Abstract
Graft-versus-host disease (GVHD) remains the major immunological complication preventing more widespread application of allogeneic hematopoietic stem cell transplants (aHSCTs). Mobilized hematopoietic stem / progenitor cells (HSPC) is the predominant choice for adult HSCT (CIBMTR, 2017), but unfortunately still frequently results in the development of GVHD. Our lab and others have been exploring the potential application of CD4+FoxP3+ T cells (Tregs) to ameliorate GVHD. A useful advance in the field would be the ability to concomitantly mobilize stem cell donors and efficiently expand the peripheral Treg compartment. We have utilized a two-pathway Treg expansion strategy previously reported from our lab (Wolf, 2017; Copsel 2018) to amplify suppressor activity in the donor inoculum to diminish GVHD. The present studies developed a protocol to concurrently induce mobilization of mouse progenitor cells and expansion of Tregs for use in pre-clinical aHSCT.
Donor mice were treated with recombinant human granulocyte colony stimulating factor (G-CSF) for 4 days (2ug/mouse) followed by plerixafor (5mg/kg) on D-1 and D0 (day of transplant). Peripheral blood was then assessed and significant elevations in granulocytes (Figure 1A) as well as multiple progenitor populations including c-Kit+ stem cells, CLP's and Lin-Sca-1ckit+ cells was observed (Figure 1B). We then employed a strategy beginning on D-6 through D-1 of transplant administering a fusion protein (TL1A-Ig) targeting TNFRSF25 and low dose IL-2 (two-pathway expansion). Peripheral blood was then assessed for Treg levels in donors on the day of transplant. We identified a marked increase in the frequency of CD4+FoxP3+ Treg cells ranging from (26-31%) within the CD4 compartment during the mobilization process (Fig. 2A). Peripheral blood was pooled from donors from both mobilized and Treg unexpanded (“TrUM”) or mobilized and Treg expanded (“TrEM”) donors and transplanted into lethally irradiated, complete MHC-mismatched BALB/c recipients. Recipients of TrUM lost weight and developed clinical signs of GVHD (Fig. 2B). In contrast, recipients of TrEM donors exhibited significantly reduced weight loss and clinical GVHD scores (Figure 2B). Transplants were also performed utilizing donors similarly prepared and transplanted into MHC-matched mice. The recipients of TrEM peripheral blood exhibited reduced GVHD compared to recipients of TrUM. Notably, mobilized donor HSPC were found to be functional as donor derived progeny (ex. CD19) were identified >1 month post-HSCT.
In total, these findings support the notion that during the donor stem / progenitor cell mobilization process, manipulation of Treg cells using our two-pathway strategy can be successfully accomplished in peripheral blood resulting in an effective and translational approach to ameliorate GVHD following allo-HSCT.