Th18 - Long Term Expanded Purified Allospecific Tr1 Cells Maintain a Suppressive Phenotype and Function in the Presence of Inflammatory Cytokines and Express a Chemokine Receptor Profile Involved in Allograft Tolerance.

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 transplantation. Recent studies have shown the need for a more exhaustive characterization to ensure a bona fide Tr1 population to be used as therapy. It has been reported that the proinflammatory environment induced by the allograft could affect the suppressive function of Treg cells, although the stability of Tr1 cells in vitro and in vivo has been poorly investigated. Another important aspect that must be considered is the homing of Tr1 cells to achieve an efficient allograft tolerance, where key chemokine receptor expression plays an important role.

Here, we established an optimized protocol that allows long term in vitro expansion of highly purified allospecific Tr1 (exp-allo-Tr1). After being expanded under proinflammatory conditions we observed that our population maintains a high co-expression of CD49b, LAG-3 (≈80%), which produce high levels of IL-10 (≈90%) and expressed co-inhibitory receptors (PD-1, CTLA-4, TIM-3, TIGIT, and CD39) and efficiently suppressed allospecific but third party T cell responses.  Additionally, we observed that exp-allo-Tr1 expressed a high percentage of chemokine receptors involved in allograft tolerance, such as CCR2 (65.5%±19.75), CCR4 (77.5%±10.45) but not CCR7 (16.9%±7.75). Moreover, exp-allo-Tr1 also expressed CXCR3 (47.3 %), which could promote their homing to the kidney allograft. 

Our data indicate that exp-allo-Tr1 are stable and express relevant homing receptors supporting their potential use in long-term transplantation tolerance.