ABSTRACT
Although T cell help for B cells was described several decades ago, it was the identification of CXCR5 expression by B follicular helper T (Tfh) cells and the subsequent discovery of their dependence on BCL6 that led to the recognition of Tfh cells as an independent helper subset and accelerated the pace of discovery. More than 20 transcription factors, together with RNA-binding proteins and microRNAs, control the expression of chemotactic receptors and molecules important for the function and homeostasis of Tfh cells. Tfh cells prime B cells to initiate extrafollicular and germinal center antibody responses and are crucial for affinity maturation and maintenance of humoral memory. In addition to the roles that Tfh cells have in antimicrobial defense, in cancer, and as HIV reservoirs, regulation of these cells is critical to prevent autoimmunity. The realization that follicular T cells are heterogeneous, comprising helper and regulatory subsets, has raised questions regarding a possible division of labor in germinal center B cell selection and elimination.
Subject(s)
B-Lymphocytes/immunology , Germinal Center/immunology , Immunity, Humoral , T-Lymphocyte Subsets/immunology , T-Lymphocytes, Helper-Inducer/immunology , Animals , Cell Differentiation , Gene Expression Regulation , Humans , Immunologic Memory , Lymphocyte Activation , MicroRNAs/genetics , Proto-Oncogene Proteins c-bcl-6/metabolism , Receptors, CXCR5/metabolismABSTRACT
Voltage-gated proton currents regulate generation of reactive oxygen species (ROS) in phagocytic cells. In B cells, stimulation of the B cell antigen receptor (BCR) results in the production of ROS that participate in B cell activation, but the involvement of proton channels is unknown. We report here that the voltage-gated proton channel HVCN1 associated with the BCR complex and was internalized together with the BCR after activation. BCR-induced generation of ROS was lower in HVCN1-deficient B cells, which resulted in attenuated BCR signaling via impaired BCR-dependent oxidation of the tyrosine phosphatase SHP-1. This resulted in less activation of the kinases Syk and Akt, impaired mitochondrial respiration and glycolysis and diminished antibody responses in vivo. Our findings identify unanticipated functions for proton channels in B cells and demonstrate the importance of ROS in BCR signaling and downstream metabolism.
Subject(s)
B-Lymphocytes/immunology , Ion Channels/immunology , Reactive Oxygen Species/immunology , Receptors, Antigen, B-Cell/immunology , Animals , B-Lymphocytes/enzymology , Enzyme Activation/immunology , Immunoblotting , Intracellular Signaling Peptides and Proteins/immunology , Mice , Mice, Knockout , Microscopy, Confocal , Mitochondria/immunology , Oncogene Protein v-akt/immunology , Protein-Tyrosine Kinases/immunology , Signal Transduction , Syk KinaseABSTRACT
Antibody-forming cells (AFCs) expressing the chemokine receptor CXCR3 are recruited to sites of inflammation where they help clear pathogens but may participate in autoimmune diseases. Here we identify a mechanism that induces CXCR3 expression by AFC and germinal center (GC) B cells. This happens when CD8 T cells are recruited into CD4 T cell-dependent B-cell responses. Ovalbumin-specific CD4 T cells (OTII) were transferred alone or with ovalbumin-specific CD8 T cells (OTI) and the response to subcutaneous alum-precipitated ovalbumin was followed in the draining lymph nodes. OTII cells alone induce T helper 2-associated class switching to IgG1, but few AFC or GC B cells express CXCR3. By contrast, OTI-derived IFN-γ induces most responding GC B cells and AFCs to express high levels of CXCR3, and diverse switching to IgG2a, IgG2b, with some IgG1. Up-regulation of CXCR3 by GC B cells and AFCs and their migration toward its ligand CXCL10 are shown to depend on B cells' intrinsic T-bet, a transcription factor downstream of the IFN-γR signaling. This model clarifies how precursors of long-lived AFCs and memory B cells acquire CXCR3 that causes their migration to inflammatory foci.
Subject(s)
B-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Cell Movement/immunology , Receptors, CXCR3/immunology , T-Box Domain Proteins/immunology , Vaccines/immunology , Adoptive Transfer , Alum Compounds , Animals , B-Lymphocytes/metabolism , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , CD4-Positive T-Lymphocytes/transplantation , CD8-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/transplantation , Cell Differentiation/immunology , Chemokine CXCL10/immunology , Chemokine CXCL10/metabolism , Flow Cytometry , Germinal Center/immunology , Germinal Center/metabolism , Immunization/methods , Interferon-gamma/immunology , Interferon-gamma/metabolism , Ligands , Mice , Mice, Inbred C57BL , Ovalbumin/immunology , Receptors, CXCR3/genetics , Receptors, CXCR3/metabolism , Reverse Transcriptase Polymerase Chain Reaction , T-Box Domain Proteins/genetics , T-Box Domain Proteins/metabolism , Th2 Cells/immunology , Th2 Cells/metabolism , Up-Regulation/geneticsABSTRACT
Vaccination with purified capsular polysaccharide Vi Ag from Salmonella typhi can protect against typhoid fever, although the mechanism for its efficacy is not clearly established. In this study, we have characterized the B cell response to this vaccine in wild-type and T cell-deficient mice. We show that immunization with typhoid Vi polysaccharide vaccine rapidly induces proliferation in B1b peritoneal cells, but not in B1a cells or marginal zone B cells. This induction of B1b proliferation is concomitant with the detection of splenic Vi-specific Ab-secreting cells and protective Ab in Rag1-deficient B1b cell chimeras generated by adoptive transfer-induced specific Ab after Vi immunization. Furthermore, Ab derived from peritoneal B cells is sufficient to confer protection against Salmonella that express Vi Ag. Expression of Vi by Salmonella during infection did not inhibit the development of early Ab responses to non-Vi Ags. Despite this, the protection conferred by immunization of mice with porin proteins from Salmonella, which induce Ab-mediated protection, was reduced postinfection with Vi-expressing Salmonella, although protection was not totally abrogated. This work therefore suggests that, in mice, B1b cells contribute to the protection induced by Vi Ag, and targeting non-Vi Ags as subunit vaccines may offer an attractive strategy to augment current Vi-based vaccine strategies.
Subject(s)
Antigens, Bacterial/immunology , B-Lymphocyte Subsets/immunology , B-Lymphocyte Subsets/microbiology , Polysaccharides, Bacterial/biosynthesis , Salmonella typhi/immunology , Animals , Antibodies, Bacterial/biosynthesis , Antibodies, Bacterial/physiology , Antigens, Bacterial/biosynthesis , B-Lymphocyte Subsets/transplantation , Mice , Mice, Inbred C57BL , Mice, Knockout , Peritoneal Cavity/cytology , Peritoneal Cavity/microbiology , Peritoneum/cytology , Peritoneum/immunology , Peritoneum/metabolism , Polysaccharides, Bacterial/immunology , Porins , Salmonella Vaccines/administration & dosage , Salmonella Vaccines/biosynthesis , Salmonella Vaccines/immunology , Typhoid Fever/immunology , Typhoid Fever/metabolism , Typhoid Fever/prevention & controlABSTRACT
Hematopoietic cells constitutively express CD31/PECAM1, a signaling adhesion receptor associated with controlling responses to inflammatory stimuli. Although expressed on CD4(+) T cells, its function on these cells is unclear. To address this, we have used a model of systemic Salmonella infection that induces high levels of T cell activation and depends on CD4(+) T cells for resolution. Infection of CD31-deficient (CD31KO) mice demonstrates that these mice fail to control infection effectively. During infection, CD31KO mice have diminished numbers of total CD4(+) T cells and IFN-γ-secreting Th1 cells. This is despite a higher proportion of CD31KO CD4(+) T cells exhibiting an activated phenotype and an undiminished capacity to prime normally and polarize to Th1. Reduced numbers of T cells reflected the increased propensity of naive and activated CD31KO T cells to undergo apoptosis postinfection compared with wild-type T cells. Using adoptive transfer experiments, we show that loss of CD31 on CD4(+) T cells alone is sufficient to account for the defective CD31KO T cell accumulation. These data are consistent with CD31 helping to control T cell activation, because in its absence, T cells have a greater propensity to become activated, resulting in increased susceptibility to become apoptotic. The impact of CD31 loss on T cell homeostasis becomes most pronounced during severe, inflammatory, and immunological stresses such as those caused by systemic Salmonella infection. This identifies a novel role for CD31 in regulating CD4 T cell homeostasis.
Subject(s)
Apoptosis/immunology , Lymphocyte Activation/immunology , Platelet Endothelial Cell Adhesion Molecule-1/immunology , Salmonella Infections/immunology , Salmonella/immunology , Th1 Cells/immunology , Adoptive Transfer , Animals , Apoptosis/genetics , Cell Survival/genetics , Cell Survival/immunology , Inflammation/genetics , Inflammation/immunology , Inflammation/microbiology , Lymphocyte Activation/genetics , Mice , Platelet Endothelial Cell Adhesion Molecule-1/genetics , Salmonella/genetics , Salmonella Infections/geneticsABSTRACT
Alum-precipitated protein (alum protein) vaccines elicit long-lasting neutralizing antibody responses that prevent bacterial exotoxins and viruses from entering cells. Typically, these vaccines induce CD4 T cells to become T helper 2 (Th2) cells that induce Ig class switching to IgG1. We now report that CD8 T cells also respond to alum proteins, proliferating extensively and producing IFN-γ, a key Th1 cytokine. These findings led us to question whether adoptive transfer of antigen-specific CD8 T cells alters the characteristic CD4 Th2 response to alum proteins and the switching pattern in responding B cells. To this end, WT mice given transgenic ovalbumin (OVA)-specific CD4 (OTII) or CD8 (OTI) T cells, or both, were immunized with alum-precipitated OVA. Cotransfer of antigen-specific CD8 T cells skewed switching patterns in responding B cells from IgG1 to IgG2a and IgG2b. Blocking with anti-IFN-γ antibody largely inhibited this altered B-cell switching pattern. The transcription factor T-bet is required in B cells for IFN-γ-dependent switching to IgG2a. By contrast, we show that this transcription factor is dispensable in B cells both for IFN-γ-induced switching to IgG2b and for inhibition of switching to IgG1. Thus, T-bet dependence identifies distinct transcriptional pathways in B cells that regulate IFN-γ-induced switching to different IgG isotypes.
Subject(s)
B-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Immunoglobulin Class Switching , Interferon-gamma/immunology , Ovalbumin/immunology , T-Box Domain Proteins/metabolism , Vaccines/immunology , Adoptive Transfer , Alum Compounds , Animals , B-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/cytology , Mice , Mice, Inbred C57BLABSTRACT
Rapid production of neutralizing antibody can be critical for limiting the spread of infection. Such early antibody results when B-cell blasts mature directly to plasmablasts without forming germinal centers. These extrafollicular responses can involve Ig class switch recombination (CSR), producing antibody that can readily disseminate through infected tissues. The present study identifies the differentiation stage where CSR occurs in an extrafollicular response induced by 4-hydroxy-3-nitrophenyl acetyl (NP) conjugated to Ficoll (NP-Ficoll). To do this, we took advantage of the antigen dose dependency of CSR in this response. Thus, while both 30 and 1 µg NP-Ficoll induce plasmablasts, only the higher antigen dose induces CSR. Activation-induce cytidine deaminase (AID) is critical for CSR and in keeping with this a proportion of NP-specific B-cell blasts induced by 30 µg NP-Ficoll express AID. None of the B blasts responding to the non-CSR-inducing 1 µg dose of NP-Ficoll express AID. We confirmed that CSR occurs in B blasts by demonstrating the presence of rearranged heavy-chain transcripts in B blasts in the 30 µg response. CSR in this extrafollicular response is confined to B blasts, because NP-specific plasmablasts, identified by expressing CD138 and Blimp-1, no longer express AID and cannot undergo CSR.
Subject(s)
B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Immunoglobulin Class Switching/genetics , Immunoglobulin Class Switching/immunology , Plasma Cells/immunology , Animals , Cytidine Deaminase/genetics , Cytidine Deaminase/immunology , Ficoll/pharmacology , Immunoconjugates/immunology , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Heavy Chains/immunology , Mice , Mice, Inbred C57BL , Nitrophenols/pharmacology , Phenylacetates/pharmacology , Positive Regulatory Domain I-Binding Factor 1 , Syndecan-1/genetics , Syndecan-1/immunology , Transcription Factors/genetics , Transcription Factors/immunologyABSTRACT
NF-κB1-dependent signaling directs the development of CD4(+) Th2 cells during allergic airway inflammation and protective responses to helminth infection. Here, we show that IL-4 and IL-13 production is NF-κB1-dependent in mouse OVA-specific CD4(+) (OTII) T cells responding to alum-precipitated OVA (alumOVA) immunization. More surprisingly, we found that NF-κB1 deficiency in OTII cells also selectively impairs their CXCR5 induction by alumOVA without affecting upregulation of BCL6, IL-21, OX40 and CXCR4 mRNA and PD-1 protein. This results in functional impairment of follicular helper T cells. Thus, fewer germinal center B cells develop in LN responses to alumOVA in T-cell-deficient mice reconstituted with NF-κB1(-/-) OTII cells as opposed to NF-κB1(+/+) OTII cells, while plasma cell numbers are comparable. Unlike CXCR5 induction in CD4(+) T cells, NF-κB1-deficient recirculating follicular B cells are shown to express normal levels of CXCR5. The selective effects of NF-κB1-deficiency on Th2 and follicular helper T cell induction do not appear to be due to altered expression of the Th2-associated transcription factors - GATA-3, c-Maf and Ikaros. Altogether, these results suggest that NF-κB1 regulates the expression of CXCR5 on CD4(+) T cells primed in vivo, and thus selectively controls the T-cell-dependent germinal center component of B-cell response to alumOVA.
Subject(s)
B-Lymphocytes/metabolism , CD4-Positive T-Lymphocytes/metabolism , NF-kappa B/metabolism , Receptors, CXCR5/metabolism , Th2 Cells/metabolism , Adoptive Transfer , Alum Compounds/administration & dosage , Animals , B-Lymphocytes/immunology , B-Lymphocytes/pathology , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/pathology , Cells, Cultured , Germinal Center/pathology , Immunization , Lymphocyte Activation/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , NF-kappa B/genetics , NF-kappa B/immunology , Ovalbumin/immunology , Receptors, CXCR5/genetics , Receptors, CXCR5/immunology , Th2 Cells/immunology , Th2 Cells/pathology , Up-Regulation/geneticsABSTRACT
Control of intracellular Salmonella infection requires Th1 priming and IFN-γ production. Here, we show that efficient Th1 priming after Salmonella infection requires CD11c(+) CD11b(hi) F4/80(+) monocyte-derived dendritic cells (moDCs). In non-infected spleens, moDCs are absent from T-cell zones (T zones) of secondary lymphoid tissues, but by 24 h post-infection moDCs are readily discernible in these sites. The accumulation of moDCs is more dependent upon bacterial viability than bacterial virulence. Kinetic studies showed that moDCs were necessary to prime but not sustain Th1 responses, while ex vivo studies showed that antigen-experienced moDCs were sufficient to induce T-cell proliferation and IFN-γ production via a TNF-α-dependent mechanism. Importantly, moDCs and cDCs when co-cultured induced superior Th1 differentiation than either subset alone, and this activity was independent of TNF-α. Thus, optimal Th1 development to Salmonella requires the rapid accumulation of moDCs within T zones and their collaboration with cDCs.
Subject(s)
Dendritic Cells/metabolism , Salmonella Infections/immunology , Salmonella/immunology , Spleen/pathology , Th1 Cells/metabolism , Animals , Antigen Presentation , Antigens, Differentiation/biosynthesis , CD11b Antigen/biosynthesis , CD11c Antigen/biosynthesis , Cell Communication , Cell Differentiation , Cell Proliferation , Cells, Cultured , Dendritic Cells/immunology , Dendritic Cells/microbiology , Dendritic Cells/pathology , Interferon-gamma/metabolism , Lymphocyte Activation , Mice , Mice, Inbred C57BL , Monocytes/pathology , Salmonella/pathogenicity , Th1 Cells/immunology , Th1 Cells/microbiology , Th1 Cells/pathology , Tumor Necrosis Factor-alpha/immunology , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Clearance of disseminated Salmonella infection requires bacterial-specific Th1 cells and IFN-γ production, and Th1-promoting vaccines are likely to help control these infections. Consequently, vaccine design has focused on developing Th1-polarizing adjuvants or Ag that naturally induce Th1 responses. In this study, we show that, in mice, immunization with soluble, recombinant FliC protein flagellin (sFliC) induces Th2 responses as evidenced by Ag-specific GATA-3, IL-4 mRNA, and protein induction in CD62L(lo) CD4(+) T cells without associated IFN-γ production. Despite these Th2 features, sFliC immunization can enhance the development of protective Th1 immunity during subsequent Salmonella infection in an Ab-independent, T-cell-dependent manner. Salmonella infection in sFliC-immunized mice resulted in augmented Th1 responses, with greater bacterial clearance and increased numbers of IFN-γ-producing CD4(+) T cells, despite the early induction of Th2 features to sFliC. The augmented Th1 immunity after sFliC immunization was regulated by T-bet although T-bet is dispensable for primary responses to sFliC. These findings show that there can be flexibility in T-cell responses to some subunit vaccines. These vaccines may induce Th2-type immunity during primary immunization yet promote Th1-dependent responses during later infection. This suggests that designing Th1-inducing subunit vaccines may not always be necessary since this can occur naturally during subsequent infection.
Subject(s)
Bacterial Vaccines , Flagellin/immunology , Salmonella Infections/immunology , Salmonella typhimurium/immunology , T-Box Domain Proteins/metabolism , Th1 Cells/metabolism , Th2 Cells/metabolism , Animals , Bacterial Load , Cells, Cultured , Gene Expression Regulation , Immunization , Interferon-gamma/metabolism , Lymphocyte Activation/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Salmonella Infections/microbiology , Salmonella typhimurium/growth & development , Salmonella typhimurium/pathogenicity , T-Box Domain Proteins/genetics , T-Box Domain Proteins/immunology , T-Cell Antigen Receptor Specificity , Th1 Cells/immunology , Th1 Cells/microbiology , Th1 Cells/pathology , Th2 Cells/immunology , Th2 Cells/microbiology , Th2 Cells/pathologyABSTRACT
Long-lived plasma cells in the bone marrow produce memory antibodies that provide immune protection persisting for decades after infection or vaccination but can also contribute to autoimmune and allergic diseases. However, the composition of the microenvironmental niches that are important for the generation and maintenance of these cells is only poorly understood. Here, we demonstrate that, within the bone marrow, plasma cells interact with the platelet precursors (megakaryocytes), which produce the prominent plasma cell survival factors APRIL (a proliferation-inducing ligand) and IL-6 (interleukin-6). Accordingly, reduced numbers of immature and mature plasma cells are found in the bone marrow of mice deficient for the thrombopoietin receptor (c-mpl) that show impaired megakaryopoiesis. After immunization, accumulation of antigen-specific plasma cells in the bone marrow is disturbed in these mice. Vice versa, injection of thrombopoietin allows the accumulation and persistence of a larger number of plasma cells generated in the course of a specific immune response in wild-type mice. These results demonstrate that megakaryocytes constitute an important component of the niche for long-lived plasma cells in the bone marrow.
Subject(s)
Bone Marrow Cells/metabolism , Megakaryocytes/metabolism , Plasma Cells/metabolism , Stem Cell Niche/metabolism , Animals , Bone Marrow Cells/cytology , Cell Communication/drug effects , Cell Count , Cells, Cultured , Female , Flow Cytometry , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Interleukin-6/genetics , Interleukin-6/metabolism , Male , Megakaryocytes/cytology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Microscopy, Confocal , Ovalbumin/immunology , Ovalbumin/pharmacology , Plasma Cells/cytology , Plasma Cells/drug effects , Receptors, Thrombopoietin/genetics , Receptors, Thrombopoietin/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Stem Cell Niche/cytology , Thrombopoietin/pharmacology , Tumor Necrosis Factor Ligand Superfamily Member 13/genetics , Tumor Necrosis Factor Ligand Superfamily Member 13/metabolismABSTRACT
Invasive nontyphoidal Salmonella (NTS), including Salmonella typhimurium (STm), are major yet poorly-recognized killers of infants in sub-Saharan Africa. Death in these children is usually associated with bacteremia, commonly in the absence of gastrointestinal symptoms. Evidence from humans and animal studies suggest that severe infection and bacteremia occur when specific Ab is lacking. Understanding how Ab responses to Salmonella are regulated will help develop vaccines against these devastating infections. STm induces atypical Ab responses characterized by prominent, accelerated, extrafollicular T-independent (TI) Ab against a range of surface antigens. These responses develop without concomitant germinal centers, which only appear as infection resolves. Here, we show STm rapidly induces a population of TI B220(+)CD5(-) B1b cells during infection and TI Ab from B1b cells targets the outer membrane protein (Omp) porins OmpC, OmpD and OmpF but not flagellin. When porins are used as immunogens they can ablate bacteremia and provide equivalent protection against STm as killed bacterial vaccine and this is wholly B cell-dependent. Furthermore Ab from porin-immunized chimeras, that have B1b cells, is sufficient to impair infection. Infecting with porin-deficient bacteria identifies OmpD, a protein absent from Salmonella Typhi, as a key target of Ab in these infections. This work broadens the recognized repertoire of TI protein antigens and highlights the importance of Ab from different B cell subsets in controlling STm infection. OmpD is a strong candidate vaccine target and may, in part, explain the lack of cross-protection between Salmonella Typhi and STm infections.
Subject(s)
Antibodies, Bacterial/biosynthesis , Porins/immunology , Salmonella/metabolism , Animals , B-Lymphocytes/cytology , Base Sequence , Blotting, Western , DNA Primers , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Mice , Peritoneal Cavity/cytology , Salmonella/immunologyABSTRACT
IL-6 and APRIL influence the growth, differentiation, and survival of normal and neoplastic Ab-forming cells (AFC). In this study, we identify two subsets of myeloid cells that associate with the AFC and are the main producers of these factors during a T-dependent Ab response to alum-precipitated protein in mouse lymph nodes. First CD11c(+)CD8alpha(-) dendritic cells located in the perivascular area of the T zone provide about half of the IL-6 mRNA produced in the node together with significant amounts of APRIL mRNA. The number of these cells increases during the response, at least in part due to local proliferation. The second subset comprises Gr1(+)CD11b(+)F4/80(+) monocyte/macrophages. These colonize the medullary cords during the response and are the other main IL-6 mRNA producers and the greatest source of APRIL mRNA. This medullary cord monocyte/macrophage subset results in local increase of APRIL mRNA that mirrors the polarity of CXCL12 expression in the node. The distribution of these myeloid cell subsets correlates with a gradient of AFC maturation assessed by progressive loss of Ki67 as AFC pass from the B cell follicle along the perivascular areas to the medullary cords.
Subject(s)
Dendritic Cells/cytology , Interleukin-6/immunology , Leukocytes, Mononuclear/cytology , Lymph Nodes/cytology , Plasma Cells/cytology , Tumor Necrosis Factor Ligand Superfamily Member 13/immunology , Adoptive Transfer , Animals , Cell Differentiation/immunology , Cell Proliferation , Dendritic Cells/immunology , Flow Cytometry , Leukocytes, Mononuclear/immunology , Lymph Nodes/immunology , Lymphocyte Subsets/cytology , Lymphocyte Subsets/immunology , Macrophages/cytology , Macrophages/immunology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microdissection , Microscopy, Confocal , Myeloid Cells/cytology , Myeloid Cells/immunology , Plasma Cells/immunology , RNA, Messenger/analysis , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Exceptionally germinal center formation can be induced without T cell help by polysaccharide-based antigens, but these germinal centers involute by massive B cell apoptosis at the time centrocyte selection starts. This study investigates whether B cells in germinal centers induced by the T cell-independent antigen (4-hydroxy-3-nitrophenyl)acetyl (NP) conjugated to Ficoll undergo hypermutation in their immunoglobulin V region genes. Positive controls are provided by comparing germinal centers at the same stage of development in carrier-primed mice immunized with a T cell-dependent antigen: NP protein conjugate. False positive results from background germinal centers and false negatives from non-B cells in germinal centers were avoided by transferring B cells with a transgenic B cell receptor into congenic controls not carrying the transgene. By 4 d after immunization, hypermutation was well advanced in the T cell-dependent germinal centers. By contrast, the mutation rate for T cell-independent germinal centers was low, but significantly higher than in NP-specific B cells from nonimmunized transgenic mice. Interestingly, a similar rate of mutation was seen in extrafollicular plasma cells at this stage. It is concluded that efficient activation of hypermutation depends on interaction with T cells, but some hypermutation may be induced without such signals, even outside germinal centers.
Subject(s)
Germinal Center/immunology , Mutation , T-Lymphocytes/immunology , Adoptive Transfer , Animals , Animals, Congenic , Antigens, T-Independent/administration & dosage , B-Lymphocytes/cytology , B-Lymphocytes/immunology , Base Sequence , DNA/genetics , Germinal Center/cytology , Immunization , Immunoglobulin Variable Region/genetics , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Mice, Transgenic , Molecular Sequence Data , Nitrophenols/administration & dosage , Nitrophenols/immunology , Phenylacetates , Signal Transduction/immunology , T-Lymphocytes/cytologyABSTRACT
This study characterizes the diversity of CD4 Th cells produced during a Th2 response in vivo. Kinetics of effector and memory cell differentiation by mouse OVA-specific CD4 T cells was followed during primary responses to alum-precipitated OVA. The complexity of the CD4 T response was assessed in nodes draining and distant from the site of immunization for phenotype, location and function. By 3 days IL-4-producing effector CD4 T cells developed in the draining node and a proportion of the responding cells had migrated to B-cell follicles, while yet others had left the draining node. Some of these early migrant cells were recirculating cells with a central memory phenotype. These had divided four or more times in the draining node before migrating to distant nodes not exposed to antigen. We questioned the responsiveness of these early central-memory-like cells on secondary antigen challenge at sites distant to the primary immunization. They re-entered cell cycle and migrated to B-cell follicles, much more rapidly than naive CD4 T cells and could still be induced to produce IL-4. Their production and survival were independent of the starting frequency of antigen-specific CD4 T cells. Thus intranodal effector cells and recirculating, rapidly responding central-memory-like cells emerged simultaneously from the third day of a primary Th2 response.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , Cell Differentiation/immunology , Immunologic Memory/immunology , Lymph Nodes/immunology , Th2 Cells/immunology , Animals , Animals, Congenic , Antigens, CD/analysis , CD4-Positive T-Lymphocytes/metabolism , CD4-Positive T-Lymphocytes/transplantation , Cell Movement/immunology , Cell Proliferation , Gene Expression/immunology , Immunization, Secondary , Immunophenotyping , Interleukin-4/genetics , Interleukin-4/metabolism , Kinetics , Leukocyte Common Antigens/genetics , Lymph Nodes/cytology , Lymphocyte Activation/immunology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Ovalbumin/immunology , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/immunology , Receptors, CXCR5/metabolism , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , T-Lymphocyte Subsets/transplantation , Th2 Cells/metabolism , Transplantation Chimera/immunology , VaccinationABSTRACT
CD4 T helper (Th) cell differentiation defined by in vitro cytokine-directed culture systems leaves major gaps in our knowledge of the mechanisms driving divergent Th differentiation. This is evident from our analysis of the response of mouse ovalbumin-specific CD4 T cells to different forms of ovalbumin that induce markedly distinct responses in vivo. We show that live attenuated ovalbumin-expressing Salmonella (SalOVA) induce Th1-associated T-bet and IFN-gamma. Conversely, alum-precipitated ovalbumin (alumOVA) induces the Th2-associated GATA-3 and IL-4. The early diversity occurring within these CD4 T cells isolated 3 days after immunization was assessed using real-time RT-PCR microfluidic cards designed with 384 selected genes. The technique was validated both at the population and single cell levels at different stages of the responses, showing beta2-microglobulin to be a more stably expressed reference mRNA than either beta-actin or 18S RNA. SalOVA was then shown selectively to induce the OVA-specific CD4 T cells to produce many chemokines and pro-inflammatory cytokines, contrasting with alumOVA-induced cells that only produced a few Th2-associated cytokines. Several cytokines and features associated with follicular helper functions were induced in the OVA-specific CD4 T cells by both antigens. Finally, IL-17RB is strongly associated with OVA-specific CD4 T cells responding to alumOVA, suggesting that alum may promote Th2 immune response through a role for the IL-25/IL-17RB pathway.
Subject(s)
Alum Compounds/pharmacology , CD4-Positive T-Lymphocytes/metabolism , Ovalbumin/immunology , Ovalbumin/metabolism , Salmonella/immunology , Animals , CD4-Positive T-Lymphocytes/drug effects , CD4-Positive T-Lymphocytes/immunology , Cell Differentiation/drug effects , Cell Differentiation/genetics , Cell Differentiation/immunology , Gene Expression Profiling , Gene Expression Regulation/immunology , Immunization , Immunoprecipitation , Interferon-gamma/metabolism , Interleukin-4/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Oligonucleotide Array Sequence Analysis , Ovalbumin/drug effects , Salmonella/metabolism , Salmonella Infections/genetics , Salmonella Infections/immunology , Salmonella Infections/metabolism , Salmonella Infections/therapy , Th1 Cells/immunology , Th1 Cells/metabolism , Th1 Cells/physiology , Th2 Cells/immunology , Th2 Cells/metabolism , Th2 Cells/physiologyABSTRACT
DNA microarray analysis of B cell subsets has identified comprehensive programs of gene expression that distinguish B cells at discrete stages of differentiation. The next task is to identify key genetic signals within these complex programs that regulate the dynamic cellular events during B cell activation in vivo. After stimulation with antigen, naïve B cells proliferate and differentiate, and then produce antibodies. Crucial qualitative differences in antibody responses are observed depending on whether or not B cells receive T cell help during activation. Proteins, lipopolysaccharides, and polysaccharides stimulate T-dependent (TD), T-independent type 1 (TI-1), and type 2 (TI-2) antibody responses, respectively. Only TD responses generate somatically mutated antibody-forming (plasma) cells and memory B cells, which produce high affinity anamnestic responses to subsequent antigen challenge. Somatic mutation of immunoglobulin genes occurs during B cell proliferation in germinal centres (GC), which are typical in TD responses but rare in TI responses. However, we have described a model, which is exceptional because numerous large GC form in response to a model TI-2 antigen, (4-hydoxy-3-nitrophenyl) acetyl (NP)-Ficoll. Significantly, these GC undergo involution before memory B cells are generated. This model provides an opportunity to investigate the genetic signals that drive memory cell formation, and we have compared global gene expression in TI and TD GC to identify a relatively small number of genes that are differentially expressed between the two prototypic B cell responses. This model demonstrates how genome-scale technology can be adapted to investigate specific aspects of B cell biology.
Subject(s)
B-Lymphocyte Subsets/immunology , Immunologic Memory , Oligonucleotide Array Sequence Analysis , Animals , B-Lymphocyte Subsets/metabolism , Gene Expression Profiling , Gene Rearrangement, B-Lymphocyte , Germinal Center/cytology , Germinal Center/immunology , Immunization , Mice , Mice, Transgenic , Models, Immunological , T-Lymphocytes/immunologyABSTRACT
BACKGROUND: Although in vitro IL-4 directs CD4 T cells to produce T helper 2 (Th2)-cytokines, these cytokines can be induced in vivo in the absence of IL-4-signalling. Thus, mechanism(s), different from the in vitro pathway for Th2-induction, contribute to in vivo Th2-differentiation. The pathway for in vivo IL-4-independent Th2-differentiation has yet to be characterized. FINDINGS: Helios (ikzf2), a member of the Ikaros transcription regulator family, is expressed in thymocytes and some antigen-matured T cells as well as in regulatory T cells. It has been proposed that Helios is a specific marker for thymus-derived regulatory T cells. Here, we show that mouse ovalbumin-specific CD4 (OTII) cells responding to alum-precipitated ovalbumin (alumOVA) upregulate Th2 features - GATA-3 and IL-4 - as well as Helios mRNA and protein. Helios is also upregulated in follicular helper T (TFh) cells in this response. By contrast, OTII cells responding to the Th1 antigen - live attenuated ovalbumin-expressing Salmonella - upregulate Th1 features - T-bet and IFN-γ - but not Helios. In addition, CD4 T cells induced to produce Th2 cytokines in vitro do not express Helios. The kinetics of Helios mRNA and protein induction mirrors that of GATA-3. The induction of IL-4, IL-13 and CXCR5 by alumOVA requires NF-κB1 and this is also needed for Helios upregulation. Importantly, Helios is induced in Th2 and TFh cells without parallel upregulation of Foxp3. These findings suggested a key role for Helios in Th2 and TFh development in response to alum-protein vaccines. We tested this possibility using Helios-deficient OTII cells and found this deficiency had no discernable impact on Th2 and TFh differentiation in response to alumOVA. CONCLUSIONS: Helios is selectively upregulated in CD4 T cells during Th2 and TFh responses to alum-protein vaccines in vivo, but the functional significance of this upregulation remains uncertain.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , Cell Differentiation/immunology , DNA-Binding Proteins/immunology , Forkhead Transcription Factors/immunology , T-Lymphocyte Subsets/immunology , T-Lymphocytes, Helper-Inducer/immunology , Th2 Cells/immunology , Transcription Factors/immunology , Adoptive Transfer , Alum Compounds/pharmacology , Animals , CD4-Positive T-Lymphocytes/cytology , Cell Line/drug effects , Cytokines/immunology , DNA-Binding Proteins/genetics , Forkhead Transcription Factors/genetics , GATA3 Transcription Factor/metabolism , Interleukin-13/immunology , Interleukin-4/genetics , Interleukin-4/immunology , Mice , Mice, Inbred C57BL , Mice, Transgenic , NF-kappa B/immunology , Ovalbumin/immunology , Ovalbumin/pharmacology , Signal Transduction/immunology , T-Lymphocyte Subsets/cytology , T-Lymphocytes, Helper-Inducer/cytology , Th2 Cells/cytology , Transcription Factors/geneticsABSTRACT
While IL-4 directs CD4 T cells to produce Th2 cytokines (including IL-4, IL-13, IL-5) in vitro it has been shown that production of these cytokines can be induced in vivo in the absence of IL-4/IL-13/STAT-6 signaling. The present report shows that CD8 as well as CD4 T cells activated through their TCR, in vitro upregulate the Th2-features - IL-4, IL-13, IL-5, and GATA-3. However, in vivo while alum-precipitated antigen strongly and selectively induces these Th2-features in CD4 T cells, CD8 T cells mount a markedly different response to this antigen. This CD8 response is associated with strong proliferation and production of IFN-gamma, but no Th2-features are induced. Alum-protein formulations are widely used in human vaccines and typically induce strong antibody responses characterized by the differentiation of IL-4-producing CD4 T cells and immunoglobulin class switching to IgG1. Nevertheless, the mechanism responsible for CD4 Th2 and follicular helper T cell commitment triggered by these alum-protein vaccines is still poorly understood. Analysis of the in vivo response to alum-precipitated protein shows that while subsets of CD4 T cells strongly upregulate Th2 and follicular helper T cell features including the surface markers OX40, CXCR5, PD-1, IL-17RB and the transcription factor c-Maf, CD8 T cells do not. These discrete differences between responding CD4 and CD8 T cells provide further insight into the differences between Th2 polarization of CD4 T cells directed by IL-4 in vitro and the induction of IL-4 production by CD4 T cells in vivo in response to alum-precipitated protein.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Cytokines/biosynthesis , Interleukin-4/immunology , Lymphocyte Activation/immunology , Alum Compounds/pharmacology , Animals , CD4-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/metabolism , Cell Separation , Cytokines/immunology , Flow Cytometry , Immunologic Factors/immunology , Immunologic Factors/metabolism , Interleukin-4/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Ovalbumin/immunology , Peptide Fragments/immunology , Reverse Transcriptase Polymerase Chain Reaction , Th2 Cells/immunologyABSTRACT
Nontyphoidal Salmonellae are a major cause of life-threatening bacteremia among HIV-infected individuals. Although cell-mediated immunity controls intracellular infection, antibodies protect against Salmonella bacteremia. We report that high-titer antibodies specific for Salmonella lipopolysaccharide (LPS) are associated with a lack of Salmonella-killing in HIV-infected African adults. Killing was restored by genetically shortening LPS from the target Salmonella or removing LPS-specific antibodies from serum. Complement-mediated killing of Salmonella by healthy serum is shown to be induced specifically by antibodies against outer membrane proteins. This killing is lost when excess antibody against Salmonella LPS is added. Thus, our study indicates that impaired immunity against nontyphoidal Salmonella bacteremia in HIV infection results from excess inhibitory antibodies against Salmonella LPS, whereas serum killing of Salmonella is induced by antibodies against outer membrane proteins.