Programming of Donor T Cells Using Allogeneic Delta-like Ligand 4-Positive Dendritic Cells to Reduce Gvhd but Retain GVL Activity

Host antigen-presenting cells (APCs) are critical for inducing a potent graft-versus-leukemia (GVL) response after allogeneic hematopoietic stem-cell transplantation (allo-HSCT). In this setting, host APCs activate donor T cells to become effector T cells that recognize and react to antigens in malignant cells. However, alloreactive T cells also mediate graft-versus-host disease (GVHD), which causes significant morbidity and mortality after allo-HSCT. Many studies suggest that if alloreactive T cells have reduced capacity to expand in local tissues, they will be unable to trigger severe GVHD. Thus, it is possible that host APC induction of qualitative changes in donor T cells can potentially modify their anti-host toxicities while retaining the GVL effect.

Here we report the establishment of a cellular programming approach that reduces the GVHD toxicity of donor T cells using host dendritic cells (DCs) that express high levels of Dll4 (named Dll4^hi DCs). We have previously identified inflammatory Dll4^hi DCs. They occurred in HSCT mice early during GVHD induction and had a greater ability than Dll4-negative DCs to induce IFN-γ and IL-17 in alloantigen-activated T cells. However, only approximately 0.03 X 10⁵ Dll4^hi DCs were recovered from one HSCT mouse. To provide adequate numbers of Dll4^hi DCs for therapeutic translation, we developed a novel culture system capable of producing large number of Dll4^hi DCs (about 100.0 X 10⁵) from the bone marrow (BM) of one mouse using Flt3L and the TLR agonists lipopolysaccharide (LPS) and R848, which activate TLR4 and TLR7/8, respectively. Dll4^hi DCs showed significantly different phenotype as compared to conventional DCs derived from GM-CSF-stimulated BM cells (named GM-DCs), as evidenced by expressing higher levels of Dll4, Ifnb, Il4, Il6 and Ido, and producing lower levels of iNOS and arginase I. When cultured with C57BL/6 (B6) mouse CD4⁺ T cells (H2^b) at a T cell : DC ratio of 4:1 for 5 days, BALB/c mouse Dll4^hi DCs (H2^d) induced 3- to 5-fold more in frequency of alloreactive effector T cells producing high levels of IFN-γ and IL-17 compared to GM-DCs. Following transfer, allogeneic Dll4^hi DC-induced CD4⁺ T cells were unable to mediate severe GVHD in BALB/c recipients, with all of them surviving 60 days after allo-HSCT. In contrast, both unstimulated B6 CD4⁺ T cells and allogeneic GM-DC-induced B6 CD4⁺ T cells caused lethal GVHD in all BALB/c recipients, indicating that GM-DCs could not be used for reducing the GVHD toxicity of donor CD4⁺ T cells. Mechanistic analysis showed that Dll4^hi DC-induced CD4⁺ T cell recipients showed 2- to 6-fold less donor CD4⁺ T cells in the spleen, liver, and intestine 12 days after transplantation compared to unstimulated CD4⁺ T cell recipients. This reduction of Dll4^hi DC-induced CD4⁺ T cells was associated with markedly increased apoptosis in recipient mice. IFN-γ production by Dll4^hi DC-induced CD4⁺ T cells was essential for their anti-GVHD effects. Absence of T cell IFN-γ led to improved survival and expansion of Dll4^hi DC-induced CD4⁺ T cells in transplant recipients and caused lethal GVHD. Finally, we demonstrated that Dll4^hi DC-induced alloreactive T cells had acquired the ability to kill A20 leukemic cells in BALB/c recipients and control growth of P815 mastocytoma cells in the second model of BDF1 recipients, leading to significantly improved survival of mice receiving allo-HSCT. Furthermore, in the third mouse model of GVHD directed against minor histocompatibility antigens, B6 Dll4^hi DC-induced C3H.SW CD8⁺T cells produced high levels of IFN-γ, had reduced capacity to mediate GVHD in B6 recipients, but preserved GVL activity against C1498 myeloid leukemic cells.

In summary, our findings demonstrate that in vitro Dll4^hi DC programming represents a novel and effective platform to reduce toxicities of donor T cells. This strategy has several potential advantages compared to current and developing methods for the modification of donor T cells to reduce GVHD, including a relatively short period of culture, no requirement for T cell subset selection and no need of viral transduction. Importantly, this method may lead to new strategies that can produce large amount of leukemic cell-reactive donor T cells with decrease capability of causing severe GVHD.