Fig 2: NK cells in the livers of NAFLD patients are less cytotoxic than those from healthy controls. (A) Immune cells were isolated from human livers. NK cells were identified by scatter, and as live CD45+ CD56+ CD3- Tbethi Eomeslo cells. Liver-resident NK cells (lrNK) were identified as live CD45+ CD56+ CD3- Tbetlo Eomeshi cells. (B,C) Intrahepatic leukocytes were cultured for 4 h in the presence of anti-CD107a and Brefeldin A. Representative CD107a staining of NK cells from a healthy (B) and a NAFLD (C) liver is shown. (D) Inverse correlation between NK cell degranulation and histological score in human livers. H, healthy; SS, simple steatosis; NAS, NASH Activity Score; C, cirrhosis. Significance was determined using Spearman's Rank Correlation. (E) CD56+ CD3- CD16+ CXCR6- NK cells were sorted from healthy (n = 4) and NAFLD (n = 3) livers and cultured for 4 h with the human NK target cell, K562. At the end of culture, target cell death was assessed using AnnexinV staining. (F) Inverse correlation between liver-resident NK cell degranulation and histological score in human livers. Significance was determined using Spearman's Rank Correlation. (G) CD56+ CD3- CXCR6+ liver-resident NK cells were sorted from healthy (n = 4) and NAFLD (n = 3) livers and cultured for 4 h with the human NK target cell, K562. At the end of culture, target cell death was assessed using AnnexinV staining. For (E,G), medians are shown. For these small datasets, no statistical testing was carried out.

Fig 3: PS3D1@DMXAA elicits TAA-specific CD8+ T cell priming.(A) Schematic illustrating the process of DC activation in the TME and DC migration to the tdLN for antigen presentation and activation of T cells. Mice bearing B16.F10 tumors were treated with PBS (1), free DMXAA (2), PS3D1 (3), a physical mixture of empty PS3D1 and DMXAA (4), or PS3D1@DMXAA (5) intravenously for five times, 2 days apart. (B) Percentages of CD103+CD11c+ DCs of CD45+ cells in tumors were measured by flow cytometry on day 14 (n = 4). (C and D) Representative flow cytometric analysis (C) and the corresponding quantification (D) of CD103+CD11c+ DCs of CD45+ cells in tdLN on day 14 (n = 4). (E) Intravenous treatment scheme for mice with established B16-OVA tumor. Mice bearing B16-OVA tumors were treated as in (A). Then, the mice were sacrificed on day 7 for different analysis. (F and G) Percentages of OVA (SIINFEKL)–specific CD8+ T cells in TME (F) and spleen (G) were measured by flow cytometry (n = 3). (H) ELISpot analysis of IFN-? spot-forming cells among splenocytes after ex vivo restimulation with OVA (SIINFEKL) peptide on day 7. (I) Flow cytometric quantification of granzyme B+CD8+ T cells among splenocytes of mice after ex vivo restimulation with SIINFEKL (OVA) peptide. (J) Granzyme B protein levels in the supernatant of splenocytes after ex vivo restimulation with OVA (SIINFEKL) peptide for 24 hours on day 7 (n = 4). (K) Flow cytometric quantification of CD107a+CD8+ T cells among splenocytes after ex vivo restimulation with SIINFEKL (OVA) peptide on day 7. Data are means ± SD, and statistical significance was calculated by one-way ANOVA with Tukey’s test. ***P < 0.001 and **P < 0.01.