Isolation of Splenic Stromal Cells

Splenic single-cell suspensions were prepared by mechanical disruption. The isolation of stromal cells from spleen has been described68 (link). Briefly, spleens were flushed with RPMI containing 3 mg ml⁻¹ Collagenase IV (Worthington), 40 μg ml⁻¹ DNAseI (Roche) and 2% (vol/vol) FCS), were cut into pieces and digested. Erythrocytes were lysed in 0.15 M NH₄Cl/10 mM KHCO₃/0.1 mM EDTA and haematopoietic CD45⁺ cells were depleted using anti-CD45 beads (Miltenyi Biotec). For TIL analysis, tumours were cut and digested with accutase (PAA), Collagenase IV (Worthington), Hyaluronidase (Sigma) and DNAseI (Roche) and red blood cells were lysed. Mononuclear cells were isolated using a Histopaque-1119 gradient.