Fig. 5: Metastatic lungs preferentially attract less differentiated NK cells while impairing the migration and cytotoxicity of the differentiated subset.

A–H PyMT cells (5 × 10⁴) were injected i.v. into Rag2^-/-Il2rg^-/- mice. 7 days post-inoculation, FACS-sorted CD45.1⁺CD27^high (gray) and CD45.2⁺CD11b^high (blue) NK cells were adoptively transferred at a 1 to 1 ratio and NK cells were analyzed 1 day and 6 days post-transfer. A Schematic illustration of experimental approach. B Bar charts depicting the frequency of CD45.1⁺CD27^high and CD45.2⁺CD11b^high NK cells in the lung comparing intravascular (iv) and extravascular (ev) NK cells at day 1 post-transfer. D Representative contour plots and (D) bar charts depicting differentiation stages of CD45.1⁺CD27^high-derived NK cells in naïve intravascular, metastatic tumor intravascular and tumor extravascular lung tissue at day 6 post transfer. E Representative histograms and bar charts depicting CD49a expression intravascular and metastatic tumor extravascular from CD27^high-derived NK cells at day 6 post transfer. F Representative histograms and (G) bar charts showing CX3CR1, KLRG1 and Gzmb expression in naïve intravascular, metastatic tumor intravascular and tumor extravascular CD11b^high NK cells at day 1 post-transfer. H–O CD45.2 CD27^high or CD11b^high NK cells from Ncr1^CreTgfr2^fl (TR2 cKO) mice or Ncr1^wtTgfbr2^fl (CTRL) littermate controls, together with the WT CD45.1 counterparts were adoptively transferred at a 1 to 1 ratio into metastatic-bearing Rag2^-/-l2rg^-/- mice and NK cells were analyzed 1 day and 5 days post-transfer. H Schematic illustration of experimental approach. I Representative histogram and (J) bar charts showing the expression of CD49a on intravascular and extravascular CD45.2⁺CD27^high NK cells from Ncr1^CreTgfr2^fl (TR2 cKO) or Ncr1^wtTgfbr2^fl (CTRL) mice day 5 post-transfer. K Representative histograms and bar charts depicting (L) CX3CR1, KLRG1 and (M) Gzmb expression in intravascular and extravascular CD45.2⁺CD11b^high NK cells from Ncr1^CreTgfr2^fl (TR2 cKO) or Ncr1^wtTgfbr2^fl (CTRL) 1 day post transfer. N Bar charts depicting number (#) of intravascular and extravascular CD45.2⁺CD11b^high NK cells per lung (TR2 cKO or WT) 1 day post transfer. O Bar charts depicting number (#) of intravascular and extravascular CD45.2⁺CD27^high NK cells per lung (TR2 cKO or WT) 5 days post transfer. Data is representative of 3 (B–G) or 2 (I–O) different experiments with n = 3–5. Error bars display means ± SD. Statistical significance was determined by two-way ANOVA with a Sidak’s post hoc test (F) or One-way ANOVA with Tukey’s multiple comparisons test (B, D, H, K, M); *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. ns, not significant.