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.     

Skin immune responses to peptide and protein antigen are TLR4 independent

Little is known about the innate immune mechanisms regulating adaptive immune responses elicited through the skin. Tissue injury is postulated to liberate Toll like receptor 4 (TLR4) ligands. In this study, we determined whether TLR4 signaling modulates the response to epidermal injury induced by tape stripping (TS) and whether it alters humoral and cellular immune responses generated through epicutaneous immunization with peptide+cholera toxin (CT). The combined use of cholera toxin and TS with antigen promoted optimal antigen-specific CD4(+) and CD8(+) T cell proliferation in Balb/c and C57BL/6 mice, respectively. TLR4 mutant mice had similar T cell responses to wild type mice. Further, OVA-protein specific IgG, IgG(1), IgG(2a), and IgE titers were similar in wild type and TLR4 mutant mice. Thus, TLR4 signaling was not required for the generation of epicutaneous T cell or antibody mediated immune responses and did not alter the quality of the immune responses elicited.     

Toll-like receptor ligands modulate dendritic cells to augment cytomegalovirus- and HIV-1-specific T cell responses

Optimal Ag targeting and activation of APCs, especially dendritic cells (DCs), are important in vaccine development. In this study, we report the effects of different Toll-like receptor (TLR)-binding compounds to enhance immune responses induced by human APCs, including CD123(+) plasmacytoid DCs (PDCs), CD11c(+) myeloid DCs (MDCs), monocytes, and B cells. PDCs, which express TLR7 and TLR9, responded to imidazoquinolines (imiquimod and R-848) and to CpG oligodeoxynucleotides stimulation, resulting in enhancement in expression of costimulatory molecules and induction of IFN-alpha and IL-12p70. In contrast, MDCs, which express TLR3, TLR4, and TLR7, responded to poly(I:C), LPS, and imidazoquinolines with phenotypic maturation and high production of IL-12 p70 without producing detectable IFN-alpha. Optimally TLR ligand-stimulated PDCs or MDCs exposed to CMV or HIV-1 Ags enhanced autologous CMV- and HIV-1-specific memory T cell responses as measured by effector cytokine production compared with TLR ligand-activated monocytes and B cells or unstimulated PDCs and MDCs. Together, these data show that targeting specific DC subsets using TLR ligands can enhance their ability to activate virus-specific T cells, providing information for the rational design of TLR ligands as adjuvants for vaccines or immune modulating therapy.     

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.     

Induction of CD4(+) T-cell-independent immunoglobulin responses by inactivated influenza virus

Through cognate interaction between antigen-specific B-cell and CD4(+) alphabeta T cells, the CD4(+) alphabeta T cells secrete cytokines that initiate immunoglobulin (Ig) class switching from IgM to IgG. In this study, we show that formalin-inactivated influenza PR8 virus induces virus-specific IgM and IgG responses in the absence of CD4(+) T cells and that all four subclasses of IgG are produced. The immunized CD4-deficient mice were also found to be completely protected against lethal infection with live, pathogenic influenza virus. The ability of CD4(+) T-cell-deficient mice to generate these IgG responses was not found to be impaired when these mice were depleted of CD8(+) T cells with an anti-CD8 monoclonal antibody. In contrast, alphabeta T-cell-deficient mice (TCRbeta(-/-)) were not found to produce significant amounts of IgG upon immunization with formalin-inactivated PR8 virus. These results suggest that CD4(-) CD8(-) double-negative alphabeta T cells are playing a role in regulating Ig class switching in the absence of CD4(+) T cells.     

Immunization with HIV-1 Gag protein conjugated to a TLR7/8 agonist results in the generation of HIV-1 Gag-specific Th1 and CD8+ T cell responses

One strategy to induce optimal cellular and humoral immune responses following immunization is to use vaccines or adjuvants that target dendritic cells and B cells. Activation of both cell types can be achieved using specific TLR ligands or agonists directed against their cognate receptor. In this study, we compared the ability of the TLR7/8 agonist R-848, which signals only via TLR7 in mice, with CpG oligodeoxynucleotides for their capacity to induce HIV-1 Gag-specific T cell and Ab responses when used as vaccine adjuvants with HIV-1 Gag protein in mice. Injection of R-848 and CpG oligodeoxynucleotides alone enhanced the innate immune responses in vivo as demonstrated by high serum levels of inflammatory cytokines, including IL-12p70 and IFN-alpha, and increased expression of CD80, CD86, and CD40 on CD11c(+) dendritic cells. By contrast, R-848 was a relatively poor adjuvant for inducing primary Th1 or CD8(+) T cell responses when administered with HIV-1 Gag protein. However, when a TLR7/8 agonist structurally and functionally similar to R-848 was conjugated to HIV-1 Gag protein both Th1 and CD8(+) T cells responses were elicited as determined by intracellular cytokine and tetramer staining. Moreover, within the population of HIV-1 Gag-specific CD8(+) CD62(low) cells, approximately 50% of cells expressed CD127, a marker shown to correlate with the capacity to develop into long-term memory cells. Overall, these data provide evidence that TLR7/8 agonists can be effective vaccine adjuvants for eliciting strong primary immune responses with a viral protein in vivo, provided vaccine delivery is optimized.     

TLR9 signaling in B cells determines class switch recombination to IgG2a

Although IgG2a is the most potent Ab isotype in the host response to viral and bacterial infections, the regulation of class switch recombination to IgG2a in vivo is not yet well understood. Recognition of pathogen-associated molecular patterns by dendritic cells expressing TLRs, like TLR7, recognizing ssRNA, or TLR9, recognizing DNA rich in nonmethylated CG motifs (CpG), favors induction of Th1 responses. It is generally assumed that these Th1 responses are responsible for the TLR-mediated induction of IgG2a. Using virus-like particles loaded with CpGs, we show here that TLR9 ligands can directly stimulate B cells to undergo isotype switching to IgG2a. Unexpectedly, TLR9 expression in non-B cells did not affect isotype switching in the Ab response against virus-like particles. Thus, TLR9 can regulate isotype switching to IgG2a directly by interacting with B cells rather than indirectly by inducing Th1 responses.     

Differential roles for RIG-I-like receptors and nucleic acid-sensing TLR pathways in controlling a chronic viral infection

The necessity for pathogen recognition of viral infection by the innate immune system in initiating early innate and adaptive host defenses is well documented. However, little is known about the role these receptors play in the maintenance of adaptive immune responses and their contribution to resolution of persistent viral infections. In this study, we demonstrate a nonredundant functional requirement for both nucleic acid-sensing TLRs and RIG-I-like receptors in the control of a mouse model of chronic viral infection. Whereas the RIG-I-like receptor pathway was important for production of type I IFNs and optimal CD8(+) T cell responses, nucleic acid-sensing TLRs were largely dispensable. In contrast, optimal anti-viral Ab responses required intact signaling through nucleic acid-sensing TLRs, and the absence of this pathway correlated with less virus-specific Ab and deficient long-term virus control of a chronic infection. Surprisingly, absence of the TLR pathway had only modest effects on Ab production in an acute infection with a closely related virus strain, suggesting that persistent TLR stimulation may be necessary for optimal Ab responses in a chronic infection. These results indicate that innate virus recognition pathways may play critical roles in the outcome of chronic viral infections through distinct mechanisms.     

Differential role of TLR- and RLR-signaling in the immune responses to influenza A virus infection and vaccination

The innate immune system recognizes influenza A virus via TLR 7 or retinoic acid-inducible gene I in a cell-type specific manner in vitro, however, physiological function(s) of the MyD88- or interferon-beta promoter stimulator 1 (IPS-1)-dependent signaling pathways in antiviral responses in vivo remain unclear. In this study, we show that although either MyD88- or IPS-1-signaling pathway was sufficient to control initial antiviral responses to intranasal influenza A virus infection, mice lacking both pathways failed to show antiviral responses, resulting in increased viral load in the lung. By contrast, induction of B cells or CD4 T cells specific to the dominant hemagglutinin or nuclear protein Ags respectively, was strictly dependent on MyD88 signaling, but not IPS-1 signaling, whereas induction of nuclear protein Ag-specific CD8 T cells was not impaired in the absence of either MyD88 or IPS-1. Moreover, vaccination of TLR7- and MyD88-deficient mice with inactivated virus failed to confer protection against a lethal live virus challenge. These results strongly suggest that either the MyD88 or IPS-1 signaling pathway is sufficient for initial antiviral responses, whereas the protective adaptive immune responses to influenza A virus are governed by the TLR7-MyD88 pathway.     

A nonredundant role for IL-21 receptor signaling in plasma cell differentiation and protective type 2 immunity against gastrointestinal helminth infection

Pathogen-specific Ab production following infection with the gut-dwelling roundworm Heligmosomoides polygyrus is critical for protective immunity against reinfection. However, the factors required for productive T cell-B cell interactions in the context of a type 2-dominated immune response are not well defined. In the present study, we identify IL-21R signaling as a critical factor in driving pathogen-specific plasma cell differentiation and protective immunity against H. polygyrus in mice. We show that B cells require direct IL-21R signals to differentiate into CD138(+) plasma cells. In contrast, IL-21R signaling is dispensable for germinal center formation, isotype class switching, and Th2 and T follicular helper cell differentiation. Our studies demonstrate a selective role for IL-21 in plasma cell differentiation in the context of protective antiparasitic type 2 immunity.     

Asparagine Endopeptidase Controls Anti-Influenza Virus Immune Responses through TLR7 Activation

Intracellular Toll-like receptors (TLRs) expressed by dendritic cells recognize nucleic acids derived from pathogens and play an important role in the immune responses against the influenza virus (IAV), a single-stranded RNA sensed by different receptors including TLR7. However, the importance of TLR7 processing in the development of anti-viral immune responses is not known. Here we report that asparagine endopeptidase (AEP) deficient mice are unable to generate a strong anti-IAV response, as demonstrated by reduced inflammation, cross presentation of cell-associated antigens and priming of CD8⁺ T cells following TLR7-dependent pulmonary infection induced by IAV. Moreover, AEP deficient lung epithelial- or myeloid-cells exhibit impaired TLR7 signaling due to defective processing of this receptor. Indeed, TLR7 requires a proteolytic cleavage by AEP to generate a C-terminal fragment competent for signaling. Thus, AEP activity is critical for TLR7 processing, opening new possibilities for the treatment of influenza and TLR7-dependent inflammatory diseases.     

MyD88 is required for the formation of long-term humoral immunity to virus infection

Development of long-term humoral immunity is a major goal of vaccination, but the mechanisms involved in the formation of long-term Ab responses are still being determined. In this study, we identify a previously unknown requirement for MyD88, an adaptor molecule that mediates signals at most TLRs, for the generation of long-term humoral immunity during live virus infection. Polyoma virus-infected MyD88 knockout mice generated strong acute T cell-dependent antiviral IgM and IgG responses and developed germinal centers. Activation-induced cytidine deaminase, an enzyme required for isotype switching and somatic hypermutation, was also induced in germinal center B cells, similar to wild-type mice. However, MyD88 knockout mice failed to develop bone marrow plasma cells and did not maintain long-term serum antiviral Ab responses. The isotype distribution of antiviral IgG responses was also altered; serum IgG2a and IgG2b levels were diminished, whereas IgG1 responses were not affected. The requirement for MyD88 for the formation of long-term humoral immunity to polyoma virus was intrinsic to B cells and was independent of IL-1R and IL-18R, cytokine receptors that also signal through MyD88. Our findings show that MyD88-dependent signaling pathways in B cells are essential for effectively generating long-term Ab responses and implicate a role for TLR in the formation of long-term humoral immunity.     

Toll-like receptor 4-dependent activation of dendritic cells by a retrovirus

Mouse mammary tumor virus (MMTV) is a milk-borne retrovirus that exploits the adaptive immune system. It has recently been shown that MMTV activates B cells via Toll-like receptor 4 (TLR4), a molecule involved in innate immune responses. Here, we show that direct virus binding to TLR4 induced maturation of bone marrow-derived dendritic cells and up-regulated expression of the MMTV entry receptor (CD71) on these cells. In vivo, MMTV increased the number of dendritic cells in neonatal Peyer's patches and their expression of CD71; both these effects were dependent on TLR4. Thus, retroviral signaling through TLRs plays a critical role in dendritic-cell participation during infection.     

Regulation of T cell activation by TLR ligands

Regulatory T cells (Treg) maintain peripheral tolerance and play a critical role in the control of the immune response in infection, tumor defense, organ transplantation and allergy. CD4(+)CD25(high) Treg suppress the proliferation and cytokine production of CD4(+)CD25(-) responder T cells. The suppression requires cell-cell-contact and/or production of inhibitory cytokines like IL-10 or TGF-β. The current knowledge about the regulation of Treg suppressive function is limited. Toll-like receptors (TLR) are widely expressed in the innate immune system. They recognize conserved microbial ligands such as lipopolysaccharide, bacterial lipopeptides or viral and bacterial RNA and DNA. TLR play an essential role in innate immune responses and in the initiation of adaptive immune responses. However, certain TLR are also expressed in T lymphocytes, and the respective ligands can directly modulate T cell function. TLR2, TLR3, TLR5 and TLR9 act as costimulatory receptors to enhance proliferation and/or cytokine production of T-cell receptor-stimulated T lymphocytes. In addition, TLR2, TLR5 and TLR8 modulate the suppressive activity of naturally occurring CD4(+)CD25(high) Treg. The direct responsiveness of T lymphocytes to TLR ligands offers new perspectives for the immunotherapeutic manipulation of T cell responses. In this article we will discuss the regulation of Treg and other T cell subsets by TLR ligands.     

Cutting edge: enhancement of antibody responses through direct stimulation of B and T cells by type I IFN

Type I IFN (IFN-alphabeta) is induced rapidly by infection and plays a key role in innate antiviral defense. IFN-alphabeta also exerts stimulatory effects on the adaptive immune system and has been shown to enhance Ab and T cell responses. We have investigated the importance of B and T cells as direct targets of IFN-alphabeta during IFN-alpha-mediated augmentation of the Ab response against a soluble protein Ag. Strikingly, the ability of IFN-alpha to stimulate the Ab response and induce isotype switching was markedly reduced in mice in which B cells were selectively deficient for the IFN-alphabetaR. Moreover, IFN-alpha-mediated enhancement of the Ab response was also greatly impaired in mice in which T cells were selectively IFN-alphabetaR-deficient. These results indicate that IFN-alphabetaR signaling in both B and T cells plays an important role in the stimulation of Ab responses by IFN-alphabeta.     

Saturated and polyunsaturated fatty acids reciprocally modulate dendritic cell functions mediated through TLR4

TLRs provide critical signals to induce innate immune responses in APCs such as dendritic cells (DCs) that in turn link to adaptive immune responses. Results from our previous studies demonstrated that saturated fatty acids activate TLRs, whereas n-3 polyunsaturated fatty acids inhibit agonist-induced TLR activation. These results raise a significant question as to whether fatty acids differentially modulate immune responses mediated through TLR activation. The results presented in this study demonstrate that the saturated fatty acid, lauric acid, up-regulates the expression of costimulatory molecules (CD40, CD80, and CD86), MHC class II, and cytokines (IL-12p70 and IL-6) in bone marrow-derived DCs. The dominant negative mutant of TLR4 or its downstream signaling components inhibits lauric acid-induced expression of a CD86 promoter-reporter gene. In contrast, an n-3 polyunsaturated fatty acid, docosahexaenoic acid, inhibits TLR4 agonist (LPS)-induced up-regulation of the costimulatory molecules, MHC class II, and cytokine production. Similarly, DCs treated with lauric acid show increased T cell activation capacity, whereas docosahexaenoic acid inhibits T cell activation induced by LPS-treated DCs. Together, our results demonstrate that the reciprocal modulation of both innate and adaptive immune responses by saturated fatty acid and n-3 polyunsaturated fatty acid is mediated at least in part through TLRs. These results imply that TLRs are involved in sterile inflammation and immune responses induced by nonmicrobial endogenous molecules. These results shed new light in understanding how types of dietary fatty acids differentially modulate immune responses that could alter the risk of many chronic diseases.     

Toll-like receptor ligands induce human T cell activation and death, a model for HIV pathogenesis

BACKGROUND: Recently, heightened systemic translocation of microbial products was found in persons with chronic HIV infection and this was linked to immune activation and CD4(+) T cell homeostasis. METHODOLOGY: We examined here the effects of microbial Toll-like receptor (TLR) ligands on T cell activation in vitro. CONCLUSIONS/FINDINGS: We show that exposure to TLR ligands results in activation of memory and effector CD4(+) and CD8(+) T cells. After exposure to each of 8 different ligands that activate TLRs 2, 3, 4, 5, 7, 8, and 9, CD8(+) T cells are activated and gain expression of the C type lectin CD69 that may promote their retention in lymphoid tissues. In contrast, CD4(+) T cells rarely increase CD69 expression but instead enter cell cycle. Despite activation and cell cycle entry, CD4(+) T cells divide poorly and instead, disproportionately undergo activation-induced cell death. Systemic exposure to TLR agonists may therefore increase immune activation, effector cell sequestration in lymphoid tissues and T cell turnover. These events may contribute to the pathogenesis of immune dysfunction and CD4+ T cell losses in chronic infection with the human immunodeficiency virus.     

Toll-like receptor ligands directly promote activated CD4+ T cell survival

Toll-like receptor (TLR) engagement by pathogen-associated molecular patterns (PAMPs) is an important mechanism for optimal cellular immune responses. APC TLR engagement indirectly enhances activated CD4(+) T cell proliferation, differentiation, and survival by promoting the up-regulation of costimulatory molecules and the secretion of proinflammatory cytokines. However, TLRs are also expressed on CD4(+) T cells, suggesting that PAMPs may also act directly on activated CD4(+) T cells to mediate functional responses. In this study, we show that activated mouse CD4(+) T cells express TLR-3 and TLR-9 but not TLR-2 and TLR-4. Treatment of highly purified activated CD4(+) T cells with the dsRNA synthetic analog poly(I:C) and CpG oligodeoxynucleotides (CpG DNA), respective ligands for TLR-3 and TLR-9, directly enhanced their survival without augmenting proliferation. In contrast, peptidoglycan and LPS, respective ligands for TLR-2 and TLR-4 had no effect. Enhanced survival mediated by either poly(I:C) or CpG DNA required NF-kappaB activation and was associated with Bcl-x(L) up-regulation. However, only CpG DNA, but not poly(I:C)-mediated effects on activated CD4(+) T cells required the TLR/IL-1R domain containing adaptor molecule myeloid differentiation factor 88. Collectively, our results demonstrate that PAMPs can directly promote activated CD4(+) T cell survival, suggesting that TLRs on T cells can directly modulate adaptive immune responses.     

Modulation of B cell responses by Toll-like receptors

B lymphocytes are well known because of their key role in mediating humoral immune responses. Upon encounter with antigen and on cognate interaction with T cells, they differentiate into antibody-secreting plasma cells, which are critical for protection against a variety of pathogens. In addition to their antibody-production function, B cells are efficient antigen-presenting cells and express a variety of pathogen recognition receptors (PRRs). Engagement of these PRRs with their respective ligands results in cytokine and chemokine secretion and the upregulation of co-stimulatory molecules. These events constitute innate immune responses. Toll-like receptor (TLR) activation provides a third signal for B cell activation and is essential for optimal antigen-specific antibody responses. In some situations, TLR activation in B cells can result in autoimmunity. The purpose of this review is to provide some insights into the way that TLRs influence innate and adaptive B cell responses.