Fig 2: Characterization of surrounding cells in dome-like structures. (A–C, E, G) Immunofluorescence images of dome-like structures at 2 weeks after tamoxifen injection. Scale bars, 20 µm. (A) The multilayered epithelium overlying basally extruded RasV12-trasformed cells. The dotted areas are shown at higher magnification in the lower panels. Dashed lines indicate the epithelium outside (1) or inside (2) the dome-like structure. Arrowheads indicate stromal cells within the dome-like structure. (B) Disrupted tight junctions in the overlying epithelium. Arrowheads or arrows indicate regions where tight junctions are absent or elongated, respectively. (C, D) Increased cell proliferation in the overlying epithelium within dome-like structures. (D) Quantification of the Ki-67-positive epithelial cells. Data are mean ± SEM n = 222 (not-extruded YFP), 191 (not-extruded RasV12), and 222 (RasV12 in dome-like structures) cells from four CK19-YFP or five CK19-RasV12 mice. n = 1124 (surrounding not-extruded YFP), 1270 (surrounding not-extruded RasV12), and 1708 (surrounding RasV12 in dome-like structures) cells from four CK19-YFP or six CK19-RasV12 mice. *p < 0.05, **p < 0.01, one-way ANOVA with Tukey's test. (E, F) Accumulation of activated fibroblasts in dome-like structures. (E) The dotted area is shown at higher magnification in the lower panels. Arrowheads indicate a-SMA-positive activated fibroblasts within the dome-like structure, whereas arrows indicate a-SMA-positive smooth muscle underneath the epithelium. Note that smooth muscle has much higher a-SMA expression compared with activated fibroblasts. (F) Quantification of a-SMA+ fibroblasts around YFP or RasV12 cells. Data are mean ± SEM n = 89 (not extruded in CK19-YFP), 77 (not extruded in CK19-RasV12), and 107 (dome-like structure in CK19-RasV12) areas from four mice. *p < 0.05, one-way anova with Tukey's test. (G, H) Accumulation of immune cells in dome-like structures. (G) Arrowheads indicate CD45-positive immune cells within the dome-like structure. (H) Quantification of CD45+ cells around YFP or RasV12 cells. Data are mean ± SEM n = 89 (not extruded in CK19-YFP), 39 (not extruded in CK19-RasV12), and 99 (dome-like structure in CK19-RasV12) areas from three mice. *p < 0.05, ****p < 0.001, one-way anova with Tukey's test.

Fig 3: Naïve T cell survival is impaired in vivo but not in vitro. Cell Trace Violet (CTV)-labeled CD4+ naïve T cells isolated from young CD45.1+ mice were adoptively transferred into sublethally-irradiated young and aged WT hosts. (A). Histogram of CTV dilution in CD45.1+CD4+CD62LhiCD44lo cells in young (black) and aged (red) hosts. (B). The percent of naïve CD45.1+ T cells that have diluted CTV during proliferation (left panel) and the percent of live host cells derived from the CD45.1+ donor (right panel). Relative fluorescence intensity (RFI) of (C). CD127 and pSTAT5 in adoptively-transferred naïve T cells in young (black) and aged (hosts). Compiled data from two experimental trials shown, with each trial consisting of two age groups of three recipients. RFI was based on normalization to the mean of the cells within the young host for the respective experimental trial. Analyzed by t tests on compiled data, p-values: ns, not significant; * < 0.05. (D). Lymphocytes from young CD45.1+ mice were added to established cultures of young or aged lymph node CD45- stromal cells, with or without supplementation with rIL-7 and a-IL-7. Numbers of congenic naïve CD4+ and CD8+ T cells was quantified after 3-day co-culture. Compiled data from three experimental trials shown, with each trial consisting of triplicate wells and cell numbers normalized to the mean of cells present when co-cultured with young stroma. Means from each experiment were analyzed by two-way ANOVA with adjustment for multiple comparisons, p-values: ns, not significant.

Fig 4: Treatment of EMT6-CDDP orthotopic primary mouse breast cancer with different CTX regimens.a Mice bearing EMT6-CDDP were treated with either saline control, MTD CTX (red arrows), LDM CTX continuous daily (green arrow start point), or CTX140 1q6d (blue arrows). b Survival data from a. Log rank (Mantel Cox) tests; CTX140 1q6d vs control **p = 0.0015, MTD CTX vs control **p = 0.0041, LDM CTX vs control *p = 0.016, n = 5 per group. c The EMT6-CDDP primary tumor treatment experiment was repeated and tumors were analyzed by CyTOF at 29 DPI (18 days after the start of treatment), i.e., after one cycle of MTD CTX and three doses of CTX140 1q6d. CyTOF analysis of four pooled tumors from each treatment group. SPADE trees of each treatment group, the size of each sphere represents number of cells, and the color represents expression level of CD45. Error bars represent SEM.

Fig 5: Alteration in immune cell subtypes following deletion of Alox15 in ß cells of NOD micepLNs were isolated from NOD-Cre+ and NOD-?ß mice at 8 weeks of age, dissociated into single cells, and analyzed by cytometry by time of flight (CyTOF) or flow cytometry. N = 3–4 individual animal replicates.(A) tSNE maps of CD45+ immune cell populations in NOD-Cre+ and NOD-?ß pancreata by CyTOF. Cells are colored by their FlowSOM-based cluster-assignment cell.(B) Heatmap of protein expression levels across all populations identified by FlowSOM.(C) Quantification of each immune cell type identified by FlowSOM-based clusters.(D) Quantification of CD11b+IFN-?+ cells.(E) Quantification of CD4+CD25+ cells.(F) Flow cytometry of pLNs of CD4+ and CD8+ T cell populations.Data are expressed as the mean ± SEM. *p < 0.05.