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1.
Nat Commun ; 14(1): 6035, 2023 09 27.
Article in English | MEDLINE | ID: mdl-37758698

ABSTRACT

γδ T cells play a pivotal role in protection against various types of infections and tumours, from early childhood on and throughout life. They consist of several subsets characterised by adaptive and innate-like functions, with Vγ9Vδ2 being the largest subset in human peripheral blood. Although these cells show signs of cytotoxicity, their modus operandi remains poorly understood. Here we explore, using live single-cell imaging, the cytotoxic functions of γδ T cells upon interactions with tumour target cells with high temporal and spatial resolution. While γδ T cell killing is dominated by degranulation, the availability of lytic molecules appears tightly regulated in time and space. In particular, the limited co-occurrence of granzyme B and perforin restrains serial killing of tumour cells by γδ T cells. Thus, our data provide new insights into the cytotoxic arsenal and functions of γδ T cells, which may guide the development of more efficient γδ T cell based adoptive immunotherapies.


Subject(s)
Antineoplastic Agents , Child, Preschool , Humans , Perforin , Immunotherapy, Adoptive , Receptors, Antigen, T-Cell, gamma-delta , Cytotoxicity, Immunologic
2.
Cell Immunol ; 375: 104516, 2022 05.
Article in English | MEDLINE | ID: mdl-35413621

ABSTRACT

Mutations causing loss of the NF-κB regulator IκBNS, result in impaired development of innate-like B cells and defective plasma cell (PC) differentiation. Since productive PC differentiation requires B cell metabolic reprogramming, we sought to investigate processes important for this transition using the bumble mouse strain, deficient for IκBNS. We report that LPS-activated bumble B cells exhibited elevated mTOR activation levels, mitochondrial accumulation, increased OXPHOS and mROS production, along with a reduced capacity for autophagy, compared to wildtype B cells. Overall, our results demonstrate that PC differentiation in the absence of IκBNS is characterized by excessive activation during early rounds of B cell division, increased mitochondrial metabolism and decreased autophagic capacity, thus improving our understanding of the role of IκBNS in PC differentiation.


Subject(s)
Lymphocyte Activation , NF-kappa B , Animals , Cell Differentiation/genetics , Mice , Mice, Knockout , NF-kappa B/metabolism , Oxidative Stress
3.
Immunol Cell Biol ; 97(5): 485-497, 2019 05.
Article in English | MEDLINE | ID: mdl-30597621

ABSTRACT

Impaired classical NF-κB pathway signaling causes reduced antibody responses to T-independent (TI) antigens. We investigated the potential reasons for defective TI responses in mice lacking the atypical inhibitory kappa B (IκB) protein of the NF-κB pathway, IκBNS. Analyses of the plasma cell compartment in vitro and in vivo after challenge with lipopolysaccharide (LPS) showed significant decreases in the frequencies of plasma cells in the absence of IκBNS. In vitro activation of B cells via the B cell receptor or via Toll-like receptor 4 revealed that early activation events were unaffected in IκBNS-deficient B cells, while proliferation was reduced compared to in similarly stimulated wildtype (wt) B cells. IκBNS-deficient B cells also displayed impaired upregulation of the transmembrane activator and calcium modulator cyclophilin ligand interactor (TACI), which is essential for TI responses, and decreased sensitivity to TACI ligands upon stimulation. Furthermore, IκBNS-deficient B cells, in contrast to wt B cells, displayed altered expression of IRF4, Blimp-1 and Pax5 upon LPS-induced differentiation, indicating impaired transcriptional regulation of plasma cell generation.


Subject(s)
Cell Differentiation , Gene Expression Regulation/immunology , I-kappa B Proteins/deficiency , Plasma Cells/immunology , Transmembrane Activator and CAML Interactor Protein/immunology , Animals , Cell Differentiation/genetics , Cell Differentiation/immunology , I-kappa B Proteins/immunology , Mice , Mice, Knockout , Plasma Cells/cytology , Toll-Like Receptor 4/genetics , Toll-Like Receptor 4/immunology , Transmembrane Activator and CAML Interactor Protein/genetics
4.
J Immunol ; 200(2): 775-787, 2018 01 15.
Article in English | MEDLINE | ID: mdl-29222168

ABSTRACT

Marginal zone (MZ) B cells reside in the splenic MZ and play important roles in T cell-independent humoral immune responses against blood-borne pathogens. IκBNS-deficient bumble mice exhibit a severe reduction in the MZ B compartment but regain an MZ B population with age and, thus, represent a valuable model to examine the biology of MZ B cells. In this article, we characterized the MZ B cell defect in further detail and investigated the nature of the B cells that appear in the MZ of aged bumble mice. Flow cytometry analysis of the splenic transitional B cell subsets demonstrated that MZ B cell development was blocked at the transitional-1 to transitional-2-MZ precursor stage in the absence of functional IκBNS. Immunohistochemical analysis of spleen sections from wild-type and bumble mice revealed no alteration in the cellular MZ microenvironment, and analysis of bone marrow chimeras indicated that the MZ B cell development defect in bumble mice was B cell intrinsic. Further, we demonstrate that the B cells that repopulate the MZ in aged bumble mice were distinct from age-matched wild-type MZ B cells. Specifically, the expression of surface markers characteristic for MZ B cells was altered and the L chain Igλ+ repertoire was reduced in bumble mice. Finally, plasma cell differentiation of sorted LPS-stimulated MZ B cells was impaired, and aged bumble mice were unable to respond to NP-Ficoll immunization. These results demonstrate that IκBNS is required for an intact MZ B cell compartment in C57BL/6 mice.


Subject(s)
B-Lymphocyte Subsets/immunology , B-Lymphocyte Subsets/metabolism , Clonal Selection, Antigen-Mediated , I-kappa B Kinase/deficiency , Spleen/immunology , Spleen/metabolism , Age Factors , Animals , Antigens, T-Independent/immunology , B-Lymphocyte Subsets/cytology , Biomarkers , Cell Differentiation , Immunoglobulin G/genetics , Immunoglobulin G/immunology , Immunophenotyping , Lipopolysaccharides/immunology , Mice , Mice, Knockout , Phenotype
5.
J Biol Chem ; 290(45): 27345-27359, 2015 Nov 06.
Article in English | MEDLINE | ID: mdl-26370074

ABSTRACT

Siglec-1 (sialoadhesin, CD169) is a surface receptor on human cells that mediates trans-enhancement of HIV-1 infection through recognition of sialic acid moieties in virus membrane gangliosides. Here, we demonstrate that mouse Siglec-1, expressed on the surface of primary macrophages in an interferon-α-responsive manner, captures murine leukemia virus (MLV) particles and mediates their transfer to proliferating lymphocytes. The MLV infection of primary B-cells was markedly more efficient than that of primary T-cells. The major structural protein of MLV particles, Gag, frequently co-localized with Siglec-1, and trans-infection, primarily of surface-bound MLV particles, efficiently occurred. To explore the role of sialic acid for MLV trans-infection at a submolecular level, we analyzed the potential of six sialic acid precursor analogs to modulate the sialylated ganglioside-dependent interaction of MLV particles with Siglec-1. Biosynthetically engineered sialic acids were detected in both the glycolipid and glycoprotein fractions of MLV producer cells. MLV released from cells carrying N-acyl-modified sialic acids displayed strikingly different capacities for Siglec-1-mediated capture and trans-infection; N-butanoyl, N-isobutanoyl, N-glycolyl, or N-pentanoyl side chain modifications resulted in up to 92 and 80% reduction of virus particle capture and trans-infection, respectively, whereas N-propanoyl or N-cyclopropylcarbamyl side chains had no effect. In agreement with these functional analyses, molecular modeling indicated reduced binding affinities for non-functional N-acyl modifications. Thus, Siglec-1 is a key receptor for macrophage/lymphocyte trans-infection of surface-bound virions, and the N-acyl side chain of sialic acid is a critical determinant for the Siglec-1/MLV interaction.


Subject(s)
Moloney murine leukemia virus/pathogenicity , Sialic Acid Binding Ig-like Lectin 1/chemistry , Sialic Acid Binding Ig-like Lectin 1/physiology , Animals , Binding Sites , Cell Line , Gangliosides/chemistry , Gangliosides/metabolism , Host-Pathogen Interactions/physiology , Humans , Interferon-alpha/physiology , Leukemia, Experimental/physiopathology , Leukemia, Experimental/virology , Lymphocytes/physiology , Lymphocytes/virology , Macrophages/physiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Models, Molecular , Moloney murine leukemia virus/genetics , Moloney murine leukemia virus/physiology , N-Acetylneuraminic Acid/chemistry , Receptors, Virus/chemistry , Receptors, Virus/physiology , Retroviridae Infections/physiopathology , Retroviridae Infections/virology , Sialic Acid Binding Ig-like Lectin 1/genetics , Tumor Virus Infections/physiopathology , Tumor Virus Infections/virology
6.
Retrovirology ; 12: 37, 2015 May 07.
Article in English | MEDLINE | ID: mdl-25947229

ABSTRACT

BACKGROUND: Myeloid cells are key players in the recognition and response of the host against invading viruses. Paradoxically, upon HIV-1 infection, myeloid cells might also promote viral pathogenesis through trans-infection, a mechanism that promotes HIV-1 transmission to target cells via viral capture and storage. The receptor Siglec-1 (CD169) potently enhances HIV-1 trans-infection and is regulated by immune activating signals present throughout the course of HIV-1 infection, such as interferon α (IFNα). RESULTS: Here we show that IFNα-activated dendritic cells, monocytes and macrophages have an enhanced ability to capture and trans-infect HIV-1 via Siglec-1 recognition of viral membrane gangliosides. Monocytes from untreated HIV-1-infected individuals trans-infect HIV-1 via Siglec-1, but this capacity diminishes after effective antiretroviral treatment. Furthermore, Siglec-1 is expressed on myeloid cells residing in lymphoid tissues, where it can mediate viral trans-infection. CONCLUSIONS: Siglec-1 on myeloid cells could fuel novel CD4(+) T-cell infections and contribute to HIV-1 dissemination in vivo.


Subject(s)
HIV-1/immunology , HIV-1/physiology , Interferon-alpha/metabolism , Myeloid Cells/virology , Sialic Acid Binding Ig-like Lectin 1/biosynthesis , Up-Regulation , Adult , CD4-Positive T-Lymphocytes/virology , Cells, Cultured , Humans , Male
7.
J Leukoc Biol ; 98(1): 5-14, 2015 Jul.
Article in English | MEDLINE | ID: mdl-25646359

ABSTRACT

The deoxynucleoside triphosphate triphosphohydrolase and 3' → 5' exonuclease SAMHD1 restricts HIV-1 infection in noncycling hematopoietic cells in vitro, and SAMHD1 mutations are associated with AGS. Little is known about the in vivo expression and functional regulation of this cellular factor. Here, we first assessed the SAMHD1 protein expression profile on a microarray of 25 human tissues from >210 donors and in purified primary cell populations. In vivo, SAMHD1 was expressed in the majority of nucleated cells of hematopoietic origin, including tissue-resident macrophages, DCs, pDCs, all developmental stages of thymic T cells, monocytes, NK cells, as well as at lower levels in B cells. Of note, SAMHD1 was abundantly expressed in HIV target cells residing in the anogenital mucosa, providing a basis for its evaluation as a cellular factor that may impact the efficiency of HIV transmission. Next, we examined the effect of the activation status and proinflammatory cytokine treatment of cells on expression and phosphorylation of SAMHD1. Activated, HIV-susceptible CD4(+) T cells carried pSAMHD1(T592), whereas resting CD4(+) T cells and macrophages expressed the unphosphorylated protein with HIV-restrictive activity. Surprisingly, stimulation of these primary cells with IFN-α, IFN-γ, IL-4, IL-6, IL-12, IL-18, IL-27, or TNF-α affected neither SAMHD1 expression levels nor threonine 592 phosphorylation. Only IL-1ß moderately down-regulated SAMHD1 in activated CD4(+) T cells. Taken together, this study establishes the first cross-sectional protein expression profile of SAMHD1 in human tissues and provides insight into its cell cycle-dependent phosphorylation and unresponsiveness to multiple proinflammatory cytokines.


Subject(s)
Gene Expression Profiling , Monomeric GTP-Binding Proteins/metabolism , Cell Line , Cytokines/metabolism , HIV-1/metabolism , HIV-1/physiology , Humans , Lymphocyte Activation , Membrane Fusion , Monomeric GTP-Binding Proteins/genetics , Phosphorylation , SAM Domain and HD Domain-Containing Protein 1 , T-Lymphocytes/immunology
8.
PLoS Biol ; 10(12): e1001448, 2012.
Article in English | MEDLINE | ID: mdl-23271952

ABSTRACT

Dendritic cells (DCs) are essential antigen-presenting cells for the induction of immunity against pathogens. However, HIV-1 spread is strongly enhanced in clusters of DCs and CD4(+) T cells. Uninfected DCs capture HIV-1 and mediate viral transfer to bystander CD4(+) T cells through a process termed trans-infection. Initial studies identified the C-type lectin DC-SIGN as the HIV-1 binding factor on DCs, which interacts with the viral envelope glycoproteins. Upon DC maturation, however, DC-SIGN is down-regulated, while HIV-1 capture and trans-infection is strongly enhanced via a glycoprotein-independent capture pathway that recognizes sialyllactose-containing membrane gangliosides. Here we show that the sialic acid-binding Ig-like lectin 1 (Siglec-1, CD169), which is highly expressed on mature DCs, specifically binds HIV-1 and vesicles carrying sialyllactose. Furthermore, Siglec-1 is essential for trans-infection by mature DCs. These findings identify Siglec-1 as a key factor for HIV-1 spread via infectious DC/T-cell synapses, highlighting a novel mechanism that mediates HIV-1 dissemination in activated tissues.


Subject(s)
Dendritic Cells/metabolism , Dendritic Cells/virology , Gangliosides/metabolism , HIV Infections/immunology , HIV-1/physiology , Lipid Bilayers/metabolism , Sialic Acid Binding Ig-like Lectin 1/metabolism , Dendritic Cells/drug effects , Exosomes/drug effects , Exosomes/metabolism , Gene Silencing/drug effects , HEK293 Cells , HIV Infections/pathology , HIV Infections/virology , Humans , Immunological Synapses/drug effects , Lipopolysaccharides/pharmacology , Liposomes/metabolism , Up-Regulation/drug effects , Virion/drug effects , Virion/metabolism
9.
Nat Med ; 18(11): 1682-7, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22972397

ABSTRACT

Unlike activated CD4(+) T cells, resting CD4(+) T cells are highly resistant to productive HIV-1 infection. Early after HIV-1 entry, a major block limits reverse transcription of incoming viral genomes. Here we show that the deoxynucleoside triphosphate triphosphohydrolase SAMHD1 prevents reverse transcription of HIV-1 RNA in resting CD4(+) T cells. SAMHD1 is abundantly expressed in resting CD4(+) T cells circulating in peripheral blood and residing in lymphoid organs. The early restriction to infection in unstimulated CD4(+) T cells is overcome by HIV-1 or HIV-2 virions into which viral Vpx is artificially or naturally packaged, respectively, or by addition of exogenous deoxynucleosides. Vpx-mediated proteasomal degradation of SAMHD1 and elevation of intracellular deoxynucleotide pools precede successful infection by Vpx-carrying HIV. Resting CD4(+) T cells from healthy donors following SAMHD1 silencing or from a patient with Aicardi-Goutières syndrome homozygous for a nonsense mutation in SAMHD1 were permissive for HIV-1 infection. Thus, SAMHD1 imposes an effective restriction to HIV-1 infection in the large pool of noncycling CD4(+) T cells in vivo. Bypassing SAMHD1 was insufficient for the release of viral progeny, implicating other barriers at later stages of HIV replication. Together, these findings may unveil new ways to interfere with the immune evasion and T cell immunopathology of pandemic HIV-1.


Subject(s)
CD4-Positive T-Lymphocytes , HIV-1 , Monomeric GTP-Binding Proteins , Autoimmune Diseases of the Nervous System/genetics , Autoimmune Diseases of the Nervous System/virology , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , HIV Infections , HIV-1/genetics , HIV-1/metabolism , HIV-2/genetics , HIV-2/metabolism , Human Immunodeficiency Virus Proteins/genetics , Human Immunodeficiency Virus Proteins/metabolism , Humans , Monomeric GTP-Binding Proteins/genetics , Monomeric GTP-Binding Proteins/metabolism , Nervous System Malformations/genetics , Nervous System Malformations/virology , Reverse Transcription/genetics , SAM Domain and HD Domain-Containing Protein 1 , Viral Regulatory and Accessory Proteins/genetics , Viral Regulatory and Accessory Proteins/metabolism , Virion/genetics , Virion/growth & development , Virus Replication
10.
Proc Natl Acad Sci U S A ; 108(33): 13688-93, 2011 Aug 16.
Article in English | MEDLINE | ID: mdl-21808013

ABSTRACT

Human CD317 is an intrinsic immunity factor that restricts the release of enveloped viruses, including the major pathogens HIV and Lassa virus, from infected cells in culture. Its importance for infection control in humans is unclear, due in part to its incompletely defined in vivo expression pattern. CD317 also has been proposed as a selective target for immunotherapy of multiple myeloma. To provide a framework for studies of the biological functions, regulation, and therapeutic potential of CD317, we performed microarray-based expression profiling in 468 tissue samples from 25 healthy organs from more than 210 patients. We found that CD317 protein was expressed to varying degrees in all organs tested and detected in a number of specialized cell types, including hepatocytes, pneumocytes, ducts of major salivary glands, pancreas and kidney, Paneth cells, epithelia, Leydig cells, plasma cells, bone marrow stromal cells, monocytes, and vascular endothelium. Although many of these cell types are in vivo targets for pathogenic viruses, restriction by CD317 or virus-encoded antagonists has been documented in only some of them. Limited cell type-dependent coexpression of CD317 with the IFN biomarker MxA in vivo and lack of responsive stimulation in organ explants suggest that interferons may only partially regulate CD317. This in vivo expression profiling sheds light on the biology and species-specificity of CD317, identifies multiple thus far unknown interaction sites of viruses with this restriction factor, and refutes the concept of its restricted constitutive expression and primary IFN inducibility. CD317's widespread expression calls into question its suitability as a target for immunotherapy.


Subject(s)
Antigens, CD/analysis , Antigens, CD/immunology , Antigens, Neoplasm/analysis , Antiviral Agents/analysis , GPI-Linked Proteins/analysis , GPI-Linked Proteins/immunology , Humans , Immunity , Interferons/analysis , Protein Array Analysis , Species Specificity , Tissue Array Analysis , Tissue Distribution
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