Fig 2: Loss of the miR-15 family results in B cell progenitor expansion in vitro. (A) Schematic illustration of the sorting strategy for the B cell progenitor in vitro culture. B220+c-KIT+NK1.1-CD19- B cell progenitors were sorted from the bone marrow of miR-15a/bfl/fl Vav-Cre and miR-15a/b+/+ Vav-Cre mice and cultured in the presence of IL-7, FLT3L and SCF. A representative FACS plot depicts the percentages of pro-B (c-KIT+CD25-), intermediate B (c-KIT-CD25-) and pre-B cells (c-KIT-CD25+) under basal conditions after 1 week. (B) Statistical analysis of the distribution of pro-, intermediate and pre-B cell progenitors derived from miR-15a/bfl/fl Vav-Cre mice and miR-15a/b+/+ Vav-Cre mice under basal conditions. (C) Representative FACS blots and statistical analysis of the progenitor B cell differentiation upon IL-7 withdrawal in vitro. (D) Progenitors from miR-15a/bfl/fl Vav-Cre mice and miR-15a/b+/+ Vav-Cre mice were labeled via stable expression of either dsRed or GFP, mixed at equal ratios at a cell density of 5 x 104 cells/ml and tracked over time by flow cytometry in the presence of all growth factors. (E) The bar graph depicts the percentages of EdU+ progenitors derived from miR-15a/bfl/fl Vav-Cre mice or miR-15a/b+/+ Vav-Cre mice after labeling with 10 ug/ml EdU for 4 hrs. (F) Quantification of the percentage of apoptotic progenitors under steady state conditions as analyzed by AnnexinV staining. Error bars depict the standard deviation of the mean. Numbers indicate the percentage of cells in the respective region. MiR-15a/bfl/fl Vav-Cre progenitors were statistically compared to miR-15a/b+/+ Vav-Cre progenitors by an unpaired two-tailed Student’s t-test. *P < 0.05.

Fig 3: The TME Is Dynamically Modified Post-dual Therapy(A–F) MC38-luc tumor-bearing mice were sacrificed 1 day after the first or second injection of CpG in different groups (n = 6 in each group), and tumor nodules on both sides were collected and digested for qRT-PCR to explore the cancer-immune set point. The expression levels of all markers are relative to the housekeeping gene, HPRT1. (A–C) Results show the levels of perforin (A), granzyme B (B), and IFN-? (C), 1 day after the first injection of CpG in the treated side. (D–F) 1 day after the second injection of CpG, the levels of CD8 (D), TGF-ß (E), and CD105 (F) from the untreated side are also presented. (G–N) MC38-luc tumor-bearing mice were euthanized 1 day after finishing the whole treatment, and tumor nodules from both sides were collected and lysed for flow cytometry to analyze the TME. Data are quantified (n = 8 in each group). (G–L) Quantities of activated TNF-a+CD8+ T cells (G) and IFN-?+CD4+ T cells (H); the ratio of more severely exhausted PD-1+CTLA-4+CD8+ T cells (I); and quantities of NKs (defined as CD45+CD3-NK1.1+) (J), macrophages (defined as CD45+CD11b+ F4/80+) (K), and Tregs (defined as CD45+CD4+FOXP3+) (L) from both sides are shown. (M and N) On the untreated side, the number of myeloid-derived suppressor cells (MDSCs) (defined as CD45+CD11b+Gr-1+) (M) and DCs (defined as CD45+CD11c+) (N) is also presented. Values are presented as mean ± SD. Two-way ANOVA was used to analyze the data from (G) to (L). One-way ANOVA was used to analyze the statistical significance in other panels (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; and ns, not significant).

Fig 4: Nlrp3 deficiency promotes tumor regression and anti-tumor immunity. (A) Tumor volume curve of WT (n = 4) and Nlrp3-/- (n = 4) mice 9–14 days after B16.F10 inoculation, tumor weight on day 14, and representative image of excised melanoma tumors. (B) Tumor volume curve of WT (n = 4) and Nlrp3-/- (n = 5) mice 11–21 days after LLC inoculation, tumor weight on day 21, and representative image of excised LLC tumors. (C) Representative fluorescence-activated cell sorting (FACS) plots and percentages of intratumoral CD45+ cells of WT (n = 9) and Nlrp3-/- (n = 7) mice 15 days after B16.F10 inoculation. (D) Gating strategy and frequencies of intratumoral CD4+, CD8+, and NK1.1+ cells in CD45+ population of WT (n = 9) and Nlrp3-/- (n = 7) mice 15 days after B16.F10 inoculation. (E) Representative FACS plots and frequencies of CD8+ IFN-?+ cells from CD45+ tumor-infiltrating cells of WT (n = 4) and Nlrp3-/- (n = 4) mice 15 days after B16.F10 inoculation, and representative overlay and mean fluorescence intensity (MFI) of IFN-? produced by CD8+ cells after in vitro activation. Data are shown as mean ( ± S.D.). Representative data from at least two independent experiments are shown. Statistical significance was obtained by unpaired Student’s t-test. Symbols: (*), p = 0.05; (**), p = 0.01, n = biologically independent mouse samples.

Fig 5: Hematopoietic deletion of miR-15a/16-1 and miR-15b/16-2 blocks NK cell development and promotes the accumulation of myeloid cells. (A) Quantitative PCR for the expression of mature miR-15a, miR-15b and miR-16 in B cells derived from miR-15a/b+/+ Vav-Cre, miR-15afl/fl Vav-Cre, miR-15bfl/fl Vav-Cre and miR-15a/bfl/fl Vav-Cre mice. Each dot represents the data derived from one mouse. n.d. = not detected. (B) NK cells defined as NK1.1+TCRbeta- were classified as stage I to stage IV based on their expression of CD27 versus CD11b (schematic left panel and contour plots, numbers indicate the percentage of cells within the respective quadrant). The line and bar graphs show the analysis of all data sets for the depicted genotypes, and again each dot represents data from one mouse. (C) Schematic illustration of the gating strategy and representative primary data for myeloid cells and neutrophils in spleens derived from all genotypes. Numbers indicate the percentage of cells within the respective gate. (D) Bar graphs show the percentages of indicated populations within total cells or CD11b+ myeloid cells for all analyzed mice. (E) Bar graphs indicate the total splenic cell numbers and spleen weight for the respective genotypes. Each dot represents the data derived from one mouse. Error bars depict the standard deviation of the mean. Sample groups were statistically compared as indicated by an unpaired two-tailed Student’s t-test. *P < 0.05; **P < 0.005; ***P < 0.0005.