Host differences in influenza-specific CD4 T cell and B cell responses are modulated by viral strain and route of immunization

The antibody response to influenza infection is largely dependent on CD4 T cell help for B cells. Cognate signals and secreted factors provided by CD4 T cells drive B cell activation and regulate antibody isotype switching for optimal antiviral activity. Recently, we analyzed HLA-DR1 transgenic (DR1) mice and C57BL/10 (B10) mice after infection with influenza virus A/New Caledonia/20/99 (NC) and defined epitopes recognized by virus-specific CD4 T cells. Using this information in the current study, we demonstrate that the pattern of secretion of IL-2, IFN-γ, and IL-4 by CD4 T cells activated by NC infection is largely independent of epitope specificity and the magnitude of the epitope-specific response. Interestingly, however, the characteristics of the virus-specific CD4 T cell and the B cell response to NC infection differed in DR1 and B10 mice. The response in B10 mice featured predominantly IFN-γ-secreting CD4 T cells and strong IgG2b/IgG2c production. In contrast, in DR1 mice most CD4 T cells secreted IL-2 and IgG production was IgG1-biased. Infection of DR1 mice with influenza PR8 generated a response that was comparable to that in B10 mice, with predominantly IFN-γ-secreting CD4 T cells and greater numbers of IgG2c than IgG1 antibody-secreting cells. The response to intramuscular vaccination with inactivated NC was similar in DR1 and B10 mice; the majority of CD4 T cells secreted IL-2 and most IgG antibody-secreting cells produced IgG2b or IgG2c. Our findings identify inherent host influences on characteristics of the virus-specific CD4 T cell and B cell responses that are restricted to the lung environment. Furthermore, we show that these host influences are substantially modulated by the type of infecting virus via the early induction of innate factors. Our findings emphasize the importance of immunization strategy for demonstrating inherent host differences in CD4 T cell and B cell responses.     

Sensing of Immature Particles Produced by Dengue Virus Infected Cells Induces an Antiviral Response by Plasmacytoid Dendritic Cells

Dengue virus (DENV) is the leading cause of mosquito-borne viral illness and death in humans. Like many viruses, DENV has evolved potent mechanisms that abolish the antiviral response within infected cells. Nevertheless, several in vivo studies have demonstrated a key role of the innate immune response in controlling DENV infection and disease progression. Here, we report that sensing of DENV infected cells by plasmacytoid dendritic cells (pDCs) triggers a robust TLR7-dependent production of IFNα, concomitant with additional antiviral responses, including inflammatory cytokine secretion and pDC maturation. We demonstrate that unlike the efficient cell-free transmission of viral infectivity, pDC activation depends on cell-to-cell contact, a feature observed for various cell types and primary cells infected by DENV, as well as West Nile virus, another member of the Flavivirus genus. We show that the sensing of DENV infected cells by pDCs requires viral envelope protein-dependent secretion and transmission of viral RNA. Consistently with the cell-to-cell sensing-dependent pDC activation, we found that DENV structural components are clustered at the interface between pDCs and infected cells. The actin cytoskeleton is pivotal for both this clustering at the contacts and pDC activation, suggesting that this structural network likely contributes to the transmission of viral components to the pDCs. Due to an evolutionarily conserved suboptimal cleavage of the precursor membrane protein (prM), DENV infected cells release uncleaved prM containing-immature particles, which are deficient for membrane fusion function. We demonstrate that cells releasing immature particles trigger pDC IFN response more potently than cells producing fusion-competent mature virus. Altogether, our results imply that immature particles, as a carrier to endolysosome-localized TLR7 sensor, may contribute to regulate the progression of dengue disease by eliciting a strong innate response.     

Modulation of α-Enolase Post-Translational Modifications by Dengue Virus: Increased Secretion of the Basic Isoforms in Infected Hepatic Cells

Hepatic cells are major sites of dengue virus (DENV) replication and liver injury constitutes a characteristic of severe forms of dengue. The role of hepatic cells in dengue pathogenesis is not well established, but since hepatocytes are the major source of plasma proteins, changes in protein secretion by these cells during infection might contribute to disease progression. Previously, we showed that DENV infection alters the secretion pattern of hepatic HepG2 cells, with α-enolase appearing as one of the major proteins secreted in higher levels by infected cells. ELISA analysis demonstrated that DENV infection modulates α-enolase secretion in HepG2 cells in a dose-dependent manner, but has no effect on its gene expression and on the intracellular content of the protein as assessed by PCR and western blot analyses, respectively. Two-dimensional western blots showed that both intracellular and secreted forms of α-enolase appear as five spots, revealing α-enolase isoforms with similar molecular weights but distinct isoeletric points. Remarkably, quantification of each spot content revealed that DENV infection shifts the isoform distribution pattern of secreted α-enolase towards the basic isoforms, whereas the intracellular protein remains unaltered, suggesting that post-translational modifications might be involved in α-enolase secretion by infected cells. These findings provide new insights into the mechanisms underlying α-enolase secretion by hepatic cells and its relationship with the role of liver in dengue pathogenesis. In addition, preliminary results obtained with plasma samples from DENV-infected patients suggest an association between plasma levels of α-enolase and disease severity. Since α-enolase binds plasminogen and modulates its activation, it is plausible to speculate the association of the increase in α-enolase secretion by infected hepatic cells with the haemostatic dysfunction observed in dengue patients including the promotion of fibrinolysis and vascular permeability alterations.     

Age-related changes in in vitro immunoglobulin secretion: Comparison of responses to T-dependent and T-independent polyclonal activators

In vitro Ig secretion by peripheral blood mononuclear cells (MNCs) from old and young donors, in response to T-dependent (TD) [pokeweed mitogen (PWM)] and T-independent (TI) [Salmonella paratyphii B (SPB)] activation were compared. In older donors, the IgG and IgA responses to PWM were comparable to those of young donors; the IgM response was reduced in the elderly. With SPB activation, IgA response was again preserved, whereas IgG response was reduced and IgM secretion was markedly decreased. These data indicate class-specific changes in Ig responsiveness to both TD and TI cell activators with age. The reduction in TI-induced IgG and IgM responses in the elderly suggest that changes in B cells themselves have occurred. The preservation of the TD IgG response in concert with reduced TI response indicates that a decline in T-suppressor influences over B cells in the elderly coupled with reduced B-cell synthesizing capacity can result in apparent “preservation” of the final Ig response. In keeping with the above postulate, analysis of individual elderly donors' responses indicated that some of the old donors responded to PWM, but not SPB; none of the old donors responded to SPB and not PWM. In contrast, some young donors did respond to SPB, but not PWM. These results also suggest that nonresponse to PWM in young donors relates to an override of functionally intact B cells by T-regulator influences.     

Differential Targeting of Viral Components by CD4+ versus CD8+ T Lymphocytes in Dengue Virus Infection

Dengue virus (DENV) is the principal arthropod-borne viral pathogen afflicting human populations. While repertoires of antibodies to DENV have been linked to protection or enhanced infection, the role of T lymphocytes in these processes remains poorly defined. This study provides a comprehensive overview of CD4⁺ and CD8⁺ T cell epitope reactivities against the DENV 2 proteome in adult patients experiencing secondary DENV infection. Dengue virus-specific T cell responses directed against an overlapping 15mer peptide library spanning the DENV 2 proteome were analyzed ex vivo by enzyme-linked immunosorbent spot assay, and recognition of individual peptides was further characterized in specific T cell lines. Thirty novel T cell epitopes were identified, 9 of which are CD4⁺ and 21 are CD8⁺ T cell epitopes. We observe that whereas CD8⁺ T cell epitopes preferentially target nonstructural proteins (NS3 and NS5), CD4⁺ epitopes are skewed toward recognition of viral components that are also targeted by B lymphocytes (envelope, capsid, and NS1). Consistently, a large proportion of dengue virus-specific CD4⁺ T cells have phenotypic characteristics of circulating follicular helper T cells (CXCR5 expression and production of interleukin-21 or gamma interferon), suggesting that they are interacting with B cells in vivo. This study shows that during a dengue virus infection, the protein targets of human CD4⁺ and CD8⁺ T cells are largely distinct, thus highlighting key differences in the immunodominance of DENV proteins for these two cell types. This has important implications for our understanding of how the two arms of the human adaptive immune system are differentially targeted and employed as part of our response to DENV infection.     

IL-7 promotes CD95-induced apoptosis in B cells via the IFN-γ/STAT1 pathway

Interleukin-7 (IL-7) concentrations are increased in the blood of CD4+ T cell depleted individuals, including HIV-1 infected patients. High IL-7 levels might stimulate T cell activation and, as we have shown earlier, IL-7 can prime resting T cell to CD95 induced apoptosis as well. HIV-1 infection leads to B cell abnormalities including increased apoptosis via the CD95 (Fas) death receptor pathway and loss of memory B cells. Peripheral B cells are not sensitive for IL-7, due to the lack of IL-7Ra expression on their surface; however, here we demonstrate that high IL-7 concentration can prime resting B cells to CD95-mediated apoptosis via an indirect mechanism. T cells cultured with IL-7 induced high CD95 expression on resting B cells together with an increased sensitivity to CD95 mediated apoptosis. As the mediator molecule responsible for B cell priming to CD95 mediated apoptosis we identified the cytokine IFN-γ that T cells secreted in high amounts in response to IL-7. These results suggest that the lymphopenia induced cytokine IL-7 can contribute to the increased B cell apoptosis observed in HIV-1 infected individuals.     

The role of CD40-CD154 interaction in antiviral T cell-independent IgG responses

Polyomavirus (PyV) infection elicits protective T cell-independent (TI) IgG responses in T cell-deficient mice. The question addressed in this report is whether CD40 signaling plays a role in this TI antiviral IgG response. Because CD40 ligand (CD40L) can be expressed on numerous cell types in addition to activated T cells, it is possible that cells other than T cells provide CD40L to signal through CD40 on B cells and hence positively influence the antiviral TI IgG responses. In this study we show, by blocking CD40-CD40L interactions in vivo with anti-CD40L Ab treatment in TCR betaxdelta-/- mice and by using SCID mice reconstituted with CD40-/- B cells, that the lack of CD40 signaling in B cells results in a 50% decrease in TI IgG secreted in response to PyV. SCID mice reconstituted with CD40L-/- B cells also responded to PyV infection with diminished IgG secretion compared with that of SCID mice reconstituted with wild-type B cells. This finding suggests that B cells may provide the CD40L for CD40 signaling in the absence of T cell help during acute virus infection. Our studies demonstrate that, although about half of the TI IgG responses to PyV are independent of CD40-CD40L interactions, these interactions occur in T cell-deficient mice and enhance antiviral TI Ab responses.     

Human cytomegalovirus differentially controls B cell and T cell responses through effects on plasmacytoid dendritic cells

Plasmacytoid dendritic cells (PDCs), the main producers of type I IFN in response to viral infection, are essential in antiviral immunity. In this study, we assessed the effect of human CMV (HCMV) infection on PDC function and on downstream B and T cell responses in vitro. HCMV infection of human PDCs was nonpermissive, as immediate-early but not late viral Ags were detected. HCMV led to partial maturation of PDCs and up-regulated MHC class II and CD83 molecules but not the costimulatory molecules CD80 and CD86. Regardless of viral replication, PDCs secreted cytokines after contact with HCMV, including IFN-alpha secretion that was blocked by inhibitory CpG, suggesting an engagement of the TLR7 and/or TLR9 pathways. In the presence of B cell receptor stimulation, soluble factors produced by HCMV-matured PDCs triggered B cell activation and proliferation. Through PDC stimulation, HCMV prompted B cell activation, but only induced Ab production in the presence of T cells or T cell secreted IL-2. Conversely, HCMV hampered the allostimulatory ability of PDCs, leading to decreased proliferation of CD4(+) and CD8(+) T cells. These findings reveal a novel mechanism by which HCMV differentially controls humoral and cell-mediate immune responses through effects on PDCs.     

B Cell Responses to HIV Antigen Are a Potent Correlate of Viremia in HIV-1 Infection and Improve with PD-1 Blockade

Infection with Human Immunodeficiency Virus Type 1 (HIV-1) induces defects of both cellular and humoral immune responses. Impaired CD4+ T cell help and B cell dysfunction may partially explain the low frequency of broadly neutralizing antibodies in HIV-infected individuals. To understand the extent of B cell dysfunction during HIV infection, we assessed the level of B cell activation at baseline and after stimulation with a variety of antigens. Increased levels of viremia were associated with higher baseline expression of the activation marker CD86 on B cells and with decreased ability of B cells to increase expression of CD86 after in vitro stimulation with inactivated HIV-1. In a series of cell isolation experiments B cell responses to antigen were enhanced in the presence of autologous CD4+ T cells. HIV infected individuals had a higher frequency of PD-1 expression on B cells compared to HIV- subjects and PD-1 blockade improved B cell responsiveness to HIV antigen, suggesting that inhibitory molecule expression during HIV-1 infection may contribute to some of the observed B cell defects. Our findings demonstrate that during chronic HIV infection, B cells are activated and lose full capacity to respond to antigen, but suppression of inhibitory pressures as well as a robust CD4+ T cell response may help preserve B cell function.     

Decreased dengue replication and an increased anti-viral humoral response with the use of combined Toll-like receptor 3 and 7/8 agonists in macaques

Background

Pathogenic versus protective outcomes to Dengue virus (DENV) infection are associated with innate immune function. This study aimed to determine the role of increased TLR3- and TLR7/8-mediated innate signaling after Dengue infection of rhesus macaques in vivo to evaluate its impact on disease and anti-DENV immune responses.

Methodology/Principal Findings

TLR3 and TLR7/8 agonists (emulsified in Montanide) were administered subcutaneously to rhesus macaques at 48 hours and 7 days after DENV infection. The Frequency and activation of myeloid dendritic cells, plasmacytoid dendritic cells, and B cells were measured by flow cytometry while the serum levels of 14 different cytokines and chemokines were quantified. Adaptive immune responses were measured by DENV-specific antibody subtype measurements. Results showed that the combined TLR agonists reduced viral replication and induced the development of a proinflammatory reaction, otherwise absent in Dengue infection alone, without any clear signs of exacerbated disease. Specifically, the TLR-induced response was characterized by activation changes in mDC subsets concurrent with higher serum levels of CXCL-10 and IL-1Ra. TLR stimulation also induced higher titers of anti-DENV antibodies and acted to increase the IgG2/IgG1 ratio of anti-DENV to favor the subtype associated with DENV control. We also observed an effect of DENV-mediated suppression of mDC activation consistent with prior in vitro studies.

Conclusions/Significance

These data show that concurrent TLR3/7/8 activation of the innate immune response after DENV infection in vivo acts to increase antiviral mechanisms via increased inflammatory and humoral responses in rhesus macaques, resulting in decreased viremia and melioration of the infection. These findings underscore an in vivo protective rather than a pathogenic role for combined TLR3/7/8-mediated activation in Dengue infection of rhesus macaques. Our study provides definitive proof-of-concept into the mechanism by which DENV evades immune recognition and activation in vivo.     

Infected Cell Killing by HIV-1 Protease Promotes NF-κB Dependent HIV-1 Replication

Acute HIV-1 infection of CD4 T cells often results in apoptotic death of infected cells, yet it is unclear what evolutionary advantage this offers to HIV-1. Given the independent observations that acute T cell HIV-1 infection results in (1) NF-κB activation, (2) caspase 8 dependent apoptosis, and that (3) caspase 8 directly activates NF-κB, we questioned whether these three events might be interrelated. We first show that HIV-1 infected T cell apoptosis, NF-κB activation, and caspase 8 cleavage by HIV-1 protease are coincident. Next we show that HIV-1 protease not only cleaves procaspase 8, producing Casp8p41, but also independently stimulates NF-κB activity. Finally, we demonstrate that the HIV protease cleavage of caspase 8 is necessary for optimal NF-κB activation and that the HIV-1 protease specific cleavage fragment Casp8p41 is sufficient to stimulate HIV-1 replication through NF-κB dependent HIV-LTR activation both in vitro as well as in cells from HIV infected donors. Consequently, the molecular events which promote death of HIV-1 infected T cells function dually to promote HIV-1 replication, thereby favoring the propagation and survival of HIV-1.     

Dengue virus-specific CD4+ and CD8+ T lymphocytes target NS1, NS3 and NS5 in infected Indian rhesus macaques

Every year, Dengue virus (DENV) infects approximately 100 million people. There are currently several vaccines undergoing clinical studies, but most target the induction of neutralizing antibodies. Unfortunately, DENV infection can be enhanced by subneutralizing levels of antibodies that bind virions and deliver them to cells of the myeloid lineage, thereby increasing viral replication (termed antibody-dependent enhancement [ADE]). T lymphocyte-based vaccines may offer an alternative that avoids ADE. The goal of our study was to describe the cellular immune response generated after primary DENV infection in Indian rhesus macaques. We infected eight rhesus macaques with 10⁵ plaque-forming units (PFU) of DENV serotype 2 (DENV2) New Guinea C (NGC) strain, and monitored viral load and the cellular immune response to the virus. Viral replication peaked at day 4 post-infection and was resolved by day 10. DENV-specific CD4⁺ and CD8⁺ T lymphocytes targeted nonstructural (NS) 1, NS3 and NS5 proteins after resolution of peak viremia. DENV-specific CD4⁺ cells expressed interferon-gamma (IFN-γ) along with tumor necrosis factor-alpha (TNF-α), interleukin-2 (IL-2), and macrophage inflammatory protein-1 beta (MIP-1β). In comparison, DENV-specific CD8⁺ cells expressed IFN-γ in addition to MIP-1β and TNF-α and were positive for the degranulation marker CD107a. Interestingly, a fraction of the DENV-specific CD4⁺ cells also stained for CD107a, suggesting that they might be cytotoxic. Our results provide a more complete understanding of the cellular immune response during DENV infection in rhesus macaques and contribute to the development of rhesus macaques as an animal model for DENV vaccine and pathogenicity studies.     

Induction of IL-6 (B cell stimulatory factor-2/IFN-beta 2) production by HIV

Polyclonal B cell activation is commonly observed in AIDS and in infection with HIV. The effect of HIV on the induction of B cell stimulatory factor 2 (BSF-2) production was examined, since BSF-2 plays an essential role in the differentiation of activated B cells to Ig-secreting cells. Increased BSF-2 mRNA levels and increased BSF-2 secretion were observed soon after exposure of mononuclear cells isolated from healthy donors to both "live" and inactivated HIV. HIV-induced BSF-2 production was seen in monocyte/macrophages, but not in T cells. These results suggest that the HIV-induced overproduction of BSF-2 might contribute to the polyclonal B cell activation seen in AIDS and in infection with HIV.     

Outer Membrane Protein A (OmpA) of Shigella flexneri 2a Induces TLR2-Mediated Activation of B Cells: Involvement of Protein Tyrosine Kinase,ERK and NF-κB

B cells are critically important in combating bacterial infections and their differentiation into plasma cells and memory cells aids bacterial clearance and long-lasting immunity conferred by essentially all vaccines. Outer membrane protein A (OmpA) of Shigella flexneri 2a has been demonstrated to induce the production of IgG and IgA in vivo following immunization of mice through intranasal route, but the direct involvement of B cells in OmpA-mediated immune regulation was not determined. Consequently, we investigated whether OmpA can modulate B cell functions and identified the molecular events involved in OmpA-induced B cell immune response in vitro. We show that OmpA of S. flexneri 2a activates B cells to produce protective cytokines, IL-6 and IL-10 as well as facilitates their differentiation into antibody secreting cells (ASCs). The immunostimulatory properties of OmpA are attributed to the increased surface expression of MHCII and CD86 on B cells. We also report here that B cell activation by OmpA is mediated strictly through recognition by TLR2, resulting in initiation of cascades of signal transduction events, involving increased phosphorylation of protein tyrosine kinases (PTKs), ERK and IκBα, leading to nuclear translocation of NF-κB. Importantly, a TLR2 antibody diminishes OmpA-induced upregulation of MHCII and CD86 on B cell surface as well as significantly inhibits B cell differentiation and cytokine secretion. Furthermore, we illustrate that B cell differentiation into ASCs and induction of cytokine secretion by OmpA are dependent on PTKs activity. Moreover, we identify that OmpA-induced B cell differentiation is entirely dependent on ERK pathway, whereas both NF-κB and ERK are essential for cytokine secretion by B cells. Overall, our data demonstrate that OmpA of S. flexneri 2a amplifies TLR signaling in B cells and triggers B cell immune response, which is critical for the development of an effective adaptive immunity to an optimal vaccine antigen.     

The type-specific capsular carbohydrate of Hemophilus influenzae B is a potent mitogen for murine B lymphocytes

In this study we investigated the in vitro mitogenic properties of the capsular carbohydrate of Hemophilus influenzae b, polyribosylribitolphosphate (PRP). PRP was found to be a potent polyclonal activator of murine B lymphocytes. PRP induced normal B cells to undergo blastogenesis, DNA synthesis, and differentiation to IgM and IgG secretion. IgG3 accounted for the majority of the IgG. No PRP-specific antibody was detectable, indicating the polyclonal origin of the secreted immunoglobulin (Ig). T lymphocytes were neither activated by PRP nor required for B cell proliferation or Ig secretion. In addition, T cell-depleted spleen cells also depleted of accessory (A) cells by passage through Sephadex G-10 retained responsiveness to PRP. Trace lipopolysaccharide (LPS) contamination was not responsible for the mitogenic effect, as shown by the ability of C3H/HeJ spleen cells to proliferate in response to PRP and by the failure of polymyxin B to inhibit PRP-induced DNA synthesis. The B cell responses induced by PRP and LPS were similar with respect to T cell and A cell independence, to the magnitude of DNA synthesis, and to Ig secretion and the Ig isotypes expressed. These data, taken with the finding that the combination of optimal doses of PRP and LPS did not give an additive DNA synthetic response, indicate that PRP and LPS were activating similar B cell populations. However, in contrast to LPS, PRP was capable of inducing significant DNA synthesis in cultures containing as few as 1,000 B cells, suggesting that PRP-driven proliferation was less dependent on cellular interactions than the response to LPS. The differential ability of PRP and LPS to stimulate C3H/HeJ B cells and to stimulate B cell proliferation at low density indicates basic differences between these two mitogens in their mechanisms of B cell activation.     

Activation of marginal zone B cells from lupus mice with type A(D) CpG-oligodeoxynucleotides

Several types of CpG-oligodeoxynucleotides (ODN) have been recently characterized. In mice, type A(D) CpG-ODNs primarily stimulate macrophages and dendritic cells, but fail to stimulate B cells. On the contrary, type B(K) CpG-ODNs are excellent B cell activators. Type C CpG-ODNs combine features of both types A(D) and B(K) CpG-ODNs. Despite cell type preferences, all CpG-ODNs require the presence of TLR9 for activation. In this study, we show that a subset of B cells from lupus mice responds to type A(D) CpG-ODN stimulation vigorously and directly with increased CD25 and CD86 expression and IL-10 secretion. Furthermore, these CpG-ODNs induce high surface IgM expression and promote 50- to 100-fold higher IgM and IgG3 secretion in lupus B cells than in controls. This response is similar to that seen with bacterial DNA stimulation of B cells. Type A(D)-responsive cells are enriched within lupus B cells with the marginal zone (MZ) phenotype. These cells are at least twice more numerous in lupus mice than in controls. The ability of lupus B cells to respond to type A(D) CpG-ODN stimulation is not due to differential TLR9 expression. Therefore, type A(D) CpG-ODNs may contribute to the lupus pathogenesis by inducing MZ-B cell activation, costimulatory molecule expression, and polyclonal Ig secretion. Through increased IL-10 secretion, MZ-B cells may also modify the activity of other cell types, particularly dendritic cells and macrophages.     

Cytomegalovirus pp71 Protein Is Expressed in Human Glioblastoma and Promotes Pro-Angiogenic Signaling by Activation of Stem Cell Factor

Glioblastoma multiforme (GBM) is a highly malignant primary central nervous system neoplasm characterized by tumor cell invasion, robust angiogenesis, and a mean survival of 15 months. Human cytomegalovirus (HCMV) infection is present in >90% of GBMs, although the role the virus plays in GBM pathogenesis is unclear. We report here that HCMV pp71, a viral protein previously shown to promote cell cycle progression, is present in a majority of human GBMs and is preferentially expressed in the CD133+, cancer stem-like cell population. Overexpression of pp71 in adult neural precursor cells resulted in potent induction of stem cell factor (SCF), an important pro-angiogenic factor in GBM. Using double immunofluorescence, we demonstrate in situ co-localization of pp71 and SCF in clinical GBM specimens. pp71 overexpression in both normal and transformed glial cells increased SCF secretion and this effect was specific, since siRNA mediated knockdown of pp71 or treatment with the antiviral drug cidofovir resulted in decreased expression and secretion of SCF by HCMV-infected cells. pp71- induced upregulation of SCF resulted in downstream activation of its putative endothelial cell receptor, c-kit, and angiogenesis as measured by increased capillary tube formation in vitro. We demonstrate that pp71 induces a pro-inflammatory response via activation of NFΚB signaling which drives SCF expression. Furthermore, we show that pp71 levels and NFKB activation are selectively augmented in the mesenchymal subtype of human GBMs, characterized by worst patient outcome, suggesting that HCMV pp71-induced paracrine signaling may contribute to the aggressive phenotype of this human malignancy.     

CD4 T cell-independent antibody response promotes resolution of primary influenza infection and helps to prevent reinfection

It is generally believed that the production of influenza-specific IgG in response to viral infection is dependent on CD4 T cells. However, we previously observed that CD40-deficient mice generate influenza-specific IgG during a primary infection, suggesting that influenza infection may elicit IgG responses independently of CD4 T cell help. In the present study, we tested this hypothesis and show that mice lacking CD40 or CD4 T cells produce detectable titers of influenza-specific IgG and recover from influenza infection in a manner similar to that of normal mice. In contrast, mice completely lacking B cells succumb to influenza infection, despite the presence of large numbers of functional influenza-specific CD8 effector cells in the lungs. Consistent with the characteristics of a T-independent Ab response, long-lived influenza-specific plasma cells are not found in the bone marrow of CD40-/- and class II-/- mice, and influenza-specific IgG titers wane within 60 days postinfection. However, despite the short-lived IgG response, CD40-/- and class II-/- mice are completely protected from challenge infection with the same virus administered within 30 days. This protection is mediated primarily by B cells and Ab, as influenza-immune CD40-/- and class II-/- mice were still resistant to challenge infection when T cells were depleted. These data demonstrate that T cell-independent influenza-specific Ab promotes the resolution of primary influenza infection and helps to prevent reinfection.     

B cell response to T helper cell subsets. II. Both the stage of T cell differentiation and the cytokines secreted determine the extent and nature of helper activity.

Helper activity of several murine CD4+ T cell subsets was examined. Effector Th, derived from naive cells after 4 days of in vitro stimulation with alloantigen, when generated in the presence of IL-4, secreted high levels of IL-4, IL-5, and IL-6, and low levels of IL-2 and IFN-gamma, and induced the secretion of all Ig isotypes particularly IgM, IgG1, IgA, and IgE from resting allogeneic B cells. Effectors generated with IL-6 secreted IL-2, IL-4, IL-5, IL-6, and IFN-gamma, and induced similar levels of total Ig, 25 to 35 micrograms/ml, but with IgM, IgG3, IgG1, and IgG2a isotypes predominating. Helper activity of these Th was significantly greater than that of effectors generated with IL-2 (10-15 micrograms/ml Ig) and of 24-h-activated naive and memory cells (2-4 micrograms/ml), both of which induced mainly IgM. Unlike other isotypes, IgE was induced only by effector Th generated with IL-4. Blocking studies showed that secretion of all isotypes in response to IL-6-primed effectors was dependent on IL-2, IL-5, and IL-6. IL-4 was required for optimal IgM, IgG1, and IgA secretion, but limited secretion of IgG2a, whereas IFN-gamma was required for optimal IgG2a secretion, and limited IgM, IgG1, and IgA. In contrast, secretion of all isotypes in response to IL-4-primed effectors was dependent on IL-5, although IL-4 and IFN-gamma were also essential for IgE and IgG2a, respectively. Addition of exogenous IL-5 to B cell cultures driven by IL-6-primed effectors did not obviate the requirement for IL-2, IL-4, and IL-6, suggesting that interaction of IL-4-primed effectors with B cells was qualitatively different from that of IL-6-primed effectors, driving B cells to a stage requiring only IL-5 for differentiation. Addition of exogenous factors to IL-2-primed effector Th, particularly IL-4 in the presence of anti-IFN-gamma, resulted in levels of Ig, including IgE, comparable to those induced with other effectors. These results show that functionally distinct Th cell subsets can be generated rapidly in vitro, under the influence of distinct cytokines, which vary dramatically in their levels of help for resting B cells. The cytokines involved in responses to distinct Th cells differ depending on the quality of interaction with the B cell, and the extent of help is strongly determined by the quantity and nature of cytokines secreted by the T cells.     

Identification of IFN-γ-producing innate B cells

Although B cells play important roles in the humoral immune response and the regulation of adaptive immunity, B cell subpopulations with unique phenotypes, particularly those with non-classical immune functions, should be further investigated. By challenging mice with Listeria monocytogenes, Escherichia coli, vesicular stomatitis virus and Toll-like receptor ligands, we identified an inducible CD11a^hiFcγRIII^hi B cell subpopulation that is significantly expanded and produces high levels of IFN-γ during the early stage of the immune response. This subpopulation of B cells can promote macrophage activation via generating IFN-γ, thereby facilitating the innate immune response against intracellular bacterial infection. As this new subpopulation is of B cell origin and exhibits the phenotypic characteristics of B cells, we designated these cells as IFN-γ-producing innate B cells. Dendritic cells were essential for the inducible generation of these innate B cells from the follicular B cells via CD40L-CD40 ligation. Increased Bruton''s tyrosine kinase activation was found to be responsible for the increased activation of non-canonical NF-κB pathway in these innate B cells after CD40 ligation, with the consequent induction of additional IFN-γ production. The identification of this new population of innate B cells may contribute to a better understanding of B cell functions in anti-infection immune responses and immune regulation.