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Departments of
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Microbiology and Immunology, and
Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA 30322
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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- and 
-chains that associate with each
other and the invariant chain
(Ii)3 trimer in the
endoplasmic reticulum. This association prevents the binding of
endogenously synthesized peptides to the MHC class II molecule. The
class II Ii complex is transported to endosomes where the Ii is
degraded by proteolytic enzymes, leaving a small portion of Ii, termed
class II-associated Ii peptide bound to the peptide-binding cleft of
the MHC class II molecule. When Ag-containing endosomes fuse with the
lysosomes, the pH drops and the class II-associated Ii peptide is
replaced by antigenic peptides with higher affinities for the
MHC-binding cleft in a manner that is dependent on the activity of
HLA-DM. The peptide-MHC complex is then transported to the plasma
membrane.
HLA-DO, a nonclassical MHC class II-like protein encoded in the class
II region of the MHC, is composed of HLA-DO (formerly HLA-DN or
HLA-DZ; Refs. 2 and 3) and HLA-DO
(4, 5). HLA-DO differs from other components of the class
II Ag presentation pathway in that it is selectively expressed in B
cells and a subset of thymic epithelial cells, but not other
professional APCs (6, 7, 8, 9, 10). Its function remains poorly
understood and controversial (11). HLA-DO binds tightly to
HLA-DM in the endoplasmic reticulum and the complex remains associated
after transport to endosomal compartments (9). Several
studies have demonstrated that HLA-DO inhibits the peptide loading
function of HLA-DM, although DO-DM complexes may retain activity in the
highly acidic environment of the lysosome-related MHC class II
compartments in B cells (12, 13, 14, 15). However, it has also
been reported that HLA-DO enhances HLA-DM activity (16).
Currently, there is no information on whether HLA-DO expression is
modulated in B cells. Whatever its function, HLA-DO plays a role in
modulating peptide loading and Ag presentation by B cells.
The MHC class II isotypes (HLA-DR, -DQ, and -DP), HLA-DM, and Ii genes
have been shown to be regulated by a common set of transcription
factors that bind to a series of promoter proximal elements termed the
W, X1, X2, and Y boxes (reviewed in Ref. 17). These
factors include RFX, X2BP/CREB, NF-Y, and class II transactivator
(CIITA). RFX is composed of the subunits RFX5, RFXAP, and RFX-B/ANK
(18, 19, 20, 21), and was shown to bind as a complex to the X1 box
of the upstream enhancer element of these genes (22, 23).
X2BP/CREB binds to the X2 box in a cooperative manner with RFX
(23, 24). NF-Y also binds cooperatively to its sequence,
the Y box, which is located downstream of the X-box region sequences
(25). CIITA functions as a coactivator by interacting with
the above factors once they are bound to their respective DNA elements
(26, 27, 28). CIITA is expressed constitutively in
Ag-presenting cells and can be induced by IFN-
in other cell types
(29). Both RFX and CIITA are required for MHC class II and
HLA-DM gene expression (30, 31). Patients carrying
mutations in their genes for CIITA or any one of the RFX components
exhibit a SCID called the bare lymphocyte syndrome (BLS; reviewed in
Ref. 32). Unlike the other MHC class II-like genes,
regulation of the HLA-DO genes by CIITA has been controversial. In
previous reports, IFN- did not induce HLA-DO in non-Ag-presenting
cells, suggesting that CIITA does not play a role in its regulation
(4). However, a recent report suggests that the two chains
of HLA-DO, HLA-DO, and HLA-DO are differentially regulated
(33). HLA-DOB was shown to be regulated by RFX, but not
regulated by CIITA, while HLA-DOA was regulated by both factors.
While performing a search for novel genes regulated by CIITA, a comparison between the transcripts produced by the B cell line Raji (wild-type for CIITA) and its irradiated mutant daughter line, RJ2.2.5, which is mutant for CIITA, was performed. More than 12,000 cDNAs were compared. In this analysis, HLA-DOB was consistently found to be expressed at a higher level in Raji cells when compared with RJ2.2.5, suggesting a role of CIITA in its expression. Because this observation was inconsistent with the above reports, a detailed analysis of the role of CIITA in HLA-DOB expression was conducted. Quantitative RT-PCR, intracellular staining and flow cytometry, HLA-DOB promoter-reporter assays, and chromatin immunoprecipitations (ChIP) were performed to confirm the DNA microarray data. The combined data suggest that HLA-DOB is in fact regulated by CIITA, which can impact the overall level of HLA-DO expression and ultimately, Ag presentation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Raji, a Burkitt’s lymphoma-derived cell line, is wild-type for CIITA and positive for MHC class II gene expression (34). The human B cell line RJ2.2.5 was derived by mutagenesis from Raji cells and selected for loss of HLA class II Ag expression (35). RJ2.2.5 is deficient for CIITA (26, 36). The SJO cell line was derived from a patient with BLS and is defective for RFX5 (18). Raji and RJ2.2.5 were grown in RPMI supplemented with 5% FBS, 5% bovine calf serum, 2 mM glutamine, 5 U/ml penicillin, and 5 µg/ml streptomycin sulfate. The RFX5-deficient cell line was grown in F12-DMEM with 20% FBS and the supplements above. A431, an epithelial carcinoma cell line was grown in DMEM with 10% FBS and the above supplements.
cRNA preparation
Total RNA was isolated from cells by the Nonidet P-40 lysis method (37) or the RNeasy method (Qiagen, Valencia, CA). Total RNA (20 µg) was used directly for the cDNA synthesis using the Super Choice system for cDNA synthesis (Life Technologies, Grand Island, NY) with an oligo(dT) primer containing a T7 phage promoter sequence. The cDNA obtained was in vitro transcribed into cRNA using T7 RNA polymerase and bioarray high yield RNA transcript labeling reagents (Enzo Diagnostics, Farmingdale, NY) with biotinylated CTP and UTP. Biotin-labeled cRNA was purified using an RNeasy column, and fragmented at 95°C for 35 min in fragmentation buffer (40 mM Tris acetate (pH 8.1), 100 mM potassium acetate, 30 mM magnesium acetate). The quality of the cRNA was verified on an agarose gel before and after fragmentation. After fragmentation, the cRNA averaged 35–100 bp in length.
DNA microarray hybridization and analysis
Test DNA microarray chips from Affymetrix (Santa Clara, CA) were used to check the cRNA for equal hybridization to 5' and 3' oligonucleotides of housekeeping genes before each experiment. Four independent experiments were conducted. The initial set used the Affymetrix Hu6800 (A-D) human microarray chips. Experiments two through four used the Affymetrix human U95A chips. All microarrays were prehybridized in hybridization solution (100 mM MES, 885 mM NaCl, 20 mM EDTA, and 0.01% Tween 20) at 45°C for 15 min. Microarray chips were then hybridized with 200 µl of hybridization solution containing 0.05 µg/µl of fragmented cRNA spiked with control cRNA, and control oligonucleotide denatured at 95°C for 5 min 38 . The arrays were hybridized at 45°C for 16 h, washed, and stained using the EukGE-WS2 protocol on an Affymetrix fluidics station with 5 µg/µl streptavidin-PE (Molecular Probes, Eugene, OR) and acetylated BSA (2 mg/ml) in the above hybridization solution. For U95A chips, an additional amplification step was conducted after primary staining by the addition of a biotinylated anti-streptavidin Ab (3 µg/ml), goat IgG (0.1 mg/ml), and acetylated BSA (2 mg/ml) in hybridization buffer followed by washes and staining with streptavidin-PE as in the first step. Arrays were scanned on a Hewlett-Packard (Palo Alto, CA) gene array scanner and the data obtained were analyzed using software obtained through Affymetrix. The GENECHIP 3.2 software was used for HU6800 arrays and Microarray Suite 4.0 was used for the U95A arrays. The fluorescence intensities of all chips were normalized using a scaling factor of 2500 so that they could be compared with each other. The results obtained using the Microarray Suite 4.0 software were published into a database using the MICRODB software followed by comparative analysis using the DMT software (Affymetrix).
Real-time RT-PCR
Multiple RNA preparations using RNeasy method (Qiagen) were prepared for quantitative RT-PCR analysis. RNA (2 µg) was reverse transcribed using Superscript II RT (Life Technologies), and buffers from a RT-PCR kit (Applied Biosystems, Branchburg, NJ) according to the manufacturer’s directions. A total of 1/20 of the reverse transcription reaction was used for quantitative PCR in a reaction containing SYBR-green buffer, 5% DMSO, 1x SYBR (BioWhittaker Molecular Applications, Rockland, ME), 0.04% gelatin, 0.3% Tween, 50 mM KCl and 20 mM Tris (pH 8.3), 3 mM MgCl2, 0.2 mM dNTP, and 50 nM of each primer. A two-step PCR with denaturation at 95°C for 15 s and annealing and extension at 60°C for 1 min for 40 cycles was conducted in a BIO-RAD I-cycler (Bio-Rad, Hercules, CA). An additional set of PCR-using primers for the GAPDH transcripts was conducted to provide a normalization reference in the reactions. The primer sets used were: HLA-DOB, 5'-CTGTGGAGTGGAGAGCTCA and 5'-CAGCTCTTGAGACCCTCATTACC; HLA-DOA, 5'-CAGGCATTGGGAGCTCCAG and 5'-GGATCATTACCTGGGGACAC; and GAPDH, 5'-CCATGGGGAAGGTGAAGGTCGGAGTC and 5'-GGTGGTGCAGGAGGCATTGCTGATG. The HLA-DRA primer sets have been previously described (27). The threshold cycle values for the HLA-DO and HLA-DRA genes were normalized to the threshold cycle of GAPDH and converted to linear scale. All real-time RT-PCR were conducted at least three times from independent RNA preparations. The average of these experiments is presented relative to the levels in Raji cells.
Plasmid construction and transient transfection
Plasmids pHACIITA and pCIITA20.2 express wild-type and dominant-negative forms of CIITA, respectively (39, 40). pGL3-DRA was a generous gift from the lab of Dr. P. van den Elsen (Leiden University Leiden, The Netherlands) (41). A 317-bp fragment upstream of HLA-DOB gene transcription start site was PCR-amplified and inserted into KpnI/XhoI-digested pGL3 to create the HLA-DOB promoter-reporter pGL3-DOB. Primers used for the PCR of the HLA-DOB promoter sequence were 5'-CCCGGTACCGTCTGCCTAGTTCTTC and GCCCTCGAGTGAGTAGTAAAATCGTC. For transient transfections, 10 µg of the luciferase reporter vector (pGL3-DOB, pGL3-DRA, or pGL3), with or without 10 µg of pHACIITA or CIITA20.2, were transfected into Raji and RJ2.2.5 cells by electroporation, as described previously (42). A total of 1 µg of pTK-RL (Promega, Madison, WI), which expresses a Renilla luciferase gene, was cotransfected to normalize for transfection efficiency, and pUC18 was added to all transfection to bring the DNA concentration to 50 µg. Cells were harvested 24 h after transfection, washed, and lysed by three rounds of freezing and thawing. Luciferase activity was measured using the Dual Luciferase Assay kit (Promega).
Stable transfections
Stable cell lines expressing the pHACIITA expression plasmid
were created in RJ2.2.5, A431, and Jurkat cells. All transformed pools
were selected for 2–3 wk in G418. The RJ2.2.5-CIITA transformant pool
was stained for MHC class II proteins using the pan murine anti-MHC
class II Ab IVA12, and the positive cells were selected by binding to
anti-mouse magnetic beads (Dynabeads, Lake Success, NY). The cells
bound to beads were washed in PBS containing 5 mM EDTA and 1% BSA, and
the cells were cultured in selection medium until they detached from
the beads. A431 cells expressing wild-type CIITA were generated by
transfecting the cells with pHACIITA using Fugene (Roche Molecular
Biochemicals, Indianapolis, IN), followed by selection in medium
containing 1 mg/ml G418. The cells were checked for class II expression
3 wk later by flow cytometry. The Jurkat-CIITA line was transformed by
electroporation and selected on G418. The pool of cells was analyzed
after 3 wk of selection. The Jurkat-CIITA lines represent pools of
CIITA-expressing and -nonexpressing cells, as noted by their HLA-DR
expression levels (see text and Fig. 7
), whereas the majority of the
transformants of the A431 cells express HLA-DR (data not shown).
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protein expression by flow cytometry
Specific (HLA-DM-PE and HLA-DR-PerCP) and isotype-matched
negative-control Abs were purchased from BD PharMingen (Franklin Lakes,
NJ). Unlabeled anti-HLA-DO mAb, a gift from Dr. H. Kropshofer
(German Cancer Research Center, Heidelberg, Germany) (16),
was FITC-conjugated as described in Coligan et al. (43).
Intracellular staining was performed using Fix & Perm kit (Caltag
Laboratories, Burlingame, CA) following the manufacturer’s
instructions. Briefly, cells were washed with PBS/1% BSA and fixed
with medium A for 15 min at room temperature. Cells were then washed
twice with PBS/1% BSA and incubated in medium B containing HLA-DM or
HLA-DO Abs for 15 min at room temperature. Cells were then washed
three times and analyzed by flow cytometry in a FACSCalibur (BD
PharMingen).
ChIP
ChIP using CIITA and RFX5 Abs were conducted as described previously (24, 27). Primer pairs used to amplify HLA-DOB promoter were: 5'-ATTGGAAACTCCTCAGATTGACAACCA and 5'-TCTGCAGGCAAACAATGGTTGAGTTGTA. PCR products were amplified for 35 cycles and analyzed by agarose gel electrophoresis.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DNA microarrays were used to identify new genes that were differentially regulated between wild-type B cells and those deficient in the master regulator of MHC class II expression, CIITA. The cell lines chosen for this analysis were the two best-matched cell lines, Raji and RJ2.2.5. Raji cells are a B lymphoblastoid cell line derived from a patient with Burkitt lymphoma (34). RJ2.2.5 cells were derived directly from Raji cells by mutagenesis and selection for the loss of MHC class II genes (35). RJ2.2.5 cells are completely negative for MHC class II and HLA-DM expression due to the complete deletion of one CIITA allele and a large internal deletion of the other allele (26, 36). Four separate RNA samples were prepared from each of these cell lines. Initially, the HuFL6800 DNA microarray series was available and was used in one set of analyses. Following this initial screen, the U95A microarrays (Affymetrix) became available and were used instead due to their higher density and greater complexity of transcripts represented. Biotinylated cRNA prepared from RNA derived from both cell types were used for hybridization to the gene chips and the results were analyzed as described in Materials and Methods. HLA-DRA, -DRB, -DQA, -DMA and -DMB, and the Ii genes were found to be expressed at high levels in Raji and absent or low (background hybridization) in RJ2.2.5. These data and their complete analysis will be presented separately. Thus, all the MHC class II genes regulated by CIITA were identified.
Consistent with previous analyses, HLA-DOA showed a 2- to 10-fold
increase in Raji cells compared with RJ2.2.5 cells on the U95A chips
(Table I). However, due to previous
reports (33), it was surprising to find that HLA-DOB
transcripts were consistently 1.8- to 2.6-fold higher in Raji cells
compared with RJ2.2.5 cells (Table I). This result suggested that CIITA
may regulate HLA-DOB, and warranted further investigation.
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protein expression is very low in CIITA- and
RFX5-deficient cells
As a first step to study the expression of HLA-DO protein, the
level of HLA-DO protein was assayed in RJ2.2.5 cells. Intracellular
staining of HLA-DO and HLA-DM was performed in Raji, RJ2.2.5, and
the RFX5-deficient, BLS-derived cell line SJO. A previous analysis
found no HLA-DOA or HLA-DOB transcription in the absence of RFX5
(33). The stained cells were analyzed by flow cytometry.
In comparison to Raji cells, RJ2.2.5 showed very low levels of
HLA-DO (Fig. 1). SJO cells also
displayed very low levels of both HLA-DM and HLA-DO (Fig. 1). These
data suggest that both CIITA and RFX are required for the expression of
the heterodimer HLA-DO. However, this interpretation does not exclude
the possibility that the HLA-DO protein is unstable in the absence
of its heteromeric partner, HLA-DO, which is also absent in both
mutant cell lines.
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Previous studies comparing RJ2.2.5 and Raji cell lines for
HLA-DOB expression used Northern blots (4, 33). While
Northern blots have been a long-standing reference for RNA levels,
quantitative comparisons to a reference RNA is difficult, such that
small changes could be missed. To verify the DNA microarray data,
quantitative RT-PCR using real-time instrumentation was used.
Additionally, a pool of RJ2.2.5 cells stably transfected with a CIITA
expression vector (termed RJ2.2.5-CIITA cells) and selected for HLA-DR
surface expression were generated and analyzed to determine whether
CIITA could have a direct effect on expression of the HLA-DO
genes. RJ2.2.5-CIITA cells expressed about one-tenth the level of
surface HLA-DR as Raji cells (data not shown). Using the reverse
transcriptase reaction from RNA prepared from these three cell lines,
quantitative PCR were performed for HLA-DOB, HLA-DOA, HLA-DRA, and
GAPDH transcripts. The results were normalized to the levels of GAPDH.
Significant levels of HLA-DOB transcripts were expressed in RJ2.2.5
cells, but this level is 
5- to 6-fold less than in Raji (Fig. 2). HLA-DOA transcripts were also
detected in RJ2.2.5 cells, although they were close to background. In
contrast, HLA-DRA expression was absent in RJ2.2.5 cells. The analysis
of the RJ2.2.5-CIITA cell line showed a 3-fold increase in the level of
HLA-DOB mRNA over that observed in RJ2.2.5 cells. Although not fully
reverted to their levels in Raji cells, both HLA-DOA and HLA-DRA mRNAs
were also substantially increased over their RJ2.2.5 levels. The level
of HLA-DRA in this stable cell line, though high, was still 10-fold
less than in Raji cells. This level of HLA-DRA mRNA is in agreement
with the level of HLA-DR expressed on the surface of this cell line
(data not shown), and may reflect the overall level of CIITA expressed
in the transfected cells. Real-time RT-PCR using RNA from SJO indicates
complete absence of HLA-DOB transcripts (data not shown). These
findings indicate that mRNA transcripts for HLA-DOB are present in
RJ2.2.5 (CIITA-/-) cells, but are up-regulated in cells
that contain CIITA. Because these results were in disagreement with
previously published Northern blot analysis (4, 33),
several stocks of RJ2.2.5 cells were analyzed with similar results.
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Analysis of 5' upstream sequence of the HLA-DOB promoter reveals a
potential X-Y box-like region. This sequence is very similar to other
class II elements (Fig. 3). However, it
has been shown previously that a 250-bp fragment of HLA-DOB promoter
was not sufficient to confer activation of a reporter gene
(44). To test whether this sequence was functionally
active in a more sensitive luciferase reporter assay, a 317-bp fragment
spanning the WXY box of the HLA-DOB promoter was cloned upstream of a
luciferase reporter gene, and the resulting construct termed pGL3-DOB.
pGL3-DOB was transiently transfected into both Raji and RJ2.2.5 cells,
and the expression of the reporter assayed. The relative activity of
the reporter was 12-fold decreased in RJ2.2.5 in comparison to Raji
(Fig. 4). The pGL3-DRA reporter,
containing the luciferase gene cloned downstream to HLA-DRA promoter,
was used as a positive control. The pGL3-DRA reporter expressed 50-fold
higher in Raji cells in comparison to RJ2.2.5 cells.
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). The
dominant-negative CIITA20.2 mutant did not have any effect on the
pGL3-DOB promoter activity, suggesting that CIITA is functioning as a
coactivator in this system. An HLA-DRA promoter reporter was assayed in
parallel and serves as a control for the system. Its response to CIITA
and CIITA20.2 were as previously published (45).
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To obtain evidence that CIITA interacts directly with the HLA-DOB
promoter in vivo, a ChIP assay was conducted in Raji cells using an Ab
directed against CIITA. Through the cross-linking of live cells with
formaldehyde, ChIP assays provide direct evidence for the association
of a factor with a given DNA sequence in vivo (46, 47).
Immunoprecipitation of CIITA- and RFX5-containing chromatin using
formaldehyde-fixed Raji cell lysates, followed by PCR, revealed
specific bands for the HLA-DOB promoter for both Abs (Fig. 6). Thus, both RFX5 and CIITA can be
found at the HLA-DOB promoter in vivo and demonstrate that CIITA can or
does play a role in the regulation of the HLA-DOB gene.
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The above results indicate that CIITA can bind to the HLA-DOB
promoter and increase its expression in B cells. To show that this
interaction results in the expression of HLA-DO protein,
RJ2.2.5-CIITA cells were analyzed for their level of HLA-DO
expression by intracellular staining and flow cytometry (Fig. 7
A). The results showed
increased levels of both HLA-DM and DO in the RJ2.2.5-CIITA cell
line compared with its parental control. To address the role of CIITA
in non-B cells, CIITA expressing non-APC lines were generated from
A431, an epithelial carcinoma cell line, and the T cell line Jurkat.
Surface expression and quantitative RT-PCR showed that HLA-DRA
expression in the A431-CIITA transfectants was 10-fold higher than in
the parental control cells treated with IFN- (data not shown).
Intracellular staining of HLA-DM and HLA-DOB in A431-CIITA cells showed
high levels of HLA-DM expression, but no HLA-DOB (Fig. 7B). Similarly, the Jurkat-CIITA transformant pool
showed significant HLA-DR expression, but no HLA-DO expression (Fig. 7C). These data demonstrate that CIITA augments HLA-DOB
expression only in B cells. These findings suggest the presence of an
additional tissue-specific factor for HLA-DOB expression in B
cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This finding was both expected and unexpected. It was expected because
the heterodimeric partner of the HLA-DO protein, HLA-DO, was
shown by others to be regulated by both RFX and CIITA
(33). Moreover, an analysis of the upstream region of the
HLA-DOB gene revealed the presence of an X-Y box regulatory sequence. X
and Y box regulatory sequences are found 5' to all MHC class II genes
and serve as the direct binding sites for RFX, X2BP/CREB, and NF-Y.
CIITA is known to interact with the DNA-bound factors
(26, 27, 28), suggesting that CIITA should regulate HLA-DOB.
The current finding was unexpected due to previous reports in which
Northern blots showed little if any difference between the levels of
HLA-DOB in RJ2.2.5 cells when compared with Raji cells (4, 33). However, these Northern blots were not intended to be
absolutely quantitative, as a dilution series of the RNA samples was
not performed, nor were the blots quantitated by other means. Thus, the
relative amounts of the HLA-DOB transcripts, when compared with the
loading control, could not be quantitatively determined. Complicating
the issue is the fact that HLA-DOB transcripts are present in RJ2.2.5
cells. Additionally, the induction of CIITA by IFN- in professional
and nonprofessional APCs (non-B cells) did not lead to an induction of
HLA-DO (4), suggesting that the HLA-DO genes were
regulated differently.
Because HLA-DOB mRNA is expressed in RJ2.2.5 cells in the absence of
CIITA, this suggests that other factors and mechanisms play a role in
controlling its expression. This notion is consistent with all the
previous data and the observation that HLA-DO expression is limited to
B cells and not to professional APCs. The analysis of intracellular
HLA-DO protein levels in Raji and RJ2.2.5 showed a clear difference.
This difference could be caused by several mechanisms. The first is
that while HLA-DOB mRNA is present in RJ2.2.5 cells, HLA-DOA mRNA
levels are very low. If the encoded proteins are expressed at the
mRNA-represented levels, then it is very possible that HLA-DO
proteins are degraded in the absence of their HLA-DO partner. The
role of CIITA here would be to increase expression of both HLA-DOA and
HLA-DOB mRNA such that maximal expression of HLA-DO could occur.
Second, because HLA-DO transport to lysosomal vesicles and to the
peptide loading compartment, the MIIC is dependent on its association
with HLA-DM (9). It is possible that HLA-DOB does not
accumulate in cells unless HLA-DM is present. This would be the case in
RJ2.2.5, where HLA-DM is absent.
The HLA-DO genes are unusual in that their expression is mostly
restricted to B cells and to thymic epithelial cells. This includes the
possibility that HLA-DOB is controlled by factors that are specific to
B cells and thymic epithelial cells, and that such factors are not
found in other APC types. This suggestion would provide an explanation
for the lack of HLA-DOB in non-B cells exposed to IFN-, and also for
the absence of HLA-DOB expression in non-APCs in the presence of
transfected CIITA, as shown here. It has been proposed by Cresswell and
colleagues (12) that nonprofessional APCs cannot express
HLA-DO in the presence of IFN- so that they can achieve their
maximal Ag-processing capacity. Furthermore, the differential
regulation of HLA-DM and HLA-DO levels by CIITA was suggested as a
mechanism for APC and non-APCs to avoid HLA-DO inhibitory activity
(48). However, this is more likely to be attributed to the
tissue-specific factor controlling HLA-DO expression than to CIITA.
Although CIITA can augment HLA-DO expression in B cells, it cannot
overcome the need for the additional tissue-specific factor in
non-APCs. Thus, in B cells, a tissue-specific factor and CIITA together
drive HLA-DOB and HLA-DOA expression, thereby regulating the function
of HLA-DM. The B cell is a unique Ag-presenting cell, as it presents Ag
only once in its lifetime. Hence, the necessity to tightly control Ag
processing and presentation may reside with HLA-DO. The enhancement of
the expression of both HLA-DOB and -DOA by CIITA may provide B cells
the greatest opportunity to control Ag presentation. In contrast to the
partial dependence of HLA-DO genes on CIITA, it appears that these
promoters are completely dependent on RFX. For HLA-DOB, RFX may play a
role in promoting the binding of the tissue-specific factor to the
HLA-DOB regulatory region.
The role of CIITA in HLA-DOB expression may also be considered similar to CIITA’s role in MHC class I expression. MHC class I expression has been shown to be induced by, but not dependent on, CIITA for its expression (49, 50). The HLA-DO genes appear to have evolved more recently from class II MHC genes (reviewed in Refs. 11 and 51). It is likely that the HLA-DOB gene has diverged further with additional elements in its promoter to allow B cell/thymus-specific expression.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jeremy M. Boss, Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 30322. E-mail address: boss{at}microbio.emory.edu
3 Abbreviations used in this paper: Ii, invariant chain; BLS, bare lymphocyte syndrome; ChIP, chromatin immunoprecipitation; CIITA, class II transactivator.
Received for publication August 28, 2001. Accepted for publication December 4, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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chain gene DZ is distinct from genes in the DP, DQ and DR subregions. EMBO J. 4:2231.[Medline]
: a new chain gene in HLA-D with a distinct regulation of expression. EMBO J. 4:2839.[Medline]
chain gene of H-2O has an unexpected location in the major histocompatibility complex. J. Exp. Med. 176:477. mediated by the transactivator gene CIITA. Science 265:106./DO heterodimer. J. Immunol. 165:1410.2-microglobulin genes. Immunity 9:531.[Medline]
-induced MHC class I transactivation: the ISRE-mediate route and a novel pathway involving CIITA. Immunity 6:601.[Medline]
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G. H. Boekhoudt, Z. Guo, G. W. Beresford, and J. M. Boss Communication Between NF-{kappa}B and Sp1 Controls Histone Acetylation Within the Proximal Promoter of the Monocyte Chemoattractant Protein 1 Gene J. Immunol., April 15, 2003; 170(8): 4139 - 4147. [Abstract] [Full Text] [PDF]
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U. M. Nagarajan, A. Bushey, and J. M. Boss Modulation of Gene Expression by the MHC Class II Transactivator J. Immunol., November 1, 2002; 169(9): 5078 - 5088. [Abstract] [Full Text] [PDF]
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