Figure 4: Endogenous CD8 T-cells induce LN-like vasculature in i.p. tumours.

(a) Infiltration of naive Thy1.1⁺ OT-I cells and CD44^lo naive polyclonal T cells purified from C57BL/6 and co-transferred into WT or Rag2^−/− mice with established i.p. B16-OVA tumours was determined after 18 h by flow cytometry. n=3 per genotype. Infiltration of naive OT-I cells into i.p. B16-OVA tumours in the indicated mice was determined. n=3 (b) or 5 (c,d). (e) Experimental protocol used for the repletion of Rag2^−/− mice with CD8 T cells before tumour implantation. On day 14 after tumour implantation, tumours from one cohort of mice were harvested for immunofluorescence and reverse transcriptase–PCR. A second cohort of mice received naive OT-I cells and tumours were processed for flow cytometry to assess infiltration into tumours. Representative images (f) and summary data (g, n=3–4) for PNAd expression in tumours in the indicated mice. (h) Ccl21 expression (n=2) in i.p. tumour lysates was quantified as in Fig. 3. (i) Infiltration of naive OT-I cells into i.p. B16-OVA tumours in the indicated mice was determined. n=3–5 per group. (j) The absolute number of endogenous (non-OT-I) CD8⁺ T cells that had accumulated in day 14 i.p. B16-OVA tumours in mice with the indicated background was determined by flow cytometry. Representative images (k) and summary data (l, n=2) for endogenous CD8 T-cell accumulation in day 14 B16-F1 or B16-OVA tumours growing in WT mice determined by immunofluorescence. (m) Expression of PNAd in either B16-F1 or B16-OVA tumours growing in the indicated mice. n=4 per group. Data (mean+s.e.m.) are representative of at least two independent experiments. ns: P>0.05, *P<0.05, **P<0.01, ***P<0.001 by unpaired t-test (a,c,d) or one-way analysis of variance with Tukey’s post test (b,g–m).