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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.
doi:10.1128/IAI.69.6.3860-3868.2001
PMCID: PMC98409  PMID: 11349052
2.  Cell-Mediated Protection against Pulmonary Yersinia pestis Infection  
Infection and Immunity  2005;73(11):7304-7310.
Pulmonary infection with the bacterium Yersinia pestis causes pneumonic plague, an often-fatal disease for which no vaccine is presently available. Antibody-mediated humoral immunity can protect mice against pulmonary Y. pestis infection, an experimental model of pneumonic plague. Little is known about the protective efficacy of cellular immunity. We investigated the cellular immune response to Y. pestis in B-cell-deficient μMT mice, which lack the capacity to generate antibody responses. To effectively prime pulmonary cellular immunity, we intranasally vaccinated μMT mice with live replicating Y. pestis. Vaccination dramatically increased survival of μMT mice challenged intranasally with a lethal Y. pestis dose and significantly reduced bacterial growth in pulmonary, splenic, and hepatic tissues. Vaccination also increased numbers of pulmonary T cells, and administration of T-cell-depleting monoclonal antibodies at the time of challenge abrogated vaccine-induced survival. Moreover, the transfer of Y. pestis-primed T cells to naive μMT mice protected against lethal intranasal challenge. These findings establish that vaccine-primed cellular immunity can protect against pulmonary Y. pestis infection and suggest that vaccines promoting both humoral and cellular immunity will most effectively combat pneumonic plague.
doi:10.1128/IAI.73.11.7304-7310.2005
PMCID: PMC1273885  PMID: 16239527
3.  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.
doi:10.1016/j.virol.2008.03.010
PMCID: PMC2431983  PMID: 18410949
HSV-2; vaginal; B cell-deficient; CD4 T cells; immunization
4.  Bovine CD4+ T-Lymphocyte Clones Specific for Rhoptry-Associated Protein 1 of Babesia bigemina Stimulate Enhanced Immunoglobulin G1 (IgG1) and IgG2 Synthesis 
Infection and Immunity  1999;67(1):155-164.
Optimal protective immunity against babesial infection is postulated to require both complement-fixing and opsonizing antibodies in addition to gamma interferon (IFN-γ)-mediated macrophage activation. The rhoptry-associated protein 1 (RAP-1) of Babesia bigemina induces partial protective immunity and is a candidate vaccine antigen. Previous studies demonstrated that cattle immunized with native protein that were subsequently protected against challenge had a strong IFN-γ and weaker interleukin-4 (IL-4) response in immune lymph node lymphocytes that reflected the cytokine profile of the majority of CD4+ T-cell clones obtained from peripheral blood. RAP-1-specific T helper (Th) cell clones that coexpress IFN-γ and IL-4 are typical of numerous parasite-specific clones examined. However, the function of such cells as helper cells to enhance immunoglobulin secretion by bovine B cells has not been reported. In cattle, both immunoglobulin G1 (IgG1) and IgG2 can fix complement, but IgG2 is the superior opsonizing subclass. Therefore, studies were undertaken to ascertain the functional relevance of RAP-1-specific, CD4+ Th0 cells as helper cells to enhance IgG1 and/or IgG2 production by autologous B lymphocytes. For comparison, Th0 clones specific for the metazoan parasite Fasciola hepatica that expressed relatively more IL-4 than the B. bigemina-specific Th cells were similarly assayed. B. bigemina RAP-1-specific clones could enhance production of both IgG1 and IgG2 by autologous B cells, whereas Th cell clones specific for F. hepatica enhanced predominantly IgG1 production. The capacity to enhance IgG2 production was associated with production of IFN-γ by Th cells cocultured with B cells, antigen, and IL-2. The in vitro helper T-cell activity of these T-cell clones was representative of the in vivo serologic responses, which were composed of a mixed IgG1-IgG2 response in B. bigemina RAP-1 immune cattle and a biased IgG1 response in F. hepatica-immune cattle.
PMCID: PMC96291  PMID: 9864210
5.  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.
doi:10.1128/IAI.70.12.6911-6918.2002
PMCID: PMC133017  PMID: 12438369
6.  NK cells influence both innate and adaptive immune responses after mucosal immunisation with antigen and mucosal adjuvant* 
NK cells were found to be recruited in a temporally controlled manner to the nasal-associated lymphoid tissue and the cervical lymph nodes of mice following intranasal immunisation with Ag85B-ESAT6 antigen from Mycobacterium tuberculosis mixed with Escherichia coli heat-labile toxin as adjuvant. These NK cells were activated and they secreted a diverse range of cytokines and other immunmodulators. Using antibody depletion targeting anti-asialo GM1, we found evidence for altered trafficking, impaired activation and cytokine secretion of dendritic cells, macrophages and neutrophils in immunised NK cell depleted mice compared to control animals. Analysis of antigen-specific immune responses revealed an attenuated antibody and cytokine response in immunised NK cell depleted animals. Systemic administration of rIL-6 but not rIFN-γ significantly restored immune responses in mice depleted of NK cells. In conclusion, cytokine production, particularly IL-6, via NK cells and NK cell activated immune populations, plays an important role in the establishment of local innate immune responses and the consequent development of adaptive immunity after mucosal immunisation.
doi:10.4049/jimmunol.0903357
PMCID: PMC3517975  PMID: 20220095
7.  Selective development of T helper (Th)2 cells induced by continuous administration of low dose soluble proteins to normal and beta(2)- microglobulin-deficient BALB/c mice 
Continuous administration of soluble proteins, delivered over a 10-d period by a mini-osmotic pump implanted subcutaneously, induces a long- lasting inhibition of antigen-specific T cell proliferation in lymph node cells from BALB/c mice subsequently primed with antigen in adjuvant. The decreased T cell proliferative response is associated with a down-regulation of the T helper cell (Th)1 cytokines interleukin (IL)-2 and interferon (IFN)-gamma and with a strong increase in the secretion of the Th2 cytokines IL-4 and IL-5 by antigen specific CD4+ T cells. This is accompanied by predominant inhibition of antigen- specific antibody production of IgG2a and IgG2b, rather than IgG1 isotype. Interestingly, inhibition of Th1 and priming of Th2 cells is also induced in beta(2) microglobulin-deficient BALB/c mice, indicating that neither CD8+ nor CD4+ NK1.1+ T cells, respectively, are required. The polarization in Th2 cells is stably maintained by T cell lines, all composed of CD4+/CD8- cells expressing T cell receptor for antigen (TCR) alpha/beta chains, derived from BALB/c mice treated with continuous antigen administration, indicating that they originate from Th2 cells fully differentiated in vivo. This polarization is induced in BALB/c mice by continuous administration of any protein antigen tested, including soluble extracts from pathogenic microorganisms. Priming of Th2 cells is dose dependent and it is optimal for low rather than high doses of protein. Blocking endogenous IL-4 in vivo inhibits expansion of antigen-specific Th2 cells, but does not restore IFN-gamma production by T cells from mice treated with soluble antigen-specific Th2 cells, but does not restore IFN-gamma production by T cells from mice treated with soluble antigen, indicating the involvement of two independent mechanisms. Consistent with this, Th2 cell development, but not inhibition of Th1 cells, depends on non-major histocompatibility complex genetic predisposition, since the Th2 response is amplified in BALB/c as compared to DBA/2, C3H, or C57BL/6 mice whereas tested. These findings support the hypothesis that continuous release of low amounts of protein antigens from pathogenic microorganisms may polarize the immune response toward a Th2 phenotype in susceptible mouse strains.
PMCID: PMC2192465  PMID: 8627161
8.  Induction of murine T-helper-cell responses to the filarial nematode Brugia malayi. 
Infection and Immunity  1993;61(3):1105-1112.
The purpose of this study was to examine the murine T-helper-cell (Th) cytokine response to the human filarial parasite Brugia malayi. In the first 14 days following intraperitoneal inoculation of live microfilariae into BALB/c mice, filarial antigen-driven splenic lymphoid cells produced gamma interferon (IFN-gamma) and little or no interleukin-5 (IL-5). After this time, IL-5 production increased (to 10 to 12 ng per 5 x 10(6) cells) coincident with a marked diminution in IFN-gamma generation. A single subcutaneous immunization with soluble microfilarial antigens also induced an IFN-gamma but no IL-5 response, whereas immunization three times elicited a predominant Th2-like reaction characterized by IL-4 and IL-5 production by CD4+ lymph node lymphocytes and a 10-fold increase in serum immunoglobulin E. The importance of IL-10 in establishing the balance between parasite-specific Th1 and Th2 responses was demonstrated by the ability of neutralizing monoclonal antibody to this cytokine to increase IFN-gamma production by splenic and lymph node cells from mice chronically exposed to live microfilariae or immunized multiple times with soluble filarial antigens.
PMCID: PMC302845  PMID: 8094378
9.  B Cells Are Essential for Vaccination-Induced Resistance to Virulent Toxoplasma gondii 
Infection and Immunity  2000;68(3):1026-1033.
T lymphocytes and gamma interferon (IFN-γ) are known mediators of immune resistance to Toxoplasma gondii infection, but whether B cells also play an important role is not clear. We have investigated this issue using B-cell-deficient (μMT) mice. If vaccinated with attenuated T. gondii tachyzoites, μMT mice are susceptible to a challenge intraperitoneal infection with highly virulent tachyzoites that similarly vaccinated B-cell-sufficient mice resist. Susceptibility is evidenced by increased numbers of parasites at the challenge infection site and by extensive mortality. The susceptibility of B-cell-deficient mice does not appear to be caused by deficient T-cell functions or diminished capacity of vaccinated and challenged B-cell-deficient mice to produce IFN-γ. Administration of Toxoplasma-immune serum, but not nonimmune serum, to vaccinated B-cell-deficient mice significantly prolongs their survival after challenge with virulent tachyzoites. Vaccinated mice lacking Fc receptors or the fifth component of complement resist a challenge infection, suggesting that neither Fc-receptor-dependent phagocytosis of antibody-coated tachyzoites nor antibody-dependent cellular cytotoxicity nor antibody-and-complement-dependent lysis of tachyzoites is a crucial mechanism of resistance. However, Toxoplasma-immune serum effectively inhibits the infection of host cells by tachyzoites in vitro. Together, the results support the hypothesis that B cells are required for vaccination-induced resistance to virulent tachyzoites in order to produce antibodies and that antibodies may function protectively in vivo by blocking infection of host cells by tachyzoites.
PMCID: PMC97244  PMID: 10678903
10.  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.
doi:10.1371/journal.pone.0036138
PMCID: PMC3338602  PMID: 22558358
11.  Diminished Primary and Secondary Influenza Virus-Specific CD8+ T-Cell Responses in CD4-Depleted Ig−/− Mice 
Journal of Virology  2000;74(20):9762-9765.
Optimal expansion of influenza virus nucleoprotein (DbNP366)-specific CD8+ T cells following respiratory challenge of naive Ig−/− μMT mice was found to require CD4+ T-cell help, and this effect was also observed in primed animals. Absence of the CD4+ population was consistently correlated with diminished recruitment of virus-specific CD8+ T cells to the infected lung, delayed virus clearance, and increased morbidity. The splenic CD8+ set generated during the recall response in Ig−/− mice primed at least 6 months previously showed a normal profile of gamma interferon production subsequent to short-term, in vitro stimulation with viral peptide, irrespective of a concurrent CD4+ T-cell response. Both the magnitude and the localization profiles of virus-specific CD8+ T cells, though perhaps not their functional characteristics, are thus modified in mice lacking CD4+ T cells.
PMCID: PMC112411  PMID: 11000251
12.  Pretreatment with Recombinant Flt3 Ligand Partially Protects against Progressive Cutaneous Leishmaniasis in Susceptible BALB/c Mice 
Infection and Immunity  2001;69(2):673-680.
Dendritic cells are potent antigen-presenting cells that also produce interleukin-12 (IL-12) during innate and adaptive cellular immune responses and that thereby promote the differentiation of gamma interferon (IFN-γ)-producing Th1-type CD4+ T lymphocytes. We hypothesized that expanded dendritic-cell populations in mice pretreated with the hematopoietic cytokine Flt3L would protect against cutaneous Leishmania major infection. Pretreatment of disease-susceptible BALB/c mice with 10 μg of recombinant Flt3L (rFlt3L) for 9 to 10 days before infection increased lymph node IL-12 p40 productive capacity 20-fold compared to that of saline-injected controls. Furthermore, 9 of 22 (40.9%) rFlt3L-pretreated BALB/c mice resolved their cutaneous infections, whereas none of the 22 control BALB/c mice healed. Healed, rFlt3L-pretreated mice did not develop disease following reinfection. Flt3L pretreatment also reduced parasite numbers 1,000-fold in the cutaneous lesions at 2 weeks after infection relative to numbers in lesions of untreated controls. However, Flt3L pretreatment did not significantly alter L. major-induced IFN-γ and IL-4 production in lymph node culture at 1, 2, and 4 weeks after infection. Despite the lack of Th immune deviation, Flt3L ligand-pretreated lymph nodes expressed up to 10-fold higher levels of IL-12 p40 and inducible (type 2) nitric oxide synthase mRNA at 7 days after infection. In contrast, treatment with rFlt3L after infection failed to protect against disease despite comparable expansions of dendritic cells and IL-12 p40 productive capacity in both infected and uninfected BALB/c mice treated with rFlt3L. We conclude that rFlt3L pretreatment before infection with L. major reduces parasite load and promotes healing of cutaneous lesions without stable cytokine deviation towards a dominant Th1 cytokine phenotype.
doi:10.1128/IAI.69.2.673-680.2001
PMCID: PMC97938  PMID: 11159954
13.  Polarization of Allogeneic T-Cell Responses by Influenza Virus-Infected Dendritic Cells 
Journal of Virology  2000;74(17):7738-7744.
The developing immune response in the lymph nodes of mice infected with influenza virus has both Th1- and Th2-type characteristics. Modulation of the interactions between antigen-presenting cells and T cells is one mechanism that may alter the quality of the immune response. We have previously shown that the ability of dendritic cells (DC) to stimulate the proliferation of alloreactive T cells is changed by influenza virus due to viral neuraminidase (NA) activity. Here we show that DC infected with influenza virus A/PR/8/34 (PR8) stimulate T cells to produce different types of cytokines in a dose-dependent manner. Optimal amounts of the Th1-type cytokines interleukin-2 (IL-2) and gamma interferon (IFN-γ) were produced from T cells stimulated by DC infected with low doses of PR8, while the Th2-type cytokines IL-4 and IL-10 were produced only in response to DC infected with high doses of PR8. IL-2 and IFN-γ levels corresponded with T-cell proliferation and were dependent on the activity of viral NA on the DC surface. In contrast, IL-4 secretion required the treatment of T cells with NA. Since viral particles were released only from DC that are infected with high doses of PR8, our results suggest that viral NA on newly formed virus particles desialylates T-cell surface molecules to facilitate a Th2-type response. These results suggest that the activity of NA may contribute to the mixed Th-type response observed during influenza virus infection.
PMCID: PMC112302  PMID: 10933679
14.  Gamma Interferon Is Not Required for Mucosal Cytotoxic T-Lymphocyte Responses or Heterosubtypic Immunity to Influenza A Virus Infection in Mice 
Journal of Virology  2000;74(12):5495-5501.
Heterosubtypic immunity (HSI) is defined as cross-protection against influenza virus of a different serotype than the virus initially encountered and is thought to be mediated by influenza virus-specific cytotoxic T lymphocytes (CTL). Since gamma interferon (IFN-γ) stimulates cytotoxic cells, including antigen-specific CTL which may control virus replication by secretion of antiviral cytokines such as tumor necrosis factor alpha and IFN-γ, we have investigated the mechanism of HSI by analyzing the role of IFN-γ for HSI in IFN-γ gene-deleted (IFN-γ−/−) mice. It has been reported that IFN-γ is not required for recovery from primary infection with influenza virus but is important for HSI. Here, we conclusively show that IFN-γ is not required for induction of secondary influenza virus-specific CTL responses in mediastinal lymph nodes and HSI to lethal influenza A virus infection. Although T helper 2 (Th2)-type cytokines were upregulated in the lungs of IFN-γ−/− mice after virus challenge, either Th1- or Th2-biased responses could provide heterosubtypic protection. Furthermore, titers of serum-neutralizing and cross-reactive antibodies to conserved nucleoprotein in IFN-γ−/− mice did not differ significantly from those in immunocompetent mice. These results indicate that lack of IFN-γ does not impair cross-reactive virus-specific immune responses and HSI to lethal infection with influenza virus. Our findings provide new insight for the mechanisms of HSI and should be valuable in the development of protective mucosal vaccines against variant virus strains, such as influenza and human immunodeficiency virus.
PMCID: PMC112034  PMID: 10823854
15.  CXCR3 chemokine receptor-ligand interactions in the lymph node optimize CD4+ T helper 1 cell differentiation 
Immunity  2012;37(6):1091-1103.
SUMMARY
Differentiation of naïve CD4+ T cells into T helper (Th) cells is a defining event in adaptive immunity. The cytokines and transcription factors that control Th cell differentiation are understood, however it is not known how this process is orchestrated within lymph nodes (LNs). Here we have shown that the CXCR3 chemokine receptor was required for optimal generation of interferon (IFN)-γ secreting Th1 cells in vivo. Using a CXCR3 ligand reporter mouse, we found that stromal cells predominately expressed the chemokine ligand CXCL9 while hematopoietic cells expressed CXCL10 in LNs. Dendritic cell (DC)-derived CXCL10 facilitated T cell-DC interactions in LNs during T cell priming while both chemokines guided intranodal positioning of CD4+ T cells to interfollicular and medullary zones. Thus, different chemokines acting on the same receptor can function locally to facilitate DC-T cell interactions and globally to influence intranodal positioning, and that both functions contribute to Th1 cell differentiation.
doi:10.1016/j.immuni.2012.08.016
PMCID: PMC3525757  PMID: 23123063
16.  An Essential Role of Antibodies in the Control of Chikungunya Virus Infection 
The Journal of Immunology Author Choice  2013;190(12):6295-6302.
In recent years, Chikungunya virus (CHIKV) was responsible for epidemic outbreaks in intertropical regions. Although acquired immunity has been shown to be crucial during CHIKV infection in both humans and mice, their exact role in the control of CHIKV infection remains unclear. In this study, wild-type (WT), CD4−/−, and B cell (μMT) knockout mice were infected with CHIKV. Sera were taken at different days postinfection and measured for anti-CHIKV Ab levels. Isotype and neutralizing capacity of these Abs were assessed in vitro, and specific linear epitopes were mapped. Viremia in CHIKV-infected μMT mice persisted for more than a year, indicating a direct role for B cells in mediating CHIKV clearance. These animals exhibited a more severe disease than WT mice during the acute phase. Characterization of CHIKV-specific Abs revealed that anti-CHIKV Abs were elicited early and targeted epitopes mainly at the C terminus of the virus E2 glycoprotein. Furthermore, CD4−/− mice could still control CHIKV infection despite having lower anti-CHIKV Ab levels with reduced neutralizing capacity. Lastly, pre-existing natural Abs in the sera of normal WT mice recognized CHIKV and were able to partially inhibit CHIKV. Taken together, natural and CHIKV infection–induced specific Abs are essential for controlling CHIKV infections.
doi:10.4049/jimmunol.1300304
PMCID: PMC3677171  PMID: 23670192
17.  CD4+ Th1 Cells Induced by Dendritic Cell-Based Immunotherapy in Mice Chronically Infected with Leishmania amazonensis Do Not Promote Healing  
Infection and Immunity  2004;72(8):4455-4463.
The susceptibility of mice to Leishmania amazonensis infection is thought to result from an inability to develop a Th1 response. Our data show that the low levels of gamma interferon (IFN-γ) produced by the draining lymph node (DLN) cells of chronically infected mice could be enhanced in vitro and in vivo with L. amazonensis antigen-pulsed bone marrow-derived dendritic cells (BM-DC) and the Th1-promoting cytokine interleukin-12 (IL-12). Given intralesionally to chronically infected mice, this treatment induced the upregulation of mRNA levels for IFN-γ, the transcription factor T-box expressed in T cells, and IL-12 receptor β2 in CD4+ T cells from the DLN and an increase in parasite-specific immunoglobulin G2a in the serum. However, this Th1 response was not associated with healing, and the antigen-specific enhancement of IFN-γ production remained impaired in the DLN. However, addition of IL-12 to the in vitro recall response was able to recover this defect, suggesting that antigen-presenting cell-derived IL-12 production may be limited in infected mice. This was supported by the fact that L. amazonensis amastigotes limited the production of IL-12p40 from BM-DC in vitro. Altogether, our data indicate that the immune response of mice chronically infected with L. amazonensis can be enhanced towards a Th1 phenotype but that the presence of Th1 CD4+ T cells does not promote healing. This suggests that the phenotype of the CD4+ T cells may not always be indicative of protection to L. amazonensis infection. Furthermore, our data support growing evidence that antigen-presenting cell function, such as IL-12 production, may limit the immune response in L. amazonensis-infected mice.
doi:10.1128/IAI.72.8.4455-4463.2004
PMCID: PMC470671  PMID: 15271903
18.  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.
doi:10.1128/IAI.00014-07
PMCID: PMC2168332  PMID: 17875636
19.  Downregulation of Key Early Events in the Mobilization of Antigen-bearing Dendritic Cells by Leukocyte Immunoglobulin-like Receptor B4 in a Mouse Model of Allergic Pulmonary Inflammation 
PLoS ONE  2013;8(2):e57007.
Leukocyte Immunoglobulin-like Receptor B4 (LILRB4) null mice have an exacerbated T helper cell type 2 (Th2) immune response and pulmonary inflammation compared with Lilrb4+/+ animals when sensitized intranasally with ovalbumin (OVA) and low-dose lipopolysaccharide (LPS) followed by challenge with OVA. Moreover, OVA-challenged Lilrb4−/− mice exhibit greater migration of antigen (Ag)-bearing dendritic cells (DCs) to lymph nodes and accumulation of interleukin 4- and interleukin 5-producing lymph node lymphocytes. The main objective of this study was to determine how the absence of LILRB4 leads to a greater number of DCs in the lymph nodes of Ag-challenged mice and increased lung Th2 inflammation. Mice were sensitized intranasally with PBS alone or containing OVA and LPS; additional cohorts were subsequently challenged with OVA. Expression of chemokine (C-C motif) ligand 21 (CCL21) in the lung was assessed immunohistologically. OVA ingestion and expression of LILRB4 and chemokine (C-C motif) receptor 7 (CCR7) were quantified by flow cytometry. Inhalation of OVA and LPS induced upregulation of LILRB4 selectively on lung Ag-bearing DCs. After sensitization and challenge, the lung lymphatic vessels of Lilrb4−/− mice expressed more CCL21, a chemokine that directs the migration of DCs from peripheral tissue to draining lymph nodes, compared with Lilrb4+/+ mice. In addition, lung DCs of challenged Lilrb4−/− mice expressed more CCR7, the CCL21 receptor. The lungs of challenged Lilrb4−/− mice also contained significantly greater numbers of CD4+ cells expressing interleukin-4 or interleukin-5, consistent with the greater number of Ag-bearing DCs and Th2 cells in lymph nodes and the attendant exacerbated Th2 lung pathology. Our data establish a new mechanism by which LILRB4 can downregulate the development of pathologic allergic inflammation: reduced upregulation of key molecules needed for DC migration leading to decreases in Th2 cells in lymph nodes and their target tissue.
doi:10.1371/journal.pone.0057007
PMCID: PMC3576413  PMID: 23431396
20.  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
21.  Resting Respiratory Tract Dendritic Cells Preferentially Stimulate T Helper Cell Type 2 (Th2) Responses and Require Obligatory Cytokine Signals for Induction of  Th1 Immunity  
The Journal of Experimental Medicine  1998;188(11):2019-2031.
Consistent with their role in host defense, mature dendritic cells (DCs) from central lymphoid organs preferentially prime for T helper cell type 1 (Th1)-polarized immunity. However, the “default” T helper response at mucosal surfaces demonstrates Th2 polarity, which is reflected in the cytokine profiles of activated T cells from mucosal lymph nodes. This study on rat respiratory tract DCs (RTDCs) provides an explanation for this paradox. We demonstrate that freshly isolated RTDCs are functionally immature as defined in vitro, being surface major histocompatibility complex (MHC) II lo, endocytosishi, and mixed lymphocyte reactionlo, and these cells produce mRNA encoding interleukin (IL)-10. After ovalbumin (OVA)-pulsing and adoptive transfer, freshly isolated RTDCs preferentially stimulated Th2-dependent OVA-specific immunoglobulin (Ig)G1 responses, and antigen-stimulated splenocytes from recipient animals produced IL-4 in vitro. However, preculture with granulocyte/macrophage colony stimulating factor increased their in vivo IgG priming capacity by 2–3 logs, inducing production of both Th1- and Th2-dependent IgG subclasses and high levels of IFN-γ by antigen-stimulated splenocytes. Associated phenotypic changes included upregulation of surface MHC II and B7 expression and IL-12 p35 mRNA, and downregulation of endocytosis, MHC II processing– associated genes, and IL-10 mRNA expression. Full expression of IL-12 p40 required additional signals, such as tumor necrosis factor α or CD40 ligand. These results suggest that the observed Th2 polarity of the resting mucosal immune system may be an inherent property of the resident DC population, and furthermore that mobilization of Th1 immunity relies absolutely on the provision of appropriate microenvironmental costimuli.
PMCID: PMC2212375  PMID: 9841916
dendritic cell; lung; function; T helper cell type 1; T helper cell type 2
22.  Effect of in vivo administration of recombinant murine gamma interferon on in vitro lymphoproliferative responses following immunization with Candida albicans. 
Infection and Immunity  1992;60(5):1927-1935.
The effect of in vivo administration of recombinant murine gamma interferon (rMuIFN-gamma) on in vitro proliferation of lymphocytes to Candida antigens and lectins was examined in naive CBA/J mice and in similar mice colonized with Candida albicans by intragastric (i.g.) intubation and/or inoculated intradermally (i.d.) with the fungus. Lymph node lymphocyte and splenic lymphocyte (splenocyte) responses to soluble cytoplasmic substances derived from C. albicans varied with the route of inoculation of the fungus, the sex of the animal, and the presence or absence of rMuIFN-gamma treatment. In the absence of rMuIFN-gamma treatment, lymphoid cells from lymph nodes draining the site of the i.d. lesion responded well to soluble cytoplasmic substances. Colonization of the gut of female mice with C. albicans either had no effect or promoted better lymph node responses when such animals were also challenged i.d., whereas gut colonization of males followed by i.d. challenge appeared to have a suppressive influence on the level of proliferation in response to antigens in vitro. Antigen-specific splenocyte responses could be detected as well, and they were best in animals inoculated i.g.-i.d. or i.d. only. With the exception of lymph node lymphocytes from male mice, treatment of infected animals, regardless of the route of infection, with rMuIFN-gamma frequently resulted in lowered responses to antigens when comparable treatment groups were examined. With respect to mitogen stimulation, infection with C. albicans, especially i.g. or i.g.-i.d., resulted in a population of lymph node lymphocytes with lower-than-normal responses to concanavalin A but higher-than-normal responses to lipopolysaccharide (LPS). Splenocyte responses to mitogens were not altered as dramatically as the responses of lymph node lymphocytes, but splenocytes from female mice had a suppressed response regardless of the route of exposure to C. albicans, and those from mice which were maximally stimulated with C. albicans, i.e., inoculated i.g.-i.d., also had a suppressed response to concanavalin A. Treatment with rMuIFN-gamma either had no effect on the subsequent splenocyte responses or boosted subnormal mitogen responses toward the normal range. Collectively, these data illustrate that exposure to both C. albicans and rMuIFN-gamma influenced the responses to mitogen and C. albicans antigen of lymph node lymphocyte and splenocyte populations, as detected in vitro by lymphoproliferation. Treatment with rMuIFN-gamma often resulted in increased responsiveness to a B cell mitogen, LPS, and decreased responsiveness to a C. albicans antigen.
PMCID: PMC257096  PMID: 1563784
23.  Antigens derived from lung-stage larvae of Schistosoma mansoni are efficient stimulators of proliferation and gamma interferon secretion by lymphocytes from mice vaccinated with attenuated larvae. 
Infection and Immunity  1995;63(5):1980-1986.
Protective immunity in C57BL/6 mice exposed to optimally irradiated larvae of Schistosoma mansoni operates against challenge parasites in the lungs and is dependent upon T-helper 1 (Th1) lymphocytes which secrete abundant gamma interferon (IFN-gamma). As an initial step in the identification of the molecules which mediate this immunity, antigenic materials released by larvae at various times during in vitro culture were compared for the ability to induce proliferation of lymph node cells recovered from mice 4 to 6 days after exposure to attenuated parasites. Cells from mice vaccinated with cercariae proliferated most strongly to larval antigens released soon after transformation. In contrast, cells from mice immunized with lung-stage schistosomula responded poorly to these early secretions but proliferated vigorously to antigens released by older larvae. In further studies on the cytokine profile of the responding lymphocytes, it was observed that the balance between IFN-gamma and interleukin-4 (IL-4) secretion depended on the source of antigen used for restimulation. Thus, material released between days 6 and 8 by in vitro-cultured larvae, and the soluble extracts of whole lung-stage larvae, induced abundant IFN-gamma but little IL-4. This finding implies that an overwhelming proportion of the lymphocytes responsive to lung-stage antigens had the Th1 phenotype. In contrast, antigens from cercariae and skin-stage larvae induced the lowest levels of IFN-gamma but higher levels of IL-4. It appears that a proportion of the cells with specificities for early antigens had the Th2 or Th0 phenotype. Our results emphasize that antigens from lung-stage larvae are an important source of potentially protective molecules.
PMCID: PMC173253  PMID: 7729911
24.  Trance, a Tumor Necrosis Factor Family Member, Enhances the Longevity and Adjuvant Properties of Dendritic Cells in Vivo 
Mature dendritic cells (DCs) are powerful antigen presenting cells that have the unique capacity to migrate to the T cell zone of draining lymph nodes after subcutaneous injection. Here we report that treatment of antigen-pulsed mature DCs with tumor necrosis factor (TNF)-related activation-induced cytokine (TRANCE), a TNF family member, before immunization enhances their adjuvant capacity and elicits improved T cell priming in vivo, such that both primary and memory T cell immune responses are enhanced. By enumerating migratory DCs in the draining lymph nodes and by studying their function in stimulating naive T cells, we show that one of the underlying mechanisms for enhanced T cell responses is an increase in the number of ex vivo antigen-pulsed DCs that are found in the T cell areas of lymph nodes. These results suggest that the longevity and abundance of mature DCs at the site of T cell priming influence the strength of the DC-initiated T cell immunity in situ. Our findings have the potential to improve DC-based immunotherapy; i.e., the active immunization of humans with autologous DCs that have been pulsed with clinically significant antigens ex vivo.
PMCID: PMC2195824  PMID: 10662795
TRANCE; dendritic cells; T cell; immunization
25.  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.
doi:10.1128/JVI.01152-06
PMCID: PMC1900320  PMID: 17376910

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