Programme - Thursday

Aims

Impaired mitophagy leads to dysfunctional mitochondria accumulation and tumor-infiltrating T lymphocytes (TILs) exhaustion. However, the mechanisms connecting the accumulation of dysfunctional mitochondria and T cell exhaustion remain poorly understood. We speculate that cells employ alternative strategies to remove depolarized mitochondria, potentially inducing CD8⁺ T cell exhaustion.

Methods

1) a degron-mCherry reporter and a regulatory heme (RH) detection assay were developed to examine protein degradation. 2) the correlation between RH and CD8⁺ T cell differentiation was investigated by detecting RH concentration and BACH2 and BLIMP1 expression in exhausted CD8⁺ T cells. 3) hemin, a heme mimic, was used to assess the effect on CD8⁺ T cell exhaustion. 4) the heme-binding site mutated BACH2 was utilized to explore the mechanism of heme-induced exhaustion. 5) PGRMC2, responsible for heme import into the nucleus, was knocked out to verify the impact on TILs' exhaustion.

Results

CD8⁺ T cells with dysfunctional mitochondria engage mitochondrial protein degradation, leading to increased RH concentration. RH correlates with TILs differentiation, and exogenous heme directly induces the BACH2-BLIMP1 transcriptional networks, which is essential for commitment of T cell exhaustion. Targeting PGRMC2 to reduce RH nuclear distribution ameliorates CD8⁺ T cell exhaustion and enhancing mitochondrial fitness.

Conclusions

Heme-mediated reprogramming guides CD8⁺ T cell exhaustion in response to mitochondrial dysfunction. Manipulating heme signaling can be exploited to restore mitochondrial fitness and tailor T cell-based cellular therapies.

Aim: Psoriasis is an autoimmune skin disease, which is driven by the IL-23/IL-17A axis. The immunoproteasome is a special form of the 26S proteasome in which the standard catalytically active β-subunits (β1c, β2c, and β5c) are replaced by low molecular mass polypeptide (LMP)2 (β1i), multicatalytic endopeptidase complex-like (MECL)-1 (β2i) and LMP7 (β5i). It has been shown that Immunoproteasome inhibition is a promising strategy in reducing IL-23 secretion and suppressing Th17 cell development. Therefore, the therapeutic potential of immunoproteasome inhibition in psoriasis pathogenesis was assessed.

Methods: The therapeutic use of ONX 0914, a selective inhibitor of the immunoproteasome, was investigated in Card14ΔE138^+/- mice, which spontaneously develop psoriasis-like symptoms, and in the imiquimod murine model.

Results: In both models, treatment with ONX 0914 significantly reduced skin thickness, inflammation scores, and pathological lesions in the analyzed skin tissue. Furthermore, immunoproteasome inhibition normalized the expression of several pro-inflammatory genes in the ear and significantly reduced the inflammatory infiltrate, accompanied by a significant alteration in the αβ⁺ and γδ⁺ T cell subsets.

Conclusions: ONX 0914 ameliorated psoriasis-like symptoms in two different murine psoriasis models, which supports the use of immunoproteasome inhibitors as a therapeutic treatment in psoriasis.

Persistent exposure to antigen during chronic infection or cancer renders T cells dysfunctional. The molecular mechanisms regulating this state of exhaustion are thought to be common in infection and cancer, despite obvious differences in their microenvironments. We discovered that NFAT5, an NFAT family member lacking an AP-1 docking site, is highly expressed in exhausted T cells responding to chronic infection and tumors but is a central player selectively in tumor-induced exhaustion. While NFAT5 overexpression in CD8+ T cells reduced tumor control, NFAT5 deletion improved tumor control by promoting the accumulation of tumor-specific CD8 T cells that expressed less TOX and PD-1 and produced more cytokines specifically among precursor exhausted cells. Conversely, NFAT5 did not promote T cell exhaustion during chronic infection. While NFAT5 expression was induced by TCR triggering, its transcriptional activity was specific to the tumor microenvironment and required hyperosmolarity. NFAT5 thus promotes CD8 T cell exhaustion in a tumor-selective fashion.

The activation and differentiation of naïve T cells into T helper (Th) cells are precisely controlled processes. The translation of peptides from short open reading frames (sORFs) on RNAs that are annotated as non-protein coding (ncRNAs) can impact cells in multiple ways. Our aim is to elucidate the role of short ORF-encoded polypeptides (SEPs) during Th1 cell differentiation and activation and to determine the effect of translational regulation.

We are using ribosome profiling and RNA sequencing of human ex vivo differentiated Th1 cells compared to unstimulated naïve T cells to detect so far unknown translation events, focusing on SEPs, and estimate translation efficiencies of RNAs during differentiation and after activation. Mass spectrometry (MS) based methods are then used to confirm our results on the protein level.

Ribosome footprints, an indicator of translation, could be detected in known coding sequences as well as on ncRNAs. Resulting promising SEPs were validated by MS. Additionally, the analysis of differential translation efficiencies between Th1 and unstimulated naïve T cells resulted in the identification of transcripts that were regulated in translation, and led us to further investigate the prenylation pathway.

The multi-omics approach presented here promises to provide valuable insights into regulatory processes in Th cell differentiation and activation. Identifying differentially expressed proteins or novel peptides, and additional mechanisms of post-transcriptional gene regulation could provide suitable targets for the treatment of immune diseases.

Atopic dermatitis (AD) is a chronic skin disease characterized by type 2 inflammation. Carbonic anhydrase (CA) an enzyme that regulates tissue acid balance is increased in keratinocytes in AD. We aimed to investigate how increasing CA enzyme affects type 2 inflammation and barrier function in AD skin. Bulk, single-cell, and spatial RNA-seq of lesional and non-lesional skin of AD patients were analysed. In addition, the expression of CA enzymes and barrier-related molecules were investigated in reconstructed epidermis (Episkin®) and ex-vivo human skin (NativeSkin®) treated with IL-4, IL-13, and IL-22. Their influence on epithelial barrier integrity was determined by electrical impedance spectroscopy. The effect of CA2 inhibitor treatment was investigated in MC903-induced AD-like model in mice and in ex-vivo samples. CA2 is increased in keratinocytes and correlate with pro-inflammatory genes in AD patients, type 2 cytokine-stimulated- Episkin and -NativeSkin. IL-4, IL-13, and IL-22 induced barrier impairment in NativeSkin and the inhibition of CA2 partially recovered the downregulation of electric impedance induced by cytokines. In line with these findings, in vivo inhibition of CA2 significantly improved AD-associated pathological features in mice. As a conclusion, we demonstrate that type 2 cytokines are potent enhancers of CA2 expression across species and specific inhibition of CA2 can suppress allergic skin inflammation. Our results thus identify the enzyme CA2 as a promising new druggable target in AD, and perhaps other skin inflammatory conditions.

Allergies rise worldwide, with peanut allergy being the major cause of fatal food-induced anaphylaxis, while pollen allergies impact up to 30% of the population. Traditional allergy treatment methods rely on desensitization to the specific allergen.

To make allergy treatment safer and faster, Mabylon developed an anti-allergen drug discovery pipeline, which allows rapid generation of effective patient-derived antibodies.

Allergen-targeting antibodies block IgE binding to the allergens and prevent effector cell activation. We have shown efficacy of such an approach for both peanut and pollen allergy in vitro and in humanized mouse models. One limitation of anti-allergen antibody therapies, as demonstrated by our research and others, is the need of antibody cocktails to be effective, which can be costly to produce and may involve additional regulatory considerations. Thus, we are developing engineered multi-specific antibodies to create highly effective, single antibody-based therapeutics. We also investigated a combinatorial approach in which allergen-targeting antibodies are combined with allergen immunotherapy. In vitro, the allergen-targeting antibodies in combination with immunotherapy are able to induce a tolerogenic cytokine milieu.

In conclusion, with the premise of good safety profile and rapid onset of protection, Mabylon’s allergen-targeting antibodies have the potential to become a novel therapy for the treatment of peanut and pollen allergies. The engineering of antibody cocktails to one multi-specific molecule allows targeting multiple allergies at once.

There is an urgent need to identify novel biomarkers to detect as early as possible severe cases of COVID-19. Extracellular vesicles (EVs) contain cargoes derived from the cell of origins that include proteins, lipids, and nucleic acids. As a consequence of cellular activation, EVs concentration is often increased during disease and specific EV biomarkers can be found in biofluids. We investigated the EV composition in the lungs and matching plasma of severe COVID-19 patients by using mass spectrometry-based proteomics. We optimized the isolation of EVs from small amount of plasma and broncho-alveolar lavage (BAL) and characterized them. Next, we performed proteomics in two independent cohorts (discovery and validation) to identify a “COVID-19 severe signature” common in BAL and plasma. Proteomics of EVs isolated from BAL showed that COVID-19 positive samples had a higher median of unique proteins detected (1518, CI 118-2027; n=49). The 12 conventional EVs markers examined were found in all our BAL samples and were present in >70% or >85% of negative and positive COVID-19 samples respectively. Overall characterization of COVID lung proteome shows infection signatures. Gene set enrichment analysis showed cellular pathways specifically involved in COVID infections including inflammation and tissue remodeling. Different machine learning models can all predict COVID infection based on proteome signatures. Next, plasma EV proteome will be analyzed to find a common signature together with the BAL, that will be validated in the second cohort.

We present an interim analysis from a pivotal phase 2/3 randomized, double-blind, placebo-controlled, case-driven clinical trial (NCT05127434) in adults aged ≥60 years assessing mRNA-1345, an investigational mRNA vaccine encoding the RSV prefusion stabilized F glycoprotein. Participants were randomized 1:1 to receive mRNA-1345 50 μg or placebo. Primary efficacy endpoints were prevention of a first episode of RSV-associated lower respiratory tract disease [RSV-LRTD] with ≥2 or ≥3 lower respiratory symptoms between 14 days and 12 months post-injection; secondary efficacy endpoints include RSV-associated acute respiratory disease [RSV-ARD] with ≥1 respiratory symptom between 14 days and 12 months post-injection. mRNA-1345 was well-tolerated; no safety concerns were identified (solicited local adverse reactions [Ars]: mRNA-1345=58.7%; placebo=16.2%; solicited systemic ARs: mRNA-1345=47.7%; placebo=32.9%). Primary efficacy endpoints were met in the per-protocol efficacy set (n=35,088), including a vaccine effectiveness [VE] of 83.7% (95.88% CI, 66.0-92.2; P<0.0001) against RSV-LRTD with ≥2 symptoms and VE of 82.4% (96.36% CI, 34.8-95.3; P=0.0078) against RSV-LRTD with ≥3 symptoms. For the secondary efficacy endpoint, VE was 68.4% (95% CI, 50.9-79.7) against RSV-ARD. Symptom distribution between mRNA-1345 and placebo recipients, and additional efficacy analyses by RSV subtype will be discussed. mRNA-1345 had a favourable safety and tolerability profile in adults aged ≥60 years and is efficacious in preventing a spectrum of symptomatic RSV disease including ARD and LRTD.

Introduction:. Monoclonal antibodies (mAbs) in cancer immunotherapy often act by Antibody-Dependent Cellular Cytotoxicity (ADCC) and are dependent of the interaction between their Fc region and the FcYRIII (CD16) on NK cells. However, increased levels of plasma IgG in cancer patients reduces the efficiency of mAbs through competitive binding of CD16. Thus, te development of CD16 BiKEs may show superior efficiency compared to mABs. Here we developed a new CD70 targeting BiKE and compared its efficacy in vitro to a CD70 targeting mAb.

Materials and Methods: Whole PBMCs and purified NK cells were co-cultured with different CD70^{WT or KO} cells lines in presence of the different antibody constructs. Expression of CD107a, CD16, INFy and TNF were determined as well as NK cell activation and cytotoxicity by flow cytometry and calcein release assay

Results: BiKE CD70 efficiently activated purified NK cells in presence of CD70^WT AML cells in vitro. In the presence of whole PBMCs, BIKE showed superior activation of NK cells compared to a CD70 mAb. Moreover, we observed low CD16 shedding upon activation of NK cells by BiKE..

Conclusions:

Our results indicate that a CD70 targeting BiKE efficiently eliminates AML cells in vitro. Persistence of CD16 expression could allow rechallenging of NK cells with BiKE.

Due to its stimulatory potential for immunomodulatory CD4⁺ regulatory T (Treg) cells, low-dose interleukin-2 (IL-2) immunotherapy has recently gained considerable attention for treatment of various autoimmune diseases. Although early-stage clinical trials have correlated expansion of circulating Treg cells with clinical response to IL-2 treatment, detailed mechanistic data on responding Treg cell subsets and effects on other immune cells are lacking. In this investigator-initiated phase-2 clinical trial of low-dose IL-2 immunotherapy in systemic lupus erythematosus (SLE) patients, we performed an in-depth study of circulating and cutaneous immune cells by imaging mass cytometry, high-parameter spectral flow cytometry, bulk and single-cell RNA sequencing with cellular indexing, and targeted serum proteomics, building a comprehensive atlas of in vivo human immune responses to IL-2. Low-dose IL-2 stimulated various circulating immune cells, including Treg cells with skin-homing properties that appeared in the skin of SLE patients in close interaction with endothelial cells, whereas other cutaneous immune cells remained unchanged. Analysis of surface proteins and transcriptomes detected different IL-2-driven Treg cell programs, including highly proliferative CD38⁺ HLA-DR⁺, activated gut-homing CD38⁺, and skin-homing HLA-DR⁺ Treg cells. These data identify functionally distinct Treg cell subsets in human blood and skin, including those most responsive to IL-2 immunotherapy, thus providing unprecedented insight into human IL-2-responsive immune cells.