Skip to main content
. Author manuscript; available in PMC: 2023 Aug 2.
Published in final edited form as: Cell Metab. 2022 Jul 11;34(8):1137–1150.e6. doi: 10.1016/j.cmet.2022.06.008

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

Figure 3.

(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.