T Cell-independent Research Articles

Limited expression of APRIL and its receptors prior to intestinal IgA plasma cell development during human infancy.

The absence of immunoglobulin A (IgA) in the intestinal tract renders young infants highly susceptible to enteric infections. However, mediators of initial IgA induction in this population are undefined. We determined the temporal acquisition of plasma cells by isotype and expression of T cell-independent (TI) and -dependent (TD) IgA class switch factors in the human intestinal tract during early infancy. We found that IgA plasma cells were largely absent in the infant intestine until after one month of age, approaching adult densities later in infancy than both IgM and IgG. The restricted development of IgA plasma cells in the first month was accompanied by reduced expression of the TI factor APRIL and its receptors TACI and BCMA within isolated lymphoid follicles (ILFs). Moreover, APRIL and BCMA expression both strongly correlated with increasing IgA plasma cell densities over time. Conversely, TD mediators (CD40L and CD40) were expressed within ILFs prior to one month and were not associated with IgA plasma cell generation. In addition, preterm infants had lower densities of IgA plasma cells and reduced APRIL expression compared with full term infants. Thus, blunted TI responses may contribute to the delayed induction of intestinal IgA during early human infancy.

T cell–independent B cell activation induces immunosuppressive sialylated IgG antibodies

Antigen-specific Abs are able to enhance or suppress immune responses depending on the receptors that they bind on immune cells. Recent studies have shown that pro- or antiinflammatory effector functions of IgG Abs are also regulated through their Fc N-linked glycosylation patterns. IgG Abs that are agalactosylated (non-galactosylated) and asialylated are proinflammatory and induced by the combination of T cell-dependent (TD) protein antigens and proinflammatory costimulation. Sialylated IgG Abs, which are immunosuppressive, and Tregs are produced in the presence of TD antigens under tolerance conditions. T cell-independent (TI) B cell activation via B cell receptor (BCR) crosslinking through polysaccharides or via BCR and TLR costimulation also induces IgG Abs, but the Fc glycosylation state of these Abs is unknown. We found in mouse experiments that TI immune responses induced suppressive sialylated IgGs, in contrast to TD proinflammatory Th1 and Th17 immune responses, which induced agalactosylated and asialylated IgGs. Transfer of low amounts of antigen-specific sialylated IgG Abs was sufficient to inhibit B cell activation and pathogenic immune reactions. These findings suggest an immune regulatory function for TI immune responses through the generation of immunosuppressive sialylated IgGs and may provide insight on the role of TI immune responses during infection, vaccination, and autoimmunity.

Long-Lived Bone Marrow Plasma Cells Are Induced Early in Response to T Cell-Independent or T Cell-Dependent Antigens

The signals required to generate long-lived plasma cells remain unresolved. One widely cited model posits that long-lived plasma cells derive from germinal centers (GCs) in response to T cell-dependent (TD) Ags. Thus, T cell-independent (TI) Ags, which fail to sustain GCs, are considered ineffective at generating long-lived plasma cells. However, we show that long-lived hapten-specific plasma cells are readily induced without formation of GCs. Long-lived plasma cells developed in T cell-deficient mice after a single immunization with haptenated LPS, a widely used TI Ag. Long-lived plasma cells also formed in response to TD Ag when the GC response was experimentally prevented. These observations establish that long-lived plasma cells are induced in both TI and TD responses, and can arise independently of B cell maturation in GCs.

Complexed BCR-TLR9 stimulation induces an activation associated cell death in B cells which is rescued by BLyS (60.12)

Abstract Combined B cell receptor (BCR) and Toll-like receptor 9 (TLR9) signaling has been associated with the T cell Independent (TI) activation of autoreactive B cells in the presence of self-DNA. However, TLR9 knockout models of humoral autoimmunity have increased disease severity, suggesting the receptor has a more complex role. We have recently found that while both WT and AM14 RF producing B cells stimulated with complexed BCR-TLR9 agonists indeed proliferate, they die rapidly within 48-72hrs of initial stimulus, implying that combined BCR:TLR9 signaling has a role in limiting the T cell independent activation of B cells. Importantly, this activation associated cell death is unique to complexed BCR-TLR stimulation, as BCR agonist (anti-IgM) alone, TLR9 agonist (CpG ODN) alone, or both BCR + TLR9 agonists uncomplexed induces cells to continue to survive and proliferate robustly within this time frame. Further, this apoptosis involves the intrinsic mitochondrial cell death pathway, as it involves cleavage of caspase 9, occurs independently of caspase 8, and is rescued by the presence of a BCLxL transgene. Finally, we have found that addition of the survival cytokine BLyS rescues B cells proliferating in response to complexed BCR-TLR9 from cell death through BLyS binding to BR3, providing insight into how increased BLyS titers contribute to the onset of autoreactive B cell activation.

The omentum is a site of IgM production during T cell-independent bacterial infection in secondary lymphoid organ-deficient mice (43.6)

Abstract Infection of mice with the bacterium Ehrlichia muris results in a T cell-independent (TI) splenic IgM response, and this antibody is secreted largely by a population of CD11c-expressing B cell plasmablasts in the spleen. However, we found that the TI IgM is maintained in the absence of a spleen; infection of splenectomized mice resulted in a modest decrease in antigen-specific serum IgM, indicating that splenic B cells are not required for the generation of the protective antibody response. To identify the source of the IgM-secreting cells, we infected secondary lymphoid organ (SLO)-deficient mice, which lack both spleen and lymph nodes. SLO-deficient mice were similarly able to produce ehrlichia-specific IgM, and both splenectomized and SLO-deficient mice were protected against challenge infection with a lethal ehrlichial species. These findings led us to investigate the omentum as a source of IgM-secreting cells during infection. We identified the omentum as a large reservoir of antigen-specific IgM-secreting cells; these B cells expressed CD11c, but lacked expression of CD138. CD11c-expressing B cells were elicited by intraperitoneal, but not intravenous infection, revealing a route-dependent response to peritoneal antigen. Our data reveal the omentum as an important site of TI IgM production against peritoneal antigens, in the absence of conventional secondary lymphoid organs.

BAFF neutralization blocks T cell-independent IgM production (70.3)

Abstract Infection of mice with the intracellular bacterium Ehrlichia muris induces a protective T cell-independent (TI) IgM response. The TI IgM produced during acute infection is secreted almost exclusively by a population of CD11c-expressing B cell plasmablasts in the spleen. We investigated the role of B cell activating factor of the TNF family (BAFF) on the generation of the IgM-secreting plasmablasts. To this end, we neutralized BAFF in vivo using a monoclonal antibody directed against murine BAFF. Blockade of BAFF in infected mice did not affect the generation of splenic plasmablasts; however, treatment nearly eliminated the number of antigen-specific IgM-secreting cells, serum IgM titers were reduced, and the plasmablasts failed to express cell-surface CD138. Analysis of mRNA expression in FACS-sorted plasmablasts from anti-BAFF and isotype control-treated mice revealed no differences in transcript levels of prdm1 and xbp1, genes encoding the transcription factors Blimp-1 and XBP-1, which are essential for plasma cell differentiation. Thus, our data indicate that BAFF signaling regulates a checkpoint in plasmablast differentiation, downstream of Blimp-1 and XBP-1 transcription, but prior to CD138 expression.

The RP105/MD-1 complex is indispensable for TLR4/MD-2-dependent proliferation and IgM-secreting plasma cell differentiation of marginal zone B cells

Marginal zone (MZ) B cells mount rapid T-cell-independent (T-I) immune responses against microbial components such as LPS. While Toll-like receptor 4 (TLR4) is essential for LPS responses, MZ B cells uniquely express high levels of another LPS sensor Radioprotective 105 (RP105). However, little is known about how RP105 is used by MZ B cells. In this study, we investigated TLR4- or RP105-dependent MZ B cell responses by utilizing agonistic monoclonal antibodies (mAbs) to each receptor. Cross-linking TLR4 and RP105 at the same time with the mAbs induced robust IgM-secreting plasma cell generation as lipid A moiety of LPS. In contrast, stimulation with either mAb alone did not elicit such responses. RP105-deficient MZ B cells failed to produce IgM-secreting plasma cells in response to lipid A. TLR4 or lipid A stimulation of MZ B cells up-regulated their B lymphocyte-induced maturation protein 1 (Blimp-1) and X-box-binding protein 1 (Xbp-1) mRNA expression. RP105 stimulation alone did not give these responses and in fact decreased TLR4-mediated their expression. Compared with wild-type (WT) MZ B cells, RP105-deficient MZ B cells exhibited increased levels of Blimp-1 and Xbp-1 mRNA expression in response to lipid A. Lipid A or TLR4 plus RP105 stimulation induced massive proliferation and expression of Bcl-xL and c-Myc in WT but not RP105-deficient MZ B cells. These responses contributed to TLR4-mediated anti-apoptotic responses in MZ B cells. Thus, RP105 contributes in a unique way to the TLR4-dependent survival, proliferation and plasma cell generation of MZ B cells.

Mouse CD11bhighLung Dendritic Cells Have More Potent Capability to Induce IgA than CD103+Lung Dendritic CellsIn Vitro

Lung dendritic cells (LDCs) are primary antigen-presenting cells that develop IgA-producing plasma cells in the lung through class switch recombination (CSR) in naive B cells. Recently, the major LDC subsets were found to comprise CD103(-)CD11b(high) LDCs (CD11b(high) LDCs) and CD103(+)CD11b(low or negative) LDCs (CD103(+) LDCs), but their abilities to induce IgA production have not been defined. Under T cell-dependent (T-D) and T cell-independent (T-ID) conditions, we compared the abilities of these two LDC populations to induce IgA. CD11b(high) or CD103(+) LDCs obtained from BALB/c mice were cocultured with naive IgD(+) B cells in the presence of LPS, with or without anti-CD40 monoclonal antibody (mAb) (i.e., T-D and T-ID coculture conditions, respectively). Under both T-D and T-ID conditions, CD11b(high) LDCs induced significantly greater amounts of IgA production, together with a significantly higher mRNA expression of activation-induced cytidine deaminase, than did CD103(+) LDCs. However, the protein expression of a proliferation-inducing ligand, B cell-activating factor of the tumor necrosis family, or retinaldehyde dehydrogenase-1 did not differ between the two LDC subsets. CD11b(high) LDCs displayed a significantly greater capacity to secrete IL-6 and IL-10 in response to LPS, with or without anti-CD40 mAb. Moreover, the IgA production induced by CD11b(high) LDCs in T-D coculture was attenuated by neutralizing both IL-6 and IL-10. These findings suggest that, of the two major LDCs, CD11b(high) LDCs more efficiently induce IgA than do CD103(+) LDCs, possibly through their potent capacity to produce IgA-inducing cytokines.

Oral-Nasopharyngeal Dendritic Cells Mediate T Cell-Independent IgA Class Switching on B-1 B Cells

Native cholera toxin (nCT) as a nasal adjuvant was shown to elicit increased levels of T-independent S-IgA antibody (Ab) responses through IL-5- IL-5 receptor interactions between CD4+ T cells and IgA+ B-1 B cells in murine submandibular glands (SMGs) and nasal passages (NPs). Here, we further investigate whether oral-nasopharyngeal dendritic cells (DCs) play a central role in the induction of B-1 B cell IgA class switch recombination (CSR) for the enhancement of T cell-independent (TI) mucosal S-IgA Ab responses. High expression levels of activation-induced cytidine deaminase, Iα-Cμ circulation transcripts and Iμ-Cα transcripts were seen on B-1 B cells purified from SMGs and NPs of both TCRβ−/− mice and wild-type mice given nasal trinitrophenyl (TNP)-LPS plus nCT, than in the same tissues of mice given nCT or TNP-LPS alone. Further, DCs from SMGs, NPs and NALT of mice given nasal TNP-LPS plus nCT expressed significantly higher levels of a proliferation-inducing ligand (APRIL) than those in mice given TNP-LPS or nCT alone, whereas the B-1 B cells in SMGs and NPs showed elevated levels of transmembrane activator and calcium modulator cyclophilin ligand interactor (TACI) expression. Interestingly, high frequencies of IgA+ B-1 B cells were induced when peritoneal IgA− IgM+ B cells were stimulated with mucosal DCs from mice given nasal TNP-LPS plus nCT. Taken together, these findings show that nasal nCT plays a key role in the enhancement of mucosal DC-mediated TI IgA CSR by B-1 B cells through their interactions with APRIL and TACI.

Role of the receptor-like protein tyrosine phosphatase CD148 in the T cell-independent antibody response (109.12)

Abstract Efficient elimination of a pathogen is dependent on both rapid and sustained antibody production by B cells. In contrast with conventional (B2) B cells, which produce antigen-specific antibody later in infection, B1 B cells generate natural antibodies and contribute to the T-cell independent (TI) antibody response. B cell activation and subsequent antibody secretion involve tyrosine phosphorylation, which is tightly regulated by protein tyrosine kinases (PTKs) and receptor-like protein tyrosine phosphatases (RPTPs). Previous studies from our lab have demonstrated that the RPTPs CD148 and CD45 have redundant positive regulatory roles in the development and antigen receptor signaling of B2 B cells by acting on on Src PTKs. However, the role of RPTPs in signal transduction in B1 B cells is not well understood. We have found that mice with a targeted deletion of the transmembrane domain of CD148 have an impaired IgM response when challenged with a TI antigen. This suggests that CD148 has a unique role in the TI response that cannot be compensated by CD45. Here, we use the mb1-cre transgenic mouse model as well as adoptive transfer experiments and in vitro analysis of B cell activation to characterize the function and specificity of CD148 in the B1 B cell response to TI antigen. These studies describe a previously unappreciated cell-autonomous role for CD148 in modulating signaling pathways that are important for the generation of TI antibody responses by B1 B cells.

Induction of a polyreactive IgM response by bacterial infection (99.26)

Abstract Infection of mice with the bacterium Ehrlichia muris induces a protective T cell-independent (TI) IgM response. A unique property of the TI IgM produced during early infection is that it is polyreactive; that is, it binds a number of unrelated foreign and self antigens, including influenza virus, Borrelia burgdorferi, ss- and dsDNA, insulin, and thyroglobulin. We hypothesized that polyreactivity results from cross-reactivity of antigen-specific IgM, or that antibodies of unrelated specificity were generated via polyclonal B cell activation. To address these hypotheses, we generated B cell hybridomas from infected spleen cells and determined whether they produced pathogen-specific IgM. Eighty percent of the IgM-producing hybridomas were ehrlichia-specific; 10% of the IgM bound an immunodominant ehrlichial antigen, OMP-19, and the remainder bound yet unidentified ehrlichial antigen(s). Polyreactivity was exclusively associated with OMP-19-specific IgM, however, indicating that the properties of the antigen (e.g., spatial distribution on the bacterium) influence the selection of polyreactive IgM. These data indicate that polyreactive IgM is generated by antigen-specific B cells. Our data support a model whereby polyreactivity among antigen-specific IgM allows for the heteroligation (Mouquet et al., 2010) of OMP-19 and additional ehrlichial antigens, and serves to increase the relative affinity of IgM.

Maintenance of serological memory in virus-infected T cell-deficient mice (105.36)

Abstract Many viruses can induce strong acute T cell-independent (TI) B cell responses due to their complex arrays of repetitive antigenic determinants on virions, coupled with the induction of innate cytokines, but T cell help is thought necessary for the development of long term humoral immunity in the form of long-lived plasma cells and memory B cells. However, we have found that T cell deficient (TCRbxd KO) mice persistently infected with polyomavirus (PyV) develop long-lasting high serum antiviral IgG responses, and we questioned whether these mice could generate TI B cell memory. TCR bxd KO PyV-infected mice had very low numbers of antibody secreting cells within the bone marrow compared to wild type (WT) mice. Moreover, by using a functional adoptive transfer assay, memory B cell responses were not detected in T cell deficient mice. In SCID mice reconstituted with TCRbxd KO mouse splenocytes, antiviral serum IgG gradually decreased, presumably due to a lack of supply of naïve B cells from bone marrow. In contrast, CD4 T cell deficient mice lack T cell help but have only short-lived antibody responses, perhaps due to control of persistent antigen by CD8 T cells. Therefore, we hypothesized that a high level of the persistent PyV antigen may subvert immunological tolerance and activate naïve B cell populations continuously, thereby maintaining the long-lasting IgG responses in T cell deficient mice. Here, we provide evidence for that hypothesis.

Breakage of B cell tolerance by antigens on follicular dendritic cells

Background and objectivesAutoimmune disorders frequently display autoreactive germinal centres (GCs) with immune complex (IC)-bearing follicular dendritic cells (FDCs) suggesting that FDCs are involved in the pathogenesis of autoimmune diseases. The...

Prominent Role for Plasmacytoid Dendritic Cells in Mucosal T Cell-Independent IgA Induction

Although both conventional dendritic cells (cDCs) and plasmacytoid dendritic cells (pDCs) are present in the gut-associated lymphoid tissues (GALT), the roles of pDCs in the gut remain largely unknown. Here we show a critical role for pDCs in Tcell-independent (TI) IgA production by B cells in the GALT. When pDCs of the mesenteric lymph nodes (MLNs) and Peyer's patches (PPs) (which are representative GALT) were cultured with naive B cells to induce TI IgA class switch recombination (CSR), IgA production was substantially higher than in cocultures of these cells with cDCs. IgA production was dependent on APRIL and BAFF production by pDCs. Importantly, pDC expression of APRIL and BAFF was dependent on stromal cell-derived type I IFN signaling under steady-state conditions. Our findings provide insight into the molecular basis of pDC conditioning to induce mucosal TI IgA production, which may lead to improvements in vaccination strategies and treatment for mucosal-related disorders.

TNFα blockade impairs T cell dependent humoural responses

Tumour necrosis factor α (TNFα) blockade in spondyloarthritis (SpA) induces antibodies specific for double stranded DNA, which is a T cell independent (TI) antigen. As these antibodies were restricted to...

Decreased Marginal Zone B-cell (MZB) Expression in Common Variable Immunodeficiency (CVID) Does Not Predict Deficienct IgM Isohemagglutinin (IgMIH) Production

RATIONALE: IgMIH are classically used to access polysaccharide antigen response. MZBs (CD19+IgD+CD27+) are thought to be important in humoral immunity against T-cell independent (TI) antigens such as polysaccharides. We examined B-cell immunophenotypes and IgMIH in patients with CVID. METHODS: CVID patients, being treated with IVIG, (n=10, 50% male, 7-82 yrs), were prospectively enrolled. B-cell immunophenotypes were analyzed by flow cytometry and analyzed by FlowJo software. IgMIH (n=10) were identified by commercial laboratories. RESULTS: 6/10 patients had decreased levels of MZB cells for age, while IgMIH (10/10) were normal. CONCLUSIONS: In CVID patients, the circulating subpopulation of MZB cells does not appear to correlate with IgMIH. IgM in IVIG is <2 mcg/ml and thus unlikely to affect IgMIH levels, as evidenced by one patient taking IVIG with AB blood type (not included in this study) which showed absent IgMIHs. Thus, a decreased MZB B-cell subpopulation in CVID does not predict the inability to express TI IgMIH. The role of the circulating MZB frequency in generating IgM and IgG anti-pneumococcal antibody responses in CVID patients remains to be determined.

CD20 deficiency in humans results in impaired T cell–independent antibody responses

CD20 was the first B cell differentiation antigen identified, and CD20-specific mAbs are commonly used for the treatment of B cell malignancies and autoantibody-mediated autoimmune diseases. Despite this the role of CD20 in human B cell physiology has remained elusive. We describe here a juvenile patient with CD20 deficiency due to a homozygous mutation in a splice junction of the CD20 gene (also known as MS4A1) that results in "cryptic" splicing and nonfunctional mRNA species. Analysis of this patient has led us to conclude that CD20 has a central role in the generation of T cell-independent (TI) antibody responses. Key evidence to support this conclusion was provided by the observation that although antigen-independent B cells developed normally in the absence of CD20 expression, antibody formation, particularly after vaccination with TI antigens, was strongly impaired in the patient. Consistent with this, TI antipolysaccharide B cell responses were severely impeded in CD20-deficient mice. Our study therefore identifies what we believe to be a novel type of humoral immunodeficiency caused by CD20 deficiency and characterized by normal development of antigen-independent B cells, along with a reduced capacity to mount proper antibody responses.

Regulation of T-cell-independent and T-cell-dependent antibody production by circadian rhythm and melatonin

Melatonin is a hormone that has immunomodulatory activity and is believed to influence the production of antibodies in mammals. The aim of the present study was to investigate the effect of suppressed melatonin synthesis on the antibody production. BALB/c mice were immunized with T-cell-dependent (TD) and T-cell-independent (TI) antigens and kept under (i) normal lighting, (ii) constant exposure to light, (iii) exposed to light and treated daily with melatonin. It was revealed that melatonin modulated TD and TI antibody production. Suppressed melatonin synthesis increased the amount of IgM, IgG1, IgG2b and IgG3 antibodies after immunization with TI antigen. The level of TD antibodies IgM, IgG2a, IgG2b and IgG3 also increased, however, the antigen-specific antibodies of IgG1 isotype significantly decreased in mice exposed to light. Daily melatonin treatment brought the antibody level back to normal. The antibody concentration in the sera of mice kept at normal lighting was significantly higher when the immunizations were performed in the evening. The action of melatonin on B cells via MT2 receptor was shown in vitro and in vivo.

Generation of Protective T Cell-Independent Antiviral Antibody Responses in SCID Mice Reconstituted with Follicular or Marginal Zone B Cells

B cells generated in the bone marrow of adult mice enter the periphery as transitional B cells and subsequently differentiate into one of two phenotypically and functionally distinct subsets, marginal zone (MZ) or follicular (Fo) B cells. Recent reports indicate, however, that in response to environmental cues, such as lymphopenia, mature Fo B cells can change to display phenotypic markers characteristic of MZ B cells. Previously, we found that splenic B cells transferred to SCID mice responded to polyoma virus (PyV) infection with T cell-independent (TI) IgM and IgG secretion, reducing the viral load and protecting mice from the lethal effect of the infection. The contribution of MZ and Fo B cell subsets to this antiviral TI-2 response, however, has not been addressed. In this study, we show that both sort-purified MZ and Fo B cells generate protective TI Ab responses to PyV infection when transferred into SCID mice. Moreover, the transferred Fo B cells in the spleens of the PyV-infected SCID mice change phenotype, with many of them displaying MZ B cell characteristics. These findings demonstrate the plasticity of the B cell subsets in virus-infected hosts and show for the first time that B cells derived exclusively from Fo B cells can effectively function in antiviral TI-2 responses.

Development of 5-valent conjugate pneumococcal protein A – Capsular polysaccharide pneumococcal vaccine against invasive pneumococcal disease

In this study, we synthesized a 5-valent pneumococcal conjugate vaccine, which was prepared with the pneumococcal capsular polysaccharides (PCPs) (from Streptococcus pneumoniae 1, 5, 6B, 19F, 23F) and pneumococcal surface protein A (PspA) mediated by 1,4-butanediol diglycidyl ether. The PspA cloned from serotype 19 strain showed good cross-immune response to 1, 5, 6B, and 23F serotypes of Streptococcus pneumonia ( S. pneumoniae). Analysis of the maturation process of conjugate polyclonal antibody showed that conjugation with the protein carrier converted the polysaccharide from a weak T cell-independent (TI) antigen to a T cell-dependent (TD) antigen, although antibodies affinity to polysaccharide was not as strong as it to PspA in conjugate. We used an invasive disease mouse model to evaluate the protective efficacy of this conjugate vaccine. Active and passive protection against intraperitoneal challenge with virulent type 6B strain showed that the median survival times for mice immunized with conjugate were significantly longer than that of mice treated with capsular polysaccharides or PspA alone. Our study's results showed that immunization of the 5-valent PspA-capsular polysaccharides conjugate vaccine could afford strong protection to mice against the invasion of 1, 5, 6B, 19F, 23F serotypes S. pneumoniae.