Revisiting the specificity of the MHC class II transactivator CIITAin vivo

Revisiting the specificity of the MHC class IItransactivator CIITA in vivo

Luc A. Otten1,2, Salom� LeibundGut-Landmann**1, Joachim Huarte3,Isabelle C. Kos-Braun***1, Christine Lavanchy2, Emmanu�le Barras1,Bettina Borisch1, Viktor Steimle4, Hans Acha-Orbea*2 and Walter Reith*1

1 Department of Pathology and Immunology, University of Geneva Medical School,Geneva, Switzerland

2 Department of Biochemistry, Faculty of Biology and Medicine, University ofLausanne, Epalinges, Switzerland

3 Department of Genetic Medicine and Development, University of Geneva MedicalSchool, Geneva, Switzerland

4 Department of Biology, University of Sherbrooke, Sherbrooke, Canada

CIITA is a master regulatory factor for the expression of MHC class II (MHC-II) andaccessory genes involved in Ag presentation. It has recently been suggested that CIITAalso regulates numerous other genes having diverse functions within and outside theimmune system. To determine whether these genes are indeed relevant targets of CIITAin vivo, we studied their expression in CIITA-transgenic and CIITA-deficient mice. Incontrast to the decisive control of MHC-II and related genes by CIITA, nine putative non-MHC target genes (Eif3s2, Kpna6, Tap1, Yars, Col1a2, Ctse, Ptprr, Tnfsf6 and Plxna1)were found to be CIITA independent in all cell types examined. Two other target genes,encoding IL-4 and IFN-c, were indeed found to be up- and down-regulated, respectively,in CIITA-transgenic CD4+ T cells. However, there was no correlation between MHC-IIexpression and this Th2 bias at the level of individual transgenic T cells, indicating anindirect control by CIITA. These results show that MHC-II-restricted Ag presentation,and its indirect influences on T cells, remains the only pathway under direct control byCIITA in vivo. They also imply that precisely regulated MHC-II expression is essential formaintaining a proper Th1-Th2 balance.

Supporting information for this article is available athttp://www.wiley-vch.de/contents/jc_2040/2006/35687_s.pdf

Introduction

The class II transactivator CIITA functions as a mastercontrol factor for the expression of genes required forMHC class II (MHC-II) mediated Ag presentation [1–5].This MHC-II-mediated Ag presentation is essential forthe development of CD4+ T cells in the thymus and for

Molecular immunology

* These two authors contributed equally to this paper.

Correspondence: Dr. Luc A. Otten, Department ofBiochemistry, Faculty of Biology and Medicine, University ofLausanne, 155 chemin des boveresses, 1066 Epalinges,SwitzerlandFax: +41-216925705e-mail: [email protected]

Received 10/11/05Revised 19/2/06

Accepted 5/4/06

[DOI 10.1002/eji.200535687]

Key words:CIITA � Gene

expression � MHCclass II � Th celldifferentiation

� Transgenic mice

Abbreviations: B2m: b2-microglobulin � BLS: bare lymphocytesyndrome � Ii: invariant chain � MEF: mouse embryonicfibroblasts � MHC-I: MHC class I � MHC-II: MHC class II �RPA: RNase protecion assay � TBP: TATA-binding protein

Luc A. Otten et al. Eur. J. Immunol. 2006. 36: 1548–15581548

f 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji.de

** Present address: Immunobiology Laboratory, Cancer Re-search UK London Research Institute, UK

*** Present address: Wellcome Trust Center for Cell Biology,University of Edinburgh, UK

the initiation, amplification and regulation of adaptiveimmune responses by Th cells in the periphery. CIITAcontrols the transcription of all MHC-II genes, as well thegenes encoding the invariant chain (Ii) and two non-classical MHC-II molecules (HLA-DM and HLA-DO inman; H2-M and H2-O in mice), which are accessorymolecules involved in the intracellular transport andpeptide loading of MHC-II molecules [2–5]. In mostsituations, the expression pattern of the gene encodingCIITA (MHC2TA in man; C2ta in mice) dictates the celltype specificity and level of MHC-II expression [2–5].Two modes of CIITA and MHC-II expression arerecognized, constitutive expression in cells specializedfor Ag presentation (B cells, macrophages, DC, thymicepithelial cells) and IFN-c-induced expression in mostother cell types [2–5].

The MHC2TA gene is mutated in certain patientssuffering from bare lymphocyte syndrome (BLS), ahereditary immunodeficiency disease characterized bythe absence of MHC-II expression [1, 2]. DefectiveMHC-II expression results in both CD4+ T cell lympho-penia and the inability to present Ag to CD4+ T cells,which severely impairs the ability to mount protectiveimmune responses. All clinical and immunologicalabnormalities in this syndrome can be explained bydeficient MHC-II-mediated Ag presentation. The ab-sence of other overt phenotypes suggested that CIITA ishighly dedicated to MHC-II expression. The subsequentdiscovery that CIITA also regulates the expression of Ii,DM and DO did not alter this view [2, 3].

BLS patients show a partial reduction in MHC class I(MHC-I) expression in certain cell types [2–4]. This wasexplained by the finding that the MHC-II-specificregulatory machinery, including CIITA, contributes tooptimal expression of MHC-I genes [6, 7]. This again didnot constitute a major challenge to the conclusion thatCIITA is specific for MHC-II and related genes.

The high specificity of CIITA was recently called intoquestion by reports implicating CIITA in the regulationof a surprising variety of unrelated genes involved inmany different functions within and outside the immunesystem [4]. The genes encoding IL-4 and Fas ligand(FasL) were proposed to be repressed by CIITA in mouseT cells [8, 9]. Those encoding cathepsin E and IL-10were suggested to be repressed by CIITA inmouse B cellsand/or DC [10, 11]. The genes encoding collagentype Ia2, thymidine kinase and cyclin D1were proposedto be repressed by CIITA in IFN-c-induced cells [12]. Amicroarray analysis identified 16 additional genes ofdiverse functions that were proposed to be down-regulated by CIITA in a human B cell line [13]. Theexpression of several other genes was reported to beenhanced by CIITA. Microarray experiments identifiedthe gene encoding Plexin-A1, which was found to beactivated by CIITA inmouse DC, and a diverse set of over

40 genes that were proposed to be up-regulated byCIITA in human B cells and IFN-c-induced cells [13, 14].

Several reasons prompted us to determine whetherthese non-MHC genes are indeed regulated by CIITA invivo. First, the control of such a functionally diverse setof genes by CIITA is at odds with the fact that all defectsexhibited by BLS patients can be attributed directly orindirectly to the absence of MHC-II expression [2].Second, none of the proposed new target genes containthe highly conserved regulatory module required forrecruiting CIITA to the promoters of MHC-II and relatedgenes [4]. Third, experimental support for the newtargets is largely limited to a correlation between theirexpression and that of CIITA, altered expression inCIITA-deficient cells and/or experiments involvingtransfections and reporter gene assays [4]. Finally,certain targets have been the subject of controversy[15–18].

We performed a rigorous analysis of the expression ofselected candidate target genes in CIITA knockout miceand in transgenic (Tg) mice showing widespread CIITAexpression. In contrast to the pivotal role of CIITA in theregulation of MHC-II genes, we found no evidence for animpact of CIITA on the expression of eight non-MHC-related genes. The expression of two genes – encodingthe Th2 cytokine IL-4 and the Th1 cytokine IFN-c – wasindeed affected in activated CIITA-Tg Th cells. However,this results from a general change in the balancebetween Th1 and Th2 differentiation rather than from adirect control by CIITA. These results argue against thenotion that CIITA is a pleiotropic factor having wide-spread functions extending beyond its well-establishedrole in the control of MHC and related genes.

Results

Transgenic mice exhibiting different levels ofubiquitous CIITA expression

Five independent lines of Tgmice –MC25, MC30, MC52,MC41 and MC42 – were generated using a constructdesigned to obtain robust and widespread expression ofCIITA (supplementary Fig.1). Tgmice obtainedwith thisconstruct exhibited no developmental defects, lethalityor pathological phenotype.

For each line, the abundance of Tg-derived CIITA (Tg-CIITA) mRNA was quantified and compared to thenumber of genomic copies of the transgene (supple-mentary Fig. 1). Tg-CIITA mRNA abundance correlatesclosely with transgene copy number when low tointermediate numbers of copies have been integrated.A plateau is reached only at very high copy numbers. Theconstruct is thus expressed in a copy number-dependentmanner.

Eur. J. Immunol. 2006. 36: 1548–1558 Molecular immunology 1549


Three Tg lines – MC25, MC52 and MC42 – werechosen for further study because they express low,intermediate and high levels of Tg-CIITA mRNA (Fig. 1).In MC25 and MC52 mice, Tg-CIITA mRNA levels inlymphoid organs (spleen and LN) are lower than thoseof the endogenous CIITA mRNA. In MC42 mice, Tg andendogenous CIITA mRNA are similar in abundance inlymphoid tissues, such that the total amount of CIITAmRNA is increased only twofold relative to normal.

Endogenous CIITA mRNA levels are low in non-lymphoid tissues of WT mice. In the Tg mice, total CIITAmRNA abundance is increased in all non-lymphoidtissues tested, including the ileum, skin, liver, kidneyand lung (Fig. 1). As in lymphoid tissues, this increase islow in MC25 mice, intermediate in MC52 mice and highin MC42 mice. In the latter, CIITA mRNA abundance isincreased to levels comparable to those found in non-Tglymphoid organs. These results show that the Tg miceexhibit widespread CIITA expression, but this ectopicexpression remains within normal physiological levels,even in the most strongly expressing MC42 mice.

Impact of the CIITA transgene on MHC-IIexpression

As a quantitative and functionally relevant readout forderegulated CIITA expression in the Tg mice, wequantified the expression of various MHC-II and relatedgenes by RNase protection assays (RPA) or quantitativeRT-PCR. Results obtained for the liver and kidney areshown in Fig. 2A. Enhanced CIITA expression leads to astrong and coordinate increase in the expression of allMHC-II and related genes examined. Only H2-DMbstands apart in that it is activated to lower levels in theliver.

The expression of MHC-II and related genes was alsoassessed in CD4+ T cells (Fig. 2B). In non-Tg cells, CIITAmRNA levels did not increase upon activation [15]. Incontrast, Tg-CIITA mRNA abundance increased duringthe activation of CD4+ T cells, reaching levels higherthan those found in control B cells (Fig. 2B) [15]. Thisenhanced CIITA expression augments the expression ofMHC-II and related genes to levels close to those foundin B cells. As in the liver, H2-DMb mRNA was inducedless than the others.

MHC-I expression is only increased by strongCIITA expression

RPA was used to quantify H2-K1 and b2-microglobulin(B2 m) mRNA in several organs of the MC25, MC52 andMC42 mice (Fig. 3A). B2 m mRNA abundance wasunaffected in all three Tg mice. Similarly, H2-K1 mRNAabundance was not affected in the MC25 and MC52mice. However, H2-K1 mRNA levels were increased inthe MC42 mice. Cell surface H2-K and H2-D expressionwas next examined by FACS in activated CD4+ T cellsfromMC42 and control mice (Fig. 3B). In contrast to thestrong induction of MHC-II expression, no increase inMHC-I was observed. Finally, to complement the Tgmouse data, we compared H2-K1 and B2 mmRNA levelsbetween CIITA–/– and CIITA+/– mice. No reduction inH2-K1 or B2 m mRNA expression was observed inCIITA–/– mice.

Impact of CIITA on new candidate target genes

To determine whether CIITA controls multiple targetgenes in vivo in addition toMHC-II and related genes, weselected candidates for which evidence supportingregulation by CIITA was the most compelling. Selectioncriteria included altered expression in CIITA-transfected

Figure 1. Characterization of CIITA-Tg mice. Endogenous CIITA (e-CIITA), Tg-CIITA, H2-Ea, H2-Aa and TBP mRNAwere quantifiedby RPA invarious tissues fromTg andnon-Tg (Tg–) littermates. A representative gel is shownon the left. Valueswerenormalized toTBP and are presented in arbitrary units. Each data point corresponds to a single mouse. The results shown are representative oftwo experiments performed with two different sets of mice.



or CIITA-mutant cells, a change in expression induced byIFN-c, studies suggesting a direct CIITA-mediatedregulation and immune-relevant functions. Among thegenes proposed to be activated by CIITA, we chose thoseencoding TGFb RIP1 (Eif3s2), Importin a7 (Kpna6),Tap1 (Tap1), and tyrosyl-tRNA synthetase (Yars) [13].Among the genes suggested to be repressed by CIITA, we

selected those encoding collagen type I a2 (Col1a2),cathepsin E (Ctse), tyrosine phosphatase receptor(Ptprr) and FasL (Tnfsf6) [9, 10, 12, 13].

We first examined whether the mRNA expressionpatterns of these genes correlate with that of CIITA invarious tissues and cell types of non-Tg mice. In contrastto MHC-II expression, there is little or no evidence for apositive or negative correlation, respectively, betweenthe abundance of CIITA mRNA and expression of thegenes that have been suggested to be activated orrepressed by CIITA (supplementary Fig. 2) [10, 12, 13].

Tap1, Yars, Eif3s2, Kpna6, Ctse and Col1a2 mRNAwere next quantified in various tissues and CD4+ T cellsfrom MC42 and control mice (Fig. 4A, B). In contrast tothe marked increase in MHC-II expression, no majorincrease or decrease in mRNA abundance was observedin the Tg mice for any of the six putative target genes.The strongest difference observed was a threefoldincrease in Tap1 mRNA abundance in the kidney. Allother changes were less than twofold. Tap1, Yars, Eif3s2,Kpna6, Ctse and Col1a2 mRNA were then quantified inresting and activated B cells, and in unstimulated andIFN-c-activated mouse embryonic fibroblasts (MEF),from CIITA p(III+IV)–/– mice (Fig. 4C, D). These miceharbor a deletion in the regulatory region of the C2tagene that abrogates CIITA expression in B cells and IFN-c-induced cells of non-hematopoietic origin [19]. Incontrast to the strong loss of MHC-II expression, nochange in expression exceeding twofold was observedfor any of the six genes. Taken together, the results showthat there is no statistically significant effect of CIITA onthe expression of these genes (Table 1).

Induction of the FasL gene (Tnfsf6) during T cellactivation has been suggested to be inhibited by CIITA[9, 20]. We therefore examined the induction of Tnfsf6mRNA in activated T cells from MC42 mice. The

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Figure 2. Expression of MHC-II and related genes is induced bythe CIITA transgene. (A) mRNA abundance for MHC-II andrelated genes in the kidney and liver of Tg and non-Tg (Tg–)littermates. Results are plotted as a function of CIITA mRNAabundance. H2-Ea and H2-Aa mRNA were quantified by RPA.The otherswere quantified by real-time RT-PCR. Similar resultswere obtained in multiple organs including those shown inFig. 1. (B) Naive CD4+ CD62Lhigh T cells from MC42 mice (Tg+)were stimulated in vitrowith anti-CD3 antibody. The indicatedmRNAwere quantified by real-time RT-PCR after 0, 48 and 96 hof activation. Non-Tg (Tg–) B cells and CD4+ T cells were usedas references. Results are plotted as a function of CIITA mRNAabundance on linear (H2-Aa, H2-DMa) or logarithmic (H2-DMb)scales. Data points correspond to a single mouse or to theaverage of two mice. The mean and standard deviation areshown for independent RT-PCR measurements. The resultsshownare representative of three experiments performedwithdifferent sets of mice. Results in (A) and (B) were obtainedwithdifferent mice.



Figure 3. Role of CIITA in MHC-I expression. (A) H2-K1 andB2 m mRNA were quantified by RPA in the indicated organsfrom non-Tg (–) or MC25, MC52 and MC42 mice. *: not tested.(B) CD4+ T cells from non-Tg (Tg–, grey histograms) or MC42mice (Tg+, black histograms) were activated for 2 days. MHC-II,H2-K and H2-D expression was analyzed by FACS. Negativecontrols (Ab–, no antibody) are represented as filled histo-grams. For H2-K and H2-D, the Tg+ histograms overlapperfectly with the Tg– histograms and the former havetherefore been shifted vertically to improve visualization.Similar results were obtained with ex vivo T cells (data notshown). (C) H2-K1 and B2 m mRNAwere quantified by RPA inthe indicated organs from CIITA+/– and CIITA–/– mice. I-Aa+/–

and I-Aa–/– mice were used as controls. Similar results wereobtained for the LN and for H2-D1 expression (data not shown).Each data point corresponds to a single mouse. Results in (A)and (B) were obtained with different mice. Independentexperiments were performed with different mice.

Figure 4. Influence of CIITA on the expression of putative non-MHC-related target genes. The indicated mRNA were quanti-fied by real-time RT-PCR or RPA (C2ta and H2-Aa in tissues).(A) Naive (ex vivo) and activated (anti-CD3 antibody) CD4+

T cells from MC42 (Tg+) and non-Tg (Tg–) mice. (B) Liver,kidney and lung fromMC42 (Tg+) and non-Tg (Tg–) mice. (C) Exvivo and activated (LPS + IL-4) B cells fromCIITA-p(III+IV)–/– andCIITA-p(III+IV)+/– mice. (D) Non-stimulated and IFN-c-inducedMEF from CIITA-p(III+IV)–/– and CIITA-p(III+IV)+/– mice. Valueswere expressed on a logarithmic scale as the ratio between Tgand non-Tg cells (A, B) or between cells fromheterozygous andhomozygous knockout mice (C, D). nt: not tested; na: notapplicable because one or both values were not significantlyabove non-specific background (see supplementary Table 1).Data points correspond to a single mouse or to the average oftwo mice. The mean and standard deviation are shown forindependent RT-PCR measurements. All results are represen-tative of at least two experiments performedwith different setsof mice. Results in (A) and (B), and in (C) and (D), were obtainedwith different mice.



induction of Il2 mRNA was used to monitor activation.We observed no significant difference in the induction ofTnfsf6 mRNA between MC42 and control T cells(Fig. 5A). No difference was observed in activatedCD8+ T cells. At best, a modest 25% decrease wasobserved in Tnfsf6 transcript levels in activated MC42CD4+ T cells (data not shown). However, cell surfaceFasL expression is not significantly altered in MC42CD4+ T cells (Fig. 5B).

We next examined expression of the Plexin-A1 gene(Plxna1), which was reported to be induced by CIITA inactivated DC [14]. In contrast to H2-Aa, Plxna1expression was not found to be increased by the CIITAtransgene in activated MC42 DC (supplementary Fig. 3).

Finally, we assessed the role of CIITA in repressingthe tyrosine phosphate receptor gene (Ptprr) (supple-mentary Table 1), which was proposed to be repressedby CIITA in a human B cell line and in IFN-c-inducedfibroblasts [13]. Significant Ptprr expression was foundto be restricted to the brain, where we could notdocument an impact of the CIITA transgene because it isexpressed at only very low levels in this tissue (data notshown). Other cell types and tissues contain PtprrmRNAlevels that are too low to allow us to document aconvincing influence of the transgene. However, no de-repression of the Ptprr gene was observed in B cells orMEF from CIITA-deficient mice.

Deregulated cytokine expression in CIITA-TgCD4+ T cells

CIITA has been shown to modulate expression of thegenes encoding IL-4 and IFN-c in activated mouse Thcells [8, 15, 16, 18]. However, this influence of CIITA oncytokine production and the mechanisms that areresponsible have been a matter of debate [8, 15, 16,18, 21–23]. We therefore addressed this question furtherusing our Tg mice.

Naive CD4+ T cells isolated from MC25, MC52,MC42 and non-Tg littermates were activated underneutral, Th1 or Th2 conditions. CIITA, MHC-II, IL-4 andIFN-c mRNA were then quantified (Fig. 6). IL-4 and

IFN-c mRNA levels were modulated as expected by thedifferent conditions: The IL-4/IFN-c mRNA ratio washigh under Th2 conditions, low under Th1 conditionsand intermediate under unbiased conditions. This wastrue for both Tg and control Th cells. However, a Th2bias – evidenced by an increase in the IL-4/IFN-cmRNAratio – was observed under all three conditions in the TgT cells. In contrast to enhanced MHC-II expression,

Table 1. The impact of the CIITA mutations on expression of CIITA, H2-Aa and proposed target genes

C2ta H2-Aa Tap1 Eif3s2 Yars Kpna6 COL1A2 Ctse

Average fold changea) 21.02 44.97 1.33 1.04 1.02 1.02 1.04 1.05

Minimum fold change 4.36 2.72 0.85 0.74 0.63 0.80 0.83 0.28

Maximum fold change 51.97 202.44 3.05 1.50 1.55 1.32 1.30 2.20

Number of conditions tested 7 8 9 9 9 9 5 6

p valueb) <0.001 <0.001 0.5 0.5 0.5 0.5 0.5 0.5

a) All results from supplementary Table 1 were pooled to calculate the ratio in expression between the C2ta high- and low-expression genotypes (Tg/non-Tg and WT/KO).

b) Chi square calculation of probability for fold change being 1.0.

Figure 5. Impact of CIITA on FasL induction in activated MC42T cells. (A) IL-2 and FasLmRNAweremeasured by real-timeRT-PCR in ex vivo and activated (2 and 6 h) spleen cells fromMC42(Tg+) and non-Tg (Tg–) littermates. Each data point corre-sponds to a single mouse. The mean and standard deviationare shown for independent RT-PCRmeasurements. The resultsshown are representative of four experiments performed withdifferent sets of mice. (B) Cell surface FasL expression wasanalyzed by FACS (Ab+, gray histograms) on ex vivo andactivated (6 h) CD4+ spleen cells from MC42 (Tg+) and non-Tg(Tg–) mice.



which was strong in T cells from all three Tg mice, theTh2 bias was mainly evident in MC42 T cells, whichexpress CIITA most strongly. Induction of the Th2 bias isthus less sensitive to an increase in CIITA than theactivation of MHC-II expression.

We next examined whether there is a correlationbetween the increase in MHC-II expression and reducedIFN-c production or enhanced IL-4 synthesis at the levelof individual cells. This experiment was based on thefollowing reasoning (Fig. 7A). If the Ifng gene isrepressed by CIITA, only cells exhibiting high MHC-IIexpression should show a concomitant reduction inIFN-c production. Similarly, if the Il4 gene is activated byCIITA, only cells exhibiting high levels of MHC-II shouldshow an increase in IL-4. On the other hand, if the Th2bias is an indirect consequence of deregulated CIITA orMHC-II expression, there might not be a correlation inindividual cells between enhanced MHC-II and alteredcytokine expression.

Naive CD4+ T cells isolated from MC25, MC52,MC42 and control mice were activated under Th1conditions to induce the synthesis of IFN-c by most cells.After 4 days, IFN-c and MHC-II expression weremeasured simultaneously by FACS (Fig. 7B). A pro-gressive increase in the number of MHC-II-positive cells(29, 58 and 91%) was observed for MC25, MC52 andMC42 cells. Cells expressing low and high amounts ofMHC-II (as a reliable read-out for cell-intrinsic CIITAactivity [15]) were then monitored separately for IFN-cproduction. No reduction in the number of IFN-c-positive cells was evident in either fraction of the MC25cells (65–82%). However, the IFN-c-positive fractionwas markedly reduced in MC52 (44–49%) and MC42(28–25%) cells. Importantly, this reduction was equallystrong in the MHC-II-low and -high fractions (44%versus 49% inMC52; 28% versus 25% inMC42).We nextquantified IL-4 and IFN-c mRNA in activated MHC-II-low and -high MC42 Th cells (Fig. 7C). Under Th1conditions, MC42 cells exhibit an increase in IL-4 mRNAand a reduction in IFN-cmRNA compared to the non-Tgcells. These changes are equally strong in MHC-II-lowand -high cells.

The lack of correlation between CIITA-drivenMHC-IIexpression and altered cytokine expression suggests thatthe two effects are independent, and argues against adirect activation of the Il4 gene and repression of the Ifnggene by CIITA. The results are more consistent with anindirect influence of ectopic CIITA or MHC-II expressionon the balance between Th1 and Th2 differentiation. Toaddress the latter possibility, we quantified the expres-sion of the genes encoding GATA-3 (Gata3) and T-bet(Tbx21), two transcription factors playing key roles inthe differentiation of Th2 and Th1 cells, respectively[24]. A Th2 bias evidenced by an increase in the ratiobetween Gata3 and Tbx21mRNAwas observed in MC42Th cells activated under unbiased and Th1 conditions(supplementary Fig. 4). This is mainly due to an increasein Gata3 expression.

Discussion

The prime importance of CIITA for regulating MHC-IIexpression is supported by strong biochemical andgenetic evidence. Particularly convincing support wasprovided by the fact that MHC-II expression is abolishedin CIITA-deficient BLS patients and CIITA knockoutmice[2, 3, 19, 25, 26]. In both cases, no pathologicalphenotypes distinct from those arising from the absenceof MHC-II-mediated Ag presentation have been docu-mented. This suggests that CIITA is highly dedicated tothe regulation of MHC-II expression. Nevertheless, aseries of reports have implicated CIITA in the regulationof a wide variety of other genes functioning within and

Figure 6. CIITA induces a Th2 bias in CIITA-Tg Th cells. Theindicated mRNA were quantified by real-time RT-PCR inactivated CD4+ T cells from MC25, MC52 or MC42 mice (Tg+,filled symbols) and non-Tg littermates (Tg–, open symbols).Activation was performed under unbiased (U), Th1 or Th2conditions. H2-AamRNA levels (top) and the ratio between IL-4and IFN-c mRNA (bottom) were plotted as a function of CIITAmRNA abundance. Each data point corresponds to a singlemouse. The mean and standard deviation are shown forindependent RT-PCR measurements. The results shown arerepresentative of three experiments performed with differentsets of mice.



outside the immune system [8–14, 20]. These reportssuggested that CIITA might play a pleiotropic role invarious biological processes. It was therefore importantto establish the biological relevance of these putativenew targets in vivo. We analyzed the specificity of CIITAin vivo in multiple mouse tissues and cell types usingcomplementary loss- and gain-of-function CIITA mu-tants. These experiments provided no evidence for a keyrole of CIITA in controlling the expression of genes otherthan MHC-II and related genes. Only an indirectinfluence of CIITA on genes involved in Th celldifferentiation (Il4, Ifng, Gata3, and Tbx21) wasrevealed. In conclusion, MHC-II-mediated Ag presenta-tion and its repercussions on the development andactivation of CD4+ T cells are so far the only biological

processes that are regulated to amajor extent by CIITA invivo. The designation of CIITA as the “master controlfactor” for MHC-II and related genes thus remainsentirely justified.

All MHC-II and related genes were readily induced ina coordinated manner in all tissues and cell types of theCIITA-Tg mice. This globally enhanced MHC-II expres-sion is very sensitive to increased CIITA abundance, as itis already evident in the MC25 mice, which express theTg at only low levels. MHC-I genes are much lesssensitive to increased CIITA expression. A clear increasein MHC-I mRNA is detected only in the MC42 mice,which express the CIITA transgene to an extent farexceeding that required to obtain strong induction ofMHC-II genes. Moreover, induction is evident for theH2-K1 heavy-chain gene but not for the B2 m gene,which may explain why CIITA-induced MHC-I mRNAexpression is not accompanied by an increase in cellsurface MHC-I molecules. The role of CIITA in activatingMHC-I genes is thus much less critical than its pivotalfunction as a transactivator of MHC-II and related genes.This conclusion is consistent with the absence of amarked reduction in MHC-I mRNA or cell surfaceexpression in CIITA-deficient mice [27]. Clearly, MHC-Iexpression is largely CIITA independent in mice.

Ectopic CIITA expression in Tg mice, or its absence inknockout mice, has no significant repercussions onexpression of the Tap1, Yars, Eif3s2, Kpna6, Ctse, Col1a2,Ptprr, Tnfsf6 and Plxna1 genes, despite previousevidence suggesting they might be regulated by CIITA[9, 10, 12–14, 20]. Several explanations could accountfor this discrepancy. First, there could be species-specificdifferences between the target gene specificity of CIITAin humans and mice. Several of the earlier studies wereperformed with human cells [12, 13]. Second, therecould be subtle differences between the specificities of

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Figure 7. Altered IL-4 and IFN-c expression in CIITA-Tg Th cellsdoes not correlate with enhanced MHC-II expression.(A) Predicted results if CIITA expression in Th cells directlyinfluences Il4 and Ifng expression. (B) Naive CD4+ T cells fromMC25, MC52 and MC42 mice, and non-Tg littermates (Tg–),were activated under Th1 conditions and analyzed by FACS forcell surfaceMHC-II expression and IFN-c secretion.MHC-II-lowand -high cells were gated separately for the analysis of IFN-cexpression and are represented as light and dark greyhistograms, respectively. The percentage of cells expressinghigh levels of MHC-II or IFN-c is indicated. The results shownare representative of two experiments. (C) MC42 CD4+ T cellswere activated under Th1 conditions, sorted into MHC-II-lowand -high fractions and analyzed for IL-4 and IFN-c mRNAexpression by real-timeRT-PCR.Non-TgT cells activatedunderTh1 and Th2 conditions were used as controls. Each data pointcorresponds to a single mouse. The mean and standarddeviation are shown for independent RT-PCR measurements.The results shown are representative of two experimentsperformed with different sets of mice.



the three CIITA isoforms [2–4, 28]. Our Tg mice expressisoform IV, whereas earlier studies may have assessedspecific functions of isoforms I or III. A third possibilityis that regulation of the non-MHC genes by CIITA couldbe restricted to specific cell types, activation states orenvironmental conditions not addressed here. In thiscontext, it is worth mentioning that our results suggestthe existence of a tissue-specific influence of CIITA onTap1 expression (Fig. 4B). Finally, it is likely that thetransfections and reporter gene assays performed withcell lines in earlier studies did not reproduce faithfullywhat occurs in vivo [8–10, 12, 14, 21].

Several of the putative target genes studied here wereidentified by microarray experiments comparing WTcells and mutant cells lacking CIITA [10, 13, 14]. Ourinability to document a control of these genes by CIITAhighlights the importance of validating the resultsobtained by microarray experiments when dealing withpotential targets of transcription factors. Expressionchanges in cells lacking a given transcription factor donot necessarily reflect direct regulation by this factor.They could instead result from indirect effects, such asthe altered expression of other regulatory factors, cross-talk between different transcription activation path-ways, or a change in the equilibrium of a population ofdiverse multi-protein complexes containing multipleshared and specific factors. Such indirect effects are notnecessarily biologically relevant. They are moreoverlikely to vary between different cell types, species andexperimental systems. To distinguish between directcontrol and indirect effects, it is therefore important tovalidate putative target genes in multiple primary celltypes and tissues in vivo using gain- and loss-of-functionmutants of the transcription factor. Cross-speciescomparisons are also valuable in this respect.

Several reports have established that deregulatedCIITA expression has an impact on cytokine geneactivation during Th1/Th2 differentiation [8, 15, 16,18]. Th1 cells from CIITA-deficient mice were found toexhibit de-repressed IL-4 expression [8, 23]. Conversely,activated Th cells from CIITA Tg mice exhibit a Th2 biascharacterized by enhanced IL-4 and reduced IFN-cexpression [15, 16, 18]. For several reasons, themechanisms that mediate these influences of CIITA oncytokine expression are far from clear. First, whetherCIITA exerts a positive or negative influence on Il4expression is debated because both the loss and gain ofCIITA expression induce a similar Th2 bias [8, 15, 16, 18,21–23]. Second, we found no positive correlationbetween MHC-II and IL-4 expression, or an inversecorrelation between MHC-II and IFN-c expression, inindividual CIITA-Tg Th cells. This should have beenobserved if the Il4 and Ifng genes were immediate anddirect targets of CIITA. Third, induction of the Th2 biasrequires higher doses of CIITA than the activation of

MHC-II genes, suggesting that a different mechanism isinvolved. Fourth, the CIITA-Tg Th cells also exhibitaltered expression of T-bet and GATA-3, two transcrip-tion factors implicated in Th cell differentiation,suggesting that CIITA has a global impact on Th celldifferentiation. Such a global change is consistent withthe observations that the expression of Th2 cytokinesother than IL-4 is also increased in CIITA-deficient andCIITA-Tgmice [8,16,18, 23] and that GATA-3 expressionis increased in CIITA-deficient mice [23]. Finally, it is notclear whether the impact of CIITA on cytokine geneexpression is actually a T cell-autonomous effect.

Our results suggest that deregulated CIITA expres-sion affects Il4 and Ifng expression by an indirectmechanism related to a general effect on Th celldifferentiation. This may result from the abnormalexpression of CIITA and/or MHC-II molecules in T cells,either in a cell-autonomous fashion or because theT cells receive signals from their interactions with otherMHC-II-positive T cells. Alternatively, the altered pat-tern of Th cell differentiation may result from the factthat the T cells have developed and evolved in anenvironment exhibiting widespread CIITA-drivenMHC-II expression. In the latter case, abnormal inter-actions of naive T cells with MHC-II-positive cells mayhave left an imprint that favors Th2 differentiation.Whatever the mechanism is, it is clear that maintaining acorrect pattern of CIITA andMHC-II expression is criticalfor ensuring a correct balance between Th1 and Th2differentiation in mice. In contrast to mouse T cells,activated human T cells express CIITA at levels suffi-ciently high to induce strong MHC-II expression.Whether this affects human Th differentiation is notknown. However, the Th2 bias induced by ectopic CIITAexpression in mouse T cells suggests that the impact ofendogenous CIITA expression on human Th celldifferentiation should be examined.

Materials and methods

Mice

The CIITA construct was generated by standard techniques andused to generate Tg mice as described in SupplementaryMethods. Mice were screened by FACS for ectopic MHC-IIexpression on CD4+ B220– PBL. Experiments were performedwith Tg mice backcrossed into a CBA/j background for overfive generations. Tg mice and littermate controls were sex-matched, over 8 wk old and housed under conventionalconditions. CIITA and H2-Aa knockout mice backcrossed ontoa C57B6/J background were provided by C. H. Chang andH. Bluethmann [25, 29]. CIITA-p(III+IV) mice were on amixed Sv129 � C57BL/6 background [19]. Experiments wereapproved by the Swiss federal and cantonal veterinaryauthorities.



Cells

For RNA analysis, LN or spleen CD4+ CD62L+ cells wereisolated using MACS CD4-beads (Miltenyi Biotech, BergischGladbach, Germany) and FACS as described [15]. For cytokinesecretion assays, CD4+ CD62L+ cells were purified by twosteps using MACS beads. Splenic B cells were purified to*90% purity using Thy1-based complement depletion andactivated with LPS and IL-4 as described [19]. Spleen cells orpurified CD4+ CD62L+ cells were activated in flat-bottom 96-well or 48-well Costar plates with immobilized anti-CD3antibody (5 lg/mL), at 105 cells/200 lL or at 0.5 � 106 cells/500 lL, respectively, as described [15].

mRNA quantification

RPA for TATA-binding protein (TBP), CIITA (nucleotides3583–3929, accession number AF042159), H2-Ea, B2 m(nucleotides 157–296, accession number X01838) and H2-K1 mRNA were done as described [30]. RPA results werequantified using a phosphorimager (Molecular Dynamics).Quantitative RT-PCR was done using SYBR Green and a LightCycler (Roche, Switzerland) or an ABI7700 platform (AppliedBiosystems) as described [15]. Results were normalized toTBP.Values were expressed relative to a standard cDNA preparationmade from splenocytes and in vitro activated lymphocytes.Primers for TBP, CIITA, IFN-c and IL-4 mRNA have beendescribed [15]. Other primers are provided in supplementaryTable 2. All PCR quantifications were done in duplicate ortriplicate. Unless indicated otherwise, each data point wasderived from a single mouse. Repeated experiments wereperformed with different sets of mice.

Flow cytometry

Cells were incubated with mAb against FcR (2.4G2) to blocknon-specific Fc-mediated binding and against B220 (RA3–6B2;Caltag, Lifebiotech AG, Z�rich, Switzerland), CD4 (L3T4), I-Aand I-E (2G9), H2-K (SF1–1.1), H2-D (15-5-5), and FasL(MFL3). Unless indicated otherwise, antibodies were fromBecton Dickinson (Basel, Switzerland). Exclusion of dead cellswas done using sorting parameters and DAPI or 7-amino-actinomycin-D (Molecular Probes, Invitrogen, Basel, Switzer-land) staining.

Cytokine detection

An IFN-c secretion assay (Miltenyi Biotech) was performedaccording to the manufacturer's instructions except that thecytokine secretion step was performed at 1 � 106 cells/50 mL.Th2 conditions and an isotype control antibody (PE-rat IgG1)were used to define IFN-c-negative cells.

Acknowledgements: We thank W. Held, H. R. MacDo-nald, F. Radtke and F. Tacchini-Cottier for helpfuldiscussions and P. Zaech for FACS sorting. We areespecially grateful to S. Lattmann, E. Sauberli, Q. G.Steiner and A. Wilson for valuable help. The work ofL.A.O., W.R. and H.A.-O. was supported by the Swiss

National Science Foundation (SNF) and the GabriellaGiorgi-Cavaglieri Foundation. Thework ofW.R. was alsosupported by the Ligue Genevoise Contre le Cancer; thatof J.H. was supported by the SNF.

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Documents

Revisiting the specificity of the MHC class II transactivator CIITAin vivo