Autologous Peripheral Blood Stem Cell Products from Patients with Hematologic Malignancies Have Increased Frequency of Regulatory T Cells (CD4+FoxP3+ Treg).

Increased numbers of regulatory T cells (Treg) with immune suppressive function have been observed in patients with hematological malignancies. Many of these patients will undergo stem cell mobilization with high dose cyclophosphamide and G-CSF to prepare for autologous stem cell transplantation. Although infusion of low dose cyclophosphamide (Cy) seems to reduce the number of circulating Tregs, mobilization with high dose Cy and G-CSF was shown to increase the frequency of immunosuppresive Treg in stem cell autografts of myeloma patients. Treg populations have been defined by different phenotypic criteria, including CD4+FoxP3+ and CD4+CD25+ CD127^lo. In this study, we compare the impact of donor types and stem cell mobilization regimens on CD4+FoxP3+ and CD4+CD25+ CD127^lo Treg frequencies in 63 apheresis products.

Methods: Apheresis products from 23 healthy donors (17 donors received G-CSF and 6 had no stem cell mobilization) and 40 patients with hematological malignancies (1 AML, 2 HD, 25 NHL, 10 MM, 2 neuroblastoma) were evaluated. Patients with malignancies were mobilized with chemotherapy (5 RICE, 25 Cy, 1 Ara-c+Etoposide) + G-CSF (N=31), AMD3100 + G-CSF (N=5), or G-CSF alone (N=2). Samples from each product were incubated with fluorochrome-conjugated antibodies against cell surface makers, CD45, CD4, CD14, CD127, and CD25, followed by intracellular staining with anti-FoxP3 antibody. Six-color flow cytometry and sequential gating was performed for each sample to determine the frequency of CD4⁺Foxp3⁺ and CD4⁺CD25⁺CD127^lo Treg population.

Results: A strong correlation was found between CD4⁺Foxp3⁺ and CD4⁺CD25⁺CD127^lo Treg subsets (r=0.67, p<0.001).The percentage of both Treg populations was not different between mobilized or non-mobilized products from healthy donors (p=0.94) or between NHL and MM patients (p=0.81). Higher CD4+FoxP3+ Treg frequency was observed in autologous grafts from patients compared to healthy donors (p<0.001). The median CD4+FoxP3+ Treg frequency among mobilized products from patients and healthy donors were 8.5% (range, 1.8–38.9) and 4.3% (range, 1.8–15.0), respectively. Autografts from patients who received AMD3100+G-CSF compared to those who received chemotherapy + G-CSF also revealed higher CD4+FoxP3+ Treg content (median 11.3 vs. 7.5, p=0.04). Similar analysis using CD4⁺CD25⁺CD127^lo Treg subset did not demonstrate any statistical significance.

Conclusions: Although correlation was observed between CD4+FoxP3+ and CD4⁺CD25⁺CD127^lo subsets, CD4+FoxP3+ is a more sensitive marker for the Treg population in stem cell products. Higher CD4+FoxP3+ Treg frequency was observed in grafts from patients with hematological malignancies compared to healthy donors. The addition of a novel mobilization agent, AMD3100, to Cy mobilization may further increase the Treg content of autologous stem cell products.