Emergence of a Specific Intrapulmonary CD4+ T Cell Subset Prior to the Onset of Lung Allograft Dysfunction

Purpose We have applied cutting-edge mass cytometry (MC) technology to serial bronchoalveolar lavage (BAL) cells obtained from lung transplant recipients (LTRs) and have used it to compare the cellular composition of the BAL compartment in patients who remain stable or go on to develop lung allograft dysfunction (LAD), defined here as a drop, from any cause, in the forced expiratory volume in 1 second of 20% from the best post-transplant baseline value. Methods We have collected and cryopreserved BAL cells from 50 consecutive LTRs at their 3-, 6- and 9-month post-transplant surveillance bronchoscopies. Cryopreserved samples were then recovered and labeled with a panel of 37 heavy metal-tagged antibodies against surface markers and intracellular cytokines before MC. We applied t-distributed stochastic neighbor embedding (tSNE; Fig1 A) which assists with better visualization of multi-dimensinal analysis to identify both common and rare cell populations in the BAL. We further applied an algorithm called cluster identification, characterization and regression (CITRUS; Fig1 B) to allow unsupervised identification of differentially represented BAL cellular subsets between stable and LAD patients. Results Longitudinal analysis of the first 25 patients demonstrated different cell subsets and cytokine-producing cells, with substantial intra- and inter-patient variation. Our data identified an elevation in the frequency of CD4 + CD57 + PD-1 + cells separates LAD (18.41±4.89%) from stable patients (4.43±2.11%; Fig1 C). Emergence of CD57 + PD-1 + events among CD4 + T cells preceded LAD by up to several weeks (Fig1 D), suggesting that these cells may contribute to LAD development rather than simply correlating with it. Conclusion Our data suggest that emergence of CD4 + CD57 + PD-1 + T cells may predict and/or contribute to allograft dysfunction. Molecular and functional studies on this cell population are underway to obtain a better understanding of their role in lung transplant immunobiology.