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Altered metabolism is a hallmark of both cell division and cancer. Chronic lymphocytic leukemia (CLL) cells circulate between peripheral blood (PB) and lymph nodes (LNs), where they receive proliferative and prosurvival signals from surrounding cells. However, insight into the metabolism of LN CLL and how this may relate to therapeutic response is lacking. To obtain insight into CLL LN metabolism, we applied a 2-tiered strategy. First, we sampled PB from 8 patients at baseline and after 3-month ibrutinib (IBR) treatment, which forces egress of CLL cells from LNs. Second, we applied in vitro B-cell receptor (BCR) or CD40 stimulation to mimic the LN microenvironment and performed metabolomic and transcriptomic analyses. The combined analyses indicated prominent changes in purine, glucose, and glutamate metabolism occurring in the LNs. CD40 signaling mostly regulated amino acid metabolism, tricarboxylic acid cycle (TCA), and energy production. BCR signaling preferably engaged glucose and glycerol metabolism and several biosynthesis routes. Pathway analyses demonstrated opposite effects of in vitro stimulation vs IBR treatment. In agreement, the metabolic regulator MYC and its target genes were induced after BCR/CD40 stimulation and suppressed by IBR. Next, 13C fluxomics performed on CD40/BCR-stimulated cells confirmed a strong contribution of glutamine as fuel for the TCA cycle, whereas glucose was mainly converted into lactate and ribose-5-phosphate. Finally, inhibition of glutamine import with V9302 attenuated CD40/BCR-induced resistance to venetoclax. Together, these data provide insight into crucial metabolic changes driven by the CLL LN microenvironment. The prominent use of amino acids as fuel for the TCA cycle suggests new therapeutic vulnerabilities.

Tumor heterogeneity complicates biomarker development and fosters drug resistance in solid malignancies. In lymphoma, our knowledge of site-to-site heterogeneity and its clinical implications is still limited. Here, we profiled 2 nodal, synchronously acquired tumor samples from 10 patients with follicular lymphoma (FL) using single-cell RNA, B-cell receptor (BCR) and T-cell receptor sequencing, and flow cytometry. By following the rapidly mutating tumor immunoglobulin genes, we discovered that BCR subclones were shared between the 2 tumor sites in some patients, but in many patients, the disease had evolved separately with limited tumor cell migration between the sites. Patients exhibiting divergent BCR evolution also exhibited divergent tumor gene-expression and cell-surface protein profiles. While the overall composition of the tumor microenvironment did not differ significantly between sites, we did detect a specific correlation between site-to-site tumor heterogeneity and T follicular helper (Tfh) cell abundance. We further observed enrichment of particular ligand-receptor pairs between tumor and Tfh cells, including CD40 and CD40LG, and a significant correlation between tumor CD40 expression and Tfh proliferation. Our study may explain discordant responses to systemic therapies, underscores the difficulty of capturing a patient’s disease with a single biopsy, and furthers our understanding of tumor-immune networks in FL.

B-cell receptor (BCR) signaling and T-cell interactions play a pivotal role in chronic lymphocytic leukemia (CLL) pathogenesis and disease aggressiveness. CLL cells can use microRNAs (miRNAs) and their targets to modulate microenvironmental interactions in the lymph node niches. To identify miRNA expression changes in the CLL microenvironment, we performed complex profiling of short noncoding RNAs in this context by comparing CXCR4/CD5 intraclonal cell subpopulations (CXCR4dimCD5bright vs CXCR4brightCD5dim cells). This identified dozens of differentially expressed miRNAs, including several that have previously been shown to modulate BCR signaling (miR-155, miR-150, and miR-22) but also other candidates for a role in microenvironmental interactions. Notably, all 3 miR-29 family members (miR-29a, miR-29b, miR-29c) were consistently down-modulated in the immune niches, and lower miR-29(a/b/c) levels associated with an increased relative responsiveness of CLL cells to BCR ligation and significantly shorter overall survival of CLL patients. We identified tumor necrosis factor receptor–associated factor 4 (TRAF4) as a novel direct target of miR-29s and revealed that higher TRAF4 levels increase CLL responsiveness to CD40 activation and downstream nuclear factor-κB (NF-κB) signaling. In CLL, BCR represses miR-29 expression via MYC, allowing for concurrent TRAF4 upregulation and stronger CD40–NF-κB signaling. This regulatory loop is disrupted by BCR inhibitors (bruton tyrosine kinase [BTK] inhibitor ibrutinib or phosphatidylinositol 3-kinase [PI3K] inhibitor idelalisib). In summary, we showed for the first time that a miRNA-dependent mechanism acts to activate CD40 signaling/T-cell interactions in a CLL microenvironment and described a novel miR-29–TRAF4–CD40 signaling axis modulated by BCR activity.

© 2017 by The American Society of Hematology. Clinical studies indicate that thrombocytopenia correlates with the development of severe falciparum malaria, suggesting that platelets either contribute to control of parasite replication, possibly as innate parasite killer cells or function in eliciting pathogenesis. Removal of platelets by anti-CD41 mAb treatment, platelet inhibition by aspirin, and adoptive transfer of wild-type (WT) platelets to CD40-KO mice, which do not control parasite replication, resulted in similar parasitemia compared with control mice. Human platelets at a physiologic ratio of 1 platelet to 9 red blood cells (RBCs) did not inhibit the in vitro development or replication of blood-stage Plasmodium falciparum. The percentage of Plasmodium-infected (iRBCs) with bound platelets during the ascending parasitemia in Plasmodium chabaudi- and Plasmodium berghei-infected mice and the 48-hour in vitro cycle of P falciparum was

Chronic graft-versus-host disease (cGVHD) is a leading cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation. Having shown that germinal center (GC) formation and immunoglobulin deposition are required for multiorgan system cGVHD and associated bronchiolitis obliterans syndrome (BOS) in a murine model, we hypothesized that T follicular helper (Tfh) cells are necessary for cGVHD by supporting GC formation and maintenance. We show that increased frequency of Tfh cells correlated with increased GC B cells, cGVHD, and BOS. Although administering a highly depletionary anti-CD20 monoclonal antibody (mAb) to mice with established cGVHD resulted in peripheral B-cell depletion, B cells remained in the lung, and BOS was not reversed. BOS could be treated by eliminating production of interleukin-21 (IL-21) by donor T cells or IL-21 receptor (IL-21R) signaling of donor B cells. Development of BOS was dependent upon T cells expressing the chemokine receptor CXCR5 to facilitate T-cell trafficking to secondary lymphoid organ follicles. Blocking mAbs for IL-21/IL-21R, inducible T-cell costimulator (ICOS)/ICOS ligand, and CD40L/CD40 hindered GC formation and cGVHD. These data provide novel insights into cGVHD pathogenesis, indicate a role for Tfh cells in these processes, and suggest a new line of therapy using mAbs targeting Tfh cells to reverse cGVHD.

Splenic marginal zone lymphoma (SMZL) is a mature B-cell neoplasm characterized by rather indolent clinical course. However, nearly one third of patients experience a rapidly progressive disease with a dismal outcome. Despite the characterization of clone geneticsandthe recognition of deregulated immunologic stimulation in the pathogenesis of SMZL, little is known about microenvironment dynamics and their potential biological influence on disease outcome. Here we investigate the effect of stroma-intrinsic features on SMZL disease progression by focusing on the microenvironment of the bone marrow (BM), which represents an elective disease localization endorsing diagnostic and prognostic relevance. We show that the quality of the BM stromal meshwork of SMZL infiltrates correlates with time to progression. In particular, we describe the unfavorable prognostic influence of dense CD40 expression by BM stromal cells, which involves the contribution of CD40 ligand (CD40L)-expressing bystander mast cells infiltrating SMZL BM aggregates. The CD40/CD40L-assisted crosstalk between mesenchymal stromal cells and mast cells populating the SMZL microenvironment finds correlation in p53-/- mice developing SMZL and contributes to the engendering of detrimental proinflammatory conditions. Our study highlights a dynamic interaction, playing between nonneoplastic elements within the SMZL niche, toward disease progression. © 2014 by The American Society of Hematology.

Clinical studies indicate that thrombocytopenia correlates with the development of severe falciparum malaria, suggesting that platelets either contribute to control of parasite replication, possibly as innate parasite killer cells or function in eliciting pathogenesis. Removal of platelets by anti-CD41 mAb treatment, platelet inhibition by aspirin, and adoptive transfer of wild-type (WT) platelets to CD40-KO mice, which do not control parasite replication, resulted in similar parasitemia compared with control mice. Human platelets at a physiologic ratio of 1 platelet to 9 red blood cells (RBCs) did not inhibit the in vitro development or replication of blood-stage

Hemodynamic forces are important effectors of endothelial cell phenotype and function. Because CD40-CD154 interactions between endothelial cells and mononuclear leukocytes or activated platelets play an important role in vascular dysfunction, we investigated the effects of cyclic stretch on CD40 expression in human cultured endothelial cells. Short-term stretch transiently up-regulated CD40 expression while long-term stretch resulted in a distinct decline in CD40 protein which was prevented by inhibition of the 20S proteasome or scavenging of peroxynitrite. Tyrosine nitration of CD40 also occurred under static conditions on addition of authentic peroxynitrite, and according to mass spectrometry analysis Tyr-82 but not Tyr-31 was its target in the native protein. Immunofluorescence analysis of endothelial cells transduced with a control or Tyr-82 to Ala mutated AAV9-CD40-eGFP expression construct confirmed a peroxynitrite-dependent redistribution of the protein from the cell membrane to the cytoplasm, which was prevented by methyl-β-cyclodextrin. Moreover, CD154-stimulated IL-12p40 and E-selectin expression markedly decreased after exposure to authentic peroxynitrite or cyclic stretch, respectively. Coimmunoprecipitation demonstrated a decreased binding of TRAF2 and TRAF6 to the CD40 protein after tyrosine nitration. Through this posttranslational oxidative modification of an important costimulatory molecule, endothelial cells are able to quickly adapt to unfavorable hemodynamics and maintain their anti-inflammatory phenotype.

CD40L on CD4+ T cells plays a vital role in the activation of antigen-presenting cells, thus catalyzing a positive feedback loop for T-cell activation. Despite the pivotal juxtaposition of CD40L between antigen-presenting cells and T-cell activation, only a T-cell receptor stimulus is thought to be required for early CD40L surface expression. We show, for the first time, that CD40L expression on peripheral blood CD4+ T cells is highly dependent on a cell-cell interaction with CD14hiCD16− monocytes. Interactions with ICAM-1, LFA-3, and to a lesser extent CD80/CD86 contribute to this enhancement of CD40L expression but are not themselves sufficient. The contact-mediated increase in CD40L expression is dependent on new mRNA and protein synthesis. Circulating myeloid dendritic cells also possess this costimulatory activity. By contrast, CD14loCD16+ monocytes, plasmacytoid dendritic cells, B-cell lymphoma lines, and resting, activated, and Epstein-Barr virus–immortalized primary B cells all lack the capacity to up-regulate early CD40L. The latter indicates that a human B cell cannot activate its cognate T cell to deliver CD40L-mediated help. This finding has functional implications for the role of biphasic CD40L expression, suggesting that the early phase is associated with antigen-presenting cell activation, whereas the late phase is related to B-cell activation.

CD40 is highly expressed on various B-lineage malignancies and represents an attractive immunotherapy target for neoplastic disease. Previous work showed that engineering the Fc domain of an antibody for increased binding to Fcγ receptors (FcγRs) significantly enhanced Fc-mediated immune effector function and antitumor activity in vitro and in vivo. We developed a humanized anti-CD40 antibody similarly Fc-engineered for increased FcγR binding (XmAbCD40) and compared its efficacy with that of an anti-CD40 native IgG1 analog and the anti-CD20 antibody rituximab. XmAbCD40 increased antibody-dependent cell-mediated cytotoxicity (ADCC) up to 150-fold relative to anti-CD40 IgG1 against B-lymphoma, leukemia, and multiple myeloma cell lines, and significantly enhanced ADCC against primary tumors. XmAbCD40 was also superior to rituximab in enhancing ADCC (both in cell lines and primary tumors) and in augmenting antibody-dependent cellular phagocytosis. XmAbCD40 significantly inhibited lymphoma growth in disseminated and established mouse xenografts and was more effective than the IgG1 analog or rituximab. An anti-CD40 antibody constructed to abrogate FcγR binding showed no reduction of tumor growth, indicating that the in vivo antitumor activity of XmAbCD40 is primarily mediated via FcγR-dependent mechanisms. These data demonstrate that XmAbCD40 displays potent antitumor efficacy and merits further evaluation for the treatment of CD40+ malignancies.

Complement receptor 2–negative (CR2/CD21−) B cells have been found enriched in patients with autoimmune diseases and in common variable immunodeficiency (CVID) patients who are prone to autoimmunity. However, the physiology of CD21−/lo B cells remains poorly characterized. We found that some rheumatoid arthritis (RA) patients also display an increased frequency of CD21−/lo B cells in their blood. A majority of CD21−/lo B cells from RA and CVID patients expressed germline autoreactive antibodies, which recognized nuclear and cytoplasmic structures. In addition, these B cells were unable to induce calcium flux, become activated, or proliferate in response to B-cell receptor and/or CD40 triggering, suggesting that these autoreactive B cells may be anergic. Moreover, gene array analyses of CD21−/lo B cells revealed molecules specifically expressed in these B cells and that are likely to induce their unresponsive stage. Thus, CD21−/lo B cells contain mostly autoreactive unresponsive clones, which express a specific set of molecules that may represent new biomarkers to identify anergic B cells in humans.

Complex molecular mechanisms control B-cell fate to become a memory or a plasma cell. Interleukin-24 (IL-24) is a class II family cytokine of poorly understood immune function that regulates the cell cycle. We previously observed that IL-24 is strongly expressed in leukemic memory-type B cells. Here we show that IL-24 is also expressed in human follicular B cells; it is more abundant in CD27+ memory B cells and CD5-expressing B cells, whereas it is low to undetectable in centroblasts and plasma cells. Addition of IL-24 to B cells, cultured in conditions shown to promote plasma cell differentiation, strongly inhibited plasma cell generation and immunoglobulin G (IgG) production. By contrast, IL-24 siRNA increased terminal differentiation of B cells into plasma cells. IL-24 is optimally induced by BCR triggering and CD40 engagement; IL-24 increased CD40-induced B-cell proliferation and modulated the transcription of key factors involved in plasma cell differentiation. It also inhibited activation-induced tyrosine phosphorylation of signal transducer and activator of transcription-3 (STAT-3), and inhibited the transcription of IL-10. Taken together, our results indicate that IL-24 is a novel cytokine involved in T-dependent antigen (Ag)–driven B-cell differentiation and suggest its physiologic role in favoring germinal center B-cell maturation in memory B cells at the expense of plasma cells.

Subjects with X-linked hyper-IgM syndrome (X-HIgM) have a markedly reduced frequency of CD27+ memory B cells, and their Ig genes have a low level of somatic hypermutation (SHM). To analyze the nature of SHM in X-HIgM, we sequenced 209 nonproductive and 926 productive Ig heavy chain genes. In nonproductive rearrangements that were not subjected to selection, as well as productive rearrangements, most of the mutations were within targeted RGYW, WRCY, WA, or TW motifs (R = purine, Y = pyrimidine, and W = A or T). However, there was significantly decreased targeting of the hypermutable G in RGYW motifs. Moreover, the ratio of transitions to transversions was markedly increased compared with normal. Microarray analysis documented that specific genes involved in SHM, including activation-induced cytidine deaminase (AICDA) and uracil-DNA glycosylase (UNG2), were up-regulated in normal germinal center (GC) B cells, but not induced by CD40 ligation. Similar results were obtained from light chain rearrangements. These results indicate that in the absence of CD40-CD154 interactions, there is a marked reduction in SHM and, specifically, mutations of AICDA-targeted G residues in RGYW motifs along with a decrease in transversions normally related to UNG2 activity.

Dendritic cells (DCs) are known to produce C1q, the initiator of the classical complement pathway. We demonstrate that murine DCs deficient in C1q (C1qa−/−) are poorer than wild-type (WT) DCs at eliciting the proliferation and Th1 differentiation of antigen-specific T cells. These defects result from decreased production of IL-12p70 by C1qa−/− DCs and impaired expression of costimulatory molecules CD80 and CD86 in response to CD40 ligation. The defective production of IL-12p70 and the reduced expression of CD80 and CD86 by C1qa−/− DCs were specifically mediated via CD40 ligation, as normal levels of IL-12p70 and CD80/86 were observed after ligation of Toll-like receptors (TLRs) on C1qa−/− DCs. CD40 ligation on C1qa−/− DCs, but not TLR ligation, results in decreased phosphorylation of p38 and ERK1/2 kinases. A strong colocalization of CD40 and C1q was observed by confocal microscopy upon CD40 ligation (but not TLR ligation) on DCs. Furthermore, human DCs from 2 C1q-deficient patients were found to have impaired IL-12p70 production in response to CD40L stimulation. Our novel data suggest that C1q augments the production of IL-12p70 by mouse and human DCs after CD40 triggering and plays important roles in sustaining the maturation of DCs and guiding the activation of T cells.

Translation of small interfering RNA (siRNA)–based approaches into practical therapeutics is limited because of lack of an effective and cell-specific delivery system. Herein, we present a new method of selectively delivering siRNA to dendritic cells (DCs) in vivo using CD40 siRNA-containing immunoliposomes (siILs) that were decorated with DC-specific DEC-205 mAb. Administration of CD40 siILs resulted in DC-specific cell targeting in vitro and in vivo. On treatment with CD40 siILs, the expression of CD40 in DCs, as well allostimulatory activity was inhibited. In vivo administration resulted in selective siRNA uptake into immune organs and functional immune modulation as assessed using a model antigen. In conclusion, this is the first demonstration of DC-specific siRNA delivery and gene silencing in vivo, which highlights the potential of DC-mediated immune modulation and the feasibility of siRNA-based clinical therapy.

Mesenchymal stem cells (MSCs), in addition to their multilineage differentiation, exert immunomodulatory effects on immune cells, even dendritic cells (DCs). However, whether they influence the destiny of full mature DCs (maDCs) remains controversial. Here we report that MSCs vigorously promote proliferation of maDCs, significantly reduce their expression of Ia, CD11c, CD80, CD86, and CD40 while increasing CD11b expression. Interestingly, though these phenotypes clearly suggest their skew to immature status, bacterial lipopolysaccharide (LPS) stimulation could not reverse this trend. Moreover, high endocytosic capacity, low immunogenicity, and strong immunoregulatory function of MSC-treated maDCs (MSC-DCs) were also observed. Furthermore we found that MSCs, partly via cell-cell contact, drive maDCs to differentiate into a novel Jagged-2–dependent regulatory DC population and escape their apoptotic fate. These results further support the role of MSCs in preventing rejection in organ transplantation and treatment of autoimmune disease.

In lymph node (LN) proliferation centers in chronic lymphocytic leukemia (CLL), the environment protects from apoptotic and cytotoxic triggers. Here, we aimed to define the molecular basis for the increased drug resistance and searched for novel strategies to circumvent it. The situation in CLL LN could be mimicked by prolonged in vitro CD40 stimulation, which resulted in up-regulation of antiapoptotic Bcl-xL, A1/Bfl-1, and Mcl-1 proteins, and afforded resistance to various classes of drugs (fludarabine, bortezomib, roscovitine). CD40 stimulation also caused ERK-dependent reduction of Bim-EL protein, but ERK inhibition did not prevent drug resistance. Drugs combined with sublethal doses of the BH3-mimetic ABT-737 displayed partial and variable effects per individual CD40-stimulated CLL. The antiapoptotic profile of CD40-triggered CLL resembled BCR-Abl–dependent changes seen in chronic myeloid leukemia (CML), which prompted application of c-Abl inhibitors imatinib or dasatinib. Both compounds, but especially dasatinib, prevented the entire antiapoptotic CD40 program in CLL cells, and restored drug sensitivity. These effects also occurred in CLL samples with dysfunctional p53. Importantly, ex vivo CLL LN samples also displayed strong ERK activation together with high Bcl-xL and Mcl-1 but low Bim levels. These data indicate that CLL cells in chemoresistant niches may be sensitive to therapeutic strategies that include c-Abl inhibitors.

CD40 signaling is critical for innate and adaptive immunity against pathogens, and the cytoplasmic domain of CD40 is highly conserved both within and between species. A novel missense single nucleotide polymorphism (SNP) in the cytoplasmic domain of CD40 at position 227 (P227A) was identified, which resides on a conserved ancestral haplotype highly enriched in persons of Mexican and South American descent. Functional studies indicated that signaling via human (h) CD40-P227A stably expressed in several B-cell lines led to increased phosphorylation of c-Jun, increased secretion of the pro-inflammatory cytokines interleukin (IL)–6 and TNF-α, and increased Ig production, compared with wild-type hCD40. Cooperation between hCD40-P227A signaling and B-cell receptor (BCR)– or Toll-like receptor 9 (TLR9)–mediated signaling was also enhanced, resulting in elevated and synergistic production of IL-6 and Ig. We have thus identified a novel genetic variant of hCD40 with a gain-of-function immune phenotype.

Germinal center (GC) B cells undergo somatic hypermutation, class switch recombination, and rapid clonal expansion to produce high-affinity antibodies. The BCL6 transcriptional repressor facilitates this phenotype because it can repress DNA damage checkpoint genes. GC B and T cells can make transient direct physical contact; T cells were observed to be associated with dead B-cell fragments. We thus hypothesized that one function of CD40 signaling from T cells within this timeframe could be to modulate BCL6 activity. CD40 signaling rapidly disrupts the ability of BCL6 to recruit the SMRT corepressor complex by excluding it from the nucleus, leading to histone acetylation, RNA polymerase II processivity, and activation of BCL6 target genes, such as CD23b, ATR, and TP53. Washout of CD40 to emulate transient T-cell contact permitted BCL6 target gene mRNA levels to return to their repressed levels, demonstrating that this is a reversible process, which could allow centroblasts that pass quality control to either continue proliferation or undergo terminal differentiation. These data suggest that transient CD40 signaling in the GC might allow T cells to weed out heavily damaged centroblasts while at the same time promoting survival of intact B cells, which could undergo differentiation or additional rounds of proliferation.

CD40 and its ligand, CD154, are major costimulatory molecules whose interactions are important in humoral and cellular immunity. We hypothesized that single nucleotide polymorphisms (SNPs) in TNFRSF5 and TNFSF5 encoding the CD40 and CD154 proteins, respectively, influence lymphoma risk, particularly a functional TNFRSF5 SNP (−1C>T, rs1883832) associated with reduced B-cell CD40 expression. TNFRSF5 and TNFSF5 SNPs were examined in a population-based case-control study of non-Hodgkin lymphoma (376 cases/801 controls with DNA), and compelling findings were followed up in 2 independent populations. Pooled analyses of all 3 case-control studies (total N = 1776 non-Hodgkin lymphoma cases, N = 2482 controls) revealed an increased risk of follicular lymphoma (FL) associated with the TNFRSF5 −1TT genotype (odds ratio = 1.6; 95% confidence interval, 1.1-2.4). In addition, among women, an inverse association was found between the variant A allele for a TNFSF5 6809G>A SNP and FL risk (OR = .61; 95% CI, 0.36-0.98). In genotype-phenotype studies, significantly reduced circulating soluble CD40 was observed in TNFRSF5 −1TT compared with −1CC carriers. Further, dendritic cells from those with −1TT versus −1CC genotypes exhibited lower CD40 cell surface expression. These results suggest that the TNFRSF5 −1C>T polymorphism may increase FL susceptibility through mechanisms that hinder cellular immune responses. Further studies are needed to explore these findings.

Identification of Toll-like receptors (TLRs) and their ligands, and tumor necrosis factor–tumor necrosis factor receptor (TNF-TNFR) pairs have provided the first logical, hypothesis-based strategies to molecularly concoct adjuvants to elicit potent cell-mediated immunity via activation of innate and adaptive immunity. However, isolated activation of one immune pathway in the absence of others can be toxic, ineffective, and detrimental to long-term, protective immunity. Effective engineered vaccines must include agents that trigger multiple immunologic pathways. Here, we report that combinatorial use of CD40 and TLR agonists as a cancer vaccine, compared with monotherapy, elicits high frequencies of self-reactive CD8+ T cells, potent tumor-specific CD8+ memory, CD8+ T cells that efficiently infiltrate the tumor-burdened target organ; therapeutic efficacy; heightened ratios of CD8+ T cells to FoxP3+ cells at the tumor site; and reduced hepatotoxicity. These findings provide intelligent strategies for the formulation of multifactorial vaccines to achieve maximal efficacy in cancer vaccine trials in humans.

CD40 is an integral plasma membrane–associated member of the TNF receptor family that has recently been shown to also reside in the nucleus of both normal B cells and large B-cell lymphoma (LBCL) cells. However, the physiological function of CD40 in the B-cell nucleus has not been examined. In this study, we demonstrate that nuclear CD40 interacts with the NF-κB protein c-Rel, but not p65, in LBCL cells. Nuclear CD40 forms complexes with c-Rel on the promoters of NF-κB target genes, CD154, BLyS/BAFF, and Bfl-1/A1, in various LBCL cell lines. Wild-type CD40, but not NLS-mutated CD40, further enhances c-Rel–mediated Blys promoter activation as well as proliferation in LBCL cells. Studies in normal B cells and LBCL patient cells further support a nuclear transcriptional function for CD40 and c-Rel. Cooperation between nuclear CD40 and c-Rel appears to be important in regulating cell growth and survival genes involved in lymphoma cell proliferation and survival mechanisms. Modulating the nuclear function of CD40 and c-Rel could reveal new mechanisms in LBCL pathophysiology and provide potential new targets for lymphoma therapy.

The recruitment of tumor necrosis factor receptor–associated factors (TRAFs) 1, 2, 3, 5, and 6 to the CD40 cytoplasmic tail upon CD40 trimerization results in downstream signaling events that ultimately lead to CD40-dependent, thymus-dependent (TD) humoral immune responses. Previously, we have shown signaling through the C-terminal tail of CD40 in the absence of canonical TRAF-binding sites is capable of signaling through an alternative TRAF2-binding site. Here, we demonstrate that B cells from mice harboring CD40 with only the C-terminal tail can activate both canonical and noncanonical NFκB signaling pathways. Moreover, while lacking germinal center formation, several hallmarks of humoral immune responses including clonal B-cell activation/expansion, antibody isotype switching, and affinity maturation remain normal. This study demonstrates a new functional domain in CD40 that controls critical aspects of B-cell immunity in an in vivo setting.

Previous studies on apoptosis defects in acute lymphoblastic leukemia (ALL) have focused on chemotherapy-induced, primarily mitochondrial death pathways. Yet, immunologic surveillance mechanisms including sensitization to apoptotic signals mediated via the death receptor CD95 might contribute to leukemic control. Here, we show that primary B-cell precursor ALL cells from children escape from receptor-dependent cell death in 2 ways: Resting ALL blasts are protected from receptor-mediated apoptosis due to the absence of CD95 surface expression. However, even though CD40 ligation results in up-regulation of CD95, ALL blasts, unlike normal B cells, remain resistant to apoptosis. We show that this apoptosis resistance involves the selective up-regulation of the short isoforms of the caspase-8 inhibitor c-FLIP acting directly at the CD95 receptor level. Treatment with cycloheximide during CD40 activation prevents up-regulation of those c-FLIP isoforms and sensitizes ALL cells toward CD95-mediated apoptosis. We therefore propose that induction of the short c-FLIP isoforms inhibits the onset of CD95-induced apoptosis in primary CD40-stimulated ALL cells despite high CD95 expression.

Growing evidence points to the potential of agonistic anti-CD40 mAbs as adjuvants for vaccination against cancer. These appear to act by maturing dendritic cells (DCs) and allowing them to prime CD8 cytotoxic T lymphocytes (CTLs). Although it is well established that optimal T-cell priming requires costimulation via B7:CD28, recent studies emphasize the contribution of TNF receptors to this process. To understand how anti-CD40 mAbs trigger effective antitumor immunity, we investigated the role of TNFR superfamily members CD27 and 4-1BB in the generation of this immunity and showed that, although partially dependent on 4-1BB:4-1BBL engagement, it is completely reliant on CD27:CD70 interactions. Importantly, blocking CD70, and to some extent 4-1BBL, during anti-CD40 treatment prevented accumulation of tumor-reactive T cells and subsequent tumor protection. However, it did not influence changes in DC number, phenotype, nor the activity of CTLs once immunity was established. We conclude that CD27:CD70 and 4-1BB:4-1BBL interactions are needed for DC-driven accumulation of antitumor CTLs following anti-CD40 mAb treatment. Finally, in support of the critical role for CD70:CD27, we show for the first time that agonistic anti-CD27 mAbs given without a DC maturation signal completely protect tumor-bearing mice and provide a highly potent reagent for boosting antitumor T-cell immunity.

Bone homeostasis is regulated by a delicate balance between osteoblastic bone formation and osteoclastic bone resorption. Osteoclastogenesis is controlled by the ratio of receptor activator of NF-κB ligand (RANKL) relative to its decoy receptor, osteoprotegerin (OPG). The source of OPG has historically been attributed to osteoblasts (OBs). While activated lymphocytes play established roles in pathological bone destruction, no role for lymphocytes in basal bone homeostasis in vivo has been described. Using immunomagnetic isolation of bone marrow (BM) B cells and B-cell precursor populations and quantitation of their OPG production by enzyme-linked immunosorbent assay (ELISA) and real-time reverse transcriptase–polymerase chain reaction (RT-PCR), cells of the B lineage were found to be responsible for 64% of total BM OPG production, with 45% derived from mature B cells. Consistently B-cell knockout (KO) mice were found to be osteoporotic and deficient in BM OPG, phenomena rescued by B-cell reconstitution. Furthermore, T cells, through CD40 ligand (CD40L) to CD40 costimulation, promote OPG production by B cells in vivo. Consequently, T-cell–deficient nude mice, CD40 KO mice, and CD40L KO mice display osteoporosis and diminished BM OPG production. Our data suggest that lymphocytes are essential stabilizers of basal bone turnover and critical regulators of peak bone mass in vivo.

Heteroclitic peptide modifications increase immunogenicity, allowing generation of cytotoxic T lymphocytes (CTLs) against weakly immunogenic tumor-associated antigens (TAAs). A critical issue is whether T cells generated against heteroclitic peptides retain the ability to recognize and kill tumor cells expressing the original weak TAAs, and whether there is a lower threshold of binding affinity of the native peptides, below which such CTLs can still kill primary tumor cells. To examine this we used a model examining the ability of native and heteroclitic immunoglobulin (Ig)–derived peptides to generate CTLs that can kill chronic lymphocytic leukemia (CLL) cells. We demonstrate that CTLs generated against heteroclitic peptides have enhanced killing of CD40-activated B cells pulsed with either heteroclitic (P < .001) or native peptide (P = .04) and primary CLL cells (P = .01). The novel finding reported here is that the rate-limiting factor appears to be the ability to generate CTLs and that once generated, CTL lysis of primary tumor cells is independent of the binding affinity of the native peptide. These findings have implications for vaccination strategies in malignancies and are currently being further examined in vivo in murine models.

Despite the importance of thymic stromal cells to T-cell development, relatively little is known about their biology. Here, we use single-cell analysis of stromal cells to analyze extensive changes in the number and composition of thymic stroma throughout life, revealing a surprisingly dynamic population. Phenotypic progression of thymic epithelial subsets was assessed at high resolution in young mice to provide a developmental framework. The cellular and molecular requirements of adult epithelium were studied, using various mutant mice to demonstrate new cross talk checkpoints dependent on RelB in the cortex and CD40 in the medulla. With the use of Ki67 and BrdU labeling, the turnover of thymic epithelium was found to be rapid, but then diminished on thymic involution. The various defects in stromal turnover and composition that accompanied involution were rapidly reversed following sex steroid ablation. Unexpectedly, mature cortical and medullary epithelium showed a potent capacity to stimulate naive T cells, comparable to that of thymic dendritic cells. Overall, these studies show that the thymic stroma is a surprisingly dynamic population and may have a more direct role in negative selection than previously thought. ispartof: Blood vol:108 issue:12 pages:3777-3785 ispartof: location:United States status: published

We have identified a population of newly formed bone marrow (BM) B cells that shares multiple characteristics with late transitional B cells in the spleen. Both late splenic transitional B cells and cells within this uncharacterized BM population expressed the cell-surface phenotype AA4+ CD23+, yet the developmental kinetics and the renewal rate of AA4+ CD23+ BM B cells mirrored recently formed BM B cells. Further, unlike the least mature B cells in the BM and spleen, AA4+ CD23+ BM B cells expressed the homing receptor CD62L, were dependent on the antiapoptotic cytokine receptor BR3 and the tec family kinase Btk, and proliferated in response to IL-4 plus CD40 stimulation. Finally, frequencies of λ light chain-positive B cells declined among AA4+ CD23+ B cells in both the BM and spleen, suggesting that V-gene selection events correlate with CD23 expression in both compartments. These observations indicate that the first step in B-cell maturation occurs in both the BM and the periphery and suggest that recently formed B cells exit the BM as a heterogeneous pool of immature and semimature B cells.

Ectodermal dysplasia with immune deficiency (EDI) is caused by alterations in NEMO (nuclear factor [NF]–κB essential modulator). Most genetic mutations are located in exon 10 and affect the C-terminal zinc finger domain. However, the biochemical mechanism by which they cause immune dysfunction remains undetermined. In this report, we investigated the effect of a cysteine-to-arginine mutation (C417R) found in the NEMO zinc finger domain on dendritic cell (DC) function. Following CD40 stimulation of DCs prepared from 2 unrelated patients with the NEMO C417R mutation, we found NEMO ubiquitination was absent, and this was associated with preserved RelA but absent c-Rel activity. As a consequence, CD40 stimulated EDI DCs failed to synthesize the c-Rel–dependent cytokine interleukin-12, had impaired up-regulation of costimulatory molecules, and failed to support allogeneic lymphocyte proliferation in vitro. In contrast, EDI DCs stimulated with the TLR4 ligand lipopolysaccharide (LPS) showed normal downstream NF-κB activity, DC maturation, and NEMO ubiquitination. These findings show for the first time how mutations in the zinc finger domain of NEMO can lead to pathway specific defects in NEMO ubiquitination and thus immune deficiency.

Germline (GL) transcription is regulated by specific promoters and immunoglobulin heavy chain (IgH) 3′ locus enhancers and is necessary for Ig class-switch recombination (CSR). We have generated different transgenic lines containing the GL ϵ promoter, switch (S) ϵ region, and constant (C) ϵ region with or without the DNase I–sensitive regions (HS) 3A-HS1,2 or HS3B-HS4 3′ IgH enhancer pairs. The enhancerless construct was expressed in B cells activated by interleukin (IL)–4 and CD40, thus resembling regulation of the endogenous gene. Both enhancer-containing transgenes efficiently increased expression in B cells and were strongly up-regulated by stimuli. In addition, Sϵ regions of the transgene containing HS3B-HS4 were mutated in activated, sorted B cells. Such mutations are known to precede CSR and are dependent on activation-induced cytidine deaminase (AID). Our findings show that all elements necessary for recruitment of the recombination machinery are present in the transgene containing HS3 and HS4. These enhancers probably provide something more specific than mere increased accessibility of switch regions. We propose that transcription factors binding the enhancers help to target the recombination machinery to the switch regions.

CD40 is expressed on various immune cells, including macrophages and microglia. Aberrant expression of CD40 is associated with autoimmune inflammatory diseases such as multiple sclerosis and rheumatoid arthritis. Interaction of Toll-like receptor-4 (TLR4) with the Gram-negative bacteria endotoxin lipopolysaccharide (LPS) results in the induction of an array of immune response genes. In this study, we describe that LPS is a strong inducer of CD40 expression in macrophages and microglia, which occurs at the transcriptional level and involves the activation of the transcription factors nuclear factor-kappaB (NF-kappaB) and signal transducer and activator of transcription 1alpha (STAT-1alpha). LPS-induced CD40 expression involves the endogenous production of the cytokine interferon-beta (IFN-beta), which contributes to CD40 expression by the activation of STAT-1alpha. Blocking IFN-beta-induced activation of STAT-1alpha by IFN-beta-neutralizing antibody reduces LPS-induced CD40 gene expression. Furthermore, LPS induces acetylation and phosphorylation of histones H3 and H4 and the recruitment of NF-kappaB, STAT-1alpha, and RNA polymerase II on the CD40 promoter in vivo in a time-dependent manner, all events important for CD40 gene transcription. These results indicate that both LPS-induced NF-kappaB activation and endogenous production of IFN-beta that subsequently induces STAT-1alpha activation play critical roles in the transcriptional activation of the CD40 gene by LPS.

Patients with chronic lymphocytic leukemia (CLL) treated with adenovirus CD154 (Ad-CD154, CD40 ligand [CD40L]) gene therapy experienced rapid reductions in leukemia cell counts and lymph node size associated with the induced expression of Fas (CD95). However, CLL cells initially resist CD95-mediated apoptosis within the first 3 days after CD40 ligation in vitro. Thereafter, they become sensitive, which is associated with the CD40-induced expression of the proapoptotic protein B-cell leukemia 2 homology 3 (BH3) interacting domain death agonist (Bid). We hypothesized that the initial resistance to CD95-mediated apoptosis may be due to the high-level expression of X-linked inhibitor of apoptosis protein (XIAP) by CLL cells. Consistent with this, CLL cells from patients 1 day after treatment with autologous Ad-CD154-transduced CLL cells became sensitive to CD95-mediated apoptosis following treatment with a novel XIAP inhibitor, 1540-14. Similarly, 1540-14 specifically enhanced CD95-mediated apoptosis of CLL cells following CD40 ligation in vitro. Immunoblot analyses demonstrated that treatment with 1540-14 allowed CD40-stimulated CLL cells to experience high-level activation of caspases-8 and -3 and cleavage of poly(adenosine diphosphate [ADP]-ribose) polymerase following CD95 ligation. This study demonstrates that distal apoptosis regulators contribute to the initial resistance of CD40-activated CLL cells to CD95-mediated apoptosis and suggests that XIAP inhibitors might enhance the effectiveness of immune-based treatment strategies that target CD40, such as CD154 gene therapy. (Blood. 2005;106:1742-1748)

The life cycle of dendritic cells (DCs) must be precisely regulated for proper functioning of adaptive immunity. However, signaling pathways actively mediating DC death remain enigmatic. Here we describe a novel mechanism of hierarchical transcriptional control of DC life and death. Ligation of tumor necrosis factor receptor superfamily (TNFR-SF) members on DCs and cognate contact with T cells resulted in quantitatively balanced nuclear factor-κB (NF-κB) and c-Jun N-terminal kinase (JNK)–mediated activator protein-1 (AP-1) induction and strongly enhanced DC longevity. Specific blockade of NF-κB in DCs induced strongly augmented JNK/AP-1 activity because of elevated levels of reactive oxygen species. In this scenario, DC activation by TNFR-SF members or T cells induced DC apoptosis. Specific inhibition of JNK/AP-1 rescued DCs from this activation-induced cell death program and restored TNFR-SF member- and T-cell–mediated survival. We conclude that JNK/AP-1 activity is under negative feedback control of NF-κB and can execute apoptosis in DCs. Thus, feedback-controlled signaling amplitudes of 2 transcriptional pathways decide the fate of a DC.

Dendritic cells (DCs) are involved in the initiation and regulation of innate and adaptive immune responses. Several molecular mechanisms regulate these diverse DC functions, and we have previously reported that mouse dendritic cells (mDCs) can produce interleukin-2 (IL-2) in vitro and in vivo, in response to microbial activation and T-cell-mediated stimuli. This property is shared by different DC subtypes, including Langerhans cells. Here we show that, on appropriate stimulation, human DCs, both plasmacytoid and myeloid subtypes, also express IL-2. Interestingly, the production of IL-2 by myeloid DCs is induced by T-cell-mediated stimuli and depends on the presence of IL-15. The key role of this cytokine in regulating IL-2 production was also confirmed in the mouse system. In particular, we could show that DCs from IL-15-deficient mice were strongly impaired in the ability to produce IL-2 after interactions with different microbial stimuli. Our results indicate that DC-produced IL-2 is tightly coregulated with the expression of IL-15.

The CD40-CD154 dyad has a central role in the development of immune-inflammatory processes. Therefore, disruption of CD40 signaling has the potential to be therapeutically useful in a number of disease indications, including autoimmune syndromes, atherosclerosis, and allograft rejection. Blocking antibodies to CD154 have been successfully employed in experimental animal models, and recently in clinical trials, to prevent or treat these immunologically induced diseases. However, the thrombotic events observed in some of these studies raise important issues regarding future use of anti-CD154 antibodies in humans. In this study, we demonstrate that a small interfering RNA (siRNA) can effectively reduce the surface expression of the human CD40 costimulatory receptor. Moreover, by rendering endothelial cells unresponsive to CD154+ Jurkat cell–mediated activation through RNA interference, induction of endothelial cell-adhesion molecule expression and leukocyte adhesion is prevented in vitro. Thus, anti-CD40 siRNA may become a safe and effective therapeutic option for interfering with CD40-CD154–mediated acute or chronic immune-inflammatory conditions.

Memory B cells are responsible for the rapidly emerging antibody response after antigen reexposure. The signals required for the restimulation of memory B cells have not been fully explained. We used a murine model of anti–factor VIII (FVIII) antibody responses in hemophilia A to study the requirements for the restimulation of FVIII-specific memory B cells and their differentiation into anti-FVIII antibody-producing cells. We were particularly interested in the significance of activated T cells and costimulatory interactions. Our results indicate that the restimulation of FVIII-specific memory B cells is strictly dependent on interactions with activated T cells. These activated T cells can be specific for either FVIII or third-party antigens. Restimulation by T cells specific for third-party antigens requires the presence of FVIII, indicating that signals induced by B-cell receptor (BCR) triggering and by interactions with activated T cells are important. The blockade of B7-1 or B7-2 as well as the blockade of CD40L inhibits the restimulation and differentiation of FVIII-specific memory B cells in vitro and in vivo. The interference with inducible costimulator–inducible costimulator ligand (ICOS-ICOSL) interactions, however, does not cause any modulation. As expected, the production of anti-FVIII antibodies by plasma cells is not dependent on any of the costimulatory interactions tested.

Vaccination with antigen-presenting cells (APCs) engineered to mimic mechanisms of immune stimulation represents a promising approach for cancer immunotherapy. Dendritic cell vaccines have entered phase 3 testing in adult malignancies, but such vaccines in children have been limited. We demonstrate that CD40-activated B cells (CD40-B) transfected with RNA may serve as an alternative vaccine that can be generated from small blood volumes regardless of patient age. CD40-B from pediatric patients are efficient APCs and can be loaded with RNA as an antigenic payload, permitting simultaneous targeting of multiple antigenic epitopes without the necessity of HLA matching. For viral and tumor antigens, CD40-B/RNA technology induced cytotoxic T lymphocytes (CTLs) from adults and children, which could be identified with peptide/major histocompatibility complex (MHC) tetramers. These CTLs secreted interferon-γ (IFN-γ) and killed targets in an MHC-restricted fashion. For pooled neuroblastoma RNA and autologous neuroblastoma RNA, CTLs that lysed neuroblastoma cell lines, including CTLs specific against the widely expressed tumor-antigen survivin, were generated. These findings support a novel platform for tumor-specific vaccine or adoptive immunotherapies in pediatric malignancies.

Imatinib mesylate (STI571) is a competitive Bcr-Abl tyrosine kinase inhibitor and has yielded encouraging results in treatment of chronic myelogenous leukemia (CML) and gastrointestinal stroma tumors (GISTs). Apart from inhibition of the Abl protein tyrosine kinases, it also shows activity against platelet-derived growth factor receptor (PDGF-R), c-Kit, Abl-related gene (ARG), and their fusion proteins while sparing other kinases. In vitro studies have revealed that imatinib mesylate can inhibit growth of cell lines and primitive malignant progenitor cells in CML expressing Bcr-Abl. However, little is known about the effects of imatinib mesylate on nonmalignant hematopoietic cells. In the current study we demonstrate that in vitro exposure of mobilized human CD34+ progenitors to therapeutic concentrations of imatinib mesylate (1-5 μM) inhibits their differentiation into dendritic cells (DCs). DCs obtained after 10 to 16 days of culture in the presence of imatinib mesylate showed concentration-dependent reduced expression levels of CD1a and costimulatory molecules such as CD80 and CD40. Furthermore, exposure to imatinib mesylate inhibited the induction of primary cytotoxic T-lymphocyte (CTL) responses. The inhibitory effects of imatinib mesylate were accompanied by down-regulation of nuclear localized RelB protein. Our results demonstrate that imatinib mesylate can act on normal hematopoietic cells and inhibits the differentiation and function of DCs, which is in part mediated via the nuclear factor κB signal transduction pathway.

Crosslinking of the antigen receptors on the immature B-cell lymphoma, WEHI-231, leads to growth arrest and apoptosis. Commitment to such B-cell receptor (BCR)–mediated apoptosis correlates with mitochondrial phospholipase A2 activation, disruption of mitochondrial function, and cathepsin B activation. CD40 signaling has been reported to rescue WEHI-231 B cells from BCR-driven apoptosis primarily via up-regulation of the antiapoptotic protein Bcl-xL. Coupling of the BCR to the mitochondrial phospholipase A2–dependent apoptotic pathway can be prevented by rescue signals via CD40. We now show that overexpression of Bcl-xL can prevent mitochondrial phospholipase A2 activation, disruption of mitochondrial potential, and postmitochondrial execution of BCR-mediated apoptosis via cathepsin B activation. Moreover, overexpression of Bcl-xL protects WEHI-231 B cells from mitochondrial disruption and apoptosis resulting from culture with exogenous arachidonic acid, the product of phospholipase A2 action, suggesting that Bcl-xL may act to antagonize arachidonic acid–mediated disruption of mitochondrial integrity. However, although Bcl-xL expression can mimic CD40-mediated rescue of BCR-driven apoptosis, it cannot substitute for CD40 signaling in the reversal of BCR-mediated growth arrest of WEHI-231 B cells. Rather, CD40 signaling additionally induces conversion of arachidonic acid to prostaglandin E2 (PGE2), which promotes WEHI-231 B-cell proliferation by restoring the sustained, cycling extracellular signal–regulated/mitogen-activated protein kinase (ErkMAPkinase) signaling required for cell cycle progression.

In B lymphocytes, induction of apoptosis or programmed cell death (PCD) by Fas (CD95/APO-1) is suppressed by the triggering of CD40. This suppression controls various aspects of the humoral immune response, including antibody affinity maturation. The opposing effects of these receptors are also crucial to B-cell homeostasis, autoimmune disease, and cancer. Cytoprotection by CD40 involves activation of protective genes mediated by NF-κB transcription factors; however, its basis remains poorly understood. Here, we report that, in B cells, Gadd45β is induced by CD40 through a mechanism that requires NF-κB and that this induction suppresses Fas-mediated killing. Importantly, up-regulation of Gadd45β by CD40 precedes Fas-induced caspase activation, as well as up-regulation of other NF-κB–controlled inhibitors of apoptosis such as Bcl-xL and c-FLIPL. In the presence of Gadd45β, the Fas-induced apoptotic cascade is halted at mitochondria. However, in contrast to Bcl-xL, Gadd45β is unable to hamper the “intrinsic” pathway for apoptosis and in fact appears to block Fas cytotoxicity herein by suppressing a mitochondria-targeting mechanism activated by this receptor. These findings identify Gadd45β as a critical mediator of the prosurvival response to CD40 stimulation and provide important new insights into the apoptotic mechanism that is triggered by Fas in B cells.

We describe large B cells, many of which have a stellate or dendritic morphology, found in the interfollicular T-cell-rich areas of human peripheral lymphoid tissue. These B cells probably require CD40/CD40 ligand signaling for their generation and have a unique phenotype and a post-germinal center pattern of immunoglobulin gene mutation. The only comparable B cell is the "asteroid" cell of the thymic medulla. Their anatomic location and pattern of gene mutation suggest that these cells may represent the cell of origin of some human large-cell lymphomas. Studies in experimental animals should help to elucidate the role of these cells in the immune response.

We provide phenotypic and functional evidence of premonocytoid dendritic cells (DCs) and preplasmacytoid DCs in blood and of corresponding DC subsets in secondary lymphoid tissue of rhesus monkeys. Subsets were identified and sorted by 4-color flow cytometry using antihuman monoclonal antibodies cross-reactive with rhesus monkey. To mobilize pre-DC subsets, fms-like tyrosine 3 kinase ligand (Flt3L; 100 μg/kg subcutaneously) was administered for 10 days. Presumptive pre-DC subsets were identified within the lineage- (Lin-) major histocompatibility complex (MHC) class II+ fraction of blood mononuclear cells. Premonocytoid DCs were CD11c+CD123- (interleukin-3Rα- [IL-3Rα-]). Preplasmacytoid DCs were characterized as CD11c-CD123++ Flt3L increased the CD11c+ pre-DC (7-fold) and CD123++ pre-DC subsets (3-fold) in blood. The freshly isolated CD11c+ pre-DC subset induced modest proliferation of naive allogeneic T cells. After overnight culture with granulocyte macro-phage-colony-stimulating factor (GMCSF) and CD40L, both subsets up-regulated surface costimulatory molecules, and CD11c+ pre-DCs became potent allostimulators. Freshly isolated CD123++ pre-DCs showed typical plasmacytoid morphology and, when cultured with IL-3 and CD40L for 72 hours, developed mature DC morphology. Following stimulation with CD40L, CD11c+ pre-DCs secreted increased levels of IL-12p40. Importantly, herpes simplex virus-stimulated CD123++ pre-DCs, but not CD11c+ pre-DCs, secreted interferon-α (IFN-α). Corresponding DC subsets were identified by flow analysis and immunohistochemistry in lymph nodes wherein both populations were increased 2- to 3-fold by Flt3L administration. CD123+ pre-DCs produced IFN-α in response to in vivo viral infection. Thus, rhesus monkeys exhibit 2 distinct DC precursor populations that closely resemble those of humans. Both are mobilized into blood and lymphoid tissue by Flt3L, offering potential for their further characterization and possible therapeutic application. (Blood. 2003;102:2513-2521)

The mechanisms of interaction between anti-CD40 monoclonal antibody (mAb) therapy and external beam irradiation were investigated in 2 syngeneic B-cell lymphoma models. We have established doses of anti-CD40 mAb and irradiation which, although ineffective when given singly, were capable of providing long-term protection when used in combination. Furthermore, such treatment was not only critically dependent on the dose of mAb and irradiation but also on tumor load, with greater efficacy only occurring at higher tumor burden. Using blocking antibody, the potency of treatment was shown to be totally dependent on CD8+ T cells, with protective levels of CD8+ cells occurring only in mice receiving the combination of anti-CD40 and irradiation. Interestingly, the ratio of T cells (CD8+) to tumor cells in mice receiving combination treatment was between 10 and 15 times that seen in animals given anti-CD40 or irradiation alone. In vivo tracking experiments revealed a 2-phase decrease in tumor burden, the first resulting directly from the external irradiation and the second, occurring 5 days later, concomitant with the rise in tumor-specific CD8+ cells. We suggest that the external irradiation induced an initial kill of lymphoma cells, probably by apoptosis, which releases tumor antigens and slows the progression of the malignancy to allow generation of a curative cytotoxic T lymphocyte (CTL) response promoted by the anti-CD40 mAb. Combining irradiation with immunomodulatory mAb as described here appears to provide a powerful new approach to the management of cancer.

B7-H2, which is expressed constitutively on B cells and binds the inducible costimulator (ICOS) on antigen-activated T cells, is a member of the B7 family of costimulatory ligands. We have inactivated B7-H2 in the mouse. B7-H2–/– mice generate normal populations of B and T cells in their various lymphoid organs but have lower basal levels of heavy chain class–switched antibodies in their sera. These mice are able to mount normal immune responses to both type I and type II T-cell–independent antigens. However, their pattern of responses to a T-cell–dependent antigen is altered, with greatly reduced production of antigen-specific heavy chain class–switched antibodies, the levels of which could not be elevated even with repeated immunizations. This suggests a critical role for B7-H2 in the recall phases of the immune response. Germinal center formation is also impaired in the mutant mice. While B cells from the mutant mice could response normally to anti-IgM, anti-CD40, and lipopolysaccharide stimulation, the production of T-helper–type II cytokines such as interleukin-4 (IL-4) and IL-10 by primed CD4+ T cells from mutant mice were reduced. This indicated that the defects in humoral responses and germinal center formation in B7-H2–deficient mice are due to the lack of T-cell–mediated help to the B cells. Hence, B7-H2 on B cells is important for recruiting T-cell help via its interaction with ICOS and plays a critical role in costimulating humoral immune responses.

The mitogen-activated protein kinase (MAPK) (also called extracellular signal–regulated kinase [ERK]) pathway has been implicated in malignant transformation and in the regulation of cellular growth and proliferation of several tumor types, but its expression and function in Hodgkin disease (HD) are unknown. We report here that the active phosphorylated form of MAPK/ERK is aberrantly expressed in cultured and primary HD cells. Inhibition of the upstream MAPK kinase (also called MEK) by the small molecule UO126 inhibited the phosphorylation of ERK and demonstrated a dose- and time-dependent antiproliferative activity in HD cell lines. UO126 modulated the levels of several intracellular proteins including B-cell lymphoma protein 2 (Bcl-2), myeloid cell leukemia–1 (Mcl-1) and caspase 8 homolog FLICE-inhibitory protein (cFLIP), and induced G2M cell-cycle arrest or apoptosis. Furthermore, UO126 potentiated the activity of apoliprotein 2/tumor necrosis factor–related apoptosis-inducing ligand (APO2L/TRAIL) and chemotherapy-induced cell death. Activation of CD30, CD40, and receptor activator of nuclear kappaβ (RANK) receptors in HD cells by their respective ligands increased ERK phosphorylation above the basal level and promoted HD cell survival. UO126 inhibited basal and ligand-induced ERK phosphorylation, and inhibited ligand-induced cell survival of HD cell lines. These findings provide a proof-of-principle that inhibition of the MEK/ERK pathway may have therapeutic value in HD.

The therapeutic effects of intravenous immunoglobulins (IVIGs) in several autoimmune diseases are characterized by a decrease in pathologic autoantibody levels. Although little direct evidence has been reported in humans, the large repertoire of natural immunoglobulin G (IgG) antibodies in IVIGs is expected to be involved in the regulation of autoreactive B lymphocytes. In normal adult mice, IVIGs have been reported to modulate immature B cells as well as peripheral B lymphocytes through V-region connections. Studies with human serum also indicated that anti-idiotypic antibodies, present in IVIG preparations, could recognize both natural and pathologic autoantibodies. We have used an in vitro culture system to characterize the direct effect of IVIGs on human B lymphocytes. This in vitro culture system involves CD40 activation of B lymphocytes by its ligand CD154 in the presence of cytokines. In this system, addition of IVIGs decreased by 50% to 80% the expansion of B lymphocytes. This reduced expansion was due to a decrease in the proliferation rate. In addition, a portion of B lymphocytes was differentiated into IgG-secreting cells in the presence of IVIGs and the secreted IgGs were reactive with antigens such as nucleoprotamine, dsDNA, tetanus toxin, and human IgG F(ab′)2 fragments. These observations indicate that IVIGs can have direct effects on B lymphocytes and suggest that such IVIG regulation of B lymphocytes could be involved in the therapeutic effects of IVIGs in autoimmune diseases.

CD40 is present on both normal and neoplastic B-lineage cells. CD40 stimulation of normal B cells has been shown to promote normal growth and differentiation, whereas aggressive histology B lymphomas are growth inhibited. The inhibition of neoplastic B-cell growth is believed to occur via activation-induced cell death in which stimuli that typically promote the growth of normal cells prevent the growth of their neoplastic counterparts. We show here that CD40 stimulation using either a soluble recombinant human CD40 ligand (srhCD40L) or anti-CD40 monoclonal antibody resulted in apoptosis of human Burkitt lymphoma cell lines. Additional studies examining the mechanism of CD40-mediated death revealed an increase in bax messenger RNA with a subsequent increase in Bax protein in the mitochondria of the treated cells. In vitro exposure of the cells to bax antisense oligonucleotides resulted in a significant decline in Bax protein levels and partial protection from CD40-mediated death, indicating that induction of Bax was at least one mechanism underlying this inhibitory effect of CD40 stimulation on lymphomas. When immunodeficient mice bearing Burkitt lymphoma were treated with srhCD40L, significant increases in survival were observed indicating a direct antitumor effect as a result of CD40 stimulation in vivo. Overall, these results demonstrate that CD40 ligation of aggressive histology B-lymphoma cells results in inhibition both in vitro and in vivo and thus may be of potential clinical use in their treatment.

We have studied the impact of B-cell receptor (BCR) or CD40 ligation on the in vitro chemotactic response of tonsillar B cells to 4 chemokines: stromal cell–derived factor (SDF)–1α, macrophage inflammatory protein (MIP)–3α, MIP-3β, and B-cell–attracting chemokine (BCA)–1. In the tonsil, SDF-1 and MIP-3α are both expressed in the crypt epithelium, while MIP-3β is found in the T zone and BCA-1 in the follicles. Resting virgin and memory B cells display a similar chemotaxis pattern, and they both have the potential to migrate in vitro to all 4 chemokines studied. This pattern of responsiveness is strongly modified by a surrogate antigen (Ag) but is not altered by CD40 ligand. We report here that surrogate Ag induces a profound and sustained suppression of the response to the crypt chemokines SDF-1α and MIP-3α, while it exacerbates the migratory response to MIP-3β. The effect of surrogate Ag on the response to BCA-1 is biphasic: After an initial phase of suppression, chemotaxis toward BCA-1 is strongly up-regulated. Our results suggest that Ag is primarily responsible for reprogramming the B-cell chemotaxis responsiveness during the humoral response. We propose that it initiates an ordered change of the chemotaxis machinery allowing Ag-activated B cells to relocate in the T zone and B-cell follicles sequentially.