PMC

All data is representative flow cytometric analysis of CD11b⁺DC1 and CD103⁺DC2 populations (gated on CD45⁺, Ly6C⁻, MHCII⁺, and CD24⁺) Data shown as mean ± SEM. Statistical significance indicated by *p<0.05, **p<0.01, ***p<0.001; ns=not statistically significant.
(A) Ectopic PyMT-VO tumors from an Irf8^−/−(KO) compared to control (WT). Relative cell proportions as a % of total MHCII⁺ cells. Data pooled from individual mice (n=6) from 2 independent experiments.
(B) Ectopic B78chOVA tumors in Irf4^f/f x CD11c-CRE⁺ host compared to Cre-negative littermates. Relative cell proportions as a % of total MHCII⁺ cells. Data pooled from individual mice (n=7) from 2 independent experiments.
(C) Ectopic B78chOVA tumors in Batf3 KO, compared to WT. Relative cell proportions graphed as a % of total MHCII⁺. Data pooled from individual mice (n=6).
(D) Ectopic B78chOVA tumors in Zbtb46-DTR mice, receiving acute 24 hour depletion with DT or PBS. Relative cell proportions graphed as a % of total MHCII⁺ cells. Data pooled from individual mice (n=6) from 2 independent experiments.

(A) Flow cytometry and gating of tumor APC populations from digested and CD45 enriched PyMTchOVA tumors. A–C: Representative of greater than 5 independent experiments.
(B) Cytometry of tumor APC populations in ectopic B78ChOVA tumors.
(C) Histogram of tumor-derived mCherry fluorescence, by tumor-infiltrating immune cells in B78chOVA.
(D) Representative cytometry of digested human melanoma metastatic biopsy identifying corollary DC and TAM populations defined by CD45⁺ Lin⁻ (CD3e, CD56, CD19) HLA-DR⁺ and split by CD14, BDCA1 and BDCA3. Double negative cells likely reflect B cells escaping lineage gate, immature monocytes or pDC.
(E) Relative proportions of tumor infiltrating myeloid cells as a % of total CD45⁺ cells for PyMTchOVA and B78chOVA models. Pooled data from individual tumors, presented as mean ± SEM from (n=5) mice.
(F) Frequency of DC and TAM populations infiltrating human metastatic melanoma presented as a % of total CD45⁺ cells. Pooled data from multiple patients, presented as mean ± SEM from (n=4) biopsies.
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All data (A–G) is from the ectopic B78chOVA tumor model. Cell lineages are defined as per .
(A) Expression of a panel of DC specific markers compared to respective isotype (grey shaded). A black box outlines the CD103⁺ DC2 population.
(B) Differential expression of Macrophage specific markers (colored) with corresponding isotypes (grey shaded). A black box outlines the CD11b⁺ DC1, TAM1, and TAM2 populations.
(C) Specific expression of DC-T_h2 makers (colored), by CD11b⁺ DC1 populations compared to respective isotype (grey shaded). A black box outlines CD11b⁺ DC1.
(D) Global transcriptional profiles revealed by RNAseq of FACS-purified populations from biological triplicates. Data displayed as a heat map of Log₂ fold change relative to the global average of the top 1000 genes by maximum variance between DC1, DC2, TAM1, and TAM2.
(E) PCA of DC1, DC2, TAM1, and TAM2 populations based on RNAseq global transcriptional profiles.
(F) qRT-PCR analysis of expression of Irf4, Irf8, Myb, and Zbtb46 (zDC) from sorted APC populations. Data presented as mean ΔCt ± SEM calculated from biological triplicates (n=3), (N.D. not detected).
(G) Intracellular staining for IRF4 and IRF8 in tumor APC populations as compared to the respective isotype (grey).
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All data (A–G) is from the ectopic B78chOVA tumor model. T cells+BMDC (shaded grey), T cells+BMDC+ SL8 (unshaded grey), T cells+tumor APCs (respective colored histograms). Plated at 20,000 T cells: 4,000 APC ratio. Representative flow plots from 4 independent experiments, unless noted.
(A) Flow cytometry of early activation markers, Nur77^GFP and CD69 (12 hr) on naïve or previously activated OT-I CD8⁺ T cells cultured on sorted APC populations directly from tumors
(B) Representative cytometry of Naïve OT-I CD8⁺ T cell proliferation, measured by dye dilution of eFluor670 plotted against Nur77^GFP (as measure of TCR triggering), at 72 hours following co-culture with tumor APC populations. Total cell yield counts listed above graphs.
(C) Histogram overlay of Naive T cell proliferation between tumor APCs.
(D) Representative cytometry of T cell proliferation, measured by dye dilution of eFluor670 plotted against Nur77^GFP, at 72 hours for previously activated OT-I CD8⁺ T cell blasts cultured on tumor APC populations. Total cell yield counts listed above graphs.
(E) Histogram overlay of previously activated OT-I CD8⁺ T cell proliferation across tumor APCs.
(F) Representative cytometry of T cell proliferation, measured by dye dilution of eFluor670, at 72 hours for Naive OT-II CD4⁺ T cells cultured on tumor APC populations. Representative flow plots from 2 independent experiments.
(G) Histogram overlay of Naïve OT-II CD4⁺ T cell proliferation across tumor APCs.
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(A) qPCR of Csf1r, Csf2rb, and Csf3r expression from sorted APCs. Data presented as mean ΔCt ± SEM calculated from biological triplicates (n=3) of individual B78chOVA tumors (N.D. not detected).
(B) Cytometry of tumor APCs after 3 days of αCSF1 (αCSF1, dotted) compared to isotype (filled) treated tumor animals. Quantified as % of total tumor CD45⁺ cells, pooled from individual mice (n=6) from 2 independent experiments shown as mean ± SEM. Statistical significance indicated by *p<0.05, **p<0.01, ***p<0.001; ns=not statistically significant
(C) Schematic of BM progenitor adoptive transfer and contributions to BM, spleen, and tumor.
(D) Representative cytometry of tumor arriving congenic cells. Gated on CD45.2 and following the gating strategy of .
(E) Competitive BM adoptive transfer of WT vs. Csf2rb KO GMP progenitors into B78chOVA tumor recipients. Repopulation efficiency plotted as % of total transferred cells. Representative gating of tumor arriving GMP cells, WT (grey), KO (purple). Quantification of tumor arriving DCs, defined by CD24⁺ CD11c⁺. Data pooled from 2 independent experiments, plotted as mean ± SEM from individual tumors (n=6).
(F) Cytometry of CD11b⁺ DC1 and CD103⁺ DC2 populations (gated on CD45⁺, Ly6C⁻ MHCII⁺, CD24⁺) between ectopic B16-F10, B16-GMCSF and B16-FLT3L cytokine expressing tumors. Populations presented as % of total MHCII⁺ cells for each tumor. Data are pooled from 3 independent experiments, plotted as mean ± SEM from individual tumors (n=6).
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All data (A–G) is from the ectopic B78chOVA tumor model.
(A) Heat map of Log₂ transformed expression from RNAseq across populations for selected genes involved in cross presenting, cytokine and chemokine production, and costimulation. Color scale defined as, green=bottom 20^th percentile, red=top 80^th percentile, with 20^th–80^th percentile graduated and centered at yellow (50^th percentile). Data from biological triplicates of sorted cells.
(B) Cytometry of surface protein levels of ligands for T cell regulatory molecules (colored) as compared to respective isotypes (grey).
(C) Cytometry of MCHI and MHCII (colored) expression compared to respective isotype (shaded).
(D) Cytometry of ex vivo dextran uptake across populations. Grey=no dextran, light histogram=dextran binding at 4° C, and dark histogram=dextran uptake at 37° C, displayed in triplicate. Delta geometric Mean Fluorescence Intensity (gMFI) for each population plotted as mean ± SEM. Data are representative of 2 independent experiments (n=6).
(E) Cytometry analysis of relative pH of endocytic compartments across populations. B78 tumor cells were transfected with the ratiometric pH construct, N1-mCherry-pHlourin. Representative histograms show florescence of pHluorin in mCherry⁺ cells, where less pH-GFP represents a more acidic environment. Grey histograms are respective populations from a non-pHluorin expressing control tumor (B78 parental). Data summarized as the ratio of gMFI between GFP and mCherry fluorescence. Data presented as mean ratio ± SEM, pooled from 3 independent experiments.
(F) Intracellular cytokine stain of IL12 in populations. % of IL12⁺ cells quantified across each population, data pooled from 2 independent experiments, (n=3), plotted as mean ± SEM. Statistical significance indicated by *p<0.05.
(G) Il12b and Il10 transcript levels, measured by qPCR. Data presented as mean ΔCt ± SEM calculated from biological triplicates (n=3) of individual tumors, (N.D. not detected).
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(A) Tumor growth curve plotted as tumor area (mm²) over time for EG7.1 in zDC-DTR hosts. Arrows indicate time of i.p. D.T./PBS administration, and i.v. transfer of 5×10⁶ previously activated OT-I CD8⁺ T cells. DT/PBS was subsequently administered every 3^rd day and FTY-720/Saline was subsequently administered every other day throughout time course. Representative data presented as mean tumor area ± SEM (n=4) from 2 independent experiments. Statistical significance indicated by *p<0.05.
(B) Comparison of prognostic value of CD103⁺/CD103⁻ Ratio Gene Signal as compared to the individual genes (either CD103⁺ specific, green, or TAM1/TAM2/CD11b DC1 specific genes, red) using TCGA datasets in a multivariate COX proportional hazards survival analysis adjusting for cancer type as a covariate. Data expressed as Hazard Ratio (HR) with 95% confidence intervals, where a value <1 means increased Overall Survival (OS); >1 means decreased OS for genes with BH p values<0.05 (bolded values).
(C) Comparison of the prognostic value of the CD103⁺/CD103⁻ Ratio Gene Signal with several published prognostic gene signatures using TCGA datasets in a multivariate COX proportional hazards survival analysis adjusting for cancer type as a covariate. Data expressed as Hazard Ratio (HR) with 95% confidence intervals, where a value <1 means increased Overall Survival (OS); >1 means decreased OS for genes with BH p values<0.05.
(D) K-M plot across all 12 cancer types in Human TCGA data sets, adjusting for cancer type based on HIGH CD103⁺/CD103⁻ gene Ratio and LOW CD103⁺/CD103⁻ Ratio expressers (median split/cancer).
(E) K-M plot for overall survival of Breast Cancer patients in TCGA data set. Data Parsed on HIGH CD103⁺/CD103⁻ gene Ratio and LOW CD103⁺/CD103⁻ Ratio expressers.
(F) K-M plot for overall survival of Head and Neck Squamous Cell Carcinoma patients in TCGA data set. Data Parsed on HIGH CD103⁺/CD103⁻ gene Ratio and LOW CD103⁺/CD103⁻ Ratio expressers.
(G) K-M plot for overall survival of Lung Adenocarcinoma patients in TCGA data set. Data Parsed on HIGH CD103⁺/CD103⁻ gene Ratio and LOW CD103⁺/CD103⁻ Ratio expressers.
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(A) Representative cytometry of tumor APCs in PyMTchOVA x Cx3cr1-eGFP x Cd11c-mCherry. Populations as previously defined, are plotted as mCherry vs. GFP. Green, Yellow and Red circles indicate the fluorescent profile each population displays in this model. Red (mCherry only cells), Yellow (mCherry and GFP double positive cells), and Green (GFP only cells). By flow cytometry DC1/DC2 populations fall in the Cherry-only population, while TAM1 and TAM2 comprise the yellow and green populations respectively.
(B) Intravital 2-photon representative still image of an early carcinoma lesion from a PyMTchOVA x Cx3cr1-eGFP x Cd11c-mCherry reporter. Regions indicated with dashed line, marked either distal or marginating to lesions, were determined with a combination of mCherry fluorescence and collagen structure. Collagen fibers marked (white) by 2^nd harmonic generation. Scale bar 50 μm. Inset: Quantification of Proximal/distal location of the APCs within the tumor. Data pooled from independent imaging runs, presented as mean ± SEM.
(C) Representative confocal still image from live tumor slices in PyMTchOVA x Cx3cr1-eGFP x Cd11cmCherry tumors, stained with CD11b-A647 antibody. mCherry only cell (arrowhead DC2, red) and mCherry⁺ CD11b⁺ cell (arrow DC1, purple) in the tumor. Scale bar 15 μm.
(D) Representative image sequence of CFP expressing OT-1 CD8⁺ T cells (blue) dynamically interacting with APC cells in the PyMTchOVA x Cx3cr1-eGFP x Cd11c-mCherry model by live slice confocal imaging 4 days after T cell transfer at 0, 30 and 60 min. Arrows indicate T cell interactions with Red (DC1/DC2), Green (TAM1) or Yellow (TAM2) cells. Scale bar 30 μm. Last panel displays time projection of CFP expressing T cells through 60 min imaging timeframe, with outline color dictated by APC of contact.
(E) APC-T cell contacts in vivo as a % of total T cell couples observed. Accumulated data of 4 different positions imaged for 30 minutes in 2 independent intravital 2 photon imaging runs. Contacts were scored manually by counting physical contact made between T cells and red, yellow and green APCs. Color of bar represents the APC of contact (Red: CD103⁺, CD11b⁺ DC1, Green: TAM1, yellow: TAM2).
(F) Ex vivo T cell coupling assay with digested tumor positively selected for CD45⁺ cells with previously activated OT-I CD8⁺ T cell. Data calculated as % of T cells couples within each of the populations (left), and as a total % of T cell couples (right). Data pooled from 2 independent experiments, plotted as mean ± SEM.
See also and .