Human LAP(+)GARP(+)FOXP3(+) regulatory T cells attenuate xenogeneic graft versus host disease

Wang, Huihui; Song, Hyo; Pham, Anha V.; Cooper, Laurence J.; Schulze, Janika J.; Olek, Sven; Tran, Dat Q.

NOD.Cg-Prkdc scid Il2rg tm1Wjl/SzJ (NSG) mice were obtained from Jackson Laboratory and maintained in specific pathogen free conditions at the CLAMC (University of Texas Health Science Center at Houston). In this protocol, the mice were not irradiated. On day 0, mice were injected with human CD25 − PBMC (30×10 6 ) with or without expanded CD25 + , LAP + Tregs and LAP‾ nonTregs (30×10 6 ). Following injection of cells, animals were monitored twice a week for weight loss and other signs of xGVHD, e.g. hunched back, ruffled fur and reduced mobility [16, 17] . Mice were euthanized if they experienced >15% weight loss or showed signs of compromised health and were considered to have human graft-related disease if an expanded population of human cells was detected simultaneously in blood. Mice were evaluated for human cells in blood by flow cytometer weekly until day 70 or until they were euthanized. To document CD25 − PBMC-associated cell expansion, the animals were bled (10-40 μl) and red blood cells were lysed with ACK Lysing Buffer (Quality Biological, Inc.). All animal protocols were approved by CLAMC at the University of Texas Health Science Center at Houston.