Cell-dependent Humoral Immune Responses Research Articles

Human IgM-expressing memory B cells.

A hallmark of T cell dependent (TD) humoral immune responses is the generation of long-lived memory B cells. The generation of these cells occurs primarily in the germinal center (GC) reaction, where antigen-activated B cells undergo affinity maturation as a major consequence of the combined processes of proliferation, somatic hypermutation of their immunoglobulin V (IgV) region genes, and selection for improved affinity of their B-cell antigen receptors. As many B cells also undergo class-switching to IgG or IgA in these TD responses, there was traditionally a focus on class-switched memory B cells in both murine and human studies on memory B cells. However, it has become clear that there is also a large subset of IgM-expressing memory B cells, which have important phenotypic and functional similarities but also differences to class-switched memory B cells. There is an ongoing discussion about the origin of distinct subsets of human IgM+ B cells with somatically mutated IgV genes. We argue here that the vast majority of human IgM-expressing B cells with somatically mutated IgV genes in adults is indeed derived from GC reactions, even though a generation of some mostly lowly mutated IgM+ B cells from other differentiation pathways, mainly in early life, may exist.

The transcription factor Mef2d regulates B:T synapse-dependent GC-TFH differentiation and IL-21-mediated humoral immunity.

Communication between CD4 T cells and cognate B cells is key for the former to fully mature into germinal center-T follicular helper (GC-TFH) cells and for the latter to mount a CD4 T cell-dependent humoral immune response. Although this interaction occurs in a B:T synapse-dependent manner, how CD4 T cells transcriptionally regulate B:T synapse formation remains largely unknown. Here, we report that Mef2d, an isoform of the myocyte enhancer factor 2 (Mef2) transcription factor family, is a critical regulator of this process. In CD4 T cells, Mef2d negatively regulates expression of Sh2d1a, which encodes SLAM-associated protein (SAP), a critical regulator of B:T synapses. We found that Mef2d regulates Sh2d1a expression via DNA binding-dependent transcriptional repression, inhibiting SAP-dependent B:T synapse formation and preventing antigen-specific CD4 T cells from differentiating into GC-TFH cells. Mef2d also impeded IL-21 production by CD4 T cells, an important B cell help signaling molecule, via direct repression of the Il21 gene. In contrast, CD4 T cell-specific disruption of Mef2d led to a substantial increase in GC-TFH differentiation in response to protein immunization, concurrent with enhanced SAP expression. MEF2D mRNA expression inversely correlates with human systemic lupus erythematosus (SLE) patient autoimmune parameters, including circulating TFH-like cell frequencies, autoantibodies, and SLEDAI scores. These findings highlight Mef2d as a pivotal rheostat in CD4 T cells for controlling GC formation and antibody production by B cells.

Neuraminidase Inhibitor Zanamivir Ameliorates Collagen-Induced Arthritis.

Altered sialylation patterns play a role in chronic autoimmune diseases such as rheumatoid arthritis (RA). Recent studies have shown the pro-inflammatory activities of immunoglobulins (Igs) with desialylated sugar moieties. The role of neuraminidases (NEUs), enzymes which are responsible for the cleavage of terminal sialic acids (SA) from sialoglycoconjugates, is not fully understood in RA. We investigated the impact of zanamivir, an inhibitor of the influenza virus neuraminidase, and mammalian NEU2/3 on clinical outcomes in experimental arthritides studies. The severity of arthritis was monitored and IgG titers were measured by ELISA. (2,6)-linked SA was determined on IgG by ELISA and on cell surfaces by flow cytometry. Zanamivir at a dose of 100 mg/kg (zana-100) significantly ameliorated collagen-induced arthritis (CIA), whereas zana-100 was ineffective in serum transfer-induced arthritis. Systemic zana-100 treatment reduced the number of splenic CD138+/TACI+ plasma cells and CD19+ B cells, which was associated with lower IgG levels and an increased sialylation status of IgG compared to controls. Our data reveal the contribution of NEU2/3 in CIA. Zanamivir down-modulated the T and B cell-dependent humoral immune response and induced an anti-inflammatory milieu by inhibiting sialic acid degradation. We suggest that neuraminidases might represent a promising therapeutic target for RA and possibly also for other antibody-mediated autoimmune diseases.

Identification and functional characterization of CD154 in T cell-dependent immune response in Nile tilapia (Oreochromis niloticus)

CD154, a member of the TNF superfamily, is a multifunctional molecule highly expressed in activated T cells, and plays important roles in T cell-dependent humoral immune response. In this study, CD154 of Nile tilapia (Oreochromis niloticus) was identified, and its functions in the T cell-dependent immune response were demonstrated. The open reading frame (ORF) of OnCD154 is 699 bp, encoding a protein of 232 amino acids with a 23 amino acid transmembrane region. Amino acid sequence of OnCD154 is highly homologous to that of other teleost fish, especially rainbow trout. Quantitative real-time PCR (qRT-PCR) demonstrated that mRNA of OnCD154 is highly expressed in immune organs, especially in spleen, thymus, gills, head kidney, etc. In addition, the anti-OnCD154 polyclonal antibody (anti-(r)OnCD154) was successfully prepared, and it can react with natural protein in head kidney leukocytes. Following two immunizations with keyhole limpet hemocyanin (KLH) in vivo, the significantly up-regulated expression level of OnCD154 mRNA appeared earlier (fifth day) and higher (42.9 folds) in the second challenge than the first on in head kidney. Further, after stimulation with KLH in vitro, the expressions of T cell-dependent immune response-related molecules (activated T cell specific surface molecules CD3ε and CD154) and B cell differentiation-related molecules (Blimp1 and sIgM) and CD40 were significantly up-regulated in head kidney leukocytes. Moreover, the up-regulated expressions of these molecules were blocked with the treatment of anti-(r)OnCD154 antibody. Taken together, these results indicate that OnCD154 might get involved in T cell-dependent immune response, and provide a new insight into the humoral immune response of teleost fish.

Recombinant Rabies Virus Overexpressing OX40-Ligand Enhances Humoral Immune Responses by Increasing T Follicular Helper Cells and Germinal Center B Cells.

Rabies, caused by the rabies virus (RABV), remains a serious threat to public health in most countries. Development of a single-dose and efficacious rabies vaccine is the most important method to restrict rabies virus transmission. Costimulatory factor OX40-ligand (OX40L) plays a crucial role in the T cell-dependent humoral immune responses through T-B cell interaction. In this work, a recombinant RABV overexpressing mouse OX40L (LBNSE-OX40L) was constructed, and its effects on immunogenicity were evaluated in a mouse model. LBNSE-OX40L-immunized mice generated a larger number of T follicular helper (Tfh) cells, germinal center (GC) B cells, and plasma cells (PCs) than the parent virus LBNSE-immunized mice. Furthermore, LBNSE-OX40L induced significantly higher levels of virus-neutralizing antibodies (VNA) as early as seven days post immunization (dpi), which lasted for eight weeks, resulting in better protection for mice than LBNSE (a live-attenuated rabies vaccine strain). Taken together, our data in this study suggest that OX40L can be a novel and potential adjuvant to improve the induction of protective antibody responses post RABV immunization by triggering T cell-dependent humoral immune responses, and that LBNSE-OX40L can be developed as an efficacious and nonpathogenic vaccine for animals.

Novel ZBP1-IRF3 Dependency in Multiple Myeloma Mediated By IRF3-Driven Regulation of Cell Cycle Genes

ZBP1 is an inducible nucleic acid (NA) sensor that is activated when pathogen NA bind to its Zα and Zβ domains. ZBP1 is required for TBK1-dependent phosphorylation of the transcription factor IRF3 (pIRF3) followed by its direct activation of type I interferon genes. However, the role, if any, of ZBP1 in tumour biology is not known. By searching for genes selectively expressed in multiple myeloma (MM) we identified ZBP1 mRNA expressed in 29 MM cell lines (MMCL) but not in >1000 other cancer cell lines (CCLE dataset); ZBP1 was expressed in all 766 patient myeloma PC (CoMMpass dataset) but not in normal blood cells (Blueprint) or 53 healthy tissues (GTex). We confirmed expression of ZBP1 mRNA and/or protein in MMCL, primary human and murine germinal centre B (GCB) and plasma cells (PC) as well as in myeloma PC. By inducing T cell-dependent humoral immune responses after ip alum-NP-KLH immunisation, we explored the role of selective and constitutive expression of Zbp1 in GCB to PC transition. We found no differences in the frequency of splenic GCB cells and PC between control WT and Zbp1-/- mice and in GCB cell frequency between immunised WT and Zbp1-/- mice. However, compared to WT, the increase in PC frequency in immunised Zbp1-/- mice was 50% lower (n=10/group, p<0.0001) commensurate with a 40% (n=6/group, p<0.01), lower increase in NP-KLH-specific IgG but not IgM levels in Zbp1-/- mice. These findings suggest that although Zbp1 is not required for GCB cell and PC development it is required for optimal, T cell-dependent humoral immune responses. To explore the function of ZBP1 in MM we depleted by 2 lentiviral shRNAs either isoform 1 (contains both Zα and Zβ domains; shRNA1) or both isoform 1 and isoform 2 (latter lacks Zα domain; shRNA2). Both shRNAs were toxic to all 5 MMCL tested suggesting that isoform 1 but not isoform 2 is essential for myeloma cell survival. This effect was specific because survival of K562 cells, which lack expression of ZBP1, was not affected by either shRNA and exogenous ZBP1 cDNA rescued cell death of ZBP1-depleted myeloma cells. Dox-induced ZBP1 depletion was toxic to MMCL in vitro and significantly inhibited myeloma cell growth in a subcutaneous NSG model of the MMCL H929 and MM.1S. Together, these findings reveal a novel myeloma cell-specific ZBP1 dependency. Transcriptome analysis of ZBP1-depleted H929 and MM.1S cells showed amongst the significantly downregulated genes enrichment for the cell cycle control and DNA repair pathways consistent with a critical role of ZBP1 in promoting myeloma cell proliferation. Flow-cytometric analysis of ZBP1-depleted MMCL as well as of patient-derived myeloma PC revealed cell cycle arrest at the G0/1 phase and increasing apoptosis. Exploring potential links with IRF3, we first observed that unlike in non-malignant cells, IRF3 was constitutively phosphorylated in MMCL. Using protein-co-immunoprecipitation we found that endogenous ZBP1 interacts with IRF3 and TBK1 while upon co-transfection with different ZBP1 deletion mutants, ZBP1-IRF3 interaction required primarily the ZBP1, RHIM domain-containing, C-terminus. Further, while in ZBP1-depleted myeloma cells total IRF3 and TBK1 levels were not altered, pIRF3 and pTBK1 levels decreased thus showing a post-translational dependency of constitutive pIRF3 and pTBK1 on ZBP1. Finally, pharmacological inhibition of TBK1 resulted in decrease of pIRF3 without affecting total IRF3. Importantly, shRNA-mediated IRF3 depletion resulted in cell cycle arrest and death of MMCL. By integrating histone mark and in-house IRF3 ChiP-seq with transcriptome of IRF3-depleted MM.1S cells we identified 770 down- and 330 up-regulated genes predicted to be directly regulated by IRF3. Pathway enrichment analysis confirmed cell cycle as the most highly regulated by IRF3. Notably, we observed no direct or indirect regulation of the interferon genes (e.g., IFNA1, IFNB1) by IRF3. As well as the IRF3 motif, IRF3 cistrome analysis revealed significant enrichment for the distinct IRF4 motif. Integration of the IRF3/IRF4 cistromes identified >80% IRF3 binding regions are co-occupied by IRF4 and co-regulation of cell cycle genes. Further we validated IRF3-IRF4 interaction at the IRF4 super-enhancer by ChIP-re-ChIP. These data show a novel dependency in MM comprising constitutive activation of the ZBP1-IRF3 pathway and regulation of cell cycle and proliferation by IRF3 thus providing opportunities for therapeutic targeting. Disclosures Caputo: GSK: Research Funding. Auner:Amgen: Other: Consultancy and Research Funding; Takeda: Consultancy; Karyopharm: Consultancy. Karadimitris:GSK: Research Funding.

\u03b3\u03b4 T cells control humoral immune response by inducing T follicular helper cell differentiation

γδ T cells have many known functions, including the regulation of antibody responses. However, how γδ T cells control humoral immunity remains elusive. Here we show that complete Freund’s adjuvant (CFA), but not alum, immunization induces a subpopulation of CXCR5-expressing γδ T cells in the draining lymph nodes. TCRγδ+CXCR5+ cells present antigens to, and induce CXCR5 on, CD4 T cells by releasing Wnt ligands to initiate the T follicular helper (Tfh) cell program. Accordingly, TCRδ−/− mice have impaired germinal center formation, inefficient Tfh cell differentiation, and reduced serum levels of chicken ovalbumin (OVA)-specific antibodies after CFA/OVA immunization. In a mouse model of lupus, TCRδ−/− mice develop milder glomerulonephritis, consistent with decreased serum levels of lupus-related autoantibodies, when compared with wild type mice. Thus, modulation of the γδ T cell-dependent humoral immune response may provide a novel therapy approach for the treatment of antibody-mediated autoimmunity.

Human regulatory B cells control the TFH cell response

Follicular helper T (TFH) cells support terminal B-cell differentiation. Human regulatory B (Breg) cells modulate cellular responses, but their control of TFH cell-dependent humoral immune responses is unknown. We sought to assess the role of Breg cells on TFH cell development and function. Human T cells were polyclonally stimulated in the presence of IL-12 and IL-21 to generate TFH cells. They were cocultured with B cells to induce their terminal differentiation. Breg cells were included in these cultures, and their effects were evaluated by using flow cytometry and ELISA. B-cell lymphoma 6, IL-21, inducible costimulator, CXCR5, and programmed cell death protein 1 (PD-1) expressions increased on stimulated human T cells, characterizing TFH cell maturation. In cocultures they differentiated B cells into CD138+ plasma and IgD-CD27+ memory cells and triggered immunoglobulin secretions. Breg cells obtained by Toll-like receptor 9 and CD40 activation of Bcells prevented TFH cell development. Added to TFH cell and B-cell cocultures, they inhibited B-cell differentiation, impeded immunoglobulin secretions, and expanded Foxp3+CXCR5+PD-1+ follicular regulatory T cells. Breg cells modulated IL-21 receptor expressions on TFH cells and B cells, and their suppressive activities involved CD40, CD80, CD86, and intercellular adhesion molecule interactions and required production of IL-10 and TGF-β. Human Breg cells control TFH cell maturation, expand follicular regulatory T cells, and inhibit the TFH cell-mediated antibody secretion. These novel observations demonstrate a role for the Breg cell in germinal center reactions and suggest that deficient activities might impair the TFH cell-dependent control of humoral immunity and might lead to the development of aberrant autoimmune responses.

Impaired T Cell-dependent Humoral Immune Response Associated with Juvenile-onset Recurrent Respiratory Papillomatosis Progression.

Whether humoral immunity plays a role in HPV type 6 or 11 virus-mediated Juvenile-onset Recurrent Respiratory Papillomatosis (JORRP) remains unknown. In the present study, serum total IgG level in 44 JORRP patients was significantly decreased compared with that in 40 healthy controls. Moreover, expanded CD3−CD19+ B cells with down-regulation of CD23, CD40, HLA-DR and up-regulation of CD86 expression were found in the peripheral blood of JORRP patients. Flow cytometry analysis of B-cell compartment showed that the frequency of both CD19+CD27hi plasma cells and CD19+CD27+ memory B cells were decreased in JORRP patients. Importantly, although the proportion of circulating CXCR5+PD1hi Tfh cells was not changed, the function of Tfh cells were greatly impaired with reduced ability of IL-21 secretion to promote B cell maturation. Association analysis by the Kaplan-Meier method revealed that IL-21 secreting Tfh cell was positively correlated to the CD27+ B cell subset frequency, the serum IgG level and the frequency of recurrence in JORRP patients, but negatively correlated to the percentage of IgD+CD27− B cell. We concluded that a reduced IL-21 secretion by Tfh cells may limit B cell maturation and antibody production in JORRP patients and Tfh cell-derived IL-21 might be associated with JORRP outcome in clinic.

Expression of CD154 (CD40L) on stimulated T lymphocytes in patients with idopathic thrombocytopenic purpura

Objectives: The role of CD40–CD154 (CD40L) interaction in T cell-dependent humoral immune response is strongly established. Increased expression of CD154 on stimulated T cells is observed in rheumatic diseases and is associated with disease activity. We investigated the expression of CD154 on T cells from idiopathic thrombocytopenic purpura (ITP) patients and assessed the significance of CD154 expression in disease status.Methods: We enrolled 59 ITP patients, 23 healthy controls, and 19 patients with non-immune thrombocytopenia. Isolated mononuclear cells were stimulated in RPMI medium containing phorbol myristate acetate (PMA) (5 ng/mL) and ionomycin (500 ng/mL) for 2 h at 37°C. The expression of CD154 on CD4+T cells was evaluated using flow cytometry and serum soluble CD40L levels were measured.Results: In ITP patients, the percentage of CD4+ CD154+ cells and mean fluorescence intensity (MFI) of CD154 on activated CD4+T cells was not different from that in the healthy controls and non-immune thrombocytopenia patients. Additionally, the percentage and expression level of CD154 was not different between ITP patients with low platelet counts (<50 000/μL) and those with 50 000/μL or more. Soluble CD40L levels were significantly lower in ITP patients with low platelet counts than in healthy controls, but were not correlated with CD154 expression.Conclusion: Increased CD154 expression on CD4+T cells was not observed in ITP patients and was not related with low platelet counts. Overexpression of CD154 on CD4+T cells is unlikely to be central to the pathogenesis of ITP, and other immune dysfunctions should be targeted for therapy purposes.

Reduced T Cell–Dependent Humoral Immune Response in Microsomal Prostaglandin E Synthase-1 Null Mice Is Mediated by Nonhematopoietic Cells

Microsomal PGE synthase-1 (mPGES-1) is an inducible enzyme that specifically catalyzes the conversion of PGH2 to PGE2. We showed that mPGES-1 null mice had a significantly reduced incidence and severity of collagen-induced arthritis compared with wild-type (WT) mice associated with a marked reduction in Abs to type II collagen. In this study, we further elucidated the role of mPGES-1 in the humoral immune response. Basal levels of serum IgM and IgG were significantly reduced in mPGES-1 null mice. Compared with WT mice, mPGES-1 null mice exhibited a significant reduction of hapten-specific serum Abs in response to immunization with the T cell-dependent (TD) Ag DNP-keyhole limpet hemocyanin. Immunization with the T cell-independent type 1 Ag trinitrophenyl-LPS or the T cell-independent type 2 Ag DNP-Ficoll revealed minimal differences between strains. Germinal center formation in the spleen of mPGES-1 null and WT mice were similar after immunization with DNP-keyhole limpet hemocyanin. To determine whether the effect of mPGES-1 and PGE2 was localized to hematopoietic or nonhematopoietic cells, we generated bone marrow chimeras. We demonstrated that mPGES-1 deficiency in nonhematopoietic cells was the critical factor for reduced TD Ab production. We conclude that mPGES-1 and PGE2-dependent phenotypic changes of nonhematopoietic/mesenchymal stromal cells play a key role in TD humoral immune responses in vivo. These findings may have relevance to the pathogenesis of rheumatoid arthritis and other autoimmune inflammatory diseases associated with autoantibody formation.

Interleukin-2 Inhibits Germinal Center Formation by Limiting T Follicular Helper Cell Differentiation

T follicular helper (Tfh) cells promote Tcell-dependent humoral immune responses by providing Tcell help to B cells and by promoting germinal center (GC) formation and long-lived antibody responses. However, the cellular and molecular mechanisms that control Tfh cell differentiation invivo are incompletely understood. Here we show that interleukin-2 (IL-2) administration impaired influenza-specific GCs, long-lived IgG responses, and Tfh cells. IL-2 did not directly inhibit GC formation, but instead suppressed the differentiation of Tfh cells, thereby hindering the maintenance of influenza-specific GC B cells. Our data demonstrate that IL-2 is a critical factor that regulates successful Tfh and B cell responses invivo and regulates Tfh cell development.

Heterozygous signal transducer and activator of transcription 3 mutations in hyper-IgE syndrome result in altered B-cell maturation

Heterozygous signal transducer and activator of transcription 3 mutations in hyper-IgE syndrome result in altered B-cell maturation

Cutting Edge: Distinct Waves of BCL6 Expression during T Follicular Helper Cell Development

T follicular helper (T(FH)) cells are central to the development and regulation of T cell-dependent humoral immune responses. The transcriptional repressor BCL6 is required for T(FH) responses, but the kinetics of BCL6 protein expression in activated CD4(+) T cells have not been established. We measured BCL6 expression during T(FH) cell development at the single-cell level using intracellular staining and YFP-BCL6 fusion protein reporter mice. BCL6 was immediately upregulated in all dividing T cells during dendritic cell-T cell interactions. A second wave of early BCL6 expression coincided with the induction of CXCR5, resulting in a distinct and stable T(FH) cell population. Cognate B cells were not required for the induction of BCL6, but supported the expansion of T(FH) cells. These data suggest that BCL6 participates in very early events in T(FH) cell development, and that repeated encounters with APCs reinforce BCL6 expression, thereby establishing the T(FH) cell phenotype.

Establishing the kinetics of TFH cell development in vivo (152.11)

Abstract T follicular helper (TFH) cells have recently emerged as the main regulators of T cell-dependent humoral immune responses. Yet both the developmental pathway and the maintenance of TFH cells are only poorly understood. In addition, it is not clear if TFH cells are a stable T helper cell lineage or if the TFH phenotype represents a transient activation state. The transcriptional repressor BCL6 has been shown to be crucial for the establishment of the TFH phenotype. However, the mechanism by which BCL6 accomplishes this task and the regulation of BCL6 in TFH cells at the pre- and post-transcriptional level remain largely unknown. In order to generate a comprehensive kinetic profile of BCL6 expression in TFH cells on the genomic and protein level, we are taking advantage of a novel murine BCL6-reporter strain and improved intracellular BCL6 staining reagents.

Detection and Functional Evaluation of −262A/T and −188A/G Polymorphisms of SLAM Gene in Patients with Systemic Lupus Erythematosus

Signaling lymphocytic activation molecule (SLAM) has been related to the pathology of systemic lupus erythematosus (SLE) through regulation of T cell-dependent humoral immune responses. We investigated the functional associations of the -262A/T and -188A/G polymorphisms of SLAM in Chinese patients with SLE. Genotyping of -262A/T (rs2295614) and -188A/G (rs2295613) in SLAM was carried out in 248 cases and 278 controls. Promoter activities of haplotypes on the SLAM gene were evaluated with the dual-luciferase reporter system. The mRNA expressions of SLAM on peripheral blood mononuclear cells (PBMC) of SLE patients with different genotypes were determined by real-time polymerase chain reaction. Frequencies of -262A allele and -188G allele were significantly higher in SLE patients than in controls. Haplotype analysis and multifactorial logistic regression analysis showed that individuals with the AG/AG haplotype had increased susceptibility to SLE (p = 0.002, OR 1.478, 95% CI 1.152-1.897). In response to PHA stimulation, the SLAM mRNA expression on PBMC of SLE patients was significantly higher in -262A-188G haplotype homozygotes compared with -262A-188G heterozygotes and individuals with other genotypes. Our findings suggest that -262A-188G haplotype in the SLAM gene promoter contributes to the risk of SLE by increasing the expression of SLAM.

Flagellin does not promote a CD8+ T cell response to the SIINFEKL epitope (130.42)

Abstract Flagellin, the ligand for TLR5, is a potent adjuvant for CD4+ T cell-dependent humoral immune responses and has been reported by several groups to promote CD8+ T cell responses in vivo. However, other groups have failed to detect an effect of flagellin on the CD8+ T cell response. In an attempt to resolve this controversy, we created a protein composed of the SIINFEKL epitope and flanking amino acids fused to the N-terminus of full length Salmonella flagellin for use with TCR transgenic OT-I mice. We found that the CD8+ T cell stimulatory activity of this protein was, as expected, dependent on proteasome processing and was markedly more potent than ovalbumin by itself. However, the adjuvant effect of the fusion protein was TLR5 and IPAF independent. A fusion peptide containing the hypervariable region Salmonella or Pseudomonas flagellin and the SIINFEKL epitope also stimulated antigen-specific CD8+ T cell activation. These results are consistent with the conclusion that flagellin does not promote antigen-specific CD8+ T cell activation. Since the flagellin fusion protein containing the SIINFEKL epitope was far superior to ovalbumin as an inducer of CD8+ T cell activation, it is likely that uptake and processing of the SIINFEKL epitope by antigen-presenting cells is enhanced outside the context of ovalbumin.

Beclin-1 is required for T cell-mediated immune responses (143.19)

Abstract Beclin 1, the mammalian homologue of the yeast Atg6, regulates macroautophagy (autophagy) in eukaryotic cells and plays a critical role in the regulation of cell death and survival in various types of cells. Beclin 1 is expressed in T cells. However, its role in T cell-mediated immune responses remains unknown. We have found that mice with Beclin-1 deletion in T cells have profound deficiencies in mounting T dependent immune responses. Autophagy is abolished in Beclin 1 deficient T cells. We observed that, upon activation, Beclin 1 deficient T cells undergo programmed cell death. Mice with Beclin 1 deletion in T cells fail to mount T cell-dependent humoral immune responses to KLH and are resistant to experimental autoimmune encephalomyelitis. Our work establishes a crucial role of Beclin 1 in autoimmunity.

Rescue of the mature B cell compartment in BAFF-deficient mice by treatment with recombinant Fc-BAFF

BAFF deficiency in mice impairs B cell development beyond the transitional stage 1 in the spleen and thus severely reduces the size of follicular and marginal zone B cell compartments. Moreover, humoral immune responses in these mice are dramatically impaired. We now addressed the question whether the decrease in mature B cell numbers and the reduced humoral immune responses in BAFF-deficient mice could be overcome by the injection of recombinant BAFF. We therefore engineered a recombinant protein containing the human IgG1 Fc moiety fused to receptor-binding domain of human BAFF (Fc-BAFF). At 1 week after the second injection of this fusion protein a complete rescue of the marginal zone B cell compartment and a 50% rescue of the follicular B cell compartment was observed. Moreover these mice mounted a T cell-dependent humoral immune response indistinguishable from wild-type mice. By day 14 upon arrest of Fc-BAFF treatment mature B cell numbers in the blood dropped by 50%, indicating that the life span of mature B cells in the absence of BAFF is 14 days or less. Collectively these findings demonstrate that injection of Fc-BAFF in BAFF-deficient mice results in a temporary rescue of a functional mature B cell compartment.

Crucial Role for BAFF-BAFF-R Signaling in the Survival and Maintenance of Mature B Cells

Defects in the expression of either BAFF (B cell activating factor) or BAFF-R impairs B cell development beyond the immature, transitional type-1 stage and thus, prevents the formation of follicular and marginal zone B cells, whereas B-1 B cells remain unaffected. The expression of BAFF-R on all mature B cells might suggest a role for BAFF-R signaling also for their in vivo maintenance. Here, we show that, 14 days following a single injection of an anti-BAFF-R mAb that prevents BAFF binding, both follicular and marginal zone B cell numbers are drastically reduced, whereas B-1 cells are not affected. Injection of control, isotype-matched but non-blocking anti-BAFF-R mAbs does not result in B cell depletion. We also show that this depletion is neither due to antibody-dependent cellular cytotoxicity nor to complement-mediated lysis. Moreover, prevention of BAFF binding leads to a decrease in the size of the B cell follicles, an impairment of a T cell dependent humoral immune response and a reduction in the formation of memory B cells. Collectively, these results establish a central role for BAFF-BAFF-R signaling in the in vivo survival and maintenance of both follicular and marginal zone B cell pools.