T-dependent Humoral Responses Research Articles

Blunting specific T-dependent antibody responses with engineered “decoy” B cells

Antibody inhibitors pose an ongoing challenge to the treatment of subjects with inherited protein deficiency disorders, limiting the efficacy of both protein replacement therapy and corrective gene therapy. Beyond their central role as producers of serum antibody, B cells also exhibit many unique properties that could be exploited in cell therapy applications, notably including antigen-specific recognition and the linked capacity for antigen presentation. Here we employed CRISPR/Cas9 to demonstrate that ex vivo antigen-primed Blimp1-knockout “decoy” B cells, incapable of differentiation into plasma cells, participated in and downregulated host antigen-specific humoral responses after adoptive transfer. Following ex vivo antigen pulse, adoptively transferred high affinity antigen-specific decoy B cells were diverted into germinal centers en masse, thereby reducing participation by endogenous antigen-specific B cells in T-dependent humoral responses and suppressing both cognate and linked antigen-specific IgG following immunization with conjugated antigen. This effect was dose-dependent and, importantly, did not impact concurrent unrelated antibody responses. We demonstrated the therapeutic potential of this approach by treating factor VIII (FVIII)-knockout mice with antigen-pulsed decoy B cells prior to immunization with a FVIII conjugate protein, thereby blunting the production of serum FVIII-specific IgG by an order of magnitude as well as reducing the proportion of animals exhibiting functional FVIII inhibition by 6-fold.

T-independent antigen induces humoral memory through germinal centers.

T-dependent humoral responses generate long-lived memory B cells and plasma cells (PCs) predominantly through germinal center (GC) reaction. In human and mouse, memory B cells and long-lived PCs are also generated during immune responses to T-independent antigen, including bacterial polysaccharides, although the underlying mechanism for such T-independent humoral memory is not clear. While T-independent antigen can induce GCs, they are transient and thought to be nonproductive. Unexpectedly, by genetic fate-mapping, we find that these GCs actually output memory B cells and PCs. Using a conditional BCL6 deletion approach, we show memory B cells and PCs fail to last when T-independent GCs are precluded, suggesting that the GC experience per se is important for programming longevity of T-independent memory B cells and PCs. Consistent with the fact that infants cannot mount long-lived humoral memory to T-independent antigen, B cells from young animals intrinsically fail to form T-independent GCs. Our results suggest that T-independent GCs support humoral memory, and GC induction may be key to effective vaccines with T-independent antigen.

Redox sensor NPGPx restrains ZAP70 activity and modulates T cell homeostasis

Emerging evidences implicate the contribution of ROS to T cell activation and signaling. The tyrosine kinase, ζ-chain-associated protein of 70 kDa (ZAP70), is essential for T cell development and activation. However, it remains elusive whether a direct redox regulation affects ZAP70 activity upon TCR stimulation. Here, we show that deficiency of non-selenocysteine containing phospholipid hydroperoxide glutathione peroxidase (NPGPx), a redox sensor, results in T cell hyperproliferation and elevated cytokine productions. T cell-specific NPGPx-knockout mice reveal enhanced T-dependent humoral responses and are susceptible to experimental autoimmune encephalomyelitis (EAE). Through proteomic approaches, ZAP70 is identified as the key interacting protein of NPGPx through disulfide bonding. NPGPx is activated by ROS generated from TCR stimulation, and modulates ZAP70 activity through redox switching to reduce ZAP70 recruitment to TCR/CD3 complex in membrane lipid raft, therefore subduing TCR responses. These results reveal a delicate redox mechanism that NPGPx serves as a modulator to curb ZAP70 functions in maintaining T cell homeostasis.

B Cell Development and T-Dependent Antibody Response Are Regulated by p38γ and p38δ.

p38MAP kinase (MAPK) signal transduction pathways are important regulators of inflammation and the immune response; their involvement in immune cell development and function is still largely unknown. Here we analysed the role of the p38 MAPK isoforms p38γ and p38δ in B cell differentiation in bone marrow (BM) and spleen, using mice lacking p38γ and p38δ, or conditional knockout mice that lack both p38γ and p38δ specifically in the B cell compartment. We found that the B cell differentiation programme in the BM was not affected in p38γ/δ-deficient mice. Moreover, these mice had reduced numbers of peripheral B cells as well as altered marginal zone B cell differentiation in the spleen. Expression of co-stimulatory proteins and activation markers in p38γ/δ-deficient B cells are diminished in response to B cell receptor (BCR) and CD40 stimulation; p38γ and p38δ were necessary for B cell proliferation induced by BCR and CD40 but not by TLR4 signaling. Furthermore, p38γ/δ-null mice produced significantly lower antibody responses to T-dependent antigens. Our results identify unreported functions for p38γ and p38δ in B cells and in the T-dependent humoral response; and show that the combined activity of these kinases is needed for peripheral B cell differentiation and function.

Assessing the role of the T-box transcription factor Eomes in B cell differentiation during either Th1 or Th2 cell-biased responses.

Successful T-dependent humoral responses require the production of antibody-secreting plasmablasts, as well as the formation of germinal centers which eventually form high-affinity B cell memory. The ability of B cells to differentiate into germinal center and plasma cells, as well as the ability to tailor responses to different pathogens, is driven by transcription factors. In T cells, the T-box transcription factors T-bet and Eomesodermin (Eomes) regulate effector and memory T cell differentiation, respectively. While T-bet has a critical role in regulating anti-viral B cell responses, a role for Eomes in B cells has yet to be described. We therefore investigated whether Eomes was required for B cell differentiation during either Th1 or Th2 cell-biased immune responses. Here, we demonstrate that deletion of Eomes specifically in B cells did not affect B cell differentiation in response to vaccination, as well as following viral or helminth infection. In contrast to its established role in CD8+ T cells, Eomes did not influence memory B cell differentiation. Finally, the use of an Eomes reporter mouse confirmed the lack of Eomes expression during immune responses. Thus, germinal center and plasma cell differentiation and the formation of isotype-switched memory B cells in response to infection are independent of Eomes expression.

CD19 cytoplasmic tyrosine residues Y482 and Y513 are sufficient to promote normal B cell development and differentiation but induce a skewed humoral immune response.

Abstract CD19 amplifies BCR signal transduction by recruiting SH2 domain-containing proteins to its nine cytoplasmic phosphorylated tyrosine residues. Previous studies have shown that Y482 and Y513, which recruit PI3K are required for normal B cell development/differentiation and a productive T-dependent humoral response. However, it has not been determined if CD19 Y482 and Y513 are sufficient to promote normal B cell differentiation or antibody response to immunization. Furthermore, the effects of upstream CD19 tyrosine-mediated Vav/PLCγ2 and Grb2/Sos signaling are not well characterized. Using mice expressing CD19 mutated to only express tyrosines Y482 and Y513, we demonstrate that CD19 mediated Lyn/PI3K signaling is sufficient for B1 B cell development, marginal zone B cell maturation, and germinal center B cell formation. However, these mice exhibit an increased antibody response against T-independent antigens and are impaired in their ability to generate class-switched germinal center B cells during a T-dependent immune response, as well as long-lived, class-switched antibody secreting cells. These findings suggest that cross-talk between signal effectors recruited to Y482 and Y513 and those recruited to other CD19 tyrosine residues is important for determining the qualitative nature of the humoral response.

Identifying the precursor of natural IgM-producing plasma cells (LYM2P.736)

Abstract Natural antibody is constitutively present in the serum, and plays an important role in the clearance of cellular debris, the early control of viral and bacterial expansions, and the initiation of T-dependent humoral responses. However, despite widespread recognition of the critical role of natural IgM in the prevention of both infectious and autoimmune diseases, the source of natural antibody long remained obscure. We have found that most serum IgM in resting mice is produced by a discrete population of IgM plasma cells in the bone marrow, a substantial fraction of which are long-lived cells that occupy a survival niche distinct from that required by IgG plasma cells. Natural IgM plasma cells originate from precursors in the peritoneal cavity, as transfer of unfractionated peritoneal washout cells completely restores serum IgM and the specific compartment of bone marrow plasma cells in Rag1-deficient mice. Surprisingly, however, transfer of purified peritoneal B1a, B1b, or B2 cells did not efficiently reconstitute serum IgM, while a distinct peritoneal B-cell population reconstituted both natural IgM and peritoneal B1a cells in Rag1-deficient mice. We propose that natural IgM plasma cells represent an “innate B-cell” population descended from yolk sac progenitors, and that natural IgM plasma cells are not the progeny of B1a cells per se but instead share an earlier common ancestor also present in the peritoneal cavity.

CD19 cytoplasmic tyrosines 482 and 513 mediating binding to PI3K are sufficient to reconstitute the T-dependent humoral immune response (IRM8P.713)

Abstract CD19 is a co-stimulatory receptor on B cells that has been shown to play a critical role in regulating B cell differentiation and the humoral immune response. In mice lacking CD19 expression, B1 and marginal zone (MZ) B cell subpopulations are absent and the T-dependent humoral response is severely impaired in association with defective GC B cell generation and GC responses resulting in loss of class switching, affinity maturation and ultimately the failure to generate Ag-specific memory B cells and Ab secreting cells (ASC) that produce class switched, high affinity Abs. Previous studies have demonstrated the selective mutation of tyrosines 482 and 513 (4/5F mice) in the cytoplasmic domain of CD19 is sufficient to recapitulate the phenotype associated with complete loss of CD19 expression. In the present study, mice were generated in which CD19 is expressed containing only Y482 and Y513 in the absence of the other seven cytoplasmic tyrosine residues (4/5Y mice). Analysis of 4/5Y mice revealed that these residues alone are sufficient to restore B1 and MZ B cell development as well as the T-dependent humoral immune response. Studies to examine mice expressing CD19 with a selective mutation to either tyrosine 482 or 513 revealed a preferential requirement for tyrosine 482 to promote a normal humoral response. These data support the conclusion that CD19 predominantly contributes to normal B cell development and the T-dependent humoral response through its recruitment of PI3K.

The origin of natural antibody (LYM6P.764)

Abstract Natural antibody is constitutively present in the serum of all mammals. This unique IgM plays an important role in the clearance of cellular debris, the early control of viral and bacterial expansion, and the initiation of T-dependent humoral responses. Despite decades of study, the origins of natural antibody have not been precisely defined. Transfer experiments have shown a close correlation between peritoneal cells, including B1a cells, and natural antibody, but peritoneal B1 cells do not secrete adequate amounts of IgM to support the levels found in mouse serum. Here, we show that a discrete population of IgM plasmacytes in the bone marrow produces all serum IgM in resting mice. These plasmacytes originate from cells in the peritoneal cavity, but not from B1a B cells, and transfer of these progenitors completely restores serum IgM and the specific compartment of bone marrow plasmacytes in Rag1 deficient mice. This plasmacyte compartment contains a substantial fraction of non-dividing, long lived cells that do not occupy the IgG plasmacyte survival niche in the bone marrow but instead depend on IL-5. Genetic analysis of these IgM plasmacytes reveals an unmutated, clonally diverse population strongly enriched for VH11 rearrangements. We propose that these natural IgM plasmacytes represent an “innate B-cell” population descended from yolk sac progenitors, and that natural IgM plasmacytes are not the progeny of B1 B cells but instead share an earlier common ancestor.

Myor/ABF-1 Mrna Expression Marks Follicular Helper T Cells but Is Dispensable for Tfh Cell Differentiation and Function In Vivo

Follicular T helper cells (Tfh) are crucial for effective antibody responses and long term T cell-dependent humoral immunity. Although many studies are devoted to this novel T helper cell population, the molecular mechanisms governing Tfh cell differentiation have yet to be characterized. MyoR/ABF-1 is a basic helix-loop-helix transcription factor that plays a role in the differentiation of the skeletal muscle and Hodgkin lymphoma. Here we show that MyoR mRNA is progressively induced during the course of Tfh-like cell differentiation in vitro and is expressed in Tfh responding to Alum-precipitated antigens in vivo. This expression pattern suggests that MyoR could play a role in the differentiation and/or function of Tfh cells. We tested this hypothesis using MyoR-deficient mice and found this deficiency had no impact on Tfh differentiation. Hence, MyoR-deficient mice developed optimal T-dependent humoral responses to Alum-precipitated antigens. In conclusion, MyoR is a transcription factor selectively up-regulated in CD4 T cells during Tfh cell differentiation in vitro and upon response to alum-protein vaccines in vivo, but the functional significance of this up-regulation remains uncertain.

SAP gene transfer restores cellular and humoral immune function in a murine model of X-linked lymphoproliferative disease

AbstractX-linked lymphoproliferative disease (XLP1) arises from mutations in the gene encoding SLAM-associated protein (SAP) and leads to abnormalities of NKT-cell development, NK-cell cytotoxicity, and T-dependent humoral function. Curative treatment is limited to allogeneic hematopoietic stem cell (HSC) transplantation. We tested whether HSC gene therapy could correct the multilineage defects seen in SAP−/− mice. SAP−/− murine HSCs were transduced with lentiviral vectors containing either SAP or reporter gene before transplantation into irradiated recipients. NKT-cell development was significantly higher and NK-cell cytotoxicity restored to wild-type levels in mice receiving the SAP vector in comparison to control mice. Baseline immunoglobulin levels were significantly increased and T-dependent humoral responses to NP-CGG, including germinal center formation, were restored in SAP-transduced mice. We demonstrate for the first time that HSC gene transfer corrects the cellular and humoral defects in SAP−/− mice providing proof of concept for gene therapy in XLP1.

B cells regulate antibody responses through the medullary remodeling of inflamed lymph nodes

Lymph node (LN) structure is remodeled during immune responses, a process which is considered to play an important role in the regulation of immune function. To date, little attention has been paid to the remodeling of the medullary region, despite its proposed role as a niche for antibody-producing plasma cells. Here, we show that B cells mediate medullary remodeling of antigen-draining LNs during inflammation. This process occurs with kinetics similar to changes in plasma cell number and is accompanied by stromal renetworking which manifests as the segregation of B cells and plasma cells. Medullary remodeling depends on signaling via the lymphotoxin-β receptor and the presence of B cells but occurs independently of T-dependent humoral responses or other immune cell subsets including T cells, monocytes and neutrophils. Moreover, reconstitution of non-cognate polyclonal B cells in B cell-deficient mice restores not only the medullary remodeling but also the antibody response by separately transferred cognate B cells, suggesting that non-cognate B cells contribute to antibody responses through medullary remodeling. We propose that non-cognate B cells mediate the expansion of the plasma cell niche in LN through medullary remodeling, thereby regulating the size of the LN plasma cell pool.

Endosomal TLR signaling is required for anti-nucleic acid and rheumatoid factor autoantibodies in lupus

Using the Unc93b1 3d mutation that selectively abolishes nucleic acid-binding Toll-like receptor (TLR) (TLR3, -7, -9) signaling, we show these endosomal TLRs are required for optimal production of IgG autoAbs, IgM rheumatoid factor, and other clinical parameters of disease in 2 lupus strains, B6-Fas(lpr) and BXSB. Strikingly, treatment with lipid A, an autoAb-inducing TLR4 agonist, could not overcome this requirement. The 3d mutation slightly reduced complete Freund's adjuvant (CFA)-mediated antigen presentation, but did not affect T-independent type 1 or alum-mediated T-dependent humoral responses or TLR-independent IFN production induced by cytoplasmic nucleic acids. These findings suggest that nucleic acid-sensing TLRs might act as an Achilles' heel in susceptible individuals by providing a critical pathway by which relative tolerance for nucleic acid-containing antigens is breached and systemic autoimmunity ensues. Importantly, this helps provide an explanation for the high frequency of anti-nucleic acid Abs in lupus-like systemic autoimmunity.

The Lupus-Related Lmb3 Locus Contains a Disease-Suppressing Coronin-1A Gene Mutation

Here, we show that a lupus-suppressing locus is caused by a nonsense mutation of the filamentous actin-inhibiting Coronin-1A gene. This mutation was associated with developmental and functional alterations in T cells including reduced migration, survival, activation, and Ca2+ flux. T-dependent humoral responses were impaired, but no intrinsic B cell defects were detected. By transfer of T cells, it was shown that suppression of autoimmunity could be accounted for by the presence of the Coro1a(Lmb3) mutation in T cells. Our results demonstrate that Coronin-1A is required for the development of systemic lupus and identify actin-cytoskeleton regulatory proteins as potential targets for modulating autoimmune diseases.

Maturation of an Antibody Response Is Governed by Modulations in Flexibility of the Antigen-Combining Site

Although affinity maturation constitutes an integral part of T-dependent humoral responses, its structural basis is less well understood. We compared the physicochemical properties of antigen binding of several independent antibody panels derived from both germline and secondary responses. We found that antibody maturation essentially reflects modulations in entropy-control of the association, but not dissociation, step of the binding. This influence stems from variations in conformational heterogeneity of the antigen-combining site, which in turn regulates both the affinity and specificity for antigen. Thus, the simple device of manipulating conformational flexibility of paratope provides a mechanism wherein the transition from a degenerate recognition capability to a high-fidelity effector response is readily achieved, with the minimum of somatic mutations.

B cell responses to a peptide epitope. X. Epitope selection in a primary response is thermodynamically regulated.

We examine the etiological basis of hierarchical immunodominance of B cell epitopes on a multideterminant Ag. A model T-dependent immunogen, containing a single immunodominant B cell epitope, was used. The primary IgM response to this peptide included Abs directed against diverse determinants presented by the peptide. Interestingly, affinity of individual monomeric IgM Abs segregated around epitope recognized and was independent of their clonal origins. Furthermore, affinity of Abs directed against the immunodominant epitope were markedly higher than that of the alternate specificities. These studies suggested that the affinity of an epitope-specific primary response, and variations therein, may be determined by the chemical composition of epitope. This inference was supported by thermodynamic analyses of monomer IgM binding to Ag, which revealed that this interaction occurs at the expense of unfavorable entropy changes. Permissible binding required compensation by net enthalpic changes. Finally, the correlation between chemical composition of an epitope, the resultant affinity of the early primary humoral response, and its eventual influence on relative immunogenicity could be experimentally verified. This was achieved by examining the effect of various amino-terminal substitutions on immunogenicity of a, hitherto cryptic, amino-terminal determinant. Such experiments permitted delineation of a hierarchy of individual amino acid residues based on their influence; which correlated well with calculated Gibbs-free energy changes that individual residue side chains were expected to contribute in a binding interaction. Thus, maturation of a T-dependent humoral response is initiated by a step that is under thermodynamic control.

OX40-Deficient Mice Are Defective in Th Cell Proliferation but Are Competent in Generating B Cell and CTL Responses after Virus Infection

OX40, a member of the TNF receptor superfamily, is expressed on activated T cells and implicated in stimulation of T cells and T-dependent humoral responses. We generated OX40 −/− mice and found that the formation of extrafollicular plasma cells, germinal centers, and antibody responses was independent of OX40. After infection with LCMV and influenza virus, OX40 −/− mice retain primary and memory cytotoxic T cell responses with normal expansion and decline of specific CTL. In contrast, CD4 + T cell proliferation and the number of IFN-γ-producing CD4 + T cells were reduced in OX40 −/− mice. Moreover, the number of CD4 + T cells infiltrating the lungs of influenza virus–infected OX40 −/− mice was reduced. These results define a unique role of OX40 in the generation of optimal CD4 + T cell responses in vivo.

Requirement for Nuclear Factor-κB Activation by a Distinct Subset of CD40-Mediated Effector Functions in B Lymphocytes

Abstract CD40 stimulation, which is crucial for generating an effective T-dependent humoral response, leads to the activation of transcription factors NF-AT (nuclear factor of activated T cells), AP-1 (activator protein-1), and NF-κB (nuclear factor-κB). However, which CD40-mediated B cell functions actually require activation of specific transcription factors is unknown. We examined the causal relationship between NF-κB activation and CD40 effector functions by evaluating CD40 functions in the presence of an inducible mutant inhibitory κBα (IκBα) superrepressor. IκBαAA inhibited nuclear translocation of multiple NF-κB dimers without the complicating effect of depriving cells of NF-κB during development. This approach complements studies that use mice genetically deficient in single or multiple NF-κB subunits. Interestingly, only a subset of CD40 effector functions was found to require NF-κB activation. Both CD40-induced Ab secretion and B7-1 up-regulation were completely abrogated by expression of IκBαAA. Surprisingly, up-regulation of Fas, CD23, and ICAM-1 was partially independent, and up-regulation of LFA-1 was completely independent, of CD40-induced NF-κB activation. For the first time, it is clear that distinct transcription factors are required for the dynamic regulation of CD40 functions.

Selection in a T-dependent primary humoral response: new insights from polypeptide models.

Regulatory mechanisms involved in the induction and progression of T-dependent humoral responses have been extensively delineated using a variety of haptens as model antigens. However, several unanswered questions remain with respect to those elicited by structurally more complex molecules. Our own laboratory has been pursuing this latter aspect using designed synthetic peptides as model systems. The cumulative results indeed support that humoral responses to such antigens involve several additional layers of regulation, beyond that identified with haptens. At the first level, the multiplicity of antigenic determinants recognized by the preimmune B-cell pool is soon subject to competitive pressures that restrict, both at the level of repertoire and epitope, fine specificities of early activated clonotypes. Selection at this stage is on the basis of affinity for epitope, which, in turn, is under thermodynamic control. This selected B-cell subset proceeds to populate germinal centers, where further optimization--by way of somatic hypermutation followed by clonal selection--is in favor of increased on-rates of antigen binding. Thus, contrary to findings with hapten antigens, maturation of antibody responses to polypeptides occurs in two discrete, but sequential, stages. The first is for B cells with optimum affinity for the corresponding epitope. This is then followed by further improvement on the basis of increased on-rates of antigen/epitope binding. It is a combination of these two processes which results in the high fidelity of antibodies produced in the secondary response.

Effect of ethanol on B cell expression of major histocompatibility class II proteins in immunized mice

Acute or chronic exposure to ethanol (EtOH), as well as other stimuli that induce a neuroendocrine stress response, can decrease the expression of MHC class II proteins (immune-associated antigens, Ia) on B cells and macrophages. In a mouse model for binge drinking, it has been shown that this decrease is caused by EtOH-induced increases in endogenous glucocorticoids. Decreased Ia expression would be expected to suppress T-dependent humoral responses, and such suppression has been noted in our model. However, it has been reported that activated B cells are much less susceptible to glucocorticoid-induced decreases in Ia expression than are resting B cells. Thus, it is not clear that the decreased Ia observed in our previous studies with non-immunized mice could account for decreased humoral responses, because it has not been directly determined that decreased Ia expression occurs in immunized mice. To examine this issue, splenocytes from mice immunized with sheep erythrocytes were studied by flow cytometry. Mice were treated with EtOH by gavage and immunized 12 h later, because our previous results indicate that this produces maximal suppression of the humoral response. In immunized mice, EtOH decreased Ia expression on B cells at 6 and 12 h after immunization, but not at 24 or 74 h. In a dose-response study, a substantial decrease in Ia expression on B cells was observed at an EtOH dosage of 6.0 or 7.0 g/kg. Thus, decreased Ia expression is a potential mechanism for EtOH-induced suppression of the humoral response. A glucocorticoid antagonist (RU 486) partially blocked the EtOH-induced decrease in Ia expression, suggesting that glucocorticoids are involved in the reduction of Ia expression in immunized mice. Direct administration of corticosterone to produce blood levels comparable to those noted in EtOH-treated mice did not significantly decrease Ia expression, but Ia expression tended to be lower in mice treated with corticosterone. Taken together, these results indicate that glucocorticoids play some role in decreasing Ia expression in immunized mice, but they are less important than in non-immunized mice.