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. 2018 Mar 5;215(3):877-893.
doi: 10.1084/jem.20171435. Epub 2018 Feb 7.

Myeloid-targeted Immunotherapies Act in Synergy to Induce Inflammation and Antitumor Immunity

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Free PMC article

Myeloid-targeted Immunotherapies Act in Synergy to Induce Inflammation and Antitumor Immunity

Curtis J Perry et al. J Exp Med. .
Free PMC article

Abstract

Eliciting effective antitumor immune responses in patients who fail checkpoint inhibitor therapy is a critical challenge in cancer immunotherapy, and in such patients, tumor-associated myeloid cells and macrophages (TAMs) are promising therapeutic targets. We demonstrate in an autochthonous, poorly immunogenic mouse model of melanoma that combination therapy with an agonistic anti-CD40 mAb and CSF-1R inhibitor potently suppressed tumor growth. Microwell assays to measure multiplex protein secretion by single cells identified that untreated tumors have distinct TAM subpopulations secreting MMP9 or cosecreting CCL17/22, characteristic of an M2-like state. Combination therapy reduced the frequency of these subsets, while simultaneously inducing a separate polyfunctional inflammatory TAM subset cosecreting TNF-α, IL-6, and IL-12. Tumor suppression by this combined therapy was partially dependent on T cells, and on TNF-α and IFN-γ. Together, this study demonstrates the potential for targeting TAMs to convert a "cold" into an "inflamed" tumor microenvironment capable of eliciting protective T cell responses.

Figures

Figure 1.
Figure 1.
Myeloid cell heterogeneity in Braf/Pten tumors. (A) Immune cellularity of tumors from flow cytometry at end point, 8 wk after tumor induction. TAMs were defined as CD45+ CD11b+ Ly6G, distinct from TANs (CD45+ CD11b+ Ly6G+), and tumor-infiltrating DCs (TIDCs; CD45+ CD11c+ CD11b Ly6G). Regulatory CD4+ T (T reg) cells were defined as CD45+ CD3+ CD4+ Foxp3+ and distinguished from non-regulatory CD4+ T cells by Foxp3 expression. CD45+ CD8+ CD3+ defined CD8 T cells. Data are from three independent experiments (n = 9–12). (B) F4/80 and CD3 IHC representative images from tumors at end point (1 cm3). Bars, 150 µm. (C) F4/80+ and CD3+ nuclei were counted per hpf from IHC images of tumors (as in B) from two independent experiments, 6–15 hpf/tumor (n = 12–30). Significance was determined by unpaired Student’s t test. (D) Contour plot shows expression of Ly6C and F4/80 on TAMs from end point (1 cm3) tumors. Plot isconcatenated (combined) from each individual mouse tumor (n = 6). The mean ± SD for each subset (Ly6C+ F4/80int, Ly6C F4/80int, and Ly6C F4/80hi) is reported. Data are from one experiment and are representative of three experiments (n = 6 each group). (E) Bar graphs show mean fluorescence intensity (MFI) or percentage expressing the indicated proteins for the three TAM subsets outlined in D from end point (1 cm3) tumors. Data are from one experiment and are representative of three experiments (n = 6 each group). Significant differences between groups were determined by one-way ANOVA with Holm-Sidak multiple comparisons correction; *, P < 0.05; **, P < 0.01; ****, P < 0.001.
Figure 2.
Figure 2.
Functional heterogeneity in TAM subsets in Braf/Pten melanomas. (A) Polyfunctionality of TAMs isolated from end point (1 cm3) tumors. Bar graphs show fraction of single cells captured cosecreting 0, 1, 2, or >3 targets. Data are pooled from two independent experiments. (B) Violin plots of single-cell secretion profiling results for sorted CD11b+ TAMs. Black bar indicates calculated threshold of detection. Data are pooled from two independent experiments. a.u., arbitrary units. (C) Fractions of cells secreting each target from sorted subpopulations. Error bars represent the 95% confidence interval calculated by bootstrapping (see Statistics in Materials and methods for details). Statistical significance determined by nonoverlapping confidence intervals. *, P < 0.05. (D) 2D t-SNE representation of single TAMs based on the secretion levels of 15 proteins. Functional TAM clusters were identified with PhenoGraph from three independent tumors (Fig. S2). TAMs were isolated from end point (1 cm3) tumors, sorted into subpopulations by surface markers (Ly6C+ F4/80int, Ly6C F4/80int, and Ly6C F4/80hi), and analyzed for secretion. TAMs that did not express any of the 15 measured targets above the detection limit were excluded from the analysis. t-SNE map shows all sorted TAM subpopulations pooled together from two independent experiments. Clusters coded by color (see key). (E) Percentage of cells in each cluster from D secreting each of the 15 measured targets. Cluster names are based on the primary secreted target(s) in each cluster. (F) Stacked bar graph shows the distribution of functional clusters in D within each sorted subpopulation (Ly6C+ F4/80int, Ly6C F4/80int, and Ly6C F4/80hi). Clusters coded by color (see key).
Figure 3.
Figure 3.
Effects of CSF-1Ri and CD40 agonist treatment alone or in combination on TAM subsets. (A) Braf/Pten mice were treated with CSF-1Ri (600 mg PLX6134/kg chow) and/or CD40 (10 mg FGK4.5/kg every 3 d) 30 d after tumor induction until end point (day 60) for a total of 10 doses of FGK4.5. Line graphs show size of Braf/Pten melanomas over time. Data are the mean ± SEM of tumor size of five independent experiments (n = 15–25). Significance was determined by one-way ANOVA with Holm-Sidak correction for multiple comparisons and post-hoc unpaired Student’s t tests at the end point (day 60). (B) Bar graphs show numbers per gram of tumor of infiltrating immune cell types (as indicated) in control tumors or tumors treated with CSF-1Ri, CD40, or the combination as measured by flow cytometry at the end point (day 60). Immune cell populations defined as in Fig. 1 A. Data are from two independent experiments (n = 6–12). Significance was determined using one-way ANOVA with Holm-Sidak multiple comparisons correction. (C–E) Flow plots show number of Ly6C+ F4/80int TAMs (C), percentage of CD206+PD-L1+ TAMs (D), and percentage of TAMs expressing TNF-α (E) from control tumors or those treated with CSF-1Ri, CD40, or the combination at the end point (day 60). Flow plots show concatenated plots of each individual mouse tumor. Data are from one experiment (n = 3–6) representative of five independent experiments (total n = 8–17). Significance was determined by one-way ANOVA with Holm-Sidak multiple comparisons correction. (F) Flow plots show infiltrating TANs in the tumors as determined by Ly6G+ CD11b+ staining and flow cytometry. Data are from three independent experiments (n = 6–11). Significance determined by one-way ANOVA with Holm-Sidak multiple comparisons correction; *, P < 0.05; **, P < 0.01; ***, P < 0.005; ****, P < 0.001.
Figure 4.
Figure 4.
Changes in myeloid cell mRNA expression patterns in response to CSF-1Ri and CD40 agonist treatment alone or in combination. (A) Principal component analysis of RNA sequencing data: all expressed genes were analyzed by principal component analysis (SIMCA). Data are from three independent experiments of CD11b+ TAMs sorted from end point (day 60)-pooled tumors treated as described in Fig. 3 A and Fig. S1 A with mRNA isolated and libraries prepared from all four groups in triplicate (n = 3 for each group). (B) RNA sequencing profile of TAMs showing the log2fold change of treatments over control. Data are from three independent experiments (n = 3 each group, with three individual tumors pooled for each sample). To find differentially regulated sets of genes for signature generation, a 1.5 log2fold change difference between samples and p-value–adjusted (Holm-Sidak) to ≤ 0.01 was used. (C) The most enriched gene set of CSF-1Ri+CD40 is positively correlated with BMDM 40 min after treatment with LPS (GSE14769). NES = 1.89, Nominal p-value = 0.0, FDR q-value = 0.055, Family-wise error rate (FWER) p-value = 0.055. FDR, false discovery rate; NES, normalized enrichment score. (D) Selected transcription factors, cytokines, and chemokines consistent with inflammatory signaling by TAMs. RNA sequencing profile of TAMs showing the log2fold change of treatments over control. Data are from three independent experiments (n = 3 each group, with three individual tumors pooled for each sample).
Figure 5.
Figure 5.
Combined CSF-1Ri and CD40 agonist treatment drives formation of a poly-functional inflammatory subset of TAMs. (A) Violin plot of single-cell secretion profiling results for control and CSF-1Ri+CD40-treated TAMs. TAMs were sorted at end point (day 60) and analyzed for single-cell secretion from mice treated as described in Fig. 3 A. a.u., arbitrary units. (B) Heat maps of the log2fold change of CSF-1Ri+CD40 treatment over control for the percentage of TAMs secreting as measured by single-cell secretion profiling (left) and gene expression as measured by RNA sequencing (right) for the indicated targets. Nonsignificant changes (P > 0.05) were set to 0. (C) 2D t-SNE representation of single TAMs from control and CSF-1Ri+CD40-treated tumors based on secretion levels of 15 proteins. Functional TAM subsets were identified by clustering with PhenoGraph. TAMs that did not express any of the 15 measured targets above the detection limit were excluded from the analysis (∼50% in control and ∼50% in treated; data not depicted). (D) Percentage of cells in each subset in C secreting each target signal. Cluster names are based on the primary secreted target(s) in each cluster. (E) Functional TAM subsets ranked by their prevalence in CSF-1Ri+CD40 treatments versus controls, normalized by cell number. (F) Fraction of cells cosecreting TNF-α, IL-6, and IL-12 in control and CSF-1Ri+CD40-treated tumors. Error bars represent a 95% confidence interval calculated by bootstrapping. Statistical significance determined by nonoverlapping confidence intervals; *, P < 0.05. (G) 2D t-SNE representation of single-cell cytokine expression from individual TAMs from control and CSF-1Ri+CD40-treated tumors as in C colored by relative expression of the indicated cytokine.
Figure 6.
Figure 6.
Antitumor immunity provided by combined CSF-1Ri, and CD40 agonist therapy is partially dependent on T cells and on the inflammatory cytokines TNF-α and IFN-γ. (A) T cell depletion interfered with tumor growth suppression from combined CSF-1Ri and CD40 agonist therapy. Braf/Pten mice were treated with CSF-1Ri (600 mg PLX6134/kg chow) and/or CD40 (10 mg FGK4.5/kg every 3 d), with or without T cell depletion (10 mg GK1.5 and TIB210/kg every 3 d), 30 d after tumor induction until end point (day 45) for a total of five treatments with FGK4.5 + GK1.5/TIB210. Line graphs show the size of Braf/Pten melanomas over time. Data are presented as mean + SEM of tumor size of two independent experiments (n = 6 for each group). Data were compared using one-way ANOVA with Holm-Sidak correction for multiple comparisons (day 45). (B) The top eight T cell pathways significantly modulated by CSF-1Ri+CD40 treatment compared with control identified by ingenuity pathway analysis (sorted by ascending p-values). Pathway analysis was performed on genes that had an absolute log2fold change >1 and P < 0.05. (C) Ifng transcription by CD3+ T cells from control and CSF-1Ri+CD40-treated tumors. For B and C, cells were isolated from end point (day 60), and sorted in parallel with the TAMs in Fig. 4 A with the sort layout outlined in Fig. S1 A. Sorted cells were CD3+/CD45+/CD11b/LiveDEAD. Data are from two to three independent experiments (n = 2–3 each group, with three individual tumors pooled for each sample). (D) Kaplan-Meier curves show time to tumor end point (1 cm3) in groups of Braf/Pten mice that were treated with CSF-1Ri chow (600 mg PLX6134/kg chow) and CD40 agonistic antibody (10 mg FGK4.5 clone/kg every 3 d i.p.) with or without TNF-α (10 mg XT3.11/kg every 3 d) and/or IFN-γ (10 mg XMG1.2/kg every 3 d) blocking antibody starting at day 30 after tumor induction. End point was tumor volume >1 cm3. Data are from one experiment (n = 3–6) representative of three independent experiments (total n = 6–15). **, P < 0.01; ****, P < 0.001.

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