A High Proportion of CD4+CD25+ Lymphocytes Associates with a Risk of aGvHD and Inversely Correlate with Proportions of CMV and EBV Specific CD8+ Cells in Blood.

Treg cells control immunoresponsiveness and it is suggested that they may be beneficial in HSCT patients by preventing aGvHD. We followed 58 patients (8 children from 2 to 13 yrs of age, 50 adults from 19 to 60 yrs of age) for the proportions of CD4+CD25+ cells in association with the clinical symptomatology. Twenty seven and 31 patients received HSCT (myeloablative:17 patients, reduced intensity conditioning: 31 patients) from sibling and unrelated donors, respectively.

CD4+CD25+ cells were enumerated in blood in HSCT receiving patients together with other lymphocyte markers (three color cytometry analysis) starting at the beginning of hematological recovery (usually from +11 to + 25 day post HSCT), then in one or two weeks intervals while the patients stay in the hospital and then one time per months. In addition skin lesions suggesting developing of aGvHD prompted us to control lymphocyte profile. In 18 patients (62 measurements) lymphocyte profiling included detection of CMV (CD8high, HLA-A*0201/NLVPMVATV) and EBV (CD8high, HLA-A*0201/GLCTLVAML) specific cytotoxic cells.

We found:

1. patients at the very first day of aGvHD manifestation (histopathology proven) had higher proportions of CD4+CD25+ cells (mean±sem: 11,95%±1,5 vs 4,59%±1,5, p=0,003), and their CD4+CD25bright+ subpopulation more frequently exceeded upper limit of normal values (11/15 vs 3/11, p=0,03) than patients lacking aGvHD

2. patients having or developing aGvHD between day 25 to 60 post transplant had higher proportions of CD4+CD25+ cells as compared to patients lacking aGvHD (6,9%±0,8 vs 3,9%±0,6, p=0,006), CD4+CD25+ cells and CD4+CD25bright+ cells were positive for FoxP3 staining in 38,4%±2,4 and 76,3%±2,0, respectively, while CD4+CD25− cells had FoxP3 in 12,9%±1,2 only.

Similarly FoxP3 transcripts were seen (Q-PCR) in purified CD4+ cells but not in CD4− cells. We can assume that CD4+CD25+ cells are equipped with a suppressor cell machinery, therefore, represent Treg cell population.

Notably, an inverse correlation was found between proportions of EBV specific CD8+ cells and proportions of CD4+CD25+ cells in blood (r=−0,38, p=0,002). CMV specific CD8+ cells were in the protective range (usually seen in patients lacking CMV reactivation) only in cases having low CD4+CD25+ proportions (<6%) in blood (10/48 vs 0/17, p=0,04). Patients with a high proportion of CD4+CD25+ cells (≥10%) in blood suffered more frequently from life threatening transplant complications including viral reactivation than their low CD4+CD25+ counter partners (16/39 vs 2/16, p=0,04)

In conclusion:

1. CD4+CD25+ cells and their CD25+bright population were frequently FoxP3 positive meeting the criteria of Treg cells,

2. Treg cells were in a high proportion in patients developing aGvHD and this may reflect the level of alloreactivity in these patients,

3. as a consequence, being furnished in suppressor function, they hampered viral response,

4. finally Treg cells unfavorably affected the outcome of transplantation.