Panel A shows a GSEA Hallmark bar graph and enrichment plots comparing WT and CD1d-knockout macrophage signatures. Panel B shows bar graphs of Nos2, Cxcl10, Il12, Arg1, and Chi3l3 expression in stimulated macrophages, measured in arbitrary units. Panel C shows a tumor study schematic, tumor growth line graph, and organ weight bar graphs. The line graph plots tumor size in square centimeters versus days after orthotopic injection. The bar graphs show tumor, spleen, tumor-draining lymph node, and non-draining lymph node weights in grams. Panel D shows flow cytometry plots and bar graphs of immune cell numbers in WT and CD1d-knockout tumors. Cell populations include major histocompatibility complex 2 positive Ly6C positive, major histocompatibility complex 2 positive Ly6C negative, cDC1, cDC2, Neu, CD11b positive, T-cell receptor beta positive, B, and natural killer cells. The y-axis represents cell numbers per cubic centimeter of tumor. Panel E shows bar graphs of CD4 positive T cells, regulatory T cells, gamma delta T cells, and natural killer T cells in tumors. The y-axis represents cell numbers per cubic centimeter of tumor. Panel F shows flow cytometry plots and bar graphs of interferon gamma positive and granzyme B positive CD8 positive T cells, displaying frequencies and geometric mean fluorescence intensity. Panel G shows a mixed bone marrow chimera schematic and paired scatter plots comparing WT and CD1d-knockout immune cell frequencies. The y-axis represents frequencies as a percentage of CD45 or CD11b cells.
CD1d deficiency remodels the immune infiltrates in the TME in a manner that supports tumor control. (A) Bulk RNA-seq for pMacs isolated from WT or CD1d-KO mice. Left: Results of GSEA Hallmark pathway analysis showing top enriched gene sets. NES values indicate enrichment (red bars, positive NES) in CD1d-KO or WT pMacs (blue bars, negative NES). Right: Enrichment plot for transcriptional signature of CD1d-KO (versus WT) pMacs compared with signatures from macrophages stimulated with LPS + IFN-γ (top) or IL-4 (bottom) (Jablonski et al., 2015). (B) WT or CD1d-KO BMDMs were stimulated with IFN-γ, IFN-γ and LPS, or IL-4 as indicated, and the expression of the depicted genes was measured by qPCR (n = 6–14; data are pooled from 3 to 10 independent experiments). (C) WT and CD1d-KO mice were orthotopically injected with EO771 cells, and tumor growth was monitored over time (left). Bar plots represent weights of tumors, spleens, TDLNs, and NDLNs in WT and CD1d-KO mice (n = 25; data are pooled from eight independent experiments). Data are shown as the mean ± SEM. (D and E) Analyses of immune infiltrates in EO771 tumors from WT or CD1d-KO mice at day 15 after tumor cell injection depicting cell numbers for the indicated cell populations (n = 9–20; data are pooled from three to seven independent experiments). Data are shown as the mean ± SEM. (F) Representative plots (left) and quantification (right) of frequencies and GeoMFI for IFN-γ+ and GzmB+ within CD8+ T cells in WT and CD1d-KO tumors (day 15) (n = 6–8; data are pooled from three independent experiments). Data are shown as the mean ± SEM. (G) Mixed BM chimeras (WT:CD1d-KO; 50:50) were generated by cotransferring WT (CD45.1+) and CD1d-KO (CD45.2+) BM into irradiated recipients (CD45.1+CD45.2+). After reconstitution, mice were orthotopically injected with EO771 cells and immune infiltrates were analyzed at day 15. Data show frequencies of the WT and CD1d-KO depicted populations in the TME as frequency of CD45 (left) or CD11b (right) (n = 8; data are pooled from two independent experiments). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, unpaired (B–F) or paired (G) t test or two-way ANOVA with Sidak’s multiple comparisons (C, tumor growth). TDLNs, tumor-draining lymph nodes; NDLNs, nondraining lymph nodes; NES, normalized enrichment score; GeoMFI, geometric mean fluorescence intensity.