Macrophages impede CD8 T cells from reaching tumor cells and limit the efficacy of anti-PD-1 treatment

Abstract

In a large proportion of cancer patients, CD8 T cells are excluded from the vicinity of cancer cells. The inability of CD8 T cells to reach tumor cells is considered an important mechanism of resistance to cancer immunotherapy. We show that, in human lung squamous-cell carcinomas, exclusion of CD8 T cells from tumor islets is correlated with a poor clinical outcome and with a low lymphocyte motility, as assessed by dynamic imaging on fresh tumor slices. In the tumor stroma, macrophages mediate lymphocyte trapping by forming long-lasting interactions with CD8 T cells. Using a mouse tumor model with well-defined stromal and tumor cell areas, macrophages were depleted with PLX3397, an inhibitor of colony-stimulating factor-1 receptor (CSF-1R). Our results reveal that a CSF-1R blockade enhances CD8 T cell migration and infiltration into tumor islets. Although this treatment alone has minor effects on tumor growth, its combination with anti-PD-1 therapy further increases the accumulation of CD8 T cells in close contact with malignant cells and delays tumor progression. These data suggest that the reduction of macrophage-mediated T cell exclusion increases tumor surveillance by CD8 T cells and renders tumors more responsive to anti-PD-1 treatment.

Keywords: T cells; cancer; immunotherapy; macrophages; migration.

Fig. 1.

CD8 T cell infiltration into tumor islets confers a good prognostic value in lung squamous-cell carcinoma and is related to lymphocyte motility. (A) Survival analysis of patients with stage I–II squamous-cell carcinomas, according to the density of CD8 T cells in tumor islets. Patients (n = 51) with tumor enriched in CD8 T cells were divided into two groups on the basis of the T:S of CD8 T cells. A CD8 T:S value of 0.16 (median) was used as the cutoff to determine high and low groups. Kaplan–Meier curves were used to estimate overall survival of the two groups, and the log-rank test was used to compare the difference between the two curves. (Right) Representative images of human lung tumors stained for cytokeratin (blue) and CD8 T cells (red) with a high and a low CD8 T:S ratio. (B and C) Migration of endogenous CD8 T cells in vibratome sections of viable human lung tumors. (B, Left) Snapshots of a time lapse with trajectories of individual endogenous CD8 T cells (red) in slices stained with anti-EpCAM (blue) for tumor cells. Tracks are color-coded according to CD8 T cell displacement length. (Scale bars: 20 µm.) Tumor #94 has a high CD8 T:S ratio (0.89), whereas tumor #64 has a low CD8 T:S ratio (0.11). (B, Right) Displacement length (Top) and average speed (Bottom) of endogenous CD8 T cells as a function of the percentage of CD8 T cells in tumor islets; n = 9. (C) Displacement length (Left) and average speed (Right) of resident CD8 T cells in the stromal and tumor cell regions of human squamous-cell non-small cell lung cancer tumor slices; n = 9; medians are shown in red. Mann–Whitney test: ***P < 0.001.

Fig. 2.

CD8 T cells are engaged in long-lasting interactions with stromal TAMs in human lung tumors. (A–E) Localization and migration of endogenous CD8 T cells in vibratome sections of viable human lung tumors. (A) Distribution of CD8 T cells, TAMs, and polymorphonuclear cells (PMNs) in the stroma and tumor islets represented as mean ± SEM; n = 8. Mann–Whitney test: **P < 0.01 and ***P < 0.001. (B, Left) Confocal image of a human lung-tumor slice stained for EpCAM (blue), CD11c (green), and CD8 (red). The boxed area is highlighted at higher magnification on the right side. (Scale bars: 100 µm.) (B, Right) Frequency of CD8 T cells in contact with TAMs; n = 7. Mann–Whitney test: *P < 0.05. (C) Tracks of individual endogenous CD8 T cells in relation to TAMs and EpCAM tumor cells. Tracks are color-coded according to CD8 T cell displacement length. (Scale bar: 30 µm.) See also Movies S1 and S2. (D) Displacement length (Left) and average speed (Right) of endogenous CD8 T cells in contact or not with TAM during a 20-min recording; n = 9; Mann–Whitney test: ***P < 0.001. (E) Displacement length (Left) and average speed (Right) of endogenous CD8 T cells in the stroma as a function of the percentage of CD8 T cells in contact with TAMs in the same region for each microscopic field; n = 9 patients, two to four videos for each patient. Each value is the average value for all stromal CD8 T cells of the same slice.

Fig. 3.

TAM depletion increases CD8 T cell number, motility, and infiltration into tumor islets of Met-1–bearing mice. FVB mice bearing Met-1 tumors were treated with PLX3397 from day 7. Between day 24 and 28 tumors were resected and analyzed by flow cytometry (A) or confocal microscopy (C–E). (A) Frequencies of myeloid subsets (Left) and lymphoid subsets (Right) among total live cells in tumors. Results are shown as mean ± SEM; n = 16 mice/group from three independent experiments; Mann–Whitney test: ***P < 0.001. Macrophages are defined as CD11b⁺Ly6G⁻Ly6C⁻F4/80⁺, monocytes as CD11b⁺Ly6G⁻Ly6C⁺, and polymorphonuclear cell (PMN) as CD11b⁺Ly6G⁺Ly6C^int. (B) Tumor volumes determined at the sacrifice of mice, n = 22 mice/group. Medians are shown. Mann–Whitney test: *P < 0.05. (C) Motility of resident CD8 T cells in Met-1 tumor slices from mice treated or not with PLX3397. (C, Left) Trajectories of individual resident CD8 T cells (red) in relation to F4/80⁺ TAMs (green) and EpCAM⁺ tumor cells (blue). (Scale bars: 25 µm.) Tracks are color-coded according to CD8 T cell displacement length. See also Movies S3 and S4. (C, Right) Displacement length (Left) and average speed (Right) of endogenous CD8 T cells in tumor slices from mice treated or not with PLX3397; n = 12 mice/group, two to four time lapses/mouse, three independent experiments; Mann–Whitney test: ***P < 0.001. (D) Distribution of resident CD8 T cells in tumor slices of mice treated or not with PLX3397. (D, Left) Confocal images. (Scale bars: 100 µm.) (D, Right) Proportion of CD8 T cells in tumor islets represented as mean ± SEM; n = 11; Mann–Whitney test: *P < 0.05. (E) Displacement length (Left) and average speed (Right) of endogenous CD8 T cells as a function of the percentage of CD8 T cells in tumor islets. Black and red points correspond to the control and to the PLX3397 conditions, respectively.

Fig. 4.

TAM depletion enhances anti–PD-1 immunotherapy in tumor-bearing mice. FVB mice bearing MET-1 tumors were treated with PLX3397 from day 7. On days 9, 12, 15, and 18, mice were treated or not with anti–PD-1 or control IgG by i.p. injection. Tumors were collected and analyzed between days 24 and 28. (A) Tumor volume for each treatment group. Results are shown as mean ± SEM; n = 21 mice/group from three independent experiments. Mann–Whitney test, isotype vs. other groups: *P < 0.05 and ***P < 0.001. (B) Frequencies of myeloid subsets (Left) and lymphoid subsets (Right) among total live cells in tumors determined by flow cytometry. Results are shown as mean ± SEM; n = 15 mice/group from three independent experiments; Mann–Whitney test, isotype vs. other groups: *P < 0.05 and ***P < 0.001. (C) Proportion of CD8 T cells among total live cells as a function of tumor volumes determined at the endpoint; n = 60. (D) Luminex analysis of inflammatory chemokines produced by fresh Met-1 tumor slices kept in culture for 18 h; n = 10 mice/group from three independent experiments. Mann–Whitney test, isotype vs. other groups: **P < 0.01 and ***P < 0.001. (E) Proportion of intratumoral CD8 T cells among total live cells as a function of CCL2 (Left) and CXCL9 (Right) produced by Met-1 tumor slices kept in culture for 18 h. (F) Chemokine mRNA levels of Met-1 cells sorted from fresh tumors. The expression levels of chemokines were analyzed by qRT–PCR; n = 6 mice/group from two independent experiments; Mann–Whitney test, isotype vs. other groups: **P < 0.01. (G) Granzyme B release in the supernatant of fresh Met-1 tumor slices kept in culture for 18 h were measured by ELISA; n = 10 mice/group from three independent experiments; Mann–Whitney test, isotype vs. other groups: **P < 0.01 and ***P < 0.001.

Fig. 5.

Combination therapy of CSF-1R inhibitor and anti–PD-1 affects CD8 T cell number and motility in tumor islets. FVB mice bearing MET-1 tumors were treated with PLX3397 from day 7. On days 9, 12, 15, and 18, mice were treated or not with anti–PD-1 or control IgG. Tumors were collected and analyzed by confocal microscopy from days 24–28. (A) Distribution of resident CD8 T cells in Met-1 tumors for each condition. (A, Left) Confocal images of Met-1 tumor slices stained for EpCAM (blue), CD8 T cells (green), and fibronectin (red). (Scale bars: 100 µm.) (A, Right) Fraction of resident CD8 T cells in tumor islets of Met-1 tumors; n = 10 mice/group from three independent experiments; Mann–Whitney test, isotype vs. other groups: *P < 0.05 and **P < 0.01. (B) Displacement length (Left) and average speed (Right) of endogenous CD8 T cells in the stroma and tumor islets of Met-1 tumor slices; n = 7 mice/group from three independent experiments, two to three time lapses/mouse. Mann–Whitney test, isotype vs. other groups: ***P < 0.001. (C) Fraction of total CD8 T cells residing in tumor islets and displaying low motility (average speed: <2 μm/min); n = 7 mice/group from three independent experiments, two to three time lapses/mouse; Mann–Whitney test: *P < 0.05.