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Genetic absence of PD-1 promotes accumulation of terminally differentiated exhausted CD8+ T cells.

Odorizzi PM, Pauken KE, Paley MA, Sharpe A, Wherry EJ - J. Exp. Med. (2015)

Bottom Line: Increased proliferation between days 8 and 14 postinfection is associated with subsequent decreased CD8(+) T cell survival and disruption of a critical proliferative hierarchy necessary to maintain exhausted populations long term.Ultimately, the absence of PD-1 leads to the accumulation of more cytotoxic, but terminally differentiated, CD8(+) TEX cells.They also highlight a novel role for PD-1 in preserving TEX cell populations from overstimulation, excessive proliferation, and terminal differentiation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Penn Institute for Immunology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104 Department of Microbiology and Penn Institute for Immunology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104.

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Diminished long-term proliferation and stability of CD8+ T cells in the absence of PD-1. WT and PD-1 KO P14 cells were mixed at a 1:1 ratio (250 cells each), adoptively transferred into naive recipient mice, and infected with LCMV clone 13. In CD4-depleted mice, αCD4 was given at days −1 and 1 p.i. P14 responses were then analyzed at the indicated time points. (A) Longitudinal analysis of the frequency (left) and absolute numbers (right) of WT and PD-1 KO P14 cells in the spleen during LCMV clone 13 infection. (B) Longitudinal analysis of the total number of WT and PD-1 KO P14 cells in the spleen of CD4-depleted mice with LCMV clone 13 infection. (C) Summary of the total numbers of WT and PD-1 KO P14 cells coproducing IFNγ and TNF (left) or Ki67+ (right) at day 300 p.i. in the spleen. (D) Longitudinal analysis of the frequency of Ki67+ WT and PD-1 KO P14 cells in the blood during LCMV clone 13 infection. (E) Protein expression of PD-1 versus Ki67 in WT and PD-1 KO P14 cells at day 42 p.i. in the spleen during infection with LCMV clone 13 with (bottom) or without (top) CD4 depletion. Values indicate the frequency of P14 cells positive for Ki67. (F) Representative histogram of protein expression of p-S6235/236 in WT and PD-1 KO P14 cells at day 21 p.i. in the spleen after stimulation with GP33 peptide for 60 min. Values indicate the MFI of expression for phospho-proteins for individual mice (top) and multiple mice (bottom). All error bars indicate ±SEM. *, P < 0.05; **, P < 0.01 (paired Student’s t test) for all graphs (A–F). All data are representative of three independent experiments with at least five mice per group.
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fig5: Diminished long-term proliferation and stability of CD8+ T cells in the absence of PD-1. WT and PD-1 KO P14 cells were mixed at a 1:1 ratio (250 cells each), adoptively transferred into naive recipient mice, and infected with LCMV clone 13. In CD4-depleted mice, αCD4 was given at days −1 and 1 p.i. P14 responses were then analyzed at the indicated time points. (A) Longitudinal analysis of the frequency (left) and absolute numbers (right) of WT and PD-1 KO P14 cells in the spleen during LCMV clone 13 infection. (B) Longitudinal analysis of the total number of WT and PD-1 KO P14 cells in the spleen of CD4-depleted mice with LCMV clone 13 infection. (C) Summary of the total numbers of WT and PD-1 KO P14 cells coproducing IFNγ and TNF (left) or Ki67+ (right) at day 300 p.i. in the spleen. (D) Longitudinal analysis of the frequency of Ki67+ WT and PD-1 KO P14 cells in the blood during LCMV clone 13 infection. (E) Protein expression of PD-1 versus Ki67 in WT and PD-1 KO P14 cells at day 42 p.i. in the spleen during infection with LCMV clone 13 with (bottom) or without (top) CD4 depletion. Values indicate the frequency of P14 cells positive for Ki67. (F) Representative histogram of protein expression of p-S6235/236 in WT and PD-1 KO P14 cells at day 21 p.i. in the spleen after stimulation with GP33 peptide for 60 min. Values indicate the MFI of expression for phospho-proteins for individual mice (top) and multiple mice (bottom). All error bars indicate ±SEM. *, P < 0.05; **, P < 0.01 (paired Student’s t test) for all graphs (A–F). All data are representative of three independent experiments with at least five mice per group.

Mentions: The increase in cell death of PD-1 KO P14 cells suggests that long-term durability of virus-specific CD8+ T cells may be compromised in the absence of PD-1. During LCMV clone 13 infection, the frequency and absolute number of WT P14 cells began to stabilize in the spleen and blood between days 22 and 42 p.i. (Fig. 5 A and not depicted), consistent with previous findings (Shin et al., 2007; Wherry et al., 2007; Blattman et al., 2009). In contrast, PD-1 KO P14 cells declined in both frequency and absolute number until at least day 42 p.i., suggesting that long-term durability of virus-specific CD8+ T cell responses may be compromised in the absence of PD-1 (Fig. 5 A). However, viremia during LCMV clone 13 infection of C57BL/6 mice gradually resolves by days 60–90 p.i, complicating the interpretation of changes in exhaustion at late time points (Matloubian et al., 1994; Blackburn et al., 2009). Therefore, we next used a model of chronic LCMV infection where transient depletion of CD4+ T cells before infection with LCMV clone 13 leads to persistent, life-long viremia (Matloubian et al., 1994). As observed in CD4+ T cell–sufficient clone 13 infection, WT P14 cell responses stabilized in frequency and absolute number after day 48 p.i. in CD4+ T cell–depleted mice (Fig. 5 B and not depicted). In contrast, the number of PD-1 KO P14 cells failed to stabilize and gradually declined over time. By day 300 p.i., the total number of PD-1 KO P14 cells was less than that of WT P14 cells (Fig. 5 B). This deterioration in the number of PD-1 KO P14 cells was also indicated by a significant decrease in the total numbers of IFNγ+TNF+ cells in the spleen at this time point (Fig. 5 C, left). The inability of PD-1 KO P14 cells to form a stable, long-term population of TEX cells in the spleen could be caused, at least in part, by the increased cell death observed in this population. However, ongoing antigen-driven proliferation is central to the durability of TEX cell populations (McCune et al., 2000; Shin et al., 2007). To interrogate how ongoing proliferation is impacted by PD-1 deficiency, we examined proliferation of WT and PD-1 KO P14 cells over the time course of LCMV clone 13 infection. Despite similar expression of Ki67 during the acute phase of infection, PD-1 KO P14 cells had decreased Ki67 expression beginning at day 27 p.i. in the blood (Fig. 5 D). By day 42 p.i., PD-1 KO P14 cells had dramatically reduced expression of Ki67 in the spleen of both CD4+ T cell–sufficient (clone 13) and CD4+ T cell–depleted (Cl-13 αCD4) mice (Fig. 5 E), and this defect in ongoing proliferation persisted until at least day 300 p.i. in CD4-depleted mice (Fig. 5 C, right). In addition, PD-1 KO P14 cells incorporated significantly less BrdU from days 35 to 42 p.i. than WT P14 cells (not depicted). Collectively, these findings indicate that increased cell death and loss of sustained proliferative ability contribute to the deceased stability of PD-1 KO P14 cells in chronic infection. This shift in the proliferative ability of PD-1 KO P14 cells during the chronic phase of infection corresponds with a loss of TCR responsiveness. At day 21 p.i., both WT and PD-1 KO P14 cells have decreased TCR signaling compared with day 8 p.i., as measured by phosphorylation of mTOR2448 and S6235/236 after brief ex vivo peptide restimulation (Fig. 5 F and not depicted). However, PD-1 KO P14 cells had significantly reduced phosphorylation of S6235/236 at day 21 p.i. compared with WT P14 cells (Fig. 5 F). Of note, by day 35 p.i., both WT and PD-1 KO P14 cells had minimal phosphorylation of S6235/236 and other signaling molecules (not depicted). Thus, the PD-1 KO P14 cells that survive into the chronic phase of infection are a less durable population and have reduced TCR responsiveness, as well as limited proliferative ability.


Genetic absence of PD-1 promotes accumulation of terminally differentiated exhausted CD8+ T cells.

Odorizzi PM, Pauken KE, Paley MA, Sharpe A, Wherry EJ - J. Exp. Med. (2015)

Diminished long-term proliferation and stability of CD8+ T cells in the absence of PD-1. WT and PD-1 KO P14 cells were mixed at a 1:1 ratio (250 cells each), adoptively transferred into naive recipient mice, and infected with LCMV clone 13. In CD4-depleted mice, αCD4 was given at days −1 and 1 p.i. P14 responses were then analyzed at the indicated time points. (A) Longitudinal analysis of the frequency (left) and absolute numbers (right) of WT and PD-1 KO P14 cells in the spleen during LCMV clone 13 infection. (B) Longitudinal analysis of the total number of WT and PD-1 KO P14 cells in the spleen of CD4-depleted mice with LCMV clone 13 infection. (C) Summary of the total numbers of WT and PD-1 KO P14 cells coproducing IFNγ and TNF (left) or Ki67+ (right) at day 300 p.i. in the spleen. (D) Longitudinal analysis of the frequency of Ki67+ WT and PD-1 KO P14 cells in the blood during LCMV clone 13 infection. (E) Protein expression of PD-1 versus Ki67 in WT and PD-1 KO P14 cells at day 42 p.i. in the spleen during infection with LCMV clone 13 with (bottom) or without (top) CD4 depletion. Values indicate the frequency of P14 cells positive for Ki67. (F) Representative histogram of protein expression of p-S6235/236 in WT and PD-1 KO P14 cells at day 21 p.i. in the spleen after stimulation with GP33 peptide for 60 min. Values indicate the MFI of expression for phospho-proteins for individual mice (top) and multiple mice (bottom). All error bars indicate ±SEM. *, P < 0.05; **, P < 0.01 (paired Student’s t test) for all graphs (A–F). All data are representative of three independent experiments with at least five mice per group.
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fig5: Diminished long-term proliferation and stability of CD8+ T cells in the absence of PD-1. WT and PD-1 KO P14 cells were mixed at a 1:1 ratio (250 cells each), adoptively transferred into naive recipient mice, and infected with LCMV clone 13. In CD4-depleted mice, αCD4 was given at days −1 and 1 p.i. P14 responses were then analyzed at the indicated time points. (A) Longitudinal analysis of the frequency (left) and absolute numbers (right) of WT and PD-1 KO P14 cells in the spleen during LCMV clone 13 infection. (B) Longitudinal analysis of the total number of WT and PD-1 KO P14 cells in the spleen of CD4-depleted mice with LCMV clone 13 infection. (C) Summary of the total numbers of WT and PD-1 KO P14 cells coproducing IFNγ and TNF (left) or Ki67+ (right) at day 300 p.i. in the spleen. (D) Longitudinal analysis of the frequency of Ki67+ WT and PD-1 KO P14 cells in the blood during LCMV clone 13 infection. (E) Protein expression of PD-1 versus Ki67 in WT and PD-1 KO P14 cells at day 42 p.i. in the spleen during infection with LCMV clone 13 with (bottom) or without (top) CD4 depletion. Values indicate the frequency of P14 cells positive for Ki67. (F) Representative histogram of protein expression of p-S6235/236 in WT and PD-1 KO P14 cells at day 21 p.i. in the spleen after stimulation with GP33 peptide for 60 min. Values indicate the MFI of expression for phospho-proteins for individual mice (top) and multiple mice (bottom). All error bars indicate ±SEM. *, P < 0.05; **, P < 0.01 (paired Student’s t test) for all graphs (A–F). All data are representative of three independent experiments with at least five mice per group.
Mentions: The increase in cell death of PD-1 KO P14 cells suggests that long-term durability of virus-specific CD8+ T cells may be compromised in the absence of PD-1. During LCMV clone 13 infection, the frequency and absolute number of WT P14 cells began to stabilize in the spleen and blood between days 22 and 42 p.i. (Fig. 5 A and not depicted), consistent with previous findings (Shin et al., 2007; Wherry et al., 2007; Blattman et al., 2009). In contrast, PD-1 KO P14 cells declined in both frequency and absolute number until at least day 42 p.i., suggesting that long-term durability of virus-specific CD8+ T cell responses may be compromised in the absence of PD-1 (Fig. 5 A). However, viremia during LCMV clone 13 infection of C57BL/6 mice gradually resolves by days 60–90 p.i, complicating the interpretation of changes in exhaustion at late time points (Matloubian et al., 1994; Blackburn et al., 2009). Therefore, we next used a model of chronic LCMV infection where transient depletion of CD4+ T cells before infection with LCMV clone 13 leads to persistent, life-long viremia (Matloubian et al., 1994). As observed in CD4+ T cell–sufficient clone 13 infection, WT P14 cell responses stabilized in frequency and absolute number after day 48 p.i. in CD4+ T cell–depleted mice (Fig. 5 B and not depicted). In contrast, the number of PD-1 KO P14 cells failed to stabilize and gradually declined over time. By day 300 p.i., the total number of PD-1 KO P14 cells was less than that of WT P14 cells (Fig. 5 B). This deterioration in the number of PD-1 KO P14 cells was also indicated by a significant decrease in the total numbers of IFNγ+TNF+ cells in the spleen at this time point (Fig. 5 C, left). The inability of PD-1 KO P14 cells to form a stable, long-term population of TEX cells in the spleen could be caused, at least in part, by the increased cell death observed in this population. However, ongoing antigen-driven proliferation is central to the durability of TEX cell populations (McCune et al., 2000; Shin et al., 2007). To interrogate how ongoing proliferation is impacted by PD-1 deficiency, we examined proliferation of WT and PD-1 KO P14 cells over the time course of LCMV clone 13 infection. Despite similar expression of Ki67 during the acute phase of infection, PD-1 KO P14 cells had decreased Ki67 expression beginning at day 27 p.i. in the blood (Fig. 5 D). By day 42 p.i., PD-1 KO P14 cells had dramatically reduced expression of Ki67 in the spleen of both CD4+ T cell–sufficient (clone 13) and CD4+ T cell–depleted (Cl-13 αCD4) mice (Fig. 5 E), and this defect in ongoing proliferation persisted until at least day 300 p.i. in CD4-depleted mice (Fig. 5 C, right). In addition, PD-1 KO P14 cells incorporated significantly less BrdU from days 35 to 42 p.i. than WT P14 cells (not depicted). Collectively, these findings indicate that increased cell death and loss of sustained proliferative ability contribute to the deceased stability of PD-1 KO P14 cells in chronic infection. This shift in the proliferative ability of PD-1 KO P14 cells during the chronic phase of infection corresponds with a loss of TCR responsiveness. At day 21 p.i., both WT and PD-1 KO P14 cells have decreased TCR signaling compared with day 8 p.i., as measured by phosphorylation of mTOR2448 and S6235/236 after brief ex vivo peptide restimulation (Fig. 5 F and not depicted). However, PD-1 KO P14 cells had significantly reduced phosphorylation of S6235/236 at day 21 p.i. compared with WT P14 cells (Fig. 5 F). Of note, by day 35 p.i., both WT and PD-1 KO P14 cells had minimal phosphorylation of S6235/236 and other signaling molecules (not depicted). Thus, the PD-1 KO P14 cells that survive into the chronic phase of infection are a less durable population and have reduced TCR responsiveness, as well as limited proliferative ability.

Bottom Line: Increased proliferation between days 8 and 14 postinfection is associated with subsequent decreased CD8(+) T cell survival and disruption of a critical proliferative hierarchy necessary to maintain exhausted populations long term.Ultimately, the absence of PD-1 leads to the accumulation of more cytotoxic, but terminally differentiated, CD8(+) TEX cells.They also highlight a novel role for PD-1 in preserving TEX cell populations from overstimulation, excessive proliferation, and terminal differentiation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Penn Institute for Immunology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104 Department of Microbiology and Penn Institute for Immunology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104.

Show MeSH
Related in: MedlinePlus