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1.  B Cells and Antibodies Are Required for Resistance to the Parasitic Gastrointestinal Nematode Trichuris muris 
Infection and Immunity  2001;69(6):3860-3868.
Previous studies using cell transfers and antibody receptor knockout mice have shown that B cells and antibodies are not essential components of the expulsion mechanism in Trichuris muris infections. Serum transfer experiments have given mixed results regarding the importance of antibodies in this infection model, and the role of B cells in initiating or maintaining T-cell responses has not been addressed. We used B-cell-deficient μMT mice to determine if B cells play a role in anti-T. muris immune responses. In contrast to wild-type C57BL/6 mice, μMT mice were susceptible to infection. Antigen-restimulated mesenteric lymph node cells from infected μMT mice produced only naive levels of Th2-associated cytokines but had increased levels of gamma interferon. However, these mice appeared capable of mounting a Th2-dependent mucosal mastocytosis, though this was significantly delayed compared to that seen in wild-type mice. Resistance to T. muris was restored following reconstitution with naive C57BL/6 splenic B cells, as was in vitro Th2 cytokine production in response to parasite antigen. Treatment of μMT mice with anti-interleukin-12 monoclonal antibody during the first 2 weeks of infection also restored immunity, suggesting that μMT mice can be manipulated to expel worms at the time of T-cell priming. Additionally, treatment of μMT mice with parasite-specific immunoglobulin G1 purified from the serum of resistant NIH mice prevented worm establishment, suggesting an important role for antibodies. Our results as a whole describe the first detailed report of a critical role for B cells in resistance to an intestinal nematode.
PMCID: PMC98409  PMID: 11349052
2.  B Cells Regulate Neutrophilia during Mycobacterium tuberculosis Infection and BCG Vaccination by Modulating the Interleukin-17 Response 
PLoS Pathogens  2013;9(7):e1003472.
We have previously demonstrated that B cells can shape the immune response to Mycobacterium tuberculosis, including the level of neutrophil infiltration and granulomatous inflammation at the site of infection. The present study examined the mechanisms by which B cells regulate the host neutrophilic response upon exposure to mycobacteria and how neutrophilia may influence vaccine efficacy. To address these questions, a murine aerosol infection tuberculosis (TB) model and an intradermal (ID) ear BCG immunization mouse model, involving both the μMT strain and B cell-depleted C57BL/6 mice, were used. IL (interleukin)-17 neutralization and neutrophil depletion experiments using these systems provide evidence that B cells can regulate neutrophilia by modulating the IL-17 response during M. tuberculosis infection and BCG immunization. Exuberant neutrophilia at the site of immunization in B cell-deficient mice adversely affects dendritic cell (DC) migration to the draining lymph nodes and attenuates the development of the vaccine-induced Th1 response. The results suggest that B cells are required for the development of optimal protective anti-TB immunity upon BCG vaccination by regulating the IL-17/neutrophilic response. Administration of sera derived from M. tuberculosis-infected C57BL/6 wild-type mice reverses the lung neutrophilia phenotype in tuberculous μMT mice. Together, these observations provide insight into the mechanisms by which B cells and humoral immunity modulate vaccine-induced Th1 response and regulate neutrophila during M. tuberculosis infection and BCG immunization.
Author Summary
Mycobacterium tuberculosis poses a serious threat to public health globally. It has been well established that T cells are critical in protection against M. tuberculosis. The role of B cells and humoral immunity in the process is less well understood. We previously showed that B cells and humoral immunity regulate the immune response against M. tuberculosis. The present study examined the mechanisms by which B cells regulate the host neutrophilic response upon exposure to mycobacteria and how neutrophilia may modulate the development of vaccine-induced protective immunity. The data reveal that B cells can regulate neutrophilia during M. tuberculosis infection and BCG vaccination by modulating the IL-17 response. Vaccination studies show that excess neutrophilia adversely affects the development of BCG-elicited Th1 response. These observations suggest that B cells can optimize the development of protective immunity upon BCG vaccination by regulating the IL-17/neutrophilic response. Understanding the mechanisms by which B cells and humoral immunity modulate the immune response during M. tuberculosis infection and BCG immunization, particularly those that regulate IL-17 levels and neutrophilia, may lead to the development of novel strategies for the control of the tubercle bacillus, including efficacious vaccines.
PMCID: PMC3708864  PMID: 23853593
3.  Synergistic Roles of Antibody and Interferon in Noncytolytic Clearance of Sindbis Virus from Different Regions of the Central Nervous System▿  
Journal of Virology  2007;81(11):5628-5636.
Sindbis virus (SINV) is an alphavirus that causes infection of neurons and encephalomyelitis in adult immunocompetent mice. Recovery can occur without apparent neurological damage. To better define the factors facilitating noncytolytic clearance of SINV in different regions of the central nervous system (CNS) and the roles of innate and adaptive immune responses at different times during infection, we have characterized SINV infection and clearance in the brain, brain stem, and spinal cords of severe combined immunodeficiency (SCID) and C57BL/6 (wild-type [WT]) mice and mice deficient in beta interferon (IFN-β) (BKO), antibody (μMT), IFN-γ (GKO), IFN-γ receptor (GRKO), and both antibody and IFN-γ (μMT/GKO). WT mice cleared infectious virus by day 8, while SCID mice had persistent virus replication at all sites. For 3 days after infection, BKO mice had higher titers at all sites than WT mice, despite similar IFN-α production, but cleared virus similarly. GKO and GRKO mice cleared infectious virus from all sites by days 8 to 10 and, like WT mice, displayed transient reactivation at 12 to 22 days. μMT mice did not clear virus from the brain, and clearance from the brain stem and lumbar spinal cord was delayed, followed by reactivation. Eighty-one days after infection, μMT/GKO mice had not cleared virus from any site, but titers were lower than for SCID mice. These studies show that IFN-β is independently important for early control of CNS virus replication, that antiviral antibody is critical for clearance from the brain, and that both antibody and IFN-γ contribute to prevention of reactivation after initial clearance.
PMCID: PMC1900320  PMID: 17376910
4.  Importance of T Cells, Gamma Interferon, and Tumor Necrosis Factor in Immune Control of the Rapid Grower Mycobacterium abscessus in C57BL/6 Mice▿  
Infection and Immunity  2007;75(12):5898-5907.
Mycobacterium abscessus is an emerging rapidly growing mycobacterium that causes tuberculous-like lesions in humans. We studied the immune control of this organism in C57BL/6 mice challenged intravenously with 107 CFU. Bacteria were eliminated from both the spleen and the liver within 90 days, and liver histology showed organized granulomatous lesions. A T- and B-cell requirement was investigated by challenging Rag2−/−, Cd3ɛ−/−, and μMT−/− mice. Rag2−/− and Cd3ɛ−/− mice were significantly impaired in the ability to clear M. abscessus from the liver and spleen, and μMT−/− mice were significantly impaired in the ability to clear M. abscessus from the liver, suggesting that infection control was primarily T cell dependent in the spleen and both T and B cell dependent in the liver. The liver granulomatous response was similar to that of wild-type controls in μMT−/− mice but completely absent in Cd3ɛ−/− and Rag2−/− mice. We studied the involvement of gamma interferon (IFN-γ) and tumor necrosis factor (TNF) by challenging C57BL/6 mice deficient in the IFN-γ receptor (Ifngr1−/−) and in TNF (Tnf−/−). Ifngr1−/− mice were significantly impaired in M. abscessus control both in the spleen and in the liver, and granulomas were profoundly altered. The effect was even more substantial in Tnf−/− mice; they failed to control M. abscessus infection in the liver and died within 20 to 25 days after infection with many hepatic inflammatory foci and major lesions of ischemic necrosis in the liver and kidney. These features were not observed with the closely related species M. chelonae. T-cell immunity, IFN-γ, and TNF are central factors for the control of M. abscessus in C57BL/6 mice, as they are for the control of pathogenic slowly growing mycobacteria.
PMCID: PMC2168332  PMID: 17875636
5.  Replication-defective virus vaccine-induced protection of mice from genital herpes simplex virus 2 requires CD4 T cells 
Virology  2008;376(1):205-210.
Replication-defective herpes simplex virus 2 (HSV-2), used as an immunization strategy, protects against HSV-2 challenge in animal models. The roles of replication-defective virus-induced T cell subsets in control of HSV-2 infection have not been established. Mice lacking B cells (μMT) were immunized, depleted of CD4 or CD8 T cells, and then challenged intravaginally with HSV-2 to elucidate T cell subset contributions in the absence of virus-specific antibody. Immunized, CD4-depleted μMT mice developed severe infection of the genital tract and nervous system. In contrast, depletion of CD8 T cells from μMT mice did not attenuate protection. Immunized wild-type mice depleted of CD4 T cells also developed more severe HSV-2 infection than mice from which CD8 T cells were depleted. Thus, immunization with replication-defective virus induces T cell responses that effectively control HSV-2 infection in the absence of HSV-immune antibody, and CD4 T cells play the predominant role in this protective effect.
PMCID: PMC2431983  PMID: 18410949
HSV-2; vaginal; B cell-deficient; CD4 T cells; immunization
6.  Evidence for an Underlying CD4 Helper and CD8 T-Cell Defect in B-Cell-Deficient Mice: Failure To Clear Persistent Virus Infection after Adoptive Immunotherapy with Virus-Specific Memory Cells from μMT/μMT Mice 
Journal of Virology  1998;72(11):9208-9216.
Adoptive transfer of virus-specific memory lymphocytes can be used to identify factors and mechanisms involved in the clearance of persistent virus infections. To analyze the role of B cells in clearing persistent infection with lymphocytic choriomeningitis virus (LCMV), we used B-cell-deficient μMT/μMT (B−/−) mice. B−/− mice controlled an acute LCMV infection with the same kinetics and efficiency as B-cell-competent (B+/+) mice via virus-specific, major histocompatibility complex (MHC) class I-restricted CD8+ cytotoxic T lymphocytes (CTL). CTL from B−/− and B+/+ mice were equivalent in affinity to known LCMV CTL epitopes and had similar CTL precursor frequencies (pCTL). Adoptive transfer of memory cells from B+/+ mice led to virus clearance from persistently infected B+/+ recipients even after in vitro depletion of B cells, indicating that B cells or immunoglobulins are not required in the transfer population. In contrast, transfer of memory splenocytes from B−/− mice failed to clear virus. Control of virus was restored neither by transferring higher numbers of pCTL nor by supplementing B−/− memory splenocytes with LCMV-immune B cells or immune sera. Instead, B−/− mice were found to have a profound CD4 helper defect. Furthermore, compared to cultured splenocytes from B+/+ mice, those from B−/− mice secreted less gamma interferon (IFN-γ) and interleukin 2, with differences most pronounced for CD8 T cells. While emphasizing the importance of CD4 T-cell help and IFN-γ in the control of persistent infections, the CD4 T-helper and CD8 T-cell defects in B−/− mice suggest that B cells contribute to the induction of competent T effector cells.
PMCID: PMC110340  PMID: 9765468
7.  B-Cell-Deficient Mice Show an Exacerbated Inflammatory Response in a Model of Chlamydophila abortus Infection  
Infection and Immunity  2002;70(12):6911-6918.
The resolution of Chlamydophila abortus (Chlamydia psittaci serotype 1) infection is dependent on gamma interferon and CD8+ T cells, and classically, B cells have been considered to play a minimal role in host defense. The role of B cells in the immune response was studied by using a model of infection in mice with genetically modified immunoglobulin M transmembrane domains (μMT). In the absence of B cells, infection with C. abortus leads to an acute severe fatal disease that involves a disseminated intravascular coagulation syndrome. μMT mice displayed an increased level of proinflammatory cytokines in serum, and an increased number of neutrophils was observed in the lesions. The possible deleterious role of neutrophils in the pathogenesis of disease in μMT mice was determined by depletion of the neutrophils with the monoclonal antibody RB6-8C5. This led to an enhancement of the bacterial burden and early mortality in both μMT and wild-type mice, while necrotic lesions remained. Analysis of the presence of immunoregulatory cytokines showed significantly lower levels of transforming growth factor β in the sera of μMT mice. However, mice lacking mature B cells were able to establish a specific immune response that protected them from a secondary challenge. Taken together, these data suggest an immunomodulatory role for B cells in the early events of C. abortus primary infection that can protect mice against an exaggerated inflammatory response.
PMCID: PMC133017  PMID: 12438369
8.  Involvement of Gonadal Steroids and Gamma Interferon in Sex Differences in Response to Blood-Stage Malaria Infection†  
Infection and Immunity  2006;74(6):3190-3203.
To examine the hormonal and immunological mechanisms that mediate sex differences in susceptibility to malaria infection, intact and gonadectomized (gdx) C57BL/6 mice were inoculated with Plasmodium chabaudi AS-infected erythrocytes, and the responses to infection were monitored. In addition to reduced mortality, intact females recovered from infection-induced weigh loss and anemia faster than intact males. Expression microarrays and real-time reverse transcription-PCR revealed that gonadally intact females exhibited higher expression of interleukin-10 (IL-10), IL-15Rα, IL-12Rβ, Gadd45γ, gamma interferon (IFN-γ), CCL3, CXCL10, CCR5, and several IFN-inducible genes in white blood cells and produced more IFN-γ than did intact males and gdx females, with these differences being most pronounced during peak parasitemia. Intact females also had higher anti-P. chabaudi immunoglobulin G (IgG) and IgG1 responses than either intact males or gdx females. To further examine the effector mechanisms mediating sex differences in response to P. chabaudi infection, responses to infection were compared among male and female wild-type (WT), T-cell-deficient (TCRβδ−/−), B-cell-deficient (μMT), combined T- and B-cell-deficient (RAG1), and IFN-γ knockout (IFN-γ−/−) mice. Males were 3.5 times more likely to die from malaria infection than females, with these differences being most pronounced among TCRβδ−/−, μMT, and RAG1 mice. Male mice also exhibited more severe weight loss, anemia, and hypothermia, and higher peak parasitemia than females during infection, with WT, RAG1, TCRβδ−/−, and μMT mice exhibiting the most pronounced sexual dimorphism. The absence of IFN-γ reduced the sex difference in mortality and was more detrimental to females than males. These data suggest that differential transcription and translation of IFN-γ, that is influenced by estrogens, may mediate sex differences in response to malaria.
PMCID: PMC1479253  PMID: 16714546
9.  B Cells and Antibody Play Critical Roles in the Immediate Defense of Disseminated Infection by West Nile Encephalitis Virus 
Journal of Virology  2003;77(4):2578-2586.
West Nile virus (WNV) causes severe central nervous system (CNS) infection primarily in humans who are immunocompromised or elderly. In this study, we addressed the mechanism by which the immune system limits dissemination of WNV infection by infecting wild-type and immunodeficient inbred C57BL/6J mice with a low-passage WNV isolate from the recent epidemic in New York state. Wild-type mice replicated virus extraneuronally in the draining lymph nodes and spleen during the first 4 days of infection. Subsequently, virus spread to the spinal cord and the brain at virtually the same time. Congenic mice that were genetically deficient in B cells and antibody (μMT mice) developed increased CNS viral burdens and were vulnerable to lethal infection at low doses of virus. Notably, a ∼500-fold difference in serum viral load was detected in μMT mice as early as 4 days after infection, a point in the infection when low levels of neutralizing immunoglobulin M antibody were detected in wild-type mice. Passive transfer of heat-inactivated serum from infected and immune wild-type mice protected μMT mice against morbidity and mortality. We conclude that antibodies and B cells play a critical early role in the defense against disseminated infection by WNV.
PMCID: PMC141119  PMID: 12551996
10.  B cells are important as antigen presenting cells for induction of MHC-restricted arthritis in transgenic mice. 
Molecular immunology  2007;44(11):2988-2996.
Rheumatoid arthritis and its animal model, collagen-induced arthritis, are known as a T and B cell dependent disease. To analyze the role of B cells in arthritis, we generated B cell deficient (muMT) mice carrying HLA-DQ8 as transgene, Aβo.DQ8.μmt mice. HLA-DQ8 transgenic mice (Aβo.DQ8) are susceptible to collagen induced arthritis, an animal model for inflammatory arthritis. Deletion of IgM gene led to the absence of B cells while T cells were comparable to Aβo.DQ8 mice. Arthritis and autoantibodies was completely abrogated in B cell deficient DQ8 mice. T cell response and proinflammatory cytokine production in response to type II collagen and its derived peptides in vitro was significantly decreased despite an increased number of Mac-1 positive cells in DQ8.μmt mice compared to DQ8 mice suggesting B cells could be important for antigen presentation as well. In vitro substitution of B cells from wild type mice restored the response in DQ8.umt mice. B cells could also present CII-derived peptides to antigen-specific DQ8-restricted hybridomas reinforcing the role of B cells in presentation of antigens to T cells. The data suggest that B cells can be involved in pathogenesis of arthritis by producing autoantibodies and antigen presentation.
PMCID: PMC1995074  PMID: 17303243
B cells; transgenic/knockout mice; antigen presentation; rheumatoid arthritis; autoantibodies
11.  Polarized Helper-T-Cell Responses against Leishmania major in the Absence of B Cells 
Infection and Immunity  1999;67(1):266-270.
B-cell-to-T-cell signaling can shape helper T (Th) cell responses. During infection with Leishmania major, Th response is critical in determining the outcome of disease. Resistance depends on the generation of a protective Th1 response, while susceptibility is mediated by the generation of a Th2 response. In this study, we determined whether B cells are required for the development of polarized Th1 and Th2 responses during infection with L. major. Mice lacking B cells due to disruption of the immunoglobulin M locus (μMT) were infected with L. major, and disease progression and Th cell development were assessed. On the genetically resistant C57BL background, both wild-type and μMT mice controlled the infection and mounted a Th1 response. On the genetically susceptible BALB/c background, both wild-type and μMT mice were susceptible to infection and generated Th2 responses. Thus, during L. major infection, neither direct antigen presentation or costimulation by B cells nor antibody-mediated effector functions are essential for the development of polarized Th responses.
PMCID: PMC96306  PMID: 9864225
12.  SLAT/Def6 plays a critical role in the pathogenic process of experimental autoimmune uveitis (EAU) 
Molecular Vision  2012;18:1858-1864.
SWAP 70-like adaptor of T cells (SLAT; aka Def6) is a recently discovered guanine nucleotide exchange factor for Rho guanosine triphosphate (GTP)ases that has been previously shown to play a role in cluster of differentiation(CD)4+ T cell activation, T-helper (Th)1/Th2/Th17 differentiation and development of experimental autoimmune encephalomyelitis. Here, we investigated the role of SLAT/Def6 in the development of experimental autoimmune uveitis (EAU), an animal model for several uveitic conditions in humans.
SLAT/Def6 deficient (“KO”) mice and C57BL/6 controls were immunized with interphotoreceptor retinoid-binding protein (IRBP), along with pertussis toxin. The development of ocular inflammation was determined by both fundoscopy and histological examination. Lymphoid cells from draining lymph nodes were cultured with IRBP to measure lymphocyte proliferation and release of cytokines. Purified dendritic cells were tested for their capacity to present antigen to responding lymphocytes. In addition, the lymphoid cells were tested for the expression of forkhead box P3 (FoxP3), using conventional methods, and the activity of T-regulatory cells was determined by their capacity to inhibit in vitro proliferative responses. Serum anti -IRBP antibody levels were measured by enzyme-linked immonosorbant assay (ELISA). quantitative polymerase chain reaction (qPCR) was used to determine the transcript levels of cytokines in inflamed eyes.
SLAT/Def6 KO mice had significantly reduced EAU compared to controls. Cells isolated from draining lymph nodes of SLAT/Def6 KO mice exhibited impaired proliferation and production of Th1 and Th17 signature cytokines (interferon [IFN]-γ and interleukin [IL]-17, respectively) when compared with cells isolated from control mice. qPCR of inflamed eyes detected similar levels of IFN-γ transcript in control and SLAT/Def6 KO mice, whereas the IL-17 transcript levels in eyes of the SLAT/Def6 KO mice were lower than in eyes of the controls. The SLAT/Def6 KO mice resembled their wild type (WT) controls, however, in the levels of their serum antibody against IRBP, the antigen presenting capacity of their dendritic cells, the proportion of cells expressing Foxp3 and the immunosuppressive activity of their T-regulatory cells.
SLAT/Def6 KO mice exhibit reduced capacity to develop ocular inflammation and cellular activity when immunized with IRBP. Our study provides new data showing that SLAT/Def6 plays a major role in the T cell-mediated autoimmune processes that bring about the inflammatory eye disease, EAU.
PMCID: PMC3398495  PMID: 22815639
13.  B Cell Subsets Contribute to Both Renal Injury and Renal Protection after Ischemia/Reperfusion 
Ischemia/reperfusion (I/R) triggers a robust inflammatory response within the kidney. Numerous components of the immune system contribute to the resultant renal injury including the complement system. We sought to identify whether natural antibodies bind to the post-ischemic kidney and contribute to complement activation after I/R. We depleted peritoneal B cells in mice by hypotonic shock. Depletion of the peritoneal B cells prevented the deposition of IgM within the glomeruli after renal I/R, and attenuated renal injury after I/R. We found that glomerular IgM activates the classical pathway of complement but does not cause substantial deposition of C3 within the kidney. Furthermore, mice deficient in classical pathway proteins were not protected from injury, indicating that glomerular IgM does not cause injury through activation of the classical pathway. We also subjected mice deficient in all mature B cells (μMT mice) to renal I/R and found that they sustained worse renal injury than wild-type controls. Serum IL-10 levels were lower in the μMT mice. Regarded together, these results indicate that natural antibody produced by peritoneal B cells binds within the glomerulus after renal I/R and contributes to functional renal injury. However, non-peritoneal B cells attenuate renal injury after I/R, possibly through the production of IL-10.
PMCID: PMC3133676  PMID: 20810984
14.  Immunoglobulin E and Eosinophil-Dependent Protective Immunity to Larval Onchocerca volvulus in Mice Immunized with Irradiated Larvae  
Infection and Immunity  2004;72(2):810-817.
Mice immunized with irradiated Onchocerca volvulus third-stage larvae developed protective immunity. Eosinophil levels were elevated in the parasite microenvironment at the time of larval killing, and measurements of total serum antibody levels revealed an increase in the immunoglobulin E (IgE) level in immunized mice. The goal of the present study was to identify the role of granulocytes and antibodies in the protective immune response to the larval stages of O. volvulus in mice immunized with irradiated larvae. Immunity did not develop in mice if granulocytes, including both neutrophils and eosinophils, were eliminated, nor did it develop if only eosinophils were eliminated. Moreover, larvae were killed in naïve interleukin-5 transgenic mice, and the killing coincided with an increase in the number of eosinophils and the eosinophil peroxidase (EPO) level in the animals. To determine if EPO was required for protective immunity, mice that were genetically deficient in EPO were immunized, and there were no differences in the rates of parasite recovery in EPO-deficient mice and wild-type mice. Two mouse strains were used to study B-cell function; μMT mice lacked all mature B cells, and Xid mice had deficiencies in the B-1 cell population. Immunity did not develop in the μMT mice but did develop in the Xid mice. Finally, protective immunity was abolished in mice treated to eliminate IgE from the blood. We therefore concluded that IgE and eosinophils are required for adaptive protective immunity to larval O. volvulus in mice.
PMCID: PMC321619  PMID: 14742524
15.  Antibody Prophylaxis and Therapy against West Nile Virus Infection in Wild-Type and Immunodeficient Mice 
Journal of Virology  2003;77(24):12941-12949.
West Nile virus (WNV) is a mosquito-borne Flavivirus that causes encephalitis in a subset of susceptible humans. Current treatment for WNV infections is supportive, and no specific therapy or vaccine is available. In this study, we directly tested the prophylactic and therapeutic efficacy of polyclonal antibodies against WNV. Passive administration of human gamma globulin or mouse serum prior to WNV infection protected congenic wild-type, B-cell-deficient (μMT), and T- and B-cell-deficient (RAG1) C57BL/6J mice. Notably, no increased mortality due to immune enhancement was observed. Although immune antibody completely prevented morbidity and mortality in wild-type mice, its effect was not durable in immunocompromised mice: many μMT and RAG1 mice eventually succumbed to infection. Thus, antibody by itself did not completely eliminate viral reservoirs in host tissues, consistent with an intact cellular immune response being required for viral clearance. In therapeutic postexposure studies, human gamma globulin partially protected against WNV-induced mortality. In μMT mice, therapy had to be initiated within 2 days of infection to gain a survival benefit, whereas in the wild-type mice, therapy even 5 days after infection reduced mortality. This time point is significant because between days 4 and 5, WNV was detected in the brains of infected mice. Thus, passive transfer of immune antibody improves clinical outcome even after WNV has disseminated into the central nervous system.
PMCID: PMC296058  PMID: 14645550
16.  Necessary and Sufficient Role for T Helper Cells To Prevent Fungal Dissemination in Allergic Lung Disease ▿ †  
Infection and Immunity  2011;79(11):4459-4471.
Mucosal immune responses to fungal infection range from T helper type 2 (Th2) cell-directed allergic inflammation to Th1-predominant neutrophilic inflammation, but the mechanisms directing these divergent mucosal immune outcomes and the role of T cells in host defense against mucosal fungal infections are not known. Here we examined the mouse mucosal immune responses to 12 filamentous environmental fungal species over a broad range of exposure doses and determined the requirement of T cells for host defense. For all tested fungi, low-grade conidium exposures induced Th2- and eosinophil-predominant allergic lung disease, whereas higher exposures led to rapid conversion to neutrophil- and Th1 cell-predominant inflammation, a phenomenon we term immune phenotype switching. All fungal exposure doses were further linked to the secretion of interleukin-17A (IL-17A). Fungal infections with Curvularia lunata and Aspergillus fumigatus were typically confined to the airway during allergic inflammation but became locally invasive and disseminated to the brain at higher conidium challenge doses, in association with predominant Th1 responses. Fungal dissemination occurred at relatively low challenge doses with the conidia of Aspergillus fumigatus administered to recombinase activating gene 1 (Rag-1)-deficient mice, which lack B and T cells, but B cell-deficient μMT mice and T helper cell-reconstituted Rag-1-deficient mice were comparable to wild-type mice in preventing fungal dissemination. Our findings demonstrate that Th2 cell-predominant allergic responses followed by immune phenotype switching and fungal dissemination are highly predictable outcomes with progressive fungal infectious burdens and that T helper cell responses are protective against lethal fungal dissemination.
PMCID: PMC3257943  PMID: 21875960
17.  Regulatory B-cells limit CNS inflammation and neurologic deficits in murine experimental stroke 
Evaluation of infarct volumes and infiltrating immune cell populations in mice after middle cerebral artery occlusion (MCAO) strongly implicates a mixture of both pathogenic and regulatory immune cell subsets in stroke pathogenesis and recovery. Our goal was to evaluate the contribution of B-cells to the development of MCAO by comparing infarct volumes and functional outcomes in WT versus B-cell deficient μMT−/− mice. The results clearly demonstrate larger infarct volumes, higher mortality, more severe functional deficits and increased numbers of activated T-cells, macrophages, microglial cells and neutrophils in the affected brain hemisphere of MCAO-treated μMT−/− vs. WT mice. These MCAO-induced changes were completely prevented in B-cell restored μMT−/− mice after transfer of highly purified WT GFP+ B-cells that were detected in the periphery, but not the CNS. In contrast, transfer of B-cells from IL-10−/− mice had no effect on infarct volume when transferred into μMT−/− mice. These findings strongly support a previously unrecognized activity of IL-10-secreting WT B-cells to limit infarct volume, mortality rate, recruitment of inflammatory cells and functional neurological deficits 48h after MCAO. Our novel observations are the first to implicate IL-10-secreting B-cells as a major regulatory cell type in stroke and suggest that enhancement of regulatory B-cells might have application as a novel therapy for this devastating neurologic condition.
PMCID: PMC3111929  PMID: 21653859
18.  Clearance of HIV Type 1 Envelope Recombinant Sendai Virus Depends on CD4+ T Cells and Interferon-γ But Not B Cells, CD8+ T Cells, or Perforin 
T cell-mediated viral clearance is classically attributed to the CD8+ T cell subset, but CD4+ T cells can sometimes assume this role. One such instance was illustrated by the immunization of C57BL/6 mice with HIV-1 envelope, followed by challenge with a recombinant Sendai virus (rSeV-env) carrying a gene for secreted HIV-1 envelope protein. Vaccinated mice that lacked both B cells (μMT) and CD8+ T cells controlled virus, but control was lost when CD4+ T cells were depleted. To explain this activity, we questioned whether CD4+ T cells might utilize perforin for killing of MHC class II-positive targets. We also asked if the process might depend on IFN-γ, which can upregulate MHC expression and enhance T cell recruitment to sites of virus challenge. To address these possibilities, we vaccinated perforin-KO mice with HIV-1 envelope and challenged them with rSeV-env. We found that perforin was not required for (1) CD4+ T cell homing to the site of virus challenge, (2) expression of Th1 and Th2 cytokines (including IFN-γ), or (3) virus clearance. To determine if IFN-γ was required for protection, we repeated experiments in IFN-γ-KO animals. In this case, significant protection was lost, although the CD4+ T cells trafficked readily to the site of infection. In fact, local CD4+ T cell numbers in vaccinated IFN-γ- KO mice exceeded those in wild type animals. In both cases, cells were αß TCR+, NK-1.1–, and CD44+, typifying an activated CD4+ T cell subset. Taken together, our results showed that HIV-1 envelope recombinant virus clearance was dependent on CD4+ T cells and IFN-γ, but occurred in the absence of B cells, CD8+ T cells, or perforin.
PMCID: PMC2932556  PMID: 20623995
19.  B-Cell Deficiency Suppresses Vaccine-Induced Protection against Murine Filariasis but Does Not Increase the Recovery Rate for Primary Infection 
Infection and Immunity  2001;69(11):7067-7073.
To establish the role of B cells and antibodies in destroying filariae, mice lacking mature B cells and therefore unable to produce antibodies were used. Litomosoides sigmodontis offers a good opportunity for this study because it is the only filarial species that completes its life cycle in mice. Its development was compared in B-cell-deficient mice (BALB/c μMT mice) and wild-type BALB/c mice in two different in vivo situations, vaccination with irradiated larvae and primary infection. In all cases, mice were challenged with subcutaneous inoculation of 40 infective larvae. Vaccine-induced protection was suppressed in B-cell-deficient mice. In these mice, eosinophils infiltrated the subcutaneous tissue normally during immunization; however, their morphological state did not change following challenge inoculation, whereas in wild-type mice the percentage of degranulated eosinophils was markedly increased. From this, it may be deduced that the eosinophil–antibody–B-cell complex is the effector mechanism of protection in vaccinated mice and that its action is fast and takes place in the subcutaneous tissue. In primary infection, the filarial survival and growth was not modified by the absence of B cells. However, no female worm had uterine microfilariae, nor did any mice develop a patent infection. In these mice, concentrations of type 1 (gamma interferon) and type 2 (interleukin-4 [IL-4], IL-5 and IL-10) cytokines in serum were lower and pleural neutrophils were more numerous. The effects of the μMT mutation therefore differ from those in B1-cell-deficient mice described on the same BALB/c background, which reveal a higher filarial recovery rate and microfilaremia. This outlines B2-cell-dependent mechanisms as favorable to the late maturation of L. sigmodontis.
PMCID: PMC100087  PMID: 11598082
20.  T cells that are naturally tolerant to cartilage-derived type II collagen are involved in the development of collagen-induced arthritis 
Arthritis Research  2000;2(4):315-326.
The immunodominant T-cell epitope that is involved in collagen-induced arthritis (CIA) is the glycosylated type II collagen (CII) peptide 256-270. In CII transgenic mice, which express the immunodominant CII 256-270 epitope in cartilage, the CII-specific T cells are characterized by a partially tolerant state with low proliferative activity in vitro, but with maintained effector functions, such as IFN-γ secretion and ability to provide B cell help. These mice were still susceptible to CIA. The response was mainly directed to the glycosylated form of the CII 256-270 peptide, rather than to the nonglycosylated peptide. Tolerance induction was rapid; transferred T cells encountered CII within a few days. CII immunization several weeks after thymectomy of the mice did not change their susceptibility to arthritis or the induction of partial T-cell tolerance, excluding a role for recent thymic emigrants. Thus, partially tolerant CII autoreactive T cells are maintained and are crucial for the development of CIA.
A discussion is ongoing regarding the possible role of cartilage-directed autoimmunity as a part of the pathogenesis of rheumatoid arthritis (RA). One possibility is that the association of RA with shared epitope-expressing DR molecules reflects a role for major histocompatibility complex (MHC) class II molecules as peptide receptors, and that the predilection of the inflammatory attack for the joint indicates a role for cartilage as a source of the antigenic peptides. A direct role for CII in the development of arthritis is apparent in the CIA model, in which a definite role for MHC class II molecules and a role for CII-derived peptides have been demonstrated [1,2,3]. Remarkably, it was found that the identified MHC class II molecule in the CIA model Aq has a structurally similar peptide binding pocket to that of the shared epitope, expressing DR4 molecules [4]. In fact, DR4 (DRB1*0401) and DR1 (DRB1*0101) transgenic mice are susceptible to CIA because of an immune response to a peptide that is almost identical to that which is involved in Aq-expressing mice [5,6]. They are both derived from position 260-273 of the CII molecule; the peptide binds to the Aqmolecule with isoleucine 260 in the P1 pocket, but with phenylalanine 263 in the P1 pocket of the DR4 and DR1 molecules.
Although these findings do not prove a role for CII in RA, they show that such recognition is possible and that there are structural similarities when comparing mouse with human. However, there are also strong arguments against such a possibility. First, arthritis can evolve without evidence for a cartilage-specific autoimmunity, as seen with various adjuvant-induced arthritis models [7,8] and in several observations using transgenic animals with aberrant immunity to ubiquitously expressed proteins [9,10,11]. Moreover, the MHC association in the adjuvant arthritis models correlates with severity of the disease rather than susceptibility [7,8], as has also been observed in RA [12]. Second, it has not been possible to identify the CII-reactive T cells from RA joints, or to achieve a strong and significant CII proliferative response from T cells derived from RA joints. Most recently these negative observations were corroborated using DR4+CII peptide tetramer reagents [13]. On the other hand, it has also been difficult to isolate autoreactive CII-specific T cells from CIA, and it can be anticipated that, even in the CIA model, T cells that are specific for CII will be hard to find in the joints [4].
We believe that the explanations for these observations in both experimental animals and humans are related to tolerance. The CIA model in the mouse is usually induced with heterologous CII, and is critically dependent on an immune response to the glycosylated CII peptide 256-270, which is bound to the MHC class II Aq molecule. In CII transgenic mice, expressing the heterologous (rat) form of the immunodominant CII 256-270 epitope in cartilage, we observed partial T-cell tolerance. This tolerance is characterized by a low proliferative activity, but with maintained effector functions such as production of IFN-γ and the ability to give help to B cells to produce anti-CII IgG antibodies [14]. Interestingly, these mice were susceptible to arthritis. However, a possibility was that T cells that had newly emerged from the thymus and that were not yet tolerized when the mice were immunized with CII led to the induction of arthritis. We have now addressed this possibility and found that induction of tolerance occurs within a few days, and that mice lacking recent thymic emigrants (ie thymectomized mice) display partially tolerant T cells and susceptibility to arthritis to the same extent as nonthymectomized mice. In addition we found that T cells that are reactive with the nonmodified peptides are relatively more affected by tolerance than T cells that are reactive with the more immunodominant glycosylated variants.
To investigate the possibility that T cells that are naturally tolerant to the cartilage protein CII are involved in the development of arthritis, and to exclude a role for nontolerized recent thymic T-cell emigrants in the development of arthritis.
Materials and methods:
A mutated mouse CII, expressing glutamic acid instead of aspartic acid at position 266, was expressed in a transgenic mouse called MMC (mutated mouse collagen) that has been described earlier [14]. The mice were thymectomized, or sham-operated, at 7 weeks of age and allowed to recover for 4 weeks before being immunized with rat CII in complete Freund's adjuvant. Arthritis development was recorded and sera analyzed for anti-CII IgG, IgG1 and IgG2a levels. To assay T-cell effector functions, other MMC and control mice were immunized in the hind footpads with rat CII in complete Freund's adjuvant, and the draining popliteal lymph nodes were taken 10 days later. The lymph node cells (LNCs) were used for proliferation assay, IFN-γ enzyme-linked immunosorbent assay (ELISA) and B-cell enzyme-linked immunospot (ELISPOT). For the proliferation assay, 106 cells were put in triplicate cultures in microtitre wells together with antigen and incubated for 72h before thymidine-labelling and harvesting 15-18h later. For IFN-γ ELISA analysis, supernatant from the proliferation plates was removed before harvesting and used in an ELISA to quantify the amount of IFN-γ produced [15]. B-cell ELISPOT was performed to enumerate the number of cells producing anti-CII IgG [16].
T-cell lines that were reactive towards rat CII were established by immunization with rat CII. An established T-cell line that was reactive with CII and specific for the CII 256-270 peptide was restimulated with freshly collected, irradiated, syngenic spleen cells and rat CII for 3 days followed by 2 weeks of IL-2 containing medium. Immediately before transfer, the cells were labelled with the cytoplasmic dye 5 (and 6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) [17]. Labelled cells (107) were injected intravenously into transgenic MMC mice and nontransgenic littermates. The mice were killed 4 days after cell transfer, and the concentration of CFSE-labelled cells was determined by flow cytometry.
Results and discussion:
To investigate whether and how quickly CII-reactive T cells will encounter CII in vivo, an established T-cell line that is reactive towards rat CII was labelled with the cytoplasmic dye CFSE and transferred into MMC-QD and control mice. Four days later the mice were killed, and it was found that MMC-transgenic mice had dramatically fewer CFSE-labelled cells in the spleen than did nontransgenic littermates (0.11% compared with 0.57%). Similarly, reduced numbers of CFSE-positive cells were observed in blood. This indicates that the T cells encountered the mutated CII that was present in the cartilage of MMC mice, but not in the nontransgenic littermates. Presumably, CII from cartilage is spread by antigen-presenting cells (APCs) to peripheral lymphoid organs. This observation also suggests that newly exported T cells from the thymus will be tolerized to CII in the periphery within less than 4 days.
To further investigate whether the MMC mice harbours naïve or tolerized T cells, the mice were immunized with CII at different time points after thymectomy that were well in excess of the times required for their encounter with CII. After 10 days, the response was analyzed in vitro towards both the nonglycosylated and the glycosylated CII 256-270 peptides as well as towards purified protein derivative. The galactosylated form of the peptide (Fig. 1) was used because this is the most immunodominant modification [18]. In contrast to control mice, LNCs from transgenic mice did not proliferate significantly towards the nonglycosylated peptide, indicating that these cells have been specifically tolerized, which is in accordance with earlier observations [14]. A reduced, but still significant proliferation was also observed toward the immunodominant glycosylated CII peptide. Most important, however, was that the proliferative response in the MMC mice did not decrease after thymectomy. Similarly, a significant IFN-γ production towards the glycosylated CII peptide was observed in the MMC mice. The response was somewhat reduced compared with that observed in nontransgenic littermates, and this was especially true for the response toward the nonglycosylated peptide. Again, no decrease in the MMC response by thymectomy was observed. Taken together, the T-cell response in transgenic mice was reduced in comparison with that in the nontransgenic littermates. Furthermore, the response in transgenic animals did not decrease by thymectomy (4 or 8 weeks before immunization), showing that autoreactive T cells are still maintained (and partially tolerized) with significant effector functions at least up to 8 weeks after thymectomy, excluding a exclusive role for recent thymic emigrants in the autoimmune response towards CII. To investigate whether thymectomized mice, lacking recent CII-specific thymic emigrants, were susceptible to CIA, mice were immunized with CII 4 weeks after thymectomy and were observed for arthritis development during the following 10 weeks. Clearly, the thymectomized MMC mice were susceptible to arthritis (five out of 18 developed arthritis; Fig. 2), and no significant differences in susceptibility between thymectomized and sham-operated mice, or between males and females, were seen. In accordance with earlier results [14], MMC transgenic mice had a significantly reduced susceptibility to arthritis as compared with the nontransgenic littermates (P < 0.0001 for arthritic scores, disease onset and incidence). All mice were bled at 35 days after immunization, and the total levels of anti-CII IgG were determined. Transgenic mice developed levels of anti-CII IgG significantly above background, but the antibody titres were lower than in nontransgenic littermates (P < 0.0001). No effect on the antibody levels by thymectomy was observed, nor did thethymectomy affect the distribution of IgG1 versus IgG2a titres,indicating that the observed tolerance is not associated with a shift from a T-helper-1- to a T-helper-2-like immune response. These findings show that T cells that are specific for a tissue-specific matrix protein, CII, are partially tolerized within a few days after thymus export and that these tolerized cells are maintained after thymectomy. Most important, mice that lack newly exported CII reactive T cells are still susceptible to CIA, suggesting that the partially tolerant T cells are involved in development of arthritis.
In the light of these data it is possible to explain some of the findings in RA. T-cell reactivity to CII has been shown in RA patients, but with a very weak proliferative activity [19,20]. This is fully compatible with observations in mouse and rat CIA when autologous CII, and not heterologous CII, are used for immunization. This is particularly true if the responses are recorded during the chronic phase of disease, in which the antigen-specific T-cell responses seem to be suppressed in both humans and experimental animals. These observations were confirmed in a recent report [21] in which it was shown that CII-reactive T-cell activity could be detected in RA patients if IFN-γ production but not proliferation was measured. In the present studies in mice the strongest response is seen towards post-translational modifications of the peptide. Because the T-cell contact points are the same whether the peptide is bound to DR4 or to Aq, it is fully possible that post-translational modifications of the peptide also plays a significant role in humans [22]. The fact that IgG antibodies specific for CII are found in many RA patients could be explained by maintained B-cell helper functions of CII-reactive T cells. In fact, it has been reported [23,24] that the occurrence of IgG antibodies to CII is associated with shared epitope DR4 molecules. These observations are thus compatible with a role for CII reactivity in RA. To avoid any confusion, it needs to be stressed that RA is a heterogeneous syndrome in which not only CII, but also other cartilage proteins and other mechanisms are of importance. Such a pathogenic heterogeneity is reflected by the multitude of experimental animal models that have demonstrated how many different pathways may lead to arthritis [25].
PMCID: PMC17814  PMID: 11056672
autoimmunity; rheumatoid arthritis; T lymphocytes; tolerance; transgenic
21.  CD4+ T Cell–mediated Granulomatous Pathology in Schistosomiasis Is Downregulated by a B Cell–dependent Mechanism Requiring Fc Receptor Signaling  
The effector functions of CD4+ T lymphocytes are generally thought to be controlled by distinct populations of regulatory T cells and their soluble products. The role of B cells in the regulation of CD4-dependent host responses is less well understood. Hepatic egg granuloma formation and fibrosis in murine schistosomiasis are dependent on CD4+ lymphocytes, and previous studies have implicated CD8+ T cells or cross-regulatory cytokines produced by T helper (Th) lymphocytes as controlling elements of this pathologic process. In this report, we demonstrate that B cell–deficient (μMT) mice exposed to Schistosoma mansoni develop augmented tissue pathology and, more importantly, fail to undergo the spontaneous downmodulation in disease normally observed during late stages of infection. Unexpectedly, B cell deficiency did not significantly alter T cell proliferative response or cause a shift in the Th1/Th2 balance. Since schistosome-infected Fc receptor–deficient (FcR γ chain knockout) mice display the same exacerbated egg pathology as that observed in infected μMT mice, the B cell– dependent regulatory mechanism revealed by these experiments appears to require receptor-mediated cell triggering. Together, the data demonstrate that humoral immune response/FcR interactions can play a major role in negatively controlling inflammatory disease induced by CD4+ T cells.
PMCID: PMC2212140  PMID: 9463412
22.  Exosomes Released from Mycoplasma Infected Tumor Cells Activate Inhibitory B Cells 
PLoS ONE  2012;7(4):e36138.
Mycoplasmas cause numerous human diseases and are common opportunistic pathogens in cancer patients and immunocompromised individuals. Mycoplasma infection elicits various host immune responses. Here we demonstrate that mycoplasma-infected tumor cells release exosomes (myco+ exosomes) that specifically activate splenic B cells and induce splenocytes cytokine production. Induction of cytokines, including the proinflammatory IFN-γ and the anti-inflammatory IL-10, was largely dependent on the presence of B cells. B cells were the major IL-10 producers. In splenocytes from B cell deficient μMT mice, induction of IFN-γ+ T cells by myco+ exosomes was greatly increased compared with wild type splenocytes. In addition, anti-CD3-stimulated T cell proliferation was greatly inhibited in the presence of myco+ exosome-treated B cells. Also, anti-CD3-stimulated T cell signaling was impaired by myco+ exosome treatment. Proteomic analysis identified mycoplasma proteins in exosomes that potentially contribute to the effects. Our results demonstrate that mycoplasma-infected tumor cells release exosomes carrying mycoplasma components that preferentially activate B cells, which in turn, are able to inhibit T cell activity. These results suggest that mycoplasmas infecting tumor cells can exploit the exosome pathway to disseminate their own components and modulate the activity of immune cells, in particular, activate B cells with inhibitory activity.
PMCID: PMC3338602  PMID: 22558358
23.  T Cells Facilitate Recovery from Venezuelan Equine Encephalitis Virus-Induced Encephalomyelitis in the Absence of Antibody▿  
Journal of Virology  2010;84(9):4556-4568.
Venezuelan equine encephalitis virus (VEEV) is a mosquito-borne RNA virus of the genus Alphavirus that is responsible for a significant disease burden in Central and South America through sporadic outbreaks into human and equid populations. For humans, 2 to 4% of cases are associated with encephalitis, and there is an overall case mortality rate of approximately 1%. In mice, replication of the virus within neurons of the central nervous system (CNS) leads to paralyzing, invariably lethal encephalomyelitis. However, mice infected with certain attenuated mutants of the virus are able to control the infection within the CNS and recover. To better define what role T cell responses might be playing in this process, we infected B cell-deficient μMT mice with a VEEV mutant that induces mild, sublethal illness in immune competent mice. Infected μMT mice rapidly developed the clinical signs of severe paralyzing encephalomyelitis but were eventually able to control the infection and recover fully from clinical illness. Recovery in this system was T cell dependent and associated with a dramatic reduction in viral titers within the CNS, followed by viral persistence in the brain. Further comparison of the relative roles of T cell subpopulations within this system revealed that CD4+ T cells were better producers of gamma interferon (IFN-γ) than CD8+ T cells and were more effective at controlling VEEV within the CNS. Overall, these results suggest that T cells, especially CD4+ T cells, can successfully control VEEV infection within the CNS and facilitate recovery from a severe viral encephalomyelitis.
PMCID: PMC2863737  PMID: 20181704
24.  B-Cell-Dependent Memory T Cells Impede Nonmyeloablative Mixed Chimerism Induction in Presensitized Mice 
Presensitization to HLA antigens limits the success of organ transplantation. The achievement of donor-specific tolerance via mixed chimerism could improve outcomes of transplantation in presensitized patients. In presensitized B-cell-deficient μMT B6 mice, we developed nonmyeloablative bone marrow transplantation (BMT) regimens that successfully tolerized presensitized T cells, achieving long-term (LT) multilineage chimerism and tolerance to donor-type skin. To apply these regimens in wild-type (WT) animals while avoiding antibody-mediated destruction of donor bone marrow cells, presensitized WT B6 mice were rested >2 years to allow alloantibody clearance. However, chimerism and tolerance were not reliably achieved in LT presensitized WT B6 mice in which alloantibody had declined to minimal or undetectable levels before BMT. Strong antidonor memory T-cell responses were detected in LT presensitized WT B6 mice after rejection of donor bone marrow (BM) occurred, whereas levels of alloantibody remained consistently low. In contrast, presensitized μMT B6 mice had diminished memory T-cell responses compared to WT B6 mice. These data implicate T-cell memory, but not alloantibody, in rejection of donor BM in LT presensitized WT mice.
PMCID: PMC3816363  PMID: 21831158
Alloantibodies; memory; mixed chimerism; presensitization; T-cell memory; tolerance
25.  Bradykinin B2 Receptors of Dendritic Cells, Acting as Sensors of Kinins Proteolytically Released by Trypanosoma cruzi, Are Critical for the Development of Protective Type-1 Responses  
PLoS Pathogens  2007;3(11):e185.
Although the concept that dendritic cells (DCs) recognize pathogens through the engagement of Toll-like receptors is widely accepted, we recently suggested that immature DCs might sense kinin-releasing strains of Trypanosoma cruzi through the triggering of G-protein-coupled bradykinin B2 receptors (B2R). Here we report that C57BL/6.B2R−/− mice infected intraperitoneally with T. cruzi display higher parasitemia and mortality rates as compared to B2R+/+ mice. qRT-PCR revealed a 5-fold increase in T. cruzi DNA (14 d post-infection [p.i.]) in B2R−/− heart, while spleen parasitism was negligible in both mice strains. Analysis of recall responses (14 d p.i.) showed high and comparable frequencies of IFN-γ-producing CD4+ and CD8+ T cells in the spleen of B2R−/− and wild-type mice. However, production of IFN-γ by effector T cells isolated from B2R−/− heart was significantly reduced as compared with wild-type mice. As the infection continued, wild-type mice presented IFN-γ-producing (CD4+CD44+ and CD8+CD44+) T cells both in the spleen and heart while B2R−/− mice showed negligible frequencies of such activated T cells. Furthermore, the collapse of type-1 immune responses in B2R−/− mice was linked to upregulated secretion of IL-17 and TNF-α by antigen-responsive CD4+ T cells. In vitro analysis of tissue culture trypomastigote interaction with splenic CD11c+ DCs indicated that DC maturation (IL-12, CD40, and CD86) is controlled by the kinin/B2R pathway. Further, systemic injection of trypomastigotes induced IL-12 production by CD11c+ DCs isolated from B2R+/+ spleen, but not by DCs from B2R−/− mice. Notably, adoptive transfer of B2R+/+ CD11c+ DCs (intravenously) into B2R−/− mice rendered them resistant to acute challenge, rescued development of type-1 immunity, and repressed TH17 responses. Collectively, our results demonstrate that activation of B2R, a DC sensor of endogenous maturation signals, is critically required for development of acquired resistance to T. cruzi infection.
Author Summary
Antibodies and IFN-γ-producing effector T cells are essential for the immune control of infection by Trypanosoma cruzi, the intracellular protozoa that causes human Chagas disease. Despite the potency of anti-parasite immunity, the parasites are not cleared from their intracellular niches. Instead, a low grade chronic infection prevails, provoking severe immunopathology in the myocardium. Although it is well established that innate sentinel cells sense T. cruzi through receptors for microbial structures, such as Toll-like receptors, it remained unclear whether endogenous inflammatory signals also contribute to the development of adaptive immunity. The present study was motivated by awareness that T. cruzi trypomastigotes (extracellular infective forms) are equipped with proteases that liberate the pro-inflammatory bradykinin peptide from an internal segment of kininogens. Here we demonstrate that splenic dendritic cells (DCs), the antigen-presenting cells that coordinate the adaptive branch of immunity in lymphoid tissues, are potently activated via G-protein-coupled bradykinin B2 receptors (B2R). Analysis of the outcome of infection in B2R-knockout mice revealed that the mutant mice developed a typical susceptible phenotype, owing to impaired development of IFN-γ-producing effector T cells. Notably, the immune dysfunction of B2R-knockout mice was corrected upon cell transfer of wild-type DCs, thus linking development of protective T cells to DCs' sensing of endogenous danger signals (kinins) released by trypomastigotes.
PMCID: PMC2098834  PMID: 18052532

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