Metabolic reprogramming of donor T cells enhances graft-versus-leukemia effects in mice and humans

Abstract

Acute myeloid leukemia (AML) relapse after allogeneic hematopoietic cell transplantation (allo-HCT) has a dismal prognosis. We found that T cells of patients relapsing with AML after allo-HCT exhibited reduced glycolysis and interferon-γ production. Functional studies in multiple mouse models of leukemia showed that leukemia-derived lactic acid (LA) interfered with T cell glycolysis and proliferation. Mechanistically, LA reduced intracellular pH in T cells, led to lower transcription of glycolysis-related enzymes, and decreased activity of essential metabolic pathways. Metabolic reprogramming by sodium bicarbonate (NaBi) reversed the LA-induced low intracellular pH, restored metabolite concentrations, led to incorporation of LA into the tricarboxylic acid cycle as an additional energy source, and enhanced graft-versus-leukemia activity of murine and human T cells. NaBi treatment of post-allo-HCT patients with relapsed AML improved metabolic fitness and interferon-γ production in T cells. Overall, we show that metabolic reprogramming of donor T cells is a pharmacological strategy for patients with relapsed AML after allo-HCT.

Fig. 1.. Characterization of CD8⁺ T cells of patients with AML during disease progression from primary diagnosis to relapse after allo-HCT.

(A) ECAR of CD8⁺ T cells isolated from the peripheral blood of patients with AML at the time of primary diagnosis compared with samples from healthy individuals [healthy control (HC)] (n = 19). n.s., not significant. (B) OCR of CD8⁺ T cells (n = 19). (C) Flow cytometry–based analysis of CD8⁺ T cells (Tc). Representative fluorescence-activated cell sorting (FACS) plots (left) and statistical analysis (right) of the percentage of cells expressing IFN-γ (HC, n = 18; AML, n = 17). (D) Representative FACS plots (left) and statistical analysis (right) of the percentage of cells expressing TNFα (HC, n = 16; AML, n = 17). (E) Representative FACS plots (left) and statistical analysis (right) of the percentage of cells expressing perforin (HC, n = 18; AML, n = 17). (F) OCR measured at baseline and in response to oligomycin, carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP), etomoxir (ETO), and rotenone + antimycin A (R/A) in CD8⁺ T cells isolated from one patient during remission and at the time of relapse after allo-HCT. (G) OCR of CD8⁺ T cells isolated from the peripheral blood of patients with AML during remission and at the time of relapse after allo-HCT (n = 21). (H) OCR of CD8⁺ T cells isolated from the peripheral blood of patients with AML at two time points (tp1 and tp2) in remission after allo-HCT (n = 17). (I) ECAR of CD8⁺ T cells isolated as described in (G) (n = 21). (J) ECAR of CD8⁺ T cells isolated as described in (H) (n = 17). (K) Representative FACS plots (left) and statistical analysis (right) of the percentage of CD8⁺ T cells expressing IFN-γ, isolated as described in (G) (n = 10). (L) Representative FACS plots (left) and statistical analysis (right) of the percentage of CD8⁺ T cells expressing IFN-γ, isolated as described in (H) (n = 18). Each data point represents the measurement of an individual patient at the indicated time point; n values represent individual patients. P values were determined using the two-sided Student’s unpaired t/Mann-Whitney (HC versus AML) or paired t/Wilcoxon (two time points of one patient) test.

Fig. 2.. Murine T cells are metabolically impaired by AML cells in vitro and in vivo.

(A) Recipient mice (BALB/c background) were transplanted with 5 × 10⁶ C57BL/6 bone marrow (BM) cells (allo BM) with or without additional 1 × 10⁵ syngeneic leukemia cells (WEHI-3B luc) and 2 × 10⁵ C57BL/6 T cells (allo Tc). Tumor growth was monitored by bioluminescence imaging (BLI). d0, day 0. (B) Analysis of ECAR of reisolated CD8⁺ T cells (n = 6, means ± SEM). (C) Analysis of OCR (n = 6, means ± SEM). The experiment was performed six times (with 18 mice in total); n values represent biologically independent experiments, pooling together three mice for each experiment. P values were determined using the one-sample Student’s unpaired t test. (D) ECAR measured at baseline and in response to glucose, oligomycin, and 2-deoxyglucose (2-DG) for CD8⁺ T cells isolated from 48-hour cell culture under CD3/CD28 stimulation either alone or in coculture with AML cells or incubated in medium originating from 48-hour AML cell culture. Graph is representative of four biologically independent experiments. (E) ECAR of CD8⁺ T cells (n = 4 to 6). (F) OCR of CD8⁺ T cells (n = 4 to 6). (G) ECAR of CD8⁺ T cells isolated from 48-hour cell culture and stimulated with CD3/CD28, from T cells incubated alone, in coculture with BM-derived dendritic cells (BMDCs), or T cells incubated in medium originating from 48-hour BMDC cell culture (n = 6). (H) OCR of CD8⁺ T cells (n = 6). The n values represent biologically independent experiments (means ± SEM). P values were determined using one-way ANOVA. (I) Microarray-based analysis of gene expression of CD8⁺ T cells isolated after 48-hour CD3/CD28 stimulation and incubated in fresh medium or medium originating from 48-hour AML or BMDC cell culture. Bar plot depicts the top 10 down-regulated gene sets in T cells cultured in AML medium compared to T cells cultured in DC medium. MTORC1, mammalian target of rapamycin complex 1. (J) Tile display shows differentially regulated OXPHOS genes as determined from linear model analysis. Only regulated genes with an adjusted P < 0.05 were selected. The color scale represents the row-wise Z score of gene expression with red being the highest and blue being the lowest. (K) Tile display shows differentially regulated glycolysis genes.

Fig. 3.. Proliferative and metabolic unfitness of T cells is accompanied by reduced antitumor activity.

(A) Microarray-based analysis of gene expression in CD8⁺ T cells cultured as in Fig. 2I. Tile display shows differentially regulated mitosis related genes as determined from linear model analysis. The color scale represents the row-wise Z score of gene expression, with red being the highest and blue being the lowest. (B) Cell cycle analysis of CD8⁺ T cells incubated in fresh medium or medium originating from 48-hour AML cell culture. Representative FACS plots (left) and statistical analysis (right) of four biologically independent experiments (means ± SEM) are shown. P value was determined using two-way ANOVA showing P value comparing G₀ phases. (C) Proliferation assay of T cells after 72 hours of culture in conditions described in (B). Representative histograms (left) and statistical analysis (right) of percentages of proliferating cells in five biologically independent experiments (means ± SEM) are shown. P value was determined using two-sided Student’s unpaired t test. (D) Recipient mice (BALB/c background) were transplanted with 5 × 10⁶ C57BL/6 BM cells (allo BM). C57BL/6 luc CD8⁺ T cells (allo Tc) were isolated from 48-hour cell culture described in (B), and 3 × 10⁵ CD8⁺ T cells were transplanted into each mouse. T cell expansion was monitored by BLI. Representative BLI images (left) and statistical analysis (right) of photon signals on day 6 after BMT pooled from two independent experiments with n = 10 per group, P value was determined using Mann-Whitney test. (E) Recipient mice (BALB/c background) were transplanted with 5 × 10⁶ C57BL/6 BM cells (allo BM) with or without additional 1 × 10⁴ syngeneic leukemia cells (WEHI-3B luc). C57BL/6 CD8⁺ T cells (allo Tc) were isolated from 48-hour cell culture described in (B), and 2 × 10⁵ CD8⁺ T cells were transplanted into each mouse. Survival was monitored. Data were pooled from two independent experiments with n = 5 mice per group in the BM group and n = 10 in the BM + AML groups. P values were determined using the log-rank (Mantel-Cox) test. (F) Recipient mice (C57BL/6 background) were transplanted with 5 × 10⁶ BALB/c BM cells (allo BM) with additional 5 × 10³ splenocytes derived from a syngeneic mouse carrying MLL^PTD/+;Flt3^ITD/+ mutated tumor. BALB/c CD8⁺ T cells (allo Tc) were isolated from 48-hour cell culture described in (B), and 2 × 10⁵ CD8⁺ T cells were transplanted into each mouse. Survival was monitored. Data were pooled from two independent experiments with n = 10 mice per group. P values were determined using the log-rank (Mantel-Cox) test. (G) Recipient mice (Rag2^−/−Il2rγ^−/− background) were transplanted with human leukemia cells (MOLM-13 luc). C57BL/6 CD8⁺ T cells (allo Tc) were isolated from 48-hour cell culture described in (B), and 2 × 10⁵CD8⁺ T cells were transplanted into each mouse. Survival was monitored. Data were pooled from two independent experiments with n = 12 mice per group. P values were determined using the log-rank (Mantel-Cox) test.

Fig. 4.. LA is increased in the AML supernatant inhibiting T cell proliferation.

(A) Relative abundance of LA in fresh RPMI compared to T cell supernatant after 48-hour cell culture in fresh medium or medium originating from 48-hour AML cell culture or in 48-hour AML cell culture supernatant determined by liquid chromatography–mass spectrometry (LC-MS). Statistical analysis of six to nine biologically independent experiments (means ± SEM) is shown. P values were determined using one-way ANOVA, ****P < 0.0001. (B) Concentration of lactate in fresh RPMI compared to T cell culture supernatants after 48-hour culture alone, with AML cells or with BMDCs, or in medium originating from 48-hour AML/BMDC cell culture, determined by NMR analysis. Statistical analysis of seven to nine biologically independent experiments (means ± SEM) using Kruskal-Wallis test is shown. (C) pH measurements of T cell culture supernatants after 48-hour culture in fresh medium or medium originating from 48-hour AML/BMDC cell culture. Statistical analysis of three to six biologically independent experiments (means ± SEM) is shown. P values were determined using one-way ANOVA. (D) Relative abundance of LA in the serum of patients with AML during remission and at the time of relapse after allo-HCT obtained by LC-MS (n = 7). (E) Relative abundance of LA in the serum of patients with AML at two time points in remission after allo-HCT obtained by LC-MS (n = 16). Each data point in (D) and (E) represents the measurement of an individual patient at the indicated time point; n values represent individual patients. P values were determined using the two-sided Student’s paired t test. (F) ECAR measured at baseline and in response to glucose, oligomycin, and 2-DG for CD8⁺ T cells isolated from 48-hour cell culture treated with indicated concentrations of LA. Graph is representative of five biologically independent experiments. (G) ECAR of CD8⁺ T cells (n = 5). The n values represent biologically independent experiments (means ± SEM). P values were determined using one-way ANOVA. (H) Cell cycle analysis of CD8⁺ T cells. Statistical analysis of five biologically independent experiments (means ± SEM) is shown. P values were determined using two-way ANOVA showing P values of G₀ phases. (I) Proliferation assay of T cells treated with LA as described in (F) or at a similar pH achieved using HCl (pH 7.0) for 72 hours. Statistical analysis (left) of percentage of proliferating cells of four biologically independent experiments (means ± SEM) and representative histograms (right) is shown. P value was determined using one-way ANOVA. ****P < 0.0001. (J) Total lysates of CD8⁺ T cells after CD3/28 stimulation and treatment with 15 mM LA for the indicated time points were analyzed by Western blotting. Graph depicts quantification of pS6K. Statistical analysis of three biologically independent experiments is shown. For quantification, all proteins were normalized to the corresponding loading control 14-3-3. P values were calculated using two-way ANOVA.

Fig. 5.. NaBi reverses metabolic and functional T cell defects caused by LA.

(A) ECAR measured at baseline and in response to glucose, oligomycin, and 2-DG for CD8⁺ T cells isolated from 48-hour cell culture under CD3/CD28 stimulation and treated with LA (15 m M) and/or NaBi (15 mM) as indicated. Graph is representative of four biologically independent experiments. (B) ECAR of CD8⁺ T cells (n = 4). The n values represent biologically independent experiments (means ± SEM). P values were determined using one-way ANOVA. (C) Representative histograms of proliferation assay of T cells for 72 hours. (D) Statistical analysis of the percentage of proliferating cells in four biologically independent experiments (means ± SEM). P values were determined using one-way ANOVA. (E) Cell cycle analysis of CD8⁺ T cells. Statistical analysis of four biologically independent experiments (means ± SEM) is shown. P values were determined using two-way ANOVA showing P value for G₀ phases. (F) Polar metabolites were extracted from CD8⁺ T cells, acquired by LC-MS, and analyzed using an untargeted approach with XCMS software. Graph shows total number of changed metabolites between T cells without treatment and T cells treated with LA. (G) Total number of changed metabolites between T cells treated with LA with or without NaBi. (H) Top five metabolic pathways and two others of interest decreased in T cells treated with LA compared to control. The number of decreased metabolites flagged in each pathway is indicated. ABC, ATP (adenosine triphosphate)–binding cassette. (I) Top five metabolic pathways and two others of interest increased in T cells treated with LA and NaBi compared to LA alone. The number of increased metabolites flagged in each pathway is indicated. (J) Targeted analysis of metabolites of interest using assayR for all four treatment groups. The values have been normalized to control without treatment; the color scale depicts the fold change. PEP, phosphoenolpyruvate; PG, phosphoglycerol; AMP, adenosine monophosphate; GMP, guanosine monophosphate; CDP, cytidine diphosphate; UDP, uridine diphosphate; ADP, adenosine diphosphate; GDP, guanosine diphosphate; CTP, cytidine triphosphate; UTP, uridine triphosphate; GTP, guanosine triphosphate.

Fig. 6.. NaBi treatment of LA-challenged T cells leads to increased LA uptake and incorporation into the TCA cycle.

(A) Metabolites were extracted from CD8⁺ T cells isolated after 48 hours of CD3/CD28 stimulation. Cells were treated with ¹³C–heavy-labeled LA (15 mM) with or without NaBi (15 mM) as indicated (n = 4), and ¹³C-labeled metabolites were identified by mass spectrometry. Average background from ¹³C labeling, detected in the unlabeled samples, was subtracted from all samples. Graph shows relative abundance of unlabeled and labeled LA. (B) Relative abundance of citrate. (C) Relative abundance of succinate. (D) Relative abundance of fumarate. (E) Relative abundance of malate. The n values represent biologically independent experiments (means ± SEM). * in black represents the significance of differences in total abundance of each metabolite, calculated by one-way ANOVA or Kruskal-Wallis test. # in purple indicates the significance of differences in ¹³C metabolite isotopolog, calculated by two-way ANOVA. */#P < 0.05, **/##P < 0.01, ***/###P < 0.001, and ****/####P < 0.0001. (F) ECAR measured at baseline and in response to glucose, oligomycin, and 2-DG for CD8⁺ T cells isolated from 48-hour cell culture under CD3/CD28 stimulation treated with LA (15 mM), NaBi (15 mM), and/or MCT1 inhibitor AZD3965 (100 nM) as indicated. Graph is representative of three biologically independent experiments. (G) ECAR of CD8⁺ T cells (n = 3 to 5). The n values represent biologically independent experiments (means ± SEM). P values were determined using one-way ANOVA.

Fig. 7.. Leukemia-derived LA induces intracellular pH changes and causes the impaired T cell phenotype.

(A) ECAR measured at baseline and in response to glucose, oligomycin, and 2-DG for CD8⁺ T cells isolated from 48-hour cell culture under CD3/CD28 stimulation incubating in fresh medium or medium originating from 48-hour AML cell culture. NaBi (15 mM) was added to T cell culture. Graph is representative of five biologically independent experiments. (B) ECAR of CD8⁺ T cells (n = 5). The n values represent biologically independent experiments (means ± SEM). P values were determined using one-way ANOVA. (C) Representative histogram of proliferation assay of T cells for 72 hours. (D) Statistical analysis of percentage of proliferating cells in four biologically independent experiments (means ± SEM). P values were determined using one-way ANOVA. (E) Cell cycle analysis of CD8⁺ T cells. Statistical analysis of five biologically independent experiments (means ± SEM) is shown. P values were determined using two-way ANOVA showing P value for G₀ phases. (F) Intracellular pH of CD8⁺ T cells after 30-min incubation with LA (15 mM), NaBi (15 mM), and/or medium originating from 48-hour AML cell culture, calculated using pH-controlled buffers (pH 5.5, pH 6.5, and pH 7.5) for calibration [shown in (G)]. Statistical analysis of four to six biologically independent experiments (means ± SEM), P values were determined using one-way ANOVA. (G) One representative of four biologically independent experiments described in (F) is shown. (H) ECAR of CD8⁺ T cells isolated from 48-hour cell culture under CD3/CD28 stimulation incubating in fresh medium or medium originated from 48-hour AML cell culture with administration of NaCl (15 mM), Hepes (15 mM), or increased pH achieved using NaOH (pH adjustment to pH 8) (n = 4). The n values represent biologically independent experiments (means ± SEM). P values were determined using one-way ANOVA. (I) Polar metabolites were extracted from CD8⁺ T cells after 30-min CD3/CD28 stimulation and treatment with LA (15 mM) and/or NaBi (15 mM), acquired by LC-MS, and analyzed using a targeted approach with assayR. Graph shows relative abundance of lactate for all four treatment groups (n = 4). The n values represent biologically independent experiments (means ± SEM). P values were determined using one-way ANOVA.

Fig. 8.. NaBi promotes the GVL effect and rescues human T cell function.

(A) Recipient mice (BALB/c background) were transplanted with 5 × 10⁶ C57BL/6 BM cells (allo BM) with additional 1 × 10⁴ syngeneic leukemia cells (WEHI-3B luc). C57BL/6 T cells (allo Tc) were transplanted 2 days later (3 × 10⁵). NaBi (200 mM) was administered for 14 days (days 2 to 16 after BMT) in the drinking water. Survival was monitored. Data were pooled from two independent experiments with n = 10 per group, and P values were determined using the log-rank (Mantel-Cox) test. (B) Recipient mice (C57BL/6 background) were transplanted with 5 × 10⁶ BALB/c BM cells (allo BM) with additional 5 × 10³ splenocytes derived from a syngeneic mouse carrying MLL^PTD/+;Flt3^ITD/+ mutated tumor. A total of 3 × 10⁵ BALB/c CD8⁺ T cells (allo Tc) were transplanted 2 days later. NaBi (200 mM) was administered as described in (A). Survival was monitored. Data were pooled from two independent experiments with n = 9 to 10 mice per group, and P values were determined using the log-rank (Mantel-Cox) test. (C) ECAR measured at baseline and in response to glucose, oligomycin, and 2-DG for human primary CD8⁺ T cells from healthy donors after 48 hours of cell culture under CD3/CD28 stimulation incubating in fresh medium or medium originating from 48-hour AML cell culture. LA (15 mM) and/or NaBi (15 mM) were added to T cell culture. Graph is representative of three biologically independent experiments. (D) ECAR after oligomycin injection of CD8⁺ T cells (n = 3). The n values represent individual healthy donors (means ± SEM). P values were determined using one-way ANOVA. (E) Metabolic analysis of patients with relapsed AML after allo-HCT receiving DLIs before and after 7-day oral treatment with bicaNorm (sodium bicarbonate). Graph shows serum pH. (F) HCO₃⁻ concentration in the peripheral blood of the patients in (E). (G) OCR measured at baseline and in response to oligomycin, FCCP, and rotenone + antimycin A in isolated CD8⁺ T cells. (H) OCR after FCCP injection. (I) Representative FACS plots (left) and statistical analysis (right) of the percentage of CD8⁺ T cells expressing IFN-γ versus side scatter (SSC) (n = 10). (J) Representative FACS plots (left) and statistical analysis (right) of the percentage of CD8⁺ T cells expressing TNFα (n = 10). Each data point represents the measurement of an individual patient at the indicated time point; n values represent individual patients. P values were determined using the two-sided Student’s paired t test or Wilcoxon test.