Fresh tonsils were obtained as discarded surgical waste from de-identified immune-competent children (n = 10) undergoing tonsillectomy to address airway obstruction or a history of recurrent tonsillitis. These studies were approved by The Children’s Hospital of Philadelphia Institutional Review Board as non-human subjects research. The mean age of donors was 5.7 years (range 2–16 years) and 50% were male. Tonsillar mononuclear cells were isolated from tissues by mechanical disruption (tonsils were minced and pressed through a 70-micron cell screen) followed by Ficoll-Paque centrifugation. CD19-positive cells were removed (StemCell) and CD4+ T cells were enriched with magnetic beads (Biolegend) prior to sorting naive T cells (CD4+CD45RO-) and T follicular helper cells (CD4+CD45RO+CD25loCXCR5hiPD1hi) on a MoFlo Astrios EQ (Beckman Coulter). The gating strategy is shown in Supplementary Fig. 12 .
Magnetic beads
Manufactured by BioLegend
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Magnetic beads are spherical particles that can be easily manipulated using a magnetic field. They are made of a magnetic core and a polymer coating, which allows for the attachment of various biomolecules. Magnetic beads are a versatile tool for a wide range of applications in research and diagnostics.
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Lab products found in correlation
Cd8 t cell enrichment kit, by BioLegend (2 mentions)
Pe labeled anti ifn γ antibody, by BioLegend (2 mentions)
Moflo astrios eq, by Beckman Coulter (1 mentions)
Cytoflex lx flow cytometer, by Beckman Coulter (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Cpt tube, by BD (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Ficoll paque plus density gradient centrifugation, by GE Healthcare (1 mentions)
Live dead stain, by Thermo Fisher Scientific (1 mentions)
Facsaria, by BD (1 mentions)
Fitc labeled anti cd8, by BioLegend (1 mentions)
Analyst 1, by Thermo Fisher Scientific (1 mentions)
Acquity uplc system, by Waters Corporation (1 mentions)
Retronectin, by Takara Bio (1 mentions)
Cd3 cd28 dynabead, by Thermo Fisher Scientific (1 mentions)
Nu nu mice, by Jackson ImmunoResearch (1 mentions)
Matrigel, by BD (1 mentions)
Anti cd3, by BD (1 mentions)
Anti cd28, by BD (1 mentions)
9 protocols using magnetic beads
1
Isolation of Naive T Cells and Tfh from Tonsils
2
Assessing Immune Cell-Epithelial Interactions
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Blood was collected from healthy donors, and peripheral blood mononuclear cells (PBMC) were separated by density gradient centrifugation. Then, CD4+ T cells were isolated with magnetic beads (Biolegend, 480009). The Caco-2 cells were plated in a 6-well plate using RPMI 1640 medium (Gibco, USA) one day ahead. After removing non-adherent cells by washing and changing with fresh medium, CD4+ T cells were added. After 48h, the cell medium was collected and centrifuged to collect CD4+ T cells, while Caco-2 cells were collected after trypsin digestion. Immune cell number and apoptosis were assessed by a CytoFLEX LX flow cytometer (Beckman Coulter) according to the manufacturer’s instructions.
3
PBMC Isolation and CD4+/CD14+ Cell Enrichment
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Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density-gradient centrifugation (4 × 8 ml, CPT tube, BD, Franklin Lakes, USA). CD4+ T cells were then negatively, and CD14+ cells positively selected via magnetic beads (BioLegend, San Diego, USA, >90.96% purity on average), and cultured under stimulatory conditions as described in the online Supplementary Material and Methods
4
Isolation of Tonsillar T-cell Subsets
5
Quantification of CD8+ T cell suppression by PMN-MDSCs
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PMN-MDSCs were separated by magnetic beads (BioLegend), and CD8+ T-cells were separated by CD8+ T Cell Enrichment Kit (BioLegend). Then, PMN-MDSCs and carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE) labeled CD8+T cells were cocultured for 72 h. To count the CD8+CFSE+ and CD8+IFN-γ+ T-cell subpopulation, T-cells were first stained with FITC-labeled anti-CD8 (Biolegend), and then stained with PE-labeled anti-IFN-γ antibody (Biolegend). The CD8+CFSE+ and CD8+IFN-γ+ T-cells were detected by flow cytometry (Becton Dickinson).
6
Lipid Metabolism in PMN-MDSCs
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PMN-MDSCs were separated by magnetic beads (BioLegend). The lipid and PGE2 concentrations in the cell supernatant were detected to evaluate the lipid metabolism ability of PMN-MDSCs. The Acquity UPLC system (Waters) was used to separate extracted samples. The concentrations of Lipids and PGE2 were detected using scheduled multiple reaction monitoring (MRM). Analyst 1.6.2 software (Applied Biosystems) was used to analyzed the LC-MS data.
7
Activation and Transduction of Primary T Cells
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Primary human T cells were isolated from leukoreduction cones21 (link) from healthy donors after approval from the Institutional Review Board at Mayo Clinic. T cells were negatively selected using magnetic beads (BioLegend), stimulated with CD3/CD28 Dynabeads (Thermo Fisher) at a 3:1 (beads to cell) ratio for 5–6 days and grown in the presence of IL-7 (5 ng/ml) and IL-15 (5 ng/ml). T cells were transduced on retronectin (Takara)-coated plates with concentrated lentivirus vector on days 2 and 3. Transduction efficiency was determined on days 7–10 with a flow cytometry using a biotinylated recombinant human CD126 and mCherry reporter genes. T cells were expanded for 2 weeks before downstream experiments.
8
Mouse Tumor Xenograft Model with CD8 T-Cell Isolation
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All animal work was approved by the OHSU Institutional Animal Use and Care Committee. Animal experiments were performed in accordance with the OHSU IACUC guidelines and regulations. Immune compromised 8–10 week old female nu/nu mice purchased from Jackson Labs were injected subcutaneously with 1 million mycoplasma-negative tumor cells in Matrigel (BD) per flank. 8–10 week old female Balb/C mice were injected subcutaneously with 1X104 CT26 cells. Tumor growth was measured with calipers, with volume computed as ½ * Length * Width2. Mice were randomized into groups once the average tumor volume reached 80mm3, approximately 6 days after injection. Mouse CD8 T-cells were purified from pooled spleen and lymph node single cell suspensions of Balb/C or C57BL/6 mice (n = 6 mice) using negative selection based magnetic beads (Biolegend) per manufacturer’s recommendations.
9
Suppression of CD8+ T Cell Proliferation by PMN-MDSCs
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CD8 + T cells were isolated from the spleen of C57BL/6 mice by CD8 + T Cell Enrichment Kit (BioLegend). PMN-MDSCs were isolated from the tumor or spleen of C57BL/6 mice by magnetic beads (BioLegend). PMN-MDSCs were then cocultured with CFDA-SE (Carboxy uorescein diacetate, succinimidyl ester) labeled CD8 + T cells in the medium containing anti-CD3 (1 µg/mL) and anti-CD28 (1 µg/mL) antibodies (BD Biosciences) for 72 hours. To enumerate the CFSE + CD8 + and IFN-γ + CD8 + T-cell subset, cells were rst stained with FITC-labeled anti-mCD8 (Biolegend), then xed or permeabilized before they were stained with PE-labeled anti-IFN-γ antibody (Biolegend). The CFSE + CD8 + and IFN-γ + CD8 + T cells were detected by ow cytometry (Becton Dickinson, San Jose, CA, USA).
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