Visual Abstracts: Thursday

Individuals on anti-CD20 therapy are susceptible to severe COVID-19, but vaccination protects them from hospitalizations and death. While the vaccine induces weaker antibody responses in anti-CD20 treated individuals than in immunocompetent controls, it elicits robust T-cell responses. We studied spike-specific CD8 T cells 4-6 months after the 2^nd and the 3^rd dose of SARS-CoV-2 mRNA vaccination in 23 uninfected anti-CD20 treated patients (18/23 on ocrelizumab for multiple sclerosis, 5/23 on rituximab for rheumatoid diseases) and 10 uninfected controls. Using activation-induced marker assay, we observed a higher frequency of spike-specific CD8 T cells in anti-CD20 treated patients than in controls after both doses. After two doses, the distribution of effector/memory subsets in spike-specific CD8 T cells was skewed in patients compared to controls with an increased frequency of terminal effector memory cells (CD45RA+CCR7-) at the expense of effector memory cells (CD45RA-CCR7-). After three doses the differences in memory subset distributions were restored. T-cell receptor (TCR) analysis of spike-specific CD8 T cells showed similar diversity and clonality in patients and controls. In contrast, the TCR repertoires after two versus three doses contained fewer overlapping clonotypes in patients than in controls. Altogether, our data suggest altered maintenance mechanisms of antigen-specific CD8 T-cell population in anti-CD20-treated individuals compared to controls and help optimizing the vaccine recommendations for immunocompromised individuals.

Aim: To assess the performance characteristics of a novel, single-use, microarray immunoassay prototype (MosaiQ Allergy Bet v 1) (Bet-v-1-MA), used with the fully automated MosaiQ^® system, for the detection of Bet v 1 specific IgE (sIgE), compared with ImmunoCAP™ Specific IgE (Phadia AB). Assay’s reproducibility and repeatability were also assessed.

Methods: 163 serum samples, characterized by the comparator method as reactive (n=63) or as non-reactive (n=100) were tested with Bet-v-1-MA. Magazine lot reproducibility was assessed over 5 days/3 lots/2 instruments; instrument reproducibility was evaluated over 5 days/1 lot/3 instruments; repeatability was assessed on 1 instrument and 1 magazine lot, 2 runs/day/5 days.

Results: After the protocol exclusion of 1 reactive sample, Bet-v-1-MA identified as reactive 60 out of 62 characterized reactive samples by the comparator and all 100 non-reactive samples; for a positive, negative and overall agreement of 96.8% (95%CI: 88.8%, 99.6%), 100% (95%CI: 96.4%, 100%) and 98.8% (95%CI: 95.6%, 99.9%), respectively. Agreement of Bet-v-1-MA with expected results in the evaluations of reproducibility (by lot and by instrument) and repeatability (869, 435 and 291 data points, respectively) were all 100%.

Conclusion: Bet-v-1-MA showed high concordance with the compared device for detecting Bet v 1 sIgE. Bet-v-1-MA demonstrated a high degree of precision in the reproducibility and repeatability evaluations. This device/platform has the potential to multiplex; further ongoing steps include the addition of other allergens to the microarray.

Guillain-Barré syndrome (GBS) is an autoimmune disorder of the peripheral nervous system (PNS) usually associated with preceding infections, which are believed to serve as a trigger for its development. We have recently revealed the presence of autoreactive memory CD4⁺ T cells targeting PNS-myelin proteins in GBS patients. Intriguingly, such cells showed a certain degree of cross-reactivity between distinct PNS-myelin antigens and, in patients associated with primary cytomegalovirus (CMV) infection, with CMV antigens (Súkeníková L et al, Nature, 2024). However, the cellular and molecular bases of such cross-reactivity remain elusive. Here, we use in vitro T cell screenings, TCR sequencing and computational tools to investigate the degree of cross-reactivity of autoreactive T cell clones in GBS patients. By screening over 200 autoreactive CD4⁺ T cell clones, encompassing over 50 different TCR Vb clonotypes, isolated from five patients, three of which had a known preceding infection (1 CMV and 2 of unknown origin), we describe that the vast majority of clonotypes displayed some degree of cross-reactivity between distinct PNS-myelin and/or CMV antigens. We are currently employing computational tools and in vitro functional screenings to predict and identify potential epitopes targeted by the cross-reactive T cell immunity in GBS patients. Overall, these results suggest that autoreactive and broadly cross-reactive T cells are a common feature of GBS patients and provide evidence that virus infections could trigger the disease through T cell cross-reactivity.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a proven therapy for hematologic diseases but it remains a challenge due to graft-versus-host disease (GvHD). Despite advancements in pharmacological and graft engineering techniques, current treatment of GvHD rely predominantly on broad-spectrum immunosuppression. This approach heightens the risk of other serious complications such as cancer relapse and systemic infections. The T-Safe project aims to develop an injectable and bioactive biomaterial scaffold to induce tolerogenic regulatory T-cells (T-regs) capable of long-term reduction or prevention of GvHD. The underlying research hypothesis is that confined uptake of apoptotic cell debris by professional phagocytes (DCs and/or macrophages) in a tolerogenic environment will lead to durable T-regs induction. Our preliminary data show that after implantation in mice, the biomaterial scaffold successfully promotes vascularization and attracts endogenous cells. In vivo loading of T-Safe with apoptotic cells shows that local APCs can phagocytize these cells, a crucial step for antigen presentation and subsequent T-reg generation. Lastly, injecting T-regs into the scaffold has shown promising results, with data indicating that these cells can migrate from the biomaterial scaffold to secondary lymphoid organs (SLOs), main site of T-reg-mediated GvHD suppression of GvHD. Altogether, our data indicate that T-Safe acts as a tolerogenic niche for T-reg generation, with adequate vascularization for immune cell entry and T-reg exit to draining lymph nodes.

Allergen immunotherapy (AIT) is the sole curative treatment for allergies, promoting the production of allergen-specific neutralizing IgG antibodies and tolerance. However, AIT is often linked with allergic adverse events. This project aims to pinpoint IgG-binding epitopes on the major birch pollen allergen Bet v 1 to generate hypoallergenic peptides, enhancing AIT's safety and efficacy.

Blood samples from 5 healthy and 30 birch-pollen allergic patients (20 with subcutaneous or sublingual immunotherapy) were collected. Rhinitis Quality of Life Questionnaires (RQLQ) quantified patient symptoms. Sera were assessed for allergen-specific IgE, IgG, and IgG4, and for basophil degranulation inhibition. IgG- and IgE-secreting B cells were analyzed via single-cell DropMap microfluidics. Linear and conformational IgG epitopes on Bet v 1 were determined using overlapping peptides and CLIPS technology. Candidate peptides were synthesized using Fmoc technology, and allergenicity was evaluated in a basophil degranulation assay.

All allergic patients had Bet v 1-specific IgE and higher RQLQ scores. AIT patients showed elevated IgG and IgG4 levels and stronger Bet v 1-specific basophil degranulation inhibition. Four non-overlapping Bet v 1-specific IgG-binding sites were identified. The peptides, designed from these regions, did not induce degranulation in sensitized basophils. 

This study utilizes a patient-derived approach to identify clinically relevant IgG-binding Bet v 1 epitopes for the design of hypoallergenic peptides. This method could pave the way to a safer and more effective AIT.

Aim: Periodic fever, aphthous stomatitis, pharyngitis, adenitis (PFAPA) is a cyclic autoinflammatory syndrome that typically manifests in young children. Our aim is to assess monocyte, dendritic cell (DC), and NK cell functionality in children with PFAPA.

Methods: Whole blood from children with PFAPA during and between flares (n=13), and age-matched healthy controls (n=10) was used for flow cytometric characterisation of innate immune cells at baseline and after six hours stimulation with LPS, a TLR7-agonist, or IFN-γ. We quantified production of IL-12/23, TNFα, IFNα, IFNγ, and IP10 upon stimuli. We also transcriptomic analysis on whole blood cells and measured serum cytokine levels by multiplex.

Results: We found decreased frequencies of most innate cells during PFAPA flares. Cell stimulations showed increased TNFα production in DCs and CD14+CD16+ monocytes after stimulation with LPS in PFAPA subjects compared to controls. TNFα and IL12/23 production was also increased between flares without stimulation. In serum, IL-1RA, IL-15, IL-6, and IL-10 levels were increased during flares, while levels between flares were similar to controls, except IL-10 and GM-CSF which were lower between flares than in controls. Blood transcriptomics showed upregulated pathways associated with innate antiviral responses during flares, while controls and between flares were similar.

Conclusion: Our initial analyses show that DC and monocytes in PFAPA may respond differently to innate stimuli. Further functional and genetic analyses of these cells could reveal pathways involved in this dysregulation.

Aim: Binding of IgE to FcεRI and CD23, contributes to different immunological processes involved in the development of allergic disease. Allergen-induced crosslinking of IgE-FcεRI complexes on allergic effector cells leads to cellular degranulation. Recently, we have described a fast acting anti-IgE molecule, termed KIH_E07_79, that rapidly disrupts IgE-FcεRI complexes, inhibits pre-activated human basophils ex vivo and stops pre-initiated systemic anaphylaxis in mice in vivo. Here, we further compared the capacity of KIH_E07_79 to neutralize IgE, inhibit receptor binding and suppress IgE production in primary human B-cells in vitro to classical anti-IgE antibodies.

Methods: IgE-receptor interactions were assessed by ELISA, SPR measurements and flow cytometry. Human B-cells were isolated from whole blood and stimulated with IL-4 and an anti-CD40 antibody. IgE production was assessed by ELISA, qPCR and flow cytometry.

Results: KIH_E07_79 efficiently inhibited IgE binding to both IgE receptors on primary human basophils and B-cells, respectively. Interestingly, KIH_E07_79 suppressed IgE production and reduced the number of IgE⁺ cells in primary human B-cells in vitro.

Conclusions: The disruptive anti-IgE molecule KIH_E07_79 efficiently inhibits IgE-binding to FcεRI and CD23. In addition to active desensitization of allergic effector cells, KIH_E07_79 inhibits IgE production and reduces IgE⁺ cells in primary human B-cells. These additional modes-of-action could potentially be of clinical relevance and might increase the efficacy of such a multi-level IgE targeting approach.

Aim: Autoinflammatory diseases (AIDs) pose diagnostic challenges due to their rarity and severity. IL-1β and IL-18 (a strong IFN-γ inducer) are pro-inflammatory cytokines of the IL-1 family that are secreted upon inflammasome/caspase pathway activation and therapeutic targets in AIDs. We aimed to measure the serum levels of IL-1β, IL-18, their inhibitors, IL-6 and IFN-γ in a large European cohort of AID patients (ImmunAID consortium).

Methods: We prospectively collected serum from 274 patients with monogenic or genetically undiagnosed AIDs, alongside 49 healthy controls. Commercial ELISAs measured IL-18, IL-18BP, IL-1Ra, IFN-γ, and IL-6, while a homemade ELISA quantified free IL-18 levels. IL-1β levels were assessed using an electrochemiluminescence assay. Correlations were established between cytokine levels and reported clinical and laboratory data. ROC curves were drawn to assess the diagnostic value of various parameters.

Results: Total IL-18 and free IL-18 were higher in Still’s disease than in other AIDs. IFN-γ was the most elevated in Still’s disease and IFN-γ levels correlated with total and free IL-18 (r=0.37 and 0.30 respectively, p<0.0001). In contrast, IL-1β, IL-1Ra, IL-18BP, and IL-6 levels did not differ between AIDs patients. Total and free IL-18 correlated strongly with ferritin (r=0.44 and 0.52 respectively, p<0.0001) and served as promising predictors of Still’s disease (AUC: 0.92 and 0.85, respectively).

Conclusion: Our findings highlight the diagnostic potential of IL-18 in Still’s disease and support targeting the IL-18-IFN-γ axis in its management.

CD19-CAR T cells are emerging as promising therapies yet exposed to on- and off-target toxicities possibly resulting from the formation of CAR-CD28 heterodimer. Modulations and functional implications of CAR-CD28 interactions have not been thoroughly studied.

We engineered 2nd generation CD19-CARs with either a CD28ζ or 4-1BBζ intracellular-domain (ICD) with selected mutations in the CD28 transmembrane-domain (TMD) targeting: (A) the CD28 evolutionary conserved YxxxxT motif (M1b=T171L), (B) the CD28-core polar amino acids (CYSxxxT->LLLxxxL=M4), (C) the GxxxA dimerization motif (to YxxxL=M5). Control wild-type CD28 TMD-CAR (WT) was used. CARs were linked to an EGFRt reporter and inserted into the TRAC locus of primary human T cells to control genomic integration.

Except for the M5 construct, all four mutants significantly reduced CAR-CD28 formation independently of the ICD assessed by the division index to anti-CD28 mAb stimulation and confirmed using a split fluorescent protein system. Mutating the threonine (M1b) strongly reduced cell-surface CAR expression and sensitivity against CD19 -low but not -high targets. CAR expression and sensitivity was progressively restored in the M4 CAR construct, although it remained inferior to WT-CAR and was ICD dependent.

Our data suggest that the TMD conformation dictates CAR expression level and its interaction with endogenous CD28. CAR T cell sensitivity can be modulated using selected TMD-ICD pairs, opening the perspectives to engineer safer CAR-T cell products for non-oncological applications.

Janus Kinase inhibitors (JAKi) are a novel class of disease-modifying antirheumatic drugs that selectively target JAK kinases to prevent/reduce the inflammation associated with the pathology. However, because JAKs are directly involved in the signalling of inflammatory cytokines needed for vaccine mode of action, their use have been associated with lower vaccine response. Our group recently showed that the cellular response to COVID-19 mRNA vaccine is impaired in patients treated with JAKi (see Madelon et al.)

Here, we investigated the impact of systemic JAKi  therapy on the local tissues targeted by the vaccines (muscle and lymph node (LN)) in mouse models. We assessed the immune response to mRNA-based and adjuvanted vaccines because, they efficiently mobilise innate immunity to promote robust and long-lasting vaccine response and this may be impacted by JAKi. We hypothesised that some tissue-resident cells, particularly those in the LN, are less affected due to limited drug accessibility to the tissue or variations in drug sensitivity at the cellular level. Our preliminary observations indicate that JAKi exert their effect mainly in blood and muscle, while the LN is relatively preserved from the inhibition of JAKI-dependent signalling. Additional research will  identify which immune cells or pathways are most affected by the JAKi in these organs and and how this translates into a weakened immune response to vaccinations. . Our data will complement observation in in vaccinated RD patients treated with JAKi and shed light on how these drugs impairs immunity in different organs.

Aim: Natural Killer (NK) cells are crucial in transplant rejection. Introducing inhibitory ligands for NK cells on pig cells could offer an effective means to suppress NK cell cytotoxicity. Cells expressing HLA-E can interact with human NK cells, inhibiting their destructive activity. Previous studies showed HLA-E transgenic pigs reduced NK cell lysis of porcine endothelial cells (PAECs). Our study examines HLA-E's protective effects in PAECs using 2D and 3D microfluidic systems.

Methods: Human NK cells were activated with IL-2 and co-cultured with WT and HLA-E/CD46 transgenic PAECs. Porcine TNF was used for endothelial activation to simulate the clinical conditions of xenografts. Live cell tracking was conducted in 2D and 3D microfluidic systems.

Results: HLA-E/CD46 transgenes reduced NK cell lysis of PAECs. Analysis of our 2D live cell tracking data revealed that NK cells killed PAECs mainly via apoptosis. NK cells exhibited greater attachment to WT PAECs, characterized by more constrained movements in single-cell trajectory analysis, whereas interactions with TG PAECs resulted in longer trajectories. In the 3D microchannel system, more attached NK cells and higher number of dead PEACs were observed in under flow conditions in WT PAEC. 

Conclusion: This study demonstrated that introducing HLA-E molecules into genetically modified porcine endothelial cells resulted in effective inhibition of human xenoreactive NK cells. These findings could inform future genetic modifications in pigs aimed at mitigating rejection mediated by human NK cells.

NOD-like receptors (NLRs) are best known for their role as innate immune sensors. Upon cellular perturbations, NLRs like NLR CARD-containing (NLRC) 4 and NLR pyrin-containing (NLRP) 3, switch from an inactive/close conformation to an open one, allowing the formation of a multimeric platform known as inflammasome. NLRC5, despite belonging to the NLR family, exerts a completely different function; it transcriptionally regulates Major Histocompatibility Complex class I (MHCI) genes, the core genes of the adaptive immune system. This NLR shuttles to the nucleus where it occupies the promoter of MHCI genes by interacting with epigenetic and transcription factors to regulate transcription. Despite its emerging role in anticancer immunity, the molecular mechanisms underlying NLRC5 function are still unknown. NLRC5 shows the same domains organization of other NLRs, which are known to undergo conformational changes once activated. It is therefore temping to hypothesize that similar structural configurations to those identified in inflammasome-forming NLRs regulate NLRC5 activity.

In this work, by using both biochemical and molecular biology approaches we aimed to identify key residues, in NLRC5, which will allow us to clarify NLRC5’s structure and function. Understanding the conformational changes of NLRC5 will shed light on its mode of action and will open new avenues for the development of modulators of NLRC5 activity and, thus, of MHCI levels, which is highly relevant to cancer immunotherapy.

Natural Killer (NK) cells patrol for virally infected and malignant cells. Clinical trials using chimeric antigen receptors (CAR)-NK therapy are showing promising results, especially in hematological tumors. This therapy is based on the NK-92 cell line expressing both CAR and the high-affinity Fc receptor 3A variant (CD16A, FCGR3A gene). The human non-Hodgkin lymphoma-derived NK-92 cell line lacks the surface expression of CD16A presumably due to a non-functional promoter; according to literature, FCGR3A mRNA is not expressed. Incidentally, by RT-PCR, we found the expression mRNA covering the extracellular domains of CD16 in NK-92 cells, yet at lower levels compared to freshly isolated human NK cells. Furthermore, we confirmed the absence of CD16A using several monoclonal antibodies by flow cytometry. This study aims to understand why CD16A surface expression is absent in NK-92 cells. We hypothesize that FCGR3A expression may be regulated at the post-transcriptional level, namely by micro-RNA (miRNA). By using miRNA-inhibiting oligonucleotides, we targeted two miRNAs overexpressed in CD16A-negative human primary NK cells. Preliminary results indicate that miRNA inhibition partially rescues CD16A expression in NK-92 cells. Additional factors potentially affecting CD16A surface expression in NK-92 cells, such as the lack of the transmembrane domain, are being addressed. This study could elucidate the mechanisms involved in the expression of CD16A and suggest the NK developmental stage from which NK-92 cells originated.

The use of pig organs is closer to become an alternative to the shortage of human organs, while the modulation of the acquired immunity is being explored. Our aim was to develop tools to assess the xenospecific responses of CD8⁺ T cells (CTL) to monolayers of porcine aortic endothelial cells (PAEC) from wild type (WT) and transgenic (TG) animals.

Human CTL were characterized and purified from PBMC that were stimulated with irradiated PAEC for 3 weeks. CTL’s specificity was tested by conventional cytotoxicity assay using primary PAEC_WT/TG as targets. In addition, cytotoxicity was analysed by live-cell imaging under static (2D) and microfluidic (3D) co-cultures at an effector to target (E:T) ratio of 1:1. Read outs were the numbers of CTL’s adhered and lysed PAEC (apoptotic bodies and necrotic Draq7⁺ cells).

After stimulation with PAEC, CTL phenotype underwent a transition from naïve to memory. In conventional assay, CTL were highly specific for PAEC_WT, and their cytotoxicity was dependent on the number of effector cells. Yet, the effect was evident only at high E:T ratios ( ≥ 5:1). In 2D live-cell imaging, CTL lysed PAEC_WT mostly via apoptotic pathways (11.26 ± 0.55%) compared to necrosis (6.02 ± 1.73%). Human PD-L1 in PAEC_TG protected them from apoptosis (0.13 ± 0.12%) and necrosis (1.17 ± 0.73%). In 3D perfusions, CTL adhered less to PAEC_TG, but the number of lysed Draq7⁺ PAEC_WT was similar.

Human CTL and PAEC interactions were assessed using a more physiological, sensitive, and reliable 2D and 3D live-cell imaging method under static and flow conditions than conventional assays.

Endosomal nucleic acid sensing by TLR7-9 plays a central role in antimicrobial immunity and in several autoimmune conditions such as systemic lupus erythematosus (SLE). We recently identified a novel innate adaptor, TASL, which controls TLR7/9-induced responses by assembling in an IRF5-activating signalling complex with the endosomal solute carrier SLC15A4 (Heinz et al, Nature 2020). We further demonstrated that this complex is “druggable” (Boeszoermenyi et al, Nat. Commun. 2023). Notably, all the components of this signaling axis are associated to SLE in GWAS. Here, we address the pathophysiological role of TASL and its murine paralogue TASL2 in vivo.

Newly generated Tasl, Tasl2 and Tasl double knockout mice were characterized with Slc15a4-deficient mice by monitoring TLR7/9 responses ex vivo and in vivo. The impact of Slc15a4 and Tasl deficiency was further investigated in chronic LCMV infection and in the pristane-induced SLE model.

Here we show that TLR7/9-induced IRF5 activation and cytokine production are impaired in Slc15a4^KO and Tasl^DKO primary immune cells. In vivo, Slc15a4^KO and Tasl^DKO show a profound defect in type I IFN and cytokine production upon stimulation with TLR7/9 agonists. Accordingly, Slc15a4^KO and Tasl^DKO mice displayed impaired antiviral responses to LCMV infection, while being strongly protected from disease in the pristane-induced SLE model.

This study demonstrates the critical role of SLC15A4 and TASL for TLR7/9-driven inflammatory responses in vivo, further supporting the therapeutic potential of targeting this complex in SLE and related diseases.

Tumor immunity arises from complex interactions between tumor cells, stromal cells and immune cells of the tumor microenvironment (TME). Tumor cells escape immunosurveillance by inhibiting directly on immune effector cells or acting on stromal cells, such as cancer-associated fibroblast, lymphatic and blood endothelial cell, playing key role in anti-tumor responses. Nociceptors are emerging in the literature as an integral part of the TME. Their main role is to convey noxious stimuli from the periphery to the central nervous system leading to pain sensation. When activated, these peripheral sensory neurons can secrete neuropeptides from their peripheral axons, known to act on stromal and immune cells in several contexts. Nociceptors, often present in solid tumor, can impact tumor growth by modifying anti-tumor immunity in different mouse model of cancer. However, the influence of nociceptors on tumor stroma is poorly described. By using a genetically-ablated nociceptor mouse model, we showed that sensory neurons promote tumor growth and alter TME (immune cell and stromal cell composition and phenotype) in orthotopic lymphangiogenic melanoma and breast cancer. The better characterization of TME modulation by nociceptors in different cancers may unveil new potential targets to enhance cancer treatments and response to immunotherapy.    

To exert their innate-like function B cells depend on pattern recognition receptors, such as Toll-like receptors (TLRs). Upon TLR stimulation and activation of downstream signaling pathways B cells undergo metabolic changes which prompt their effector maturation. c-MYC is a known driver of metabolic reprogramming in lymphocytes. Here we investigate the role of c-MYC in TLR stimulated B cells, particularly in regards to its impact on metabolism, cell activation, and cytokine production with the help of transcriptomics, metabolomics, and biochemical studies.

To elucidate how TLR engagement prompts B cells stimulation, we performed transcriptomics and metabolomics analysis of primary B cells following treatment with a TLR-ligand. Integration of transcriptomics and metabolomics analysis highlighted glucose metabolism as the most enriched pathway, with targets of the transcription factor MYC being the most prominently altered gene sets. Using mitochondrial stress assays we observed that the increase in both mitochondrial respiration and glycolysis observed after TLR stimulation of B cells, is limited when cells are additionally treated with the c-MYC inhibitor 10058-F4 for 48 hours. Similarly, inhibition of c-MYC lead to a decrease in the expression of the activation marker CD71 and in cytokines TNF and IL-6 compared to controls.

These findings position c-MYC as a significant contributor of B cell metabolism and effector function, especially with regards to cell activation and cytokine production, and provide new perspectives on the regulation of innate-like functions of B cells.

Background: In recent years new medications have improved the treatment options for patients affected by HAE. The Swiss HAE Cohort study started in March 2023 and we aim to assess the clinical characteristics and quality of life of patients with HAE living in Switzerland.

Methods/Design: This prospective, longitudinal, multi-center cohort study includes ten centers in Switzerland. Patients affected by hereditary angioedema Type 1, Type 2, HAE with normal C1-Inhibitor (nC1-INH HAE) and acquired angioedemas are included. Clinical data including attack-rate, medication use, comorbidities and quality of life with the Angioedema Control Test (AECT) and Angioedeme Quality of Life test (AeQoL) are collected at inclusion and at annual follow-ups.

Results: Mean age of the 62 patients (42 women and 20 men) is 45.9 [SD 20.39] years. Fourty-eight patients are under prophylactic therapy and 14 have therapy on demand. The AECT score for women is 13.5, for men 13.3. The AeQoL score for women is 16.5 and 11.5 for men. The overall attack rate in the last 6 months before inclusion is 1.5 [SD 1.9], with 1.8 for women and 1.0 for men. The most common comorbidities are arterial hypertension, cancer, dyslipidemia, cardiopathy, and depression/anxiety.

Discussion: HAE is well controlled in women and men. Quality of life in women is slightly decreased compared with men, this may reflect the fact that women are more affected by HAE. In the international context Swiss HAE patients showed the best results for AE-QoL, which could be associated with the high availability of the newest treatments.

Mechanisms and molecules associated with the evasion of the immune response by parasitic helminths can be exploited for the treatment of type 2 immune response disorders. Key mediators in type 2 inflammatory diseases are bioactive metabolites of arachidonic acid. Here, we identified the immune-modulatory glutamate dehydrogenase (GDH) in the larval extract of the helminth Heligmosomoides polygyrus bakeri (Hpb). We particularly assessed whether helminthic GDH (heGDH) regulates allergy or anti-helminth immunity by modulating immune cell metabolism.

Effects of heGDH on the metabolism of monocyte derived macrophages (MDM) were quantified by LC-MS/MS (eicosanoids, TCA metabolites) and seahorse analysis. Furthermore, heGDH treated MDM were subjected to RNA- and ChIP sequencing to assess effects on gene expression profiles and epigenetic changes. 

In macrophages, heGDH induced the production of prostanoids and 2-hydroxyglutarate, which contributed to the suppression of pro-inflammatory cysteinyl leukotrienes. Moreover, heGDH treated MDM showed an induction of regulatory genes, which depended on histone acetylation via p300. Treatment of mice with heGDH attenuated house dust mite- induced allergic airway inflammation in mice, while the treatment during Hpb infection resulted in chronicity and PGE₂ production in host macrophages.

Our findings thus suggest that heGDH mediates immune evasion by inducing the p300-prostaglandin axis. Thus, anti-inflammatory modulation of macrophages by heGDH may be translated into new immunomodulatory strategies for the treatment of inflammatory diseases.

Aim: Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths, with chronic inflammation in the intestine being a prominent catalyst. Interleukin-33 (IL-33) signaling plays a controversial role in intestinal inflammation and CRC, and its exact mode of action in the tumor environment is unclear. Here, we want to investigate the dual role (nuclear/ST2-independent vs soluble) of IL-33 in CRC.

Methods: CRC was induced by treating WT, St2^-/-, Il33^-/- and St2^-/-;Il33^-/- mice with azoxymethane (AOM) and dextran sodium sulfate (DSS). Bulk RNA sequencing has been performed on tumor vs. adjacent tumor-free intestinal tissue of these mice. MC38 cell lines transduced with different IL-33 isoforms were injected s.c. in WT, St2^-/-, Il33^-/-, St2^-/-;Il33^-/- mice to observe tumor growth.

Results: St2^-/- mice treated with AOM/DSS were protected from intestinal tumors, whereas Il33^-/- and St2^-/-;Il33^-/- mice developed colorectal tumors with similar numbers and load to WT controls. RNA sequencing revealed a distinct patternfor St2^-/- mice. In MC38 tumors, nuclear IL-33 showed a pro-tumorigenic effect, while soluble IL-33 acted anti-tumorigenic. Similar to the AOM/DSS model, St2^-/- mice injected with MC38 cells showed reduced tumor growth compared to Il33^-/- and WT mice.

Conclusion: IL-33 has a dual role in the AOM/DSS CRC model: pro-tumorigenic when soluble (ST2-dependent) and anti-tumorigenic in the nucleus (ST2-independent). In a model of s.c. injected MC38 cell lines, nuclear IL-33 has a pro-tumorigenic effect in tumors and an anti-tumorigenic effect in non-tumor tissue.

Aim : Myasthenia gravis (MG) is an autoimmune disease of multifactorial etiology, results from the deleterious effects of autoantibodies directed against components of the postsynaptic membrane of the neuromuscular junction, in which genetic factors and cytokines seem to play an important role. The aim of this study was to investigate potential associations of cytokines (SNPs) and MG in Algerian patients.

Materiel : We performed a case–control study that included 27 patients and 74 healthy subjects. Genomic DNA  was extracted using the salting out method. DNA quality was assessed on agarose gel, and its concentration was adjusted  using a Nanodrop spectrophotometer. Cytokines SNPs genotyping was performed by the polymerase chain reaction sequence–specific primers (PCR-SSP) method and the amplification bands were visualized using a gel imager.

Results : Our results showed that the TNF-α −308G/A (P < 0.005) and TGF-β1 +869T/T (P < 0.05) genotypes were more frequent among patients with MG compared with healthy individuals, whereas TNF-α −308G/G (P < 0.0001), TGF-β1 +869T/C (P < 0.05), and IFN-γ +874A/A (P < 0.05) were less frequent. Our results  showed that IL-10 and IL-6 SNPs did not show any significant difference in distribution between patients and healthy individuals.

Conclusion : We report the existence of associations between the polymorphisms of  cytokines (TNF-a, TGF-b1, and IFN-g) and myasthenia gravis in  Algerian patients.More comprehensive studies involving a larger number of patients and cytokines levels measurements will be required to further validate our observations

Abstract

Aim: Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by chronic systemic inflammation and the production of autoantibodies against nucleus. The Matrix metalloproteinases (MMP) and oxidative stress have been demonstrated to be essential mediators of SLE pathogenesis. The aim of this study was to analyze the activity of MMP-2 and MMP-9 in SLE patients and to investigate the effects of oxidative stress by analyzing the production of nitric oxide (NO) and malondialdehyde (MDA).

Methods: Activity levels of MMP-2 and MMP-9 were measured by zymography in plasma from 30 SLE patients and 50 healthy subjects. Plasma MDA and NO concentrations were determined using the thiobarbituric acid reactive substances technique and the Griess method.

Results: Our results showed a significant increase in MMP-9, NO, and MDA serum levels in lupus patients compared to controls (p=0.01; p <10^-3). However, no significant difference was found in MMP2 levels between the two groups (p >0.05). Furthermore, our results showed a remarkable increase in the plasma expression level of MMP-9 in patients with joint, skin, and kidney involvement. Furthermore, SLE patients with anti-RNP antibodies had higher serum concentrations of MMP-2 than anti-RNP-negative patients. The high MMP-9 levels correlated with the disease activity indice SLEDAI. No correlation was found between our patients' NO, MDA, and MMP production.

Conclusion: This study suggests that oxidative stress markers (NO, MDA) and MMP-9 could be useful inflammatory biomarkers for the progression and exacerbation of SLE.

Aim: The impairment of the epithelial barrier function is associated with various allergic and inflammatory skin disorders. The electrical impedance spectroscopy (EIS, Nevisense®) is a noninvasive tool to detect skin barrier integrity. We recently developed a method to evaluate skin barrier integrity in ex-vivo human skin (NativeSkin®) that exhibits normal skin barrier with almost all cell types. We investigated the mechanisms of epithelial barrier dysfunction and inflammation in human skin caused by surfactants which is an active ingredients of house cleaning products. Methods: NativeSkins were treated with different types of surfactants, sodium dodecyl sulfate (SDS), cocoyl methyl glucamide (CMG), and cocamidopropyl betaine (CAPB) at several dilutions. Skin barrier function was assessed by EIS. RNA sequencing and targeted multiplex proteomics analyses in skin lysate samples were performed. Treatment effects of an antioxidant, N-acetylcysteine (NAC), were investigated in the air-liquid interface cultured normal human epidermal keratinocytes. Results: The epicutaneous application of SDS/CMG/CAPB demonstrated the downregulations of EIS in a dose-dependent manner. Even CMG and CAPB showed a disruptive effect on the skin barrier integrity. SDS activated the pathways related to Oxidative stress and Endoplasmic reticulum stress. NAC treatment mitigated the increased permeability caused by surfactants. Conclusions: We demonstrated that exposure to surfactants can impair the skin barrier. Oxidative stress may play a key role in the skin barrier damage induced by surfactants.

The tumor-draining lymph nodes (tdLN) are the initial site of metastases and are the prime site for generating robust antitumor responses. In this study, we explored the efficacy of a universal immune activator (ImmAct) targeted to the tdLN. This approach can be viewed as an attempt to turn a cold unresponsive tdLN, into a hot, responsive site. The adjuvant antitumor efficacy of our novel intranodal injection was evaluated in an aggressive metastatic mammary carcinoma murine model. The cancer cells were inoculated subcutaneously in the lower quadrant of the mouse to provoke the tdLN (inguinal lymph node). The study encompasses a range of methodologies, including in vivo and in vitro assays and high-dimensional flow cytometry analysis. Our findings demonstrated that intranodal administration of ImmAct following the dissection of the primary tumor led to improved tumor-free survival and minimized weight loss. ImmAct led to both local and systemic alterations in the cellular and humoral immunity. Additionally, after ImmAct treatment, non-responders showed a higher rate of exhausted CD8⁺ T cells compared to responders. Indeed, our innovative approach surpassed the gold standard surgery of sentinel lymph node excision. Overall, intranodal administration of ImmAct yielded a robust antitumor immune response, offering protection against micrometastases and relapse.