Signal peptide cleavage is essential for surface expression of a regulatory T cell surface protein, leucine rich repeat containing 32 (LRRC32)

Background: Elevated numbers of regulatory T cells (T-regs) have been implicated in certain cancers. Depletion of T-regs has been shown to increase anti-tumor immunity. T-regs also play a critical role in the suppression of autoimmune responses. The study of T-regs has been hampered by a lack of adequate surface markers. Leucine Rich Repeat Containing 32 (LRRC32), also known as Glycoprotein A Repetitions Predominant (GARP), has been postulated as a novel surface marker of activated T-regs. However, there is limited information regarding the processing of LRRC32 or the regulatory phenotype and functional activity of T-regs expressing LRRC32. Results: Using naturally-occurring freshly isolated T-regs, we demonstrate that low levels of LRRC32 are present intracellularly prior to activation and that freshly isolated LRRC32(+) T-regs are distinct from LRRC32(-)T(regs) with respect to the expression of surface CD62L. Using LRRC32 transfectants of HEK cells, we demonstrate that the N-terminus of LRRC32 is cleaved prior to expression of the protein at the cell surface. Furthermore, we demonstrate using a construct containing a deleted putative signal peptide region that the presence of a signal peptide region is critical to cell surface expression of LRRC32. Finally, mixed lymphocyte assays demonstrate that LRRC32(+) T-regs are more potent suppressors than LRRC32(-)T(regs). Conclusions: A cleaved signal peptide site in LRRC32 is necessary for surface localization of native LRRC32 following activation of naturally-occurring freshly-isolated regulatory T cells. LRRC32 expression appears to alter the surface expression of activation markers of T cells such as CD62L. LRRC32 surface expression may be useful as a marker that selects for more potent T-reg populations. In summary, understanding the processing and expression of LRRC32 may provide insight into the mechanism of action of T-regs and the refinement of immunotherapeutic strategies aimed at targeting these cells.