Critical Role for Mouse Marginal Zone B Cells in PF4/Heparin Antibody Production

Abstract Abstract 1175 Heparin-induced thrombocytopenia (HIT) is an immune-mediated disorder that can cause fatal arterial or venous thrombosis/thromboembolism. Immune complexes consisting of platelet factor 4 (PF4), heparin and PF4/heparin-reactive antibodies are central to the pathogenesis of HIT. However, the B-cell origin of HIT antibody production is not known. Here we show that upon challenge with PF4/heparin complexes, anti-PF4/heparin antibody production is severely impaired in B cell-specific Notch2-deficient mice (CD19CreNotch2fl/fl) that specifically lack marginal zone (MZ) B cells, and that antibody production is readily generated in wild-type mice (CD19CreNotch2+/+). As expected, Notch2-deficient mice responded normally to challenge with T cell-dependent antigen NP-CGG but not T cell-independent antigen TNP-Ficoll, in agreement with the lack of MZ B cells in the mutant mice. PF4/heparin-specific antibodies produced by wild-type mice on a C57BL/6 background were IgG2b and IgG3 isotypes. An in vitro class-switching assay showed that MZ B cells from wild-type C57BL/6 mice were capable of producing antibodies of IgG2b and IgG3 isotypes. Lastly, MZ, but not follicular (FO), B cells adoptively transferred into B cell-deficient muMT mice responded to PF4/heparin complex challenge by producing PF4/heparin-specific antibodies of IgG2b and IgG3 isotypes. Taken together, these data demonstrate that MZ B cells play a critical role in production of PF4/heparin-specific antibodies. Disclosures: Arepally: Teva Pharmaceuticals: Research Funding.

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Critical Role of T Cells in PF4/Heparin Antibody Production

Blood ◽

10.1182/blood.v124.21.1554.1554 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1554-1554

Author(s):

Yongwei Zheng ◽

Mei Yu ◽

Anand Padmanabhan ◽

Richard H. Aster ◽

Renren Wen ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

B Cell ◽

Antibody Production ◽

Cd4 T Cells ◽

Wild Type ◽

Specific Antibodies ◽

Deficient Mice

Abstract Heparin-induced thrombocytopenia (HIT) is an antibody-mediated disorder that can cause arterial or venous thrombosis/thromboembolism, and platelet factor 4 (PF4)/ heparin-reactive antibodies are essential to the pathogenesis of HIT. Our recent studies have demonstrated that marginal zone (MZ) B cells play a major role in production of PF4/heparin-specific antibodies. However, the role of T cells in production of these pathogenic antibodies is not clear. Here we showed that PF4/heparin complex-induced production of PF4/heparin-specific antibodies was markedly impaired in mice, in which CD4 T cells were depleted by administration of GK1.5 anti-CD4 monoclonal antibody. As expected, the CD4 T cell-depleted mice responded normally to T cell-independent antigen TNP-Ficoll but not T cell-dependent antigen NP-CGG, in agreement with the lack of CD4 T cells in these GK1.5-treated mice. Further, following adoptive transfer of a mixture of wild-type splenic B cells and splenocytes from B cell-deficient μMT mice, T and B cell-deficient Rag1 knockout mice responded to PF4/heparin complex challenge to produce PF4/heparin-specific antibodies. In contrast, Rag1-deficient mice that received a mixture of wild-type splenic B cells and splenocytes from Rag1-deficient mice barely produced PF4/heparin-specific antibodies upon PF4/heparin complex challenge. These data suggest that T cells are required for production of PF4/heparin-specific antibodies. Consistent with this concept, mice with B cells lacking CD40 molecule, a B cell costimulatory molecule that helps T cell-dependent B cell responses, displayed a marked reduction of PF4/heparin-specific antibody production following PF4/heparin complex challenge. Also as expected, mice with CD40-deficient B cells were able to respond to T cell-independent antigen TNP-Ficoll but not T cell-dependent antigen NP-CGG, consistent with the lack of T-cell help in these mice. Taken together, these findings demonstrate that T cells play an essential role in production of PF4/heparin-specific antibodies by MZ B cells. Disclosures No relevant conflicts of interest to declare.

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Critical role for mouse marginal zone B cells in PF4/heparin antibody production

Blood ◽

10.1182/blood-2013-01-477091 ◽

2013 ◽

Vol 121 (17) ◽

pp. 3484-3492 ◽

Cited By ~ 32

Author(s):

Yongwei Zheng ◽

Mei Yu ◽

Andrew Podd ◽

Liudi Yuan ◽

Debra K. Newman ◽

...

Keyword(s):

B Cells ◽

Antibody Production ◽

Marginal Zone ◽

Critical Role ◽

Marginal Zone B Cells ◽

Specific Antibodies ◽

Key Points

Key PointsMZ B cells play a critical role in the production of PF4/heparin-specific antibodies.

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Formalin-Inactivated Coxiella burnetii Phase I Vaccine-Induced Protection Depends on B Cells To Produce Protective IgM and IgG

Infection and Immunity ◽

10.1128/iai.00297-13 ◽

2013 ◽

Vol 81 (6) ◽

pp. 2112-2122 ◽

Cited By ~ 12

Author(s):

Guoquan Zhang ◽

Ying Peng ◽

Laura Schoenlaub ◽

Alexandra Elliott ◽

William Mitchell ◽

...

Keyword(s):

T Cell ◽

B Cells ◽

Phase I ◽

B Cell ◽

Coxiella Burnetii ◽

Critical Role ◽

Partial Protection ◽

Content Type ◽

Deficient Mice

ABSTRACTTo further understand the mechanisms of formalin-inactivatedCoxiella burnetiiphase I (PI) vaccine (PIV)-induced protection, we examined if B cell, T cell, CD4+T cell, or CD8+T cell deficiency in mice significantly affects the ability of PIV to confer protection against aC. burnetiiinfection. Interestingly, compared to wild-type (WT) mice, PIV conferred comparable levels of protection in CD4+T cell- or CD8+T cell-deficient mice and partial protection in T cell-deficient mice but did not provide measurable protection in B cell-deficient mice. These results suggest that PIV-induced protection depends on B cells. In addition, anti-PI-specific IgM was the major detectable antibody (Ab) in immune sera from PIV-vaccinated CD4+T cell-deficient mice, and passive transfer of immune sera from PIV-vaccinated CD4+T cell-deficient mice conferred significant protection. These results suggest that T cell-independent anti-PI-specific IgM may contribute to PIV-induced protection. Our results also suggested that PIV-induced protection may not depend on complement activation and Fc receptor-mediated effector functions. Furthermore, our results demonstrated that both IgM and IgG from PIV-vaccinated WT mouse sera were able to inhibitC. burnetiiinfectionin vivo, but only IgM from PIV-vaccinated CD4+T cell-deficient mouse sera inhibitedC. burnetiiinfection. Collectively, these findings suggest that PIV-induced protection depends on B cells to produce protective IgM and IgG and that T cell-independent anti-PI-specific IgM may play a critical role in PIV-induced protection againstC. burnetiiinfection.

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T Cell-Independent Gamma Interferon and B Cells Cooperate To Prevent Mortality Associated with DisseminatedChlamydia muridarumGenital Tract Infection

Infection and Immunity ◽

10.1128/iai.00143-18 ◽

2018 ◽

Vol 86 (7) ◽

pp. e00143-18 ◽

Cited By ~ 6

Author(s):

Taylor B. Poston ◽

Catherine M. O'Connell ◽

Jenna Girardi ◽

Jeanne E. Sullivan ◽

Uma M. Nagarajan ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

Tract Infection ◽

B Cell ◽

Gamma Interferon ◽

Wild Type ◽

Content Type ◽

Ifn Γ ◽

Deficient Mice

ABSTRACTCD4 T cells and antibody are required for optimal acquired immunity toChlamydia muridarumgenital tract infection, and T cell-mediated gamma interferon (IFN-γ) production is necessary to clear infection in the absence of humoral immunity. However, the role of T cell-independent immune responses during primary infection remains unclear. We investigated this question by inoculating wild-type and immune-deficient mice withC. muridarumCM001, a clonal isolate capable of enhanced extragenital replication. Genital inoculation of wild-type mice resulted in transient dissemination to the lungs and spleen that then was rapidly cleared from these organs. However, CM001 genital infection proved lethal forSTAT1−/−andIFNG−/−mice, in which IFN-γ signaling was absent, and forRag1−/−mice, which lacked T and B cells and in which innate IFN-γ signaling was retained. In contrast, B cell-deficient muMT mice, which can generate a Th1 response, and T cell-deficient mice with intact B cell and innate IFN-γ signaling survived. These data collectively indicate that IFN-γ prevents lethal CM001 dissemination in the absence of T cells and suggests a B cell corequirement. Adoptive transfer of convalescent-phase immune serum but not naive IgM toRag1−/−mice infected with CM001 significantly increased the survival time, while transfer of naive B cells completely rescuedRag1−/−mice from CM001 lethality. Protection was associated with a significant reduction in the lung chlamydial burden of genitally infected mice. These data reveal an important cooperation between T cell-independent B cell responses and innate IFN-γ in chlamydial host defense and suggest that interactions between T cell-independent antibody and IFN-γ are essential for limiting extragenital dissemination.

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Wiskott-Aldrich syndrome protein (WASP) and N-WASP are critical for peripheral B-cell development and function

Blood ◽

10.1182/blood-2010-09-308197 ◽

2012 ◽

Vol 119 (17) ◽

pp. 3966-3974 ◽

Cited By ~ 51

Author(s):

Lisa S. Westerberg ◽

Carin Dahlberg ◽

Marisa Baptista ◽

Christopher J. Moran ◽

Cynthia Detre ◽

...

Keyword(s):

T Cell ◽

B Cells ◽

B Cell ◽

Marginal Zone ◽

Cell Development ◽

B Cell Development ◽

Wild Type ◽

And Function ◽

Syndrome Protein

Abstract The Wiskott-Aldrich syndrome protein (WASP) is a key cytoskeletal regulator of hematopoietic cells. Although WASP-knockout (WKO) mice have aberrant B-cell cytoskeletal responses, B-cell development is relatively normal. We hypothesized that N-WASP, a ubiquitously expressed homolog of WASP, may serve some redundant functions with WASP in B cells. In the present study, we generated mice lacking WASP and N-WASP in B cells (conditional double knockout [cDKO] B cells) and show that cDKO mice had decreased numbers of follicular and marginal zone B cells in the spleen. Receptor-induced activation of cDKO B cells led to normal proliferation but a marked reduction of spreading compared with wild-type and WKO B cells. Whereas WKO B cells showed decreased migration in vitro and homing in vivo compared with wild-type cells, cDKO B cells showed an even more pronounced decrease in the migratory response in vivo. After injection of 2,4,6-trinitrophenol (TNP)–Ficoll, cDKO B cells had reduced antigen uptake in the splenic marginal zone. Despite high basal serum IgM, cDKO mice mounted a reduced immune response to the T cell–independent antigen TNP-Ficoll and to the T cell–dependent antigen TNP–keyhole limpet hemocyanin. Our results reveal that the combined activity of WASP and N-WASP is required for peripheral B-cell development and function.

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Impaired T- and B-cell development in Tcl1-deficient mice

Blood ◽

10.1182/blood-2004-04-1453 ◽

2005 ◽

Vol 105 (3) ◽

pp. 1288-1294 ◽

Cited By ~ 29

Author(s):

Sang-Moo Kang ◽

Maria Grazia Narducci ◽

Cristina Lazzeri ◽

Adriana M. Mongiovì ◽

Elisabetta Caprini ◽

...

Keyword(s):

T Cell ◽

B Cells ◽

B Cell ◽

Marginal Zone ◽

Cell Development ◽

B Cell Development ◽

Cell Stage ◽

Marginal Zone B Cells ◽

Induced Apoptosis ◽

Deficient Mice

AbstractTCL1, the overexpression of which may result in T-cell leukemia, is normally expressed in early embryonic tissues, the ovary, and lymphoid lineage cells. Our analysis of mouse B-lineage cells indicates that Tcl1 expression is initiated in pro-B cells and persists in splenic marginal zone and follicular B cells. T-lineage Tcl1 expression begins in thymocyte progenitors, continues in CD4+CD8+ thymocytes, and is extinguished in mature T cells. In Tcl1-deficient mice, we found B lymphopoiesis to be compromised at the pre-B cell stage and T-cell lymphopoiesis to be impaired at the CD4+CD8+ thymocyte stage. A corresponding increase was observed in thymocyte susceptibility to anti-CD3ϵ–induced apoptosis. Reduced numbers of splenic follicular and germinal center B cells were accompanied by impaired production of immunoglobulin G1 (IgG1) and IgG2b antibodies in response to a T-dependent antigen. The marginal zone B cells and T-cell–independent antibody responses were also diminished in Tcl1-/- mice. This analysis indicates a significant role for Tcl1, a coactivator of Akt signaling, in normal T- and B-cell development and function.

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RhoH-Deficient Mice Show Altered B Cell Populations In Vivo.

Blood ◽

10.1182/blood.v110.11.2307.2307 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2307-2307

Author(s):

Abel Sanchez-Aguilera ◽

Jose Cancelas ◽

David A. Williams

Keyword(s):

Bone Marrow ◽

T Cell ◽

B Cells ◽

B Cell ◽

Marginal Zone ◽

Cell Receptor ◽

Wild Type ◽

Marginal Zone B Cells

Abstract RhoH is a GTPase-deficient, hematopoietic-specific member of the family of Rho GTPases (Li et al, 2002). RhoH has been described as regulating proliferation and engraftment of hematopoietic progenitor cells (Gu et al, 2005) and integrin-mediated adhesion in T cells (Cherry et al, 2004). Additionally, RhoH plays a critical role in T-cell development and T-cell receptor signaling (Gu et al, 2006; Dorn et al, 2007). However, the potential role of RhoH in the differentiation and biological functions of B cells are unknown. To answer these questions, we analyzed the B-cell phenotype of RhoH−/− mice and the in vitro properties of RhoH-deficient splenic B cells compared to their wild-type counterparts. RhoH−/− mice showed increased B-cell numbers in the bone marrow, mainly due to an increase in the number of pro-B, pre-B and immature B cells. In the spleen, lymph nodes and peripheral blood, RhoH−/− mice showed a significant decrease in the number of follicular (B-2) cells (B220+ CD93– IgDhigh CD21low). The number of splenic marginal zone B cells (B220+ CD93– IgDlow CD21high), plasma cells (CD93– CD38+ CD138+) in bone marrow and spleen, and B-1 cells (IgM+ CD5+) in peritoneal cavity were not significantly different from those in wild-type animals. These alterations have functional significance, since the serum concentrations of IgM and IgG1 were significantly lower in RhoH−/− mice. However, splenic B cells isolated from RhoH−/− mice did not show any significant differences in their in vitro activation by anti-IgM, CD40 ligation or IL-4 stimulation, nor did they differ in their proliferative response to lipopolysaccharide. In vitro migration of RhoH-deficient B cells in response to CXCL12 or CXCL13 was similar to that of wild-type B cells. Given the important role of RhoH in signal transduction downstream the T cell receptor, we investigated the possible role of RhoH in B cell receptor signaling. Although total splenic B cells from RhoH−/− mice showed markedly increased phosphorylation of SYK and ERK after anti-IgM stimulation compared to wild-type B cells, sorted populations of splenic B-2 and marginal zone B cells from RhoH−/− and wild-type animals did not differ in the activation of these kinases, suggesting that the observed difference can be attributed to the different cellular composition of the B cell compartment (i.e. B-2 vs marginal zone B cells) in RhoH−/− mice. These data imply that the phenotype observed in RhoH−/− mice may not reflect an intrinsic defect in B cells but may be attributed to crosstalk between B cells and other hematopoietic cell populations. Composition of B cell subsets in wild-type and RhoH−/− mice (total cell number ×106, ± standard deviation, N=9) Bone marrow Spleen (*) indicates p<0.05; (**), p<0.01; (***), p<0.005 RhoH+/+ RhoH−/− RhoH+/+ RhoH−/− total B cells 7.8±1.8 11.0±2.4 (**) total B cells 31.7±10.1 25.4±8.8 pro-B 0.12±0.03 0.15±0.04 (*) transitional 8.7±1.2 8.6±2.8 pre-B 2.6±0.6 3.8±0.8 (***) B-2 11.6±4.1 7.6±2.5 (*) immature 1.5±0.4 2.1±0.5 (*) marginal 3.2±1.1 3.9±1.6 mature 1.4±0.7 1.7±0.9

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Immunoglobulin D (IgD)-deficient mice reveal an auxiliary receptor function for IgD in antigen-mediated recruitment of B cells.

Journal of Experimental Medicine ◽

10.1084/jem.177.1.45 ◽

1993 ◽

Vol 177 (1) ◽

pp. 45-55 ◽

Cited By ~ 161

Author(s):

J Roes ◽

K Rajewsky

Keyword(s):

T Cell ◽

B Cells ◽

B Cell ◽

Primary Response ◽

Affinity Maturation ◽

Resting Cells ◽

Wild Type ◽

Normal Numbers ◽

Immunoglobulin D ◽

Deficient Mice

To assess the role of immunoglobulin D (IgD) in vivo we generated IgD-deficient mice by gene targeting and studied B cell development and function in the absence of IgD expression. In the mutant animals, conventional and CD5-positive (B1) B cells are present in normal numbers, and the expression of the surface markers CD22 and CD23 in the compartment of conventional B cells indicates acquisition of a mature phenotype. As in wild-type animals, most of the peripheral B cells are resting cells. The IgD-deficient mice respond well to T cell-independent and -dependent antigens. However, in heterozygous mutant animals, B cells expressing the wild type IgH locus are overrepresented in the peripheral B cell pool, and T cell-dependent IgG1 responses are further dominated by B cells expressing the wild-type allele. Similarly, in homozygous mutant (IgD-deficient) animals, affinity maturation is delayed in the early primary response compared to control animals, although the mutants are capable of generating high affinity B cell memory. Thus, rather than being involved in major regulatory processes as had been suggested, IgD seems to function as an antigen receptor optimized for efficient recruitment of B cells into antigen-driven responses. The IgD-mediated acceleration of affinity maturation in the early phase of the T cell-dependent primary response may confer to the animal a critical advantage in the defense against pathogens.

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Vav proteins regulate peripheral B-cell survival

Blood ◽

10.1182/blood-2004-12-4894 ◽

2005 ◽

Vol 106 (7) ◽

pp. 2391-2398 ◽

Cited By ~ 33

Author(s):

Elena Vigorito ◽

Laure Gambardella ◽

Francesco Colucci ◽

Simon McAdam ◽

Martin Turner

Keyword(s):

B Cells ◽

B Cell ◽

Marginal Zone ◽

Cell Receptor ◽

Cross Linking ◽

Marginal Zone B Cells ◽

Survival Signal ◽

B Cell Survival ◽

Follicular B Cells ◽

Deficient Mice

AbstractMice lacking all 3 Vav proteins fail to produce significant numbers of recirculating follicular or marginal zone B cells. Those B cells that do mature have shortened lifespans. The constitutive nuclear factor-kappaB (NF-κB) activity of resting naive B cells required Vav function and expression of cellular reticuloendotheliosis (c-Rel). Rel-A was reduced in Vav-deficient B cells. Furthermore, expression of the NF-κB-regulated antiapoptotic genes A1 and Bcl-2 was reduced in mature Vav-deficient B cells. Overexpression of Bcl-2 restored the number of mature follicular B cells in the spleens of Vav-deficient mice. When activated by B-cell receptor (BCR) cross-linking, Vav-deficient B cells failed to activate NF-κB. Vav proteins thus regulate an NF-κB-dependent survival signal in naive B cells and are required for NF-κB function after BCR cross-linking.

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Marginal Zone B Cells Mediate a CD4 T Cell Dependent Extrafollicular Antibody Response Following RBC Transfusion in Mice

Blood ◽

10.1182/blood.2020009376 ◽

2021 ◽

Author(s):

Patricia E Zerra ◽

Seema R Patel ◽

Ryan Philip Jajosky ◽

Connie M Arthur ◽

James W McCoy ◽

...

Keyword(s):

T Cell ◽

B Cells ◽

Antibody Response ◽

B Cell ◽

T Cell Activation ◽

Antibody Formation ◽

Marginal Zone ◽

Cd4 T Cell ◽

Marginal Zone B Cells ◽

Rbc Transfusion

Red blood cell (RBC) transfusions can result in alloimmunization toward RBC alloantigens that can increase the probability of complications following subsequent transfusion. An improved understanding of the immune mechanisms that underlie RBC alloimmunization is critical if future strategies capable of preventing or even reducing this process are to be realized. Using the HOD (hen egg lysozyme and ovalbumin fused to human Duffy) model system, we aimed to identify initiating immune factors that may govern early anti-HOD alloantibody formation. Our findings demonstrate that HOD RBCs continuously localize to the marginal sinus following transfusion, where they co-localize with marginal zone (MZ) B cells. Depletion of MZ B cells inhibited IgM and IgG anti-HOD antibody formation, while CD4 T cell depletion only prevented IgG anti-HOD antibody development. HOD-specific CD4 T cells displayed similar proliferation and activation following transfusion of HOD RBCs into wild type or MZ B cell deficient recipients, suggesting that IgG formation is not dependent on MZ B cell-mediated CD4 T cell activation. Moreover, depletion of follicular B cells failed to substantially impact the anti-HOD antibody response and no increase in antigen specific germinal center B cells was detected following HOD RBC transfusion, suggesting that antibody formation is not dependent on the splenic follicle. Despite this, anti-HOD antibodies persisted for several months following HOD RBC transfusion. Overall, these data suggest MZ B cells can initiate and then contribute to RBC alloantibody formation, highlighting a unique immune pathway that can be engaged following RBC transfusion.

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