Influence of G-CSF on the Expression of IL-2 and IL-7 Cytokine Receptor α-Chain in T Regulatory Cells from Healthy Donors and Possible Relation to GvHD

Background: G-CSF-mobilization of hematopoietic stem cells to blood followed by apheresis is now commonly used instead of bone marrow harvests to increase the stem cell yield for clinical transplantation, leading to an enhanced engraftment. Blood harvests also contain more T-cells, and result in slightly more chronic graft-versus-host disease. G-CSF modulates T-cells, but its effect on regulatory T-cells is unknown. FoxP3 expressing CD4⁺ T cells have been shown to inhibit active effector cells and limit immune reactions to autologous and allogeneic antigens. These T cells derive from thymus and are characterized to have high/medium CD25 and low CD127 expression levels in healthy volunteers, and may reflect steady state. We hypothesized, that G-CSF treatment may affect the functional state of T regulatory cells by modulating the surface expression of these two cytokine receptors.

Experimental setup: Blood from healthy stem cell donors were sampled before and after 5 days of G-CSF mobilization (n=58) and analyzed with 5 color flow cytometry. FoxP3⁺/CD4⁺ cells were analyzed for expression of CD25 and CD127. CD45RA was used to distinguish naïve versus antigen experienced T cells.

Results: Previously we found that the level of CD127 receptor on the surface increases upon G-CSF treatment, measured by mean fluorescence intensities (MFI) in FoxP3 positive (FP) and negative (FN) cell populations. Additionally, G-CSF up-regulates CD25 expression measured by MFI selectively in the FN population, no significant changes were observed in the FP population. In this study we extended sample number to 58 and studied T regulatory cell populations more extensively for CD25, CD127 expression. Previous studies showed that the CD127^lo CD25^med/hi population identifies and better correlates to the T regulatory population than CD25 alone. We examined the FoxP3 positive population and found that while CD127^lo/CD25^med/hi cells were indeed FoxP3 positive, the fraction of FoxP3 positive cells that were CD127^hi/CD25^medcoincided graphically with the conventional T cell population. This population on the average represented 21,8%±SEM3,9 (range 2,6–71,8 %) of FoxP3 positive cells in healthy unmobilized donor blood. Comparison G-CSF mobilized vs unmobilized donors did not show any difference. CD45RA analysis in unmobilized donors revealed that some donors have significant numbers of naïve T regulatory cells (12,5%± 1,89 SEM). We examined if G-CSF has any effect on the aforementioned population and did not find significant differences in overall group comparisons. However, in individual paired samples we observed a clear induction of CD45RA⁺/FoxP3⁺ positive cells, and hypothesized that this might influence the risk for GvHD. The CD45RA fraction of FoxP3 cells were significantly larger in donors whose recipients developed GvHD requiring systemic steroid treatment (n=11) as compared to donors whose recipients did not develop GvHD or developed grade I GvHD (n=13) requiring only local treatment, 15,5%±2,8SEM vs. 7.9%±1.8 SEM of CD45RA expressing cells. These findings may be important and require further more thorough characterization of CD127hi/CD25med and CD45RAhi populations. These studies are ongoing in our laboratory.

Conclusions: GCSF influence T-reg populations, and T-reg subsets may be correlated with GvH