Characterization of B Cells in Lungs of Chronic Lung Allograft Dysfunction Patients by Single-Cell RNA Sequencing

Abstract

<h3>Purpose</h3> The primary factor limiting long-term survival in lung transplant recipients is chronic lung allograft dysfunction (CLAD), which manifests as fibrosis of the airways and/or lung parenchyma. The high incidence of CLAD despite the use of T cell-targeted immunosuppressive therapies suggests that other immune cells may contribute to its development. While multiple studies point towards a role of B cells in CLAD, knowledge regarding the specific populations of B cells prevalent in lungs of CLAD patients is lacking. We aimed to define the transcriptional signatures of B cell populations in explanted CLAD lungs by single-cell RNA sequencing (scRNAseq). <h3>Methods</h3> Single cell suspensions were made from 4 samples of CLAD lung explants. B cells were isolated by flow sorting and transcriptionally profiled using 10X genomics (10,000 cells, 50,000 reads/cell) within 2 hours. The transcriptional heterogeneity of B cells in CLAD lungs was analyzed using the R package Seurat. <h3>Results</h3> Based on flow cytometry, B cells (CD45+/CD3-/CD19+) were present in the lungs of all four CLAD patients, consistent with previously published reports of B cell-rich tertiary lymphoid organs in CLAD lungs. ScRNAseq analysis of expression levels of immunoglobulins, transcription factors, and surface markers of conventional B cells subsets revealed the presence of both naïve and antigen-experienced B cells in CLAD lungs. We identified populations of transitional B cells, naïve follicular B cells, germinal center cells, plasmablasts, and memory B cells. Notably, we identified a distinct population of class-switched B cells (IgM/IgD low, IgG/IgA high, and CD38+). In addition, our analysis revealed that many B cells expressed markers of antigen presentation including CD74, CIITA, and HLA-DMA, suggesting that B cells in CLAD lungs may function as antigen-presenting cells. <h3>Conclusion</h3> Our scRNAseq results show the prevalence of both naïve and antigen-experienced B cells in the lungs of CLAD patients. Further analyses will aim to identify specific B cell populations that may be associated with allograft dysfunction.