Regulatory T cells from patients with end-stage organ disease can be isolated, expanded and cryopreserved according good manufacturing practice improving their function

Francesca Ulbar, Tiziana Montemurro, Tatiana Jofra, Miriam Capri, Giorgia Comai, Valentina Bertuzzo, Cristiana Lavazza, Alessandra Mandelli, Mariele Viganò, Silvia Budelli, Maria Giulia Bacalini, Chiara Pirazzini, Paolo Garagnani, Valeria Giudice, Daria Sollazzo, Antonio Curti, Mario Arpinati, Gaetano La Manna, Matteo Cescon, Antonio Daniele PinnaClaudio Franceschi, Manuela Battaglia, Rosaria Giordano, Lucia Catani, Roberto Massimo Lemoli

Research output: Contribution to journalArticle

Abstract

Background: Here, we isolated, expanded and functionally characterized regulatory T cells (Tregs) from patients with end stage kidney and liver disease, waiting for kidney/liver transplantation (KT/LT), with the aim to establish a suitable method to obtain large numbers of immunomodulatory cells for adoptive immunotherapy post-transplantation. Methods: We first established a preclinical protocol for expansion/isolation of Tregs from peripheral blood of LT/KT patients. We then scaled up and optimized such protocol according to good manufacturing practice (GMP) to obtain high numbers of purified Tregs which were phenotypically and functionally characterized in vitro and in vivo in a xenogeneic acute graft-versus-host disease (aGVHD) mouse model. Specifically, immunodepressed mice (NOD-SCID-gamma KO mice) received human effector T cells with or without GMP-produced Tregs to prevent the onset of xenogeneic GVHD. Results: Our small scale Treg isolation/expansion protocol generated functional Tregs. Interestingly, cryopreservation/thawing did not impair phenotype/function and DNA methylation pattern of FOXP3 gene of the expanded Tregs. Fully functional Tregs were also isolated/expanded from KT and LT patients according to GMP. In the mouse model, GMP Tregs from LT or KT patient proved to be safe and show a trend toward reduced lethality of acute GVHD. Conclusions: These data demonstrate that expanded/thawed GMP-Tregs from patients with end-stage organ disease are fully functional in vitro. Moreover, their infusion is safe and results in a trend toward reduced lethality of acute GVHD in vivo, further supporting Tregs-based adoptive immunotherapy in solid organ transplantation.

Original languageEnglish
Article number250
JournalJournal of Translational Medicine
Volume17
Issue number1
DOIs
Publication statusPublished - Aug 5 2019

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T-cells
Regulatory T-Lymphocytes
Adoptive Immunotherapy
Liver
Transplantation (surgical)
Inbred NOD Mouse
End Stage Liver Disease
Thawing
SCID Mice
Cryopreservation
Organ Transplantation
Graft vs Host Disease
DNA Methylation
Grafts
Liver Transplantation
Kidney Transplantation
Chronic Kidney Failure
Blood
Cell Count
Genes

Keywords

  • End stage organ disease
  • Murine model
  • Regulatory T cells
  • Safety

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Regulatory T cells from patients with end-stage organ disease can be isolated, expanded and cryopreserved according good manufacturing practice improving their function. / Ulbar, Francesca; Montemurro, Tiziana; Jofra, Tatiana; Capri, Miriam; Comai, Giorgia; Bertuzzo, Valentina; Lavazza, Cristiana; Mandelli, Alessandra; Viganò, Mariele; Budelli, Silvia; Bacalini, Maria Giulia; Pirazzini, Chiara; Garagnani, Paolo; Giudice, Valeria; Sollazzo, Daria; Curti, Antonio; Arpinati, Mario; La Manna, Gaetano; Cescon, Matteo; Pinna, Antonio Daniele; Franceschi, Claudio; Battaglia, Manuela; Giordano, Rosaria; Catani, Lucia; Lemoli, Roberto Massimo.

In: Journal of Translational Medicine, Vol. 17, No. 1, 250, 05.08.2019.

Research output: Contribution to journalArticle

Ulbar, Francesca ; Montemurro, Tiziana ; Jofra, Tatiana ; Capri, Miriam ; Comai, Giorgia ; Bertuzzo, Valentina ; Lavazza, Cristiana ; Mandelli, Alessandra ; Viganò, Mariele ; Budelli, Silvia ; Bacalini, Maria Giulia ; Pirazzini, Chiara ; Garagnani, Paolo ; Giudice, Valeria ; Sollazzo, Daria ; Curti, Antonio ; Arpinati, Mario ; La Manna, Gaetano ; Cescon, Matteo ; Pinna, Antonio Daniele ; Franceschi, Claudio ; Battaglia, Manuela ; Giordano, Rosaria ; Catani, Lucia ; Lemoli, Roberto Massimo. / Regulatory T cells from patients with end-stage organ disease can be isolated, expanded and cryopreserved according good manufacturing practice improving their function. In: Journal of Translational Medicine. 2019 ; Vol. 17, No. 1.
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abstract = "Background: Here, we isolated, expanded and functionally characterized regulatory T cells (Tregs) from patients with end stage kidney and liver disease, waiting for kidney/liver transplantation (KT/LT), with the aim to establish a suitable method to obtain large numbers of immunomodulatory cells for adoptive immunotherapy post-transplantation. Methods: We first established a preclinical protocol for expansion/isolation of Tregs from peripheral blood of LT/KT patients. We then scaled up and optimized such protocol according to good manufacturing practice (GMP) to obtain high numbers of purified Tregs which were phenotypically and functionally characterized in vitro and in vivo in a xenogeneic acute graft-versus-host disease (aGVHD) mouse model. Specifically, immunodepressed mice (NOD-SCID-gamma KO mice) received human effector T cells with or without GMP-produced Tregs to prevent the onset of xenogeneic GVHD. Results: Our small scale Treg isolation/expansion protocol generated functional Tregs. Interestingly, cryopreservation/thawing did not impair phenotype/function and DNA methylation pattern of FOXP3 gene of the expanded Tregs. Fully functional Tregs were also isolated/expanded from KT and LT patients according to GMP. In the mouse model, GMP Tregs from LT or KT patient proved to be safe and show a trend toward reduced lethality of acute GVHD. Conclusions: These data demonstrate that expanded/thawed GMP-Tregs from patients with end-stage organ disease are fully functional in vitro. Moreover, their infusion is safe and results in a trend toward reduced lethality of acute GVHD in vivo, further supporting Tregs-based adoptive immunotherapy in solid organ transplantation.",
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T1 - Regulatory T cells from patients with end-stage organ disease can be isolated, expanded and cryopreserved according good manufacturing practice improving their function

AU - Ulbar, Francesca

AU - Montemurro, Tiziana

AU - Jofra, Tatiana

AU - Capri, Miriam

AU - Comai, Giorgia

AU - Bertuzzo, Valentina

AU - Lavazza, Cristiana

AU - Mandelli, Alessandra

AU - Viganò, Mariele

AU - Budelli, Silvia

AU - Bacalini, Maria Giulia

AU - Pirazzini, Chiara

AU - Garagnani, Paolo

AU - Giudice, Valeria

AU - Sollazzo, Daria

AU - Curti, Antonio

AU - Arpinati, Mario

AU - La Manna, Gaetano

AU - Cescon, Matteo

AU - Pinna, Antonio Daniele

AU - Franceschi, Claudio

AU - Battaglia, Manuela

AU - Giordano, Rosaria

AU - Catani, Lucia

AU - Lemoli, Roberto Massimo

PY - 2019/8/5

Y1 - 2019/8/5

N2 - Background: Here, we isolated, expanded and functionally characterized regulatory T cells (Tregs) from patients with end stage kidney and liver disease, waiting for kidney/liver transplantation (KT/LT), with the aim to establish a suitable method to obtain large numbers of immunomodulatory cells for adoptive immunotherapy post-transplantation. Methods: We first established a preclinical protocol for expansion/isolation of Tregs from peripheral blood of LT/KT patients. We then scaled up and optimized such protocol according to good manufacturing practice (GMP) to obtain high numbers of purified Tregs which were phenotypically and functionally characterized in vitro and in vivo in a xenogeneic acute graft-versus-host disease (aGVHD) mouse model. Specifically, immunodepressed mice (NOD-SCID-gamma KO mice) received human effector T cells with or without GMP-produced Tregs to prevent the onset of xenogeneic GVHD. Results: Our small scale Treg isolation/expansion protocol generated functional Tregs. Interestingly, cryopreservation/thawing did not impair phenotype/function and DNA methylation pattern of FOXP3 gene of the expanded Tregs. Fully functional Tregs were also isolated/expanded from KT and LT patients according to GMP. In the mouse model, GMP Tregs from LT or KT patient proved to be safe and show a trend toward reduced lethality of acute GVHD. Conclusions: These data demonstrate that expanded/thawed GMP-Tregs from patients with end-stage organ disease are fully functional in vitro. Moreover, their infusion is safe and results in a trend toward reduced lethality of acute GVHD in vivo, further supporting Tregs-based adoptive immunotherapy in solid organ transplantation.

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KW - Murine model

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KW - Safety

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