An Increasing Severity of Chronic GvHD Is Associated to an Activated and Cytotoxic T Cell Mediated Immune-Phenotype

Introduction. Chronic graft-versus-host-disease (cGvHD) is a late complication (>90 days) after allogeneic hematopoietic stem cell transplantation (HSCT). Symptoms might occur in multiple organs and vary from mild to severe. In this study, the NIH criteria were used to group the patients. We performed an extensive immune-phenotypic analysis using cells and plasma from patients diagnosed with and without cGvHD. The aim of the study is to gain information on possible markers that could be used by clinicians to aid in the diagnosis of cGvHD.

Methods. Peripheral blood samples were obtained from patients ≥ 1 year post-HSCT with an acute GvHD no higher than grade I. Patients were divided into 4 groups: no (N=10), mild (N=6), moderate (N=5) and severe cGvHD (N=8). Cytokine levels were measured by Luminex and an immune-phenotypic analysis was done by multicolour flow cytometry. Univariate analysis comparing the different patient groups was done with the Mann-Whitney U test.

Results. Due to usage of immunosuppressive treatments in the moderate and severe cGvHD groups, we chose to compare patients with no versus mild cGvHD and moderate versus severe cGvHD. Expression of the cell activation marker CD38 was higher on total T cells and CD8+ T cells (p=0.005 for both subsets) in mild cGvHD patients compared to patients without cGVHD. Furthermore, severe cGvHD patients had higher levels of CD8+ T cells (p=0.03) than patients with moderate cGvHD. Additionally, several Th2-associated cytokines were down regulated in severe cGvHD patients, compared to the other patient groups. An analysis was also done based on the presence of cGvHD in specific organs (lung, mouth and skin) within the moderate and severe cGvHD group. In patients with lung cGvHD; both the expression of degranulation marker CD107a (p=0.003) on T cells and the expression of KIR receptor CD158b on NK cells negative for the Fc receptor CD16 (p=0.035) were found to be lower. Anti-inflammatory cytokine IL-10 was decreased (p=0.018) in patients with mouth cGvHD. Lastly, CD158b+ CD16+ NK cells and total CD158b+ NK cells were found to be less frequent in patients with skin cGvHD (p=0.017 for both subsets).

Discussion. Mild and severe cGvHD patients appear to have a more activated and cytotoxic immune-phenotype compared to patients with no and moderate cGvHD, respectively. Additionally, patients with specific organ-oriented cGvHD seem to have a lower percentage of NK cell subsets. In conclusion, it appears that patients with an increasing severity of cGvHD have a more activated cytotoxic phenotype, which seems to mostly depend on CD8+ T cells and not on NK cells. By studying the phenotype of the immune system in these cGvHD patients, we can gain new insights in the pathogenesis behind cGvHD. Our findings, i.e. distorted immune cell phenotypes, could potentially be used as biomarkers in the diagnostics of cGvHD.