Wolfgang Merkt

Impaired immune clearance of senescent fibroblasts is a putative driver of pulmonary fibrosis. Exhausted natural killer (NK) cells have been implicated in this process, yet the underlying immune evasion mechanisms remain poorly understood. Using single-cell RNA sequencing (scRNA-seq) and spectral flow cytometry, we identified natural killer group 2 member A (NKG2A) as the predominant inhibitory checkpoint receptor expressed on NK cells in fibrotic lung diseases. Mechanistic in vitro coculture studies showed that NK cell suppression was mediated by senescent fibroblasts expressing human leukocyte antigen-E (HLA-E), the high-affinity ligand for NKG2A. scRNA-seq analysis of lungs from patients with idiopathic pulmonary fibrosis (IPF) further identified selective HLA-E expression in senescent HAS1 ⁺ fibroblast subsets. Further, spatial transcriptomics and multiplex immunofluorescence of patient lungs demonstrated that HLA-E ⁺ fibroblasts were positioned at the periphery of fibroblast foci adjacent to NKG2A ⁺ NK cells, establishing an immune-privileged niche. In contrast, extracellular matrix–producing myofibroblasts at the core of fibrotic foci lacked HLA-E and exhibited minimal NK engagement. In vivo, therapeutic blockade of NKG2A restored NK cell function, promoted clearance of senescent fibroblasts, and promoted fibrosis resolution in the bleomycin-induced mouse model. Monalizumab, a clinical-grade NKG2A inhibitor, reactivated patient-derived NK cells and enhanced lysis of human senescent fibroblasts in vitro. Together, these findings uncover a spatially restricted immune checkpoint axis that allows senescent fibroblasts to evade immune NK surveillance. Targeting the HLA-E/NKG2A axis represents a promising therapeutic strategy to restore NK cell–mediated immune clearance of senescent fibroblasts and reverse pulmonary fibrosis.

Autoimmune-mediated connective tissue diseases such as antisynthetase syndrome (ASyS) and systemic sclerosis (SSc) have a high unmet medical need. Here we report on treatment under compassionate use with the CD19×CD3 T cell engager (TCE) blinatumomab and the BCMA×CD3 TCE teclistamab in five patients with treatment-refractory ASyS and in five patients with treatment-refractory SSc, respectively. Induction therapy with blinatumomab or teclistamab reduced target cells in affected muscle and skin, respectively, and decreased autoantibody titers. Blinatumomab induced rapid clinical, serological and histological improvement of myositis and stabilization of interstitial lung disease (ILD) in patients with ASyS. Teclistamab improved skin fibrosis, stabilized ILD and resolved tendon friction rubs in patients with SSc. Inhibition of B cell redifferentiation by maintenance therapy with rituximab (RTX) enabled prolonged disease control, even for patients previously unresponsive to RTX. Treatment was associated with adverse events including cytokine release syndrome (CRS) up to grade 3, in two patients with ASyS and in all patients with SSc. No immune effector cell-associated neurotoxicity syndrome (ICANS) occurred. Respiratory infections treated with antibiotics occurred in six patients. Blinatumomab and teclistamab may offer potential as rescue therapies for patients with treatment-refractory ASyS and SSc.

Objectives: The role of autoantibody-producing B cells in connective tissue diseases (CTD) has recently been highlighted by the successful treatment with CD19-targeting CAR T cells. Detrimental effects of autoantibodies are linked to the formation of deposited IgG complexes and the activation of immune cells via Fcγ receptors (FcγRs). The role of circulating immune complexes (cICs) as a link between adaptive and innate immunity has remained understudied. Clinical testing of cICs has been hindered by the lack of reliable detection methods. The aim of this study was to determine the potential of IgG-containing cICs to activate FcγRs (their bioactivity) using a new detection method. Methods: A reporter cell platform was used to assess the presence and bioactivity of cICs in IgG-autoantibody-positive CTD patients (cross-section analysis) and in patients treated with CD19-CAR T cells (longitudinal analysis). Results: The bioactivity of cICs in the cohort of patients with CTDs was significantly higher compared with healthy controls and patients with IgG-autoantibody-negative systemic inflammatory disease (psoriatic arthritis). Analyses of individual diseases revealed the presence of cICs in the sera of all CTDs, including systemic sclerosis (SSc) and primary Sjögren’s syndrome, although there was significant heterogeneity among individuals. Within SSc, patients positive for anti-topoisomerase-I (Scl70) autoantibodies, diffuse cutaneous and lung involvement had significantly enhanced cIC bioactivity. Finally, the bioactivity of cICs was significantly reduced in CTD patients after CD19-CAR T cell therapy. Conclusions: Our study reveals the presence of FcγR-engaging cICs in CTDs and demonstrates that the bioactivity of cICs is correlated with clinical phenotypes and treatment outcomes.

Objective: Musculoskeletal diseases such as osteoarthritis (OA) are leading causes of pain, physical inactivity and disability worldwide. Synovial fibroblasts (SFs) expressing fibroblast activation protein (FAP) are crucial for OA progression. Positron emission tomography (PET) with ⁶⁸ Gallium-labelled FAP-inhibitors ( ⁶⁸ Ga-FAPI) visualizes FAP-positive activated fibroblasts. Here, we systematically analyze FAPI-uptake respecting OA and joint degeneration in a cohort of 268 patients. Methods: ⁶⁸ Ga-FAPI-PET-scans of 268 oncological patients were analyzed for increased joint-associated FAPI-uptake, quantified by maximal, mean standardized uptake values (SUVmax/mean) and target to blood ratios (TBR) and compared with computed tomography-based OA-classification according to Kellgren and Lawrence. FAP-expression of SF from OA and rheumatoid arthritis (RA) (15 OA- and 26 RA-patients) were analyzed by single-cell cytometry by time of flight (cyTOF). Results: 148 of 268 patients (55.2%) showed increased joint-associated FAPI-uptake (average SUVmax/mean of 3.25 ± 1.28/ 1.94 ± 0.70). The most frequent FAPI-positive joints were acromioclavicular and shoulder joints followed by sternoclavicular, lumbar facet and hip joints. FAPI-uptake was frequently asymmetric whereas OA-scoring displayed a symmetric allocation of degenerative changes suggesting discordance between FAPI-uptake and OA-scoring. Only in end-stage-OA (grade 4 according to Kellgren and Lawrence) FAPI-uptake was concordantly and significantly elevated. Abundant FAP-positive fibroblasts in end-stage-OA were confirmed by CyTOF . Conclusion: FAPI-uptake in joints is frequent and can occur in presence as well as in absence of joint degeneration. We suggest that FAPI-PET/CT complements conventional musculoskeletal imaging by providing information on synovial activity, the exact triggers of which remain to be elucidated.

Objective: The high potential of CD19.CAR-T cells to treat autoimmune diseases such as Systemic Sclerosis (SSc) supposedly relies on the disappearance of autoantibodies. Here we investigated effects of CAR-T cells on the innate immune system which is an important contributor to pathology in SSc. Methods: Longitudinal analysis of peripheral blood mononuclear cells from an Scl70 + SSc patient treated with CAR-T cells sampled over 18 months by 29-color spectral flow cytometry, in vitro experiments using sera from patient cohorts. Results: In the patient treated with CAR-T cells, the substantial clinical improvement was paralleled by dynamic changes in innate lymphoid cells, namely Fcγ-receptor IIIA-expressing natural killer (NK) cells. NK cells adopted a more juvenile, less activated, and less differentiated phenotype. In parallel, the potency of serum to form Scl70-containing immune complexes that activate Fcγ-receptor IIIA decreased over time. These observations suggested a mechanistic link between reversal of adaptive autoimmunity and recovering Fcγ-receptor IIIA-expressing innate immune cells after CAR-T cell therapy via regressing immune complex activity. Experiments with sera from the non-CAR-T-treated SSc cohort confirmed that Scl70-containing immune complexes activate Fcγ-receptor IIIA-expressing NK cells in a dose-dependent manner, substantiating the relevance of this link between adaptive and innate immunity in SSc. Conclusion: This report describes for the first time the phenotypic recovery of innate Fcγ-receptor-expressing cells in an SSc patient treated with CAR-T cells. Decreasing autoantibody levels associated with a reduced ability to form functional immune complexes, the latter appearing to contribute to pathology in SSc via activation of Fcγ receptor IIIA + cells such as NK cells. Graphical Proposed mechanism of involvement of NK cells and soluble Immune Complexes (sICs) in disease progression during active autoimmunity in SSc (left) and resolution of fibrosis after deep B cell depletion by CD19.CAR-T cells and disappearance of autoantibodies (right).

Wolfgang Merkt, Manuel Röhrich; Comment on: Fibroblast activation protein inhibitor-positron emission tomography in aortitis: fibroblast pathology in activ