Myasthenia gravis (MG) is a chronic autoimmune disease mediated by autoantibodies targeting the neuromuscular junction and leading to muscle weakness. Although there are autoantibodies of different specificities, most MG patients have autoantibodies directed against the nicotinic acetylcholine receptor (AChR), in particular against the extracellular domain of the α1 subunit (αECD), containing the main immunogenic region (MIR). Here, we demonstrate an original approach to selectively deplete plasma cells secreting autoantibodies targeting αECD. An antibody-mediated cytotoxicity-engager (ACE) consisting of an anti-hCD38-antibody conjugated to hAChR αECD (αECD) was used to deplete hAChR αECD-specific cells selectively in vivo. The reduction of pathogenic cells was accompanied by lower antibody titers, a reduction of MG disease score, protection of grip strength, and maintenance of body weight. Notably, antibody-secreting cells that are nonspecific for hAChR αECD were not affected. The resulting amelioration of MG pathology in ACE-treated animals highlights the decisive role of αECD-antibodies in the pathogenesis of MG and the clinical relevance of the novel therapeutic strategy.

Proliferative lupus nephritis (LN) is triggered by deposition of autoantibodies in glomeruli and paralleled by a T cell-rich kidney infiltrate. Although these T cells have been attributed to the propagation of tissue injury, it is unclear how they are activated and whether T cell autoreactivity drives local inflammation. Kidney-infiltrating T cells are also observed in urine, where they have high resemblance to interstitial T cells. Therefore, urinary T cells are a proxy for investigating tissue pathogenesis. Here, we analyzed urinary T cells to elucidate whether a kidney-specific T cell autoimmune reaction contributes to tubulointerstitial inflammation in LN. Using single-cell RNA sequencing, we compared transcriptomes and clonotypes of T cells from the blood and urine of patients with active LN and showed that urinary T cells were mostly activated CD8 effector memory cells recruited from a circulating CX3CR1+ subset. Several urinary CD8 T cell clones were expanded. However, upon in vitro testing of their T cell receptors, we did not observe autoreactivity against autologous tubular epithelial cells. Instead, ~20% of expanded clonotypes were Epstein-Barr virus-specific or cytomegalovirus-specific, but respective viral antigens were undetectable in kidney biopsies or urine. Conversely, kidney-infiltrating T cells had access to interleukin-15 and interferon-β (IFN-β), and stimulation with these cytokines was sufficient to trigger degranulation and production of tumor necrosis factor, IFN-γ, and granzyme K. Together, these results show that CD8+CX3CR1+ T cells are recruited into the kidney in LN, where they are activated by cytokines, enabling them to contribute to local inflammation.

Abstract Persistence of memory T lymphocytes, in the apparent absence of antigen, is a hallmark of immune memory and key to adaptive immunity to recurrent infections. The signaling pathways ensuring survival and quiescence of the memory T cells are largely enigmatic. Here we show, by inhibition in vivo , that persistence of surface CD69+KLF2-tissue-resident memory T cells of murine bone marrow and spleen is blocked by antibodies to the integrins VLA-4 and LFA-1, connecting the memory T cells to VCAM1 and ICAM1 of stromal cells. Persistence requires the PI3K/AKT signaling pathway, since it is blocked by Wortmannin, and it involves PI3K-dependent survival genes. Surface CD69-KLF2+ memory T cells of the bone marrow are also dependent on integrin-mediated contact to stromal cells. Their persistence critically depends on the NF-kB pathway, their PI3K signaling pathway is not relevant. Blocking Jak1 and 3 of the interleukin-7 and -15 signaling pathways does affect memory T cells of the spleen, but not those of the bone marrow. Thus, tissue-resident KLF2+ and KLF2-memory T cells, and memory T cells of spleen and bone marrow, use different signaling pathways, adapting them to their respective tissues and reflecting an unexpected heterogeneity in the molecular mechanisms of persistence.

Adaptive immunity relies on antibodies and memory B and T cells, with memory T cells providing "reactive memory". These cells either circulate in the blood or remain as tissue-resident memory T cells, yet the epigenetic mechanisms underlying their recall function and maintenance are not well understood. Here, we present a comprehensive analysis of 56 reduced representation bisulfite sequencing (RRBS) datasets from 22 memory CD4 and CD8 T-cell populations isolated from human bone marrow, intestine, spleen, lung, skin, and peripheral blood, including surface CD69-positive and CD69-negative cells. Our study reveals unique DNA hypomethylation patterns in tissue-resident memory T cells, particularly in regions associated with genes involved in tissue homing, residency, and transcription factors regulating recall effector memory. The methylomes and differential methylation signatures identified here serve as a valuable resource for understanding the epigenetic program of memory T lymphocytes, their roles in immunological recall, and their maintenance within specific tissues.

In a subset of children and adolescents, SARS-CoV-2 infection induces a severe acute hyperinflammatory shock1 termed multisystem inflammatory syndrome in children (MIS-C) at four to eight weeks after infection. MIS-C is characterized by a specific T cell expansion2 and systemic hyperinflammation3. The pathogenesis of MIS-C remains largely unknown. Here we show that acute MIS-C is characterized by impaired reactivation of virus-reactive memory T cells, which depends on increased serum levels of the cytokine TGFβ resembling those that occur during severe COVID-19 (refs. 4,5). This functional impairment in T cell reactivity is accompanied by the presence of TGFβ-response signatures in T cells, B cells and monocytes along with reduced antigen-presentation capabilities of monocytes, and can be reversed by blocking TGFβ. Furthermore, T cell receptor repertoires of patients with MIS-C exhibit expansion of T cells expressing TCRVβ21.3, resembling Epstein-Barr virus (EBV)-reactive T cell clones capable of eliminating EBV-infected B cells. Additionally, serum TGFβ in patients with MIS-C can trigger EBV reactivation, which is reversible with TGFβ blockade. Clinically, the TGFβ-induced defect in T cell reactivity correlates with a higher EBV seroprevalence in patients with MIS-C compared with age-matched controls, along with the occurrence of EBV reactivation. Our findings establish a connection between SARS-CoV-2 infection and COVID-19 sequelae in children, in which impaired T cell cytotoxicity triggered by TGFβ overproduction leads to EBV reactivation and subsequent hyperinflammation.

IgA-coated fractions of the intestinal microbiota of Crohn’s disease (CD) patients have been shown to contain taxa that hallmark the compositional dysbiosis in CD microbiomes. However, the correlation between other cellular properties of intestinal bacteria and disease has not been explored further, especially for features that are not directly driven by the host immune-system, e.g. the expression of surface sugars by bacteria. By sorting and sequencing IgA-coated and lectin-stained fractions from CD patients microbiota and healthy controls, we found that lectin-stained bacteria were distinct from IgA-coated bacteria, but still displayed specific differences between CD and healthy controls. To exploit the discriminatory potential of both, immunoglobulin coated bacteria and the altered surface sugar expression of bacteria in CD, we developed a multiplexed single cell-based analysis approach for intestinal microbiota. By multi-parameter microbiota flow cytometry (mMFC) we characterized the intestinal microbiota of 55 CD patients and 44 healthy controls for 11-parameters in total, comprising host-immunoglobulin coating and the presence of distinct surface sugar moieties. The data were analyzed by machine-learning to assess disease-specific marker patterns in the microbiota phenotype. mMFC captured detailed characteristics of CD microbiota and identified patterns to classify CD patients. In addition, we identified phenotypic signatures in the CD microbiota which not only reflected remission after 6 weeks of anti-TNF treatment, but were also able to predict remission before the start of an adalimumab treatment course in a pilot study. We here present the proof-of-concept demonstrating that multi-parameter single-cell bacterial phenotyping by mMFC could be a novel tool with high translational potential to expand current microbiome investigations by phenotyping of bacteria to identify disease- and therapy-associated cellular alterations and to reveal novel target properties of bacteria for functional assays and therapeutic approaches.

Across tissues, tissue-resident memory T cells have been defined as cells that express CD69 on their cell surface but not sphingosine-1-phosphate receptor 1 (S1PR1), the receptor for the tissue-egress signal sphingosine-1-phosphate (S1P). It is less clear whether CD69-negative memory T cells are also tissue-resident. Here, we compare transcriptomes and T cell receptor repertoires of individual CD4 and CD8 memory T cells from paired blood and bone marrow samples from three human donors. CD69- memory T cells of blood and bone marrow share transcriptionally defined clusters, characterized by signature genes and reflecting their imprinting during original activation. However, cells of related clusters from blood and bone marrow have different TCR repertoires, evidence that they represent distinct compartments of memory and indicating that the CD69- memory T cells are residents of the bone marrow. Interestingly, the surface CD69- memory T cells of bone marrow do transcribe the CD69 gene and express S1PR1, suggesting that they are blindfolded to the perception of the egress signal sphingosine-1-phosphate by dimerization and internalization of CD69 and S1PR1, maintaining them in the bone marrow.

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(1) Background: The basophil activation test (BAT) is a functional whole blood-based ex vivo assay to quantify basophil activation after allergen exposure by flow cytometry. One of the most important prerequisites for the use of the BAT in the routine clinical diagnosis of allergies is a reliable, standardized and reproducible data analysis workflow. (2) Methods: We re-analyzed a public mass cytometry dataset from peanut (PN) allergic patients (n = 6) and healthy controls (n = 3) with our binning approach “pattern recognition of immune cells” (PRI). Our approach enabled a comprehensive analysis of the dataset, evaluating 30 markers to achieve optimal basophil identification and activation through multi-parametric analysis and visualization. (3) Results: We found FcεRIα/CD32 (FcγRII) as a new marker couple to identify basophils and kept CD63 as an activation marker to establish a modified BAT in combination with our PRI analysis approach. Based on this, we developed an algorithm for automated raw data processing, which enables direct data analysis and the intuitive visualization of the test results including controls and allergen stimulations. Furthermore, we discovered that the expression pattern of CD32 correlated with FcεRIα, anticorrelated with CD63 and was detectable in both the re-analyzed public dataset and our own flow cytometric results. (4) Conclusions: Our improved BAT, combined with our PRI procedure (bin-BAT), provides a reliable test with a fully reproducible analysis. The advanced bin-BAT enabled the development of an automated workflow with an intuitive visualization to discriminate allergic patients from non-allergic individuals.

Bone marrow plasma cells (BMPC) are the correlate of humoral immunity, consistently releasing antibodies into the bloodstream. It remains unclear if BMPC reflect different activation environments or maturation of their precursors. Here we define human BMPC heterogeneity and track the recruitment of antibody-secreting cells (ASC) from SARS-CoV-2 vaccine immune reactions to the bone marrow (BM). Trajectories based on single-cell transcriptomes and repertoires of peripheral and BM ASC reveal sequential colonisation of BMPC compartments. In activated B cells, IL-21 suppresses CD19 expression, indicating that CD19low-BMPC are derived from follicular, while CD19high-BMPC originate from extrafollicular immune reactions. In primary immune reactions, both CD19low- and CD19high-BMPC compartments are populated. In secondary immune reactions, most BMPC are recruited to CD19high-BMPC compartments, reflecting their origin from extrafollicular reactivations of memory B cells. A pattern also observable in vaccinated-convalescent individuals and upon diphtheria/tetanus/pertussis recall-vaccination. Thus, BMPC diversity reflects the evolution of a given humoral immune response.

Immunological memory provided by memory B cells, memory T cells and long-lived (memory) plasma cells yields long-lasting, effective cellular and humoral immune protection to previously encountered pathogens and vaccines. Memory B cells provide a fast and potent anamnestic antibody response following a reencounter with antigen, thereby providing an extra arm of immunity to pathogens that are not cleared by pre-existing antibodies. In addition, they qualify as potent antigen-presenting and immunoregulatory cells in secondary immune reactions. We are only beginning to understand the heterogeneity of memory B cells and their compartmentalisation. Plasma cells persist, potentially for a lifetime, in specialised niches in the tissues, especially in bone marrow and gut, but also in inflamed tissue. The molecular mechanisms governing the long-term persistence of memory B and plasma cells, in the apparent absence of antigen, are only now beginning to be unravelled. While long-lived plasma cells provide essential protection against pathogens, their longevity and resistance to irradiation, immunosuppression and therapies targeting B cells can be problematic for the treatment of chronic antibody-mediated diseases.