Dendritic Cells Promote the Differentiation of ILCs into NCR-ILC3s in the Lungs of Mice Exposed to Cigarette Smoke.

The involvement of Group 3 innate lymphoid cells (ILC3s) and dendritic cells (DCs) in chronic lung inflammation has been increasingly regarded as the key to understand the inflammatory mechanisms of smoke-related chronic obstructive pulmonary disease (COPD). However, the mechanism underlying the engagement of both remains unclear. Our study aimed to explore NCR-ILC3 differentiation in the lungs of mice exposed to cigarette smoke (CS) and to further investigate whether DCs activated by CS exposure contribute to the differentiation of ILCs into NCR-ILC3s. The study involved both in vivo and in vitro experiments. In the former, the frequencies of lung NCR-ILC3s and NKp46-IL-17A+ ILCs and the expression of DCs, CD40, CD86, IL-23, and IL-1ß quantified by flow cytometry were compared between CS-exposed mice and air-exposed mice. In the latter, NKp46-IL-17A+ ILC frequencies quantified by flow cytometry were compared after two cocultures, one involving lung CD45+Lin-CD127+ ILCs sorted from air-exposed mice and DCs sifted by CD11c magnetic beads from CS-exposed mice and another including identical CD45+Lin-CD127+ ILCs and DCs from air-exposed mice. The results indicated significant increases in the frequencies of NCR-ILC3s and NKp46-IL-17A+ ILCs; in the expression of DCs, CD40, CD86, IL-23, and IL-1ß in CS-exposed mice; and in the frequency of NKp46-IL-17A+ ILCs after the coculture with DCs from CS-exposed mice. In conclusion, CS exposure increases the frequency of lung ILCs and NCR-ILC3s. CS-induced DC activation enhances the differentiation of ILCs into NCR-ILC3s, which likely acts as a mediating step in the involvement of NCR-ILC3s in chronic lung inflammation.

Bacteriophages from treatment-naïve type 2 diabetes individuals drive an inflammatory response in human co-cultures of dendritic cells and T cells.

Individuals with type 2 diabetes (T2D) show signs of low-grade inflammation, which is related to the development of insulin resistance and beta-cell dysfunction. However, the underlying triggers remain unknown. The gut microbiota is a plausible source as it comprises pro-inflammatory bacteria, bacterial metabolites and viruses, including (bacterio)phages. These prokaryotic viruses have been shown to mediate inflammatory responses in gastrointestinal disease. Given the differences in phage populations in healthy individuals versus those with cardiometabolic diseases such as T2D, we here questioned whether phages from T2D individuals would have increased immunogenic potential. To address this, we isolated intestinal phages from a fresh stool sample of healthy controls and individuals with newly diagnosed, treatment-naive T2D. Phages were purified using cesium chloride ultracentrifugation and incubated with healthy donor dendritic cells (DCs) and autologous T cells. Donors with T2D had slightly higher free viral particle numbers compared to healthy controls (p = .1972), which has been previously associated with disease states. Further, phages from T2D induced a higher inflammatory response in DCs and T cells than phages from HC. For example, the expression of the co-stimulatory molecule CD86 on DCs (p < .001) and interferon-y secretion from T cells (p < .01) were increased when comparing the two groups. These results suggest that phages might play a role in low-grade inflammation in T2D individuals.

Targeting dendritic cell activation: the therapeutic impact of paeoniflorin in cortosteroid-dependent dermatitis management.

This study investigates the mechanism through which paeoniflorin inhibits TSLP expression to regulate dendritic cell activation in corticosteroid-dependent dermatitis treatment. Utilizing databases like TCMSP, we identified paeoniflorin's components, targets, and constructed networks. Molecular docking and gene enrichment analysis helped pinpoint key targets and pathways affected by paeoniflorin. In vitro and in vivo models were used to study CD80, CD86, cytokines, T-cell activation, skin lesions, histopathological changes, TSLP, CD80, and CD86 expression. Our study revealed paeoniflorin's active constituent targeting IL-6 in corticosteroid-dependent dermatitis. In vitro experiments demonstrated reduced TSLP expression, CD80, CD86, and cytokine secretion post-paeoniflorin treatment. In vivo, paeoniflorin significantly decreased skin lesion severity, cytokine levels, TSLP, CD80, and CD86 expression. The study highlights paeoniflorin's efficacy in inhibiting TSLP expression and suppressing dendritic cell activation in corticosteroid-dependent dermatitis, suggesting its potential as a therapeutic intervention. Additionally, it offers insights into the complex molecular mechanisms underlying paeoniflorin's anti-inflammatory properties in treating corticosteroid-dependent dermatitis.

Blockade of CD80/CD86-CD28 co-stimulation augments the inhibitory function of peptide antigen-specific regulatory T cells.

Mixed lymphocyte culture under the blockade of CD80/CD86-CD28 co-stimulation induces anergic (completely hyporesponsive) T cells with immune suppressive function (inducible suppressing T cells: iTS cells). Previously, iTS cell therapy has demonstrated outstanding benefits in clinical trials for organ transplantation. Here, we examined whether peptide antigen-specific iTS cells are inducible. DO 11.10 iTS cells were obtained from splenocytes of BALB/c DO 11.10 mice by stimulation with OVA peptide and antagonistic anti-CD80/CD86 mAbs. When DO 11.10 iTS or Foxp3- DO 11.10 iTS cells were stimulated with OVA, these cells produced IL-13, but not IL-4. DO 11.10 iTS cells decreased IL-4 and increased IL-13 production from OVA-stimulated naïve DO 11.10 splenocytes. When Foxp3+ DO 11.10 iTS cells were prepared, these cells significantly inhibited the production of IL-4 and IL-13 compared with freshly isolated Foxp3+ DO 11.10 T cells. Moreover, an increase in the population expressing OX40, ICOS, and 4-1BB suggested activation of Foxp3+ DO 11.10 iTS cells. Thus, blockade of CD80/CD86-CD28 co-stimulation during peptide antigen stimulation augments the inhibitory function of Foxp3+ regulatory T cells, and does not induce anergic Foxp3- conventional T cells. Peptide-specific Foxp3+ regulatory iTS cells could be useful for the treatment of allergic and autoimmune diseases without adverse effects.

Saponin Esculeoside A and Aglycon Esculeogenin A from Ripe Tomatoes Inhibit Dendritic Cell Function by Attenuation of Toll-like Receptor 4 Signaling.

Dendritic cells (DCs) can initiate immune response through the presenting antigens to naïve T lymphocytes. Esculeoside A (EsA), a spirosolane glycoside, is reported as a major component in the ripe fruit of tomato. Little is known about the effect of tomato saponin on mice bone marrow-derived DCs. This study revealed that EsA and its aglycon, esculeogenin A (Esg-A), attenuated the phenotypic and functional maturation of murine DCs stimulated by lipopolysaccharide (LPS). We found that EsA/Esg-A down-regulated the expression of major histocompatibility complex type II molecules and costimulatory molecule CD86 after LPS stimulation. It was also determined that EsA-/Esg-A-treated DCs were poor stimulators of allogeneic T-cell proliferation and exhibited impaired interleukin-12 and TNF-α production. Additionally, EsA/Esg-A was able to inhibit TLR4-related and p-NFκB signaling pathways. This study shows new insights into the immunopharmacology of EsA/Esg-A, and represents a novel approach to controlling DCs for therapeutic application.

Obesity induces PD-1 on macrophages to suppress anti-tumour immunity.

Obesity is a leading risk factor for progression and metastasis of many cancers1,2, yet can in some cases enhance survival3-5 and responses to immune checkpoint blockade therapies, including anti-PD-1, which targets PD-1 (encoded by PDCD1), an inhibitory receptor expressed on immune cells6-8. Although obesity promotes chronic inflammation, the role of the immune system in the obesity-cancer connection and immunotherapy remains unclear. It has been shown that in addition to T cells, macrophages can express PD-19-12. Here we found that obesity selectively induced PD-1 expression on tumour-associated macrophages (TAMs). Type I inflammatory cytokines and molecules linked to obesity, including interferon-γ, tumour necrosis factor, leptin, insulin and palmitate, induced macrophage PD-1 expression in an mTORC1- and glycolysis-dependent manner. PD-1 then provided negative feedback to TAMs that suppressed glycolysis, phagocytosis and T cell stimulatory potential. Conversely, PD-1 blockade increased the level of macrophage glycolysis, which was essential for PD-1 inhibition to augment TAM expression of CD86 and major histocompatibility complex I and II molecules and ability to activate T cells. Myeloid-specific PD-1 deficiency slowed tumour growth, enhanced TAM glycolysis and antigen-presentation capability, and led to increased CD8+ T cell activity with a reduced level of markers of exhaustion. These findings show that obesity-associated metabolic signalling and inflammatory cues cause TAMs to induce PD-1 expression, which then drives a TAM-specific feedback mechanism that impairs tumour immune surveillance. This may contribute to increased cancer risk yet improved response to PD-1 immunotherapy in obesity.

Development of Hsp90 inhibitor to regulate cytokine storms in excessive delayed- and acute inflammation.

BACKGROUND: The surplus cytokines remaining after use in the early stages of the inflammatory response stimulate immune cells even after the response is over, causing a secondary inflammatory response and ultimately damaging the host, which is called a cytokine storm. Inhibiting heat shock protein 90 (Hsp90), which has recently been shown to play an important role in regulating inflammation in various cell types, may help control excessive inflammatory responses and cytokine storms. METHODS: We discovered an anti-inflammatory compound by measuring the inhibitory effect of CD86 expression on spleen DCs (sDCs) using the chemical compounds library of Hsp90 inhibitors. Subsequently, to select the hit compound, the production of cytokines and expression of surface molecules were measured on the bone marrow-derived DCs (BMDCs) and peritoneal macrophages. Then, we analyzed the response of antigen-specific Th1 cells. Finally, we confirmed the effect of the compound using acute lung injury (ALI) and delayed-type hypersensitivity (DTH) models. RESULTS: We identified Be01 as the hit compound, which reduced CD86 expression the most in sDCs. Treatment with Be01 decreased the production of pro-inflammatory cytokines (IL-6, TNF-α, and IL-1ß) in BMDC and peritoneal macrophages stimulated by LPS. Under the DTH model, Be01 treatment reduced ear swelling and pro-inflammatory cytokines in the spleen. Similarly, Be01 treatment in the ALI model decreased neutrophil infiltration and lower levels of secreted cytokines (IL-6, TNF-α). CONCLUSIONS: Reduction of CD80 and CD86 expression on DCs by Be01 indicates reduced secondary inflammatory response by Th1 cells, and reduced release of pro-inflammatory cytokines by peritoneal macrophages may initially control the cytokine storm.

Development of a Protocol for Obtaining a Homologous Cell Product of Animal Origin Intended for Preclinical Studies of Antitumor Vaccine CaTeVac.

When developing a program of preclinical studies of human cell-based drugs intended for adoptive immunotherapy of cancer patients, the biological effect should be substantiated by data describing their immunological action. Administration and study of human autologous dendritic cell vaccine to immunocompetent animals are not adequate in terms of immunological compatibility. It is possible to use immunocompromised, knockout, or transgenic animals or to obtain a homologous cellular product, namely, a preparation based on animal cells using a technology similar to obtaining the original preparation for clinical practice in humans. Within the framework of this study, we have developed a protocol for obtaining a homologous cell product based on animal dendritic cells (mice, rats) according to a similar technology for obtaining human vaccine dendritic cells, and demonstrated the comparability of morphological characteristics and expression of differentiation antigens of dendritic cells (CD11c, CD80, CD86, and CD83) of animals (mice) and humans.

K33 only mutant ubiquitin augments bone marrow-derived dendritic cell-mediated CTL priming via PI3K-Akt pathway.

To explore the effect of K33 only mutant ubiquitin (K33O) on bone marrow-derived dendritic cells' (BMDCs') maturity, antigen uptake capability, surface molecule expressions and BMDC-mediated CTL priming, and further investigate the role of PI3K-Akt engaged in K33O-increased BMDC maturation, antigen uptake and presentation, surface molecule expressions and BMDC-based CTL priming. BMDCs were conferred K33O and other ubiquitin mutants (K33R, K48R, K63R-mutant ubiquitin) incubation or LY294002 and wortmannin pretreatment. PI3K-Akt phosphorylation, antigen uptake, antigenic presentation and CD86/MHC class I expression in BMDC were determined by western blot or flow cytometry. BMDC-based CTL proliferation and priming were determined by in vitro mixed lymphocyte reaction (MLR), ex vivo enzyme-linked immunospot assay (Elispot) and flow cytometry with intracellular staining, respectively. The treatment with K33O effectively augmented PI3K-Akt phosphorylation, BMDCs' antigen uptake, antigenic presentation, CD86/MHC class I and CD11c expressions. MLR, Elispot and flow cytometry revealed that K33O treatment obviously enhanced CTL proliferation, CTL priming and perforin/granzyme B expression. The pretreatment with PI3K-Akt inhibitors efficiently abrogated K33O's effects on BMDC. The replenishment of K33 only mutant ubiquitin augments BMDC-mediated CTL priming in bone marrow-derived dendritic cells via PI3K-Akt signalling.

Optimal conditions for adenoviral transduction of immature dendritic cells without affecting the tolerogenic activity of DC-based immunotherapy.

Dendritic cells (DCs) play a pivotal role in maintaining immune tolerance. Using recombinant adenovirus (rAd) to deliver vectors to immature dendritic cells (imDCs) is an important method for studying the tolerogenic function of DCs. We found that using RPMI medium and a higher MOI during transduction increased the expression of CD80, CD86, and MHC-II on the surface of imDCs. Our data reveal a significant increase in the secretion of the pro-inflammatory cytokine IL-6 in the group showing the most pronounced phenotypic changes. In the mouse heart transplant model, imDCs with unstable phenotype and function due to adenoviral transduction resulted in an increased proportion of Th1 and Th17 cells in recipients. However, these effects can be managed, and our proposed optimized transduction strategy significantly minimizes these adverse effects. Our study holds significant implications for the development and optimization of immunotherapy utilizing tolerogenic dendritic cells.

An integrated in silico-in vitro investigation to assess the skin sensitization potential of 4-Octylphenol.

One of the major challenges in chemical toxicity testing is the possibility to protect human health against adverse effects with non-animal methods. In this paper, 4-Octylphenol (OP) was tested for skin sensitization and immunomodulatory effects using an integrated in silico-in vitro test approach. In silico tools (QSAR TOOLBOX 4.5, ToxTree and VEGA) were used together with several in vitro tests including HaCaT cells (quantification of IL-6; IL-8; IL-1α and IL-18 by ELISA and expression of genes TNF, IL1A, IL6 and IL8 by RT- qPCR), RHE model (quantification of IL-6; IL-8; IL-1α and IL-18 by ELISA) and THP-1 activation assay (CD86/CD54 expression and IL-8 release). Additionally, the immunomodulatory effect of OP was investigated using lncRNAs MALAT1 and NEAT1 expression and LPS-induced THP-1 activation (CD86/CD54 expression and IL-8 release). The in silico tools predicted OP as a sensitizer. In vitro tests are also concordant with the in silico prediction. OP increased IL-6 expression (HaCaT cells); IL-18 and IL-8 expressions (RHE model). An irritant potential was also shown by a great expression of IL-1α (RHE model); and increased expression of CD54 marker and IL-8 in THP-1 cells. Immunomodulatory effects of OP were demonstrated by the downregulation of NEAT1, MALAT1 (epigenetic markers), IL6 and IL8; and an increase in LPS-induced CD54 and IL-8 expressions. Overall, results indicate that OP is a skin sensitizer, being positive in three key events of the AOP for skin sensitization, also showing immunomodulatory effects.

Endothelial mechanical stretch regulates the immunological synapse interface of renal endothelial cells in a sex-dependent manner.

Increased mechanical endothelial cell stretch contributes to the development of numerous cardiovascular and renal pathologies. Recent studies have shone a light on the importance of sex-dependent inflammation in the pathogenesis of renal disease states. The endothelium plays an intimate and critical role in the orchestration of immune cell activation through upregulation of adhesion molecules and secretion of cytokines and chemokines. While endothelial cells are not recognized as professional antigen-presenting cells, in response to cytokine stimulation, endothelial cells can express both major histocompatibility complex (MHC) I and MHC II. MHCs are essential to forming a part of the immunological synapse interface during antigen presentation to adaptive immune cells. Whether MHC I and II are increased under increased mechanical stretch is unknown. Due to hypertension being multifactorial, we hypothesized that increased mechanical endothelial stretch promotes the regulation of MHCs and key costimulatory proteins on mouse renal endothelial cells (MRECs) in a stretch-dependent manner. MRECs derived from both sexes underwent 5%, 10%, or 15% uniaxial cyclical stretch, and immunological synapse interface proteins were determined by immunofluorescence microscopy, immunoblot analysis, and RNA sequencing. We found that increased endothelial mechanical stretch conditions promoted downregulation of MHC I in male MRECs but upregulation in female MRECs. Moreover, MHC II was upregulated by mechanical stretch in both male and female MRECs, whereas CD86 and CD70 were regulated in a sex-dependent manner. By bulk RNA sequencing, we found that increased mechanical endothelial cell stretch promoted differential gene expression of key antigen processing and presentation genes in female MRECs, demonstrating that females have upregulation of key antigen presentation pathways. Taken together, our data demonstrate that mechanical endothelial stretch regulates endothelial activation and immunological synapse interface formation in renal endothelial cells in a sex-dependent manner.NEW & NOTEWORTHY Endothelial cells contribute to the development of renal inflammation and have the unique ability to express antigen presentation proteins. Whether increased endothelial mechanical stretch regulates immunological synapse interface proteins remains unknown. We found that antigen presentation proteins and costimulatory proteins on renal endothelial cells are modulated by mechanical stretch in a sex-dependent manner. Our data provide novel insights into the sex-dependent ability of renal endothelial cells to present antigens in response to endothelial mechanical stimuli.

Common fragrance chemicals activate dendritic cells in coculture with keratinocytes.

BACKGROUND: Fragrances are important contact allergens; however, investigation of their skin sensitization potency has been challenging in new approach methods (NAMs). Many fragrance chemicals are susceptible to autoxidation or can be metabolized by enzymes constitutively expressed in skin keratinocytes. Strong sensitizers can be formed in both of these processes. Further, keratinocytes can modulate the dendritic cell (DC) activation and maturation potential, a key event in the acquisition of contact allergy. OBJECTIVES: To evaluate the 2D coculture model consisting of keratinocytes and DCs using different weak to moderate sensitizing fragrance chemicals. Further, to investigate fragrances and related oxidation products in the in vitro model and compare to in vivo data. METHODS: Chemicals were tested in the coculture activation test (COCAT), consisting of HaCaT keratinocytes and THP-1 cells. THP-1 cell surface expression of costimulatory and adhesion molecules (CD86 and CD54) collected after 24 h incubation with the chemicals was analysed using flow cytometry. RESULTS: Twenty-four molecules were tested positive, three were negative (n = 27). Four pairs were evaluated, with aldehydes showing a 6- to 13-fold stronger responses compared to their corresponding alcohols. CONCLUSIONS: Results provide insight into the activation of DC in their natural environment of keratinocytes. α,ß-Unsaturated alcohols were classified as weaker sensitizers compared to their corresponding aldehydes. In sum, testing of fragrances retrieved results in good agreement with in vivo data.

TRAM deletion attenuates monocyte exhaustion and alleviates sepsis severity.

Monocyte exhaustion characterized by immune-suppressive features can develop during sepsis and contribute to adverse patient outcomes. However, molecular mechanisms responsible for the establishment of immune-suppressive monocytes with reduced expression of immune-enhancing mediators such as CD86 during sepsis are not well understood. In this study, we identified that the TLR4 intracellular adaptor TRAM plays a key role in mediating the sustained reduction of CD86 expression on exhausted monocytes and generating an immune-suppressive monocyte state. TRAM contributes to the prolonged suppression of CD86 through inducing TAX1BP1 as well as SARM1, collectively inhibiting Akt and NFκB. TRAM deficient mice are protected from cecal slurry-induced experimental sepsis and retain immune-competent monocytes with CD86 expression. Our data reveal a key molecular circuitry responsible for monocyte exhaustion and provide a viable target for rejuvenating functional monocytes and treating sepsis.

Therapeutic gene editing of T cells to correct CTLA-4 insufficiency.

Heterozygous mutations in CTLA-4 result in an inborn error of immunity with an autoimmune and frequently severe clinical phenotype. Autologous T cell gene therapy may offer a cure without the immunological complications of allogeneic hematopoietic stem cell transplantation. Here, we designed a homology-directed repair (HDR) gene editing strategy that inserts the CTLA-4 cDNA into the first intron of the CTLA-4 genomic locus in primary human T cells. This resulted in regulated expression of CTLA-4 in CD4+ T cells, and functional studies demonstrated CD80 and CD86 transendocytosis. Gene editing of T cells isolated from three patients with CTLA-4 insufficiency also restored CTLA-4 protein expression and rescued transendocytosis of CD80 and CD86 in vitro. Last, gene-corrected T cells from CTLA-4-/- mice engrafted and prevented lymphoproliferation in an in vivo murine model of CTLA-4 insufficiency. These results demonstrate the feasibility of a therapeutic approach using T cell gene therapy for CTLA-4 insufficiency.

The Role of CD40, CD86, and Glutathione S-Transferase Omega 1 in the Pathogenesis of Chronic Obstructive Pulmonary Disease.

Objective: The aim of the study was to explore the relevance of CD40, CD86, and GSTO1 with the pathogenesis of COPD. Methods: Patients with acute exacerbation of COPD were contrasted with the healthy and nonsmoking ones and smoking but without COPD ones. The changes of CD40, CD86, and GSTO1 in the peripheral blood, collected from different groups, were detected by flow cytometry and western blotting, respectively. Results: Compared with the nonsmoking group and smoking but without the COPD group, the expression of CD40 and CD86 of the patients with COPD increased significantly, but the expression of GSTO1 decreased. CD40 and CD86 were negatively correlated with FEV1%, while GSTO1 was positively correlated with FEV1% and negatively correlated with CD40 and CD86. Conclusion: CD40, CD86, and GSTO1 may play a role in the pathogenesis of COPD, and they are related to the severity of COPD and the degree of changes in the lung function.

Differential regulation of CD8+ CD86+ Vγ1.1+ γδT cell responses in skin barrier tissue protection and homeostatic maintenance.

Compared to αßT cells, γδT cells are more innate-like and preferentially function as the first line of defense in barrier tissues. Certain populations of γδT cells possess adaptive immune cell properties but their regulation is not well understood. We herein report that while innate-like γδT17 cells dominated in the skin of WT mice, Vγ1.1+ γδT cells with adaptive T cell-like properties predominantly expanded in the skin of TCRß-/- and B2m-/- mice. Commensal bacteria drove expansion of Vγ1.1+ skin γδT cells, functional properties of which correlated with local immune requirements. That is, Vγ1.1+ skin γδT cells in TCRß-/- mice were a heterogeneous population; while Vγ1.1+ skin γδT cells in B2m-/- mice were mostly CD8+ CD86+ cells that had a similar function of CD8+ CD86+ skin αßT cells in supporting local Treg cells. We also found that intrinsic TGF-ß receptor 2-derived signals in skin CD8+ αßT and γδT cells are required for their expression of CD86, a molecule important in supporting skin Treg cells. Our findings reveal broad functional potentials of γδT cells that are coordinately regulated with αßT cells to help maintain local tissue homeostasis.

PDZ Proteins SCRIB and DLG1 Regulate Myeloma Cell Surface CD86 Expression, Growth, and Survival.

Despite advances in the treatment of multiple myeloma in the past decades, the disease remains incurable, and understanding signals and molecules that can control myeloma growth and survival are important for the development of novel therapeutic strategies. One such molecule, CD86, regulates multiple myeloma cell survival via its interaction with CD28 and signaling through its cytoplasmic tail. Although the CD86 cytoplasmic tail has been shown to be involved in drug resistance and can induce molecular changes in multiple myeloma cells, its function has been largely unexplored. Here, we show that CD86 cytoplasmic tail has a role in trafficking CD86 to the cell surface. This is due in part to a PDZ-binding motif at its C-terminus which is important for proper trafficking from the Golgi apparatus. BioID analysis revealed 10 PDZ domain-containing proteins proximal to CD86 cytoplasmic tail in myeloma cells. Among them, we found the planar cell polarity proteins, SCRIB and DLG1, are important for proper CD86 surface expression and the growth and survival of myeloma cells. These findings indicate a mechanism by which myeloma cells confer cellular survival and drug resistance and indicate a possible motif to target for therapeutic gain. IMPLICATIONS: These findings demonstrate the importance of proper trafficking of CD86 to the cell surface in myeloma cell survival and may provide a new therapeutic target in this disease.

Ubiquitin-like protein 3 (UBL3) is required for MARCH ubiquitination of major histocompatibility complex class II and CD86.

The MARCH E3 ubiquitin (Ub) ligase MARCH1 regulates trafficking of major histocompatibility complex class II (MHC II) and CD86, molecules of critical importance to immunity. Here we show, using a genome-wide CRISPR knockout screen, that ubiquitin-like protein 3 (UBL3) is a necessary component of ubiquitination-mediated trafficking of these molecules in mice and in humans. Ubl3-deficient mice have elevated MHC II and CD86 expression on the surface of professional and atypical antigen presenting cells. UBL3 also regulates MHC II and CD86 in human dendritic cells (DCs) and macrophages. UBL3 impacts ubiquitination of MARCH1 substrates, a mechanism that requires UBL3 plasma membrane anchoring via prenylation. Loss of UBL3 alters adaptive immunity with impaired development of thymic regulatory T cells, loss of conventional type 1 DCs, increased number of trogocytic marginal zone B cells, and defective in vivo MHC II and MHC I antigen presentation. In summary, we identify UBL3 as a conserved, critical factor in MARCH1-mediated ubiquitination with important roles in immune responses.

Aqueous extract of Passiflora alata leaves modulates in vitro the indoleamine 2,3-dioxygenase (IDO) and CD86 expression in bone marrow-derived professional antigen-presenting cells polarizing NOD mice T cells to a Treg profile.

Dendritic cells (DCs) and macrophages are professional antigen-presenting cells (pAPCs), numerous in the pancreas of nonobese diabetic (NOD) mice and playing an essential role in the autoimmune response of type 1 diabetes. The expression of the enzyme indoleamine 2,3-dioxygenase (IDO) is a critical factor for the tolerogenic activity of pAPCs, acting in the catabolism of tryptophan, providing metabolites that suppress the T cell effectors and induce T regulatory cells differentiation. Here we investigated the in vitro mechanisms of lyophilized aqueous extract from Passiflora alata leaves (LAEPAL) that modulates bone marrow-derived professional antigen-presenting cells (BM-pAPCs), affecting their ability to polarize T cells. A cell culture model was defined using mixed cultures of BM-pAPCs and T lymphocytes NOD mice with stressed MIN-6 cells as a source of pancreatic ß cells antigens. We showed that the treatment with 300 µg/mL of LAEPAL induces a significant decrease in the CD4 and CD8 T effector lymphocytes proliferation from diabetic but not in non-diabetic mice, followed by a reduction of the IL-6 and IFN-γ cytokines release in the cell cultures supernatants. Moreover, we observed an increase of CD4+CD25+FoxP3+ Tregs in the cell cultures from diabetic mice. These results could be partially explained by the LAEPAL modulatory effects in BM-pAPCs, downregulating the CD86 co-stimulatory molecule expression, and increasing IDO-1 expression in F4/80+ BM-pAPCs. These results contribute to a better understanding of the polyphenols' immunomodulatory properties, meaning they could induce tolerogenic antigen-presenting cells, which could polarize T cells to a Treg profile and decrease the activity of CD4+ and CD8+ T effector cells.