Fig 2: Flow cytometry analysis from approximately day 31 of co-cultureCells were harvested after 3 weeks of MS5 co-culture and stained for surface markers.(A) Cells were gated for singlets and scatter and then for viability (7AAD) and CD45 expression as indicated in the figure (top panel). Expression of CD19 and CD34 is shown within the 7AAD-CD45+ fraction (second panel, left). The CD19 positive fraction is further gated for expression of CD10 and CD20 (right). Counting beads were used in this experiment and can be seen in the scatter plot in the top middle panel.(B) Cells were gated for singlets and scatter (as in Figure 8A, plots not shown). Contour plot of viability (7AAD) and CD45 expression as shown in the figure (left). Further gating on 7AAD-CD45+ cells for B lymphoid (CD19) and, within the CD19- gate, myeloid (CD11b and CD14) markers are shown to the right.

Fig 3: Systemic collagen-binding domain IL-12 (CBD-IL-12) cytokine fusion therapy inhibits prostate cancer tumor growth and reprograms the tumor immune microenvironment.a Schematic of CBD-IL-12, composed of the p35 and p40 subunits fused to the CBD from von Willebrand factor domain A3. b Volumes of RM9 subcutaneous tumors in sygeneic C57Bl/6 mice over time with treatment with vehicle, anti-PD-1 (clone 29 F.1A12) 200 ?g by intraperitoneal injection every 5 days, or CBD-IL-12 25 ?g by intravenous injection every 5 days starting on day 0. n = 7 mice in vehicle and CBD-IL-12 treated groups and n = 8 mice in anti-PD1 treated group. p < 0.0001 at day 9 and 12. Bars represent mean with SEM. P-values are derived from two-way ANOVA with Dunnett’s multiple comparisons test, ns not significant. c Uniform Manifold Approximation and Projection (UMAP) plots of different immune cell subsets (top) from single-cell RNA-seq (scRNA-seq) analysis of five RM9 tumors each aggregated from mice treated with vehicle or CBD-IL-12. UMAP plots colored with gene expression of immune cell subset-specific markers for pan-monocytes/macrophages, M1 and M2 polarized marcophages, conventional type 1 dendritic cells (cDC1), natural killer (NK) cells, and T helper type 1 (Th1) cells. d Plots showing the frequency of specific immune cell populations (relative to CD45 + immune cells) identified by scRNA-seq analysis including CD4+ and CD8+ T cells, Th1 (Infg+Tbx21+) and Th2 (cMAF+Gata3+) cells, Ly6C+/- monocytes/macrophages (Ly6C+/-Adgre1+), M1 macrophages (CD80+CD86+INOS2+), M2 macrophages (CD163+Mrc1+cMAF+), cDC1 (XCR1+IRF8+), conventional type 2 dendritic cells (cDC2, CD1+IRF4+), migratory CD103+ dendritic cells (Itgae+), eosinophils (SiglecF+), neutrophils (Ly6G+), and NK cells (Klrb1c+Ncr1+) in tumors treated with vehicle or CBD-IL-12. Volcano plots showing differential gene expression in (e) tumor cells and (f) T cells from RM9 tumors of mice treated CBD-IL-12 relative to those treated with vehicle. FC fold change, FDR false discovery rate. Source data are provided in the Source Data file.

Fig 4: Combining CBD-IL-12 with STEAP1-mBB? CAR T cell therapy reprograms the tumor immune microenvironment and promotes antigen presentation and epitope spreading.a Photomicrographs of STEAP1 (top), B2m (middle), and CD3 (bottom) IHC staining of RM9-hSTEAP1 lung tumors after treatment with mouse untransduced T cells or STEAP1-mBB? CAR T cells with or without CBD-IL-12 treatment. Scale bars = 50 µm. b Bar plot showing IHC quantification of CD3 positive cells infiltrating the metastatic lung tumors (n = 4 tumors per group). One-way ANOVA p = 0.0021. Error bar represents mean with SD. Plots showing the frequencies of (c) Ly6C-F4/80+MHC-II+ (left, one-way ANOVA p = 0.0005) and Ly6C-F4/80+iNOS2+ (right, one-way ANOVA p = 0.0017) macrophages, (d) Ly6C+F4/80+MHC-II+ (left, one-way ANOVA p = 0.0038) and Ly6C+F4/80+iNOS2+ (right, one-way ANOVA p = ns) macrophages, (e) CD11b+XCR1+ cDC1 (one-way ANOVA p = 0.0003), (f) F4/80+SiglecF+ eosinophils (one-way ANOVA p = 0.0008), and (g) Ly6G+ neutrophils (one-way ANOVA p = 0.0035) normalized to total CD45+ cells as determined by multiparametric flow cytometry after treatment with untransduced T cells, STEAP1-mBB? CAR T cells, untransduced T cells and CBD-IL-12, and STEAP1-mBB? CAR T cells and CBD-IL-12 at maximal treatment response (nadir) and tumor relapse (relapse). h Bar plots representing Simpson clonality as a measure of ‘evenness’ of the TCR repertoire analyzed by TCRB sequencing on tumor infiltrating cells collected from mice in (a). n = 4 tumors per group. P-values for untransduced T cels + CBD-IL-12 compared to untransduced T cells and STEAP1-mBB? CAR T cells are 0.008 and 0.02, respectively; and STEAP1-mBB? CAR T cells + CBD-IL-12 compared to untransduced and STEAP1-mBB? CAR T cells are 0.004 and 0.01, respectively. Error bar represents mean with SD. For panels (c–g) n = 3 tumors per group, *p < 0.05; **p < 0.01, ***p < 0.001, p-values in panels (b–h) are from one-way ANOVA with Dunn’s multiple comparisons test. Source data are provided in the Source Data file.

Fig 5: Human brain metastases are well infiltrated by PD-1+ CD8+ T cells(A) Experimental schema and distribution of samples among primary tumor histologies.(B and C) Density of CD45+ lymphocytes (B) and CD8+ T cells (C) for all 31 brain metastases grouped by primary tumor type.(D and E) Density of CD45+ lymphocytes (D) and CD8+ T cells (E) for all 31 brain metastases grouped by patient disease status at time of tumor resection.(F) Frequency of CD8+ cells among lymphocytes grouped by tumor type for all 31 patients.(G and H) Density of CD4+ T cells (G) and B cells (H) for 20 and 16 of the tumors, respectively.(I) Percentage of CD8+ T cells expressing PD-1 in 21 tumors.(J) Phenotype of PD-1- versus PD-1+ CD8+ T cells within brain metastases (n = 12–14 for each marker).(K) Percentage of tumor-infiltrating TCF-1+ CD8+ T cells expressing PD-1, TOX, or both (n = 14).Bars on graphs indicate median. In (B)–(E) and (G), statistics show variance among primary tumor types with the Kruskal-Wallis test. In (F), (H), and (I), statistics show one-way ANOVA. In (J), statistics show a mixed-effects model analysis with Sidak’s multiple comparisons test.See also Figure S1.