Plasmacytoid dendritic cells, effector T-lymphocytes and inflammatory cytokines in graft-versus-host disease

Perić, Zinaida (2014) Plasmacytoid dendritic cells, effector T-lymphocytes and inflammatory cytokines in graft-versus-host disease. PhD thesis, Sveučilište u Zagrebu.

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Abstract

GVHD continues to be a major source of morbidity and mortality following allogeneic stem cell transplantation (allo-SCT). Previous studies established the role of Th17 subpopulation of lymphocytes and the plasmacytoid dendritic cells (pDC) in the pathophysiology of several autoimmune diseases. The aim of this research was to evaluate the role of pDC and Th17 in the peripheral blood of patients at day 100 after allo-SCT in acute and chronic GVHD and GVL effect. The other aim of the study was to expand the search for chronic GVHD biomarkers by validating cytokines in the blood of patients at day 100 after allo-SCT and to determine a composite prognostic score for prediction of chronic GVHD. This study included peripheral blood mononuclear cells (PBMC) of 79 patients taken at day 100 after allo-SCT and 152 serums from patients who underwent allo-SCT between 2005 and 2011 in Centre Hospitalier Universitaire de Nantes, France. For functional analysis of pDC, PBMC from 79 blood samples were stimulated with TLR7 and TLR9 ligands in the presence of IL-3 over 6 hours, and then stained for surface markers and intracellular cytokines (IFN alpha, TNF alpha and IL6). Th17 were evaluated quantitatively on the same PBMC by staining for specific surface markers. Forty-one serum cytokine and chemokine was studied in all 152 serums using Luminex xMAP technology. We observed a significant decrease of total and activated pDC, as well as Th17 and IFN gamma producing cells in the blood of patients with grade 2-4 acute GVHD as compared to patients with grade 0-1 acute GVHD. After dividing patients into 2 distinct groups, using the median value of pDC and Th17, we observed that a low pDC count predicts relapse and worse overall survival and low Th17 predicts more extensive chronic GVHD. In the multivariate analysis, low pDC count retained it’s predictive value for worse overall survival together with the older age of the patients while low Th17 count stayed the only independent predictor of extensive chronic GVHD. Independently from all relevant clinical factors, 10 cytokines were found to be significantly correlated with the development of extensive chronic GVHD. Based on the significant cytokines and clinical factors, we established a practical prognostic score using the traditional multivariate Cox model combined with“time-dependent ROC curves”. The area under the time-dependent ROC curve of 0.80 indicated that such composite score is a powerful predictor of the risk of extensive chronic GVHD. In concordance with previous GVHD studies, the significant decrease of both pDC and Th17 cells in peripheral blood on day 100 after allo-SCT in patients with clinically severe acute GVHD could be the result of the migration of these cells to target tissues of GVHD. Therefore, this study provides evidence for a potential new pathophysiological link between pDC and Th17 in human acute GVHD, and identifies these cells as potential new targets for prophylaxis and treatment of GVHD. Moreover, we established pDC and Th17 counts in the peripheral blood of patients at day 100 after allo-SCT as valuable predictors of overall survival, relapse and chronic GVHD and have proposed that these cells, have an important role in both GVHD and GVL effect. Monitoring pDC and Th17 count could allow for early classification of patients according to the risk for adverse events and allow for potential early therapeutical interventions to improve their clinical outcomes. Furthermore, new noninvasive score developed in this study to accurately predict the risk of extensive chronic GVHD after allo-SCT could be used as a decision tool in the clinical management of allo-SCT.

Abstract in Croatian

GVHD je vrlo česta komplikacija transplantacije alogenične koštane srži (alo-TKS), i dalje povezana uz visoku smrtnost. Prijašnje studije su utvrdile ulogu Th17 populacije i plazmacitoidnih dendritičkih stanica (pDC) u mnogim autoimunim bolestima. Cilj ovog istraživanja je bio procijeniti ulogu pDC i Th17 u perifernoj krvi bolesnika stoti dan nakon alo-TKS, u akutnom i kroničnom GVHD-u te GVL učinku. Također, cilj ove studije je bio procijeniti ulogu citokina kao potencijalnih biomarkera za kronični GVHD u krvi bolesnika stoti dan nakon alo-TKS kao i načiniti praktičan prognostički skor za pojavu kroničnog GVHD-a. U ovo je istraživanje uključeno 79 bolesnika kojima su periferne mononuklearne matične stanice (PMMC) uzete stoti dan nakon alo-TKS i 152 seruma uzetih stoti dan nakon alo-TKS od bolesnika koji su alotransplantirani između 2005. i 2011. u Centre Hospitalier Universitaire de Nantes, u Francuskoj. Za analizu funkcije pDC, PMMC izolirane iz 79 uzoraka krvi, stimulirane su ligandima za TLR7 i TLR9 receptore u prisutnosti IL3 kroz 6 sati, a potom bojane za površinske markere i unutarstanične citokine (IFN alfa, TNF alfa i IL6). Th17 su kvantitativno određene na istim uzorcima bojanjem na specifične površinke markere. Četrdeset i jedan citokin i kemokin je određivan u serumu 152 bolesnika korištenjem Luminex xMAP tehnologije. Primijetili smo značajno smanjenje aktiviranih pDC, Th17 te stanica koje proizvode IFN gama u bolesnika koji su stoti dan nakon alo-TKS imali razvijen akutni GVHD stadija II-IV u odnosu na bolesnike bez ili sa stadijem I akutnog GVHD-a. Kada smo bolesnike podijelili u dvije skupine, uzimajući kao razdjelnicu medijan vrijednosti postotka pDC i Th17, primijetili smo da niže vrijednosti pDC u krvi predviđaju relaps i lošije ukupno preživljenje bolesnika, a niže vrijednosti Th17 predviđaju razvoj ekstenzivnog kroničnog GVHD-a. U multivarijatnoj analizi, niže vrijednosti pDC su zadržale prediktivnu vrijednost za lošije preživljenje bolesnika, kao i starija dob bolesnika, dok su niže vrijednosti Th17 bile jedini neovisni čimbenik za predviđanje ekstenzivnog kroničnog GVHD-a. Deset se citokina, neovisnih o važnim kliničkim faktorima, pokazalo značajnima za razvoj ekstenzivnog kroničnog GVHD-a. Pomoću značajnih citokina i kliničkih čimbenika, stvorili smo praktičan prognostički skor na temelju Coxove multivarijatne analize i vremenski ovisnih ROC krivulja. Prema AUC vrijednosti od 0.8, radi se o relativno moćnom predskazatelju rizika ekstenzivnog kroničnog GVHD-a. U skladu s našom prijašnjom studijom uloge pDC i Th17 u GVHD-u, značajno sniženje ovih populacija stanica stoti dan u perifernoj krvi bolesnika s teškim akutnim GVHD-om bi moglo označiti migraciju aktiviranih stanica u ciljna tkiva GVHD-a. U svakom slučaju, istovremeno značajno sniženje broja obiju populacija u krvi bolesnika može ukazivati na njihovu povezanost u patofiziologiji GVHD-a, te ih identificirati kao potencijalne nove ciljne molekule u profilaksi i liječenju GVHD-a. Također, utvrdili smo da su brojevi ovih stanica u perifernoj krvi stoti dan nakon alo-TKS vrijedni prediktori za ukupno preživljenje, relaps i kronični GVHD, te tako pretpostavili da ove stanice imaju značajnu ulogu i u GVHD-u i GVL-u. Monitoriranje broja ovih stanica bi moglo omogućiti klasifikaciju bolesnika prema riziku za razvoj neželjenih događaja te potencijalnu ranu terapijsku intervenciju za poboljšanje njihovog kliničkog ishoda. Novi prediktivni skor za razvoj kroničnog ekstenzivnog GVHD-a mogao bi postati vrijedan alat u kliničkom odlučivanju nakon alo-TKS.

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