Visual Abstracts: Friday

Aim: To assess the analytical performance of a novel, single-use, multiplexed microarray immunoassay prototype (MosaiQ AiPlex CTDplus; AliveDx, Eysins, Switzerland), used with the fully automated MosaiQ^® system, for the simultaneous detection of fifteen autoantibodies associated with CTD, compared with selected CE-marked devices.

Methods:

Serum samples characterized as reactive to ≥1 autoantibody (n= between 23 and 101 per analyte) or non-reactive (n=100) by CE-marked devices were included. For CCP, only reactive samples were included. Each individual non-reactive sample was tested with the investigational device once; reactive samples were tested in duplicates. Positive percentage agreement (PPA) and negative percentage agreement (NPA) for individual analytes were calculated.

Results:

Compared with relevant CE-marked devices, the investigational prototype showed, respectively, the following positive (PPA) and negative (NPA) percent agreement per analyte: CCP 86%/NA, chromatin 75%/95%, CENP-B 98%/97%, DFS70 94%/100%, dsDNA 95%/97%, Jo-1 93%/96%, Ribosomal P 98%/96%, RNAP3 76%/95%, Scl-70 81%/95%, Sm 88%/95%, Sm/RNP 98%/98%, SSA-60 99%/100%, SSB 93%/97%, TRIM21 99%/100%, U1RNP 97%/98%.

Conclusions:

The investigational prototype demonstrated substantial agreement with the compared CE-marked devices. Further studies will allow for expanded performance assessment of the investigational device. This fully-automated multiplexed platform has the potential to contribute to optimizing CTD evaluation by simplifying complex testing pathways and analyzing large number of samples per day.

Background: Effective management of primary and secondary immunodeficiency disorders requires experienced centers which are limited in number and funding. Synergizing specialized private practices and university centers enhances patient care, expedites access to specialized care and comprehensive laboratory analysis, and facilitates participation in prospective patient cohorts including genetic investigations.

Methods: From an initial cohort of 18 patients (median age 52 yrs, 8 females), we present two cases illustrating the collaborative care model. Case 1 showcases a syndromal immunodeficiency with musculoskeletal involvement, emphasizing the role of private practitioners in diagnosis and management. Case 2 depicts late-onset antibody deficiency with pulmonary manifestations, highlighting university resources for advanced diagnostics and genetic analysis.

Results: Collaboration reduces diagnostic delay (median 4.5 yrs), tailors treatment plans, and provides access to cutting-edge diagnostics and research. Integrating clinical immunologists in private practices and university centers optimizes patient care, increases local awareness, fosters ongoing research and monitoring for immunodeficiency disorders.

Conclusion: Integrating specialized clinical immunologists in private practices and university centers optimizes patient care for immunodeficiency disorders. This seamless collaboration not only addresses immediate clinical needs but also ensures inclusion in prospective cohorts for ongoing research and long-term monitoring, ultimately improving outcomes for patients.

MHCI molecules play a central role in adaptive immunity. They sample peptides generated within the cell and signal if the cell is transformed or infected to effector cells of the immune system. Studies are lacking on how cellular metabolism might further impact antigen presentation.

We thus investigated the effect of altered metabolism on MHCI levels using mouse bone marrow-derived dendritic cells (BMDCs). Mouse BMDCs grown in galactose-containing medium, or in the presence of inhibitors of the glycolysis pathway exhibited higher MHCI surface expression as compared to the levels of BMDCs cultured in glucose-containing medium. As the total bolus of MHCI molecules was not increased, we assessed the kinetics of MHCI surface export. BMDCs cultured in the presence of galactose and inhibitors of glycolysis recruited MHCI faster to the surface. Notably, RNA-sequencing results highlighted a significantly higher expression of genes related to the anterograde transport from the endoplasmic ER to the Golgi apparatus and downregulation of genes related to ER-Golgi retrograde transport in BMDCs grown in galactose, in line with the altered trafficking of these molecules between the Golgi and cell surface. This increased surface expression of MHCI is correlated with the enhanced activation potential of CD8+ T cells in vitro.

Collectively, these results show that the metabolic state of DCs regulates the MHCI through post-translational mechanisms and influences the activation of T cells. These findings might be highly relevant to diseases such as metabolic disorders, cancer, and infections

The clinical indications for Intravenous immunoglobulins G (IVIG) are steadily increasing. However, there is a limited supply of human plasma to produce it. Alternatives include engineered recombinant molecules, e.g. hexamer (HEX) composed of Fc from IgG1 fused to IgM μ-tailpiece. One of the mechanisms of action of IVIG is blocking Fc-gamma receptors (FcγRs) thereby inhibiting antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by natural killer (NK) cells.

The goal of this study was to measure the effect of IVIG and HEX-variants on NK cell viability and function. NK cell viability was tested by vital cell dye exclusion and Annexin V apoptosis assay (flow cytometry). The cytokine release by NK cells was measured after culture with IVIG or HEX-variants. NK cytotoxicity (ADCC and direct cytotoxicity) were assessed by non-radioactive release assays after NK culture in the presence of IVIG or HEXs at variable times. Targets consisted in Daudi plus anti-CD20 or K562 cells for ADCC and direct cytotoxicity, respectively.

HEX was the most potent inhibitor of ADCC (90% inhibition), after both 1h and 16h of culture with human NK cells, followed by IVIG. Remarkably, direct cytotoxicity was also inhibited by the tested molecules, with a higher inhibition mediated by IVIG (55% inhibition). Both IVIG and HEX induced limited cell death (<20%). All molecules but not Fc monomer triggered the release of TNFα and IFNγ by NK cells.

In conclusion, HEX inhibit NK cell effector functions and may have the potential to substitute IVIG for the treatment of auto-antibody-mediated diseases.

Introduction: Lymphocyte function-associated antigen 1 (LFA-1) is a T cell integrin regulating adhesion, immune synapse formation and effector function. LFA-1 is increasingly selected as target for cancer immunotherapy. The affinity and avidity of LFA-1 are regulated by interactions with lipid rafts stabilised by sphingomyelin complexed cholesterol.

Hypothesis: We hypothesise that perturbation of plasma membrane (PM) cholesterol alters integrin mediated T cell adhesion and function.

Aim: Establish PM cholesterol perturbation models to study the biophysical mechanisms by which PM cholesterol controls LFA-1 mediated T cell function.

Methods: PM cholesterol distribution and content were perturbed in Jurkat and primary effector T cells using autogramin-2, a sterol-derived synthetic compound, and methyl-b-cyclodextrin complexed cholesterol (MbCD-chol) respectively. The effect on T cell adhesion, and effector function was assessed.

Results: Increasing total PM cholesterol content with MbCD-chol altered neither LFA-1 mediated adhesion nor anti-tumour cytotoxic killing. Yet, perturbing PM cholesterol distribution with autogramin-2 significantly reduced adhesion and LFA-1 mediated immune cell function. This phenotype was recapitulated through liberation of sphingomyelin complexed cholesterol.

Conclusion: These results suggest that stabilisation by sphingomyelin-complexed cholesterol is necessary for LFA-1 dependent effector T cell function. The implications of these findings for cancer-immunotherapy are subject of further study.

Background: Omalizumab, a therapeutic monoclonal anti-IgE antibody, is successfully used in several allergic conditions. Its moderate affinity for IgE and weak disruptive efficacy, however, necessitate frequent dosing over prolonged periods. Here we characterized two new high-affinity omalizumab variants with improved disruptive efficacy, which could potentially optimize treatment outcome.

Methods: We compared binding characteristics and treatment efficacy of the new anti-IgE antibodies C03-H1L2 and C03-H2L2 with omalizumab. Surface plasmon resonance was used to measure binding kinetics and stoichiometry was assessed by size exclusion chromatography. In vitro assays were employed to evaluate receptor inhibition profiles, disruptive efficacy and anaphylactogenicity. In vivo efficacy was explored in a passive systemic anaphylaxis mouse model.

Results: Both omalizumab variants demonstrated increased affinity to human IgE. While immune complex formation and receptor inhibition profiles remained similar to omalizumab, the variants showed significantly improved disruptive efficacy. Both C03 antibodies removed IgE with faster kinetics from its high-affinity receptor FcεRI than omalizumab. In the systemic anaphylaxis mouse model, the C03 antibodies reached complete desensitization within 36 hours of single dose application.

Conclusions: These findings demonstrate that successfully established therapeutic anti-IgE biologicals, like omalizumab, can be optimized. Such antibody variants could yield novel and potentially more efficient therapeutic options for the treatment of allergies.

JAK inhibitors (JAKi) target inflammatory cytokine signaling, potentially reducing efficacy of vaccines and the induction of persistent cellular immunity. We performed a comprehensive analysis of the innate and adaptive immune responses in 32 healthy participants and 22 rheumatic disease (RD) patients treated with JAKi who received a third dose of mRNA COVID-19 vaccine.

Levels of SARS-CoV-2 spike-specific antibodies were significantly lower in JAKi-treated patients as compared to controls before and 1 month after the 3rd dose, although the overall response remained high in both groups 6 months later. In contrast, spike-specific CD8+ T cell responses in JAKi-treated patients were either no detectable or lower than controls at all time points, correlating with lower serum levels of IP10, IL1Ra, MCP1 measured 1 day after vaccination. Gene-set enrichment analysis of gene expression in whole blood at the same time point revealed the absence of JAK-STAT and interferon-related pathways in patients. Potential defects in innate cell function were assessed at time of vaccination by stimulating PBMC with different TLRs or cytokines in vitro. Interferon-gamma stimulation of NK cells from JAKi-treated RD patients was impaired, suggesting that these cells play a critical role to CD8+ T cell response induced by mRNA vaccine in vivo.

In conclusion, CD8+ T cell response to mRNA vaccine is impaired in JAKi-treated patients. Our in-depth immune profiling identified key innate pathways associated with immune response to mRNA vaccines.

Bladder cancer was ranked as the 4^th most frequent cancer in males and 14^th in females in America and Europe in 2022. Among all the patients, 20% is diagnosed with muscle-invasive bladder cancer (MIBC), characterized by disruption of the urothelium and invasion of the muscle layer by tumor cells. The gold standard treatment is cisplatin-based chemotherapy followed by radical cystectomy, but it confers patients a survival of only 15 months. The use of immunotherapy, including antibodies targeting the PD-1/PD-L1 axis, has been approved but the response rate is ranging from 15% to 24%, which highlights the urgent need to develop new therapeutic options. Radiotherapy (RT) is used in BC for bladder-sparing protocol, which include very few patients who will undergo high dose RT combined with chemotherapy. However, low-dose RT has been investigated for its capacity to stimulate the TME and could be used in combination with immunotherapy.

Our project aims to detail the effects of different doses of RT on the TME of MIBC. We used a mouse model of MIBC that recapitulates features of the human disease, presents a highly suppressive TME and resistance to anti-PD-1 treatment. MIBC-bearing mice were irradiated with different doses of X-rays and the immune compartment was characterized by flow cytometry 7 days post-RT. Preliminary data showed an increase of anti-tumor immune cells, especially CD8 T cells, 7 days post-RT with low dose of X-rays compared to untreated mice. The beneficial effect of RT can lead to the development of novel therapeutic solution for MIBC patients, especially combined with immunotherapy treatments.

The success of allogeneic hematopoietic stem cell transplantation (alloHSCT) partly relies on the beneficial graft-versus-leukemia effect, mediated by donor-derived alloreactive NK cells through their killer-cell Immunoglobulin-like receptor (KIR). Conflicting results have been reported with a scarcity of data interrogating the effect of KIR allelic polymorphism. With the aim to fill this gap, donor KIR genes derived from a national cohort of 1292 donor/recipient alloHSCT pairs were genotyped at a high-resolution and combined with the recipient HLA genotype to test their association with various transplant outcomes. In univariate analysis, we observed a lower progression-free survival (PFS) (p=0.01) and higher transplant-related mortality (TRM) (p=0.012) in donor/recipient pairs bearing KIR2DS4*00101_D - HLA-C2/A11_R interactions, which was confirmed by multivariate analysis (PFS: hazard ratio [HR], 1.38, p=0.0025; TRM: HR = 1.53, p=0.018). PFS was also significantly influenced by the number of KIR2DL3_D – HLA-C1_R interactions (HR=1.08, p=0.02). In addition, these recipients showed a higher risk of developing chronic graft-versus-host disease with KIR2DS4*00101_D - HLA-C2/A11_R interactions (HR=1.31, p=0.021) or by the strength of KIR2DL2/L3_D - HLA-C1_R interactions (HR=1.21, p=0.009). Recipients lacking a KIR2DS2_D – HLA-C16_R interaction had a lower rate of relapse (p=0.0023), although the significance was lost in multivariate analysis. Our study indicates the potential detrimental effect of KIR activating interactions, especially KIR2DS4, potentially due to its sustained expression in an overactivated environment.

Aims

Mast cells (MCs) are long-lived tissue resident cells capable of storing a wide range of biologically active mediators in their granules. Recent studies have uncovered the antigen storage capabilities of MCs, which, coupled with their extended lifespan, suggest a potential immunological antigen storage function similar to that observed in lymphocytes.

Methods

Antigen storage capabilities of mouse and human MCs were investigated through flow cytometric analysis. In vitro cultured MCs were tested for the uptake and storage of Fel d 1, Fel d 1 coupled to Qb Virus-like particles (VLPs; Qb-Fel d 1), and immune complexes of Fel d 1/Qb-Fe l d 1 with IgG as well as IgE. Additionally, we investigate antigen-transfer from MCs to other immune cells, such as Dendritic Cells (DCs), and lymphocytes. Antigen localization will furthermore be investigated through imaging flow cytometry.

Results

Our data confirms the antigen storage capabilities of MCs up to 21 days. Storage duration was significantly enhanced in presence of IgE antibodies. Moreover, the role of MCs as antigen storage cells is currently being investigated in vivo to discern their impact on shaping the immune response. Preliminary results show in vitro MC antigen storage. Furthermore, in vitro antigen transfer from antigen-bearing MCs to DCs was observed.

Conclusion

The role of MCs as long-term antigen reservoirs, along with their ability to transfer antigens to DCs, implies a novel contribution to immune mechanisms that has yet to be fully understood, potentially impacting various diseases, including autoimmune conditions.

Ferroptosis is a form of regulated cell death induced by iron-dependent lipid peroxidation. Alternatively activated macrophages (AAM) play key roles in type 2 immunity against helminth parasites, despite being highly susceptible to lipid peroxidation and ferroptotic cell death. Here, we identify transglutaminase-2 (TG2), a conserved marker of human and murine AAM, as a central driver of ferroptosis susceptibility. When infected with a parasitic nematode, wildtype mice show a profound increase of lipid peroxidation and ferroptotic cell death in the small intestine, while mice lacking hematopoietic TG2 are protected from ferroptosis. While products of arachidonic acid oxidation (eicosanoids and 4-hydroxynenal (4-HNE)) as well as membrane lysophospholipids are reduced in the absence of hematopoietic TG2, oxidized PE species accumulate, indicative of a TG2-dependent pro-ferroptotic lipid remodeling. In line, AAM from TG2 deficient mice show an increased PUFA/ MUFA ratio in membrane phospholipids as well as exaggerated accumulation of oxidized PE species and reduced cell death upon ferroptosis induction. Thus, TG2 drives ferroptosis susceptibility in type 2 immunity by remodeling the phospholipid metabolism of a key immune cell type.

When immune cells infiltrate the tumor, their functions can be altered. For example, T cells can reach a state of “exhaustion”, characterized by expression of inhibitory receptors, such as programmed cell death protein 1 (PD-1). SH-2 domain-containing phosphatase (SHP)-2 is reported to be an important component of the inhibitory effects of PD-1 by interacting with it. Paradoxically, SHP-2 is best known as a positive regulator downstream of growth factor receptors and inhibitors targeting this phosphatase are currently under clinical evaluation to dampen cancer progression.

To date, the biological meaning of the interplay between SHP-2 and PD-1 and their downstream signalling remains an open question. Whereas their interaction is thought to be essential for T cell exhaustion, in vivo data from our lab indicate that Shp-2 is dispensable for PD-1 signaling in T cells and that its homologous phosphatase SHP-1 does not play a redundant function.

This brought us to:

1. generate mice lacking PD-1 in the T cells, which better control tumor and will enable us to further study the underlying molecular mechanism;
2. evaluate the role of the PD-1/SHP-2 axis in other immune cells mediating anticancer responses; single deletion of these proteins in myeloid cells delays tumor growth, supporting their function in establishing tumor suppression.

We are currently investigating how PD-1 and Shp-2 diverge or converge into a tumor-supportive role of lymphoid and myeloid cells, hoping that our results will highlight the potential benefits and risks for future anti-cancer therapies.

Alzheimer’s disease (AD) is the most common form of dementia and may contribute to 60–70% of cases. Accumulation of extracellular plaques containing amyloid-b peptide characterizes one feature of the neuropathology of AD.

The pathogenesis of the disease appears 10–20 years before AD's clinical manifestation. Thus, the goal of innovative treatments should be to postpone or stop the disease before it reaches the preclinical stage.

Recently, 2 mAbs have been approved for the treatment of AD, namely Aducanumab and Lecanemab. In the current project, we design vaccines to induce polyclonal antibodies of the same specificity as the approved mAbs. We developed a vaccine based on virus-like particles (VLP) derived from the cucumber mosaic virus (CuMV_TT) fused with different epitopes of Abeta_1-42. Our experiments have now shown that genetic fusion of different epitopes of Ab (Abeta_3-6, Abeta_1-6 and Abeta_1-7) to the surface of CuMV_TT resulted in three vaccine candidates which are highly immunogenic and induced IgG antibodies against the full-length Abeta_1-42. Moreover, IgG generated by the CuMV_TT-Abeta_3-6 vaccine, showed the same recognition profile as approved mAb Aducanumab, preferably binding oligomers. Additionally, the generated antibodies specifically bound Abeta plaques in human brains as well as in mouse brain tissue. Treatment with the CuMV_TT-Abeta_3-6 vaccine, in transgenic mice prone to the disease, resulted in a decrease in the amount of Abeta plaques in the brains of these mice. In addition, we are now testing CuMV_TT-Abeta_1-16 for the induction of Lecanemab type antibodies.

Trained immunity of innate immune cells confers protection against pathogens, however, the contribution of innate immune memory to type 2 immune responses such as allergy or helminth infection is only beginning to emerge. We previously showed that intranasal house dust mite (HDM) exposure alters bone marrow (BM) progenitor cells implicating central trained immunity in a murine model of allergic airway inflammation (AAI).

To assess the reprogramming of innate immune cells in BM progenitors as well as locally in the lung in two settings of type 2 immunity.

We studied the transcriptional reprogramming of bone marrow-derived macrophages (BMDM) and alveolar macrophages (AMs) in in vivo models of AAI and helminth (Heligmosomoides polygyrus bakeri (Hpb)) infection in wildtype and transgenic (Ifnar^fl/fl x Vav1^Cre) mice using RNA seq and qPCR.

RNA seq analysis of BMDM from HDM-sensitized WT mice revealed a strong induction of type 1 IFN signaling. Global Ifnar KO mice showed no reduction of AAI whereas the deficiency in type 1 IFN signaling in the hematopoetic compartent resulted in reduced inflammation. Ifnar^fl/fl x Vav1^Cre mice displayed lower BAL cell counts and reduced airway eosinophils compared to WT during AAI. RNA seq data of AMs from Ifnar KO mice indicated that type 1 IFN might contribute to airway remodeling. We further observed an upregulation of several markers of type 2 immunity in BMDMs from Ifnar^fl/fl x Vav1^Cre mice infected with Hpb.

Type 1 IFN signaling drives type 2 inflammation and regulates the reprogramming of innate immune cells during AAI and helminth infection.

Aim

Double negative (DN) T cells are a subpopulation of T lymphocytes involved in immune response and regulation lacking the surface expression of the CD4 and CD8 co-receptors. They normally express the T cell receptor (TCR)-γδ or, in small proportion, -αβ.

Method

We report the case of a 4-year-old boy with an aberrant T lymphocyte population of CD3+/DN/TCRαβ-/TCRγδ- and IgG2 deficiency.

Results

The patient came to our attention at the age of 4 months, when he was hospitalized for an incomplete Kawasaki disease and a post-natal CMV infection treated with Valganciclovir.

A complete immunological work-up showed normal serum immunoglobulin levels but an IgG2 deficiency. Lymphocyte subpopulations presented an increased percentage of DN T cells (10% of lymphocytes), with an aberrant TCRαβ-/TCRγδ- population, equal to about 30% of DN cells and 4% of total CD3+. Further investigation on this subset showed a proper expression of the other T cell lineage markers (CD2,CD5,CD7), the absence of CD56, and a polyclonal rearrangement of TCR gene. A deeper characterization excluded an expansion of the recent thymic emigrant lymphocytes or of activated T cell fraction. 

Conclusions

We are dealing with a case of a patient with IgG2 deficiency and a CD3+/DN/TCRαβ-/γδ- population, stable in percentage, and where the normal expression of T cell markers together with the absence of a clonal TCR rearrangement seems to exclude an immuno- or onco-hematological pathology. This population of unknown origin and significance is still under investigation and further analysis are scheduled.

Lymphatic endothelial cells (LECs) form lymphatic vessels (LVs) that expand upon inflammation, a process called lymphangiogenesis. They play an important role in cancer as they can drain immune cells to the tumor and have been shown to be beneficial for immunotherapy. However, LECs can also promote metastasis and inhibit anti-tumor immune response. This project aims at harnessing LECs to improve anti-tumor activity in lymphangiogenic tumors. The laboratory engineered several mouse tumor cell lines to overexpress the lymphangiogenic factor VEGF-C (VC) to increase the lymphangiogenesis associated to the tumor growth. Preliminary data have shown that some molecules that might be implicated in the ability of LECs to regulate metastasis or immune cells are upregulated in LECs from highly immune infiltrated tumors such as the colorectal model MC38ovaVC, while they are downregulated in less immune infiltrated tumor models such as the B16ovaVC melanoma model. CSF-1 and IL33 are 2 of these molecules that are being studied. CSF-1, colony stimulating factor 1, is a hematopoietic growth factor. Proliferation, survival, and differentiation of macrophages depend on this cytokine. IL33 is a cytokine expressed by endothelial cells, epithelial cells, and fibroblastic reticular cells (FRCs). Known as an alarmin, its role can be very interesting in the modulation of the immune response. Harnessing the LEC functions by modifying the expression of these molecules could improve anti-tumor activity and/or decrease metastasis. 

Aim: The importance of immunomodulation in bone healing is widely recognized, and the field of osteoimmunology is rapidly growing in significance. Large bone defects often need additional material to fill voids and support bone formation. In this study, we investigate the immunomodulatory properties of novel bone-forming materials using high-throughput techniques.

Methods: The response of PBMCs to hydrogels based on tyramine-modified hyaluronic acid gel (THA), agarose, and fibrin sealant, as well as beta-tricalcium phosphate and hyaluronic acid particles, was examined in monolayer and transwell cultures. Our experiments included measuring cell proliferation using thymidine assay, cell viability with propidium iodide in flow cytometry, and targeted proteomics using Proximity Extension Assay.

Results: The data showed a slight increase in cell proliferation when exposed to THA gels, agarose, or fibrin sealant. In the targeted proteomics data, fibrin sealant and bone particle combinations led to upregulated proteins associated with bone remodeling and low inflammatory response. Conditions related to agarose and bone particles did not induce significant alterations in our biomarker panels. We identified the matrisome, plasmacytoma, apoptosis, and immune response-related pathways for the fibrin sealant as well as its combination with filtrated bone particles.

Conclusions: PBMC response to fibrin sealant and its combination with filtered bone particles point out reduced bone resorption and potentially increased bone formation.

Background/Aim: Sensitizations to pollen or house dust mites can directly impact atopic dermatitis (AD). Some patients report an AD exacerbation upon exposure. This study aims to gain more insights into the sensitization patterns to pollen allergens of AD patients in Europe compared to sub-Saharan Africa (SSA).

Methods: We included 20 AD patients and 10 healthy controls (HC) from each center in Switzerland (CH), Tanzania (TZ) and Madagascar (MD) in this case-control study. We analyzed the sera with the ALEX² Allergy Explorer, measuring total IgE and 300 specific IgE antibodies.

Results: The prevalence of ARC and allergic asthma in AD patients was similar in all countries (RCA: 60% in TZ, 70% in CH, 75% in MD; asthma: 25% in TZ, 30% in CH, 20% in MD). Total IgE levels were significantly lower in the CH than in both SSA HC groups (TZ vs. CH: p=0.03, MD vs. CH: p=0.04).

We found major differences in sensitization patterns to inhalative allergens, especially to grass pollen allergens. Swiss AD patients were sensitized to various grass pollen such as bahia grass (Pas n), bermuda grass (Cyn d, Cyn d 1), common reed (Phr c), perennial ryegrass (Lol p 1), rye (Sec c_pollen), and timothy grass (Phl p 1, Phl p 2). However, no AD patient or HC subject from the SSA cohort was sensitized to the tested grass pollen.

Conclusions: The absence of grass pollen sensitizations in SSA is most likely due to a lack of commercially available sIgEs tailored to the African environment. More research is needed to identify local types and counts of pollen to develop suitable diagnostics and therapies.

The sympathetic nervous system (SNS) has been shown to strongly impact immune responses. SNS fibers innervate all tissues and release the catecholamine noradrenaline locally, which binds cell surface alpha- or beta-adrenergic receptors. The skin is densely innervated by SNS fibers and represents one of the first lines of defense of an organism against pathogens. It harbors numerous immune cells that act as sentinels, reacting to pathogens or malignant cell proliferation by mounting a robust inflammatory response. Leukocyte migration through the lymphatic vessels network is crucial to mount an efficient adaptive immune response. The aim of this project is to dissect how dendritic cell (DC) trafficking in the skin is governed by the SNS.

To study the impact of the noradrenergic signaling on DC trafficking, both pharmacological inhibition of the B2 adrenergic receptor (B2AR), as well as a B2AR full KO (B2KO) mouse model were used, as this is the main adrenergic receptor expressed on leukocytes. Different ex vivo and in vivo cell trafficking methods were employed to study DC draining into lymphatic vessels.

Our data point to a role of noradrenergic signaling in modulating DCs migration, as the pharmacological inhibition or the lack of B2AR, induces a higher DC migration capacity towards lymphatic vessels and draining lymph nodes, compared to controls.

This study will gain fundamental insights into how the immune system is tuned by the SNS, which should lead to the pharmacological optimization of immune responses to infections, vaccination regimes as well as anti-tumor therapies.

Aim : Rejection events (REs) are commonly caused by acute and chronic injuries that alter graft function, leading to kidney allograft rejection (KAR) and graft failure.  REs are triggered following allorecognition of the alloantigen (donor human leukocyte antigens [HLA]) by T cells. Our study aimed to investigated the association between the TNFA-308A/G SNP  and KAR in Algerian patients who underwent kidney transplantation and to evaluate the possible associations of SNP with PG-a-HLA-Ab.

Methods : Genotyping of the TNFA -308A/G SNP was performed for 116 patients  selected randomly  using a case- control strategy from transplantation centers in Algeria  and 197 healthy individuals (HI).  All patients received transplants from living donors. Genomic DNA (gDNA) was extracted using the salting out method. The TNFA -308A/G SNP genotyping was performed using real-time polymerase chain reaction (PCR) with a TaqMan 5′ nuclease assay. 

Results : The frequencies of TNFA -308A allele and AA genotype were higher in the PWR than in the HI groups (p = 0.001, OR = 2.26, p = 0.0004, OR = 5.53, CI- 1.89-16.6 respectively)., particularly among PWR patients with de novo anti-HLA  antibodies. However, the frequency of TNFA -308G allele was lower in the PWR than in the PWoR (p = 0.001, OR = 0.3, and the HI group (p = 0.001, OR = 0.44) 

Conclusion : Our results suggest an association of the TNFA -308A allele who have PG-a-HLA-Ab might have a higher risk. Thus, therapeutic strategies can be adapted to minimize KAR risk in patients who have a genetic proclivity for increased pro-inflammatory TNF-α activity.

Vaccines are powerful tools against various immune-regulated diseases, including cancer. The magnitude of vaccine-induced immune responses is known to vary with the time of day (TOD) of vaccination in both mice and humans. However, the mechanism behind these TOD-dependent responses remains to be elucidated. In this study, we investigated how different immunological adjuvants affect TOD-dependent vaccination responses and whether we can manipulate them to increase vaccine efficacy. To identify adjuvants that elicit strong time-dependent responses, we screened for receptors with time-dependent gene expression and stimulated them with their ligands at different times of the day. Preliminary results show that the adjuvants LPS and Poly I:C induce a stronger immune response on primary mouse bone marrow-derived dendritic cells (mBMDCs) when administered at a specific time of day, measured by qPCR analysis. Together, we aim to uncover TOD-dependent immune responses to adjuvants and assess a comprehensive network of genes and proteins that drive this response. When completed, this work will provide a mechanistic understanding of TOD-dependent vaccine responses that could be used to improve vaccination strategies in human trials.

Aim

In celiac disease, HLA-DQ2.5 facilitates the presentation of deamidated gluten-derived peptides to antigen-specific CD4+ T-cells, triggering immune activation and enteropathy. The adoptive transfer of engineered (e) gluten-specific regulatory T-cells (Tregs) may suppress the effector function of pathogenic T cells.

Methods

Five TCRs recognizing the immunodominant DQ2.5-glia-a1a and glia-a2 epitopes were tested in a TCR-deficient NFAT-luciferase cell line. We next replaced the endogenous TCR of human primary T-cells and Tregs through homology-directed repair targeting the TCR alpha and beta constant loci using AAV and CRISPR-Cas9. The same strategy was applied to murine T-cells and Tregs, and evaluated in HLA-DQ2.5+ C57BL/6 transgenic mice exposed to gliadin via oral gavage.

Results

Jurkat cells transfected with the mRNA of any of the five TCRs exhibited peptide-specific NFAT activity. Human primary eCD4+ T cells displayed a similar mean functional avidity (EC50) for either specific peptide but not for glia-a1a/a2 overlapping epitopes. Human eTregs demonstrated superior suppressive activity compared to polyclonal Tregs in an EC50-dependent manner. In vivo, eCD4+ T-cells migrated and proliferated in the small intestine and draining lymph nodes. This proliferation was suppressed only in the presence of gluten-specific eTregs.

Conclusion

Redirecting Tregs to a single immunodominant gliadin-derived peptide could be sufficient for selective trafficking into the gut and draining lymph nodes. These cells hold therapeutic potential for restoring gluten tolerance in celiac patients.

Aim: Recent research demonstrates specific variants of gamma delta (γδ) T cells possess innate properties, previously deemed only adaptative. Our understanding of the innate-like behavior of γδ T cells upon encountering virus-like nanoparticles (VLPs) remains limited. Exploring the interaction between γδ T cells and VLPs presents an intriguing avenue for research, offering insights into how VLPs can serve as an effective platform to enhance their expansion and activation.

Methods: We created novel plant-derived VLPs, engineered to incorporate TLR ligands to effectively stimulating the innate immune system. The study encompasses in vivo and in vitro assays, FC analysis and RNA sequencing.

Results: Our findings demonstrate robust uptake of our novel VLPs by γδ T cells, leading to significant expansion in draining lymph nodes upon subcutaneous injection. Interestingly, in mice lacking TLR7 or C3, γδ T cell expansion was reduced, suggesting their involvement in the immune response triggered by our VLPs. Subsequent analysis of γδ T cell Vγ1 and Vγ4 subtypes post-VLP administration revealed substantial expansion and distinct activation profiles. Ongoing RNA sequencing will unveil activation pathways, shedding light on the molecular mechanisms underlying their response to our innovative VLPs.

Conclusions: Our novel data reveals the innate-like response of γδ T cells to our VLPs, loaded with different innate stimuli. This highlights the rapid and crucial role of γδ T cells in early immune responses, offering insights for potential immunotherapeutic strategies against cancer.

Monogenic primary immunodeficiencies (PIDs) exist, yet more often pathogenesis is polygenic in nature. Unravelling how polygenic defects interrelate and contribute to clinical phenotypes is challenging, and key to fully understanding pathogenesis, nonetheless. Here we explore a pathway-centric interrogation to uncover the contribution of distinct, but functionally interlinked, genes to PID pathogenesis and clinical heterogeneity.

Testing this hypothesis in a cohort of >900 PID patients led us to identify a family characterized by Mendelian inheritance of a gain-of-function mutation in CXCR4 and a rare, uncharacterized mutation in LFA-1. Specifically, pathway interrogation flagged LFA-1 inside-out signalling as a downstream event of CXCR4 activation.

The identified CXCR4 mutation (L321Pfs*1) causes a syndrome variably characterized by a combination of warts, hypogammaglobulinemia, infections and myelokathexis (WHIM syndrome). Cell-surface expression of the identified LFA-1 variant (αL434P) was abolished in T-cell lines, highlighting its functional significance. Within the affected family, disease manifestation is heterogeneous and, intriguingly, segregates with co-inheritance of wild type vs. mutated LFA-1 – the latter being associated with reduced severity. Pathway interrogation thus identified genetic variations in two functionally-linked genes, associating with distinct clinical phenotypes.

Pathway-centric interrogation of genomic data may emerge as a novel approach to disentangling the polygenic complexity of PID, with the potential to define unexpected and novel therapies.

Aim: 10-20% of all prosthesis patients suffer from implant-related complications within 1-2 years after surgery. Our study aimed to explore the characteristics of metal allergies and potential associated risk factors in prosthesis patients.

Methods: We conducted a retrospective monocentric study at the University Hospital of Zürich. We identified patients who underwent patch testing for metal/additive allergies between 2011 and 2021 and had a joint or dental prosthesis.

Results: A total of 225 patients with a mean age of 72 were included, with 34.2% being male and 65.8% female. Pain was the most common symptom (84.8%), followed by eczematous skin lesions and joint instability (both around 20%), and redness/swelling (13-16%).

In 67.1% of the patients undergoing patch testing, an allergy was diagnosed, with nickel being the most common allergen (31.5%), followed by vanadium chloride (13.7%) and gentamycin (an antibiotic that is often added to bone cement).

60.3% of the metal/additive-allergic patients had a previously known contact allergy to metals (25.7%) or other substances (34.6%). Nickel was the most common allergen (19.5%), followed by silver (3.1%).

Conclusion: A previous contact allergy may be an indicator of a prosthesis-related metal/additive allergy. In contrast, the clinical presentation or consideration of atopic/non-atopic comorbidities do not seem to provide helpful clues for the diagnosis. Our findings suggest that metal/additive allergies might be an underestimated cause of postoperative complaints.