Gsk3 is a metabolic checkpoint regulator in B cells.

B cells predominate in a quiescent state until an antigen is encountered, which results in rapid growth, proliferation and differentiation of the B cells. These distinct cell states are probably accompanied by differing metabolic needs, yet little is known about the metabolic control of B cell fate. Here we show that glycogen synthase kinase 3 (Gsk3) is a metabolic sensor that promotes the survival of naive recirculating B cells by restricting cell mass accumulation. In antigen-driven responses, Gsk3 was selectively required for regulation of B cell size, mitochondrial biogenesis, glycolysis and production of reactive oxygen species (ROS), in a manner mediated by the co-stimulatory receptor CD40. Gsk3 was required to prevent metabolic collapse and ROS-induced apoptosis after glucose became limiting, functioning in part by repressing growth dependent on the myelocytomatosis oncoprotein c-Myc. Notably, we found that Gsk3 was required for the generation and maintenance of germinal center B cells, which require high glycolytic activity to support growth and proliferation in a hypoxic microenvironment.

Plasmacytoid Dendritic Cells and Type I Interferon Promote Extrafollicular B Cell Responses to Extracellular Self-DNA.

Class-switched antibodies to double-stranded DNA (dsDNA) are prevalent and pathogenic in systemic lupus erythematosus (SLE), yet mechanisms of their development remain poorly understood. Humans and mice lacking secreted DNase DNASE1L3 develop rapid anti-dsDNA antibody responses and SLE-like disease. We report that anti-DNA responses in Dnase1l3-/- mice require CD40L-mediated T cell help, but proceed independently of germinal center formation via short-lived antibody-forming cells (AFCs) localized to extrafollicular regions. Type I interferon (IFN-I) signaling and IFN-I-producing plasmacytoid dendritic cells (pDCs) facilitate the differentiation of DNA-reactive AFCs in vivo and in vitro and are required for downstream manifestations of autoimmunity. Moreover, the endosomal DNA sensor TLR9 promotes anti-dsDNA responses and SLE-like disease in Dnase1l3-/- mice redundantly with another nucleic acid-sensing receptor, TLR7. These results establish extrafollicular B cell differentiation into short-lived AFCs as a key mechanism of anti-DNA autoreactivity and reveal a major contribution of pDCs, endosomal Toll-like receptors (TLRs), and IFN-I to this pathway.

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.).

Alloreactive memory CD4 T cells promote transplant rejection by engaging DCs to induce innate inflammation and CD8 T cell priming.

Alloreactive memory T cells have been implicated as central drivers of transplant rejection. Perplexingly, innate cytokines, such as IL-6, IL-1ß, 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.

Mechanics reveals the role of peristome geometry in prey capture in carnivorous pitcher plants (Nepenthes).

Carnivorous pitcher plants (Nepenthes) are a striking example of a natural pitfall trap. The trap's slippery rim, or peristome, plays a critical role in insect capture via an aquaplaning mechanism that is well documented. While the peristome has received significant research attention, the conspicuous variation in peristome geometry across the genus remains unexplored. We examined the mechanics of prey capture using Nepenthes pitcher plants with divergent peristome geometries. Inspired by living material, we developed a mathematical model that links the peristomes' three-dimensional geometries to the physics of prey capture under the laws of Newtonian mechanics. Linking form and function enables us to test hypotheses related to the function of features such as shape and ornamentation, orientation in a gravitational field, and the presence of "teeth," while analysis of the energetic costs and gains of a given geometry provides a means of inferring potential evolutionary pathways. In a separate modeling approach, we show how prey size may correlate with peristome dimensions for optimal capture. Our modeling framework provides a physical platform to understand how divergence in peristome morphology may have evolved in the genus Nepenthes in response to shifts in prey diversity, availability, and size.

Self-affirmation increases reemployment success for the unemployed.

Losing a job is one of life's most stressful events. Furthermore, maladaptive reactions to unemployment can trap people in a vicious cycle that derails their reemployment efforts. The current research tested whether a brief values-based self-affirmation intervention increases the odds of reemployment after a job loss and during unemployment, which presumably breaks this vicious cycle. Two field experiments, including one with a governmental employment agency, found that a 15-min self-affirmation exercise-i.e., reflecting on one's most important values-increased key employment-related outcomes after 4 wk, including the probability and speed of reemployment and the number of job offers. Because the ordeal of job loss and the probability of reemployment may be particularly challenging for individuals above the age of 50 y, we also explored whether the intervention was equally effective for those above and below 50 y of age. Demonstrating the generality of this effect, the efficacy of the intervention did not differ between individuals below and above the age of 50, and it was also effective for both recently unemployed and chronically unemployed individuals. Because self-affirmations have more typically been tested in educational contexts, the current research demonstrates the wide-ranging value of this intervention. By diminishing the vicious cycle of unemployment, the present studies show how a simple self-affirmation intervention can help individuals succeed in the labor market.

B cell peripheral tolerance is promoted by cathepsin B protease.

B cells that bind soluble autoantigens receive chronic signaling via the B cell receptor (signal-1) in the absence of strong costimulatory signals (signal-2), and this leads to their elimination in peripheral tissues. The factors determining the extent of soluble autoantigen-binding B cell elimination are not fully understood. Here we demonstrate that the elimination of B cells chronically exposed to signal-1 is promoted by cathepsin B (Ctsb). Using hen egg lysozyme-specific (HEL-specific) immunoglobulin transgenic (MD4) B cells and mice harboring circulating HEL, we found improved survival and increased proliferation of HEL-binding B cells in Ctsb-deficient mice. Bone marrow chimera experiments established that both hematopoietic and nonhematopoietic sources of Ctsb were sufficient to promote peripheral B cell deletion. The depletion of CD4+ T cells overcame the survival and growth advantage provided by Ctsb deficiency, as did blocking CD40L or removing CD40 from the chronically antigen-engaged B cells. Thus, we suggest that Ctsb acts extracellularly to reduce soluble autoantigen-binding B cell survival and that its actions restrain CD40L-dependent pro-survival effects. These findings identify a role for cell-extrinsic protease activity in establishing a peripheral self-tolerance checkpoint.

The emerging roles of MARCH8 in viral infections: A double-edged Sword.

The host cell membrane-associated RING-CH 8 protein (MARCH8), a member of the E3 ubiquitin ligase family, regulates intracellular turnover of many transmembrane proteins and shows potent antiviral activities. Generally, 2 antiviral modes are performed by MARCH8. On the one hand, MARCH8 catalyzes viral envelope glycoproteins (VEGs) ubiquitination and thus leads to their intracellular degradation, which is the cytoplasmic tail (CT)-dependent (CTD) mode. On the other hand, MARCH8 traps VEGs at some intracellular compartments (such as the trans-Golgi network, TGN) but without inducing their degradation, which is the cytoplasmic tail-independent (CTI) mode, by which MARCH8 hijacks furin, a cellular proprotein convertase, to block VEGs cleavage. In addition, the MARCH8 C-terminal tyrosine-based motif (TBM) 222YxxL225 also plays a key role in its CTI antiviral effects. In contrast to its antiviral potency, MARCH8 is occasionally hijacked by some viruses and bacteria to enhance their invasion, indicating a duplex role of MARCH8 in host pathogenic infections. This review summarizes MARCH8's antiviral roles and how viruses evade its restriction, shedding light on novel antiviral therapeutic avenues.

Poorly Differentiated Hepatocellular Carcinoma Cells Avoid Apoptosis by Interacting with T Cells via CD40-CD40 Ligand Linkage.

Hepatocellular carcinoma (HCC) is associated with increased soluble CD40 levels. This study aimed to investigate CD40's role in liver tumor progression. CD40 levels were examined in HCC patient tissues and various HCC cell lines, and their interaction with CD4+T cells was studied. RNA sequencing analysis was performed to explore the mechanisms of CD40 induction. Poorly differentiated HCC tumor tissues exhibited high membrane-bound CD40 expression, in contrast to nontumor areas. Poorly differentiated HCC cell lines showed high expression of membrane-bound CD40 with low CD40 promoter methylation, which was the opposite of that observed in the well-differentiated HCC cell lines. Solely modulating CD40 expression in HCC cells exerted no direct consequences on cell growth or appearance. Interestingly, the human hepatoma cell line HLF co-cultured with activated (CD40 ligand+) CD4+ T cells had increased CD40 levels and a modest 3.2% dead cells. The percentage of dead cells increased to 10.9% and underwent preneutralizing CD40 condition, whereas preblocking both CD40 and integrin α5ß1 concomitantly caused only 1.9% cell death. RNA sequencing of co-cultured HLFs with activated CD4+ T cells revealed the up-regulation of interferon and immune-response pathways. Increased interferon-γ levels in the activated T-cell media stimulated the Janus kinase/signal transducer and activator of transcription 3 pathway, resulting in increased CD40 expression in HLF. Collectively, CD40 expression in poorly differentiated HCC cells prevented cell death by interacting with CD40 ligand in activated T cells. Targeting CD40 may represent a promising anticancer therapy.

Targeting the immune microenvironment in Waldenström macroglobulinemia via halting the CD40/CD40-ligand axis.

Recent investigations have improved our understanding of the molecular aberrations supporting Waldenström macroglobulinemia (WM) biology; however, whether the immune microenvironment contributes to WM pathogenesis remains unanswered. First, we showed how a transgenic murine model of human-like lymphoplasmacytic lymphoma/WM exhibits an increased number of regulatory T cells (Tregs) relative to control mice. These findings were translated into the WM clinical setting, in which the transcriptomic profiling of Tregs derived from patients with WM unveiled a peculiar WM-devoted messenger RNA signature, with significant enrichment for genes related to nuclear factor κB-mediated tumor necrosis factor α signaling, MAPK, and PI3K/AKT, which was paralleled by a different Treg functional phenotype. We demonstrated significantly higher Treg induction, expansion, and proliferation triggered by WM cells, compared with their normal cellular counterpart; with a more profound effect within the context of CXCR4C1013G-mutated WM cells. By investigating the B-cell-to-T-cell cross talk at single-cell level, we identified the CD40/CD40-ligand as a potentially relevant axis that supports WM cell-Tregs interaction. Our findings demonstrate the existence of a Treg-mediated immunosuppressive phenotype in WM, which can be therapeutically reversed by blocking the CD40L/CD40 axis to inhibit WM cell growth.

Combination CTLA4Ig and Anti-CD40 Ligand Treatment Modifies T and B Cell Metabolic Profiles and Promotes B Cell Receptor Remodeling in a Mouse Model of Systemic Lupus Erythematosus.

Systemic lupus erythematosus is a complex autoimmune disease with significant morbidity that demands further examination of tolerance-inducing treatments. Short-term treatment of lupus-prone NZB/WF1 mice with combination CTLA4Ig and anti-CD40 ligand, but not single treatment alone, suppresses disease for >6 mo via modulation of B and T cell function while maintaining immune responses to exogenous Ags. Three months after a 2-wk course of combination costimulatory blockade, we found a modest decrease in the number of activated T and B cells in both combination and single-treatment cohorts compared with untreated controls. However, only combination treatment mice showed a 50% decrease in spare respiratory capacity of splenic B and T cells. RNA sequencing and gene set enrichment analysis of germinal center (GC) B cells confirmed a reduction in the oxidative phosphorylation signature in the combination treatment cohort. This cohort also manifested increased expression of BCR-associated signaling molecules and increased phosphorylation of PLCγ in GC B cells after stimulation with anti-IgG and anti-CD40. GC B cells from combination treatment mice also displayed a signature involving remodeling of GPI-linked surface proteins. Accordingly, we found a decrease in cell surface expression of the inhibitory molecule CD24 on class-switched memory B cells from aged NZB/W mice that corrected in the combination treatment cohort. Because both a profound decrease in BCR signaling and remodeled immune cell metabolism enhance loss of tolerance in lupus-prone mice, our findings help to explain the restoration of tolerance observed after short-term combination costimulatory blockade.

Follicular CD8+ T Cells Are Elevated in HIV Infection and Induce PD-L1 on B Cells.

Follicular CD8+CXCR5+ T cells are a specialized CD8+ T cell subset with unique follicular-homing capabilities that have been reported to display effector functions in viral immunity, tumor immunity, and autoimmunity. CD8+CXCR5+ T cells exhibit B cell helper functions and express CD40L, ICOS, programmed cell death protein 1 (PD-1), and BCL-6, the transcriptional regulator of CD4+CXCR5+ T follicular helper cells and of germinal center B cells. HIV is known to be sequestered in lymphoid follicles, and CD8+CXCR5+ T cell frequency is a marker for disease severity, given that HIV-infected patients with lower numbers of circulating CD8+CXCR5+ T cells display lower CD4+ T cell counts. Likewise, several groups have reported a direct correlation between the quantity of CD8+CXCR5+ T cells and suppression of HIV viral load. In this study, we observed elevated absolute numbers of CD8+CXCR5+ and CD8+CXCR5+BCL-6+PD-1+ T cells in the blood of HIV-infected participants of the Multicenter AIDS Cohort Study. We further demonstrated in vitro that activated human CD8+CXCR5+ T cells isolated from peripheral blood and tonsil from healthy donors show increased CD40L expression and induce the production of PD ligand 1 (PD-L1)+IgG+ B cells. Moreover, absolute numbers of CD8+CXCR5+ T cells significantly and positively correlated with numbers of PD-L1+ B cells found in blood of HIV-infected individuals. Altogether, these results show that activated CD8+CXCR5+ T cells have the ability to activate B cells and increase the percentage of PD-L1+ and PD-L1+IgG+ B cells, which provides insights into the early events of B cell activation and differentiation and may play a role in disease progression and lymphomagenesis in HIV-infected individuals.

CD40-CD154: A perspective from type 2 immunity.

The interaction between CD40 and CD154 (CD40 ligand) is central in immunology, participating in CD4+ T cell priming by dendritic cells (DC), CD4+ T cell help to B cells and classical macrophage activation by CD4+ T cells. However, its role in the Th2 side of immunology including helminth infection remains incompletely understood. Contrary to viral and bacterial stimuli, helminth products usually do not cause CD40 up-regulation in DC, and exogenous CD40 ligation drives Th2-biased systems towards Th1. On the other hand, CD40 and CD154 are necessary for induction of most Th2 responses. We attempt to reconcile these observations, mainly by proposing that (i) CD40 up-regulation in DC in Th2 systems is mostly induced by alarmins, (ii) the Th2 to Th1 shift induced by exogenous CD40 ligation is related to the capacity of such ligation to enhance IL-12 production by myeloid cells, and (iii) signals elicited by endogenous CD154 available in Th2 contexts and by exogenous CD40 ligation are probably different. We stress that CD40-CD154 is important beyond cognate cellular interactions. In such a context, we argue that the proliferation response of B-cells to IL-4 plus CD154 reflects a Th2-specific mechanism for polyclonal B-cell amplification and IgE production at infection sites. Finally, we argue that CD154 is a general immune activation signal across immune polarization including Th2, and propose that competition for CD154 at tissue sites may provide negative feedback on response induction at each site.

CD40 stimulation via CD40 ligand enhances adenovirus-mediated tumour immunogenicity including 'find-me', 'eat-me', and 'kill-me' signalling.

Immunostimulatory gene therapy using oncolytic viruses is currently evaluated as a promising therapy for cancer aiming to induce anti-tumour immunity. Here, we investigate the capacity of oncolytic adenoviruses (LOAd) and their transgenes to induce immunogenicity in the infected tumour cells. Oncolysis and death-related markers were assessed after infection of eight human solid cancer cell lines with different LOAd viruses expressing a trimerized, membrane-bound (TMZ)-CD40L, TMZ-CD40L and 41BBL, or no transgenes. The viruses induced transgene expression post infection before they were killed by oncolysis. Death receptors TRAIL-R1, TRAIL-R2 and Fas as well as immunogenic cell death marker calreticulin were upregulated in cell lines post infection. Similarly, caspase 3/7 activity was increased in most cell lines. Interestingly, in CD40+ cell lines there was a significant effect of the TMZ-CD40L-encoding viruses indicating activation of the CD40-mediated apoptosis pathway. Further, these cell lines showed a significant increase of calreticulin, and TRAIL receptor 1 and 2 post infection. However, LOAd viruses induced PD-L1 upregulation which may hamper anti-tumour immune responses. In conclusion, LOAd infection increased the immunogenicity of infected tumour cells and this was potentiated by CD40 stimulation. Due to the simultaneous PD-L1 increase, LOAd viruses may benefit from combination with antibodies blocking PD1/PD-L1.

Platelet transfusion enhances pro-aggregatory status shortly after coronary artery bypass grafting (CABG while modulating platelet pro-inflammatory state 1-week post-surgery.

During coronary artery bypass grafting (CABG), the surgical procedure, particularly the manipulation of the major arteries of the heart, induces a significant inflammatory state that may compromise platelet function to the extent that platelet transfusion is required. Given stored platelets as a major source of biological mediators, this study investigates the effects of platelet transfusion on the major pro-aggregatory, pro-inflammatory and immunomodulatory markers of platelets. Platelets from 20 patients, 10 who received platelet transfusion and 10 without, were subjected to flow cytometery where P-selectin and CD40 ligand (CD40L) expressions and PAC-1 binding (activation-specific anti GPIIb/GPIIIa antibody) analysed at five-time points of 24 h before surgery, immediately, 2 h, 24 h and 1 week after surgery. Analysis of intra-platelet transforming growth factor-beta-1 (TGF-ß1) was also conducted using western blotting. Patients with platelet transfusion showed increased levels of P-selectin, CD40L and intra-platelet TGF-ß1 2-h after surgery compared to those without transfusion (p < 0.05). PAC-1 binding was increased 24 h after surgery in transfused patients (p < 0.05). Given the significant post-transfusion elevation of platelet TGF-ß1, P-sel/CD40L reduction in transfused patients a week after was of much interest. This study showed for the first time the significant effects of platelet transfusion on the pro-inflammatory, pro-aggeregatory and immunomodulatory state of platelets in CABG patients, which manifested with immediate, midterm and delayed consequences. While the increased pro-inflammatory conditions manifested as an immediate effect of platelet transfusion, the pro-aggregatory circumstances emerged 24 h post-transfusion. A week after surgery, attenuations of pro-inflammatory markers of platelets in transfused patients were shown, which might be due to the immunomodulatory effects of TGF-ß1.

Modulating CD40 and integrin signaling in the proinflammatory nexus using a 15-amino-acid peptide, KGYY15.

CD40 signaling has long been a target in autoimmunity. Attempts to block signaling between CD40 and CD154 during clinical trials using monoclonal antibodies suffered severe adverse events. Previously, we developed a peptide, KGYY15, that targets CD40 and, in preclinical trials, prevents type 1 diabetes in >90% of cases and reverses new-onset hyperglycemia in 56% of cases. It did so by establishing normal effector T-cell levels rather than ablating the cells and causing immunosuppression. However, the relationship between KGYY15 and other elements of the complex signaling network of CD40 is not clear. Studying interactions between proteins from autoimmune and nonautoimmune mice, we demonstrate interactions between CD40 and integrin CD11a/CD18, which complicates the understanding of the inflammatory nexus and how to prevent autoinflammation. In addition to interacting with CD40, KGYY15 interacts with the integrins CD11a/CD18 and CD11b/CD18. We argue that modulation of CD40-CD154 signaling may be more advantageous than complete inhibition because it may preserve normal immunity to pathogens.

Fibroblast-like synoviocytes preferentially induce terminal differentiation of IgD+ memory B cells instead of naïve B cells.

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.

Differential induction of donor-reactive Foxp3+ regulatory T cell via blockade of CD154 vs CD40.

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)-1ß from CD11b+ and CD11c+ dendritic cells, and blockade of IL-1ß 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-1ß-dependent mechanism by which CD154:CD11b interactions prevent the generation of donor-reactive Foxp3+ iTreg during transplantation.

The diagnostic value and validation of IL-22 combimed with sCD40L in tuberculosis pleural effusion.

BACKGROUND: There is substantial evidence indicating that cytokines play a role in the immune defense against tuberculosis. This study aims to evaluate the levels of various cytokines in pleural effusion to ditinguish between tuberculosis pleurisy and malignant pleurisy. METHODS: A total of 82 participants with pleural effusion were included in the training cohort, and 76 participants were included in the validation cohort. The individuals were divided into tuberculosis and malignant pleurisy groups. The concentrations of interleukin-1ß (IL-1ß), IL-4, IL-6, IL-10, IL-17 A, IL-17 F, IL-21, IL-22, IL-25, IL-31, IL-33, interferon-γ (IFN-γ), soluble CD40 ligand (sCD40L) and tumor necrosis factor-α (TNF-α) in pleural effusion were measured using a multiplex cytokine assay. The threshold values were calculated according to the receiver operating characteristic (ROC) curve analysis to aid in diagnosing tuberculosis pleurisy. Furthermore, the combined measure was validated in the validation cohort. RESULTS: The levels of all 14 cytokines in pleural effusion were significantly higher in participants with tuberculosis compared to those with malignant pleurisy (all P < 0.05). The area under the curve (AUC) was ≥ 0.920 for the IL-22, sCD40L, IFN-γ, TNF-α and IL-31, which were significantly increased in tuberculous pleural effusion (TPE) compared to MPE in the training cohort. Threshold values of 95.80 pg/mL for IFN-γ, 41.80 pg/mL for IL-31, and 18.87 pg/mL for IL-22 provided ≥ 90% sensitivity and specificity in distinguishing between tuberculosis pleurisy and malignant pleurisy in the training cohort. Among these, IL-22 combined with sCD40L showed the best sensitivity and specificity (94.0% and 96.9%) for diagnosing tuberculosis pleurisy, and this finding was validated in the validation cohort. CONCLUSION: We demonstrated that the levels of IL-1ß, IL-4, IL-6, IL-10, IL-17 A, IL-17 F, IL-21, IL-22, IL-25, IL-31, IL-33, IFN-γ, sCD40L and TNF-α in pleural effusion had significant difference between tuberculosis pleurisy and malignant pleurisy. Specifically, IL-22 ≥ 18.87 pg/mL and sCD40L ≥ 53.08 pg/mL can be clinically utilized as an efficient diagnostic strategy for distinguishing tuberculosis pleurisy from malignant pleurisy.

Role of Skewed X-Chromosome Inactivation in Common Variable Immunodeficiency.

The term common variable immunodeficiency (CVID) encompasses a clinically diverse group of disorders, mainly characterized by hypogammaglobulinemia, insufficient specific antibody production, and recurrent infections. The genetics of CVID is complex, and monogenic defects account for only a portion of cases, typically <30%. Other proposed mechanisms include digenic, oligogenic, or polygenic inheritance and epigenetic dysregulation. In this study, we aimed to assess the role of skewed X-chromosome inactivation (XCI) in CVID. Within our cohort of 131 genetically analyzed CVID patients, we selected female patients with rare variants in CVID-associated genes located on the X-chromosome. Four patients harboring heterozygous variants in BTK (n = 2), CD40LG (n = 1), and IKBKG (n = 1) were included in the study. We assessed XCI status using the HUMARA assay and an NGS-based method to quantify the expression of the 2 alleles in mRNA. Three of the 4 patients (75%) exhibited skewed XCI, and the mutated allele was predominantly expressed in all cases. Patient 1 harbored a hypomorphic variant in BTK (p.Tyr418His), patient 3 had a pathogenic variant in CD40LG (c.288+1G>A), and patient 4 had a hypomorphic variant in IKBKG (p.Glu57Lys) and a heterozygous splice variant in TNFRSF13B (TACI) (c.61+2T>A). Overall, the analysis of our cohort suggests that CVID in a small proportion of females (1.6% in our cohort) is caused by skewed XCI and highly penetrant gene variants on the X-chromosome. Additionally, skewed XCI may contribute to polygenic effects (3.3% in our cohort). These results indicate that skewed XCI may represent another piece in the complex puzzle of CVID genetics.