CD36-mediated ferroptosis dampens intratumoral CD8+ T cell effector function and impairs their antitumor ability

Abstract

Understanding the mechanisms underlying how T cells become dysfunctional in a tumor microenvironment (TME) will greatly benefit cancer immunotherapy. We found that increased CD36 expression in tumor-infiltrating CD8⁺ T cells, which was induced by TME cholesterol, was associated with tumor progression and poor survival in human and murine cancers. Genetic ablation of Cd36 in effector CD8⁺ T cells exhibited increased cytotoxic cytokine production and enhanced tumor eradication. CD36 mediated uptake of fatty acids by tumor-infiltrating CD8⁺ T cells in TME, induced lipid peroxidation and ferroptosis, and led to reduced cytotoxic cytokine production and impaired antitumor ability. Blocking CD36 or inhibiting ferroptosis in CD8⁺ T cells effectively restored their antitumor activity and, more importantly, possessed greater antitumor efficacy in combination with anti-PD-1 antibodies. This study reveals a new mechanism of CD36 regulating the function of CD8⁺ effector T cells and therapeutic potential of targeting CD36 or inhibiting ferroptosis to restore T cell function.

Keywords: CD36; CD8(+) T cells; ferroptosis; lipid peroxidation.

Figure 1.. Increased CD36 expression on tumor-infiltrating CD8⁺ T cells is associated with tumor progression and poor survival in human and murine cancers

(A) IPA analysis of canonical pathway changes and (B) CD36 expression in tumor-infiltrating CD8⁺ T cells from long- and short-survival melanoma patients. (C) CD8⁺ T cells from MM and MGUS patient’s bone marrow analyzed for the expression of CD36. (D) CD36 expression of CD8⁺ T cells from MM patient’s bone marrow and blood. (E) Tumor volume of the mice and CD36 expression on tumor-infiltrating CD8⁺ T cells at days 7 and 14 after tumor injection. (F) Tumor burden of the mice and CD36 expression on tumor-infiltrating CD8⁺ T cells after Vk*MYC MM cell injection. (G and H) CD36 expression on cultured CD8⁺ T cells with tumor tissue (20 or 50 μl at 1 mg/ml), βCD, or cholesterol treatment. (I and J) Tumor growth curves, tumor burden at week 3, and survival of B16 bearing WT and CD36^−/− B6 mice. (K and L) WT and CD36^−/− B6 mice were injected with anti-CD8 antibodies 1 day before B16 tumor injection and every 3 days for a total of 5 injections. Tumor growth curves, tumor volumes at week 4, and mouse survival are shown. Differences in survival curves between the groups were analyzed by log-rank (Mantel–Cox) test. Data are presented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001. See also Figure S1.

Figure 2.. CD36 expression decreases cytotoxic cytokine production and impairs antitumor function of human and murine CD8⁺ T cells

(A) Correlation between CD36 and cytotoxic cytokine expressions in melanoma patient’s tumor-infiltrating CD8⁺ T cells. (B) Expression of IFNγ and TNFα by tumor-infiltrating CD8⁺ T cells at days 7 and 14 after B16 tumor injection. (C-E) Tumor-infiltrating (C), spleen (D), draining lymph node and lymph node (E) CD8⁺ T cells were analyzed for the expression of IFNγ or TNFα at day 14 after tumor injection in WT and CD36^−/− B6 mice. (F-H) Tumor-infiltrating lung (F), spleen (G), draining lymph node and lymph node (H) CD8⁺ T cells were analyzed for the expression of IFNγ and TNFα at day 14 after tumor injection in WT and CD36^−/− B6 mice. (I) Tumor foci in the lung were counted after 2 weeks of B16 tumor injection in WT and CD36^−/− B6 mice. (J) Expression of IFNγ and TNFα at day 28 by tumor-infiltrating CD8⁺ T cells after Vk*MYC cell injection in WT or CD36^−/− B6 mice. (K) Tumor development at day 28 in WT and CD36^−/− B6 mice after Vk*MYC cell injection. (L) At day 16 after lung B16 tumor inoculation, adoptively transferred WT or CD36^−/− CD8⁺ Pmel-1 T cells in tumor, lymph nodes, and spleen were analyzed for IFNγ expression. (M) Adoptively transferred WT or CD36^−/− CD8⁺ Pmel-1 T cells in s.c. B16 tumor were analyzed for IFNγ and TNFα expression at day 17 after tumor inoculation. (N) B16 tumor burden and survival of mice treated with WT or CD36^−/− CD8⁺ Pmel-1 T cells. Data are presented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001. See also Figure S2.

Figure 3.. CD36 regulates transcriptional and metabolic programs, including lipid peroxidation, in tumor-infiltrating human and murine CD8⁺ T cells

(A and B) Pathway analysis of changes between tumor-infiltrating CD36^−/− CD8⁺ and WT CD8⁺ T cells. (C) Heatmap and (D) gene-set enrichment analysis of lipid peroxidation- and ferroptosis-related genes. (E-G) Single cell sequencing analysis of signaling pathway (E), gene set enrichment (F) and gene expression of melanoma patient’s tumor-infiltrating CD8⁺ T cells (G). (H-K) Tumor-infiltrating CD8⁺ T cells from WT or CD36^−/− mice were analyzed for lipid peroxidation (H), cell death (I), iron (J) and cytosolic ROS (K) 2 weeks after tumor inoculation. Data are presented as mean ± SEM. **p < 0.01; ***p < 0.001. See also Figure S3 and Table S1.

Figure 4.. CD36 mediates ferroptosis and reduces cytotoxic cytokine production in human and murine CD8⁺ T cells

(A and B) Tumor-infiltrating CD8⁺ T cells were analyzed for CD36 expression, lipid peroxidation and 7-AAD 12 days after tumor inoculation. (C-F) Tumor-infiltrating CD8⁺ T cells were analyzed for lipid peroxidation and cell death at days 7 and 14 after tumor injection (C and D). Spleen and tumor-infiltrating CD8⁺ T cells were analyzed for CD36 expression (E), lipid peroxidation (F) and cell death (G) at day 14 after tumor injection. (H-J) Cultured CD8⁺ T cells were treated with tumor tissue (50 μl at 1 mg/ml) for 1 day before analyzed for CD36 expression (H), lipid peroxidation (I), and IFNγ or TNFα expression (J). (K and L) Cultured WT or CD36^−/− CD8⁺ T cells were analyzed for lipid peroxidation, cell death (K) and IFNγ expression (L) after culture with tumor tissue for 1 day (50 μl at 1 mg/ml). (M) Cultured T cells were analyzed for lipid peroxidation and IFNγ or TNFα expression with or without tumor tissue (50 μl at 1 mg/ml) and ferrostatin-1 (5 μM) treatment. (N) Bone marrow tumor-infiltrating CD8⁺ T cells were analyzed for lipid peroxidation and cell death at days 10, 20 or 30 after Vk*MYC cell injection. (O and P) Tumor-infiltrating CD8⁺ T cells from MM patients were analyzed for CD36 expression, lipid peroxidation and cell death. (Q-S) CD8⁺ T cells from MM or MGUS patient’s bone marrow and blood were analyzed for lipid peroxidation and cell death. (T and U) Cultured human CD8⁺ T cells were treated with MM patient bone marrow plasma (100 μl) and analyzed for CD36 expression (T), and lipid peroxidation (U). (V) Cultured human CD8⁺ T cells were treated with MM patients’ bone marrow plasma (100 μl) and CD36-blocking antibodies (15 μg/ml) before analyzed for lipid peroxidation and cell death. Ferro: ferrostatin-1; S: supernatant. Data are presented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001. See also Figure S4.

Figure 5.. CD36 mediates ferroptosis and reduces cytotoxic cytokine production in CD8⁺ T cells through uptake of fatty acid

(A-C) Fatty acid contents of tumor, spleen and normal adjacent skin 2 weeks after tumor inoculation (A), tumor-bearing lung (2 weeks after tumor inoculation) and normal lung tissues (B), tumor-bearing bone marrow (4 weeks after tumor inoculation) and normal bone marrows from tumor free B6 mice (C). (D-F) Lipid peroxidation, cell death (D and E), IFNγ or TNFα expression (F) of the T cells measured after fatty acid (μl) or RSL-3 (10 μM) treatment during T cell differentiation. RSL-3 (10 μM) served as a positive control for inducing ferroptosis (E). (G-I) Cultured T cells were analyzed for lipid peroxidation, cell death (G) and IFNγ or TNFα expression (H and I) after treatment with fatty acid (5 μl) or ferrostatin-1 (μM). (J and K) Lipid peroxidation, cell death (J), and IFNγ or TNFα expression (K) of human T cells measured after treatment with fatty acid (5 μl). (L and M) Lipid peroxidation (L) and IFNγ or TNFα expression (M) of human T cells measured after treatment with fatty acid (5 μl) and ferrostatin-1 (5 μM). (N) Fatty acid uptake assay was performed on cultured WT or CD36^−/− CD8⁺ T cells using fatty acid uptake kit. (O-R) Lipid peroxidation (O), cell death (P), and IFNγ or TNFα expression (Q and R) of WT or CD36^−/− CD8⁺ T cells measured after treatment with fatty acid (5 μl). (S and T) Lipid peroxidation, cell death (S), and IFNγ or TNFα expression (T) of human T cells measured after treatment with fatty acid (5 μl) and CD36-blocking antibodies (10 μg/ml). FA: fatty acid; Ferro: ferrostatin-1. Data are presented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001. See also Figure S5 and S6.

Figure 6.. Targeting ferroptosis or CD36 enhances the efficacy of CD8⁺ T cell- and ICB-based cancer immunotherapy

Fatty acid (5 μl), RSL-3 (10 μM), or ferrostatin-1 (5 μM) was added during T cell differentiation. (A-D) Relative numbers of transferred T cells measured by flow cytometry on day 7 (A) or 14 (B) after T cell transfer. Lipid peroxidation and 7-AAD of transferred T cells were measured by flow cytometry on day 14 (B). IFNγ or Ki67 expression of transferred T cells were measured by flow cytometry on day 7 (C and D). (E and F) B16 tumor growth and survival of mice treated with adoptive transfer of control CD8⁺ Pmel-1 T cells, fatty acid-, RSL-3-, or ferrostatin-1- treated Pmel-1 T cells (E), and tumor growth and survival of mice treated with IgG control, anti-PD-1 antibodies, CD8⁺ Pmel-1 T cells, CD36^−/− CD8⁺ Pmel-1 T cells, CD8⁺ Pmel-1 T cells in combination with anti-PD-1 antibodies, or CD36^−/− CD8⁺ Pmel-1 T cells in combination with anti-PD-1antibodies (F). (G) Schematic diagram showing the mechanism underlying fatty acid-induced dysfunction in CD8⁺ effector T cells in TME. FA: fatty acid; Ferro: ferrostatin-1. Differences in survival curves between the groups were analyzed by log-rank (Mantel–Cox) test. Data are presented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.