Background: We previously identified xeno-antigen reactive human HLADR+CD27+Tregs with enhanced suppressive function in vitro and more capable of suppressing islet xenograft rejection in a humanised mouse model of neonatal porcine islet cell clusters (NICC) transplantation.

Aims: 1) Phenotyping HLADR+CD27+Tregs and 2) evaluating possible pathways through which they exert a suppressive activity.

Methods: Human CD4+CD25+hiCD127-Tregs isolated from peripheral-blood-mononuclear-cells (PBMCs) were co-cultured with irradiated porcine PBMCs for three subsequent cycles in the presence of IL-2/rapamycin and anti-CD3/CD28 beads. At day 21, phenotyping HLADR+CD27+Tregs of expanded Tregs was performed using flow cytometry for both surface- and intracellular expression (MFI) of the transmembrane proteins CD95, ICOS, CTLA-4, GITR; surface expression of CD39 and CD62L; and intracellular expression of FOXP3 and Helios compared to CD27-, HLADR-, HLADR-CD27-,  HLADR+CD27-, HLADR-CD27+, depleted-HLADR+CD27+Treg subsets or all expanded Tregs.

Results: There are no differences of FOXP3 and CD39 expression on HLADR+CD27+Tregs compared to all other subsets. CD62L increases significantly on HLADR+CD27+Tregs when compared to HLADR-CD27-, CD27- and HLADR-Tregs. HLADR+CD27+Tregs have an increasing trend of Helios expression compared to all other subsets, with a significant increase when compared to HLADR-CD27- and HLADR-Tregs. An increasing trend can also be observed in HLADR+CD27+Tregs CTLA-4 expression when compared to all other subsets. Furthermore, HLADR+CD27+Tregs show significant enhanced total-expression of CD95 (P <0.01) compared to all other subsets (except HLA-DR+CD27-Tregs), suggesting that the enhanced expression of CD95 is associated with HLA-DR expression. HLADR+CD27+ Tregs also show significant enhanced surface-expression of ICOS (P <0.05) compared to depleted-HLADR+CD27+Tregs subset. At the same time, both surface-expression and total-expression of GITR is decreased significantly in HLADR+CD27+Tregs compared to all other subsets.

Conclusion: The enhanced suppressive function of xeno-antigen reactive HLADR+CD27+Tregs is associated with enhanced expression of CD95 apoptotic antigen and ICOS costimulatory molecule. HLADR and CD27 are the important immune checkpoints for xeno-antigen specific Tregs.