CD40 Ligand Knockout Mice Research Articles

CD40 ligand deficiency causes functional defects of peripheral neutrophils that are improved by exogenous IFN-γ

Patients with X-linked hyper-IgM syndrome caused by CD40 ligand (CD40L) deficiency often present with episodic, cyclic, or chronic neutropenia, suggesting abnormal neutrophil development in the absence of CD40L-CD40 interaction. However, even when not neutropenic and despite immunoglobulin replacement therapy, CD40L-deficient patients are susceptible to life-threatening infections caused by opportunistic pathogens, suggesting impaired phagocyte function and the need for novel therapeutic approaches. We sought to analyze whether peripheral neutrophils from CD40L-deficient patients display functional defects and to explore the invitro effects of recombinant human IFN-γ (rhIFN-γ) on neutrophil function. We investigated the microbicidal activity, respiratory burst, and transcriptome profile of neutrophils from CD40L-deficient patients. In addition, we evaluated whether the lack of CD40L in mice also affects neutrophil function. Neutrophils from CD40L-deficient patients exhibited defective respiratory burst and microbicidal activity, which were improved invitro by rhIFN-γ but not soluble CD40L. Moreover, neutrophils from patients showed reduced CD16 protein expression and a dysregulated transcriptome suggestive of impaired differentiation. Similar to CD40L-deficient patients, CD40L knockout mice were found to have impaired neutrophil responses. In parallel, we demonstrated that soluble CD40L induces the promyelocytic cell line HL-60 to proliferate and mature by regulating the expression of genes of the same Gene Ontology categories (eg, cell differentiation) when compared with those dysregulated in peripheral blood neutrophils from CD40L-deficient patients. Our data suggest a nonredundant role of CD40L-CD40 interaction in neutrophil development and function that could be improved invitro by rhIFN-γ, indicating a potential novel therapeutic application for this cytokine.

Immune responses toPneumocystis murinaare robust in healthy mice but largely absent in CD40 ligand-deficient mice

Pneumocystis is a pathogen of immunocompromised hosts but can also infect healthy hosts, in whom infection is rapidly controlled and cleared. Microarray methods were used to examine differential gene expression in the lungs of C57BL/6 and CD40 ligand knockout (CD40L-KO) mice over time following exposure to Pneumocystis murina. Immunocompetent C57BL/6 mice, which control and clear infection efficiently, showed a robust response to infection characterized by the up-regulation of 349 primarily immune response-associated genes. Temporal changes in the expression of these genes identified an early (Week 2), primarily innate response, which waned before the infection was controlled; this was followed by primarily adaptive immune responses that peaked at Week 5, which coincided with clearance of the infection. In conjunction with the latter, there was an increased expression of B cell-associated (Ig) genes at Week 6 that persisted through 11 weeks. In contrast, CD40L-KO mice, which are highly susceptible to developing severe Pneumocystis pneumonia, showed essentially no up-regulation of immune response-associated genes at Days 35-75. Immunohistochemical staining supported these observations by demonstrating an increase in CD4+, CD68+, and CD19+ cells in C57BL/6 but not CD40L-KO mice. Thus, the healthy host demonstrates a robust, biphasic response to infection by Pneumocystis; CD40L is an essential upstream regulator of the adaptive immune responses that efficiently control infection and prevent development of progressive pneumonia.

B cells and T cells are critical for the preservation of bone homeostasis and attainment of peak bone mass in vivo

AbstractBone 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.

Signaling through CD40 ligand decreases CD80 expression on murine Langerhans cells and enhances IL-12 p40 production

We previously showed that murine Langerhans cells (LC) express CD40 ligand (CD40L). In this study, we further investigated the function of CD40L on LC using agonistic antibodies and CD40L knockout (KO) mice. Signaling through CD40L decreased CD80 expression on LC 48 h after stimulation and the decrease was more remarkable in the presence of interferon-γ (IFN-γ). Signaling through CD40 enhanced the production of IL-12 p40 from LC, and simultaneous signaling through CD40L slightly augmented this effect. Addition of IFN-γ further enhanced IL-12 p40 production. LC from CD40L KO mice expressed similar levels of surface molecules such as CD40, CD80, CD86, and MHC class II, compared with those from wild-type mice. However, they produced less amount of IL-12 p40 during 48 h after purification. These results suggest that signaling through CD40L on LC is important in regulating IL-12 production, which is critical for Th1 type immune responses.

CD28, TNF Receptor, and IL-12 Are Critical for CD4-Independent Cross-Priming of Therapeutic Antitumor CD8+ T Cells

Previously, we have shown that priming of therapeutic CD8(+) T cells in tumor vaccine-draining lymph nodes of mice vaccinated with GM-CSF secreting B16BL6 melanoma cells occurs independent of CD4 T cell help. In this study, we examined the contribution of the major costimulatory molecules, CD40 ligand (CD40L), CD80, and CD86, in the priming of CD8(+) T cells. Priming of therapeutic CD8(+) T cells by a GM-CSF-transduced tumor vaccine did not require CD40 and CD40L interactions, as therapeutic T cells could be generated from mice injected with anti-CD40L Ab and from CD40L knockout mice. However, costimulation via either CD80 or CD86 was required, as therapeutic T cells could be generated from mice injected with either anti-CD80 or anti-CD86 Ab alone, but administration of both Abs completely inhibited the priming of therapeutic T cells. Blocking experiments also identified that priming of therapeutic T cells in MHC class II-deficient mice required TNFR and IL-12 signaling, but signaling through CD40, lymphotoxin-betaR, or receptor activator of NF-kappaB was not essential. Thus, cross-priming of therapeutic CD8(+) T cells by a tumor vaccine transduced with GM-CSF requires TNFR, IL-12, and CD28 signaling.

Anti-CD40 antibody induces antitumor and antimetastatic effects: the role of NK cells.

We assessed the effect of the stimulatory anti-CD40 Ab on NK cell activation in vivo and the therapeutic potential of activated NK cells in tumor-bearing mice. Single-dose i.p. injection of the anti-CD40 Ab resulted in production of IL-12 and IFN-gamma in vivo, followed by a dramatic increase in NK cell cytolytic activity in PBLs. NK cell activation by anti-CD40 Ab was also observed in CD40 ligand knockout mice. Because NK cells express CD40 ligand but not CD40, our results suggest that NK activation is mediated by increased cytokine production upon CD40 ligation of APCs. Treatment of tumor-bearing mice with anti-CD40 Ab resulted in substantial antitumor and antimetastatic effects in three tumor models. Depletion of NK cells with anti-asialo GM1 Ab reduced or abrogated the observed antitumor effects in all the tested models. These results indicate that a stimulatory CD40 Ab indirectly activates NK cells, which can produce significant antitumor and antimetastatic effects.

CD40-ligand stimulates myelopoiesis by regulating flt3-ligand and thrombopoietin production in bone marrow stromal cells

CD40 ligand (CD40L)/CD40 interactions play a central role in T-cell–dependent B-cell activation as previously shown by in vitro studies, the phenotype of CD40L knockout mice and the defective expression of CD40L in patients who have X-linked immunodeficiency with hyper-IgM. The distribution of CD40 in cells other than of myeloid and lymphoid lineages has suggested additional functions for this receptor/ligand couple. Here we show that CD40L stimulates myelopoiesis with a noticeable effect on megakaryocytopoiesis in cocultures of hematopoietic progenitor cells and bone marrow stromal cells. These results suggest a mechanism by which T-cell or platelet-associated or soluble CD40L may regulate myelopoiesis.

Disruption of antigen-induced inflammatory responses in CD40 ligand knockout mice.

The objective of this study was to investigate the contribution of the interaction between CD40 and its ligand (CD40L) to antigen-induced airways inflammatory responses. To this end, we used a model involving ovalbumin (OVA) sensitization followed by OVA aerosol challenge in CD40L knockout (KO) mice. OVA-specific IgE and IgG1 were detected in the serum of the sensitized control, but not in CD40L-KO mice. After antigen challenge, sensitized control mice developed airway inflammation that was primarily eosinophilic. This inflammatory response was dramatically reduced in CD40L-KO mice. In contrast, similar numbers of eosinophils were observed in both the bone marrow and the peripheral blood in the sensitized controls and mutant strains after antigen challenge. To investigate the mechanisms underlying these findings, we examined levels of the cytokines IL-5, IL-4, and TNFalpha in both bronchoalveolar lavage (BAL) and serum. Similar levels of IL-5 were detected in BAL and serum of control and CD40L-KO mice; however, negligible levels of IL-4 in BAL and serum and of TNFalpha in BAL were detected in CD40L-KO mice when compared with control mice. Furthermore, we demonstrated that endothelial cell expression of vascular cell adhesion molecule 1 in OVA-sensitized and -challenged CD40L-KO mice was, as detected by immunohistochemistry, markedly decreased compared with that observed in similarly treated control mice. In addition, we locally overexpressed IL-4 and TNFalpha by using an adenoviral (Ad)-mediated gene transfer approach. Intranasal administration of either Ad/TNFalpha or Ad/IL-4 into OVA-sensitized and -challenged CD40L-KO mice did not reconstitute airway eosinophilia. However, concurrent administration of Ad/TNFalpha and Ad/IL-4 upregulated endothelial expression of vascular cell adhesion molecule 1, and resulted in full reconstitution of the inflammatory response in the airways. Together, these findings demonstrate the importance of the CD40-CD40L costimulatory pathway in the full expression of the inflammatory response in the airways.

CD40 Ligand Is Required for Protective Cell-Mediated Immunity to Leishmania major

The CD40–CD40 ligand (CD40L) signaling process is a pivotal component of multiple immunoregulatory pathways. Although the role that CD40L plays in humoral immune responses is fairly well defined, its function(s) in cell-mediated responses in vivo has not been established. We investigated this issue by assessing the course of Leishmania major infection in CD40L knockout (CD40LKO) mice that were generated on a resistant background. In response to parasite challenge, CD40LKO mice developed ulcerating cutaneous lesions and failed to mount a vigorous Th1-like response. The impaired Th1-like response appears to be related to a defect in the ability of CD40LKO T cells to induce the production of IL-12 from macrophages. Treatment with exogenous IL-12 prevented disease progression in CD40LKO mice, and administration of recombinant CD40L provided partial protection against infection. Thus, a protective cell-mediated immune response to L. major appears to be dependent upon CD40L-induced IL-12 secretion by antigen-presenting cells.