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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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CD8+ T cell exhaustion develops 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. For some experiments, 500 WT or 500 PD-1 KO P14 cells were transferred into separate naive recipient mice. P14 responses were then analyzed during the chronic phase of infection (day 42 p.i.) in the spleen. (A) Intracellular cytokine staining for IFNγ and TNF after stimulation with GP33 peptide (left). Values indicate the frequency of P14 cells producing IFNγ and/or TNF for individual mice at day 42 p.i. (B) Summary of the frequency of P14 cells producing IFNγ for multiple mice. (C) Summary of the frequency (left) and total number (right) of P14 cells coproducing IFNγ and TNF for multiple mice. (D) Protein expression of the indicated inhibitory receptors on naive CD8+ T cells, WT P14 cells, and PD-1 KO P14 cells of individual mice. Values indicate MFI of expression by FACS. (E) Boolean gating analysis of the simultaneous protein expression of multiple inhibitory receptors (Lag-3, 2B4, CD160, and Tim-3) on WT and PD-1 KO P14 cells. Pie charts display individual populations grouped according to total number of inhibitory receptors expressed. (F) Intracellular cytokine staining for IFNγ and TNF after stimulation with GP33 peptide for mice with separate transfer of WT or PD-1 KO P14 cells compared with mice with co-transferred WT and PD-1 KO P14 cells (left). Values indicate the frequency of P14 cells coproducing IFNγ and TNF for individual (left) and multiple mice (right). All error bars indicate ±SEM. **, P < 0.01 (all paired Student’s t test [A–E] except unpaired Student’s t test for separate transfer of P14 cells [F]). All data are representative of three to five independent experiments with at least five mice per group.
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fig2: CD8+ T cell exhaustion develops 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. For some experiments, 500 WT or 500 PD-1 KO P14 cells were transferred into separate naive recipient mice. P14 responses were then analyzed during the chronic phase of infection (day 42 p.i.) in the spleen. (A) Intracellular cytokine staining for IFNγ and TNF after stimulation with GP33 peptide (left). Values indicate the frequency of P14 cells producing IFNγ and/or TNF for individual mice at day 42 p.i. (B) Summary of the frequency of P14 cells producing IFNγ for multiple mice. (C) Summary of the frequency (left) and total number (right) of P14 cells coproducing IFNγ and TNF for multiple mice. (D) Protein expression of the indicated inhibitory receptors on naive CD8+ T cells, WT P14 cells, and PD-1 KO P14 cells of individual mice. Values indicate MFI of expression by FACS. (E) Boolean gating analysis of the simultaneous protein expression of multiple inhibitory receptors (Lag-3, 2B4, CD160, and Tim-3) on WT and PD-1 KO P14 cells. Pie charts display individual populations grouped according to total number of inhibitory receptors expressed. (F) Intracellular cytokine staining for IFNγ and TNF after stimulation with GP33 peptide for mice with separate transfer of WT or PD-1 KO P14 cells compared with mice with co-transferred WT and PD-1 KO P14 cells (left). Values indicate the frequency of P14 cells coproducing IFNγ and TNF for individual (left) and multiple mice (right). All error bars indicate ±SEM. **, P < 0.01 (all paired Student’s t test [A–E] except unpaired Student’s t test for separate transfer of P14 cells [F]). All data are representative of three to five independent experiments with at least five mice per group.

Mentions: Previous studies demonstrating potent inhibition of CD8+ T cells by the PD-1 pathway suggest that PD-1 may be essential for the development of T cell exhaustion (Barber et al., 2006; Keir et al., 2008; Wherry, 2011; Frebel et al., 2012). Thus, we tested the hypothesis that PD-1 KO P14 cells would not become exhausted during chronic LCMV infection. First, we examined the ability of WT versus PD-1 KO P14 cells to produce cytokines at day 42 p.i. after ex vivo peptide restimulation. At this time point, WT P14 cells displayed characteristic features of exhaustion. Compared with control memory P14 cells generated after acute LCMV infection, exhausted WT P14 cells less efficiently produced IFNγ (mean of 86% vs. 51%) or coproduced IFNγ and TNF (mean of 76% vs. 8%; Fig. 2 A). Surprisingly, production of IFNγ was even more severely reduced in the PD-1 KO P14 population, with a mean of only 39% of cells producing IFNγ (Fig. 2, A and B). PD-1 KO P14 cells also developed a significant reduction in IFNγ production per cell, as indicated by lower mean fluorescence intensity (MFI) of IFNγ expression compared with WT P14 cells in the same mice (Fig. 2 A and not depicted). In addition, PD-1 KO P14 cells exhibited reduced poly-functionality compared with WT P14 cells, with minimal coproduction of IFNγ and TNF (Fig. 2, A and C). There was a similar trend toward decreased total numbers of PD-1 KO IFNγ+TNF+ cells compared with WT in the spleen (Fig. 2 C). In contrast, the ability to degranulate, as measured by LAMP-1/CD107a staining, was retained (not depicted). Reduced cytokine production in the absence of PD-1 also corresponded with higher expression of multiple other inhibitory receptors, a second key feature of exhaustion. PD-1 KO P14 cells expressed higher Lag-3, 2B4/CD244, CD160, and Tigit than WT P14 cells at day 42 p.i. (Fig. 2 D). A higher frequency of PD-1 KO P14 cells also simultaneously coexpressed Lag-3, 2B4, Tim-3, and CD160 compared with WT cells (Fig. 2 E). Analysis of WT and PD-1 KO P14 cells in separate mice resulted in similar findings, confirming the CD8+ T cell–intrinsic nature of this effect (Fig. 2 F and not depicted). These findings indicate that CD8+ T cell exhaustion can develop in the absence of PD-1. In fact, two defining features of CD8+ TEX cells, loss of cytokine poly-functionality, and elevated inhibitory receptor coexpression, were more severe when CD8+ T cell–intrinsic PD-1 signals were absent.


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)

CD8+ T cell exhaustion develops 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. For some experiments, 500 WT or 500 PD-1 KO P14 cells were transferred into separate naive recipient mice. P14 responses were then analyzed during the chronic phase of infection (day 42 p.i.) in the spleen. (A) Intracellular cytokine staining for IFNγ and TNF after stimulation with GP33 peptide (left). Values indicate the frequency of P14 cells producing IFNγ and/or TNF for individual mice at day 42 p.i. (B) Summary of the frequency of P14 cells producing IFNγ for multiple mice. (C) Summary of the frequency (left) and total number (right) of P14 cells coproducing IFNγ and TNF for multiple mice. (D) Protein expression of the indicated inhibitory receptors on naive CD8+ T cells, WT P14 cells, and PD-1 KO P14 cells of individual mice. Values indicate MFI of expression by FACS. (E) Boolean gating analysis of the simultaneous protein expression of multiple inhibitory receptors (Lag-3, 2B4, CD160, and Tim-3) on WT and PD-1 KO P14 cells. Pie charts display individual populations grouped according to total number of inhibitory receptors expressed. (F) Intracellular cytokine staining for IFNγ and TNF after stimulation with GP33 peptide for mice with separate transfer of WT or PD-1 KO P14 cells compared with mice with co-transferred WT and PD-1 KO P14 cells (left). Values indicate the frequency of P14 cells coproducing IFNγ and TNF for individual (left) and multiple mice (right). All error bars indicate ±SEM. **, P < 0.01 (all paired Student’s t test [A–E] except unpaired Student’s t test for separate transfer of P14 cells [F]). All data are representative of three to five independent experiments with at least five mice per group.
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fig2: CD8+ T cell exhaustion develops 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. For some experiments, 500 WT or 500 PD-1 KO P14 cells were transferred into separate naive recipient mice. P14 responses were then analyzed during the chronic phase of infection (day 42 p.i.) in the spleen. (A) Intracellular cytokine staining for IFNγ and TNF after stimulation with GP33 peptide (left). Values indicate the frequency of P14 cells producing IFNγ and/or TNF for individual mice at day 42 p.i. (B) Summary of the frequency of P14 cells producing IFNγ for multiple mice. (C) Summary of the frequency (left) and total number (right) of P14 cells coproducing IFNγ and TNF for multiple mice. (D) Protein expression of the indicated inhibitory receptors on naive CD8+ T cells, WT P14 cells, and PD-1 KO P14 cells of individual mice. Values indicate MFI of expression by FACS. (E) Boolean gating analysis of the simultaneous protein expression of multiple inhibitory receptors (Lag-3, 2B4, CD160, and Tim-3) on WT and PD-1 KO P14 cells. Pie charts display individual populations grouped according to total number of inhibitory receptors expressed. (F) Intracellular cytokine staining for IFNγ and TNF after stimulation with GP33 peptide for mice with separate transfer of WT or PD-1 KO P14 cells compared with mice with co-transferred WT and PD-1 KO P14 cells (left). Values indicate the frequency of P14 cells coproducing IFNγ and TNF for individual (left) and multiple mice (right). All error bars indicate ±SEM. **, P < 0.01 (all paired Student’s t test [A–E] except unpaired Student’s t test for separate transfer of P14 cells [F]). All data are representative of three to five independent experiments with at least five mice per group.
Mentions: Previous studies demonstrating potent inhibition of CD8+ T cells by the PD-1 pathway suggest that PD-1 may be essential for the development of T cell exhaustion (Barber et al., 2006; Keir et al., 2008; Wherry, 2011; Frebel et al., 2012). Thus, we tested the hypothesis that PD-1 KO P14 cells would not become exhausted during chronic LCMV infection. First, we examined the ability of WT versus PD-1 KO P14 cells to produce cytokines at day 42 p.i. after ex vivo peptide restimulation. At this time point, WT P14 cells displayed characteristic features of exhaustion. Compared with control memory P14 cells generated after acute LCMV infection, exhausted WT P14 cells less efficiently produced IFNγ (mean of 86% vs. 51%) or coproduced IFNγ and TNF (mean of 76% vs. 8%; Fig. 2 A). Surprisingly, production of IFNγ was even more severely reduced in the PD-1 KO P14 population, with a mean of only 39% of cells producing IFNγ (Fig. 2, A and B). PD-1 KO P14 cells also developed a significant reduction in IFNγ production per cell, as indicated by lower mean fluorescence intensity (MFI) of IFNγ expression compared with WT P14 cells in the same mice (Fig. 2 A and not depicted). In addition, PD-1 KO P14 cells exhibited reduced poly-functionality compared with WT P14 cells, with minimal coproduction of IFNγ and TNF (Fig. 2, A and C). There was a similar trend toward decreased total numbers of PD-1 KO IFNγ+TNF+ cells compared with WT in the spleen (Fig. 2 C). In contrast, the ability to degranulate, as measured by LAMP-1/CD107a staining, was retained (not depicted). Reduced cytokine production in the absence of PD-1 also corresponded with higher expression of multiple other inhibitory receptors, a second key feature of exhaustion. PD-1 KO P14 cells expressed higher Lag-3, 2B4/CD244, CD160, and Tigit than WT P14 cells at day 42 p.i. (Fig. 2 D). A higher frequency of PD-1 KO P14 cells also simultaneously coexpressed Lag-3, 2B4, Tim-3, and CD160 compared with WT cells (Fig. 2 E). Analysis of WT and PD-1 KO P14 cells in separate mice resulted in similar findings, confirming the CD8+ T cell–intrinsic nature of this effect (Fig. 2 F and not depicted). These findings indicate that CD8+ T cell exhaustion can develop in the absence of PD-1. In fact, two defining features of CD8+ TEX cells, loss of cytokine poly-functionality, and elevated inhibitory receptor coexpression, were more severe when CD8+ T cell–intrinsic PD-1 signals were absent.

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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Related in: MedlinePlus