Recombinant human il 7

Sepsis induction in mice was performed as previously described [21 (link)]. Briefly, human stool samples were collected and stored at -80°C. Animals were randomly allocated to the sepsis or sham group. Sepsis was induced by intraperitoneal (i.p.) injection of 1.75 ml/kg body weight stool suspension, diluted (1:4) in saline. Sham mice received the equivalent volume of saline (i.p.). The septic mice received antibiotic treatment (meropenem 12 mg/kg, administered subcutaneously). The first antibiotic injection was performed 7 h post sepsis induction, after which it was given every 12 h for the next 3 days. Mice were monitored for symptoms including conjunctivitis, diarrhea, weakness and lack of movement. On average 50% of the mice died during the acute phase of sepsis (days 1–5). Surviving mice were used for the analysis of long-term sequelae following sepsis. The experimental scheme is depicted in S1A Fig.
From day 5–9 septic mice were either subcutaneously injected with PBS or recombinant human IL-7 (R&D Systems, 2.5 μg/mouse/day). Human IL-7 can bind and signal via the murine IL-7 receptor [26 (link)]. In order to stabilize the cytokine, IL-7 was mixed with a ten-fold higher concentration of an anti-human IL-7 antibody (clone M25; BioXCell) [27 (link),28 (link)].

Purified CD8⁺ T cells were stimulated with recombinant human IL-7 (10 ng/mL; R&D Systems, Minneapolis, Minnesota, USA) [22 (link)] for 48 h in the presence of anti-CD3/CD28, along with either JAK inhibitor (10 µmol/L; Sigma-Aldrich, Temecula, California, USA), STAT5 inhibitor (250 µmol/L; Merck Millipore), or PI3K inhibitor (LY294002) (25 µmol/L; Sigma-Aldrich) as previously described [20 (link)]. Control cells were only stimulated with anti-CD3/CD28 for maintenance of CD8⁺ T cell survival. In certain experiments, stimulated CD8⁺ T cells were washed twice. 10⁴ of CD8⁺ T cells were co-cultured with 10⁵ of melanoma cell line SK-MEL-5 cells for 48 h.

Peptide epitope mapping was done either by ELISpot, using an additional blood sample if the volunteer was available, or by expanding short term T cell lines (TCL) using left over PBMC. For cross-reactivity assays, short-term T cell lines (TCL) were always used for reasons of consistency. PBMC (2 x 10⁶) were cultured with 5 μg/ml JEV peptide pools, or 10 μg/ml individual peptide, to which responses had been detected in ELISpot assays, in 1 ml sRPMI supplemented with 10% human serum, 10% Natural T cell growth factor/IL2 (“T-stim,” Helvetica Healthcare) and 20 ng/ml recombinant human IL7 (R&D systems). TCLs were expanded for 7–10 days in culture, rested overnight in R10 without peptide, then stimulated with peptides for six hours in the presence of 10 μg/ml brefeldin A (Sigma).

CM, TM, and EM CD4 T-cell subsets were sorted from thawed PBMCs as explained above. Sorted cells were washed and resuspended (1 × 10⁶ cells per ml) in R10 with 30 ng/ml recombinant human IL-15 (R&D systems), and 30 ng/ml recombinant human IL-7 (R&D systems). Before and after five days of stimulation, cells were washed and stained with the following combination of monoclonal antibodies: anti-CCR7-BB700 (clone 3D12), anti-Ki67-AL700 (B56), anti-CD3-BUV395 (clone SP34-2), anti-CD45RA-BUV737 (clone HI100), anti-CD27-BV605 (clone L128), anti-CD8-BUV496 (clone RPA-T8), anti-granzyme-B-TRPE (clone GB11) all from BD Biosciences; anti-CD4-BV650 (clone OKT4), anti-HLA-DR-BV750 (clone L243), anti-PD-1-BV785 (clone EH12.2H7) all from Biolegend; anti-CD38-APC (clone OKT10) from Ray Biotech; anti-CD127-PE-Cy5 (clone EBIORDR5) from Scientific Thermofisher; anti-Perforin-FITC (clone Pf344) from MAbtech; LIVE/ DEAD Fixable Near-IR Dead Cell Stain from Thermo Fisher Scientific. Samples were collected on a FACSymphony system (BD Biosciences) and analyzed in FlowJo (version 9.9.6, TreeStar).

Human TLR1-9 Agonist Kit, TLR1/TLR2 agonist Pam3CSK4, TLR5 agonist flagellin (FLA), and TLR2/TLR6 agonist FSL-1 were purchased from InvivoGen. Recombinant human IL-7 and recombinant human IL-15 were products of R&D Systems.

PBMCs were cultured in 10 ng/ml recombinant human IL-7 (R&D Systems), IL-12 (10 ng/ml), IL-23 (5 ng/ml), IL-18(10 ng/ml), or a combination of all cytokines with LPS as positive control (100 ng/ml) for 5 days as indicated or left untreated at 37°C and 5% CO₂ in RPMI medium supplemented with 10% fetal calf serum and 50 μg/ml gentamicin (Gibco) (RF10 medium). Following 6 days of culture, appropriate surface (CD3, CD8, CD4, TCR7.2, and CD161) staining and intracellular staining with Ki-67 and Granzyme-B were performed as described earlier (61 (link)).

GPC3-CAR T cells for both the protocols were generated using peripheral blood mononuclear cells (PBMCs) from patients first stimulated with CD3 and CD28 (Miltenyi Biotec) mAbs in the presence of recombinant human IL-7 (10 ng/ml) and IL-15 (5 ng/mL, both from R&D Systems) on day 1 and transduced with retroviral particles encoding the GPC3-CAR construct in 24-well, RetroNectin-coated plates (Takara Bio) on day 3. Next, T cells were washed and replated on day 5, expanded and tested followed by cryopreservation on Day 8. 15.GPC3-CAR T cells were also generated using PBMCs stimulated with CD3 and CD28 mAbs in the presence of recombinant human IL-7 and IL-15 on day 1. They were transduced with retroviral particles encoding the iC9.NGFR.IL15 construct in RetroNectin-coated plates on day 3, resuspended and transduced with retroviral particles encoding the GPC3-CAR construct on day 4.