Encoding CD40 Ligand Research Articles

Preclinical development of a first-in-class vaccine encoding HER2, Brachyury and CD40L for antibody enhanced tumor eradication

The induction of antiviral innate immunity by systemic immunization with live virus can be employed to positively impact the response to therapeutic vaccination. We previously demonstrated that systemic immunization with a non-replicating MVA encoding CD40 ligand (CD40L) enhances innate immune cell activation and function, and triggers potent antitumor CD8+ T cell responses in different murine tumor models. Antitumor efficacy was increased when combined with tumor targeting antibodies. Here we report the development of TAEK-VAC-HerBy (TVH), a first-in-class human tumor antibody enhanced killing (TAEK) vaccine based on the non-replicating MVA-BN viral vector. It encodes the membrane bound form of human CD40L, HER2 and the transcription factor Brachyury. TVH is designed for therapeutic use in HER2- or Brachyury-expressing cancer patients in combination with tumor targeting antibodies. To preclude possible oncogenic activities in infected cells and to prevent binding of vaccine-encoded HER2 by monoclonal antibodies trastuzumab and pertuzumab, genetic modifications of HER2 were introduced in the vaccine. Brachyury was genetically modified to prevent nuclear localization of the protein thereby inhibiting its transcriptional activity. CD40L encoded in TVH enhanced human leukocyte activation and cytokine secretion in vitro. Lastly, TVH intravenous administration to non-human primates was proven immunogenic and safe in a repeat-dose toxicity study. Nonclinical data presented here highlight TVH as a first-in-class immunotherapeutic vaccine platform currently under clinical investigation.

An mRNA mix redirects dendritic cells towards an antiviral program, inducing anticancer cytotoxic stem cell and central memory CD8+ T cells.

Dendritic cell (DC)-maturation stimuli determine the potency of these antigen-presenting cells and, therefore, the quality of the T-cell response. Here we describe that the maturation of DCs via TriMix mRNA, encoding CD40 ligand, a constitutively active variant of toll-like receptor 4 and the co-stimulatory molecule CD70, enables an antibacterial transcriptional program. Besides, we further show that the DCs are redirected into an antiviral transcriptional program when CD70 mRNA in TriMix is replaced with mRNA encoding interferon-gamma and a decoy interleukin-10 receptor alpha, forming a four-component mixture referred to as TetraMix mRNA. The resulting TetraMixDCs show a high potential to induce tumor antigen-specific T cells within bulk CD8+ T cells. Tumor-specific antigens (TSAs) are emerging and attractive targets for cancer immunotherapy. As T-cell receptors recognizing TSAs are predominantly present on naive CD8+ T cells (TN), we further addressed the activation of tumor antigen-specific T cells when CD8+ TN cells are stimulated by TriMixDCs or TetraMixDCs. In both conditions, the stimulation resulted in a shift from CD8+ TN cells into tumor antigen-specific stem cell-like memory, effector memory and central memory T cells with cytotoxic capacity. These findings suggest that TetraMix mRNA, and the antiviral maturation program it induces in DCs, triggers an antitumor immune reaction in cancer patients.

Intranodal vaccination with mRNA-optimized dendritic cells in metastatic melanoma patients

Autologous dendritic cell (DC) therapy is an experimental cellular immunotherapy that is safe and immunogenic in patients with advanced melanoma. In an attempt to further improve the therapeutic responses, we treated 15 patients with melanoma, with autologous monocyte-derived immature DC electroporated with mRNA encoding CD40 ligand (CD40L), CD70 and a constitutively active TLR4 (caTLR4) together with mRNA encoding a tumor-associated antigen (TAA; respectively gp100 or tyrosinase). In addition, DC were pulsed with keyhole limpet hemocyanin (KLH) that served as a control antigen. Production of this DC vaccine with high cellular viability, high expression of co-stimulatory molecules and MHC class I and II and production of IL-12p70, was feasible in all patients. A vaccination cycle consisting of three vaccinations with up to 15×106 DC per vaccination at a biweekly interval, was repeated after 6 and 12 months in the absence of disease progression. mRNA-optimized DC were injected intranodally, because of low CCR7 expression on the DC, and induced de novo immune responses against control antigen. T cell responses against tyrosinase were detected in the skin-test infiltrating lymphocytes (SKIL) of two patients. One mixed tumor response and two durable tumor stabilizations were observed among 8 patients with evaluable disease at baseline. In conclusion, autologous mRNA-optimized DC can be safely administered intranodally to patients with metastatic melanoma but showed limited immunological responses against tyrosinase and gp100.

Clinical trials with MRNA electroporated dendritic cells for stage III/IV melanoma patients

Meeting abstracts TriMixDC-MEL consists of autologous monocyte-derived DC that are electroporated with synthetic mRNA encoding CD40 ligand, a constitutively active TLR4, CD70 and fusion proteins of DC.LAMP with 4 melanoma associated antigens (MAGE-A3, MAGE-C2, tyrosinase and gp100). TriMixDC-MEL

Induced pluripotent stem cell-derived neural stem cells transduced with baculovirus encoding CD40 ligand for immunogene therapy in mouse models of breast cancer.

The interaction between CD40 ligand (CD40L) and CD40 can directly inhibit growth of CD40-positive carcinoma cells and may indirectly inhibit tumor growth through coordination of immune responses. Many efforts in CD40L cancer gene therapy have been focused on direct CD40L gene transfer into malignant target cells. This in vivo gene therapy approach relies on high-efficiency gene transfer and could be technically challenging for the treatment of certain cancers, especially multisite metastases. We report herein an alternative means of using the tumor-homing property of neural stem cells (NSCs) to deliver CD40L molecules into tumor tissues. NSCs were derived from human induced pluripotent stem cells, transduced in vitro with a baculoviral vector encoding CD40L, and intravenously injected into immunocompetent mice with orthotopic and metastatic breast cancers. Through a bystander mechanism of intercellular transfer of CD40L from the donor NSCs to tumor target cells, the treatment impeded tumor growth, leading to prolonged survival of the tumor-bearing mice. We further showed that compared with the stem cell-based gene therapy that employed a suicide gene, the CD40L immunogene therapy did not cause liver and kidney injury in the treated mice. This new approach may be particularly valuable for metastatic cancer treatments after systemic stem cell administration.

Safety and biodistribution of a double-deleted oncolytic vaccinia virus encoding CD40 ligand in laboratory Beagles

We evaluated adverse events, biodistribution and shedding of oncolytic vaccinia virus encoding CD40 ligand in two Beagles, in preparation for a phase 1 trial in canine cancer patients. Dog 1 received one dose of vaccinia virus and was euthanized 24 hours afterwards, while dog 2 received virus four times once weekly and was euthanized 7 days after that. Dogs were monitored for adverse events and underwent a detailed postmortem examination. Blood, saliva, urine, feces, and organs were collected for virus detection. Dog 1 had mild fever and lethargy while dog 2 experienced a possible seizure 5.5 hours after first virus administration. Viral DNA declined quickly in the blood after virus administration in both dogs but was still detectable 1 week later by quantitative polymerase chain reaction. Only samples taken directly after virus infusion contained infectious virus. Small amounts of viral DNA, but no infectious virus, were detected in a few saliva and urine samples. Necropsies did not reveal any relevant pathological changes and virus DNA was detected mainly in the spleen. The dogs in the study did not have cancer, and thus adverse events could be more common and viral load higher in dogs with tumors which allow viral amplification.

Modulation of Regulatory T Cell Function by Monocyte-Derived Dendritic Cells Matured through Electroporation with mRNA Encoding CD40 Ligand, Constitutively Active TLR4, and CD70

Regulatory T cells (Tregs) counteract anticancer immune responses through a number of mechanisms, limiting dendritic cell (DC)-based anticancer immunotherapy. In this study, we investigated the influence of various DC activation stimuli on the Treg functionality. We compared DCs activated by electroporation with mRNA encoding constitutively active TLR4 (caTLR4) and CD40 ligand (DiMix-DCs), or these factors together with mRNA encoding the costimulatory molecule CD70 (TriMix-DCs) with DCs maturated in the presence of a mixture of inflammatory cytokines (DCs maturated with a combination of the cytokines IL-1β, IL-6, TNF-α, and PGE2) for their ability to counteract Tregs on different levels. We first demonstrated that there was no difference in the extent of Treg induction starting from CD4(+)CD25(-) T cells under the influence of the different DC maturation stimuli. Second, we showed that both DiMix- and TriMix-DCs could partly alleviate Treg inhibition of CD8(+) T cells. Third, we observed that CD8(+) T cells that had been precultured with DiMix-DCs or TriMix-DCs were partially protected against subsequent Treg suppression. Finally, we showed that Tregs cocultured in the presence of TriMix-DCs, but not DiMix-DCs, partially lost their suppressive capacity. This was accompanied by a decrease in CD27 and CD25 expression on Tregs, as well as an increase in the expression of T-bet and secretion of IFN-γ, TNF-α, and IL-10, suggesting a shift of the Treg phenotype toward a Th1 phenotype. In conclusion, these data suggest that TriMix-DCs are not only able to suppress Treg functions, but moreover could be able to reprogram Tregs to Th1 cells under certain circumstances.

X-linked Hyper IgM Syndrome

X-linked hyper IgM syndrome is associated with abnormalities in the gene encoding CD40 ligand (CD40LG). A typical phenotype evolves during infancy in affected males. This phenotype includes neutropenia, dysgammaglobulinemia, bacterial sinopulmonary infections, and opportunistic infections. In the absence of the typical phenotypic features, clinicians must maintain a high level of suspicion for X-linked hyper IgM syndrome. We describe a unique hemizygous CD40LG mutation which was discovered in a 12-year-old boy with chronic severe neutropenia, a normal IgG level, and absence of sinopulmonary or opportunistic infections. The clinical implications of this mutation and associated atypical phenotype are discussed.

Phase IB study on combined intradermal (ID) and intravenous (IV) administration of autologous mRNA electroporated dendritic cells (DC) as a single-agent cellular immunotherapy or combined with ipilimumab.

2507 Background: Autologous monocyte-derived DC electroporated with synthetic mRNA encoding CD40 ligand, a constitutively active TLR4, and CD70 (TriMix-DC) have superior T-cell stimulatory capacity. In a pilot clinical trial, ID administration of TriMixDC-MEL (a mixture of TriMix-DC co-electroporated with mRNA encoding a fusion of DC.LAMP and 1 of 4 melanoma associated antigens) was immunogenic but resulted in limited anti-tumor activity in patients (pts) with advanced melanoma. Ipilimumab (IPI) is a CTLA-4 blocking mAb with established activity in pts with advanced melanoma. Methods: TriMixDC-MEL by the IV and ID-route was investigated as a single-agent or in combination with IPI (10 mg/kg q3wks x4, allowing for maintenance with IPI-alone q12wks in pts PFS at >24 wks). Ratio of ID/IV administered DC: Cohort-1: 20.106/4.106 DC [2pts], -2: 12.106/12.106 DC [3pts], -3: 4.106/20.106 [6pts], and -4: 0/24.106 DC [4pts]; DC were administered 4x q2w, and a 5th administration on w16. in cohort-5, DC (4.106-ID/20.106-IV) were first administered alone and 2w thereafter in combination with IPI for a total of 4 administrations [6pts]. Results: Local skin reactions (gr1-2) were observed in all pts receiving DC ID, flu-like symptoms (< gr2) were observed in 12/21 pts, post IV-infusion chills (gr2) in 7/21 pts. Cytokines release (including a >2-fold rise in the serum levels of IL-1RA, IL-6, IL-8, IL-17, G-CSF, IFN-g, MIP-1a, MIP-1b, TNF-a) was documented during chills. Best objective tumor response: 2x PR + 2x CR (by RECIST) out of 15 pts (27%) treated with DC-only (ongoing after 10+, 14+, 19+, and 20+ mths) and 3 PR out of 6 pts (all stage IV-M1c) treated with DC+IPI (ongoing after 5+, 5+, 6+ mths). Conclusions: ID/IV-administration of TriMixDC-MEL as a single-agent cellular immunotherapy or combined with IPI is associated with distinct but manageable side-effects and has clinical activity against pretreated advanced melanoma. Activity compares favorably with TriMixDC-MEL by ID-administration-only. Combined ID/IV administration of TriMixDC-MEL with IPI is currently under further evaluation in a phase II trial.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Preclinical Evaluation of TriMix and Antigen mRNA-Based Antitumor Therapy

The use of tumor-associated antigen (TAA) mRNA for therapeutic purposes is under active investigation. To be effective, mRNA vaccines need to deliver activation stimuli in addition to TAAs to dendritic cells (DC). In this study, we evaluated whether intranodal delivery of TAA mRNA together with TriMix, a mix of mRNA encoding CD40 ligand, constitutive active Toll-like receptor 4 and CD70, results in the in situ modification and maturation of DCs, hence, priming of TAA-specific T cells. We showed selective uptake and translation of mRNA in vivo by lymph node resident CD11c(+) cells. This process was hampered by codelivery of classical maturation stimuli but not by TriMix mRNA. Importantly, TriMix mRNA induced a T-cell-attracting and stimulatory environment, including recruitment of antigen-specific CD4(+) and CD8(+) T cells and CTLs against various TAAs. In several mouse tumor models, mRNA vaccination was as efficient in CTL induction and therapy response as vaccination with mRNA-electroporated DCs. Together, our findings suggest that intranodal administration of TAA mRNA together with mRNA encoding immunomodulating molecules is a promising vaccination strategy.

Therapeutic Vaccination With an Autologous mRNA Electroporated Dendritic Cell Vaccine in Patients With Advanced Melanoma

The immunostimulatory capacity of dendritic cells is improved by co-electroporation with mRNA encoding CD40 ligand, constitutively active toll-like receptor 4, and CD70 (TriMix-DC). This pilot clinical trial evaluated the feasibility, safety, and immunogenicity of a therapeutic vaccination containing autologous TriMix-DC co-electroporated with mRNA encoding a human leukocyte antigen class II-targeting signal linked to 1 of 4 melanoma-associated antigens (MAGE-A3, MAGE-C2, tyrosinase, and gp100) in patients with advanced melanoma. Thirty-five American Joint Committee on Cancer stage III/IV melanoma patients received autologous TriMix-DC (4 administrations 2 weeks apart). Immune monitoring was performed by evaluating skin biopsies of delayed type IV hypersensitivity (DTH) reactions for presence of vaccinal antigen-specific DTH-infiltrating lymphocytes (DIL). Thereafter, patients could receive interferon-alpha-2b (IFN-α-2b) 5 MU subcutaneously 3 times weekly and additional TriMix-DC every 8 weeks. TriMix-DC-related adverse events comprised grade 2 local injection site reactions (all patients), and grade 2 fever and lethargy (2 patients). Vaccinal antigen-specific DIL were found in 0/6 patients tested at vaccine initiation and in 12/21 (57.1%) assessed after the fourth vaccine. A positive postvaccination DTH test correlated with IL-12p70 secretion capacity of TriMix-DC. No objective responses to TriMix-DC alone were seen according to RECIST. Twenty-nine patients received IFN-α-2b after the fourth vaccine without unexpected adverse events. During TriMix-DC/IFN-α-2b combination therapy, 1 partial response and 5 stable disease (disease control of >6 months with regression of metastases) were observed in 17 patients with evaluable disease at baseline. In conclusion, this study demonstrated that therapeutic vaccination with autologous TriMix-DC is feasible, safe, and immunogenic and can be combined with sequential IFN-α-2b.

A phase I clinical trial on the combined intravenous (IV) and intradermal (ID) administration of autologous TriMix-DC cellular therapy in patients with pretreated melanoma (TriMixIDIV).

2519 Background: Autologous monocyte derived dendritic cells (DC) electroporated with synthetic messenger RNA (smRNA) encoding CD40 ligand, a constitutively active TLR4, and CD70 (TriMixDC), togeth...

Therapeutic vaccination with an autologous TriMix-Dendritic cell vaccine combined with sequential interferon alfa-2b in patients with advanced melanoma

9024 Background: Electroporation of dendritic cells (DC) with mRNA encoding fusion-proteins of a HLA-class II targeting signal and a melanoma associated antigen (MAA) together with mRNA encoding CD40 ligand, a constitutively active TLR4 and CD70 (TriMix) improves the immunostimulatory capacity of autologous DC. Methods: Following leukapheresis, immature DCs (derived from adherent PBMC cultured for 6 days in IL-4 / GM-CSF supplemented medium) are electroporated with mRNA encoding MAGE-A3, MAGE-C2, Tyrosinase and gp100 linked to DC-LAMP, and TriMix mRNA. TriMix-DC (12.5 106/antigen) are cryopreserved and administered by 4 ID-injections q2w, and q8w thereafter. After the 4th vaccination, interferon alfa-2b (IFN- a2b, 5 MIU TIW) is initiated. Immune monitoring is performed by skin biopsy of a vaccine injection site. Biopsies are investigated by IHC and by analyzing the activation (CD137+), cytolytic capacity (CD107a+), and cytokine release (IFN-γ and TNF-α) of DTH infiltrating T-cells in response to autologous EBV-B cells expressing MAA. Results: 29 pts (17M/12F; med age 49, range 28–75) with stage III/IV melanoma, nl LDH, and no CNS metastases were recruited. Vaccine related AE's (first 24 pts): gr2 local injection site reactions (all pts); fever & lethargy (gr2, 1 pt). Pts (20) who initiated IFN-a2b experienced constitutional side effects (gr3, 1 pt). Vaccine-specific DTH infiltrating T cells were documented post-vaccination in 13/17 pts (10/13 pts had a CD137+CD8+ and 2/13 pts a CD4+ response). Out of the 11 pts without evaluable disease, 2 had a local recurrence (salvaged by surgery). After a mFU of 7.8 mths (range 4.3–13.7) all pts remain disease-free. Out of the 13 pts with measurable disease, BOR (RECIST) was 8 SD and 5 PD; 1 pt with initial PD subsequently obtained a PR. Regression of metastases occurred in lung- (2 pts), orbita- (1 pt) and lymph node metastases (3 pts). After a mFU of 7 mths (range 1–14), the mPFS is 3,1 mths (95% CI 2,29–4,08); 4 pts remain progression-free after respectively 5, 8, 10 and 11 mths of follow-up. Conclusions: Therapeutic vaccination with TriMix-DC combined with sequential IFN-a2b is feasible, safe, immunogenic and associated with anti-tumor activity in patients with advanced melanoma. [Table: see text]

Single-Step Antigen Loading and Activation of Dendritic Cells by mRNA Electroporation for the Purpose of Therapeutic Vaccination in Melanoma Patients

A critical factor determining the effectiveness of currently used dendritic cell (DC)-based vaccines is the DC activation or maturation status. We have recently shown that the T-cell stimulatory capacity of DCs pulsed with tumor-antigen-derived peptides can be considerably increased by activating the DCs through electroporation with mRNA encoding CD40 ligand, CD70, and a constitutively active Toll-like receptor 4 (TriMix DCs). Here, we investigate whether TriMix DCs can be coelectroporated with whole tumor-antigen-encoding mRNA. The T-cell stimulatory capacity of TriMix DCs pulsed with the immunodominant MelanA-A2 peptide and that of TriMix DCs coelectroporated with MelanA mRNA were compared in vitro. TriMix DCs were also coelectroporated with mRNA encoding Mage-A3, Mage-C2, tyrosinase, or gp100. The capacity of these DCs to stimulate tumor-antigen-specific T cells in melanoma patients was investigated both in vitro before vaccination and after DC vaccination. Like peptide-pulsed TriMix DCs, TriMix DCs coelectroporated with MelanA mRNA are very potent in inducing MelanA-specific CD8(+) T cells in vitro. These T cells have an activated phenotype, show cytolytic capacity, and produce inflammatory cytokines in response to specific stimulation. TriMix DCs coelectroporated with tyrosinase are able to stimulate tyrosinase-specific CD8(+) T cells in vitro from the blood of nonvaccinated melanoma patients. Furthermore, TriMix DCs coelectroporated with Mage-A3, Mage-C2, or tyrosinase are able to induce antigen-specific CD8(+) T cells through therapeutic DC vaccination. TriMix DCs coelectroporated with whole tumor-antigen mRNA stimulate antigen-specific T cells in vitro and induce antigen-specific T-cell responses in melanoma patients through vaccination. Therefore, they represent a promising new approach for antitumor immunotherapy.

Extracellular vesicles containing virus-encoded membrane proteins are a byproduct of infection with modified vaccinia virus Ankara

Vaccinia virus is a structurally complex virus that multiplies in the cell cytoplasm. The assembly of Vaccinia virus particles and their egress from infected cells exploit cellular pathways. Most notably, intracellular mature viral particles are enwrapped by Golgi-derived or endosomal vesicles. These enveloped particles, enriched in virus-encoded proteins, migrate to the cell surface where they are released into the extracellular space through fusion of their outer envelope with the cell membrane. We report that baby hamster kidney cells productively infected with the modified vaccinia virus Ankara strain (MVA) also release extracellular vesicles containing virus-encoded envelope proteins but devoid of any virus cargo. Such vesicles were visualized on the cell surface by electron microscopy and immunogold labelling of the B5 envelope protein. A portion of the B5 protein was found to be associated with non-viral material in high speed ultracentrifugation pellets and displayed a buoyant density characteristic of exosomes released by some cell types. An unrelated transmembrane protein (CD40 ligand) encoded by the MVA genome was also incorporated into extracellular vesicles but not into the envelopes that surround extracellular enveloped virus. High speed pellets obtained by centrifugation of culture medium from cells infected with MVA encoding CD40 ligand displayed the ability to induce dendritic cell maturation suggesting that the ligand is on the outer surface of the extracellular vesicles. We propose that the formation of extracellular vesicles after vaccinia virus infection is a byproduct of the pathway leading to the formation of extracellular enveloped virus.

Therapeutic Vaccination against Murine Lymphoma by Intratumoral Injection of Recombinant Fowlpox Virus Encoding CD40 Ligand

The interaction between CD40 ligand (CD40L, CD154) and its receptor CD40 on antigen-presenting cells is essential for the initiation of cell-mediated and humoral immune responses. Malignant B cells also express CD40 and respond to CD40L by enhancing expression of costimulatory molecules. In this study, we investigated the therapeutic antitumor effect of intratumoral administration of recombinant fowlpox virus encoding murine CD40L (rF-mCD40L) in a murine B-cell lymphoma model. BALB/c mice with established s.c. and widely metastatic A20 lymphoma tumors were treated with intratumoral injections of rF-mCD40L together with systemic chemotherapy. This combined chemoimmunotherapy resulted in complete tumor regression and long-term survival of the mice. Some tumor cells in the injected sites expressed the CD40L transgene and had increased expression of the CD80 and CD86 costimulatory molecules. The therapeutic effect was dependent on CD8 but not on CD4 T cells. Moreover, there was a requirement that the recombinant CD40L virus be injected directly into the tumor, as opposed to peritumoral or distant sites. Thus, rF-mCD40L injected directly into the tumor microenvironment enhances the immunogenicity of tumor B cells. The results support future plans for intratumoral injection of rF-mCD40L in patients with lymphoma.

DEC-205 antigen targeting at a dendritic cell-enriched site and CD40L-directed dendritic cell activation enhances CD4+ T cell priming. (47.12)

Abstract To enhance DNA vaccine efficacy, we tested the hypothesis that inoculation of calves with a single dose of a DNA vaccine capable of DC recruitment, DC antigen-targeting and activation will prime significant CD4+ T cell responses. To test this hypothesis, we developed a DNA construct, pCC98MSP1, in which a DEC-205-specific single chain antibody was used for DC-targeting of an Anaplasma marginale MSP1a DR-restricted CD4+ T cell antigen. A DNA construct, pCD40L-ED, encoding CD40 ligand was used for DC activation and DNA-encoded Flt3L/GM-CSF were used for DC recruitment. Recombinant CC98MSP1 binds to DEC-205 in vitro and to skin DCs in situ. In a dose-escalation experiment, DR-matched calves were immunized with a single dose of pCC98MSP1 (0.25–3mg) mixed with pCD40L-ED, pFlt-3L and pGM-CSF (0.5mg each). Immunization with 0.25mg pCC98MSP1 primed CD4+ T cell responses detectable in fresh PBMC 1wk post-immunization (PI). A dose dependent CD4+ T cell response was detected and surprisingly, at 2wks PI, the response induced by 0.25mg was not statistically different from the responses induced by 0.5–3mg pCC98MSP1. The responses were maintained for >9wks PI and underwent rapid recall 4 days post-boost with 0.25mg pCC98MSP1. This data supports the hypothesis that immunization with a single dose of a DNA vaccine capable of DC recruitment, DC-antigen targeting and activation enhances vaccine efficacy. Funding: USDA-CSREES 2005-01693

Osteopenia in X-linked hyper-IgM syndrome reveals a regulatory role for CD40 ligand in osteoclastogenesis

We report that osteopenia is a prominent and previously unappreciated clinical feature of patients with X-linked hyper-IgM syndrome, an inherited immune deficiency disorder caused by mutations in the gene encoding CD40 ligand (CD40L). We therefore conducted studies to determine the relationship between CD40L and osteoclastogenesis. Recognizing that activated T cells express surface receptor activator of NF-kappaB ligand (RANKL) and can induce osteoclast differentiation of myeloid cells expressing RANK, we assessed the capacity of wild-type T cells and CD40L(-/-) T cells to induce osteoclastogenesis in vitro. Relative to wild-type T cells, activated CD40L(-/-) T cells from both humans and mice promoted robust osteoclast differentiation of myeloid cells. Whereas activated CD40L(-/-) T cells had normal expression of RANKL, they were deficient in IFN-gamma production. In subsequent studies, we cultured activated CD40L(-/-) T cells in the presence of IFN-gamma, and we found that the osteoclastic capacity of CD40L(-/-) T cells could be greatly diminished. These results show that CD40L can influence RANKL signaling through T cell priming, and thus they demonstrate a regulatory role for CD40L in bone mineralization that is absent in patients with X-linked hyper-IgM syndrome.

Transcription factors TFE3 and TFEB are critical for CD40 ligand expression and thymus-dependent humoral immunity

TFE3 and TFEB are broadly expressed transcription factors related to the transcription factor Mitf. Although they have been linked to cytokine signaling pathways in nonlymphoid cells, their function in T cells is unknown. TFE3-deficient mice are phenotypically normal, whereas TFEB deficiency causes early embryonic death. We now show that combined inactivation of TFE3 and TFEB in T cells resulted in a hyper-immunoglobulin M syndrome due to impaired expression of CD40 ligand by CD4(+) T cells. Native TFE3 and TFEB bound to multiple cognate sites in the promoter of the gene encoding CD40 ligand (Cd40lg), and maximum Cd40lg promoter activity and gene expression required TFE3 or TFEB. Thus, TFE3 and TFEB are direct, physiological and mutually redundant activators of Cd40lg expression in activated CD4(+) T cells critical for T cell-dependent antibody responses.