Anti cd4 gk1

Spleen cells were collected from mice (n = 8 per group) euthanized at 30 days after the last vaccine dose (before infection) and at 45 days post-infection (after infection). Splenocytes (5 × 10⁶ cells/mL) were plated in 24-well plates (Nunc) in complete RPMI 1640 medium (non-stimulated, control) or stimulated with ChimeraT or L. infantum SLA (10.0 and 25.0 μg/mL, respectively) for 48 h at 37 °C in 5% (v/v) CO₂. IFN-γ, IL-4, IL-10, IL-12p70, and GM-CSF levels were quantified in spleen cell culture supernatants by capture ELISA (BD OptEIA TM set mouse kits; Pharmingen, USA), following the manufacturer’s instructions. The presence of nitrite in cell supernatants was quantified by Griess reaction [56 (link)]. In addition, the IFN-γ-producing T-cell profile in the ChimeraT/Saponin and ChimeraT/Liposome groups was evaluated by the addition of anti-CD4 (GK 1.5) or anti-CD8 (53-6.7) monoclonal antibodies into the in vitro cultures (5 μg of each antibody; Pharmingen^®, San Diego, CA, USA), for a period of 48 h at 37 °C with 5% CO₂. Anti-IL-12 monoclonal antibody (C17.8; 5 μg; Pharmingen) was included as a positive control. Appropriate isotype-matched controls (rat IgG2a (R35-95) and rat IgG2b (95-1)) were used.

To isolate CLPs, pre-NKPs, NKPs, mNK and eNK cells, Tibiae and femora were flushed with PBS and erythrocytes were lysed. We enriched for NK cells and their precursors by depletion of CD3⁺, F4/80⁺, Gr-1⁺ (anti-CD3-Bio (145-2C11, 1:100, BioLegend), anti-F4/80-Bio (CI:A3-1, 1:200, Serotec), anti-Gr-1-Bio (RB6-8C4, 1:400, BD Biosciences), Streptavidin Microbeads, Milteny Biotec.) and CD19⁺ cells (CD19 Microbeads, Milteny Biotec.) using an autoMACS pro-separator (Miltenyi Biotec). CLPs (live, CD45⁺Lin⁻CD122⁻CD127⁺CD135⁺CD244⁺), pre-NKPs (live, CD45⁺Lin⁻CD122⁻CD127⁺CD135⁻CD244⁺), NKPs (live, CD45⁺Lin⁻CD122⁺CD244⁺CD27⁺CD49b⁻NKp46⁻), mNK (live, CD45⁺Lin⁻CD122⁺CD244⁺CD49b⁺NKp46⁺) and eNK cells (live, CD45⁺Lin⁻CD122⁺CD244⁺CD49b⁺ NKp46⁻) were sorted with an AriaIII Sorter directly into RLT lysis buffer (Qiagen) or into MyeloCult M5300 media if further in vitro culturing was required. The following markers were included in the lineage staining (anti-CD3 (17A2, 1:100, BioLegend); anti-CD19 (1D3, 1:200, BD Biosciences); anti-CD14 (mC5-3, 1:400, BD Biosciences); anti-Gr-1 (RB6-8C4, 1:400, eBioscience), anti-CD8 (53-6.7, 1:400, BD Biosciences); anti-CD4 (GK1.5, 1:400, BD Biosciences). The characterization of NKPs was performed according to Fathman et al.30 (link)

LSEC were cultured overnight on gelatine-coated transwell membranes with a 5 μm pore size (Corning, Sigma-Aldrich) to form confluent cell layers [18 (link)]. LSEC were treated with the specific clathrin inhibitor CPZ (30 μM) [25 (link)], the caveolae-specific inhibitors nystatin (10 μM) or filipin (15 μM) [26 (link)] or the CXCR4 antagonist AMD3100 (10 μM) [27 (link)] added to the lower chamber of the transwell for 10 min (CPZ, nystatin, filipin) or 60 min (AMD3100). After washing, LSEC layers were pre-incubated with the chemokine (CXCL12, 50 nM; CXCL9, CXCL10, both 100 nM; all R&D Systems) applied to the lower chamber of the transwell for 120 min. After removal of the chemokine, 5x105 total CD4+ T cells were added to the upper chamber of the transwell and were allowed to transmigrate across the LSEC layer for 90 min. Transmigrated cells from the lower chamber or from the input were mixed with Fluoresbrite beads (Polysciences, Eppelheim, Germany), stained with anti-CD4 (GK1.5) and anti-CD45RB antibody (16A; both BD Biosciences) and analyzed by flow cytometry. Absolute cell numbers were determined by gating on CD4+ for total or CD45RBlow CD4+ for effector/memory CD4+ T cells in relation to defined numbers of beads.

For human tonsils, frozen sections of tonsils, 6 μm in thickness, were fixed with cold acetone and stained with anti-CD3 (UCHT1l, BD Biosciences) and anti-Tox2 (LS-C29895, LSBio) followed by Alexa Fluor 568–conjugated anti-mouse IgG1 (A-21124, Molecular Probes) and Alexa Fluor 488–conjugated anti-rabbit (A-11070, Molecular Probes). Last, sections were counterstained for 2 min with 3 μM DAPI (4′,6-diamidino-2-phenylindole). For mice spleen, frozen sections, 6 μm in thickness, were fixed with True-Nuclear buffer (BioLegend) and stained with anti-CD4 (GK 1.5) and anti-Bcl6 (K112-91, BD Biosciences) at 4°C overnight followed by 20 min at room temperature for anti-IgD (11-26c.2a, BioLegend) in Perm/Wash buffer (BioLegend). Sections were mounted with ProLong Glass Antifade Mountant with NucBlue (Invitrogen) for DAPI counterstaining and sealing. Slide images were observed under a Leica SP5 confocal microscope with a PlanApo 20× objective (numerical aperture, 0.7) and a PlanApo 40× objective (numerical aperture, 1.25).