Adoptive Immunotherapy with Regulatory and Conventional T Cells in Haploidentical Transplantation Primes Dendritic Cells to Promote T Cell Alloreactivity in the Bone Marrow and Tolerance in the Periphery

In high-risk acute leukemia patients undergoing HLA haploidentical T cell-depleted tranplantation, we demonstrated that adoptive immunotherapy with donor T regulatory cells (Tregs; 2x10⁶/kg) co-infused with conventional T cells (Tcon; 1x10⁶/kg ) provided significant protection from acute graft-versus-host disease (aGvHD) and was associated with an almost complete control of leukemia relapse (graft versus leukemia effect, GvL) (Di Ianni et al., Blood 2011; Martelli et al., Blood 2014; Ruggeri et al., ASH 2018). In the present study we investigated whether Tregs interact with bone marrow (BM) and peripheral blood (PB) dendritic cells (DCs) and whether such interaction is responsible for GvHD protection and GvL effect. Twenty six patients (median age 54 ; 20 AML; 4 ALL; 2 MDS) transplanted between July 2016 and April 2019 were evaluated up to one year after the transplant. BM and PB DCs (using CD123 for plasmocitoid DC-pDC; CD11c for myeloid DC-mDC; CD80/CD86 for costimulatory molecules) and T cells (CD3/CD4/CD8; CD4/CD25/CD127; CD28/PD-1/TIM3) were analysed by flow-cytometry. DCs were also sorted and analysed by RT-PCR for a panel of genes involved in activation (IL-6; TNF-a; IL-12; CCR7; NOTCH ligands) vs tolerigenic (TGF-beta; PD-1/PDL1; IDO; IL-10; ICOS) pathways. To study the effects of DCs on T cell proliferation, pre-activated (with GM-CSF at 50 ng/ml, IL-4 at 800 U/ml and TNF-a at 50 ng/ml for 18 hrs) BM and PB CD1c⁺ DCs were co-cultured for 96 hrs with autologous CFSE labelled BM and PB CD3+ cells at a DC:CD3 ratio of 1:10. mDC numbers were significantly higher in BM than PB during the first 6 months after transplant. BM-derived mDCs expressed higher levels of the co-stimulatory receptor CD86. No differences emerged in pDCs. RT-PCR showed an activation signature in BM-DCs (significantly higher IL-6 level) and a tolerigenic signature in PB-DCs (significantly higher TGF-beta and PDL-1 levels). BM-derived CD8⁺ T cells displayed a higher expression of the co-stimulatory receptor CD28 than PB-derived CD8⁺ T cells (30.3±18.8 vs 9.2±4.9; p<0.05 ). In contrast, the expression of the immune checkpoint inhibitor PD-1 was significantly higher in both PB-derived CD4 (69%±29 vs 24±11) and CD8 (65±25 vs 4±3; p<0.05) T cells than BM-derived T lymphocytes. T cells from both BM and PB did not express the T cell exhaustion marker TIM-3. CD3/CFSE⁺-DCs co-cultures showed a T cell proliferation rate that was significantly higher in BM than in PB (25±7.2 vs 6.7±8.7; p<0.05). These data show that haploidentical transplantation with Treg/Tcon immunotherapy promotes the reconstitution of DCs with an activating signature in the BM and a tolerigenic signature in the PB. Human peripheral blood Tregs that are used for adoptive immunotherapy are largely CD45RO+ and express low level of CxCR4 bone marrow homing receptor. When infused in immunodeficient mice they migrate to the periphery (spleen, gut, liver) but are unable to home to the bone marrow (Ruggeri et al., ASH 2018). In conclusion, Tregs/DC interaction induce tolerance in the periphery (and may protect from GvHD). In the BM, in the absence of Tregs, DCs activate alloreactive Tcon and may favour killing of the leukemic targets. Therefore, Tregs/DC interactions may contribute to the separation between GvL effect and GvHD in the Treg based haploidentical transplantation.