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 pro-inflammatory, 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 oxidised 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 pro-inflammatory. 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 characterise 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, IL-6 and IFN-γ production. LDN, but not NDN, CD10 expression was inversely correlated to LOX-1, which correlated with IL-6 production. LDNs resisted apoptosis and differentiated into antigen-presenting/neutrophil-hybrid-like cells, which co-expressed MHC-Class II HLA-DR and immuno-inhibitory PD-L1, but did not suppress T-cell proliferation. These data suggest LDNs in cGVHD are predominantly immature, pro-inflammatory and may have pathogenic potential.
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