Vascular leukocytes contribute to tumor vascularization

Abstract There is no proof that hematopoietic cells contribute significantly to vasculogenesis in postnatal life. Here we report a novel leukocyte subset within ovarian carcinoma that coexpresses endothelial and dendritic cell markers. Fluorescence-activated cell sorter (FACS) analysis identified a high frequency of VE-cadherin+ CD45+ leukocytes (39% of host cells) in 10 of 10 solid tumors evaluated. This population represented less than 1% of nontumor cells in ascites and peripheral blood. At the protein level, more than 86% of these cells expressed the endothelial markers P1H12, CD34, and CD31 and leukocyte markers CD11c and major histocompatibility complex (MHC) class II. At the mRNA level, we detected TEM1, TEM7, and Thy-1, specific markers of angiogenic endothelium. Finally, this population has the capacity to generate functional blood vessels in vivo. Because of its mixed phenotype, we named this population vascular leukocytes (VLCs). Our data provide an important link between hematopoietic endothelial precursors and vascular development in postnatal life and a possible novel therapeutic target.

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Hierarchy of Susceptibility of Dendritic Cell Subsets to Infection by Leishmania major: Inverse Relationship to Interleukin-12 Production

Infection and Immunity ◽

10.1128/iai.70.7.3874-3880.2002 ◽

2002 ◽

Vol 70 (7) ◽

pp. 3874-3880 ◽

Cited By ~ 31

Author(s):

Sandrine Henri ◽

Joan Curtis ◽

Hubertus Hochrein ◽

David Vremec ◽

Ken Shortman ◽

...

Keyword(s):

Immune Responses ◽

Mhc Class Ii ◽

Leishmania Major ◽

Parasitophorous Vacuole ◽

Class Ii ◽

Host Cells ◽

Interleukin 12 ◽

Histocompatibility Complex ◽

T Cell Immune Responses ◽

Antigen Presenting

ABSTRACT Dendritic cells (DCs) are professional antigen-presenting cells which initiate and regulate T-cell immune responses. Here we show that murine splenic DCs can be ranked on the basis of their ability to phagocytose and harbor the obligately intracellular parasite Leishmania major. CD4+ CD8− DCs are the most permissive host cells for L. major amastigotes, followed by CD4− CD8− DCs; CD4− CD8+ cells are the least permissive. However, the least susceptible CD4− CD8+ DC subset was the best interleukin-12 producer in response to infection. Infection did not induce in any DC subset production of the proinflammatory cytokine gamma interferon and nitric oxide associated with the induction of Th1 responses. The number of parasites phagocytosed by DCs was low, no more than 3 organisms per cell, compared to more than 10 organisms per macrophage. In infected DCs, the parasites are located in a parasitophorous vacuole containing both major histocompatibility complex (MHC) class II and lysosome-associated membrane protein 1 molecules, similar to their location in the infected macrophage. The parasite-driven redistribution of MHC class II to this compartment indicates that infected DCs should be able to present parasite antigen.

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

Molecular Biology of the Cell ◽

10.1091/mbc.10.9.2891 ◽

1999 ◽

Vol 10 (9) ◽

pp. 2891-2904 ◽

Cited By ~ 35

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.

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Virulence protein VirD5 ofAgrobacterium tumefaciensbinds to kinetochores in host cells via an interaction with Spt4

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1706166114 ◽

2017 ◽

Vol 114 (38) ◽

pp. 10238-10243 ◽

Cited By ~ 9

Author(s):

Xiaorong Zhang ◽

G. Paul H. van Heusden ◽

Paul J. J. Hooykaas

Keyword(s):

Chromosome Segregation ◽

High Frequency ◽

Chromosomal Instability ◽

Plant Cells ◽

Tumor Formation ◽

Host Cells ◽

Infection Site ◽

Virulence Proteins ◽

Virulence Protein

The bacteriumAgrobacterium tumefacienscauses crown gall tumor formation in plants. During infection the bacteria translocate an oncogenic piece of DNA (transferred DNA, T-DNA) into plant cells at the infection site. A number of virulence proteins are cotransported into host cells concomitantly with the T-DNA to effectuate transformation. Using yeast as a model host, we find that one of these proteins, VirD5, localizes to the centromeres/kinetochores in the nucleus of the host cells by its interaction with the conserved protein Spt4. VirD5 promotes chromosomal instability as seen by the high-frequency loss of a minichromosome in yeast. By using both yeast and plant cells with a chromosome that was specifically marked by alacOrepeat, chromosome segregation errors and the appearance of aneuploid cells due to the presence of VirD5 could be visualized in vivo. Thus, VirD5 is a prokaryotic virulence protein that interferes with mitosis.

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Dendritic cell subsets in blood and lymphoid tissue of rhesus monkeys and their mobilization with Flt3 ligand

Blood ◽

10.1182/blood-2002-09-2929 ◽

2003 ◽

Vol 102 (7) ◽

pp. 2513-2521 ◽

Cited By ~ 97

Author(s):

P. Toby H. Coates ◽

Simon M. Barratt-Boyes ◽

Linyou Zhang ◽

Vera S. Donnenberg ◽

Peta J. O'Connell ◽

...

Keyword(s):

Mhc Class Ii ◽

Lymphoid Tissue ◽

Rhesus Monkeys ◽

Mononuclear Cells ◽

Flow Analysis ◽

Interferon Α ◽

Color Flow ◽

Histocompatibility Complex ◽

Simplex Virus

Abstract We provide phenotypic and functional evidence of premonocytoid dendritic cells (DCs) and preplasmacytoid DCs in blood and of corresponding DC subsets in secondary lymphoid tissue of rhesus monkeys. Subsets were identified and sorted by 4-color flow cytometry using antihuman monoclonal antibodies cross-reactive with rhesus monkey. To mobilize pre-DC subsets, fms-like tyrosine 3 kinase ligand (Flt3L; 100 μg/kg subcutaneously) was administered for 10 days. Presumptive pre-DC subsets were identified within the lineage- (Lin-) major histocompatibility complex (MHC) class II+ fraction of blood mononuclear cells. Premonocytoid DCs were CD11c+CD123- (interleukin-3Rα- [IL-3Rα-]). Preplasmacytoid DCs were characterized as CD11c-CD123++ Flt3L increased the CD11c+ pre-DC (7-fold) and CD123++ pre-DC subsets (3-fold) in blood. The freshly isolated CD11c+ pre-DC subset induced modest proliferation of naive allogeneic T cells. After overnight culture with granulocyte macro-phage-colony-stimulating factor (GMCSF) and CD40L, both subsets up-regulated surface costimulatory molecules, and CD11c+ pre-DCs became potent allostimulators. Freshly isolated CD123++ pre-DCs showed typical plasmacytoid morphology and, when cultured with IL-3 and CD40L for 72 hours, developed mature DC morphology. Following stimulation with CD40L, CD11c+ pre-DCs secreted increased levels of IL-12p40. Importantly, herpes simplex virus-stimulated CD123++ pre-DCs, but not CD11c+ pre-DCs, secreted interferon-α (IFN-α). Corresponding DC subsets were identified by flow analysis and immunohistochemistry in lymph nodes wherein both populations were increased 2- to 3-fold by Flt3L administration. CD123+ pre-DCs produced IFN-α in response to in vivo viral infection. Thus, rhesus monkeys exhibit 2 distinct DC precursor populations that closely resemble those of humans. Both are mobilized into blood and lymphoid tissue by Flt3L, offering potential for their further characterization and possible therapeutic application. (Blood. 2003;102:2513-2521)

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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

Molecular and Cellular Biology ◽

10.1128/mcb.20.20.7716-7725.2000 ◽

2000 ◽

Vol 20 (20) ◽

pp. 7716-7725 ◽

Cited By ~ 70

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.

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Ex vivo depletion of alloreactive cells based on CFSE dye dilution, activation antigen selection, and dendritic cell stimulation

Blood ◽

10.1182/blood-2003-04-1098 ◽

2004 ◽

Vol 103 (3) ◽

pp. 1158-1165 ◽

Cited By ~ 67

Author(s):

Wayne R. Godfrey ◽

Mark R. Krampf ◽

Patricia A. Taylor ◽

Bruce R. Blazar

Keyword(s):

T Cells ◽

Mhc Class Ii ◽

High Efficiency ◽

Ex Vivo ◽

Peripheral Blood Mononuclear ◽

Flow Cytometric ◽

Histocompatibility Complex ◽

Cell Transplants ◽

Activation Antigen

AbstractEliminating alloreactive cells from T-cell populations would enable the transfer of immune function to patients who receive stem cell transplants. However, high-efficiency depletion has proved difficult to achieve. We sought to develop ex vivo approaches for the maximal depletion of alloreactive CD4+ T cells. Using a flow cytometric cell sorting approach after mixed lymphocyte reaction (MLR) culture, we have found that sorted CFSEbright (5-(and-6)-carboxyfluorescein diacetate succinmidyl ester) (nondivided) and activation antigen-negative cells are markedly depleted of alloreactivity. With HLA-mismatched peripheral blood mononuclear cell (PBMC) stimulators we have consistently attained (90%-95%) depletion of alloreactivity. Importantly, when purified matured monocyte-derived dendritic cells (DCs) are used as stimulators, a 100-fold (99%) reduction in alloreactivity was attained, resulting in abrogation of the secondary MLR. Significantly, the CFSEbright CD25- cells recovered from these cultures retained general immunoreactivity, including responses to Candida and cytomegalovirus (CMV) antigens. In addition, a CFSE-based approach was tested and found to be sufficient for graft-versus-host disease (GVHD) prevention in vivo, in a major histocompatibility complex (MHC) class II disparate murine model. This efficient approach to selectively deplete mature alloantigen-specific T cells may permit enhanced immune reconstitution without GVHD. (Blood. 2004;103:1158-1165)

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Endogenous human cytomegalovirus gB is presented efficiently by MHC class II molecules to CD4+ CTL

Journal of Experimental Medicine ◽

10.1084/jem.20050162 ◽

2005 ◽

Vol 202 (8) ◽

pp. 1109-1119 ◽

Cited By ~ 61

Author(s):

Nagendra R. Hegde ◽

Claire Dunn ◽

David M. Lewinsohn ◽

Michael A. Jarvis ◽

Jay A. Nelson ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Mhc Class Ii ◽

Glial Cells ◽

Human Cytomegalovirus ◽

Cd4 T Cells ◽

Class Ii ◽

Host Cells ◽

Cd4 T Cell

Human cytomegalovirus (HCMV) infects endothelial, epithelial, and glial cells in vivo. These cells can express MHC class II proteins, but are unlikely to play important roles in priming host immunity. Instead, it seems that class II presentation of endogenous HCMV antigens in these cells allows recognition of virus infection. We characterized class II presentation of HCMV glycoprotein B (gB), a membrane protein that accumulates extensively in endosomes during virus assembly. Human CD4+ T cells specific for gB were both highly abundant in blood and cytolytic in vivo. gB-specific CD4+ T cell clones recognized gB that was expressed in glial, endothelial, and epithelial cells, but not exogenous gB that was fed to these cells. Glial cells efficiently presented extremely low levels of endogenous gB—expressed by adenovirus vectors or after HCMV infection—and stimulated CD4+ T cells better than DCs that were incubated with exogenous gB. Presentation of endogenous gB required sorting of gB to endosomal compartments and processing by acidic proteases. Although presentation of cellular proteins that traffic into endosomes is well known, our observations demonstrate for the first time that a viral protein sorted to endosomes is presented exceptionally well, and can promote CD4+ T cell recognition and killing of biologically important host cells.

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Identification of ACE2 modifiers by CRISPR screening

10.1101/2021.06.10.447768 ◽

2021 ◽

Author(s):

Emily J Sherman ◽

Carmen Mirabelli ◽

Vi T Tang ◽

Taslima G Khan ◽

Andrew A Kennedy ◽

...

Keyword(s):

Cell Lines ◽

Individual Cell ◽

Mrna Level ◽

Host Factors ◽

Host Cells ◽

Functional Networks ◽

Huh7 Cells ◽

Therapeutic Development ◽

Immortalized Cell

SARS-CoV-2 infection is initiated by binding of the viral spike protein to its receptor, ACE2, on the surface of host cells. ACE2 expression is heterogeneous both in vivo and in immortalized cell lines, but the molecular pathways that govern ACE2 expression remain unclear. We now report high-throughput CRISPR screens for functional modifiers of ACE2 surface abundance. We identified 35 genes whose disruption was associated with a change in the surface abundance of ACE2 in HuH7 cells. Enriched among these ACE2 regulators were established transcription factors, epigenetic regulators, and functional networks. We further characterized individual cell lines with disruption of SMAD4, EP300, PIAS1, or BAMBI and found these genes to regulate ACE2 at the mRNA level and to influence cellular susceptibility to SARS-CoV-2 infection. Collectively, our findings clarify the host factors involved in SARS-CoV-2 entry and suggest potential targets for therapeutic development.

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

Journal of Experimental Medicine ◽

10.1084/jem.184.6.2153 ◽

1996 ◽

Vol 184 (6) ◽

pp. 2153-2166 ◽

Cited By ~ 156

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.

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Differential regulation of Igf1 and Igf2 mRNA levels in tilapia hepatocytes: effects of insulin and cortisol on GH sensitivity

Journal of Endocrinology ◽

10.1530/joe-10-0456 ◽

2011 ◽

Vol 211 (2) ◽

pp. 201-210 ◽

Cited By ~ 44

Author(s):

Andrew L Pierce ◽

Jason P Breves ◽

Shunsuke Moriyama ◽

Tetsuya Hirano ◽

E Gordon Grau

Keyword(s):

Growth And Development ◽

Mrna Level ◽

Differential Regulation ◽

Postnatal Life ◽

Mrna Levels ◽

Primary Cultured Hepatocytes ◽

Mammalian Liver ◽

Growth Promoting ◽

Stimulation Of

Igf1 and Igf2 stimulate growth and development of vertebrates. In mammals, liver-derived endocrine Igf1 mediates the growth promoting effects of GH during postnatal life, whereas Igf2 stimulates placental and fetal growth and is not regulated by GH. Insulin enhances Igf1 production by the mammalian liver directly, and by increasing hepatocyte sensitivity to GH. We examined the regulation ofigf1andigf2mRNA levels by GH, insulin, and cortisol, and the effects of insulin and cortisol on GH sensitivity in primary cultured hepatocytes of tilapia, a cichlid teleost. GH increased mRNA levels of bothigf1andigf2in a concentration-related and biphasic manner over the physiological range, with a greater effect onigf2mRNA level. Insulin increased basaligf2mRNA level, and strongly increased GH-stimulatedigf2mRNA level, but slightly reduced basaligf1mRNA level and did not affect GH-stimulatedigf1mRNA level. Cortisol inhibited GH stimulation ofigf1, but increased GH stimulation ofigf2mRNA level. The synergistic effect of insulin and GH onigf2mRNA level was confirmedin vivo. These results indicate that insulin and cortisol differentially modulate the response ofigf1andigf2mRNA to GH in tilapia hepatocytes, and suggest that the regulation of liver Igf2 production differs between fish and mammals. Regulation of liver Igf2 production in fish appears to be similar to regulation of liver Igf1 production in mammals.

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