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Autophagy is essential for effector CD8+ T cell survival and memory formation


The importance of autophagy in the generation of memory CD8+ T cells in vivo is not well defined. We report here that autophagy was dynamically regulated in virus-specific CD8+ T cells during acute infection of mice with lymphocytic choriomeningitis virus. In contrast to the current paradigm, autophagy decreased in activated proliferating effector CD8+ T cells and was then upregulated when the cells stopped dividing just before the contraction phase. Consistent with those findings, deletion of the gene encoding either of the autophagy-related molecules Atg5 or Atg7 had little to no effect on the proliferation and function of effector cells, but these autophagy-deficient effector cells had survival defects that resulted in compromised formation of memory T cells. Our studies define when autophagy is needed during effector and memory differentiation and warrant reexamination of the relationship between T cell activation and autophagy.

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Figure 1: Analysis of autophagy in virus-specific CD8+ T cells during a viral infection.
Figure 2: Autophagic flux in virus-specific CD8+ T cells is inversely correlated with cell proliferation status.
Figure 3: Autophagic flux is inhibited during the T cell clonal expansion phase via impairment of autophagosome maturation into autophagolysosomes.
Figure 4: Atg7 deficiency results in survival defects in effector CD8+ T cells during the effector-to-memory transition.
Figure 5: Survival defects of Atg5-deficient T cells during the contraction phase following infection with LCMV Armstrong strain.
Figure 6: Antigen-specific CD8+ T cells lacking Atg7 exhibit cell-intrinsic defects in the development into long-term memory cells in chimeras.
Figure 7: Metabolomic and transcriptomic analysis of Atg7-deficient CD8+ T cells.
Figure 8: Autophagy in CD8+ T cells is essential for regulating chronic infection with LCMV.

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We thank A. Rao (La Jolla Institute for Allergy and Immunology) for the MSCV-IRES-Thy-1.1 retroviral vector; J. Jacob (Emory University) for Gzmb-Cre transgenic mice; V. Tran for the LC-MS experiments; R. Karaffa and S. Durham for sorting cells by flow cytometry at the Emory Flow Cytometry Core Facility; and A. Rae for assistance in the use of ImageStream. Supported by the US National Institutes of Health (R01 AI030048 to R.A. and R01 AI084887 to H.W.V.), the Mérieux Foundation (R.A.) and the Crohn's and Colitis Foundation (H.W.V.).

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Authors and Affiliations



X.X., K.A., S.L., M.W.B., J.M., D.R.G., D.P.J., H.W.V. and R.A. designed the research; X.X., K.A., S.L., J.-H.H., L.Y., W.G.T. and B.T.K. performed experiments. X.X., K.A., S.L., M.W.B., E.L.P., D.R.G., D.P.J., H.W.V. and R.A. analyzed data; and X.X., K.A., S.L., E.L.P., H.W.V. and R.A. wrote the manuscript.

Corresponding authors

Correspondence to Koichi Araki, Herbert W Virgin or Rafi Ahmed.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Dynamic regulation of autophagy in antigen-specific CD8+ T cells.

(a) A cartoon illustration of autophagy pathway highlighting molecules that are used to assess autophagy activity in this study. LC3b and p62 are both targeted to autophagosomes, which subsequently fuse with lysosomes for degradation. (b and c) mRNA levels of LC3b (b) and p62 (c) in P14 at different time points post LCMV Armstrong infection. (d) Protein expression levels of LC3b and p62 at early stages of P14 cell activation. GP33 peptide (200 µg) was intraveneously injected into P14 transgenic mice. The corresponding mRNA levels are show in (e). Errors bars in (b), (c) and (e) represent SEM. (b) - (e) are representative of at least two independent experiments.

Supplementary Figure 2 Autophagy activity during the early expansion phase of CD8+ T cells.

(a) Experimental set-up. Retrovirus transduced P14 cells were adoptively transferred into B6 mice, followed by LCMV Armstrong infection. (b-e) Flow cytometry plots of adoptively transferred P14 cells in spleens. P14 cells transduced with MIT retrovirus harboring GFP-LC3b (either wild type or G120A mutant) are positive for the congenic marker Thy1.1. Day 2 (b) and day 3 (d) p.i. splenocytes were used for the analysis. The percentage of GFP-negative cells out of the transduced P14 (Thy1.1+) cells from each group is summarized in (c) and (e). Errors bars in (c) and (e) represent SEM. (b) and (d) are representative of two independent experiments, n3 in each group.

Supplementary Figure 3 Autophagy activity measured in endogenous CD8+ T cells using retrovirus carrying the reporter gene encoding GFP-LC3b.

(a) Experimental set-up. Hematopoietic stem cells transduced with MIT retrovirus harboring GFP-LC3b (either wild type or G120A mutant) were introduced into irradiated host mice, which were left for 8-10 weeks for reconstitution of the hematopoietic system. (b) Flow plots showing changes in GFP intensity over the course of LCMV Armstrong infection in MIT-GFP-LC3b and MIT-G120A groups. Summary plots were shown on the right. (b) is representative of two independent experiments, n2 in each group.

Supplementary Figure 4 Characterization of Atg7-deficient H-2Db–gp33–specific CD8+ T cells.

(a) DbGP33-specific CD8 T cells were purified from day 8 mice infected with LCMV Armstrong (2x105 pfu). Representative plots of target cells before and after cell sorting, showing a typical > 95% purity. (b) Flow cytometry plot of total activated CD8 T cells (CD44hiCD62Llo) before and after cell sorting. Spleens were collected from mice infected with LCMV Armstrong (2x105 pfu) at day 8 p.i.. (c) Phenotypic properties of Atg7-deficient DbGP33-specific T cells at day 8 p.i (black outlined histograms). Solid gray histograms represent wild-type DbGP33-specific T cells. (d) Numbers of tetramer-positive cells in spleens 5 days p.i. (e) Flow plots showing percent of cells showing Brdu+ tetramer+ CD8+ T cells 5 hr after Brdu i.p. injection at day 5 p.i.; data summarized in (f). (g) Flow plots showing percent of cells are Annexin V+ tetramer+ CD8+ T cells at day 5 p.i; data summarized in (h). Errors bars in (d), (f) and (h) represent SEM. (c), (e) and (g) are representative of at least two independent experiments, n3 in each group.

Supplementary Figure 5 Bone marrow chimera reconstitution and T cell response following infection with LCMV Armstrong strain.

(a) Experimental set-up for generating Atg7fl/fl plus C57BL/6 (CD45.2/CD45.1) control and Atg7fl/fl Gzmb-Cre plus C57BL/6 (CD45.2/CD45.1) experimental mixed bone marrow chimera mice. CD8 T cell response was evaluated following the LCMV Armstrong infection. (b) Peripheral blood mononuclear cells were used to assess the level of reconstitution in the bone marrow chimeras prior to infection. Gated on total CD8 T cells. Plots to the right of the arrows indicate the level of reconstitution 30 days p.i in CD44lo populations. Gated on total CD8 T cells. Number on each quadrant represents the percentage in each. (c) Summary plot of percentage of DbGP33- and DbNP396-specific T cells from day 8 to day 30 in the peripheral blood of the chimeric mice. The number of tetramer-positive cells on day 8 p.i. is normalized to 100%. The dashed line represents tetramer-positive cell of C57BL/6 origin (CD45.1+) and the solid black and red lines of Atg7fl/fl and Atg7fl/fl Gzmb-Cre origin (CD45.2+), respectively. Each line represents data from one experimental mouse. Data are representative of two independent experiments.

Supplementary Figure 6 Metabolic activity network of Atg7-deficient H-2Db–gp33+ CD8+ T cells.

Prediction directly from m/z feature tables (Supplementary Table 1) by method previously described1. Metabolites are colored according to log2 fold change.

1. Li, S. et al. Predicting network activity from high throughput metabolomics. PLoS Comput Biol 9, e1003123 (2013).

Supplementary Figure 7 Virus-specific CD8+ T cell response during chronic infection with LCMV in the absence of Atg7.

Atg7fl/fl Gzmb-Cre and control mice were infected with LCMV Clone-13. (a) and (c) Flow cytometric analysis of LCMV-specific T cells post LCMV Clone 13 infection in PBMC, spleen, liver and lung on days 8 and 15 p.i., respectively. The numbers on FACS plots indicate the percentage of DbGP276-specific T cells on the gated CD8 T cells in each sample examined. (b) and (d) The total numbers of DbGP276-specific T cells in each tissue are plotted at days 8 and 15 p.i., respectively. n=3-5 in each group. Data are representative of two independent experiments. Error bars indicate SEM. *, p0.05. ***, p0.0005

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Supplementary Text and Figures

Supplementary Figures 1–7 (PDF 1226 kb)

Supplementary Table 1

List of metabolites that were significantly different between Atg7f/f and Atg7f/f Gzmb-Cre (XLS 27 kb)

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Xu, X., Araki, K., Li, S. et al. Autophagy is essential for effector CD8+ T cell survival and memory formation. Nat Immunol 15, 1152–1161 (2014).

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