Clinical Correlation of Foxp3 Regulatory Gene Expressions and NK Cells in Early Engraftment after Myeloablative Allogeneic Stem Cell Transplantation.

Regulatory T cells (Tregs) have been established as a key role of self tolerance and preventing proliferation of auto- and alloantigen-reactive T cells. CD4+CD25+ Tregs suppress GVHD in animal models, but in human data on GVHD following stem cell transplantation (SCT) is conflicting. We postulate that CD4+Foxp3+Tregs not only reduce the incidence of acute graft-versus host disease (aGVHD) but inhibit the NK cell functions in patients who received allogeneic SCT. CD4+Foxp3+Tregs also may adversely affect the GVL effect and cause the relapse of disease.

Patients and Methods: 25 patients (AML:13, ALL:9, CML:2, NHL:1) were undergone allogeneic SCT. Nine patients were infused from unrelated donor and five patients were from HLA one-mismatch. Three patients added alemtuzumab to myeloablative conditioning regimen due to HLA mismatch. Peripheral blood mononuclear cell (PBMC) were separated 3 or 4 weeks after SCT when absolute neutrophil count reached above 1000 × 10⁹/L. NK cells were phenotypically analyzed by flow cytometry using directly conjugated antibodies to CD3 and CD56. CD4+ cells were isolated from PBMC using micro-bead (MACS) and the expression levels of Foxp3 mRNA were assessed by quantitative real-time PCR.

Results: 12 patients developed grade 2–4 acute GVHD and 10 patients relapsed. Patients who experienced Gr2-4 aGVHD had significantly lower the relapse rate than those who Gr0-1 aGVHD (P=0.002). Foxp3 gene expressions within CD4+ T cells were significantly lower in Gr2-4 aGVHD patients (median, 4.278 ng/μl) than in Gr0-1 aGVHD patients (median 7.914 ng/μl) except three patients treated with alemtuzumab conditioning (P=0.016). All of three patients used alemtuzumab experienced the relapse and no aGVHD. They also had very lower Foxp3 gene expressions (median, 4.760 ng/μl) than those in Gr0-1 aGVHD patients. Without alemtuzumab used patients, the levels of Foxp3 expression in relapsed patients were significantly higher than those in non-relapsed patients (median, 11.684 ng/μl and 2.031 ng/μl, respectively) (P=0.001). However, we could not find an inverse correlation between NK cell expressions and CD4+Foxp3+Tregs and a positive correlation between infused T cell doses and CD4+Foxp3+Tregs expression.

Conclusion: The levels of CD4+Foxp3+Tregs affect the incidence of aGVHD and predict the risk of the relapse. Alemtuzumab may influence the T cell recovery including regulatory T cells after early post-SCT.