Human Mesenchymal Cells Control T Regulatory Phenotypes and Functions.
Authors: Di Ianni Mauro , Papa Beatrice Del , Moretti Lorenzo , De Ioanni Maria , Bonifacio Elisabetta , Falzetti Franca , Tabilio Antonio 
Published: April 2020

Abstract
Despite much investigation into T regulatory cells (Tregs), little is known about the mechanism controlling their recruitment and function. Since mesenchymal cells (MSCs) exert an immune regulatory function and suppress T cell proliferation, this in vitro study investigated their role in Treg recruitment and function. hMSCs and different T cell populations (CD3+, CD3+/CD45RA+, CD3+/CD45RO+, CD4+/CD25+, CD4+/CD25+/CD45RO+, CD4+/CD25+/CD45RA+) from healthy donors were co-cultured for up to 15 days. Harvested lymphocytes were analysed by flow-cytometry; FoxP3 and CD127 expression were measured by real time PCR; regulatory activity was assessed. Within CD3+ fraction, percentages of CD4+/CD25bright and CD4+/CD25bright/Foxp3+ cells were higher in the presence of hMSCs (42% vs 34% for the CD4+/CD25bright and 20.5 vs 3.5 for the CD4+/CD25bright/Foxp3+). Within CD3+/CD45RA+ and CD3+/CD45RO+ fractions, the T reg starting fraction of the naïve population rose from 0.05% ± 0.01 CD4/CD25 positive cells to 0.2% ± 0.14 and the T reg starting fraction of the memory population rose from 0.3% ± 0.05 CD4/CD25 positive cells to 1.5% ± 0.9. Within CD4+/CD25+, CD4+/CD25+/CD45RA+, CD4+/CD25+/CD45RO+, FoxP3 expression did not change in CD4+/CD25+ cells. It increased 5.38±2.3 fold in CD4+/CD25+/CD45RA+ and 7.98±1.9 fold in CD4+/CD25+/CD45RO+ cells. CD127 expression decreased by -582±29.2 fold in CD4+/CD25+ cells, by -216±17.5 fold in CD4+/CD25+/CD45RA+ and by -71.95±12.6 fold in CD4+/CD25+/CD45RO+ cells. Cytofluorimetric analysis showed CD4+/CD25+/FoxP3+ was up-regulated to 14%±4 and CD127 down-regulated to 4.4%±1.5 vs respectively 0.2%±0.28 and 21.9%±3.5 in controls without MSCs (CD4+/CD25+ alone). FoxP3 up-regulation was more marked in CD4+/CD25bright cells (51%±10 vs 0.1%±0.7 in controls). Sorted Tregs exert potent immunosuppressive activity on CD4+/CD25- cell populations. Inhibitory activity was lost within 15 days when sorted T regs were cultured without hMSC. On day +15 proliferation using CD4+/CD25+ cells as suppressor cells was 1.7±0.8 vs 45.2±12 in controls; proliferation using CD4/CD25+/CD45RA+ as suppressor cells was 5.4±3.1, vs 21.3±11.2 in controls, and proliferation with CD4+/CD25+/RO+ as suppressor cells was 2.3±2 vs 33.5±8.7 in controls. We demonstrate MSC recruit Tregs from a fraction of CD3+ and from immunoselected CD3+/CD45RA+ and CD3+/CD45RO+ fractions. After culture with MSCs both immunoselected fractions registered increases in the CD4+/CD25bright/FoxP3 subset and CD127 expression was down regulated. When purified Treg populations (CD4/CD25+, CD4/CD25+/CD45RA+ and CD4/CD25+/CD45RO+) were used as fraction for hMSC co-cultures, they maintain FoxP3 expression and CD127 expression is down-regulated. Treg suppressive capacity was maintained in Treg populations that were layered on MSC for up to 15 days while control Tregs lost all suppressive activity after 5 days culture. In conclusion, our study demonstrates that MSCs recruit, regulate and maintain T regulatory phenotype and function over time.