Molecular monitoring of acute graft-versus-host disease after allogenic stem cell transplantation

Sammanfattning: Acute graft-versus-host disease (GVHD) remains a major barrier to the wider application of allogeneic stem cell transplantation (SCT) for a variety of diseases. GVHD occurs when the transplanted donor Tlymphocytes react to host antigens on antigen-presenting cells and attack host tissues. The donor lymphocytes are introduced into a milieu that promotes their direct attack on target cells, causing tissue damage through perform, granzyme B, and Fas/Fas ligand (FasL) interactions. In addition, the dysregulated production of inflammatory cytokines, such as TNF-alpha, IFN-gamma, IL-1, and others, may cause direct tissue damage. The immunobiology of acute GVHD is complex and the precise mechanisms by which host tissues are damaged remain unclear. Despite progress in understanding the mediators involved in acute GVHD, treatment has remained frustrating; most patients who develop the severe manifestations of GVHD succumb to it or to complications of its treatment. Today, the only available method of diagnosing acute GVHD relies on clinical observations and clinical judgments. Since plenty of other problems can influence the symptoms seen in patients after SCT and further confuse the picture of the patient's disease, diagnosing acute GVHD can be rather difficult. It is well known that predicting the risk of acute GVHD before its clinical manifestation and early administration of additional therapy may result in less incidence of severe GVHD. Hence, new methods to diagnose acute GVHD are desired. Therefore, the major aim of this thesis has been to find new reliable molecular methods to diagnose acute GVHD after allogeneic SCT. The gene expression of T-cell effector molecules, granzym B, perform, FasL, and TNF-alpha, was evaluated as diagnostic markers for acute GVHD after SCT. Peripheral blood samples from patients were analysed by competitive or real-time polymerase chain reaction (PCR). An up-regulation of the gene expression of was seen in association with acute GVHD diagnosis in 23 of 27, 26 of 27, and 24 of 27 patients diagnosed with acute GVHD for granzyme B, perform and FasL, respectively. The gene expression of TNF-alpha did not show the same pattern. Interestingly, we also found that patients with increasing levels during steroid treatment, 2 of 3 in Paper I and 10 of 10 in Paper II, showed persistent or deteriorating acute GVHD. Using VNTR analysis, chimerism patterns were investigated in 34 patients during the early posttransplantation period. The difference in the clearance rate of host T-cells between day 7 and day 10 was compared. In this study, we found that there was a significantly higher risk for patients with complete donor chimerism on day 7, together with patients with an increase of more than 50% in the donor CD4+ T-cell population between day 7 and 10, to develop grades II-IV acute GVHD. Our data indicate that early monitoring of T-cell chimerism after SCT may be of use for early identification of patients at risk of developing moderate to severe acute GVHD after SCT. We hypothesized that the gene expression of chemokine receptors in peripheral blood from patients after SCT would reflect the development of acute GVHD since these molecules are involved in the recruitment of activated T-lymphocytes during acute GVHD. Quantitative real-time PCR was used to assess the gene expression of the chemokine receptors CCR5, CXCR3, CCR1, and CCR2. In this study we found that increasing levels of these receptors were associated with acute GVHD. We found increasing levels in connection to acute GVHD diagnosis of all four markers in 35, 33, 35, and 35 of 46 occasions of acute GVHD for CCR5, CXCR3, CCR1, and CCR2, respectively. Another important finding was that the gene expression levels increased before acute GVHD was diagnosed clinically with the median number of days before diagnosis ranging from 3 to 5 days. In conclusion, although the pathophysiology of acute GVHD still remains complex, this study shows that different molecular markers involved in this complicated disorder may be used to diagnose and predict acute GVHD and to monitor steroid treatment. This may prevent life-threatening complications and improve the outcome for patients after allogeneic SCT.