Ex Vivo Expansion of Canine CD4⁺CD25⁺ Regulatory T Cells (T_reg) with Artificial Antigen Presenting Cells (aAPC).

We hypothesize that immune tolerance after allogeneic hematopoietic cell transplantation (HCT) is maintained by an active cellular mechanism that includes regulators of immune function, CD4⁺CD25⁺ T_reg cells. Furthermore, infusion of patient-specific T_reg cells might provide therapeutic benefit, improving the control of graft-versus-host (GVH) and host-versus-graft (HVG) reactions after HCT. In preparation for large-scale in vivo studies with T_reg in the well-established canine model of HCT, we characterized the in vitro function of canine CD4⁺CD25⁺ T_reg cells and asked if canine-specific aAPC could expand T_reg cells ex vivo. Responder peripheral blood mononuclear cells (PBMC) were obtained by leukapheresis and ficoll-hypaque separation (n = 7 dogs) and cultured in bulk mixed leukocyte culture (MLC) conditions with 3^rd party dog leukocyte antigen (DLA)-mismatched, unrelated and irradiated (22 Gy) CD34⁺ derived dendritic cells (10:1 responder: stimulator ratio) or PBMC (1:1). The starting number of responder cells ranged from 120 – 555 x10⁶ CD3⁺ cells. On day 4 of MLC, cells were incubated with anti-CD25 monoclonal antibody (ACT-1). CD25⁺ cells were isolated by positive immunomagnetic selection (Miltenyi), and assessed for phenotype and in vitro function. After selection, cells were 83% – 97% CD3⁺CD4⁺CD25⁺ by flow cytometry. The CD4⁺CD25⁺ T cells expressed the forkhead/winged helix transcription factor foxP3, assessed by Western blot with polyclonal anti-human foxP3 antibody, while CD25⁻ T cells did not express foxP3. In addition, the CD4⁺CD25⁺ T_reg inhibited proliferation of fresh primary MLCs a median of 79% (range, 54% – 88%) when CD4⁺CD25⁺ cells were added at 1:5 or 1:25 ratio (T_reg: responder) and pulsed with ³H-thymidine after 6 days. CD4⁺CD25⁺ T_reg inhibited proliferation of primary MLCs in a cell contact-dependent manner if T_reg shared the DLA-type of the responder cells. Addition of CD25⁻ T cells did not inhibit MLCs. The yield of CD4⁺CD25⁺ T_reg after bulk culture and immunomagnetic selection ranged from 2.2 – 24 x10⁶ cells. On day 5 of cell culture, aAPC were added to stimulate and expand the CD4⁺CD25⁺ T_reg cells. The aAPC (KT32) expressing the Fcγ receptor CD32, canine CD86, and human IL-15, were loaded with the canine-specific mitogenic anti-CD3ε antibody 17.6F9 (0.5 mg/mL) and irradiated (100 Gy) prior to stimulation of CD4⁺CD25⁺ T_reg (at 1:1 or 1:10 aAPC:T_reg ratio). Seven days later, T_reg cells had expanded a median of 23-(range, 8–36) fold. The expanded cells maintained the phenotype and in vitro function as T_reg prior to expansion. The expanded T_reg were 44–76% (median, 59%) CD4⁺CD25⁺, expressed foxP3, and inhibited primary MLCs a median of 85% (range, 64%–97%) at a 1:5 ratio. Bulk culture expansion generated a median of 105 (range, 52–135) x10⁶ T_reg, cell doses that are potentially sufficient for assessment of in vivo function in the dog model of HCT. In summary, canine T_reg can be isolated from PBMC following short-term allogeneic stimulation, immunomagnetic selection and expansion with canine-specific aAPC. The expanded T_reg maintain the cell phenotype and functional characteristics of allo-stimulated CD4⁺CD25⁺ T_reg cells. In future studies, expanded T_reg cells will be evaluated for in vivo function to inhibit GVH and HVG reactions in the canine model of allogeneic HCT.