Fig 2: Human CD8+ and γδ T cell proliferation and activation relies on three signals. Proliferation of CD8+ T cells in the presence of serial dilutions of peptide P26‐35, and (A) either CD86/4‐1BBL/HLA‐A*02‐expressing (red squares) or CD86/4‐1BBL‐expressing (blue circles) K562 cells and cytokines (IL‐7 + IL‐15 + IL‐21) (n = 5); (B) either CD86/4‐1BBL/HLA‐A*02‐expressing (red squares) or HLA‐A*02‐expressing (blue circles) K562 cells and cytokines (n = 5); and (C) CD86/4‐1BBL/HLA‐A*02‐expressing K562 cells, and the absence (open black squares) or presence of IL‐2 (red circles), IL‐7 (blue squares), IL‐15 (red triangles), IL‐21 (blue circles), or IL‐7 + IL‐15 + IL‐21 (red squares) (n = 5). Proliferation of γδ T cells in the presence of serial dilutions of an anti‐human TCR γδ antibody, and (D) either the absence (blue circles) or presence (red squares) of K562‐derived aAPC expressing CD86 and 4‐1BBL and cytokines (n = 6); (E) either parental (blue circles) or CD86/4‐1BBL‐expressing (red squares) K562 cells and cytokines (n = 6); and (F) K562‐derived aAPC expressing CD86 and 4‐1BBL, and either the absence (open black squares) or presence of IL‐2 (red circles), IL‐7 (blue squares), IL‐15 (red triangles), IL‐21 (blue circles), IL‐15 + IL‐21 (open red squares) or IL‐7 + IL‐15 + IL‐21 (solid red squares) (n = 6). Data is shown as the mean ± SEM. Normalization of the data and statistical analysis were performed as described in the Section Methods, 2.

Fig 3: CRISPR-Cas9-mediated gene editing of tonsil CD4 T cells can be used as a tool to mechanistically dissect T cell activation and CD4 T cell helper function(A) Schematic of the experimental setup to assess activation response of CD4 T cells after nucleofection of bulk or isolated CD4 T cells. Briefly, bulk cells or positively isolated CD4 T cells after bead removal were either left untreated or nucleofected with NT RNP complex prior to seeding, i.e., merging with non-CD4 T cells for the reconstituted culture. One day after seeding, cultures were stimulated with SEB or left untreated and activation response was assessed on subsequent days by flow cytometry.(B and C) Representative histograms showing CD69, CD25, CD38, and HLA-DR expression on tonsil CD4 T cells on day 7 post nucleofection of bulk cells (B) or isolated CD4 T cells (C). Superimposed histograms show expression profiles of the non-nucleofected culture (gray) and cultures nucleofected with non-targeting control (NT Ctrl) RNP (red). Dashed lines represent unstimulated conditions while solid lines represent SEB-stimulated cultures.(D and E) Quantification of (B) and (C) for day 1, day 8, and day 13 post SEB stimulation for non-nucleofected or NT conditions with and without stimulation after bulk (D) or isolated CD4 T cell (E) nucleofection. Each symbol represents one donor. Shown are means with SD. Tonsil cells were cultured in 96-well F-bottom plates.(F) Schematic of the experimental setup for assessing immunocompetency of tonsil bulk or CD4 T cells after nucleofection. Briefly, bulk cells or positive isolated CD4 T cells after bead removal were either left untreated or nucleofected with NT RNP complex prior to seeding, i.e., merging with non-CD4 T cells for the reconstituted culture. Seven days after seeding, EBV peptide pool was added together with interleukin (IL)-7. On day 7 and day 14, baseline and expanded EBV-specific CD8 T cells, respectively, were identified by production of IFNγ following stimulation with the EBV peptide pool.(G) Representative dot plots showing IFNγ+ CD8 T cells in tonsil cultures for bulk or isolated CD4 T cell nucleofection (NT Ctrl.) in comparison with untreated (no nucl.) conditions on day 7 or day 14 of culture upon restimulation with EBV PP, respectively.(H and I) Quantification of (G) for five tonsils, showing frequency of IFNγ+ CD8 T cells on day 14 in cultures were either bulk cells or isolated CD4 T cells were left untreated (no nucl) or nucleofected with NT Ctrl. Shown are means with SD. Each symbol represents one donor.(J and M) Characterization of memory subsets based on expression of CD45RO and CD62L among total CD8 (J) or CD4 (M) T cells (gray) or superimposed IFNγ+ CD8 T cells (J) (red) on day 14 following peptide stimulation of tonsil cells cultured with EBV peptide pool for 7 days (K) and (L). Quantification of (J) for total CD8 T cells in cultures where bulk cells (left) or isolated CD4 T cells (right) were nucleofected (NT Ctrl.) or left untreated (no nucl.).(N and O) Quantification of (M) as described above. TEff = T effector cells, TEM = effector memory cells, TCM = central memory cells, Tnaive = naive T cells. Shown are means with SD for five tonsils. Tonsil cells were cultured in 96-well F-bottom plates. Statistical significance was assessed by repeated-measures one-way ANOVA for data in (D) and (E), except for CD38 expression after bulk nucleofection and HLA-DR expression after CD4 nucleofection where data were not normally distributed and Friedman test was used instead. For (H) and (I), Friedman test was used for comparison of more than two groups and Wilcoxon test was used for data in (K), (L), (N), and (O) for comparison of two groups, ns, p > 0.05; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.

Fig 4: 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 5: Characterization of IL-7 cleavage by MMP-9. (A) Recombinant IL-7 produced in E. coli (left), insect cells (middle) or human HEK-293 cells (right) were incubated with different concentrations of active MMP-9 at the indicated molar ratios (MMP-9/IL-7), for a period of 2h. Fragments were separated by SDS-PAGE under reducing conditions followed by silver staining. MMP-9, intact IL-7 (IL-7i) and two cleavage products (IL-7* & IL-7**) are indicated with arrowheads, respectively, in black and green color. inh; negative control in the presence of 500 µM of the MMP inhibitor SB-3CT (inh). (B) Recombinant IL-7 produced in E. coli (top, non-glycosylated), insect cells (middle, partially glycosylated) or human HEK-293 cells (bottom, fully glycosylated) were incubated with active MMP-9 at a molar ratio of 1:100 (MMP-9/IL-7) and samples were taken at the indicated time-points. Fragments were resolved by SDS-PAGE under reducing conditions. Intact IL-7 (IL-7i) and two cleavage products (IL-7* & IL-7**) are indicated with arrowheads, respectively, in black and green color. inh; negative control in the presence of 500 µM of the MMP inhibitor SB-3CT. (C), Edman sequencing data. Identification of the neo-N-termini generated upon digestion of IL-7 with MMP-9 (1:100, MMP-9:IL-7 for 6h). Identification of the LGEAQ N-terminus from fragment IL-7**, indicates an MMP-9 cleavage site between the amino acid residues A128 and L129. Variations in N-terminal amino acids of fragments IL-7* are due to the different IL-7 expression systems ( Supplementary Table 1 ). (D) Amino acid sequence of human IL-7 with indication of glycosylation sites (blue), disulfide bridges (yellow), MMP-9 cleavage site (green triangle) and IL-7 fragments (green arrows). (E) 3D model of IL-7 [PDB structure 3DI3 (29)] with indication of the location of the MMP-9 cleavage site (green triangle), glycosylation sites (blue hexagons) and disulfide bridges (yellow).