Publications by authors named "Ian C M MacLennan"

39 Publications

Follicular Helper T Cells.

Annu Rev Immunol 2016 05 22;34:335-68. Epub 2016 Feb 22.

School of Immunity and Infection, University of Birmingham, Birmingham B15 2TT, United Kingdom.

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.
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http://dx.doi.org/10.1146/annurev-immunol-041015-055605DOI Listing
May 2016

CD8 T cells induce T-bet-dependent migration toward CXCR3 ligands by differentiated B cells produced during responses to alum-protein vaccines.

Blood 2012 Nov 11;120(23):4552-9. Epub 2012 Oct 11.

Medical Research Council Centre for Immune Regulation, University of Birmingham Medical School, Birmingham, United Kingdom.

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.
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http://dx.doi.org/10.1182/blood-2012-03-417733DOI Listing
November 2012

Early B blasts acquire a capacity for Ig class switch recombination that is lost as they become plasmablasts.

Eur J Immunol 2011 Dec 10;41(12):3506-12. Epub 2011 Nov 10.

MRC Centre for Immune Regulation, School of Immunity and Infection, University of Birmingham, Birmingham, UK.

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.
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http://dx.doi.org/10.1002/eji.201141762DOI Listing
December 2011

CD31 is required on CD4+ T cells to promote T cell survival during Salmonella infection.

J Immunol 2011 Aug 6;187(4):1553-65. Epub 2011 Jul 6.

Medical Research Council Centre for Immune Regulation, School of Immunity and Infection, Institute for Biomedical Research, University of Birmingham, Birmingham B15 2TT, United Kingdom.

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.
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http://dx.doi.org/10.4049/jimmunol.1000502DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3160468PMC
August 2011

Helios is associated with CD4 T cells differentiating to T helper 2 and follicular helper T cells in vivo independently of Foxp3 expression.

PLoS One 2011 3;6(6):e20731. Epub 2011 Jun 3.

School of Immunity and Infection, MRC Centre for Immune Regulation, Institute for Biomedical Research, University of Birmingham, Birmingham, England, United Kingdom.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0020731PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3108993PMC
October 2011

T-zone localized monocyte-derived dendritic cells promote Th1 priming to Salmonella.

Eur J Immunol 2011 Sep 4;41(9):2654-65. Epub 2011 Aug 4.

Medical Research Council Centre for Immune Regulation, School of Immunity and Infection, Institute of Biomedical Research, University of Birmingham, Birmingham, UK.

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.
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http://dx.doi.org/10.1002/eji.201141440DOI Listing
September 2011

Selective effects of NF-κB1 deficiency in CD4⁺ T cells on Th2 and TFh induction by alum-precipitated protein vaccines.

Eur J Immunol 2011 Jun 25;41(6):1573-82. Epub 2011 May 25.

MRC Centre for Immune Regulation, IBR, School of Immunity and Infection, University of Birmingham, Birmingham, UK.

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.
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http://dx.doi.org/10.1002/eji.201041126DOI Listing
June 2011

Soluble flagellin, FliC, induces an Ag-specific Th2 response, yet promotes T-bet-regulated Th1 clearance of Salmonella typhimurium infection.

Eur J Immunol 2011 Jun 10;41(6):1606-18. Epub 2011 May 10.

MRC Centre for Immune Regulation, University of Birmingham, Birmingham, UK.

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.
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http://dx.doi.org/10.1002/eji.201041089DOI Listing
June 2011

IFN-{gamma} produced by CD8 T cells induces T-bet-dependent and -independent class switching in B cells in responses to alum-precipitated protein vaccine.

Proc Natl Acad Sci U S A 2010 Oct 20;107(40):17292-7. Epub 2010 Sep 20.

Medical Research Council Centre for Immune Regulation, School of Immunity and Infection, University of Birmingham, Birmingham B15 2TT, United Kingdom.

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.
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http://dx.doi.org/10.1073/pnas.1004879107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2951392PMC
October 2010

Megakaryocytes constitute a functional component of a plasma cell niche in the bone marrow.

Blood 2010 Sep 10;116(11):1867-75. Epub 2010 Jun 10.

Institute for Systemic Inflammation Research, University of Lübeck, Ratzeburger Allee 160, Lübeck, Germany.

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.
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http://dx.doi.org/10.1182/blood-2009-12-259457DOI Listing
September 2010

Dysregulated humoral immunity to nontyphoidal Salmonella in HIV-infected African adults.

Science 2010 Apr;328(5977):508-12

Medical Research Council Centre for Immune Regulation and Clinical Immunology Service, Institute of Biomedical Research, School of Immunity and Infection, University of Birmingham, Birmingham, UK.

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.
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http://dx.doi.org/10.1126/science.1180346DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3772309PMC
April 2010

IL-4 directs both CD4 and CD8 T cells to produce Th2 cytokines in vitro, but only CD4 T cells produce these cytokines in response to alum-precipitated protein in vivo.

Mol Immunol 2010 Jun 13;47(10):1914-22. Epub 2010 Apr 13.

MRC Centre for Immune Regulation, the IBR, School of Immunity and Infection, University of Birmingham, Birmingham B15 2TT, United Kingdom.

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.
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http://dx.doi.org/10.1016/j.molimm.2010.03.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3826121PMC
June 2010

HVCN1 modulates BCR signal strength via regulation of BCR-dependent generation of reactive oxygen species.

Nat Immunol 2010 Mar 7;11(3):265-72. Epub 2010 Feb 7.

Medical Research Council Toxicology Unit, University of Leicester, Leicester, UK.

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.
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http://dx.doi.org/10.1038/ni.1843DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3030552PMC
March 2010

The porin OmpD from nontyphoidal Salmonella is a key target for a protective B1b cell antibody response.

Proc Natl Acad Sci U S A 2009 Jun 1;106(24):9803-8. Epub 2009 Jun 1.

School of Immunity and Infection and Medical Research Council Centre for Immune Regulation, University of Birmingham, Birmingham B15 2TT, United Kingdom.

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.
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http://dx.doi.org/10.1073/pnas.0812431106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2701014PMC
June 2009

Early simultaneous production of intranodal CD4 Th2 effectors and recirculating rapidly responding central-memory-like CD4 T cells.

Eur J Immunol 2009 Jun;39(6):1573-86

MRC Centre for Immune Regulation, the IBR, Department of Immunology, University of Birmingham, Birmingham, UK.

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.
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http://dx.doi.org/10.1002/eji.200838922DOI Listing
June 2009

Dendritic cells and monocyte/macrophages that create the IL-6/APRIL-rich lymph node microenvironments where plasmablasts mature.

J Immunol 2009 Feb;182(4):2113-23

Medical Research Council Centre for Immune Regulation, University of Birmingham Medical School, Birmingham, United Kingdom.

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.
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http://dx.doi.org/10.4049/jimmunol.0802771DOI Listing
February 2009

Molecular differences between the divergent responses of ovalbumin-specific CD4 T cells to alum-precipitated ovalbumin compared to ovalbumin expressed by Salmonella.

Mol Immunol 2008 Aug 25;45(13):3558-66. Epub 2008 Jun 25.

MRC Centre for Immune Regulation, University of Birmingham, Medical School, Birmingham, UK.

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.
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http://dx.doi.org/10.1016/j.molimm.2008.05.010DOI Listing
August 2008

B cells: the follicular dimension of the marginal zone.

Immunol Cell Biol 2008 Mar-Apr;86(3):219-20. Epub 2008 Feb 26.

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http://dx.doi.org/10.1038/icb.2008.2DOI Listing
July 2008

Critical synergy of CD30 and OX40 signals in CD4 T cell homeostasis and Th1 immunity to Salmonella.

J Immunol 2008 Mar;180(5):2824-9

Medical Research Council Centre for Immune Regulation, Division of Immunity and Infection, University of Birmingham, Birmingham, United Kingdom.

CD30 and OX40 (CD134) are members of the TNFR superfamily expressed on activated CD4 T cells, and mice deficient in both these molecules harbor a striking defect in the capacity to mount CD4 T cell-dependent memory Ab responses. This article shows that these mice also fail to control Salmonella infection because both CD30 and OX40 signals are required for the survival but not commitment of CD4 Th1 cells. These signals are also needed for the survival of CD4 T cells activated in a lymphopenic environment. Finally, Salmonella and lymphopenia are shown to act synergistically in selectively depleting CD4 T cells deficient in OX40 and CD30. Collectively these findings identify a novel mechanism by which Th1 responses are sustained.
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http://dx.doi.org/10.4049/jimmunol.180.5.2824DOI Listing
March 2008

Levels of autoantibodies, unlike antibodies to all extrinsic antigen groups, fall following B cell depletion with Rituximab.

Eur J Immunol 2008 Jan;38(1):292-8

MRC Centre for Immune Regulation, Medical School, University of Birmingham, UK.

Many autoantibodies have variable-region sequences indicating their production in an affinity-matured antibody response involving germinal centers (GC). Plasma cells from GC can be long-lived, do not express CD20 and thus should not be depleted by a therapeutic monoclonal Ab against human CD20 - Rituximab. Nevertheless, autoantibody titers often fall following Rituximab treatment. To test if this reflects exclusive production by short-lived plasma cells in extrafollicular Ab responses, we monitored, after Rituximab treatment, levels of natural Ab and Ab against extrinsic antigens that do not induce productive GC. Eleven patients with active vasculitis and anti-proteinase-3 (PR3) Ab were assessed before and during 5 months after Rituximab therapy. Blood B cells were undetectable within 2 wk, and all patients achieved clinical remission. Levels of natural Ab - isohemagglutinins and anti-phosphorylcholine Ab - and Ab levels against thymus-independent and thymus-dependent extrinsic antigens were little affected. By contrast, 5 months after Rituximab, IgG autoantibody against PR3 had fallen to a median of 22% of pretreatment values. While the kinetics of this fall do not suggest an intrinsically short lifespan of autoantibody-producing cells, the data are consistent with Rituximab causing loss of sites within inflammatory tissues that selectively sustain autoantibody-producing cells.
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http://dx.doi.org/10.1002/eji.200737557DOI Listing
January 2008

microRNA-155 regulates the generation of immunoglobulin class-switched plasma cells.

Immunity 2007 Dec 6;27(6):847-59. Epub 2007 Dec 6.

Laboratory of Lymphocyte Signalling and Development, The Babraham Institute, Cambridge, CB22 3AT, UK.

microRNA-155 (miR-155) is expressed by cells of the immune system after activation and has been shown to be required for antibody production after vaccination with attenuated Salmonella. Here we show the intrinsic requirement for miR-155 in B cell responses to thymus-dependent and -independent antigens. B cells lacking miR-155 generated reduced extrafollicular and germinal center responses and failed to produce high-affinity IgG1 antibodies. Gene-expression profiling of activated B cells indicated that miR-155 regulates an array of genes with diverse function, many of which are predicted targets of miR-155. The transcription factor Pu.1 is validated as a direct target of miR155-mediated inhibition. When Pu.1 is overexpressed in wild-type B cells, fewer IgG1 cells are produced, indicating that loss of Pu.1 regulation is a contributing factor to the miR-155-deficient phenotype. Our results implicate post-transcriptional regulation of gene expression for establishing the terminal differentiation program of B cells.
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http://dx.doi.org/10.1016/j.immuni.2007.10.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4135426PMC
December 2007

Axon growth and guidance genes identify T-dependent germinal centre B cells.

Immunol Cell Biol 2008 Jan 16;86(1):3-14. Epub 2007 Oct 16.

Division of Immunology and Genetics, John Curtin School of Medical Research, The Australian National University, Canberra, Australia.

Selection of B cells subjected to hypermutation in germinal centres (GC) during T cell-dependent (TD) antibody responses yields memory cells and long-lived plasma cells that produce high affinity antibodies biased to foreign antigens rather than self-antigens. GC also form in T-independent (TI) responses to polysaccharide antigens but failed selection results in GC involution and memory cells are not generated. To date there are no markers that allow phenotypic distinction of T-dependent and TI germinal centre B cells. We compared the global gene expression of GC B cells purified from mice immunized with either TD or TI antigens and identified eighty genes that are differentially expressed in TD GC. Significantly, the largest cluster comprises genes involved in growth and guidance of neuron axons such as Plexin B2, Basp1, Nelf, Shh, Sc4mol and Sult4alpha. This is consistent with formation of long neurite (axon and dendrite)-like structures by mouse and human GC B cells, which may facilitate T:B cell interactions within GC, affinity maturation and B cell memory formation. Expression of BASP1 and PLEXIN B2 protein is very low or undetectable in resting and TI GC B cells, but markedly upregulated in GC B cells induced in the presence of T cell help. Finally we show some of the axon growth genes upregulated in TD-GC B cells including Basp1, Shh, Sult4alpha, Sc4mol are also preferentially expressed in post-GC B cell neoplasms.
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http://dx.doi.org/10.1038/sj.icb.7100123DOI Listing
January 2008

Recirculating CD4 memory T cells mount rapid secondary responses without major contributions from follicular CD4 effectors and B cells.

Eur J Immunol 2007 Jun;37(6):1476-84

Department of Biochemistry, University of Lausanne, Epalinges, Switzerland.

For weeks after primary immunization with thymus-dependent antigens the responding lymph nodes contain effector CD4 T cells in T zones and germinal centers as well as recirculating memory T cells. Conversely, remote nodes, not exposed to antigen, only receive recirculating memory cells. We assessed whether lymph nodes with follicular effector CD4 T cells in addition to recirculating memory CD4 T cells mount a more rapid secondary response than nodes that only contain recirculating memory cells. Also, the extent to which T cell frequency governs accelerated CD4 T cell recall responses was tested. For this, secondary antibody responses to a superantigen, where the frequency of responding T cells is not increased at the time of challenge, were compared with those to conventional protein antigens. With both types of antigens similar accelerated responses were elicited in the node draining the site of primary immunization and in the contralateral node, not previously exposed to antigen. Thus recirculating memory cells are fully capable of mounting accelerated secondary responses, without the assistance of CD4 effector T cells, and accelerated memory responses are not solely dependent on higher T cell frequencies. Accelerated memory CD4 T cell responses were also seen in B cell-deficient mice.
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http://dx.doi.org/10.1002/eji.200636573DOI Listing
June 2007

Salmonella induces a switched antibody response without germinal centers that impedes the extracellular spread of infection.

J Immunol 2007 May;178(10):6200-7

Medical Research Council Centre for Immune Regulation, University of Birmingham, Birmingham, United Kingdom.

T-dependent Ab responses are characterized by parallel extrafollicular plasmablast growth and germinal center (GC) formation. This study identifies that, in mice, the Ab response against Salmonella is novel in its kinetics and its regulation. It demonstrates that viable, attenuated Salmonella induce a massive early T-dependent extrafollicular response, whereas GC formation is delayed until 1 mo after infection. The extrafollicular Ab response with switching to IgG2c, the IgG2a equivalent in C57BL/6 mice, is well established by day 3 and persists through 5 wk. Switching is strongly T dependent, and the outer membrane proteins are shown to be major targets of the early switched IgG2c response, whereas flagellin and LPS are not. GC responses are associated with affinity maturation of IgG2c, and their induction is associated with bacterial burden because GC could be induced earlier by treating with antibiotics. Clearance of these bacteria is not a consequence of high-affinity Ab production, for clearance occurs equally in CD154-deficient mice, which do not develop GC, and wild-type mice. Nevertheless, transferred low- and high-affinity IgG2c and less efficiently IgM were shown to impede Salmonella colonization of splenic macrophages. Furthermore, Ab induced during the infection markedly reduces bacteremia. Thus, although Ab does not prevent the progress of established splenic infection, it can prevent primary infection and impedes secondary hemogenous spread of the disease. These results may explain why attenuated Salmonella-induced B cell responses are protective in secondary, but not primary infections.
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http://dx.doi.org/10.4049/jimmunol.178.10.6200DOI Listing
May 2007

B cell clones that sustain long-term plasmablast growth in T-independent extrafollicular antibody responses.

Proc Natl Acad Sci U S A 2006 Apr 3;103(15):5905-10. Epub 2006 Apr 3.

Medical Research Council Centre for Immune Regulation, University of Birmingham, Birmingham B15 2TT, United Kingdom.

Some antigens induce Ab responses without T lymphocyte help. Among these, many polysaccharide-based antigens cause marginal zone B cells to proliferate and differentiate into plasma cells. B1 cells also respond to some of these antigens. In this article, we report that antigen-specific B1b cells, in response to the T-independent antigen (4-hydroxy-3-nitrophenyl)-acetyl (NP)-Ficoll, develop into clones that sustain Ab production for months with continued production of plasma cells and the accumulation of antigen-specific B cells in follicles. The persistence of this T-independent plasmablast response contrasts with the short-term plasmablast growth associated with T-dependent extrafollicular responses. The nature of the cells responding to NP-Ficoll was probed by using chimeras that have B1 cells but lack primary B lymphopoietic capacity and have very few B2 cells or T cells. The chimeras were constructed by transferring 10(5) IgM(+) IgD(-) peritoneal exudate cells into mice unable to produce their own T and B cells because of deficiency in recombinase-activating gene 1 (RAG-1). The chimeras mounted sustained IgM and IgG3 anti-NP Ab responses to NP-Ficoll. This finding was associated with continued NP-specific extrafollicular plasmablast growth and the accumulation of NP-specific B cells in follicles. B cells were not found in the marginal zones of chimeras, and they also lacked recirculating IgD(+) cells and CD3(+) cells. The absence of B2 and T cells confirms that hemopoietic cell chimerism leading to primary lymphopoiesis had not been established.
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http://dx.doi.org/10.1073/pnas.0601502103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1424660PMC
April 2006

Germinal centers still hold secrets.

Immunity 2005 Jun;22(6):656-7

MRC Centre for Immune Regulation, University of Birmingham, United Kingdom.

There has been recent progress in understanding the mechanism of somatic hypermutation in germinal centers. By contrast, there is more theory than certainty about the way those cells that acquire heightened affinity for antigen are subsequently selected. Wang and Carter (2005) highlight this in the current issue of Immunity in a study of germinal center development in mice with impaired CD19 signaling.
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http://dx.doi.org/10.1016/j.immuni.2005.06.002DOI Listing
June 2005

Homeostatic cell-cycle control by BLyS: Induction of cell-cycle entry but not G1/S transition in opposition to p18INK4c and p27Kip1.

Proc Natl Acad Sci U S A 2004 Dec 10;101(51):17789-94. Epub 2004 Dec 10.

Department of Pathology and Graduate Program in Immunology and Microbial Pathogenesis, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021, USA.

Cell-cycle entry is critical for homeostatic control in physiologic response of higher organisms but is not well understood. The antibody response begins with induction of naive mature B cells, which are naturally arrested in G(0)/G(1) phase of the cell cycle, to enter the cell cycle in response to antigen and cytokine. BLyS (BAFF), a cytokine essential for mature B cell development and survival, is thought to act mainly by attenuation of apoptosis. Here, we show that BLyS alone induces cell-cycle entry and early G(1) cell-cycle progression, but not S-phase entry, in opposition to the cyclin-dependent kinase inhibitors p18(INK4c). Independent of its survival function, BLyS enhances the synthesis of cyclin D2, in part through activation of NF-kappaB, as well as CDK4 and retinoblastoma protein phosphorylation. By convergent activation of the same cell-cycle regulators in opposition to p18(INK4c), B cell receptor signaling induces cell-cycle entry and G(1) progression in synergy with BLyS, but also DNA replication. The failure of BLyS to induce S-phase cell-cycle entry lies in its inability to increase cyclin E and reduce p27(Kip1) expression. Antagonistic cell-cycle regulation by BLyS and p18(INK4c) is functionally linked to apoptotic control and conserved from B cell activation in vitro to antibody response in vivo, further indicating a physiologic role in homeostasis.
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http://dx.doi.org/10.1073/pnas.0406111101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC535425PMC
December 2004

Responses to the soluble flagellar protein FliC are Th2, while those to FliC on Salmonella are Th1.

Eur J Immunol 2004 Nov;34(11):2986-95

MRC Centre for Immune Regulation, University of Birmingham, Birmingham, UK.

Features of the Th1 or Th2 phenotype start to develop during CD4 T cell priming. This study of the response to the bacterial flagellar protein FliC shows that either Th1 or Th2 responses can be induced in mice depending upon how FliC is presented. This is shown by assessing the cytokine mRNA and class of FliC-specific plasma cells induced in situ. Soluble recombinant (r)FliC and polymerized FliC are strongly Th2 polarizing, inducing IL-4, NIP45 and c-Maf mRNA as well as epsilon and gamma1 switch transcripts and switching to IgG1. CD28-requirement for this switching shows its T cell dependence. rFliC was unable to induce markers of Th1 activity including IL-12, T-bet and IFN-gamma. Conversely, when FliC is presented in its native context surface-bound on live, flagellated Salmonella, switching is predominantly to IgG2a (IgG2c in C57BL/6 mice), reflecting Th1 activity. The development of divergent FliC-specific polarization to either Th1 or Th2 indicates that the context in which this antigen is encountered rather than its intrinsic immunostimulatory properties determines the direction of Th polarization.
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http://dx.doi.org/10.1002/eji.200425403DOI Listing
November 2004

Pinpointing IL-4-independent acquisition and IL-4-influenced maintenance of Th2 activity by CD4 T cells.

Eur J Immunol 2004 Mar;34(3):686-694

The Medical Research Council Centre for Immune Regulation, University of Birmingham, Birmingham, GB.

Naive CD4 T cells develop Th2 activity early in primary responses to alum-precipitated proteins by producing IL-4 mRNA and inducing B cells to produce gamma1 and epsilon switch transcripts. Both IL-4-dependent and IL-4-independent pathways for IL-4 induction are recognized, but their relative contribution to the different phases of primary Th2 responses in vivo is uncertain. We show the primary induction of IL-4 synthesis in lymph nodes responding to alum-precipitated protein is overwhelmingly in antigen-specific CD4 T cells and is unimpaired in IL-4Ralpha(-/-) mice, which can produce but do not respond to IL-4 and IL-13. Ig class-switching in extra-follicular responses, reflecting Th2 activity, is also unimpaired in these mice. By contrast, 7 days after immunization--when T cells are selecting B cells in germinal centers and T cell priming has occurred--non-responsiveness to IL-4 is associated with smaller germinal centers, increased levels of T-bet and gamma2a switch transcripts and reduced gamma1 and epsilon transcripts. These data indicate that Th2 characteristics acquired during T cell priming and the initial CD4 T cell interaction with B cells are largely IL-4-independent, whereas IL-4 production induced during priming has a significant role in maintaining the Th2 phenotype as T cells select B cells in germinal centers.
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http://dx.doi.org/10.1002/eji.200324510DOI Listing
March 2004

NF-kappa B1 p50 is required for BLyS attenuation of apoptosis but dispensable for processing of NF-kappa B2 p100 to p52 in quiescent mature B cells.

J Immunol 2003 Jul;171(2):761-8

Department of Pathology, Cornell-Rockefeller University-Sloan-Kettering Institute Tri-Institutional MD-PhD Program, Weill Medical College of Cornell University, New York, NY, 10021, USA.

B lymphocyte stimulator (BLyS), a TNF family protein essential for peripheral B cell development, functions primarily through attenuation of B cell apoptosis. In this study, we show that BLyS activates NF-kappaB through both classical and alternative pathways with distinct kinetics in quiescent mature B cells. It rapidly and transiently enhances the p50/p65 DNA binding activity and induces phosphorylation of IkappaBalpha characteristic of the classical NF-kappaB pathway, albeit maintaining IkappaBalpha at a constant level through ongoing protein synthesis and proteasome-mediated destruction. With delayed kinetics, BLyS promotes the processing of p100 to p52 and sustained formation of p52/RelB complexes via the alternative NF-kappaB pathway. p50 is dispensable for p100 processing. However, it is required to mediate the initial BLyS survival signals and concomitant activation of Bcl-x(L) in quiescent mature B cells ex vivo. Although also a target of BLyS activation, at least one of the A1 genes, A1-a, is dispensable for the BLyS survival function. These results suggest that BLyS mediates its survival signals in metabolically restricted quiescent B cells, at least in part, through coordinated activation of both NF-kappaB pathways and selective downstream antiapoptotic genes.
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http://dx.doi.org/10.4049/jimmunol.171.2.761DOI Listing
July 2003