Lrrc32 mRNA is preferentially expressed in naturally-occurring freshly-isolated non-expanded human T
but is not observed on the surface of these cells. a) T cell sorting gates based upon CD25 surface expression. b) Lrrc32 mRNA expression comports with Foxp3 mRNA expression and CD25 surface expression, and Lrrc32 mRNA is preferentially expressed in Tregs, compared to Teffs. Relative expressions of 18SrRNA-normalized Foxp3 and Lrrc32 genes were determined using real-time PCR. Data summarize four independent experiments. Results are expressed as the mean ± SEM. *p = 0.01 compared to CD25mid cells, **p = 0.005 compared to CD25mid cells. c) Flow cytometric analysis of freshly-isolated activated CD4+ human T cells shows that the CD25hi subgroup (composed of the CD25hi+ and CD25hi++ subgroups denoted in figure 1b) demarcating Tregs expresses LRRC32 on the cell surface (top right panel) but that the CD25- subgroup demarcating Teffs expresses negligible amounts of LRRC32 on the cell surface (bottom right panel). Analysis strategy is indicated via the arrows. d) Flow cytometric analysis of sorted resting CD4+ human Tregs shows that they lack surface expression of LRRC32 protein (top left panel). However, after permeabilization of the cell membrane, a low expression of LRRC32 protein can be observed intracellularly (top right panel). After activation of the sorted Tregs with a Treg expansion kit, LRRC32 can be seen on the surface of these activated Tregs (bottom left panel), and evidence of intracellular expression of LRRC32 can also be observed after permeabilization of the cell membrane (bottom right panel).