Evaluation of the Phenotype and GVHD-Inducing Potential of Splenic T Cells Isolated from G-CSF, AMD3100, or G-CSF and AMD3100 Pretreated Allogeneic Donors.
Authors: Rettig Michael , Holt Matthew , Liu Fulu , Ritchey Julie , Link Daniel , Bridger Gary , DiPersio John 
Published: April 2020

Abstract
Recent data suggest that the incidence and severity of acute graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell (HSC) transplantation may depend on the agent used to mobilize HSC from the bone marrow (BM) to the peripheral blood. AMD3100 is a bicyclam that selectively and reversibly blocks CXCL12 (SDF-1) binding to, and signaling through, CXCR4. We and others have previously demonstrated that AMD3100 induces the rapid mobilization of both murine and human HSCs, causing up to a 10-fold increase in CD34+ cells in the peripheral blood within 4–6 hours of a single injection. Furthermore, a single dose of AMD3100 can be combined with a well-tolerated dose of G-CSF on the fifth day of HSC mobilization to markedly increase the mobilization of CD34+ cells. Since T cells from donors treated with G-CSF reportedly have a reduced capacity to induce GVHD relative to those from control-treated donors, we analyzed the phenotype and GVHD potential of T cells isolated from the spleens of donors that were unmobilized or treated with G-CSF, AMD3100, or G-CSF and AMD3100. C57BL/6 mice were treated with G-CSF (250 μg/kg/d for 5 days), AMD3100 (single injection of 5 mg/kg), or G-CSF + AMD3100 (AMD3100 only given on the final day of G-CSF). Following mobilization, we purified splenic T-lymphocytes to &gt;95% by negative immunoselection and examined expression of CD4, CD8, CD25, CD69, CD30, α4β7, CD44, and CD62L on CD3+ cells by flow cytometry. Although mobilization did not significantly alter the percentage of CD4+, CD8+ or CD4+CD8+ T cells in the spleen, there was a trend toward a lower percentage of splenic CD4+CD25+CD62L+ naturally occurring T regulatory cells following G-CSF and/or AMD3100 administration. Similar to previous reports by others, we observed that the percentage of CD4+ and CD8+ cells that expressed CD62L and the level of CD62L expression was significantly decreased in G-CSF-treated donors (both with and without AMD3100) compared with the untreated control. This decrease in the expression of CD62L was not observed on T cells isolated from donors that were treated with AMD3100 alone. Furthermore, the reduction in CD62L expression on T cells isolated from G-CSF-treated donors did not coincide with an increase in the expression of CD44, suggesting that the loss of CD62L was not due to an expansion of memory T cells. In fact, all three mobilization regimens induced a significant decrease in the percentage of CD44hi expressing splenic T cells. Consistent with this observation, G-CSF and/or AMD3100 administration did not induce T cell activation as assessed by CD25, CD30 and CD69 expression. Similarly, we found no alterations in expression of the intestinal homing receptor, α4β7, following donor mobilization with G-CSF and/or AMD3100. To evaluate the GVHD-inducing potential of the purified T cells, we injected 5e5 or 2e6 total T cells, along with T cell depleted C57BL/6 BM, into lethally irradiated BALB/c recipients. Surprisingly, GVHD-related weight loss, median survivals and lymphoid reconstitution were identical in each group using Kaplan-Meier and Mantel/Cox log rank analyses. These data clearly suggest that AMD3100 alone or when combined with G-CSF as a mobilizing agent has no significant effect on T cell function as measured by GVHD studies. These studies further support the use of AMD3100 for the mobilization of allogeneic stem cells.