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PubMed2000

Role for CD40-CD40 ligand interactions in the immune response to solid tumours.

CD40-mediated interactions play an important role in the response to infections, transplantation, and cancer by affecting the development, activation, proliferation and differentiation of a variety of immune cells. In the current study we examined the role of CD40-mediated interactions in immune responses to bladder, pancreatic and breast carcinomas as well as melanoma cell lines using soluble human CD40L (rhCD40L) or anti-CD40 mAb in vitro. CD40 expression was readily detected in a large proportion of the cell lines and was augmented but not induced de novo by treatment with IFNgamma. Treatment of CD40-positive cell lines with rhCD40L or anti-CD40mAb enhanced cell surface expression of ICAM-1 and FAS and stimulated the production of IL-6, IL-8, GROalpha, GM-CSF and TNFalpha but not IL-4, IL-10, TGFbeta, MCP-1, RANTES, MIP-1beta, or IP-10. In addition, incubation of CD40+ tumour cell lines with immobilised rhCD40L or anti-CD40 mAb in vitro resulted in significant inhibition of proliferation and a corresponding decrease in viability. This CD40-mediated inhibition of cell growth was due, at least in part, to alterations in cell cycle and the induction of apoptosis. Transfection of CD40-negative tumour cell lines with the cDNA for CD40 conferred responsiveness to rhCD40L and anti-CD40 antibody. Finally, the presence of CD40 on the surface of carcinoma lines was found to be an important factor in the generation of tumour-specific T cell responses.

3T3 Cells↗
PubMed1997

Modulation of soluble CD40 ligand bioactivity with anti-CD40 antibodies.

The B cell surface molecule CD40 may be activated either by its ligand CD40L or by anti-CD40 antibodies. In this study, five new anti-CD40 monoclonal antibodies (MAb) were characterized. Bioactivity of the MAb was assessed using a receptor hybrid consisting of the extracellular domain of CD40 and the intracellular domain of the p55 TNF receptor as a model for CD40 activation. Two agonistic MAb were able to enhance the activation of this CD40 hybrid CD40L. These MAb bound to an epitope that was not located within the CD40L-binding region indicating that activation of CD40 occurs epitope-independent. A second pair of ligand mimetic anti-CD40 MAb which appeared to bind to the CD40L binding site decreased CD40L bioactivity. With regard to ligand mimetic effects binding of the CD40L epitope was not of advantage. Combining anti-CD40 MAb with different epitope specificities or cross linking anti-CD40 MAB with secondary antibodies enhanced ligand mimetic effects. These data clearly show that ligand or antibody-mediated receptor aggregation is the major mechanism by which CD40 is activated. Furthermore, our data support that an aggregate of activated receptors is favorable in regard to CD40 activation.

Animals↗
PubMed2004

Cd40 but not CD154 knockout mice have reduced inflammatory response in polymicrobial sepsis: a potential role for Escherichia coli heat shock protein 70 in CD40-mediated inflammation in vivo.

The CD40-CD154 system controls various aspects of the host inflammatory response in models of cellular and humoral immunity. Recently, we described a role for CD40 in the innate immune response in polymicrobial sepsis. However, recent data suggests that CD40 maybe activated by CD154 or directly via bacterial heat shock protein (HSP) 70. Therefore, we decided to test the mechanism of CD40 activation in murine polymicrobial sepsis. Wild-type (WT), CD40, and CD154 underwent cecal ligation and puncture (CLP). Compared with WT mice, CD40 had improved survival in association with attenuated production of IL-12, TNF-alpha, and IL-6. In contrast, CD154 mice behaved similar to WT mice with regard to mortality and cytokine production. The differential response of CD40 and CD154 mice to CLP was not due to a general attenuated response to inflammatory stimuli, as all three strains had similar survival after LPS administration, and CD40 macrophages had normal production of IL-12 in response to lipopolysaccharide. In contrast, CD40 macrophages had attenuated IL-12 production in response to Escherichia coli HSP70 (DnaK). Furthermore, intraperitoneal administration of DnaK resulted in a 4-fold increase in IL-12 in WT mice, which was absent in CD40 mice. This data demonstrates CD154-independent CD40 activation in polymicrobial sepsis and suggests that bacterial HSP70 is capable of stimulating CD40 in vitro and in vivo.

Animals↗
PubMed2003

[Functional significance of CD40 and CD40 ligand linking on human lung cells].

OBJECTIVE: To determine the possible functional significance of CD40 and CD40 ligand (CD154) linking on human lung carcinomas and to assess the potential of CD40 as a therapeutic target. METHODS: We evaluated the effect of CD40L on the surface expression of major histocompatibility complex class I (MHC-I), Fas, bcl2, CD54, epidermal growth factor receptor (EGFR), p-glucoprotein (PGP), lung related protein (LRP), cell cycle, cell apoptosis and growth kinetics of 7 lung cancer cell lines (including 1 CD40-transfected cell line GLC-82/CD40) by gene cloning, Western blotting, and flow cytometry etc. RESULTS: Significant increased expression of MHC-I, CD54 and Fas was observed in 4 tumor lines expressing high levels of CD40 after CD40L (0.1 micro g/ml) linked to CD40. CD40L (0.1 micro g/ml or greater) in the fifth day was found to significantly inhibit the proliferation of 4 cell lines expressing high levels of CD40, decreased the percentage of aneuploid cell, and inhibited S-phase cells entering G2/M phase. The effect of CD40 cross-linking was reversible. CD40-moderate and low and negative tumor did not respond to CD40L. All of 7 cell lines show no significant changes in apoptosis in either the experimental (CD40L pulsed) or control (medium only) cell cultures. CONCLUSION: Expression of CD40 on lung tumors cells expressing high level of CD40 (including CD40-transfected cells) may represent a potential therapeutic target.

CD40 Antigens↗
PubMed1994

The role of CD40-CD40 ligand interaction in human T cell-B cell collaboration.

Interactions between CD40 on B cells and its ligand on activated T cells have been reported to play an important role in T cell-B cell collaboration. In this current study, a mAb against the human CD40 ligand (5c8) was used to investigate the impact of CD40-CD40 ligand interactions in the initial activation of normal human peripheral blood B cells and in subsequent proliferation and differentiation. B cells were activated by co-culture with anti-CD3-stimulated normal T cells. mAb against CD40 ligand blocked initial T cell-dependent B cell activation, as assessed by [3H]uridine incorporation and IL-2R expression. Subsequent B cell proliferation and differentiation were also inhibited by this mAb. In addition to its effect on B cell activation, 5c8 also inhibited the capacity of B cells to augment IL-2 production by anti-CD3 activated T cells, implying a role for CD40-CD40 ligand interactions in the accessory function of B cells. Despite the importance of CD40-CD40 ligand interactions in T cell-B cell collaboration, CD40 ligand-deficient T cell clones were found to induce initial activation of B cells and support Ig production. This effect was only marginally effected by mAb to LFA-1 and ICAM-1, suggesting that additional interaction molecules play a role in T cell-B cell collaboration. Taken together, the data indicate that CD40-CD40 ligand interactions plays an important role in T cell-dependent B cell activation and subsequent differentiation but additional interaction structures are also involved in T cell-B cell collaboration.

Adult↗
PubMed1997

Bidirectional regulation of human B cell responses by CD40-CD40 ligand interactions.

Positive and negative effects of CD40 ligation on human B cell function were suggested by the observation that mAb to CD40 ligand partially blocked the suppressive influences of anti-CD3-stimulated control CD4+ T cells, as well as the B cell stimulatory effects of anti-CD3 activated mitomycin C-treated CD4+ T cells. To examine the negative effects of CD40 ligation in greater detail, B cells were cultured with anti-CD3 activated mitomycin C-treated CD4+ T cells that expressed optimal levels of CD40 ligand; additional recombinant human CD40 ligand significantly suppressed Ig production, but not proliferation. In contrast, when B cells were stimulated with SAC (formalinized Cowan I strain Staphylococcus aureus) and IL-2 in the absence of T cells, small amounts of recombinant CD40 ligand-stimulated Ig production, whereas larger quantities directly suppressed Ig secretion. The suppressive action of CD40 ligation on Ig production was most apparent after initial B cell activation. Moreover, IgD-memory B cells were significantly more sensitive to inhibition by CD40 ligation than IgD+ naive B cells. Engagement of CD40 not only suppressed Ig secretion by IgD- memory B cells, but also expression of CD38. Finally, activated B cells acquired the capacity to down-regulate CD40 ligand expression by stimulated CD4+ T cells more effectively than resting B cells. These results indicate that during T cell-B cell collaboration, engagement of CD40 can influence Ig production both positively and negatively, depending on the density of CD40 ligand as well as the stage of B cell activation and differentiation.

ADP-ribosyl Cyclase↗
PubMed1998

CD40-tumor necrosis factor receptor-associated factor (TRAF) interactions: regulation of CD40 signaling through multiple TRAF binding sites and TRAF hetero-oligomerization.

CD40 is a TNF receptor superfamily member that provides activation signals in antigen-presenting cells such as B cells, macrophages, and dendritic cells. Multimerization of CD40 by its ligand initiates signaling by recruiting TNF receptor-associated factors (TRAFs) to the CD40 cytoplasmic domain. Recombinant human TRAF proteins overexpressed in insect cells were biochemically characterized and used to finely map TRAF binding regions in the human CD40 cytoplasmic domain. TRAF1, TRAF2, TRAF3, and TRAF6, but not TRAF4 or TRAF5, bound directly to the CD40 cytoplasmic domain. CD40 interactions with TRAF2 and TRAF3 were stronger than the interactions with TRAF1 and TRAF6. Full-length TRAF3 and TRAF5 formed hetero-oligomers, presumably through their predicted isoleucine zippers. TRAF3-TRAF5 hetero-oligomers interacted with CD40, indicating that TRAF5 can be indirectly recruited to the CD40 cytoplasmic domain. Overlapping peptides synthesized on cellulose membranes were used to map each TRAF interaction region. TRAF1, TRAF2, and TRAF3 interacted with the same region. The recognition site for TRAF6 was a nonoverlapping membrane proximal region. Using peptides with progressive deletions, a minimal TRAF1, TRAF2, and TRAF3 binding region was mapped to the PVQET sequence in the CD40 cytoplasmic domain. The minimal region for TRAF6 binding was the sequence QEPQEINF. These studies demonstrate that the CD40 cytoplasmic domain contains two nonoverlapping TRAF binding regions and suggest that TRAF1, TRAF2, and TRAF3 could bind competitively to one site. Relative affinities and competition of individual and hetero-oligomeric TRAF proteins for CD40 binding sites may contribute to receptor specificity and cell-type selectivity in CD40-dependent signaling.

Amino Acid Sequence↗
PubMed2003

CD40-deficient, influenza-specific CD8 memory T cells develop and function normally in a CD40-sufficient environment.

Two models have been proposed to explain the requirement for CD40 signaling in CD8 T cell responses. The first model suggests that CD4 T cells activate antigen-presenting cells (APCs) through CD40 signaling (APC licensing). In turn, licensed APCs are able to prime naive CD8 T cells. The second model suggests that CD154-expressing CD4 T cells activate CD40-bearing CD8 T cells directly. Although the requirement for CD40 in APC licensing can be bypassed by inflammatory responses to pathogens that activate APCs directly, the second model predicts that CD8 responses to all antigens will be dependent on CD40 signaling. Here we determined which model applies to CD8 responses to influenza. We demonstrate that optimal CD8 T cell responses to influenza are dependent on CD40 signaling, however both primary and secondary responses to influenza require CD40 expression on non-T cells. Furthermore, CD40-/- CD8 T cells proliferate and differentiate to the same extent as CD40+/+ CD8 T cells in response to influenza, as long as they have equal access to CD40+/+ APCs. Thus, CD4 T cells do not activate influenza-specific CD8 cells directly through CD40 signaling. Instead, these data support the classical model, in which CD4 T cells provide help to CD8 T cells indirectly by activating APCs through CD40.

Animals↗
PubMed1999

Rapid induction of CD40 on a subset of granulocyte colony-stimulating factor-mobilized CD34(+) blood cells identifies myeloid committed progenitors and permits selection of nonimmunogenic CD40(-) progenitor cells.

CD40 antigen is a costimulatory molecule highly expressed on dendritic cells (DC) and activated B cells, which induces T-cell proliferation through the binding with CD40L receptor. In this study, we evaluated CD40 expression on normal CD34(+) blood cells and functionally characterized CD34(+)CD40(+) and CD34(+)CD40(-) cell subsets. CD40, CD80, and CD86 antigens were constitutively expressed on 3.2% +/- 4.5%, 0%, and 1.8% +/- 1.2% CD34(+) blood cells, respectively. However, after 24 hours in liquid culture with medium alone, or with tumor-necrosis-factor-alpha (TNF-alpha), or with allogeneic mononuclear cells 10.8% +/- 3.8%, 75.3% +/- 15.0% and 53. 7% +/- 17.0% CD34(+) blood cells, respectively, became CD40(+). After incubation for 24 hours with TNF-alpha CD34(+)CD40(+) blood cells expressed only myeloid markers and contained less than 5% CD86(+) and CD80(+) cells. Also, a 24-hour priming with TNF-alpha or ligation of CD40 significantly increased the CD34(+) blood cells alloantigen presenting function. Finally, purified CD34(+)CD40(+) blood cells stimulated an alloreactive T-cell response in MLC, were enriched in granulocytic, monocytic, and dendritic precursors, and generated high numbers of DC in 11-14 d liquid cultures with GM-CSF, SCF, TNF-alpha and FLT-3L. In contrast, CD34(+)CD40(-) cells were poorly immunogenic, contained committed granulocytic and erythroid precursors and early progenitors, and differentiated poorly toward the DC lineage. In conclusion, a short incubation with TNF-alpha allows the selection of CD40(+) blood progenitors, which may be a useful source of DC precursors for antitumor vaccine studies, and also a CD34(+)CD40(-) blood cell fraction that could be exploited in innovative strategies of allogeneic transplantation across HLA barriers.

Adult↗
PubMed1995

CD40-CD40 ligand interactions stimulate B cell antigen processing.

The interactions between B cell CD40 and T cell CD40 ligand (CD40L) have been shown recently to play an important role in T cell-dependent activation of B cells. Here, we show that the ligation of CD40 stimulates the processing of antigen by B cells. The activation of an antigen-specific T cell hybrid by B cells co-cultured with insect cells expressing recombinant CD40L or with a CD40-specific monoclonal antibody requires less antigen and fewer B cells compared to control cells. The augmentation was observed both for processing initiated by antigen binding to and cross-linking the surface immunoglobulin, and processing of antigen taken up by fluid-phase pinocytosis. CD40 appears to affect a step in the intracellular processing of antigen, as CD40 has no effect on the presentation of an antigenic peptide which does not require processing. In addition, the CD40-induced augmentation of processing is not attributable to the effect of CD40 ligation on the cell surface expression of B7, LFA-1 or CD23. CD40 ligation does not affect the biosynthesis of the class II EK molecules, and although ligation of CD40 induces B cell proliferation, the augmentation of processing does not require proliferation. The ability of CD40 to stimulate B cell antigen processing has the potential to influence significantly the outcome of antigen-dependent T cell-B cell interactions.

Adjuvants, Immunologic↗
PubMed2003

The transmembranous domain of CD40 determines CD40 partitioning into lipid rafts.

Stimulation of CD40 has been previously shown to result in a release of ceramide in small sphingolipid-enriched rafts in the cell membrane [Grassmé et al., J. Immunol. 168 (2002) 298-307]. Those rafts fused to larger signaling platforms that served to cluster CD40. Here, we defined molecular mechanisms of CD40 clustering in membrane platforms. To this end, we replaced the transmembranous domain of CD40 with that of CD45, a molecule known to be excluded from lipid rafts. Murine T cells were stably transfected with wild-type CD40 or chimeric CD40/CD45. Flow cytometry confirmed normal binding properties of the mutant to CD40 ligand. Stimulation with CD40 ligand resulted in clustering of wild-type CD40, while the chimeric CD40/45 receptor failed to cluster. This correlated with a deficiency of the chimeric receptor to activate JNK, p38 MAP kinase and SAPK, known signaling molecules of the intracellular pathway initiated by CD40. Forced crosslinking of the CD40/45 chimeric receptor restored, at least partially, these signaling events. The results suggest that the transmembranous domain of CD40 is central for the recruitment to and clustering of CD40 in membrane platforms.

Amino Acid Sequence↗
PubMed1996

CD40-CD40 ligand interactions are critical in T-B cooperation but not for other anti-viral CD4+ T cell functions.

CD40-CD40 ligand (CD40L) interaction is required for the generation of antibody responses to T-dependent antigens as well as for the development of germinal centers and memory B cells. The role of the CD40-CD40L interaction in the induction of antigen-specific. Th cells and in mediating Th cell effector functions other than cognate help for B cells is less well understood. Using CD40- and CD40L-deficient mice together with lymphocytic choriomeningitis virus and vesicular stomatitis virus as viral model antigens, this study corroborates earlier findings that no lg isotype switching of virus-specific antibodies was measurable upon infection of CD40- or CD40L-deficient mice. In contrast, in vivo induction of virus-specific CD4+ T cells measured by proliferation and cytokine secretion of primed virus-specific Th cells in vitro was not crucially dependent on the CD40-CD40L interaction. In addition, virus-specific Th cells primed in a CD40-deficient environment, adoptively transferred into CD40-competent recipients, were able to mediate lg isotype switch. Th-mediated effector functions distinct from and in addition to T-B collaboration were analyzed in CD40- and CD40L-deficient and normal mice: (a) local inflammatory reactions upon LCMV infection mediated by LCMV-specific Th cells were not dependent on a functional CD40-CD40L interaction, (b) cytokine-mediated protection by CD4+ T cells primed by vesicular stomatitis virus against a challenge infection with recombinant vaccinia virus expressing the glycoprotein of vesicular stomatitis virus was found to be equivalent in CD40L-deficient and normal mice. Thus, CD40-CD40L interaction plays a crucial role in T-B interactions for Th-dependent activation of B cells but not, or to a much lesser extent, in T cell activation, antigen-specific Th cell responses in vitro, and for interleukin-mediated Th cell effector functions in vivo.

Animals↗
PubMed2000

Expression of CD40 and CD40 ligand in the human conjunctival epithelium.

PURPOSE: CD40 antigen is a membrane receptor that plays a role in the regulation of immune reactions. The expressions of CD40 and CD40 ligand (CD40L) were investigated ex vivo and in vitro in conjunctival epithelial cells, in correlation with HLA DR class H antigen, previously shown to be upregulated in conjunctival inflammatory conditions. METHODS: Impression cytology specimens were collected in 186 patients: 52 normal ones, 65 with keratoconjunctivitis sicca, and 69 with chronic conjunctivitis. Cells were processed for flow cytometry, by using monoclonal antibodies to CD40, CD40L, and HLA DR antigens. Chang conjunctival cells were also used and treated with human recombinant interferon (IFN)-gamma or tumor necrosis factor (TNF)-alpha. CD40, CD40L, and HLA DR expressions were studied by flow cytometry after 24 and 48 hours of treatment. RESULTS: CD40 was found in both normal and pathologic eyes. Quantitation of levels of fluorescence showed a significantly higher expression in pathologic eyes than in normal ones (P < 0.0001). CD40L was variably and inconstantly expressed by conjunctival cells. A strong expression of HLA DR was observed in pathologic eyes, whereas normal eyes showed very low levels (P < 0.0001). Significantly positive correlations were found among CD40, CD40L, and HLA DR levels. Chang conjunctival cells expressed CD40 in basal conditions, whereas CD40L and HLA DR were negative. CD40 expression significantly increased after 24 hours of IFNgamma treatment and after 48 hours' exposure to TNFalpha. These cytokines had no effect on CD40L expression. HLA DR was upregulated after 24 hours of treatment with IFNgamma but remained negative after exposure to TNFalpha. CONCLUSIONS: Human conjunctival epithelial cells normally express CD40 antigen, and, more inconsistently, CD40L. Flow cytometry showed higher expression of these molecules in inflammatory eyes than in normal ones in correlation with class II antigen expression, as well as CD40 and HLA DR upregulation after treatment with proinflammatory cytokines in vitro.

Adult↗
PubMed1996

CD40 and the effect of anti-CD40-binding on human multiple myeloma clonogenicity.

CD40 is a 48 kDa glycosylated phospoprotein that is a member of the tumor necrosis factor receptor (TNF-R) superfamily. CD40 was originally identified in B lymphocytes, and is found on monocytes, dendritic cells, some carcinoma cell lines, and the thymic epithelium. CD40 is expressed on normal pre-B through mature B stages of differentiation. For normal B cells, the cross-linking of CD40 induces cell cycle progression, long-term proliferation in vitro, IgE secretion, increased adhesion molecule (LFA-1) expression, and low level IL-6 secretion. The natural ligand of CD40 (CD40L, gp39, or T-BAM, for T-B cell activating molecule) was recently identified as an inducible molecule expressed transitionally on activated T cells. Although originally believed to be absent in normal and malignant plasma cells, CD40 has been demonstrated on the majority of myeloma cell lines and myeloma cells from plasma cell dyscrasia (PCD) patient specimens tested. CD40 activation modulated myeloma cell proliferation and clonogenicity in vitro, suggesting that the CD40 pathway is active in myeloma cell growth. For the IL-6 dependent cell line ANBL-6, CD40 activation was associated with autocrine IL-6 production. However, the IL-6 pathway does not appear to play a predominant role in CD40 activation of non-IL-6-dependent MM cell lines and patient primary bone marrow cultures. The possible pathophysiologic role of the CD40 receptor in human multiple myeloma is discussed.

Animals↗
PubMed2000

Proinflammatory and Th2-derived cytokines modulate CD40-mediated expression of inflammatory mediators in airway epithelia: implications for the role of epithelial CD40 in airway inflammation.

Cytokines produced by activated macrophages and Th2 cells within the lung play a key role in asthma-associated airway inflammation. Additionally, recent studies suggest that the molecule CD40 modulates lung immune responses. Because airway epithelial cells can act as immune effector cells through the expression of inflammatory mediators, the epithelium is now considered important in the generation of asthma-associated inflammation. Therefore, the goal of the present study was to examine the effects of proinflammatory and Th2-derived cytokines on the function of CD40 in airway epithelia. The results show that airway epithelial cells express CD40 and that engagement of epithelial CD40 induces a significant increase in expression of the chemokines RANTES, monocyte chemoattractant protein (MCP-1), and IL-8 and the adhesion molecule ICAM-1. Cross-linking epithelial CD40 had no effect on expression of the adhesion molecule VCAM-1. The proinflammatory cytokines TNF-alpha and IL-1beta and the Th2-derived cytokines IL-4 and IL-13 modulated the positive effects of CD40 engagement on inflammatory mediator expression in airway epithelial cells. Importantly, CD40 ligation enhanced the sensitivity of airway epithelial cells to the effects of TNF-alpha and/or IL-1beta on expression of RANTES, MCP-1, IL-8, and VCAM-1. In contrast, neither IL-4 nor IL-13 modified the effects of CD40 engagement on the expression of RANTES, MCP-1, IL-8, or VCAM-1; however, both IL-4 and IL-13 attenuated the effects of CD40 cross-linking on ICAM-1 expression. Together, these findings suggest that interactions between CD40-responsive airway epithelial cells and CD40 ligand+ leukocytes, such as activated T cells, eosinophils, and mast cells, modulate asthma-associated airway inflammation.

Adjuvants, Immunologic↗
PubMed2006

Stimulation through CD40 on mouse and human renal cell carcinomas triggers cytokine production, leukocyte recruitment, and antitumor responses that can be independent of host CD40 expression.

CD40, a member of the TNFR superfamily, is expressed on a variety of host immune cells, as well as some tumors. In this study, we show that stimulation of CD40 expressed on both mouse and human renal carcinoma cells (RCCs) triggers biological effects in vitro and in vivo. Treatment of the CD40+ Renca mouse RCC tumor cells in vitro with an agonistic anti-CD40 Ab induced strong expression of the genes and proteins for GM-CSF and MCP-1, and induced potent chemotactic activity. Similarly, administration of alphaCD40 to both wild-type and CD40-/- mice bearing Renca tumors resulted in substantial amounts of TNF-alpha and MCP-1 in the serum, increased the number of total splenocytes and MHC class II+ CD11c+ leukocytes, and when combined with IFN-gamma, inhibited the progression of established Renca tumors in vivo in both wild-type and CD40-/- mice. Similarly, treatment of CD40+ A704 and ACHN human RCC lines with mouse anti-human CD40 Ab induced strong expression of genes and proteins for MCP-1, IL-8, and GM-CSF in vitro and in vivo. Finally, in SCID mice, the numbers of ACHN pulmonary metastases were dramatically reduced by treatment with species-specific human CD40 Ab. These results show that CD40 stimulation of CD40+ tumor cells can enhance immune responses and result in antitumor activity.

Animals↗
PubMed2002

Inhibition of CD40 expression and CD40-mediated dendritic cell function by tumor-derived IL-10.

As CD40 plays a key role in both antitumor immunity and DC maturation, we have studied the regulation of its expression during DC hematopoiesis (dendropoiesis) in vitro and in vivo in the tumor microenvironment. Using MC38 colon adenocarcinoma tumor models, we have demonstrated that DCs generated in vitro from bone marrow precursors obtained from tumor-bearers have significantly lower expression of CD40 molecules compared to DCs generated from tumor-free mice. Furthermore, CD40 expression on DCs isolated from the spleens of tumor-bearing mice was also significantly reduced, suggesting that tumor-derived factors inhibit CD40 expression on DCs during dendropoiesis both in vitro and in vivo. Interestingly, CD40 ligation on DCs generated from tumor-bearers did not result in inducible expression of IL-12 protein or IL-12 p40 mRNA. However, Staphylococcus aureus-induced IL-12 production by DCs was not altered in tumor-bearers, confirming that inhibition of IL-12 production by DCs generated in vitro from tumor-bearing mice was due to reduced expression of CD40 on DCs. We have also shown that MC38 tumor cells produce IL-10 and that exogenous IL-10 causes downregulation of CD40 expression on DCs. In addition, endogenous IL-10 produced by colon carcinoma cells inhibited CD40-dependent IL-12 production by DCs since tumor-induced inhibition of IL-12 production was abrogated by neutralizing anti-IL-10 antibody. Finally, systemic administration of FLT3L and/or CD40L reversed CD40 and IL-12 (p40) deficiency of DCs in tumor-bearing mice in vivo. These findings thus demonstrate that tumor-derived factors, including IL-10, inhibit CD40 expression on DCs and DC precursors and suppress their maturation and function.

Animals↗
PubMed2004

In situ demonstration of CD40- and CD154-positive cells in psoriatic lesions and keratinocyte production of chemokines by CD40 ligation in vitro.

In psoriatic lesions, T cells and keratinocytes are in an activated state. Ligation of CD40 expressed on activated keratinocytes with CD154 expressed on activated T cells is thought to be involved in the pathogenesis of psoriasis. However, the presence of CD40(+) and CD154(+) cells in psoriatic skin has not been thoroughly studied. The present study has therefore examined their presence by immunohistochemistry in the lesional and non-lesional skin of ten patients. The influence of CD154-CD40 ligation on the release of chemokines (IL-8, RANTES, and MCP-1) and complement components (C3 and factor B) from keratinocytes was also investigated in vitro. Studies using single and double staining showed that clusters of CD40(+) keratinocytes were present in both lesional and non-lesional skin; CD40(+)CD1a(+) Langerhans cells in lesional, non-lesional, and normal skin; and numerous CD40(+)CD83(+) cells in lesional skin. CD1a(+) and CD83(+) cells always expressed CD40 strongly. Numerous T cells were seen in lesional skin. A small number of T cells expressed CD154. CD154(+) T cells were seen in the lesional epidermis of seven of ten patients-in six, in juxtaposition to CD40(+) cells including keratinocytes. In non-lesional epidermis, CD154(+) T cells were seen in two patients-in one, in juxtaposition to CD40(+) keratinocytes. In vitro studies showed that IFN-gamma-treated keratinocytes released small amounts of IL-8, RANTES, and MCP-1; ligation of these cells with CD154-transfected J558 cells or soluble CD154 greatly enhanced the release. This ligation did not enhance the release of C3 and factor B. These results warrant further studies on the role of CD40 ligation in the pathogenesis of psoriasis.

Adult↗