Role of ADAM10 and ADAM17 in Regulating CD137 Function

Abstract

Human CD137 (4-1BB), a member of the TNF receptor family, and its ligand CD137L (4-1BBL), are expressed on immune cells and tumor cells. CD137/CD137L interaction mediates bidirectional cellular responses of potential relevance in inflammatory diseases, autoimmunity and oncology. A soluble form of CD137 exists, elevated levels of which have been reported in patients with rheumatoid arthritis and various malignancies. Soluble CD137 (sCD137) is considered to represent a splice variant of CD137. In this report, however, evidence is presented that A Disintegrin and Metalloproteinase (ADAM)10 and potentially also ADAM17 are centrally involved in its generation. Release of sCD137 by transfected cell lines and primary T cells was uniformly inhibitable by ADAM10 inhibition. The shedding function of ADAM10 can be blocked through inhibition of its interaction with surface exposed phosphatidylserine (PS), and this effectively inhibited sCD137 generation. The phospholipid scramblase Anoctamin-6 (ANO6) traffics PS to the outer membrane and thus modifies ADAM10 function. Overexpression of ANO6 increased stimulated shedding, and hyperactive ANO6 led to maximal constitutive shedding of CD137. sCD137 was functionally active and augmented T cell proliferation. Our findings shed new light on the regulation of CD137/CD137L immune responses with potential impact on immunotherapeutic approaches targeting CD137.

Keywords: ADAM10; ADAM17; Anoctamin-6; CD137; T cell proliferation; cancer.

Figure 1

Release of sCD137 is mediated by ADAM10 in HT29 cells. (A,B) Cells were analyzed for the relative amount of shedding products in the supernatant in relation to total CD137 by ELISA shown as fold change compared to mock-treated cells. (A) Incubation with ADAM10 inhibitor GI (3 µM), ADAM10/17 inhibitor GW (3 µM) or broad-spectrum metalloprotease inhibitor marimastat (MM, 10 µM) for 24 h resulted in significantly reduced amounts of sCD137. (B) Cells were stimulated with ionomycin (IO; 1 µM), melittin (Mel; 1 µM) for 30 min or PMA (200 ng/mL) for 2 h. Stimulation with IO and Mel resulted in significantly increased CD137 shedding in HT29 cells. * indicates significant increase, # indicates significant decrease (*/# p < 0.05 (A) n = 4; (B) n = 3; ± s.e.m.) and ns = no significant difference compared to mock-treated cells (Ø). Data were analyzed by one-way analysis of variance and Bonferroni multiple comparison post hoc test.

Figure 2

ADAM10 and ADAM17 mediate CD137 release in HEK cells. (A,B) Cells were analyzed for the relative amount of shedding products in the supernatant in relation to total CD137 by ELISA shown as fold change compared to mock-treated cells. (A) Release of CD137 was significantly decreased in mock-treated HEK293T dKO A10/17 cells compared to WT cells. Incubation with metalloprotease inhibitors GI (3 µM), GW (3 µM) and MM (10 µM) for 24 h resulted in a significantly reduced amount of sCD137 in WT cells but not in HEK293T A10/A17 dKO cells. (B) Cells were stimulated with ionomycin (IO, 1 µM), melittin (Mel; 1 µM) for 30 min or PMA (200 ng/mL) for 2 h. Stimulation with ionomycin resulted in significantly increased shedding of CD137 in HEK293T WT cells. No significant difference was observed in HEK293T dKO A10/A17 cells. (C) HEK293T dKO A10/A17 cells were co-transfected with CD137 and ADAM10, ADAM17 or mock vector and analyzed by CD137 ELISA. Re-Transfection of ADAM10 and ADAM17 resulted in a significant increase in CD137 shedding in dKO A10/A17 cells. * indicates significant increase, # indicates significant decrease compared to mock-transfected cells (Ø) (*/# p < 0.05 (A–C) n = 3; ± s.e.m.). ns = no significant difference. Data were analyzed by one-way (C) or two-way (A,B) analysis of variance and Bonferroni multiple comparison post hoc test.

Figure 3

Ionomycin-induced shedding of CD137 depends on ADAM-phosphatidylserine (PS) interaction. CD137-transfected HT29 cells (A) or HEK293T WT cells (B) were stimulated with ionomycin (IO; 1 µM) for 30 min in the presence of OPS or lactadherin (LA, 2 µM) and analyzed for the relative amount of shedding products in the supernatant by ELISA. OPS and lactadherin significantly reduced CD137-shedding. * indicates significant increase compared to mock-treated cells, # indicates significant decrease (*/# p < 0.05 (A) n = 4; (B) n = 5; ± s.e.m.) or ns = no significant difference compared to IO-stimulated cells. Data were analyzed by one-way analysis of variance and Holm-Sidak multiple comparison post hoc test.

Figure 4

Anoctamin-6 (ANO6) modulates CD137 shedding. ANO6 and mock-transfected (A) HT29 cells or (B) HEK293T WT cells were stimulated with ionomycin (IO; 1 µM) for 30 min. In addition, cells co-transfected with CD137 and hyperactive ANO6 were analyzed in the absence of any stimulus for 30 min. The relative amount of shedding products in the supernatant was determined in relation to total CD137 by ELISA. IO-induced shedding was significantly increased upon overexpression of ANO6. Shedding of CD137 was significantly increased upon overexpression of hyperactive ANO6 compared to mock-transfected non-stimulated cells. * indicates significant increase (* p < 0.05 (A) n = 5; (B) n = 3; ± s.e.m.). Data were analyzed by one-way analysis of variance and Holm–Sidak multiple comparison post hoc test.

Figure 5

ADAM10 is the major sheddase of endogenously expressed CD137 in T cells. (A/C) Primary CD4⁺ T cells or (B/D) CD8⁺ T cells were treated with ADAM10 inhibitor GI (3 µM), mixed ADAM10/ADAM17 inhibitor GW (3 µM) or broad-spectrum metalloprotease inhibitor marimastat (MM, 10 µM) for 6, 24, 72 or 96 h. (A/B) Supernatants were analyzed for soluble shedding products of CD137 with ELISA. Treatment with GI, GW and MM resulted in a strikingly reduced amount of sCD137. One representative experiment out of five experiments is shown with mean ± SD. (C/D) Expression of cell surface CD137 on CD4⁺ and CD8⁺ T cells was analyzed using FITC labeled anti-CD137 mAb 72 h after activation. Incubation with MM (blue), GW (orange) and GI (green) increased the amount of cell bound CD137 compared to untreated cells (red). Black line: mock-stained control. One representative out of three experiments is shown.

Figure 6

Shed sCD137 augments proliferation of CD4+ and CD8+ T cells. Proliferation assay of primary (A) CD4⁺ T cells or (B) CD8⁺ T cells. WT HEK293T cells were either co-transfected with CD137 and mock vector, CD137 and hyperactive ANO6 (ANO6H), or ANO6H and mock vector. Then, 24 h after transfection, medium was changed and supernatants were harvested after further 24 h. Pre-activated T cells were cultured in the presence of the precleared conditioned HEK cell medium for 72 h and proliferation was analyzed. Untreated control cells were standardized to baseline. * indicates significant increase compared to mock-treated cells, # indicates significant decrease (*/# p < 0.05 (A,B) n = 4; ± s.e.m.). Data were analyzed by one-way analysis of variance and Sidak multiple comparison post hoc test.