Fig. 7

BTNL8 is expressed by miTregs. a Discriminatory gene analysis identified genes upregulated in Mreg-cocultured CD4⁺ T cells compared to controls. The 10 most highly upregulated reporters in allogeneic Mreg-cocultured CD4⁺ T cells compared to IFN-γ Mφ-cocultured CD4⁺ T cells are shown (n = 5; median ± MADM). b qPCR for BTNL8 mRNA in CD4⁺ T cells cocultured with allogeneic Mregs or IFN-γ Mφ, CD4⁺ T cells cultured alone and freshly isolated CD4⁺ T cells (n = 9; mean ± SEM; KW). c Human peripheral blood T cells were flow-sorted for CD3⁺CD4⁺CD25⁺CD127^int Tregs and CD3⁺CD4⁺CD25⁻CD127⁺ non-Tregs. BTNL8 mRNA expression was not detected in unactivated Treg or conventional T cells, but was induced by stimulation with αCD3/αCD28 beads for 5 days (n = 4; MW). d Western blot for BTNL8 using goat α-BTNL8 (Santa Cruz, sc-245053) to compare T cells cocultured with allogeneic Mregs or IFN-γ Mφ. A band corresponding to BTNL8 was detected at 37 kD. e BTNL8 mRNA was more highly expressed by GARP⁺ miTregs than GARP⁻ non-Tregs (n = 6). f Detection of BTNL8 expression in Mreg-cocultured T cells by flow cytometry after intracellular staining with rabbit α-BTNL8 pAb (LSBio #C453018) against aa 161–210. Histogram is gated on live CD3⁺ CD4⁺ CD8⁻ CD25⁺ FoxP3⁺ single events. g Pre-incubation of rabbit α-BTNL8 pAb with specific peptide (LSBio #E18020) blocked detection of BTNL8, whereas a non-specific peptide corresponding to the N terminus of BTNL8 (#E18019) had no effect. h By flow cytometry, BTNL8 appeared to be expressed by all Mreg-cocultured CD4⁺ T cells, but miTregs expressed higher levels of BTNL8 than CD25⁺ FoxP3⁻ or CD25⁻ FoxP3⁻ non-Tregs (n = 3). i Compared to recombinant human Fc (rhFc), addition of 10 μg ml⁻¹ BTNL8-Fc fusion protein enhanced miTreg generation (n = 8; MW). j A concentration of 20 μg ml⁻¹ α-BTNL8 rabbit mAb (R&D #2187B) enhanced miTreg generation compared to isotype control (n = 9; MW). k NOD-scid-gamma (NSG) mice were reconstituted with 5 × 10⁶ naive human CD4⁺ T cells and then treated with either 5 × 10⁶ allogeneic Mregs (n = 3) or vehicle only (n = 3). Five days after cell transfer, splenic human T cells were recovered by bead-sorting, and end-point PCR for BTNL8 mRNA was performed