ABSTRACT
MHC class II molecules function to present exogenous antigen-derived peptides to CD4 T cells to both drive T cell activation and to provide signals back into the class II antigen-presenting cell. Previous work established the presence of multiple GxxxG dimerization motifs within the transmembrane domains of MHC class II α and ß chains across a wide range of species and revealed a role for differential GxxxG motif pairing in the formation of two discrete mouse class II conformers with distinct functional properties (i.e., M1-and M2-paired I-Ak class II). Biochemical and mutagenesis studies detailed herein extend this model to human class II by identifying an anti-HLA-DR mAb (Tü36) that selectively binds M1-paired HLA-DR molecules. Analysis of the HLA-DR allele reactivity of the Tü36 mAb helped define other HLA-DR residues involved in mAb binding. In silico modeling of both TM domain interactions and whole protein structure is consistent with the outcome of biochemical/mutagenesis studies and provides insight into the possible structural differences between the two HLA-DR conformers. Cholesterol depletion studies indicate a role for cholesterol-rich membrane domains in the formation/maintenance of Tü36 mAb reactive DR molecules. Finally, phylogenetic analysis of the amino acid sequences of Tü36-reactive HLA-DR ß chains reveals a unique pattern of both Tü36 mAb reactivity and key amino acid polymorphisms. In total, these studies bring the paradigm M1/M2-paired MHC class II molecules to the human HLA-DR molecule and suggest that the functional differences between these conformers defined in mouse class II extend to the human immune system.
Subject(s)
Amino Acid Motifs , HLA-DR Antigens , Histocompatibility Antigens Class II , Animals , Humans , Mice , CD4-Positive T-Lymphocytes/metabolism , Dimerization , Histocompatibility Antigens Class II/metabolism , HLA-DR Antigens/genetics , HLA-DR Antigens/metabolism , Phylogeny , Amino Acid Motifs/physiologyABSTRACT
Group 2 innate lymphoid cells (ILC2) are tissue-resident, long-lived innate effector cells implicated in allergy and asthma. Upon activation, mature ILC2 rapidly secrete large amounts of type-2 cytokines and other effector molecules. The molecular pathways that drive ILC2 activation are not well understood. In this study, we report that the transcriptional controller core binding factor ß (CBFß) is required for ILC2 activation. Deletion or inhibition of CBFß did not impair the maintenance of ILC2 at homeostasis but abolished ILC2 activation during allergic airway inflammation. Treatment with CBFß inhibitors prevented ILC2-mediated airway hyperresponsiveness in a mouse model of acute Alternaria allergen inhalation. CBFß promoted expression of key ILC2 genes at both transcriptional and translational levels. CBF transcriptional complex directly bound to Il13 and Vegfa promoters and enhancers, and controlled gene transcription. CBFß further promoted ribosome biogenesis and enhanced gene translation in activated ILC2. Together, these data establish an essential role for CBFß in ILC2 activation.
Subject(s)
Core Binding Factor beta Subunit/immunology , Gene Expression Regulation/immunology , Immunity, Innate/immunology , Lymphocyte Activation/immunology , Lymphocytes/immunology , Animals , Hypersensitivity/immunology , Mice , Mice, KnockoutABSTRACT
Antigen processing and MHC class II-restricted antigen presentation by antigen-presenting cells such as dendritic cells and B cells allows the activation of naïve CD4+ T cells and cognate interactions between B cells and effector CD4+ T cells, respectively. B cells are unique among class II-restricted antigen-presenting cells in that they have a clonally restricted antigen-specific receptor, the B cell receptor (BCR), which allows the cell to recognize and respond to trace amounts of foreign antigen present in a sea of self-antigens. Moreover, engagement of peptide-class II complexes formed via BCR-mediated processing of cognate antigen has been shown to result in a unique pattern of B cell activation. Using a combined biochemical and imaging/FRET approach, we establish that internalized antigen-BCR complexes associate with intracellular class II molecules. We demonstrate that the M1-paired MHC class II conformer, shown previously to be critical for CD4 T cell activation, is incorporated selectively into these complexes and loaded selectively with peptide derived from BCR-internalized cognate antigen. These results demonstrate that, in B cells, internalized antigen-BCR complexes associate with intracellular MHC class II molecules, potentially defining a site of class II peptide acquisition, and reveal a selective role for the M1-paired class II conformer in the presentation of cognate antigen. These findings provide key insights into the molecular mechanisms used by B cells to control the source of peptides charged onto class II molecules, allowing the immune system to mount an antibody response focused on BCR-reactive cognate antigen.
Subject(s)
Histocompatibility Antigens Class II/metabolism , Receptors, Antigen, B-Cell/metabolism , Animals , Antigen Presentation , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Cell Line , Fluorescence Resonance Energy Transfer , Histocompatibility Antigens Class II/chemistry , Humans , Mice , Multiprotein Complexes/chemistry , Multiprotein Complexes/metabolism , Protein Binding , Receptors, Antigen, B-Cell/chemistry , Receptors, Antigen, B-Cell/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolismABSTRACT
Major histocompatibility complex (MHC) class II molecules exhibit conformational heterogeneity, which influences their ability to stimulate CD4 T cells and drive immune responses. Previous studies suggest a role for the transmembrane domain of the class II αß heterodimer in determining molecular structure and function. Our previous studies identified an MHC class II conformer that is marked by the Ia.2 epitope. These Ia.2(+) class II conformers are lipid raft-associated and able to drive both tyrosine kinase signaling and efficient antigen presentation to CD4 T cells. Here, we establish that the Ia.2(+) I-A(k) conformer is formed early in the class II biosynthetic pathway and that differential pairing of highly conserved transmembrane domain GXXXG dimerization motifs is responsible for formation of Ia.2(+) versus Ia.2(-) I-A(k) class II conformers and controlling lipid raft partitioning. These findings provide a molecular explanation for the formation of two distinct MHC class II conformers that differ in their inherent ability to signal and drive robust T cell activation, providing new insight into the role of MHC class II in regulating antigen-presenting cell-T cell interactions critical to the initiation and control of multiple aspects of the immune response.
Subject(s)
Histocompatibility Antigens Class II/immunology , Amino Acid Motifs , Amino Acid Sequence , Cell Line , Dimerization , Endoplasmic Reticulum/immunology , Histocompatibility Antigens Class II/chemistry , Molecular Sequence Data , Molecular Structure , Sequence Homology, Amino AcidABSTRACT
BACKGROUND: Antigen-specific CD4 T cells are activated by small numbers of antigenic peptide-MHC class II (pMHC-II) complexes on dendritic cells (DCs). RESULTS: Newly generated pMHC-II complexes are present in small clusters on the DC surface. CONCLUSION: pMHC-II clusters permit efficient T cell activation. SIGNIFICANCE: The appearance of clustered pMHC-II reveals the organization of the T cell antigen receptor ligand on the DC surface. Dendritic cells (DCs) function by stimulating naive antigen-specific CD4 T cells to proliferate and secrete a variety of immunomodulatory factors. The ability to activate naive T cells comes from the capacity of DCs to internalize, degrade, and express peptide fragments of antigenic proteins on their surface bound to MHC class II molecules (MHC-II). Although DCs express tens of thousands of distinct MHC-II, very small amounts of specific peptide-MHC-II complexes are required to interact with and activate T cells. We now show that stimulatory MHC-II I-A(k)-HEL(46-61) complexes that move from intracellular antigen-processing compartments to the plasma membrane are not randomly distributed on the DC surface. Confocal immunofluorescence microscopy and quantitative immunoelectron microscopy reveal that the majority of newly generated MHC-II I-A(k)-HEL(46-61) complexes are expressed in sub-100-nm microclusters on the DC membrane. These microclusters are stabilized in cholesterol-containing microdomains, and cholesterol depletion inhibits the stability of these clusters as well as the ability of the DCs to function as antigen-presenting cells. These results demonstrate that specific cohorts of peptide-MHC-II complexes expressed on the DC surface are present in cholesterol-dependent microclusters and that cluster integrity is important for antigen-specific naive CD4 T cell activation by DCs.
Subject(s)
Cell Membrane/metabolism , Cholesterol/metabolism , Histocompatibility Antigens Class II/metabolism , Membrane Microdomains/metabolism , Animals , Antigen Presentation , Antigens/immunology , CD4-Positive T-Lymphocytes/immunology , Cells, Cultured , Dendritic Cells/immunology , Dendritic Cells/metabolism , Lymphocyte Activation , Membrane Lipids/metabolism , Mice , Mice, Transgenic , Protein TransportABSTRACT
Activation of the FcγR via antigen containing immune complexes can lead to the generation of reactive oxygen species, which are potent signal transducing molecules. However, whether ROS contribute to FcγR signaling has not been studied extensively. We set out to elucidate the role of NADPH oxidase-generated ROS in macrophage activation following FcγR engagement using antigen-containing immune complexes. We hypothesized that NOX2 generated ROS is necessary for propagation of downstream FcγR signaling and initiation of the innate immune response. Following exposure of murine bone marrow-derived macrophages (BMDMs) to inactivated Francisella tularensis (iFt)-containing immune complexes, we observed a significant increase in the innate inflammatory cytokine IL-6 at 24 h compared with macrophages treated with Ft LVS-containing immune complexes. Ligation of the FcγR by opsonized Ft also results in significant ROS production. Macrophages lacking the gp91(phox) subunit of NOX2 fail to produce ROS upon FcγR ligation, resulting in decreased Akt phosphorylation and a reduction in the levels of IL-6 compared with wild type macrophages. Similar results were seen following infection of BMDMs with catalase deficient Ft that fail to scavenge hydrogen peroxide. In conclusion, our findings demonstrate that ROS participate in elicitation of an effective innate immune in response to antigen-containing immune complexes through FcγR.
Subject(s)
Bone Marrow Cells/metabolism , Hydrogen Peroxide/metabolism , Interleukin-6/metabolism , Macrophages/metabolism , Membrane Glycoproteins/metabolism , NADPH Oxidases/metabolism , Receptors, IgG/metabolism , Animals , Bacterial Proteins/genetics , Bacterial Proteins/immunology , Bacterial Proteins/metabolism , Bone Marrow Cells/immunology , Catalase/genetics , Catalase/immunology , Catalase/metabolism , Francisella tularensis/enzymology , Francisella tularensis/genetics , Francisella tularensis/immunology , Hydrogen Peroxide/immunology , Immunity, Innate/physiology , Interleukin-6/genetics , Interleukin-6/immunology , Macrophages/immunology , Membrane Glycoproteins/genetics , Membrane Glycoproteins/immunology , Mice , Mice, Knockout , NADPH Oxidase 2 , NADPH Oxidases/genetics , NADPH Oxidases/immunology , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/immunology , Proto-Oncogene Proteins c-akt/metabolism , Receptors, IgG/genetics , Receptors, IgG/immunology , Signal Transduction/genetics , Signal Transduction/immunologyABSTRACT
The BCR allows for Ag-driven B cell activation and subsequent Ag endocytosis, processing, and presentation to recruit T cell help. Core drivers of BCR signaling and endocytosis are motifs within the receptor's cytoplasmic tail (primarily CD79). However, BCR function can be tuned by other proximal cellular elements, such as CD20 and membrane lipid microdomains. To identify additional proteins that could modulate BCR function, we used a proximity-based biotinylation technique paired with mass spectrometry to identify molecular neighbors of the murine IgM BCR. Those neighbors include MHC class II molecules, integrins, various transporters, and membrane microdomain proteins. Class II molecules, some of which are invariant chain-associated nascent class II, are a readily detected BCR neighbor. This finding is consistent with reports of BCR-class II association within intracellular compartments. The BCR is also in close proximity to multiple proteins involved in the formation of membrane microdomains, including CD37, raftlin, and Ig superfamily member 8. Known defects in T cell-dependent humoral immunity in CD37 knockout mice suggest a role for CD37 in BCR function. In line with this notion, CRISPR-based knockout of CD37 expression in a B cell line heightens BCR signaling, slows BCR endocytosis, and tempers formation of peptide-class II complexes. These results indicate that BCR molecular neighbors can impact membrane-mediated BCR functions. Overall, a proximity-based labeling technique allowed for identification of multiple previously unknown BCR molecular neighbors, including the tetraspanin protein CD37, which can modulate BCR function.
Subject(s)
Immunity, Humoral , Membrane Proteins , Animals , Mice , Cell Line , Lymphocyte Activation , Mice, Knockout , Receptors, Antigen, B-CellABSTRACT
B cell receptor (BCR)-mediated antigen (Ag) processing and presentation lead to B cell-T cell interactions, which support affinity maturation and immunoglobulin class switching. These interactions are supported by generation of peptide-MHC class II complexes in multivesicular body-like MIIC compartments of B cells. Previous studies have shown that trafficking of Ag·BCR complexes to MVB-like MIIC occurs via an ubiquitin-dependent pathway and that ubiquitination of Ag·BCR complexes occurs by an Src family kinase signaling-dependent mechanism that is restricted to lipid raft-resident Ag·BCR complexes. This study establishes that downstream Syk-dependent BCR signaling is also required for BCR ubiquitination and BCR-mediated antigen processing and presentation. Knockdown studies reveal that of the two known Syk-binding E3 ubiquitin ligases c-Cbl and Cbl-b, only c-Cbl appears to have a central role in BCR ubiquitination, trafficking to MIIC, and ubiquitin-dependent BCR-mediated antigen processing and presentation. These results establish the novel role for Syk signaling and the Syk-binding ubiquitin ligase c-Cbl in the BCR-mediated processing and presentation of cognate antigen and define one mechanism by which antigen-induced BCR ubiquitination is modulated to impact the initiation and maturation of the humoral immune response.
Subject(s)
Antigen Presentation/physiology , Intracellular Signaling Peptides and Proteins/immunology , Protein-Tyrosine Kinases/immunology , Proto-Oncogene Proteins c-cbl/immunology , Receptors, Antigen, B-Cell/immunology , Signal Transduction/physiology , Ubiquitination/immunology , Animals , Cell Line , Histocompatibility Antigens Class II/genetics , Histocompatibility Antigens Class II/immunology , Histocompatibility Antigens Class II/metabolism , Humans , Immunity, Humoral/physiology , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Mice , Protein Binding/physiology , Protein Transport/physiology , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins c-cbl/genetics , Proto-Oncogene Proteins c-cbl/metabolism , Receptors, Antigen, B-Cell/genetics , Receptors, Antigen, B-Cell/metabolism , Syk Kinase , T-Lymphocytes/immunology , T-Lymphocytes/metabolismABSTRACT
We have previously demonstrated that immunization with the inactivated Francisella tularensis, a Category A intracellular mucosal pathogen, combined with IgG2a anti-F. tularensis monoclonal antibody (Ab), enhances protection against subsequent F. tularensis challenge. To understand the mechanism(s) involved, we examined the binding, internalization, presentation, and in vivo trafficking of inactivated F. tularensis in the presence and absence of opsonizing monoclonal Ab. We found that when inactivated F. tularensis is combined with anti-F. tularensis monoclonal Ab, presentation to F. tularensis-specific T cells is enhanced. This enhancement is Fc receptor (FcR)-dependent, and requires a physical linkage between the monoclonal Ab and the inactivated F. tularensis immunogen. This enhanced presentation is due, in part, to enhanced binding and internalization of inactivated F. tularensis by antigen(Ag)-presenting cells, and involves interactions with multiple FcR types. Furthermore, targeting inactivated F. tularensis to FcRs enhances dendritic cell maturation and extends the time period over which Ag-presenting cells stimulate T cells. In vivo trafficking studies reveal enhanced transport of inactivated F. tularensis immunogen to the nasal-associated lymphoid tissue in the presence of monoclonal Ab, which is FcRn-dependent. In summary, these are the first comprehensive studies using a single-vaccine protection model/immunogen to establish the array of mechanisms involved in enhanced immunity/protection mediated by an FcR-targeted mucosal immunogen. These results demonstrate that multiple cellular/immune mechanisms contribute to FcR-enhanced immunity.
Subject(s)
Antibodies, Bacterial/immunology , Antibodies, Monoclonal/immunology , Antigens, Bacterial/immunology , Francisella tularensis/immunology , Immunity/immunology , Microbial Viability/immunology , Administration, Intranasal , Animals , Antibody Specificity/immunology , Antigen Presentation/immunology , Antigen-Antibody Complex/immunology , Antigen-Presenting Cells/immunology , Dendritic Cells/immunology , Endocytosis/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Protein Binding , Receptors, Fc/metabolism , Species Specificity , T-Lymphocytes/immunologyABSTRACT
Previous work established that binding of the 11-5.2 anti-I-A(k) mAb, which recognizes the Ia.2 epitope on I-A(k) class II molecules, elicits MHC class II signaling, whereas binding of two other anti-I-A(k) mAbs that recognize the Ia.17 epitope fail to elicit signaling. Using a biochemical approach, we establish that the Ia.2 epitope recognized by the widely used 11-5.2 mAb defines a subset of cell surface I-A(k) molecules predominantly found within membrane lipid rafts. Functional studies demonstrate that the Ia.2-bearing subset of I-A(k) class II molecules is critically necessary for effective B cell-T cell interactions, especially at low Ag doses, a finding consistent with published studies on the role of raft-resident class II molecules in CD4 T cell activation. Interestingly, B cells expressing recombinant I-A(k) class II molecules possessing a ß-chain-tethered hen egg lysosome peptide lack the Ia.2 epitope and fail to partition into lipid rafts. Moreover, cells expressing Ia.2(-) tethered peptide-class II molecules are severely impaired in their ability to present both tethered peptide or peptide derived from exogenous Ag to CD4 T cells. These results establish the Ia.2 epitope as defining a lipid raft-resident MHC class II conformer vital to the initiation of MHC class II-restricted B cell-T cell interactions.
Subject(s)
Antigen Presentation/immunology , Epitopes/immunology , Histocompatibility Antigens Class II/immunology , Membrane Microdomains/immunology , Animals , B-Lymphocytes/immunology , Cell Communication/immunology , Humans , Lymphocyte Activation , Membrane Microdomains/chemistry , Mice , T-Lymphocytes/immunologyABSTRACT
BCR-mediated Ag processing and presentation is critical to the initiation and control of a humoral immune response. Trafficking of internalized Ag-BCR complexes to intracellular Ag processing compartments is driven by ubiquitination of the cytoplasmic domain of the BCR. Using a biochemical approach, it is here established that ubiquitinated Ag-BCR complexes are formed via a signaling-dependent mechanism and restricted to plasma membrane lipid rafts. Because the structure of lipid rafts is temperature sensitive, the impact of physiological-range temperature changes (PRTCs; 33-39°C) on lipid raft-dependent and -independent BCR functions was investigated. Whereas the kinetics of lipid raft-independent BCR internalization is unaffected by temperature changes within this range, raft-dependent BCR signaling and ubiquitination as well as BCR-mediated Ag processing are significantly affected. The extent and duration of Ag-BCR ubiquitination is increased and prolonged at 37-39°C (normal to febrile temperature) compared with that at 33°C (peripheral body temperature). As might be expected, increased temperature also accelerates the overall kinetics of Ag-BCR degradation. Notably, at 33°C the expression of peptide-MHC class II complexes derived from the BCR-mediated processing of cognate Ag is profoundly slowed, whereas the kinetics of expression of peptide-MHC class II complexes derived from fluid-phase Ag processing remains unchanged. These results establish the effect of PRTCs on multiple lipid raft-dependent BCR functions including the processing and presentation of cognate Ag, suggesting one mechanism by which PRTCs, such as fever, may impact the initiation and/or maturation of a humoral immune response.
Subject(s)
Antigen Presentation/immunology , B-Lymphocytes/immunology , Membrane Microdomains/immunology , Receptors, Antigen, B-Cell/immunology , Animals , B-Lymphocytes/chemistry , B-Lymphocytes/metabolism , Blotting, Western , Humans , Membrane Microdomains/chemistry , Membrane Microdomains/metabolism , Mice , Protein Transport/immunology , Receptors, Antigen, B-Cell/chemistry , Receptors, Antigen, B-Cell/metabolism , Temperature , Ubiquitinated Proteins/chemistry , Ubiquitinated Proteins/immunology , Ubiquitinated Proteins/metabolism , UbiquitinationABSTRACT
The intracellular bacterium Francisella tularensis survives and replicates within macrophages, ultimately killing the host cell. Resolution of infection requires the development of adaptive immunity through presentation of F. tularensis antigens to CD4+ and CD8+ T cells. We have previously established that F. tularensis induces macrophage prostaglandin E2 (PGE2) production, leading to skewed T-cell responses. PGE2 can also downregulate macrophage major histocompatibility complex (MHC) class II expression, suggesting that F. tularensis-elicited PGE2 may further alter T-cell responses via inhibition of class II expression. To test this hypothesis, gamma interferon (IFN-gamma)-activated reporter macrophages were exposed to supernatants from F. tularensis-infected macrophages, and the class II levels were measured. Exposure of macrophages to infection supernatants results in essentially complete clearance of surface class II and CD86, compromising the macrophage's ability to present antigens to CD4 T cells. Biochemical analysis revealed that infection supernatants elicit ubiquitin-dependent class II downregulation and degradation within intracellular acidic compartments. By comparison, exposure to PGE2 alone only leads to a minor decrease in macrophage class II expression, demonstrating that a factor distinct from PGE2 is eliciting the majority of class II degradation. However, production of this non-PGE2 factor is dependent on macrophage cyclooxygenase activity and is induced by PGE2. These results establish that F. tularensis induces the production of a PGE2-dependent factor that elicits MHC class II downregulation in IFN-gamma-activated macrophages through ubiquitin-mediated delivery of class II to lysosomes, establishing another mechanism for the modulation of macrophage antigen presentation during F. tularensis infection.
Subject(s)
Francisella tularensis/immunology , Histocompatibility Antigens Class II/immunology , Macrophages/microbiology , Tularemia/immunology , Animals , Antigen Presentation/immunology , Blotting, Western , Dinoprostone/biosynthesis , Dinoprostone/immunology , Down-Regulation , Flow Cytometry , Francisella tularensis/metabolism , Histocompatibility Antigens Class II/metabolism , Immunoprecipitation , Interferon-gamma/immunology , Macrophage Activation/immunology , Macrophages/immunology , Macrophages/pathology , Mice , Ubiquitination/immunologyABSTRACT
In addition to functioning as a ligand to engage TCRs and drive TCR signaling, MHC class II molecules are signaling molecules that generate a number of signals within APCs, such as B lymphocytes. Moreover, MHC class II signaling is critical for B cell activation and development of a robust humoral immune response. Murine class II molecules exist in two distinct conformational states, based primarily on the differential pairing of transmembrane domain GxxxG dimerization motifs (i.e., M1- and M2-paired class II). This laboratory has previously reported that the binding of a multimerized form of an anti-class II mAb that selectively recognizes M1-paired I-Ak class II drives intracellular calcium signaling in resting murine B cells and that this signaling is dependent on both src and Syk protein tyrosine kinase activity. In contrast, multimerized forms of two different anti-I-Ak mAbs that bind both M1- and M2-paired class II fail to elicit a response. In this report, a flow cytometry-based calcium flux assay is used to demonstrate that coligation of M1- and M2-paired I-Ak class II results in the active and selective inhibition of M1-paired I-Ak class II B cell calcium signaling by M2-paired class II molecules. Because M1- and M2-paired class II can be loaded with different sets of peptides derived from Ags acquired by distinct pathways of endocytosis, these findings suggest an MHC class II signaling-based mechanism by which CD4 T cells of differing specificities can either enhance or suppress B cell activation.
Subject(s)
B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Calcium Signaling/immunology , Histocompatibility Antigens Class II/metabolism , Lymphocyte Activation/immunology , Animals , Antibodies, Monoclonal/metabolism , Antigen-Presenting Cells/metabolism , CD4-Positive T-Lymphocytes/metabolism , Cell Communication/immunology , Cross Reactions/immunology , Epitopes, B-Lymphocyte/metabolism , Histocompatibility Antigens Class II/immunology , Immunity, Humoral , Immunoreceptor Tyrosine-Based Activation Motif , Immunoreceptor Tyrosine-Based Inhibition Motif , Mice , Peptides/immunology , Protein Tyrosine Phosphatases/metabolism , Protein-Tyrosine Kinases/metabolism , Spleen/cytologyABSTRACT
MHC class II-restricted antigen presentation by dendritic cells is necessary for activation of naïve CD4 T cells, whereas class II-restricted antigen presentation by B lymphocytes and macrophages is important for the recruitment of CD4+ helper and regulatory T cells. Antigen presentation by B cells is also important for induction of T cell tolerance. B cells are unique among these three types of MHC class II-expressing antigen presenting cells (APC) as they constitutively express high levels of cell surface class II molecules and express a clonally restricted antigen specific receptor, the B cell receptor (BCR). Here, I review our current understanding of three major steps that underlie the processing and presentation of BCR-bound cognate antigen: (1) endocytosis of antigen-BCR (Ag-BCR) complexes, (2) Ag-BCR trafficking to intracellular antigen processing compartments and (3) generation of antigenic peptide-MHC class II complexes, with a particular focus on the role of BCR ubiquitination in each. I will highlight potential topics for future research and briefly discuss the impact of the cell biology of BCR-mediated antigen processing on the response of the B cell and T cell to the cell-cell interactions mediated by B cell-expressed peptide-class II complexes.
Subject(s)
Receptors, Antigen, B-Cell/immunology , Ubiquitin/metabolism , Amino Acid Sequence , Animals , Histocompatibility Antigens Class II/metabolism , Humans , Peptides/metabolism , Protein Transport , Receptors, Antigen, B-Cell/chemistryABSTRACT
Macrophages (MØs) are sentinels of the immune system that use pattern recognition receptors such as Toll-like receptors (TLR) to detect invading pathogens and immune receptors such as FcγR to sense the host's immune state. Crosstalk between these two signaling pathways allows the MØ to tailor the cell's overall response to prevailing conditions. However, the molecular mechanisms underlying TLR-FcγR crosstalk are only partially understood. Therefore, we employed an immunologically-relevant MØ stimulus, an inactivated gram-negative bacterium that bears TLR2 agonists but no TLR4 agonist (iBTLR2) opsonized with a monoclonal antibody (mAb-iBTLR2), as a tool to study FcγR regulation of TLR2-driven production of IL-6, a key inflammatory cytokine. We chose this particular agonist as an investigational tool because MØ production of any detectable IL-6 in response to mAb-iBTLR2 requires both TLR2 and FcγR signaling, making it an excellent system for the study of receptor synergy. Using genetic, pharmacological and immunological approaches, we demonstrate that the murine MØ IL-6 response to mAb-iBTLR2 requires activation of both the TLR/NF-κB and FcγR/ITAM signaling pathways. mAb-iBTLR2 engagement of TLR2 drives NF-κB activation and up-regulation of IL-6 mRNA but fails to result in IL-6 cytokine production/release. Here, Src family kinase-driven FcγR ITAM signaling is necessary to enable IL-6 mRNA incorporation into polysomes and translation. These results reveal a novel mechanism by which FcγR ITAM signaling synergizes with TLR signaling, by "licensing" cytokine mRNA ribosome binding/translation to drive a strong murine MØ cytokine response.
Subject(s)
Interleukin-6/metabolism , RNA, Messenger/metabolism , Receptors, IgG/metabolism , Toll-Like Receptor 2/metabolism , Animals , Female , Macrophages/metabolism , Mice , Mice, Inbred C57BL , NF-kappa B/metabolism , Protein Binding , Ribosomes/metabolism , Signal Transduction , Toll-Like Receptor 4/metabolismABSTRACT
MHC class II molecules present antigen-derived peptides to CD4T cells to drive the adaptive immune response. Previous work has established that class II αß dimers can adopt two distinct conformations, driven by the differential pairing of transmembrane domain GxxxG dimerization motifs. These class II conformers differ in their ability to be loaded with antigen-derived peptide and to effectively engage CD4T cells. Motif 1 (M1) paired I-A(k) class II molecules are efficiently loaded with peptides derived from the processing of B cell receptor-bound antigen, have unique B cell signaling properties and high T cell stimulation activity. The 11-5.2mAb selectively binds M1 paired I-A(k) class II molecules. However, the molecular determinants of 11-5.2 binding are currently unclear. Here, we report the ability of a human class II transmembrane domain to drive both M1 and M2 class II conformer formation. Protease sensitivity analysis further strengthens the idea that there are conformational differences between the extracellular domains of M1 and M2 paired class II. Finally, MHC class II chain alignments and site directed mutagenesis reveals a triad of molecular regions that contributes to 11-5.2mAb binding. In addition to transmembrane GxxxG motif domain pairing, 11-5.2 binding is influenced directly by α chain residue Glu-71 and indirectly by the region around the inter-chain salt bridge formed by α chain Arg-52 and ß chain Glu-86. These findings provide insight into the complexity of 11-5.2mAb recognition of the M1 paired I-A(k) class II conformer and further highlight the molecular heterogeneity of peptide-MHC class II complexes that drive T cell antigen recognition.
Subject(s)
Histocompatibility Antigens Class II/chemistry , Histocompatibility Antigens Class II/immunology , Animals , Antibodies, Monoclonal , Antigen Presentation/immunology , Blotting, Western , CD4-Positive T-Lymphocytes/immunology , Humans , Lymphocyte Activation/immunology , Mice , Mice, Inbred BALB C , Molecular Conformation , Mutagenesis, Site-DirectedABSTRACT
IgG (mAb)-opsonized, inactivated Francisella tularensis LVS (iFt-mAb) enhances TLR2-dependent IL-6 production by macrophages via Fcγ receptors (FcγR). In mice, vaccination with iFt-mAb provides IgA-dependent protection against lethal challenge with Ft LVS. Because inflammasome maturation of IL-1ß is thought important for antibody-mediated immunity, we considered the possibility that iFt-mAb elicits an FcγR-dependent myeloid cell inflammasome response. Herein, we find that iFt-mAb enhances macrophage and dendritic cell IL-1ß responses in a TLR2- and FcγR-dependent fashion. Although iFt-mAb complexes bind FcγR and are internalized, sensing of cytosolic DNA by absent in melanoma 2 (AIM2) is not required for the IL-1ß response. In contrast, ASC, caspase-1, and NLR family pyrin domain-containing 3 (NLRP3) are indispensable. Further, FcγR-mediated spleen tyrosine kinase (Syk) signaling is required for this NLRP3-dependent IL-1ß response, but the alternative IL-1ß convertase caspase-8 is insufficient. Finally, iFt-mAb-vaccinated wild-type mice exhibit a significant delay in time to death, but IL-1R1- or Nlrp3-deficient mice vaccinated in this way are not protected and lack appreciable Francisella-specific antibodies. This study demonstrates that FcγR-mediated Syk activation leads to NLRP3 inflammasome-dependent IL-1ß production in macrophages and suggests that an Nlrp3- and IL-1R-dependent process contributes to the IgA response important for protection against Ft LVS. These findings extend our understanding of cellular responses to inactivated pathogen-opsonized vaccine, establish FcγR-elicited Syk kinase-mediated NLRP3 inflammasome activation, and provide additional insight toward understanding crosstalk between TLR and FcγR signals.
Subject(s)
Antibodies, Bacterial/immunology , Antigen-Antibody Complex/immunology , Antigens, Bacterial/immunology , Francisella tularensis/immunology , Inflammasomes/immunology , NLR Family, Pyrin Domain-Containing 3 Protein/immunology , Receptors, IgG/immunology , Syk Kinase/immunology , Toll-Like Receptor 2/immunology , Animals , Antibodies, Monoclonal/immunology , Bacterial Vaccines , Caspase 8/immunology , Cell Line , Dendritic Cells/immunology , Dendritic Cells/metabolism , Interleukin-1beta/immunology , Macrophages/immunology , Macrophages/metabolism , Mice, Inbred C57BL , Mice, Knockout , Monocytes/immunology , Monocytes/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/deficiency , Signal Transduction , VaccinationABSTRACT
Following antigen recognition, B cell receptor (BCR)-mediated endocytosis is the first step of antigen processing and presentation to CD4+ T cells, a crucial component of the initiation and control of the humoral immune response. Despite this, the molecular mechanism of BCR internalization is poorly understood. Recently, studies of activated B cell-like diffuse large B cell lymphoma (ABC DLBCL) have shown that mutations within the BCR subunit CD79b leads to increased BCR surface expression, suggesting that CD79b may control BCR internalization. Adaptor protein 2 (AP2) is the major mediator of receptor endocytosis via clathrin-coated pits. The BCR contains five putative AP2-binding YxxØ motifs, including four that are present within two immunoreceptor tyrosine-based activation motifs (ITAMs). Using a combination of in vitro and in situ approaches, we establish that the sole mediator of AP2-dependent BCR internalization is the membrane proximal ITAM YxxØ motif in CD79b, which is a major target of mutation in ABC DLBCL. In addition, we establish that BCR internalization can be regulated at a minimum of two different levels: regulation of YxxØ AP2 binding in cis by downstream ITAM-embedded DCSM and QTAT regulatory elements and regulation in trans by the partner cytoplasmic domain of the CD79 heterodimer. Beyond establishing the basic rules governing BCR internalization, these results illustrate an underappreciated role for ITAM residues in controlling clathrin-dependent endocytosis and highlight the complex mechanisms that control the activity of AP2 binding motifs in this receptor system.
Subject(s)
Adaptor Protein Complex 2/metabolism , Endocytosis , Receptors, Antigen, B-Cell/chemistry , Receptors, Antigen, B-Cell/metabolism , Amino Acid Motifs , Amino Acid Sequence , Animals , CD79 Antigens/chemistry , CD79 Antigens/metabolism , Coated Pits, Cell-Membrane/metabolism , Coated Pits, Cell-Membrane/ultrastructure , Mice , Molecular Sequence Data , Protein BindingABSTRACT
Francisella tularensis is a bacterial pathogen that uses host-derived PGE2 to subvert the host's adaptive immune responses in multiple ways. Francisella-induced PGE2 acts directly on CD4 T cells to blunt production of IFN-γ. Francisella-induced PGE2 can also elicit production of a >10 kDa soluble host factor termed FTMØSN (F. tularensismacrophage supernatant), which acts on IFN-γ pre-activated MØ to down-regulate MHC class II expression via a ubiquitin-dependent mechanism, blocking antigen presentation to CD4 T cells. Here, we report that FTMØSN-induced down-regulation of MØ class II is the result of the induction of MARCH1, and that MØ expressing MARCH1 "resistant" class II molecules are resistant to FTMØSN-induced class II down-regulation. Since PGE2 can induce IL-10 production and IL-10 is the only reported cytokine able to induce MARCH1 expression in monocytes and dendritic cells, these findings suggested that IL-10 is the active factor in FTMØSN. However, use of IL-10 knockout MØ established that IL-10 is not the active factor in FTMØSN, but rather that Francisella-elicited PGE2 drives production of a >10 kDa host factor distinct from IL-10. This factor then drives MØ IL-10 production to induce MARCH1 expression and the resultant class II down-regulation. Since many human pathogens such as Salmonella typhi, Mycobacterium tuberculosis and Legionella pneumophila also induce production of host PGE2, these results suggest that a yet-to-be-identified PGE2-inducible host factor capable of inducing IL-10 is central to the immune evasion mechanisms of multiple important human pathogens.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , Francisella tularensis/immunology , Histocompatibility Antigens Class II/biosynthesis , Interleukin-10/biosynthesis , Macrophages/immunology , Ubiquitin-Protein Ligases/biosynthesis , Dinoprostone/physiology , Down-Regulation , Humans , Interleukin-10/genetics , Macrophages/metabolism , Ubiquitin-Protein Ligases/physiology , Up-RegulationABSTRACT
The high rate of HIV-1 mutation and the frequent sexual transmission highlight the need for novel therapeutic modalities with broad activity against both CXCR4 (X4) and CCR5 (R5)-tropic viruses. We investigated a large number of natural products, and from Sargassum fusiforme we isolated and identified palmitic acid (PA) as a natural small bioactive molecule with activity against HIV-1 infection. Treatment with 100 microM PA inhibited both X4 and R5 independent infection in the T cell line up to 70%. Treatment with 22 microM PA inhibited X4 infection in primary peripheral blood lymphocytes (PBL) up to 95% and 100 microM PA inhibited R5 infection in primary macrophages by over 90%. Inhibition of infection was concentration dependent, and cell viability for all treatments tested remained above 80%, similar to treatment with 10(-6)M nucleoside analogue 2', 3'-dideoxycytidine (ddC). Micromolar PA concentrations also inhibited cell-to-cell fusion and specific virus-to-cell fusion up to 62%. PA treatment did not result in internalization of the cell surface CD4 receptor or lipid raft disruption, and it did not inhibit intracellular virus replication. PA directly inhibited gp120-CD4 complex formation in a dose-dependent manner. We used fluorescence spectroscopy to determine that PA binds to the CD4 receptor with K(d) approximately 1.5 +/- 0.2 microM, and we used one-dimensional saturation transfer difference NMR (STD-NMR) to determined that the PA binding epitope for CD4 consists of the hydrophobic methyl and methelene groups located away from the PA carboxyl terminal, which blocks efficient gp120-CD4 attachment. These findings introduce a novel class of antiviral compound that binds directly to the CD4 receptor, blocking HIV-1 entry and infection. Understanding the structure-affinity relationship (SAR) between PA and CD4 should lead to the development of PA analogs with greater potency against HIV-1 entry.