Fig 2: 90Y-NM600 increased CD8 +T cell infiltration into prostate tumors but also increased checkpoint molecule expression. TRAMP-C1 tumors were collected from mice (n=5 per group) at several time points following no treatment (control), low-dose TRT, or high-dose TRT, digested, and evaluated for cellular composition by flow cytometry. Shown are: (A) CD3 +CD8+T cells as a percentage of CD45 +cells. (B) CD69 (left) and CD137 (right) median fluorescence intensity (MFI) on CD8 +T cells. (C) Percent of CD8 +T cells bearing markers for effector memory (CD44 +CD27-CD62L-), central memory (CD44 +CD27+CD62L+), resident memory (CD44 +CD69+CD103+) or short-lived effector (KLRG-1 +CD127-) cell phenotypes. (D) MFI of PD-1, CTLA-4, LAG-3, and VISTA on CD8 +T cells (E) PD-L1 MFI on dendritic cells (CD11b-CD11c+MHCII+ cells), myeloid-derived suppressor cells (MDSCs; CD11b+Gr-1+), and tumor cells (CD45-). *P<0.05, **p<0.01, *** p<0.001 compared with untreated controls via two-way ANOVA with Benjamini-Hochberg adjustment of pairwise tests. To perform pairwise testing, untreated controls were pooled then compared with each TRT-treated group. Error bars represent mean±SE. Results shown are from one experiment and are representative of two independent experiments. ANOVA, analysis of variance; DCs, dendritic cells; TRT, targeted radionuclide therapy.

Fig 3: MiR-34a drives DC activation.(a) miR-34a-/- DCs show reduced production of pro-inflammatory cytokines. Bone marrow DCs from WT and miR-34a-/- mice (n=6 pooled) were stimulated with LPS or CL097 for 24 h. (b,c) miR-34a-/- DCs show reduced expression of MHC class II. WT and miR-34a-/- DCs were stimulated as in a for 24 h and the expression of co-stimulatory molecules was evaluated as median fluorescent index (MFI) by flow cytometry. Representative MFI expression of MHC class II (b), and quantitative expression of MHC class II, co-stimulatory (CD40 and CD86), and inhibitory (PD-L1) molecules is shown (c). *P<0.05 Mann–Whitney u-test between genotypes. Data are presented as mean±s.e.m. of 4–6 replicates from two independent experiments. (d,e) miR-34a-/- DCs show a reduced interaction with antigen-specific T cells. CMTPX-labelled WT and miR-34a-/- DCs were co-cultured with CFSE-labelled OT-II CD4+ T cells at 1:1 or 1:3 ratios in the presence of OVA peptide or OVA plus LPS for 24. The contact area was analysed with In Cell Analyzer 2000. Representative pictures of 1:1 cultures (d) and quantitative evaluation (e) presented as mean±s.e.m. of % of overlapping areas between DC and T cells compared to the total cell surface area (six technical replicates in total) are shown. *P<0.05 between conditions, &P<0.05 between genotypes, two-way ANOVA followed by Tukey’s multiple comparison test or unpaired t-test. (f,g) miR-34a regulates pro-inflammatory cytokine production by human DCs. (f–h) Healthy donor monocyte-derived inflammatory DCs (n=4–5) were transfected with miR-34a mimic (34am) or miR-34a inhibitor (34i) or appropriate controls (Cm=mimic or Ci=inhibitor); and (g) DCs (conventional CD1c+ DCs; n=7) were sorted from RA patients’ blood and transfected with miR-34a inhibitor (34i) or control inhibitor (Ci). The DCs were then stimulated with LPS or CL097 for 24 h. (f) Enforced expression of miR-34a increased TNF production; *P<0.05, Kruskal–Wallis followed with Dunn’s multiple comparison tests or paired t-test. (g,h) miR-34a inhibitor reduced the production of cytokines by healthy donor monocyte-derived DCs (g) and sorted PB RA CD1c+ DCs (h). *P<0.05 paired t-test. (f,g) Data are presented as mean±s.e.m of 4–7 biological replicates obtained in separate experiments. ANOVA, analysis of variance.

Fig 4: VISTA Expression Promotes an Immunosuppressive Microenvironment, but Does Not Alter Response to PD-1(A) Tumors were analyzed for tumor-infiltrating lymphocytes 7 days after injection. The presence of FOXP3+CD4+CD3+ T regulatory cells was determined by flow cytometry as a percentage of cells gated as Live and CD45+. Data were collected from 9 mice per group, combined from 2 independent experiments. *p < 0.05.(B) As in (A), dendritic cells (gated as Live F4/80-CD11c+MHCNhiCD3-CD45+) were analyzed for MHC II levels by flow cytometry, and mean fluorescence intensity (MFI) was quantified. *p < 0.05.(C) As in (A), tumor-associated macrophages (TAMs) (gated as Live CD11b+F4/80+CD3-CD45+) were analyzed for PD-L1 positivity. MFI of PD-L1+ cells was quantified in the TAM immune cell population. **p < 0.01.(D) As in (A), myeloid-derived suppressor cells (MDSCs) (gated as Live CD11b+GR-1+CD3-CD45+) were analyzed for PD-L1 positivity. MFI of PD-L1+ cells was quantified in the MDSC immune cell population. *p < 0.05.(E) D4M UV2 VISTA cells were injected into C57BL/6 mice. When tumors reached ~50 mm3, animals were treated with either anti-PD-1 antibody or the corresponding isotype control (rat IgG2a) every 2–3 days. Data were collected from 5 mice treated with isotype and 6 mice treated with anti-PD-1. One animal in the anti-PD-1 treatment group with zero tumor volume was excluded from the statistical analyses of tumor growth curves. For the remaining animals, the fitted group average tumor growth curves are depicted by a bold line. Anti-PD-1 treatment delayed tumor growth on average by 12.2 days (95% CI 6.5–17.8, p = 0.001) as compared to the control group.(F) D4M UV2 VISTA cells were injected into C57BL/6 mice. When tumors reached ~50 mm3, animals were treated with either anti-VISTA antibody or the corresponding isotype control (hamster polyclonal IgG) every 2–3 days. Data were collected from 7 mice treated with isotype and 8 mice treated with anti-VISTA.(G) The time to doubling was defined as the first observation day when the tumor volume exceeded twice the volume at day 0. Animals with tumor volumes that did not double at any time were censored on the last day of observation. The time to doubling was analyzed using the Kaplan-Meier estimator of the survival curves and Cox proportional hazards model.See also Figures S4 and S5.