Long-term antigen exposure irreversibly modifies metabolic requirements for T cell function

Abstract

Energy metabolism is essential for T cell function. However, how persistent antigenic stimulation affects T cell metabolism is unknown. Here, we report that long-term in vivo antigenic exposure induced a specific deficit in numerous metabolic enzymes. Accordingly, T cells exhibited low basal glycolytic flux and limited respiratory capacity. Strikingly, blockade of inhibitory receptor PD-1 stimulated the production of IFNγ in chronic T cells, but failed to shift their metabolism towards aerobic glycolysis, as observed in effector T cells. Instead, chronic T cells appeared to rely on oxidative phosphorylation (OXPHOS) and fatty acid oxidation (FAO) to produce ATP for IFNγ synthesis. Check-point blockade, however, increased mitochondrial production of superoxide and reduced viability and effector function. Thus, in the absence of a glycolytic switch, PD-1-mediated inhibition appears essential for limiting oxidative metabolism linked to effector function in chronic T cells, thereby promoting survival and functional fitness.

Keywords: PD-1; T lymphocytes; immunology; inflammation; metabolism; mouse.

Figure 1.. Prolonged chronic alloantigen stimulation alters functional metabolic requirements in alloreactive CD4 +T cells.

(A) Frequencies and numbers of CD3⁺ T cells in the spleen of non-irradiated B6 (H-2b) Rag2^-/- Il2rg^-/- recipients 6 and 21 days after reconstitution with purified BALB/c (H-2d) CD4⁺ T cells. Represented data are means ± SEM. Data presented are representative of two independent experiments with 4–5 mice in each experimental group. *indicates p=0.0079 by the Mann-Whitney test. NS indicates non-significant. (B) Phenotype of chronic alloreactive CD4⁺ T cells. Spleen CD3⁺ cells from mice as described in (A) were analyzed for their expression of CD44, CD62L and PD-1 by flow cytometry. Represented data are means ± SEM. Data presented are representative of two independent experiments with 4–5 mice in each experimental group. *indicates p=0.0079 (compared to naive) by the Mann-Whitney test. (C) IFNγ production by alloreactive CD4⁺ T cells purified from mice as described in (A) and stimulated by irradiated B6 splenocytes in the presence of control or neutralizing anti-PD-L1 antibodies. Represented data are means ± SEM of five replicates and are representative of 2 independent experiments. * indicates p=0.0079 and ** indicate p<0.0286 by the Mann-Whitney test. (D) Inhibition (%) of IFNγ production by alloreactive CD4⁺ T cells purified and stimulated as in (C) with different doses of 2-Deoxy-D-glucose (2-DG). Represented data are means ± SEM of five replicates and are representative of 2 independent experiments.

Figure 2.. PD-1 controls the activity of chronic anti-male CD4 +T cells.

(A) Phenotype of chronic anti-male CD4⁺ T cells. Spleen CD3⁺ cells from normal Marilyn mice (naive) or from non-irradiated B6 male (H-2b) Rag2^-/- Il2rg^-/- recipients 21 days after reconstitution with purified Marylin CD4⁺ TCRBV6⁺ T cells (chronic), were analyzed for their expression of CD44, CD62L and PD-1 by flow cytometry. Represented data are means ± SEM. Data presented are representative of two independent experiments with 4–5 mice in each experimental group. * indicates p=0.0079 (compared to naive) by the Mann-Whitney test. (B) IFNγ production in the culture supernatant of spleen cells isolated from non-irradiated male B6 (H-2b) Rag2^-/- Il2rg^-/- recipients 21 days after reconstitution with purified Marylin CD4⁺ TCRBV6⁺ T cells. Control, anti-PD-L1 and/or anti-I-A/I-E neutralizing antibodies were added to the cultures. Represented data are means ± SEM of five replicates. Data presented are representative of three independent experiments. ND indicates below detection limits. (C) IFNγ production by CD4⁺ T cells purified from non-irradiated B6 male (H-2b) Rag2^-/- Il2rg^-/- recipients 21 days after reconstitution with purified Marylin CD4⁺ TCRBV6⁺ T cells. Cells were stimulated with anti-CD3/CD28 antibodies in the presence of immobilized Ctrl Ig or PD-L1-Ig fusion proteins. Represented data are means ± SEM of five replicates. Data presented are representative of three independent experiments. * indicates p=0.0079 (compared to Ctrl) by the Mann-Whitney test. (D) IFNγ production by CD4⁺ T cells purified as in C and stimulated with male peptide-loaded B6 Cd3^-/- bone marrow-derived dendritic cells in the presence of Ctrl or anti-PD-L1 neutralizing antibodies. Represented data are means ± SEM of three replicates. Data presented are representative of two independent experiments. (E) Cell counts of GFP⁺ T cells infiltrating the liver of non-irradiated male B6 (H-2b) Rag2^-/- Il2rg^-/- recipients 21 days after reconstitution with purified Marylin GFP⁺ CD4⁺ TCRBV6⁺ T cells and that received i.p. Ctrl or anti-PD-L1 neutralizing antibodies. * indicates p=0.0079 (compared to Ctrl antibody group) by the Mann-Whitney test. Represented data are means ± SEM. Data presented are representative of two independent experiments with 4–5 mice in each experimental group. (F) In vitro proliferation is assessed by fluorescent decay in CFSE-stained chronic or effector T cells after anti-CD3/CD28 stimulation. (G) IFNγ mRNA relative expression by anti-CD3/CD28-stimulated CD4⁺ T cells purified from non-irradiated B6 male (H-2b) Rag2^-/- Il2rg^-/- recipients 21 days after reconstitution with purified Marylin CD4⁺ TCRBV6⁺ T cells. Purified T cells were stimulated in the presence of anti-PD-L1 or control antibodies for 2, 4 and 6 hr. No significant difference was found between the two conditions. (H) IFNγ production by anti-CD3/CD28-stimulated CD4⁺ T cells purified from non-irradiated B6 male (H-2b) Rag2^-/- Il2rg^-/- recipients 21 days after reconstitution with purified Marylin CD4⁺ TCRBV6⁺ T cells. Purified T cells were stimulated in the presence of anti-PD-L1 or control antibodies. No significant difference was found between the two conditions.

Figure 3.. Long-term chronic anti-male CD4⁺T cells exhibit a low metabolism and are unable to shift their metabolism to produce IFNγ after activation.

(A) Basal ECAR (left) and basal and maximal OCR (right) of naive, effector or chronic CD4⁺ T cells. Data (means ± SEM of five replicated cultures) are representative of two independent experiments. * indicates p=0.0079 by the Mann-Whitney test. (B) Mitochondria mass of effector or chronic CD4⁺ T cells labeled with MitoTracker Green FM. Means ± SEM of MFI from T cells isolated from 4 mice per group is shown. Data are representative of two independent experiments. * indicates p=0.0079 by the Mann-Whitney test. NS is non-significant. (C) ATP in effector or chronic CD4⁺ T cells before and after anti-CD3/CD28 antibody stimulation. Data (means ± SEM of five replicated cultures) are representative of two independent experiments. (D) Purified naive, effector or chronic CD4⁺ T cells were stimulated with anti-CD3/CD28 antibodies or left unstimulated. After 12, 24, 48 and 72 hr, culture supernatants were harvested and glucose, lactate, IL-2 and IFNγ concentrations were determined as described in Material and methods. Data (means ± SEM from five replicated cultures) are representative of two independent experiments. (E) GLUT1 mRNA expression was assessed by quantitative PCR in effector and chronic CD4⁺ T cells after activation with PMA/ionomycin. Data (mean ± SEM) are from 4 to 5 mice per group and are representative of two independent experiments. * indicates p=0.0079 by the Mann-Whitney test. (F) Flow cytometry analysis of effector or chronically stimulated (chronic) CD4⁺ T cells labeled with 2-NBDG ex vivo (higher panel) or following anti-CD3/CD-28 antibody stimulation (lower panel). Means ± SEM of MFI from five animals per group and are representative of three independent experiments. (G) Mitostress assay on chronic CD4⁺ T cells isolated 7 or 21 days isolated from non-irradiated B6 male (H-2b) Rag2^-/- Il2rg^-/- recipients 21 days after reconstitution with purified Marylin CD4⁺ TCRBV6⁺ T cells. Data are representative of two independent experiments. (H) 2-DG-mediated inhibition of IFNγ production by anti-CD3/CD28-stimulated chronic CD4⁺ T cells. Data (mean ±SEM) were calculated from five replicated cultures and are representative of two independent experiments. (I) Ex vivo expression of inhibitory receptors 2B4, LAG3 and PD-1 by naive Marilyn CD4⁺ T cells or chronic CD4⁺ T cells purified from non-irradiated B6 male (H-2b) Rag2^-/- Il2rg^-/- recipients 7 or 21 days after reconstitution with purified Marylin CD4⁺ TCRBV6⁺ T cells. Data are representative of two independent experiments.

Figure 4.. Fatty acid oxidation supports the energy demand required for IFNγ production in long-term chronic CD4⁺ T cells.

(A) Effector or chronic CD4⁺ T cells were stimulated with anti-CD3/CD28 antibodies with or without 2-DG, DON, Etomoxir or oligomycin. After 48 hr, culture supernatants were harvested and IFNγ concentrations were determined by ELISA. Percent inhibition were calculated as described in Materials and methods. Data (mean ± SEM) were calculated from five replicated cultures and are representative of three independent experiments. (B) IFNγ production by effector (left) and chronic (right) T cells stimulated with anti-CD3/CD28 antibodies for 48 hr in medium containing glucose or galactose, in the presence of oligomycin. Data (mean ± SEM) were calculated from five replicated cultures and are representative of three independent experiments. (C) Glucose consumption (left) and lactate production (right) in cultures of effector or chronic T cells stimulated with anti-CD3/CD28 antibodies for 48 hr in the presence of oligomycin. Data (mean ± SEM) were calculated from five replicated cultures and are representative of three independent experiments. (D) Ex vivo relative expression of FATP4 and CPT1a mRNA in chronic or effector T cells. Data (mean ± SEM) were calculated from five mice per group and are representative of two independent experiments. * indicates p=0.0079 by the Mann-Whitney test. (E) Bodipy-C₁₆ uptake by chronic or effector T cells. Data (mean ± SEM) were calculated from five replicated cultures and are representative of two independent experiments. * indicates p=0.0079 by the Mann-Whitney test. (F) Glucose consumption (left panel), lactate (middle panel) and IFNγ production (right panel) by chronic CD4⁺ T cells stimulated for 48 hr by anti-CD3/CD28 antibodies in the presence of FAO inhibitor Etomoxir. Data were calculated from five replicated cultures and are representative of two independent experiments.

Figure 5.. PD-1 is not required for the induction and maintenance of the metabolic profile developed by chronic CD4⁺ T cells.

(A) IFNγ production in cultures of whole spleen cells isolated from male Rag2^-/- Il2rg^-/- B6 recipents adoptively transferred with anti-male Pdcd1^+/+ or Pdcd1^-/- Rag2^-/- Marilyn T cells for 21 days. Anti-I-A/I-E antibodies were added as indicated. Data (mean ± SEM of five replicated cultures) are representative of three independent experiments. * indicates p=0.0079 (compared to Pdcd1^+/+) by the Mann-Whitney test. NS indicates non-significant. (B) Basal ECAR of chronic Pdcd1^+/+ or Pdcd1^-/- CD4⁺ T cells and effector CD4⁺ T cells. Data (mean ± SEM of five replicated cultures) are representative of two independent experiments. * indicates p=0.0079 and NS indicates non-significant by the Mann-Whitney test. (C) Basal and maximal OCR of chronic Pdcd1^+/+ or Pdcd1^-/- CD4⁺ T cells and effector CD4⁺ T cells. (left panel). Maximal/basal OCR ratios (right panel). Data (mean ± SEM of five replicated cultures) are representative of two independent experiments. * indicates p=0.0079 by the Mann-Whitney test. (D) Chronic Pdcd1^+/+ or Pdcd1^-/- CD4 +T cells or effector CD4⁺ T cells were stimulated with anti-CD3/CD28 antibodies. After 48 hr, culture supernatants were harvested and lactate, IL-2 and IFNγ concentrations were determined as described in Material and methods. Data (mean ± SEM from five replicated cultures) are representative of three independent experiments. (E) Chronic Pdcd1^+/+ or Pdcd1^-/- CD4⁺ T cells or effector CD4⁺ T cells were stimulated with anti-CD3/CD28 antibodies in the presence of 2-DG, DON, Etomoxir or oligomycin. After 48 hr, culture supernatants were harvested and IFNγ concentrations were determined by ELISA. Percent inhibition was calculated as described in Materials and methods. Data (mean ± SEM) were calculated from five replicated cultures and are representative of three independent experiments. (F) Ex vivo relative expression of FATP4 and CPT1a mRNA in Pdcd1^+/+ or Pdcd1^-/- chronic T cells. Data (mean ± SEM) were calculated from five replicated cultures and are representative of three independent experiments. * indicates p=0.0079. (G) Bodipy-C₁₆ uptake by in Pdcd1^+/+ or Pdcd1^-/- chronic T cells. Data are representative of two independent experiments.

Figure 6.. By limiting T cell metabolism, PD-1 prevents activation-dependent mitochondrial ROS production and cell death.

(A) Flow cytometry analysis of chronic Pdcd1^+/+ or Pdcd1^-/- CD4⁺ T cells for intra-mitochondrial ROS production. Ex vivo spleen cells were labeled with MitoSOX Red and analyzed by flow cytometry. Gating was carried out on TCRBV6⁺ CD4⁺ cells as indicated. Mean ± SEM of percentage of positive cells from TCRBV6⁺ CD4⁺ T cells isolated from 5 mice is shown. Data are representative of two independent experiments. * indicates p=0.0079. (B) Flow cytometry analysis of chronic Pdcd1^+/+ or Pdcd1^-/- CD4⁺ T cells for their mitochondrial membrane potential. Ex vivo spleen cells were labeled with MitoTracker Red CMXRos and analyzed by flow cytometry. Analysis was gated on TCRBV6⁺ CD4⁺ cells as in (A). Mean ± SEM of MFI from TCRBV6⁺ CD4⁺ T cells isolated from 5 mice per group is shown. Data are representative of two independent experiments. * indicates p=0.0079 by the Mann-Whitney test. (C) Cell viability of purified chronic Pdcd1^+/+ or Pdcd1^-/- CD4⁺ T cells after anti-CD3/CD28 stimulation for 24 hr culture with or without NAC. Mean ± SEM of percentage of viable cells (DyeFL-negative) in TCRBV6⁺ CD4⁺ T cells isolated from 5 mice per group is shown. Data are representative of two independent experiments. * indicates p=0.0079 by the Mann-Whitney test. NS indicates non-significant. (D) Frequencies (%) of anti-male CD4⁺ TCRBV6⁺ T cells in the spleen of lymphopenic male recipients. Cells were analyzed 7 and 28 days after adoptive transfer. Mean ± SEM of percentage of TCRBV6⁺ CD4⁺ T cells isolated from 6 mice per group is shown. Data are representative of two independent experiments. * indicates p=0.0079 by the Mann-Whitney test. NS indicates non-significant.

Figure 7.. By limiting ROS production, PD-1 maintains functional fitness in chronically stimulated CD4⁺ T cells.

(A) Frequencies (%) of IFNγ-producing cells among purified chronic Pdcd1^+/+ or Pdcd1^-/- CD4⁺ T cells stimulated with anti-CD3/CD28 antibodies for 24 hr with or without NAC. After adding Brefeldin for the last 4 hr, cells were labelled for intracellular IFNγ. Mean ± SEM from T cells isolated from 5 mice per group is shown. Data are representative of two independent experiments. * and ** indicate p=0.0079 and p=0.0286, respectively, by the Mann-Whitney test. NS indicates non-significant. (B) Relative IFNγ production among purified chronic Pdcd1^+/+ or Pdcd1^-/- CD4⁺ T cells stimulated with anti-CD3/CD28 antibodies for 24 hr with or without NAC as in D. After adding Brefeldin for the last 4 hr, cells were labelled for intracellular IFNγ. Mean of MFI ± SEM from T cells isolated from 5 mice per group is shown. Data are representative of two independent experiments. * and ** indicate p=0.0079 and p=0.0286, respectively, by the Mann-Whitney test. NS indicates non-significant. (C) Western blotting analysis of mTORC1-dependent control of protein translation. Phosphorylation status of mTORC1 targets S6 protein and 4EBP1 was analysed in chronic Pdcd1^+/+ or Pdcd1^-/- CD4⁺ T cells ex vivo or after stimulation with anti-CD3/CD28 antibodies for 48 hr with or without mTOR inhibitor PP242. Analysis of EIf2 phosphorylation was used as a specificity control. Data are representative of three independent experiments. (D) Relative expression of IFNγ mRNA in chronic Pdcd1^+/+ or Pdcd1^-/- CD4⁺ T cells stimulated by anti-CD3/CD28 antibodies for the indicated periods of time. Data (mean ± SD) are representative of two independent experiments including 4 mice per group. * and ** indicate p=0.0079 and p=0.0286, respectively. (E) Analysis of IFNγ mRNA decay in chronic Pdcd1^+/+ or Pdcd1^-/- CD4⁺ T cells stimulated by anti-CD3/CD28 antibodies. Data (mean ± SD) are one experiment including 4 mice per group.

Figure 8.. Different outcome from PD-1-mediated regulation of T cell metabolism in early or late chronic antigen stimulation.

In chronic T cells responsible for early GVHD, PD-1-mediated inhibition of T cell metabolism promotes mitochondrial dysfunction and cell death. In T cells responsible for late GVHD, failure to engage glycolysis after PD-1 blockade stimulates OXPHOS. This leads to overload of the electron transport chain (ETC), ROS production, mRNA decay and cell death.