Cd4 l3t4

8–10 week old C57BL/6 mice were injected i.v. with LLC exosomes (10μg, in 100μl PBS) every 3 days for 2 weeks. Mice were fasted for 12 hours prior to intraperitoneal injection with ¹⁸FDG. One hour after injection, the mice were euthanized, lungs were harvested and measured on a Biodex Atomlab^™ 500 for radioactivity. Lung tissue was enzymatically digested (collagenase (5g/L), Hyaluronidase (0.4g/L), DNAse I (0.15g/L)) for 20 minutes with rotation at 37°C. Following digestion, RBC’s were lysed using ACK. Cells were stained with primary Biotin anti-mouse CD19 (Biolegend,115504), secondary Streptavidin MicroBeads (Miltenyl Biotec,130-048-101), CD8a (Ly-2) MicroBeads (Miltenyl Biotec,130-049-401), CD4 (L3T4) (Miltenyl Biotec,130-049-201) for 15 minutes. Cells were then magnetically separated on an autoMACS Pro separator (Miltenyl Biotec) using the positive selection protocol. The negative fraction was then collected and read on a Biodex Atomlab^™ 500 for radioactivity. Cells were then resuspended in Trizol and saved in the −80°C for RT-PCR.

Flow cytometry analysis was performed as described previously(17 (link)), using a FACScan (BD Pharmingen) instrument. The antibodies used to characterize leukemic cells are indicated in supplementary Table S2. 4′,6-diamidino-2-phenylindole (DAPI) (BD Pharmingen, Cat#564907) or propidium iodide (PI) (Invitrogen, Cat# P3566) were used for viability staining.
For sorting DN cells; DN thymocytes were enriched by staining with CD4 (L3T4)(Cat# 130–117-043) and CD8a (Ly-2)(Cat#130–117-044) Microbeads (Miltenyi biotec), and depletion of the stained cells using MACS LD column (Cat# 130–042-901, Miltenyi biotec), according to the manufacturer’s recommended protocol. The depleted cells were then stained with mouse anti CD4, CD8, CD25, CD44 specific antibodies and sorted with an Aria FACS Aria II, FACS instrument.
Tissues were fixed in 10% Formalin, paraffin-embedded, and sections stained with hematoxylin and eosin, CD3 (MCA1477, AbD Serotec, Raleigh, NC, USA), B220 (553086, BD Pharmingen), or Myeloperoxidase (1:1000, DAKO-A0398). Tissue histology was analyzed as described previously(18 (link)).

Splenic CD3⁺ T cells or NK cells were isolated from naïve mice using anti-CD90.2 magnetic beads (Miltenyi Biotec) or NK cell isolation kit (Miltenyi Biotec) according to the manufacturer’s instructions. Splenic CD4⁺ or CD8⁺ T cells were isolated from naïve OT-II or OT-I mice by using CD4 (L3T4) or CD8a (Ly-2) microbeads (Miltenyi Biotec), respectively. BM-derived monocytes were isolated from naïve mice using the Monocyte Isolation Kit (Miltenyi Biotec) following the manufacturer’s protocol. Neutrophils were isolated from lung tissues of naïve or tumor-bearing mice using anti-Ly6G microbeads (Miltenyi Biotech). Viability and purity of isolated cells were assessed with flow cytometry to be above 90%.
For the primary culture of lung fibroblasts, sorted CD45⁻CD31⁻CD326⁻CD140a⁺ fibroblasts were cultured in the MesenCult^™ Basal Medium (STEMCELL Technologies). The first to third passages of the primary fibroblasts were used for the ex vivo experiments.
To prepare fibroblast-derived conditioned medium (CM), ex vivo cultured lung fibroblasts or freshly sorted lung CD140a⁺ fibroblasts were cultured in RPMI-1640 medium at a density of 5×10⁵ cells/ml for 16 hours. Then the supernatant was collected after centrifuging at 1, 000g for 20 min to remove cells and debris. The freshly collected supernatant was used as CM for further experiments.