Tumor cells dictate anti-tumor immune responses by altering pyruvate utilization and succinate signaling in CD8+ T cells

Abstract

The tumor microenvironment (TME) is a unique metabolic niche that can inhibit T cell metabolism and cytotoxicity. To dissect the metabolic interplay between tumors and T cells, we establish an in vitro system that recapitulates the metabolic niche of the TME and allows us to define cell-specific metabolism. We identify tumor-derived lactate as an inhibitor of CD8⁺ T cell cytotoxicity, revealing an unexpected metabolic shunt in the TCA cycle. Metabolically fit cytotoxic T cells shunt succinate out of the TCA cycle to promote autocrine signaling via the succinate receptor (SUCNR1). Cytotoxic T cells are reliant on pyruvate carboxylase (PC) to replenish TCA cycle intermediates. By contrast, lactate reduces PC-mediated anaplerosis. The inhibition of pyruvate dehydrogenase (PDH) is sufficient to restore PC activity, succinate secretion, and the activation of SUCNR1. These studies identify PDH as a potential drug target to allow CD8⁺ T cells to retain cytotoxicity and overcome a lactate-enriched TME.

Keywords: T cells; cancer metabolism; lactate; pyruvate; succinate; tumor immunity.

Figure 1.. TME mimicking nutrient availability impairs CD8⁺ T cell cytotoxicity and PC activity.

(A) Schematic representation of CD8⁺ T cell activation and co-culture with tumor cells in RPMI and CM. T indicates T cells in mono-culture and T co-c indicates T cells in co-culture with tumor cells. (B) Cell number of B16-OVA tumor cells in RPMI and CM. Analysis was performed after 24 hours. (C) Cell number of CD8⁺ T cells in mono-culture in RPMI and CM. Analysis was performed after 24 hours. (D-F) Killing and geometric mean of GzmB and IFNγ expression by CD8⁺ T cells in co-culture with B16-OVA tumor cells in RPMI and CM. Analysis was performed after 24 hours. (G) Secreted IFNγ levels by CD8⁺ T cells in co-culture with B16-OVA tumor cells in RPMI and CM. Analysis was performed after 24 hours. (H) Geometric mean of Ki67 expression by CD8⁺ T cells in co-culture with B16-OVA tumor cells in RPMI and CM. Analysis was performed after 24 hours. (I) Schematic representation of cell-size-based separation of B16 tumor cells and CD8⁺ T cells. (J) Flow cytometric measurement of T cell populations before and after filtration. (K) Schematic representation of ¹³C₆-glucose contribution to TCA cycle intermediates via PDH or PC activity. Grey arrow depicts low activity. White circles indicate carbon-12 (¹²C) while black circles indicate ¹³C atoms from glucose. (L-M) Contribution of ¹³C₆-glucose to malate and fumarate M+2 (derived from PDH activity) and M+3 (derived from PC activity) in CD8⁺ T cells in mono-culture and in co-culture with B16-OVA tumor cells in RPMI and CM. Analysis was performed after 6 hours. The number of biological replicates for each experiment was n=3. Error bars represent s.d. Two-tailed unpaired Student’s T-test was performed. *P<0.05; **P<0.01; ***P<0.001. See also Figure S1.

Figure 2.. Lactate secretion by tumor cells rewires CD8⁺ T cell metabolism.

(A) Heatmap representing metabolite changes in tumor CM compared to RPMI. CM was obtained after 24 hours culture of tumor cells. (B) Absolute extracellular lactate levels in RPMI and CM. CM was obtained after 24 hours culture of tumor cells. (C) Schematic representation of plasma and TIF isolation from mice bearing B16-OVA tumors. Analysis was performed at day 18 after tumor implantation. (D) Absolute lactate levels in TIF of mice with B16-OVA tumors. (E) PC activity of CD8⁺ T cell in co-culture with B16-OVA tumor cells in CM, in CM obtained from tumor cells cultured with LDH inhibitor or in CM obtained from tumor cells cultured with LDH inhibitor + supplementation of lactate. Analysis was performed after 6 hours. (F) PC activity of CD8⁺ T cell in co-culture with B16-OVA tumor cells in RPMI and RPMI + lactate (10mM). Analysis was performed after 6 hours. (G-I) Killing and and geometric mean of GzmB and IFNγ expression by CD8⁺ T cells in co-culture with B16-OVA tumor cells in RPMI or RPMI + lactate (10mM). Analysis was performed after 24 hours. (J) Secreted IFNγ levels by CD8⁺ T cells in co-culture with B16-OVA tumor cells in RPMI or RPMI + lactate (10mM). Analysis was performed after 24 hours. The number of biological replicates for each experiment was n≥3. Error bars represent s.d. Two-tailed unpaired Student’s T-test was performed. *P<0.05; **P<0.01; ***P<0.001. See also Figure S2.

Figure 3.. Inhibition of PDH increases PC activity and CD8⁺ T cell cytotoxicity through TCA cycle anaplerosis.

(A) Schematic representation of PC and PDH activity. Inhibition of PDH is achieved by using the inhibitor CPI-613 at the concentration of 100μM. (B-D) Killing and geometric mean of GzmB and IFNγ expression by CD8⁺ T cells transduced with shRNA for PC or CTRL and cultured with B16-OVA tumor cells in RPMI. Analysis was performed after 24 hours. (E) Secreted IFNγ levels by CD8⁺ T cells transduced with shRNA for PC or CTRL and cultured with B16-OVA tumor cells in RPMI. Analysis was performed after 24 hours. (F) PC activity of CD8⁺ T cells in co-culture with B16-OVA tumor cells pre-treated with or without the PDH inhibitor CPI-613 in RPMI + lactate (10mM). CD8⁺ T cells were pre-treated with the PDH inhibitor for 24 hours (prior co-culture). Analysis was performed after 6 hours. (G) Killing by CD8⁺ T cells in co-culture with B16-OVA tumor cells pre-treated with or without the PDH inhibitor CPI-613 in RPMI + lactate (10mM). CD8⁺ T cells were pre-treated with the PDH inhibitor for 24 hours (prior co-culture). Analysis was performed after 24 hours. (H) PC activity of CD8⁺ T cells in co-culture with B16-OVA tumor cells pre-treated with or without the PDH inhibitor CPI-613 in RPMI. CD8⁺ T cells were pre-treated with the PDH inhibitor for 24 hours (prior co-culture). Analysis was performed after 6 hours. (I-J) Killing and geometric mean of IFNγ expression by CD8⁺ T cells in co-culture with B16-OVA tumor cells pre-treated with or without the PDH inhibitor CPI-613 in RPMI. CD8⁺ T cells were pre-treated with the PDH inhibitor for 24 hours (prior co-culture). Analysis was performed after 24 hours. (K) Secreted IFNγ levels by CD8⁺ T cells in co-culture with B16-OVA tumor cells pre-treated with or without the PDH inhibitor CPI-613 in RPMI. CD8⁺ T cells were pre-treated with the PDH inhibitor for 24 hours (prior co-culture). Analysis was performed after 24 hours. (L-M) Killing and and geometric mean of IFNγ expression by CD8⁺ T cells transduced with shRNA for PDH or CTRL in co-culture with B16-OVA tumor cells in RPMI. Analysis was performed after 24 hours. (N) Secreted IFNγ levels by CD8⁺ T cells transduced with shRNA for PDH or CTRL in co-culture with B16-OVA tumor cells in RPMI. Analysis was performed after 24 hours. (O) Killing by CD8⁺ T cells transduced with shRNA for PC and in co-culture with B16-OVA tumor cells in RPMI +/− supplementation with OAA, malate, or fumarate. Analysis was performed after 24 hours. (P) Killing by CD8⁺ T cells in co-culture with B16-OVA tumor cells in RPMI + lactate (10mM) +/− supplementation of OAA, malate, or fumarate. Analysis was performed after 24 hours. The number of biological replicates for each experiment was n≥3. Error bars represent s.d. Two-tailed unpaired Student’s T-test was performed. *P<0.05; **P<0.01; ***P<0.001. See also Figure S2 and S3.

Figure 4.. PC activity enables succinate secretion and CD8⁺ T cell function.

(A) Metabolite secretion in RPMI and CM from CD8⁺ T cells in mono-culture or in co-culture with B16-OVA tumor cells. Analysis was performed after 6 hours. (B) SDHA activity of CD8⁺ T cells in mono-culture and CD8⁺ T cells in co-culture with B16-OVA tumor cells in RPMI. Analysis was performed after 6 hours. To determine SDHA activity, oxygen consumption rate was measured after pre-treatment with rotenone and treatment with succinate. (C) Killing by CD8⁺ T cells in co-culture with B16-OVA tumor cells in RPMI, CM and RPMI + lactate (10mM) +/− supplementation of dimethyl-succinate. Analysis was performed after 24 hours. (D) Secreted IFNγ levels by CD8⁺ T cells in co-culture with B16-OVA tumor cells in RPMI, CM and RPMI + lactate (10mM) +/− supplementation of dimethyl-succinate. Analysis was performed after 24 hours. (E) Succinate secretion by CD8⁺ T cells in RPMI + lactate (10mM) upon pre-treatment with the PDH inhibitor. CD8⁺ T cells were pre-treated with the PDH inhibitor for 24 hours (prior co-culture). Analysis was performed after 6 hours. (F) Schematic of ¹³C₄-succinate labeling into the TCA cycle. (G) ¹³C₄-succinate contribution to malate M+4 in CD8⁺ T cells transduced with shRNA for PC or CTRL and co-cultured with B16-OVA tumor cells in RPMI. Analysis was performed after 6 hours. The number of biological replicates for each experiment was n≥3. Error bars represent s.d. Two-tailed unpaired Student’s T-test was performed. *P<0.05; **P<0.01; ***P<0.001. See also Figure S4.

Figure 5.. Extracellular succinate activates SUCNR1 and promotes CD8⁺ T cell cytotoxicity.

(A) Killing by CD8⁺ T cells transduced with shRNA for SUCNR1 or CTRL and co-cultured with B16-OVA tumor cells in RPMI +/− addition of dimethyl-succinate. Analysis was performed after 24 hours. (B) Geometric mean of IFNγ expression by CD8⁺ T cells transduced with shRNA for SUCNR1 or CTRL and co-cultured with B16-OVA tumor cells in RPMI. Analysis was performed after 24 hours. (C) Secreted IFNγ levels by CD8⁺ T cells transduced with shRNA for SUCNR1 or CTRL and co-cultured with B16-OVA tumor cells in RPMI. Analysis was performed after 24 hours. (D) Killing by CD8⁺ T cells transduced with shRNA for SUCNR1 or CTRL and co-cultured with B16-OVA tumor cells in RPMI upon pre-treatment with the PDH inhibitor. CD8⁺ T cells were pre-treated with the PDH inhibitor for 24 hours (prior co-culture). Analysis was performed after 24 hours. (E) Killing by CD8⁺ T cells in co-culture with B16-OVA tumor cells in RPMI + lactate (10mM) upon pre-treatment with the succinate receptor agonist (SRA) cis-epoxysuccinic acid. CD8⁺ T cells were pre-treated with SRA for 24 hours (prior co-culture). Analysis was performed after 24 hours. (F-G) Killing and geometric mean of IFNγ expression by CD8⁺ T cells in co-culture with B16-OVA tumor cells in RPMI following pre-treatment with the SRA cis-epoxysuccinic acid. CD8⁺ T cells were pre-treated with SRA for 24 hours (prior co-culture). Analysis was performed after 24 hours. (H) Secreted IFNγ levels by CD8⁺ T cells in co-culture with B16-OVA tumor cells in RPMI following pre-treatment with the SRA cis-epoxysuccinic acid. CD8⁺ T cells were pre-treated with SRA for 24 hours (prior to co-culture). Analysis was performed after 24 hours. The number of biological replicates for each experiment was n=3. Error bars represent s.d. Two-tailed unpaired Student’s T-test was performed. *P<0.05; **P<0.01; ***P<0.001. See also Figure S5.

Figure 6.. Inhibition of PDH in vivo increases succinate levels in the TME and promotes anti-tumor immunity.

(A) Schematic representation of treatment with the PDH inhibitor CPI-613 (25mg/kg) or vehicle +/− combination with isotype control or αPD-1 in mice with B16 OVA tumors. Isotype and αPD-1 were used at 100μg dose (three doses in total). (B) Absolute succinate levels in tumor interstitial fluid from mice treated with either vehicle or the PDH inhibitor CPI-613 with or without antibodies to specifically deplete CD8⁺ T cells. Analysis was performed at day 18. n=5. Two-tailed unpaired Student’s T-test was performed. (C) % of OT-1 CD8⁺ T cells of CD45⁺ with the congenic markers 45.1 and 45.2 transduced with a shRNA for PDH and CTRL in B16-OVA tumors. OT-1 shPDH 45.1 and OT-1 shCTRL 45.2 were adoptively transferred using a ratio 1:1, one day prior B16-OVA tumor implantation. Analysis was performed at day 16. n=5. Two-tailed unpaired Student’s T-test was performed. (D) Average tumor growth curves showing tumor volume in mice upon adoptive transfer of OT-1 CD8⁺ T cells transduced with a shRNA for PDH or CTRL. Adoptive transfer was performed 9 days after tumor implantation. n=8. A two-way Anova with Sidak’s multiple comparison test was performed. (E) Average tumor growth curves showing tumor volume in mice treated with either vehicle or the PDH inhibitor CPI-613. Vehicle: n=7; PDH inh.: n=7. A two-way Anova with Sidak’s multiple comparison test was performed. (F) Average tumor growth curves showing tumor volume in mice treated with either vehicle or the PDH inhibitor CPI-613 with antibodies to specifically deplete CD8⁺ T cells. Vehicle αCD8: n=9; PDH inh.: n=9. A two-way Anova with Sidak’s multiple comparison test was performed. (G-H) Geometric mean of GzmB and IFNγ expression by CD8⁺ T cells of tumors in mice treated with either vehicle or the PDH inhibitor CPI-613. Analysis was performed at day 15. n=10. Two-tailed unpaired Student’s T-test was performed. (I) Average tumor growth curves showing tumor volume in mice treated with either vehicle or the PDH inhibitor CPI-613 and isotype control or αPD-1. Vehicle + isotype: n=9; PDH inh. + isotype: n=8; Vehicle + αPD1 : n=8; PDH inh. + αPD1: n=9. A two-way Anova with Sidak’s multiple comparison test was performed. (J) % of OT-1 CD8⁺ T cells of CD45⁺ with the congenic markers 45.1 and 45.2 transduced with a shRNA for SUCNR1 and CTRL in B16-OVA tumors. OT-1 shSUCNR1 45.1 and OT-1 shCTRL 45.2 were adoptively transferred using a ratio 1:1, one day prior B16-OVA tumor implantation. Analysis was performed at day 16. n=5. Two-tailed unpaired Student’s T-test was performed. (K) Average tumor growth curves showing tumor volume in mice treated with either vehicle or the SRA cis-epoxysuccinic acid. Vehicle: n=5; SRA: n=5. A two-way Anova with Sidak’s multiple comparison test was performed. This is a representative experiment of two independent experiments. (L) Average tumor growth curves showing tumor volume in mice treated with either vehicle or the SRA cis-epoxysuccinic acid with antibodies to specifically deplete CD8⁺ T cells. Vehicle αCD8: n=5; PDH inh.: n=5. A two-way Anova with Sidak’s multiple comparison test was performed. This is a representative experiment of two independent experiments. (M-N) Geometric mean of GzmB and IFNγ expression by CD8⁺ T cells of tumors in mice treated with either vehicle or the SRA cis-epoxysuccinic acid. Analysis was performed at day 15. n=7. Two-tailed unpaired Student’s T-test was performed. (O) Schematic representation CD8⁺ T cell-tumor cell metabolic circuit. On the left the metabolic activity of a metabolically fit CD8⁺ T cell is depicted. On the right, tumor cell metabolism impairs CD8⁺ T cell activity causing decreased CD8⁺ T cell effector function. Overall, the figure shows extracellular lactate to modulate TCA cycle entry of pyruvate through either PC or PDH, and to dictate the capacity of T cells to secrete succinate to promote cytolytic activity through the SUCNR1. Green indicates up-regulation. Grey and red indicate down-regulation. Error bars represent s.e.m. *P<0.05; **P<0.01; ***P<0.001. See also Figure S6.