Patient Characteristic Autologous Peripheral Blood Stem Cell Products from Patients with Hematologic Malignancies Have Increased Frequency of Regulatory T Cells (CD4+FoxP3+ Treg) Grace Kao, MD, Kristen Stevenson, MS, Elliot Kim, Elena Spanjaard, PhD, Haesook T Kim, PhD, and Jerome Ritz, MD Allogeneic Autologous Abstract Patient Characteristic Results 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+ CD127lo. In this study, we compare the impact of donor types and stem cell mobilization regimens on CD4+FoxP3+ and CD4+CD25+CD127lo Treg frequencies in 98 apheresis products. Methods: Apheresis products from 29 healthy donors (23 donors received G-CSF and 6 had no stem cell mobilization) and 69 patients with malignancies (1 AML, 2 HD, 25 NHL, 10 MM, 2 neuroblastoma) were evaluated. Patients with malignancies were mobilized with chemotherapy (7 RICE, 2 ICE, 41 Cy, 1 Cy+Topotecan, 1 Arac+ Etoposide) + G-CSF (N=52), AMD3100 + G-CSF (N=9), or G-CSF alone (N=8). 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+CD127lo Treg population. Results: A strong correlation was found between CD4+Foxp3+ and CD4+CD25+CD127lo Treg subsets (r=0.47, p<0.001).The percentage of both Treg populations was not different between mobilized or non-mobilized products from healthy donors (p=0.81) or between NHL and MM patients (p=0.93). 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 9.7% (range, 1.8- 38.9) and 4.6% (range, 1.8-15.0), respectively. Higher CD4+FoxP3+ Treg content was also observed in autografts of patients who received AMD3100+G-CSF compared to those who received chemotherapy + G-CSF (p=0.05). Similar analysis using CD4+CD25+CD127lo Treg subset demonstrate a marginal elevation in AMD3100 group (p=0.08). Conclusions: Although correlation was observed between CD4+FoxP3+ and CD4+CD25+CD127lo 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 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. Figure 1 Correlation between CD4+Foxp3+ and CD4+CD25+CD127lo contents for both allogeneic and autologous apheresis products P=0.93 CD4+Foxp3+ Figure 2 Comparison of CD4+Foxp3+ Treg content within mobilized apheresis products by donor and disease type Methods Fresh apheresis samples were treated with IOTest 3 Lysing Solution (Beckman Coulter) to remove red blood cells. 2x106 cells were stained for flow cytometry using anti-CD45 (BD), anti-CD4 (BD), anti-CD14 (BD), anti-CD127 (eBioscience), anti-CD25 (BD, Beckman Coulter or Miltenyi) and anti-Foxp3 antibody (eBioscience or BD), then acquired using a FACSAria or FACSCanto (BD) and analyzed using FACSDiva (BD). Sequential gating was used to identify Treg populations; CD45+ events, then lymphocytes based on forward and side scatter, and 2x104-2x105 CD45+CD4+ gated events were collected for each sample. CD4+Foxp3+ and CD4+CD25+CD127lo Treg populations were further analyzed to identified cells within each population that were CD127lo or Foxp3+ for the first 39 samples. Figure 3 Higher CD4+Foxp3+ Treg frequency was observed in mobilized apheresis products from autologous donors Figure 4 No difference in Treg frequency was seen between non- mobilized and G-CSF mobilized apheresis products from allogenic donors P=0.05 P=0.08 Figure 5 Higher CD4+Foxp3+ Treg frequency was observed in apheresis products from autologous donors mobilized with AMD3100 + G-CSF when compared to donors mobilized with chemotherapy + G-CSF CD4+Foxp3+ Population 64.3% 3.9% 32.4% Background Regulatory T Cells (Treg) are an important component of self-tolerance and influence of Treg cells in suppressing pathologic immune responses in autoimmune diseases, transplantation, and graft-versus-host disease are reported. The transcription factor, FoxP3, was recently discovered as the most specific intracellular marker and regulator of Treg cell development and function. Major obstacle to the study and application of Treg cells in the human setting has been the lack of specific cell surface biomarkers. Surface IL-7 receptor (CD127) is down-regulated on a subset of CD4+ T cells in peripheral blood that contents high concentration of FoxP3 + cells. These cells are as suppressive as the “classic” CD4+CD25hi Treg subset. We previously showed that Treg population defined by CD4+Foxp3+ and CD4+CD25+CD127lo populations are not exactly the same and CD4+CD25+CD127lo population contains a significant subset of cells which lack Foxp3 expression. Chemotherapy or growth factors used for stem cell mobilization may affect the Treg content of apheresis products used for hematopoietic stem cell transplantation. The major goal of this study is to Compare intracelluar staining with convenient cell surface markers to quantify Treg content in apheresis products used for hematopoietic stem cell transplantation and adoptive immunotherapy. Compare the Treg content in apheresis products obtained from healthy donors and patients with malignancies. Evaluate the impact of mobilization strategy on Treg cell content of apheresis products Conclusion CD4+CD25+CD127lo Population 4.2% 59.1% The percentage of CD4+CD25+CD127lo cells is a comparable but less sensitive biomarker than CD4+Foxp3+ cells for Treg content estimation. The CD4+CD25+CD127lo population contains a significant subset of cells which lack Foxp3 expression. G-CSF mobilization did not affect the Treg content of apheresis allografts when compared to non-mobilized grafts from healthy donors. There was no difference in Treg content in grafts obtained from myeloma or lymphoma donors. Higher CD4+Foxp3+ Treg content within the CD4+ population was found in apheresis products from donors with malignancies when compared to ones from healthy donors. Autologous apheresis products obtained from patients who underwent salvage mobilizaion with AMD3100 and G-CSF was found to contain higher frequency of CD4+Foxp3+ Treg cells within the CD4+ population than patients who were successfully mobilized with cyclophasphamide and G-CSF. The high Treg frequency found in this donor population may be a reflection of the donor disease status or as the result of new mobilization regimen. Our findings warrant further investigation to determine whether graft CD4+CD25+CD127lo cell content is an effective biomarker for clinical outcome for patients who undergo either allogeneic or autologous hematopoietic stem cell transplants. if the new mobilization agent, AMD3100, can affect the Treg content of apheresis autografts obtained from patients with malignancies and allografts from healthy donors. There are no relevant conflicts of interest to disclose for all authors.