A family of trans-acting factors with distinct regulatory functions control expression of MHC class...

Irnmunol Res 1990;9:20-33 " 1990 S. Karger AG, Basel

0257-277X '90/0091-002052.7510

A Family of Trans-Acting Factors with Distinct Regulatory Functions Control Expression of MHC Class II Genes

Roberro S. Accolh~ a, Paolo Dellabona b, Leonardo Scarpellino a, Giuseppe Carra c, Silvia Sarloris a

~lstitmo di Scienze Immunologiche, Universith di Verona. Policlinico di Borgo Roma, Verona. Italy; b Laboratoire de G6n6tique Mol6culaire des Eucaryotes du CNRS, Institut dc Chimie Biologique, Faculte de M6decine, Strasbourg, France; qst i tuto di Chimica Biologica, Universith di Verona, Italy

Introduction

Genes of the major histocompatibility complex (MItC) encode for products which play a key role in the homeostasis of the immune system. Among them, the class II MHC products are cel[ surface glycoproteins required for a correct antigen recognition by T cells of helper (TH) type [1 ]. Class II glycoproteins are heterodimers composed of an a- and a 13-subunit of 34 and 28 kilodallons, respectively. The vast majority, if not all, of class II molecules and corresponding genes have been identified and biochemically characterized. These studies have provided di- rect evidence of the high degree of structural polymorphism and heterogeneity of class II genes. In man, MHC class II genes are located in a region spanning at least 1,200 kilo- base of DNA in chromosome 6. The esti- mated length of the mouse class II region is not available but probably is shorter than the corresponding human region. Human class II genes are divided in at least three subregions: DP. DQ and DR, each including sev-

eral genes (2 a- and 2 {3-genes in DP and DQ subregions, 2-3 13- and a single a-gene in the DR subregion). Additional genes have been described as DOI3 and DZct. Both genes map between DP and DQ subregions, DZ being closely associated to DP. At least one a and one 13 DP gene are expressed to form the clas- sical class II heterodimer. The remaining DP genes are pseudogenes and thus are not expressed. The four DQ genes are all expressable, although controversy still exists whether only one or both a-13 pairs can be expressed as mature proteins. Of the three possible DR13 genes only two are expressed in associa- tion with the corresponding ct-gene. The third DR13 gene is a pseudogene. In certain haplotypes like DR1 a single expressable [~- gene has been found, mRNA for DO13 and DZct have been detected although no corresponding protein products have yet been described. The routine class II region is relatively simpler than the human class II region. Two ct- and two ~3-genes have been identified which code for the corresponding subunit of the two class II heterodimers, I-A

Transcriptional Regulation of MHC Class II Genes 21

and I-E, found on the cell surface. I-Act and 13 genes reside in the I-A subregion; I-Ect is located in the I-E subregion, whereas the l- Et3 gene resides in the I-A subregion. Other genes have been identified as I-AJ33, which is a pseudogene, and I-A132 (homologue to DO[3), which is expressed at the mRNA level but of which, like human DOI3 and DZcg no corresponding protein product has been isolated. For a comprehensive review on MHC class I[ genes and their products see Guille- mot et al. [2] and the literature cited therein. Beside polymorphism and heterogeneity, an other element of complexity, namely regulation of expression, makes the study of the MHC class II system a matter of intensive investigation. MHC gene products exert their immune-related function only if expressed on the cell surface and only if they are expressed in a 'sufficient quantity'. Thus any biological mechanism affecting the expression of MHC genes and/or their products is likely to produce an imbalance on MHC-related functions.

For several years our laboratory has de- voted particular attention to the study of human and mouse class II gene expression. In this report we will review the work performed by our group and in collaboration with several colleagues. We will try to frame it within the context of the present knowl- edge of class 11 gene regulation as it stems from the work of many laboratories involved in this exciting aspect of immunogenetics.

The Two Basic Ways of Expressing Class II Genes

If one analyzes a wide variety of tissues and organs, it clearly appears that class II gene expression is not a peculiarity of all tis-

sues. It is found mainly in cells of the immune system, as well as in certain epithelial cells of the thymus and in the basal mem- brane of the kidney. However. under the influence of certain physiological and pathological stimuli a wide variety of cells from different tissues acquire the capacity to express class II gene products. Thus at least two modes of expression can be de- fined: (1) constitutive expression, that is ac- quisition and maintenance during ontogeny of the relevant phenotype, where B cells can be considered as the prototype of constitutive class II gene expression, and (2) inducible expression, that is capacity to tran- siently express the relevant phenotype. Cases of inducible expression may include stages of ontogenetic development or states of activation by a variety of physiological or pathological stimuli. Indeed, the vast majority of cell types expressing class II genes are included in this category. During hemato- poiesis (fig. 1) class II gene expression is de- tectable in early precursors of the lymphoid, granulocyte-macrophage and erythroid lineages [reviewed in 3]. As differentiation proceeds, some of the above lineages main- tain the capacity to express class II genes, whereas others lose it. It has been demonstrated that cells expressing class lI genes de novo can perlbrm immune-related /'unc- tions like antigen presentation to T cells [4]. It is known that a wide variety of immune disorders as autoimmune diseases [5] or im- munodeficiencies [6] are characterized by an altered expression of these genes. Thus it is fundamental to study the biological mechanisms implicated in the control of class II gene expression and particularly the genetic mechanisms which dictate the distinct modes of expression of these genes in various cell types.

22 Accolla/Dellabona/Scarpellino/Carra/Sartoris

S'I" E M CELL

�9 , "i" L-PROG

�9 0 0 " ' 0 BFU-E CFU-E " E-BLAST E

0 . . . 0 , 0 .... : : :- . CFU-ME ME P L A T

0 , 0 ~ 0 " " ' CFU.EO$ EOS.BLAST " . EOS , " " : ' i , ' '

. �9 O �9 i : - Y : i ' ' "

c F U ~ M I . �9 " : " , o ~. ' ~ � 9

M-BLAb'T ": MONO " - 5 . . . �9 .

�9 �9 �9 O PRE-B IMM-B B PLAS

0 ,0 0 0 = PRE-T CORTEX-T M E D - T T

Fig. 1. MHC class II gene expression in hemato- poiesis. Circles represent stages of development in which class II antigen expression is present (e) or absent (o). CFU = Colony-forming unit; BFU = burst-forming unit; GEMM = granulocyte, er)'thro- cyte, macrophage, megakaryocyte progenitors: L- PROG = lymphocyte progenilor; E-BLAST = ery- throblast; E = mature erythrocyte; ME = megakaryocyte: PLAT = platelet: CFU-EOS = precursors of eosinophils; EOS-BLAST = eosinophiloblast; EOS =

mature eosinophiI; CFU-GM = common granulocyte-monocyte/macrophage precursor; MYE-BLAST = myeloblast; NEUTR = neutrophil: M-BLAST = monoblast; MONO = monocyte/macrophage; Pre-B = B cell precursor; IMM-B = immature B cell; B = mature B cell; PLAS = plasma cell; PRE-T = T lymphocyte precursor, CORTEX-T and MED-T = immature 1" lymphocyte of the thymus cortex and thymus medulla, respectively; T = mature peripheral T cell.

T h e G e n e t i c Con t ro l of Cons t i tu t ive

C l a s s II E x p r e s s i o n

As m e n t i o n e d a b o v e , c o n s t i t u t i v e class II

gene e x p r e s s i o n is r e s t r i c t ed to ve ry few cell

types. T h e p r o t o t y p e o f th is exp re s s ion is

f ound in B cells, the c o m p a r t m e n t o f h e m o -

p o i e t i c cells p r o g r a m m e d to syn thes ize a n d

sec re te spec i f i c a n t i b o d i e s . In m a n all assess-

ab le B cell p recur so r s , B cell blasts a n d ma-

tu re B cel ls exp res s class II an t igens [7]. In

c e r t a i n c i r c u m s t a n c e s class II gene expres -

s ion in B cells can be i n c r e a s e d b u t n e v e r

i n d u c e d ex novo . W e h a v e s t u d i e d t h e ge-

ne t i c c o n t r o l o f the c o n s t i t u t i v e class II gene

e x p r e s s i o n in B cells by t a k i n g a d v a n t a g e o f

e x p e r i m e n t a l l y i n d u c e d B cell m u t a n t s tha t

h a v e lost e x p r e s s i o n o f th is gene fami ly .

Class II n e g a t i v e B cells w e r e d e r i v e d f r o m

the B u r k i t t ' s l y m p h o m a l ine Ra j i by y-ray

m u t a g e n e s i s f o l l owed by i m m u n o s e l e c t i o n

w i t h m o n o c l o n a l a n t i b o d i e s and c o m p l e -


meat. Interestingly, immunoselection performed with either anti-DR, anti-DR/DP or anti-DQ monomorphic antibodies gave rise to very similar variants characterized bv a loss of expression of the entire family of class II molecules [8] generally attributable to the absence of specific mRNA of both ct- and 13- subunits [9]. Similar variants have been originated also by other investigators [10-12]. The relatively high frequency of these variants in the mutagenized and immunose- lected population (1:500,000) and the apparent integrity of the various class II genes strongly suggested that the class II negative phenotype arose from a defect in the mechanism controlling the expression of class II genes and not from mutations or deletions of the structural genes. Furthermore, the absence of expression of both class II hapfo- types in the variants without an apparent loss of expression of MHC class I, invariant (In) chain and of all other assessable B cell markers was an indication of the exquisite specificity of such a control mechanism. A more refined molecular analysis of these variants demonstrated that the transcription of class II genes was abolished, The basic question was then to assess whether the class II negative phenotype of the Raji variants was the result of the loss of a positive regulatory control or the activation of a suppressor mechanism. In order to answer this question, cell hybridization experiments between the human class II negative RJ2.2.5 variant and class lI positive B cells were performed. As partner of the human variant, we chose mouse B cells Ibr several reasons. First, interspecies somatic ceil hybrids are easier to study as compared to intraspecies hybrids, since reagents, as monoclonal antibodies, that can easily distinguish human markers from routine markers and vice versa, are

available; these reagents could then be used for rapid screening of the hybrids by immu- nofluorescence and cell sorter analyses or by radioimmunoassay. Second, interspecies somatic cell hybrids tend to preferentially seg- regate chromosomes of one partner; thus the loss or gain of a specific phenotype should be easily correlated to a specific chromosome. Third, mouse and human class II gene expression may be under the control of similar genetic mechanisms (this was indeed our strong bias at the time we initiated these experiments), and therefore complementation experiments could be possible. In a first series of experiments we fused RJ2,2.5 human cells with mouse M12.4.1 B lymphoma cells. We were extremely happy to find that certain hybrids reexpressed the human class tl positive phenotvpe [13]. Reexpression of human class lI genes associated to the stabil- ity of murine class lI gene expression suggested that the phenotype of the RJ2.2.5 human variant was the result of a loss of a positive regulator whose function can be complemented by the analogue of the murine B cells. However, the chromosome segregation pattern of the hybrids (preferential segregation of human vs. mouse chromosomes) could not rule out the possibility that a suppressor factor of human origin and species-specific was operating in RJ2.2.5 cells and was segregating preferentially in the hybrids. The activator nature of the trans-acting regulatory factor was proved by a further series of experiments in which normal murine spleen cells were used as fusion partners of RJ2.2.5 cells [14]. Again, hybrids were generated that reexpressed the human class I1 genes. Furthermore and more importantly, micromaniputated hybrid clones selected on the basis of a human class II positive phenotype spontaneously reverted, with time in

2~ Accolla/Deltabona/Scarpellino/Carra/Sartoris

culture, versus a negative phenotype as a function of loss of routine genetic material. Moreover the number of cells loosing mu- fine markers such as MHC class I and class II antigens was clearly different from the number of cells loosing reexpressability of human class II genes, indicating that the two events were dissociated. This promptcd us to physically enrich distinct populations of cloned hybrids by cell-sorting procedures. Karyotype and molecular analyses of these enriched populations demonstrated that reexpression of human class II genes as well as maintenance of routine class II gene expression in the hybrids was under the control of a positive trans-acting activator factor encoded by routine chromosome 16 and thus not only MHC-unlinked but also located on a distinct chromosome. The locus encoding the trans-acting activator has been designated as air-I (activator of immune response genes number I) [15]. alr-I represents the first identified locus controlling the expression of a family of MHC genes.

In the course of these experiments we observed that not all human class II genes of RJ2.2.5 could be reexpressed at levels comparable to those found in the parental Raji cells. In particular, DQct and 13 genes were either not expressed or expressed at very low levels. Of course it is very difficult to establish general rules for the physiology of gene expression on the basis of r egu la to r events taking place in somatic cett hybrids and particularly in interspecies hybrids. However, the fact that some precursors of hemopoietic cells [ 161 as well as some human tumors [ 17, 18] express DR in the absence of expression of DQ genes suggests that the various human class II genes may be under the control of various factors, themselves developmentally regulated. Expression of the gene encoding

the In chain, a non-MHC-encoded glycopro- tein associated to class II heterodimers intra- cellularly, is also developmentally regulated [19]. In cells like fibroblasts and endothelial cells, the de novo induction of class II gene expression is accompanied by a corresponding induction of In chain gene expression [20]. Both events seem to be due to the con- comitant activation of gene transcription. However in certain cases the In chain can be expressed in the absence of class II gene expression. For example In chain mRNA and corresponding proteins are present in RJ2.2.5 class II negative variant although at reduced levels as compared to wild-type parental Raji cells [9, 2i]. After hybridization and positive complementat ion by a mouse B cell, reexpression of human class II genes was accompanied by a strong increase in the level of In-chain-specific mRNA comparable to the levels observed in the parental Raji cells [14]. Recent experiments performed in our laboratory on isolated nuclei of such somatic cell hybrids indicate that increased levels of mRNA are due to the increased transcription of In chain gene [Deltabona and Accolla, unpubl, results]. Thus, although In chain expression can be found in the absence of class II gene expression, the level of transcription of this gene in B cells is somehow correlated with the expression of class II genes, suggesting the existence of shared mechanisms of control of gene expression.

From the above considerations one can anticipate that aIr-I is not the unique locus controlling class II gene expression in B cells. Recent experiments conducted in collaboration with J. Strominger at Harvard and D. Pious at the University of Seattle confirm this expectation. We will briefly discuss the experimental approach and the results dem-

Transcriptional Regulation of MHC Class i[ Genes 25

onstrating the existence of a heterogeneous family of B-cell-specific trans-acting activators.

A severe combined immunodeficiency syndrome, congenital in nature and affecting neonates and very young children, designated bare-lymphocyte syndrome (BLS) has been described. BLS is characterized by severe opportunistic infections caused by the incapacity of the individual to mount a normal immune response [6]. The analysis of blood cells from BES patients shows that B cells and monocytes do not express at all class II antigens. The activation of monocytes by interferon-y ([FN-y) does not result in the induction of class II expression in these cells. Similarly, T cells activated by mitogens do not express class II products. Thus the immunodeficiency is brought about by the incapacity of accessory cells and in general of antigen-presenting cells to perform their function. When B cells are analyzed at molecular level, a very peculiar aspect is observed: lack of class II mRNA of all identifiable class I[ genes and presence, although at reduced levels, of In chain mRNA [22]. This pattern is highly reminis- cent of the pattern observed in our class II negative mutants. It was therefore pertinent to ask whether the genetic defect of RJ2.2.5 was similar to the one observed in BLS. Somatic cell fusions between RJ2.2.5 and TF, a cell line originated from a patient su f feting from BLS. were carried out. In addi- tion somatic cell fusions were also performed by using as partner cells the 6.1.6 B cells originated in the laboratory of D. Pious and displaying characteristics similar to RJ2.2.5 cells [10, 11, 23]. Heterokaryons were analyzed soon after fusion by several assays including cell surface immunofluores- cence and RNA typing to assess the class I1

phenotype of fused cells. Results clearly demonstrated that reexpression of class 1I genes both at mRNA and protein level and from both parental cells can be obtained in hybrids between RJ2.2.5 and TF or RJ2.2.5 and 6.1.6 but not in hybrids between 6.1.6 and TF, suggesting that the genetic defects implicated in this form of BLS and in 6.1.6 B cell variant do not affect, and are distinct from, the air-1 locus function [24]. The locus (or loci) encoding the activator factors com- plementing the defect of TF and 6.1.6 and present in RJ2.2.5 cells may be operationally designated as air-2. The existence of more than one factor regulating the expression of MHC class II genes in B cells is supported also by findings of other investigators who have used similar somatic cell genetics ap- proaches to complement the defect of 6.1.6 cells [25] and other class II negative B cell variants [261.

It has been recently shown that the promoter region of class II genes contains DNA sequence motifs highly conserved between mouse and man that are absolutely necessa~" for the expression of the class It gene family [27, 28]. These sequence motifs, designated X and Y boxes, respectively, located in the 5' flanking portion of the class II genes behave as enhancer elements and can modulate expression of heterologous genes. X and Y boxes bind specific DNA-binding proteins which may have functional importance [28- 31]. Since we knew that the alr- l -encoded trans-acting factor was implicated in the control of class II gene transcription we first asked whether the 5' enhancer region of RJ2.2.5 cells was anatomically similar to the equivalent region of the wild-type Raft parental cells. In experiments conducted in collaboration with the group of C. Benoist and D. Mathis in Strasbourg, a series of plasmid

26 Acco[la/Dellabona/Scarpellino/Carra/Sartoris

constructs containing a rabbit [3-globin re- porter gene, an SV40 enhancerless promoter and distinct portions of the X-Y enhancer region were transfected into several cell lines. These cells included Raji, RJ2.2.5 and RJ2.2.5 derivatives, either somatic cell hybrids or transfectants originated in our laboratory by introducing mouse DNA into R J2.2.5 and selecting for stable expression of human class II genes [32]. The enhancer function of the routine Ea X and Y boxes, could be easily shown in Raji cells when both X and Y regions were present. In contrast the enhancer function was totally abolished in RJ2.2.5 cells; it was however restored in RJ2.2.5 derivatives reexpressing class II genes. Furthermore DNA-binding proteins present in nuclear extracts from either Raji or RJ2.2.5 ceils were absolutely undistin- guishable in their number and binding pattern for DNA sequences such as isolated X- or Y-box-specific oligonucleotides as assessed by gel retardation and methylation interference assays [33]. Taken together, these results establish that the aIr-I gene function is primarily implicated in an enhancer function ~hich can be precisely map- ped at the DNA level. In this context RJ2.2.5 represents the first mammalian cell mutant with a documented defect in an enhancer factor (excluding steroid hormone recep- tors). However. the subtle molecular defect affecting class II gene transcription in the mutant is still unclear. It might be a subtle mutation of either X- or Y-binding proteins which does not affect DNA-binding capacity, an alteration in another DNA-binding protein, a mutation in a factor that does not bind DNA but interacts with the nuclear factors binding to X or Y boxes or an incorrect posttranslational modification of one of these factors. Relevant to this aspect is the

recent finding that in some B cell lines derived from patients with BLS, alterations of some DNA-binding proteins specific for the 5' conserved enhancer region have been observed. In one case the absence of a specific X-binding protein was demonstrated [34]. The recent isolation of cDNAs specific for nuclear proteins binding to X [35] and Y [36] DNA sequences will certainly contrib- ute to the definition of their role in the control of class II gene transcription.

Terminal Differentiation of B Cells Is Accompanied by Active Suppression of Class II Gene Transcription

During ontogeny and differentiation of the B cell lineage, class II gene expression is Ibund in the early precursors and is main- tained up to the stage of mature B cells. However, terminally differentiated B cells, that is plasma cells, do not express this MHC gene family [7]. In an at tempt to clari/), the genetic and molecular mechanisms responsi- ble for this developmentally regulated event we made use again of a somatic cell genetics approach consisting in creating hybrids between human class II positive B cells and murine class II negative plasmocytoma cells. The basic question was whether the extinc- tion of class II antigen expression in plasma cells is caused by a simple silencing of the activator mechanisms or by an active suppression of the class II positive phenotype. A ve~' large number of somatic cell hybrids were analyzed; it was found that none of the hybrids reexpressed mouse class II antigens which could have been an indication of a trans-acting dominant alr gene function. More important, none of the above hybrids retained expression of human class II anti-


gens even in the presence of human chromosome 6 as assessed by the retention of MHC class I antigen expression. A carefuI karyotype analysis of an informative family of hybrids demonstrated that mouse chromosomes were all retained, whereas a preferential segregation of human chromosomes was observed. However, the lack of expression of human class II antigens did not correlate with the segregation of any particular human chromosome, clearly indicating that an active suppression brought about by mouse plasmocytoma-specific factors was responsi- hie for the absence of class II antigen expression in the human 13 ceil • mouse plasmocytoma cell hybrids [37]. Furthermore, bio- chemical studies demonstrated that the lack of expression of class II antigens was due to the absence o f specific mRNA as a result of the lack of transcription of specific class II genes as assessed by nuclear analysis of pri- maD" transcripts [38]. Thus terminal differentiation of B cells in plasma cells is accompanied by a developmentally regulated active suppression of MHC class II genes be- having as dominant trait over the class II positive phenotype of a B cell. Taken together these data demonstrate the existence of at least two families of trans-acting thctors with opposite function for class II gene expression: the first (including air- l- and air- 2-encoded factors) operating as activators and the second as suppressors of transcription. By similarity with the air loci designa- tion, we may operationally define as sir (suppressor of immune response genes) the locus(i) encoding the putative plasma-cell-specific transcriptional suppressor factor(s). At the present time we ignore the precise mechanism through which slr-encoded factors operate. They might directty inhibit transcription of aIr-I or alr-2 genes or function-

ally inactivate the trans-acting activators either by interacting with them or by com- peting with them for regulatory DNA sequences such as those involved in the class- II-specific enhancer function.

Inducible Expression of Class II Genes

We have discussed so far a number of experiments focussing on the understanding of the genetic and molecular mechanisms implicated in the constitutive control of class It gene expression in the B cell lineage. We will try now to address our attention to experimental models possibly useful to in- vestigate the inducible class II gene expression, its molecular and genetic constraints and the possibte relationships with mechanisms of constitutive class II gene expression. In our laboratory, we took advantage of the availability of the murine P388 D 1 macrophage cell line that has capacity to express class II antigens upon induction by exogenous stimuli like IFN-3, [39]. Molecular studies performed in our as well as other laboratories have shown that the class II negative phenotype of uninduced cells correlates primarily with the absence, or highly reduced accumulation, of specific mRNA [40, 41]. It was important, therefore, to assess whether the control of class II gene expression in macrophages induced by IFN-y was mediated by similar trans-acting factors as those operating in B cells like the air-I- encoded trans-activator. In an at tempt to answer this question, somatic cell hybrids between P388 DI and RJ2.2.5 cells were constructed and then analyzed for their MHC phenotype under different conditions. The results of this approach were interesting in several respects. First, we found that the

28 Accolla/Dellabona/Scarpellino/Carra/Sa rtoris

hybrids invariably displayed a class II negative phenotype indicating the lack of genetic complementation between the two cell types. In this respect, therefore, macrophages differ from BLS B cells which can complement, and can be complemented by, RJ2.2.5 cells. Treatment with recombinant murine IFN-y was effective in modifying the MHC phenotype of the hybrids. Indeed we observed an increased expression of murine and human class I antigens, attributable to an increased accumulation of specific mRNAs, and a de novo induction of murine class II gene expression similar to, if not higher than, the one observed in parental P388 D1 cells. However, in no case did we observe an induction of human class II gene expression [40]. Therefore, the induction of class II gene expression by IFN-? in macrophages is not sufficient to complement the defect of R.I2.2.5 celIs. These results indicate that air- I gene products are either inactive or absent in P388 D1 macrophage cells stimulated with IFNq,. Thus at least in this experimental system we can firmly establish a first relevant distinction between constitutive and inducible class II gene expression.

The fact however that the B cell human partner used for fusion was a class II negative cell did not allow to discriminate whether the constitutive class II positive phenotype is dominant or recessive versus the class II negative phenotype observed in macrophages. In order to answer this question somatic cell hybrids have been recently constructed between class II positive B cells and class II negative macrophages [Sartoris, Scarpellino and Accolla, unpubt, data]. Pre- liminary results show that in certain hybrids the B cell class II phenotype is dominant versus the macrophage phenotype in that de novo and stable expression ofmur ine class II

genes is observed. Murine class I1 gene expression is remarkably high and cannot be further modified by treatment with IFN-?. Thus it would appear that intracellular trans- acting factors involved in the constitutive class II gene expression can influence in a positive way the expression of class II genes of a cell which can express such genes only under the effect of exogenous stimuli.

A Model for the Developmental Regulation of M H C Class II Gene Expression

On the basis of the experimental results reviewed in this report we would like to propose a minimal model for the transcriptional control of class II gene expression during ontogeny and differentiation. This does not imply, of course, that class II antigen expression is controlled exclusively at the transcriptional level. Posttranscriptional and posttranslational mechanisms of control exist and certainly play an important role in MHC class-ll-related functions [reviewed in 2].

The basic features of the proposed model are depicted in figure 2 and wilt be briefly discussed. The following assumptions are critical for this model: (1)the functional and anatomical distinction of the class II promoter region in two districts. 'c' and 'i', each sensitive to constitutive and inducible transcription factors, respectively, and (2) the developmentally controlled generation of tissue-specific transcriptional regulators.

During ontogeny precursors of B cells rapidly and irreversibly lose the function of promoter region i possibly as result of the interaction with specific DNA-binding proteins affecting the local chromatin structure.

Transcri.ptionat Regulation of MHC (?lass II Genes 29

Fig. 2. A model for the transcriptional control of MHC class II gone expression. _c and i = Class [I promoter regions interacting with, or functionally dependent from, constitutive and inducible trans- acting factors, respectively; + and - = positive and negative control, re- spectivel~r. For complete descrip- tion of the model see the text.

sir " a|r-2 air-1 promoter clan fi

cTSF c T A F J - Expression fQonst i tut ive}

s i t " a i r -2 a i r -1 promoter class I I "

! U t eTSF ~ T A F ~ Suppr~.sion

�9 sir " air-2 air-1 promoter class i l

J / Expression

exogenous s t imu t i ( reducib le)

inactive element active element

The model does not exclude that some of these DNA-binding factors may even be

part. or under the control, of the air gone products which at the same time are actively produced as constitutive trans-acting activator factors (cTAF). These in turn positively influence the activity of class II promoter region c c_ either by binding directly to DNA,

by modifying the binding of preexisting factors or by both actions. At this stage constitutive suppressor factors (cTSF) encoded by

an sir locus (or loci) are either absent or functionally inactive. The terminal differen-

tiation of B cells in plasma cells is accompanied by the activation of the sir locus and/or its products and by the consequent suppression of class II gone transcription. Several mechanisms can be envisaged: ( l ) cTSF may

compete with cTAF for binding to the pro-

moter region _c; (2) cTSF may inactivate cTAF by binding to them and thus prevent-

ing cTAF to bind to promoter region c, or (3) cTSF are transcriptional inactivators of

the air gene family. Whatever the mechanism, it is clear that the plasma-cell-derived cTSF function behaves as a dominant trait

versus the B-cell-specific cTAF function. Thus positive and negative signals regulating class II gone expression may behave as dominant or recessive traits, depending upon the particular developmental stage of the cell in

which they operate. The differentiation of the monocyte-mac-

rophage cell lineage is accompanied by a distinct series of events related to MHC class II gene expression. First, the promoter region i

30 Accolla/Deltabona/Scarpellino/Carra/Sartoris

is functionally 'open', unblocked; so is the promoter region c. Under uninduced conditions class II gene transcription does not take place or is highly reduced, more because of a lack of transcriptional activators than because of the presence of constitutive transcriptional suppressors. The model implies that in monocyte-macrophage cell lineage eTAF as well as cTSF are not present because of the developmentally controlled silencing of air and sir gene families. After activation with exogenous stimuli and particularly with IFN-u a series of events take place some of which directly relate to the de novo activity of tissue-specific inducible transcription activator factors (iTAF) specific for class II genes. These factors may be either synthesized ex novo or simply functionally activated, iTAF are absent or functionally irrelevant in B cells undergoing similar treatment. Their molecular target is the promoter region i, and the corresponding interactions have very. little influence, if any, on the function of promoter region c. Several lines of evidence are in agreement with the above predictions: (1) Induction with IFN-7 never results in a permanent, constitutive expression of class I1 genes. (2) Class II gene expression in B cells is not modulated by IFN-7 treatment. This is so also for B cells defective in class 1I gene expression because of cTAF defects [22. 40. 42] and, more importantly, even when these defective B cells are artificially provided with iTAF by means of somatic cell fusion with macmphages and subsequent induction with IFN-y [40]. (3) Class II positive B cells, on the other hand, can constitutively restore the expression of macrophage class II genes as demonstrated by our recent data. thus suggesting that B-cell-specific cTAF may interact with the 'open' promoter region c of the macrophage.

The proposed model has several implica- tions. First, a profound difference between constitutive and inducible class II gene expression is envisaged with a dominance of the constitutive versus the inducible phenotype. This in turn implies that the developmentally controlled class II gene expression in B cells is probably the result of a more heterogeneous series of events as compared to macrophages. Second, and relevant to the general problem of control of gene expression, attention should be given to the definition of dominance and recessiveness of a genetic trait. Indeed class II gene expression is a paradigmatic example of dominant or recessive phenotypes depending upon the developmental stage of the cell lineage considered. Third, the various mechanisms controlling class I1 gene expression in distinct cell types may have evolved tbr specific functional requirements. Thus, B cells must express constitutively class II genes to be able to present to the appropriate T cell clones only and always the processed anti- genic peptides derived from the antigen cap- tured through their immunoglobulin recep- tor. The following T cell activation will in turn result in the production of B-cell-specific proliferation and differentiation factors that will eventually 'drive' the B cell to the antibody-secreting plasma cell stage. At this point, antigen-presenting function is not necessary any more, and class II gene expression is suppressed. Constitutive class II gene expression is not required for macrophages and similar antigen-presenting cells, since they have no immune specialized functions like B cells. On the contrary, continuous and undiscriminate antigen-presenting functions may be dangerous for the individual, as poly- clonal T cell activation generated via this process may generate immune dysfunctions

Transcriptional Regulation of MHC Class It Genes 31

l ike a u t o i m m u n e d iseases . T h u s m a c r o -

phages h a v e e v o l v e d to exp res s class II genes

on ly in i n d u c i b l e f a s h i o n a n d p a r t i c u l a r l y

u p o n a c t i v a t i o n by e x o g e n o u s s t imu l i .

H o w e v e r , b e y o n d the o b v i o u s i m p o r -

t ance for i m m u n e r egu la t ion , t h e c o n t r o l o f

class l I gene e x p r e s s i o n m a y be i m p l i c a t e d in

o t h e r f u n d a m e n t a l b io log i ca l p rocesses .

W i t h i n th is f r a m e we b e l i e v e t h a t p a r t i c u l a r

a t t e n t i o n wil l be a d d r e s s e d to t he pos s ib l e

i n f l u e n c e o f class II gene e x p r e s s i o n in t he

p roces s o f cell d i f f e r e n t i a t i o n a n d m a t u r a -

t i o n d u r i n g o n t o g e n y .

Acknowledgements

This work was supported by grants from: M.P.I. Aspetti Clinico-Sperimentali della Risposta Immune; Associazione Italiana Ricerca sul Cancro (AIRC), and Associazione per la Ricerca Biomedica (ARBI). Silvia Sartoris was supported by a fellowship from AIRC.

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Prof. R.S. Accolla Istituto di Scienze Immunologiche Universit'h di Verona Poticlinico di Borgo Roma 1-37100 Verona (Italy)

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