Clinical-Grade Expanded Regulatory T Cells Are Enriched with Highly Suppressive Cells Producing IL-10, Granzyme B, and IL-35

Francesca Ulbar , Ida Villanova , Raffaella Giancola , Stefano Baldoni , Francesco Guardalupi , Bianca Fabi , Paola Olioso , Anita Capone , Rosaria Sola , Sara Ciardelli , Beatrice Del Papa , Antonello Brattelli , Ilda Ricciardi , Stefano Taricani , Giulia Sabbatinelli , Ornella Iuliani , Cecilia Passeri , Paolo Sportoletti , Stella Santarone , Antonio Pierini , Mauro Di Ianni
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引用次数: 8

Abstract

In the setting of T cell-depleted, full-haplotype mismatched transplantation, adoptive immunotherapy with regulatory T cells (Tregs) and conventional T cells (Tcons) can prevent graft-versus-host disease (GVHD) and improve post-transplantation immunologic reconstitution and is associated with a powerful graft-versus-leukemia effect. To improve the purity and the quantity of the infused Tregs, good manufacturing practices (GMP)-compatible expansion protocols are needed. Here we expanded Tregs using an automated, clinical-grade protocol. Cells were extensively characterized in vitro, and their efficiency was tested in vivo in a mouse model. Tregs were selected by CliniMacs (CD4+CD25+, 94.5 ± 6.3%; FoxP3+, 63.7 ± 11.5%; CD127+, 20 ± 3%; suppressive activity, 60 ± 7%), and an aliquot of 100 × 106 was expanded for 14 days using the CliniMACS Prodigy System, obtaining 684 ± 279 × 106 cells (CD4+CD25+, 99.6 ± 0.2%; FoxP3+, 82 ± 8%; CD127+, 1.1 ± 0.8%; suppressive activity, 75 ± 12%). CD39 and CTLA4 expression levels increased from 22.4 ± 12% to 58.1 ± 13.3% (P < .05) and from 20.4 ± 6.7% to 85.4 ± 9.8% (P < .01), respectively. TIM3 levels increased from .4 ± .05% to 29 ± 16% (P < .05). Memory Tregs were the prevalent population, whereas naive Tregs almost disappeared at the end of the culture. mRNA analysis displayed significant increases in CD39, IL-10, granzyme B, and IL-35 levels at the end of culture period (P < .05). Conversely, IFNγ expression decreased significantly by day +14. Expanded Tregs were sorted according to TIM3, CD39, and CD62L expression levels (purity >95%). When sorted populations were analyzed, TIM3+ cells showed significant increases in IL-10 and granzyme B (P < .01) .When expanded Tregs were infused in an NSG murine model, mice that received Tcons only died of GVHD, whereas mice that received both Tcons and Tregs survived without GVHD. GMP grade expanded cells that display phenotypic and functional Treg characteristics can be obtained using a fully automated system. Treg suppression is mediated by multiple overlapping mechanisms (eg, CTLA-4, CD39, IL-10, IL-35, TGF-β, granzyme B). TIM3+ cells emerge as a potentially highly suppressive population.

© 2020 American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc.

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临床级扩增调节性T细胞富含产生IL-10、颗粒酶B和IL-35的高抑制性细胞
在T细胞衰竭、全单倍型错配移植的情况下,调节性T细胞(Tregs)和常规T细胞(Tcons)的过继免疫治疗可以预防移植物抗宿主病(GVHD)和改善移植后免疫重建,并与强大的移植物抗白血病效应相关。为了提高注射Tregs的纯度和数量,需要符合良好生产规范(GMP)的扩增协议。在这里,我们使用自动化的临床级方案扩展Tregs。在体外对细胞进行了广泛的表征,并在小鼠模型中对其效率进行了体内测试。CliniMacs筛选treg (CD4+CD25+, 94.5±6.3%;FoxP3+, 63.7±11.5%;Cd127 +, 20±3%;使用CliniMACS Prodigy系统扩增14天,获得684±279 × 106个细胞(CD4+CD25+, 99.6±0.2%;FoxP3+, 82±8%;Cd127 +, 1.1±0.8%;抑制活性,75±12%)。CD39和CTLA4的表达水平从22.4±12%增加到58.1±13.3% (P <0.05),从20.4±6.7%上升到85.4±9.8% (P <. 01),分别。TIM3水平从0.4±0.05%上升至29±16% (P <. 05)。记忆型treg是普遍存在的群体,而幼稚型treg在培养结束时几乎消失了。mRNA分析显示CD39、IL-10、颗粒酶B和IL-35水平在培养期结束时显著升高(P <. 05)。相反,IFNγ的表达在第14天显著下降。扩增treg根据TIM3、CD39和CD62L表达水平进行分类(纯度>95%)。对分选群体进行分析时,TIM3+细胞IL-10和颗粒酶B显著升高(P <在NSG小鼠模型中注入扩增的Tregs,接受Tcons的小鼠仅死于GVHD,而同时接受Tcons和Tregs的小鼠则存活,无GVHD。使用全自动系统可以获得显示表型和功能Treg特征的GMP级扩增细胞。Treg抑制是由多种重叠机制介导的(如CTLA-4、CD39、IL-10、IL-35、TGF-β、颗粒酶B)。TIM3+细胞是一种潜在的高抑制群体。©2020美国移植和细胞治疗学会。Elsevier Inc.出版。
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来源期刊
CiteScore
6.60
自引率
0.00%
发文量
1061
审稿时长
3-6 weeks
期刊介绍: Biology of Blood and Marrow Transplantation publishes original research reports, reviews, editorials, commentaries, letters to the editor, and hypotheses and is the official publication of the American Society for Transplantation and Cellular Therapy. The journal focuses on current technology and knowledge in the interdisciplinary field of hematopoetic stem cell transplantation.
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