Jes6 1a12

IL-2/α-IL-2 complexes were previously described to induce proliferation of T cells and NK cells in vivo in mice after intraperitoneally (i.p.) injection^{30 (link),31 (link)}. Complexes were prepared by mixing 2.5 μg recombinant murine IL-2 (0.67 mg/ml) and 7.5 μg anti-IL-2 mAb JES6-1A12 (3 mg ml, BioXCell) per mouse. The IL-2/α-IL-2 mixture was incubated at 37 °C for 30 min, adjusted to 200 μl/mouse with sterile PBS and injected into mice. Mice were sacrificed 1 day post injection.

For a single injection, 2.5 µg rIL-2 and 7.5 µg anti-IL-2 mAb JES6.1A12 (BioXCell, USA) or S4B6 (purified from hybridoma culture supernatant by standard procedure) were mixed to prepare the IL-2 complexes. After a 30 min incubation at 37°C, the volume was adjusted to 200 µl with sterile PBS and injected i.p. into mice. Control mice were left untreated.

CD4⁺ T cells were purified from spleens of uninfected BALB/c CD45.1⁺ (CByJ.SJL(B6)-Ptprc^a/J), BALB/c and Chi3l1^-/- mice using CD4 MACS L3T4 beads (Miltenyi Biotech) and stimulated for 48 hours as previously described (41 (link)) with 2.5 μg/mL plate-bound anti-CD3 (145-2C11, BioXcell) and 2.5μg/ml plate-bound anti-CD28 (37.51, eBioscience). To mimic early T_FH development cells were cultured in the presence of 10 μg/ml rIL-6 (R&D) and neutralizing antibodies to IL-2 (JES6-1A12, BioXcell, 5 μg/ml and S4B6-1 BioXcell, 5 μg/mL). For CD4 T cell co-cultures, BALB/c or Chi3l^-/- CD45.2⁺ T cells were mixed at a 1:1 ratio with CD45.1⁺ T cells and then stimulated for 48 hours with CD3 and CD28 antibodies.

Mice were treated for the indicated days with injections of cytokine or cytokine: anti-cytokine monoclonal antibody complexes. For IL-2 complexes (IL-2C), mice received 2 μg per day of recombinant IL-2 (eBioscience) premixed with 20 μg of anti-mouse IL-2 monoclonal antibody clone S4B6-1 (S4B6) (BD Pharmingen). In certain experiments, the amount of IL-2 in the complexes was varied, as indicated. Complexes were incubated at room temperature for 20 minutes (min.) before intraperitoneal (i.p.) injection in 200 μL of PBS. IL-2C in 50 μL of PBS were also administered intranasally (i.n.). When IL-2 was administered as free cytokine, animals were treated with 20 μg per day in 200 μL of PBS injected i.p.
For some experiments, mice were treated as indicated with 0.25 mg per day of anti-CD122 (IL-2 Rβ) antibody (5H4) to block IL-2 signaling, 0.25 mg per day anti-IL-2 antibodies (S4B6 and JES6-1A12) to neutralize IL-2, 0.5 mg per day of anti-CD70 antibody (FR-70) to block CD70 signaling, 0.25 mg per day of anti-NK1.1 (PK136) to deplete NK cells, 0.5 mg of anti Ly-6G Ab to deplete neutrophils (1A8), or with appropriate isotype control antibody (all Bioxcell). Antibody was delivered by i.p. injection in 200 μL of PBS.

The following anti-mouse mAbs were used for surface staining during flow cytometry: CD3-eFluor 450, CD8-PerCP-Cy5.5, CD11b-AlexaFluor 700, CD11b-eFluor 450, CD14-FITC, CD25-APC, CD25-eFluor 450, CD45.1-APC, Ly6C-APC-eFluor 780, I-A/E-FITC (eBioscience, San Diego, CA, USA), CD45.1-eFluor 450, CD45R-Horizont V500, CD3-Horizont V500, CD4-Horizont V500, CD4-PerCP, CD8-Horizont V500, Ly6G-AlexaFluor 700, Siglec F-PE (BD Biosciences, San Jose, California, USA) and TLR4-APC (BioLegend, San Diego, Ca, USA). For intracellular staining, the following anti-mouse mAbs were used: TNFα-PE, Foxp3- and IFNγ-PE (eBioscience; San Diego, California, USA). Fc-block (anti-CD16/CD32 mAbs; eBioscience, San Diego, California, USA) was used both in surface and intracellular staining. For ELISA, matched anti-mouse capture mAb and biotinylated detection mAb against TNF-α, IFN-γ, IL-1β, IL-12 and IL-6 were used (R&D System, Minneapolis, Minnesota, USA). Blocking anti-mouse CD25 (PC61.5), CD4 (GK1.5) and anti-IFN-γ (XMG1.2) mAbs, as well as anti-mIL-2 mAbs for preparing IL-2 complexes (S4B6, JES6-1A12), were obtained from BioXcell (Lebanon, New Hampshire, USA).