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: Rescued Treg cells in inflamed mice are activated and potently suppressive in inflammatory settings.a-c, Male Foxp3DTR-GFPCd4creERT2 and Foxp3LSLCd4creERT2 mice were treated with 4-OHT on postnatal day 14. Data are pooled from three independent experiments. a, Percentages of splenic Treg cells at indicated time points following 4-OHT treatment. Two-tailed unpaired t-tests with Holm-Sidak multiple comparison. b, c, representative histograms (b) and combined data (c) showing expression of Ki67, CTLA4 and GITR by rescued and control splenic Treg cells on day 7 post 4-OHT treatment. Two-tailed unpaired t-tests. d, e, RNA-seq analysis of rescued Treg cells, control Treg cells, and Treg “wannabes” from male mice of indicated genotypes treated with 4-OHT on postnatal day 14 and analyzed 7 days afterwards. d, Principal component analysis of gene expression in the three indicated cell populations (top), and empirical cumulative distribution function plots showing gene signatures of activated Treg cells (bottom left) and Treg cells expressing a constitutively active form of STAT5b (bottom right). Two-sided Kolmogorov-Smirnov test. e, Heatmaps showing expression of curated genes in rescued and control Treg cells from mice of indicated genotypes. f, Expression of Ki67, CTLA4 and GITR by indicated splenic Treg cell subsets from mosaic heterozygous Foxp3DTR-GFP/WTCd4creERT2 and Foxp3LSL/WTCd4creERT2 female mice treated with 4-OHT on postnatal day 14 and analyzed after 7 days. One-way ANOVA with Tukey’s multiple comparison test. g, Suppression of in vitro proliferation of conventional CD4 T cells induced by a-CD3 antibody and antigen-presenting cells by control or rescued Treg cells (GFP+) or Treg “wannabes” (Thy1.1+) from indicated strains of mice on day 7 post 4-OHT treatment. Two-way ANOVA with Tukey’s multiple comparison test. h-j, Adult mosaic heterozygous female Foxp3DTR-GFP/WTCd4creERT2 and Foxp3DTR-GFP/LSLCd4creERT2 mice were treated as in Figure 2a. h, Percentages of splenic Treg cells at indicated time points following tamoxifen treatment. Multiple t-tests with Holm-Sidak multiple comparison. i, j, Representative histograms (i) and combined data (j) showing expression of indicated markers by Treg cells at week 1. Two-tailed unpaired t-tests. All error bars denote mean ± s.e.m. ns, non-significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.