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Rheumatoid arthritis (RA) is a systemic autoimmune disease driven by highly active autoantibody-producing B cells. Activation of B cells is maintained within ectopic germinal centres found in affected joints. Fibroblast-like synoviocytes (FLS) present in inflamed joints support B-cell survival, activation, and differentiation. CD27+ memory B cells and naive B cells show very different responses to activation, particularly by CD40 ligand (CD40L). We show that FLS-dependent activation of human B cells is dependent on interleukin-6 (IL-6) and CD40L. FLS have been shown to activate both naive and memory B cells. Whether the activating potential of FLS is different for naive and memory B cells has not been investigated. Our results suggest that FLS-induced activation of B cells is dependent on IL-6 and CD40L. While FLS are able to induce plasma cell differentiation, isotype switching, and antibody production in memory B cells, the ability of FLS to activate naive B cells is significantly lower., (© 2024 The Author(s). Immunology published by John Wiley & Sons Ltd.)

CD154, also known as CD40 ligand, is a costimulatory molecule involved in humoral and adaptive immune responses upon pairing with its classical receptor, CD40. The CD154/CD40 dyad is a key participant in the pathogenesis of many autoimmune diseases, including systemic lupus erythematosus (SLE). In SLE, the major cells at play, T and B lymphocytes, are shown to overexpress CD154 and CD40, respectively. Subsequently, these cells and other CD40-positive cells engage in numerous effector functions contributing to SLE development. With the recent identification of additional receptors for CD154, all belonging to the integrin family, the role of CD154 in SLE is more complex and calls for deeper investigation into its biological significance. Many therapeutic strategies directed against the CD154/CD40 couple have been deployed for the treatment of SLE and proved efficient in animal models and human studies. However, the incidence of thromboembolic complications in patients treated with these anti-CD154/CD40 antibodies halted their further clinical assessments and called for another class of therapies targeting these molecules. Second-generation antibodies directed against CD154 or CD40 are showing promising results in the advanced stages of clinical testing. Our review presents a thorough description of CD154 and its receptors, CD40 and the integrin family members in SLE pathogenesis. All these elements of the CD154 system represent important therapeutic targets for the treatment of SLE.

Colorectal cancer (CRC) is the second common cause of cancer mortality worldwide, and it still lacks effective approaches for relapsed and metastatic CRC. Recently, oncolytic virus has been emerged as a promising immune therapeutic strategy. In this study, we develop a novel oncolytic adenovirus, rAd.mDCN.mCD40L, which drive oncolytic activity by telomerase reverse transcriptase promoter (TERTp). rAd.mDCN.mCD40L expressed both mouse genes of decorin (mDCN) and CD40 ligand (mCD40L), and produced effective cytotoxicity in both human and mouse CRC cells. Moreover, oncolytic adenovirus mediated mDCN over-expression inhibited Met expression in vitro. In CT26 subcutaneous tumor model, intratumorally delivery of oncolytic adenoviruses could inhibit tumor growth and liver metastasis, while mDCN and/or mCD40L armed oncolytic adenoviruses produced much more impressive responses. No obvious toxicity was detected in lung, liver and spleen. Moreover, mDCN and/or mCD40L armed oncolytic adenoviruses altered the immune state to activate anti-tumor responses, including increasing CD8 + T effector cells and CD4 + memory T cells, reducing MDSCs and Tregs in peripheral blood. Furthermore, mDCN and/or mCD40L armed oncolytic adenoviruses mediated mDCN and/or mCD40L expression in tumors, and up-regulated Th1 cytokines and reduced Th2 cytokines in tumors, which will be benefit for remodeling tumor microenvironment. Importantly, rAd.mDCN.mCD40L and rAd.mCD40L prevented tumor liver metastasis much more effectively than rAd.Null and rAd.mDCN. Therefore, rAd.mDCN.mCD40L and rAd.mCD40L are promising approaches for CRC therapy., (© 2024. The Author(s).)

Alloreactive memory T cells have been implicated as central drivers of transplant rejection. Perplexingly, innate cytokines, such as IL-6, IL-1β, and IL-12, are also associated with rejection of organ transplants. However, the pathways of innate immune activation in allogeneic transplantation are unclear. While the role of microbial and cell death products has been previously described, we identified alloreactive memory CD4 T cells as the primary triggers of innate inflammation. Memory CD4 T cells engaged MHC II-mismatched dendritic cells (DCs), leading to the production of innate inflammatory cytokines. This innate inflammation was independent of several pattern recognition receptors and was primarily driven by TNF superfamily ligands expressed by alloreactive memory CD4 T cells. Blocking of CD40L and TNFα resulted in dampened inflammation, and mice genetically deficient in these molecules exhibited prolonged survival of cardiac allografts. Furthermore, myeloid cell and CD8 T cell infiltration into cardiac transplants was compromised in both CD40L- and TNFα-deficient recipients. Strikingly, we found that priming of naive alloreactive CD8 T cells was dependent on licensing of DCs by memory CD4 T cells. This study unravels the key mechanisms by which alloreactive memory CD4 T cells contribute to destructive pathology and transplant rejection., Competing Interests: Competing interests statement:The authors declare no competing interest.

Recently published studies in both murine models and a meta-analysis of non-human primate renal transplant studies showed that anti-CD154 reagents conferred a significant survival advantage over CD40 blockers in both animal models and across multiple organs. Here we sought to compare the induction of donor-reactive forkhead box P3+-induced regulatory T cells (Foxp3 + iTreg) in mice treated with anti-CD154 versus anti-CD40 monoclonal antibodies (mAbs). Results indicated that while treatment with anti-CD154 mAb resulted in a significant increase in the frequency of donor-reactive CD4 + Foxp3 + iTreg following transplantation, treatment with anti-CD40 or Cd40 deficiency failed to recapitulate this result. Because we recently identified CD11b as an alternate receptor for CD154 during alloimmunity, we interrogated the role of CD154:CD11b interactions in the generation of Foxp3 + iTreg and found that blockade of CD11b in Cd40 -/- recipients resulted in increased donor-reactive Foxp3 + iTreg as compared with CD40 deficiency alone. Mechanistically, CD154:CD11b inhibition decreased interleukin (IL)-1β from CD11b + and CD11c + dendritic cells, and blockade of IL-1β synergized with CD40 deficiency to promote Foxp3 + iTreg induction and prolong allograft survival. Taken together, these data provide a mechanistic basis for the observed inferiority of anti-CD40 blockers as compared with anti-CD154 mAb and illuminate an IL-1β-dependent mechanism by which CD154:CD11b interactions prevent the generation of donor-reactive Foxp3 + iTreg during transplantation., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Mandy L. Ford reports financial support was provided by National Institutes of Health. Mandy L. Ford reports a relationship with Veloxis Pharmaceuticals Inc that includes: consulting or advisory. I am a deputy editor at the American Journal of Transplantation. I am on the Board of Directors of the American Society of Transplantation. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 American Society of Transplantation & American Society of Transplant Surgeons. Published by Elsevier Inc. All rights reserved.)

Memory B cells (mBCs) are characterized by their long-term stability, fast reactivation, and capability to rapidly differentiate into antibody-secreting cells (ASCs). However, the role of T cells in the differentiation of mBCs, in contrast to naive B cells, remains to be delineated. We study the role of T cells in mBC responses, using CD40L stimulation and autologous T-B co-cultures. Our results showed that increased CD40L levels led to a selective increased proliferation of IgM+ mBC, which did not class-switched, resulting in higher frequencies of IgM+ ASCs and a lower frequency of IgG+ ASCs. The IgG+/IgA+ mBCs were unaffected. We further compared the transcription of immune-related genes in IgM+ and IgG+ pre-plasmablasts cultured at high (500 ng/mL) and low (50 ng/mL) CD40L levels. In response to increased CD40L levels, both populations exhibited a core response to genes related to activation (TRAF1, AKT3, CD69, and CD80). However, they differed in genes related to cytokine/chemokine/homing interactions (CCL3/4/17, LTA, NKX2-3, BCL2 and IL21R) and cell-cell interactions (HLADR, CD40, and ICOSL), which were largely confined to IgG+ cells. Our findings revealed that in co-cultures with a high T-ratio, the response was similar to that found in cultures with high CD40L levels. These results suggest that IgG+ mBCs have a greater capacity for proliferation and T cell interaction, and weaker migration capabilities, leading to a preference for an IgG response over IgM in the short term. This adaptable response could fine-tune the memory repertoire with different functions of IgG versus IgM mBCs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Rincon-Arevalo, Stefanski, Le, Cases, Wiedemann, Szelinski, Ritter, Dang, Lino, Dörner and Schrezenmeier.)

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., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Although Multiple Sclerosis (MS) is primarily thought to be an autoimmune condition, its possible viral etiology must be taken into consideration. When mice are administered neurotropic viruses like mouse hepatitis virus MHV-A59, a murine coronavirus, or its isogenic recombinant strain RSA59, neuroinflammation along with demyelination are observed, which are some of the significant manifestations of MS. MHV-A59/RSA59 induced neuroinflammation is one of the best-studied experimental animal models to understand the viral-induced demyelination concurrent with axonal loss. In this experimental animal model, one of the major immune checkpoint regulators is the CD40-CD40L dyad, which helps in mediating both acute-innate, innate-adaptive, and chronic-adaptive immune responses. Hence, they are essential in reducing acute neuroinflammation and chronic progressive adaptive demyelination. While CD40 is expressed on antigen-presenting cells and endothelial cells, CD40L is expressed primarily on activated T cells and during severe inflammation on NK cells and mast cells. Experimental evidences revealed that genetic deficiency of both these proteins can lead to deleterious effects in an individual. On the other hand, interferon-stimulated genes (ISGs) possess potent antiviral properties and directly or indirectly alter acute neuroinflammation. In this review, we will discuss the role of an ISG, ISG54, and its tetratricopeptide repeat protein Ifit2; the genetic and experimental studies on the role of CD40 and CD40L in a virus-induced neuroinflammatory demyelination model., (© 2023 International Union of Biochemistry and Molecular Biology.)

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., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Weakened germinal center responses by the aged immune system result in diminished immunity against pathogens and reduced efficacy of vaccines. Prolonged contacts between activated B cells and CD4 + T cells are crucial to germinal center formation and T follicular helper cell (Tfh) differentiation, but it is unclear how aging impacts the quality of this interaction. Peptide immunization confirmed that aged mice have decreased expansion of antigen-specific germinal center B cells and reduced antibody titers. Furthermore, aging was associated with accumulated Tfh cells, even in naïve mice. Despite increased numbers, aged Tfh had reduced expression of master transcription factor BCL6 and increased expression of the ectonucleotidase CD39. In vitro activation revealed that proliferative capacity was maintained in aged CD4 + T cells, but not the costimulatory molecule CD40L. When activated in vitro by aged antigen-presenting cells, young CD4 + naïve T cells generated reduced numbers of activated cells with upregulated CD40L. To determine the contribution of cell-extrinsic influences on antigen-specific Tfh induction, young, antigen-specific B and CD4 + T cells were adoptively transferred into aged hosts prior to peptide immunization. Transferred cells had reduced expansion and differentiation into germinal center B cell and Tfh and reduced antigen-specific antibody titers when compared to young hosts. Young CD4 + T cells transferred aged hosts differentiated into Tfh cells with reduced PD-1 and BCL6 expression, and increased CD39 expression, though they maintained their mitochondrial capacity. These results highlight the role of the lymphoid microenvironment in modulating CD4 + T cell differentiation, which contributes to impaired establishment and maintenance of germinal centers., (© 2024 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

Sjögren's disease (SjD) is a chronic, systemic autoimmune disease with no approved disease-modifying therapies. Dazodalibep (DAZ), a novel nonantibody fusion protein, is a CD40 ligand antagonist that blocks costimulatory signals between T and B cells and antigen-presenting cells, and therefore may suppress the wide spectrum of cellular and humoral responses that drive autoimmunity in SjD. This study was a phase 2, randomized, double-blinded, placebo (PBO)-controlled trial of DAZ with a crossover stage in two distinct populations of participants with SjD. Population 1 had moderate-to-severe systemic disease activity and population 2 had an unacceptable symptom burden and limited systemic organ involvement. All participants had a diagnosis of SjD, with 21.6% and 10.1% having an associated connective tissue disease (rheumatoid arthritis or systemic lupus erythematosus) in populations 1 and 2, respectively. The remaining participants would be considered as having primary Sjögren's syndrome. The primary endpoint for population 1 (n = 74) was the change from baseline in the European League Against Rheumatism Sjögren's Syndrome Disease Activity Index at day 169. The primary endpoint for population 2 (n = 109) was the change from baseline in the European League Against Rheumatism Sjögren's Syndrome Patient Reported Index at day 169. The primary endpoints (least squares mean ± standard error) were achieved with statistical significance for both population 1 (DAZ, -6.3 ± 0.6; PBO, -4.1 ± 0.6; P = 0.0167) and population 2 (DAZ, -1.8 ± 0.2; PBO, -0.5 ± 0.2; P = 0.0002). DAZ was generally safe and well tolerated. Among the most frequently reported adverse events were COVID-19, diarrhea, headache, nasopharyngitis, upper respiratory tract infection, arthralgia, constipation and urinary tract infection. In summary, DAZ appears to be a potential new therapy for SjD and its efficacy implies an important role for the CD40/CD40 ligand pathway in its pathogenesis. ClinicalTrials.gov identifier: NCT04129164 ., (© 2024. The Author(s).)

Soluble components secreted by Tfh cells are critical for the germinal center responses. In this study, we investigated whether Tfh cells could regulate the B-cell response by releasing small extracellular vesicles (sEVs). Our results showed that Tfh cells promote B-cell differentiation and antibody production through sEVs and that CD40L plays a crucial role in Tfh-sEVs function. In addition, increased Tfh-sEVs were found in mice with collagen-induced arthritis (CIA). Adoptive transfer of Tfh cells significantly exacerbated the severity of CIA; however, the effect of Tfh cells on exacerbating the CIA process was significantly diminished after inhibiting sEVs secretion. Moreover, the levels of plasma Tfh-like-sEVs and CD40L expression on Tfh-like-sEVs in RA patients were significantly higher than those in healthy subjects. In summary, Tfh cell-derived sEVs can enhance the B-cell response, and exacerbate the procession of autoimmune arthritis., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental contaminant and high affinity ligand for the aryl hydrocarbon receptor (AhR). In animal models, AhR activation by TCDD generally inhibits antibody secretion. However, it is less clear if this translates to human antibody production. Using a human Burkitt lymphoma B-cell line (CL-01) that can be stimulated to secrete Ig and undergo class switch recombination to other Ig isotypes, the current study evaluated the effects of AhR activation or antagonism on the human Ig isotypic expression profile with CD40L+IL-4 stimulation. Our results suggest that AhR agonists (TCDD and indirubin) have little to no effect on IgM or IgA secretion, which were also not induced with stimulation. However, AhR activation significantly inhibited stimulation-induced IgG secretion, an effect reversed by the AhR antagonist CH223191. Evaluation of Ig heavy chain (IgH) constant region gene expression (ie Cμ, Cγ1-4, Cα1-2, and Cε that encode for IgM, IgG1-4, IgA1-2, and IgE, respectively) demonstrated differential effects. While Cμ and Cα2 transcripts were unaffected by stimulation or AhR agonists, AhR activation significantly inhibited stimulation-induced Cγ2-4 and Cε mRNA transcripts, which was reversed by AhR antagonism. Notably, AhR antagonism in the absence of exogenous AhR ligands significantly increased IgG and IgA secretion as well as the expression of Cγ2-4 and Cε. These results suggest that modulation of AhR activity differentially alters the IgH isotypic expression profile and antibody secretion that may be partly dependent on cellular stimulation. Since a variety of chemicals from anthropogenic, industrial, pharmaceutical, dietary, and bacterial sources bind the AhR, the ability of environmental exposures to alter AhR activity (i.e. activate or inhibit) may have a direct influence on immune function and antibody-relevant disease conditions., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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 (B RM ) 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 B RM 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 B RM 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 B RM cells, which subsequently protect the airways and parenchyma against future potential infections., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Etesami, Barker, Shenoy, De Ana, Arafa, Grifno, Matschulat, Vannini, Pihl, Breen, Soucy, Goltry, Ha, Betsuyaku, Browning, Varelas, Traber, Jones, Quinton, Maglione, Nia, Belkina and Mizgerd.)

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 H 2 O 2 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.

The oral mucosa is constantly exposed to a plethora of stimuli including food antigens, commensal microbiota and pathogens, requiring distinct immune responses. We previously reported that human oral epithelial cells (OECs) suppress immune responses to bacteria, using H413 and TR146 OEC lines and primary OECs in co-culture with dendritic cells (DCs) and T cells (OEC-conditioned cells). OECs reduced DCs expression of CD80/CD86 and IL-12/TNFα release and impaired T cell activation. Here, we further evaluated the immunosuppression by these OECs and investigated the underlying mechanisms. OEC-conditioned DCs did not induce CD4 T cell polarization towards Treg, judging by the absence of FoxP3 expression. OECs also repressed T-bet/IFNγ expression in CD4 and CD8 T cells activated by DCs or anti-CD3/CD28 antibodies. This inhibition depended on OEC:T cell ratio and IFNγ repression occurred at the transcriptional level. Time-lapse experiments showed that OECs inhibited early steps of T cell activation, consistent with OECs inability to suppress T cells stimulated with PMA/ionomycin. Blocking CD40/CD40L, CD58/CD2 and PD-L1/PD-1 interactions with specific antibodies did not disrupt T cell suppression by OECs. However, preventing prostaglandin E2 (PGE2) synthesis or blocking PGE2 binding to the cognate EP2/EP4 receptors, restored IFNγ and TNFα production in OEC-conditioned T cells. Finally, treating OECs with poly(I:C), which simulates viral infections, limited T cell suppression. Overall, these results point to an inherent ability of OECs to suppress immune responses, which can nonetheless be eluded when OECs are under direct assault., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Sanchez-Trincado, Pelaez-Prestel, Lafuente and Reche.)

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., (Copyright © 2021. Published by Elsevier B.V.)

CD40 is a potent activating receptor expressed on antigen-presenting cells (APCs) of the immune system. CD40 regulates many aspects of B and T cell immunity via interaction with CD40L expressed on activated T cells. Targeting antigens to CD40 via agonistic anti-CD40 antibody fusions promotes both humoral and cellular immunity, but current anti-CD40 antibody-antigen vaccine prototypes require co-adjuvant administration for significant  in vivo  efficacy. This may be a consequence of dulling of anti-CD40 agonist activity via antigen fusion. We previously demonstrated that direct fusion of CD40L to anti-CD40 antibodies confers superagonist properties. Here we show that anti-CD40-CD40L-antigen fusion constructs retain strong agonist activity, particularly for activation of dendritic cells (DCs). Therefore, we tested anti-CD40-CD40L antibody fused to antigens for eliciting immune responses  in vitro  and  in vivo . In PBMC cultures from HIV-1-infected donors, anti-CD40-CD40L fused to HIV-1 antigens preferentially expanded HIV-1-specific CD8 +  T cells versus CD4 +  T cells compared to analogous anti-CD40-antigen constructs. In normal donors, anti-CD40-CD40L-mediated delivery of Influenza M1 protein elicited M1-specific T cell expansion at lower doses compared to anti-CD40-mediated delivery. Also, on human myeloid-derived dendritic cells, anti-CD40-CD40L-melanoma gp100 peptide induced more sustained Class I antigen presentation compared to anti-CD40-gp100 peptide. In human CD40 transgenic mice, anti-CD40-CD40L-HIV-1 gp140 administered without adjuvant elicited superior antibody responses compared to anti-CD40-gp140 antigen without fused CD40L. In human CD40 mice, compared to the anti-CD40 vehicle, anti-CD40-CD40L delivery of Eα 52-68 peptide elicited proliferating of TCR I-Eα 52-68 CD4 +  T cells producing cytokine IFNγ. Also, compared to controls, only anti-CD40-CD40L-Cyclin D1 vaccination of human CD40 mice reduced implanted EO771.LMB breast tumor cell growth. These data demonstrate that human CD40-CD40L antibody fused to antigens maintains highly agonistic activity and generates immune responses distinct from existing low agonist anti-CD40 targeting formats. These advantages were  in vitro  skewing responses towards CD8 +  T cells, increased efficacy at low doses, and longevity of MHC Class I peptide display; and in mouse models, a more robust humoral response, more activated CD4 +  T cells, and control of tumor growth. Thus, the anti-CD40-CD40L format offers an alternate DC-targeting platform with unique properties, including intrinsic adjuvant activity., Competing Interests: The authors VC, SZ, YL, and GZ are inventors on patent application WO2020193718. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ceglia, Zurawski, Montes, Kroll, Bouteau, Wang, Ellis, Igyártó, Lévy and Zurawski.)

NFAT activating protein with ITAM motif 1 (NFAM1) is an ITAM bearing-transmembrane receptor that has been reported to play a role in B cell signaling and development. We performed expression analysis of NFAM1 using publicly available gene expression data sets and found that NFAM1 expression is significantly induced in intestinal biopsies from Crohn's disease (CD) and ulcerative colitis (UC) patients. At the cellular level, we further observed high expression of NFAM1 in monocytes and neutrophils, and low expression in B and T cells. To explore the role of NFAM1 in multiple immune cells and its potential role in IBD, we generated NFAM1 -/- mice. In contrast with previous reports using NFAM1-transgenic mice, NFAM1 -/- mice have no obvious defects in immune cell development, or B cell responses. Interestingly, NFAM1 -/- monocytes produce reduced levels of TNF-α in response to activation by multiple IBD-relevant stimuli, including CD40L, TLR ligands and MDP. Additional cytokines and chemokines such as IL-6, IL-12, CCL3 and CCL4 are also reduced in CD40L stimulated NFAM1 -/- monocytes. Collectively, these findings indicate that NFAM1 promotes monocyte activation, thereby amplifying the response to diverse stimuli. Similarly, we observed that deletion of NFAM1 in human monocytes reduces expression of CD40L-induced CCL4. Lastly, to assess the role of NFAM1 in IBD, we compared development of anti-CD40 induced colitis in NFAM1 +/+ and NFAM1 -/- mice. We found that although NFAM1 deletion had no impact on development of gut pathology, we did observe a decrease in serum TNF-α, confirming that NFAM1 promotes pro-inflammatory cytokine production in vivo . Taken together, we conclude that NFAM1 functions to amplify cytokine production and should be further evaluated as a therapeutic target for treatment of autoimmune disease., Competing Interests: SC served as Principal Investigator of the contract. KWJ, JPG, ES, ACM, SEF, DS, SSW, ERAG, IY, JR, LL, JZ, MLM and JL were employees of Boehringer Ingelheim. The authors disclose that financial support for this project was provided by Boehringer Ingelheim via a research contract to Sanford Burnham Prebys Medical Discovery Institute. Boehringer Ingelheim had the following involvement with the study: experimental design, data acquisition, analysis and writing the paper., (Copyright © 2022 Juchem, Gounder, Gao, Seccareccia, Yeddula, Huffmaster, Côté-Martin, Fogal, Souza, Wang, Glynn, Yung, Ritchie, Li, Zheng, Mbow, Li and Chanda.)

SARS-CoV-2 infections present a tremendous threat to public health. Safe and efficacious vaccines are the most effective means in preventing the infections. A variety of vaccines have demonstrated excellent efficacy and safety around the globe. Yet, development of alternative forms of vaccines remains beneficial, particularly those with simpler production processes, less stringent storage conditions, and the capability of being used in heterologous prime/boost regimens which have shown improved efficacy against many diseases. Here we reported a novel DNA vaccine comprised of the SARS-CoV-2 spike protein fused with CD40 ligand (CD40L) serving as both a targeting ligand and molecular adjuvant. A single intramuscular injection in Syrian hamsters induced significant neutralizing antibodies 3-weeks after vaccination, with a boost substantially improving immune responses. Moreover, the vaccine also reduced weight loss and suppressed viral replication in the lungs and nasal turbinates of challenged animals. Finally, the incorporation of CD40L into the DNA vaccine was shown to reduce lung pathology more effectively than the DNA vaccine devoid of CD40L. These results collectively indicate that this DNA vaccine candidate could be further explored because of its efficacy and known safety profile., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Tamming, Duque, Tran, Zhang, Pfeifle, Laryea, Wu, Raman, Gravel, Russell, Hashem, Alsulaiman, Alhabbab, Gao, Safronetz, Cao, Wang, Chen, Johnston, Sauve, Rosu-Myles and Li.)

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., Competing Interests: The authors have declared that no competing interests exist.

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., Competing Interests: EN declares financial relationship due to patent WO2017/162797. The remaining author(s) declare(s) that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Olguín-Contreras, Mendler, Popowicz, Hu and Noessner.)

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 EC 50 , 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.

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., (© 2021. The Author(s).)

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., (Copyright © 2021 by The American Association of Immunologists, Inc.)

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., (Copyright © 2021 by The American Association of Immunologists, Inc.)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Antigen-specific lymphocytes are increasingly investigated in autoimmune diseases and immune therapies. We sought to identify thyrotropin receptor (TSHR)-specific lymphocytes in mouse models of Graves' disease, including Graves' patient-specific immunotype human leukocyte antigen (HLA)-DR3, and in frozen and thawed Graves' patient blood samples. Methods and Results: Splenic lymphocytes of adenovirus (Ad)-TSHR-immunized BALB/c mice were stimulated with TSHR-specific peptides C, D, or J. Furthermore, CD154-expressing cells were enriched, expanded in vitro , and analyzed for binding of peptide-major histocompatibility complex (MHC) II multimers ("tetramers," immunotype H2-IA d ). Only peptides C and J were able to elicit increased expression/secretion of CD154 and interferon-γ, and tetramers which were loaded with peptide C resulted in antigen-specific signals in splenic lymphocytes from Ad-TSHR-immunized mice. Accordingly, TSHR-specific HLA-DR3-MHC class II tetramers loaded with peptide p10 specifically bound to human HLA-DR3-(allele B1*03:01)-transgenic Bl/6 mouse splenic T lymphocytes. In addition, we fine-tuned a protocol to reliably measure thawed human peripheral blood mononuclear cells (PBMCs), which resulted in reliable recovery after freezing and thawing with regard to vitality and B and T cell subpopulation markers including regulatory T cells (CD3, CD4, CD25, FoxP3, CD25 high , CD127 low ). TSHR-specific HLA-DR3-MHC class II tetramers loaded with peptide p10 identified antigen-specific T cells in HLA-DR3-positive Graves' patients' thawed PBMCs. Moreover, stimulation-dependent release of interleukin (IL)-1beta, IL-6, tumor necrosis factor-alpha from thawed PBMCs occurred at the expected levels. Conclusions: Novel MHC II tetramers identified TSHR-specific T lymphocytes in Ad-TSHR-immunized hyperthyroid BALB/c or HLA-DR3-transgenic mice and in thawed human PBMCs from patients with Graves' disease. These assays may contribute to measure both disease severity and effects of novel immune therapies in future animal studies and clinical investigations of Graves' disease.

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., (Copyright © 2021 by The American Association of Immunologists, Inc.)

CD154, a member of the TNF superfamily, is a multifunctional molecule highly expressed in activated T cells, and plays important roles in T cell-dependent humoral immune response. In this study, CD154 of Nile tilapia (Oreochromis niloticus) was identified, and its functions in the T cell-dependent immune response were demonstrated. The open reading frame (ORF) of OnCD154 is 699 bp, encoding a protein of 232 amino acids with a 23 amino acid transmembrane region. Amino acid sequence of OnCD154 is highly homologous to that of other teleost fish, especially rainbow trout. Quantitative real-time PCR (qRT-PCR) demonstrated that mRNA of OnCD154 is highly expressed in immune organs, especially in spleen, thymus, gills, head kidney, etc. In addition, the anti-OnCD154 polyclonal antibody (anti-(r)OnCD154) was successfully prepared, and it can react with natural protein in head kidney leukocytes. Following two immunizations with keyhole limpet hemocyanin (KLH) in vivo, the significantly up-regulated expression level of OnCD154 mRNA appeared earlier (fifth day) and higher (42.9 folds) in the second challenge than the first on in head kidney. Further, after stimulation with KLH in vitro, the expressions of T cell-dependent immune response-related molecules (activated T cell specific surface molecules CD3ε and CD154) and B cell differentiation-related molecules (Blimp1 and sIgM) and CD40 were significantly up-regulated in head kidney leukocytes. Moreover, the up-regulated expressions of these molecules were blocked with the treatment of anti-(r)OnCD154 antibody. Taken together, these results indicate that OnCD154 might get involved in T cell-dependent immune response, and provide a new insight into the humoral immune response of teleost fish., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

The therapeutic applications of regulatory T cells (T regs ) include treating autoimmune diseases, graft-versus-host disease and induction of transplantation tolerance. For ex-vivo expanded T regs to be used in deceased donor transplantation, they must be able to suppress T cell responses to a broad range of human leukocyte antigen (HLA). Here, we present a novel approach for the expansion of polyspecific T regs in cynomolgus macaques that was adapted from a good manufacturing practice-compliant protocol. T regs were isolated by fluorescence-activated cell sorting (FACS) and expanded in the presence of a panel of CD40L-stimulated B cells (CD40L-sBc). Prior to T reg culture, CD40L-sBc were expanded in vitro from multiple major histocompatibility complex (MHC)-disparate macaques. Expanded T regs expressed high levels of forkhead box protein 3 (FoxP3) and Helios, a high percentage of T reg -specific demethylated region (TSDR) demethylation and strong suppression of naïve T cell responses in vitro. In addition, these T regs produced low levels of inflammatory cytokines and were able to expand post-cryopreservation. Specificity assays confirmed that these T regs were suppressive upon activation by any antigen-presenting cells (APCs) whose MHC was shared by CD40L-sBc used during expansion, proving that they are polyspecific. We developed an approach for the expansion of highly suppressive cynomolgus macaque polyspecific T regs through the use of a combination of CD40L-engineered B cells with the potential to be translated to clinical studies. To our knowledge, this is the first report that uses a pool of MHC-mismatched CD40L-sBc to create polyspecific T regs suitable for use in deceased-donor transplants., (© 2020 British Society for Immunology.)

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., Competing Interests: The authors declare that no competing interests exist. The authors, TS and SK, are affiliated with Wakunaga Pharmaceutical Co., Ltd. (Hiroshima, Japan) and were responsible for measurement of anti-HLA antibodies using WAKFlow developed by this company. This was a scientific collaboration, and there is no commercial relationship between our laboratory in Hiroshima University and Wakunaga Pharmaceutical Co., Ltd. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.

Decreasing graft rejection and increasing graft and patient survival are great challenges facing liver transplantation (LT). Different T cell subsets participate in the acute cellular rejection (ACR) of the allograft. Cell-mediated immunity markers of the recipient could help to understand the mechanisms underlying acute rejection. This study aimed to analyse different surface antigens on T cells in a cohort of adult liver patients undergoing LT to determine the influence on ACR using multi-parametric flow cytometry functional assay. Thirty patients were monitored at baseline and during 1 year post-transplant. Two groups were established, with (ACR) and without (NACR) acute cellular rejection. Leukocyte, total lymphocyte, percentages of CD4 + CD154 + and CD8 + CD154 + T cells, human leukocyte antigen (HLA) mismatch between recipient-donor and their relation with ACR as well as the acute rejection frequencies were analysed. T cells were stimulated with concanavalin A (Con-A) and surface antigens were analysed by fluorescence activated cell sorter (FACS) analysis. A high percentage of CD4 + CD154 + T cells (P = 0·001) and a low percentage of CD8 + CD154 + T cells (P = 0·002) at baseline were statistically significant in ACR. A receiver operating characteristic analysis determined the cut-off values capable to stratify patients at high risk of ACR with high sensitivity and specificity for CD4 + CD154 + (P = 0·001) and CD8 + CD154 + T cells (P = 0·002). In logistic regression analysis, CD4 + CD154 + , CD8 + CD154 + and HLA mismatch were confirmed as independent risk factors to ACR. Post-transplant percentages of both T cell subsets were significantly higher in ACR, despite variations compared to pretransplant. These findings support the selection of candidates for LT based on the pretransplant percentages of CD4 + CD154 + and CD8 + CD154 + T cells in parallel with other transplant factors., (© 2020 British Society for Immunology.)

Chronic inflammation drives the development of atherosclerosis. Despite optimal treatment of classical cardiovascular risk factors, a substantial portion of the population has elevated inflammatory biomarkers and develops atherosclerosis-related complications, indicating that a residual inflammatory risk drives atherosclerotic cardiovascular disease in these patients. Additional anti-inflammatory therapeutic strategies are therefore required. The co-stimulatory molecule CD40 and its ligand CD40L (CD154) have a central role in the regulation of the inflammatory response during the development of atherosclerosis by modulating the interaction between immune cells and between immune cells and non-immune cells. In this review, we discuss the role of the CD40-CD40L dyad in atherosclerosis, and we discuss recent studies on the therapeutic potential of novel CD40-CD40L targeting strategies in cardiovascular medicine.

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.

Calcitriol and 9-cis retinoic acid (9cRA) play a fundamental role in shaping the adaptive immune response by altering the Ig profile and the differentiation of B cells, controlled by their corresponding nuclear receptors, VDR and RAR. Herein, after the establishment of a plasmablast differentiation culture, we investigated how both ligands modulate human naïve B cell differentiation and to which extent VDR/RXR and RAR/RXR signaling interferes. Calcitriol and 9cRA mediated activation of purified naïve B cells resulted in a strong differentiation of CD27 + CD38 + plasmablasts and antibody secretion. The significant IgA response was preceded by a strong induction of α-germline transcription (GLT). Induction of αGLT and consecutively IgA secretion driven by calcitriol is a novel observation and we show by magnetic chromatin IP that this was mediated by recruitment of the VDR to the TGF-β promoter thus inducing TGF-β expression. Finally, as revealed by transcriptomic profiling calcitriol and 9cRA modulate several signals required for differentiation and isotype switching in a noncompeting but rather additive manner. Calcitriol and 9cRA participate in the control of the IgA response in human activated naïve B cells. The balance between both ligands may be an important factor in channeling humoral immune responses toward a protective direction., (© 2020 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

CD40L is a member of the TNF superfamily that participates in immune cell activation. It binds to and signals through several integrins, including αvβ3 and α5β1, 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 α4β1, which is prevalently expressed on the same CD4+ T cells as CD40L, is another receptor for CD40L. Soluble (s)CD40L activated soluble integrins αvβ3, α5β1, and α4β1 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., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

While effective in specific settings, adoptive chimeric antigen receptor (CAR) T cell therapy for cancer requires further improvement and optimization. Our previous results show that CD40L-overexpressing CAR T cells mobilize endogenous immune effectors, resulting in improved antitumor immunity. However, the cell populations required for this protective effect remain to be identified. Here we show, by analyzing Batf3 -/- mice lacking the CD103 + conventional dendritic cell type 1 (cDC1) subpopulation important for antigen cross-presentation, that CD40L-overexpressing CAR T cells elicit an impaired antitumor response in the absence of cDC1s. We further find that CD40L-overexpressing CAR T cells stimulate tumor-resident CD11b - CD103 - double-negative (DN) cDCs to proliferate and differentiate into cDC1s in wild-type mice. Finally, re-challenge experiments show that endogenous CD8 + T cells are required for protective antitumor memory in this setting. Our findings thus demonstrate the stimulatory effect of CD40L-overexpressing CAR T cells on innate and adaptive immune cells, and provide a rationale for using CD40L-overexpressing CAR T cells to improve immunotherapy responses.

Background: Autologous monocyte-derived mRNA co-electroporated dendritic cells with mRNA encoding CD40 ligand (CD40L), CD70 and a constitutively activated TLR4 (caTLR4) (referred to as TriMixDC-MEL) have anti-tumor activity in advanced melanoma patients. We investigated the safety and activity of adjuvant TriMixDC-MEL in stage III/IV melanoma patients., Materials and Methods: Forty-one patients were randomly assigned to treatment with TriMixDC-MEL (n = 21) and standard follow-up (n = 20). "Cross-over" was allowed at the time of non-salvageable recurrence. The primary endpoint was the percentage of patients alive and disease-free at 1-year. For a subset of patients, (formalin-fixed paraffin-embedded), tumor tissue samples were available for mRNA expression profiling and PD-L1 immunohistochemical staining., Results: Baseline characteristics were well balanced. One-year after randomization, 71% of patients in the study arm were alive and free of disease compared to 35% in the control arm. After a median follow-up of 53 months (range 3-67), 23 patients experienced a non-salvageable melanoma recurrence (TriMixDC-Mel arm n = 9 and control arm n = 14).The median time to non-salvageable recurrence was superior in the TriMixDC-MEL arm (median 8 months (range 1-6) vs. not reached; log-rank p 0.044). TriMixDC-MEL-related adverse events (AE) consisted of transient local skin reactions, flu-like symptoms and post-infusion chills. No grade ≥ 3 AE's occurred. The mRNA expression profiling revealed four genes (STAT2, TPSAB1, CD9 and CSF2) as potential predictive biomarkers., Conclusion: TriMixDC-MEL id/iv as adjuvant therapy is tolerable and may improve the 1-year disease-free survival rate. Combination of optimized autologous monocyte-derived DC-formulations warrants further investigation in combination with currently approved adjuvant therapy options.

Lupus nephritis (LN) is a significant complication of systemic lupus erythematosus (SLE), increasing its morbidity and mortality. Although the current standard of care helps suppress disease activity, it is associated with toxicity and ultimately does not cure SLE. At present, there are no therapies specifically indicated for the treatment of LN and there is an unmet need in this disease where treatment remains a challenge. The CD40-CD40L pathway is central to SLE pathogenesis and the generation of autoantibodies and their deposition in the kidneys, resulting in renal injury in patients with LN. CD40 is expressed on immune cells (including B cells, monocytes and dendritic cells) and also non-haematopoietic cells. Interactions between CD40L on T cells and CD40 on B cells in the renal interstitium are critical for the local expansion of naive B cells and autoantibody-producing B cells in LN. CD40L-mediated activation of myeloid cells and resident kidney cells, including endothelial cells, proximal tubular epithelial cells, podocytes and mesangial cells, further amplifies the inflammatory milieu in the interstitium and the glomeruli. Several studies have highlighted the upregulated expression of CD40 in LN kidney biopsies, and preclinical data have demonstrated the importance of the CD40-CD40L pathway in murine SLE and LN. Blocking this pathway is expected to ameliorate inflammation driven by infiltrating immune cells and resident kidney cells. Initial experimental therapeutic interventions targeting the CD40-CD40L pathway, based on CD40L antibodies, were associated with an increased incidence of thrombosis. However, this safety issue has not been observed with second-generation CD40/CD40L antibodies that have been engineered to prevent platelet activation. With these advancements, together with recent preclinical and clinical findings, it is anticipated that selective blockade of the CD40-CD40L pathway may address the unmet treatment needs in SLE, LN and other autoimmune diseases., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Granzyme B-expressing B cells have been shown to be an important regulatory B cell subset in humans. However, it is unclear which subpopulations of B cells express GZMB under normal conditions and which protocols effectively induce ex vivo expansion of GZMB + B cells. We found that in the peripheral blood of normal individuals, plasmablasts were the major B cell subpopulation that expressed GZMB. However, when using an in vitro plasmablast differentiation protocol, we obtained only 2% GZMB + B cells. Nevertheless, using an expansion mixture containing IL-21, anti-BCR, CpG oligodeoxynucleotide, CD40L, and IL-2, we were able to obtain more than 90% GZMB + B cells after 3 d culture. GZMB + B cells obtained through this protocol suppressed the proliferation of autologous and allogenic CD4 + CD25 - effector T cells. The suppressive effect of GZMB + B cells was partially GZMB dependent and totally contact dependent but was not associated with an increase in effector T cell apoptosis or uptake of GZMB by effector T cells. Interestingly, we showed that GZMB produced by B cells promoted GZMB + B cell proliferation in ERK1/2-dependent manner, facilitating GZMB + B cell expansion. However, GZMB + B cells tended to undergo apoptosis after prolonged stimulation, which may be considered a negative feedback mechanism to limit their uncontrolled expansion. Finally, we found that expanded GZMB + B cells exhibited a regulatory phenotype and were enriched in CD307b hi , CD258 hi CD72 hi , and CD21loPD-1 hi B cell subpopulations. Our study, to our knowledge, provides new insight into biology of GZMB + B cells and an efficient method to expand GZMB + B cells for future cell therapy applications., (Copyright © 2020 by The American Association of Immunologists, Inc.)

CD11c + T-bet + B cells generated during ehrlichial infection require CD4 + T cell help and IL-21 signaling for their development, but the exact T cell subset required had not been known. In this study, we show in a mouse model of Ehrlichia muris that type 1 T follicular helper (T FH1 ) cells provide help to CD11c + T-bet + B cells via the dual secretion of IL-21 and IFN-γ in a CD40/CD40L-dependent manner. T FH1 cell help was delivered in two phases: IFN-γ signals were provided early in infection, whereas CD40/CD40L help was provided late in infection. In contrast to T-bet + T cells, T-bet + B cells did not develop in the absence of B cell-intrinsic Bcl-6 but were generated in the absence of T-bet. T-bet-deficient memory B cells were largely indistinguishable from their wild-type counterparts, although they no longer underwent switching to IgG2c. These data suggest that a primary function of T-bet in B cells during ehrlichial infection is to promote appropriate class switching, not lineage specification. Thus, CD11c + memory B cells develop normally without T-bet but require Bcl-6 and specialized help from dual cytokine-producing T FH1 cells., (Copyright © 2020 by The American Association of Immunologists, Inc.)

CD4 + T lymphocytes consist of naïve, antigen-specific memory, and memory-phenotype (MP) cell compartments at homeostasis. We recently showed that MP cells exert innate-like effector function during host defense, but whether MP CD4 + T cells are functionally heterogeneous and, if so, what signals specify the differentiation of MP cell subpopulations under homeostatic conditions is still unclear. Here we characterize MP lymphocytes as consisting of T-bet high , T-bet low , and T-bet - subsets, with innate, Th1-like effector activity exclusively associated with T-bet high cells. We further show that the latter population depends on IL-12 produced by CD8α + type 1 dendritic cells (DC1) for its differentiation. Finally, our data demonstrate that this tonic IL-12 production requires TLR-MyD88 signaling independent of foreign agonists, and is further enhanced by CD40-CD40L interactions between DC1 and CD4 + T lymphocytes. We propose that optimal differentiation of T-bet high MP lymphocytes at homeostasis is driven by self-recognition signals at both the DC and Tcell levels.

Background: Tolerogenic vaccines represent antigen-specific interventions designed to re-establish self-tolerance and thereby alleviate autoimmune diseases, which collectively comprise over 100 chronic inflammatory diseases afflicting more than 20 million Americans. Tolerogenic vaccines comprised of single-chain GM-CSF-neuroantigen (GMCSF-NAg) fusion proteins were shown in previous studies to prevent and reverse disease in multiple rodent models of experimental autoimmune encephalomyelitis (EAE) by a mechanism contingent upon the function of CD4 + CD25 + FOXP3 + regulatory T cells (Tregs). GMCSF-NAg vaccines inhibited EAE in both quiescent and inflammatory environments in association with low-efficiency T cell receptor (TCR) signaling events that elicited clonal expansion of immunosuppressive Tregs., Methods: This study focused on two vaccines, including GMCSF-MOG (myelin oligodendrocyte glycoprotein 35-55/MOG 35-55 ) and GMCSF-NFM (neurofilament medium peptide 13-37/NFM 13-37 ), that engaged the transgenic 2D2 TCR with either low or high efficiencies, respectively. 2D2 mice were crossed with FOXP3 IRES eGFP (FIG) mice to track Tregs and further crossed with Rag -/- mice to reduce pre-existing Treg populations., Results: This study provided evidence that low and high efficiency TCR interactions were integrated via CD40L expression levels to control the Treg/Tcon balance. The high-efficiency GMCSF-NFM vaccine elicited memory Tcon responses in association with activation of the CD40L costimulatory system. Conversely, the low-efficiency GMCSF-MOG vaccine lacked adequate TCR signal strength to elicit CD40L expression and instead elicited Tregs by a mechanism that was impaired by a CD40 agonist. When combined, the low- and high-efficiency GMCSF-NAg vaccines resulted in a balanced outcome and elicited both Tregs and Tcon responses without the predominance of a dominant immunogenic Tcon response. Aside from Treg expansion in 2D2-FIG mice, GMCSF-MOG caused a sustained decrease in TCR-β, CD3, and CD62L expression and a sustained increase in CD44 expression in Tcon subsets. Subcutaneous administration of GMCSF-MOG without adjuvants inhibited EAE in wildtype mice, which had a replete Treg repertoire, but was pathogenic rather than tolerogenic in 2D2-FIG-Rag1 -/- mice, which lacked pre-existing Tregs., Conclusions: This study provided evidence that the GMCSF-MOG vaccine elicited antigenic responses beneath the CD40L triggering threshold, which defined an antigenic niche that drove dominant expansion of tolerogenic myelin-specific Tregs that inhibited EAE.

Vascularized composite allotransplantation (VCA) allows tissue replacement after devastating loss but is currently limited in application and may be more widely performed if maintenance immunosuppression was not essential for graft acceptance. We tested whether peri-transplant costimulation blockade could prolong VCA survival and required donor bone-marrow cells, given that bone-marrow might promote graft immunogenicity or graft-versus-host disease. Peritransplant CD154 mAb/rapamycin (RPM) induced long-term orthotopic hindlimb VCA survival (BALB/c->C57BL/6), as did CTLA4Ig/RPM. Surprisingly, success of either protocol required a bone-marrow-associated, radiation-sensitive cell population, since long-bone removal or pre-transplant donor irradiation prevented long-term engraftment. Rejection also occurred if Rag1-/- donors were used, or if donors were treated with a CXCR4 inhibitor to mobilize donor BM cells pre-transplant. Donor bone-marrow contained a large population of Foxp3+ T-regulatory (Treg) cells, and donor Foxp3+ Treg depletion, by diphtheria toxin administration to DEREG donor mice whose Foxp3+ Treg cells expressed diphtheria toxin receptor, restored rejection with either protocol. Rejection also occurred if CXCR4 was deleted from donor Tregs pre-transplant. Hence, long-term VCA survival is possible across a full MHC disparity using peritransplant costimulation blockade-based approaches, but unexpectedly, the efficacy of costimulation blockade requires the presence of a radiation-sensitive, CXCR4+ Foxp3+ Treg population resident within donor BM.