Antigen-independent, autonomous B cell receptor signaling drives activated B cell DLBCL.

Diffuse large B cell lymphoma of activated B cell type (ABC-DLBCL), a major cell-of-origin DLBCL subtype, is characterized by chronic active B cell receptor (BCR) signaling and NF-κB activation, which can be explained by activating mutations of the BCR signaling cascade in a minority of cases. We demonstrate that autonomous BCR signaling, akin to its essential pathogenetic role in chronic lymphocytic leukemia (CLL), can explain chronic active BCR signaling in ABC-DLBCL. 13 of 18 tested DLBCL-derived BCR, including 12 cases selected for expression of IgM, induced spontaneous calcium flux and increased phosphorylation of the BCR signaling cascade in murine triple knockout pre-B cells without antigenic stimulation or external BCR crosslinking. Autonomous BCR signaling was associated with IgM isotype, dependent on somatic BCR mutations and individual HCDR3 sequences, and largely restricted to non-GCB DLBCL. Autonomous BCR signaling represents a novel immunological oncogenic driver mechanism in DLBCL originating from individual BCR sequences and adds a new dimension to currently proposed genetics- and transcriptomics-based DLBCL classifications.

Autonomous B-cell receptor signaling and genetic aberrations in chronic lymphocytic leukemia-phenotype monoclonal B lymphocytosis in siblings of patients with chronic lymphocytic leukemia.

Clonal expansion of CD5-expressing B cells, commonly designated as monoclonal B lymphocytosis (MBL), is a precursor condition for chronic lymphocytic leukemia (CLL). The mechanisms driving subclinical MBL B-cell expansion and progression to CLL, occurring in approximately 1% of affected individuals, are unknown. An autonomously signaling B-cell receptor (BCR) is essential for the pathogenesis of CLL. The objectives of this study were functional characterization of the BCR of MBL in siblings of CLL patients and a comparison of genetic variants in MBL-CLL sibling pairs. Screening of peripheral blood by flow cytometry detected 0.2-480 clonal CLL-phenotype cells per microliter (median: 37/µL) in 34 of 191 (17.8%) siblings of CLL patients. Clonal BCR isolated from highly purified CLL-phenotype cells induced robust calcium mobilization in BCR-deficient murine pre-B cells in the absence of external antigen and without experimental crosslinking. This autonomous BCR signal was less intense than the signal originating from the CLL BCR of their CLL siblings. According to genotyping by single nucleotide polymorphism array, whole exome, and targeted panel sequencing, CLL risk alleles were found with high and similar prevalence in CLL patients and MBL siblings, respectively. Likewise, the prevalence of recurrent CLL-associated genetic variants was similar between CLL and matched MBL samples. However, copy number variations and small variants were frequently subclonal in MBL cells, suggesting their acquisition during subclinical clonal expansion. These findings support a stepwise model of CLL pathogenesis, in which autonomous BCR signaling leads to a non-malignant (oligo)clonal expansion of CD5+ B cells, followed by malignant progression to CLL after acquisition of pathogenic genetic variants.

Acquired N-Linked Glycosylation Motifs in B-Cell Receptors of Primary Cutaneous B-Cell Lymphoma and the Normal B-Cell Repertoire.

Primary cutaneous follicle center lymphoma (PCFCL) is a rare mature B-cell lymphoma with an unknown etiology. PCFCL resembles follicular lymphoma (FL) by cytomorphologic and microarchitectural criteria. FL B cells are selected for N-linked glycosylation motifs in their B-cell receptors (BCRs) that are acquired during continuous somatic hypermutation. The stimulation of mannosylated BCR by lectins on the tumor microenvironment is therefore a candidate driver in FL pathogenesis. We investigated whether the same mechanism could play a role in PCFCL pathogenesis. Full-length functional variable, diversity, and joining gene sequences of 18 PCFCL and 8 primary cutaneous diffuse large B-cell lymphoma, leg-type were identified by unbiased Anchoring Reverse Transcription of Immunoglobulin Sequences and Amplification by Nested PCR and BCR reconstruction from RNA sequencing data. Low BCR variation demonstrated negligible ongoing somatic hypermutation in PCFCL and primary cutaneous diffuse large B-cell lymphoma, leg-type, and indicated that the PCFCL microarchitecture does not act as a functional germinal center. Similar to FL but in contrast to primary cutaneous diffuse large B-cell lymphoma, leg-type, BCR genes of 15 PCFCLs (83%) had acquired N-linked glycosylation motifs. These motifs were located at the BCR positions converted to N-linked glycosylation motifs in normal B-cell repertoires with low prevalence but mostly at different positions than those found in FL. The cutaneous localization of PCFCL might suggest a role for lectins from commensal skin bacteria in PCFCL lymphomagenesis.

Retinal Pigment Epithelial Cells Control Early Mycobacterium tuberculosis Infection via Interferon Signaling.

Purpose: Mycobacterium tuberculosis (Mtb) bacilli have been found in retinal pigment epithelial (RPE) cells from uveitis patients without signs of systemic tuberculosis (TB) infection. RPE cells are important for ocular immune privilege and uveitis development. Methods: To address a potential role for Mtb-infected RPE cells in the development of uveitis, we delineated the response to Mtb infection in human RPE cells and primary human macrophages, the main target cell of Mtb. Primary human RPE cells, the human RPE cell line ARPE-19, and monocyte-derived proinflammatory M1 and anti-inflammatory M2 macrophages were infected with DsRed-expressing Mtb strain H37Rv. Infection rates and clearance were addressed along with RNA sequencing analysis, a confirmation analysis by dual-color reverse-transcriptase multiplex ligation-dependent probe amplification (dcRT-MLPA) and cytokine secretion. Results: RPE cells robustly controlled intracellular outgrowth of Mtb early after infection. The response in RPE cells to control Mtb survival was dominated by interferon (IFN) signaling and further characterized by prominent regulation of cell death/survival-associated genes and low-level production of Th1-associated cytokines. In contrast, macrophages engaged a plethora of responses including IFN signaling and communication between innate and adaptive immune cells to induce granuloma formation. Conclusions: Together, our data demonstrate that RPE cells display a strong response to Mtb infection that appears, however, incomplete in comparison to the macrophage response to Mtb. The RPE response might reflect a balance between mechanisms aimed at Mtb eradication and mechanisms that limit retinal inflammation.

A dose-dependent plasma signature of the safety and immunogenicity of the rVSV-Ebola vaccine in Europe and Africa.

The 2014-2015 Ebola epidemic affected several African countries, claiming more than 11,000 lives and leaving thousands with ongoing sequelae. Safe and effective vaccines could prevent or limit future outbreaks. The recombinant vesicular stomatitis virus-vectored Zaire Ebola (rVSV-ZEBOV) vaccine has shown marked immunogenicity and efficacy in humans but is reactogenic at higher doses. To understand its effects, we examined plasma samples from 115 healthy volunteers from Geneva who received low-dose (LD) or high-dose (HD) vaccine or placebo. Fifteen plasma chemokines/cytokines were assessed at baseline and on days 1, 2 to 3, and 7 after injection. Significant increases in monocyte-mediated MCP-1/CCL2, MIP-1ß/CCL4, IL-6, TNF-α, IL-1Ra, and IL-10 occurred on day 1. A signature explaining 68% of cytokine/chemokine vaccine-response variability was identified. Its score was higher in HD versus LD vaccinees and was associated positively with vaccine viremia and negatively with cytopenia. It was higher in vaccinees with injection-site pain, fever, myalgia, chills, and headache; higher scores reflected increasing severity. In contrast, HD vaccinees who subsequently developed arthritis had lower day 1 scores than other HD vaccinees. Vaccine dose did not influence the signature despite its influence on specific outcomes. The Geneva-derived signature associated strongly (ρ = 0.97) with that of a cohort of 75 vaccinees from a parallel trial in Lambaréné, Gabon. Its score in Geneva HD vaccinees with subsequent arthritis was significantly lower than that in Lambaréné HD vaccinees, none of whom experienced arthritis. This signature, which reveals monocytes' critical role in rVSV-ZEBOV immunogenicity and safety across doses and continents, should prove useful in assessments of other vaccines.

Host Immune Responses Differ between M. africanum- and M. tuberculosis-Infected Patients following Standard Anti-tuberculosis Treatment.

Epidemiological differences exist between Mycobacterium africanum (Maf)- and Mycobacterium tuberculosis (Mtb)-infected patients, but to date, contributing host factors have not been characterised. We analysed clinical outcomes, as well as soluble markers and gene expression profiles in unstimulated, and ESAT6/CFP-10-, whole-Maf- and Mtb-stimulated blood samples of 26 Maf- and 49 Mtb-HIV-negative tuberculosis patients before, and after 2 and 6 months of anti-tuberculosis therapy. Before treatment, both groups had similar clinical parameters, but differed in few cytokines concentration and gene expression profiles. Following treatment the body mass index, skinfold thickness and chest X-ray scores showed greater improvement in the Mtb- compared to Maf-infected patients, after adjusting for age, sex and ethnicity (p = 0.02; 0.04 and 0.007, respectively). In addition, in unstimulated blood, IL-12p70, IL12A and TLR9 were significantly higher in Maf-infected patients, while IL-15, IL-8 and MIP-1α were higher in Mtb-infected patients. Overnight stimulation with ESAT-6/CFP-10 induced significantly higher levels of IFN-γ and TNF-α production, as well as gene expression of CCL4, IL1B and TLR4 in Mtb- compared to Maf-infected patients. Our study confirms differences in clinical features and immune genes expression and concentration of proteins associated with inflammatory processes between Mtb- and Maf-infected patients following anti-tuberculosis treatment These findings have public health implications for treatment regimens, and biomarkers for tuberculosis diagnosis and susceptibility.

Short-term high-fat diet increases macrophage markers in skeletal muscle accompanied by impaired insulin signalling in healthy male subjects.

Macrophage markers in skeletal muscle of obese subjects are elevated and inversely relate to insulin sensitivity. The present study aimed to investigate whether short-term high-fat high-calorie (HFHC) diet already increases macrophage markers and affects glucose metabolism in skeletal muscle of healthy lean subjects. Muscle biopsies were obtained from 24 healthy lean young men before and after a 5-day HFHC-diet. mRNA expression levels of relevant genes in muscle and glucose, insulin, C-peptide and cholesteryl ester transfer protein (CETP) levels in plasma were measured. In addition, we assessed hepatic triacylglycerol ('triglyceride') (HTG) content by magnetic resonance spectroscopy and subcutaneous white adipose tissue (sWAT) biopsies were analysed histologically from a subset of subjects (n=8). A 5-day HFHC-diet markedly increased skeletal muscle mRNA expression of the general macrophage markers CD68 (3.7-fold, P<0.01) and CD14 (3.2-fold, P<0.01), as well as the M1 macrophage markers MARCO (11.2-fold, P<0.05), CD11c (1.8-fold, P<0.05) and MRC1 (1.7-fold, P<0.05). This was accompanied by down-regulation of SLC2A4 and GYS1 mRNA expression, and elevated plasma glucose (+4%, P<0.001) and insulin (+55%, P<0.001) levels together with homoeostasis model assessment of insulin resistance (HOMA-IR) (+48%, P<0.001), suggesting development of insulin resistance (IR). Furthermore, the HFHC-diet markedly increased HTG (+118%, P<0.001) and plasma CETP levels (+21%, P<0.001), a marker of liver macrophage content, whereas sWAT macrophage content remained unchanged. In conclusion, short-term HFHC-diet increases expression of macrophage markers in skeletal muscle of healthy men accompanied by reduced markers of insulin signalling and development of IR. Therefore, recruitment of macrophages into muscle may be an early event in development of IR in response to short-term HFHC-feeding.

Longitudinal immune responses and gene expression profiles in type 1 leprosy reactions.

PURPOSE: Leprosy, a chronic disease initiated by Mycobacterium leprae, is often complicated by acute inflammatory reactions. Although such episodes occur in at least 50% of all leprosy patients and may cause irreversible nerve damage, no laboratory tests are available for early diagnosis or prediction of reactions. Since immune- and genetic host factors are critical in leprosy reactions, we hypothesize that identification of host-derived biomarkers correlated to leprosy reactions can provide the basis for new tests to facilitate timely diagnosis and treatment thereby helping to prevent tissue damage. METHODS: The longitudinal host response of a leprosy patient, who was affected by a type 1 reaction (T1R) after MDT-treatment, was studied in unprecedented detail, measuring cellular and humoral immunity and gene expression profiles to identify biomarkers specific for T1R. RESULTS: Cytokine analysis in response to M. leprae revealed increased production of IFN-γ, IP-10, CXCL9, IL-17A and VEGF at diagnosis of T1R compared to before T1R, whereas a simultaneous decrease in IL-10 and G-CSF was observed at T1R. Cytokines shifts coincided with a reduction in known regulatory CD39(+)CCL4(+) and CD25(high) T-cell subsets. Moreover, RNA expression profiles revealed that IFN-induced genes, (V)EGF, and genes associated with cytotoxic T-cell responses (GNLY, GZMA/B, PRF1) were upregulated during T1R, whereas expression of T-cell regulation-associated genes were decreased. CONCLUSIONS: These data show that increased inflammation, vasculoneogenesis and cytotoxicity, perturbed T-cell regulation as well as IFN-induced genes play an important role in T1R and provide potential T1R-specific host biomarkers.

Structural and functional analysis of the TAP-inhibiting UL49.5 proteins of varicelloviruses.

Viral infections are counteracted by virus-specific cytotoxic T cells that recognize the infected cell via MHC class I (MHC I) molecules presenting virus-derived peptides. The loading of the peptides onto MHC I molecules occurs in the endoplasmic reticulum (ER) and is facilitated by the peptide loading complex. A key player in this complex is the transporter associated with antigen processing (TAP), which translocates the viral peptides from the cytosol into the ER. Herpesviruses have developed many strategies to evade cytotoxic T cells. Several members of the genus Varicellovirus encode a UL49.5 protein that prevents peptide transport through TAP. These include bovine herpesvirus (BoHV) 1, BoHV-5, bubaline herpesvirus 1, cervid herpesvirus 1, pseudorabies virus, felid herpesvirus 1, and equine herpesvirus 1 and 4. BoHV-1 UL49.5 inhibits TAP by preventing conformational changes essential for peptide transport and by inducing degradation of the TAP complex. UL49.5 consists of an ER luminal N-terminal domain, a transmembrane domain and a cytosolic C-terminal tail domain. In this study, the following features of UL49.5 were deciphered: (1) chimeric constructs of BoHV-1 and VZV UL49.5 attribute the lack of TAP inhibition by VZV UL49.5 to its ER-luminal domain, (2) the ER-luminal and TM domains of UL49.5 are required for efficient interaction with and inhibition of TAP, (3) the C-terminal RXRX sequence is essential for TAP degradation by BoHV-1 UL49.5, and (4) in addition to the RXRX sequence, the cytoplasmic tail of BoHV-1 UL49.5 carries a motif that is required for efficient TAP inhibition by the protein. A model is presented depicting how the different domains of UL49.5 may block the translocation of peptides by TAP and target TAP for proteasomal degradation.

Discovery of low-affinity preproinsulin epitopes and detection of autoreactive CD8 T-cells using combinatorial MHC multimers.

Autoreactive cytotoxic CD8 T-cells (CTLs) play a key pathogenic role in the destruction of insulin-producing beta-cells resulting in type 1 diabetes. However, knowledge regarding their targets is limited, restricting the ability to monitor the course of the disease and immune interventions. In a multi-step discovery process to identify novel CTL epitopes in human preproinsulin (PPI), PPI was digested with purified human proteasomes, and resulting COOH-fragments aligned with algorithm-predicted HLA-binding peptides to yield nine potential HLA-A1, -A2, -A3 or -B7-restricted candidates. An UV-exchange method allowed the generation of a repertoire of multimers including low-affinity HLA-binding peptides. These were labeled with quantum dot-fluorochromes and encoded in a combinatorial fashion, allowing parallel and sensitive detection of specific, low-avidity T-cells. Significantly increased frequencies of T-cells against four novel PPI epitopes (PPI(4-13)/B7, PPI(29-38)/A2, PPI(76-84)/A3 and PPI(79-88)/A3) were detected in stored blood of patients with recent onset diabetes but not in controls. Changes in frequencies of circulating CD8 T-cells against these novel epitopes were detected in blood of islet graft recipients at different time points after transplantation, which correlated with clinical outcome. In conclusion, our novel strategy involving a sensitive multiplex detection technology and requiring minimal volumes of stored blood represents a major improvement in the direct ex-vivo characterization and enumeration of immune cells in the pathogenesis of type 1 diabetes.

Inhibition of mouse TAP by immune evasion molecules encoded by non-murine herpesviruses.

Herpesviruses escape elimination by cytotoxic T lymphocytes through specific interference with the antigen-presenting function of MHC class I (MHC I) molecules. The transporter associated with antigen processing (TAP) forms a bottleneck in the MHC I antigen presentation pathway. The fact that multiple viruses, especially herpesviruses, encode molecules blocking TAP function is a case in point. The action of these viral immuno evasins is usually potent and very specific, making these proteins valuable tools for studying the cell biology of antigen presentation, including alternative antigen processing pathways. Yet, no dedicated TAP inhibitor has been described for any of the mouse herpesviruses. To permit the use of immuno evasins derived from non-mouse herpesviruses in mouse models, we assessed the cross-species activity of four TAP inhibitors and one tapasin inhibitor in the context of three different mouse haplotypes, H-2(b), H-2(d), and H-2(k). Two of the four TAP inhibitors, the bovine herpesvirus 1-encoded UL49.5 and the human cytomegalovirus (HCMV)-encoded US6 protein, potently inhibited mouse TAP. ICP47 and BNLF2a, encoded by herpes simplexvirus 1 and Epstein-Barr virus, respectively, failed to inhibit TAP in all mouse cells tested. Previous work, however, demonstrated that US6 did not cross the mouse species barrier. We now show that substitution of the cysteine residue at position 108 was responsible for this lack of activity. The HCMV-encoded tapasin inhibitor US3 efficiently downregulated H-2(d) molecules on 3T3 cells, but not in other cell lines tested. Finally, we show that synthetic peptides comprising the functional domain of US6 can be exploited as a versatile TAP inhibitor. In conclusion, a complete overview is presented of the applicability of herpesvirus-encoded TAP and tapasin inhibitors in mouse cells of different genetic background.

Effective CD8(+) T cell priming and tumor protection by enterotoxin B subunit-conjugated peptides targeted to dendritic cells.

In our previous studies we have shown that bacterial enterotoxin B subunits are effective vehicles to deliver antigen into the MHC class I processing route. Here we have used the non-toxic Escherichia coli heat labile enterotoxin B subunit (EtxB) conjugated to OVA peptide (EtxB-peptide) to address the impact on induction of specific CD8(+) T cells in vivo. Although incubation of DCs with these EtxB-peptide conjugates as such did not induce DC maturation in vitro MHC class I antigen presentation was much more efficient as compared to peptide alone. Antigen presentation was further enhanced upon DC maturation with the TLR-4 ligand LPS. Injection of matured DCs incubated with EtxB-peptide conjugates lead to strong induction of OVA-specific CD8(+) T lymphocytes and fully prevented the outgrowth of lethal B16 melanoma in wild type mice. Our data demonstrate that bacterial non-toxic B subunit-peptide conjugates are potent vaccine vehicles for induction of protective CD8(+) T cell responses.

The varicellovirus UL49.5 protein blocks the transporter associated with antigen processing (TAP) by inhibiting essential conformational transitions in the 6+6 transmembrane TAP core complex.

TAP translocates virus-derived peptides from the cytosol into the endoplasmic reticulum, where the peptides are loaded onto MHC class I molecules. This process is crucial for the detection of virus-infected cells by CTL that recognize the MHC class I-peptide complexes at the cell surface. The varicellovirus bovine herpesvirus 1 encodes a protein, UL49.5, that acts as a potent inhibitor of TAP. UL49.5 acts in two ways, as follows: 1) by blocking conformational changes of TAP required for the translocation of peptides into the endoplasmic reticulum, and 2) by targeting TAP1 and TAP2 for proteasomal degradation. At present, it is unknown whether UL49.5 interacts with TAP1, TAP2, or both. The contribution of other members of the peptide-loading complex has not been established. Using TAP-deficient cells reconstituted with wild-type and recombinant forms of TAP1 and TAP2, TAP was defined as the prime target of UL49.5 within the peptide-loading complex. The presence of TAP1 and TAP2 was required for efficient interaction with UL49.5. Using deletion mutants of TAP1 and TAP2, the 6+6 transmembrane core complex of TAP was shown to be sufficient for UL49.5 to interact with TAP and block its function. However, UL49.5-induced inhibition of peptide transport was most efficient in cells expressing full-length TAP1 and TAP2. Inhibition of TAP by UL49.5 appeared to be independent of the presence of other peptide-loading complex components, including tapasin. These results demonstrate that UL49.5 acts directly on the 6+6 transmembrane TAP core complex of TAP by blocking essential conformational transitions required for peptide transport.

Varicellovirus UL 49.5 proteins differentially affect the function of the transporter associated with antigen processing, TAP.

Cytotoxic T-lymphocytes play an important role in the protection against viral infections, which they detect through the recognition of virus-derived peptides, presented in the context of MHC class I molecules at the surface of the infected cell. The transporter associated with antigen processing (TAP) plays an essential role in MHC class I-restricted antigen presentation, as TAP imports peptides into the ER, where peptide loading of MHC class I molecules takes place. In this study, the UL 49.5 proteins of the varicelloviruses bovine herpesvirus 1 (BHV-1), pseudorabies virus (PRV), and equine herpesvirus 1 and 4 (EHV-1 and EHV-4) are characterized as members of a novel class of viral immune evasion proteins. These UL 49.5 proteins interfere with MHC class I antigen presentation by blocking the supply of antigenic peptides through inhibition of TAP. BHV-1, PRV, and EHV-1 recombinant viruses lacking UL 49.5 no longer interfere with peptide transport. Combined with the observation that the individually expressed UL 49.5 proteins block TAP as well, these data indicate that UL 49.5 is the viral factor that is both necessary and sufficient to abolish TAP function during productive infection by these viruses. The mechanisms through which the UL 49.5 proteins of BHV-1, PRV, EHV-1, and EHV-4 block TAP exhibit surprising diversity. BHV-1 UL 49.5 targets TAP for proteasomal degradation, whereas EHV-1 and EHV-4 UL 49.5 interfere with the binding of ATP to TAP. In contrast, TAP stability and ATP recruitment are not affected by PRV UL 49.5, although it has the capacity to arrest the peptide transporter in a translocation-incompetent state, a property shared with the BHV-1 and EHV-1 UL 49.5. Taken together, these results classify the UL 49.5 gene products of BHV-1, PRV, EHV-1, and EHV-4 as members of a novel family of viral immune evasion proteins, inhibiting TAP through a variety of mechanisms.

Constitutive and IFNgamma-induced activation of MHC2TA promoter type III in human melanoma cell lines is governed by separate regulatory elements within the PIII upstream regulatory region.

Cell lines established from tumor tissue of cutaneous melanoma biopsies often display constitutive and IFNgamma-inducible expression of MHC class II molecules. The expression of MHC class II molecules in melanoma is associated with an overall poor prognosis and unfavorable clinical outcome. We have analyzed the DNA elements and interacting transcription factors that control the constitutive and IFNgamma-inducible expression of the class II transactivator (CIITA), a co-activator essential for transcription of all MHC class II genes. Our studies reveal the activation of multiple CIITA promoter regions (CIITA-PII, -PIII and -PIV) in melanoma cell lines for both the constitutive and IFNgamma-inducible expression of MHC class II molecules. Furthermore, we show that constitutive and IFNgamma-inducible expression of the CIITA-PIII isoform is governed by separate regulatory elements within the PIII upstream regulatory region (PURR). Similarly constitutive activation in melanoma of CIITA-PII, CIITA-PIII, and CIITA-PIV does not require components of the IFNgamma signaling pathway. However, these components are readily recruited to the PURR and CIITA-PIV after exposure of cells to IFNgamma and account for the IFNgamma-induced expression of CIITA. Together, our data reveal the contribution of distinct elements and factors in the constitutive and IFNgamma-inducible expression of CIITA in melanoma cell lines of the skin.

E47, IRF-4, and PU.1 synergize to induce B-cell-specific activation of the class II transactivator promoter III (CIITA-PIII).

In B cells, expression of CIITA and resulting major histocompatibility complex II (MHCII) is mediated exclusively by promoter III (CIITA-PIII) activation. Recent studies have established that CIITA-PIII also participates in the expression of CIITA in activated human T cells, dendritic cells, and monocytes. In this study we characterized the various regulatory elements and interacting factors of CIITA-PIII that account for specific activation in B lymphocytes. We identified 2 E-box motifs and an Ets/ISRE-consensus element (EICE) in CIITA-PIII as playing a crucial role in the B-cell-specific transcriptional regulation of CIITA. Abolishment of factor binding to these elements resulted in a strong reduction of CIITA-PIII activation in B cells only, whereas it did scarcely affect or not affect the activity of CIITA-PIII in activated T cells and monocytes. We show that in B cells, E47 and PU.1/IRF-4 interact with the E-box motifs and the EICE, respectively, and act synergistically in the activation of CIITA-PIII. Moreover, functional inhibition of either E47 or IRF-4 resulted in strong reduction of CIITA-PIII activity in B lymphocytes only. The finding that PU.1, IRF-4, and E47 play an important role in the B-cell-mediated activation of CIITA-PIII provides a link between antigen presentation functions and activation and differentiation events in B lymphocytes.

Transcriptional regulation of the MHC class II trans-activator (CIITA) promoter III: identification of a novel regulatory region in the 5'-untranslated region and an important role for cAMP-responsive element binding protein 1 and activating transcription factor-1 in CIITA-promoter III transcriptional activation in B lymphocytes.

The class II trans-activator (CIITA), which acts as a master regulator for expression of MHC class II genes, is expressed constitutively in mature B cells. This constitutive expression of CIITA is driven by CIITA promoter III (CIITA-PIII). However, little is known about the factors that control the B cell-mediated trans-activation of CIITA-PIII. In this study using B cells we have identified several cAMP-responsive elements (CREs) in the proximal promoter and in the 5'-untranslated region (5'-UTR) that are involved in the activation of CIITA-PIII. We show that activating transcription factor (ATF)/CRE binding protein (CREB) factors bind to the CREs in vitro and in vivo. Notably, our results also reveal that the 5'-UTR of CIITA-PIII functions as an important regulatory region in B lymphocytes. Furthermore, transient cotransfections of a CIITA-PIII luciferase reporter construct with either CREB-1 or ATF-1 boost CIITA-PIII trans-activation in a dose-dependent manner, which was further enhanced by addition of general coactivator CREB-binding protein. Transient transfections using mutant CIITA-PIII luciferase reporter constructs that either lack the (5'-UTR) or abolish binding of CREB-1 and ATF-1 to the CRE located in activation response element-2, displayed severely reduced promoter activity in B cells. A similar successive deletion of the CREs resulted in a subsequent reduction of CREB-1-induced activity of CIITA-PIII in B cells. Together our results argue for an important role of ATF/CREB factors and the 5'-UTR of CIITA-PIII in the trans-activation of CIITA-PIII in B cells.

Lack of IFN-gamma-mediated induction of the class II transactivator (CIITA) through promoter methylation is predominantly found in developmental tumor cell lines.

Downregulation of major histocompatibility complex (MHC) molecules by tumor cells impairs cellular immune recognition and contributes to inefficient cell-mediated tumor eradication. Low or lack of expression of MHC molecules is frequently observed in early developmental or embryonically derived tumor cells. Considering the central role of the class II transactivator (CIITA) in MHC class II- and class I-mediated antigen presentation, we compared the induction of CIITA by interferon-gamma (IFN-gamma) in a diverse panel of developmental and more differentiated tumor cell lines. In contrast to the more differentiated tumor cell lines, none of the developmental tumor cell lines were capable of expressing CIITA after treatment with IFN-gamma. Remarkably, in transient transfection assays, CIITA promoter IV (CIITA-PIV) was found to be induced by IFN-gamma. Southern blot analysis of genomic DNA obtained from the developmental tumor cell lines indicated that the absence of endogenous CIITA induction was due to methylation of the CIITA-PIV region. Exposure to 5-azacytidine restored induction of CIITA and congruent HLA-DRA expression in these cells. The observation that only developmental tumor cell lines, originating from various tissues, employ methylation to silence CIITA expression may reflect the natural status of CIITA expression during early development rather than oncogenic transformation.