Background By targeted DNA sequencing of 305 diagnostic follicular lymphoma (FL) biopsies, we identified somatic mutations of Cathepsin S (CTSS) in 8% of cases (24/305), mostly clustered at Y132 (19/24) converting Y to D (16/19). Another 13% of FL had CTSS amplifications (37/286), associated with higher CTSS expression (P= 0.05). CTSS is a cysteine protease that is highly expressed in endolysosomes of antigen presenting cells and malignant B-cells. CTSS is involved in proteolytical processing of antigenic peptides for presentation on MHC-II to be recognized by antigen specific CD4+ T-cells.1 CTSS is synthesized as an inactive zymogen, which is converted to its active form by autocatalytic cleavage of the autoinhibitory propeptide (pro-CTSS).
Materials and Methods We used CRISPR/Cas9 to introduce CTSS Y132D into Karpas422, a B-cell lymphoma cell line that harbors the FL hallmark translocation t(14;18). We purified pro-CTSS WT and Y132D and assayed the in vitro autocatalytic cleavage over time. We then tested the impact of CTSS on CD4+ T-cell activation in co-culture assays, in a previously described in vivo model2 which we slightly modified to reflect FL-like conditions, and in primary patient samples.
Results Single-cell derived Y132D mutant Karpas422 clones showed >3-fold higher ratios of active CTSS to pro-CTSS (N=4, P= 0.0003). Immunoprecipitated CTSS Y132D had >3-fold higher in vitro substrate cleavage activity compared to CTSS wild type (WT) (N=6, P= 0.001) which was mediated by an accelerated conversion from pro-CTSS to active CTSS (11 minutes for CTSS Y132D vs 17 minutes for CTSS WT; N=3, P=0.04). Molecular dynamics simulations showed that the Y132D mutation shortens the distances by ~2Å between the catalytic triad of active CTSS (C139, H278, N298) and a stretch of amino acids from the proform (L80, G81, D82, S94), which could facilitate intramolecular cleavage. The higher substrate cleavage activity of CTSS Y132D came along with a high capacity to stimulate antigen specific CD4+ T cell responses in vitro and in vivo. Additionally, CTSS overexpression could phenocopy this high CD4+ T cell activation. Lastly, we aimed to correlate CTSS aberrations with clinical outcome in patients who received standard immunochemotherapy (R-CHOP) for advanced FL (N=51 with available CTSS mutation and gene expression data). Compared to all other patients (N=34), patients with CTSS Y132 mutations or CTSS overexpression (N=17) had longer failure free survival (P=0.012).
Conclusions Here, we provide biochemical, structural, functional and clinical evidence that aberrant CTSS activity induces a supportive immune microenvironment in FL. We propose that aberrant CTSS activity can elicit a CD4+ T-cell driven tumor-promoting immune response, which could be amplified within the microenvironment and substantially impact the biology and clinical course of the disease. Thus, aberrant CTSS activity is a promising biomarker and therapeutic target in FL and potentially also other tumors.
Riese, R.J., et al., Essential role for cathepsin S in MHC class II-associated invariant chain processing and peptide loading. Immunity 1996; 4(4): p. 357–66.
Kim, K.J., et al., Establishment and characterization of BALB/c lymphoma lines with B cell properties. J Immunol 1979; 122(2): p. 549–54.
Disclosure Information J.A. Hildebrand: None. D. Bararia: None. S. Stolz: None. S. Häbe: None. F. Osorio-Barrios: None. M.D. Bartoschek: None. E. Gaitzsch: None. V. Jurinovic: None. K. Rautter: None. C. Ludwig: None. S. Bultmann: None. H. Leonhardt: None. S. Eustermann: None. K. Hopfner: None. W. Hiddemann: None. M. Bergwelt: None. M. Schmidt-Supprian: None. M.B. Sárosi: None. M. Rudelius: None. V. Passerini: None. J. Mautner: None. O. Weigert: None.
Statistics from Altmetric.com
If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.