MDCs are located in all nonhematopoietic tissues in immature form and in marginal zones of secondMicrobes and Infection 1999, 879-885
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ary lymphoid tissue. LDCs and MDCs share a number of properties including dendritic morphology and elevated surface expression of a range of molecules required for the stimulation of naive T cells including MHC class I and II [58]. They differ in expression of CD8 and the ligands recognized by monoclonal antibodies 33D1 and NLDC145, which bind the DEC-205 scavenger receptor [2, 58, 61]. Thus, LDCs are CD8α+ 33D1–DEC-205hi, whereas MDCs are CD8α–33D1+ DEC-205–/lo. The phenotype of the majority of interdigitating DCs (IDCs) is consistent with that of LDCs rather than MDCs. It also has been reported that there is heterogeneity in the DC populations in their developmental dependence on transcription factors [62]. However, it is not yet clear whether DCs of different origins perform distinct functions which require different sets of transcriptional machinery to express MHC class II. It will be interesting to see whether the difference of DCs that has been observed correlates with other functional or phenotypic distinctions. It is of interest to note that a subset of DCs from mice deficient in RFX5 also express MHC class II on the cell surface [11]. However, there was no clear correlation between the pattern of MHC class II expression on RFX5deficient DCs and the origin of the DCs, as distinguished by surface expression of CD8. Rather, the RFX5 independent expression of MHC class II is restricted to the final stages of DC maturation. The identify of DCs expressing MHC class II in the absence of CIITA is not yet known. MHC class-II-positive cells are also present in the germinal centers of spleens and lymph nodes of CIITA–/– mice. Since MHC class-II-positive cells are in both the ‘light’ and ‘dark’ areas it seems likely that B cells in GCs express a low level of MHC class II. Consistent with this observation, BLS-2 cells derived from patients in complementation group A with the defect in the CIITA gene also express a very low level of MHC class II [4]. Thus, MHC class II expression is not completely abolished in B cells by CIITA deficiency. The residual expression of MHC class II is unlikely due to an unidentified CIITA homologue since B cells in GC but not other areas of spleen express MHC class II. A similar pattern of expression has been observed in mice carrying the MHC class II transgene with promoter region deletions, particularly ‘Sma’ mutants, in which deletion of the far upstream enhancer element of the Eα gene-restricted expression in the B-cell lineage to those residing in GCs [57]. In addition, activated but not resting B cells of RFX5–/– mice express intermediate levels of MHC class II [11]. Therefore, activation of B cells by cytokines or T-cell help may change the status or lead to a particular combination of transcription factors binding to the S-X-Y motif of the class II promoter. This will allow assembly of a functional preinitiation complex; the lack of one transcription factor, either CIITA or RFX5, can be compensated by others. It should be noted that RJ2.2.5 cells deficient in the CIITA gene expressed MHC class II when cells were introduced with activated ras genes [23] or treated with phorbol esters (J. Lee, personal communication). These results suggest a possible role for the ras signal in class II gene expression in the absence of CIITA. In the thymus of both CIITA–/– lines, there was weak expression of MHC class II in a subset of epithelial cells 881
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characterized by a low level of keratin synthesis. These cells may potentially represent a different lineage or maturation stage; there was stronger and patchy expression in the medulla, on a rather rare population of cells that has not been identified [6, 59]. In contrast, in the thymus of RFX5–/– mice, the medulla stained strongly and homogeneously for MHC class II whereas the cortex was almost completely negative for MHC class II. Although thymic epithelial cells in the thymus of CIITA–/– mice express MHC class II the level is lower than that of the normal mice. As a consequence, very few mature CD4 T cells are present in peripheral lymphoid organs but above they are still those of fully class-II-deficient Aβ–/– mice. CIITA–/– mice are impaired in T-dependent antigen responses and MHC class-II-mediated allogeneic responses. However, GCs do form, suggesting that some B-T interactions occur, but yet they are functional, as reflected by the presence of GCs in the spleen [59]. Since all of these observations were made in two independently derived CIITA–/– lines, this provides strong evidence for a CIITA-independent mode of MHC class II gene expression.
4. CIITA and MHC class II expression in mouse T cells Human T cells express MHC class II upon activation and can present antigen to helper T cells, thereby playing a role in the cellular immune response [18, 27, 43]. However, it is widely believed that activated mouse T cells do not express MHC class II molecules [30, 40]. Previously, we also reported that mouse T cells do not express MHC class II molecules upon stimulation with concanavalin A (ConA) and this was attributed to the lack of CIITA gene expression [7]. Our recent data, however, demonstrates that the endogenous CIITA gene can be expressed in mouse T cells, and the expression of CIITA confers MHC class II gene expression (Gourley et al., unpublished). Interestingly, the expression of CIITA and thus MHC class II is restricted to Th1 but not Th2 cells during differentiation. Naive CD4 T cells differentiate to Th1 and Th2 effector cells and this process depends on the environment, particularly in the presence of interleukins (ILs). IL-12 or IL-4 is a critical determinant to drive Th1 or Th2 respectively [12]. When naive CD4 T cells were stimulated with anti-CD3 in the presence of IL-12 or IL-4, the CIITA gene was expressed in cells stimulated with IL-12 but not IL-4 (Gourley et al., unpublished). Since the stimulation of T cells with IL-12 results in IFN-γ production, the induction of CIITA gene transcripts by IL-12 may be mediated by IFN-γ rather than direct activation of the CIITA gene promoter by IL-12. This was tested by adding anti-IFN-γ antibody in the culture to block endogenously produced IFN-γ. CIITA gene transcripts were greatly reduced when IFN-γ was limited, demonstrating that IFN-γ is the principal activator of CIITA gene transcription. MHC class II molecules were not detectable by flow cytometry suggesting that the level of MHC class II transcripts is too low, while detectable by RT-PCR, to result in protein expression on the cell surface. The study by Thomas et al., however, demonstrated that mouse T-cell clones can express MHC class II on the cell 882
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surface upon induction with a viral peptide [56]. Surface expression of MHC class II by T cells required de novo synthesis in the presence of the stimulatory peptide and was inhibited by coculture with TCR-specific antibody, brefeldin A, or cycloheximide. Control studies indicated that class II was not passively acquired from APCs. Interestingly, MHC class-II-expressing clones are of the Th1 type. A Th2-type clone with the same MHC restriction did not express MHC class II in the presence of stimulatory peptide. Furthermore, extinction of MHC class II expression was evident in the hybridomas generated from the same Th1 clone, which might account for the failure to report class II expression by mouse T cells. Taking these two studies together, they indicate that mouse T cells can express MHC class II under the right environment, and that the expression is restricted in T cells responsive to IFN-γ signaling. The consequence of class II expression by Th1 cells in vivo remains elusive.
5. Concluding remarks Studies to date indicate that there are two distinct mechanisms for expressing MHC class II genes; CIITAdependent and -independent. Although CIITA may not be the ‘master’ control gene for the MHC class II locus based on the findings described above, CIITA is required for the optimum level of MHC class II expression in all cells tested. CIITA is a critical transcription factor for most of the constitutive expression of MHC class II genes. Furthermore, CIITA expression is required for IFN-γ-inducible MHC class II expression. In this respect, it is not surprising to observe CIITA and thus MHC class II expression in Th1 cells. It is evident that mouse T cells can express MHC class II like human T cells if they are stimulated with proper stimuli such as IFN-γ. Therefore, cells capable of transducing the IFN-γ signal, such as mouse T cells undergoing Th1 differentiation, are able to express CIITA as well as MHC class II. Despite all the progress that has been made in understanding the regulation of MHC class II gene expression, many questions still remain unanswered. i) Which subset of DCs and epithelial cells do express MHC class II in the absence of CIITA or RFX5? Do they reflect different lineages? What is the biological consequence of MHC class II expression on these particular cell types? Do they perform different functions as APC? Do they share a common regulatory mechanism to express MHC class II and if so, what is it? ii) What is the signaling event inducing MHC class II expression upon B-cell activation in the absence of CIITA? Does the ras signaling pathway play a role in MHC class II gene expression? Does CIITA has anything to do with ras signaling transduction pathway? iii) Is the level of CIITA and MHC class II RNA high enough under any condition to lead to MHC class II expression on the surface of Th1 cells? Do MHC class-II-expressing Th1 cells exist and present antigen in vivo? What is the biological significance of MHC class II expression by Th1 cells? How does the MHC class II gene become extinguished following immortalization of T-cell hybridoma and what are the regulatory elements in this process? Microbes and Infection 1999, 879-885
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Acknowledgments We are grateful to Drs Lathe Claflin, Wes Dunnick, and Dennis Thiele for their discussion and the critical reading of the manuscript.
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[41] Osborne A., Tschickardt M., Blanck G., Retinoblastoma protein expression facilitates chromatin remodeling at the HLA-DRA promoter, Nucleic Acids Res. 25 (1997) 5095–5102. [42] Peterson C.L., Tamkun J.W., The SWI-SNF complex: a chromatin remodeling machine?, Trends in Biochem. Sci. 20 (1995) 143–146. [43] Pichler W.J., Wyss-Coray T., T cells as antigen-presenting cells, Immunol. Today 315 (1994) 312–315. [44] Piskurich J.F., Wang Y., Linhoff M.W., White L.C., Ting J.P.Y., Identification of distinct regions of 5’ flanking DNA that mediate constitutive IFN-γ, Stat1, and TGF-αregulated expression of the class II transactivator gene, J. Immunol. 160 (1998) 233–240. [45] Reith W., Satola S., Herrero Sanchez C., Lisowskagrospeire B., Griscelli C., Hadam M.R., Mach B., Congenital immunodeficiency with a regulatory defect in MHC class II gene expression lacks a specific HLA-DR promoter binding protein RF-X, Cell 53 (1988) 897–906. [46] Reith W., Siegrist C.A., Durand B., Barras E., Mach B., Function of major histocompatibility complex class II promoters requires cooperative binding between factors RFX and NF-Y, Proc. Natl. Acad. Sci. USA 91 (1994) 554–558. [47] Riley J.L., Westerheide S.D., Price J.A., Brown J.A., Boss J.M., Activation of class II MHC genes requires both the X box region and the class II transactivator (CIITA), Immunity 2 (1995) 533–543. [48] Scholl T., Mahanta S.K., Strominger J.L., Specific complex formation between the type II bare lymphocyte syndromeassociated transactivators CIITA and RFX5, Proc. Natl. Acad. Sci. USA 94 (1997) 6330–6334. [49] Seidl C., Saraiya C., Osterweil Z., Fu Y.P., Lee J.S., Genetic complexity of regulatory mutants defective for HLA class II gene expression, J. Immunol. 148 (1992) 1576–1584. [50] Shikama N., Lyon J., LaThangue N., The p300/CBP family: integrating signals with transcription factors and chromatin, Trends Cell Biol. 7 (1997) 230–236. [51] 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 (1994) 1329–1336. [52] Singh P., Coe J., Hong W., A role for retinoblastoma protein in potentiating transcriptional activation by the glucocorticoid receptor, Nature 374 (1995) 562–565. [53] Steimle V., Durand B., Barras E., Zufferey M., Hadam M., Mach B., Reith W., A novel DNA-binding regulatory factor is mutated in primary MHC class II deficiency (bare lymphocyte syndrome), Genes Dev. 9 (1995) 1021–1032. [54] Steimle V., Otten L.A., Zufferey M., Mach B., Complementation cloning of an MHC class II transactivator mutated in hereditary MHC class II deficiency (or Bare Lymphocyte Syndrome), Cell 75 (1993) 135–146. [55] 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 (1994) 106–109. [56] Thomas D.B., Smith C.A., Graham C.M., Viral peptide specific induction of MHC class II expression by murine T cell clones, J. Immunol. 157 (1996) 2386–2394. Microbes and Infection 1999, 879-885
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[57] Van Ewijk W., Ron Y., Monaco J., Kappler J., Marrack P., LeMeur M., Gerlinger P., Durand B., Benoist C., Mathis D., Compartmentalization of MHC class II gene expression in transgenic mice, Cell 53 (1988) 357–370. [58] Vremec D., Shortman K., Dendritic cell subtypes in mouse lymphoid organs: cross-correlation of surface markers changes with incubation and differences among thymus spleen and lymph nodes, J. Immunol. 159 (1997) 565–573. [59] Williams G.S., Malin M., Chang C.H., Boyd R., Benoist C., Mathis D., Mice lacking the transcription factor CIITA a second look, Int. Immunol. 10 (1998) 1957. [60] Wright K.L., Chin K.C., Linhoff M., Skinner C., Brown J.A., Boss J.M., Stark G.R., Ting J.P.Y., CIITA stimulation of transcription factor binding to major histocompatibility
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complex class II and associated promoters in vivo, Proc. Natl. Acad. Sci. USA 95 (1998) 6267–6272. [61] Wu L., Li C.L., Shortman K., Thymic dendritic cell precursors: relationship to the T lymphocyte lineage and phenotype of the dendritic cell progeny, J. Exp. Med. 184 (1996) 903–911. [62] Wu L., Nichogiannopoulou A., Shortman K., Georgopoulos K., Cell-autonomous defects in dendritic cell populations of Ikaros mutant mice point to a developmental relationship with the lymphoid lineage, Immunity 7 (1997) 483–492. [63] Zhou H., Glimcher L.H., 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 2 (1995) 545–553.
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Class II transactivator: is it a master switch for MHC class II gene expression? Cheong-Hee Chang*, Stacey Roys, Tania Gourley Department of Microbiology and Immunology, The University of Michigan Medical School, Ann Arbor, MI 48109-0620, USA
1. Introduction Major histocompatibility complex (MHC) class II molecules are heterodimeric cell surface glycoproteins whose expression is critical for the development of CD4 T cells and the ability of vertebrates to mount an immune response. These molecules are expressed on antigenpresenting cells (APCs) such as B cells, macrophages, and dendritic cells which take up, process, and present antigens to CD4 T cells. The expression of MHC class II on B cells is required for the collaboration between B and T cells which in turn is necessary for an efficient antibody response. These molecules are also expressed on epithelial cells of the thymus, where thymocytes go through positive and negative selection to generate the mature T-cell repertoire. The proper expression of MHC class II molecules is therefore crucial to regulate immune responses. Specifically, the lack of MHC class II expression can cause immunodeficiency whereas aberrant expression might result in autoimmune responses (reviewed in [33]). The expression of MHC class II genes is regulated primarily at the transcriptional level. The molecular mechanisms responsible for the regulation of MHC class II gene expression have been extensively studied (reviewed in [33]). Many cis-regulatory elements have been identified and DNA binding proteins have been characterized that could potentially be involved in the regulation of MHC class II gene expression. The conserved promoter elements, S, X, and Y boxes are present in all class II genes of both human and mouse and these elements are necessary and sufficient to confer both constitutive and inducible expression in transient transfection experiments. Multiple DNA binding proteins which recognize these cis elements, particularly X and Y boxes, have been characterized and cloned [21, 37, 45]. RFX and X2BP, ubiquitously expressed proteins, bind to the X box and X2 box of MHC class II promoter, respectively. The Y box is recognized by another ubiquitously expressed protein NFY [22, 46]. It has been observed that X2BP and NF-Y bind cooperatively with RFX, suggesting that they form a higher order complex [37]. MHC class II genes are inducible
* Correspondence and reprints Microbes and Infection 1999, 879-885
upon IFN-γ treatment and the proximal promoter elements including S, X, and Y boxes are sufficient to mediate the induction [33]. However, the proximal promoter does not contain any conventional IFN-γ responsive element. Rather, IFN-γ induction is mediated indirectly by a transcription factor, class II transactivator (CIITA), the expression of which is IFN-γ-inducible. Bare lymphocyte syndrome (BLS) is a hereditary severe combined immunodeficiency disease. It is characterized by the lack of HLA class II gene expression and a reduced number of mature CD4 T cells in the periphery of BLS patients (reviewed in [33]). There is genetic heterogeneity among BLS patients, and studies demonstrated the existence of at least four distinct complementation groups (group A-D) suggesting that there are four different regulatory genes that are essential for MHC class II gene expression [3, 49]. Cells derived from patients in group A and the in vitro-generated mutant Raji cell line RJ2.2.5 show intact MHC class II structural genes and the normal profile of DNA binding proteins to the MHC class II promoter elements [25]. These results suggest the requirement of other factor(s) necessary to activate class II genes. It has been shown that CIITA can complement MHC class II expression in these cells and that the defect in BLS-2 cells and RJ2.2.5 is caused by a mutation in the CIITA gene [54]. A deficiency in the binding of RFX to the X box of MHC class II promoters is observed in the complementation groups B, C, and D. Successful complementation of the cell line SJO which belongs to the complementation group C led to the identification of RFX5, a new family member of RFX [53]. RFX-associated protein RFXAP, a subunit of RFX, is responsible for the defect of complementation group D [14]. RFXAP interacts with RFX5 but it does not contain the characteristic RFX DNA binding motif. The defect in complementation group B has not yet been identified but it is believed to be a deficiency in an unidentified subunit of RFX.
2. CIITA CIITA was initially identified as a critical transcription factor which is required for both constitutive and IFN-γinducible expression of MHC class II genes [5, 54, 55]. CIITA also activates other genes involved in antigen pre879
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sentation such as invariant chain and H2-M genes [4, 9, 26]. In addition, introduction of the CIITA gene driven by a constitutive promoter is sufficient to activate MHC class II genes in plasmacytoma cells or mouse T cells where MHC class II genes are not normally expressed, suggesting that CIITA is a potent transcriptional activator regulating APC function [7, 51]. However, expression of CIITA in mouse T cells or melanoma cells, which are not professional APCs, is not sufficient to present protein antigens although they can present peptide antigens. Therefore, an additional molecule(s) which is not under the control of CIITA is required to either uptake or process protein antigens. The expression of the CIITA gene is controlled at least by four promoters (PI to PIV), leading to multiple CIITA transcripts with different first exons [39]. PI and PIII direct dendritic cell and B-cell-specific expression respectively, whereas PIV, residing downstream of PIII, mediates IFN-γinducible expression. Like many other IFN-γ-inducible genes CIITA cannot be induced in either JAK1 [5] or STAT-1-deficient cells [29, 36]. It has been shown that STAT-1 binding to the GAS site of the PIV and the subsequent interaction of STAT-1 and USF-1 dictates IFN-γinducible expression [38]. The study by Piskurich et al., however, demonstrated that IFN-γ-inducible expression of CIITA requires 4 kb upstream DNA in addition to sequences between nucleotides -545 to -113 relative to the PIII transcriptional start site [44]. Thus, it is not clear whether the CIITA gene has two independent promoters which respond to IFN-γ induction. It has also been shown that TGF-β inhibits IFN-γ-mediated induction of CIITA expression through the same promoter indicating the promoter is a target for TGF-β. Although CIITA is a potent transcriptional activator, it is not known to bind directly to DNA and its mode of action is not yet completely understood. It has been shown that acidic, proline/serine/threonine-rich, and GTP-binding regions of CIITA are critical for its function in MHC class II gene transactivation [8, 47, 63]. Although the role of the carboxyl-terminus of CIITA is not well characterized, the carboxyl-terminal 41 amino acids of CIITA are indispensable for the activation of MHC class II gene expression [10]. CIITA may function as a transcriptional coactivator interacting with the transcription factors bound to the MHC class II promoter. Studies have shown that the interaction between CIITA and multiple regions of the TATA box binding protein (TBP) and the TBP-associated factor (TAF) is critical to CIITA-mediated transactivation [17, 34]. Furthermore, CIITA interacts with the B-cellspecific transcription factor BOB [16] and RFX which is required for MHC class II gene expression [48]. CIITA is also required for the assembly of transcription factors specifically on the MHC class II and associated genes although this effect is limited in IFN-γ-inducible cell types [60]. It is not surprising, therefore, that CIITA might interact with many different proteins; the nature of the interaction between CIITA and specific DNA binding proteins or coactivators dictates the outcome. Recently, it has been demonstrated that CIITA interacts with the cAMP responsive element binding (CREB) binding protein (CBP) [28]. The acidic domain of CIITA has 880
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been shown to interact with two regions of CBP, 770 N-terminal amino acids of CBP, and the CH3 domain of CBP [28]. The CH3 domain of CBP/p300 interacts with E1A which is known to downregulate MHC class II expression [1, 20, 24]. Thus, E1A competes with CIITA for CBP, resulting in downregulation of MHC class II expression. CBP and p300 are the members of histone acetyltransferases (HATs) and a novel class of transcriptional coactivators that are suspected of being fundamentally important in various signal-modulating transcriptional events. HAT and histone deacetyltransferases have been clearly implicated in the mechanism of transcriptional activation and repression of many genes [50]. p300/CBP can also recruit other coactivators such as SRC-1, p/CIP, and P/CAF [50]. The ability of p300/CBP to interact with multiple, signal-dependent transcription factors has led to the proposal that these coactivators function as signal integrators by coordinating complex signal transduction events at the transcriptional level. Depending on the context, specific transcription factors can either cooperate or interfere with each other. This intriguing observation is consistent with the study of Wright et al., who showed that CIITA is required for the assembly of transcription factors on the MHC class II promoter in IFN-γ-inducible cell types [60]. Thus, it is tempting to speculate that upon IFN-γ signaling p300/CBP integrates signals with transcription factors and with chromatin. This can be carried out by their intrinsic acetyltransferase activities and their ability to associate with acetyltransferase, such as P/CAF. Upon acetylation of histones, the integrity of chromatin is altered and this in turn facilitates the assembly of general transcription factors, such as TATA-binding protein and TAFs, which can associate with p300/CBP into a preinitiation complex. CIITA therefore may play a critical role in assembly of a transcriptional unit by bringing necessary transcription factors, RFX5, BOB, TAFs, and CBP, to the MHC class II promoter. Since the CIITA gene itself has to be transcribed by IFN-γ induction in IFN-γ-inducible cells, the promoter cannot be assembled in the absence of CIITA or IFN-γ signaling. In contrast, in B cells with constitutive transcription of MHC class II, the promoter is already assembled as an active configuration. Thus a loss of CIITA causes only a loss of transcription but not a loss of promoter assembly. Retinoblastoma tumor suppresser protein (RB) can rescue IFN-γ induction of MHC class II gene transcription in noninducible breast carcinoma cells [32]. Furthermore, RB is required for MHC class II inducibility in nonsmall cell lung carcinoma line [31]. Another study revealed that RB facilitates promoter occupancy in the absence of IFN-γ, indicating that RB modifies the chromatin structure independently of transcription [41]. This notion is further supported by the fact that RB interacts with BRG-1 and hBRM which contain bromodomains like that of p300/CBP. This interaction is thought to potentiate the activity of certain sequence-specific DNA binding transcription factors, by disrupting nucleosome structure [13, 42, 52]. Taken together, these observations suggest that the MHC class II promoter, like many other promoters, depends on chromatin remodeling, such as histone acetylation which can increase the accessibility of transcriptional Microbes and Infection 1999, 879-885
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regulatory proteins to chromatin templates. CIITA is a critical mediator of this process to activate MHC class II gene expression both in B cells and IFN-γ-inducible cells.
3. CIITA-independent expression of MHC class II genes The significance of CIITA function in vivo was further confirmed by CIITA–/– mice [6, 59]. Two lines of CIITA–/– mice were generated independently using different strategies; the conventional gene targeting [6] and the cre-loxP recombination system [59]. Both lines show a similar phenotype. CIITA–/– mice do not express conventional MHC class II molecules on the surface of splenic B cells and dendritic cells. In addition, macrophages resident in the peritoneal cavity do not express MHC class II molecules upon IFN-γ stimulation nor do somatic tissues of mice injected with IFN-γ. The levels of Ii and H-2M gene transcripts are substantially decreased but not absent in CIITA–/– mice. The transcription of nonconventional MHC class II genes is, however, not affected by CIITA deficiency. Furthermore, in contrast to recent results on human cell lines [19, 35], CIITA is not critically involved in the IFNγ-induced upregulation of MHC class I genes. Interestingly, a substantial number of cells can express MHC class II molecules in CIITA–/– mice, and MHC classII-positive cells are present in both lines of CIITA–/– mice. When splenic B cells in suspension from CIITA–/– mice were stained with anti-MHC class II antibody, they failed to display class II molecules although MHC class I molecules were present at normal levels. Furthermore, activation of B cells in vitro with lipopolysaccharide (LPS) and IL-4 did not induce MHC class II expression [6]. However, when frozen sections of lymph nodes were examined for the expression of MHC class II molecules, staining was clearly present in the lymph nodes from mutant mice. These MHC class II molecules are properly paired for transport since the staining was detected with the conformation-specific mAb Y3P. Staining was restricted to the paracortex, and the vast majority of B cells in the follicles were negative, in agreement with the flow cytometry analysis. Their location suggests that the class IIpositive cells are dendritic cells. However, most dendritic cells in the corresponding T-cell areas of spleen (the peri-arteriolar lymphoid sheath) expressed very few MHC class II molecules in the same CIITA–/– mice. Thus, dendritic cells (DCs) residing in the spleen and subcutaneous lymph nodes appear to represent distinct subpopulations, differing at the molecular level in their ability to express MHC class II genes independently of CIITA. It is known that there are two distinct ontogenic origins for DCs in mouse: lymphoid and myeloid DCs which induce tolerance and immunity, respectively (reviewed in [15]). Lymphoid DCs (LDCs) originate from a lymphoid precursor present in the thymus and LDCs are located in the thymic medulla and the T-cell zones of the spleen and lymph nodes [2, 61]. In contrast, the precursor of myeloid DCs (MDCs) is shared with that of macrophages. MDCs are located in all nonhematopoietic tissues in immature form and in marginal zones of secondMicrobes and Infection 1999, 879-885
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ary lymphoid tissue. LDCs and MDCs share a number of properties including dendritic morphology and elevated surface expression of a range of molecules required for the stimulation of naive T cells including MHC class I and II [58]. They differ in expression of CD8 and the ligands recognized by monoclonal antibodies 33D1 and NLDC145, which bind the DEC-205 scavenger receptor [2, 58, 61]. Thus, LDCs are CD8α+ 33D1–DEC-205hi, whereas MDCs are CD8α–33D1+ DEC-205–/lo. The phenotype of the majority of interdigitating DCs (IDCs) is consistent with that of LDCs rather than MDCs. It also has been reported that there is heterogeneity in the DC populations in their developmental dependence on transcription factors [62]. However, it is not yet clear whether DCs of different origins perform distinct functions which require different sets of transcriptional machinery to express MHC class II. It will be interesting to see whether the difference of DCs that has been observed correlates with other functional or phenotypic distinctions. It is of interest to note that a subset of DCs from mice deficient in RFX5 also express MHC class II on the cell surface [11]. However, there was no clear correlation between the pattern of MHC class II expression on RFX5deficient DCs and the origin of the DCs, as distinguished by surface expression of CD8. Rather, the RFX5 independent expression of MHC class II is restricted to the final stages of DC maturation. The identify of DCs expressing MHC class II in the absence of CIITA is not yet known. MHC class-II-positive cells are also present in the germinal centers of spleens and lymph nodes of CIITA–/– mice. Since MHC class-II-positive cells are in both the ‘light’ and ‘dark’ areas it seems likely that B cells in GCs express a low level of MHC class II. Consistent with this observation, BLS-2 cells derived from patients in complementation group A with the defect in the CIITA gene also express a very low level of MHC class II [4]. Thus, MHC class II expression is not completely abolished in B cells by CIITA deficiency. The residual expression of MHC class II is unlikely due to an unidentified CIITA homologue since B cells in GC but not other areas of spleen express MHC class II. A similar pattern of expression has been observed in mice carrying the MHC class II transgene with promoter region deletions, particularly ‘Sma’ mutants, in which deletion of the far upstream enhancer element of the Eα gene-restricted expression in the B-cell lineage to those residing in GCs [57]. In addition, activated but not resting B cells of RFX5–/– mice express intermediate levels of MHC class II [11]. Therefore, activation of B cells by cytokines or T-cell help may change the status or lead to a particular combination of transcription factors binding to the S-X-Y motif of the class II promoter. This will allow assembly of a functional preinitiation complex; the lack of one transcription factor, either CIITA or RFX5, can be compensated by others. It should be noted that RJ2.2.5 cells deficient in the CIITA gene expressed MHC class II when cells were introduced with activated ras genes [23] or treated with phorbol esters (J. Lee, personal communication). These results suggest a possible role for the ras signal in class II gene expression in the absence of CIITA. In the thymus of both CIITA–/– lines, there was weak expression of MHC class II in a subset of epithelial cells 881
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characterized by a low level of keratin synthesis. These cells may potentially represent a different lineage or maturation stage; there was stronger and patchy expression in the medulla, on a rather rare population of cells that has not been identified [6, 59]. In contrast, in the thymus of RFX5–/– mice, the medulla stained strongly and homogeneously for MHC class II whereas the cortex was almost completely negative for MHC class II. Although thymic epithelial cells in the thymus of CIITA–/– mice express MHC class II the level is lower than that of the normal mice. As a consequence, very few mature CD4 T cells are present in peripheral lymphoid organs but above they are still those of fully class-II-deficient Aβ–/– mice. CIITA–/– mice are impaired in T-dependent antigen responses and MHC class-II-mediated allogeneic responses. However, GCs do form, suggesting that some B-T interactions occur, but yet they are functional, as reflected by the presence of GCs in the spleen [59]. Since all of these observations were made in two independently derived CIITA–/– lines, this provides strong evidence for a CIITA-independent mode of MHC class II gene expression.
4. CIITA and MHC class II expression in mouse T cells Human T cells express MHC class II upon activation and can present antigen to helper T cells, thereby playing a role in the cellular immune response [18, 27, 43]. However, it is widely believed that activated mouse T cells do not express MHC class II molecules [30, 40]. Previously, we also reported that mouse T cells do not express MHC class II molecules upon stimulation with concanavalin A (ConA) and this was attributed to the lack of CIITA gene expression [7]. Our recent data, however, demonstrates that the endogenous CIITA gene can be expressed in mouse T cells, and the expression of CIITA confers MHC class II gene expression (Gourley et al., unpublished). Interestingly, the expression of CIITA and thus MHC class II is restricted to Th1 but not Th2 cells during differentiation. Naive CD4 T cells differentiate to Th1 and Th2 effector cells and this process depends on the environment, particularly in the presence of interleukins (ILs). IL-12 or IL-4 is a critical determinant to drive Th1 or Th2 respectively [12]. When naive CD4 T cells were stimulated with anti-CD3 in the presence of IL-12 or IL-4, the CIITA gene was expressed in cells stimulated with IL-12 but not IL-4 (Gourley et al., unpublished). Since the stimulation of T cells with IL-12 results in IFN-γ production, the induction of CIITA gene transcripts by IL-12 may be mediated by IFN-γ rather than direct activation of the CIITA gene promoter by IL-12. This was tested by adding anti-IFN-γ antibody in the culture to block endogenously produced IFN-γ. CIITA gene transcripts were greatly reduced when IFN-γ was limited, demonstrating that IFN-γ is the principal activator of CIITA gene transcription. MHC class II molecules were not detectable by flow cytometry suggesting that the level of MHC class II transcripts is too low, while detectable by RT-PCR, to result in protein expression on the cell surface. The study by Thomas et al., however, demonstrated that mouse T-cell clones can express MHC class II on the cell 882
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surface upon induction with a viral peptide [56]. Surface expression of MHC class II by T cells required de novo synthesis in the presence of the stimulatory peptide and was inhibited by coculture with TCR-specific antibody, brefeldin A, or cycloheximide. Control studies indicated that class II was not passively acquired from APCs. Interestingly, MHC class-II-expressing clones are of the Th1 type. A Th2-type clone with the same MHC restriction did not express MHC class II in the presence of stimulatory peptide. Furthermore, extinction of MHC class II expression was evident in the hybridomas generated from the same Th1 clone, which might account for the failure to report class II expression by mouse T cells. Taking these two studies together, they indicate that mouse T cells can express MHC class II under the right environment, and that the expression is restricted in T cells responsive to IFN-γ signaling. The consequence of class II expression by Th1 cells in vivo remains elusive.
5. Concluding remarks Studies to date indicate that there are two distinct mechanisms for expressing MHC class II genes; CIITAdependent and -independent. Although CIITA may not be the ‘master’ control gene for the MHC class II locus based on the findings described above, CIITA is required for the optimum level of MHC class II expression in all cells tested. CIITA is a critical transcription factor for most of the constitutive expression of MHC class II genes. Furthermore, CIITA expression is required for IFN-γ-inducible MHC class II expression. In this respect, it is not surprising to observe CIITA and thus MHC class II expression in Th1 cells. It is evident that mouse T cells can express MHC class II like human T cells if they are stimulated with proper stimuli such as IFN-γ. Therefore, cells capable of transducing the IFN-γ signal, such as mouse T cells undergoing Th1 differentiation, are able to express CIITA as well as MHC class II. Despite all the progress that has been made in understanding the regulation of MHC class II gene expression, many questions still remain unanswered. i) Which subset of DCs and epithelial cells do express MHC class II in the absence of CIITA or RFX5? Do they reflect different lineages? What is the biological consequence of MHC class II expression on these particular cell types? Do they perform different functions as APC? Do they share a common regulatory mechanism to express MHC class II and if so, what is it? ii) What is the signaling event inducing MHC class II expression upon B-cell activation in the absence of CIITA? Does the ras signaling pathway play a role in MHC class II gene expression? Does CIITA has anything to do with ras signaling transduction pathway? iii) Is the level of CIITA and MHC class II RNA high enough under any condition to lead to MHC class II expression on the surface of Th1 cells? Do MHC class-II-expressing Th1 cells exist and present antigen in vivo? What is the biological significance of MHC class II expression by Th1 cells? How does the MHC class II gene become extinguished following immortalization of T-cell hybridoma and what are the regulatory elements in this process? Microbes and Infection 1999, 879-885
Regulation of MHC class II expression
Acknowledgments We are grateful to Drs Lathe Claflin, Wes Dunnick, and Dennis Thiele for their discussion and the critical reading of the manuscript.
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