Identification and characterisation of Asparaginyl Endopeptidase expression and function in T regulatory cells

Abstract

The cysteine protease asparaginyl endopeptidase (AEP) is expressed in antigen presenting cells (APCs) and is involved in activation of proteolytic enzymes and antigen processing. Mice with a global deletion of AEP (Lgmn- /- ) develop a lysosomal disorder characterised by the accumulation of cathepsins but otherwise do not exhibit an immune phenotype. However, an increased frequency of regulatory T cells (Treg) in the periphery has been reported suggesting that AEP expression regulates Treg cell stability through Foxp3 degradation. To study the induction of AEP expression in mouse Treg cells, naïve and CD4+CD25+ T cells were cultured under Treginducing (iTreg) conditions which include TGF-β1. Freshly isolated CD4+ T cells did not express any AEP while cells cultured under iTreg conditions expressed the highest levels of AEP. To test the function of AEP in activated peripheral Treg cells, a mouse model of melanoma was used. Lgmn-/- mice with melanoma showed an increased frequency of tumour infiltrating Treg cells compared to controls although tumour growth was similar between the two groups. Next, the expression profile of AEP in human T cells was investigated as the mechanisms regulating AEP expression and function in these cells remain unclear. To study the induction of AEP expression in human Treg cells, naïve and CD4+CD25+ T cells were cultured under iTreg conditions. AEP was expressed in freshly isolated populations. Also, cells cultured under iTreg conditions expressed the highest levels of AEP. Blocking TGF-β1 signalling with a TGFBR-I inhibitor led to a decrease in active AEP expression within the cells. Stimuli that could block AEP activity were also tested. In keeping with previously published work, blockade of PD-1 led to an increase of AEP activity levels in human lymphocytes. These findings demonstrate that AEP expression is inversely regulated by TGF-β1 and PD-1 signalling in human T cells with implications for immunotherapy.