Abstract The tendency of isolated germinal center (GC) B cells to undergo apoptosis was suppressed by recombinant cell‐bound CD40 ligand (CD40L): after 2 days at 37°C, > 80 % of cells remained viable in the presence of CD40L as compared to < 1 % in control cultures. CD40L sustained a high rate of DNA synthesis in GC cells and was more effective than monoclonal antibody to CD40 in this regard. Group I Burkitt lymphoma (BL) cell lines induced to undergo apoptosis with anti‐immunoglobulin or calcium ionophore were also protected by CD40L. In BL cells, this route of rescue was not accompanied by induction of Bcl‐2 protein, the expression of which has been linked to hemopoietic cell survival. Bcl‐2 was induced in GC cells responding to CD40L, but its appearance was a relatively late event not reaching significant levels over controls until day 2 of culture. Thus induction of Bcl‐2 appears to be secondary to the survival signal imparted by CD40L. These findings are discussed in relation to a potential role for CD40L in supporting B cell tumors in vivo and the discovery that the molecular defect in the X‐linked Hyper‐IgM syndrome is targeted to the CD40L gene.
Recombinant human CD40 ligand (hCD40L) was expressed on the surface of CV1/EBNA cells and examined for its ability to induce proliferation and Ig secretion from human B cells in the presence or absence of soluble cytokines. hCD40L was directly mitogenic in a dose-dependent fashion for purified tonsil B cells with maximal proliferation occurring at days 5 to 7. Proliferation induced by CD40L was significantly enhanced in the presence of IL-2, IL-4, or IL-10 and strongly suppressed by transforming growth factor-beta. Although IL-5, TNF-alpha, and IFN-gamma had no stimulatory effect in the presence of hCD40L alone, if IL-4 was also present in cultures, these cytokines enhanced the proliferative response above that seen with IL-4 alone. Interestingly, in the absence of IL-4, IFN gamma had an inhibitory effect on hCD40L-induced proliferation. Although CD40L alone did not enhance Ig secretion, addition of IL-2 or IL-10 to the cultures significantly elevated the levels of IgM, IgG1, and IgA that were observed. Addition of IL-4 to the cultures did not enhance secretion of these isotypes but had a weak inhibitory effect. However, CD40L-mediated induction of IgG4 and IgE was dependent on the presence of IL-4. Of the cytokines examined, only IL-10 enhanced IgE secretion under these conditions. Although transforming growth factor-beta only partially inhibited secretion of IgM, IgG1, and IgA, it was strongly suppressive for IgG4 and IgE production. Our data demonstrate that proliferation and Ig secretion induced in the presence of CD40L can be modulated in a positive and negative fashion by soluble cytokines. IL-2 and IL-10 specifically enhance IgM, IgG1, and IgA production although IL-4, despite costimulating B cell proliferation, does not augment secretion of these isotypes but provided an essential cosignal with CD40L for the production of IgG4 and IgE.
Abstract Daily treatment of mice with fms-like tyrosine kinase 3 ligand (Flt3L) leads to a significant increase in the number of dendritic cells and induces antitumor immunity. Here, we show that Flt3L and CD40 ligand (CD40L) synergize in the generation of immune responses against two poorly immunogenic tumors, leading to complete tumor rejection in a high proportion of mice. Rechallenge of the Flt3L + CD40L-treated mice with the immunizing tumor resulted in complete inhibition of tumor growth, indicating that these animals had developed long-lasting antitumor immunity. In addition, we demonstrate that endogenous CD40L plays a critical role in antitumor immunity, since blockade of CD40-CD40L interactions in vivo prevents the generation of antitumor immunity in therapeutic and vaccination protocols. Dendritic cells generated in mice treated with Flt3L alone or in combination with CD40L were equally potent in stimulating allogeneic T cells and expressed similar levels of MHC class II, CD80, and CD86. However, mice treated with Flt3L + CD40L had significantly more dendritic cells than mice treated with either of the cytokines alone, suggesting that CD40L promotes the proliferation and/or survival of dendritic cells generated by Flt3L treatment. Dendritic cells generated in this manner are likely to be involved in the priming of antitumor immune responses.
The stimulatory requirements for T cells bearing gamma delta T cell receptors are distinct from those of alpha beta T cells. We have analyzed the ability of the CD40 ligand (CD40L) to activate neonatal thymic gamma delta T cells. CD40L is expressed on activated T cells and has been shown to induce B cell proliferation and Ig secretion as well as monocyte activation. We now demonstrate that, in the presence of an anti-TCR-gamma delta Ab, CD40L is able to induce the proliferation of neonatal thymic gamma delta cells. The presence of CD40L also leads to enhanced expression of a variety of activation-associated Ag including CD25, CD69, CD44, and Ly6C. In addition to proliferation, CD40L induces lectin-mediated cytolytic activity in thymic gamma delta T cells as well as the production of IFN-gamma and TNF-alpha. We were unable to detect IL-2 or IL-4 production in response to CD40L, and Ab-blocking studies indicate that the mechanism of activation appears to involve IL-1 but is independent of IL-2, IL-4, and IL-7. These results suggest that, in addition to its effects on B cells and monocytes, CD40L can costimulate the activation of thymic gamma delta T cells.
We have identified the murine thymoma line EL4 as a source of biologically active CD40 ligand. Using a biotin-labeled soluble CD40.Fc fusion protein, consisting of the extracellular domain of human CD40 and the Fc region of human IgG1, EL4 cells were subjected to repeated flow cytometric cell sorting to select for cells with enhanced biotinylated CD40.Fc binding. After nine rounds of sorting, the number of CD40.Fc binding sites/cell had risen from 450 on the unsorted parental EL4 cells to 15,000 on EL40.9 cells (EL4 cells sorted with biotinylated CD40.Fc for nine rounds). Scatchard analysis of radiolabeled CD40.Fc binding revealed that the surface-expressed CD40 ligand on parental EL4 and EL40.9 cells bound its receptor with a single class of high-affinity sites (Kd = 0.5 nM). Supernatant (SN) from the sorted EL40.9 cells was found to contain human and murine B cell stimulatory activity which could be removed by preclearing with immobilized CD40.Fc, confirming the presence of soluble CD40 ligand in the preparations. EL40.9 supernatant enhanced soluble CD23 (sCD23) release and induced IgE secretion from interleukin 4-stimulated human B cells. In addition, EL40.9 SN contained proliferative activity for anti-IgM-activated murine B cells which could be removed by treatment with immobilized CD40.Fc. However, the same SN had no demonstrable activity on the proliferation of human B cells. The results presented here describe, for the first time, a source of membrane-bound and soluble CD40 ligand. The soluble form of this murine ligand has activity on murine and human B cells and induces some of the functional responses predicted for the ligand based on the action of stimulatory antibodies directed against the human CD40 surface molecule.
We have reported previously that activation of human umbilical vein endothelial cells (HUVECs) through CD40, using a recombinant soluble form of trimerized CD40 ligand, leads to induction of E-selectin, vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1). Here, we compare the effects of CD40 ligand with those of tumor necrosis factor (TNF) and interleukin 1 (IL-1). All three ligands induce transient increases in E-selectin (peak 4 h) and VCAM-1 (peak 8-24 h), as well as sustained increases in ICAM-1 (plateau 24 h). Quantitatively, TNF is more potent than IL-1, which is much more potent than CD40 ligand. The same hierarchy is observed for transcriptional activation of an E-selectin promoter reporter gene construct in transiently transfected HUVECs. TNF and CD40 ligand each induced activation of the transcription factors NF-kappa B, IRF-1, and ATF-2/c-Jun, measured by electrophoretic mobility shift assays, but this response appeared quantitatively similar. All three agents transiently (peak 30 min) activated Jun NH2-terminal kinase (JNK), which has been implicated in transcription of E-selectin through its actions on ATF-2/c-Jun. Activation of JNK again showed a hierarchy of potency (TNF > IL-1 > CD40 ligand), although the time course of induction was similar for all three agents. After 44 h of pretreatment, TNF, IL-1, and CD40 ligand each display homologous desensitization for reinduction of surface expression of E-selectin. A similar pattern of homologous desensitization for reactivation of JNK was observed. We conclude that TNF, IL-1, and CD40 ligand all activate similar responses in ECs, and that homologous desensitization of JNK may explain the inability of individual cytokines to reinduce E-selectin expression.
Abstract Here we describe a novel platform that enables the production of two full length IgG molecules from a single production cell line. This technology was achieved using a molecular engineering approach to precisely control the cognate pairing of antibody heavy chains and light chains during the assembly of two antibodies in the same cell line. This technology platform, which has been designated as MabPair, can be applied for the development of therapeutic antibody products that contain a mixture of two different antibodies. The antibody cocktail is produced by the same cell line in a predefined ratio and can be manufactured using a conventional antibody production process. When addressing multiple targets, MabPair products can offer significant advantages and flexibility over a bispecific antibody. MabPair technology represents a novel approach for developing antibody combination products. This approach is exemplified by the development of PSB205, our lead MabPair product candidate. PSB205 targets two immune checkpoint pathways, PD-1 and CTLA-4. The development of PSB205, as a potential anti-cancer immunotherapeutic, is discussed. Citation Format: William C. Fanslow, Zhi Liu, Zhonghua Hu, Hua Liu, Min Shen, Yufeng Peng, Cristina Domeier, David Treiber, Ron Schoner, Wei Yan. Development of PSB205, a bifunctional MabPair product that targets PD-1 and CTLA-4 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4069.
