B cells in the pneumococcus-infected lung are heterogeneous and require CD4+ T cell help including CD40L to become resident memory B cells.

Recovery from respiratory pneumococcal infections generates lung-localized protection against heterotypic bacteria, mediated by resident memory lymphocytes. Optimal protection in mice requires re-exposure to pneumococcus within days of initial infection. Serial surface marker phenotyping of B cell populations in a model of pneumococcal heterotypic immunity revealed that bacterial re-exposure stimulates the immediate accumulation of dynamic and heterogeneous populations of B cells in the lung, and is essential for the establishment of lung resident memory B (BRM) cells. The B cells in the early wave were activated, proliferating locally, and associated with both CD4+ T cells and CXCL13. Antagonist- and antibody-mediated interventions were implemented during this early timeframe to demonstrate that lymphocyte recirculation, CD4+ cells, and CD40 ligand (CD40L) signaling were all needed for lung BRM cell establishment, whereas CXCL13 signaling was not. While most prominent as aggregates in the loose connective tissue of bronchovascular bundles, morphometry and live lung imaging analyses showed that lung BRM cells were equally numerous as single cells dispersed throughout the alveolar septae. We propose that CD40L signaling from antigen-stimulated CD4+ T cells in the infected lung is critical to establishment of local BRM cells, which subsequently protect the airways and parenchyma against future potential infections.

Crystal structures of human CD40 in complex with monoclonal antibodies dacetuzumab and bleselumab.

CD40 is a member of the tumor necrosis factor receptor superfamily, and it is widely expressed on immune and non-immune cell types. The interaction between CD40 and the CD40 ligand (CD40L) plays an essential function in signaling, and the CD40/CD40L complex works as an immune checkpoint molecule. CD40 has become a therapeutic target, and a variety of agonistic/antagonistic anti-CD40 monoclonal antibodies (mAbs) have been developed. To better understand the mode of action of anti-CD40 mAbs, we determined the X-ray crystal structures of dacetuzumab (agonist) and bleselumab (antagonist) in complex with the extracellular domain of human CD40, respectively. The structure reveals that dacetuzumab binds to CD40 on the top of cysteine-rich domain 1 (CRD1), which is the domain most distant from the cell surface, and it does not compete with CD40L binding. The binding interface of bleselumab spread between CRD2 and CRD1, overlapping with the binding surface of the ligand. Our results offer important insights for future structural and functional studies of CD40 and provide clues to understanding the mechanism of biological response. These data can be applied to developing new strategies for designing antibodies with more therapeutic efficacy.

Inhibition of CD40L with Frexalimab in Multiple Sclerosis.

BACKGROUND: The CD40-CD40L costimulatory pathway regulates adaptive and innate immune responses and has been implicated in the pathogenesis of multiple sclerosis. Frexalimab is a second-generation anti-CD40L monoclonal antibody being evaluated for the treatment of multiple sclerosis. METHODS: In this phase 2, double-blind, randomized trial, we assigned, in a 4:4:1:1 ratio, participants with relapsing multiple sclerosis to receive 1200 mg of frexalimab administered intravenously every 4 weeks (with an 1800-mg loading dose), 300 mg of frexalimab administered subcutaneously every 2 weeks (with a 600-mg loading dose), or the matching placebos for each active treatment. The primary end point was the number of new gadolinium-enhancing T1-weighted lesions seen on magnetic resonance imaging at week 12 relative to week 8. Secondary end points included the number of new or enlarging T2-weighted lesions at week 12 relative to week 8, the total number of gadolinium-enhancing T1-weighted lesions at week 12, and safety. After 12 weeks, all the participants could receive open-label frexalimab. RESULTS: Of 166 participants screened, 129 were assigned to a trial group; 125 participants (97%) completed the 12-week double-blind period. The mean age of the participants was 36.6 years, 66% were women, and 30% had gadolinium-enhancing lesions at baseline. At week 12, the adjusted mean number of new gadolinium-enhancing T1-weighted lesions was 0.2 (95% confidence interval [CI], 0.1 to 0.4) in the group that received 1200 mg of frexalimab intravenously and 0.3 (95% CI, 0.1 to 0.6) in the group that received 300 mg of frexalimab subcutaneously, as compared with 1.4 (95% CI, 0.6 to 3.0) in the pooled placebo group. The rate ratios as compared with placebo were 0.11 (95% CI, 0.03 to 0.38) in the 1200-mg group and 0.21 (95% CI, 0.08 to 0.56) in the 300-mg group. Results for the secondary imaging end points were generally in the same direction as those for the primary analysis. The most common adverse events were coronavirus disease 2019 and headaches. CONCLUSIONS: In a phase 2 trial involving participants with multiple sclerosis, inhibition of CD40L with frexalimab had an effect that generally favored a greater reduction in the number of new gadolinium-enhancing T1-weighted lesions at week 12 as compared with placebo. Larger and longer trials are needed to determine the long-term efficacy and safety of frexalimab in persons with multiple sclerosis. (Funded by Sanofi; ClinicalTrials.gov number, NCT04879628.).

In silico designing of novel epitope-based peptide vaccines against HIV-1.

The HIV-1 virus has been regarded as a catastrophe for human well-being. The global incidence of HIV-1-infected individuals is increasing. Hence, development of effective immunostimulatory molecules has recently attracted an increasing attention in the field of vaccine design against HIV-1 infection. In this study, we explored the impacts of CD40L and IFN-γ as immunostimulatory adjuvants for our candidate HIV-1 Nef vaccine in human and mouse using immunoinformatics analyses. Overall, 18 IFN-γ-based vaccine constructs (9 constructs in human and 9 constructs in mouse), and 18 CD40L-based vaccine constructs (9 constructs in human and 9 constructs in mouse) were designed. To find immunogenic epitopes, important characteristics of each component (e.g., MHC-I and MHC-II binding, and peptide-MHC-I/MHC-II molecular docking) were determined. Then, the selected epitopes were applied to create multiepitope constructs. Finally, the physicochemical properties, linear and discontinuous B cell epitopes, and molecular interaction between the 3D structure of each construct and CD40, IFN-γ receptor or toll-like receptors (TLRs) were predicted. Our data showed that the full-length CD40L and IFN-γ linked to the N-terminal region of Nef were capable of inducing more effective immune response than multiepitope vaccine constructs. Moreover, molecular docking of the non-allergenic full-length- and epitope-based CD40L and IFN-γ constructs to their cognate receptors, CD40 and IFN-γ receptors, and TLRs 4 and 5 in mouse were more potent than in human. Generally, these findings suggest that the full forms of these adjuvants could be more efficient for improvement of HIV-1 Nef vaccine candidate compared to the designed multiepitope-based constructs.

3-hydroxy butyrate dehydrogenase 2 deficiency aggravates systemic lupus erythematosus progression in a mouse model by promoting CD40 ligand demethylation.

The implications of the CD40-CD40 ligand (CD40L) signaling pathway in systemic lupus erythematosus (SLE) were well documented, due to its important role among immune cells. Previous research found that 3-hydroxy butyrate dehydrogenase 2 (BDH2), a modulator of intracellular iron homeostasis and iron transportation promoted the pathogenic process of SLE by regulating the demethylation of cd70, cd11a, and cd40l genes among CD4 + T cells. The purpose of this study was to explore the role of BDH2 in oxidative damage-induced SLE. First, CD4 + T cells treated with H2O2 were injected into the tail vein of mice to establish a lupus model. CD40L knockdown significantly decreased CD40L expression on CD4 + T cells in the spleen of SLE mice. Compared with SLE model mice, the levels of serum anti-dsDNA antibody and urinary protein in the CD40L interference group were significantly decreased. CD40L knockdown alleviated the immune complex glomerulonephritis in syngeneic SLE mice. Moreover, the levels of IFN-γ and IL-2 were decreased. However, IL-4 and IL-10 levels were significantly upregulated in the serum of CD40L knockdown SLE mice, compared with SLE model mice. Accordingly, CD40L knockdown reduced Th1/Th2 percentage in SLE mice. Inhibiting the expression of BDH2 of CD4 + T cells promoted the demethylation of CD40L, while it inhibited cell proliferation, elevated oxidative stress through increased expression of CD40L, and thus, promoted the progress of SLE. Our results demonstrate that BDH2 aggravates the pathologic progression of SLE in mice, by increasing the demethylation level of CD40L among CD4 + T cells.

Dysregulated CD40 and CD40 ligand expression in anti-N-methyl-d-aspartate receptor encephalitis.

Anti-N-methyl-d-aspartate receptor encephalitis (anti-NMDARE) is a B cell- and antibody-mediated autoimmune disease which may be regulated by CD40/CD40L signaling pathway. we enrolled anti-NMDARE patients and measured the serum CD40 and CD40L concentrations. The serum concentration of CD40 was decreased, while CD40L was increased in anti-NMDARE patients compared with that of healthy controls. The concentrations of CD40 and CD40L were both elevated in the acute stage of anti-NMDARE and were reduced during remission. Serum CD40L levels were positively correlated with serum CD40 levels. These results revealed that the CD40/CD40L signaling pathway might contribute to the pathogenesis of anti-NMDARE.

CD40L protects against mouse hepatitis virus-induced neuroinflammatory demyelination.

Neurotropic mouse hepatitis virus (MHV-A59/RSA59) infection in mice induces acute neuroinflammation due to direct neural cell dystrophy, which proceeds with demyelination with or without axonal loss, the pathological hallmarks of human neurological disease, Multiple sclerosis (MS). Recent studies in the RSA59-induced neuroinflammation model of MS showed a protective role of CNS-infiltrating CD4+ T cells compared to their pathogenic role in the autoimmune model. The current study further investigated the molecular nexus between CD4+ T cell-expressed CD40Ligand and microglia/macrophage-expressed CD40 using CD40L-/- mice. Results demonstrate CD40L expression in the CNS is modulated upon RSA59 infection. We show evidence that CD40L-/- mice are more susceptible to RSA59 induced disease due to reduced microglia/macrophage activation and significantly dampened effector CD4+ T recruitment to the CNS on day 10 p.i. Additionally, CD40L-/- mice exhibited severe demyelination mediated by phagocytic microglia/macrophages, axonal loss, and persistent poliomyelitis during chronic infection, indicating CD40-CD40L as host-protective against RSA59-induced demyelination. This suggests a novel target in designing prophylaxis for virus-induced demyelination and axonal degeneration, in contrast to immunosuppression which holds only for autoimmune mechanisms of inflammatory demyelination.

Double Strike Approach for Tumor Attack: Engineering T Cells Using a CD40L:CD28 Chimeric Co-Stimulatory Switch Protein for Enhanced Tumor Targeting in Adoptive Cell Therapy.

Activation of co-stimulatory pathways in cytotoxic T lymphocytes expressing chimeric antigen receptors (CARs) have proven to boost effector activity, tumor rejection and long-term T cell persistence. When using antigen-specific T cell receptors (TCR) instead of CARs, the lack of co-stimulatory signals hampers robust antitumoral response, hence limiting clinical efficacy. In solid tumors, tumor stroma poses an additional hurdle through hindrance of infiltration and active inhibition. Our project aimed at generating chimeric co-stimulatory switch proteins (CSP) consisting of intracellular co-stimulatory domains (ICD) fused to extracellular protein domains (ECD) for which ligands are expressed in solid tumors. The ECD of CD40L was selected for combination with the ICD from the CD28 protein. With this approach, it was expected to not only provide co-stimulation and strengthen the TCR signaling, but also, through the CD40L ECD, facilitate the activation of tumor-resident antigen-presenting cells (APCs), modulate activation of tumor endothelium and induce TCR-MHC independent apoptotic effect on tumor cells. Since CD28 and CD40L belong to different classes of transmembrane proteins (type I and type II, respectively), creating a chimeric protein presented a structural and functional challenge. We present solutions to this challenge describing different CSP formats that were successfully expressed in human T cells along with an antigen-specific TCR. The level of surface expression of the CSPs depended on their distinct design and the state of T cell activation. In particular, CSPs were upregulated by TCR stimulation and downregulated following interaction with CD40 on target cells. Ligation of the CSP in the context of TCR-stimulation modulated intracellular signaling cascades and led to improved TCR-induced cytokine secretion and cytotoxicity. Moreover, the CD40L ECD exhibited activity as evidenced by effective maturation and activation of B cells and DCs. CD40L:CD28 CSPs are a new type of switch proteins designed to exert dual beneficial antitumor effect by acting directly on the gene-modified T cells and simultaneously on tumor cells and tumor-supporting cells of the TME. The observed effects suggest that they constitute a promising tool to be included in the engineering process of T cells to endow them with complementary features for improved performance in the tumor milieu.

Simultaneous Inhibition of PD-1 and Stimulation of CD40 Signaling Pathways by Anti-PD-L1/CD40L Bispecific Fusion Protein Synergistically Activate Target and Effector Cells.

Bispecific antibodies (BsAbs) or fusion proteins (BsAbFPs) present a promising strategy for cancer immunotherapy. Numerous BsAbs targeting coinhibitory and costimulatory pathways have been developed for retargeting T cells and antigen presenting cells (APCs). It is challenging to assess the potency of BsAb that engages two different signaling pathways simultaneously in a single assay format, especially when the two antigen targets are expressed on different cells. To explore the potency of anti-PD-L1/CD40L BsAbFP, a fusion protein that binds to human CD40 and PD-L1, we engineered CHO cells as surrogate APCs that express T cell receptor activator and PD-L1, Jurkat cells with PD-1 and NFAT-luciferase reporter as effector T cells, and Raji cell with NFkB-luciferase that endogenously expresses CD40 as accessory B cells. A novel reporter gene bioassay was developed using these cell lines that allows anti-PD-L1/CD40L BsAbFP to engages both PD-1/PD-L1 and CD40/CD40L signaling pathways in one assay. As both reporters use firefly luciferase, the effects of activating both signaling pathways is observed as an increase in luminescence, either as a higher upper asymptote, a lower EC50, or both. This dual target reporter gene bioassay system reflects potential mechanism of action and demonstrated the ability of anti-PD-L1/CD40L BsAbFP to synergistically induce biological response compared to the combination of anti-PD-L1 monovalent monoclonal antibody and agonist CD40L fusion protein, or either treatment alone. The results also showed a strong correlation between the drug dose and biological response within the tested potency range with good linearity, accuracy, precision, specificity and stability indicating properties, suggesting that this "three-cell-in-one" dual target reporter gene bioassay is suitable for assessing potency, structure-function and critical quality attributes of anti-PD-L1/CD40L BsAbFP. This approach could be used for developing dual target bioassays for other BsAbs and antibodies used for combination therapy.

Soluble FAS Ligand Enhances Suboptimal CD40L/IL-21-Mediated Human Memory B Cell Differentiation into Antibody-Secreting Cells.

Differentiation of Ag-specific B cells into class-switched, high-affinity, Ab-secreting cells provides protection against invading pathogens but is undesired when Abs target self-tissues in autoimmunity, beneficial non-self-blood transfusion products, or therapeutic proteins. Essential T cell factors have been uncovered that regulate T cell-dependent B cell differentiation. We performed a screen using a secreted protein library to identify novel factors that promote this process and may be used to combat undesired Ab formation. We tested the differentiating capacity of 756 secreted proteins on human naive or memory B cell differentiation in a setting with suboptimal T cell help in vitro (suboptimal CD40L and IL-21). High-throughput flow cytometry screening and validation revealed that type I IFNs and soluble FAS ligand (sFASL) induce plasmablast differentiation in memory B cells. Furthermore, sFASL induces robust secretion of IgG1 and IgG4 Abs, indicative of functional plasma cell differentiation. Our data suggest a mechanistic connection between elevated sFASL levels and the induction of autoreactive Abs, providing a potential therapeutic target in autoimmunity. Indeed, the modulators identified in this secretome screen are associated with systemic lupus erythematosus and may also be relevant in other autoimmune diseases and allergy.

CD40L-Stimulated B Lymphocytes Are Polarized toward APC Functions after Exposure to IL-4 and IL-21.

B lymphocytes have multiple functions central to humoral immunity, including Ag presentation to T cells, cytokine secretion, and differentiation into Ab-secreting plasma cells. In vitro expansion of human B cells by continuous IL-4 stimulation and engagement of their CD40 receptor by CD40L has allowed the use of these IL-4-CD40-B cells in research for the induction of Ag-specific T cell immune responses. However, in vivo, follicular helper T cells also influence B cell activity through the secretion of IL-21. The impact of both cytokines on multiple B cell functions is not clearly defined. To further understand these cytokines in CD40-B cell biology, we stimulated CD40-B cells with IL-4 or IL-21 or both (Combo) and characterized the proliferation, subsets, and functions of these cells. We demonstrate that IL-21- and Combo-CD40-B cells are highly proliferative cells that can be rapidly expanded to high numbers. We show that IL-21-CD40-B cells polarize to Ab-secreting plasma cells, whereas IL-4- and Combo-CD40-B cells are mostly activated mature B cells that express molecules associated with favorable APC functions. We further demonstrate that both IL-4- and Combo-CD40-B cells are efficient in promoting T cell activation and proliferation compared with IL-21-CD40-B cells. Thus, our study provides a better appreciation of CD40-B cell plasticity and biology. In addition, the stimulation of B cells with CD40L, IL-4, and IL-21 allows for the fast generation of high numbers of efficient APC, therefore providing a prospective tool for research and clinical applications such as cancer immunotherapy.

Memory B cells targeting SARS-CoV-2 spike protein and their dependence on CD4+ T cell help.

Memory B cells seem to be more durable than antibodies and thus crucial for the long-term immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Here we investigate SARS-CoV-2 spike-specific memory B cells and their dependence on CD4+ T cell help in different settings of coronavirus disease 2019 (COVID-19). Compared with severely ill individuals, those who recovered from mild COVID-19 develop fewer but functionally superior spike-specific memory B cells. Generation and affinity maturation of these cells is best associated with IL-21+CD4+ T cells in recovered individuals and CD40L+CD4+ T cells in severely ill individuals. The increased activation and exhaustion of memory B cells observed during COVID-19 correlates with CD4+ T cell functions. Intriguingly, CD4+ T cells recognizing membrane protein show a stronger association with spike-specific memory B cells than those recognizing spike or nucleocapsid proteins. Overall, we identify CD4+ T cell subsets associated with the generation of B cell memory during SARS-CoV-2 infection.

A Posttranscriptional Pathway of CD40 Ligand mRNA Stability Is Required for the Development of an Optimal Humoral Immune Response.

CD40 ligand (CD40L) mRNA stability is dependent on an activation-induced pathway that is mediated by the binding complexes containing the multifunctional RNA-binding protein, polypyrimidine tract-binding protein 1 (PTBP1) to a 3' untranslated region of the transcript. To understand the relationship between regulated CD40L and the requirement for variegated expression during a T-dependent response, we engineered a mouse lacking the CD40L stability element (CD40LΔ5) and asked how this mutation altered multiple aspects of the humoral immunity. We found that CD40LΔ5 mice expressed CD40L at 60% wildtype levels, and lowered expression corresponded to significantly decreased levels of T-dependent Abs, loss of germinal center (GC) B cells and a disorganized GC structure. Gene expression analysis of B cells from CD40LΔ5 mice revealed that genes associated with cell cycle and DNA replication were significantly downregulated and genes linked to apoptosis upregulated. Importantly, somatic hypermutation was relatively unaffected although the number of cells expressing high-affinity Abs was greatly reduced. Additionally, a significant loss of plasmablasts and early memory B cell precursors as a percentage of total GL7+ B cells was observed, indicating that differentiation cues leading to the development of post-GC subsets was highly dependent on a threshold level of CD40L. Thus, regulated mRNA stability plays an integral role in the optimization of humoral immunity by allowing for a dynamic level of CD40L expression on CD4 T cells that results in the proliferation and differentiation of pre-GC and GC B cells into functional subsets.

Flow Cytometric Characterization of Human Antigen-Reactive T-Helper Cells.

The detection and functional characterization of antigen-reactive T helper (Th) cells has been challenging due to their low frequency and functional heterogeneity. Antigen-reactive T cell enrichment (ARTE) allows the in-depth characterization of antigen-specific Th lymphocytes as a prerequisite for better understanding the role of adaptive immune responses in health and disease. ARTE is based on detection of the activation markers CD154 (CD40L) (expressed on all conventional Th cell subsets, Tcons) and CD137 (4-1BB) (expressed on regulatory T cells, Tregs), which are upregulated on the surface of CD4+ T cells upon short-term (7 h) in vitro stimulation with antigens in the presence of antigen-presenting cells (APCs). To substantially increase the sensitivity for the detection of antigen-specific Th cells, ARTE combines magnetic pre-enrichment of rare antigen-reactive T cells with multiparameter flow cytometry. Using CD154 and CD137 in combination allows the parallel detection of reactive Tcons and Tregs, after stimulation with the antigen. Thus, the ARTE technology now enables to characterize antigen-specific T cells with increased sensitivity of detection allowing even the investigation of antigen-specific Th cells in the naive T cell repertoire and regardless of prior knowledge of MHC alleles or antigenic epitopes.

Boosting CAR T-cell responses in lymphoma by simultaneous targeting of CD40/4-1BB using oncolytic viral gene therapy.

Pretreatment of B-cell lymphoma patients with immunostimulatory gene therapy using armed oncolytic viruses may prime tumor lesions for subsequent chimeric antigen receptor (CAR) T-cell therapy, thereby enhancing CAR T-cell functionality and possibly increasing response rates in patients. LOAd703 (delolimogene mupadenorepvec) is an oncolytic adenovirus (serotype 5/35) that encodes for the transgenes CD40L and 4-1BBL, which activate both antigen-presenting cells and T cells. Many adenoviruses failed to demonstrate efficacy in B-cell malignancies, but LOAd703 infect cells via CD46, which enables B cell infection. Herein, we investigated the therapeutic potential of LOAd703 in human B-cell lymphoma models, alone or in combination with CAR T-cell therapy. LOAd703 could infect and replicate in B-cell lymphoma cell lines (BC-3, Karpas422, Daudi, DG-75, U-698) and induced an overall enhanced immunogenic profile with upregulation of co-stimulatory molecules CD80, CD86, CD70, MHC molecules, death receptor Fas and adhesion molecule ICAM-1. Further, CAR T-cell functionality was boosted by stimulation with lymphoma cells infected with LOAd703. This was demonstrated by an augmented release of IFN-γ and granzyme B, increased expression of the degranulation marker CD107a, fewer PD-1 + TIM-3+ CAR T cells in vitro and enhanced lymphoma cell killing both in in vitro and in vivo xenograft models. In addition, LOAd703-infected lymphoma cells upregulated the secretion of several chemokines (CXCL10, CCL17, CCL22, CCL3, CCL4) essential for immune cell homing, leading to enhanced CAR T-cell migration. In conclusion, immunostimulatory LOAd703 therapy is an intriguing approach to induce anti-lymphoma immune responses and to improve CAR T-cell therapy in B-cell lymphoma.

Interrogation of the cellular immunome of cancer patients with regard to the COVID-19 pandemic.

While vaccines directed against the SARS-CoV-2 spike protein will have varying degrees of effectiveness in preventing SARS-CoV-2 infections, the severity of infection will be determined by multiple host factors including the ability of immune cells to lyse virus-infected cells. This review will discuss the complexity of both adaptive and innate immunomes and how a flow-based assay can detect up to 158 distinct cell subsets in the periphery. This assay has been employed to show the effect of age on differences in specific immune cell subsets, and the differences in the immunome between healthy donors and age-matched cancer patients. Also reviewed are the numerous soluble factors, in addition to cytokines, that may vary in the pathogenesis of SARS-CoV-2 infections and may also be employed to help define the effectiveness of a given vaccine or other antiviral agents. Various steroids have been employed in the management of autoimmune adverse events in cancer patients receiving immunotherapeutics and may be employed in the management of SARS-CoV-2 infections. The influence of steroids on multiple immune cells subsets will also be discussed.

Development of a humanized mouse model to analyze antibodies specific for human leukocyte antigen (HLA).

In organ transplantation, human leukocyte antigen (HLA)-mismatch grafts not only induce the activation of cellular mediated immune response but also the development of chronic antibody-mediated rejection due to the donor-specific anti-HLA antibody (DSA) produced by B cells and plasma cells interacting with the graft endothelium. Significant improvement in long-term survival after transplantation can be expected if antibody-mediated rejection due to the DSA can be overcome. However, the mechanism of producing or controlling the DSA remains to be elucidated. In recent decades, "humanized" mouse models have been widely used for the basic research of human immune systems, but a humanized mouse model to analyze the mechanism of DSA production has not been established yet. Thus, we aimed to create a humanized mouse using a severe immunodeficiency mouse (NSG mouse) administered with human peripheral blood mononuclear cells (PBMCs). Initially, we detected a very low level of human total-IgG and no anti-HLA antibodies (Abs) in these mice. In our next attempt, we mixed PBMCs of various HLA antigenic combinations with or without regulatory T cells and preconditioned them by culturing on feeder cells stably transfected with human CD40 ligand (h-CD40L) alone or with h-CD40L and human B cell activating factor (h-BAFF). They were subsequently co-cultured with the corresponding irradiated stimulator PBMCs, and all cells were administered into naïve NSG mice. Although all three humanized models had sufficient human total-IgG and anti-HLA antibody production, allospecific anti-HLA Ab production was prominently suppressed whereas non-specific anti-HLA Abs were sufficiently detected. Therefore, this novel humanized mouse model might be useful for analyzing the mechanism of anti-allogeneic human B cell tolerance induction.

Soluble CD40L activates soluble and cell-surface integrin αvß3, α5ß1, and α4ß1 by binding to the allosteric ligand-binding site (site 2).

CD40L is a member of the TNF superfamily that participates in immune cell activation. It binds to and signals through several integrins, including αvß3 and α5ß1, which bind to the trimeric interface of CD40L. We previously showed that several integrin ligands can bind to the allosteric site (site 2), which is distinct from the classical ligand-binding site (site 1), raising the question of if CD40L activates integrins. In our explorations of this question, we determined that integrin α4ß1, which is prevalently expressed on the same CD4+ T cells as CD40L, is another receptor for CD40L. Soluble (s)CD40L activated soluble integrins αvß3, α5ß1, and α4ß1 in cell-free conditions, indicating that this activation does not require inside-out signaling. Moreover, sCD40L activated cell-surface integrins in CHO cells that do not express CD40. To learn more about the mechanism of binding, we determined that sCD40L bound to a cyclic peptide from site 2. Docking simulations predicted that the residues of CD40L that bind to site 2 are located outside of the CD40L trimer interface, at a site where four HIGM1 (hyper-IgM syndrome type 1) mutations are clustered. We tested the effect of these mutations, finding that the K143T and G144E mutants were the most defective in integrin activation, providing support that this region interacts with site 2. We propose that allosteric integrin activation by CD40L also plays a role in CD40L signaling, and defective site 2 binding may be related to the impaired CD40L signaling functions of these HIGM1 mutants.