Isolation and expansion of thymus‐derived regulatory T cells for use in pediatric heart transplant patients

Abstract

Regulatory T‐cells (Tregs) are a subset of T cells generated in the thymus with intrinsic immunosuppressive properties. Phase I clinical trials have shown safety and feasibility of Treg infusion to promote immune tolerance and new studies are ongoing to evaluate their efficacy. During heart transplantation, thymic tissue is routinely discarded providing an attractive source of Tregs. In this study, we developed a GMP‐compatible protocol for expanding sorted thymus‐derived CD3+CD4+CD25+CD127– (Tregs) as well as CD3+CD4+CD25+CD127–CD45RA+ (RA+Tregs) cells. We aimed to understand whether thymic RA+Tregs can be isolated and expanded offering an advantage in terms of stability as it has been previously shown for circulating adult CD45RA+ Tregs. We show that both Tregs and RA+Tregs could be expanded in large numbers and the presence of rapamycin is essential to inhibit the growth of IFN‐γ producing cells. High levels of FOXP3, CTLA4, and CD25 expression, demethylation of the FOXP3 promoter, and high suppressive ability were found with no differences between Tregs and RA+Tregs. After freezing and thawing, all Treg preparations maintained their suppressive ability, stability, as well as CD25 and FOXP3 expression. The number of thymic Tregs that could be isolated with our protocol, their fold expansion, and functional characteristics allow the clinical application of this cell population to promote tolerance in pediatric heart transplant patients.

A Novel GMP Protocol to Produce High-Quality Treg Cells From the Pediatric Thymic Tissue to Be Employed as Cellular Therapy

“…Indeed, Dijke and collaborators ( 27 ) showed that a large amount of stable, long-lived and potent FOXP3 + Treg could be isolated and expanded from a single thymus. Furthermore, Romano and colleagues ( 28 ) have recently reported a good manufacturing practice (GMP) compliant protocol to isolate and expand thymus-derived Treg cells, confirming the feasibility of the strategy. This is a revolutionary approach since children with heart diseases requiring cardiac surgery often undergo thymectomy to clear the surgical field.…”

“…Therefore, the search for an alternative source of Treg with a predominantly naïve state that allows obtaining enough quantity of cells is crucial to overcome the limitations encountered in peripheral blood and umbilical cord blood. In this sense, the thymus, a primary lymphoid organ responsible for the maturation and differentiation of T and Treg cells, which is located above the heart and discarded to gain access to the heart during pediatric cardiac surgeries, could be employed as a new source of highly undifferentiated Treg ( 27 , 28 ).…”

Reduced incidence of cardiac rejection in multi‐organ transplants: A propensity matched study

“…Thymic tissue is routinely discarded during heart transplant, however it contains regulatory T-cells with intrinsic immunosuppressive properties which might promote tolerance in transplant recipients. 14,15 Similar opportunities to influence the donor's immune system might also exist by electively including splenic or lymphatic tissue at the time of heart transplant.…”

“…While it would clearly be unreasonable to contend that a patient should receive a vital organ if they do not need it, these findings do suggest that increased attention should be paid toward the potential benefits of including additional donor tissue at the time of heart transplant. Thymic tissue is routinely discarded during heart transplant, however it contains regulatory T‐cells with intrinsic immunosuppressive properties which might promote tolerance in transplant recipients 14,15 . Similar opportunities to influence the donor's immune system might also exist by electively including splenic or lymphatic tissue at the time of heart transplant.…”

Clinical adoptive regulatory T Cell therapy: State of the art, challenges, and prospective

“…It is widely accepted that CD4, CD25 and FOXP3 are key Treg markers and are partially associated with their functional properties. Tregs can be isolated from the peripheral blood (PB) ( Landwehr-Kenzel et al, 2018 ; Roemhild et al, 2020 ), umbilical cord ( Brunstein et al, 2011 ; Brunstein et al, 2016 ), thymus ( Romano et al, 2021 ) or leukapheresis products ( Peters et al, 2008 ; Wiesinger et al, 2017 ; Mathew et al, 2018 ). Of the available options PB is the least invasive, well tolerated by the patient and routinely collected in the clinics.…”