Fig 2: Mouse IL-7 lacks the protease-susceptible loop region and is not cleaved by MMP-9. (A) Incubation of recombinant human (hu, insect cell-derived) and (mo, E. coli-derived) IL-7 with active MMP-9 and analysis of the reaction products by reducing SDS-PAGE. (B) Alignment of the amino acid sequence of human, bonobo, pig, bovine, sheep, rabbit, rat, mouse and chicken IL-7 with indication of the MMP-9 (green triangle) and serine protease (white triangle) cleavage sites in human IL-7. Multiple sequence alignment was performed with ClustalO, with relative amino acid sequence conservations indicated in various shades of blue and conserved cysteine residues in yellow. Notice the missing sequences in mouse and rat IL-7 and a different sequence in chicken IL-7 in the area which represents the protease-sensitive loop.

Fig 3: IL-7 signaling through the IL-7 receptor remains intact upon cleavage by neutrophil proteases. (A) In silico prediction of IL-7 proteolysis by neutrophil proteases (iProt-Sub tool). Separation of proteases based on their place in neutrophil granules including gelatinase (green), specific (orange) and azurophil granules (blue). (B) 3D model of IL-7 [PDB structure 3DI3 (29)] with indication of the location of the predicted cleavage sites for neutrophil proteases. (C) Digestion of IL-7 with a selection of metalloproteinases. Digestions at a molar ratio of 1/100 (MMP/IL-7) and for 4h. IL-7i, intact IL-7; fragments are indicated by asterisks (*). (D) Digestion of IL-7 with the serine proteases catG, P3 and NE. Digestions at a molar ratio of 1/100 (protease/IL-7) and for 4h. (E) Edman sequencing data. Identification of the neo-N-termini generated upon digestion of IL-7 with NE or P3 (1:100, protease:IL-7 for 4h). Identification of the ALGEAQ N-terminus from fragment IL-7**SP, indicates a shared cleavage site between the amino acid residues A127 and A128. (F) Analysis of cell-surface IL-7R, 5 minutes after stimulation of HPB-ALL cells with 50 ng/ml IL-7, cIL-7 (1/100 molar ratio MMP-9/IL-7, 4h at 37°C) or the equivalent of the indicated protease. Data represent three independent experiments, with all experimental replicates shown in the same color. Histograms represent median values and error bars represent the IQR. Data were normalized to the unstimulated condition, representing steady-state quantities of cell surface IL-7R (100% IL-7Rα). Statistical analysis done by Kruskal-Wallis test with Dunn’s correction for multiple comparisons and calculated on mean values of each experiment. (G) Analysis of pSTAT3 (Tyr705) in HPB-ALL cells, 15 minutes after stimulation with 50 ng/ml IL-7, cIL-7 (1/100 molar ratio protease/IL-7, 4h at 37°C). Each data point indicates an independent experiment. Histograms represent median values and error bars represent the IQR. Data were normalized to β-actin. (H) Growth of HBP-ALL cells, 4 days upon stimulation with 50 ng/ml IL-7, cIL-7 (1/100 molar ratio protease/IL-7, 4h at 37°C) or the equivalent of MMP-9. Data represent four independent experiments, with all experimental replicates shown in the same color. Histograms represent medians and error bars represent the IQR. Data were normalized to cells stimulated with IL-7 alone, representing 100% growth. Statistical analysis done by Kruskal-Wallis test with Dunn’s correction for multiple comparisons and calculated on mean values of each experiment. ns, not significant.

Fig 4: Functional effects of IL-7 and IL-7-receptor internalization in HPB-ALL cells and human CD8+ PBMCs remain intact upon cleavage by MMP-9. (A) Analysis of cell-surface IL-7R, 5 minutes after stimulation of HPB-ALL cells with 50 ng/ml IL-7, cIL-7 (1/100 molar ratio MMP-9/IL-7, 4h at 37°C) or the equivalent of MMP-9. Data represent four independent experiments, with experimental replicates shown in the same color. Histograms represent median values and error bars represent the IQR. Data were normalized to the unstimulated condition, representing steady-state quantities of cell surface IL-7R (100% IL-7Rα). *p < 0.05, as determined by Kruskal-Wallis test with Dunn’s correction for multiple comparisons and calculated on mean values of each experiment. (B) Analysis of pSTAT3 (Tyr705) in HPB-ALL cells, 15 minutes after stimulation with 50 ng/ml IL-7, cIL-7 (1/100 molar ratio MMP-9/IL-7, 4h at 37°C) or the equivalent of MMP-9. Each data point indicates an independent experiment. Histograms represent median values and error bars represent the IQR. Data were normalized to β-actin. (C) Growth of HBP-ALL cells, 4 days upon stimulation with 50 ng/ml IL-7, cIL-7 (1/100 molar ratio MMP-9/IL-7, 4h at 37°C) or the equivalent of MMP-9 (right panel). Data represent six independent experiments, with all experimental replicates shown in the same color. Histograms represent medians and error bars represent the IQR. Data were normalized to cells stimulated with IL-7 alone, representing 100% growth. ***p ≤ 0.001, as determined by Kruskal-Wallis test with Dunn’s correction for multiple comparisons and calculated on the mean values of each experiment. (Left panel) representative images of HPB-ALL cells with the indicated stimulations. Scale bar = 200 µm. (D) Analysis of cell-surface IL-7R, 24h after stimulation of human CD8+ PBMCs with 15 ng/ml IL-7, cIL-7 (1/100 molar ratio MMP-9/IL-7, 4h at 37°C) or the equivalent of MMP-9. Histograms represent median values and error bars represent the IQR. *p < 0.05, as determined by Kruskal-Wallis test with Dunn’s correction for multiple comparisons. ns, not significant.

Fig 5: IL-7 interaction with the IL-7 receptor remains intact upon cleavage by MMP-9. (A) His-tagged IL-7 (produced in insect cells) was subjected to histidine tag pull-down. Bead-bound proteins and non-bound proteins were resolved by reducing SDS-PAGE. (B) IL-7 in the presence (+) or absence (-) of MMP-9, subjected to non-reducing or reducing electrophoretic separation. (C) 3D model of the interaction between IL-7 and the IL-7Rα (blue) [based on PDB structure 3DI3 (29)] with indication of the IL-7 fragments generated by MMP-9 [IL-7* (orange) and IL-7** (red)]. (D) Binding of IL-7 [from E. coli (top) or mammalian cells (bottom)] and MMP-9-cleaved IL-7 (cIL-7, 1/100 molar ratio MMP-9/IL-7, 4h at 37°C) to rhIL-7Rα, as analyzed by SPR (results representative for 2 experiments, see Supplementary Figure 3 ). Colors represent different concentrations of IL-7 as indicated and black lines represent curve fits with a two-state reaction model (IL-7-IL-7Rα) according to McElroy et al. (29).