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Autophagy is a critical regulator of memory CD8(+) T cell formation.

Puleston DJ, Zhang H, Powell TJ, Lipina E, Sims S, Panse I, Watson AS, Cerundolo V, Townsend AR, Klenerman P, Simon AK - Elife (2014)

Bottom Line: Interestingly, autophagy levels were diminished in CD8(+) T cells from aged mice.We could rejuvenate CD8(+) T cell responses in elderly mice in an autophagy dependent manner using the compound spermidine.This study reveals a cell intrinsic explanation for poor CD8(+) T cell memory in the elderly and potentially offers novel immune modulators to improve aged immunity.

View Article: PubMed Central - PubMed

Affiliation: MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom.

ABSTRACT
During infection, CD8(+) T cells initially expand then contract, leaving a small memory pool providing long lasting immunity. While it has been described that CD8(+) T cell memory formation becomes defective in old age, the cellular mechanism is largely unknown. Autophagy is a major cellular lysosomal degradation pathway of bulk material, and levels are known to fall with age. In this study, we describe a novel role for autophagy in CD8(+) T cell memory formation. Mice lacking the autophagy gene Atg7 in T cells failed to establish CD8(+) T cell memory to influenza and MCMV infection. Interestingly, autophagy levels were diminished in CD8(+) T cells from aged mice. We could rejuvenate CD8(+) T cell responses in elderly mice in an autophagy dependent manner using the compound spermidine. This study reveals a cell intrinsic explanation for poor CD8(+) T cell memory in the elderly and potentially offers novel immune modulators to improve aged immunity.

No MeSH data available.


Related in: MedlinePlus

Normal Bcl-2 levels and altered GLUT-1 expression on antigen-specific Atg7−/− CD8+ T cells.(A) MCMV-immunized WT and T-Atg7−/− mice were assessed for Bcl-2 expression in splenic m45-specific CD8+ T cells on day 9 and 22 post-infection. As a control, Bcl-2 was measured in CD44lo CD8+ T cells from unimmunized mice (naïve). Quantification shows Bcl-2 mean fluorescence intensity. Statistics—Student's t test (n = 4–5). (B) GLUT-1 antibody staining on day 9 and day 22 post-infection in MCMV-immunized WT and T-Atg7−/− mice. As a control, GLUT-1 was assessed in CD44lo CD8+ T cells from unimmunized mice (naïve). Bar graphs indicate the frequency of m45-tetramer+ CD8+ T cells and CD44lo CD8+ T cells (naïve) that express GLUT-1. Statistics—Mann Whitney U-test (n = 4–5).DOI:http://dx.doi.org/10.7554/eLife.03706.011
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fig4s1: Normal Bcl-2 levels and altered GLUT-1 expression on antigen-specific Atg7−/− CD8+ T cells.(A) MCMV-immunized WT and T-Atg7−/− mice were assessed for Bcl-2 expression in splenic m45-specific CD8+ T cells on day 9 and 22 post-infection. As a control, Bcl-2 was measured in CD44lo CD8+ T cells from unimmunized mice (naïve). Quantification shows Bcl-2 mean fluorescence intensity. Statistics—Student's t test (n = 4–5). (B) GLUT-1 antibody staining on day 9 and day 22 post-infection in MCMV-immunized WT and T-Atg7−/− mice. As a control, GLUT-1 was assessed in CD44lo CD8+ T cells from unimmunized mice (naïve). Bar graphs indicate the frequency of m45-tetramer+ CD8+ T cells and CD44lo CD8+ T cells (naïve) that express GLUT-1. Statistics—Mann Whitney U-test (n = 4–5).DOI:http://dx.doi.org/10.7554/eLife.03706.011

Mentions: To understand the mechanism that leads to the failure to maintain a memory compartment in the absence of autophagy, we next checked if Atg7−/− CD8+ T cells were undergoing more cell death than wild-type cells. While this is well described for naïve autophagy deficient T cells (Puleston and Simon, 2014), it had not been analyzed in antigen-specific T cells. As expected, in wild-type mice challenged with MCMV, we found highest levels of cell death among antigen-specific CD8+ T cells just after the peak of the effector phase on day 9, whereas in T-Atg7−/− mice, cell death was found to increase over time (Figure 4A). This could not be explained by a change in levels of the anti-apoptotic protein Bcl-2 (Figure 4—figure supplement 1A). The control of mitochondrial quality and reactive oxygen species (ROS) via mitophagy, the degradation of mitochondria, has been found to prevent cell death in T cells (Pua et al., 2009). In keeping with this, mitochondrial content (Figure 4B) and mitochondrial ROS (Figure 4C) were significantly increased in Atg7−/− antigen-specific CD8+ T cells. While this likely explains the increased cell death, as shown for other hematopoietic cells (Mortensen et al., 2010) and T cells (Pua et al., 2009), mitophagy is also thought to be essential for maintenance of healthy mitochondrial energy generation. This notion is consistent with our data in other autophagy-deficient hematopoietic cell types (macrophages and primary leukemic lines, submitted) demonstrating increased glycolytic enzymes by proteomic analysis, increased lactate production and decreased oxygen consumption using metabolic seahorse measurements. Indeed, studies by Pearce et al showed that formation of the CD8+ Tmem pool is accompanied by a switch to mitochondrial respiration (Pearce et al., 2009; Sukumar et al., 2013). However, in the physiological viral infection models used here, aiming to mimic human infection, the scarcity of antigen-specific CD8+ T cells prevented us from performing these metabolic measurements. In addition, due to the loss of CD8+ Tmem in the absence of Atg7 by day 30, any metabolic analysis was restricted to early time points when antigen-specific CD8+ T cells are still present in T-Atg7−/− mice. We resorted to the measurement of a surrogate marker of glycolysis, the glucose transporter GLUT-1 on antigen-specific CD8+ T cells by two different techniques, (a) the fluorescently labeled GLUT-1 binding domain of HTLV (human T cell leukemia virus), which specifically detects GLUT-1 (Manel et al., 2003; Kinet et al., 2007), and (b) a GLUT-1 antibody staining. As expected, GLUT-1 is upregulated on CD8+ Teff (day 9) and then downregulated on CD8+ Tmem (day 22) in wild-type mice as CD8+ T cells switch to mitochondrial respiration (Figure 4D). However, antigen-specific Atg7−/− CD8+ Teff expresses more GLUT-1 and downregulation does not occur to the same extent at the later time points compared to WT cells (day 22) (Figure 4D). This suggests that autophagy supports the metabolic switch during the differentiation from Teff to Tmem. This was confirmed using the GLUT-1 antibody (Figure 4—figure supplement 1B). However, treatment with the anti-diabetic drug metformin that induces mitochondrial β-oxidation metabolism in T cells (Pearce et al., 2009) did not rescue the memory T cell compartment in this model (data not shown).10.7554/eLife.03706.010Figure 4.Atg7−/− memory CD8+ T cells show increased mitochondrial content, reactive oxygen species, apoptosis and fail to down-regulate GLUT-1.


Autophagy is a critical regulator of memory CD8(+) T cell formation.

Puleston DJ, Zhang H, Powell TJ, Lipina E, Sims S, Panse I, Watson AS, Cerundolo V, Townsend AR, Klenerman P, Simon AK - Elife (2014)

Normal Bcl-2 levels and altered GLUT-1 expression on antigen-specific Atg7−/− CD8+ T cells.(A) MCMV-immunized WT and T-Atg7−/− mice were assessed for Bcl-2 expression in splenic m45-specific CD8+ T cells on day 9 and 22 post-infection. As a control, Bcl-2 was measured in CD44lo CD8+ T cells from unimmunized mice (naïve). Quantification shows Bcl-2 mean fluorescence intensity. Statistics—Student's t test (n = 4–5). (B) GLUT-1 antibody staining on day 9 and day 22 post-infection in MCMV-immunized WT and T-Atg7−/− mice. As a control, GLUT-1 was assessed in CD44lo CD8+ T cells from unimmunized mice (naïve). Bar graphs indicate the frequency of m45-tetramer+ CD8+ T cells and CD44lo CD8+ T cells (naïve) that express GLUT-1. Statistics—Mann Whitney U-test (n = 4–5).DOI:http://dx.doi.org/10.7554/eLife.03706.011
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fig4s1: Normal Bcl-2 levels and altered GLUT-1 expression on antigen-specific Atg7−/− CD8+ T cells.(A) MCMV-immunized WT and T-Atg7−/− mice were assessed for Bcl-2 expression in splenic m45-specific CD8+ T cells on day 9 and 22 post-infection. As a control, Bcl-2 was measured in CD44lo CD8+ T cells from unimmunized mice (naïve). Quantification shows Bcl-2 mean fluorescence intensity. Statistics—Student's t test (n = 4–5). (B) GLUT-1 antibody staining on day 9 and day 22 post-infection in MCMV-immunized WT and T-Atg7−/− mice. As a control, GLUT-1 was assessed in CD44lo CD8+ T cells from unimmunized mice (naïve). Bar graphs indicate the frequency of m45-tetramer+ CD8+ T cells and CD44lo CD8+ T cells (naïve) that express GLUT-1. Statistics—Mann Whitney U-test (n = 4–5).DOI:http://dx.doi.org/10.7554/eLife.03706.011
Mentions: To understand the mechanism that leads to the failure to maintain a memory compartment in the absence of autophagy, we next checked if Atg7−/− CD8+ T cells were undergoing more cell death than wild-type cells. While this is well described for naïve autophagy deficient T cells (Puleston and Simon, 2014), it had not been analyzed in antigen-specific T cells. As expected, in wild-type mice challenged with MCMV, we found highest levels of cell death among antigen-specific CD8+ T cells just after the peak of the effector phase on day 9, whereas in T-Atg7−/− mice, cell death was found to increase over time (Figure 4A). This could not be explained by a change in levels of the anti-apoptotic protein Bcl-2 (Figure 4—figure supplement 1A). The control of mitochondrial quality and reactive oxygen species (ROS) via mitophagy, the degradation of mitochondria, has been found to prevent cell death in T cells (Pua et al., 2009). In keeping with this, mitochondrial content (Figure 4B) and mitochondrial ROS (Figure 4C) were significantly increased in Atg7−/− antigen-specific CD8+ T cells. While this likely explains the increased cell death, as shown for other hematopoietic cells (Mortensen et al., 2010) and T cells (Pua et al., 2009), mitophagy is also thought to be essential for maintenance of healthy mitochondrial energy generation. This notion is consistent with our data in other autophagy-deficient hematopoietic cell types (macrophages and primary leukemic lines, submitted) demonstrating increased glycolytic enzymes by proteomic analysis, increased lactate production and decreased oxygen consumption using metabolic seahorse measurements. Indeed, studies by Pearce et al showed that formation of the CD8+ Tmem pool is accompanied by a switch to mitochondrial respiration (Pearce et al., 2009; Sukumar et al., 2013). However, in the physiological viral infection models used here, aiming to mimic human infection, the scarcity of antigen-specific CD8+ T cells prevented us from performing these metabolic measurements. In addition, due to the loss of CD8+ Tmem in the absence of Atg7 by day 30, any metabolic analysis was restricted to early time points when antigen-specific CD8+ T cells are still present in T-Atg7−/− mice. We resorted to the measurement of a surrogate marker of glycolysis, the glucose transporter GLUT-1 on antigen-specific CD8+ T cells by two different techniques, (a) the fluorescently labeled GLUT-1 binding domain of HTLV (human T cell leukemia virus), which specifically detects GLUT-1 (Manel et al., 2003; Kinet et al., 2007), and (b) a GLUT-1 antibody staining. As expected, GLUT-1 is upregulated on CD8+ Teff (day 9) and then downregulated on CD8+ Tmem (day 22) in wild-type mice as CD8+ T cells switch to mitochondrial respiration (Figure 4D). However, antigen-specific Atg7−/− CD8+ Teff expresses more GLUT-1 and downregulation does not occur to the same extent at the later time points compared to WT cells (day 22) (Figure 4D). This suggests that autophagy supports the metabolic switch during the differentiation from Teff to Tmem. This was confirmed using the GLUT-1 antibody (Figure 4—figure supplement 1B). However, treatment with the anti-diabetic drug metformin that induces mitochondrial β-oxidation metabolism in T cells (Pearce et al., 2009) did not rescue the memory T cell compartment in this model (data not shown).10.7554/eLife.03706.010Figure 4.Atg7−/− memory CD8+ T cells show increased mitochondrial content, reactive oxygen species, apoptosis and fail to down-regulate GLUT-1.

Bottom Line: Interestingly, autophagy levels were diminished in CD8(+) T cells from aged mice.We could rejuvenate CD8(+) T cell responses in elderly mice in an autophagy dependent manner using the compound spermidine.This study reveals a cell intrinsic explanation for poor CD8(+) T cell memory in the elderly and potentially offers novel immune modulators to improve aged immunity.

View Article: PubMed Central - PubMed

Affiliation: MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom.

ABSTRACT
During infection, CD8(+) T cells initially expand then contract, leaving a small memory pool providing long lasting immunity. While it has been described that CD8(+) T cell memory formation becomes defective in old age, the cellular mechanism is largely unknown. Autophagy is a major cellular lysosomal degradation pathway of bulk material, and levels are known to fall with age. In this study, we describe a novel role for autophagy in CD8(+) T cell memory formation. Mice lacking the autophagy gene Atg7 in T cells failed to establish CD8(+) T cell memory to influenza and MCMV infection. Interestingly, autophagy levels were diminished in CD8(+) T cells from aged mice. We could rejuvenate CD8(+) T cell responses in elderly mice in an autophagy dependent manner using the compound spermidine. This study reveals a cell intrinsic explanation for poor CD8(+) T cell memory in the elderly and potentially offers novel immune modulators to improve aged immunity.

No MeSH data available.


Related in: MedlinePlus