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. 2016 Feb 16;7(7):7563-77.
doi: 10.18632/oncotarget.6927.

Successful expansion of functional and stable regulatory T cells for immunotherapy in liver transplantation

Niloufar Safinia  1 , Trishan Vaikunthanathan  1 , Henrieta Fraser  1 , Sarah Thirkell  1 , Katie Lowe  1 , Laura Blackmore  2 , Gavin Whitehouse  2 , Marc Martinez-Llordella  2 , Wayel Jassem  2 , Alberto Sanchez-Fueyo  2 , Robert I Lechler  1 , Giovanna Lombardi  1
Affiliations

V体育2025版 - Successful expansion of functional and stable regulatory T cells for immunotherapy in liver transplantation

Niloufar Safinia et al. Oncotarget. .

Abstract

Strategies to prevent organ transplant rejection whilst minimizing long-term immunosuppression are currently under intense investigation with regulatory T cells (Tregs) nearing clinical application. The clinical trial, ThRIL, recently commenced at King's College London, proposes to use Treg cell therapy to induce tolerance in liver transplant recipients, the success of which has the potential to revolutionize the management of these patients and enable a future of drug-free transplants. This is the first report of the manufacture of clinical grade Tregs from prospective liver transplant recipients via a CliniMACS-based GMP isolation technique and expanded using anti-CD3/CD28 beads, IL-2 and rapamycin. We report the enrichment of a pure, stable population of Tregs (>95% CD4(+)CD25(+)FOXP3(+)), reaching adequate numbers for their clinical application VSports手机版. Our protocol proved successful in, influencing the expansion of superior functional Tregs, as compared to freshly isolated cells, whilst also preventing their conversion to Th17 cells under pro-inflammatory conditions. We conclude with the manufacture of the final Treg product in the clinical research facility (CRF), a prerequisite for the clinical application of these cells. The data presented in this manuscript together with the much-anticipated clinical results from ThRIL, will undoubtedly inform the improved management of the liver transplant recipient. .

Keywords: Immune response; Immunity; Immunology and Microbiology Section; immunosuppression; immunotherapy; liver transplantation; regulatory T cells; tolerance. V体育安卓版.

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare no financial or commercial conflicts of interest.

Figures

Figure 1
Figure 1. GMP Treg Isolation
A. Numbers of cells isolated from 150ml of blood by CD8+ cell depletion and CD25+ cell enrichment compared between 9 ARC patients and 9 HCs. ***P < 0.001. B. Representative dot plot and graph denoting the circulating percentage of CD4+CD25Hi Tregs of 5 ARC patients and 5 HCs. Abbreviation: n.s- not significant. Data are represented as mean +/− SEM.
Figure 2
Figure 2. GMP Compatible Treg isolation and expansion
Two step strategy for Treg isolation: A. CD8+ cell depletion. B. CD25+ cell enrichment. In each case dots plots are representative of 9 ARC patients. Graphs denote the purity of the culture throughout the expansion period in both rapamycin and untreated cultures C. Tregs from ARC patients and HCs were expanded over 36 days ± rapamycin. Fold expansion was calculated from Treg numbers at each stimulation. D. Predicted Treg numbers over the 36 day expansion period calculated based on fold expansion and the assumption that all cells were expanded at each stimulation. n = 9 ARC, n = 9HCs. Abbreviation: S, stimulation; S1, day 0; S2, day 12; S3, day 24; final, day 36; n.s, not significant. **P < 0.05, ***P < 0.001. Data are represented as mean +/− SEM.
Figure 3
Figure 3. Expression of regulatory markers and homing receptor expression by Tregs throughout culture
A. Graph shows the mean percentage of CD4+CD25+ cells expressing FOXP3+ in both untreated and rapamycin treated cultures over 36 days of culture (n = 9 ARC). B. Graph shows the frequency of FOXP3Hi Tregs, from 9 ARC patients, throughout culture ± rapamycin. C. MFI of FOXP3 expression by Tregs. D. Graph shows the frequency CD127lo Tregs, from 9 ARC patients, throughout culture ± rapamycin. E. Dot plot details the frequency of CTLA-4 expression on CD4+CD25+ Tregs from one representative sample. F. The graph depicts the frequency of CD4+CD25+CTLA-4+ Tregs throughout culture ± rapamycin. G. MFI of CTLA-4 expression at S1 and day 36 of culture ± rapamycin. H. Expression of CD62L and CXCR3 on CD4+CD25+ on day 0 and at day 36 Tregs cultured ± rapamycin. Abbreviations: Mean fluorescent intensity (MFI) S- stimulation, n.s.-not significant. **P < 0.01, ***P < 0.001. Data are represented as mean +/− SEM. n = 9 ARC.
Figure 4
Figure 4. Assessement of Treg suppressive function
Representative histogram and graph from 9 ARC patients upon assessment of Treg suppressor function. The suppressive function of Tregs cultured ± rapamycin was assesed by CFSE dilution assay at day 0 and throughout the 36 day culture period. n.s.- not significant. *P < 0.05, ***P < 0.001. Data are represented as mean +/− SEM.
Figure 5
Figure 5. Intracellular expression of IL-17 and IFN-γ and production of IL-17 by ex vivo expanded Tregs
A. Frequency of IL-17+ in CD4+CD25+FOXP3+ Tregs upon isolation and post culture in the presence of rapamycin (day 36) when exposed to two separate mixes of pro-inflammatory cytokines (Mix 1: IL-2, IL1β, IL-6 and TGF-β and Mix 2: IL-2, IL-21, IL-23 and TGF-). B. IL-17 (pg/ml) concentration in a 5-day culture supernatant of the rapamycin expanded Tregs in the presence of Mix1 and Mix2. C. Frequency of IFN-γ+ in CD4+CD25+FOXP3+ Tregs upon isolation and post ex vivo expansion in the presence of rapamycin (day 36) when exposed to Mix 1 and Mix 2. D. Gating strategy and Frequency of CD161+ Tregs throughout culture. Dot plot depicts expression of CD161 on CD4+CD25+ Tregs from a representative sample of 9 ARC patients. Graph shows the dynamics of CD161 expressing Tregs throughout culture. E. Gating strategy and Frequency of CCR6+CD161+ Tregs throughout culture. Dot plot of CD161+CCR6+ co-expression on CD4+CD25+ Tregs from a representative sample of 9 ARC patients and graph of percentage CD161+CCR6+ co-expression throughout culture. n.s.-not significant. *P < 0.05,**P < 0.01. ***P < 0.001. n = 9 ARC patients. Data are represented as mean +/− SEM.
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