Original Article

Genes and Immunity (2012) 13, 299–310; doi:10.1038/gene.2011.86; published online 5 January 2012

CIITA promoter I CARD-deficient mice express functional MHC class II genes in myeloid and lymphoid compartments

W M Zinzow-Kramer1, A B Long2, B A Youngblood1,3, K M Rosenthal1, R Butler1, A-U-R Mohammed3, I Skountzou1,3, R Ahmed1,3, B D Evavold1 and J M Boss1,3

  1. 1Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA, USA
  2. 2Department of Human Genetics, Emory University School of Medicine, Atlanta, GA, USA
  3. 3Emory Vaccine Center, Emory University School of Medicine, Atlanta, GA, USA

Correspondence: Dr JM Boss, Department of Microbiology and Immunology, Emory Vaccine Center, Emory University School of Medicine, Atlanta, GA 30322, USA. E-mail: jmboss@emory.edu

Received 26 September 2011; Revised 23 November 2011; Accepted 28 November 2011
Advance online publication 5 January 2012

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Abstract

Three distinct promoters control the master regulator of major histocompatibility complex (MHC) class II expression, class II transactivator (CIITA), in a cell type-specific manner. Promoter I (pI) CIITA, expressed primarily by dendritic cells (DCs) and macrophages, expresses a unique isoform that contains a caspase-recruitment domain (CARD). The activity and function of this isoform are not understood, but are believed to enhance the function of CIITA in antigen-presenting cells. To determine whether isoform I of CIITA has specific functions, CIITA mutant mice were created in which isoform I was replaced with isoform III sequences. Mice in which pI and the CARD-encoding exon were deleted were also created. No defect in the formation of CD4 T cells, the ability to respond to a model antigen or bacterial or viral challenge was observed in mice lacking CIITA isoform I. Although CIITA and MHC-II expression was decreased in splenic DCs, pI knockout animals expressed CIITA from downstream promoters, suggesting that control of pI activity is mediated by unknown distal elements that could act at pIII, the B-cell promoter. Thus, no critical function is linked to the CARD domain of CIITA isoform I with respect to basic immune system development, function and challenge.

Keywords:

MHC class II; gene expression; CIITA; antigen presentation

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Introduction

The major histocompatibility complex class II (MHC-II) region encodes genes required for the presentation of antigenic peptide to CD4 T cells.1 The process results in various immune responses that range from direct effector responses to those that are regulatory. Constitutive expression of MHC-II is restricted to antigen-presenting cells (macrophages, B cells and dendritic cells (DCs)) and thymic epithelial cells. MHC-II expression can also be induced in most cell types by exposure to interferon-γ (IFN-γ). The expression of MHC-II must be tightly regulated, as aberrant expression can result in immune system dysfunctions, including autoimmunity, increased susceptibility to cancer and decreased resistance to infectious organisms.2, 3, 4 Loss of MHC-II gene expression results in severe immunodeficiency, as evidenced by patients with bare lymphocyte syndrome, a disease resulting from mutations within transcription factors that regulate MHC-II expression.5, 6 One bare lymphocyte syndrome complementation group was found to be deficient for the class II transactivator (CIITA), a factor that is essential for MHC-II expression.7

CIITA functions as a transcriptional coactivator interacting with other MHC-II-specific transcription factors bound to regions upstream of each MHC-II gene (reviewed in the study by Choi et al.8). The recruitment of CIITA to MHC-II promoters orchestrates a set of chromatin modifications and rearrangements that are associated with and are required for MHC-II expression. The gene encoding human CIITA contains four promoters, three of which are conserved in mice (Ciita). Ciita promoters function in a cell type-specific manner.9 Ciita promoter I (pI) is used by DCs and macrophages exposed to IFN-γ,10 and seems to be myeloid specific. Ciita pIII is expressed in cells of the lymphoid lineage (B cells, human T cells and plasmacytoid DCs (pDCs)), whereas pIV is primarily expressed in non-hematopoietic cells on exposure to IFN-γ.11 Each of the Ciita promoters contains a unique first exon, which splices into a common second exon, resulting in three distinct CIITA isoforms. Isoform I, derived from pI, is particularly intriguing because its unique exon encodes an N-terminal domain of 93 aa that bears homology to a caspase-recruitment domain (CARD).12 Such domains have been shown for other proteins to be important for protein–protein interactions.13, 14 The presence of the CARD domain in addition to other domains led to CIITA being the cardinal member of the family known as nucleotide-binding domain and leucine-rich repeat-containing (NLR) proteins.15, 16, 17 This family of proteins is related to disease resistance R genes in plants, and a number of NLR family members have functions in pathogen sensing, inflammation, cell signaling and cell death.15, 18, 19 Increasing evidence suggests that many NLRs are cytoplasmic pathogen-recognition receptors, activating immune responses to intracellular pathogens.19 Despite being a member of the NLR family, to date, no function outside transcriptional activation has been ascribed to CIITA. Previous studies that address cell type-specific function of CIITA have focused on promoters III and IV using a knockout (KO) strategy to create mice lacking either pIV or both pIII and pIV.11, 20 Using the Ciita pIV-targeted KO mouse, it was observed that cells of a non-hematopoietic lineage, but not macrophages or microglia, lost the ability to induce Ciita after exposure to IFN-γ, demonstrating a need for pIV in the expression of Ciita in non-bone marrow-derived cells.11 In addition, positive selection of CD4 T cells was severely impaired because of the loss of expression of MHC-II on cortical thymic epithelial cells, although MHC-II expression on cells of the thymic medulla was unchanged.11, 21 A deletion of the regions encompassing both pIII and pIV displayed all of the phenotypes observed in the pIV KO, and in addition, resulted in loss of MHC-II expression from B cells and pDCs, whereas conventional DCs (cDCs) and macrophages induced with IFN-γ retained MHC-II expression.20 These data point towards the necessity of pI for expression of CIITA and MHC-II in cells of the myeloid lineage.

To address a role for pI and the CARD-containing isoform in regulating CIITA expression and activity, a set of mice were constructed that replaced isoform I of CIITA with the 17-aa exon of isoform III. Effectively, this CIITApI right arrow III knock-in (KI) was designed to create a mouse that would express isoform III CIITA from pI and pIII. Using flippase (FLP) mediated recombination, an additional mouse line was created in which pI and its surrounding upstream and downstream DNA were deleted, creating a CIITApI right arrow 0 KO. Thus, two novel Ciita mouse lines were created. These mice were extensively characterized for their ability to express Ciita and MHC-II gene products and response to pathogen challenge. The results showed that KI mice expressed MHC-II at levels comparable to wild-type (WT) mice. Surprisingly, KO mice still retained Ciita expression in all cell types examined, including splenic DCs (spDCs), which typically use pI nearly exclusively. This was due to redirection of transcript initiation from pI to pIII. Thus, both KI and KO mice lack isoform I CIITA and instead express isoform III in the myeloid and lymphoid compartments in which CIITA isoform I is normally expressed. T-cell development and activation seemed normal in both KI and KO mice. KO mice were also able to mount normal immune responses to Listeria monocytogenes and lymphocytic choriomeningitis virus (LCMV) infection and rechallenge, suggesting that the isoform I CIITA does not provide an advantage in these settings. Taken together, these data demonstrate that pI of CIITA and its corresponding CARD-containing isoform are not required for proper immune system development or function, and suggest that isoform III is capable of substituting for isoform I.

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Results

Generation of Ciita pI isoform III KI and Ciita pI KO mice

To examine the function of the CARD domain-containing pI isoform in vivo, a targeting vector that replaced the first exon of Ciita's isoform I with isoform III's first exon was created using a gene targeting strategy that used both Cre-loxP and FLP-frt-recombination target site mediated recombination. To maximize the utility of this animal, the replaced exon/isoform segment was flanked by FRTs, which would allow deletion of the entire pI promoter and its first exon in vivo. This includes 298bp upstream of the transcription start site through 145bp downstream of pI exon 1 for a total of 798bp. Thus, the targeting vector contained a 2.5-kb fragment upstream of Ciita pI; an HA-tagged pIII exon 1 coding sequence under control of the endogenous isoform I promoter and flanked by FRT sites; a lox-P-flanked neomycin-resistance cassette (neo); and a 2.5-kb fragment of Ciita pI intron 1 (Figure 1a). Homologous recombination was carried out in 129SvEv embryonic stem (ES) cells. Correctly targeted ES cells were injected into C57BL/6 blastocysts and three chimeric males were obtained. These chimeras were crossed to C57BL/6J females, and the agouti F1 heterozygote offspring were examined for transmission of the targeted allele by PCR. To delete the neo cassette, mice containing the targeted allele were crossed to EIIa-Cre mice (The Jackson Laboratory, Bar Harbor, ME, USA). Offspring of these crosses were analyzed by PCR for transmission of the targeted allele and for Cre-mediated deletion of the floxed DNA (Figure 1b). Mice carrying the Cre-deleted locus were then backcrossed to C57BL/6J to generate CIITApI right arrow III (KI) mice or crossed to ACT-FLPe mice (The Jackson Laboratory) to delete pI. Offspring of these crosses were analyzed by PCR for FLP-mediated deletion of the DNA between the FRT sites (Figure 1). Mice carrying the Cre/FLP-deleted locus were then crossed to C57BL/6J to generate CIITApI right arrow 0 (KO) mice. An example of the PCR assays used to genotype the mice are shown in Figure 1b.

Figure 1.
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Ciita targeting construct. (a) Schematic of the targeting construct used to generate CiitapI right arrow III (KI) and CiitapI right arrow 0 (KO). Transgene positive mice were bred to a Cre-expressing mouse to delete the neo selective marker. KI mice were further crossed to a Flp-expressing mouse to generate the KO line. (b) PCR genotyping results for transgene positive mice, KI and KO lines.

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Speed congenic backcrosses to C57BL/6J females using the male carrying the largest number of C57BL/6J loci were carried out for four generations. Using this speed congenic strategy, only four to five backcrosses are required to obtain a line that is 98–99% C57BL/6J.22 After two backcrosses, at least three markers on each chromosome were of the C57BL/6J origin and the CIITA containing chromosome 16 was at least partially C57BL/6J (data not shown). Mice backcrossed to C57BL/6J for four to six generations were used in this study.

CIITA-targeted mice are not defective in MHC class II expression

As stated above, Ciita is expressed from three promoters in mice. PI has been shown to be specific to cells of the myeloid lineage and specific DC compartments. To determine whether Ciita-targeted mice showed defects in the expression of MHC-II genes, the levels of I-Ab were examined by flow cytometry on thioglycolate-elicited macrophages treated with IFN-γ (Figure 2a) and spDCs (Figures 2b and c). For each strain, WT control mice were compared with either their KI or KO littermates. No difference in MHC-II expression was observed between KO and KI macrophages (CD11b+) compared with littermate controls.

Figure 2.
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MHC-II surface expression on KI and KO antigen-presenting cells is similar to WT. (a) Histogram analysis of flow-cytometry data for I-Ab expression in KI and KO samples compared with WT littermate controls. Thioglycolate-elicited peritoneal macrophages were stimulated in vitro 24h with 500μml IFN-γ. Live cells were gated on by side scatter properties; macrophages were identified as CD11b+. (b) Flt3L-elicited, CD11c+-sorted spleen cells were stimulated with 1μgml LPS for 8h to induce translocation of MHC-II molecules to the surface and analyzed by flow cytometry. Total dendritic cells were identified as CD11c+ and further divided into CD45RA+CD11cint plasmacytoid (pDC) and conventional DCs (cDCs). cDCs were divided into CD11b+ myeloid (myeDC) and CD8a+ lymphoid (lymDC) sub-populations. (c) Dendritic cell subsets identified in panel b were analyzed by flow cytometry for I-Ab expression. The data generated for this figure are representative of four mice for each strain. In panels a and c, WT littermate controls are shown overlaid on KI and KO samples. In panel b, WT littermate controls are shown directly above KI and KO samples.

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Splenic DCs were separated into two classes: conventional and pDCs (CD11c+ or CD11cdim, CD45RA+, respectively). cDCs were further divided into myeloid (CD11b+) and lymphoid (CD8a+) compartments23 (Figure 2b) and analyzed for MHC-II surface expression (Figure 2c). In addition, fluorescence intensity was calculated in molecules of soluble fluorochrome for each subset of DCs and the values were compared between groups of mice (data not shown). In cases in which two MHC-II peaks were observed, each peak was quantitated separately (Figure 2c). Unlike cDCs, pDCs use Ciita pIII;20 therefore, it was expected that pI mutations would not affect Ciita expression in this subclass of cells. Indeed, neither KI nor KO strains exhibited changes in MHC-II surface expression from their littermate controls in pDC (Figure 2c). For KI animals, this was also true of the myeloid and lymphoid DC populations. However, the myeloid and lymphoid populations from KO cells consistently displayed MHC-II at levels that were 70–76% of that seen on WT (Figure 2c).

MHC-II expression is correlated with Ciita expression in targeted mice

The expression of MHC-II on the surfaces of lymphoid and myeloid DC subsets, as well as on macrophages in the KO strain was unexpected as 798bp surrounding and including pI was deleted and this promoter was reported to control the expression of Ciita in cells derived from the myeloid lineage.20 Isoform-specific quantitative real-time RT-PCR (qRT-PCR) using WT mice confirmed that the major Ciita transcript expressed in spDCs and macrophages treated with IFN-γ was isoform I (Figure 3a). Isoform III was expressed at significant levels in spDC but not in macrophages, whereas some isoform IV was detected in macrophages, but only after treatment with IFN-γ (Figure 3a).

Figure 3.
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Ciita isoform I is not required for expression of CIITA or MHC-II in cells of the myeloid lineage. Ciita isoform-specific, total Ciita mRNA or I-Aα mRNAs were quantified in Flt3L-elicited splenic DCs (left panels) or thioglycolate-elicited peritoneal macrophages (+IFN-γ) (right panels) were analyzed by qRT-PCR with the primer sets indicated. Flt3L-elicited spDCs were purified with CD11c+MACs beads and were >95% CD11c+. Thioglycolate-elicited, peritoneal cavity macrophages were purified by adherence to tissue culture plates and were >95% CD11b+. (a) Isoform-specific PCR of CIITA in WT splenic dendritic cells (spDCs) and macrophages treated for 24h with IFN-γ. (b) Total Ciita mRNA levels in KI, KO and WT littermate controls. (c) I-Aα mRNA levels in KI, KO and WT (littermate controls) spDCs or macrophages treated with IFN-γ. (d) Isoform-specific expression of Ciita. HA-III represents primers specific for the knock-in allele, HA-tagged isoform III derived from promoter I. Data were averaged from four mice for each genotype, with each representing one biological replicate. Significance was calculated using Student's t-test. *P<0.05, **P<0.005.

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To investigate the transcriptional basis for cell surface MHC-II gene expression observed on the KI and KO spDC and macrophage cell populations presented in Figure 2, RNA was also isolated from these samples to perform analyses of Ciita transcripts by qRT-PCR. Cell purity was >95% as measured by CD11c expression on spDC and CD11b expression on macrophages (Figure 2b and data not shown). Except in the case of KO spDC, in which Ciita expression was reduced by 30%, Ciita levels were similar in both WT and mutant cells (Figure 3b). This correlated with the observed decrease in surface MHC-II expression observed only on cDC from the KO line (Figure 2c). I-Aα mRNA levels were also similar to WT, except in the case of KO spDC, in which I-Aα was decreased by 55% (Figure 3c, left panel).

Isoform-specific qRT-PCR was used to determine the origin of the Ciita transcripts that were being detected (Figure 3d). Again, WT cells principally use pI and produce isoform I Ciita transcripts. KI cells were found to express isoform III CIITA; however, this could be a combination of expression from both pI and pIII as the primers cannot distinguish between the two. Using a primer specific for the HA-tag confirmed that KI spDC and macrophages express the KI, HA-tagged-isoform III CIITA allele. Unexpectedly, spDC derived from KO cells expressed high levels of transcripts containing pIII exon 1, producing CIITA isoform III, and low, but detectable levels of isoform IV. Furthermore, in the case of KO-derived macrophages, CIITA isoform III, and isoform IV, were expressed at significant levels in response to IFN-γ (Figure 3d, right panel). These results suggest that there may be a change in promoter utilization in Ciita pI KO mice.

CIITA has been found to be responsible for regulation of multiple MHC-II-associated genes, including H2-D1, H2-DMa, H2-DMb, H2-DOa, H2-DOb and Ii,24, 25 as well as genes outside the MHC locus. ChIP (chromatin immunoprecipitation)–chip experiments on human dendritic and B cells identified the genes Kiaa0841, PSMD3, RAB4B, Tpp1 and TRIM26,24 and microarray analysis of human B cells25 and mouse DCs26 identified KPNA6, RAB4B and Plxna1 as additional potential targets of Ciita.24, 25 Additional genes potentially regulated by CIITA are Cste and Col1A1.27, 28 To determine whether the CARD-containing isoform of CIITA was differentially influential in the expression of these genes, qRT-PCR was carried out on RNA isolated from spDCs and macrophages treated with IFNγ. For the most part, no significant differences in expression for the above genes were observed between KO and WT macrophages or DCs (Supplementary Figures 1a and b). However, there were two exceptions. In concordance with the level of total CIITA mRNA expressed in KO-derived spDCs, Ii was reduced to a similar extent as I-Ab (Supplementary Figures 1a and Figure 3c). The second exception involved H2-DOb expression. In contrast to the other CIITA-regulated genes in which IFN-γ treatment of WT macrophages either had limited effect or induced their expression, IFN-γ treatment of WT macrophages reduced the levels of H2-DOb mRNA by ~7-fold (Supplementary Figure 1c). Intriguingly, H2-DOb expression only decreased by about half in IFN-γ treated KO-derived macrophages (Supplementary Figure 1c). As a point of reference, H2-Ob was expressed at very low levels in both stimulated and unstimulated macrophages, whereas the expression was much higher in spDCs (data not shown). This agrees with previous data that H2-Ob is expressed primarily by B cells, subsets of thymic epithelial cells and subsets of DCs, but not by monocytes or macrophages.29, 30 Therefore, for the most part, expression of non-MHC, as well as that of MHC genes regulated by CIITA, was not dependent on the CARD domain of CIITA for induction.

spDC use alternate promoters in the absence of pI

As cells from KO mice expressed transcripts that appeared to derive from promoters III and IV, 5′ RACE (rapid amplification of cDNA ends) was conducted to determine the transcriptional start sites of the CIITA transcripts in spDCs from KO and KI mice (Figure 4 and Supplementary Figure 2). Overall, 100% of the transcripts from WT spDCs originated at pI. In KI spDCs, 77, 15.9 and 6.8% initiated their Ciita transcripts from pI, pIII and pIV, respectively. The majority of transcripts in KO-derived spDCs were initiated in the proximity of pIII (96.3%), with the remaining transcripts being derived from pIV. Thus, spDCs derived from the KO mouse have switched the utilization of their Ciita promoter from pI to pIII.

Figure 4.
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5′RACE maps Ciita expression to promoters III and IV in KI and KO spDC. 5′RACE was carried out on mRNA from WT, KI and KO splenic DCs. Wild-type spDC Ciita transcripts initiated from pI, exclusively. Both KI and KO spDC expressed Ciita transcripts from promoters III and IV. The majority of Ciita transcripts in KI cells were the HA-tagged isoform III, and were expressed from promoter I (HA-pI). N refers to the number of cloned sequences analyzed.

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CIITA isoform I is dispensable for proper T-cell development

Positive and negative thymic selection of the naive T-cell repertoire is in part controlled by the specific expression of peptide–MHC complexes by specific cell types in the thymus.31 Negative T-cell selection is maintained in both Ciita pIV and pIII/pIV KO mouse models, suggesting that bone marrow-derived thymic medullary cells, which are required for negative selection, express CIITA from pI and therefore CIITA isoform I.11, 20 Thus, any specific need for CIITA isoform I's CARD domain might be revealed by a change in the percentage of single positive CD4 T cells when comparing KI and KO with WT thymocytes. It has been shown that impairing negative selection, either by deletion of DCs or knocking down CIITA in mTECs, results in an increased percentage of CD4 T cells in the thymus.32, 33, 34 Flow cytometry of CD4 and CD8 single- and double-positive populations in the thymi of WT and Ciita-targeted mice showed that there was no perturbation of the CD4+, CD8+ or CD4+CD8+ T-cell compartments when CIITA isoform I was lacking (Figure 5). These data suggest that CIITA isoform I is not required for regulation of MHC-II expression to the appropriate levels to ensure proper T-cell development in the thymus. As the KI line has a higher percentage of transcripts derived from pIII and pIV (Figure 4) and the fact that the HA-tagged allele may add to the stability of the CIITA protein produced,35 the studies described below will use the KO model to assess the role of the pI isoform on various immune responses.

Figure 5.
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Ciita isoform I is not required for T-cell development in the thymus. Thymocytes from 5-week old mice were analyzed for CD4+, CD8+ and CD4+CD8+ populations using flow cytometry. (a) Representative dot plots from individual thymi, gated on live cells and plotted as CD4 vs CD8. Gates used to define CD4+, CD8+ and CD4+CD8+ populations are shown. (b) The results from three experiments with 3–4 mice per group are combined (N=10 WT, N=9 KI and KO). None of the samples were found to differ from the wild type by a P-value<0.05 using Student's t-test.

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The CARD domain is not required for antigen presentation in vitro

To determine whether CIITA isoform I's CARD domain is required in antigen presentation, a lymphocyte proliferation assay was used to compare T-cell activation between WT and KO mice. Lymph nodes were collected from mice primed with the LCMV, CD4 T cell-specific antigen, peptide GP61−80.36 Mice were immunized containing either 1mgml GP61−80 in complete Freund's adjuvant or a 10-fold dilution of peptide to determine whether immunization with suboptimal doses would reveal subtle differences in T-cell response. Single-cell suspensions were incubated with increasing concentrations of peptide ex vivo, and the proliferation of T cells was measured by incorporation of [3H]-thymidine. CD4 T cells derived from the draining lymph nodes of WT and KO mice displayed similar responses to the peptide antigen when immunized with either dose (Figure 6). Thus, despite loss of isoform I CIITA, there was no defect in activation of T cells in vitro, indicating no defect in antigen presentation in KO antigen-presenting cells.

Figure 6.
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CiitapI KO T cells proliferate normally in response to antigen. Mice were immunized in the footpad and at the base of the tail with the 150μl peptide/CFA emulsion containing (a) 1mgml or (b) 0.1mgml of the peptide GP61−80. After 12 days, draining lymph nodes were isolated and T-cell proliferation was measured by incorporation of 3H-thymidine in response to increasing concentrations of the antigenic peptide. Averaged data from multiple independent experiments are graphed as normalized counts per minute (CPM) as a function of increasing concentration of peptide. (Panel a: n=9 WT and n=8 KO, panel b: n=6 WT and n=7 KO).

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CIITA isoform I does not contribute to the development of EAE

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory demyelinating disease that is used as a model of multiple sclerosis. EAE is mediated by CD4 Th1 cells that recognize self-antigens associated with MHC-II molecules. CIITA isoforms I and IV were found in the brain and spinal cord of mice with acute EAE, along with infiltrating DCs.37 Suter et al. found the majority of Ciita was expressed from pI in infiltrating DCs, whereas astrocytes express isoform IV in response to IFN-γ, and microglia express low levels of both isoforms I and IV in response to IFN-γ. To examine whether this model could reveal a need for isoform I, EAE was induced in WT and KO mice, and disease scores were recorded for 4 weeks. Disease severity was compared between the two groups over the entire 4 weeks, as well as in the last week alone, as this was when the disease scores stabilized. KO mice appeared slightly more sensitive than did WT mice, with a maximum disease score of 2.5 vs 2.0 for the WT group, although overall disease severity (combined disease index), disease severity in the fourth week only, and day of onset, were not found to be statistically different between the two groups (Figure 7).

Figure 7.
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CiitapI KO mice are not more susceptible to experimentally induced autoimmune disease. Experimental autoimmune encephalomyelitis was induced in groups of three to five females by injecting an emulsion of MOG and CFA. The experiment was performed three times and results were combined (n=11 WT and n=10 KO). No difference was observed in the onset or severity of disease. Severity was measured both for the entire 4 weeks of observation and for week 4 only. The max score was 2.0 for WT mice and 2.5 for the KO group (P<0.05). Error bars represent s.e. Statistics were performed using Student's t-test (onset) or Mann–Whitney (total disease score, max score) using Prism software (Graph Pad Software, Inc, La Jolla, CA, USA).

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Ciita KO mice are not more susceptible to L. monocytogenes

Listeria monocytogenes is an intracellular pathogen that targets macrophages. Several members of the NOD family have roles in L. monocytogenes recognition, including NOD1, NOD2 and NLRP3.15, 19 To determine whether the CARD-containing CIITA has a role in resistance to L. monocytogenes, mice were infected and bacterial colony-forming units (CFUs) were counted in the spleen and liver 5 days after primary infection, or 7 days after secondary infection (Figure 8a). No difference was seen in bacterial burden in the spleens or livers of KO mice compared with WT littermate controls after either primary or secondary infection, and all mice cleared the primary infection by day 10 (data not shown). To further dissect the immune response to L. monocytogenes, splenocytes were collected at the peak of the primary and secondary T-cell response (10 or 6 days after infection or rechallenge, respectively), and intracellular cytokine staining for tumor necrosis factor-α, interleukin-2 and IFN-γ was performed in CD4+ T cells. It has previously been shown that CD4 lymphocytes express a Th1-type cytokine profile after infection with L. monocytogenes.38 There was no difference in cytokine expression in KO CD4+ spleen cells compared with WT controls (Figures 8b and c). These results indicate that KO mice are not deficient in immune response to L. monocytogenes infection and that the other CIITA isoforms can substitute for isoform I.

Figure 8.
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Ciita isoform I is not required for resistance to the intracellular bacteria Listeria monocytogenes. WT and CIITA KO mice were infected with LM, and colony-forming units (CFUs) were determined in the spleens and livers. (a) For primary infection, mice were infected with 0.5 LD50 LM and CFU were determined at day 5 after infection. For secondary infection, mice were immunized with a low-dose (0.1 LD50), then re-infected 4 weeks later with a high dose (10 LD50) of LM, CFU was determined 3 days after re-challenge. Data represent two independent experiments. N=10 WT, N=11 KO primary, N=7 WT, N=6 KO secondary. (b) Representative flow cytometry plots of intracellular cytokine expression from CD4 T cells in the peritoneum. In primary infection, cells were collected 10 days after infection with 0.8 LD50. For secondary infection, mice were infected with 0.1 LD50, and then challenged 4 weeks later with 6 LD50. Peritoneal exudate cells were collected 6 days later and CD4 T-cell intracellular cytokine levels were measured by flow cytometry. (c) Average with s.d. of the percentage of CD4 T cells (from panel b) expressing cytokines TNFα, IL-2 and IFN-γ. Each graph is representative of two independent experiments using three to four mice per group. Un, unimmunized.

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Ciita pI KO mice mount an effective immune response to LCMV

To examine whether the CARD domain containing CIITA isoform was required for specific responses to a viral infection, mice were infected with the Armstrong strain of LCMV. This viral strain generates an acute infection that is cleared after 8 days.39 After infection, WT and KO mice were analyzed for a number of parameters depicting a successful immune response to virus challenge and memory. The percentages of virus-specific CD8 or CD4 T cells after initial infection were similar between WT and KO mice (Figures 9a and e). Virus-specific CD8 T cells from the spleen were analyzed for T cell-specific memory markers CD44, CD25, CD62L, CD127, KLrg1 and PD-1 (Figure 9b). No difference was observed in the expression of these markers as well. When CD4 and CD8 T memory cells were stimulated ex vivo for the production of cytokines, tumor necrosis factor-α and IFN-γ were also found to be similar (Figure 9c). The LCMV-specific memory B-cell pool, as measured by an antigen-specific ELISPOT assay revealed higher values for Ciita pI KO mice, but this value was not found to be statistically significant (Figure 9d). Taken together, these data indicate that an effective adaptive immune response is generated in response to infection with LCMV in the absence of CIITA isoform I.

Figure 9.
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The adaptive immune response to an acute LCMV infection in KO mice is similar to WT mice. (a) Virus-specific CD8 T cells were analyzed using fluorescently labeled tetramer specific for the GP33−41 epitope of LCMV. Mice were serially bled and the percentage of LCMV-specific CD8 T cells in PBMCs was determined by flow cytometry analysis at the given time points. (b, c) Acutely infected mice were killed at the maintenance stage of T-cell memory differentiation, and phenotypic and functional analyses were performed on LCMV-specific lymphocytes. (b) Tetramer-specific (GP31−40) CD8 T cells were analyzed by flow cytometry for the T-cell markers CD44, CD25, CD28L, CD127, Klrg1 and PD-1. Red-filled histograms represent wild-type, blue histograms represent KO CD8 T cells. (c) T cell receptor-mediated cytokine expression was assessed by culturing splenocytes for 5h in the presence of peptides for the CD8 (GP33 and GP276)- and CD4 (GP61)-dominant epitopes of LCMV. (d) The absolute number of LCMV-specific memory B cells was determined using an LCMV-specific ELISPOT analysis of memory B-cell responses. (e) Virus-specific CD4 peripheral blood mononuclear T cells were analyzed for CD44 expression and with tetramer specific for the GP66−77 epitope of LCMV.

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Both LCMV and L. monocytogenes primarily elicit a T cell-mediated response. To test the B-cell response in the CIITApI null model, mice were immunized with influenza, and antibody titers were measured at various time points after immunization. No differences were seen between WT and KO mice in antibody responses as measured by hemagglutinin inhibition titers or IgG, IgG1 or IgG2a titers in the serum at 8, 14 or 28 days after infection (Supplementary Figure 3).

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Discussion

The expression of MHC-II genes is regulated at the level of transcription by the presence or absence of the master regulator CIITA. The expression of CIITA from pI was of particular interest because this isoform has homology to a CARD domain12 and its use is restricted to the myeloid and DC compartments.20 Thus, it seemed that this isoform would have unique properties that might be revealed by either substitution or deletion of the isoform. The KI/KO models created here allowed this hypothesis to be tested. Surprisingly, no gross defect in immune system function or development in either CIITApI right arrow III KI or CIITApI right arrow 0 KO was observed. In the case of the KO model, total levels of Ciita transcript were not reduced in macrophages treated with IFN-γ, and were not reduced by >30% compared with WT in spDCs. Surface MHC-II expression reflected Ciita mRNA levels. No obvious defect in T-cell development was observed either, as the percentage of CD4+ T cells in the thymus was normal. Moreover, T cells from mutant mice responded normally to in vitro stimulation and showed no significant differences to lower doses of antigen. KO mice were only marginally more susceptible than WT in one model for autoimmunity (EAE), but all mice stabilized with the same level of disease. KO mice were equally immune competent (compared with WT) when challenged with a viral or bacterial agent (LCMV or L. monocytogenes, respectively), and produced antibodies at levels comparable to WT after vaccination to influenza. It remains possible that the decreased levels of I-A and Ii observed in KO spDCs have a subtle effect on immune system function not detected by the experiments presented here. Thus, we conclude that in the models tested, no unique function can be ascribed to the CARD-containing isoform of CIITA.

Interestingly, the expression of MHC-II was decreased to a greater extent than Ciita. Nickerson et al.12 suggested that pI is a more potent transcriptional activator than the other isoforms; however, this was not confirmed by Buttice et al.,28 who found no difference in transactivation activity between the various isoforms. The reason Nickerson et al. observed greater activity from isoform I may be related to the CARD sequence, which could potentially provide increased stability of the protein.35 The half-lives of isoforms III and IV have been reported to be the same,35 but to date, there are no data on the half-life of isoform I. Data presented here argue that equal levels of CIITA either from pI (endogenous) or from the KI lead to similar levels of I-A transcripts, suggesting that CIITA levels are the limiting determinant. Similarly, isoform I is not specifically required for induction of a number of genes identified as CIITA targets, including I-Ab, H2-D1, H2-DOa, H2-DM, Ii, Col1A1, Cste, Kiaa00841, Kpna6, Plxna1, Psmd3, Rab4b, Tpp1 and Trim26, as differences in expression of these genes were not observed in mice expressing only isoform III. With the exception of Plxna1,26 which is specific to DCs, all of these genes are also expressed in B cells. KO macrophages treated with IFN-γ had higher levels of H2-DOb mRNA than did WT macrophages. This result was unexpected for a few reasons. First, H2-DO is strongly expressed in B cells, but it is poorly expressed by macrophages.29, 30, 40 Second, the levels of H2-DOb mRNA were reduced in WT but not in KO IFN-γ-treated macrophages. Finally, the regulation of HLA-DOB/H2-DOb in B cells by CIITA has been controversial; some data suggest that HLA-DOB/H2-DOb is not regulated by CIITA in B cells,41, 42 whereas other data suggest the opposite.24, 25, 43 These data may indicate that CIITA isoform III, but not isoform I, is important for the proper regulation of H2-Ob. Thus, with the possible exception of H2-DOb, the CARD domain of isoform I does not appear to be required for expression of any of the genes identified as targets of CIITA regulation.

Previous mouse KO models showed no evidence for cross-talk between the promoters. In the Ciita pIV KO mouse, expression of Ciita from myeloid and lymphoid cells was not affected.11 Similarly, in double pIII/pIV KO in which a large region was deleted, expression of Ciita in cDCs appeared normal.20 Therefore, it was unexpected that deletion of pI would have an effect on downstream promoters. The fact that deletion of the 798bp surrounding and including pI alters expression from other promoters suggests that the mechanisms that control expression from pI also operate on alternate promoters, but are prevented from doing so in cells of the myeloid lineage. Thus, it is most likely that there is an unidentified regulatory region that normally promotes expression of Ciita from pI in cells of the myeloid lineage. Expression of Ciita from pIII in DCs of mice in which pI was deleted suggests that Ciita's ‘myeloid-specific’ regulatory region can direct expression from any (or perhaps the nearest available) promoter. Alternatively, pI and pIII may share transcription factors necessary for expression and that some other mechanism prevents pIII expression in spDC. Previous models that deleted pIV or pIII/pIV failed to identify this possibility, as there was no promoter switching observed in these models and little is known about the regulation of Ciita pI. Recently, Smith et al.44 characterized the promoter proximal region and identified binding sites for the positive regulatory factors Pu.1 and IRF8, and PRDM1 as a negative regulatory factor. Choi et al.45 identified two signal transducer and activator of transcription 5-binding sites upstream of pI. Our data demonstrate that deletion of the proximal promoter region, which includes the regions studied in these reports, is insufficient to silence Ciita expression in cells that primarily use pI.

Transcription factors regulating Ciita pIII and pIV have been well characterized,46, 47, 48, 49, 50, 51, 52 and using these as a guide, some suggestions of what may occur at pI and promoter choice can be hypothesized. Several years ago, it was found that DNA methylation of the CpG dinucleotides encompassing pIV prevented expression in response to IFN-γ in tumors or cell types refractory to this induction.53, 54 Analyses of the chromatin structure of pIV in human cells showed that other mechanisms may be at play and, by analogy such mechanisms may function with pI as well. In Hela cells, five regions spread across a 110-kb span that contained the CIITA gene were identified that could influence expression of pIV in response to IFN-γ.55 These regions were shown to interact with one another through chromatin conformation capture assays.55 Intriguingly, one of these regions was upstream of pI and was also identified as a DNase-hypersensitive site in B cells. This region, termed ‘HSS1’, binds PU.1,49 a critical regulator of B-cell and myeloid cell fate, as well as signal transducer and activator of transcription 1,55 a factor activated when cells are induced by IFN-γ. HSS1 lies 3kb upstream of pI and was not deleted from our constructed mouse strains. Thus, the possibility exists that HSS1 could be a pI and pIII control region. However, it was only one of several elements in the long-range control of pIV and one of the other elements, or an as yet undiscovered element could control the use of pI. Thus, whichever element(s) controls expression in myeloid cells, our data suggest that this element targets pI first as opposed to the other Ciita promoters.

Thus, although CIITA in cells of the myeloid lineage express a unique isoform with a CARD homologous domain, this specific domain does not appear to have a unique function with respect to MHC-II expression and the ability of cells using this promoter to mount successful immune responses. Instead, the system appears to be designed to specifically control CIITA and subsequently MHC-II gene expression in a strict tissue-specific manner by having distinct promoters and regulatory mechanisms that restrict expression to each of the promoters.

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Materials and methods

Targeting vector and generation of Ciita mutant mice

A Ciita site-specific targeting construct (see Figure 1a) was generated in PGKneobpA (a gift from Dr Grant MacGregor, University of California, Irvine, CA, USA) by standard PCR and cloning techniques. It contains, in order: a 2.8-kb fragment upstream of Ciita pI (3.1–0.3kb upstream of the TSS of pI); a FLP1 recombinase target (FRT) site; upstream sequences −298 through +85 of the pI 5′-untranslated region; an AUG codon and HA-epitope-tag fused to Ciita pIII exon 1 coding sequence; 145bp from intron 1 of pI; and a second FRT site. A lox-P-flanked neomycin-resistant (neo) cassette and 3.0kb of Ciita pI intron 1 (0.5–3.5kb downstream of the TSS) completes the vector and provides the 3′ targeting arm. ClaI and SalI sites were introduced into the construct to facilitate cloning. The linearized targeting vector was transfected into 129 SvEv ES cells by electroporation, and G418-resistant clones were selected by inGenious Targeting Labs Inc. (Stony Brook, NY, USA) Homologous recombinants were screened by Southern blotting using HindIII, BamHI or EcoRI digestion in combination with probes either internal or 3′ external to the targeting construct. The presence of the upstream FRT site was determined by PCR, using primers 1 and 2 (Figure 1 and Supplementary Table 1). Correctly targeted ES cells were injected into C57BL/6 blastocysts and three chimeric males were obtained. These chimeras were crossed to C57BL/6J (The Jackson Laboratory) females and the agouti F1 heterozygote offspring were tested for the transgenic allele by PCR using primers 1 and 2 (Figure 1). Mice containing the targeted allele were crossed to EIIa-cre mice (The Jackson Laboratory). Offspring of these crosses were analyzed by PCR for transmission of the targeted allele and for Cre-mediated deletion of the floxed neo cassette (primer sets 1–2 and 3–4, Figure 1 and Supplementary Table 1). Mice carrying the Cre-deleted locus were then backcrossed to C57BL/6J to generate CIITApI right arrow III KI mice or crossed to ACT-FLPe mice (The Jackson Laboratory). Offspring of the ACT-FLPe crosses were analyzed by PCR for FLPe-mediated deletion of the DNA between the FRT sites (primer set 1–4, Supplementary Table 1). Mice carrying the Cre/FLP-deleted locus were then crossed to C57BL/6J to generate CIITApI right arrow 0 KO mice. Where indicated, C57BL/6J mice from The Jackson Laboratory aged 6–8 weeks were used. Animals were housed under standard conditions in a conventional mouse facility, where they remained healthy. The Emory University Institutional Animal Care and Utilization Committee approved all animal experiments and protocols.

To generate the targeting construct, we designed primers (D5′d, XhoI and D3′, ClaI) to amplify the upstream flanking region for homologous recombination, introducing XhoI and ClaI sites for cloning into the targeting vector. Another primer pair was designed to introduce a FRT site upstream of pI and a SalI site at the start of translation (C5′, ClaI and C3′, SalI). The next fragment inserted into the targeting vector was generated by overlap-PCR using primers that introduced a consensus Kozak sequence and HA tag and amplified the coding region of promoter III exon 1, with the intronic sequence of pI exon 1, followed by a FRT site and XhoI for cloning purposes. Primers for this step were B5′, SalI, B2-5′OL, B1-3′OL and B2-3′. Finally, A5′, NotI and A3′, SacII were designed to amplify the downstream flanking region, intron 1 of pI, for homologous recombination, introducing NotI and SacII sites for cloning into the targeting vector. All primers are listed in Supplementary Table 1. PCR was performed using genomic DNA isolated from strain 129 mouse tissues. The PCR amplicons were initially cloned into the pCR4-BluntTOPO vector (Invitrogen, Carlsbad, CA, USA) for DNA sequence confirmation and large-scale preparation, then subcloned into PGKneobpA at the XhoI site. The resulting vector was digested with NotI and SacII, and ligated with the downstream flanking region PCR fragment.

Speed congenics

We used a marker-assisted selection procedure, aka ‘speed congenic’ strategy, to construct a C57BL/6 congenic strain containing our targeted locus from a 129/SvEv:C57BL/6J mixed strain background.22, 56 We selected a genome-wide set of markers, spaced ~20cM apart, from the MIT/Whitehead mouse map that was reportedly polymorphic between C57BL/6J and 129/SvEv.22, 56 PCR primers used are available on the Mouse Genome Database at the Mouse Genome Informatics website.57 PCR products were run on polyacrylamide gels and compared with C57BL/6J and 129SvEv control DNA run in parallel. The ‘best’ breeder of each generation was heterozygous by locus-specific PCR for our targeted mutation, and carried the most C57BL/6J alleles by genome-wide SSLP PCR analysis. By the fourth backcross, all markers tested except the one next to the targeted locus were of C57BL/6J origin (data not shown).

Markers used were D1Mit211, D1Mit19, D1Mit215, D1Mit139, D1Mit150, D2Mit5, D2Mit61, D2Mit13, D2Mit206, D2Mit304, D2Mit213, D3Mit21, D3Mit40, D3Mit351, D3Mit163, D4Mit89, D4Mit275, D4Mit31, D4Mit204, D4Mit254, D5Mit148, D5Mit20, D5Mit163, D6Mit86, D6Mit33, D6Mit146, D6Mit199, D6Mit15, D7Mit310, D7Mit237, D7Mit31, D7Mit189, D8Mit155, D8Mit191, D8Mit263, D8Mit211, D8Mit93, D9Mit2, D9Mit21, D9Mit306, D9Mit279, D10Mit51, D10Mit40, D10Mit7, D10Mit162, D10Mit180, D11Mit226, D11Mit270, D11Mit5, D11Mit38, D11Mit199, D11Mit184, D12Mit153, D12Mit3, D12Mit231, D13Mit16, D13Mit179, D13Mit193, D13Mit76, D14Mit99, D14Mit60, D14Mit192, D14Mit107, D15Mit13, D15Mit46, D15Mit29, D15Mit42, D16Mit182, D16Mit103, D16Mit64, D16Mit152, D17Mit164, D17Mit51, D17Mit205, D17Mit123, D18Mit19, D18Mit23, D18Mit33, D18Mit25, D19Mit128, D19Mit119, D19Mit91, D19Mit71, DXMit136, DXMit143, DXMit213, DXMit130 and DXMit186.

5′RACE

To determine the 5′ ends of CIITA transcripts of KO and KI mice, RACE was performed using the FirstChoice RLM-RACE Kit from Applied Biosystems (Carlsbad, CA, USA) following the manufacturer's instructions. Primers used are listed in Supplementary Table 1.

Cell Collection and treatments

Dendritic cells were collected from the spleen as described in Current Protocols.58 In brief, mice were injected intraperitoneally with 30ng Flt3 Ligand-Ig (Flt3-L) for 9 days.59 Flt3-L was generously provided by Dr R Mittler (Emory University). After CO2 asphyxiation, spleens were removed and injected with Dulbeco's modified Eagle's media (Cellgro, Manassas, VA, USA) containing 10% fetal bovine serum (Sigma-Aldrich, St Louis, MO, USA) (Dulbeco's modified Eagle's mediun-10), 1 × non-essential amino acids (HyClone Laboratory, Logan, VT, USA), 1M HEPES (HyClone Laboratory), 1mM sodium pyruvate (HyClone Laboratory), 0.292mgml L-glutamine, 100unitsml penicillin, 100μgml streptomycin (Invitrogen) and 1mgml collagenase D (Roche, Indianapolis, IN, USA), cut into pieces and incubated at 37°C for 25min. A single-cell suspension was generated by forcing cells through a 40-μm cell strainer (BD Biosciences, San Jose, CA, USA). Red blood cells were lysed with ammonium-chloride potassium-chloride (ACK) lysing buffer. The CD11c+ DC population was purified using CD11c MACS beads (Miltenyi Biotech Inc., Auburn, CA, USA) according to the manufacturer's protocol. Peritoneal exudate cells were isolated in 10ml Hanks’ buffered saline solution containing 10unitsml heparin. For peritoneal macrophages, mice were injected intraperitoneal with 2.5ml 2% solution of thioglycolate 4 days before collection of the peritoneal fluid. Peritoneal exudate cells were plated 0.8–1 × 106 cells per ml in Dulbeco's modified Eagle's medium-10 containing 0.292mgml L-glutamine, 100unitsml penicillin, 100μgml streptomycin (Invitrogen) and 50μM 2-mercaptoethanol (Sigma-Aldrich) and allowed to adhere for 2h. Non-adherent cells were washed off, and adherent cells were provided fresh Dulbeco's modified Eagle's medium-10 and treated with 500unitsml IFN-γ (Peprotech Inc., Rocky Hill, NJ, USA) for the indicated time. Total primary peritoneal exudate cells and spleen cells were incubated in RPMI 1640 medium containing 10% fetal bovine serum, 0.292mgml L-glutamine, 100unitsml penicillin, 100μgml streptomycin, 50μM 2-mercaptoethanol, 1mM sodium pyruvate and 10mM HEPES buffer. For collection of B cells and T cells from the spleen or lymph nodes, a single-cell suspension was generated as described above.

Flow cytometry

For cell surface staining, 0.5 × 106 cells were treated as described previously.38 In brief, 0.5 × 106 cells were incubated on ice for 5min with anti-CD16/32 (FC block clone 2.4G2) before addition of antibodies or isotype control for 30min. Antibodies used for surface staining were purchased from BD Biosciences: anti-CD4 (clone RM4-5 PerCP, GK1.5 PE), anti-CD8 (clone 53-6.7) anti-CD11b (clone M1/70), anti-CD11c (clone HL3), anti-CD45R/B220 (clone RA3-6B2), anti-CD45RA (clone 14.8), anti-I-Ab (clone AF6-120.1) and appropriate isotype controls (A95-1, R35-95, G235-2356, G155-178). For secondary labeling, cells were washed once, followed by 20min incubation on ice with Pacific Blue-labeled streptavidin (Molecular Probes, Eugene, OR, USA). Flow cytometry was performed using a BD FACSCalibur or LSRII flow cytometer (BD Biosciences). Fluorescence intensity was calculated in molecules of soluble fluorochrome units using Quantum molecules of soluble fluorochrome beads (Bangs Laboratories Inc., Fishers, IN, USA) according to the manufacturer's protocol. All data were analyzed using FlowJo (TreeStar Inc., Ashland, OR, USA).

For intracellular cytokine staining after infection with L. monocytogenes, 3 × 106 peritoneal exudate cells were cultured for 5h at 37°C in 1ml media either without treatment, with 10ngml phorbol 12-myristate 13-acetate (Sigma-Aldrich) plus 1μM ionomycin (Sigma-Aldrich) (positive control), or with 107 cells heat-killed L. monocytogenes. Brefeldin A (Sigma-Aldrich) was added (10μgml) after the first 30min. After LCMV infection, 106 splenocytes were cultured 5–6h in the absence or presence of the indicated peptide (GP33, GP276 or GP61) plus Brefeldin A. For analysis by flow cytometry, extracellular antigens were stained as indicated above, followed by fixation and permeabilization using Fix and Perm cell permeabilization kit (Invitrogen) for intracellular antigens, according to the manufacturer's directions. Antibodies used for intracellular cytokine detection were anti-IFN-γ (clone XMG1.2), anti-interleukin-2 (clone JES6-5H4), anti-tumor necrosis factor-α (clone MP6-XT22), and appropriate isotype controls (R3-34 and A95-1), staining was performed on ice for 30min. Live cells were gated based on light scatter parameters. The lymphocyte population was gated based on forward and side scatter parameters. All antibodies used were purchased from BD Pharmingen (Franklin Lakes, NJ, USA).

T-cell proliferation

To measure MHC-II antigen- presentation efficiency, mice were injected in the footpad and base of the tail with an emulsion of complete Freund's adjuvant containing 1mgml heat-killed Mycobacterium tuberculosis (Difco, Detroit, MI, USA) and the I-Ab-specific epitope of LCMV GP61−80 peptide.60 Popliteal and inguinal lymph nodes were collected 12 days later. A single-cell suspension was generated, and 5 × 105 cells were incubated in a 96-well plate with increasing concentrations of peptide for 3 days, with 0.4μCi per well [3H]thymidine added during the last 24h. Plates were harvested using a FilterMate harvester (Packard Instrument, Meriden, CT, USA), and 3H-thymidine incorporation was measured on a 1450 LSC Microbeta TriLux counter (Perkin-Elmer, Waltham, MA, USA).

Induction of EAE

Experimental autoimmune encephalomyelitis was induced in female mice 6–8 weeks of age by subcutaneous injection in the hind flank with 200μg MOG35−55 peptide emulsified in 200μl complete Freund's adjuvant (2.5mgml M. tuberculosis) on days 0 and 7, as described previously.60, 61 On days 0 and 2 mice were administered 250ng pertussis toxin intraperitoneally (List Biological Laboratories Inc., Campbell, CA, USA). Disease severity was scored according to the following scale: 0, no disease, 1, flaccid tail, 2, hind limb weakness, 3, hind limb paralysis, 4, failure to right (forelimb weakness), 5, moribund.

Peptides

Myelin oligodendrocyte glycoprotein peptide 35–55 (MOG35−55) (MEVGWYRSPFSRVVHLYRNGK),62 LCMV epitopes GP33−41 (KAVYNFATC), GP276−286 (SGVENPGGYCL) and GP61–80 (GLNGPDIYKGVYQFKSVEFD)63 peptides were synthesized using standard 9-fluorenylmethyloxycarbonyl chemistry on a Prelude peptide synthesizer (Protein Technologies Inc.).

L. monocytogenes infection

A culture of L. monocytogenes (strain 10403s) was grown in brain heart infusion to OD=0.2, and then diluted in phosphate-buffered saline, the indicated dose was administered in 200μl. The LD50 for C57BL/6J mice was determined to be 6 × 106CFU (data not shown). Infection dose was verified by plating dilutions of the inoculum and enumerating CFU. Mice were monitored daily for morbidity and mortality. Mice were killed when moribund or weight loss was >25% of initial weight. For enumeration of CFU in organs, spleens and livers were aseptically removed and placed in phosphate-buffered saline. Tissue homogenizers were used to generate a single-cell suspension, and 1% Triton- × 100 in phosphate-buffered saline was added to final concentration of 0.5% to lyse the cells. Serial dilutions were plated on brain heart infusion plates containing 25μgml streptomycin. Colonies were counted the next day, and total CFU/organ was calculated based on the dilution plated.

Lymphocytic choriomeningitis virus

Acute LCMV infection and cellular analysis were performed as described previously.39 In brief, mice were infected intraperitoneally with 2 × 105p.f.u. LCMV-Armstrong. Mice were serially bled and killed as described in the figure legends. Staining with antibodies and MHC class I and II tetramers complexed with LCMV GP33−41, GP276−286 or GP66−77 for flow cytometric analysis of antigen-specific T cells were performed as described previously.64, 65, 66

Influenza immunization

Here, CIITA KO and WT littermate control mice received a single immunization of 5μg whole inactivated A/California/04/09 influenza virus and they were bled on days 7, 14 and 28. Sera were collected for enzyme-linked immunosorbent assay and hemagglutinin inhibition assays. As a negative control group, we included three naive WT and two CIITA KO mice. Sera were individually collected, and anti-influenza-specific antibody levels were determined quantitatively by enzyme-linked immunosorbent assay as described previously.67, 68 Hemagglutination inhibition titers based on the World Health Organization protocol69 was determined as described previously.67 The hemagglutinin inhibition titer was read as the reciprocal of the highest dilution of serum that conferred inhibition of hemagglutination. The values were expressed as the geometric mean±s.e.m.

Quantitative RT-PCR

Total RNA was isolated from cells using the RNeasy kit (Qiagen, Valencia, CA, USA). Reverse transcriptase was performed using 1μg of RNA using SuperScrpit II (Invitrogen), RNAse inhibitor (Roche), RQ1 DNase (Promega, Madison, WI, USA), oligo d(T)16 and random hexamers (Applied Biosystems). Real-time PCR using 1/33 of the cDNA product was used to quantify the amount of mRNA. Results were normalized to 18S rRNA or GAPDH mRNA levels and represented using the comparative CT method.70 Data presented represents the average of at least three independent biological replicates, and error bars represent s.e.m. Primers used are listed in Supplementary Table 1.

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Conflict of interest

The authors declare no conflict of interest.

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Acknowledgements

We thank Dr Joseph Sabatino for help with some of the experiments presented. We also thank the Emory University School of Medicine Core Facility for Flow Cytometry and the NIH tetramer core. This study was supported by the National Institutes of Health grants RO1AI43000 and R56AI34000 (to JMB), PO1 AI080192-01 (to JMB and RA), HHSN266 200700006CMD8 (to IS) and the American Cancer Society postdoctoral fellowship PF-09-134-01-MPC (to BY).

Supplementary Information accompanies the paper on Genes and Immunity website