Bendamustine Conditioning Alters Host Dendritic Cell Composition and Function

Background

A largely understudied strategy for reducing graft-versus-host disease (GvHD) is the modification of conditioning regimens. We have demonstrated that pre-transplant bendamustine (BEN) used in combination with total body irradiation (TBI) significantly reduces GvHD compared to the more commonly used cyclophosphamide (CY) + TBI. The short half-lives of BEN/CY (<4h), which are given 48h prior to transplant, indicate that these drugs do not directly affect the donor cells. Therefore, the striking improvement in GvHD by substituting CY with BEN must be due to differential effects on the host cells. Conditioned host immune cells diminish over time, but are the majority early after transplant and are able to critically interact with the infused donor cells at the earliest stages of GvHD pathogenesis.

Methods

Using an MHC-mismatched murine model of bone marrow transplantation (BMT) with C57BL/6 donors into BALB/c recipients (H2K^b into H2K^d), we evaluated the differential effects of BEN conditioning on host dendritic cells (DCs) early after transplant, as compared to CY conditioning. Transplant recipient mice were conditioned with BEN or CY on day -2 pre-BMT, and supplemented with 400cGy of total body irradiation on day -1. On days 1, 3, and 5 post-BMT, splenic pan-DCs were isolated and analyzed by flow cytometry to evaluate host (H2K^b-) DC phenotype and activation state. Further, on day 0 (without BMT), circulating leukocytes and splenic pan-DCs were isolated from conditioned mice and analyzed by flow cytometry, qPCR and allogeneic mixed leukocyte reactions.

Results

We found that BEN-conditioned pan DCs are less stimulatory of allogeneic T-cells than CY-treated DCs. BEN conditioning significantly alters host DC composition compared to CY conditioning, resulting in a significantly higher frequency and number of host CD8α⁺ DCs on days 1 and 3 post-BMT. BEN-conditioned CD8α⁺ DCs also display a highly regulatory phenotype (CD80^highCD86^low) and have greater suppressive ability compared to CY-conditioned CD8α⁺ DCs. Analysis of host DCs on day 0 (without BMT) revealed a significantly greater percentage of BEN-conditioned DCs positively expressing the Flt3 receptor compared to CY.

Conclusions

These findings advocate a potential role of Flt3L-dependent DCs, particularly CD8α⁺ DCs, in the mechanism by which BEN alters GvHD pathogenesis to limit morbidity and mortality. Thus, BEN may have advantages as an alternative agent to CY for pre-transplant conditioning in allogeneic BMT.