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. 2013 Feb 21;38(2):225-36.
doi: 10.1016/j.immuni.2012.10.020. Epub 2013 Feb 15.

Mitochondria are required for antigen-specific T cell activation through reactive oxygen species signaling

Laura A Sena  1 , Sha Li, Amit Jairaman, Murali Prakriya, Teresa Ezponda, David A Hildeman, Chyung-Ru Wang, Paul T Schumacker, Jonathan D Licht, Harris Perlman, Paul J Bryce, Navdeep S Chandel
Affiliations

Mitochondria are required for antigen-specific T cell activation through reactive oxygen species signaling

Laura A Sena et al. Immunity. .

Abstract

It is widely appreciated that T cells increase glycolytic flux during activation, but the role of mitochondrial flux is unclear. Here, we have shown that mitochondrial metabolism in the absence of glucose metabolism is sufficient to support interleukin-2 (IL-2) induction. Furthermore, we used mice with reduced mitochondrial reactive oxygen species (mROS) production in T cells (T-Uqcrfs(-/-) mice) to show that mitochondria are required for T cell activation to produce mROS for activation of nuclear factor of activated T cells (NFAT) and subsequent IL-2 induction. These mice could not induce antigen-specific expansion of T cells in vivo, but Uqcrfs1(-/-) T cells retained the ability to proliferate in vivo under lymphopenic conditions. This suggests that Uqcrfs1(-/-) T cells were not lacking bioenergetically but rather lacked specific ROS-dependent signaling events needed for antigen-specific expansion. Thus, mitochondrial metabolism is a critical component of T cell activation through the production of complex III ROS.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Mitochondrial metabolism is sufficient to support CD4+ T cell activation
a, Extracellular acidification rate (ECAR) and b, mitochondrial oxygen consumption rate (mitoOCR) of freshly isolated CD4+ T cells and 24h CD3 and CD28-stimulated CD4+ T cells (n=3 ± SEM). c, Cell viability of CD4+ T cells cultured in indicated media with and without CD3 and CD28 stimulation at 24h (n=3 ± SEM). d, Surface expression of CD69 and CD25 of CD4+ T cells cultured in indicated media at 24h (n=3 ± SEM). e, Relative IL-2 mRNA expression of CD4+ T cells cultured in indicated media at 24h, normalized to ML-19 expression (n=3 ± SEM). f, Cell growth (48h) and cell proliferation (96h) of CD4+ T cells cultured in indicated media (n=3). *p<0.05, **p<0.01
Figure 2
Figure 2. CD3-dependent calcium influx induces mROS, which are required for T cell activation
a, Mitochondrial ROS production measured by percent oxidized mito-roGFP probe of CD4+ T cells isolated from Mito-roGFP transgenic mice following CD3 and CD28 stimulation (n=3 ± SEM). b, Relative IL-2 mRNA expression of CD4+ T cells cultured with MVE and TPP (1.5uM each) at 8h, normalized to ML-19 expression (n=3 ± SEM). c, Mitochondrial ROS production measured by percent oxidized mito-roGFP probe of CD4+ T cells isolated from Mito-roGFP transgenic mice following indicated stimulation (n=3 ± SEM). # indicates a significant difference from column 1, * indicates a significant difference from column 2. d, Mitochondrial ROS production measured by percent oxidized mito-roGFP probe of CD4+ T cells isolated from Mito-roGFP transgenic mice following CD3 and CD28 stimulation with calcium inhibitors (EDTA 1mM, BTP2 200nM, Ruthenium Red (RR) 5uM, and Ru360 5uM) (n=3 ± SEM). e, Relative IL-2 mRNA expression of CD4+ T cells cultured with FCCP (1uM) and GaO (0.045U/ml) & Gal (500uM) at 4h, normalized to ML-19 expression (n=3 ± SEM). */#p<0.05, **p<0.01
Figure 3
Figure 3. Uqcrfs1fl/fl; Cd4-cre mice have reduced expression of RISP in T cells
a, Total number of cells isolated from mouse thymuses (n=5 ± SEM). b, Surface protein expression of CD4 and CD8 in mouse thymus (n=5 ± SEM). c, RISP protein expression in purified CD4+ splenic T cells and total splenocytes from Uqcrfs1fl/fl and Uqcrfs1fl/fl; Cd4-cre mice (n=4). d, Total number of splenocytes (following RBC lysis), B cells (CD45+B220+), macrophages (CD45+F4/80+), CD4+ T cells (CD45+CD4+), and CD8+ T cells (CD45+CD8+) in Uqcrfs1fl/fl and Uqcrfs1fl/fl; Cd4-cre spleens (n=5–7 ± SEM). *p<0.05, **p<0.01, ***p<0.001
Figure 4
Figure 4. Mitochondrial complex III reactive oxygen species are required for antigen-specific CD4+ T cell expansion
a, Mitochondrial ROS production measured by MitoSOX Red fluorescence following CD3 and CD28 stimulation (n=3 ± SEM). b, Surface protein expression of CD69 and CD25 in CD4+ T cells isolated from Uqcrfs1fl/fl and Uqcrfs1fl/fl; Cd4-cre mice (n=3 ± SEM). c, Relative IL-2 mRNA expression of CD4+ T cells isolated from Uqcrfs1fl/fl and Uqcrfs1fl/fl; Cd4-cre mice stimulated with anti-CD3 and anti-CD28, with and without treatment with GaO (0.045U/ml) and Gal (500uM), normalized to ML-19 expression (n=2 ± SEM). d, Relative IL-2 mRNA expression of CD4+ T cells isolated from Uqcrfs1fl/fl and Uqcrfs1fl/fl; Cd4-cre mice stimulated with PMA/ionomycin, with and without treatment with MVE (1.5uM), normalized to ML-19 expression (n=3 ± SEM). e, Mitochondrial ROS production measured by MitoSOX Red fluorescence (n=4 ± SEM). e, Nuclear and cytosolic NFAT1 levels measured by isolation of nuclear and cytosolic lysates, then immunoblot (n=3). g, Intracellular calcium concentration measured by Fura2-AM. Cells were transferred from 2mM calcium Ringer solution to 0mM calcium solution with 10ug/ml anti-CD3 antibody to stimulate intracellular store release (arrow). Cells were then transferred to 2mM calcium to allow for store-operated calcium influx (CRAC channel influx) (n=3). *p<0.05, **p<0.01, ***p<0.001
Figure 5
Figure 5. Mitochondrial complex III ROS are required for CD4+ antigen-specific CD4+ T cell expansion
a, Cell proliferation on day 4 of CD4+ T cells isolated from RISPfl/fl or RISP T KO mice and transferred into Rag1 KO mice (n=3). b, Cell proliferation on day 4 of CD4+ T cells isolated from RISPfl/fl or RISP T-KO mice and co-transferred with Thy1.1 CD4+ T cells into Rag1 KO mice (n=3). c, Surface protein expression of CD62L and CD44 in CD4+ populations from RISPfl/fl or RISP T KO spleens (n=5 ± SEM). d, Expansion of GP61-specific CD4+ T cells 6 days following IP injection of GP61 peptide in CFA into Uqcrfs1fl/fl or Uqcrfs1fl/fl; Cd4-cre mice (n=4 ± SEM). *p<0.05, **p<0.01
Figure 6
Figure 6. Mitochondrial complex III reactive oxygen species are required for antigen-specific CD4+ T cell-dependent inflammation in vivo
a–d, Ova-immunized (OVA) or sham-immunized (PBS) Uqcrfs1fl/fl and Uqcrfs1fl/fl; Cd4-cre mice were exposed to OVA by inhalation and airway inflammation was assessed (n=4 ± SEM, representative of 2 experiments). a, Cellular composition of BAL fluid. b, Representative histological lung sections stained with H&E or PAS. Bars, 100um. c, Relative IL-4, IL-5, IL-13 mRNA expression from homogenized lungs, normalized to β-actin expression. d, Serum ova-specific IgE antibody level determined by ELISA. e, Five micrograms of recombinant mouse IL-4 or PBS was administered intranasally for 3 days and airway inflammation was assessed. Number of eosinophils in BAL fluid (n=5 ± SEM). *p<0.05, **p<0.01
Figure 7
Figure 7. Mitochondrial complex III reactive oxygen species are required for antigen-specific CD8+ T cell expansion
a, Frequency of OVA/Kb-specific CD8+ T cells in LM-infected Uqcrfs1fl/fl and Uqcrfs1fl/fl; Cd4-cre mice. RISPfl/fl and RISP T KO mice were infected with rLM-OVA. On day 7 post-infection, splenocytes and hepatic leukocytes were stained with anti-TCRb, anti-CD8, and OVA/Kb dimer and analyzed by flow cytometry (n=5 ± SEM). b, Frequency of OVA/Kb-specific, IFN-g–producing CD8+ T cells in LM-infected Uqcrfs1fl/fl and Uqcrfs1fl/fl; Cd4-cre mice. Splenocytes from indicated mice were harvested on day 7 after LM infection and stimulated with either OVA peptide or HKLM. Cells were stained for CD8 expression and intracellular IFN-g, and analyzed by flow cytometry (n=5 ± SEM). c, Frequency of OVA/Kb-specific CD8+ T cells in Uqcrfs1fl/fl and Uqcrfs1fl/fl; Cd4-cre mice following a second LM-infection. One month after primary LM infection (2×103 CFU), mice were re-challenged with 4 × 104 CFU of rLM-OVA. On day 3 after secondary infection, splenocytes and hepatic leukocytes were harvested and stained with anti-TCRb, anti-CD8, and OVA/Kb dimer and analyzed by flow cytometry (n=5 ± SEM). d, Bacterial burden in spleens of LM-infected Uqcrfs1fl/fl and Uqcrfs1fl/fl; Cd4-cre mice on day 3 following secondary infection (n=5 ± SEM). *p<0.05, **p<0.01
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