Sajad Moshkelgosha

Rationale: Lymphangioleiomyomatosis (LAM) is a rare disease characterized by the proliferation of neoplastic smooth muscle-like cells in women. The role of the immune system in LAM progression is unclear. Objectives: Investigate the influence of T cells on TSC2null smooth muscle cell behaviour at cellular and transcriptome levels. Methods: Archival LAM and control lung tissues were compared by gene profiling with NanoString analyses where elevated CD8 T cell accumulation was observed in LAM vs. control tissues. Acknowledging the limited translational potential of animal models, a human T cell-TSC2null smooth muscle cell co-culture system was established and cell phenotype and proliferation over time using flow cytometry was examined. Main Results: TSC2null smooth muscle cells adopt cancer-associated fibroblast (CAF)-like phenotypes, which could be regulated by IFN-γ. TSC2null cells suppressed T cell proliferation and induced T cell anergic phenotypes, which could not be reversed by antigen receptor and co-stimulation. TSC2null cells decreased expression of co-stimulatory molecule, CD40, and increased proliferation in the presence of T cells, yet underwent apoptosis in the presence of activated T cells. Conclusions: Our findings suggest a contact-dependent feedback loop between TSC2null smooth muscle cells and T cells, which may both promote immune dysregulation in LAM and explain the invasive behavior and tissue remodeling observed clinically.

Chronic lung allograft dysfunction (CLAD) is a major cause of death after lung transplantation and manifests principally as bronchiolitis obliterans syndrome (BOS) or restrictive allograft syndrome (RAS). Although pulmonary macrophages are key regulators of lung injury and repair, their contributions to CLAD pathogenesis remain under-examined. We performed single-cell RNA sequencing-based transcriptomic analysis of CLAD (7 BOS, 7 RAS) and control lung tissue (n = 6), complemented by comparative and functional analyses. Two distinct macrophage populations were identified in CLAD: inflammatory macrophages expressing leukocyte immunoglobulin-like receptors (LILR), and fibrotic macrophages characterized by expression of osteopontin (SPP1). LILR macrophages were present in both BOS and RAS, whereas SPP1 macrophages were selectively enriched in RAS. Both populations were identified in idiopathic pulmonary fibrosis and cross-tissue comparisons. Surface marker-based sorting strategies were developed to isolate both populations. Functional studies demonstrated that LILR macrophages exhibited enhanced phagocytic activity, promoted T cell chemotaxis and secreted inflammatory mediators, whereas SPP1 macrophages produced soluble factors that drove fibroblast activation and contraction. These findings identify distinct inflammatory and fibrotic macrophage programs associated with CLAD, link RAS to conserved fibrotic macrophage states observed in pulmonary fibrosis and highlight macrophage populations as potential therapeutic targets for inflammatory and fibrotic allograft injury.

Survival after lung transplantation is limited by chronic lung allograft dysfunction (CLAD), an alloimmune fibrotic process leading to death or retransplantation after a median of 6 years. Immunosuppression fails to prevent CLAD, suggesting the existence of drug-resistant alloimmune pathways. We used time-of-flight mass cytometry to identify cells enriched in the bronchoalveolar lavage of patients with subsequent acute lung allograft dysfunction (ALAD), a risk factor for CLAD. We show that CD4 ⁺ CD57 ⁺ PD1 ⁺ T cells emerge in stable patients, conferring risks for ALAD, CLAD, and death. These cells are senescent, secrete inflammatory cytokines, and fall into two oligoclonal subsets with putative cytotoxic and follicular helper functions. Last, they are associated with fibrosis in mouse and human lung allografts, where they localize near airway epithelium and B cells. Together, our findings reveal an inflammatory T cell population that predicts future lung allograft dysfunction and may represent a rational therapeutic target.

The human lung is a complex organ that comprises diverse populations of epithelial, mesenchymal, vascular, and immune cells, which gains even greater complexity during disease states. To effectively study the lung at a single-cell level, a dissociation protocol that achieves the highest yield of viable cells of interest with minimal dissociation-associated protein or transcription changes is key. Here, we detail a rapid collagenase-based dissociation protocol (Col-Short) that provides a high-yield single-cell suspension that is suitable for a variety of downstream applications. Diseased human lung explants were obtained and dissociated through the Col-Short protocol and compared with four other dissociation protocols. Resulting single-cell suspensions were then assessed with flow cytometry, differential staining, and quantitative real-time PCR to identify major hematopoietic and nonhematopoietic cell populations, as well as their activation states. We observed that the Col-Short protocol provides the greatest number of cells per gram of lung tissue, with no reduction in viability when compared with previously described dissociation protocols. Col-Short had no observable surface protein marker cleavage as well as lower expression of protein activation markers and stress-related transcripts compared with four other protocols. The Col-Short dissociation protocol can be used as a rapid strategy to generate single cells for respiratory cell biology research.

Lung transplantation is a life-saving therapy for end-stage pulmonary disease, but its long-term outlook is poor due to a high incidence of chronic lung allograft dysfunction (CLAD). CLAD results from alloimmune-mediated progressive fibrosis and culminates in death or the need for re-transplantation after a median of 6 years. Existing immunosuppression fails to prevent CLAD, suggesting the existence of alloimmune pathways resistant to these drugs. Here, we used mass cytometry to identify cell populations enriched in the bronchoalveolar lavage (BAL) of patients with subsequent allograft dysfunction. We show that CD4 ⁺ CD57 ⁺ PD1 ⁺ T cells emerge in stable lung transplant recipients in the first year post-transplant, conferring heightened risks for CLAD and death or re-transplantation. CD4 ⁺ CD57 ⁺ PD1 ⁺ T cells display features of senescence and secrete inflammatory cytokines. Cellular indexing by transcriptomes and epitopes (CITE-Seq) on BAL CD4 ⁺ T cells revealed the existence of 2 oligoclonal CD57 ⁺ subsets with putative cytotoxic and follicular helper functions. Finally, we observed that CD4 ⁺ CD57 ⁺ PD1 ⁺ T cells are associated with lung allograft fibrosis in a mouse model and in human explanted CLAD lungs, where they localize near airway epithelium and B cells. Together, our findings reveal the existence of an inflammatory T cell population that predicts future lung allograft dysfunction and may represent a rational therapeutic target in lung transplant recipients.

Background: Bronchoalveolar lavage (BAL) is a key tool in respiratory medicine for sampling the distal airways. BAL bile acids are putative biomarkers of pulmonary microaspiration, which is associated with poor outcomes after lung transplantation. Compared to BAL, large airway bronchial wash (LABW) samples the tracheobronchial space where bile acids may be measurable at more clinically relevant levels. We assessed whether LABW bile acids, compared to BAL bile acids, are more strongly associated with poor clinical outcomes in lung transplant recipients.Methods: Concurrently obtained BAL and LABW at 3 months post-transplant from a retrospective cohort of 61 lung transplant recipients were analyzed for taurocholic acid (TCA), glycocholic acid (GCA), and cholic acid by mass spectrometry and 10 inflammatory proteins by multiplex immunoassay. Associations between bile acids with inflammatory proteins and acute lung allograft dysfunction were assessed using Spearman correlation and logistic regression, respectively. Time to chronic lung allograft dysfunction and death were evaluated using multivariable Cox proportional hazards and Kaplan–Meier methods.Results: Most bile acids and inflammatory proteins were higher in LABW than in BAL. LABW bile acids correlated with inflammatory proteins within and between sample type. LABW TCA and GCA were associated with acute lung allograft dysfunction (OR = 1.368; 95%CI = 1.036–1.806;P = 0.027, OR = 1.064; 95%CI = 1.009–1.122;P = 0.022, respectively). No bile acids were associated with chronic lung allograft dysfunction. Adjusted for risk factors, LABW TCA and GCA predicted death (HR = 1.513; 95%CI = 1.014–2.256;P = 0.042, HR = 1.597; 95%CI = 1.078–2.366;P = 0.020, respectively). Patients with LABW TCA in the highest tertile had worse survival compared to all others.Conclusions: LABW bile acids are more strongly associated than BAL bile acids with inflammation, acute lung allograft dysfunction, and death in lung transplant recipients. Collection of LABW may be useful in the evaluation of microaspiration in lung transplantation and other respiratory diseases.

Lung transplant (LT) recipients experience a low survival rate due to chronic lung allograft dysfunction (CLAD). Acute lung allograft dysfunction (ALAD) is a risk factor for CLAD. The contribution of lung macrophages (Macs) to ALAD and CLAD is not clear. Determine the role of Macs in dysfunctional lung allografts using single-cell RNA sequencing (scRNAseq) during quiescence, ALAD, and CLAD. Fresh BAL cells from 6 LT patients - 3 stable and 3 ALAD - and cells from 4 explanted CLAD lungs underwent scRNAseq. R Bioconductor and Seurat were used to perform QC, annotation, and pathway analysis. Donor and recipient deconvolution was performed using single nucleotide variations. We identified two Mac subsets uniquely present in ALAD compared to stable BAL (Fig 1A). Using pathway analysis, we annotated these as pro-inflammatory interferon-stimulated gene (ISG) and metallothionein-mediated inflammatory (MT) Macs. Using publicly available BAL scRNAseq datasets, we found that ISG and MT Macs are associated with severe inflammation in COVID-19 patients (Fig 1B). Analysis of cells from CLAD lungs revealed similar Mac populations (Fig 1C). Deconvolution demonstrated that donor-derived Macs are lost with time post-transplant (Fig 1D). Using scRNAseq, we identified specific Macs that may be associated with allograft dysfunction, raising the possibility that these cells may represent important therapeutic targets.