Targeting CD206+ macrophages disrupts the establishment of a key antitumor immune axis

Abstract

CD206 is a common marker of a putative immunosuppressive "M2" state in tumor-associated macrophages (TAMs). We made a novel conditional CD206 (Mrc1) knock-in mouse to specifically visualize and/or deplete CD206+ TAMs. Early depletion of CD206+ macrophages and monocytes (Mono/Macs) led to the indirect loss of conventional type I dendritic cells (cDC1), CD8 T cells, and NK cells in tumors. CD206+ TAMs robustly expressed CXCL9, contrasting with stress-responsive Spp1-expressing TAMs and immature monocytes, which became prominent with early depletion. CD206+ TAMs differentially attracted activated CD8 T cells, and the NK and CD8 T cells in CD206-depleted tumors were deficient in Cxcr3 and cDC1-supportive Xcl1 and Flt3l expressions. Disrupting this key antitumor axis decreased tumor control by antigen-specific T cells in mice. In human cancers, a CD206Replete, but not a CD206Depleted Mono/Mac gene signature correlated robustly with CD8 T cell, cDC1, and NK signatures and was associated with better survival. These findings negate the unqualified classification of CD206+ "M2-like" macrophages as immunosuppressive.

Graphical abstract

Figure 1.

Genetic myeloid-specific labeling of CD206⁺ macrophages in tumors. (A) Pseudotime plots of select Mono/Mac subsets in B16F10 tumors from Mujal et al. (2022). (B and C) (B) Gating on the equivalent subsets in B78chOVA tumors by flow cytometry and (C) CD206 expression in each of these subsets. (D) Schematic representation of the Mrc1^{LSL-Venus-DTR} knock-in construct before (WT) and after (DTR) Cre-mediated recombination by crossing to the Csf1r^Cre allele. (E and F) (E) Flow cytometry plots showing reporter (Venus) and CD206 expression in different immune cells in d18 B78chOVA tumors in WT (red) and DTR (blue) mice with (F) quantification of relative reporter expression (DTR – WT) in the different immune cells, segregated by CD206 expression. Fold change of relative reporter expression between the CD206⁺ populations of the different myeloid subsets are noted. (G) Reporter expression of the monocyte and TAM subsets shown in B. (H and I) (H) Average distribution of Venus reporter expressing cells in a typical B78chOVA tumor at d18 and (I) corresponding distribution segregated by CD206 expression. Bar graphs show mean ± SEM (F, G, and I); data are representative of at least two independent experiments, each with at least three biological replicates; WT levels averaged from two biological replicates in F; *P < 0.05, **P < 0.01 by paired t test and RM ANOVA and post-hoc t tests in G.

Figure S1.

Conditional CD206 reporter expression in various cell populations with distinct Cre drivers. (A–C) Representative flow cytometry gating scheme to identify myeloid cells and lymphocytes from (A) tumor and tdLN; Flow cytometry plots showing reporter (Venus) and CD206 expression in different immune cell subsets in B d18 B78chOVA tdLN in (red; Mrc1(CD206)^{LSL-Venus-DTR}) and DTR (blue; Csf1r^Cre; Mrc1^{LSL-Venus-DTR}) mice and (C) B78chOVA tumor in WT (red: Mrc1^{LSL-Venus-DTR}) and DTR^L (blue: Lyz2(LysM)^Cre; Mrc1^{LSL-Venus-DTR}) mice. Data are representative of at least two independent experiments.

Figure S2.

Relative abundance of immune cells in control and reporter-expressing mice with DTx administration in different contexts. (A) Relative abundance of different immune populations as a percentage of CD45⁺ cells in Mrc1^{LSL-Venus-DTR} (WT; red) and Csf1r^Cre; CD206^{LSL-Venus-DTR} (DTR; blue) mice with B78chOVA tumor injection (day −5), OT-I adoptive transfer (day 0) and harvest (day 14) without DTx administration. (B and C) Relative abundance of different immune populations as a percentage of live cells with (B) late and (C) early depletion regimens by DTx treatment in B78chOVA tumors. (D) Schematic representation of the experimental setup for analysis of skin in Mrc1^{LSL-Venus-DTR} (WT; red) and Csf1r^Cre; Mrc1^{LSL-Venus-DTR} (DTR; blue) mice with DTx administration. (E) Relative abundance of different immune populations in the skin as a percentage of live cells. (F) Schematic representation of the experimental setup for B78chOVA tumor injection, OT-I T cell adoptive transfer, and early DTx administration with either isotype control or anti-Ly6G antibody treatment and analysis. (G and H) Abundance of different immune populations as (G) cells per g of tumor and (H) percentage of CD45⁺ cells in WT and DTR mice in the above experiment. (I) Abundance of CD44⁺CD103⁺CD69⁺ tissue-resident memory-like cells in B78chOVA tumors at day 18 in the early depletion setting. (J–M) In early DTx administration setting in WT (red; Mrc1^{LSL-Venus-DTR}) and DTR^L (blue; Lyz2(LysM)^Cre; Mrc1^{LSL-Venus-DTR}) mice, relative abundance of (J) TAMs and CD206⁺ TAMs, (K) cDC2, (L) neutrophils, and (M) monocytes. (N) Number of CD8 T cells and NK cells per B78chOVA tdLN from WT and DTR mice at endpoint of early DTx treatment. Bar graphs show mean ± SEM; data are representative of at least two independent experiments, each with at least three biological replicates per group. ****P < 0.0001, **P < 0.01, *P < 0.05, ns = no significance by Student’s t tests or Mann–Whitney test (A–C, E, and I–N), or Kruskal–Wallis test with post-hoc test correcting for false discovery (G and H).

Figure 2.

Early CD206⁺ TAM depletion leads to a coordinated and indirect loss of NK, cDC1, and CD8 T cells in tumors. (A) Schematic representation of the experimental setup for early and late CD206⁺ TAM depletion in B78chOVA tumors using Mrc1(CD206)^{LSL-Venus-DTR} (WT) and Csf1r^Cre; CD206^{LSL-Venus-DTR} (DTR) mice. (B–H and J–P) Relative abundance of different immune populations in B78chOVA tumors as a percentage of CD45⁺ cells with (B–H) late and (J–P) early depletion regimens. (I and Q) Abundance of TAMs gated by CD206 expression, showing percentage depletion of CD206⁺ TAMs in (I) late and (Q) early depletion setting. (R–U) Representative flow cytometry plots showing CD206 versus MHCII expression in different intratumoral myeloid subsets in WT (red) and DTR (blue) mice in the (R) late and (S) early depletion regimens; relative abundance of (T) neutrophils, (U) cDC1, NK cells, and OT-I T cells in the early DTx administration setting with additional anti-Ly6G or isotype control treatment. (V–X) Relative abundance of (V) CD206⁺ cDC2, (W) cDC1, and (X) CD44⁺ CD8 T cells in the early DTx administration setting in WT and DTR^L (Lyz2(LysM)^Cre; CD206^{LSL-Venus-DTR}) mice. Bar graphs show mean ± SEM; data are representative of at least two independent experiments, each with at least three biological replicates per group; ***P < 0.001, **P < 0.01, *P < 0.05, ns = no significance by unpaired Student’s t tests or Mann–Whitney U test (B–Q and V–X) and Kruskal–Wallis test with post-hoc test correcting for false discovery rate (T and U).

Figure 3.

Loss of CXCL9-positive TAMs and CXCR3-expressing, cDC1 supportive lymphocytes with CD206⁺ TAM depletion. (A) Two-photon imaging of representative control and early DTx treated B78chOVA tumors day 12 after adoptive transfer of CD2dsRed; OT-I CD8 T cells showing three zones of Venus-expressing macrophage and associated OT-I T cell localization—edge, mid, and interior (Int.) mapped by spatial transcriptomic barcoding ZipSeq. Boxed regions are magnified (right). Scale bars are 100 and 300 µm in the control (inset) and DTx-treated images respectively. (B) UMAP representation of major immune cell populations obtained from Control and early DTx treated B78chOVA tumors on day 12 after OT-I injection aggregated across all three regions. (C) Summary heatmap showing relative log fold change of the abundance (calculated as the percent of the total number of cells recovered within that region) of each major cluster in Ctrl/DTx treated conditions, split by region of tumor. (D) Flow cytometry data showing an abundance of C1q TAMs and MHCII⁺ monocytes in Ctrl and DTx-treated conditions. (E) Distribution of C1q and Stress-responsive TAMs in the three spatial regions in control B78chOVA tumors. (F and G) Cxcl9 expression (F) aggregated across treatment conditions by cluster and (G) aggregated across clusters by condition. (H and I) (H) Representative flow cytometry plots showing CXCL9 expression in TAMs in early DTx-treated WT and DTR B78chOVA tumors and (I) corresponding quantification. (J and K) (J) Representative flow cytometry plot showing intracellular CXCL9 versus surface CD206 expression in TAMs in B78chOVA tumors at day 14 after OT-I adoptive transfer without depletion, and (K) the same CXCL9 expression split by CD206 positivity. (L) In vitro–activated CD8 T cell migration at 3 h through a 5-µm transwell insert in the presence of sorted CD206⁺ versus CD206⁻ TAMs from B78chOVA tumors, normalized to migration with no TAMs. (M and N) (M) Cxcr3, (N) Flt3l, and Xcl1 expression in the lymphocyte subset (CD8 T cell, NK cell, and CD4 T cell) by treatment group. Bar graphs show mean ± SEM; data are representative of at least two independent experiments, each with at least three biological replicates, except the spatial transcriptomics data (B, C, E–G, M, and N), from one control and one DTx-treated tumor; ***P < 0.001, **P < 0.01, *P < 0.05, ns = no significance by Mann–Whitney test (D), unpaired t test (I), and ratio paired t test (K and L).

Figure S3.

Changes in chemokine expression and immune cell abundance in CD206-depleted tumors, and associated survival data in humans. (A) Dotplot representing top five DEGs and select other genes in each immune cell cluster identified from a harmonized dataset of spatially barcoded Control and DTx treated B78chOVA tumors on day 12 after adoptive transfer of CD2dsRed; OT-I cells. (B and C) Cxcl10 expression (B) aggregated across treatment conditions by cluster and (C) aggregated across clusters by treatment. (D) CXCL9 expression in PyMTchOVA and MC38chOVA (both without OT-I adoptive transfer) TAMs split by their CD206 expression. (E and F) (E) CXCL9 expression in B78chOVA intratumoral monocytes split by CD206 expression and (F) relative abundance of CXCL9⁺ TAMs and monocytes in the same tumors. (G) Flow cytometry plot showing CD206 and CXCL9 expression in cDC2s and TAMs in B78chOVA tumors. (H) Percent of CXCR3⁺ among in vitro activated CD8 T cells prior to transmigration in the presence of CD206⁻ and CD206⁺ TAMs. (I and J) Violin plot representing (I) Cxcr3 and (J) Xcl1 and Flt3l expression in the lymphoid compartment in Control and DTx treated conditions. (K) Tumor growth curves of B78chOVA tumors with OT-I adoption and early and late DTx administration. (L and M) (L) Overlaid flow cytometry plots showing reporter (Venus) and CD206 expression in different immune cells in MC38chOVA tumors in WT (red; Mrc1^{LSL-Venus-DTR}) and DTR (blue; Csf1r^Cre; Mrc1^{LSL-Venus-DTR}) mice and (M) quantification of relative reporter expression (DTR – WT) in the different subsets, split by CD206 expression. (N) Schematic representation of the experimental setup for early and late CD206⁺ TAM depletion in MC38chOVA tumors using Mrc1^{LSL-Venus-DTR} (WT) and Csf1r^Cre; Mrc1^{LSL-Venus-DTR} (DTR) mice. (O and P) Relative abundance of different immune populations as a percentage of CD45⁺ cells with (O) late and (P) early depletion regimens. (Q) Abundance of different immune populations as total number of cells per g of MC38chOVA tumor in WT and DTR mice in the early DTx administration regimen. (R) Number of CD8 T cells and NK cells per tdLN of WT and DTR mice with MC38chOVA tumors and treated with the early DTx regimen. (S) Scatter plots of the CD206^Replete and CD206^Depleted Mono/Mac score per patient with the NK cell score (Pearson R and P value for the null hypothesis that there is not a correlation are noted). (T) Kaplan–Meier survival curves of patients grouped by the value of the CD206^Replete: CD206^Depleted signature ratio (top and bottom 20%) from TCGA split by indications, number of patients per group and P values for the log-rank test are noted for each curve in T. Bar graphs show mean ± SEM; data are representative of at least two independent experiments, each with at least three biological replicates, except the spatial transcriptomics data (A–C, I, and J), from one control and one DTx-treated tumor. ***P < 0.0001, **P < 0.01, *P < 0.05, ns = no significance by paired ratio t tests (D and E) or unpaired t tests or Mann–Whitney test in F and O–Q. n = 203 patients in S.

Figure 4.

CD206⁺ TAM depletion attenuates T cell–mediated tumor control in an immune-responsive tumor model. (A) Representative time course of MC38chOVA tumor size with or without adoptive transfer of OT-I T cells. (B) CD206 expression in monocytes gated by MHCII expression and TAMs gated by VCAM1 and CD127, as shown in Fig. 1 B, but in MC38chOVA tumors. (C and D) (C) Distribution of Venus reporter expressing cells in a typical MC38chOVA tumor at day 18 (d18) and (D) corresponding distribution segregated by CD206 expression. (E) Schematic representation of the experimental setup for early and late CD206⁺ TAM depletion in MC38chOVA tumors using Csf1r^Cre; Mrc1^{LSL-Venus-DTR} mice. (F–H and J–L) Relative abundance of (F and J) CD206⁺ and CD206⁻ TAMs, (G and K) cDC1s. and (H and L) CD8 T cells as a percentage of CD45⁺ cells with late and early depletion regimens respectively. (I and M) Representative flow cytometry plots showing CD206 versus MHCII expression in different myeloid subsets in WT (red) and DTR (blue) mice in the (I) late and (M) early depletion regimens. (N and O) %CXCL9⁺ of TAMs and (O) %C1q TAMs (gated as VCAM1^hiCD127^lo) of CD45 in WT and DTR mice with early depletion. (P) Tumor growth kinetics of MC38chOVA tumors in WT and DTR mice with DTx treatment beginning 2 days after OT-I adoptive transfer at day 0; bar graphs show mean ± SEM; data are representative of at least two independent experiments, each with at least three biological replicates per group for B–O, and with at least five biological replicates per group for A and P. **P < 0.01, *P < 0.05, ns = no significance by Student’s t test or Mann–Whitney U test or unpaired t tests.

Figure 5.

CD206^Replete Mono/Mac signature associates with antitumor immunity in human cancers. (A) Kaplan–Meier survival curves of patients in TCGA grouped by the expression of MRC1 gene. (B and C) (B) UMAP representation of the Mono/Mac subsets and (C) overlay of the CD206 Replete (Ctrl) and depleted (DTx) groups on the UMAP from spatial scSeq described in Fig. 3. (D) Top 10 genes from DGE of Mono/Macs in the Ctrl versus DTx treated conditions, which were used to generate CD206 Replete and CD206 Depleted Mono/Mac signature scores. (E) Expression of select genes defining the CD206 replete (C1qa, Cxcl9, H2-Ab1) and the CD206 depleted (Il1b, S100a8, Spp1) signatures overlaid on the same UMAPYes, those stray dots can go. There are two more encroached into panel D (next to Cxcl9, Apoe) - those can also go. (F) Heatmap of z-scored CD206^Replete, CD8, NK, and SDC score, calculated from sorted immune and live compartments, as previously described (Combes et al., 2022) in colorectal (CRC), gynecological (GYN), head and neck (HNS), kidney (KID), and lung (LUN) cancer patients. (G and H) Scatter plots of the myeloid-specific CD206 Replete and Depleted score per patient with the (G) SDC score and (H) CD8 T cell score (Pearson R and P value for the null hypothesis that there is not a correlation are noted). (I and J) Kaplan-Meier survival curves of patients grouped by the value of the (I) CD206^Replete:CD206^Depleted signature ratio and (J) CD206^Replete signature in TCGA. (K) Expression score for TAMs associated with ICB responsiveness derived from Li et al. (2024) in the CD206^Replete versus CD206^Depleted Mono/Macs; n = 205 patients in G and H; and n = 861 patients per group in I and J; P values for the log-rank test are noted in A, I, and J, and that for the Wilcoxon test is shown in K.