CD40 ligation triggers COX-2 expression in endothelial cells: evidence that CD40-mediated IL-6 synthesis is COX-2-dependent.

OBJECTIVE: To investigate whether CD40-CD154 interactions on HUVEC can trigger COX-2 synthesis as well as PGE2 and PGI2 secretion in vitro and explore whether the CD40-triggered prostanoids provide costimulatory signals for IL-6 secretion in this cell type. MATERIALS AND METHODS: COX-2 protein expression was examined in HUVEC using Western blot analysis. ELISAs were employed to assess PGE2, PGI2 and IL-6 synthesis. RESULTS: We found that COX-2 expression is upregulated when HUVEC are cultured with CD154+ D1.1 cells but not CD154- B2.7 cells. This effect was specifically inhibited by anti-CD154 mAb, and was amplified by the presence of IFNgamma. Analysis of cell supernatants showed a concomitant rise in PGE2 and PGI2 secretion triggered by CD154+ D1.1 cells, or rsCD154. Use of selective (NS-398) and non-selective (ibuprofen) COX-2 inhibitors effectively inhibited prostanoid synthesis triggered by CD40 ligation. Reduction in prostanoid levels by NS-398 was accompanied by a reduction in IL-6 secretion levels triggered by CD40 ligation. Furthermore, exogenously added PGE2 triggered a dose-dependent IL-6 secretion, which was unaffected by NS-398. CONCLUSIONS: These studies demonstrate that CD40 ligation upregulates HUVEC COX-2 expression and function. Moreover, the data strongly suggest that CD154-induced IL-6 secretion in HUVEC is dependent on COX-2 activity.

T cells in the pathogenesis of systemic lupus erythematosus: potential roles of CD154-CD40 interactions and costimulatory molecules.

CD154 is an activation-induced CD4+ T cell surface molecule that interacts with CD40 on antigen-presenting cells (APC) and upregulates the key costimulatory molecules, CD80 and CD86. Bidirectional intercellular signaling mediated by CD40 ligation and CD80/CD86 interactions with counter-receptors on T cells play central roles in regulating the survival and outgrowth of pathogenic autoreactive T cells and B cells in systemic lupus erythematosus (SLE). CD40 is also expressed on a variety of other cells, including endothelial cells and renal tubule epithelial cells. CD154 activation of APCs, endothelial cells, and renal tubular epithelial cells have proinflammatory or procoagulant effects that may contribute to the pathogenesis of lupus. This review will focus on the immunobiology of CD154-CD40 interactions and the costimulatory functions of CD80 and CD86. The experimental evidence suggesting roles for these molecules in the immunopathogenesis of SLE will be reviewed.

Analysis of CD154 and CD40 expression in native coronary atherosclerosis and transplant associated coronary artery disease.

T cells have roles in the pathogenesis of native coronary atherosclerosis (CA) and transplant-associated coronary artery disease (TCAD). The mechanisms by which T cells interact with other cells in these lesions are not fully known. CD154 is an activation-induced CD4+ T cell surface molecule that interacts with CD40+ target cells, including macrophages and endothelial cells, and induces the production of pro-inflammatory molecules, including CD54 (ICAM-1) and CD106 (VCAM-1). To investigate whether CD154-CD40 interactions might be involved in the pathogenesis of CA or TCAD we performed immunohistochemical studies of CD154 and CD40 expression on frozen sections of coronary arteries obtained from cardiac allograft recipients with CA (n=10) or TCAD (n=9). Utilizing four different anti-CD154 mAb we found that CD154 expression was restricted to infiltrating lymphocytes in CA and TCAD. CD40 expression was markedly up-regulated on intimal endothelial cells, foam cells, macrophages and smooth muscle cells in both diseases. Dual immunolabeling demonstrated many CD40+ cells co-expressed CD54 and CD106. The extent of CD40, CD54 and CD106 expression showed statistical significant correlation with the severity of disease and the amount of intimal lymphocytes. Together these studies demonstrate the presence of activated CD154+ and CD40+ cells in both CA and TCAD lesions and suggest that CD154-mediated interactions with CD40+ macrophages, foam cells, smooth muscle cells and/or endothelial cells may contribute to the pathogenesis of these diseases.

CD154 (CD40L) induces human endothelial cell chemokine production and migration of leukocyte subsets.

CD154-CD40 interactions play key roles in humoral and cellular immune responses. With regard to the latter, ligation of CD40 on endothelial cells upregulates important intercellular adhesion molecules. Activated endothelial cells also regulate leukocyte trafficking into inflammatory sites by secreting chemokines. In this study we asked whether CD154 mediated signals induce human umbilical vein endothelial cells (HUVEC) to secrete neutrophil or peripheral blood mononuclear cell (PBMC) chemoattractants. HUVEC were cocultured with CD154(+) Jurkat D1.1 cells in the presence or in the absence of anti-CD154 mAb or control mAb. Additionally, HUVEC were cocultured with control CD154(-) Jurkat B2.7 cells. Supernatants were harvested after 24 h and chemotaxis assays performed. Supernatants derived from Jurkat cells did not induce either neutrophil or PBMC chemotaxis. Resting endothelial cells produce at baseline some neutrophil and PBMC chemoattractants. However, there was significantly enhanced neutrophil and PBMC chemoattractant activity in supernatants derived from CD154 stimulated HUVEC. The enhanced leukocyte migration was specifically inhibited by anti-CD154 mAb. Anti-chemokine mAbs were used to identify specific chemokines mediating the enhanced leukocyte chemotaxis activity in CD154 stimulated HUVEC supernatants. There was complete or near complete inhibition of enhanced neutrophil and PBMC migration by anti-IL-8 and anti-monocyte chemoattractant protein-1 (MCP-1) mAbs, respectively. Anti-RANTES mAb partially blocked the enhanced PBMC migration, whereas anti-macrophage inflammatory protein-1alpha (MIP-1alpha) mAb had no effect. Utilizing specific ELISAs, we confirmed that CD40 ligation induces HUVEC to secrete IL-8, MCP-1, and RANTES, but not MIP-1alpha. Finally, we present evidence that the effects of CD154-CD40 interactions on HUVEC chemokine production are independent of IL-1beta production. These findings demonstrate that CD154-CD40 interactions induce endothelial cells to produce specific neutrophil and mononuclear cell chemoattractants.

TNF-alpha, not CD154 (CD40L), plays a major role in SEB-dependent, CD4(+) T cell-induced endothelial cell activation in vitro.

CD4(+) T cell effector molecules, in particular TNF-alpha and CD154, activate endothelial cells. However, the relative contributions of TNF-alpha and CD154 in mediating endothelial cell activation during complex Ag-driven CD4(+) T cell-endothelial cell interactions are not known. We utilized an in vitro model of CD4(+) T cell-endothelial cell interactions to characterize the contributions of TNF-alpha and CD154 in mediating upregulation of adhesion molecules CD54, CD62E, and CD106 on human umbilical vein endothelial cells (HUVEC). HUVEC were first treated with IFN-gamma to upregulate MHC Class II expression. IFN-gamma minimally effects HUVEC adhesion molecule expression but renders them capable of MHC class II restricted interactions with CD4(+) T cells. Coculturing MHC class II+ HUVEC and CD4(+) T cells with the superantigen SEB induces a rapid and marked upregulation of CD54, CD62E, and CD106 expression on HUVEC, as shown by FACS analysis. To study the effector molecules mediating SEB-driven, CD4(+) T cell-dependent endothelial cell activation, similar experiments were performed in the presence of neutralizing anti-CD154, anti-TNF-alpha, or anti-IL1 antibodies, as well as combinations of these antibodies. In contrast to the anti-CD154 or anti-IL-1 antibodies, the anti-TNF-alpha mAb markedly inhibited SEB-dependent, CD4(+) T cell-induced HUVEC activation. We conclude that TNF-alpha, not CD154, plays the major role in SEB-driven, CD4(+) T cell-induced endothelial cell activation in vitro.

CD40L-CD40 interactions regulate endothelial cell surface tissue factor and thrombomodulin expression.

During immune responses, activated endothelial cells down-regulate thrombomodulin and up-regulate tissue factor expression leading to the development of a procoagulant surface. CD4+ T cells are known to promote endothelial cell procoagulant activity, however, the molecular interactions that mediate this effect are not completely known. CD40L is an activation-induced CD4+ T cell surface molecule that functionally interacts with CD40 expressed on endothelial cells. In this study we ask if CD40L-CD40 interactions modulate endothelial cell surface tissue factor or thrombomodulin expression in vitro. Human umbilical vein endothelial cells (HUVEC) were cocultured with control cells or CD40L+ Jurkat T cells in the presence or absence of anti-CD40L mAb. By two-color FACS analysis we demonstrated that CD40 ligation induces HUVEC tissue factor expression and thrombomodulin down-regulation. Utilizing neutralizing antibodies, we show that CD40L-mediated tissue factor and thrombomodulin modulation, as well as E-selectin and VCAM-1 upregulation, is independent of tumor necrosis factor alpha, interleukin-1alpha, or interleukin-1beta production. Together these data suggest that CD40L-CD40 interactions may directly regulate endothelial cell procoagulant activity during inflammatory responses.

Human vascular endothelial cells are a rich and regulatable source of secretory sphingomyelinase. Implications for early atherogenesis and ceramide-mediated cell signaling.

We recently reported that macrophages and fibroblasts secrete a Zn2+-dependent sphingomyelinase (S-SMase), which, like lysosomal SMase, is a product of the acid SMase gene. S-SMase may cause subendothelial retention and aggregation of lipoproteins during atherogenesis, and the acid SMase gene has been implicated in ceramide-mediated cell signaling, especially involving apoptosis of endothelial cells. Because of the central importance of the endothelium in each of these processes, we now sought to examine the secretion and regulation of S-SMase by vascular endothelial cells. Herein we show that cultured human coronary artery and umbilical vein endothelial cells secrete massive amounts of S-SMase (up to 20-fold more than macrophages). Moreover, whereas S-SMase secreted by macrophages and fibroblasts is almost totally dependent on the addition of exogenous Zn2+, endothelium-derived S-SMase was partially active even in the absence of added Zn2+. Secretion of S-SMase by endothelial cells occurred both apically and basolaterally, suggesting an endothelial contribution to both serum and arterial wall SMase. When endothelial cells were incubated with inflammatory cytokines, such as interleukin-1beta and interferon-gamma, S-SMase secretion by endothelial cells was increased 2-3-fold above the already high level of basal secretion, whereas lysosomal SMase activity was decreased. The mechanism of interleukin-1beta-stimulated secretion appears to be through increased routing of a SMase precursor protein through the secretory pathway. In summary, endothelial cells are a rich and regulatable source of enzymatically active S-SMase, suggesting physiologic and pathophysiologic roles for this enzyme.

Immunohistologic analysis of renal CD40 and CD40L expression in lupus nephritis and other glomerulonephritides.

OBJECTIVE: To investigate potential mechanisms by which CD40L-mediated signals may be involved in the pathogenesis of lupus glomerulonephritis (GN). METHODS: Renal in situ CD40L and CD40 expression was examined in patient biopsy specimens. Immunohistochemical studies were performed on frozen sections utilizing anti-CD40L monoclonal antibody (MAb), anti-CD40 MAb, or control MAb. As controls, we analyzed normal kidney specimens and specimens obtained from patients with IgA nephropathy, focal segmental glomerulosclerosis, minimal change disease, idiopathic membranous GN, and antineutrophil cytoplasmic antibody-positive pauci-immune GN. Staining distribution was noted and staining intensity scored on a semiquantitative scale of 0 (no staining) to 3+ (intense staining). RESULTS: In normal kidney, CD40 was expressed on parietal epithelial cells, mesangial cells, endothelial cells, and distal tubules but not proximal tubules. Glomerular and tubular CD40 expression was markedly up-regulated in class III and class IV lupus GN, where there was intense staining of crescents, proximal and distal tubules, and interstitial mononuclear cells. In contrast, CD40 expression in class V lupus GN was similar to that in normal kidney. Interstitial mononuclear cells expressing CD40L were present in class IV lupus GN. However, these findings were not unique to lupus GN: up-regulation of CD40 and CD40L expression was similarly observed in other inflammatory renal diseases. CONCLUSION: This study shows that CD40 is expressed on a variety of renal parenchymal and non-parenchymal cells in normal kidney. Renal CD40 expression is up-regulated in class III and class IV lupus nephritis, as well as in other inflammatory renal diseases, and is associated with the presence of CD40L+ mononuclear cells.

Herpesvirus saimiri-transformed human CD4+ T cells can provide polyclonal B cell help via the CD40 ligand as well as the TNF-alpha pathway and through release of lymphokines.

We have developed human CD4+ T cell lines from the PBL of normal donors by infection with Herpesvirus saimiri (HVS), to evaluate functional properties of these immortalized lymphocytes. In this report, we characterize two such CD4+ T cell lines, CHCD4 and MHCD4, which were derived from two different donors. These cells grew independent of exogenous IL-2 stimulation for over 1 yr, and expressed surface markers (CD25+, CD69+, HLA-DR+, and B7+) associated with an activated T cell phenotype. Both lines constitutively produced and released IFN-gamma, but no IL-2 or IL-4. However, the surface expression of the two cell lines differed in that CHCD4 constitutively expressed CD40 ligand (CD40L) and membrane TNF-alpha, but MHCD4 did not. Also, CHCD4, but not MHCD4, potently induced polyclonal B cell activation and differentiation in the absence of PWM, in an MHC-unrestricted fashion. The B cell help afforded by CHCD4 included contact-dependent and soluble components. Contact-dependent help was strongly inhibited by mAb against CD40L (5C8) and to a lesser extent, by anti-TNF-alpha Ab. The CD40L-dependent helper function of CHCD4 contrasts with the recent description of other HVS-transformed CD4+ T cells that provide B cell help primarily via the membrane TNF-alpha and TNF-alphaR pathways. Furthermore, CHCD4 cells also secreted soluble factors that could mediate CD40-linked B cell differentiation into Ab-producing cells. Interestingly, this factor is not likely to be IL-2, IL-4, IL-6, IL-10, IL-15, TNF-alpha, or IFN-gamma as Abs against these cytokines were not able to inhibit the contact-independent B cell help by CHCD4. These results indicate that HVS-immortalization of CD4+ lymphocytes may produce T cell clones with a spectrum of important contact-dependent, as well as contact-independent, B cell helper function capacities.

The central role of the CD40-ligand and CD40 pathway in T-lymphocyte-mediated differentiation of B lymphocytes.

This review summarizes recent findings concerning the role of CD40-ligand and CD40 interactions in B-cell differentiation. CD40-ligand on helper CD4+ T lymphocytes interacts with CD40 on B cells and directs the selection and differentiation of clones of B lymphocytes to generate specific antibody-dependent immune responses. CD40-ligand is necessary for normal B-cell differentiation and plays several distinctive roles in this multistage process. The CD40 signaling pathway that normally regulates B-cell death appears to be usurped by the Epstein-Barr virus to mediate B-cell transformation.

Functional interactions of T cells with endothelial cells: the role of CD40L-CD40-mediated signals.

CD40 is expressed on a variety of cells, including B cells, monocytes, dendritic cells, and fibroblasts. CD40 interacts with CD40L, a 30-33-kD activation-induced CD4+ T cell surface molecule. CD40L-CD40 interactions are known to play key roles in B cell activation and differentiation in vitro and in vivo. We now report that normal human endothelial cells also express CD40 in situ, and CD40L-CD40 interactions induce endothelial cell activation in vitro. Frozen sections from normal spleen, thyroid, skin, muscle, kidney, lung, or umbilical cord were studied for CD40 expression by immunohistochemistry. Endothelial cells from all tissues studied express CD40 in situ. Moreover, human umbilical vein endothelial cells (HUVEC) express CD40 in vitro, and recombinant interferon gamma induces HUVEC CD40 upregulation. CD40 expression on HUVEC is functionally significant because CD40L+ Jurkat T cells or CD40L+ 293 kidney cell transfectants, but not control cells, upregulate HUVEC CD54 (intercellular adhesion molecule-1), CD62E (E-selectin), and CD106 (vascular cell adhesion molecule-1) expression in vitro. Moreover, the kinetics of CD40L-, interleukin 1-, or tumor necrosis factor alpha-induced CD54, CD62E, and CD106 upregulation on HUVEC are similar. Finally, CD40L-CD40 interactions do not induce CD80, CD86, or major histocompatibility complex class II expression on HUVEC in vitro. These results demonstrate that CD40L-CD40 interactions induce endothelial cell activation in vitro. Moreover, they suggest a mechanism by which activated CD4+ T cells may augment inflammatory responses in vivo by upregulating the expression of endothelial cell surface adhesion molecules.

Opposing roles of CD95 (Fas/APO-1) and CD40 in the death and rescue of human low density tonsillar B cells.

The regulation of B cell death plays roles in the selection of Ag-specific B cells in humoral immune responses, controlling B cell homeostasis and perhaps limiting transformation. The present work addresses whether CD95 induces tonsillar B cells to undergo apoptosis and, if so, whether contact-dependent CD40-L:CD40 signaling can rescue tonsillar B cells from CD95-induced apoptosis. CD95 triggering by anti-CD95 mAb (APO-1) was studied in human tonsillar B cell populations that were separated by density centrifugation into fractions enriched for either low density, CD38+ B cells or high density, resting B cells. Low density tonsillar B cells express CD95 and undergo anti-CD95-mediated apoptosis by analysis of cellular morphology or DNA fragmentation by TUNEL assay. The induction of apoptosis in low density tonsillar B cells by anti-CD95 mAb is inhibited by CD40 signals provided by stably transfected CD40-L+ 293 cells, but not by control transfected 293 cells (expressing CD8). In addition, the rescuing effect of CD40-L+ cells is inhibited specifically by anti-CD40-L (mAb 5c8). The counteracting effects of CD95 and CD40 signaling were also studied in Ramos 2G6, a homogeneous B cell tumor line of germinal center phenotype that expresses CD95 and CD40. Similar to the behavior of low density tonsillar B cells, Ramos 2G6 undergoes anti-CD95-mediated apoptosis, which is prevented by CD40-mediated rescue. These data show that CD95 induces apoptosis in low density tonsillar B cells and that CD40-L:CD40 interactions rescue low density tonsillar B cells or the B cell tumor Ramos 2G6 from CD95-induced apoptosis, and suggest roles for CD95 and CD40 in B cell death and selection, respectively.

Ligation of CD40 on fibroblasts induces CD54 (ICAM-1) and CD106 (VCAM-1) up-regulation and IL-6 production and proliferation.

CD40 was originally described as a functionally significant B cell surface molecule. However, CD40 is also expressed on monocytes, dendritic cells, epithelial cells, and basophils. We now report that synovial membrane (SM) or dermal fibroblasts also express cell surface CD40 in vitro. Fibroblast CD40 expression declines with increasing time in culture and recombinant interferon-gamma (rINF-gamma) induces fibroblast CD40 up-regulation. This effect of rINF-gamma is augmented by recombinant interleukin-1 alpha or recombinant tumor necrosis factor-alpha. CD40 expression on fibroblasts is functionally significant because CD40L-CD40 interactions induce SM fibroblast CD54 (intercellular adhesion molecule-1) and CD106 (vascular cell adhesion molecule-1) up-regulation. Moreover, ligation of CD40 augments IL-6 production by SM fibroblasts and induces fibroblasts to proliferate. In addition, rINF-gamma enhances the effect of CD40L-CD40 interactions on fibroblast proliferation. Taken together, these studies show that fibroblasts can express CD40, cytokines can regulate fibroblast CD40 expression, and CD40 ligation induces fibroblast activation and proliferation.

Phenotypic and functional characterization of T-BAM (CD40 ligand)+ T-cell non-Hodgkin's lymphoma.

The precise mechanisms regulating T-helper function have been intensively investigated. We and others have recently identified a new T-cell-B-cell-activating molecule called T-BAM that directs B-cell differentiation by interacting with the CD40 molecule on B cells. Using a specific monoclonal antibody against T-BAM (5C8), we have previously shown that T-BAM expressing T cells are predominantly CD4+CD8- and in normal lymphoid tissue have a unique distribution. However, no information has been obtained regarding the phenotype and functional properties of human neoplastic T cells. Therefore, we investigated T-BAM expression immunohistochemically in 87 well-characterized T-cell non-Hodgkin's lymphomas and lymphoid leukemias (LL). We found that 21/81 neoplasms expressed detectable T-BAM and these positive tumors belong almost exclusively to the CD4+CD8- subtype. In addition, to determine whether T-BAM expression could be induced on T-BAM-LL cells, we activated T-BAM-LLs in vitro and showed that T-BAM could be upregulated only in CD4+CD8- tumors. Our studies clearly show that T-BAM is constitutively expressed in a large number of T-cell neoplasms with a relative mature phenotype (CD4+CD8-) and that only CD4+ neoplastic T cells can be induced in vitro to express this molecule. Additional studies are necessary to identify the biologic significance of T-BAM expression and its potential and clinical implications.

T lymphocyte T cell-B cell-activating molecule/CD40-L molecules induce normal B cells or chronic lymphocytic leukemia B cells to express CD80 (B7/BB-1) and enhance their costimulatory activity.

Activation-induced cell surface molecules are involved in mediating bidirectional T-B lymphocyte signaling that is important in the induction of T or B lymphocyte effector functions. In this regard, T-BAM/CD40-L is an activation-induced CD4+ T cell surface molecule known to be important in inducing B cell effector functions. This report demonstrates that T-BAM/CD40-L molecules on a Jurkat T cell leukemia subclone (D1.1) or nonlymphoid 293 kidney cell transfectants induce B cells or B-CLL cells to express CD80 (B7/BB-1) in a manner that is specifically inhibited by anti-T-BAM/CD40-L mAb 5C8. Because activation-induced B cell surface molecules, such as CD80, deliver costimulatory signals to T cells that augment T cell proliferation, the functional costimulatory capacity of T-BAM/CD40-L-primed B cells and B-CLL cells was studied. T-BAM/CD40-L-primed B cells or B-CLL cells augment the proliferative responses of allogenic T cells. Furthermore, T-BAM/CD40-L priming is specifically inhibited by mAb 5C8. Together, these studies demonstrate that T-BAM/CD40-L induces CD80 expression on resting B cells or B-CLL cells. Moreover, T-BAM/CD40-L signaling enhances B cell costimulatory capacity. These studies suggest that T-BAM/CD40-L molecules not only induce B cell differentiative processes that result in Ab secretion, but also enable B cells to prime Ag-specific T cells for subsequent clonal expansion.

Isolation of cDNAs encoding T-BAM, a surface glycoprotein on CD4+ T cells mediating contact-dependent helper function for B cells: identity with the CD40-ligand.

"T-cell B-cell Activating Molecule" (T-BAM) is an activation-induced surface protein on CD4+ T cells that mediates a contact-dependent signal for B cell differentiation and immunoglobulin (Ig) secretion. The T-BAM protein on a helper clone of Jurkat (D1.1) was affinity purified using the anti-T-BAM mAb, 5c8. The NH2-terminal amino acid sequence of purified T-BAM was determined and found to be highly homologous to the predicted NH2-terminal sequence of a T cell ligand to the B cell CD40 molecule (CD40-L). From a D1.1 cDNA library, a clone was isolated that encodes CD40-L by sequence and drives expression of T-BAM protein on transfected cells, demonstrating that the T-BAM and CD40-L genes and proteins are identical. Moreover, transfection of T-BAM was shown to confer to non-lymphoid cells, the ability to induce B cells to upregulate the expression of surface CD23 molecules. In previous studies we showed that T-BAM was expressed predominantly on activated CD4+ and on few if any CD8+ cells. Although the current work confirms that T-BAM is largely restricted to activated CD4+ T cells, we now provide definitive evidence that T-BAM can be expressed by a small population of CD8+ T cells after activation. Importantly, a subset of CD8+ T cells do not express T-BAM after activation and this T-BAM- phenotype is maintained on certain CD8+ T cell clones. Taken together, these data unify the biology and structure of T-BAM and CD40-L and this synthesis has implications for understanding the T cell regulation of the humoral immune response.

T-BAM/CD40-L on helper T lymphocytes augments lymphokine-induced B cell Ig isotype switch recombination and rescues B cells from programmed cell death.

An important component of T cell help for B lymphocyte differentiation is the contact-dependent signaling mediated by the T cell-B cell activating molecule (T-BAM/CD40-L), an activation-induced surface membrane protein on CD4+ T helper cells in lymphoid follicles that interacts with the B cell surface molecule, CD40. The present study dissects the roles of T-BAM/CD40-L in helper function by means of a neutralizing anti-T-BAM/CD40-L mAb (5c8), a T-BAM/CD40-L-expressing T cell tumor subclone (Jurkat D1.1), and a T-BAM/CD40-L-responsive IgM+ B cell tumor of germinal center origin (RAMOS 266). Like activated T cells, D1.1 cells induce B cells to synthesize IgG, IgA, and IgE in a process that is specifically inhibited by the mAb 5c8. Although rIL-4 alone, but not Jurkat D1.1, induces IgH C gamma mRNA transcripts in RAMOS 266, the T-BAM/CD40-L molecule on D1.1 acts on rIL-4-primed RAMOS B cells to augment expression of C gamma transcripts. In addition, IgG+ RAMOS 266 clones were expanded from D1.1- and rIL-4-stimulated cultures that had undergone deletional IgH isotype switch recombination events. Furthermore, T-BAM/CD40-L signals delivered by the D1.1 clone dramatically rescue RAMOS 266 from mAb anti-IgM-induced apoptosis. Taken together, these data support the idea that T-BAM/CD40-L plays important roles in inducing Ig isotype switch recombination and the clonal selection of isotype-switched B cells.

CD40 molecules induce down-modulation and endocytosis of T cell surface T cell-B cell activating molecule/CD40-L. Potential role in regulating helper effector function.

The T-BAM/CD40-L molecule on CD4+ T cells interacts with B cell CD40 molecules to deliver contact-dependent signals that drive B cell activation and Ig secretion. Cell surface T-BAM/CD40-L expression is transient and may be closely regulated in order to limit the activation and clonal selection of noncognate B cells. We demonstrate that B cells, but not non-B cells, rapidly and specifically down-modulate surface T-BAM/CD40-L expression in a contact-dependent and temperature-sensitive manner that renders T cells unable to activate resting bystander B cells. Because the ability to down-modulate T-BAM/CD40-L correlated with CD40 expression, the role of CD40 molecules in down-modulating its ligand was directly assessed. Anti-CD40 mAb, but not control mAb, block B cell-induced T-BAM/CD40-L down-modulation. Furthermore, CD40+ nonlymphoid transfectants specifically down-modulate surface T-BAM/CD40-L expression. B cells induce T-BAM/CD40-L internalization into cytoplasmic compartments in a process that is inhibited by cytochalasin B. Pretreatment of activated T cells with lysosomotropic agents does not affect CD40-induced down-modulation of surface T-BAM/CD40-L but results in a marked accumulation of T-BAM/CD40-L in cytoplasmic vesicles. Together, these studies strongly suggest that CD40 induced T-BAM/CD40-L down-modulation occurs, in part, by receptor-mediated endocytosis followed by lysosomal degradation and may represent a mechanism to regulate CD4+ T cell helper effector functions.

Deletions in the ligand for CD40 in X-linked immunoglobulin deficiency with normal or elevated IgM (HIGMX-1).

Patients with X-linked Ig deficiency with normal or elevated IgM (HIGMX-1) fail to switch from IgM/IgD to other Ig isotypes. Interaction between the B cell antigen CD40 and the CD40 ligand expressed on activated T cells is critical for T cell driven isotype switching. We have reported that T lymphocytes from three unrelated male patients with HIGMX-1 failed to express CD40 ligand on their surface, but the mRNA for CD40 ligand was of an apparently normal size and level. Analysis of CD40 ligand cDNA from two of the patients revealed deletions that alter the reading frame. Patient 1 displayed two mutations: a C-->A transversion at nucleotide 590 and the deletion of an adjacent C nucleotide. The second patient had a 58 bp deletion from nucleotides 289-346. Furthermore, neither patient expressed a protein product detectable by the CD40L mAb, 5c8.