Fig 2: Circulating cNK cells enter the skin grafts and give rise to trNK cells.(A) Schematic of parabiosis between WT and Ncr1iCre,ROSAmT-mG B6 mice followed by m157tg,B2m-/- skin transplantation onto the WT parabiont. Representative dot plots of the spleen and donor skin from the WT parabiont gating on CD3-CD4-CD8- cells and showing CD45 intravenously (I.V.) and NKp46-GFP at day 20 after skin transplant. (B) Representative IF images of m157tg,B2m-/- skin graft from the WT parabiont showing the presence of NKp46-GFP+ (green, white arrows), CD4+ (purple), and CD8+ (red) cells. Dotted lines indicate the epidermal basement membrane. n = 5 parabiosis pairs. (C) Sorted splenic cNK cells from Ncr1iCre,ROSAmT-mG mice were adoptively transferred into WT mice 1 day after skin transplantation with m157tg,B2m-/- skin grafts. Representative dot plots of transferred NK cells (defined as CD3-CD4-CD8-NKp46-GFP+) in liver and skin grafts of mice that did not (left) and did (right) receive NKp46-GFP+ NK cells. n = 2 mice per +NK group from two independent experiments. (D) Representative images of m157tg,B2m-/- skin grafts transplanted onto WT, Ncr1iCre,Tgfbr2fl/fl, and Hobit-/- recipient mice at day 20 after transplant. n = 4 to 7 mice per group. (E) Frequencies of cNK and trNK cells in the recipient liver (top) and donor skin (bottom) at day 20 after transplant. Ncr1iCre,ROSAmT-mG mice transplanted with B6 albino WT, m157tg, B2m-/-, and m157tg,B2m-/- donor skin from Fig. 1 (n = 9 to 10 mice per group) and Ncr1iCre,Tgfbr2fl/fl and Hobit-/- recipients transplanted with m157tg,B2m-/- donor skin at day 20 after transplant (n = 4 to 7 mice per group). (E) Graphs are shown as means ± SD. Mann-Whitney U test; *P < 0.05 and **P < 0.01. (B) Scale bar, 100 µm. (D) Scale bar, 1 cm. Photo Credit: Mark D. Bunting, Massachusetts General Hospital.

Fig 3: aCXCR2-Mediated TAMs Reprogramming Induces Tumor Regression and Modulates T Cell Response and Vessel Size.Mice were treated with aCXCR2 (AZD5069 100 mg/kg) or vehicle, starting at the age of tumor formation (8 weeks), for 3 weeks.(A) Representative images of H&E (right panel) and Ki-67 IHC staining (right panel). Original magnification, 20×; scale bar, 50 µm.(B) pHp1? IF staining. Original magnification, 20×; scale bar, 25 µm.(C) Volume of anterior prostate lobes (see STAR Methods); n = 7.(D) and E) Ki-67 (D) and pHp1? quantification (E) in Ptenpc-/-; p53pc-/- prostatic tumors upon aCXCR2 treatment (n = 7 and n = 3, respectively).(F and G) Representative immunophenotyping plot (F) and quantification (G) of CD11c+CD206- pro-inflammatory and CD11c+/-CD206+ tumor-promoting macrophages (Mazzieri et al., 2011) infiltrating the tumor upon administration of aCXCR2. Events are gated on CD45+CD11b+F4/80+ cells (n = 5 mice per group).(H) RT-qPCR gene expression analysis on CD45+/F4/80+ macrophages sorted from tumors (n = 4).(I) Unbiased systematic analyses of coordinated alterations following gene expression profiling in TAMs isolated from the prostate of untreated and aCXCR2-treated mice (n = 3 per group). Graph shows significantly altered clusters of immunity-associated effector processes. Bars represent the average log2FDRq values of the gene sets included in each cluster.(J) Representative images of CD31 IHC staining on anterior lobe prostate of Ptenpc-/-; Trp53pc-/-mice upon aCXCR2 treatment. Original magnification 20×.(K and L) Quantification of area (K) and perimeter (L) of vessels in the tumor area (n = 3).(M and N) Quantification of the frequency of infiltrating CD4+CD25- (M) and CD8+ T cells (N) in prostatic tumors before and after aCXCR2 treatment. Events are gated on CD45+ cells (n = 5 mice per group).(O) Volume of anterior prostate lobes (see STAR Methods) at the end of the treatments.(P) Quantification of CD45+CD11b+F4/80+CD206+ anti-inflammatory macrophages infiltrating the tumor upon treatments. Ptenpc-/-; Trp53pc-/-mice were treated with aCXCR2 (AZD5069 100 mg/kg), 1A8 (aLy6G antibody), or aCXCR2/1A8 for 3 weeks, starting at the age of tumor formation (8 weeks). Events are gated on CD45+CD11b+ cells (n = 4 mice per group).Error bars are mean ± SEM. *p < 0.05, *p < 0.01, and ***p < 0.001.

Fig 4: CXCR2-Depleted Monocytes Infusion Drives TAM Re-education and Senescence Induction in Ptenpc-/-; p53pc-/- Tumors(A) Schedule of treatment used in the pre-clinical trial with CXCR2 WT or CXCR2-KO infused monocytes (n = 6 mice per group).(B and C) Representative FACS plots of immunophenotyping (B) and quantification (C) showing macrophages ratio of CD11c+CD206- pro-inflammatory and CD11c+/- CD206+ anti-inflammatory macrophages infiltrating the tumor upon infusion of CXCR2 WT or CXCR2-KO monocytes. Events are gated on CD45+CD11b+F4/80+ cells (n = 4 mice per group).(D and E) Representative confocal immunofluorescence image (D) and quantification (E) of F4/80-positive (red) and Inos-positive (green) tumor-infiltrating macrophages. Cells were counterstained with the nuclear marker DAPI (blue). Scale bar, 15 µM.(F) RT-qPCR gene expression analysis on CD45+F4/80+ macrophages sorted from tumors.(G–J) H&E (G), Ki-67 IHC staining (original magnification, 20×) (H), relative anterior prostate lobe volume compared with untreated Ptenpc-/-; p53pc-/- mice (I), and Ki-67 quantification in Ptenpc-/-; p53pc-/- prostatic tumors upon infusion of CXCR2 WT or CXCR2-KO monocytes (J).(K) Immunofluorescence staining of e-Cadherin-positive (red) and pHP1?-positive (green) epithelial cells in Ptenpc-/-; p53pc-/- prostatic tumors upon infusion of CXCR2 WT or CXCR2-KO monocytes. Cells were counterstained with the nuclear marker DAPI (blue). Scale bar, 20 µm.(L) Representative images of SA-ßgalactosidase IHC staining on Ptenpc-/-; p53pc-/- prostatic tumors. Original magnification, 40×. Scale bar, 100 µm.Error bars are mean ± SEM. **p < 0.01 and ***p < 0.001.

Fig 5: NK Cells Contribute to Both Innate and Adaptive Immune Control of Ptgs-/- Tumors(A) Growth profile of Ptgs+/+ and Ptgs-/- melanoma cells implanted into Rag1-/- or wild-type mice untreated or depleted of NK, CD4+, and/or CD8+ cells.(B and C) Frequency of CD8+ T cells (B); representative plots and frequency of CD8+ CD44+, CD8+ IFN?+, and CD4+ IFN?+ T cells (C) gated on live, CD45+, CD3e+ cells in Ptgs+/+ and Ptgs-/- untreated or NK cell-depleted wild-type mice analyzed 7 days post-implantation.(D) Individual growth profiles of Ptgs-/- melanoma cells in wild-type mice depleted of NK cells from the day before or a week after cancer cell implantation.(E) Growth profile of Ptgs+/+ and Ptgs-/- cells in wild-type mice or Ptgs+/+ cells in GPP mice.(F) Tumor weight, NK cell frequency, and number per g of tumor analyzed 4 days after implantation of Ptgs+/+ and Ptgs-/- cells in wild-type or Ptgs+/+ cells in GPP mice.(G) Growth profile of Ptgs+/+ and Ptgs-/- melanoma cells in wild-type or Ptgs+/+ melanoma cells in Ptger2-/- or Gzmb-Cre Ptger4floxed/floxed mice. Data are expressed as mean ± SEM, one-way (B–C and F) or two-way ANOVA (A).