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

Xu X, Araki K, Li S, Han JH, Ye L, Tan WG, Konieczny BT, Bruinsma MW, Martinez J, Pearce EL, Green DR, Jones DP, Virgin HW, Ahmed R - Nat. Immunol. (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Emory Vaccine Center and Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia, USA.

ABSTRACT
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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Antigen-specific CD8 T cells lacking the Atg7 gene exhibit cell-intrinsic defects in developing into long-term memory cells in bone marrow chimeric mice. Atg7fl/fl plus C57BL/6 (CD45.2/CD45.1) control and Atg7fl/flGzmb-Cre plus C57BL/6 (CD45.2/CD45.1) experimental mixed bone marrow chimera mice were generated. CD8 T cell response was evaluated following the LCMV Armstrong infection according to the scheme in Supplementary Fig. 5a. (a) and (b) Flow cytometric analysis of DbGP33- and DbNP396-specific T cells on day 8, 15 and 30 p.i. Gated on tetramer-positive CD8 T cells as indicated. Number on each quadrant represents the percentage of tetramer-positive cells that are either CD45.1+ or CD45.2+ as indicated on the axis. Average percentages of DbGP33- and DbNP396-specific T cells from day 8 to day 30 in the peripheral blood of both groups of chimeric mice are plotted to the right of the flow plots in (a) and (b). The black line represents tetramer-positive cell of Atg7fl/fl origin and the red line of Atg7fl/flGzmb-Cre origin. The number of tetramer-positive cells on day 8 p.i. from each mouse is normalized to 100%. (c) Percent of CD45.2+ antigen-specific cells in tissues on day 30 post-infection. The error bars in (a)–(c) indicate SEM. n=3–4 mice in each group. Data in (a)–(c) are representative of two independent experiments.
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Figure 6: Antigen-specific CD8 T cells lacking the Atg7 gene exhibit cell-intrinsic defects in developing into long-term memory cells in bone marrow chimeric mice. Atg7fl/fl plus C57BL/6 (CD45.2/CD45.1) control and Atg7fl/flGzmb-Cre plus C57BL/6 (CD45.2/CD45.1) experimental mixed bone marrow chimera mice were generated. CD8 T cell response was evaluated following the LCMV Armstrong infection according to the scheme in Supplementary Fig. 5a. (a) and (b) Flow cytometric analysis of DbGP33- and DbNP396-specific T cells on day 8, 15 and 30 p.i. Gated on tetramer-positive CD8 T cells as indicated. Number on each quadrant represents the percentage of tetramer-positive cells that are either CD45.1+ or CD45.2+ as indicated on the axis. Average percentages of DbGP33- and DbNP396-specific T cells from day 8 to day 30 in the peripheral blood of both groups of chimeric mice are plotted to the right of the flow plots in (a) and (b). The black line represents tetramer-positive cell of Atg7fl/fl origin and the red line of Atg7fl/flGzmb-Cre origin. The number of tetramer-positive cells on day 8 p.i. from each mouse is normalized to 100%. (c) Percent of CD45.2+ antigen-specific cells in tissues on day 30 post-infection. The error bars in (a)–(c) indicate SEM. n=3–4 mice in each group. Data in (a)–(c) are representative of two independent experiments.

Mentions: The conditional knockout system is a powerful tool to investigate functions of a gene within a specific cell population. However, one caveat of using this system is that the profound depletion of antigen-specific CD8 T cells observed in Atg7fl/flGzmb-Cre and Atg5fl/flGzmb-Cre mice during the course of LCMV infection (Figs. 4 and 5) may impact host environmental factors such as inflammatory cytokines that could exacerbate T cell survival and differentiation. Furthermore, other cell types, such as natural killer (NK) cells, also express granzyme B, introducing an additional level of complexity when comparing experimental and control mice. Recent work showed that NK cells were implicated in modulating T cell immunity26, 27. To address these issues, we generated mixed bone marrow chimeras that were reconstituted from an mixture of congenitally marked Atg7fl/flGzmb-Cre (CD45.2) and wild-type C57BL/6 (CD45.1) donor bone marrow cells into wild-type C57BL/6 (CD45.1) recipient mice (Supplementary Fig. 5a). This system allowed us to compare changes in number between wild type and Atg7fl/flGzmb-Cre antigen-specific CD8 T cells in the same environment (in the same mouse), therefore excluding factors that only act on one population of these cells. As a control, we also generated mixed bone marrow chimera mice that were reconstituted with Atg7fl/fl (CD45.2) (no cre) and wild-type (CD45.1) donor mice. After reconstitution, these animals were challenged with the LCMV Armstrong, and virus-specific CD8 T cells were examined over time (Supplementary Fig. 5a). At day 8 p.i., there was a significant expansion of antigen-specific T cells from both CD45.2 (knock-out) and CD45.1 (wild type) donor populations. The DbGP33 and DbNP396-specific CD8 T cell responses were longitudinally analyzed during the effector and memory stages (Fig. 6a and b, Supplementary Fig. 5c). Similar to the observation in the conditional knockout mice, we observed a more than 90% decline in Atg7-deficient LCMV-specific CD8 T cells (CD45.2+) from day 8 to day 15 in Atg7fl/flGzmb-Cre plus C57BL/6 chimeric mice, compared to a moderate decline of 50%–60% in the corresponding wild-type (CD45.2+) population in the Atg7fl/fl plus C57BL/6 controls. Four weeks after infection there were virtually no antigen-specific CD8 T cells of Atg7fl/flGzmb-Cre origin (CD45.2+) throughout the peripheral blood, lymphoid and non-lymphoid tissues in Atg7fl/flGzmb-Cre plus C57BL/6 chimeric mice (Fig. 6a–c). A significant proportion of naive CD8 T cells (CD44lo) from the Atg7fl/flGzmb-Cre donor persisted within the chimeric mice, indicating specific loss of the antigen-experienced effector CD8 T cells from Atg7fl/flGzmb-Cre mice throughout the progression of infection (Supplementary Fig. 5b). Since the cells from WT (CD45.1+) origin made up the majority of the reconstituted population within the chimeric mice (Supplementary Fig. 5b), the physiological and pathological conditions closely resembled those of the WT mice. Hence, in the absence of potential external variables, such as changes introduced in other cell types or any subtle variations in viral clearance, the defects rising from Atg7-deficiency were cell-intrinsic properties that affected survival of the antigen-specific effector CD8 T cells to the memory phase. Thus, these data confirmed that the autophagy pathway is critical for memory T cell formation.


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

Xu X, Araki K, Li S, Han JH, Ye L, Tan WG, Konieczny BT, Bruinsma MW, Martinez J, Pearce EL, Green DR, Jones DP, Virgin HW, Ahmed R - Nat. Immunol. (2014)

Antigen-specific CD8 T cells lacking the Atg7 gene exhibit cell-intrinsic defects in developing into long-term memory cells in bone marrow chimeric mice. Atg7fl/fl plus C57BL/6 (CD45.2/CD45.1) control and Atg7fl/flGzmb-Cre plus C57BL/6 (CD45.2/CD45.1) experimental mixed bone marrow chimera mice were generated. CD8 T cell response was evaluated following the LCMV Armstrong infection according to the scheme in Supplementary Fig. 5a. (a) and (b) Flow cytometric analysis of DbGP33- and DbNP396-specific T cells on day 8, 15 and 30 p.i. Gated on tetramer-positive CD8 T cells as indicated. Number on each quadrant represents the percentage of tetramer-positive cells that are either CD45.1+ or CD45.2+ as indicated on the axis. Average percentages of DbGP33- and DbNP396-specific T cells from day 8 to day 30 in the peripheral blood of both groups of chimeric mice are plotted to the right of the flow plots in (a) and (b). The black line represents tetramer-positive cell of Atg7fl/fl origin and the red line of Atg7fl/flGzmb-Cre origin. The number of tetramer-positive cells on day 8 p.i. from each mouse is normalized to 100%. (c) Percent of CD45.2+ antigen-specific cells in tissues on day 30 post-infection. The error bars in (a)–(c) indicate SEM. n=3–4 mice in each group. Data in (a)–(c) are representative of two independent experiments.
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Figure 6: Antigen-specific CD8 T cells lacking the Atg7 gene exhibit cell-intrinsic defects in developing into long-term memory cells in bone marrow chimeric mice. Atg7fl/fl plus C57BL/6 (CD45.2/CD45.1) control and Atg7fl/flGzmb-Cre plus C57BL/6 (CD45.2/CD45.1) experimental mixed bone marrow chimera mice were generated. CD8 T cell response was evaluated following the LCMV Armstrong infection according to the scheme in Supplementary Fig. 5a. (a) and (b) Flow cytometric analysis of DbGP33- and DbNP396-specific T cells on day 8, 15 and 30 p.i. Gated on tetramer-positive CD8 T cells as indicated. Number on each quadrant represents the percentage of tetramer-positive cells that are either CD45.1+ or CD45.2+ as indicated on the axis. Average percentages of DbGP33- and DbNP396-specific T cells from day 8 to day 30 in the peripheral blood of both groups of chimeric mice are plotted to the right of the flow plots in (a) and (b). The black line represents tetramer-positive cell of Atg7fl/fl origin and the red line of Atg7fl/flGzmb-Cre origin. The number of tetramer-positive cells on day 8 p.i. from each mouse is normalized to 100%. (c) Percent of CD45.2+ antigen-specific cells in tissues on day 30 post-infection. The error bars in (a)–(c) indicate SEM. n=3–4 mice in each group. Data in (a)–(c) are representative of two independent experiments.
Mentions: The conditional knockout system is a powerful tool to investigate functions of a gene within a specific cell population. However, one caveat of using this system is that the profound depletion of antigen-specific CD8 T cells observed in Atg7fl/flGzmb-Cre and Atg5fl/flGzmb-Cre mice during the course of LCMV infection (Figs. 4 and 5) may impact host environmental factors such as inflammatory cytokines that could exacerbate T cell survival and differentiation. Furthermore, other cell types, such as natural killer (NK) cells, also express granzyme B, introducing an additional level of complexity when comparing experimental and control mice. Recent work showed that NK cells were implicated in modulating T cell immunity26, 27. To address these issues, we generated mixed bone marrow chimeras that were reconstituted from an mixture of congenitally marked Atg7fl/flGzmb-Cre (CD45.2) and wild-type C57BL/6 (CD45.1) donor bone marrow cells into wild-type C57BL/6 (CD45.1) recipient mice (Supplementary Fig. 5a). This system allowed us to compare changes in number between wild type and Atg7fl/flGzmb-Cre antigen-specific CD8 T cells in the same environment (in the same mouse), therefore excluding factors that only act on one population of these cells. As a control, we also generated mixed bone marrow chimera mice that were reconstituted with Atg7fl/fl (CD45.2) (no cre) and wild-type (CD45.1) donor mice. After reconstitution, these animals were challenged with the LCMV Armstrong, and virus-specific CD8 T cells were examined over time (Supplementary Fig. 5a). At day 8 p.i., there was a significant expansion of antigen-specific T cells from both CD45.2 (knock-out) and CD45.1 (wild type) donor populations. The DbGP33 and DbNP396-specific CD8 T cell responses were longitudinally analyzed during the effector and memory stages (Fig. 6a and b, Supplementary Fig. 5c). Similar to the observation in the conditional knockout mice, we observed a more than 90% decline in Atg7-deficient LCMV-specific CD8 T cells (CD45.2+) from day 8 to day 15 in Atg7fl/flGzmb-Cre plus C57BL/6 chimeric mice, compared to a moderate decline of 50%–60% in the corresponding wild-type (CD45.2+) population in the Atg7fl/fl plus C57BL/6 controls. Four weeks after infection there were virtually no antigen-specific CD8 T cells of Atg7fl/flGzmb-Cre origin (CD45.2+) throughout the peripheral blood, lymphoid and non-lymphoid tissues in Atg7fl/flGzmb-Cre plus C57BL/6 chimeric mice (Fig. 6a–c). A significant proportion of naive CD8 T cells (CD44lo) from the Atg7fl/flGzmb-Cre donor persisted within the chimeric mice, indicating specific loss of the antigen-experienced effector CD8 T cells from Atg7fl/flGzmb-Cre mice throughout the progression of infection (Supplementary Fig. 5b). Since the cells from WT (CD45.1+) origin made up the majority of the reconstituted population within the chimeric mice (Supplementary Fig. 5b), the physiological and pathological conditions closely resembled those of the WT mice. Hence, in the absence of potential external variables, such as changes introduced in other cell types or any subtle variations in viral clearance, the defects rising from Atg7-deficiency were cell-intrinsic properties that affected survival of the antigen-specific effector CD8 T cells to the memory phase. Thus, these data confirmed that the autophagy pathway is critical for memory T cell formation.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Emory Vaccine Center and Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus