Automacs

For naïve CD4 T cell purification, CD4 T cells isolated from lymph nodes and/or spleen were first enriched with anti-CD4 microbeads (Miltenyi Biotec) followed by positive selection using autoMACS (Miltenyi Biotec), and then naïve CD44^lo CD62L^hi CD4 T cells were sorted by FACSAria (BD). The purity was more than 98%. T cell-depleted splenocytes were prepared by incubation with anti-Thy1.2 FITC monoclonal antibody (mAb) (BioLegend) and anti-FITC microbeads (Miltenyi Biotec), followed by negative selection using autoMACS. T cell-depleted splenocytes were irradiated at 30.67 Gy and used as antigen-presenting cells, as previously described [24 (link)].

To examine LN cells by flow cytometry, axillary, brachial, inguinal and popliteal LNs were collected. To obtain FACS-purified γδ T cells, LN and/or spleen cells were first incubated with biotin-conjugated anti-mouse γδ TCR mAb (GL3; #13-5711, eBioscience, San Diego, CA, USA) (1/100 diluted) and then with MicroBeads conjugated to anti-biotin mAb (#130-090-485, Miltenyi Biotec, Bergisch Gladbach, Germany) (1/8 diluted). Labelled cells were positively selected twice using autoMACS (Miltenyi Biotec). To purify whole-T cells and CD4⁺ T cells, LN and/or spleen cells were stained with Microbeads conjugated to anti-mouse Thy1.2 mAb (#130-049-101, Miltenyi Biotec) and CD4 mAb (#130-049-201, Miltenyi Biotec), respectively (1/8 diluted), and separated using an autoMACS. MACS-purified γδ T (γδ TCR⁺CD3ɛ⁺) cells and GFP⁺ γδ T (GFP⁺ γδ TCR⁺ CD3ɛ⁺) cells were further purified using a FACSAria (Becton Dickinson). FITC-anti-mouse γδ TCR mAb (UC7-13D5; eBioscience), PE-anti-mouse γδ TCR mAb (GL3; BioLegend, San Diego, CA, USA), and APC/Cy7-anti-mouse CD3ɛ mAb (145-2C11; BioLegend) (1/100 diluted) were used for labelling. The efficiency of these cell purifications was determined by flow cytometry.

The remaining elutriated fraction, containing the larger cells (20 ml/min), was used to isolate DC and monocytes. The large cell fraction was first stained with antibodies specific for the DC subsets, which included CD1c-APC (Miltenyi), CD141-VioBlue (Miltenyi), CD123-PE (BD BioSciences, Franklin Lakes, NJ, USA) and SLAN-FITC (Miltenyi), and labeled with anti-IgG beads (Miltenyi). DC were then isolated using an AutoMACS (Miltenyi) into positive and negative fractions. The positive fraction (DC enriched) was further sorted into four DC subsets: CD1c⁺ mDC, SLAN⁺ DC, CD141⁺ mDC and CD123⁺ pDC, using a FACSAria (BD BioSciences). The negative fraction (DC depleted/mono) was stained with anti-CD14-FITC and anti-CD16-PE (BD Biosciences) antibodies, labeled with IgG beads (Miltenyi) and a positive selection performed using an AutoMACS (Miltenyi) to obtain a bulk monocyte population. These cells were further sorted to obtain the CD14⁺CD16⁻ (CD14⁺) and CD16⁺CD14^lo (CD16⁺) monocyte subsets using a FACSAria. Cell populations with a purity ≥90 % were used, as determined by flow cytometry (LSR II or FACSAria; BD Bioscience). In the event of low yields of some APC subpopulation, the experiment was continued without that population. In these experiments the missing data was omitted from the plots and therefore not every donor has data shown for all conditions tested.

Mouse lung or spleen single-cell suspensions were prepared by mincing whole organs through a 40-μm cell strainer (BD Falcon, San Jose, CA) followed by red blood cell (RBC) lysis (ACK lysis buffer, Sigma–Aldrich) for 3 min. For isolation of lung APCs, RBC-free whole lung cells were labeled with anti-CD11c-conjugated magnetic beads (Miltenyi Biotec, San Diego, CA) and then isolated by autoMACS (Miltenyi Biotec). For isolation of spleen CD4 T cells, RBC-free whole splenocytes were labeled with anti-CD4 conjugated magnetic beads (Miltenyi Biotec) and then isolated by autoMACS. Mouse BMDCs were prepared as previously described with some modification (Lutz et al., 1999 (link)) Femurs and tibias of 4- to 8-week-old female were isolated and freed from the surrounding tissue. Intact bones were kept in 70% ethanol for 3 min followed by a PBS wash. Both ends of the bones were cut with scissors, and the marrow was flushed out with RPMI-1640 medium through a syringe with 26.5 needle. RBCs were then removed by ACK lysis buffer and cell debris or tissue clusters were filtered out. Cells from bone marrow were cultured in a 6-well plate with 20 ng/ml mouse GM-CSF and 10 ng/ml mouse IL-4 (R&D Systems, Minneapolis, MN) for 5 to 6 days.