CIITA Research Articles

MHC-II Deficiency Among Egyptians: Novel Mutations and Unique Phenotypes

MHC class II deficiency leads to defective CD4+ T-cell function that results from impaired antigen presentation. A genetic disorder in 1 of 4 genes results in this syndrome that is associated with the clinical phenotype of combined immunodeficiency. To describe the clinical, immunological, and molecular characteristics of 10 Egyptian patients from 9 different families having presented with MHC class II deficiency between 2012 and2017. An initial diagnosis based on the combination of clinical features and low HLA-DR expression by flow cytometry was confirmed by genetic analyses. Symptoms included failure to thrive (n= 9), persistent diarrhea (n= 5), and pneumonia (n= 8). Septicemia due to coagulase-negative staphylococci (n= 1) and Candida krusei (n= 1) was diagnosed. Nine patients orally received the live attenuated polio vaccine, of whom 3 developed acute flaccid paralysis thereafter. Nine patients received the BCG vaccine and none developed obvious signs of BCGitis. Four patients carried RFXANK gene mutations, 3 carried RFX5 gene mutations, 1 carried a CIITA gene mutation, and none carried RFXAP gene mutations. Six of the 7 detected mutations were previously unreported mutations: c.431T>C, c.247_250delTCAG, and c.600delG in the RFXANK gene; c.116+1G>A and c.715C>T in the RFX5 gene; and c.929delA in the CIITA gene. Given that Egypt is a North African country with a high rate of consanguinity, MHC class II deficiency is not rare. However, the molecular defects differ from those reported in nearby countries. Early diagnosis must be based on suspicious clinical signs and laboratory diagnosis because the defect can be missed by T-cell receptor excision circles based on neonatal screening.

Clinical, Immunological, and Molecular Findings in Five Patients with Major Histocompatibility Complex Class II Deficiency from India

Major histocompatibility complex (MHC) class II deficiency is a rare autosomal recessive form of primary immunodeficiency disorder (PID) characterized by the deficiency of MHC class II molecules. This deficiency affects the cellular and humoral immune response by impairing the development of CD4+ T helper (Th) cells and Th cell-dependent antibody production by B cells. Affected children typically present with severe respiratory and gastrointestinal tract infections. Hematopoietic stem cell transplantation (HSCT) is the only curative therapy available for treating these patients. This is the first report from India wherein we describe the clinical, immunological, and molecular findings in five patients with MHC class II deficiency. Our patients presented with recurrent lower respiratory tract infection as the most common clinical presentation within their first year of life and had a complete absence of human leukocyte antigen-antigen D-related (HLA-DR) expression on B cells and monocytes. Molecular characterization revealed novel mutations in RFAXP, RFX5, and CIITA genes. Despite genetic heterogeneity, these patients were clinically indistinguishable. Two patients underwent HSCT but had a poor survival outcome. Detectable level of T cell receptor excision circles (TRECs) were measured in our patients, highlighting that this form of PID may be missed by TREC-based newborn screening program for severe combined immunodeficiency.

FoxO1 is a regulator of MHC-II expression and anti-tumor effect of tumor-associated macrophages.

Macrophages are a critical component in host immune responses against tumor. In this work we investigated the role of forkhead box O1 (FoxO1) in the transcriptional regulation in macrophages, which affects the anti-tumor functions of tumor-associated macrophages (TAMs). First, we showed that TAMs expressed reduced levels of FoxO1, which was associated with their protumoral M2 polarization state. The suppression of FoxO1 expression in TAM was induced by the hypoxic condition in the tumor microenviroment. Next, we confirmed that FoxO1 positively regulates MHC-II genes by binding to the promoter region of Ciita gene, the master activator of multiple MHC-II genes. Loss of FoxO1 in TAMs resulted in reduced MHC-II expression. Furthermore, we used FoxO1 conditional knockout mice to show that FoxO1 deficiency in myeloid cells exacerbates tumor growth. These results demonstrate that the protumoral property of TAMs is induced by the hypoxia-triggered FoxO1 deficiency, which could be a potential target of novel anti-tumor therapies.

Association of Genetic Variation of CIITA and NTCP with Chronic Hepatitis B Virus Infection in Han Chinese Populations

Background: The CIITA plays a pivotal role in immune response by controlling HLA class II gene expression, and NTCP is a functional receptor for HBV. These variants may affect outcomes of HBV infection. Objectives: The aim of this study was to determine the association of CIITA and NTCP gene variants with chronic HBV infection and disease progression. Methods: Based on serological and clinical characteristics, 671 unrelated Han Chinese individuals were divided into three major groups: healthy subjects (170 cases), clearance subjects (199 cases), and subjects with chronic HBV infection (305 cases) consisted of 169 chronic hepatitis B, 68 liver cirrhosis, and 68 hepatocellular carcinoma patients. By logistic regression analysis, the rs2296651 AG + AA genotype decreased significantly in the chronic HBV infection group when compared to healthy subjects in dominant genetic models (OR = 0.41, 95%CI: 0.23 - 0.74). The rs9302456 CT + TT genotype and rs12882299 CT + CC significantly increased the risk of chronic HBV infection when compared to healthy subjects in dominant genetic models (rs9302456: OR = 2.24, 95%CI: 1.17 - 4.29; rs12882299: OR = 1.97, 95%CI: 1.27 - 3.07). Using the chronic hepatitis B patients as control group, our study showed that there was no association between CIITA and NTCP gene variants and HBV progression. Conclusions: Our study suggested that genetic variations in CIITA and NTCP were significantly associated with chronic HBV infection in Han Chinese populations, but not with HBV progression.

Characterization of Genomic Rearrangements Involving CIITA and SOCS1 Using Targeted Capture Sequencing of Archival Tissue Specimens

Introduction: Malignant lymphomas account for 5% of all newly diagnosed cancer cases per year and affect patients of all ages. Genomic rearrangements represent a pathogenic hallmark of most B-cell lymphoma entities and some are associated with an unfavorable clinical outcome. Recurrent structural genomic aberrations involving the MHC class II transactivator CIITA (located on chromosome 16p13) have been identified in multiple lymphoma subtypes in which they contribute to an immune escape phenotype. Moreover, inactivating mutations of the tumor suppressor gene SOCS1, located in close proximity to CIITA on chromosome 16, result in constitutively active JAK-STAT-signaling across a spectrum of lymphomas. Preliminary data from our group indicate that SOCS1 is also involved in recurrent rearrangements. However, more detailed study of these rearrangements was hampered due to the lack of analytic methods and platforms applicable to archival formalin-fixed and paraffin-embedded tissue (FFPET) specimens. Here, using FFPET biopsies, we sought to characterize the comprehensive landscape of CIITA and SOCS1rearrangement partner genes, determine the exact breakpoint anatomy, and study the functional impact of individual alterations.Methods: In order to select cases for DNA extraction and subsequent sequencing analysis we revisited the results of previously conducted fluorescence in-situ hybridization (FISH) experiments performed on lymphoma cases arranged on tissue microarrays. Cases were included based on the presence of rearrangements in CIITA, SOCS1 and the 9p24.1 locus. 92 specimens met these criteria and DNA was extracted using the Qiagen AllPrep DNA/RNA FFPE kit. An optimized 96-well-plate protocol was used for whole genome library construction (FFPET small gap protocol) and 16 to 18 libraries were multiplexed and pooled prior to capture using a custom Agilent SureSelect design targeting the aforementioned loci. Sequencing was performed on an Illumina HiSeq 2500. Predicted structural variants (SV) were generated with the computational tools DELLY and deStruct, and subsequently filtered based on read support. High confidence predictions were validated by either PCR-amplification of the genomic DNA spanning the breakpoint sequence followed by Sanger Sequencing, or customized FISH assays.Results: Library construction was successful in 68 cases (74%) and the mean coverage across the target regions for these libraries was above 600x with 97.7% of bases having an average depth of > 100x. After filtering, a list of 82 rearrangement events was generated consisting of 16 translocations (12 affecting CIITA and 4 affecting SOCS1), 44 deletions, 19 inversions and 3 duplications. Mapping of the translocation breakpoints revealed two distinct breakpoint cluster regions within intron 1 of CIITA and exon 2 of the SOCS1 gene. None of the exact breakpoints were recurrent and identification of rearrangement partner genes confirmed that CIITA and SOCS1 rearrange promiscuously. Furthermore, we demonstrate that some translocation events result in the generation of novel in-frame fusion transcripts (e.g. CIITA-PRDM16), warranting further functional characterization. Consistent with previous studies, intra-chromosomal alterations were detected in intron 1 of CIITA in48% of the cases while SOCS1 was shown to be frequently deleted (20% of cases).Overall, the concordance between FISH and targeted capture sequencing was 63%, reflecting the differences in sensitivity to detect large- and small-scale genomic events.Conclusions: Targeted capture sequencing, in conjunction with FISH, represents a valuable tool to explore the spectrum of genomic structural variants and rearrangement partner genes in archival lymphoma FFPET specimens. Future studies are required to address the functional impact of specific alterations and the utility of capture-sequencing-based assays for clinical decision-making. DisclosuresScott:NanoString Technologies: Patents & Royalties: named inventor on a patent for molecular subtyping of DLBCL that has been licensed to NanoString Technologies.

Genotypic and phenotypic predictors of inflammation in patients with chronic kidney disease.

In complex diseases such as chronic kidney disease (CKD), the risk of clinical complications is determined by interactions between phenotypic and genotypic factors. However, clinical epidemiological studies rarely attempt to analyse the combined effect of large numbers of phenotype and genotype features. We have recently shown that the relaxed linear separability (RLS) model of feature selection can address such complex issues. Here, it is applied to identify risk factors for inflammation in CKD. The RLS model was applied in 225 CKD stage 5 patients sampled in conjunction with dialysis initiation. Fifty-seven anthropometric or biochemical measurements and 79 genetic polymorphisms were entered into the model. The model was asked to identify phenotypes and genotypes that, when combined, could separate inflamed from non-inflamed patients. Inflammation was defined as a high-sensitivity C-reactive protein concentration above the median (5 mg/L). Among the 60 genotypic and phenotypic features predicting inflammation, 31 were genetic. Among the 10 strongest predictors of inflammation, 8 were single nucleotide polymorphisms located in the NAMPT, CIITA, BMP2 and PIK3CB genes, whereas fibrinogen and bone mineral density were the only phenotypic biomarkers. These results indicate a larger involvement of hereditary factors in inflammation than might have been expected and suggest that inclusion of genotype features in risk assessment studies is critical. The RLS model demonstrates that inflammation in CKD is determined by an extensive panel of factors and may prove to be a suitable tool that could enable a much-needed multifactorial approach as opposed to the commonly utilized single-factor analysis.

Repression of CIITA by the Epstein-Barr virus transcription factor Zta is independent of its dimerization and DNA binding.

Repression of the cellular CIITA gene is part of the immune evasion strategy of the γherpes virus Epstein-Barr virus (EBV) during its lytic replication cycle in B-cells. In part, this is mediated through downregulation of MHC class II gene expression via the targeted repression of CIITA, the cellular master regulator of MHC class II gene expression. This repression is achieved through a reduction in CIITA promoter activity, initiated by the EBV transcription and replication factor, Zta (BZLF1, EB1, ZEBRA). Zta is the earliest gene expressed during the lytic replication cycle. Zta interacts with sequence-specific elements in promoters, enhancers and the replication origin (ZREs), and also modulates gene expression through interaction with cellular transcription factors and co-activators. Here, we explore the requirements for Zta-mediated repression of the CIITA promoter. We find that repression by Zta is specific for the CIITA promoter and can be achieved in the absence of other EBV genes. Surprisingly, we find that the dimerization region of Zta is not required to mediate repression. This contrasts with an obligate requirement of this region to correctly orientate the DNA contact regions of Zta to mediate activation of gene expression through ZREs. Additional support for the model that Zta represses the CIITA promoter without direct DNA binding comes from promoter mapping that shows that repression does not require the presence of a ZRE in the CIITA promoter.

Systemic delivery of C II TA siRNA reduces disease severity in collagen-induced arthritis

Objective To examine the therapeutic effect of C Ⅱ TA inhibition in collagen-induced arthritis(CIA),using a delivery system tailored to target C ⅡTA gene by small interfering RNA (siRNA).Methods Mice with collagen-induced arthritis were injected intravenously with C Ⅱ TA siRNA.The clinical score was monitored for up to 4 weeks after treatment.The severity of inflammation of mouse joint was evaluated by histological examination.Real-time PCR was used to determine the cytokine mRNA expression.Cytokine production was measured by ELISA from serum.T cell proliferation was examined by MTT method.Results IFN-γ and IL-17 were elevated in CIA mice,but were iuhibited significantly by C Ⅱ TA siRNA either prevention or intervention of autoimmune arthritis.Collagen specific T cell proliferation was significantly suppressed.Increased level of IL-4 by T cells was observed in C Ⅱ TA siRNA treated group compared with that of control group.Conclusion Our findings indicate that systemic RNAi-mediated C Ⅱ TA gene silencing is effective in the treatment of CIA and regulateds the balance of Th1/Th2 differentiation. Key words: Rheumatoid arthritis; siRNA; Dendritic cell; IFN-γ; IL-4; IL-7

Novel Mutation in a Patient with MHC Class II Deficiency

Major Histocompatibility Complex Class II molecules (MHCII) are required for CD4+ T-cell development, peripheral T-cell tolerance, and the initiation of cellular and humoral immune responses. Congenital immunodeficiency resulting from functional MHCII deficiency has been described from germ line mutations within trans regulatory elements controlling MHCII transcription. Here, we present a child with congenital MHCII deficiency arising from a novel mutation within the CIITA gene.

Intracellular delivery of CII TA genes by polycationic liposomes for suppressed immune response of dendritic cells

Construction of an effective nanocomplex for suppression of CII TA proteins can be a potential strategy for inhibiting unwanted immune response.

Epigenetically modified pancreatic carcinoma PANC-1 cells can act as cancer vaccine to enhance antitumor immune response in mice.

Pancreatic cancer is characterized as a type of gastrointestinal tumor with a poor prognosis and high degree of malignancy. CIITA gene was found highly methylated in pancreatic carcinoma cell line PANC-1 and responsible for the low expression of major histocompatability complex II (MHC-II) that may lead to immune evasion. Here, we prepared pancreatic cancer vaccine with PANC-1 cells via epigenetic modification to enhance the MHC-II expression. Then the vaccine was injected into C57BL/6J mice and the effect was examined. Our study found that the vaccine could promote the proliferation of antigen-specific T cells, enhance the killing activity of cytotoxic T lymphocytes (CTLs), promote Th1-type cell-mediated secretion of cytokines IFN-γ and IL-2 while inhibiting Th2-type cell-mediated secretion of IL-4, and inhibit the secretion of TGF-β. Generally, the epigenetically modified vaccine could enhance the body's antitumor immune response, providing feasibility research on cancer vaccine for therapy of pancreatic cancer.

Multiple sclerosis susceptibility in a Brazilian sample, HLA and CIITA genes

Introduction: The multiple sclerosis (MS) is a chronic disease of the central nervous system that affects mainly young adults. This disease is characterized by the spread of demyelinating lesions in time and space. This condition may be influenced by genetic factors as heterogeneity, incomplete penetrance, polygenic inheritance and epigenetic factors, which makes this complex disease a challenge for geneticists. Objectives: The aim of this study was to investigate the association between HLA alleles from DQA1, DQB1 and DRB1 loci (6p21.3), genetic polymorphisms -168A/G (rs3087456) and +1614 G/C (rs4774) in the CIITA gene (16p13) and susceptibility to MS in a miscegenated sample from Rio de Janeiro state, RJ, Brazil. Method: DNA samples from 52 patients with relapsing remitting multiple sclerosis (MSRR) [21 males (40.38%) and 31 females (59.62%)] and 116 healthy controls [46 males (39.65%) and 70 females (60.35%)] matched by race, sex and age were analyzed by techniques of PCR, SSP-PCR, electrophoresis and DNA sequencing. Results: A significant association between MS and HLA-DRB1*15:01 allele was observed [p value=.002; Odds Ratio (OR)=3.2], especially in women (p=.001; OR=4.9), which remained statistically significant after Bonferroni correction. Furthermore, it was observed that the polymorphism +1614 G/C, “C/C” profile, in association with the allele DRB1*15:01 influences the increased susceptibility to MS in women (p=.029; OR=5.6). In addition, it was observed that the "G/G" profile in CIITA polymorphism +1614G/C may be associated with the resistance to MS (p=.02; OR=0.23), as well as HLA-DRB1*11:02 (p=.02, OR=0.5). The HLA-DQB1*06:02 allele also has been implicated as a possible susceptibility factor for MS (p=.02; OR=1.8, data not confirmed after Bonferronís correction). Conclusion: Together, these results reinforce the polygenic nature of MS, and proposed that the CIITA gene, which is the regulator of the expression of HLA-D, is an additive factor to DRB1*15:01 allele, previously described as a genetic susceptibility factor to MS in Brazilians.

The 26S proteasome in transcription elongation (561.2)

Accumulating evidence shows the 26S proteasome is involved in the regulation of gene expression. We and others have demonstrated that proteasome components bind to sites of gene transcription, regulate covalent modifications to histones, and are involved in the assembly of activator complexes in mammalian cells. The mechanisms by which the proteasome influences transcription remain unclear. Here, we define novel roles for each of the three 19S proteasome heterodimers, represented by the ATPases Sug1, S7, and S6a, in mammalian gene expression using the inflammatory gene CIITApIV. These ATPases are recruited to induced CIITApIV promoter and also associate with the coding regions. Additionally, these ATPases interact with elongation factor PTEFb complex members CDK9, Hexim and with Ser5 phosphorylated RNA Pol II. Both the generation of CIITA transcripts and efficient recruitment of RNA Pol II to CIITApIV are negatively impacted by siRNA mediated knockdown of these ATPases. Further, the histone H3 accumulation at CIITApIV promoter in the absence of ATPases, indicates failure in activation induced nucleosome eviction. It remains to be determined how these ATPases are utilized to drive requisite conformational changes in histones, transcription factors to regulate their interactions with CIITA gene. Together, these results define novel roles for 19S ATPases in promoting mammalian gene expression.

Variability in the CIITA gene interacts with HLA in multiple sclerosis

The human leukocyte antigen (HLA) is the main genetic determinant of multiple sclerosis (MS) risk. Within the HLA, the class II HLA-DRB1*15:01 allele exerts a disease-promoting effect, whereas the class I HLA-A*02 allele is protective. The CIITA gene is crucial for expression of class II HLA molecules and has previously been found to associate with several autoimmune diseases, including MS and type 1 diabetes. We here performed association analyses with CIITA in 2000 MS cases and up to 6900 controls as well as interaction analysis with HLA. We find that the previously investigated single-nucleotide polymorphism rs4774 is associated with MS risk in cases carrying the HLA-DRB1*15 allele (P=0.01, odds ratio (OR): 1.21, 95% confidence interval (CI): 1.04-1.40) or the HLA-A*02 allele (P=0.01, OR: 1.33, 95% CI: 1.07-1.64) and that these associations are independent of the adjacent confirmed MS susceptibility gene CLEC16A. We also confirm interaction between rs4774 and HLA-DRB1*15:01 such that individuals carrying the risk allele for rs4774 and HLA-DRB1*15:01 have a higher than expected risk for MS. In conclusion, our findings support previous data that variability in the CIITA gene affects MS risk, but also that the effect is modulated by MS-associated HLA haplotypes. These findings further underscore the biological importance of HLA for MS risk.

ZBTB32 Is an Early Repressor of the CIITA and MHC Class II Gene Expression during B Cell Differentiation to Plasma Cells

CIITA and MHC class II expression is silenced during the differentiation of B cells to plasma cells. When B cell differentiation is carried out ex vivo, CIITA silencing occurs rapidly, but the factors contributing to this event are not known. ZBTB32, also known as repressor of GATA3, was identified as an early repressor of CIITA in an ex vivo plasma cell differentiation model. ZBTB32 activity occurred at a time when B lymphocyte-induced maturation protein-1 (Blimp-1), the regulator of plasma cell fate and suppressor of CIITA, was minimally induced. Ectopic expression of ZBTB32 suppressed CIITA and I-A gene expression in B cells. Short hairpin RNA depletion of ZBTB32 in a plasma cell line resulted in re-expression of CIITA and I-A. Compared with conditional Blimp-1 knockout and wild-type B cells, B cells from ZBTB32/ROG-knockout mice displayed delayed kinetics in silencing CIITA during ex vivo plasma cell differentiation. ZBTB32 was found to bind to the CIITA gene, suggesting that ZBTB32 directly regulates CIITA. Lastly, ZBTB32 and Blimp-1 coimmunoprecipitated, suggesting that the two repressors may ultimately function together to silence CIITA expression. These results introduce ZBTB32 as a novel regulator of MHC-II gene expression and a potential regulatory partner of Blimp-1 in repressing gene expression.

Contrasting effects of IFNα on MHC class II expression in professional vs. nonprofessional APCs: Role of CIITA type IV promoter

We previously demonstrated that, in ex vivo cultures, IFNα downregulates the expression of MHC class II (MHCII) genes in human non-professional APCs associated with pancreatic islets. IFNα has an opposing effect on MHCII expression in professional APCs. In this study, we found that the mechanism responsible for the IFNα-mediated MHCII's downregulation in human MHCII-positive non-professional antigen presenting human non-hematopoietic cell lines is the result of the negative feedback system that regulates cytokine signal transduction, which eventually inhibits promoters III and IV of CIITA gene. Because the CIITA-PIV isoform is mostly responsible for the constitutive expression of MHCII genes in non-professional APCs, we pursued and achieved the specific knockdown of CIITA-PIV mRNA in our in vitro system, obtaining a partial silencing of MHCII molecules similar to that obtained by IFNα. We believe that our results offer a new understanding of the potential significance of CIITA-PIV as a therapeutic target for interventional strategies that can manage autoimmune disease and allograft rejection with little interference on the function of professional APCs of the immune system.

Inactivating Gene Alterations of MHC Class II Transactivator CIITA Are Recurrent in Primary Mediastinal B Cell Lymphoma and Hodgkin Lymphoma

Abstract 437 INTRODUCTION:Escape from immune surveillance has been proposed to play an important role in cancer and in particular in the pathogenesis of a subgroup of lymphomas. Using massively parallel sequencing, we have recently identified recurrent rearrangements of the CIITA gene in classical Hodgkin lymphoma (HL) and primary mediastinal B cell lymphoma (PMBCL) (Steidl et al, Nature 2011). Specifically, a gene fusion in the HL cell line KM-H2, involving the genes CIITA and FLJ27352, suppressed expression of CIITA-regulated MHC class II genes in a dominant-negative manner. Therefore, we sought to further investigate if gene alterations targeting the CIITA locus are recurrent somatic events in HL and PMBCL that lead to loss of gene function and thereby reduce immunogenicity of the malignant cells. PATIENTS AND METHODS: To characterize CIITA gene alterations, we comprehensively studied the cell lines DEV (nodular lymphocyte predominance HL-derived) and KARPAS1106P (PMBCL-derived) by whole-transcriptome paired-end sequencing (RNA-seq) and single nucleotide polymorphism arrays (Affymetrix SNP 6.0). To identify single nucleotide mutations in the CIITA coding sequence we studied additional 7 mediastinal biopsy specimens of PMBCL by RNA-seq. Fluorescence in-situ hybridization (FISH) was used to determine and validate structural and copy number alterations of the CIITA locus. 23 samples of PMBCL, 3 PMBCL-derived cell lines and 9 HL cell lines were studied for mutations and small deletions in CIITA intron 1 by long-range PCR and direct sequencing. CIITA and HLA-DR expression levels were determined by quantitative reverse transcriptase PCR. RESULTS: We identified chromosomal rearrangements affecting both alleles of CIITA in DEV cells resulting in expression of CIITA-PDL2, CIITA-SOCS1 and SOCS1-CIITA fusion transcripts. In KARPAS1106P cells chromosome 16 deletions result in the loss of one entire CIITA allele and partial loss of the other allele. As a consequence, in both cell lines wildtype CIITA and HLA-DR expression was undetectable. Genomic breakpoints within the CIITA rearrangements, together with previously identified translocations, defined a 1.6Kb breakpoint cluster region in CIITA Intron 1. Further analysis of this breakpoint cluster region revealed small intronic deletions in 10 of 23 (43%) PMBCL cases while no such deletions were detected in 18 diffuse large B cell lymphoma and 15 reactive lymph node samples. Furthermore, sequencing revealed multiple deletions ranging from 1–1948 bp and a high incidence of single nucleotide mutations in the breakpoint cluster region of the deleted alleles. The base pair changes were enriched for C to T and G to A transitions over other transitions and transversions, indicative of a somatic hypermutation process. We found significantly lower CIITA expression in cases with CIITA rearrangement and/or small intronic deletions compared to cases without CIITA alterations (p=0.044). RNAseq revealed three cases with non-synonymous coding-sequence mutations including a truncating mutation in exon 4. These changes were somatic in 3 cases with available matching normal DNA. DISCUSSION:CIITA is the target of multiple genomic hits including chromosomal translocation, deletion and coding sequence mutations in HL and PMBCL. In two HL and PMBCL cell lines this leads to complete gene inactivation and loss of HLA class II expression. These data characterize CIITA alterations as a recurrent underlying genomic event of the previously described immunophenotype of reduced HLA class II expression in a subset of PMBCL cases. Our data show that an immune escape mechanism utilized by tumor cells can be directly linked to somatically acquired alterations in cancer genomes. Disclosures:Siebert:Abbott/Vysis: .

MHC class II transactivator CIITA is a recurrent gene fusion partner in lymphoid cancers.

Chromosomal translocations are critically involved in the molecular pathogenesis of B-cell lymphomas, and highly recurrent and specific rearrangements have defined distinct molecular subtypes linked to unique clinicopathological features. In contrast, several well-characterized lymphoma entities still lack disease-defining translocation events. To identify novel fusion transcripts resulting from translocations, we investigated two Hodgkin lymphoma cell lines by whole-transcriptome paired-end sequencing (RNA-seq). Here we show a highly expressed gene fusion involving the major histocompatibility complex (MHC) class II transactivator CIITA (MHC2TA) in KM-H2 cells. In a subsequent evaluation of 263 B-cell lymphomas, we also demonstrate that genomic CIITA breaks are highly recurrent in primary mediastinal B-cell lymphoma (38%) and classical Hodgkin lymphoma (cHL) (15%). Furthermore, we find that CIITA is a promiscuous partner of various in-frame gene fusions, and we report that CIITA gene alterations impact survival in primary mediastinal B-cell lymphoma (PMBCL). As functional consequences of CIITA gene fusions, we identify downregulation of surface HLA class II expression and overexpression of ligands of the receptor molecule programmed cell death 1 (CD274/PDL1 and CD273/PDL2). These receptor-ligand interactions have been shown to impact anti-tumour immune responses in several cancers, whereas decreased MHC class II expression has been linked to reduced tumour cell immunogenicity. Thus, our findings suggest that recurrent rearrangements of CIITA may represent a novel genetic mechanism underlying tumour-microenvironment interactions across a spectrum of lymphoid cancers.

Abstract B29: Role of inflammatory-related gene expression in clear cell renal cell carcinoma

Abstract Renal cell carcinoma (RCC) is the 10th most common cancer in United States with clear-cell renal carcinoma (ccRCC) being the most common. Many genes and signaling pathways have been implicated in ccRCC development, including the inflammation pathway. However, less is known about how gene expression variation within this pathway influences ccRCC development and clinical outcomes. Gene expression in tumor and adjacent normal tissues from 93 patients with ccRCC was detected using a genome-wide expression array and a panel of 661 inflammation-related genes was then analyzed. Differential expression patterns between tumor and adjacent normal tissue were observed. Association between expression and recurrence or survival was evaluated with those genes showing significant association further tested in a validation set of 258 ccRCC tumor samples. A total of 151 genes with at least a 2-fold change in gene expression between adjacent normal and tumor were identified, with a majority of the genes being upregulated in the tumors. Eleven inflammation-related genes (BLNK, APP, IL8, CARD9, SOD2, IL10RA, GADD45G, ADORA3, CXCL1, CIITA, and NCF2) were found to be significantly associated with recurrence and/or overall survival in 93 ccRCC tumors and selected for further validation. Expression of CIITA and NCF2 was significantly associated with overall survival in the replication dataset with high expression levels associated with significant 2.65-fold (95% CI=1.28-5.49, P=0.0088) and a 2.26-fold (95% CI=1.12-4.58, P=0.023) increased risk of dying, respectively. Our results suggest expression of CIITA and NCF2 genes may be significant prognostic biomarkers for risk of dying in patients with ccRCC. Citation Information: Cancer Prev Res 2010;3(12 Suppl):B29.

Both MHC and non-MHC genes regulate inflammation and T-cell response after traumatic brain injury

Genetic regulation of autoimmune neuroinflammation is a well known phenomenon, but genetic influences on inflammation following traumatic nerve injuries have received little attention. In this study we examined the inflammatory response in a rat traumatic brain injury (TBI) model, with a particular focus on major histocompatibility class II (MHC II) presentation, in two inbred rat strains that have been extensively characterized in experimental autoimmune encephalomyelitis (EAE); DA and PVG. In addition, MHC and Vra4 congenic strains on these backgrounds were studied to give information on MHC and non-MHC gene contribution. Thus, allelic differences in Vra4, harboring the Ciita gene, was found to regulate expression of the invariant chain at the mRNA level, with a much smaller effect exerted by the MHC locus itself. Notably, however, at the protein level the MHC congenic PVG-RT1 av1 strain displayed much stronger MHCII + presentation, as shown both by immunolabeling and flow cytometry, than the PVG strain, dwarfing the effect of Ciita. The PVG-RT1 av1 strain had significantly more T-cell influx than both DA and PVG, suggesting regulation both by MHC and non-MHC genes. Finally, in terms of outcome, the EAE susceptible DA strain displayed a significantly smaller resulting lesion volume than the resistant PVG-RT1 av1 strain. These results provide additional support for a role of adaptive immune response after neurotrauma and demonstrate that outcome is significantly affected by host genetic factors.