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.     

CD40/CD40 homodimers are required for CD40-induced phosphatidylinositol 3-kinase-dependent expression of B7.2 by human B lymphocytes

Preformed CD40/CD40 homodimers were initially observed on human Burkitt lymphoma cell lines, normal B cells, and transitional bladder carcinoma cell lines. However, the nature and the biological relevance of these homodimers have not yet been investigated. In the present study, we demonstrated that Epstein-Barr virus-transformed B cells and CD40-transfected HEK 293 cells constitutively expressed disulfide-linked CD40/CD40 homodimers at low levels. Oligomerization of CD40 leads to a rapid and significant increase in the disulfide-linked CD40/CD40 homodimer formation, a response that could be prevented using a thiol-alkylating agent. Formation of CD40/CD40 homodimers was found to be absolutely required for CD40-mediated activation of phosphatidylinositol 3-kinase, which, in turn regulated B7.2 expression. In contrast, CD40 monomers provided the minimal signal emerging from CD40, activating p38 MAP kinase and inducing homotypic B cell adhesion. CD40/CD40 homodimer formation was totally independent of TRAF1/2/3/5 associations with the threonine at position 254 in the cytoplasmic tail of the CD40 molecules. This finding may be vital to better understanding the molecular mechanisms that govern cell signaling triggered by CD40/CD154 interactions.     

CD30/CD30 ligand and CD40/CD40 ligand in malignant lymphoid disorders

CD30 and CD40 are members of the tumor necrosis factor (TNF) receptor family. These two receptors have pleiotropic biologic functions including induction of apoptosis and enhancing cell survival. This review will discuss the pattern of expression of these receptors in malignant lymphoid disorders and their prospective ligands. Understanding issues related to these two ligands and their receptors in lymphoid malignancies may help to improve the classification of these diseases and could open the doors for new treatment strategies.     

Cutting edge: distinct roles for T help and CD40/CD40 ligand in regulating differentiation of proliferation-competent memory CD8+ T cells

Murine primary antiviral cytotoxic T cell (CTL) responses are often induced in the absence of Th cells. In this study, we show that virus-like particles, if combined with DNA rich in CpG motifs, efficiently trigger primary CTL responses and comparable frequencies of memory CTLs in the presence or absence of T help. However, memory CTLs primed in the absence of T help failed to proliferate upon viral challenge. Nevertheless, they were efficiently recruited to sites of inflammation, indicating that T help may regulate the balance between proliferation-competent and migration-competent memory CTLs. Surprisingly, generation of proliferation-competent memory CTLs was completely independent of CD40 or CD40L, molecules commonly assumed to be central for mediating the beneficial effects of Th cells on CTL development. Thus, Th cells but not CD40/CD40L are key for the differentiation of proliferation-competent central memory CD8(+) T cells.     

In vivo suppression of naive CD4 T cell responses by IL-2- and antigen-stimulated T lymphocytes in the absence of APC competition

After stimulation, T cells enter a transient refractory period, promoted by IL-2, during which they are resistant to re-stimulation. We previously demonstrated that these IL-2- and Ag-stimulated refractory T cells are able to suppress the Ag-induced proliferation of naive T cells in vitro. We show here that, after adoptive transfer, these T cells are also able to suppress naive T cell proliferation in vivo. More interestingly, potently suppressive T cells can be generated directly in vivo by stimulation with Ag and supplemental IL-2. The activity of the suppressive cells is dose dependent, and the suppressor and suppressed T cells need not be restricted to the same MHC or Ag. Similar to its role in promoting T cell-mediated suppression in vitro, IL-2 is critical for the induction of suppressive activity in activated T cells in vivo. Supplemental IL-2, however, cannot overcome the suppressive activity in target T cells, indicating that suppression is not mediated by competition for this cytokine. Although the activated T cells block naive T cell proliferation, the naive cells do engage Ag and up-regulate the CD25 and CD69 activation markers after stimulation. Therefore, activated T cells stimulated in the presence of IL-2 develop MHC- and Ag-unrestricted suppressive activity. These results provide a new mechanism for competition among CD4(+) T lymphocytes, in which initial waves of responding T cells may inhibit subsequently recruited naive T cells. They further suggest a novel negative feedback loop limiting the expansion of T cell responses that may be present during vigorous immune responses or after IL-2 immunotherapy.     

CD28-independent costimulation of T cells by OX40 ligand and CD70 on activated B cells.

OX40 and its ligand (OX40L) have been implicated in T cell-dependent humoral immune responses. To further characterize the role of OX40/OX40L in T-B cell interaction, we newly generated an anti-mouse OX40L mAb (RM134L) that can inhibit the costimulatory activity of OX40L transfectants for anti-CD3-stimulated T cell proliferation. Flow cytometric analyses using RM134L and an anti-mouse OX40 mAb indicated that OX40 was inducible on splenic T cells by stimulation with immobilized anti-CD3 mAb in a CD28-independent manner, while OX40L was not expressed on resting or activated T cells. OX40L was inducible on splenic B cells by stimulation with anti-IgM Ab plus anti-CD40 mAb, but not by either alone. These activated B cells exhibited a potent costimulatory activity for anti-CD3-stimulated T cell proliferation and IL-2 production. Anti-CD80 and anti-CD86 mAbs partially inhibited the costimulatory activity, and further inhibition was obtained by their combination with RM134L and/or anti-CD70 mAb. We also found the anti-IgM Ab- plus anti-CD40 mAb-stimulated B cells exhibited a potent costimulatory activity for proliferation of and IL-2 production by anti-CD3-stimulated CD28- T cells from CD28-deficient mice, which was substantially inhibited by RM134L and/or anti-CD70 mAb. These results indicated that OX40L and CD70 expressed on surface Ig- and CD40-stimulated B cells can provide CD28-independent costimulatory signals to T cells.     

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.     

Ectopic CD40 ligand expression on B cells triggers intestinal inflammation

Several studies indicate that CD4(+) T cells, macrophages, and dendritic cells initially mediate intestinal inflammation in murine models of human inflammatory bowel disease. However, the initial role of B cells in the development of intestinal inflammation remains unclear. In this study we present evidence that B cells can trigger intestinal inflammation using transgenic (Tg) mice expressing CD40 ligand (CD40L) ectopically on B cells (CD40L/B Tg). We demonstrated that CD40L/B Tg mice spontaneously developed severe transmural intestinal inflammation in both colon and ileum at 8-15 wk of age. In contrast, CD40L/B TgxCD40(-/-) double-mutant mice did not develop colitis, indicating the direct involvement of CD40-CD40L interaction in the development of intestinal inflammation. The inflammatory infiltrates consisted predominantly of massive aggregated, IgM-positive B cells. These mice were also characterized by the presence of anti-colon autoantibodies and elevated IFN-gamma production. Furthermore, although mice transferred with CD4(+) T cells alone or with both CD4(+) T and B220(+) B cells, but not B220(+) cells alone, from diseased CD40L/B Tg mice, develop colitis, mice transferred with B220(+) B cells from diseased CD40L/B Tg mice and CD4(+) T cells from wild-type mice also develop colitis, indicating that the Tg B cells should be a trigger for this colitis model, whereas T cells are involved as effectors. As it has been demonstrated that CD40L is ectopically expressed on B cells in some autoimmune diseases, the present study suggests the possible contribution of B cells in triggering intestinal inflammation in human inflammatory bowel disease.     

CD40-CD40 ligand costimulation is required for generating antiviral CD4 T cell responses but is dispensable for CD8 T cell responses.

This study documents a striking dichotomy between CD4 and CD8 T cells in terms of their requirements for CD40-CD40 ligand (CD40L) costimulation. CD40L-deficient (-/-) mice made potent virus-specific CD8 T cell responses to dominant as well as subdominant epitopes following infection with lymphocytic choriomeningitis virus. In contrast, in the very same mice, virus-specific CD4 T cell responses were severely compromised. There were 10-fold fewer virus-specific CD4 T cells in CD40L-/- mice compared with those in CD40L+/+ mice, and this inhibition was seen for both Th1 (IFN-gamma, IL-2) and Th2 (IL-4) responses. An in vivo functional consequence of this Th cell defect was the inability of CD40L-/- mice to control a chronic lymphocytic choriomeningitis virus infection. This study highlights the importance of CD40-CD40L interactions in generating virus-specific CD4 T cell responses and in resolving chronic viral infection.     

Increased T cell autoreactivity in the absence of CD40-CD40 ligand interactions: a role of CD40 in regulatory T cell development

Mutations in the CD40 ligand (CD40L) gene lead to X-linked immunodeficiency with hyper-IgM, which is often associated with autoimmune diseases. To determine the contribution of defective CD40-CD40L interactions to T cell autoreactivity, we reconstituted CD40-CD40L interactions by transferring T cells from CD40-deficient mice to syngenic athymic nude mice and assessed autoimmunity. T cells from CD40-deficient mice triggered autoimmune diseases accompanied with elevations of various autoantibodies, while those from wild-type mice did not. In CD40-deficient mice, the CD25(+) CD45RB(low) CD4(+) subpopulation which regulates T cell autoreactivity was markedly reduced. CD40-deficient APCs failed to induce T regulatory cells 1 producing high levels of an inhibitory cytokine, IL-10 in vitro. Furthermore, autoimmune development was inhibited when T cells from CD40-deficient mice were cotransferred with CD45RB(low) CD4(+) T cells from wild-type mice or with T regulatory cells 1 induced on CD40-expressing APCs. Collectively, our results indicate that CD40-CD40L interactions contribute to negative regulation of T cell autoreactivity and that defective interactions can lead to autoimmunity.     

CD40 ligand mutants responsible for X-linked hyper-IgM syndrome associate with wild type CD40 ligand

CD40 ligand (CD40L) is a 33-kDa type II membrane glycoprotein mainly expressed on activated CD4(+) T cells in trimeric form. When it is mutated, the clinical consequences are X-linked hyper-IgM syndrome (XHIM), a primary immunodeficiency disorder characterized by low levels of IgG, IgA, and elevated or normal levels of IgM. Mutated CD40L can no longer bind CD40 nor provide signals for B cells to proliferate and to switch from IgM to other immunoglobulin isotypes. When considering gene therapy for XHIM, it is important to address the possibility that the mutated CD40L associates with transduced wild type CD40L, and as a consequence, immune reconstitution is not attained. In this study, we demonstrate that the various mutated CD40L species we have identified in patients with XHIM, including both full-length and truncated mutants, associate with wild type CD40L on the cell surface of co-transfected COS cells. The association between wild type and mutated CD40L was also observed in CD4(+) T cell lines established from XHIM patients with leaky splice site mutations. The clinical phenotype of these patients suggests that this association between wild type and mutated CD40L species may result in less efficient cross-linking of CD40.     

CD40/CD40 ligand interactions are critical for elicitation of autoimmune-mediated fibrosis in the lung

Pulmonary interstitial fibrosis (PIF), associated with persistent inflammation and increased collagen deposition in the interstitium, is often considered an autoimmune disease. Hapten immune PIF (HIPIF), a model for PIF, is elicited in the lung by a single intratracheal (i.t.) challenge in mice sensitized with hapten (2,4,6-trinitrobenzene sulfonic acid, TNBS). In this study, we characterized the role of CD40/CD40 ligand (CD40L) interactions in the elicitation of secondary cell-mediated immune responses that lead to development of fibrosis in the lung using an adoptive transfer model of HIPIF. The expression of CD40 was detected on bronchoalveolar lavage (BAL) cells 1-3 days after i.t. challenge with hapten in the HIPIF lung, but not lungs from the control mice. The CD40(bright) BAL cells morphologically resembled infiltrating monocytes. Furthermore, blocking CD40/CD40L interactions with blocking Ab decreased BAL production of Th1-mediators (IL-12 and TNF-alpha). Moreover, either blocking CD40/CD40L interactions with the Ab or using IL-12 knockout recipient mice prevented the increased collagen deposition (accumulation of hydroxyproline) in the lungs during HIPIF induction. We conclude that second signals (CD40/CD40L interactions) are required for elicitation of secondary immune responses that lead to PIF in vivo. The results support the notion that CD40/CD40L interactions are involved in the pathogenesis of an ongoing autoimmune disease.     

CD43 functions as a ligand for E-Selectin on activated T cells

E-selectin, an inducible cell adhesion molecule expressed on endothelial cells, mediates the rolling on endothelium of leukocytes expressing E-selectin ligands, such as neutrophils and activated T cells. Although previous studies using mice lacking P-selectin glycoprotein ligand-1 (PSGL-1) have indicated that PSGL-1 on Th1 cells functions as an E-selectin ligand, the molecular nature of E-selectin ligands other than PSGL-1 remains unknown. In this study, we show that a 130-kDa glycoprotein was precipitated by an E-selectin-IgG chimera from mouse Th1 cells. This protein was cleaved by O-sialoglycoprotein endopeptidase and required sialic acid for E-selectin binding. The mAb 1B11, which recognizes the 130-kDa glycoform of CD43, recognized the 130-kDa band in the E-selectin-IgG precipitate. In addition, immunoprecipitation of the E-selectin-IgG precipitate with 1B11 depleted the 130-kDa protein, further confirming its identity as CD43. CD43 was also precipitated with E-selectin-IgG from cultured human T cells. E-selectin-dependent cell rolling on CD43 was observed under flow conditions using a CD43-IgG chimera generated in Chinese hamster ovary cells expressing alpha-1,3-fucosyltransferase VII and a core 2 beta-1,6-N-acetylglucosaminyltransferase. These results suggest that CD43, when modified by a specific set of glycosyltranferases, can function as an E-selectin ligand and therefore potentially mediate activated T cell migration into inflamed sites.     

Differences in signaling molecule organization between naive and memory CD4+ T lymphocytes

The immunological synapse is a highly organized complex formed at the junction between Ag-specific T cells and APCs as a prelude to cell activation. Although its exact role in modulating T cell signaling is unknown, it is commonly believed that the immunological synapse is the site of cross-talk between the T cell and APC (or target). We have examined the synapses formed by naive and memory CD4 cells during Ag-specific cognate interactions with APCs. We show that the mature immunological synapse forms more quickly during memory T cell activation. We further show that the composition of the synapse found in naive or memory cell conjugates with APCs is distinct with the tyrosine phosphatase, CD45, being a more integral component of the mature synapses formed by memory cells. Finally, we show that signaling molecules, including CD45, are preassociated in discrete, lipid-raft microdomains in resting memory cells but not in naive cells. Thus, enhanced memory cell responses may be due to intrinsic properties of signaling molecule organization.     

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.     

Multimeric soluble CD40 ligand and GITR ligand as adjuvants for human immunodeficiency virus DNA vaccines

For use in humans, human immunodeficiency virus (HIV) DNA vaccines may need to include immunostimulatory adjuvant molecules. CD40 ligand (CD40L), a member of the tumor necrosis factor (TNF) superfamily (TNFSF), is one candidate adjuvant, but it has been difficult to use because it is normally expressed as a trimeric membrane molecule. Soluble trimeric forms of CD40L have been produced, but in vitro data indicate that multimeric, many-trimer forms of soluble CD40L are more active. This multimerization requirement was evaluated in mice using plasmids that encoded either 1-trimer, 2-trimer, or 4-trimer soluble forms of CD40L. Fusion with the body of Acrp30 was used to produce the 2-trimer form, and fusion with the body of surfactant protein D was used to produce the 4-trimer form. Using plasmids for secreted HIV-1 antigens Gag and Env, soluble CD40L was active as an adjuvant in direct proportion to the valence of the trimers (1 < 2 < 4). These CD40L-augmented DNA vaccines elicited strong CD8(+) T-cell responses but did not elicit significant CD4(+) T-cell or antibody responses. To test the applicability of the multimeric fusion protein approach to other TNFSFs, a 4-trimer construct for the ligand of glucocorticoid-induced TNF family-related receptor (GITR) was also prepared. Multimeric soluble GITR ligand (GITRL) augmented the CD8(+) T-cell, CD4(+) T-cell, and antibody responses to DNA vaccination. In summary, multimeric CD40L and GITRL are new adjuvants for DNA vaccines. Plasmids for expressing multimeric TNFSF fusion proteins permit the rapid testing of TNFSF molecules in vivo.