High-dimensional flow cytometry reveals lymphocyte subset populations predictive of chronic lung allograft dysfunction.

OBJECTIVES: Despite cellular and antibody-mediated rejection being clinically relevant drivers of chronic lung allograft dysfunction (CLAD), there are few studies describing the T- and B-cell dynamics inherent to such alloreactive responses. We conducted a longitudinal immunophenotyping study of B- and T-cell subsets from pre- to 12 months post-lung transplant, focussing on patients who subsequently developed either donor specific antibodies to human leukocyte antigen class II (HLA-DSA) or CLAD within 3 years. METHODS: In a single centre, comparative study, we used high-dimensional flow cytometry clustering analysis to assess the B- and T-cell populations in blood from lung allograft recipients prior to transplantation and at 0.5, 1.5, 3, 6, 9 and 12 months post-transplantation. Recipients who developed de novo HLA-DSA at 3 months post-transplantation (n = 18) and those in whom CLAD was diagnosed within 3 years post-transplantation (n = 13) were compared to matched, DSA-negative (n = 15) or CLAD-free recipients (n = 26), respectively. RESULTS: This longitudinal study provided a detailed analysis of B- and T-cell lineage subsets, including both cell frequencies and cell counts. There were no statistically significant differences in lymphocyte populations between graft recipients with and without HLA-DSA. However, patients that developed CLAD had a mean threefold deficit in the absolute number of B cells and had significantly fewer T regulatory cells than CLAD-free patients. Strikingly, these differences existed prior to and persisted post-transplantation. CONCLUSIONS: Utilising high-dimensional flow cytometry, a new putative association was identified between two peripheral blood lymphocyte populations and the subsequent development of CLAD.