Fig. 4. Multiple mechanisms in the TME drive the conversion of cNK cells to a tumor-retained CD49a+ state.

The role of TGFβ, PGE₂ and HIF-1α were investigated in vitro and in vivo as mechanisms promoting NK cell differentiation to the tumor retained state characterized by CD49a expression and disrupted core functions. A Bar chart showing proportion of CD11b+ and CD49a+ NK cells after 48 h in culture in RPMI with IL-2/IL-15 further supplemented with TGF-β and/or DiPGE₂. B Cartoon summarizing experimental design for impeding tumor produced PGE₂. Kaede mice were grafted with either MC38 (n = 4) or MC38^{ptgs1-/- ptgs2-/-} (MC38^ptgs-/-, n = 4) cells, photoconverted on D12 and analyzed 48 h later. C Tumor weight. D Enumeration of NK cells per mg tumor. E Flow cytometry plots showing Kaede Green versus Kaede Red expression by NK cells. F Proportion of NK cells expressing Kaede Red label 48 h post photoconversion. G Proportion of NK cells in CD11b+ CD49a-, CD11b- CD49a- and CD11b- CD49a+ subsets. H Proportion of NK cells producing CCL5, IFNγ, granzyme A, granzyme C after ex vivo restimulation. I Cartoon summarizing experimental design for targeting Hif1a within NK cells, using Ncr1^cre x Hif1a^f/f x Kaede mice (NK-HIF1α KO n = 5) versus cre-negative littermates (NK-HIF1α WT n = 7) grafted with MC38 tumors, photoconverted on D12 and analyzed 48 h later. J Tumor weight. K Enumeration of NK cells per mg tumor. L Flow cytometry plots showing Kaede Green versus Kaede Red expression by NK cells. M Proportion of NK cells expressing Kaede Red label 48 h post photoconversion. N Proportion of NK cells in CD11b+ CD49a-, CD11b- CD49a- and CD11b- CD49a+ subsets. O Proportion of NK cells producing CCL5, IFNγ, granzyme A, granzyme C after ex vivo restimulation. Statistical significance was determined by unpaired t tests (C, D, F, J, K, M) or two-way ANOVA with Šidák’s multiple comparison test comparing group means (H, O). Data are presented showing all individual data points as well as the mean value +/- SD.