T cell expression of CIITA represses Th1 immunity
Weon Seo Park1,
Youngmee Bae1,
Doo Hyun Chung3,
Yoon-La Choi3,4,
Byoung Kwon Kim3,
Young Chul Sung5,
Eun Young Choi3,4,
Seong Hoe Park3,4 and
Kyeong Cheon Jung2
1 Department of Pathology, Kangwon University College of Medicine, Chunchon, Korea
2 Department of Pathology, Hallym University College of Medicine, Chunchon, Korea
3 Department of Pathology, College of Medicine and 4 Research Division for Human Life Science, Seoul National University, Seoul, Korea
5 Department of Life Science, Pohang University of Science and Technology, Pohang, Korea
Correspondence to: S. H. Park; E-mail: pshoe{at}plaza.snu.ac.kr
Correspondence to: K. C. Jung; E-mail: jungkc66{at}snu.ac.kr
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Abstract
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Despite the fact that major histocompatibility complex class II transactivator (CIITA) has been known to be involved in Th1/Th2 balance in addition to its major role as a master regulator for the expression of MHC class II genes, the exact role of CIITA in Th1/Th2 balance is still controversial. To investigate whether the Th1/Th2 balance could be modulated by T cell specific expression of CIITA, we generated CIITA-transgenic mice, in which the CIITA expression is controlled by the distal promoter of p56lck, resulting in constitutive expression of CIITA predominantly in peripheral T cells. Naive CD4+ T cells from CIITA-transgenic mice exhibited a low level of IFN-
secretion as well as impaired Th1 polarization in vitro, while IL-4 secretion was enhanced under Th2 condition. In addition, the development of experimental autoimmune encephalomyelitis (EAE), a prototype of Th1-mediated disease, was repressed in CIITA-transgenic mice. Resistance to EAE was correlated with reduced production of IFN-
in response to MOG3555, while the proliferation of MOG3555-specific T cells was not affected in CIITA-transgenic mice. Together, these data demonstrate that overexpression of CIITA in T cells inhibits Th1 differentiation and function, suggesting that the expression of CIITA in T cells might play a role in the regulation of the Th1/Th2 balance during the T cell lineage commitment.
Keywords: experimental autoimmune encephalomyelitis, IFN-
, MHC class II, transgenic mouse
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Introduction
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Major histocompatibility complex class II transactivator (CIITA) is initially identified as a factor responsible for bare lymphocyte syndrome (1) and is now considered as a critical transactivator required for the expression of MHC class II genes in both conventional and non-conventional antigen-presenting cells (APCs) (2,3). Upon binding of IFN-
, its receptor promotes transcription of CIITA gene through signaling from JAK1 to Stat1 transcription factor. Then, CIITA leads to the expression of MHC class II molecules in IFN-
-activated macrophages and non-professional APCs (4). Although CIITA is a potent transcriptional activator, it is known to exert its function through interaction with other transcription factors bound to MHC class II promoters, such as NF-Y and RFX (5). It has also been demonstrated that CIITA could interact with other transcriptional coactivators, such as Bob1 and CREB binding protein (CBP)/p300 (68). Since CBP and its close homologue, p300, have been found to be involved in the transcription of various genes, it is therefore conceivable that CIITA can compete with other transcription factors for binding to CBP/p300 (5). To date, it has been described that promoters for IL-4, thymidine kinase, cyclin D1 and collagen could be suppressed by CIITA via competition for the limited amount of CBP (8,9). Thus, it seems that the CIITA protein interacts with a variety of transcription factors in the cells and would be involved in diverse functional aspects of immune responses in vivo.
The most convincing findings regarding the physiological function of CIITA in murine T cells have been obtained from CIITA/ I-E-transgenic mice (10). CD4+ T cells from CIITA/ I-E-transgenic mice produced a high level of IL-4 even in the presence of IL-12 (10), suggesting that CIITA suppresses the transcription of IL-4 genes in activated Th1 cells. A subsequent study has demonstrated that the competition between CIITA and NF-AT for binding to CPB/p300 is critical for the regulation of IL-4 secretion by T cells (9). All these results suggest that the expression of CIITA in CD4+ T cells can cause downregulation of IL-4 production and thus lead to an enhanced Th1 population in vivo (10). However, recent results obtained from CIITA-transgenic mice have been contradictory to this view (11). In this experiment, the constitutive expression of CIITA in all organs, including T cells, resulted in a severe Th2 bias during Th cell activation and differentiation.
Numerous studies have described that the activation of human and rat T cells leads to the expression of MHC class II molecules on the cell surfaces (12,13) and that this event is driven by the induction of CIITA gene (14,15). In contrast, all the mouse T cells, regardless of developmental stage or subpopulation, do not express MHC class II molecules on their surfaces (16). Only a minimal amount of CIITA and MHC class II transcripts were demonstrated in murine T cells by RTPCR (10,11).
In this study, we examined the effects of CIITA on the regulation of T cell activation and helper cell differentiation, using CIITA transgenic mice with restricted expression of this gene on T cells, and show that Th1 differentiation of CD4+ T cells and Th1-dependent immunity are suppressed in the transgenic mice.
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Methods
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Mice
A fragment including a full-length human CIITA cDNA (a generous gift from Cheong-Hee Chang, Indiana University, USA) was introduced at the BamHI site of pw120 (a generous gift from Jae Kyun Shin, Sungkyunkwan University, Korea) vector containing the distal promoter of lck (17) (Fig. 1A). The transgene was injected into the pronuclei of fertilized eggs of FVB mice. We confirmed the presence of the inserted CIITA sequence by PCR and Southern blotting of genomic DNA of transgenic mice. Transgene-positive founders were backcrossed to C57BL/6 (B6) mice (Daehan Biolink, Chungbuk, Korea) for more than 15 generations and then maintained in a mouse facility in Hallym University. The transgenic mice used in the experiments were all on B6 background and the induction of I-Ab protein by CIITA on peripheral T cells was confirmed using flow cytometry. The CIITA-transgenic mice on B6 background were also crossed with OT-II mice to generate CIITAxOT-II double transgenic mice. The OT-II mice were a gift from Frank R. Carbone (University of Melbourne, Australia).

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Fig. 1. The generation of CIITA-Tg mice and the expression of transgene CIITA and MHC class II molecules in the transgenic mice. (A) Schematic diagram of the transgene construct. The human CIITA gene is placed under control of the distal promoter of murine lck and fused with human growth hormone poly A signal region. (B) Expression of the transgenic human (hu) CIITA was checked in various kinds of tissues from Tg mice by RTPCR (WT, wild-type littermate; Tg, CIITA-transgenic mice; lane 1, thymocytes; 2, splenocytes; 3, liver; 4, kidney; 5, lung; 6, brain and spinal cord). (C) Surface expression of I-Ab molecules on the thymocytes and lymphocytes from the spleen and lymph node of the T10-originated CIITA-Tg mice was confirmed by flow cytometry. Representative figures are shown.
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Reagents
The rat MOG3555 (MEVGWYRSPFSRVVHLYRNGK) and chicken OVA323339 (ISQAVHAAHAEINEAGR) peptides were synthesized in Peptron (Taejon, Korea). Incomplete Freund's adjuvant (IFA) and pertussis toxin were obtained from Sigma Chemical Company (St Louis, MO). Heat killed Mycobacterium tuberculosis, strain H37Ra, was purchased from Difco Laboratory (Detroit, MI). Purified protein derivative (PPD) was obtained from Statens Serum Institute (Copenhagen, Denmark). Anti-mouse antibodies used in this study are as follows: anti-I-Ab (AF6-120.1), anti-I-A/I-E (2G9), anti-CD3 (145-2C11), anti-CD28 (37.51), anti-IFN-
(XMG1.2) and anti-IL-4 (11B11) mAbs were purchased from Pharmingen (San Diego, CA), and anti-CD4 (GK 1.5), anti-CD8a (53-6.7), anti-B220 (RA3-B2) and anti-Mac-1 (M1/70) mAbs were purchased from DiNonA Inc. (Seoul, Korea).
RNA preparation, PCR and semiquantitative PCR
Total RNA was isolated using an RNA extraction kit (Bioneer, Taejon, Korea) and cDNA was first prepared by reverse transcription of 2 µg RNA in a 50 µl RT PreMix (Bioneer) containing 200 pmol oligo-dT18, followed by PCR amplification in a PCR master mix composed of 10 mM TrisHCl (pH 9.0), 2.5 U Taq DNA polymerase, 250 µU dNTP, 40 mM KCl and 1.5 mM MgCl2 (Bioneer). For semiquantitative PCR, serial dilution (3-fold) of the cDNA reaction mixtures was subjected to PCR amplification using the primers for HPRT, human-CIITA, mouse distal Lck and mouse CIITA. Primers and PCR cycles were as follows: human CIITA, 5'-CCACTTGTACGACCAGATGGAC-3' and 5'-CGTGGACAGTGAATCCACTG-3' (35 cycles); mouse CIITA, 5'-ACGCTTTCTGGCTGGATTAGT-3' and 5'-TCAACGCCAGTCTGACGAAGG-3' (35 cycles); mouse distal lck, 5'-TGGTGACTACGACGGCGAGG-3' and 5'-GGATGCTGGTGGGAGAGA-3' (32 cycles); mouse HPRT, 5'-GTTGGATACAGGCCAGACTTTGTTG-3' and 5'-GAGGGTAGGCTGGCCTATGGCT-3' (30 cycles).
Flow cytometric analysis
Fresh cell suspensions of splenocytes, lymph node cells and peripheral blood lymphocytes were prepared in phosphate-buffered saline (PBS) solution and erythrocytes were lysed by RBC lysis buffer (DiNonA Inc.). Cells were resuspended in PBS and incubated with antibodies for 30 min at 4°C and washed with PBS. The flow cytometric analysis was performed using FACSCalibur (Becton-Dickinson, Mountain View, CA).
Preparation, in vitro stimulation and induction of Th1 and Th2 differentiation of CD4+ T cells
Mouse CD4+ T cells were harvested from spleens and lymph nodes of CIITA-transgenic or littermate mice by Ficoll-Paque (Amersham Bioscience, Buckinghamshire, UK) gradient centrifugation of lymphocytes and magnetic cell sorting using MACS with anti-CD4 (GK 1.5) microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) as recommended by the manufacturer. The purity of collected CD4+ T cells was determined by flow cytometry using anti-CD4 mAb (GK 1.5) and ranged from 95 to 97%. The purified CD4+ T cells were stimulated with immobilized anti-CD3 mAb (2C11) or specific antigens and 50 µg/ml mitomycin C (Sigma)-treated splenocytes in RPMI medium (Amersham Bioscience) supplemented with 10% fetal bovine serum (HyClone, Logan, Utah) and 50 µM ß-mercaptoethanol for 3 days. The culture supernatants were used for cytokine assay by ELISA.
For the purification of naive CD4+ T cells, total CD4+ T cells were initially prepared from spleen and lymph node cells by magnetic sorting using FITC-labeled anti-CD4 mAb (GK 1.5) and anti-FITC multisort kits (Miltenyi Biotec) and then further purified by magnetic sorting of CD62L+ populations with anti-CD62L (MEL-14) microbeads (Miltenyi Biotec). Naive CD4+ T cells (1 x 106 cells/ml) were cultured in the presence of 5 µg/ml immobilized anti-CD3 (2C11) and 20 U/ml IL-2 (KOMA biotech, Seoul, Korea) and supplemented with 100 U/ml IL-12 (KOMA biotech) and 15 µg/ml anti-IL-4 mAb for Th1 condition or with 1000 U/ml IL-4 (KOMA biotech) and 15 µg/ml anti-IFN-
mAb for Th2 condition. After 4 days, cells were washed extensively, counted and restimulated with immobilized anti-CD3 mAb overnight.
Cytokine ELISA
Quantitative analysis of cytokines in cell culture supernatants was performed by sandwich ELISA using the OptEIA kits (Pharmingen).
Intracellular cytokine assay
Activated CD4+ T cells were restimulated with specific antigen and APCs for 8 h in the presence of monensin (5 ng/ml; Sigma) during the last 4 h. The cultured cells were harvested, fixed with 2% paraformaldehyde and permeabilized with 0.3% (w/v) saponin in PBS. The cells were incubated for 30 min with FITC-conjugated anti-mouse INF-
and PE-conjugated anti-mouse IL-4 mAbs (Pharmingen), washed with permeabilization buffer and analyzed by flow cytometry.
Induction of experimental autoimmune encephalomyelitis
For the induction of active experimental autoimmune encephalomyelitis (EAE), mice were injected in the bilateral hind footpads with a total of 100 µl of emulsion containing 200 µg MOG3555 in IFA supplemented with 250 µg of M. tuberculosis. They were injected intraperitonealy with 500 ng of pertussis toxin in 500 µl of PBS shortly after, and 48 h after the immunization. For the study of T cell response, mice were immunized only once with 200 µg MOG3555 in IFA and M. tuberculosis without subsequent injection of pertussis toxin. Mice were observed daily for clinical signs of EAE for up to 35 days and scored on a scale of 05 with 0.5 increments: 0, no disease; 0.5, partial loss of tail tonicity, assessed by inability to curl distal end of tail; 1, limp tail; 1.5, limp tail and unable to turn over when placed on back; 2, hind limb weakness and abnormal gait; 3, hind limb paralysis; 4, hind and fore limb paralysis; and 5, moribund state. Mean clinical score was calculated by averaging the scores of all mice in each group, including animals that did not develop EAE.
Recall response of T cells to antigens
The inguinal lymph nodes were taken 14 days after immunization with MOG3555 and CD4+ T cells were purified as described previously and cultured in the presence of mitomycin C-treated splenocytes from B6 mice. CD4+ T cells were incubated with various concentrations of MOG3555 peptide, PPD or anti-CD3 mAb and APCs for 3 days. Cultures were pulsed with 1 µCi/well [3H]thymidine (Amersham Bioscience) for the final 16 h and the mean incorporation of thymidine in DNA was measured in quadruplicate wells by liquid scintillation counting.
Histology
Brains and spinal cords were removed and fixed in 10% formalin. Paraffin-embedded sections were stained with Luxol fast blue-hematoxylin and eosin.
Statistical analysis
Comparisons of the mean cytokine secretion and mean peak disease severity between wild-type and CIITA-transgenic mice were analyzed by the MannWhitney's rank sum test. Comparison of the percentage of animals showing clinical disease between two groups was done by
2 using Fisher's exact probability. P-values < 0.05 were considered statistically significant.
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Results
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CIITA-dependent expression of MHC class II antigen in T cells
To explore the effects of CIITA on cytokine production by peripheral T cells, we generated transgenic mice that express CIITA only in T cells under the control of the distal lck promoter (Fig. 1A) which directs high expression of a target protein predominantly on peripheral T cells (17,18). By doing this, we were able to focus on the effect of CIITA expression in T cells on Th differentiation and exclude any possible influences from non-professional APC that are not supposed to express MHC class II. The transgenic mice on B6 background (CIITA-Tg hereafter) were used for all the experiments. The expression of CIITA transgene was particularly predominant in the spleen and lymph nodes, but not in the liver, kidney, lung, brain or spinal cord, as confirmed by RTPCR (Fig. 1B). The expression of the transgene was quite minimal in the thymus.
Since the major function of CIITA is the induction of expression of MHC class II, we analyzed the MHC class II expression in these mice by flow cytometry to test whether the transgenic CIITA gene functions properly in vivo. Most of the CD4+ and CD8+ T cells from the spleen and lymph node of CIITA-Tg mice expressed the I-Ab molecules on their surfaces (Fig. 1C). The I-Ab molecules were also expressed in minor subsets of thymocytes of the transgenic mice, while all types of T cells from wild-type littermates did not express the I-Ab molecules. The expression level of I-Ab in B cells and macrophages of CIITA-Tg mice was comparable to that of littermates.
CIITA-Tg mice appeared normal and throve inside both conventional and barrier animal facilities. Gross examination of organs from the transgenic mice revealed no apparent abnormalities. Moreover, flow cytometric analysis of thymocytes and splenocytes demonstrated that CIITA-Tg mice had normal profiles of lymphocytes in these organs (Table 1). Initially, three independent mouse lines, referred to as T10, T30 and T73, were generated on FVB background and then two of them (T10 and T30) were backcrossed to B6 mice. As the three lines of FVB background (data not shown) and the two lines on B6 background showed similar phenotypes, and as transgene expression was between 1.5- and 2-fold higher in the line originated from T10 than that from T30, we report here analyses made with T10-originated mice.
Effects of CIITA on the development of T helper cells
First, we analyzed the cytokine production, using total CD4+ T cells from spleens and lymph nodes of CIITA-Tg mice and wild-type control littermates. CD4+ T cells were enriched by magnetic sorting and stimulated with anti-CD3 mAb and mitomycin-treated APCs. The proliferation of CD4+ T cells was not affected by the CIITA expression (Fig. 2A). However, CIITA-transgenic CD4+ T cells secreted a lower level of IFN-
, but a higher level of IL-4, as compared to the control cells, although the IL-2 and IL-5 levels were comparable (Fig. 2B). The altered cytokine secretion was augmented by the costimulation of CD28.
Naive CD4+ T cells activated by APCs undergo clonal proliferation and then differentiation into Th1 or Th2 effector cells (19). In order to examine whether the expression of transgenic CIITA in T cells provides any influence on the cytokine production via modulation of Th effector differentiation, naive CD4+ T cells purified from the spleen of CIITA-Tg mice were induced to differentiate into either Th1 or Th2 cells in vitro and checked for their ability to produce IFN-
and IL-4. The CIITA-Tg naive CD4+ T cells stimulated under Th1 condition produced 48% lower amounts of IFN-
, while those under Th2 condition produced 2-fold higher amounts of IL-4, compared to the cells from control animals under the same conditions (Fig. 2C). This result indicates that the Th1 differentiation is suppressed, while the Th2 differentiation is enhanced in CIITA-Tg CD4+ T cells.
Since it has been reported that endogenous CIITA is not differentially regulated in Th1 and Th2 cells of both human and mouse (11), we asked whether the imbalanced secretion of Th1/Th2 cytokine by transgenic CD4+ T cells might be due to dissimilar expression of the transgenic CIITA gene between Th1 and Th2 cells. To address this issue, the levels of transgenic CIITA transcripts were compared between Th1 and Th2 CD4+ T cells using semi-quantitative RTPCR. The test on the endogenous CIITA transcripts was included as control. The amount of endogenous CIITA transcripts in CD4+ T cells was too low to be detected in the scope of our semi-quantitative PCR (Fig. 2D) without nested-second PCR in all the cases (data not shown), in contrast to readily detectable levels in B cells, suggesting that the endogenous CIITA levels in the CD4+ T cells are almost negligible, probably detectable only by sensitive methods like real-time PCR (11). In terms of unequal regulation of transgenic CIITA, it could hardly be said that there were differences in the level of transgenic CIITA transcripts between Th1 and Th2 cells (Fig. 2D), which was the case for the expression level of MHC class II between Th1 and Th2 cells (Fig. 2E). However, >9-fold decreases of the transgenic CIITA transcripts in the activated Th1 and Th2 cells were observed, compared to the level in naive CIITA-Tg CD4+ T cells (Fig. 2D). Such changes in transgenic CIITA transcription after activation were reflected in the decrease of MHC class II levels on the activated Tg CD4+ T cells along the duration of in vitro stimulation (Fig. 2E). Then, we asked if the reduced levels of CIITA transcript in the activated cells were related to the physiologic activity of lck distal promoter itself. Since lck distal promoters acting in our system are both the endogenous one and the one incorporated by transgenic construct, physiological change of lck distal promoter itself could be monitored indirectly by the change in the amount of distal promoter-driven lck transcripts. Based on this rationale, we performed a semi-quantitative RTPCR of distal lck promoter-driven lck transcripts, but the result was that lck distal promoter activities were steady, irrespective of activation statuses of CD4+ T cells. Thus, the reduced CIITA transcripts and MHC class II levels after activation could not be ascribed to original physiological characteristics of distal lck promoter. Therefore, the decrease in transgenic CIITA transcripts and MHC class II expression after cell activation was explainable only as a phenomenon noted in vitro. And we kept detecting MHC class II positive CD4+ T cells with activation phenotype (CD44hiMel-14low) from the old CIITA-Tg mice housed in conventional room or those immunized with MOG3555 peptide with pertussis toxin (data not shown). Whatever the reason for the decreased expression of transgenic CIITA in activated CD4 T cells, our results demonstrate that the Th1/Th2 imbalance in the transgenic CD4 T cells was not due to differential regulation of CIITA transgene in the Th1 and Th2 cells.
Effects of CIITA on Th differentiation of OT-II T cell receptor (TCR) transgenic CD4+ T cells
In order to test whether the repressed Th1 response recurs when the diversity of T cells is restricted to a known specificity, we established a double transgenic mouse for CIITA and OT-II TCR (20) (CIITAxOT-II dTg mice) by crossing the two transgenic mice on B6 background. OT-II TCR is composed of V
2 and Vß5 and recognizes chicken OVA323339 in the context of I-Ab (21). CD4+ T cells from the dTg mice were tested for their differentiation into each type of Th cell. Most of the CD4+ T cells from the dTg mice had V
2Vß5 TCR and showed naive phenotype (CD44lowMel-14high) and an almost identical level of MHC class II expression to that of CIITA-Tg mice (data not shown). The curve of [3H]thymidine incorporation after stimulation of dTg CD4+ T cells with APC and chicken OVA323339 peptide was superimposed on that of OT-II Tg CD4+ T cells (Fig. 3A). However, even though overall differentiation of the activated CD4+ T cells was skewed to IFN-
-producing Th1 cells in both animals, the amount of IFN-
secretion was again lower in the dTg CD4+ T cells (44% reduction) than in the CD4+ T cells from OT-II Tg mice (Fig. 3B). The reduced secretion of IFN-
was due to reduction of the number of cells expressing IFN-
as well as the decrease in the amount of IFN-
produced per cell in dTg CD4+ T cells (10% lower MFI), as compared to OT-II Tg T cells (Fig. 3C). Together, these results indicate that the forced expression of CIITA in CD4+ T cells rendered the T cells to have reduced potential to develop into Th1 type of cells.

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Fig. 3. Decreased IFN- secretion in CIITAxOT-II dTg mice. CIITA-Tg mice were crossed with OT-II mice to generate CIITAxOT-II dTg mice. Purified CD4+ T cells from OT-II Tg littermates (OT-II) and CIITAxOT-II dTg mice (dTg) were stimulated with mitomycin-treated splenocytes and chicken OVA323339 for 3 days. (A) Proliferation was measured by [3H]thymidine incorporation assay. (B) The levels of cytokines secreted in culture supernatant were determined by ELISA. (C) Purified CD4+ T cells were stimulated with OVA323339 for 2 days and then restimulated with OVA323339 in the presence of momensin, followed by intracellular cytokine staining and flow cytometry. The data represent mean ± SEM in one representative experiment of three. *P < 0.05.
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Suppression of the development of EAE in CIITA-transgenic mice
We tested whether the reduced Th1 effector function of CIITA-Tg CD4+ T cells shown in vitro is also the case in the in vivo model where Th1-mediated immunity is induced. EAE is a typical Th1-mediated inflammatory response elicited by immunizing with MOG3555 peptide and pertussis toxin into B6 mice (22,23). EAE was developed only in 6 out of 16 CIITA-Tg mice (37.5%) with a mean maximal disease score of 0.59, whereas most of the wild-type B6 mice (84.2%) developed the disease and reached maximal severity at a disease score of 0.53.0 (mean maximal disease score = 1.32; Fig. 4). Histopathologic examination revealed an even milder degree of cellular infiltration and myelin degradation in the brains and spinal cords of CIITA-Tg mice than in littermates (Fig. 5).

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Fig. 4. Low severity and incidence of Th1-dependent autoimmune disease in the CIITA-Tg mice. EAE was induced in wild-type (WT) and CIITA-Tg (Tg) mice by immunization with MOG3555 as described in Methods. Data are pooled from four independent experiments and clinical scores (mean ± SEM) are plotted as a function of days after immunization, with disease incidence indicated in parentheses (A). Representative plot of maximum disease scores for individual mice is shown (B). Statistical analysis of cumulative disease incidence (Fisher's exact probability) and maximum disease scores (MannWhitney rank-sum test) revealed the difference between the CIITA-Tg and littermates (P < 0.05).
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Fig. 5. Photomicrographs of histopathology of central nervous system stained with Luxol fast bluehematoxylin and eosin (x200). Representative fields from the lumbar spinal cord of wild-type (A) and CIITA-Tg mice (B) from day 30 after immunization. There are many mononuclear inflammatory cells infiltrating the parenchyma, and destruction of myelin sheath in control mice (A: disease score 3), while small inflammatory focus and well-preserved myelin sheath are detected in CIITA Tg mice (B: disease score 0.5).
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To investigate whether the reduced severity and low incidence of EAE was correlated with the suppression of Th1 immune reaction in the CIITA-Tg mice, regional lymph node CD4+ T cells from the mice sensitized with MOG3555 peptide and M. tuberculosis were restimulated in vitro and then tested for their secretion of INF-
and IL-4. CD4+ T cells from immunized CIITA-Tg mice and control mice proliferated at a similar level after restimulation with MOG3555 peptide, PPD, or anti-CD3 mAb as shown by the overlapping [3H]thymidine incorporation curve (Fig. 6A). However, the production of IFN-
in CIITA-Tg CD4+ T cells was significantly lower (49% decrease) than in control cells, and the CIITA-Tg CD4+ T cells produced slightly more IL-4 after restimulation than did normal CD4+ T cells. This finding suggests that the resistance of CIITA-Tg mice to the development of EAE was due to compromised Th1 differentiation and IFN-
secretion in CIITA-Tg CD4+ T cells.

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Fig. 6. Repressed development into Th1 immunity is the basis for the resistance of CIITA-Tg mice to induction of EAE. Fourteen days after immunization with 200 µg of MOG3555 in IFA and M. tuberculosis, CD4+ T cells from draining lymph nodes of the immunized CIITA-Tg (Tg) or control wild-type (WT) mice were restimulated in vitro with a titration of MOG3555, PPD (1 µg/ml) or anti-CD3 mAb (2C11; 10 µg/ml) and APCs. Proliferation was measured by [3H]thymidine incorporation (A), and cytokine secretion was determined by ELISA after 3 day culture in vitro (B). The data represent mean ± SEM in one representative experiment of three. *P < 0.05.
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Discussion
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CIITA is a key regulator for the expression of MHC class II genes (2,3). However, a number of studies have suggested its roles in Th differentiation and activation-induced cell death (9,10,24,25), extending its biological function to more than that related to MHC class II expression. Taking advantage of the CIITA-Tg mice developed in this study, we were able to analyze the effect of CIITA on Th differentiation per se and show that forced expression of CIITA in T cells suppressed Th1 differentiation and encouraged Th2 differentiation in vitro. The biological relevance of this in vitro result could be confirmed by the resistance of CIITA-Tg mice to the development of Th1-dependent autoimmune disease, EAE.
The function of CIITA in Th differentiation was first demonstrated by the observation of increased generation of IL-4-secreting CD4+ T cells in CIITA/ I-E-transgenic mice (10). A transfection study done by the same group has also shown that CIITA is capable of repressing the IL-4 promoter activity in T cell line, implying its inductive role in Th1 differentiation (9). These data predict that overexpression of CIITA in CD4+ T cells leads to an enhanced Th1 differentiation of the CIITA-Tg cells upon activation (10). However, the present data were in sharp contrast with this prediction, in that CIITA expression restrained activated CD4+ T cells from producing IFN-
under Th1 condition in vitro and Th1 immunity in the CIITA Tg mice, but encouraged IL-4 production under Th2 condition. A more recent study has shown that ectopic expression of CIITA in all types of cells in the body rendered the CIITA transgenic CD4+ T cells to differentiate preferentially into IL-4 secreting Th2-type cells (11), which is consistent with our results. It is not possible, at present, to make a clear explanation for these contradictory roles of CIITA in Th differentiation. It can only be suggested that the mechanism for the regulation of cytokine secretion by CIITA in terms of Th1/Th2 balance might be more complicated than the simple sequestration of CBP/p300 by CIITA from NF-AT binding (9). This idea of complexity of Th1/Th2 balance is supported by the studies using NF-AT1 knockout mice (2628), in which the early transcription of IL-4 gene was suppressed (26) but the production of IL-4 and Th2 bias were enhanced in the long run (2628). Moreover, it has been shown that NF-AT might positively regulate not only IL-4 but also IFN-
gene transcription, and thus NF-AT/ T cells showed marked impairment of IFN-
production even in IL-4/ background (29). Therefore, the understanding of the mechanism for Th1/Th2 balance in vivo might take one or two additional factors into consideration such as temporal and quantitative aspects of CIITA expression, as Chang et al. suggested the importance of proper expression of CIITA for the maintenance of proper Th differentiation (30).
Otten et al. (11) raised another possibility that Th2 bias observed in their system where CIITA was expressed in all types of cells and organs in the body might be from an indirect consequence of the interaction between TCRs on CD4+ cells and MHC/peptides on the cells that ectopically express CIITA. This proposition might also be an adequate explanation for the Th2 bias and repressed Th1 response observed in our system, where expression of CIITA, thereby surface expression of MHC class II, is almost completely restricted to T cells and professional APCs. This idea led us to speculate that Th2 bias observed in these two independent studies might be a result of putative TT interaction through TCRMHC present on T cells. As Otten et al. pointed out (30), since CIITA controls the expression of other accessory molecules required for functional MHC class II presentation, reconstitution of CIITA-deficient B6 mice just with transgenic I-E MHC class II is unable to recapitulate the antigen presentation by non-conventional APCs including T cells, but is still expected to have general defects in MHC class II restricted antigen presentation. On this basis, consistent Th2 bias observed in the I-E-reconstituted CIITA deficient mice (10,30) does not serve as evidence against our hypothesis. To resolve the conflicts over the key player, ectopic CIITA expression itself or deregulated MHC class II expression by non-professional APCs, for the Th2 bias in CIITA deficient and CIITA transgenic systems, requires other animal models such as a mouse system expressing our CIITA transgene on MHC class II deficient background, focus thereby being restricted to the action mechanism of CIITA on the regulation of Th1/Th2 development.
Previous studies on the function of CIITA in peripheral CD4+ T cells using CIITA knock-out and CIITA-transgenic mice focused on the Th2 bias in vitro (10,11). In our system we rather focused more on the repression of Th1 immunity. IFN-
production from CIITA-Tg CD4+ T cells was still decreased in Th1-polarizing condition in which IL-4 was neutralized by addition of anti-IL-4 antibody (Fig. 2C). In addition, the CIITA-Tg CD4+ T cells stimulated with OVA323339 (Fig. 3B), MOG3555, or PPD (Fig. 6B) produced lower levels of IFN-
(2000
15 000 pg/ml differences) than the control CD4+ T cells, even when their IL-4 secretion was minimal (<20 pg/ml). Thus, these findings suggest that the reduced incidence/severity of EAE in CIITA-Tg mice may be the result of the suppressed Th1 immunity in CIITA-Tg mice. However, in addition to the fact that Th1 and Th2 responses are mutually regulated (19,31,32), the result that the secretion of IL-4, known to down-regulate the expression of IFN-
and possibly other Th1 cytokines (3233), was enhanced by CD4+ T cells from CIITA-Tg (Fig. 6B) does not exclude the possibility that the mild EAE severity observed in the CIITA-Tg mice in this study may be a result of enhanced Th2 response. We a lack molecular explanation of how CIITA-expression in T cells caused Th1/T2 imbalance in this study. Understanding of the mechanism for this phenomenon in CIITA-Tg mice needs molecular dissections on the Th-related gene expression and further evaluation of Th2-mediated immune reaction in the transgenic mice.
In conclusion, we suggest that the constitutive expression of CIITA in T cells inhibits IFN-
secretion and Th1 differentiation both in vitro and in vivo, and CIITA and thereby MHC class II expression by T cells deregulates Th1/Th2 balance during the course of T cell immune response.
 |
Acknowledgements
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This work was supported by a Korea Research Foundation Grant (2000-042-F00017) and grant for the Molecular Medicine Research Group Program (00-J-MM-01-B-02) from the Ministry of Science and Technology of Korea.
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Abbreviations
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APC | antigen-presenting cell |
B6 mice | C57BL/6 mice |
CBP | CREB binding protein |
CIITA | MHC class II transactivator |
dTg | double transgenic |
EAE | experimental autoimmune encephalomyelitis |
IFA | incomplete Freund's adjuvant |
PBS | phosphate-buffered saline |
PPD | purified protein derivative |
TCR | T cell receptor |
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Notes
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Transmitting editor: E. Simpson
Received 11 February 2004,
accepted 17 June 2004.
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