CD40-CD154: A perspective from type 2 immunity.

The interaction between CD40 and CD154 (CD40 ligand) is central in immunology, participating in CD4+ T cell priming by dendritic cells (DC), CD4+ T cell help to B cells and classical macrophage activation by CD4+ T cells. However, its role in the Th2 side of immunology including helminth infection remains incompletely understood. Contrary to viral and bacterial stimuli, helminth products usually do not cause CD40 up-regulation in DC, and exogenous CD40 ligation drives Th2-biased systems towards Th1. On the other hand, CD40 and CD154 are necessary for induction of most Th2 responses. We attempt to reconcile these observations, mainly by proposing that (i) CD40 up-regulation in DC in Th2 systems is mostly induced by alarmins, (ii) the Th2 to Th1 shift induced by exogenous CD40 ligation is related to the capacity of such ligation to enhance IL-12 production by myeloid cells, and (iii) signals elicited by endogenous CD154 available in Th2 contexts and by exogenous CD40 ligation are probably different. We stress that CD40-CD154 is important beyond cognate cellular interactions. In such a context, we argue that the proliferation response of B-cells to IL-4 plus CD154 reflects a Th2-specific mechanism for polyclonal B-cell amplification and IgE production at infection sites. Finally, we argue that CD154 is a general immune activation signal across immune polarization including Th2, and propose that competition for CD154 at tissue sites may provide negative feedback on response induction at each site.

Update on Recurrent Focal Segmental Glomerulosclerosis in Kidney Transplantation.

BACKGROUND: Focal segmental glomerulosclerosis (FSGS) is a clinicopathological syndrome characterized by nephrotic-range proteinuria with high incidence of progression to end-stage renal disease (ESRD). In primary FSGS, 40-60% of patients develop ESRD within 10-20 years. SUMMARY: Recurrence of FSGS after kidney transplantation is frequent and is associated with poor allograft survival. The risk factors for recurrent FSGS include onset of FSGS during childhood, rapid progression of primary FSGS to ESRD, history of recurrent FSGS in previous allograft, and diffuse mesangial hypercellularity or collapsing variant of FSGS in the native kidney. The early histological findings of recurrent FSGS consist of unremarkable glomerular changes on light microscopy but significant podocyte effacement on electron microscopy; the loss of foot processes with eventual dropout of podocytes leads to the development of segmental lesions in the glomerulus. Experimental and clinical data suggest the existence of circulating permeability factors, such as soluble urokinase-type plasminogen activator receptor (suPAR), cardiotrophin-like cytokine factor-1 (CLCF-1), CD40 axis, and apolipoprotein A-Ib (ApoA-Ib), in the pathogenesis of recurrent FSGS. These biomarkers including circulating permeability factors may facilitate earlier diagnosis of FSGS posttransplant and may guide in the development of novel therapies that may be more effective and improve long-term outcomes in kidney transplantation. Key Messages: Several studies have suggested the possible circulating permeability factors, such as suPAR, CLCF-1, CD40 axis, and ApoA-Ib, in the pathogenesis and disease progression of FSGS and recurrent FSGS. Further studies should be performed to elucidate the true essential biomarker(s) associated with the onset and progression of FSGS as well as recurrent FSGS.

Targeting the CD40-CD154 Signaling Pathway for Treatment of Autoimmune Arthritis.

Full activation of T lymphocytes requires signals from both T cell receptors and costimulatory molecules. In addition to CD28, several T cell molecules could deliver costimulatory signals, including CD154, which primarily interacts with CD40 on B-cells. CD40 is a critical molecule regulating several B-cell functions, such as antibody production, germinal center formation and cellular proliferation. Upregulated expression of CD40 and CD154 occurs in immune effector cells and non-immune cells in different autoimmune diseases. In addition, therapeutic benefits have been observed by blocking the CD40-CD154 interaction in animals with collagen-induced arthritis. Given the therapeutic success of the biologics abatacept, which blocks CD28 costimulation, and rituximab, which deletes B cells in the treatment of autoimmune arthritis, the inhibition of the CD40-CD154 axis has two advantages, namely, attenuating CD154-mediated T cell costimulation and suppressing CD40-mediated B-cell stimulation. Furthermore, blockade of the CD40-CD154 interaction drives the conversion of CD4+ T cells to regulatory T cells that mediate immunosuppression. Currently, several biological products targeting the CD40-CD154 axis have been developed and are undergoing early phase clinical trials with encouraging success in several autoimmune disorders, including autoimmune arthritis. This review addresses the roles of the CD40-CD154 axis in the pathogenesis of autoimmune arthritis and its potential as a therapeutic target.

Human Germinal Center B Cells Differ from Naïve and Memory B Cells in CD40 Expression and CD40L-Induced Signaling Response.

CD40 expression is required for germinal center (GC) formation and function, but the kinetics and magnitude of signaling following CD40 engagement remain poorly characterized in human B cells undergoing GC reactions. Here, differences in CD40 expression and signaling responses were compared across differentiation stages of mature human tonsillar B cells. A combination of mass cytometry and phospho-specific flow cytometry was used to quantify protein expression and CD40L-induced signaling in primary human naïve, GC, and memory B cells. Protein expression signatures of cell subsets were quantified using viSNE and Marker Enrichment Modeling (MEM). This approach revealed enriched expression of CD40 protein in GC B cells, compared to naïve and memory B cells. Despite this, GC B cells responded to CD40L engagement with lower phosphorylation of NFκB p65 during the first 30 min following CD40L activation. Before CD40L stimulation, GC B cells expressed higher levels of suppressor protein IκBα than naïve and memory B cells. Following CD40 activation, IκBα was rapidly degraded and reached equivalently low levels in naïve, GC, and memory B cells at 30 min following CD40L. Quantifying CD40 signaling responses as a function of bound ligand revealed a correlation between bound CD40L and degree of induced NFκB p65 phosphorylation, whereas comparable IκBα degradation occurred at all measured levels of CD40L binding. These results characterize cell-intrinsic signaling differences that exist in mature human B cells undergoing GC reactions. © 2019 International Society for Advancement of Cytometry.

Deficiency of T cell CD40L has minor beneficial effects on obesity-induced metabolic dysfunction.

Objective: Obesity-associated metabolic dysfunction increases the risk of multiple diseases such as type 2 diabetes and cardiovascular disease. The importance of the co-stimulatory CD40-CD40L dyad in diet-induced obesity (DIO), with opposing phenotypes arising when either the receptor (aggravating) or the ligand (protective) is deleted, has been described previously. The functions of CD40 and CD40L are cell type dependent. As co-stimulation via T cell-mediated CD40L is essential for driving inflammation, we here investigate the role of T cell CD40L in DIO. Research design and methods: CD4CreCD40Lfl/fl mice on a C57BL/6 background were generated and subjected to DIO by administration of 15 weeks of high fat diet (HFD). Results: HFD-fed CD4CreCD40Lfl/fl mice had similar weight gain, adipocyte sizes, plasma cholesterol and triglyceride levels as their wild-type (WT) counterparts. Insulin and glucose tolerance were comparable, although CD4CreCD40Lfl/fl mice did have a decreased plasma insulin concentration, suggesting a minor improvement of insulin resistance. Furthermore, although the degree of hepatosteatosis was similar in both genotypes, the gene expression of fatty acid synthase 1 and ATP-citrate lyase had decreased, whereas expression of peroxisome proliferator-activated receptor-α had increased in livers of CD4CreCD40Lfl/fl mice, suggesting decreased hepatic lipid uptake in absence of T cell CD40L.Moreover, CD4CreCD40Lfl/fl mice displayed significantly lower numbers of effector memory CD4+ T cells and regulatory T cells in blood and lymphoid organs compared with WT. However, immune cell composition and inflammatory status of the adipose tissue was similar in CD4CreCD40Lfl/fl and WT mice. Conclusions: T cell CD40L deficiency results in a minor improvement of insulin sensitivity and hepatic steatosis in DIO, despite the strong decrease in effector T cells and regulatory T cells in blood and lymphoid organs. Our data indicate that other CD40L-expressing cell types are more relevant in the pathogenesis of obesity-associated metabolic dysfunction.

Macrophage CD40 signaling drives experimental autoimmune encephalomyelitis.

The costimulatory CD40L-CD40 dyad plays a major role in multiple sclerosis (MS). CD40 is highly expressed on MHCII+ B cells, dendritic cells and macrophages in human MS lesions. Here we investigated the role of the CD40 downstream signaling intermediates TNF receptor-associated factor 2 (TRAF2) and TRAF6 in MHCII+ cells in experimental autoimmune encephalomyelitis (EAE). Both MHCII-CD40-Traf2-/- and MHCII-CD40-Traf6-/- mice showed a reduction in clinical signs of EAE and prevented demyelination. However, only MHCII-CD40-Traf6-/- mice displayed a decrease in myeloid and lymphoid cell infiltration into the CNS that was accompanied by reduced levels of TNF-α, IL-6 and IFN-γ. As CD40-TRAF6 interactions predominantly occur in macrophages, we subjected CD40flfl LysMcre mice to EAE. This myeloid-specific deletion of CD40 resulted in a significant reduction in EAE severity, reduced CNS inflammation and demyelination. In conclusion, the CD40-TRAF6 signaling pathway in MHCII+ cells plays a key role in neuroinflammation and demyelination during EAE. Concomitant with the fact that CD40-TRAF6 interactions are predominant in macrophages, depletion of myeloid CD40 also reduces neuroinflammation. CD40-TRAF6 interactions thus represent a promising therapeutic target for MS. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

CD40 Enhances Sphingolipids in Orbital Fibroblasts: Potential Role of Sphingosine-1-Phosphate in Inflammatory T-Cell Migration in Graves' Orbitopathy.

Purpose: Graves' orbitopathy (GO) is an autoimmune orbital disorder associated with Graves' disease caused by thyrotropin receptor autoantibodies. Orbital fibroblasts (OFs) and CD40 play a key role in disease pathogenesis. The bioactive lipid sphingosine-1-phosphate (S1P) has been implicated in promoting adipogenesis, fibrosis, and inflammation in OFs. We investigated the role of CD40 signaling in inducing S1P activity in orbital inflammation. Methods: OFs and T cells were derived from GO patients and healthy control (Ctl) persons. S1P abundance in orbital tissues was evaluated by immunofluorescence. OFs were stimulated with CD40 ligand and S1P levels were determined by ELISA. Further, activities of acid sphingomyelinase (ASM), acid ceramidase, and sphingosine kinase were measured by ultraperformance liquid chromatography. Sphingosine and ceramide contents were analyzed by mass spectrometry. Finally, the role for S1P in T-cell attraction was investigated by T-cell migration assays. Results: GO orbital tissue showed elevated amounts of S1P as compared to control samples. Stimulation of CD40 induced S1P expression in GO-derived OFs, while Ctl-OFs remained unaffected. A significant increase of ASM and sphingosine kinase activities, as well as lipid formation, was observed in GO-derived OFs. Migration assay of T cells in the presence of SphK inhibitor revealed that S1P released by GO-OFs attracted T cells for migration. Conclusions: The results demonstrated that CD40 ligand stimulates GO fibroblast to produce S1P, which is a driving force for T-cell migration. The results support the use of S1P receptor signaling modulators in GO management.

Distinct CD40L receptors mediate inflammasome activation and secretion of IL-1ß and MCP-1 in cultured human retinal pigment epithelial cells.

CD40L signaling occurs in several diseases with inflammatory components, including ocular and retinal diseases. However, it has never been evaluated as a pathogenic mechanism in age-related macular degeneration (AMD) or as an inducer of inflammasome formation in any cell type. mRNA and protein levels of CD40, IL-1ß, NALP1, NALP3, caspase-1, and caspase-5 were determined by RT-PCR, qPCR, and Western blot. CD40L receptor (CD40, α5ß1, and CD11b) expression was determined by Western and immunofluorescent staining. IL-1ß, IL-18, and MCP-1 secretions were determined by ELISA. NALP1 and NALP3 inflammasome formation were determined by Co-IP. Experiments were conducted on primary human retinal pigment epithelial (hRPE) cells from four different donors. Human umbilical vein endothelial (HUVEC) and monocytic leukemia (THP-1) cells demonstrated the general applicability of our findings. In hRPE cells, CD40L-induced NALP1 and NALP3 inflammasome activation, cleavage of caspase-1 and caspase-5, and IL-1ß and IL-18 secretion. Interestingly, neutralizing CD11b and α5ß1 antibodies, but not CD40, reduced CD40L-induced IL-1ß secretion in hRPE cells. Similarly, CD40L treatment also induced HUVEC and THP-1 cells to secret IL-1ß through CD11b and α5ß1. Additionally, the CD40L-induced IL-1ß secretion acted in an autocrine/paracrine manner to feed back and induce hRPE cells to secrete MCP-1. This study is the first to show that CD40L induces inflammasome activation in any cell type, including hRPE cells, and that this induction is through CD11b and α5ß1 cell-surface receptors. These mechanisms likely play an important role in many retinal and non-retinal diseases and provide compelling drug targets that may help reduce pro-inflammatory processes.

Toll-like receptor agonists Porphyromonas gingivalis LPS and CpG differentially regulate IL-10 competency and frequencies of mouse B10 cells.

OBJECTIVE: This study is to determine the effects of P. gingivalis LPS and CpG on B10 cell expansion and IL-10 competency in vitro. MATERIAL AND METHODS: Spleen B cells were isolated from C57BL/6J mice with or without formalin-fixed P. gingivalis immunization. B cells were cultured for 48 hours under the following conditions: CD40L, CD40L+LPS, CD40L+CpG, and CD40L+LPS+CpG in the presence or absence of fixed P. gingivalis. Percentages of CD1dhiCD5+ B cells were measured by flow cytometry. IL-10 mRNA expression and secreted IL-10 were measured by real-time quantitative PCR and by ELISA respectively. RESULTS: P. gingivalis LPS plus CD40L significantly increased CD1dhiCD5+ B cell percentages and secreted IL-10 levels in both immunized and non-immunized mice B cells in the presence or absence of P. gingivalis, compared with control group. Secreted IL-10 levels were significantly increased in CD40L+LPS treated group compared with CD40L treatment group in the absence of P. gingivalis. CpG plus CD40L significantly decreased CD1dhiCD5+ B cell percentages, but greatly elevated secreted IL-10 levels in immunized and non-immunized mice B cells in the absence of P. gingivalis, compared with CD40L treatment group. CONCLUSIONS: P. gingivalis LPS and CpG differentially enhance IL-10 secretion and expansion of mouse B10 cells during innate and adaptive immune responses.

Toll-like receptor agonists Porphyromonas gingivalis LPS and CpG differentially regulate IL-10 competency and frequencies of mouse B10 cells

Abstract IL-10 expressing regulatory B cells (B10) play a key role in immune system balance by limiting excessive inflammatory responses. Effects of toll-like receptor signaling and co-stimulatory molecules on B10 activity during innate and adaptive immune responses are not fully understood. Objective This study is to determine the effects of P. gingivalis LPS and CpG on B10 cell expansion and IL-10 competency in vitro. Material and Methods Spleen B cells were isolated from C57BL/6J mice with or without formalin-fixed P. gingivalis immunization. B cells were cultured for 48 hours under the following conditions: CD40L, CD40L+LPS, CD40L+CpG, and CD40L+LPS+CpG in the presence or absence of fixed P. gingivalis. Percentages of CD1dhiCD5+ B cells were measured by flow cytometry. IL-10 mRNA expression and secreted IL-10 were measured by real-time quantitative PCR and by ELISA respectively. Results P. gingivalis LPS plus CD40L significantly increased CD1dhiCD5+ B cell percentages and secreted IL-10 levels in both immunized and non-immunized mice B cells in the presence or absence of P. gingivalis, compared with control group. Secreted IL-10 levels were significantly increased in CD40L+LPS treated group compared with CD40L treatment group in the absence of P. gingivalis. CpG plus CD40L significantly decreased CD1dhiCD5+ B cell percentages, but greatly elevated secreted IL-10 levels in immunized and non-immunized mice B cells in the absence of P. gingivalis, compared with CD40L treatment group. Conclusions P. gingivalis LPS and CpG differentially enhance IL-10 secretion and expansion of mouse B10 cells during innate and adaptive immune responses.

Macrophage effector responses of horses are influenced by expression of CD154.

Reactive intermediates contribute to innate immunity by providing phagocytes with a mechanism of defense against bacteria, viruses and parasites. To better characterize the role of CD154 in the production of reactive intermediates, we cloned and expressed recombinant equine CD154 (reqCD154) in Chinese Hamster Ovary (CHO). In co-culture experiments, CHO cells ectopically expressing reqCD154 elicited superoxide production in monocyte-derived macrophages (MDM). Collectively, our results indicate that regulation of CD154 expression plays a role in innate host defenses.

Dendritic cells induce Tc1 cell differentiation via the CD40/CD40L pathway in mice after exposure to cigarette smoke.

Dendritic cells and CD8(+) T cells participate in the pathology of chronic obstructive pulmonary disease, including emphysema, but little is known of the involvement of the CD40/CD40L pathway. We investigated the role of the CD40/CD40L pathway in Tc1 cell differentiation induced by dendritic cells in a mouse model of emphysema, and in vitro. C57BL/6J wild-type and CD40(-/-) mice were exposed to cigarette smoke (CS) or not (control), for 24 wk. In vitro experiments involved wild-type and CD40(-/-) dendritic cells treated with CS extract (CSE) or not. Compared with the control groups, the CS mice (both wild type and CD40(-/-)) had a greater percentage of lung dendritic cells and higher levels of major histocompatability complex (MHC) class I molecules and costimulatory molecules CD40 and CD80. Relative to the CS CD40(-/-) mice, the CS wild type showed greater signs of lung damage and Tc1 cell differentiation. In vitro, the CSE-treated wild-type cells evidenced more cytokine release (IL-12/p70) and Tc1 cell differentiation than did the CSE-treated CD40(-/-) cells. Exposure to cigarette smoke increases the percentage of lung dendritic cells and promotes Tc1 cell differentiation via the CD40/CD40L pathway. Blocking the CD40/CD40L pathway may suppress development of emphysema in mice exposed to cigarette smoke.

Rosuvastatin Attenuates CD40L-Induced Downregulation of Extracellular Matrix Production in Human Aortic Smooth Muscle Cells via TRAF6-JNK-NF-κB Pathway.

CD40L and statins exhibit pro-inflammatory and anti-inflammatory effects, respectively. They are both pleiotropic and can regulate extracellular matrix (ECM) degeneration in an atherosclerotic plaque. Statins can decrease both the CD40 expression and the resulting inflammation. However, the effects of CD40L and stains on atherosclerotic plaque ECM production and the underlying mechanisms are not well established. Moreover, prolyl-4-hydroxylase α1 (P4Hα1) is involved in collagen synthesis but its correlations with CD40L and statins are unknown. In the present study, CD40L suppressed P4Hα1 expression in human aortic smooth muscle cells (HASMCs) in a dose- and time-dependent manner, with insignificant changes in MMP2 expression and negative enzymatic activity of MMP9. CD40L increased TRAF6 expression, JNK phosphorylation, NF-κB nuclear translocation as well as DNA binding. Furthermore, silencing TRAF6, JNK or NF-κB genes abolished CD40L-induced suppression of P4Hα1. Lower NF-κB nuclear import rates were observed when JNK or TRAF6 silenced HASMCs were stimulated with CD40L compared to HASMCs with active JNK or TRAF6. Together, these results indicate that CD40L suppresses P4Hα1 expression in HASMCs by activating the TRAF6-JNK- NF-κB pathway. We also found that rosuvastatin inhibits CD40L-induced activation of the TRAF6-JNK- NF-κB pathway, thereby significantly rescuing the CD40L stimulated P4Hα1 inhibition. The results from this study will help find potential targets for stabilizing vulnerable atherosclerotic plaques.

Alcohol stimulates macrophage activation through caspase-dependent hepatocyte derived release of CD40L containing extracellular vesicles.

BACKGROUND & AIMS: The mechanisms by which hepatocyte exposure to alcohol activates inflammatory cells such as macrophages in alcoholic liver disease (ALD) are unclear. The role of released nano-sized membrane vesicles, termed extracellular vesicles (EV), in cell-to-cell communication has become increasingly recognized. We tested the hypothesis that hepatocytes exposed to alcohol may increase EV release to elicit macrophage activation. METHODS: Primary hepatocytes or HepG2 hepatocyte cell lines overexpressing ethanol-metabolizing enzymes alcohol dehydrogenase (HepG2(ADH)) or cytochrome P450 2E1 (HepG2(Cyp2E1)) were treated with ethanol and EV release was quantified with nanoparticle tracking analysis. EV mediated macrophage activation was monitored by analysing inflammatory cytokines and macrophage associated mRNA expression, immunohistochemistry, biochemical serum alanine aminotransferase and triglycerides analysis in our in vitro macrophage activation and in vivo murine ethanol feeding studies. RESULTS: Ethanol significantly increased EV release by 3.3-fold from HepG2(Cyp2E1) cells and was associated with activation of caspase-3. Blockade of caspase activation with pharmacological or genetic approaches abrogated alcohol-induced EV release. EV stimulated macrophage activation and inflammatory cytokine induction. An unbiased microarray-based approach and antibody neutralization experiments demonstrated a critical role of CD40 ligand (CD40L) in EV mediated macrophage activation. In vivo, wild-type mice receiving a pan-caspase, Rho kinase inhibitor or with genetic deletion of CD40 (CD40(-/-)) or the caspase-activating TRAIL receptor (TR(-/-)), were protected from alcohol-induced injury and associated macrophage infiltration. Moreover, serum from patients with alcoholic hepatitis showed increased levels of CD40L enriched EV. CONCLUSION: In conclusion, hepatocytes release CD40L containing EV in a caspase-dependent manner in response to alcohol exposure which promotes macrophage activation, contributing to inflammation in ALD.

Endothelial Dysfunction and Altered Coagulation As Mediators of Thromboembolism in Behçet Disease.

Behçet disease (BD) is a rare multisystem, inflammatory disease of unknown etiology with vascular involvement and associated thrombogenicity. This review aims to describe the involvement of various mediators in endothelial cell damage and in the hypercoagulable state of BD. The scenario of the chronic inflammation present in BD shows an increased oxidative condition that contributes to endothelial cell damage and induces platelet, leukocyte, and endothelial cell activation through the release of proinflammatory cytokines and chemokines. These factors, together with the increased levels of homocysteine observed in BD patients, induce the endothelial cell expression of adhesion molecules (VCAM-1 and ICAM-1) and tissue factor; the release of cytokines, soluble CD40L (sCD40L), matrix metalloproteinase-9, and blood coagulation factor V; the inhibition of fibrinolysis; the disruption of nitric oxide metabolism; and the increase in platelet reactivity and lipid peroxidation. Endothelial cell dysfunction leads to a prothrombotic and antifibrinolytic phenotype in BD patients. Increased levels of homocysteine, fibrinogen, and plasminogen activator inhibitor type 1 seem to be involved in the procoagulant condition of this pathology that has been verified by end-point tests as well as by global coagulation tests.

Regulated expression of murine CD40L by a lentiviral vector transcriptionally targeted through its endogenous promoter.

BACKGROUND: Targeted lentiviral vectors may contribute to circumventing genotoxicity associated with uncontrolled transcription of therapeutic genes. Some vectors replacing strong viral sequences for gene promoters such as ß-globin, CD4, CD19 or Igκ were able to drive tissue-specific expression of the transgene. Gene therapy, however, faces even greater hurdles when the therapeutic transgene is subject to strict regulatory mechanisms. This is the case of the CD40LG gene, which encodes for the CD154 (also known as CD40L) molecule, transiently expressed upon activation on CD4(+) T cells. Mutations in this gene cause the X-linked hyper IgM syndrome (HIGM1) in humans because the interaction of CD40L with its ligand CD40 triggers signals that are critical for the immunobiology of B lymphocytes. METHODS: We developed a lentiviral vector containing the murine Cd40lg cDNA under the control of its endogenous promoter. RESULTS: The CD4(+) BW5147 T cells transduced with the pCd40lg-Cd40lg lentiviral vector express CD40L only upon stimulation. The intensity of the expression correlates with the number of vector integrations per cell and detected molecules rapidly decay after removing the stimulating agent. The tissue-specific, activation-dependent and reversible expression of CD40L fully mimics the physiological induction and disappearance of the molecule from the surface of murine T lymphocytes. The functional activity of the regulated lentiviral vector is demonstrated by the ability of transduced BW5147 cells to promote the proliferation of purified B cell splenocytes. CONCLUSIONS: We have developed a fine-regulated lentiviral vector that can be a model for expressing molecules subject to stringent regulatory mechanisms.

RIP1 Cleavage in the Kinase Domain Regulates TRAIL-Induced NF-κB Activation and Lymphoma Survival.

Although TRAIL is considered a potential anticancer agent, it enhances tumor progression by activating NF-κB in apoptosis-resistant cells. Cellular FLICE-like inhibitory protein (cFLIP) overexpression and caspase-8 activation have been implicated in TRAIL-induced NF-κB activation; however, the underlying mechanisms are unknown. Here, we report that caspase-8-dependent cleavage of RIP1 in the kinase domain (KD) and intermediate domain (ID) determines the activation state of the NF-κB pathway in response to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) treatment. In apoptosis-sensitive cells, caspase-8 cleaves RIP1 in the KD and ID immediately after the recruitment of RIP1 to the receptor complex, impairing IκB kinase (IKK) recruitment and NF-κB activation. In apoptosis-resistant cells, cFLIP restricts caspase-8 activity, resulting in limited RIP1 cleavage and generation of a KD-cleaved fragment capable of activating NF-κB but not apoptosis. Notably, depletion of the cytoplasmic pool of TRAF2 and cIAP1 in lymphomas by CD40 ligation inhibits basal RIP1 ubiquitination but does not prompt cell death, due to CD40L-induced cFLIP expression and limited RIP1 cleavage. Inhibition of RIP1 cleavage at the KD suppresses NF-κB activation and cell survival even in cFLIP-overexpressing lymphomas. Importantly, RIP1 is constitutively cleaved in human and mouse lymphomas, suggesting that cFLIP-mediated and caspase-8-dependent limited cleavage of RIP1 is a new layer of mechanism that promotes NF-κB activation and lymphoma survival.

Rapamycin and CTLA4Ig synergize to induce stable mixed chimerism without the need for CD40 blockade.

The mixed chimerism approach achieves donor-specific tolerance in organ transplantation, but clinical use is inhibited by the toxicities of current bone marrow (BM) transplantation (BMT) protocols. Blocking the CD40:CD154 pathway with anti-CD154 monoclonal antibodies (mAbs) is exceptionally potent in inducing mixed chimerism, but these mAbs are clinically not available. Defining the roles of donor and recipient CD40 in a murine allogeneic BMT model, we show that CD4 or CD8 activation through an intact direct or CD4 T cell activation through the indirect pathway is sufficient to trigger BM rejection despite CTLA4Ig treatment. In the absence of CD4 T cells, CD8 T cell activation via the direct pathway, in contrast, leads to a state of split tolerance. Interruption of the CD40 signals in both the direct and indirect pathway of allorecognition or lack of recipient CD154 is required for the induction of chimerism and tolerance. We developed a novel BMT protocol that induces mixed chimerism and donor-specific tolerance to fully mismatched cardiac allografts relying on CD28 costimulation blockade and mTOR inhibition without targeting the CD40 pathway. Notably, MHC-mismatched/minor antigen-matched skin grafts survive indefinitely whereas fully mismatched grafts are rejected, suggesting that non-MHC antigens cause graft rejection and split tolerance.

B cell activating factor (BAFF) and a proliferation inducing ligand (APRIL) mediate CD40-independent help by memory CD4 T cells.

Donor-reactive memory T cells undermine organ transplant survival and are poorly controlled by immunosuppression or costimulatory blockade. Memory CD4 T cells provide CD40-independent help for the generation of donor-reactive effector CD8 T cells and alloantibodies (alloAbs) that rapidly mediate allograft rejection. The goal of this study was to investigate the role of B cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL) in alloresponses driven by memory CD4 T cells. The short-term neutralization of BAFF alone or BAFF plus APRIL synergized with anti-CD154 mAb to prolong heart allograft survival in recipients containing donor-reactive memory CD4 T cells. The prolongation was associated with reduction in antidonor alloAb responses and with inhibited reactivation and helper functions of memory CD4 T cells. Additional depletion of CD8 T cells did not enhance the prolonged allograft survival suggesting that donor-reactive alloAbs mediate late graft rejection in these recipients. This is the first report that targeting the BAFF cytokine network inhibits both humoral and cellular immune responses induced by memory CD4 T cells. Our results suggest that reagents neutralizing BAFF and APRIL may be used to enhance the efficacy of CD40/CD154 costimulatory blockade and improve allograft survival in T cell-sensitized recipients.

Loss of cooperativity of secreted CD40L and increased dose-response to IL4 on CLL cell viability correlates with enhanced activation of NF-kB and STAT6.

Chronic lymphocytic leukemia (CLL) cells fail to enter apoptosis in vivo as opposed to their non-malignant B-lymphocyte counterparts. The ability of CLL cells to escape apoptosis is highly dependent on their microenvironment. Compared to non-malignant B cells, CLL cells are more responsive to complex stimuli that can be reproduced in vitro by the addition of cytokines. To understand the molecular mechanism of the environment-dependent anti-apoptotic signaling circuitry of CLL cells, we quantified the effect of the SDF-1, BAFF, APRIL, anti-IgM, interleukin-4 (IL4) and secreted CD40L (sCD40L) on the survival of in vitro cultured CLL cells and found IL4 and sCD40L to be most efficient in rescuing CLL cells from apoptosis. In quantitative dose-response experiments using cell survival as readout, the binding affinity of IL4 to its receptor was similar between malignant and non-malignant cells. However, the downstream signaling in terms of the amount of STAT6 and its degree of phosphorylation was highly stimulated in CLL cells. In contrast, the response to sCD40L showed a loss of cooperative binding in CLL cells but displayed a largely increased ligand binding affinity. Although a high-throughput microscopy analysis did not reveal a significant difference in the spatial CD40 receptor organization, the downstream signaling showed an enhanced activation of the NF-kB pathway in the malignant cells. Thus, we propose that the anti-apoptotic phenotype of CLL involves a sensitized response for IL4 dependent STAT6 phosphorylation, and an activation of NF-kB signaling due to an increased affinity of sCD40L to its receptor.