scholarly journals RACK7 recognizes H3.3G34R mutation to suppress expression of MHC class II complex components and their delivery pathway in pediatric glioblastoma

2020 ◽  
Vol 6 (29) ◽  
pp. eaba2113
Author(s):  
Fangfang Jiao ◽  
Ze Li ◽  
Chen He ◽  
Wenqi Xu ◽  
Gensheng Yang ◽  
...  
Keyword(s):  
Mhc Class Ii ◽  
Point Mutations ◽  
Class Ii ◽  
Chromatin Binding ◽  
Histocompatibility Complex ◽  
Pediatric Glioblastoma ◽  
Pediatric Brain ◽  
Patient Mutation

Histone H3 point mutations have been identified in incurable pediatric brain cancers, but the mechanisms through which these mutations drive tumorigenesis are incompletely understood. Here, we provide evidence that RACK7 (ZMYND8) recognizes the histone H3.3 patient mutation (H3.3G34R) in vitro and in vivo. We show that RACK7 binding to H3.3G34R suppresses transcription of CIITA, which is the master regulator of MHC (major histocompatibility complex) class II molecules and genes involved in vesicular transport of MHC class II molecules to the cell surface, resulting in suppression of MHC class II molecule expression and transport. CRISPR-based knock-in correction of the H3.3G34R mutation in human pediatric glioblastoma (pGBM) cells significantly reduces overall RACK7 chromatin binding and derepresses the same set of genes as does knocking out RACK7 in the H3.3G34R pGBM cells. By demonstrating that H3.3G34R and RACK7 work together, our findings suggest a potential molecular mechanism by which H3.3G34R promotes cancer.

scholarly journals HLA-DM Interactions with Intermediates in HLA-DR Maturation and a Role for HLA-DM in Stabilizing Empty HLA-DR Molecules

1996 ◽  
Vol 184 (6) ◽  
pp. 2153-2166 ◽  
Author(s):  
Lisa K. Denzin ◽  
Craig Hammond ◽  
Peter Cresswell
Keyword(s):  
Mhc Class Ii ◽  
Antigen Processing ◽  
Class Ii ◽  
Physical Interaction ◽  
Antigenic Peptides ◽  
Histocompatibility Complex ◽  
Chain Complexes ◽  
Hla Dr

Major histocompatibility complex (MHC) class II–positive cell lines which lack HLA-DM expression accumulate class II molecules associated with residual invariant (I) chain fragments (class II–associated invariant chain peptides [CLIP]). In vitro, HLA-DM catalyzes CLIP dissociation from class II–CLIP complexes, promoting binding of antigenic peptides. Here the physical interaction of HLA-DM with HLA-DR molecules was investigated. HLA-DM complexes with class II molecules were detectable transiently in cells, peaking at the time when the class II molecules entered the MHC class II compartment. HLA-DR αβ dimers newly released from I chain, and those associated with I chain fragments, were found to associate with HLA-DM in vivo. Mature, peptide-loaded DR molecules also associated at a low level. These same species, but not DR-I chain complexes, were also shown to bind to purified HLA-DM molecules in vitro. HLA-DM interaction was quantitatively superior with DR molecules isolated in association with CLIP. DM-DR complexes generated by incubating HLA-DM with purified DR αβCLIP contained virtually no associated CLIP, suggesting that this superior interaction reflects a prolonged HLA-DM association with empty class II dimers after CLIP dissociation. Incubation of peptide-free αβ dimers in the presence of HLA-DM was found to prolong their ability to bind subsequently added antigenic peptides. Stabilization of empty class II molecules may be an important property of HLA-DM in facilitating antigen processing.

scholarly journals Early Endosomes Are Required for Major Histocompatiblity Complex Class II Transport to Peptide-loading Compartments

1999 ◽  
Vol 10 (9) ◽  
pp. 2891-2904 ◽  
Author(s):  
Valérie Brachet ◽  
Gérard Péhau-Arnaudet ◽  
Catherine Desaymard ◽  
Graça Raposo ◽  
Sebastian Amigorena
Keyword(s):  
Mhc Class Ii ◽  
Class Ii ◽  
Endocytic Pathway ◽  
Cross Linking ◽  
Histocompatibility Complex ◽  
Early Endosomes ◽  
Peptide Loading ◽  
Golgi Network ◽  
Mhc Class Ii Molecules

Antigen presentation to CD4+ T lymphocytes requires transport of newly synthesized major histocompatibility complex (MHC) class II molecules to the endocytic pathway, where peptide loading occurs. This step is mediated by a signal located in the cytoplasmic tail of the MHC class II-associated Ii chain, which directs the MHC class II-Ii complexes from the trans-Golgi network (TGN) to endosomes. The subcellular machinery responsible for the specific targeting of MHC class II molecules to the endocytic pathway, as well as the first compartments these molecules enter after exit from the TGN, remain unclear. We have designed an original experimental approach to selectively analyze this step of MHC class II transport. Newly synthesized MHC class II molecules were caused to accumulate in the Golgi apparatus and TGN by incubating the cells at 19°C, and early endosomes were functionally inactivated by in vivo cross-linking of transferrin (Tf) receptor–containing endosomes using Tf-HRP complexes and the HRP-insoluble substrate diaminobenzidine. Inactivation of Tf-containing endosomes caused a marked delay in Ii chain degradation, peptide loading, and MHC class II transport to the cell surface. Thus, early endosomes appear to be required for delivery of MHC class II molecules to the endocytic pathway. Under cross-linking conditions, most αβIi complexes accumulated in tubules and vesicles devoid of γ-adaptin and/or mannose-6-phosphate receptor, suggesting an AP1-independent pathway for the delivery of newly synthesized MHC class II molecules from the TGN to endosomes.

scholarly journals Regulation of a Graft-Versus-Leukemia Effect by Major Histocompatibility Complex Class II Molecules on Leukemia Cells: HLA-DR1 Expression Renders K562 Cell Tumors Resistant to Adoptively Transferred Lymphocytes in Severe Combined Immunodeficiency Mice/Nonobese Diabetic Mice

Blood ◽  
1997 ◽  
Vol 90 (11) ◽  
pp. 4553-4558 ◽  
Author(s):  
Frank F. Weichold ◽  
Yin-zheng Jiang ◽  
Daniel E. Dunn ◽  
Michael Bloom ◽  
Vera Malkovska ◽  
...  
Keyword(s):  
Mhc Class Ii ◽  
Nk Cell ◽  
Severe Combined Immunodeficiency ◽  
Nod Mice ◽  
Class Ii ◽  
Leukemia Cells ◽  
Combined Immunodeficiency ◽  
Histocompatibility Complex ◽  
Proliferation In Vitro

Abstract To understand the role of key molecules in determining the strength and nature of allogeneic T-cell response to leukemia, we transfected HLA-DR1 into the major histocompatibility complex (MHC)-deficient, natural killer (NK)-cell sensitive K562 leukemia cell line. Untransfected K562 cells stimulated NK proliferation in vitro and formed subcutaneous tumors in severe combined immunodeficiency/non-obese diabetic (SCID/NOD) mice. Tumor growth was inhibited by adoptive intravenous transfer of fresh unprimed peripheral blood mononuclear cells (PBMC). In contrast, HLA-DR1 transfected cells stimulated CD4+ T cells, but not NK-cell proliferation in vitro and formed tumors resistant to fresh PBMC in SCID/NOD mice. Tumors not expressing MHC were infiltrated with CD16+CD56+ lymphocytes whereas nonregressing HLA-DR1 expressing tumors showed only a scanty infiltration with both T-cell and NK-cell subsets. The results indicate that MHC class II expression by leukemia cells can determine the effector cell type that it engages. In vivo MHC class II expression rendered K562 cell tumors resistant to NK-cell mediated antitumor reactivity.

Erythropoietin as a Novel Pro-Inflammatory Mediator of Macrophages.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3596-3596
Author(s):  
Lilach Lifshitz ◽  
Galit Tabak ◽  
Max Gassman ◽  
Moshe Mittelman ◽  
Drorit Neumann
Keyword(s):  
Mhc Class Ii ◽  
Phagocytic Activity ◽  
Surface Expression ◽  
Class Ii ◽  
Experimental Models ◽  
Splenic Macrophage ◽  
And Function ◽  
Inflammatory Macrophages

Abstract Abstract 3596 Poster Board III-533 The immunomodulatory effects of erythropoietin (EPO) on the cellular and humoral compartments of the immune system were originally described by our group in multiple myeloma patients and have been further elucidated in murine experimental models (Mittelman, 2001; Katz 2005; 2007; Prutchi-Sagiv, 2006). However, the mechanisms of action by which EPO affects lymphocyte number and function are still unknown, particularly since lymphocytes do not carry EPO receptors (EPO-R). We thus set to unravel mechanisms underlying the anti-neoplastic immunomodulatory action of EPO. These studies led us to the novel discovery that dendritic cells (DCs) express EPO-R, and that EPO enhances their survival and function (Prutchi-Sagiv, 2008; Lifshitz, 2009). Here we focus on macrophages as an additional EPO target, since in analogy to DCs, macrophages are also antigen presenting cells, and serve as key effectors of the innate immune response. Using murine models, we first explored the in-vivo effects of EPO using recombinant human EPO (rHuEPO, EPREXR, JC)-injected mice, as well as transgenic mice over-expressing human EPO (termed tg6). EPO treatment was associated with an increased splenic macrophage population, detected by F4/80 expression, and an increased number of macrophages expressing CD11b, CD80 and MHC class II. We further explored the effect of in-vivo EPO administration in an inflammatory model exploiting thioglygollate injection to induce recruitment of peritoneal inflammatory macrophages. The inflammatory macrophages obtained from both EPO injected and from tg6 mice displayed increased expression of F4/80, CD11b, CD80 and MHC class II and augmented phagocytic activity, as compared to the control counterparts. These results are supported by in-vitro studies in bone marrow derived macrophages (BMDMs). We show that BMDMs express EPO-R mRNA, as detected by RT-PCR. In-vitro stimulation of the BMDMs with rHuEPO activated multiple signaling pathways including STAT1, STAT5, MAPK, AKT and NFkB indicating macrophage activation via surface EPO-R. EPO treatment of the BMDMs up-regulated their surface expression of CD11b, F4/80 and CD80, as well as enhanced their phagocytic activity. EPO treatment of LPS-stimulated BMDMs augmented IL-12 secretion, and decreased IL-10 secretion. In conclusion our results show that macrophages are direct targets of EPO and that EPO treatment enhances their pro-inflammatory activity and function. These findings point to the multifunctional role of EPO and may advance its clinical applications as an anti-neoplastic immunomodulator. Disclosures: Mittelman: BioGAL- Start up (inactive): Equity Ownership, Patents & Royalties. Off Label Use: Non erythroid effects: immune, anti-cancer (all under investigation).

Absence of STAT1 Signaling in Host Hematopoietic Cells Leads to Enhanced Gvhd Induction and Involves Increased Expression of MHC Class II and Reduced PD-L1 Expression on Dendritic Cells,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4026-4026
Author(s):  
Caisheng Lu ◽  
Huihui Ma ◽  
Ailing Liu ◽  
MeiHua Jin ◽  
Shirong Li ◽  
...  
Keyword(s):  
T Cells ◽  
Mhc Class Ii ◽  
Cell Activation ◽  
Hematopoietic Cells ◽  
Class Ii ◽  
Cfse Dilution ◽  
Stat1 Signaling ◽  
Donor Lymphocytes

Abstract Abstract 4026 Interferon-g/STAT1 signaling plays a critical role in regulating dendritic cell activation and function. Blockade of IFN-g signaling leads to reduced DC activation and impaired anti-tumor and acquired adaptive immunity. We recently reported that lack of IFN-g/STAT1 in donor lymphocytes leads to reduced GVHD induction in both MHC- and mHA-mismatched mouse BMT models. In this study, we addressed the role of host STAT1 in the regulation of GVHD. Wildtype or STAT1-deficient 129 mice (H2b) underwent allogeneic Bone Marrow Transplantation (BMT) following lethal irradiation (1044 rad). GVHD was induced using either BALB/c or B6 donor spleen cells. We unexpectedly observed that absence of STAT1 in recipient mice led to increased GVHD-associated mortality in both MHC-mismatched (MST 5 vs. 8, p=0.01) and mHA-mismatched (MST 11 vs. 23, p<0.01) BMT settings. The enhanced GVHD induction was found to be associated with increased activation (expression of CD69 and CD25) and allo-antigen driven proliferation of donor CD4 and CD8 T cells as determined by CFSE-dilution. As host APCs have been reported to being crucial for induction of GVHD, we phenotypically and functionally characterized STAT1 deficient DCs. Our studies revealed that STAT1-deficient bone marrow-derived dendritic cells (BMDCs) which were maturated in the presence of LPS showed significantly increased MHC class II, CD86, CD80 and CD40 expression compared with wildtype BMDCs. Furthermore, STAT1-deficient BMDC showed significantly increased direct allo-stimulatory capacity resulting in increased responder cell proliferation as determined by standard MLR assays using 3H-Thymidine uptake assays as well as CFSE-dilution studies. STAT1−/− BMDCs significantly promoted CD44+CD62L- expression in responder CD4 and CD8 T cells compared to wild type BMDCs (all p<0.001). The increased MHC II expression in STAT1-deficient DC was further confirmed in host CD11b+ and CD11c+ cells following GVHD induction in vivo. To determine whether non-hematopoietic cells in STAT1−/− host contribute to the increased GVHD induction, we created radiation chimeras in which STAT1 was only deficient in the hematopoietic compartment by transplanting 129.STAT1−/− BMC into 129.STAT1+/+ recipients following lethal irradiation. 120 days later GVHD was induced using fully MHC-mismatched BALB/c donor splenocytes. Similar to STAT1-deficient recipients STAT1−/− ®WT chimeras showed enhanced GVHD induction compared to STAT1+/+®WT chimeras (MST 11 vs. 5, p<0.05). To determine the mechanism underlying the enhanced expansion of donor T cells in response to stimulation with STAT1-deficient APC, we hypothesized that STAT-deficiency may impair expression of the T cell inhibitory molecules Programed Cell Death-Ligand1 or-2 (PD-L1,-L2) on APC. We therefore studied the expression of PD-L1 and PD-L2 expression on wildtype and STAT1-deficient DC. Indeed, were able to demonstrate that absence of STAT1 significantly suppressed PD-L1 expression on BMDCs upon in vitro LPS stimulation (Mean Fluorescence Intensity 167.2± 15.9 vs. 532.5±7.6, p<0.001) and also in vivo tested on day+ 6 post-BMT in the mHA-mismatched setting. In line with these results using in vitro stimulation we could demonstrate significantly reduced Activation Induced Cell Death (AICD) in activated B6.SJL CD69+ CD4 and CD8 cells stimulated with 129.STAT1−/− BMDCs compared to cells stimulated with 129.STAT1+/+ BMDCs (10.6±1.5% vs. 28.2±1.9 % for CD4; 13.0±0.7% vs. 30.5±1.1% for CD8 respectively, p<0.001 for all). Importantly, blocking IFN-g with neutralizing antibodies significantly increased MHC class II, CD86 expression and reduced reduced PD-L1 expression on BMDCs upon LPS stimulation. In summary, our data suggest two mechanisms how the absence of STAT1 signaling in host hematopoietic cells may promote the development of GVHD: First, increased expression of MHC II and co-stimulatory molecule in STAT1-deficient APC may lead to enhanced activation and proliferation of donor lymphocytes. Second, absence of STAT1 in maturated host DC inhibits PD-L1 expression thus leading to reduced AICD of activated donor lymphocytes. These findings suggest that STAT1-signaling modulates host APC function and shapes the GVH-response by causing increased allo-antigen-specific donor T cell activation, survival and proliferation. Disclosures: Lentzsch: Centocor Ortho Biotech: Research Funding; Genzyme: Consultancy; Onyx: Consultancy; Celgene: Consultancy, Research Funding.

scholarly journals CIITA Leucine-Rich Repeats Control Nuclear Localization, In Vivo Recruitment to the Major Histocompatibility Complex (MHC) Class II Enhanceosome, and MHC Class II Gene Transactivation

2000 ◽  
Vol 20 (20) ◽  
pp. 7716-7725 ◽  
Author(s):  
Sandra B. Hake ◽  
Krzysztof Masternak ◽  
Claudia Kammerbauer ◽  
Christian Janzen ◽  
Walter Reith ◽  
...  
Keyword(s):  
Major Histocompatibility Complex ◽  
Mhc Class Ii ◽  
Nuclear Localization ◽  
Class Ii ◽  
Dominant Negative ◽  
Major Histocompatibility ◽  
Major Function ◽  
Histocompatibility Complex ◽  
Leucine Rich Repeats

ABSTRACT The major histocompatibility complex (MHC) class II transactivator CIITA plays a pivotal role in the control of the cellular immune response through the quantitative regulation of MHC class II expression. We have analyzed a region of CIITA with similarity to leucine-rich repeats (LRRs). CIITA LRR alanine mutations abolish both the transactivation capacity of full-length CIITA and the dominant-negative phenotype of CIITA mutants with N-terminal deletions. We demonstrate direct interaction of CIITA with the MHC class II promoter binding protein RFX5 and could also detect novel interactions with RFXANK, NF-YB, and -YC. However, none of these interactions is influenced by CIITA LRR mutagenesis. On the other hand, chromatin immunoprecipitation shows that in vivo binding of CIITA to the MHC class II promoter is dependent on LRR integrity. LRR mutations lead to an impaired nuclear localization of CIITA, indicating that a major function of the CIITA LRRs is in nucleocytoplasmic translocation. There is, however, evidence that the CIITA LRRs are also involved more directly in MHC class II gene transactivation. CIITA interacts with a novel protein of 33 kDa in a manner sensitive to LRR mutagenesis. CIITA is therefore imported into the nucleus by an LRR-dependent mechanism, where it activates transcription through multiple protein-protein interactions with the MHC class II promoter binding complex.

scholarly journals Paracrine Transfer of Mouse Mammary Tumor Virus Superantigen

1997 ◽  
Vol 185 (3) ◽  
pp. 471-480 ◽  
Author(s):  
Marc Delcourt ◽  
Jacques Thibodeau ◽  
Francois Denis ◽  
Rafick-Pierre Sekaly
Keyword(s):  
Mhc Class Ii ◽  
Class Ii ◽  
Cell Contact ◽  
Molecular Evidence ◽  
In Vitro System ◽  
Hla Dr ◽  
Mouse Mammary Tumor ◽  
Tumor Virus

Transfer of vSAG7, the endogenous superantigen encoded in the Mtv7 locus, from MHC class II− to MHC class II+ cells has been suggested to occur both in vivo and in vitro. This transfer usually leads to the activation and deletion of T cells expressing responsive Vβs. However, there is no direct molecular evidence for such a transfer. We have developed an in vitro system which confirms this property of vSAGs. vSAG7 was transfected into a class II− murine fibroblastic line. Coculture of these cells with class II+ cells and murine T cell hybridomas expressing the specific Vβs led to high levels of IL-2 production which was specifically inhibited by vSAG7- and MHC class II–specific mAbs. Moreover, injection of vSAG7+ class II− cells in mice led to expansion of Vβ6+ CD4+ cells. We show that this transfer activity is paracrine but does not require cell-to-cell contact. Indeed, vSAG7 was transferred across semi-permeable membranes. Transfer can occur both from class II− and class II+ cells, indicating that MHC class II does not sequester vSAG7. Finally, competition experiments using bacterial toxins with well defined binding sites showed that the transferred vSAG7 fragment binds to the α1 domain of HLA-DR.

scholarly journals ITAM signaling in dendritic cells controls T helper cell priming by regulating MHC class II recycling

Blood ◽  
2010 ◽  
Vol 116 (17) ◽  
pp. 3208-3218 ◽  
Author(s):  
Daniel B. Graham ◽  
Holly M. Akilesh ◽  
Grzegorz B. Gmyrek ◽  
Laura Piccio ◽  
Susan Gilfillan ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Mhc Class Ii ◽  
Autoimmune Encephalitis ◽  
Class Ii ◽  
T Helper Cell ◽  
Helper Cell ◽  
Nucleotide Exchange ◽  
T Helper

Abstract Immature dendritic cells (DCs) specialize in antigen capture and maintain a highly dynamic pool of intracellular major histocompatibility complex class II (MHCII) that continuously recycles from peptide loading compartments to the plasma membrane and back again. This process facilitates sampling of environmental antigens for presentation to T helper cells. Here, we show that a signaling pathway mediated by the DC immunoreceptor tyrosine-based activation motif (ITAM)–containing adaptors (DAP12 and FcRγ) and Vav family guanine nucleotide exchange factors controls the half-life of surface peptide-MHCII (pMHCII) complexes and is critical for CD4 T-cell triggering in vitro. Strikingly, mice with disrupted DC ITAMs show defective T helper cell priming in vivo and are protected from experimental autoimmune encephalitis. Mechanistically, we show that deficiency in ITAM signaling results in increased pMHCII internalization, impaired recycling, and an accumulation of ubiquitinated MHCII species that are prematurely degraded in lysosomes. We propose a novel mechanism for control of T helper cell priming.

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