Decrease of FoxP3 Gene Expression Precedes and Inversely Correlates with Onset of Acute GVHD.

Background: Regulatory CD4+CD25+FoxP3+ T-cells are hypothesized to play a role in modulating the severity of acute GVHD and induction of non-responsiveness or tolerance after hematopoietic cell transplantation. Recent studies in patients post transplant demonstrated association between decrease of FoxP3 and development of acute GVHD. However sequential changes of FoxP3 expression prior to onset of clinical GVHD have not been reported.

Methods: Thirteen patients who received myeloablative-conditioning regimen for hematological malignancies and cyclosporine and methotrexate for GVHD prophylaxis have been studied on two or more occasions prior to onset of clinically significant grade II-IV acute GVHD. Six of these patients developed GVHD by day 30 (range, 29–31 days), 4 patients by day 37 (range, 35–39 days), and 3 patients showed no evidence of GHVD during the first 100 days post-transplant. Sequential measurements of FoxP3 gene expression by real time PCR were made at day 21, day 28 and day 35 post-HSCT. All samples analyzed were collected before initiation of systemic steroid therapy. The 6 patients with onset by day 30 and 1 patient without GVHD were studied at days 21 and 28. RNA was extracted from white blood cells and cDNA templates were synthesized using 1μg RNA. The standard curve method was used for relative quantitation of Foxp3.

Results: Using the paired t test, we found a significant decrease of FoxP3 gene expression between day 21 (median 0.3; range 0.01–1.9) and day 28 (median 0.13; range 0.003–0.321) (p=0.05). Additionally, a clear trend for a decreasing expression of Foxp3 was found between day 28 and day 35 (median 0.1; range 0.06–0.54). Two of the 3 patients who remained GVHD free the first 100 days showed either an increase or no change in FoxP3 expression between days 21, 28 and 35 and between days 21 and 28 respectively.

Conclusion: These preliminary results indicate a pattern of decreasing FoxP3 gene expression in patients preceding the development of acute GVHD independent of the day of onset of this disease. These studies are being extended in a new cohort of transplant patients, and monitoring of patients without GVHD is continuing to determine if there are any patterns of FoxP3 expression associated with the onset of chronic GVHD. The available preliminary data suggest that sequential measurements of FoxP3 may predict onset of clinically significant acute GVHD.