CIITA Research Articles

Curcumin Inhibits IFN‐γ Signaling in Colonic Epithelial Cells

IFN‐γ is a proinflammatory cytokine with profound effects on colonic epithelial cells. Elevated IFN‐γ disrupts the epithelial barrier function, prevents epithelial cell migration and wound healing, and primes IEC cells to express MHC class II molecules and to serve as non‐professional antigen presenting cells (APC). In this report we demonstrate that curcumin inhibits IFN‐γ signaling in colonic epithelial T‐84 and YAMC cells. Curcumin significantly inhibited IFN‐γ induced GAS element‐driven luciferase activity. IFN‐γ dramatically stimulated expression of MHCII genes, HLA‐DRα and HLA‐DPα1, while curcumin dose‐dependently prevented this increase. Curcumin inhibited IFN‐γ stimulated expression of CII TA, a master regulator of MHCII class genes. EMSA analysis showed curcumin‐mediated inhibition of STAT‐1 binding to both GAS and ISRE cis‐elements. Immunofluorescence studies demonstrated that curcumin inhibited IFN‐γ‐stimulated nuclear translocation of Stat‐1. IFN‐γ induced and JAK‐dependent phosphorylation of Stat‐1 at Tyr701, a residue critical in Stat‐1 dimerization and nuclear translocation, was also inhibited. In summary, curcumin acts as a potent inhibitor of Jak‐Stat signaling pathway in the colonic epithelial cells, a phenomenon which may partially account for the beneficial effects of curcumin in experimental colitis as well as in human IBD.Grant support: 5R01DK067286 (P.R.K.)

Ikaros, transcription factor, regulates CIITA gene expression in vascular smooth muscle cells and macrophages

Ikaros, transcription factor, regulates CIITA gene expression in vascular smooth muscle cells and macrophages

The inhibitory effect of MHC class ii transactivator gene silencing on acute rejection reaction in rat liver transplantation

Objective To investigate the inhibitory effects of MHC class Ⅱ transactivator(C ⅡTA)gene silencing in liver graft on acute rejection reaction in rat liver transplantation.Methods The eukaryotic expression vector of short hairpin RNA(shRNA)targeting rat C Ⅱ TA gene was constructed.Allogeneic liver transplantation was stably established between DA donors and Lewis recipients.There were normal saline(NS)group,control group and shRNA group(n = 16 each).The donor received pretreatment by portal vein injection of NS,control vector and shRNA vetor correspondingly 72 h before liver transplantation.Eight of the recipients were sacrificed for detection 5 days after transplantation.Pathological rejection grading was examined,and expression levels of CⅡTA and MHC-Ⅱ were detected by real time RT-PCR and immunohistochemical staining.The remaining recipients were monitored for survival time.Results The mean survival time in NS,control vector and shRNA groups was 7.75,7.25 and 15.75 days respectively.shRNA group showed obviously longer survival time(P ＜ 0.01).Compared to NS and vector control groups,pathological rejection grading in shRNA group was significantly depressed(P ＜ 0.01).The transcription levels of C Ⅱ TA and MHC-Ⅱ and the expression level of MHC-Ⅱ were also significantly inhibited in shRNA group(P ＜0.01).Conclusion Acute rejection reaction can be significandy relieved in rat liver transplantation by C Ⅱ TA gene silencing in liver graft.It might be related with depression of immunological recognition. Key words: Liver transplantation; Gene silencing; Acute rejection reaction

The influence of class II transactivator and interleukin‐6 polymorphisms on the production of antibodies to donor human leucocyte antigen mismatches in renal allograft recipients

The influence of polymorphisms in the CIITA and IL-6 genes on donor-specific human leucocyte antigen antibody production was investigated in a cohort of renal transplant recipients and their donors. CIITA and IL-6 single nucleotide polymorphisms were found to be associated with donor-specific human leucocyte antigen antibody production post-transplantation.

Functional study of the different haplotypes cDNA in the coding region of CIITA gene

Objective To study the function of 4 different haplotypes cDNA which are constructed by two non-homonymy single nueleotide polymorphism (SNP) sites C19170G (Leu45Val) and C30799G (Ala500Gly) in the coding region of human CⅡTA gene. Methods HeLa cells were transfeeted with eu-karyotic expression vectors containing four different haplotypes cDNA. C Ⅱ TA mRNA and HLA classⅡanti-gen (HLA-DR, DP, DQ) were respectively detected by RT-PCR and indirect cell immunofluoreseence tech-nique in the untransfected and transfeeted with four eukaryotic expression vectors and empty vectors HeLa cells. The quantity of HLA classⅡ antigen were analyzed by flow eytometry. Results No expression of CⅡTA mRNA and HLA class Ⅱ antigen were observed on original HeLa cells and empty vector transfected cells. CⅡTA mRNA expression was emerged, and the expression of HLA class Ⅱ antigen were observed in the HeLa cells transfected with eukaryotic expression vectors containing four different haplotypes cDNA. And there were not significantly different with the levels of HLA class Ⅱ antigen expression among HeLa cells transfected with eukaryotic expression vectors containing four different haplotypes cDNA ( P > 0.05 ). Con-dusion The SNP of Chinese at the sites C19170G(Leu45Val) and C30799G(Ala500Gly) in the coding site of C Ⅱ TA gene did not influence capability of CⅡTA trans-aetivating HLA class Ⅱgene expression. Key words: MHC classⅡtransaetivator ( CⅡTA) ; Single nueleotide polymorphism(SNP) ; Hap-lotype; HLA classⅡantigen

The inhibiting effect on rat C II TA and MHC II gene expression by plasmid vector-based RNAi technulogy

Objective To investigate the inhibitory effect on rat MHC Ⅱ transactivator (C Ⅱ TA) and MHC Ⅱ gene expression by plasmid vector-based RNAi technology. Methods According to C Ⅱ TA ge-netic information, three short hairpin RNA (shRNA) sequences were designed and the corresponding plas- mid vectors were constructed. Rat dendritic cell (DC) was transfected in vitro and rat spleen was transfected in vivo. Real time RT-PCR and flow cytometry were used to detect the expression of C Ⅱ TA and MHC Ⅱ in DC and spleen after transfection. Results C Ⅱ TA-shRNA plasmids were successfully constructed. Com-pared with control groups, the mRNA transcription levels of C Ⅱ TA and MHC Ⅱ and the expression level of MHC H were significantly inhibited in all three pC Ⅱ TA-shRNA experimental groups ( P < 0.01 ). There was positive correlation between the expression of C Ⅱ TA and MHC Ⅱ. Among the three shRNA groups, the first one showed the strongest inhibitory effect. Conclusion The expression of rat C Ⅱ TA and MHC Ⅱ can be obviously inhibited by plasmid vector of shRNA targeting C Ⅱ TA gene, which can be used for further investi-gation of gene therapy. Key words: RNA interference; C Ⅱ TA ; MHC Ⅱ ; Plasmid vector

Tumor‐mediated down‐regulation of MHC class II in DC development is attributable to the epigenetic control of the CIITA type I promoter

In patients with cancer, DC express significantly lower amounts of MHC class II compared with those of normal individuals. However, the underlying mechanisms for this have not yet been fully defined. In the present study, we found that IL-10 plays a major role in the tumor-conditioned medium (TCM)-mediated decrease of MHC class II expression on BM-derived DC. IL-10 inhibited the expression of type I CIITA during DC differentiation. GM-CSF-mediated histone (H3 and H4) acetylation at the type I promoter (pI) locus of the CIITA gene was markedly increased during DC differentiation and this increase was blocked by IL-10. We also found that STAT5 bound to the CIITA pI locus during DC differentiation, and the binding was markedly attenuated by TCM or IL-10. Altogether, these findings suggest that (i) the down-regulation of MHC class II in tumor microenvironments is most likely attributable to IL-10 in the TCM and (ii) IL-10-mediated MHC class II down-regulation results from the inhibition of type I CIITA expression. This inhibition is most likely due to blocking of the STAT5-associated epigenetic modifications of the CIITA pI locus during the entire period of DC differentiation from BM cells, as opposed to a simple inhibition of MHC class II expression at the DC stage.

Abstract 4303: Heme Oxygenase 1 Expression in Dendritic Cells Drives Cd4+ T Cell Activation in Transplantation Atherosclerosis

Although over-expression of heme oxygenase-1 (HO-1) attenuates transplantation arteriosclerosis, the mechanism by which HO-1 exerts its protective effect remains unclear. In order to investigate the effect of HO-1 expression specifically in the dendritic cell (DC) in the context of transplantation atherosclerosis, we studied the effect of HO1-deficient versus wildtype (WT) DCs on the T-cell priming response and outcome in a murine transplant arteriosclerosis model. At day 0 C57bl6 mice received either WT (N=7) or HO1-knockout DCs (N=7) pre-sensitized with Balb/c splenocytes lysate. At day 10 an allogenic aorta segment derived from Balb/c mice was transplanted into the carotid artery position of C57Bl6 mice. 14 days post transplantation, the grafts were harvested and analyzed by imumnohistology. Adoptive transfer of HO1-deficient DCs significantly increased neointimal hyperplasia as compared to WT DCs (116995 versus 38428 μm2 P&lt;0.05). HO-1 deficient DCs also increased medial thickeness (15936 versus 12034 μm2 P&lt;0.05), reduced intimal VSMCs content (46 versus 75% P&lt;0.05) and resulted in more prominent medial cell infiltration (461 versus 232 μm2 P&lt;0.05). In the transplanted aorta of the with HO-1 nullizygous DCs treated recipient group, an increase in CD4+ T-cell infiltration (9.5 versus 0.2% in WT P&lt;0.05) and IgG deposition, concomitant to a decrease of CD8+ T cell infiltration (8.1 versus 14.3%, P&lt;0.05) was observed. In line with these observations, in vitro analysis of HO-1 deficiency in DC function showed an increased priming potential for CD4+ T cells, thereby increasing the CD4+/CD8+ T cell ratio. Increased efficiency in CD4+ T cell priming by HO-1 deficient DCs was facilitated by a higher cell surface level of MHC II. Further studies indicated that HO-1 deficiency increased STAT1 phosphorylation, thereby enhancing CIITA gene expression that lead to increased MHC II levels on the DCs cell surface. HO-1 deficiency in dendritic cells increases vascular cell infiltration with a higher CD4+/CD8 T-cell ratio in vascular allografts resulting in an augmented form of transplantation arteriosclerosis. This effect is facilitated by a STAT1-CIITA-MHCII dependent intracellular pathway.

195 HLA Class II Genotype Predicts Increased Risk for Ulcerative Colitis-Associated Colorectal Cancer and Correlates with Methylation of the MHC Class II Transactivator (Ciita) Gene

195 HLA Class II Genotype Predicts Increased Risk for Ulcerative Colitis-Associated Colorectal Cancer and Correlates with Methylation of the MHC Class II Transactivator (Ciita) Gene

Vra4 Congenic Rats with Allelic Differences in the Class II Transactivator Gene Display Altered Susceptibility to Experimental Autoimmune Encephalomyelitis

Presentation of Ag bound to MHC class II (MHC II) molecules to CD4+ T cells is a key event in adaptive immune responses. Genetic differences in MHC II expression in the rat CNS were recently positioned to allelic variability in the CIITA gene (Mhc2ta), located within the Vra4 locus on rat chromosome 10. In this study, we have examined reciprocal Vra4-congenic strains on the DA and PVGav1 backgrounds, respectively. After experimental nerve injury the strain-specific MHC II expression on microglia was reversed in the congenic strains. Similar findings were obtained after intraparenchymal injection of IFN-gamma in the brain. Expression of MHC class II was also lower on B cells and dendritic cells from the DA.PVGav1-Vra4- congenic strain compared with DA rats after in vitro stimulation with IFN-gamma. We next explored whether Vra4 may affect the outcome of experimental autoimmune disease. In experimental autoimmune encephalomyelitis induced by immunization with myelin oligodendrocyte glycoprotein, DA.PVGav1-Vra4 rats displayed a lower disease incidence and milder disease course compared with DA, whereas both PVGav1 and PVGav1.DA-Vra4 rats were completely protected. These results demonstrate that naturally occurring allelic differences in Mhc2ta have profound effects on the quantity of MHC II expression in the CNS and on immune cells and that this genetic variability also modulates susceptibility to autoimmune neuroinflammation.

MicroRNA targets in immune genes and the Dicer/Argonaute and ARE machinery components

We studied 613 genes which regulate immunity and, utilizing predictive algorithms, identified 285 genes as microRNA (miRNA or miR) targets. Of these, ∼250 are newly predicted gene–miR interactions. The frequency of predicted miRNA binding sites in immune gene 3′UTRs indicated preferential targeting of immune genes compared to the genome. Major targets include transcription factors, cofactors and chromatin modifiers whereas upstream factors, such as ligands and receptors (cytokines, chemokines and TLRs), were, in general, non-targets. About 10% of the immune genes were ‘hubs’ with eight or more different miRNAs predicted to target their 3′UTRs. Hubs were focused on certain key immune genes, such as BCL6, SMAD7, BLIMP1, NFAT5, EP300 and others. NF-κB and p53 do not themselves have binding sites for miRNAs but rather these pathways are targeted by miRNAs at downstream sites. MHC class II genes lacked miRNA targets but binding sites were identified in the CIITA gene and were shown experimentally to repress IFN-γ-induced MHC class II activation. Unexpectedly, factors involved in regulating message stability via AU-rich elements (ARE) were heavily targeted. Moreover, multiple components involved in the generation and effector functions of miRNAs (Dicer and Argonautes) were themselves miRNA targets suggesting that a subset of miRNAs may indirectly control their own production as well as other miRNAs.

The role of the 19S ATPase S6a in the transcriptional regulation of major histocompatibility class II (MHC II) genes (35.26)

Abstract MHC II molecules are glycoproteins that present intracellular antigens to CD4+ T cells and play an important role in induction and regulation of adaptive immune responses. MHC II molecules are regulated at the level of transcription by a master regulator, the class II transcriptional activator, CIITA, whose association with the MHC promoter is necessary for initiation of transcription. It is well established that one mechanism of regulating transcription is through degradation of factors by the 26S proteasome. The proteasome is composed of a 19S regulatory particle that recognizes ubiquitinated proteins and a 20S proteolytic core where the tagged proteins are degraded. Previous studies in yeast have demonstrated that the S6a ATPase of the 19S associates with actively transcribed genes, suggesting a non-degradative role for the ubiquitin-proteasome machinery. To further understand these roles in mammalian cells, we have investigated the role of S6a in regulating CIITA mediated MHC II transcription. Our research indicates that S6a is recruited to actively transcribing MHC II and CIITA genes and that RNAi mediated S6a knockdown correlates with decreased expression of MHC II and CIITA. Our study demonstrates a novel mechanism of regulating MHC II expression and will enhance our understanding of mammalian transcription. Research supported by the NMSS, the Georgia Cancer Coalition and GSU.

Inhibitory effects of anti-CII TA M1-RNA on IFN-γ induced major histocompatibility complex class II antigens expression on cultured human chondrocytes

Major histocompatibility complex class II (MHC-II) trans-activator (CII TA) has been shown to be required for constitutive and IFN-γ-induced MHC-II transcription. This study investigated the inhibitory effect of anti-CII TA M1-RNA on expression of MHC-II in chondrocytes in response to IFN-γ. M1-RNAs with different guide sequence (GS) recognizing 452 or 3408 sites in CII TA (M1-452-GS and M1-3408-GS, respectively) were cloned into pUC19 vector. Target mRNA (3176–3560) in CII TA was obtained from Raji cell and inserted into pGEM-7zf(+) plasmid. The recombinant M1-RNAs and their target mRNA were incubated in a cell-free condition. It showed that only M1-3408-GS could cleave the target mRNA exclusively. M1-3408-GS was also cloned into psNAV vector (named pA3408). Chondrocytes was stably transfected with pA3408 and expressions of classical MHC-II (HLA-DR, -DP, -DQ) were analyzed by Flow Cytometry. The level of CII TA mRNA was measured by RT-PCR. Peripheral blood mono-nucleated cells (PBMNCs) were stimulated by pA3408-positive chondrocytes in mixed lymphocyte reaction, and proliferation of PBMNCs and IL-2 mRNA were detected. The expression of HLA-DR and HLA-DP on pA3408-positive chondrocytes in response to IFN-γ decreased 73.00% ± 5.24%, 88.47% ± 2.02%, respectively ( P < 0.05); So did the content of CII TA mRNA (70.11% ± 5.79%, P < 0.05). Proliferation of PBMNCs and production of IL-2 mRNA were both inhibited by pA3408 in mixed lymphocyte reaction. This is the first description that anti-CII TA M1-RNA could prevent IFN-γ-induced CII TA transcription and results in a decreased MHC-II expression in chondrocytes.

Loss of Major Histocompatibility Class II (MHCII) Gene Expression in Diffuse Large B Cell Lymphoma (DLBCL) Is Not Generally Due to Genetic Mutations in the Coding Regions of CIITA or RFX.

Loss of expression of the MHCII in a subset of DLBCL cases studied by the Leukemia and Lymphoma Molecular Profiling Project (LLMPP) has been previously associated with extremely poor prognosis. Large genetic deletions of the MHC II loci were not seen in these cases. Furthermore, gene expression profiling analysis demonstrated that the MHCII gene expression was coordinated and most likely suppressed through altered transcription. We therefore investigated the possibility of small deletions or genetic mutations of 2 key transcriptional regulators of MHCII, CIITA and RFX, as possible causes of decreased MHCII expression in DLBCL. These transcription factors were chosen because mutations in the coding region of these proteins have been shown to cause the rare genetic disease, bare lymphocyte syndrome, in which MHCII expression is lost. We designed primers to amplify all coding exons of CIITA and RFX (a multimer containing RFX5, RFXB, and RFXAP), including internal splicing regions, in a minimal number of amplifications. DNA samples were amplified by 6 multiplex PCRs of genomic DNA, then the products sequenced with separate sequencing primers and compared to NCBI curated sequences. DLBCL DNA samples for which gene expression profiling data on MHCII expression was available, were obtained from the LLMPP research group. 23 of these samples were from the lowest 10% de novo untreated average MHCII expressers, 4 from non-de novo samples expressing MHCII in the same range, 4 from low MHCII expressers in the lowest 10–25% range, and 15 were primary mediastinal B cell lymphoma (PMBL) samples. The PMBL subset of DLBCL expresses MHCII at a lower range than other DLBCL. A number of other MHCII positive and negative lymphoma samples and cell lines were also sequenced. Although various SNPs and silent changes were noted, there were few small point mutations, deletions, or splicing mutations in the low MHCII DLBCL expressers that would explain loss of MHCII expression. In RJ2.2.5, an MHCII negative Burkitts lymphoma cell line derived from Raji, which is known to have only one CIITA allele expressing RNA lacking exons 11 and 12, the genomic deletion was sequenced. One of the lowest 10% MHCII expressing LLMPP samples had an insertional duplication that caused a frameshift in the C-terminus of all copies of the CIITA gene. Another tumor sample showing functional mutations was an MHCII negative T cell lymphoma (non-LLMPP), which had nonsense mutations in both RFXAP and RFX5, all copies. In conclusion, critical deletions or mutations were not common in the studied samples. These results confirmed previous data implying loss of MHCII expression in DLBCL was most likely due to altered transcriptional regulation, and indicate that this unfortunate circumstance may frequently be amenable to therapeutic intervention to upregulate the MHCII pathway.

Protein Kinase Cδ Is Essential to Maintain CIITA Gene Expression in B Cells

Expression of MHC class II genes requires CIITA. Although the transactivation function of CIITA is well characterized, the signaling events that regulate CIITA expression are less understood. In this study, we report that CIITA expression in B cells depends on protein kinase Cdelta (PKCdelta). PKCdelta controls CIITA gene transcription mainly via modulating CREB recruitment to the CIITA promoter without affecting CIITA mRNA stability. Inhibition of PKCdelta by a pharmacological inhibitor or knocking down of endogenous PKCdelta expression by small interfering RNA reduced CREB binding to the CIITA promoter. The decrease of CIITA gene expression in the presence of the PKCdelta inhibitor was prevented by ectopically expressing a constitutively active form of CREB. In addition, histone acetylation of the CIITA promoter is regulated by PKCdelta since the PKCdelta inhibitor treatment or PKCdelta small interfering RNA resulted in decreased histone acetylation. Taken together, our study reveals that PKCdelta is an important signaling molecule necessary to maintain CIITA and MHC class II expression in B cells.

ERK and p38 MAPK Signaling Pathways Negatively Regulate CIITA Gene Expression in Dendritic Cells and Macrophages

The CIITA is a master regulator for MHC class II expression, but the signaling events that control CIITA expression remain poorly understood. In this study, we report that both constitutive and IFN-gamma-inducible expression of CIITA in mouse bone marrow-derived dendritic cells (DC) and macrophages, respectively, are regulated by MAPK signals. In DC, the inhibitory effect of LPS on CIITA expression was prevented by MyD88 deficiency or pharmacological MAPK inhibitors specific for MEK (U0126) and p38 (SB203580), but not JNK (SP600125). In macrophages, LPS inhibited IFN-gamma-inducible CIITA and MHC class II expression without affecting expression of IFN regulatory factor-1 and MHC class I. Blocking ERK and p38 by MAPK inhibitors not only rescued LPS-mediated inhibition, but also augmented IFN-gamma induction of CIITA. Moreover, the induction of CIITA by IFN-gamma was enhanced by overexpressing MAPK phosphatase-1 that inactivates MAPK. Conversely, CIITA expression was attenuated in the absence of MAPK phosphatase-1. The down-regulation of CIITA gene expression by ERK and p38 was at least partly due to decreased histone acetylation of the CIITA promoter. Our study indicates that both MAPK and phosphatase play an important role for CIITA regulation in DC and macrophages.

Direct role of NF‐κB activation in Toll‐like receptor‐triggered HLA‐DRA expression

Microbial components, such as DNA containing immunostimulatory CpG motifs (CpG-DNA) and lipopolysaccharides (LPS), elicit the cell surface expression of MHC class II (MHC-II) through Toll-like receptor (TLR)/IL-1R. Here, we show that CpG-DNA and LPS induce expression of the HLA-DRA in the human B cell line, RPMI 8226. Ectopic expression of the dominant negative mutant of CIITA and RNA interference targeting the CIITA gene indicate that CIITA activation is not enough for the maximal MHC-II expression induced by CpG-DNA and LPS. Additionally, nuclear factor (NF)-kappaB activation is required for the CpG-DNA-activated and LPS-activated HLA-DRA expression, whereas IFN-gamma-induced MHC-II expression depends on CIITA rather than on NF-kappaB. Comprehensive mutant analyses, electrophoretic mobility shift assays and chromatin immunoprecipitation assays, reveal that the functional interaction of NF-kappaB with the promoter element is necessary for the TLR-mediated HLA-DRA induction by CpG-DNA and LPS. This novel mechanism provides the regulation of MHC-II gene expression with complexity and functional diversity.

Differential expression of MHC class II molecules in highly metastatic breast cancer cells is mediated by the regulation of the CIITA transcription: Implication of CIITA in tumor and metastasis development

We analyzed the differential gene expression between variants of MDA-MB-435 human breast cancer cell line that share an identical genetic background but have different metastatic ability. The major histocompatibility complex class II was found down-regulated in highly metastatic cells and correlated with MHC transactivator (CIITA) expression. Constitutive CIITA expression observed in poorly metastatic is driven by promoters III and IV of CIITA gene. Conversely, both promoters were ineffective in highly metastatic cells. The MHC class II and CIITA expression was restored in these cells upon stimulation with IFNγ or by the treatment with a hypomethylating agent. Both treatments induced USF-1 and IRF binding complexes to promoter IV but only IFNγ induced the binding of 435-Lung2 nuclear proteins to an ARE-1 site at the promoter III. Neither Southern blot nor bisulfite sequencing of promoter IV demonstrated strong hypermethylation of this promoter at the IFNγ-responsive elements such as GAS, E-box or IRF-1. We suggest that partial or hemimethylation of promoter IV is sufficient to silence the CIITA expression in highly metastatic cells and that this epigenetic mechanism is responsible for the lack of MHC-II expression. Forced CIITA expression restored the MHC-II antigen expression in 435-Lung2 cells and abrogates spontaneous lung metastasis in both SCID and nude mice but also affected the tumorigenicity in nude mice. The increase in NK cell infiltration in nude mice bearing CIITA-tumors correlated with sign of tumor cell apoptosis and the increase in the number of NK cells in the spleens, suggesting that NK cells might be responsible for the observed antitumor activity.

Different levels of control prevent interferon-gamma-inducible HLA-class II expression in human neuroblastoma cells.

The HLA class II expression is controlled by the transcriptional activator CIITA. The transcription of CIITA is controlled by different promoters, among which promoter-IV is inducible by IFN-gamma. We analysed the regulation of HLA class II molecules by IFN-gamma in a large series of human neuroblastoma cell lines. No induction of surface or intracellular HLA class II molecules and of specific mRNA was observed, in all neuroblastomas, with the exception of a nonprototypic cell line, ACN. In a large subset of neuroblastomas IFN-gamma induced expression of CIITA mRNA, derived from promoter-IV, which was not methylated. In contrast, in another subset of neuroblastomas, CIITA was not inducible by IFN-gamma and CIITA promoter-IV was either completely or partially methylated. Interestingly, the use of DNA demethylating agents restored CIITA gene transcriptional activation by IFN-gamma, but not HLA class II expression. The defect of HLA class II was not related to alterations in RFX or NF-Y transcription factors, as suggested by EMSA or RFX gene transfection experiments. In addition, the transfection of a functional CIITA cDNA failed to induce HLA class II expression in typical neuroblastoma cells. Confocal microscopy and Western blot analysis suggested a defective nuclear translocation and/or reduced protein synthesis in CIITA-transfected NB cells. Altogether, these data point to multiple mechanisms preventing HLA class II expression in the neuroblastoma, either involving CIITA promoter-IV silencing, or acting at the CIITA post-transcriptional level.

Generating MHC Class II+/Ii- phenotype after adenoviral delivery of both an expressible gene for MHC Class II inducer and an antisense Ii-RNA construct in tumor cells.

Tumor cells engineered by gene transduction to be MHC Class II+/Ii- are novel APCs capable of presenting endogenous tumor antigen epitopes to activate T helper cells. The MHC Class II+/Ii- tumor cell phenotype is created by transfecting genes for either CIITA or IFN-gamma, and inhibiting induced Ii mRNA by an Ii reverse gene construct (Ii-RGC). Adenoviral vectors are preferred for the delivery of such genes because of high infection efficiency and ubiquity of the adenoviral receptor on many cell types and tumors. Here we show that at 5 MOI (multiplicity of infection), recombinant adenoviruses with CIITA or IFN-gamma genes converted virtually all MC-38 colon adenocarcinoma cells and Renca renal carcinoma cells in culture to MHC Class II+/Ii+ cells. A single recombinant adenovirus with both genes for IFN-gamma and Ii-RGC (rAV/IFN-gamma/Ii-RGC) efficiently induced the MHC Class II+/Ii- phenotype. Injection of tumor nodules with rAV/Ii-RGC and rAV/CIITA/IFN-gamma combined with a suboptimal dose of rAV/IL-2 induced a potent antitumor immune response. The methods are adaptable for producing enhanced genetic vaccines, attenuated virus vaccines (eg, vaccinia), and ex vivo cell-based vaccines (dendritic and tumor cells).