Generation, cryopreservation, function and in vivo persistence of ex vivo expanded cynomolgus monkey regulatory T cells

Abstract

We expanded flow-sorted Foxp3+ cynomolgus monkey regulatory T cells (Treg) >1000-fold after three rounds of stimulation with anti-CD3 mAb-loaded artificial antigen-presenting cells, rapamycin (first round only) and IL-2. The expanded Treg maintained their expression of Treg signature markers, CD25, CD27, CD39, Foxp3, Helios, and CTLA-4, as well as CXCR3, which plays an important role in T cell migration to sites of inflammation. In contrast to expanded effector T cells (Teff), expanded Treg produced minimal IFN-γ and IL-17 and no IL-2 and potently suppressed Teff proliferation. Following cryopreservation, thawed Treg were less viable than their freshly-expanded counterparts, although no significant changes in phenotype or suppressive ability were observed. Additional rounds of stimulation/expansion restored maximal viability. Furthermore, adoptively-transferred autologous Treg expanded from cryopreserved second round stocks and labeled with CFSE or VPD450 were detected in blood and secondary lymphoid tissues of normal or immunosuppressed recipients at least two months after their systemic infusion.

Regulatory T Cell Infusion Can Enhance Memory T Cell and Alloantibody Responses in Lymphodepleted Nonhuman Primate Heart Allograft Recipients

“…The phenotype of the expanded infused Treg is shown in Figure S1. Their ability to potently suppress T cell proliferative responses was confirmed (Figure A) as described previously in detail . To confirm the functional stability of the expanded Treg in vitro , we examined their response to stimulation under culture conditions that promote Th1, Th2, and Th17 cell polarization.…”

“…Briefly, cynomolgus CD4 + T cells were negatively enriched from fresh PBMCs using NHP CD4 + T cell isolation kits (Miltenyi Biotech, Auburn, CA). CD4 + CD25 + CD127 − Treg were then flow‐sorted (Figure S1) using a BD FacsAria (BD Biosciences, San Jose, CA) and expanded over 3 weeks using artificial antigen‐presenting cells (L‐32), as described . The L‐32 cells expressing CD32, CD80, and CD58 were kindly provided by Dr. M. K. Levings, University of British Columbia, Vancouver, Canada .…”

Applicability, safety, and biological activity of regulatory T cell therapy in liver transplantation

“…In murine transplant models Tregs tend to accumulate in the graft and draining lymph nodes, although Lee et al were not able to detect transferred Tregs beyond 14 days after infusion into murine islet transplant recipients . In nontransplanted nonhuman primates, adoptively transferred Tregs were shown to be short‐lived, as their numbers declined rapidly during the first week after infusion, albeit a small number of cells were still detected both in blood and in secondary lymphoid tissues for >50 days . In human hematopoietic stem cell transplantation, infused Tregs could be tracked in blood using HLA markers up to 14 days .…”

“…Tregs are long-lived and tend to migrate to the sites of inflam- into murine islet transplant recipients. 28 In nontransplanted nonhuman primates, adoptively transferred Tregs were shown to be short-lived, as their numbers declined rapidly during the first week after infusion, 29,30 albeit a small number of cells were still detected both in blood and in secondary lymphoid tissues for >50 days. 29 In human hematopoietic stem cell transplantation, infused Tregs Although it is not possible to formally establish a causal link between the development of donor hyporesponsiveness and Treg infusion, our findings could be explained by the preferential survival and/or proliferation after infusion of Treg clones with anti-donor alloreactivity, which is an observation that has been documented in experimental animal models.…”

Adoptive Cell Therapy with Tregs to Improve Transplant Outcomes: the Promise and the Stumbling Blocks

“…In the other study by Zhang et al, autologous versus allogeneic fluorochrome dye-labeled Treg were infused into either normal or ATG lymphodepleted cynomolgus monkeys. Ex vivo–expanded MHC-mismatched allogeneic Treg did not persist in the peripheral circulation as long as autologous Treg,that were detected in peripheral blood, inguinal LN, mesenteric LN and spleen for >50 days post-infusion [90]. Also, there was a gradual loss of Foxp3 expression following infusion.…”