Ex Vivo Expansion of Canine CD4+CD25+ Regulatory T Cells (Treg) 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+ Treg cells. Furthermore, infusion of patient-specific Treg 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 Treg in the well-established canine model of HCT, we characterized the in vitro function of canine CD4+CD25+ Treg cells and asked if canine-specific aAPC could expand Treg 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 3rd 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+ Treg 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 (Treg: responder) and pulsed with 3H-thymidine after 6 days. CD4+CD25+ Treg inhibited proliferation of primary MLCs in a cell contact-dependent manner if Treg shared the DLA-type of the responder cells. Addition of CD25− T cells did not inhibit MLCs. The yield of CD4+CD25+ Treg 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+ Treg 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+ Treg (at 1:1 or 1:10 aAPC:Treg ratio). Seven days later, Treg cells had expanded a median of 23-(range, 8–36) fold. The expanded cells maintained the phenotype and in vitro function as Treg prior to expansion. The expanded Treg 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 Treg, cell doses that are potentially sufficient for assessment of in vivo function in the dog model of HCT. In summary, canine Treg can be isolated from PBMC following short-term allogeneic stimulation, immunomagnetic selection and expansion with canine-specific aAPC. The expanded Treg maintain the cell phenotype and functional characteristics of allo-stimulated CD4+CD25+ Treg cells. In future studies, expanded Treg cells will be evaluated for in vivo function to inhibit GVH and HVG reactions in the canine model of allogeneic HCT.

Retention of quiescent hematopoietic cells with high proliferative potential during ex vivo stem cell culture

Human CD34+/Thy-1+/Lin- hematopoietic cells purified from bone marrow (BM) or mobilized peripheral blood (MPB) are highly enriched for pluripotent stem cells. Ex vivo expansion of this population is proposed as a means of providing accelerated short-term, as well as long-term, engraftment after myeloablative therapy. Here we demonstrate that primitive quiescent cells are retained in bulk expansion cultures of CD34+/Thy-1+/Lin- cells and that the cell production capacity of the expanded cell product can largely be attributed to cells exhibiting quiescent behavior during culture. CD34+/Thy-1+/Lin- cells from adult BM or MPB were labeled with the fluorescent membrane dye PKH26, followed by in vitro culture of 10(4) cells on a murine stromal layer in the presence of interleukin (IL)-3, IL-6, c-kit ligand (KL), and leukemia inhibitory factor (LIF). With each subsequent cell division, PKH26 fluorescence is reduced by roughly half, which allows tracking of the number of cell divisions. Progenitor cells present after a 2-week expansion period were sorted into CD34+/Lin-/dyebright and CD34+/Lin- /dyedim fractions and then cultured in a 4-week single-cell proliferation assay to characterize the proliferative capacity of each group. Fifty-nine percent of progenitors remaining dyebright after bulk culture (four or fewer cell divisions) were observed to proliferate in single cell culture, and produced an average of 1,780 cells per plated cell. In contrast, only 26% of dyedim (more than four divisions) progenitors were observed to proliferate and displayed a lower average proliferative capacity of 225 cells per plated cell. Similar behaviors were observed after a second consecutive cycle of bulk culture, indicating that quiescent cells with high proliferative capacity existed in culture for at least 4 weeks. Single CD34+/Lin-/dyebright progenitors purified from bulk cultures were observed to produce as many as 1,000 CD34 positive progeny during single cell culture, and these progeny included multilineage colony forming cells. These data demonstrate that among CD34 positive cells recovered after in vitro bulk culture, a higher proliferative capacity correlated with quiescent behavior. The described culture method provides quantitation of the cell producing capacity of individual cells in hematopoietic cell mixtures and may prove useful for predicting engrafting potential in products intended for cellular therapy.