Fig 2: Immune evasive NPE-IE tumors possess a highly immunosuppressive TME, related to Figure 3(A) & (B) Gating strategy to define myeloid (A) and lymphoid (B) populations in NPE and NPE-IE tumors.(C) Representative histograms of PD-L1 expression on microglia and macrophages in NPE-IE tumors (linked to Figure 3E). Control represents fully stained sample minus anti-PD-L1 antibody.(D) Representative histograms of CD206, CD86 and CD11c expression on macrophages from NPE and NPE-IE tumors. Data derived from 3 tumors randomly down sampled for 50,000 live cells each. Control represents fully stained sample minus either anti-CD206, anti-CD86 or anti-CD11c antibodies.(E) Flow cytometry quantification of the frequency of macrophages, M-MDSC and microglia positive for expression of PD-L1(F) Flow cytometry quantification of the frequency of NK cells as a percentage of live cells.(G) Flow cytometry quantification of the frequency of CD11b+ DCs as a percentage of live cells.(H) Flow cytometry quantification of CD11b+ DCs positive for expression of PD-L1.(I) Survival analysis of NSG mice orthotopically injected with NPE-IE cells subjected to IP injection of aCSF-1R or PBS (n = 5 mice + ?CSF-1R; n = 3 mice +PBS; p = 0.2367).

Fig 3: Total-Seq-A antibody validationSplenocytes were incubated with flow cytometry live-dead stain, Fc-block and antibodies against MHCII and CD11c, and each Total-Seq-A antibody which was conjugated to a dT-oligonucleotide tagged fluorescence dye. Flow cytometry and Total-Seq-A antibodies against CD11c were specific to different epitopes.

Fig 4: Expected outcome of a bioinformatic unbiased analysis of the 16-color murine myeloid flow cytometry panel(A) Single alive CD45+ CD3- CD19- cells were subjected to bioinformatic FlowSOM analysis based on expression of Ly6G, Ly6C, PDCA1, CD11b, CD11c, F4/80, MHCII, CD172a, CD24, XCR1, and CD4 with a metacluster number of 40. User-determined thresholds for positive signal were applied to all features. The analysis identified 40 distinct clusters of cells.(B) Clusters were annotated based on key myeloid lineage features.

Fig 5: DC sortingMyeloid enriched splenocytes were incubated with live/dead stain, Fc-block and antibodies against CD11c and MHCII. DCs were sorted as alive single CD11c+MHCII+ cells to a purity >90%.