Project description:Patients with severe uncontrolled asthma represent a distinct endotype with persistent airway inflammation and remodeling refractory to corticosteroid treatment. CD4+ T cells and their canonical effector molecules, like type 2 cytokines, play a central role in orchestrating asthma pathogenesis, and biologic therapies targeting these cytokine pathways have had promising outcomes. However, not all patients respond well to such treatment, and their effects are not always durable nor reverse airway remodeling. This observation raises the possibility that other CD4+ T cell subsets and their effector molecules may drive airway inflammation and remodeling. To address this issue in a hypothesis-free manner, we performed single-cell transcriptome analysis of >50,000 airway CD4+ T cells isolated from bronchoalveolar lavage (BAL) samples from 30 patients with mild and severe asthma. We observed striking heterogeneity in the nature of CD4+ T cells present in asthmatics' airways with tissue-resident memory (TRM) cells making a dominant contribution. Notably, a subset of CD4+ TRM cells (CD103-expressing) was significantly increased, comprising nearly 65% of all CD4+ T cells in the airways of male patients with severe asthma when compared to mild asthma. This subset was enriched for transcripts linked to T cell receptor (TCR) activation (HLA-DRB1, HLA-DPA1, CD40LG) and cytotoxicity (GZMB, GZMH), and following stimulation expressed high levels of transcripts encoding for pro-inflammatory non-TH2 cytokines (CCL3, CCL4, CCL5, TNF, IL-17A, CSF2, LIGHT) that could fuel persistent airway inflammation and remodeling. Furthermore, we found that CD4+ T cells isolated ex vivo from airways of severe asthmatics displayed signatures linked to corticosteroid resistance (FKBP5, DDIT4), as well as reduced expression of transcripts encoding for molecules that dampen TCR signaling and T cell effector functions (CREM, DUSP1, TNFAIP3). Overall, our single-cell transcriptomic analysis highlights the pathogenic properties and clinical associations of airway CD4+ T cell subsets in severe asthmatics, features that can potentially trigger their unrestrained activation, and that these findings were more pronounced in males. These pathways may therefore be crucial for driving disease severity in adult male severe asthmatics.

Project description:Purpose: Production of broad-spectrum antibodies against T cell-dependent antigens requires marginal zone (MZ) B cells to present antigenic peptides complexed to MHC II. Here we describe the trogocytic acquisition of these complexes from dendritic cells (DCs). Complement C3 (C3) binds to MHC II on murine and human DCs. Recognition of C3 by MZ B cell complement receptor 2 triggers trogocytosis of DC plasma membrane and its receptors, which become exposed on the MZ B cells. Excessive trogocytosis is deleterious but prevented by membrane ubiquitin ligase MARCH1, which limits the number of MHC II–C3 complexes displayed on the DC surface. MHC II molecules are thus receptors for constitutively activated and potentially harmful C3, an activity that enables acquisition of DC-like functions by trogocytic MZ B cells. Methods: mRNA profiles of splenic cDC1s, B cells and CD11cintCD24+CD8int cells of wild type (WT) and March1-/- mice mice were generated by deep sequencing, in triplicate, using Illumina HiSeq 2500. All 100-base-pair single-end reads were mapped to the reference mouse genome (GRCm38/mm10) using STAR aligner, and gene-wise counts were obtained using Subread package. Differential expression analysis of RNA sequencingdata was carried out using the edgeR and limma-voom packages in Bioconductor. Results: Using an optimized data analysis workflow, we mapped about 14 million sequence reads per sample to the mouse genome (build mm10) and identified 16,182 genes in the different cell types from WT and March1−/− mice.Transcriptomic profiling revealed over 6500 genes differentially expressed between cDC1s and B cells. Principle component analyses revealed very few differences between WT and March1−/− cDC1s, and between WT and March1−/− B cells. Notably, the transcriptome of CD11cintCD24+CD8int cells was similar to those of WT or March1−/− B cells, indicating they belonged to the B cell lineage. Conclusions: In our study we have described that MHC II ubiquitination by MARCH1 plays two important roles: first, to enhance the removal of surface MHC II–C3 complexes on all APCs; and second, to limit MZ B cell trogocytosis and elimination of cDCs. It is conceivable these two functions, more than the regulation of MHC II antigen presentation, have been the major drivers for the conservation of MHC II ubiquitination through evolution.

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MARCH1 controls an exhaustion-like program of effector CD4+ T cells promoting allergic airway inflammation

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