Regulatory Dendritic Cells Induced by Bendamustine Are Associated With Enhanced Flt3 Expression and Alloreactive T-Cell Death

Abstract

The growth factor Flt3 ligand (Flt3L) is central to dendritic cell (DC) homeostasis and development, controlling survival and expansion by binding to Flt3 receptor tyrosine kinase on the surface of DCs. In the context of hematopoietic cell transplantation, Flt3L has been found to suppress graft-versus-host disease (GvHD), specifically via host DCs. We previously reported that the pre-transplant conditioning regimen consisting of bendamustine (BEN) and total body irradiation (TBI) results in significantly reduced GvHD compared to cyclophosphamide (CY)+TBI. Pre-transplant BEN+TBI conditioning was also associated with greater Flt3 expression among host DCs and an accumulation of pre-cDC1s. Here, we demonstrate that exposure to BEN increases Flt3 expression on both murine bone marrow-derived DCs (BMDCs) and human monocyte-derived DCs (moDCs). BEN favors development of murine plasmacytoid DCs, pre-cDC1s, and cDC2s. While humans do not have an identifiable equivalent to murine pre-cDC1s, exposure to BEN resulted in decreased plasmacytoid DCs and increased cDC2s. BEN exposure and heightened Flt3 signaling are associated with a distinct regulatory phenotype, with increased PD-L1 expression and decreased ICOS-L expression. BMDCs exposed to BEN exhibit diminished pro-inflammatory cytokine response to LPS and induce robust proliferation of alloreactive T-cells. These proliferative alloreactive T-cells expressed greater levels of PD-1 and underwent increased programmed cell death as the concentration of BEN exposure increased. Alloreactive CD4+ T-cell death may be attributable to pre-cDC1s and provides a potential mechanism by which BEN+TBI conditioning limits GvHD and yields T-cells tolerant to host antigen.

Commentary: Post-Transplantation Cyclophosphamide Uniquely Restrains Alloreactive CD4+ T-Cell Proliferation and Differentiation After Murine MHC-Haploidentical Hematopoietic Cell Transplantation

“…A body of literature has only recently started to emerge, in large part from our group ( 2 , 10 – 14 ), regarding the immunomodulatory properties of BEN on MDSCs, DC subsets, and T cells ( 10 – 14 ). In all our murine models, whether BEN is given pre- or post-transplant, it has been associated with decreased GvHD and increased GvL.…”

Partially replacing cyclophosphamide with bendamustine in combination with cyclosporine A improves survival and reduces xenogeneic graft-versus-host-disease

“…However, as viral reactivation due to delayed immune reconstitution and relapse often lead to morbidity and mortality of patients ( 3 , 6 , 28 – 30 ), some investigators have begun evaluating a reduction in the dose of post-transplant CY to limit these complications ( 8 , 10 , 11 ). Our laboratory has taken a different approach in evaluating whether partially replacing PT-CY with post-transplant bendamustine (PT-BEN) would be advantageous, which is based on our extensive research that has delineated several immunomodulatory properties of BEN on myeloid derived suppressive cells (MDSCs) and dendritic cell (DC) subsets ( 14 – 16 ). Moreover, we have demonstrated that BEN yields tolerant T-cells with a striking absence of GvHD, while preserving T-cell dependent graft-versus-tumor (GvT) effects ( 12 ).…”

“…We have shown that using BEN as either a component of a pre-transplant conditioning regimen or when used in a post-transplant setting led to equal or improved survival in mice undergoing major histocompatibility complex (MHC) mismatched or haploidentical bone marrow transplants (haplo-BMT), respectively ( 12 , 13 ). BEN was shown to modulate immune cell number and function in transplanted mice, with increases in regulatory-like dendritic cells (DCs) ( 14 , 15 ) and myeloid-derived suppressor cells (MDSCs) ( 16 ), reduced proliferation of B and T cells, and a higher presence of T cells that are tolerized towards host MHC molecules ( 12 ). Importantly, BEN treated animals, in both pre-transplant and post-transplant settings, had superior GvT effects with reduced tumor burden and improved survival compared to those treated with CY ( 12 , 13 ).…”

Bendamustine: a review of pharmacology, clinical use and immunological effects (Review)

“…The same group exposed murine bone marrow-derived dendritic cells (BMDC) to bendamustine and incubated them with allogenic T-cells. Upon incubation, T-cells exhibited an increase in markers of T-cell exhaustion, as well as markers of activated T-cells, ICOS and CD69, and an increase in PD-1, negative regulator of immune response, followed by allogenic CD4 + cell death (96). Since PD-1 is so far considered to be crucial in T-cell exhaustion, combining bendamustine with anti-PD-1 antibody could be beneficial in both anticancer and anti-microbe setting (97).…”