Suppression of FIP200 and autophagy by tumor-derived lactate promotes naïve T cell apoptosis and affects tumor immunity

Abstract

Naïve T cells are poorly studied in cancer patients. We report that naïve T cells are prone to undergo apoptosis due to a selective loss of FAK family-interacting protein of 200 kDa (FIP200) in ovarian cancer patients and tumor-bearing mice. This results in poor antitumor immunity via autophagy deficiency, mitochondria overactivation, and high reactive oxygen species production in T cells. Mechanistically, loss of FIP200 disables the balance between proapoptotic and antiapoptotic Bcl-2 family members via enhanced argonaute 2 (Ago2) degradation, reduced Ago2 and microRNA1198-5p complex formation, less microRNA1198-5p maturation, and consequently abolished microRNA1198-5p-mediated repression on apoptotic gene Bak1 Bcl-2 overexpression and mitochondria complex I inhibition rescue T cell apoptosis and promoted tumor immunity. Tumor-derived lactate translationally inhibits FIP200 expression by down-regulating the nicotinamide adenine dinucleotide level while potentially up-regulating the inhibitory effect of adenylate-uridylate-rich elements within the 3' untranslated region of Fip200 mRNA. Thus, tumors metabolically target naïve T cells to evade immunity.

Fig. 1. FIP200 loss links to poor autophagy and high apoptosis in naïve T cells in tumor

(A) Phenotype of naïve T cells in blood and cancer tissues in ovarian cancer (OC) patients. Peripheral blood mononuclear cells and ovarian cancer tissue single cells were stained with antibodies against CD3, CD7, CD45RA, and CD45RO and analyzed with LSR II (n = 5). (B to E) Apoptotic naïve T cells in peripheral blood and ovarian cancer tissues in ovarian cancer patients. (B) Numbers on the dot plots represent the percentage of AnnexinV⁺CD45RA⁺CD45RO⁻CD3⁺ naïve T cells. The percentages of Annexin V⁺CD4⁺ (C) and Annexin V⁺CD8⁺ (D) naïve T cells are shown (n = 5, means ± SEM). *P < 0.05 (Mann-Whitney U tests) compared with control group. (E) Human naïve T cells were cultured with anti-CD3 and anti-CD28 antibodies for 48 hours. Numbers on the dot plots represent the percentage of Annexin V⁺ T cells of activated naïve T cells (n = 5). (F and G) Apoptotic naïve T cells in healthy mice and ID8 tumor–bearing mice. (F) Numbers on the dot plots represent the percentage of Annexin V⁺CD44⁻CD3⁺ fresh naïve T cells in mesenteric lymph nodes (n = 13, means ± SEM). *P < 0.05 (Mann-Whitney U tests). (G) T cells were cultured with anti-CD3 and anti-CD28 antibodies for 48 hours. Numbers on the dot plots represent the percentage of Annexin V⁺ T cells of activated naïve T cells (n = 7). (H) Autophagy component proteins in fresh peripheral blood naïve T cells in healthy human donors and ovarian cancer patients (n = 6). (I) Autophagy component proteins in fresh mouse naïve lymph node T cells from normal, ID8-, and LLC-bearing mice (n = 3).

Fig. 2. Genetic FIP200 deletion impairs autophagy induction and causes T cell apoptosis

(A) FIP200 protein expression in T cell subsets and non–T cells from Fip200^flox/floxCd4^+/+ (WT), Cd4^c/+, and Cd4^c/c (KO) mice (n = 3). (B and C) Spontaneous apoptosis of naïve T cells in WT and Fip200^−/− mice. (B) Numbers on the dot plots represent the percentage of Annexin V⁺ naïve T cells in lymph nodes. (C) The percentages of CD4⁺ and CD8⁺ Annexin V⁺ naïve T cells are shown (n = 5, means ± SEM). *P < 0.05 (Mann-Whitney U tests). (D and E) Apoptosis of cultured or activated T cells in WT and Fip200^−/− mice. Naïve lymph node T cells were cultured for 24 and 48 hours without (D) or with (E) anti-CD3 and anti-CD28 antibodies. Numbers on the dot plots represent the percentage of Annexin V⁺ T cells (n = 4, means ± SEM). *P < 0.05 (Mann-Whitney U tests). (F) The percentage of T cell subsets in lymph nodes (LNs; n = 6), spleen (SP; n = 5 to 7), bone marrow (BM; n = 3), and peripheral blood (PB; n = 3) in WT and Fip200^−/− mice. *P < 0.05 (Mann-Whitney U tests). (G) Absolute numbers of T cell subsets in spleen from WT and Fip200^−/− mice (n = 13, means ± SEM). *P < 0.05 (Mann-Whitney U tests). (H and I) WT CD45.1⁺ T cells and Fip200^−/− CD45.2⁺ T cells were transferred into Rag1^−/− mice with a 1:1 ratio. Live (H) and apoptotic (I) T cells were analyzed on day 14 in the lymph nodes and spleen in Rag1^−/− mice. Numbers on the dot plots represent the percentage of CD45.1⁺ and CD45.2⁺ T cells in total T cells (H) and the percentage of Annexin V⁺ T cells in WT and Fip200^−/− (KO) T cells (I) (n = 7). (J) Autophagy flux in T cells isolated from lymph nodes of WT and KO mice with or without TCR engagement for 24 hours. These T cells were treated with Baf for the last 4 hours to inhibit the autophagosome degradation (n = 3). (K) Expression of autophagy receptor p62 and NDP52 in fresh WT and Fip200^−/− naïve T cell subsets (n = 3).

Fig. 3

FIP200 deficiency alters mitochondria activation and ROS produc- G tion in T cells. (A and B) The mitochondria contents (A) and ROS production (B) were analyzed by FACS in naïve T cell subsets in WT and Fip200^−/− mice (n = 4 to 5, means ± SEM). *P < 0.05 (Mann-Whitney U tests). MFI, mean fluorescence intensity. (C) Effects of NAC on WT and Fip200^−/− T cell apoptosis in vitro. T cell subsets were treated with 0.1 mM NAC for 12 hours. Numbers on the dot plots represent the percentage of Annexin V⁺ T cells (n = 3). (D and E) Effects of NAC on WT and Fip200^−/− T cell apoptosis in vivo. Mice were treated daily with NAC for 4 weeks. (D) Apoptosis was determined by Annexin V staining in activated T cells. (E) The absolute numbers of naïve CD4⁺ and CD8⁺ T cells were quantified in lymph nodes in WT and Fip200^−/− mice without or with NAC treatment (n = 4 to 5, means ± SEM). *P < 0.05 (Mann-Whitney U tests) between KO mice without and with NAC treatment. (F and G) Effects of metformin on WT and Fip200^−/− naïve T cell ROS and apoptosis in vivo. Mice were treated daily with metformin for 2 weeks. (F) The ROS production was analyzed by FACS in naïve T cell subsets in WT and Fip200^−/− mice. (G) The percentage of Annexin V⁺ T cells in lymph node and spleen from WT and Fip200^−/− mice (n = 3, means ± SEM). *P < 0.05 (Mann-Whitney U tests) between KO mice without and with metformin treatment.

Fig. 4. Impaired antitumor immunity in Fip200^−/− mice

(A to D) Effect of T cell FIP200 deficiency on tumor growth in vivo. MC38 (A and B) and B16 cells (C and D) were subcutaneously inoculated into WT and Fip200^−/− mice. Tumor volume (A and C) was monitored, and tumor weight (B and D) was recorded at the end of the experiments (n = 5, means ± SEM). *P < 0.05 (Mann-Whitney U tests) between WT and Fip200^−/− tumor size at the indicated time. (E) Effect of T cell FIP200 deficiency on T cell tumor infiltration. CD4⁺ and CD8⁺ cells were analyzed by FACS in MC38 tumor tissues. Numbers on the dot plots represent the percentage of CD4⁺ and CD8⁺ T cells in CD45⁺ cells (n = 3). (F and G) Effect of metformin on MC38 tumor growth in vivo. MC38-bearing mice were treated with metformin or control for 18 days from day 7. Tumor images (F) and volume (G) are shown (n = 14 to 15, means ± SEM). *P < 0.05 (Mann-Whitney U tests). (H) Effect of metformin on ROS production in naïve T cell in MC38-bearing mice treated with metformin and control (n = 5, means ± SEM). *P < 0.05 (Mann-Whitney U tests). (I and J) Effect of metformin on naïve T cell apoptosis in MC38-bearing mice treated with metformin and control. (I) Numbers on the dot plots represent the percentage of Annexin V⁺CD44⁻CD3⁺CD62L⁺ naïve T cells (n = 5). (J) The percentages of Annexin V⁺CD44⁻CD3⁺CD62L⁺ naïve T cells are shown (n = 5, means ± SEM). *P < 0.05 (Mann-Whitney U tests).

Fig. 5. FIP200 controls Bak expression via maintaining microRNA1198-5p expression

(A) Bcl-2 protein was detected in T cell subsets in WT and Fip200^−/− mice (n = 3). (B) Bak and Bim proteins were detected in T cell subsets in WT and Fip200^−/− mice (n = 3). (C) T cell apoptosis in WT, Fip200^−/−, Bcl-2 transgenic (Bcl-2–WT) and Fip200^−/− Bcl-2 transgenic mice (Bcl-2–KO; n = 3 to 5, means ± SEM). *P < 0.05 between KO and Bcl-2–KO (Mann-Whitney U tests). (D) Absolute numbers of T cell subsets in Fip200^−/− and Bcl-2–KO spleen (n = 7, means ± SEM). *P < 0.05 (Mann-Whitney U tests). (E) Heat map of microRNAs in Fip200^−/− naïve CD4⁺ T cells. MicroRNA arrays of naïve CD4⁺ T cells in WT and Fip200^−/− mice. A fold change of >0.25 is shown. The red color represents high levels of microRNA expression in WT naïve CD4⁺ T cells as compared with Fip200^−/− naïve CD4⁺ T cells. (F) Expression of three microRNAs in Fip200^−/− naïve CD4⁺ T cells (n = 4, means ± SEM). *P < 0.05 (Mann-Whitney U tests). (G) Effect of microRNA1198-5p mimics on WT 3′UTR-Bak1 luciferase activity. The luciferase activity was measured in 293FT cells transfected with WT or mutant 3′UTR sequence of Bak1 (n = 3 in triplicate, means ± SEM). *P < 0.05 between WT and mutant (Student’s t tests). (H) Effects of microRNA1198-5p inhibitors and mimics on proapoptotic gene expression. WT and Fip200^−/− T cells from lymph nodes were transfected with microRNA1198-5p inhibitors or mimics for 48 hours. Proapoptotic proteins were detected by Western blotting (n = 3).

Fig. 6. FIP200 maintains microRNA1198-5p expression via Ago2 in naïve T cells

(A) Expression of Ago2 protein in WT and Fip200^−/− naïve T cells (n = 3). (B) Effect of Ago2 siRNAs on microRNA1198-5p expression. WT naïve CD4⁺ T cells were transfected with Ago2 siRNAs (#1 and #2) for 48 hours. The expression of micorRNA1198-5p and microRNA342-5p was quantified by real-time PCR (n = 3 to 5, means ± SEM). *P < 0.05 (Student’s t tests). (C and D) Expression of microRNA1198-5p (C) and Bak1 mRNA (D) in the Ago2 complex (n = 3, means ± SEM). *P < 0.05 (Student’s t tests). (E) Contents of Bak1 and Bax mRNAs in the Ago2 complex in WT and Fip200^−/− naïve T cells. The data are shown as the percentage of Bak1 and Bax mRNA expression compared with input (n = 3, means ± SEM). *P < 0.05 of Bak1 expression between WT and Fip200^−/− T cells (Mann-Whitney U tests). (F) Ago2 ubiquitination in WT and Fip200^−/− naïve T cells. Naïve T cells were treated with or without proteasome inhibitor MG132 (10 µM) for 2 hours (n = 3). IP, immunoprecipitation; IB, immunoblotting. (G) Expression of Ago2 and Bak protein in Atg3-WT and Atg3-KO naïve T cells (n = 3).

Fig. 7. Tumor-derived lactate suppresses FIP200 expression in naïve T cells

(A) Effect of lactate on FIP200 protein expression in human naïve T cells treated with lactate for 48 hours (n = 3). (B) Effect of lactate on human naïve T cells apoptosis treated with 20 mM lactate for 48 hours. Numbers on the dot plots represent the percentage of Annexin V⁺ naïve T cells (n = 3). (C) Lactate levels in the plasma and ascites of ovarian cancer patients. Lactate concentrations were measured in 20 plasma samples and 27 ascites. The normal range of lactate plasma levels determined in healthy individuals (0 to 2.2 mM) is indicated below the red line. (D) Effect of ascites on FIP200 and LC3-II protein in human naïve T cells treated with ascites for 24 hours (n = 3). (E) Effect of CHC on the role of lactate and ascites in FIP200 expression in human naïve T cells treated with different conditions for 48 hours (n = 3). (F) Effect of lactate on FIP200, Ago2, and Bak protein expression in mouse naïve T cells treated with different conditions for 24 hours in the presence of interleukin-7 (IL-7; n = 3). (G) Effect of lactate on ROS expression in mouse naïve T cells treated with 20 mM lactate for 48 hours with IL-7 (n = 3). (H) Effect of AREs in 3′UTR-Fip200 on luciferase activity. The luciferase activity was measured in 293FT cells transfected with WT or mutant mouse Fip200 3′UTR vectors for 48 hours. (n = 3 in triplicate, means ± SEM). *P < 0.05 between WT and mutant (Student’s t tests). (I) Effect of lactate on NAD level in mouse naïve T cells treated with 20 mM lactate for 24 hours. The total NAD in 10⁶ T cells was measured (n = 5, means ± SEM). *P < 0.05 between control and lactate treatment (Student’s t tests). (J) Effect of lactate on FIP200 expression in mouse naïve T cells treated with 20 mM lactate in the presence of 5 mM NAM for 24 hours (n = 3). (K) Effect of NAM on the inhibitory role of the AREs in 3′UTR-Fip200. 293FT cells were transfected with mouse WT or mutant 3′UTR-Fip200 plasmids for 24 hours and were treated with NAM for an additional 24 hours. Results are shown as the decreased percentage of WT luciferase activity to that of the mutant [(mutant luciferase − WT luciferase)/mutant luciferase] (n = 3 in triplicate, means ± SEM). *P < 0.05 between control and NAM treatment (Student’s t tests). (L) Effect of NAM on naïve T cell apoptosis induced by lactate. Mouse naïve T cells were treated with lactate in the presence of NAM for 24 hours. Numbers on the dot plots represent the percentage of Annexin V⁺ naïve T cells (n = 3).