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ARTICLE: Contrôle quantitatif de l’expression des molécules CMH-II par CIITA

3. ACTION QUANTITATIVE DE CIITA SUR LES GENES CMH-II

3.1. ARTICLE: Contrôle quantitatif de l’expression des molécules CMH-II par CIITA

Quantitative control of MHC class II expression by the transactivator CIITA Otten,L.A.; Steimle,V.; Bontron,S.; Mach,B.

Eur J Immunol, VOL 28, 473-478

3.1.1 Résumé de l’article

La réponse spécifique contre les antigènes ou la tolérance envers le soi par le système immunitaire adaptatif dépend de facteurs qualitatifs comme la structure de l’antigène et de facteurs quantitatifs comme la quantité d’antigène. Dans ce contexte, le niveau d’expression des molécules CMH et des peptides présentés est fonctionnellement important. A cause du rôle essentiel de CIITA dans l’expression des gènes CMH-II, nous avons étudié l’aspect quantitatif de l’action de CIITA sur le niveau d’expression des ces gènes. Un modèle alternatif aurait été une action de CIITA sur les gènes CMH-II de type

“blanc/noir” CIITA et une régulation fine médiée par d’autres facteurs.

Les données, présentées dans cet article, sont basées sur une modulation artificielle in vitro du niveau d’expression de CIITA, une corrélation in vivo entre l’expression de CIITA et celle des gènes CMH-II et la mise en évidence d’une régulation fine du niveau d’expression de CIITA. Ainsi est mise en évidence la régulation quantitative de l’expression des molécules CMH-II par CIITA. Ces données posent un jalon vers une meilleure compréhension du mode d’action de CIITA et les bases expérimentales pour moduler in vivo la présentation d’antigène dépendant des molécules CMH-II.

0014-2980/98/0202-473$17.50 + .50/0

© WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1998

Quantitative control of MHC class II expression by the transactivator CIITA

Luc Alain Otten, Viktor Steimle, S ´everine Bontron and Bernard Mach

L. Jeantet Laboratory of Molecular Genetics, Department of Genetics and Microbiology, University of Geneva Medical School, Geneva, Switzerland

Activation of T lymphocytes is quantitatively controlled by the level of expression of MHC class II molecules. Both constitutive and inducible expression of MHC class II genes is regul-ated by the transactivator CIITA, which is itself tightly regulregul-ated. Since the level of MHC class II molecules expressed is a functionally essential parameter, it was of interest to explore whether MHC class II expression is quantitatively controlled by the level of the transactivator.

This report shows that in a variety of experimental conditions one does indeed observe, in both mouse and man, a quantitative control of MHC class II expression by the level of CIITA.

This relationship between the regulator gene, which behaves as a rate-limiting factor, and its target genes clarifies our understanding of the quantitative modulation of MHC class II expression, and thus of T lymphocyte activation.

Key words: MHC class II transactivator / MHC class II / Transactivator / Quantitative control / Gene regulation

Received 18/8/97 Revised 23/10/97 Accepted 29/10/97

[I 17454]

Abbreviations: AU: Arbitrary units CIITA: MHC class II transactivator dox: Doxycicline hydrochloride RPA:

RNase protection assay

1 Introduction

MHC class II genes are expressed constitutively in APC, in epithelial of various organs and in some endothelia [1, 2]. Inducible expression is triggered in many other cells by various soluble factors, among which IFN-+ is the best characterized [2]. RFX5, RFXAP and CIITA are three recently cloned transacting factors that are essential for all forms of activation of MHC class II genes [3–7]. RFX5 and RFXAP are subunits of the RFX complex, which binds to the MHC class II promoters [5, 6]. CIITA is thought to function as a transcriptional co-activator interacting with factors bound to MHC class II promoters [4, 8]. While RFX and other transcription factors are expressed ubiquitously, CIITA is differentially expressed in a pattern correlated with that of MHC class II genes [4, 9, 10]. Regulation of CIITA expression is under develop-mental and cytokine control [9–12] and involves the dif-ferential usage of multiple CIITA promoters [13].

The cell surface concentration of MHC class II molecules directly influences the magnitude and the nature of the T lymphocyte response [14, 15]. In view of the functional importance of the quantitative control of MHC class II expression and of the key role of CIITA in the control MHC class II genes [4, 9], it was of interest to establish whether CIITA itself is a rate-limiting factor controlling this expression in a quantitative manner. An alternative mechanism would imply that CIITA acts as an on/off switch and that the rate of transcription is limited by other factors, such as those binding to the MHC class II promoters [8]. We demonstrate here that, in multiple cell types and tissues, the level of expression of CIITA is the major, and perhaps the sole, MHC class II transcription rate-determining parameter.

2 Results and discussion

2.1 CIITA regulates the level of MHC class II expression in a quantitative manner

The tetracycline-regulated expression system was used to modulate the level of CIITA produced over a wide range and thus to observe the effect of very different levels of CIITA on MHC class II expression [16]. We stably transfected a tetracycline-regulated CIITA con-struct (tet-CIITA) into the HeLa-derived HtTA cell line Eur. J. Immunol. 1998. 28: 473–478 Quantitative control of MHC class II expression by CIITA 473

Figure 1. Quantitative control of MHC class II expression by CIITA in the tetracycline-regulated CIITA-transfected clone 4.13. (A) Cells were incubated with dox at various concentrations (experiment B) and analyzed by FACS for expression of the three MHC class II isotypes. The filled profiles show 4.13 cells incubated without dox and stained with the second antibody only. (B) Analysis of CIITA and DRA mRNA levels in the following samples: clone 4.13 experiment A (diamond), clone 4.13 experiment B (square), bulk transfectants ± dox (triangle), Raji (left arrow) and IFN-+-induced HtTA cells (1000 U/ml for 48 h) analyzed at day 7 (right arrow). Expression of the transfected mouse CIITA and DRA was analyzed by RPA and PhosphorImager. The CIITA/DRA curve of transfectant 4.13 is presented in two graphs covering low and high AU values (insert) respectively. The coefficient of determina-tion (r2) is derived from the polynomial regression shown in the large graph. In IFN-+-induced HtTA and Raji no CIITA is detected because the probe is specific for the transfected mouse CIITA.

and isolated clone 4.13, in which tetracycline [or doxy-cycline hydrochloride (dox)] represses expression of the transfected CIITA cDNA. Dox does not affect cell surface MHC class II expression in non-transfected cells or in cells transfected with a CIITA constitutive expression vector (data not shown). At high concentrations of dox, clone 4.13 presents a fully negative phenotype of MHC class II expression by FACS analysis (Fig. 1A). At low concentrations of dox, induction of CIITA takes place, leading to very high levels of MHC class II expression.

Intermediate levels of MHC class II expression observed at intermediate concentrations of dox suggest a causal quantitative relationship between the level of CIITA expressed and that of MHC class II expression.

At low levels of induction of the tet-CIITA construct, the transfectants showed clear HLA-DR and -DP cell surface expression with only weak HLA-DQ expression (150 pg/

ml of dox in Fig. 1A). This MHC class II isotype-specific dissociation at the level of cell surface expression disap-peared at high levels of CIITA induction. It is of interest that such a MHC class II isotype-specific dissociation has been generally observed only at the cell surface but not at the RNA level [2, 17–19]. This suggests that this weak HLA-DQ expression is due to a post-transcriptional regulation.

The steady state mRNA level of CIITA and HLA-DRA were quantified by RNase protection assay (RPA) and PhosphorImager analysis in clone 4.13 treated with vari-ous concentrations of dox (Fig. 1B). The CIITA/MHC

474 L. A. Otten et al. Eur. J. Immunol. 1998. 28: 473–478

Figure 2. Quantitative correlation of CIITA and MHC class II mRNA expression in multiple mouse tissues. CIITA and I-E§ expression were analyzed in total RNA by RPA with a TBP probe as internal control (top) and quantified by Phosphor Imager (bottom). The coefficient of determination (r2) is derived from the linear regression. U: undigested probes, Y:

yeast, 1: spleen, 2: thymus, 3: heart, 4: lung, 5: liver, 6: stom-ach, 7: small intestine, 8: colon, 9: kidney, 10: testis, 11:

brain, 12: skin, 13: muscle.

class II dose response curve was established from the measurements derived from two different induction experiments of clone 4.13. In the presence of a high con-centration of dox, mRNA levels of CIITA and DRA are at a background level similar to those of non-transfected cells. No lag was observed between the lowest detect-able induction of CIITA (0.08 AU at 150 pg/ml of dox in Fig. 1A) and the first increase of the level of DRA mRNA (0.09 AU) and HLA-DR cell surface level. This positive correlation exists between the lowest levels of CIITA and DRA mRNA up to levels of expression of DRA which are well above those found in the IFN-+-induced HtTA cells and the strongly MHC class II-positive B lymphocyte Raji cell line (Fig. 1B). Upon further increase of the CIITA level, a plateau is reached for the level of DRA mRNA (Fig. 1B, inserted graph). These results demonstrate that the quantitative control of MHC class II expression by CIITA is relevant from very low levels of DRA mRNA to clear overexpression. This also indicates that CIITA is the rate-limiting factor for MHC class II expression over a wide range of experimental expression levels.

2.2 CIITA and MHC class II expression correlate both qualitatively and quantitatively in multiple tissues

In order to test the validity of our observations in vivo, we examined the quantitative correlation between CIITA and MHC class II RNA isolated from multiple mouse tissues.

The pattern of expression of CIITA and I-E§ was ana-lyzed by RPA and PhosphorImager (Fig. 2). Strict co-expression between CIITA and MHC class II was observed without exception. A positive and linear corre-lation between CIITA and I-E§ was observed with a coefficient of determinantion of 0.91. This observation suggests that, in multiple cell lineages, MHC class II pro-moters respond to the same extent to different levels of CIITA. Thus, neither the essential ubiquitous transcrip-tion factors that bind to MHC class II promoters nor other factors acting downstream of CIITA action seem to play a major role in the quantitative control of MHC class II expression in vivo [3, 20]. CIITA is, therefore, not only an absolute requisite for MHC class II expression but is also the major limiting factor for constitutive MHC class II transcription in multiple cell types in vivo [4, 21].

2.3 The level of CIITA is itself regulated by different amounts of IFN-q

The quantitative control of MHC class II expression by CIITA leads to the issue of the quantitative control of CIITA expression itself. We therefore examined the effect of two different concentrations of IFN-+ on the CIITA

mRNA level in HtTA cells. Expression of CIITA peaked at day 1 and then decreased during the following days with both concentrations of IFN-+ (Fig. 3). The ratio between the two CIITA inductions is about 2 :1 and is conserved throughout the whole time course. A similar ratio is observed between the two DRA mRNA inductions (data not shown). The quantitative correlation between CIITA and MHC class II expression is thus observed again.

Taken together, these data show that the level of endog-enous CIITA expression can itself be quantitatively re-gulated. Thus, across the entire cascade of MHC class II induction, from ligation of IFN-+ to its receptor to surface expression of MHC class II molecules, CIITA action has been identified as a key rate-limiting step.

Eur. J. Immunol. 1998. 28: 473–478 Quantitative control of MHC class II expression by CIITA 475

Figure 3. CIITA expression is quantitatively controlled by IFN-+. HtTA cells were incubated with two concentrations of IFN-+ for 2 days. Expression of human CIITA RNA was ana-lyzed by RPA and PhosphorImager at the days indicated.

Each point of the time course represents the mean ± SEM of three or four independant inductions (n).

3 Concluding remarks

Our present observations of the CIITA-mediated quanti-tative control of both constitutive and inducible MHC class II expression has implications for novel experimen-tal strategies of immunomodulation of T lymphocytes. It has been shown that T cell selection and activation can be significantly influenced by the level of MHC class II expression [14, 15]. Experimental modulation of the level of MHC class II expression in vivo can now be envisaged in transgenic mice or via any agent that can modulate either the expression or the activity of CIITA.

4 Materials and methods 4.1 DNA constructs

The tet-CIITA expression vector is based on pBluescript and contains the tetracycline operator PhCMV*-1 (provided by H.

Bujard, Heidelberg), the mouse CIITA cDNA form IV which is functionally equivalent to the human CIITA form III (Otten et al., manuscript in preparation), and the SV40 small T intron containing an internal SAU 3A deletion (provided by M. Bra-del, Munich) and a poly A signal [13, 16].

4.2 Cell culture, transfectants and FACS analysis The HtTA cell line (provided by H. Bujard) was grown in DMEM supplemented with 10 % FCS, penicillin/streptomy-cin and glutamine at 37 °C in 5 % CO2. The HtTA cells were electroporated with tet-CIITA and the hygromycin resistence

conferring plasmid pTG76 [4], selected for hygromycin resistence at 100 ?g/ml, enriched for HLA class II DR expression with immunomagnetic beads, and cloned. Anti-bodies and FACS procedure were as described in [4].

4.3 Induction of transfected CIITA in HtTA cells

Bulk tet-CIITA transfectants and several clones showed inducible MHC class II expression upon dox (Sigma) removal. After 2 weeks of dox treatment (10 ng/ml renewed every 2 days), the tet-CIITA transfectants were negative for MHC class II and CIITA expression as determined by FACS and RPA. In such MHC class II-negative cells, maximal and intermediate cell surface expression of DR and HLA-DQ was stably induced within 4 days by removal of dox or exposure to dox at 80 pg/ml, respectively. In clone 4.13, two independent inductions of CIITA expression (experiments A and B) were performed at 12 concentrations of dox (10 000, 2000, 600, 300, 150, 80, 40, 20, 10, 3, 1, 0 pg/ml). FACS and RPA analysis were carried out on day 4.

4.4 RPA analysis

Total RNA from BALB/c mouse organs and cytoplasmic RNA from cell lines and transfectants were prepared as described [4, 22]. The human CIITA (position 2913–3263) [4], DRA [9], and human TBP probes [13] have been described else-where. The mouse CIITA probe protects a fragment at posi-tions 3277–3623 (Otten et al., manuscript in preparation).

The I-E§d probe protects a 238-bp fragment (codons 28–139). The mouse TBP probe protects a 160-bp fragment (codons 263–317). RPA procedure was as described [4]. The I-E§ and DRA probes were generated at a 25-fold reduced specific activity as compared to the others. Murine CIITA, human CIITA, DRA and I-E§ expression was analyzed with either human TBP or mouse TBP as the internal control. The PhosphorImager signals from each sample are expressed as relative values using TBP as a reference in AU. Sensitivity and linearity of the procedure was verified by analyzing serial dilutions of RNA isolated from the bulk tet-CIITA transfec-tants and Raji. A linear input/signal ratio was observed for both CIITA and DRA mRNA, starting from 0.03 AU. Error in quantification was estimated to be around 20 %.

Acknowledgments: We thank Dr. C. Ucla, M. Zufferey and D. Wohlwend for technical help, Dr. S. Izui and Dr. D. Belin for helpful discussion and advice, and Dr. W. Reith, A.

Muhlethaler-Mottet and other members of the Mach lab for critical reading of the manuscript. This work was supported by the Swiss National Science Foundation and the Louis Jeantet Foundation.

476 L. A. Otten et al. Eur. J. Immunol. 1998. 28: 473–478

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Eur. J. Immunol. 1998. 28: 473–478 Quantitative control of MHC class II expression by CIITA 477

Correspondence: Bernard Mach, L. Jeantet Laboratory of Molecular Genetics, Department of Genetics and Microbiol-ogy, University of Geneva Medical School, 9 avenue de Champel, CH-1211 Geneva 4, Switzerland

Fax: +41-22-7025669

e-mail: Bernard.Mach@medecine.unige.ch

V. Steimle’s present address: Max-Planck-Institut für Im-munbiologie, Hans-Spemann-Laboratories, Freiburg, Ger-many

478 L. A. Otten et al. Eur. J. Immunol. 1998. 28: 473–478