Biology subjects

Keshavjee, S.

Publications and source records attributed to Keshavjee, S..

6 recordsLinked to original sources

bioRxiv2026

Distinct fibrotic, epithelial and immune transcriptomic programs in phenotypes of chronic lung allograft dysfunction

BackgroundChronic lung allograft dysfunction (CLAD) is the major cause of late mortality after lung transplantation and includes two principal phenotypes, bronchiolitis obliterans syndrome (BOS) and restrictive allograft syndrome (RAS). RAS and other phenotypes with RAS-like opacities (RLO) on chest imaging have a poorer prognosis. Despite clear clinical and pathological differences, molecular distinctions between phenotypes remain poorly defined. We aimed to explore gene transcriptional profiles across CLAD phenotypes and relevant controls. MethodsWe performed bulk RNA sequencing on explanted lung tissue from 45 lung transplant recipients with end-stage CLAD (20 with RLO and 25 without RLO). Samples from twenty-seven control donor and lobectomy lungs and sixteen idiopathic pulmonary fibrosis (IPF) lungs served as comparators. Non-negative matrix factorization (NMF) was used to identify latent transcriptomic signatures, which were correlated with clinical, radiologic, and histopathologic features. ResultsNMF identified seven distinct gene signatures that segregated CLAD phenotypes. RLO-CLAD lungs were enriched for extracellular matrix remodeling and B-cell/plasma cell-associated signatures, overlapping partly with IPF, whereas non-RLO-CLAD showed relative enrichment of epithelial injury and surfactant-response pathways. Signatures related to epithelial homeostasis and ciliary/microtubule function were progressively reduced from control lungs to non-RLO-CLAD and were most suppressed in RLO-CLAD. ConclusionsRLO-CLAD and non-RLO-CLAD, aligning with RAS and BOS phenotypes, show distinct transcriptomic signatures. RLO-CLAD is characterized by profibrotic and humoral immune signatures with profound epithelial dysfunction, whereas non-RLO-CLAD shows relative enrichment of epithelial injury responses. These data provide molecular stratification of CLAD and support the development of phenotype-specific biomarkers and targeted therapies.

bioinformatics↗

bioRxiv2023

Emergence of a senescent and inflammatory pulmonary CD4+ T cell population prior to lung allograft failure

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.

immunology↗

bioRxiv2021

Interferon-stimulated and metallothionein-expressing macrophages are associated with acute and chronic allograft dysfunction after lung transplantation

Lung transplant (LT) recipients experience episodes of immune-mediated acute lung allograft dysfunction (ALAD). ALAD episodes are a risk factor for chronic lung allograft dysfunction (CLAD), the major cause of death after LT. We have applied single-cell RNA sequencing (scRNAseq) to bronchoalveolar lavage (BAL) cells from stable and ALAD patients and to cells from explanted CLAD lung tissue to determine key cellular elements in dysfunctional lung allografts, with a focus on macrophages. We identified two alveolar macrophage (AM) subsets uniquely represented in ALAD. Using pathway analysis and differentially expressed genes, we annotated these as pro-inflammatory interferon-stimulated gene (ISG) and metallothionein-mediated inflammatory (MT) AMs. Functional analysis of an independent set of AMs in vitro revealed that ALAD AMs exhibited a higher expression of CXCL10, a marker of ISG AMs, and increased secretion of pro-inflammatory cytokines compared to AMs from stable patients. Using publicly available BAL scRNAseq datasets, we found that ISG and MT AMs are associated with more severe inflammation in COVID-19 patients. Analysis of cells from four explanted CLAD lungs revealed similar macrophage populations. Using a single nucleotide variation calling algorithm, we also demonstrated contributions of donor and recipient cells to all AM subsets early post-transplant, with loss of donor-derived cells over time. Our data reveals extensive heterogeneity among lung macrophages after LT and indicates that specific sub-populations may be associated with allograft dysfunction, raising the possibility that these cells may represent important therapeutic targets.

immunology↗

bioRxiv2021

Pro-inflammatory alveolar macrophages associated with allograft dysfunction after lung transplantation

PurposeLung transplant (LT) recipients experience episodes of immune-mediated acute lung allograft dysfunction (ALAD). We have applied single-cell RNA sequencing (scRNAseq) to bronchoalveolar lavage (BAL) cells of stable and ALAD patients to determine key cellular elements in dysfunctional lung allografts. Our particular focus here is on studying alveolar macrophages (AMs) as scRNAseq enables us to elucidate their heterogeneity and possible association with ALAD where our knowledge from cytometry-based assays is very limited. MethodsFresh bronchoalveolar lavage (BAL) cells from 6 LT patients, 3 with stable lung function (3044 {+/-} 1519 cells) and 3 undergoing an episode of ALAD (2593 {+/-} 904 cells) were used for scRNAseq. R Bioconductor and Seurat were used to perform QC, dimensionality reduction, annotation, pathway analysis, and trajectory. Donor and recipient deconvolution was performed using single nucleotide variations. ResultsOur data revealed that AMs are highly heterogeneous (12 transcriptionally distinct subsets in stable). We identified two AM subsets uniquely represented in ALAD. Based on pathway analysis and the top differentially expressed genes in BAL we annotated them as pro-inflammatory interferon-stimulated genes (ISG) and metallothioneins-mediated inflammatory (MT). Pseudotime analysis suggested that ISG AMs represent an earlier stage of differentiation which may suggest them as monocyte drive macrophages. Our functional analysis on an independent set of BAL samples shows that ALAD samples have significantly higher expression of CXCL10, a marker of ISG AM, as we as higher secretion of pro-inflammatory cytokines. Single nucleotide variation calling algorithm has allowed us to identify macrophages of donor origin and demonstrated that donor AMs are lost with time post-transplant. ConclusionUsing scRNAseq, we observed AMs heterogeneity and identified specific subsets that may be associated with allograft dysfunction. Further exploration with scRNAseq will shed light on LT immunobiology and the role of AMs in allograft injury and dysfunction.

immunology↗

bioRxiv2021

Ex vivo delivery of regulatory T cells for control of alloimmune priming in the donor lung

Survival after lung transplantation (LTx) is hampered by uncontrolled inflammation and alloimmunity. Regulatory T cells (Tregs) are being studied for post-implantation cell therapy in solid organ transplantation. Whether these systemically administered Tregs can function at the appropriate location and time is an important concern. We hypothesized that in vitro expanded, recipient-derived Tregs can be delivered to donor lungs prior to LTx via ex vivo lung perfusion (EVLP), maintaining their immunomodulatory ability. In a rat model, Wistar Kyoto (WKy) CD4+CD25high Tregs were expanded in vitro prior to EVLP. Expanded Tregs were administered to Fisher 344 (F344) donor lungs during EVLP; left lungs were transplanted into WKy recipients. Treg localization and function post-transplant were assessed. In a proof-of-concept experiment, cryopreserved expanded human CD4+CD25+CD127low Tregs were thawed and injected into discarded human lungs during EVLP. Rat Tregs entered the lung parenchyma and retained suppressive function. Expanded Tregs had no adverse effect on donor lung physiology during EVLP; lung water as measured by wet- to-dry weight ratio was reduced by Treg therapy. The administered cells remained in the graft at 3 days post-transplant where they reduced activation of intragraft effector CD4+ T cells; these effects were diminished by day 7. Human Tregs entered the lung parenchyma during EVLP where they expressed key immunoregulatory molecules (CTLA4+, 4-1BB+, CD39+, and CD15s+). Pre-transplant Treg administration can inhibit alloimmunity within the lung allograft at early time points post- transplant. Our organ-directed approach has potential for clinical translation.

immunology↗

bioRxiv2020

Connective-Tissue Growth Factor (CTGF/CCN2) Contributes to TGF-β1-Induced Lung Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by progressive and excessive accumulation of myofibroblasts and extracellular matrix in the lung. Connective-tissue growth factor (CTGF) is known to exacerbate pulmonary fibrosis in radiation-induced lung fibrosis, and in this study, we show the upregulation of CTGF from a rat lung fibrosis model induced by adenovirus vector encoding active TGF-β1 (AdTGF-β1), and also in patients with IPF. The expression of CTGF was upregulated in vascular smooth muscle cells cultured from fibrotic lungs on days 7 or 14 as well as endothelial cells sorted from fibrotic lungs on day 14 or 28 respectively. These findings suggest the role of different cells in maintaining the fibrotic phenotype during fibrogenesis. Treatment of fibroblasts with recombinant CTGF along with TGF-β increases pro-fibrotic markers in fibroblasts, confirming the synergistic effect of recombinant CTGF with TGF-β in inducing pulmonary fibrosis. Also, fibrotic extracellular matrix upregulated the expression of CTGF, as compared to normal extracellular matrix, suggesting that not only profibrotic mediators but also a profibrotic environment contributes to fibrogenesis. We also showed that pamrevlumab, a CTGF inhibitory antibody, partially attenuates fibrosis in the model. These results suggest that pamrevlumab could be an option for the treatment of pulmonary fibrosis.Competing Interest StatementT. Yanagihara was funded by the Uehara Memorial Foundation Research Fellowship and Mitacs Canada, and the research institute of St Joseph Hospital, Hamilton, ON, Canada (Post-doctoral Fellowship Award). K. Ask reports grants and personal fees from Boehringer Ingelheim, grants from Canadian Pulmonary Fibrosis Foundation, Synairgen, Alkermes, GlaxoSmitheKline, Pharmaxis, Unity, Avalyn, Canadian Institutes of Health Research, Ceapro, Pieris, outside the submitted work. M. Kolb reports grants from the Canadian Institute for Health Research and grants/ personal fees from Roche, Boehringer Ingelheim, Prometic, Respivert, Alkermes, and Pharmaxis and personal fees from Genoa. K.E. Lipson is an employee and shareholder of FibroGen, Inc. J. Tikkanen reports personal fees from CSL Behring, outside the submitted work. S.G. Chong, M. Gholiof, Q. Zhou, C. Scallan, C. Upagupta, and S. Keshavjee report no conflict of interest.View Full Text

cell biology↗