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).

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).

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