Cd31 microbead

We isolated CD31+ ECs from mouse lung by magnetic sorting using CD31 MicroBeads (1:10, 130-097-418; Miltenyi Biotec GmbH, Bergisch-Gladbach, Germany). Briefly, we anesthetized the mice (6–10 weeks old) and harvested lung tissue samples. We incubated lung tissues minced in PBS in collagenase A (10103586001; Roche Diagnostics GmbH, Mannheim, Germany) and deoxyribonuclease I (LS002138; Worthington Biochemical, Lakewood, NJ) at final concentrations of 0.5% and 20 unit/ml. We incubated the mixture for 1 h at 37 °C under gentle agitation. We stopped digestion using FBS, after which we washed the cell suspension and then passed it through a 100-μm mesh nylon screen. We incubated the cells with CD31 MicroBeads for 15 min at 4 °C and loaded them onto a MidiMACS kit (LS) (130-042-301; Miltenyi Biotec GmbH) according to the manufacturer’s instructions.

Endothelial cells were isolated from the mouse tumors following the CD31 MicroBead protocol (Miltenyi Biotec, ref. 130-097-418).
CD4 and CD8 T cells were magnetically isolated from the spleen using EasySep mouse isolation kits (StemCell Technologies, ref. 19852 and 19853, respectively) according to the manufacturer’s protocol. T lymphocytes were labeled with Carboxyfluorescein Succinimidyl Ester (CFSE; Biolegend, 422701) at 1μM for 6 minutes, resuspended in the RPMI media containing 10%FBS, 1%PenStrep, NEAA and β-mercaptoethanol, and activated with the CD3/CD28 Dynabeads (ThermoFisher, 11456D). T cells were then plated at 200,000 cells/well in a 96-well plate.
CD11b cells were isolated from tumors by following the tissue harvesting and digestion protocol, as described in the sections on harvesting of mouse tissues and flow cytometry. Myeloid cells were then isolated following the protocol for the EasySep Mouse CD11b Positive Selection Kit II (StemCell Technologies, ref. 18970). After isolation, cells were plated with CD4 or CD8 T cells at a 1:1 ratio. After 72 hours of coculture, the CFSE-low T lymphocytes were stained with the live/dead cell viability reagent (LIVE/DEAD Fixable Violet Dead Cell Stain Kit, Invitrogen) for 10 minutes on ice and counted using flow cytometry (Gallios, Beckman Coulter).

To isolated TECs, testes from 3–4-week-old mice were harvested and minced as fine as possible. Testes tissues were digested in Hanks' balanced salt solution (HBSS) supplemented with 10 mg ml⁻¹ of type collagenase (Worthington) and 20 µg ml⁻¹ of DNase I (Sigma) for 35 min at 37 ^oC with shaking over 250 rpm. Digested tissues were collected by spinning down 1500 rpm for 5 min at 4 ^oC and tissue pellets were resuspended in 0.25% tyrpsin added with 7 mg ml⁻¹ of DNase I (Worthington), then incubated at 37 ^oC for 5 min. Separated cells were then strained sequentially through a 100 µm and a 40 µm strainer and trypsin activity was quenched with equal volume of fetal bovine serum (FBS). Isolated cells were washed once with 10 ml of HBSS and spindown at 1000 rpm for 10 min at 4 ^oC. The cells were resuspended in 100–200 µl of MACS buffer and mixed with 10–20 µl of CD31 microbead (Miltenyi Biotec: Cat: 130-097-418) and 10–20 µl of mouse Fc receptor blocker and then incubation for 15 min at 4 ^oC. Antibody binding cells were isolated by MACS cell separation following the manufacturer’s instruction. Purified cells were confirmed by immunostaining with CD31 (1:50, BD; Cat: 553370), VEGFR2 (1:100; Cell signaling; Cat: 55B11) and VE-cadherin (1:100, Santa Cruz; Cat: SC-9989), which were specifically expressed on ECs and Dil-Ac-LDL uptake assay (Alfa Aescar).