Department of Immunology, Biomedical Research Institute, Mexico City, Mexico.
The National Laboratory of Flow Cytometry, Biomedical Research Institute, National Autonomous University of Mexico, Mexico City, Mexico.
Front Immunol. 2023 Feb 24;14:1062456. doi: 10.3389/fimmu.2023.1062456. eCollection 2023.
The development of new strategies based on the use of Tr1 cells has taken relevance to induce long-term tolerance, especially in the context of allogeneic stem cell transplantation. Although Tr1 cells are currently identified by the co-expression of CD49b and LAG-3 and high production of interleukin 10 (IL-10), recent studies have shown the need for a more exhaustive characterization, including co-inhibitory and chemokines receptors expression, to ensure bona fide Tr1 cells to be used as cell therapy in solid organ transplantation. Moreover, the proinflammatory environment induced by the allograft could affect the suppressive function of Treg cells, therefore stability of Tr1 cells needs to be further investigated. Here, we establish a new protocol that allows long-term expansion of highly purified expanded allospecific Tr1 ( Tr1). Our expanded Tr1 cell population becomes highly enriched in IL-10 producers (> 90%) and maintains high expression of CD49b and LAG-3, as well as the co-inhibitory receptors PD-1, CTLA-4, TIM-3, TIGIT and CD39. Most importantly, high dimensional analysis of Tr1 demonstrated a specific expression profile that distinguishes them from activated conventional T cells (T conv), showing overexpression of IL-10, CD39, CTLA-4 and LAG-3. On the other hand, Tr1 expressed a chemokine receptor profile relevant for allograft homing and tolerance induction including CCR2, CCR4, CCR5 and CXCR3, but lower levels of CCR7. Interestingly, Tr1 efficiently suppressed allospecific but not third-party T cell responses even after being expanded in the presence of proinflammatory cytokines for two extra weeks, supporting their functional stability. In summary, we demonstrate for the first time that highly purified allospecific Tr1 ( Tr1) cells can be efficiently expanded maintaining a stable phenotype and suppressive function with homing potential to the allograft, so they may be considered as promising therapeutic tools for solid organ transplantation.
基于 Tr1 细胞的新策略的发展已变得相关，以诱导长期耐受，特别是在异基因干细胞移植的背景下。尽管 Tr1 细胞目前通过共表达 CD49b 和 LAG-3 以及高水平的白细胞介素 10（IL-10）来鉴定，但最近的研究表明需要更详尽的表征，包括共抑制和趋化因子受体表达，以确保真正的 Tr1 细胞作为实体器官移植的细胞治疗。此外，同种异体移植物诱导的促炎环境可能会影响 Treg 细胞的抑制功能，因此需要进一步研究 Tr1 细胞的稳定性。在这里，我们建立了一种新的方案，允许长期扩增高度纯化的同种异体特异性 Tr1（Tr1）。我们扩增的 Tr1 细胞群体在 IL-10 产生物中高度富集（>90%），并保持高水平的 CD49b 和 LAG-3 表达，以及共抑制受体 PD-1、CTLA-4、TIM-3、TIGIT 和 CD39。最重要的是，Tr1 的高维分析显示出一种特定的表达谱，将其与激活的常规 T 细胞（Tconv）区分开来，表现出 IL-10、CD39、CTLA-4 和 LAG-3 的过度表达。另一方面，Tr1 表达了与同种异体归巢和诱导耐受相关的趋化因子受体谱，包括 CCR2、CCR4、CCR5 和 CXCR3，但 CCR7 水平较低。有趣的是，即使在存在促炎细胞因子的情况下再扩增两周，Tr1 也能有效地抑制同种异体特异性但不能抑制第三方 T 细胞反应，支持其功能稳定性。总之，我们首次证明，高度纯化的同种异体特异性 Tr1（Tr1）细胞可以在保持稳定表型和抑制功能的情况下有效地扩增，具有同种异体归巢的潜力，因此它们可能被视为实体器官移植的有前途的治疗工具。
