CD40 stimulation of human peripheral B lymphocytes: distinct response from naive and memory cells

During secondary immune response, memory B lymphocytes proliferate and differentiate into Ig-secreting cells. In mice, the binding of CD40 by CD154 clearly enhances the activation and differentiation of memory B lymphocytes. In humans, the role of CD40-CD154 in the stimulation of memory B lymphocytes is not as obvious since in vitro studies reported positive and negative effects on their proliferation and differentiation in Ig-secreting cells. In this study, we examine the response of peripheral memory and naive cells in relation to the duration of CD40-CD154 interaction. We measured the proliferation and differentiation of both subsets stimulated with CD154 and IL-4 for short- (4-5 days) and long-term (>7 days) periods. Following short-term stimulation, memory B lymphocytes did not expand but represented the only subset differentiating into IgG- and IgM-secreting cells. A longer stimulation of this population led to cell death, while promoting naive B lymphocyte proliferation, expansion, and differentiation into IgM- or IgG-secreting cells. This prolonged CD40 stimulation also triggered naive B lymphocytes to switch to IgG and to express CD27 even in absence of somatic hypermutation, suggesting that these latter events could be independent. This study suggests that naive and memory B lymphocytes have distinct requirements to engage an immune response, reflecting their different roles in humoral immunity.     

Scaffold Protein JLP Is Critical for CD40 Signaling in B Lymphocytes

CD40 expression on the surface of B lymphocytes is essential for their biological function and fate decision. The engagement of CD40 with its cognate ligand, CD154, leads to a sequence of cellular events in B lymphocytes, including CD40 cytoplasmic translocation, a temporal and spatial organization of effector molecules, and a cascade of CD40-induced signal transduction. The JLP scaffold protein was expressed in murine B lymphocytes. Using B lymphocytes from jlp-deficient mice, we observed that JLP deficiency resulted in defective CD40 internalization upon CD154/CD40 engagement. Examination of interactions and co-localization among CD40, JLP, dynein, and Rab5 in B lymphocytes suggested that CD40 internalization is a process of JLP-mediated vesicle transportation that depends on Rab5 and dynein. JLP deficiency also diminished CD40-dependent activation of MAPK and JNK, but not NF-κB. Inhibiting vesicle transportation from the direction of cell periphery to the cell center by a dynein inhibitor (ciliobrevin D) impaired both CD154-induced CD40 internalization and CD40-dependent MAPK activities in B lymphocytes. Collectively, our data demonstrate a novel role of the JLP scaffold protein in the bridging of CD154-triggered CD40 internalization and CD40-dependent signaling in splenic B lymphocytes.     

Effects of tobacco glycoprotein (TGP) on the immune system. II. TGP stimulates the proliferation of human T cells and the differentiation of human B cells into Ig secreting cells

We have been studying the effects of tobacco glycoprotein (TGP), a polyphenol-rich glycoprotein isolated from cured tobacco leaves, on the immune system. We have shown previously that mice immunized with TGP produce preferentially antibodies of the IgE isotype and that TGP is a T cell-independent B cell mitogen for mice, which stimulates B cell proliferation and B cell differentiation into Ig-secreting cells. We report herein that TGP stimulates a significant increase in [3H]TdR incorporation by human PBL and by human cord blood lymphocytes. The magnitude of the proliferative response of PBL to TGP does not correlate with the donor's titer of IgE antibodies to TGP, as assayed by a wheal and flare response after an i.d. injection of TGP, neither does it correlate with the donor's smoking history. [3H]TdR uptake is not observed before day 5 of culture, and the response peaks between days 5 and 10 of culture. Analysis of the cellular basis for the proliferative response suggests that T cells are proliferating. Two-parameter analysis by flow cytometry shows that CD3+, CD4+, and CD8+ cells are in the S + G2 + M phases, but not Ig-bearing cells or monocytes. A significant increase in HLA-DR (Ia)-bearing cells is observed on cells in all of the cell cycle phases. This increase coincides with cells entering the S phase. No increase is observed in the expression of the IL-2-R as assayed by the anti-Tac antibody. TGP also stimulates human PBL to differentiate and to produce Ig of the IgM, IgG, and IgA isotypes, without stimulating a detectable B cell proliferative response. The proliferative response of PBL is clearly due to TGP and not to contamination with LPS, because by the limulus amebocyte assay the TGP preparation contains less than 2% LPS, which could not account for the stimulation observed.     

Human B cell activation by autologous NK cells is regulated by CD40-CD40 ligand interaction: role of memory B cells and CD5+ B cells.

NK cells are a subpopulation of lymphocytes characterized primarily by their cytolytic activity. They are recognized as an important component of the immune response against virus infection and tumors. In addition to their cytolytic activity, NK cells also participate either directly or indirectly in the regulation of the ongoing Ab response. More recently, it has been suggested that NK cells have an important role in the outcome of autoimmune diseases. Here, we demonstrate that human NK cells can induce autologous resting B cells to synthesize Ig, including switching to IgG and IgA, reminiscent of a secondary Ab response. B cell activation by the NK cell is contact-dependent and rapid, suggesting an autocrine B cell-regulated process. This NK cell function is T cell-independent, requires an active cytoplasmic membrane, and is blocked by anti-CD40 ligand (anti-CD154) or CD40-mIg fusion protein, indicating a critical role for CD40-CD40 ligand interaction. Depletion studies also demonstrate that CD5+ B cells (autoreactive B-1 cells) and a heterogeneous population of CD27+ memory B cells play a critical role in the Ig response induced by NK cells. The existence of this novel mechanism of B cell activation has important implications in innate immunity, B cell-mediated autoimmunity, and B cell neoplasia.     

CD4+ T lymphocytes expressing CD40 ligand help the IgM antibody response to soluble pneumococcal polysaccharides via an intermediate cell type

Streptococcus pneumoniae causes serious infections in children, the elderly, and immunocompromised patients. Protection against infections with S. pneumoniae is mediated through Abs against the capsular polysaccharides (caps-PS). We previously showed that the murine Ab response to caps-PS is dependent on CD40-CD40L interaction. In the present paper, we addressed the question of whether the CD40-CD40L-mediated modulation of the anti-caps-PS immune reaction is the result of a direct interaction between B lymphocytes and T lymphocytes or of an indirect interaction. SCID/SCID mice reconstituted with B lymphocytes from wild-type mice did not mount anti-caps-PS Abs. SCID/SCID mice reconstituted with B lymphocytes from wild-type mice and CD4+ T lymphocytes from wild-type mice but not CD4+ T lymphocytes from CD40L knockout mice stimulated the anti-caps-PS Ab response. This indicated that CD4+ T lymphocytes stimulated the anti-caps-PS Ab response in a CD40L-dependent manner. SCID/SCID mice reconstituted with B lymphocytes from CD40 knockout mice and CD4+ T lymphocytes from wild-type mice generated an anti-caps-PS Ab response that could be inhibited by MR1, a blocking anti-CD40L Ab. These data indicated that CD4+ T lymphocytes stimulated the anti-caps-PS Ab response in an indirect way. Finally, lethally irradiated CD40 knockout mice reconstituted with bone marrow from wild-type mice mounted an anti-caps-PS Ab response that was comparable to the Ab response in wild-type mice, revealing that the required CD40 was on hemopoietic cells. In conclusion, we provide evidence that CD4+ T lymphocytes expressing CD40L stimulate the Ab response to soluble caps-PS by interacting with CD40-expressing non-B cells.     

The MS Risk Allele of CD40 Is Associated with Reduced Cell-Membrane Bound Expression in Antigen Presenting Cells: Implications for Gene Function

Human genetic and animal studies have implicated the costimulatory molecule CD40 in the development of multiple sclerosis (MS). We investigated the cell specific gene and protein expression variation controlled by the CD40 genetic variant(s) associated with MS, i.e. the T-allele at rs1883832. Previously we had shown that the risk allele is expressed at a lower level in whole blood, especially in people with MS. Here, we have defined the immune cell subsets responsible for genotype and disease effects on CD40 expression at the mRNA and protein level. In cell subsets in which CD40 is most highly expressed, B lymphocytes and dendritic cells, the MS-associated risk variant is associated with reduced CD40 cell-surface protein expression. In monocytes and dendritic cells, the risk allele additionally reduces the ratio of expression of full-length versus truncated CD40 mRNA, the latter encoding secreted CD40. We additionally show that MS patients, regardless of genotype, express significantly lower levels of CD40 cell-surface protein compared to unaffected controls in B lymphocytes. Thus, both genotype-dependent and independent down-regulation of cell-surface CD40 is a feature of MS. Lower expression of a co-stimulator of T cell activation, CD40, is therefore associated with increased MS risk despite the same CD40 variant being associated with reduced risk of other inflammatory autoimmune diseases. Our results highlight the complexity and likely individuality of autoimmune pathogenesis, and could be consistent with antiviral and/or immunoregulatory functions of CD40 playing an important role in protection from MS.     

B-cell expansion in the presence of the novel 293-CD40L-sCD40L cell line allows the generation of large numbers of efficient xenoantigen-free APC

BACKGROUND: CD40-activated B lymphocytes have been used successfully as potent APC for the induction of T-cell responses. However, the 3T3-CD40L cell line, regularly used for engagement of CD40 on the B-cell surface, is a potential source of xenoantigens. This may affect the specificity of T cells stimulated with CD40-activated B cells, especially when generation of T-cell lines specific for endogenously processed Ag is desired. METHODS: To develop a system that allows efficient expansion of B cells in the absence of sources of xenoantigens, we created a human 293-CD40L-sCD40L cell line that produces soluble CD40L and expresses CD40L on the cell surface. B cells from patients with hematologic malignancies were expanded on the 293-CD40L-sCD40L cells and used for stimulation of either naive or in vivo primed donor T cells in three HLA-identical patient-donor combinations. RESULTS: The 293-CD40L-sCD40L cell line was able to stimulate B-cell growth with an efficiency superior to that of the commonly used 3T3-CD40L cell line. In all cases T-cell lines and, subsequently, T-cell clones were generated that showed reactivity against patient and not donor B cells, suggesting their specificity for minor histocompatibility antigens (mHAg). DISCUSSION: B cells activated with GMP grade 293-CD40L-sCD40L can be used in a variety of applications. In particular, they may be suitable for ex vivo stimulation of T cells prior to donor lymphocyte infusion (DLI), which may enhance its graft versus leukemia (GvL) effect.     

T cell polarity at the immunological synapse is required for CD154-dependent IL-12 secretion by dendritic cells

Ag-specific interaction between T lymphocytes and dendritic cells (DCs) leads to both T cell and DC activation. CD154 (CD40 ligand)/CD40 interactions have been shown to play a major, although not exclusive, role in this functional cross-talk. Interactions between T cells and DCs are structured by an immunological synapse (IS), characterized by polarization of the T cell microtubule cytoskeleton toward the interacting DCs. Yet the role T cell polarization may play in T cell-induced DC activation is mostly unknown. In this study, we address the role of T cell polarity in CD154-dependent activation of DCs in a human model, using two different tools to block T cell polarity (i.e., a microtubule depolymerizing drug and an inhibitor of atypical protein kinase C). We show that CD154 is recruited and concentrated at the IS formed between human primary T cells and autologous DCs and that this recruitment requires T cell polarity at the IS. Moreover, we show that T cell polarization at the IS controls T cell-dependent CD154-CD40 signaling in DCs as well as CD154-dependent IL-12 secretion by DCs. This study shows that T cell polarity at the IS plays a key role in CD154/CD40-dependent cross-talk between CD4(+) T cells and DCs.     

B lymphocytes as antigen-presenting cells for CD4+ T cell priming in vivo

The contribution of B lymphocytes as APCs for CD4+ T cell priming remains controversial, based on findings that B cells cannot provide the requisite ligating and costimulatory signals for naive T cells to be activated. In the current study, we have examined Ag-specific T:B cell collaboration under circumstances in which B cells take up Ag through Ig receptors in vivo. This results in their activation and an ability to effectively stimulate naive CD4+ T cells both in vitro and in vivo. The aim of this work was to establish some of the key molecular interactions, as well as kinetics, between Ag-specific T and B cells that enable this priming to take place. Our approach was to amplify the starting pools of both Ag-specific T and B cell populations in vivo to track directly the events during initial T:B cell collaborations. We show that the induction of optimal levels of T cell priming to a protein Ag requires the involvement of Ag-specific B cells. The interaction that results between Ag-specific T and B cells prevents the down-modulation of B7 costimulatory molecules usually observed in the absence of appropriate T cells. Moreover, this prevention in down-modulation is independent of CD40:CD40 ligand contact. Finally, we present data suggesting that once Ag-specific T and B cells interact, there is a rapid (1-2-h) down-regulation of antigenic complexes on the surface of the B lymphocytes, possibly to prevent them from engaging other T cells in the vicinity and therefore focus the initial interaction.     

CD40-mediated activation of NF-kappa B in airway epithelial cells

We have reported previously that airway epithelial cells (AEC) express CD40 and that activation of this molecule stimulates the expression of inflammatory mediators, including the chemokine RANTES (regulated on activation normal T cell expressed and secreted). Because NF-kappaB regulates the expression of many inflammatory mediators, such as RANTES, we utilized CD40-mediated induction of RANTES expression to investigate the mechanisms that underlie CD40-mediated activation of NF-kappaB in AEC. Results demonstrate that, in AEC, intact NF-kappaB sites were required for CD40-mediated activation of the RANTES promoter. To examine activation of NF-kappaB binding directly, electrophoretic mobility shift analyses were performed. These analyses revealed that CD40 ligation stimulated NF-kappaB binding and that the activated NF-kappaB complexes were composed of p65 subunits. Additional studies focused on the CD40-triggered signaling pathways that facilitate NF-kappaB activation. Findings show that CD40 engagement activated the IkappaB kinases IKK-alpha and IKK-beta and stimulated IkappaBalpha phosphorylation. Analyses also examined the role of tumor necrosis factor-associated factor (TRAF) molecules in CD40-mediated NF-kappaB activation within AEC. Stable transfectants expressing wild-type or mutant forms of the cytoplasmic domain of CD40 suggested that TRAF3, but not TRAF2, binding was essential for CD40-mediated RANTES expression. Further studies indicated that exogenous expression of wild-type TRAF3 enhanced activation of the RANTES promoter, whereas exogenous expression of wild-type TRAF2 inhibited this activation; TRAF3-mediated enhancement was dependent upon NF-kappaB. Together, these findings suggest that, in AEC, ligation of CD40 regulates the expression of inflammatory mediators, such as RANTES, via activation of NF-kappaB. Moreover, these results suggest that CD40-mediated signaling in AEC differs with previously reported findings observed in other cell models, such as B lymphocytes.     

Cultured human follicular dendritic cells. Growth characteristics and interactions with B lymphocytes.

Minced human tonsils were digested with DNase and collagenase, and lymphoid cell-depleted low density cells were cultured and grown in granulocyte-macrophage-CSF. Large, morphologically homogenous adherent cells with elongated extensions grew continuously in culture. These nonphagocytic cells appear to be related to follicular dendritic cell (FDC) as they do not have properties of monocytic lineage cells or dendritic cells and because, like FDC, 1) they express CD11b, CD14, CD29, CD40, CD54, CD73, CD74, and VCAM-1, and do not express CD11c, CD22, T cell markers, CD18, CD25 and CD45; and 2) they bind human B lymphocytes and B cell lines, but not T lymphocytes by an adhesion blocked in part by mAb to VLA-4 (CD49d). The cultured FDC also augmented B cell proliferation stimulated by anti-mu sera and/or CD40 mAb. Cultured FDC spontaneously produced low levels of IL-6, but did not produce IL-1 alpha or TNF-alpha; however, after treatment with either IFN-gamma or LPS, they produced more IL-6. The expression of CD54 (ICAM-1) was elevated by treating the cultured FDC with either TNF-alpha, IL-1 beta, IFN-gamma or granulocyte-macrophage-CSF; in contrast, IL-4 had no effect on CD54 but rather up-regulated expression of VCAM-1. IFN-gamma, unlike the other cytokines tested, increased expression of a set of markers on cultured FDC (CD54, VCAM-1, and CD14) and converted these class II-negative cells into class II+ cells. The fact that various T cell-derived cytokines have different effects on FDC suggests that the T cell products may influence the manner by which FDC stimulate B cell proliferation and maturation.     

CD40-induced aggregation of MHC class II and CD80 on the cell surface leads to an early enhancement in antigen presentation

Ligation of CD40 on B cells increases their ability to present Ag and to activate MHC class II (MHC-II)-restricted T cells. How this occurs is not entirely clear. In this study we demonstrate that CD40 ligation on Ag-presenting B cells (APC) for a short period between 30 min and 3 h has a rapid, augmenting effect on the ability of a B cell line and normal B cells to activate T cells. This is not due to alterations in Ag processing or to an increase in surface expression of CD80, CD86, ICAM-1, or MHC-II. This effect is particularly evident with naive, resting T lymphocytes and appears to be more pronounced under limiting Ag concentrations. Shortly after CD40 ligation on a B cell line, MHC-II and CD80 progressively accumulated in cholesterol-enriched microdomains on the cell surface, which correlated with an initial enhancement in their Ag presentation ability. Moreover, CD40 ligation induced a second, late, more sustained enhancement of Ag presentation, which correlates with a significant increase in CD80 expression by APC. Thus, CD40 signaling enhances the efficiency with which APC activate T cells by at least two related, but distinct, mechanisms: an early stage characterized by aggregation of MHC-II and CD80 clusters, and a late stage in which a significant increase in CD80 expression is observed. These results raise the possibility that one important role of CD40 is to contribute to the formation of the immunological synapse on the APC side.     

CD4+ T lymphocytes with constitutive CD40 ligand in preautoimmune (NZB x NZW)F1 lupus-prone mice: phenotype and possible role in autoreactivity

Lupus disease is marked by B lymphocyte hyperactivity and the production of Abs to dsDNA. The production of these anti-dsDNA Abs is T lymphocyte dependent. However, it is not clear how CD4+ T lymphocytes provide help for B lymphocytes to produce IgG anti-dsDNA Abs. One possible mechanism is suggested by studies showing that human patients with systemic lupus erythematosus and lupus mice have increased numbers of CD40 ligand (CD40L)+ T and B lymphocytes. The results described in this study reveal that young, clinically healthy lupus-prone New Zealand Black x New Zealand White F1 (BWF1) mice have naive CD4+ T cells with preformed CD40L. These cells contribute to a brisk response to immunization and to the production of anti-dsDNA Abs. In vitro experiments revealed that CD4+ T cells with preformed CD40L could, upon stimulation, provide antiapoptotic signals for B cells but could not induce proliferation or reduce activation threshold. These results suggest that the direct target cells for the effect of T cells with preformed CD40L in lupus may not be B lymphocytes.     

Essential role for CD40 ligand interactions in T lymphocyte-mediated modulation of the murine immune response to pneumococcal capsular polysaccharides

Protection against infection with pneumococci is provided by anti-capsular polysaccharide (caps-PS) Abs. We investigated whether CD40 ligand (CD40L) plays a role in T lymphocyte-mediated regulation of the immune response to caps-PS, which are considered thymus-independent Ags. Administration of MR1, an antagonist mAb against murine CD40L, in BALB/c mice immunized with Pneumovax resulted in an inhibition of the IgM and IgG Ab response for various caps-PS serotypes. Evidence for the involvement of CD4(+) T lymphocytes in the Ab response to caps-PS was obtained in SCID/SCID mice that, when reconstituted with B lymphocytes and CD4(+) T lymphocytes, mounted a higher specific IgM response compared with SCID/SCID mice reconstituted with only B lymphocytes. This helper effect of CD4(+) T lymphocytes was abrogated by MR1. Blocking CD40L in vitro decreased the IgM response to caps-PS and abolished the helper effect of CD4(+) T lymphocytes. CD8(+) T lymphocyte-depleted murine spleen cells mounted a higher in vivo immune response than total murine spleen cells, which provided evidence for a suppressive role of CD8(+) T lymphocytes on the anti-caps-PS immune response. CD4(+) T lymphocyte-depleted murine spleen cells, leaving a B and CD8(+) T lymphocyte fraction, elicited only a weak in vivo and in vitro Ab response, which was enhanced after MR1 administration. In summary, our data provide evidence that T lymphocytes contribute to the regulation of the anti-caps-PS immune response in a CD40L-dependent manner.