Fever supports CD8+ effector T cell responses by promoting mitochondrial translation

Abstract

Fever can provide a survival advantage during infection. Metabolic processes are sensitive to environmental conditions, but the effect of fever on T cell metabolism is not well characterized. We show that in activated CD8⁺ T cells, exposure to febrile temperature (39 °C) augmented metabolic activity and T cell effector functions, despite having a limited effect on proliferation or activation marker expression. Transcriptional profiling revealed an up-regulation of mitochondrial pathways, which was consistent with increased mass and metabolism observed in T cells exposed to 39 °C. Through in vitro and in vivo models, we determined that mitochondrial translation is integral to the enhanced metabolic activity and function of CD8⁺ T cells exposed to febrile temperature. Transiently exposing donor lymphocytes to 39 °C prior to infusion in a myeloid leukemia mouse model conferred enhanced therapeutic efficacy, raising the possibility that exposure of T cells to febrile temperatures could have clinical potential.

Keywords: T cell; fever; immunology; metabolism; mitochondria.

Fig. 1.

Exposure to 39 °C during activation promotes CD8⁺ T_E cell function and anabolic metabolism. (A) Naïve CD8⁺ T cells were activated with anti-CD3/CD28 at 37 or 39 °C for 24 h and then subsequently cultured at 37 °C. (B and C) Representative histogram and bar graph of mean fluorescent intensity (MFI) of IFN-γ production assessed 72 h postactivation. (D) Percent of CD69⁺ cells 24 h after activation. (E) Representative histogram of proliferation as assessed by CellTrace Violet (CTV) 24 to 72 h after activation. (F and G) Representative histogram and bar graphs of the MFI of CD69⁺ cells, surface-expressed CD25 and CD98, and forward scatter (FSC) cell size 24 h postactivation. (H) Representative Western blots of two biological replicates showing phosphorylated (p) AKT, S6, and 4E-BP1 24 h postactivation. (I) Potential glucose uptake measured by 2-NBDG uptake 24 h postactivation. (J) Lactate production 24 h postactivation. (K) ECAR measured at baseline and at the maximal rate following addition of rotenone and antimycin. n ≥ 3 biological replicates/group as indicated by individual data points and shown as mean ± SEM (except for the Western blots, n = 2). *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 2.

Exposure to 39 °C augments mitochondrial metabolism. (A) Principal component analysis plot of RNA expression in naïve 37 or 39 °C CD8⁺ T_E cells. (B) Gene expression of significantly increased transcripts relative to naïve cells. (C) Top eight up-regulated pathways (Metascape gene analysis) in 39 °C T_E cells using data derived from the same experiments as A and B. (D) Western blot of two biological replicates of mitochondrial respiratory components 24 h postactivation. (E and F) Mean fluorescent expression (MFI) of ATP5a and MTCO1 expression 24 h postactivation measured by flow cytometry. (G) Representative electron microscope images and mitochondrial area measured in arbitrary units (AU) from electron micrographs of cells 24 h postactivation. Each point represents an individual averaged mitochondrial area. (H) Representative histogram and bar graph of mitochondrial mass as measured by MitoTracker Green 24 h postactivation. (I) Mitochondrial mass measured by MitoTracker Green MFI 24 h postactivation at 35, 37, or 39 °C. (J) A representative plot of OCR at baseline and following administration of oligomycin (oligo), fluoro-carbonyl cyanide phenylhydrazone (FCCP), and rotenone and antimycin (R/A) of T cells 24 h postactivation. (K) Basal oxygen consumption and SRC 24 h postactivation. (L) Confocal images of mitochondria (green) in CD8⁺ OT-I PhAM GFP cells counterstained with nuclear stain DAPI (blue) in 37 or 39 °C T_E cells 24 h postactivation. The white dividing line indicates an overlaid cropped image. (I) For all experiments, n ≥ 3 biological replicates/group and shown as mean ± SEM (except for L, n = 1). *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 3.

Exposure to 39 °C improves T cell metabolism and function in vivo. (A) Naïve OT-I T cells were injected (i.v) into congenic hosts, and then mice were injected s.c. in the hind flanks with OVA peptide emulsified in IFA. Mice were then either subjected to WBH to raise their temperature to 39 °C or were treated to CTRL at AT. After 8 h, all mice were housed at AT, and then cells were harvested from the mice at the 24-h timepoint. (B) Activation state of donor OT-I cells measured as percent CD69⁺. (C) Cells from the iLN were incubated ex vivo in the presence of 2-NBDG to measure glucose uptake. (D) Cells from the iLN were incubated with MitoTracker Green to assess mitochondrial mass. (A–D) Data from two independent experiments, n = 7 mice/group. (E) To induce a polyclonal T cell response, anti-CD3/CD28 was injected into the hind footpad of mice. Mice were then either subjected to WBH for 8 h or treated to CTRL at AT. Cells were harvested from the mice 24 h after anti-CD3/CD28 injection. (F) CD8⁺ cells were isolated from the popliteal LN and (G) iLN , and the mitochondrial mass was measured by MitoTracker Green. Data from two independent experiments, n = 6 mice/group. (H) Western blot of mitochondrial respiratory chain components in CD8⁺ T cells isolated from iLN 24 h after injection of anti-CD3/CD28 (each column represents cells pooled from two mice). (I) Representative histogram of MTCO1 expression in CD8⁺ T cells from iLN 24 h after injection of anti-CD3/CD28 measured by flow cytometry (each condition represents cells pooled from three mice). (J) OCR and (K) ECAR at baseline and following administration of oligomycin (oligo), fluoro-carbonyl cyanide phenylhydrazone (FCCP), and rotenone and antimycin (R/A) of CD8⁺ T cell from iLN 24 h after activation (each group represents cells pooled from three mice). Data from one of two independent experiments. (L) To model ML, irradiated recipient mice were injected with allogeneic BM cells and leukemic (ML: WEHI 3B) cells. After 2 d, 37 or 39 °C T_E cells were injected, and survival was monitored. (M). Survival of mice following BMT and leukemia induction. Data from two independent experiments, n = 11 mice/group. **P < 0.01, ***P < 0.001.

Fig. 4.

Mitochondrial translation is enhanced by febrile temperature. (A) Heatmap showing mRNA expression of genes associated with mitochondrial biogenesis in naïve or activated CD8⁺ T cells. (B) MFI of MitoTracker Green 24 h postactivation in wild-type (CTRL) and RAPTOR-deficient (RAP KO) CD8⁺ T cells. (C) Heatmap showing mRNA expression of a selection of genes that encode mitochondrial ribosomal proteins (MRPL) in naïve or activated CD8⁺ T cells. (D) MFI of MitoTracker Green of CD8⁺ T cells targeted with CRISPR/Cas9 guides against MRPL39 (gMRPL), OXA1L (gOXA1L), or a nontargeting control (gCTRL) 24 h postactivation. (E) Western blot of mitochondrial respiratory components and (F and G) flow cytometry analysis of MTCO1 and SDHA in cells targeted with CRISPR/Cas9 guides against MRPL39 (gMRPL), OXA1L (gOXA1L), or a nontargeting control (gCTRL) 24 h postactivation. (H) Cells were treated with TIG (a mitochondrial-specific translation inhibitor) or CTRL during activation, and MitoTracker Green was measured 24 h postactivation. (I) Representative Western blot of mitochondrial respiratory components in CTRL- or TIG-treated groups. (J and K) OCR and ECAR at baseline and following administration of oligomycin (oligo), fluoro-carbonyl cyanide phenylhydrazone (FCCP), and rotenone and antimycin (R/A) of T cells 24 h after activation grouped from three replicates per condition. (L) Representative histogram and bar graph showing mean fluorescent expression (MFI) of IFN-γ production measured as expression of IFN-γ-YFP in CD8⁺ cells isolated from IFN-γ reporter (GREAT) mice and assessed 72 h postactivation. n ≥ 3 biological replicates/group as indicated by individual data points and shown as mean ± SEM (except E, n = 1). *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 5.

Superior antitumor activity of 39 °C primed T cells is dependent on enhanced mitochondrial translation. (A and B) Cultures of 37 or 39 T_E cells were incubated with TIG or CTRL for 48 h then adoptively transferred into leukemic mice (using the same model as Fig. 3I). n = 4 per group (except no T cell group n = 1). (C and D) To model melanoma, recipient mice were injected s.c. with B16 melanoma cells in the flank. After 5 d, 37 or 39 °C T_E cells previously cultured with TIG or CTRL for 72 h were injected i.v., and survival was monitored. n = 4 per group and shown as mean ± SEM. (E) Cultures of 37 or 39 °C T_E cells were incubated with TIG or CTRL and then adoptively transferred into congenic recipient mice; 2 d later, donor cells were isolated. (F) Representative histogram and bar graph showing percentage of donor cells isolated from the LNs of recipient mice. n = 3 per group shown as mean ± SEM. *P < 0.05, ***P < 0.001.