Class II Transactivator Research Articles

Selective modulation of MHC class II chaperons by a novel IFN-γ-inducible class II transactivator variant in lung adenocarcinoma A549 cells

Class II transactivator (CIITA) plays a critical role in controlling major histocompatibility complex (MHC) class II gene expression. In this study, two novel alternatively spliced variants of human interferon (IFN)-γ-inducible CIITA, one missing exon 7 (CIITAΔE7), the other with TAG inserted at exon 4/5 junction (CIITA-TAG), were identified and characterized. Both variants are naturally occurring since they are present in primary cells. Unlike CIITA-TAG, CIITAΔE7 is expressed more abundantly in lung adenocarcinoma A549 cells than in the non-transformed counterpart BEAS-2B cells following IFN-γ stimulation. Transfection experiments showed that CIITAΔE7 induced a markedly lower level of surface HLA-DR, -DP, -DQ expression than CIITA-TAG in A549 cells but not in BEAS-2B cells, although both variants elicited similar amounts of total DR, DP, and DQ proteins. This differential effect was correlated with, in A549 cells, decreased expression of Ii and HLA-DM genes, along with increased expression of HLA-DO genes. Ii and HLA-DM are chaperons assisting in HLA class II assembly, while HLA-DO functions to inhibit endosomal peptide loading and HLA class II membrane transport. These findings raise the possibility that CIITAΔE7 interacts with unknown cancer-associated factors to selectively modulate genes involved in the assembly and transport of HLA class II molecules.

Transcriptional coactivator CIITA, a functional homolog of TAF1, has kinase activity

The Major Histocompatibility Complex (MHC) class II transactivator (CIITA) mediates activated immune responses and its deficiency results in the Type II Bare Lymphocyte Syndrome. CIITA is a transcriptional co-activator that regulates γ-interferon-activated transcription of MHC class I and class II genes. It is also a functional homolog of TAF1, a component of the general transcription factor complex TFIID. TAF1 and CIITA both possess intrinsic acetyltransferase (AT) activity that is required for transcription initiation. In response to induction by γ-interferon, CIITA and it's AT activity bypass the requirement for TAF1 AT activity. TAF1 also has kinase activity that is essential for its function. However, no similar activity has been identified for CIITA thus far.Here we report that CIITA, like TAF1, is a serine–threonine kinase. Its substrate specificity parallels, but does not duplicate, that of TAF1 in phosphorylating the TFIID component TAF7, the RAP74 subunit of the general transcription factor TFIIF and histone H2B. Like TAF1, CIITA autophosphorylates, affecting its interaction with TAF7. Additionally, CIITA phosphorylates histone H2B at Ser36, a target of TAF1 that is required for transcription during cell cycle progression and stress response. However, unlike TAF1, CIITA also phosphorylates all the other histones. The identification of this novel kinase activity of CIITA further clarifies its role as a functional homolog of TAF1 which may operate during stress and γ-IFN activated MHC gene transcription.

Histone deacetylase inhibitors activate CIITA and MHC class II antigen expression in diffuse large B‐cell lymphoma

Diffuse large B-cell lymphoma (DLBCL), the most common form of non-Hodgkin's lymphoma (NHL) diagnosed in the USA, consists of at least two distinct subtypes: germinal centre B (GCB) and activated B-cell (ABC). Decreased MHC class II (MHCII) expression on the tumours in both DLBCL subtypes directly correlates with significant decreases in patient survival. One common mechanism accounting for MHCII down-regulation in DLBCL is reduced expression of the MHC class II transactivator (CIITA), the master regulator of MHCII transcription. Furthermore, reduced CIITA expression in ABC DLBCL correlates with the presence of the transcriptional repressor positive regulatory domain-I-binding factor-1 (PRDI-BF1). However, the mechanisms underlying down-regulation of CIITA in GCB DLBCL are currently unclear. In this study, we demonstrate that neither PRDI-BF1 nor CpG hypermethylation at the CIITA promoters are responsible for decreased CIITA in GCB DLBCL. In contrast, histone modifications associated with an open chromatin conformation and active transcription were significantly lower at the CIITA promoters in CIITA(-) GCB cells compared with CIITA(+) B cells, which suggests that epigenetic mechanisms contribute to repression of CIITA transcription. Treatment of CIITA(-) or CIITA(low) GCB cells with several different histone deacetylase inhibitors (HDACi) activated modest CIITA and MHCII expression. However, CIITA and MHCII levels were significantly higher in these cells after exposure to the HDAC-1-specific inhibitor MS-275. These results suggest that CIITA transcription is repressed in GCB DLBCL cells through epigenetic mechanisms involving HDACs, and that HDACi treatment can alleviate repression. These observations may have important implications for patient therapy.

MHC Class II Transactivator Is an In Vivo Regulator of Osteoclast Differentiation and Bone Homeostasis Co-opted From Adaptive Immunity

The molecular networks controlling bone homeostasis are not fully understood. The common evolution of bone and adaptive immunity encourages the investigation of shared regulatory circuits. MHC Class II Transactivator (CIITA) is a master transcriptional co-activator believed to be exclusively dedicated for antigen presentation. CIITA is expressed in osteoclast precursors, and its expression is accentuated in osteoporotic mice. We thus asked whether CIITA plays a role in bone biology. To this aim, we fully characterized the bone phenotype of two mouse models of CIITA overexpression, respectively systemic and restricted to the monocyte-osteoclast lineage. Both CIITA-overexpressing mouse models revealed severe spontaneous osteoporosis, as assessed by micro-computed tomography and histomorphometry, associated with increased osteoclast numbers and enhanced in vivo bone resorption, whereas osteoblast numbers and in vivo bone-forming activity were unaffected. To understand the underlying cellular and molecular bases, we investigated ex vivo the differentiation of mutant bone marrow monocytes into osteoclasts and immune effectors, as well as osteoclastogenic signaling pathways. CIITA-overexpressing monocytes differentiated normally into effector macrophages or dendritic cells but showed enhanced osteoclastogenesis, whereas CIITA ablation suppressed osteoclast differentiation. Increased c-fms and receptor activator of NF-κB (RANK) signaling underlay enhanced osteoclast differentiation from CIITA-overexpressing precursors. Moreover, by extending selected phenotypic and cellular analyses to additional genetic mouse models, namely MHC Class II deficient mice and a transgenic mouse line lacking a specific CIITA promoter and re-expressing CIITA in the thymus, we excluded MHC Class II expression and T cells from contributing to the observed skeletal phenotype. Altogether, our study provides compelling genetic evidence that CIITA, the molecular switch of antigen presentation, plays a novel, unexpected function in skeletal homeostasis, independent of MHC Class II expression and T cells, by exerting a selective and intrinsic control of osteoclast differentiation and bone resorption in vivo.

Nitric Oxide and KLF4 Protein Epigenetically Modify Class II Transactivator to Repress Major Histocompatibility Complex II Expression during Mycobacterium bovis Bacillus Calmette-Guérin Infection

Pathogenic mycobacteria employ several immune evasion strategies such as inhibition of class II transactivator (CIITA) and MHC-II expression, to survive and persist in host macrophages. However, precise roles for specific signaling components executing down-regulation of CIITA/MHC-II have not been adequately addressed. Here, we demonstrate that Mycobacterium bovis bacillus Calmette-Guérin (BCG)-mediated TLR2 signaling-induced iNOS/NO expression is obligatory for the suppression of IFN-γ-induced CIITA/MHC-II functions. Significantly, NOTCH/PKC/MAPK-triggered signaling cross-talk was found critical for iNOS/NO production. NO responsive recruitment of a bifunctional transcription factor, KLF4, to the promoter of CIITA during M. bovis BCG infection of macrophages was essential to orchestrate the epigenetic modifications mediated by histone methyltransferase EZH2 or miR-150 and thus calibrate CIITA/MHC-II expression. NO-dependent KLF4 regulated the processing and presentation of ovalbumin by infected macrophages to reactive T cells. Altogether, our study delineates a novel role for iNOS/NO/KLF4 in dictating the mycobacterial capacity to inhibit CIITA/MHC-II-mediated antigen presentation by infected macrophages and thereby elude immune surveillance.

Adenosine signaling inhibits CIITA‐mediated MHC class II transactivation in lung fibroblast cells

Efficient antigen presentation by major histocompatibility complex (MHC) molecules represents a critical process in adaptive immunity. Class II transactivator (CIITA) is considered the master regulator of MHC class II (MHC II) transcription. Previously, we have shown that CIITA expression is upregulated in smooth muscle cells deficient in A2b adenosine receptor. Here, we report that treatment with the adenosine receptor agonist adenosine-5'N-ethylcarboxamide (NECA) attenuated MHC II transcription in lung fibro-blast cells as a result of CIITA repression. Further analysis revealed that NECA preferentially abrogated CIITA transcription through promoters III and IV. Blockade with a selective A2b receptor antagonist MRS-1754 restored CIITA-dependent MHC II transactivation. Forskolin, an adenylyl cyclase activator, achieved the same effect as NECA. A2b signaling repressed CIITA transcription by altering histone modifications and recruitment of key factors on the CIITA promoters in a STAT1-dependent manner. MRS-1754 blocked the antagonism of transforming growth factor beta (TGF-β) in CIITA induction by interferon gamma (IFN-γ), alluding to a potential dialogue between TGF-β and adenosine signaling pathways. Finally, A2b signaling attenuated STAT1 phosphorylation and stimulated TGF-β synthesis. In conclusion, we have identified an adenosine-A2b receptor-adenylyl cyclase axis that influences CIITA-mediated MHC II transactivation in lung fibroblast cells and as such have provided invaluable insights into the development of novel immune-modulatory strategies.

Both qualitative and quantitative genetic variation of MHC class II molecules may influence susceptibility to autoimmune diseases: The case of endemic pemphigus foliaceus

The MHC class II transactivator (CIITA) is a key regulator in expression of the HLA class II genes. It is well known that HLA-DRB1 genotypes have a strong influence on the risk of multifactorial autoimmune diseases, but the effect of CIITA genotypes remains controversial. We tested in a case-control study whether CIITA polymorphisms influence the risk of developing endemic pemphigus foliaceus (EPF) and whether CIITA and HLA-DRB1 interact as regards susceptibility to the disease. The rs4774 SNP is not associated to EPF, while rs3087456 in the CIITA gene promoter is associated with susceptibility [odds ratio (OR) = 2.6, p < 0.001 and OR = 2.0 p = 0.003 for genotypes G/G and G/A, respectively]. We suggest that the associations result from the effect of genetically controlled levels of CIITA on expression of the susceptible and protective HLA class II molecules. Remarkably, the interaction between CIITA and HLA-DRB1 genotypes is strong and additive. The OR for individuals having two susceptible HLA-DRB1 alleles is 14.1 in presence of the susceptible CIITA G/G or G/A genotypes and much lower (2.2) in presence of the protective CIITA A/A genotype. We conclude that quantitative as well as qualitative variation of HLA class II molecules have an effect on the risk of an individual developing EPF.

Abstract 563: Axl Regulates Major Histocompatibility Complex Class II (MHC-II) Expression through STAT1 Pathway in Smooth Muscle Cells

Introduction Major histocompatibility complex class II (MHC-II) is upregulated in vascular smooth muscle cells (VSMCs) in response to injury. Previously we found that Axl, a receptor tyrosine kinase, is important for vascular remodeling via regulation of MHC-II in vivo . The aim of this study was to identify Axl-mediated intracellular signaling that is responsible for upregulation of MHC-II in VSMCs. Methods and Results Mouse aortic smooth muscle cells (MASMCs) were isolated from Axl wild type (Axl+/+) or knockout (Axl-/-) littermates from our colony. Cells were treated with interferon gamma (IFNγ; 1,000U/ml) or PBS for various times (10min-24hrs). There was no difference in apoptosis as measured by cleaved caspase-3 in Axl-/- vs. Axl+/+ after treatment with IFNγ. Likewise, phosphorylation levels of Akt were similar between the groups. Expression of MHC-II mRNA and protein was significantly lower in Axl-/- vs. Axl+/+ MASMCs. Levels of major histocompatibility class II transactivator (CIITA) mRNA was also significantly reduced in Axl-/- MASMCs. Reduced expression of number of cytokines and chemokines was observed in Axl-/- vs. Axl+/+ MASMCs after treatment with IFNγ. Similar to our in vivo results, a complement component 3 (C3) expression was drastically reduced in Axl-/- vs. Axl+/+ MASMCs. We found that STAT1, a major regulator for CIITA-MHC-II and C3 pathways, was not phosphorylated in Axl-/- vs. Axl+/+ MASMCs upon IFNγ treatment. Conclusions This is the first study that demonstrates that Axl affects STAT1-signaling in VSMCs. Our findings suggest that Axl contributes to vascular remodeling via enhancement of pro-inflammatory signals in smooth muscle cells.

CIITA rs4774 and rs6498122 polymorphisms are associated with oral lichen planus in Chinese people: a case–control study

Oral lichen planus (OLP) is a common autoimmune inflammatory disorder that is difficult to cure, and its pathogenesis is still largely unknown. The major histocompatibility complex (MHC) class II transactivator (CIITA) gene has been reported to be an important candidate in some classical autoimmune diseases, and certain single nucleotide polymorphisms (SNPs) in CIITA have been confirmed to be associated with susceptibility to some autoimmune diseases. We conducted this research to investigate the existence of any correlation between OLP and SNPs in CIITA. A case-control study was performed to genotype 15 SNPs in the CIITA gene from 42 patients with OLP and from 86 controls; this was carried out by the PCR and then by a locus-specific single-base extension reaction. Allele detection was performed using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The SNP rs4774 variant in exon 11 (+1614G/C, Gly500Ala) of CIITA is significantly associated with OLP in healthy persons, both in genotype frequency and in allele frequency. Another intronic SNP, rs6498122, showed significant differences only in allele frequency. In conclusion, our data show that the two SNPs rs4774 and rs6498122 are associated with OLP and could also indicate the autoimmune characteristics of OLP.

Exosomes from CIITA-Transfected CT26 Cells Enhance Anti-tumor Effects

To study anti-tumor effects of exosomes from class II transactivator (CIITA) gene transfected CT26 cells. In this study, we established an MHC class II molecule-expressing murine colon cancer cell line (CT26-CIITA) by transduction of the CIITA gene. Immune effects in vitro and tumor protective results in vivo were tested and monitored. Exosomes from CT26-CIITA cells were found to contain a high level of MHC class II protein. When loaded on dendritic cells (DCs), exosomes from CT26-CIITA cells significantly increased expression of MHC class II molecules, CD86 and CD80, as compared to exosomes from CT26 cells. In vitro assays using co-culture of immunized splenocytes and exosome-loaded DCs demonstrated that CIITA- Exo enhanced splenocyte proliferation and IFN-γ production of CD4+T cells, while inhibiting IL-10 secretion. In addition, compared to exosomes from CT26 cells, CT26-CIITA-derived exosomes induced higher TNF-α and IL-12 mRNA levels. A mouse tumour preventive model showed that CT26-CIITA derived exosomes significantly inhibited tumour growth in a dose-dependent manner and significantly prolonged the survival time of tumour- bearing mice. Our findings indicate that CT26-CIITA-released exosomes are more efficient to induce anti-tumour immune responses, suggesting a potential role of MHC class II-containing tumour exosomes as cancer vaccine candidates.

Downregulating immunogenicity of Schwann cells via inhibiting a potential target of class II transactivator (CIITA) gene

Immunological rejection induced by allogeneic Schwann cells remains a problem for construction of artificial nerves. Class II transactivator (CIITA) gene is a chief regulator of major histocompatibility complex class II (MHC II) molecules which contributes to the immunogenicity of Schwann cells. This study aimed to downregulate MHC II expression by suppressing CIITA expression, therefore reducing the immunogenicity of Schwann cells. Recombinant siRNA expression vectors targeting the CIITA gene were produced and subsequently transfected into rat RSC96 Schwann cells. Interferon (IFN)-γ was used to augment immunological rejection of RSC96 cells. The mRNA levels of CIITA and MHC II were assessed by fluorescence quantitative PCR. The protein levels of MHC II were determined using flow cytometry assays. Finally, the immunogenicity of RSC96 cells was analyzed using mixed lymphocytes reactions. Results indicated the expression of MHC II molecules was at a low level in cultured RSC96 cells, while significantly elevated after treatment with IFN-γ. Concurrent treatment with the constructed CIITA siRNAs efficiently downregulated the mRNA levels of CIITA and MHC II in RSC96 cells at 48 h post-transfection. MHC II protein levels were also significantly reduced after CIITA siRNAs transfection. Correspondingly, the immunogenicity of RSC96 cells was significantly downregulated post-transfection. These studies suggest suppressing CIITA gene was efficient in reducing MHC II expression and thus decreasing the immunogenicity of rat Schwann cells.

CIITA and Its Dual Roles in MHC Gene Transcription

Class II transactivator (CIITA) is a transcriptional coactivator that regulates γ-interferon-activated transcription of Major Histocompatibility Complex (MHC) class I and II genes. As such, it plays a critical role in immune responses: CIITA deficiency results in aberrant MHC gene expression and consequently in autoimmune diseases such as Type II bare lymphocyte syndrome. Although CIITA does not bind DNA directly, it regulates MHC transcription in two distinct ways – as a transcriptional activator and as a general transcription factor. As an activator, CIITA nucleates an enhanceosome consisting of the DNA binding transcription factors RFX, cyclic AMP response element binding protein, and NF-Y. As a general transcription factor, CIITA functionally replaces the TFIID component, TAF1. Like TAF1, CIITA possesses acetyltransferase (AT) and kinase activities, both of which contribute to proper transcription of MHC class I and II genes. The substrate specificity and regulation of the CIITA AT and kinase activities also parallel those of TAF1. In addition, CIITA is tightly regulated by its various regulatory domains that undergo phosphorylation and influence its targeted localization. Thus, a complex picture of the mechanisms regulating CIITA function is emerging suggesting that CIITA has dual roles in transcriptional regulation which are summarized in this review.

The MHC-II transactivator CIITA, a restriction factor against oncogenic HTLV-1 and HTLV-2 retroviruses: similarities and differences in the inhibition of Tax-1 and Tax-2 viral transactivators.

The activation of CD4+ T helper cells is strictly dependent on the presentation of antigenic peptides by MHC class II (MHC-II) molecules. MHC-II expression is primarily regulated at the transcriptional level by the AIR-1 gene product CIITA (class II transactivator). Thus, CIITA plays a pivotal role in the triggering of the adaptive immune response against pathogens. Besides this well known function, we recently found that CIITA acts as an endogenous restriction factor against HTLV-1 (human T cell lymphotropic virus type 1) and HTLV-2 oncogenic retroviruses by targeting their viral transactivators Tax-1 and Tax-2, respectively. Here we review our findings on CIITA-mediated inhibition of viral replication and discuss similarities and differences in the molecular mechanisms by which CIITA specifically counteracts the function of Tax-1 and Tax-2 molecules. The dual function of CIITA as a key regulator of adaptive and intrinsic immunity represents a rather unique example of adaptation of host-derived factors against pathogen infections during evolution.

Preliminary results in quantitation of HLA-DRA by real-time PCR: a promising approach to identify immunosuppression in sepsis

IntroductionReduced monocyte human leukocyte antigen (mHLA)-DR surface expression in the late phase of sepsis is postulated as a general biomarker of sepsis-induced immunosuppression and an independent predictor of nosocomial infections.However, traditional monitoring of mHLA-DR by flow cytometry has disadvantages due to specific laboratory requirements. An mRNA-based HLA-DR monitoring by polymerase chain reaction (PCR) would improve the clinical usage and facilitate conduction of large multicenter studies. In this study, we evaluated an mRNA-based HLA-DR monitoring by quantitative real-time PCR (qRT-PCR) as an alternative method to traditional flow cytometry.MethodsFifty-nine patients with sepsis and blood culture growing pathogenic bacteria were studied. Blood samples were collected at day 1 or 2 after admission, for measurement of mHLA-DR by flow cytometry and mRNA expression of HLA-DRA and class II transactivator (CIITA) by qRT-PCR. Blood samples from blood donors were used as controls (n = 30).ResultsA significant reduced expression of mHLA-DR, HLA-DRA, and CIITA was seen in septic patients compared with controls. HLA-DRA mRNA level in whole blood was highly correlated with surface expression of mHLA-DR.ConclusionsPatients with sepsis display a diminished expression of HLA-DR at the monocyte surface as well as in the gene expression at the mRNA level. The mRNA expression level of HLA-DRA monitored by qRT-PCR correlates highly with surface expression of HLA-DR and appears to be a possible future biomarker for evaluation of immunosuppression in sepsis.

NLRC5: a key regulator of MHC class I-dependent immune responses

The expression of MHC class I molecules is crucial for the initiation and regulation of adaptive immune responses against pathogens. NOD-, LRR- and CARD-containing 5 (NLRC5) was recently identified as a specific transactivator of MHC class I genes (CITA). NLRC5 and the master regulator for MHC class II genes, class II transactivator (CIITA), interact with similar MHC promoter-bound factors. Here, we provide a broad overview of the molecular mechanisms behind MHC class I transcription and the role of the class I transactivator NLRC5 in MHC class I-dependent immune responses.

Identification of a Novel Protein-Coding Gene (TIHL) and Its Functional Relevance in Myeloid Cells.

AbstractAbstract 2333The gene locus BX648577 (FLJ27352/hypothetical LOC145788) was recently identified as part of a gene fusion with Class II Transactivator (CIITA) in Hodgkin's lymphoma using whole-transcriptome paired-end sequencing (Steidl C. et al., Nature 2011). While CIITA has been extensively studied, no function is known for BX648577. We therefore sought to study its expression and biological function in normal and malignant hematopoietic cells, including its effects on cellular proliferation, clonogenicity and cell death. Here, we report the endogenous expression of functional mRNA and protein encoded by the BX648577 locus, thereby establishing it as a novel protein-coding gene, which we named TIHL(translocated in Hodgkin's lymphoma).Initial comparative analysis of the 13–16kDa predicted BX648577 (TIHL) protein sequences in various species revealed high evolutionary conservation (≥54%), including invertebrates (e.g. Saccoglossus kowalevskii). We generated a TIHL(BX648577) -specific antibody and identified endogenous protein expression of TIHL by western blot analysis in several cell lines, including 293T cells, and myeloid NB4 and KG1a cells. Expression analysis was performed in several leukemia and lymphoma cell lines by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). And we found that TIHL mRNA was widely and variably expressed, with particularly high expression in NB4 and THP1 myeloid leukemia cells, 5 fold (p=2.02e-6) and 9 fold (p=6.96e-7) higher, respectively, when compared to healthy donor peripheral blood CD14+ monocytes. In addition, TIHL mRNA expression was detectable in sorted, primary human and murine bone marrow derived hematopoietic stem cells (Lin−CD34+CD38− (human) or Lin−c-Kit+Sca-1+ (mouse)) and progenitor cells (Lin−CD34+CD38+ (human) or Lin−c-Kit+Sca-1−(mouse)), as well as human healthy donor mature peripheral blood mononuclear cells with the most prominent expression in Glycophorin A and CD56 positive cells by qRT-PCR.Utilizing primary immature murine bone marrow cells (sorted Lin−c-Kit+ cells) as well as acute myeloid leukemia cell lines (NB4, KG1a); we assessed the biological consequences of modulating TIHL expression. Knockdown of TIHL by two independent shRNAs with knockdown efficiencies of 77% and 50% respectively, led to significantly decreased leukemic cell growth in suspension culture. We additionally observed decreased clonogenic capacity in both cell lines when the cells were transduced with either sh#1 or sh#2, with 76% (p =1.19e-3) and 77% (p=8.36e-4) inhibition in clonogenicity in NB4 cells when compared to cells transduced with a non-silencing control vector. Similarly, we observed 67% (p=3.28e-3) and 72% (p=4.9e-4) inhibition of clonogenicity in KG1a cells. In addition, knockdown of TIHL led to a 7–14 fold increase in specific apoptosis. We did not find changes indicative of differentiation when we analyzed cellular morphology and surface protein expression. We cloned the full length cDNA of TIHL of human and mouse into a lentiviral expression vector. While ectopic expression of human or murine TIHL in sorted Lin−c-Kit+cells did not lead to a change in clonogenicity when compared to the empty control vector, we found a 1.5 fold increase in colony forming capacity (p=0.0161), and a 2 fold increase in cell growth (p<0.05) in NB4 cells upon TIHL overexpression.Finally, in silico domain analysis suggests that TIHL may function as an adaptor protein and may be involved in facilitating previously established signal transduction pathways.In summary, we here identify TIHL (former gene locus BX648577) as a novel protein-coding gene. We show that TIHL is expressed at the mRNA and protein level with wide expression in both leukemia cell lines and normal hematopoietic cells, including stem cells. We demonstrate that TIHL is functionally relevant and that it is involved in the regulation of growth in myeloid cells. Further studies are warranted to better understand the biological role of TIHL, to examine its precise mechanisms of action, and to delineate the biological pathways implicated in its function. Disclosures:No relevant conflicts of interest to declare.

Mutations of STAT6 DNA Binding Domain Are Characteristic of PMBL and Belong to the Genetic Signature of This Entity.

AbstractAbstract 2645Primary mediastinal B-cell lymphoma (PMBL) is a separate entity of aggressive B-cell lymphoma and accounts for about 5% of aggressive lymphomas according to the World Health Organization (WHO) classification. This lymphoma affects young patients and presents as a anterior mediastinal mass consisting of large B-cells that usually express little if any surface or cytoplasmic immunoglobulin and major histocompatibility complex class I and/or class II molecules. PMBL display constitutive activation of janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway, also observed in Hodgkin lymphoma and is characterized by heterozygous missense mutations of STAT6 DNA binding domain that are not observed in diffuse large B-cell lymphoma.To determine whether these mutations could represent a diagnostic or prognostic marker useful in the clinic, we developed a PCR HRM (High Resolution Melting) assay to screen for mutations in STAT6 exon 12, which had been previously identified as a hotspot mutation. We analyzed a series of 80 patients with a mediastinal biopsy and/or histological review of PMBL, enrolled in clinical trials of the GELA (LNH-03) or the GOELAMS (075). DNAs were extracted from FFPE (formalin-fixed and paraffin-embedded) samples with QIAmp DNA mini kit (QIAGEN®) for GELA samples and fully automated Siemens Healthcare Diagnostics proprietary experimental method based on magnetic beads for GOELAMS samples. Sixteen DNAs were not analyzed due to poor amplification in PCR and four DNAs were not analyzed due to other reasons (refused protocol, inclusion at relapse, not included in the tissue microarray/TMA). All cases with abnormal denaturation curves were analyzed by Sanger sequencing. Of the remaining 60 DNA samples, we detected 20 mutated tumors in exon 12 (30%). These mutations were heterozygous single nucleotide variants, often multiple (10 cases, 8 double hit mutations and 2 triple hit mutations) and targeted mostly amino acids 417 (9 hits) and 419 (7 hits). Immunohistochemical analysis of TMA showed that the mutated tumors expressed CD23 (14/20) and MAL proteins (13/15), more frequently than non-mutated ones (10/33, p= 0.009 and 15/27, p=0.049, respectively). Interestingly, these proteins are encoded by genes belonging to the transcriptional signature of PMBL as defined by gene expression profiling in previous studies, There were no significant difference in the clinical presentation between patients with a STAT6 mutation and non-mutated cases regarding age, sex, performance status, Ann Arbor stage, LDH, IPI, treatment response, progression free survival and overall survival. However we noted an absence of elevated β2-microglobulin (1 case elevated/18) and an absence of albumin < 35 g/dl (0 case/8) in the mutated tumors compared with non-mutated tumors (11/31, p= 0.003 and 9/21, p< 0.05, respectively). Finally, we analyzed the correlations of these mutations with cytogenetic abnormalities including CIITA (Class II Transactivator) rearrangement, PD-L1/2 (Programmed death ligand) rearrangement and PD-L1/2 copy number alterations. Indeed, CIITA rearrangements have been described in 38% of PMBL and PD-L1/2 amplification were reported in 63% of PMBL. In this series of patients, CIITA rearrangements were present in 15/49 (31%) and PD-L1/2 low (3–4 copies) and high (>4 copies) level amplification observed in 33/52 (63%) of cases. It is interesting to note that STAT6-mutated cases frequently harbor CIITA rearrangement compared to STAT6-non-mutated cases (9/18 vs 6/31, p= 0.05) as well as PD-L1/2 low level copy number gains (12/19 vs 10/32, p= 0.02), suggesting that the combination of specific genomic alterations cooperate during PMBL oncogenesis.In conclusion, the frequency of this mutation suggests an in vivo selection during PMBL lymphomagenesis. The correlations with the clinico-biological data suggest that STAT6 mutations may be searched for in routine FFPE samples and may help to establish a molecular definition of PMBL that is characterized by alterations of the JAK-STAT pathway. Further investigations are required to identify the mutational mechanisms involved and the oncogenic function associated with these mutations. Disclosures:No relevant conflicts of interest to declare.

PML promotes MHC class II gene expression by stabilizing the class II transactivator

Promyelocytic leukemia (PML) nuclear bodies selectively associate with transcriptionally active genomic regions, including the gene-rich major histocompatibility (MHC) locus. In this paper, we have explored potential links between PML and interferon (IFN)-γ-induced MHC class II expression. IFN-γ induced a substantial increase in the spatial proximity between PML bodies and the MHC class II gene cluster in different human cell types. Knockdown experiments show that PML is required for efficient IFN-γ-induced MHC II gene transcription through regulation of the class II transactivator (CIITA). PML mediates this function through protection of CIITA from proteasomal degradation. We also show that PML isoform II specifically forms a stable complex with CIITA at PML bodies. These observations establish PML as a coregulator of IFN-γ-induced MHC class II expression.

Essential Cell-Autonomous Role for Interferon (IFN) Regulatory Factor 1 in IFN-γ-Mediated Inhibition of Norovirus Replication in Macrophages

Noroviruses (NVs) cause the majority of cases of epidemic nonbacterial gastroenteritis worldwide and contribute to endemic enteric disease. However, the molecular mechanisms responsible for immune control of their replication are not completely understood. Here we report that the transcription factor interferon regulatory factor 1 (IRF-1) is required for control of murine NV (MNV) replication and pathogenesis in vivo. This led us to studies documenting a cell-autonomous role for IRF-1 in gamma interferon (IFN-γ)-mediated inhibition of MNV replication in primary macrophages. This role of IRF-1 in the inhibition of MNV replication by IFN-γ is independent of IFN-αβ signaling. While the signal transducer and activator of transcription STAT-1 was also required for IFN-γ-mediated inhibition of MNV replication in vitro, class II transactivator (CIITA), interferon regulatory factor 3 (IRF-3), and interferon regulatory factor 7 (IRF-7) were not required. We therefore hypothesized that there must be a subset of IFN-stimulated genes (ISGs) regulated by IFN-γ in a manner dependent only on STAT-1 and IRF-1. Analysis of transcriptional profiles of macrophages lacking various transcription factors confirmed this hypothesis. These studies identify a key role for IRF-1 in IFN-γ-dependent control of norovirus infection in mice and macrophages.

Variable Resistance of RMS to InterferonγSignaling

Aims. Chimeric T cells directed to the γ-subunit of the fetal acetylcholine receptor (fAChR) produce large amounts of interferon-γ (IFNγ) on coculture with fAChR-expressing rhabdomyosarcoma (RMS) cells prior to RMS cell death. The aim of this study was to elucidate whether IFNγ blocks proliferation and survival of RMS cells and modulates expression of genes with relevance for cytotoxicity of chimeric T cells. Methods. Expression levels of IFNγ receptor (IFNGR), AChR, MHCI, MHCII, and CIITA (class II transactivator) by RMS were checked by flow cytometry, qRT-PCR, and western blot. Proliferation and cell survival were investigated by annexin V and propidium iodide staining and MTT (thiazolyl-blue-tetrazolium-bromide) assay. Key phosphorylation and binding sites of IFNGRs were checked by DNA sequencing. Results. IFNγ treatment blocked proliferation in 3 of 6 RMS cell lines, but reduced survival in only one. IFNGR was expressed at levels comparable to controls and binding sites for JAK and STAT1 were intact. Induction of several target genes (e.g., AChR, MHCI, and MHCII) by IFNγ was detected on the RNA level but not protein level. Conclusions. IFNγ does not significantly contribute to the killing of RMS cells by fAChR directed chimeric T cells. Signalling downstream of the IFNR receptor, including the posttranscriptional level, is impaired in most RMS cell lines.