Human cathepsin S, but not cathepsin L, degrades efficiently MHC class II-associated invariant chain in nonprofessional APCs

Abstract

MHC class II-restricted antigen presentation plays a central role in the immune response against exogenous antigens. The association of invariant (Ii) chain with MHC class II dimers is required for proper antigen presentation to CD4+ T cells by antigen-presenting cells. MHC class II complexes first traffic through the endocytic pathway to allow Ii chain degradation and antigenic peptide loading before their arrival at the cell surface. In recent years, a considerable effort has been directed toward the identification of proteases responsible for Ii chain degradation. Targeted gene deletion in mice has allowed a precise description of the cysteine proteases involved in the last step of Ii chain degradation. By using nonspecialized cellular models expressing MHC II molecules, we are now exploring the contribution of known cysteine proteases to human Ii chain processing. Surprisingly and contrary to the situation in mouse, cathepsin S was found to be the only human cysteine protease able to efficiently degrade the Ii-p10 fragment in epithelial cells. This selectivity has implications for thymic selection and indicates that differences between man and mice are probably more profound at this level than expected.

Fig. 1.

Invariant chain degradation. (a) Immunofluorescence microscopy of HeLa-CIITA stained for HLA-DR (Left, red) and Ii chain (Center, green). Colocalization is shown in the merged picture (Right, yellow). (b) [³⁵S]Met pulse–chase experiment was performed on HeLa-CIITA before HLA-DR immunoprecipitation. (c) HeLa-CIITA membrane (Left) and MelJuSo cells (Right) were immunoblotted for invariant chain. INF-γ stimulation (24 h) induces total Ii chain fragment disappearance, a phenomenon inhibited with 5 nM LHVS. INF-γ treatment does not increase MHC class II expression in HeLa-CIITA (β chain, Bottom), whereas incubation of MelJuSo cells with leupeptin induces p22 accumulation without affecting p10.

Fig. 2.

Transcription of cysteine proteases. (a) RT-PCR with primers designed to amplify human Cat K, V/L2, L, Z, S, and F was performed on HeLa and MelJuSo cells treated or not with INF-γ. (b) RT-PCR for the human AEP was performed on HeLa-CIITA and MelJuSo cells.

Fig. 3.

Cat S is induced by INF-γ. (a) Immunofluorescence microscopy of HeLa-CIITA stained for Cat S with (Right) or without (Left) INF-γ treatment. (b) Cat S was immunodetected in HeLa-CIITA membranes. (c) Confocal microscopy of HeLa-CIITA (Upper) and MelJuSo (Lower) cells transfected with human Cat S cDNA and stained for Cat S (Right) and Ii chain (Left). Neighboring nontransfected cells provide the control for Ii chain levels.

Fig. 4.

Cathepsins are active in HeLa-CIITA. (a) Immunodetection of Cat K, V/L2, and L in HeLa-CIITA. (b) Confocal microscopy of HeLa-CIITA stained for Cat L (green) and HLA-DR (red). Colocalization is shown in the merged picture (yellow). (c) CBZ-[¹²⁵I]Tyr-Ala-CN₂ was used to label active Cat L, Cat B, and Cat S in the endocytic pathway of HeLa-CIITA cells. Labeling of purified Cat L and Cat S use in the in vitro assay is also shown. (d) Transfected human Ii chain p41 is immunodetected in HeLa-CIITA membranes. (e) HeLa-CIITA transfected with p41 cDNA were labeled with CBZ-[¹²⁵I]Tyr-Ala-CN₂ to monitor Cat L activity.

Fig. 5.

In vitro degradation of Ii chain. (a) HeLa-CIITA membranes were incubated alone or with purified human Cat L (1–4 μM) or Cat S (0.4 μM) for 2 h at 37°C. (b) Mouse IgG (detected by immunoblot) was added as a control to demonstrate Cat L activity. In contrast to Ii chain, an IgG fragment is resistant to Cat S activity, whereas it is degraded by Cat L. (c) Human TEC cells were tested by RT-PCR for cathepsin transcription. Cat S transcripts were detected both in freshly isolated tumor cells and in a long-term established TEC cell line (P1.4D6).