Low-density neutrophils in chronic graft versus host disease (cGVHD) are primarily immature CD10^- and enhance T cell activation

Chronic graft-versus-host disease (cGVHD) is a frequent complication of allogeneic haematopoietic stem cell transplantation. Low density neutrophils (LDNs) in autoimmunity, which shares disease features with cGVHD, are proinflammatory, whereas those in cancer and sepsis suppress T cell immunity. Mature LDNs can be distinguished from immature LDNs on the basis of expression of CD10 and suppressive neutrophils can be identified using lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) expression. The functionality of LDNs in cGVHD has not been specifically investigated. Here, we have determined the relative contribution of immature and mature neutrophils to LDNs in cGVHD and assessed whether these were suppressive or potentially proinflammatory. Peripheral blood LDNs and normal density neutrophils (NDNs) from 30 cGVHD patients and NDNs from 10 healthy controls (HCs) were immunophenotyped by flow cytometry. The ability of LDNs and NDNs to influence T cell proliferation and cytokine production in co-cultures was quantified. To further characterize LDNs, their propensity to undergo constitutive apoptosis and differentiate ex vivo was assessed. LDNs were elevated in cGVHD versus HCs, heterogeneous in phenotype, with a predominance of immature CD10^- cells in most patients, but some mature CD10⁺ LOX-1⁺ LDNs were also detected. LDNs enhanced autologous T cell proliferation, interleukin (IL)-6 and interferon (IFN)-γ production. LDN, but not NDN, CD10 expression was inversely correlated with LOX-1, which correlated with IL-6 production. LDNs resisted apoptosis and differentiated into antigen-presenting/neutrophil-hybrid-like cells, which co-expressed major histocompatibility complex (MHC) class II HLA-DR and immuno-inhibitory programmed cell death ligand 1 (PD-L1), but did not suppress T cell proliferation. These data suggest LDNs in cGVHD are predominantly immature, proinflammatory and may have pathogenic potential.