Expression Of Exosomal PD-L1 In Gastric Diffuse Large B-cell Lymphoma And Its Role In The Immune Microenvironment

Objective:This study explores the role of programmed cell death ligand 1(PD-L1)in exosomes in the diagnosis and treatment of gastric diffuse large B-cell lymphoma(GDLBCL)by studying the expression level of exosomal PD-L1 in culture supernatant of normal gastric mucosa and GDLBCL cells as well as in plasma of normal healthy people and GDLBCL patients,Furthermore,though overexpressing and silencing exosomal PD-L1 in GDLBCL cells to study its role in immune microenvironment.Methods: We collected plasma from GDLBCL patients and healthy people as well as culture normal gastric mucosal cells and GDLBCL cells,and extraction exosomes ultracentrifugeby Exosomes 4.g ExoQuick assay and ultracentrifuge.Exosomes were identified by transmission electron microscopy,nanoparticle tracking analysis and western blot analysis.The expression of exosomal PD-L1 in cell culture supernatant and plasma was detected by western blot.And the correlation between the clinicopathological parameters and PD-L1 level in the plasma exosomes of GDLBCL patients was analyzed.Finally,exosomal PD-L1 of GDLBCL cells was overexpressed and silenced by transfection and exosomes was co-cultured with T lymphocyte to study its role in immune microenvironment.Results: 1.Exposure of CD9 and CD63 showed successful extraction of exosomes.Exosome nanoparticle tracking analysis(NTA)showed that the diameter of the exosomes presented a normal distribution and mainly concentrated in the range of 70 to 120 nm.Expression of CD9 and CD63,successful electron microscopy and exosomal nanoparticle tracking analysis suggested successful extraction of exosomes from cell culture supernatant and plasma.2.The results of western blot experiment showed that the expression level of exosomal PD-L1 in the supernatant of GDLBCL cells culture was higher than that in the supernatant of normal gastric mucosa cells.The expression level of exosomal PD-L1 in plasma of patients with GDLBCL was higher than that in normal healthy people.The expression level of exosomal PD-L1 in plasma of patients with stage III to IV GDLBCL was lower than patients with stage I to II GDLBCL;the expression level of exosomal PD-L1 in plasma of patients with pathological type GCB was lower than that with pathological type ABC;the expression level of exosomal PD-L1 in plasma of patients with IPI 3-5 scores was higher than that with 0-2 scores.(P < 0.05 for all).3.Construction of exosomes with different expression levels of PD-L1 in GDLBCL cells,After transfecting GDLBCL cells with Over expression,silencing plasmids or Negative control for 48 hours,the expression levels of PD-L1 in exosomes of GDLBCL cell culture supernatants was changed: Compared with the Negative control group,PD-L1 expression was significantly inhibited in exosomes after transfection with silencing plasmids;In exosomes transfected with over expression plasmids,PD-L1 expression was significantly up-regulated.4.MTT showed that compared with the Negative control group,the proliferation ability of T lymphocyte decreased after co-cultivation with the exosomes with PD-L1 overexpression,and increased after co-cultivation with the exosomes with PD-L1 inhibition.Conclusion:1.Over expression of exosomal PD-L1 in GDLBCL cells culture supernatant,exosomal PD-L1 is overexpressed in peripheral blood of GDLBCL patients.The expression level of exosomal PD-L1 in peripheral blood of patients with GDLBCL is significantly different from that of normal people,suggesting that exosomal PD-L1 in peripheral blood may be a new diagnostic indicators for GDLBCL.2.There were significant correlations on the expression level of exosomal PD-L1 in peripheral blood with tumor stage and pathological classification.High expression may indicate poor prognosis.3.Exosomal PD-L1 overexpression in patients with GDLBCL can inhibit the proliferation of T lymphocyte and inhibit the formation of the immune microenvironment.Exosomal PD-L1 may be used as a clinical anti-PD-1 predictors for immunotherapy.