Fig 2: Tumoricidal effect of BCG lysate is dependent on TAM and DC. (A) Growth curve of treated and untreated B16F10 melanoma with and without MΦ depletion. Data are represented as mean±SEM using two-way-ANOVA followed by Šidák’s multiple comparisons test (n=12 per group except BCG +anti-CSF1R, n=11; two independent experiments). (B–H) Relative frequencies of cell subsets measured by flow cytometry: (B) CD45+ cells, (C) CD8+ T cells, (D) CD4+ T cells, (E) NKT cells, (F) NK cells, (G) MΦ and (H) DC. (I) BCG lysate-induced fold change of the indicated cytokines and chemokines from tumor lysates on day 12 after tumor injection. Data are shown as box-and-whisker plot; the box extends between 25% and 75% and the whiskers extend to the minimum and maximum values (BCG, n=11; BCG +anti-CSF1R, n=9; two independent experiments). Statistical significance was determined using two way-ANOVA followed by Šidák’s multiple comparisons test. (J) Growth curves of BCG lysate-treated or PBS-treated B16F10 tumors in Batf3–/– mice, represented as mean±SEM using two-way-ANOVA followed by Šidák’s multiple comparisons test (n=12 per group, two independent experiments). (K–O) Relative frequencies of cell subsets measured by flow cytometry: (K) CD45+ cells, (L) CD8+ T cells, (M) CD4+ T cells, (N) NKT cells and (O) NK cells. Data are represented as mean±SD using one-way ANOVA followed by Tukey’s multiple comparisons test (n=12 per group, two independent experiments). p≤0.05 (*), p≤0.01 (**), p≤0.001 (***), p≤0.0001 (****). ANOVA, analysis of variance; DC, dendritic cell; MΦ, macrophage; TAM, tumor-associated microphage.

Fig 3: Tolerogenic nanoparticle (tNP) treatment increases antigen-specific endogenous Foxp3+ Tregs that withstand antigen challenge in the presence of a TLR agonist or complete Freund’s adjuvant (CFA). (A) WT mice were treated once i.v on day 0 with PBS, NP[2W] at a 5µg dose of 2W peptide, nanoparticles containing rapamycin alone (NP[Rapa]) at a 50µg dose of rapamycin and tNP at a 5µg dose of 2W peptide and 50µg dose of rapamycin. Mice were then challenged i.p. with 100µg 2W peptide and 20µg R848 on day 7, and spleens were assessed for 2W+CD4+ T cells on day 12. MACS-enriched 2W+ cells were Live/Dead Fixable Aqua−, B220−, CD11c−, CD11b−, CD4+, CD44+, 2W:Iab+ and then probed for Foxp3 expression. (B) Total numbers (#) of 2W+CD4+ T cells from the spleen. (C) % and (D) total # of 2W+CD4+ T cells that are Foxp3+. (E) WT mice were treated three times weekly, i.v. on days 0, 7, and 14 with PBS, NP[2W] at a 5µg dose of 2W peptide, NP[Rapa] at a 50µg dose of rapamycin, and tNP at a 5µg dose of 2W peptide and 50µg dose of rapamycin. Mice were then challenged i.p. with 100µg 2W peptide admixed 1:1 with CFA on day 28, and spleens were assessed for 2W+CD4+ T cells on day 33. (F) total # of 2W+CD4+ cells from the spleen. (G) % and (H) total # of 2W+CD4+ cells that are Foxp3+. The results from (B–D) represent an N = 3 from one experiment. The results from (F–H) represent an N = 4 from one experiment.

Fig 4: IL-32 induces a systemic CD8+ T cell–mediated tumor-specific immune response.(A) Experimental setup for in vivo tumor treatments. MC38 and B16F10 were inoculated in C57BL/6J mice, and 4T1 tumors in BALB/c mice (A–O). (B) Growth curves of IL-32–, cGAMP-, or PBS-treated primary and (C) contralateral, nontreated B16F10 melanomas. *P < 0.05, **P < 0.01, ***P < 0.001. Data are representative of 4 independent experiments, with n = 6 mice per group. (D) Kaplan-Meier survival curves of B16F10-bearing mice treated with IL-32 (n = 6) or PBS (n = 8). (E) Representative growth curves of IL-32–treated and untreated MC38 colon adenocarcinoma (n = 6) and (F) orthotopic 4T1 breast tumors (n = 10). (G–O) On day 14, the primary treated tumors and spleens were harvested for flow cytometric analyses, IHC or TCRβ chain sequencing. (G) Representative flow cytometry plots displaying frequencies of CD45+ immune cells for each treatment group and (H) their quantification shown as relative frequencies (n = 18). (I) Relative frequencies of CD8+ and CD4+ T cells as a percentage of live cells (n = 18). (J) Representative immunohistochemical staining and (K) morphometric enumeration as cells/mm2 of CD8+ T cells (n = 3). Scale bar: 20 μm. (L) Relative frequencies of IFN-γ+ cells as percentage of CD8+ T cells (n = 12). (M) Frequencies of Nur77-GFP+ cells of CD8+ T cells, as determined by flow cytometry in B16F10-inoculated and PBS- (n = 14) and IL-32–treat (n = 13) Nur77 mice. (N) TCR clonality in tumors (left, PBS, n = 7; IL-32, n = 5) and spleens (right, PBS, n = 7; IL-32, n = 6), representative of 2 independent experiments. (O) Relative frequencies of TRP-2 tetramer–positive cells, as a proportion of CD8+ T cells in tumors (left, n = 6) and spleens (right, n = 18). (P) Growth curves of IL-32–treated and untreated B16F10 primary tumors (PBS, IL-32, PBS + aCD8, n = 18; IL-32 + aCD8, n = 16) and (Q) untreated contralateral tumors with or without CD8 depletion (n = 16–18). *P = 0.0171; **P = 0.003; ****P < 0.0001. (R) Growth curves of IL-32–treated and untreated B16F10 primary tumors with or without CD4 depletion (n = 11) or (S) NK depletion (PBS, n = 12; IL-32, n = 18). (B, C, E, F, and P–S) Growth curves are shown as mean ± SEM, using 2-way ANOVA followed by Šidák’s multiple comparisons test. (D) Comparison of survival curves was performed using a log-rank (Mantel-Cox) test. (H, I, K–M, and O) Data are displayed as mean ± SEM. Statistical significance was determined by unpaired, 2-tailed Student’s t test. ****P < 0.0001.

Fig 5: CXCL9/10+ DCs occupy distinct regions in the spleen(A) Spleens from REX3 mice were imaged 6 weeks after infection with T. gondii. Sections were stained with F4/80 to highlight the RP and B220 to highlight B cell follicles. The mean fluorescence intensity was measured for CXCL9-RFP and CXCL10-BFP in each splenic region. Data were normalized to the B cell zone to adjust for background fluorescence.(B and C) Spleens from WT mice infected for 6 weeks were stained with XCR1 (B) or 33D1 (C) together with CD11c to identify cDC-1s and cDC-2s, respectively. Summary data show mean fluorescence for the indicated marker in each splenic region. Data were normalized to the B cell follicle as in (A).(D and E) WT (D) and CXCR3 KO (E) TG6-GFP neonatal chimeras were stained with 33D1 to identify cDC-2 clusters. The density of GFP cells within cDC-2 clusters was then measured and normalized to the total density of GFP cells in the slice.Data in (A) are representative of three mice, and data in (B)–(E) are representative of two mice.