Generation, cryopreservation, function and in vivo persistence of ex vivo expanded cynomolgus monkey regulatory T cells.
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.
Thomas E. Starzl Transplantation Institute, Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
Hao
H
Guo
Thomas E. Starzl Transplantation Institute, Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
Hong
H
Zhang
Thomas E. Starzl Transplantation Institute, Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
Lien
L
Lu
Thomas E. Starzl Transplantation Institute, Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
Mohamed B
MB
Ezzelarab
Thomas E. Starzl Transplantation Institute, Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA; Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA. Electronic address: thomsonaw@upmc.edu.
Angus W
AW
Thomson
D019264:Adoptive Transfer / Q000379:methods; D000818:Animals; D000938:Antigen-Presenting Cells / Q000276:immunology; D018929:Cell Culture Techniques / Q000379:methods*; D049109:Cell Proliferation; D002478:Cells, Cultured; D015925:Cryopreservation / Q000379:methods*; D005434:Flow Cytometry; D005452:Fluoresceins / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D051858:Forkhead Transcription Factors / Q000276:immunology / Q000378:metabolism; D016130:Immunophenotyping; D007371:Interferon-gamma / Q000276:immunology / Q000378:metabolism; D020381:Interleukin-17 / Q000276:immunology / Q000378:metabolism; D053645:Interleukin-2 Receptor alpha Subunit / Q000276:immunology / Q000378:metabolism; D053699:Interleukin-7 Receptor alpha Subunit / Q000276:immunology / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000276:immunology / Q000378:metabolism; D008252:Macaca fascicularis; D013388:Succinimides / Q000378:metabolism; D050378:T-Lymphocytes, Regulatory / Q000276:immunology* / Q000378:metabolism / Q000637:transplantation; D013997:Time Factors; D014182:Transplantation, Autologous / Q000379:methods
C087165:5-(6)-carboxyfluorescein diacetate succinimidyl ester; D005452:Fluoresceins; D005456:Fluorescent Dyes; D051858:Forkhead Transcription Factors; D020381:Interleukin-17; D053645:Interleukin-2 Receptor alpha Subunit; D053699:Interleukin-7 Receptor alpha Subunit; D013388:Succinimides; D007371:Interferon-gamma
Cryopreservation; Cynomolgus macaques; Ex vivo expansion; In vivo monitoring; Regulatory T cells