Abstract
<h3>Purpose</h3> CLAD, the main limitation to long-term survival after a lung transplantation (LTx), has two main phenotypes: bronchiolitis obliterans syndrome (BOS) and restrictive allograft syndrome (RAS). There is great need for better characterization of cells within the lung allograft tissue. We aimed to assess CLAD lung allografts using imaging mass cytometry (IMC), a high dimensional tissue imaging system allowing a comprehensive and multiparametric in situ exploration of tissue microenvironments at a single cell level, using heavy metal-tagged antibodies. <h3>Methods</h3> Explanted lung tissue was obtained at the time of retransplantation from 4 BOS patients, 4 RAS patients, and 4 control donor lungs. Paraffin-embedded lung tissue was sectioned and stained with 35 heavy-metal-tagged antibodies selected to identify structural and immune proteins of interest. For each sample, three 1 mm<sup>2</sup> regions of interest, centered on an airway, were ablated and data was analyzed using MCD viewer and HistoCAT software. <h3>Results</h3> To identify specific cell subsets identified by combinations of markers, we used tSNE to create a 2D representation of cells organised in 50 immune and non-immune clusters according to the similarity of their antibody-binding in 35-dimensional space (Figure 1A). We present several key examples of our findings: CLAD lungs had significantly reduced club cells (Figure 1B-D, H) and a trend towards reduced basal cells (Figure 1B-D, I). A Ki67-high basal cell population was mostly present in RAS samples (Figure 1B-D, I-J) and in close proximity to memory T cells (neighborhood analysis not shown, Figure 1L). Memory CD8+ T cells were more frequent in CLAD lungs (Figure 1E-G, K) with prominent regulatory T cells in RAS (Figure 1E-G). <h3>Conclusion</h3> IMC allowed us to identify distinct basal cells and their proximity to cytotoxic memory T cells in CLAD lungs. IMC is a powerful technology for detailed cellular analysis within intact organ structures that may shed further light on CLAD mechanisms.
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