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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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Early changes in proliferation and functionality of PD-1 KO P14 cells. For in vivo experiments, 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. P14 responses were then analyzed at the indicated time points. For early time points (days 1–4), a 1:1 ratio of mixed WT and PD-1 KO P14 cells (2.6 × 106 cells each) was adoptively transferred before infection. (A) Intracellular cytokine staining for IFNγ and TNF after stimulation with GP33 peptide (left). Values indicate the frequency of P14 cells producing IFNγ and TNF for individual (left) and multiple mice (right) at day 8 p.i. in the spleen. (B) Total number of WT and PD-1 KO P14 cells (left) and IFNγ-producing cells (right) at day 8 p.i. in the spleen. (C) Protein expression of the indicated inhibitory receptors by naive CD8+ T cells, WT P14 cells, and PD-1 KO P14 cells at day 8 p.i. in the spleens of individual mice. Values indicate MFI of expression by FACS. (D) Boolean gating analysis of the simultaneous protein expression of multiple inhibitory receptors (Lag-3, 2B4, CD160, and Tim-3) by WT and PD-1 KO P14 cells at day 8 p.i. in the spleen. Pie charts display individual populations grouped according to total number of inhibitory receptors expressed. (E) Expression of CTV as a measure of proliferation on WT and PD-1 KO P14 cells on the indicated days p.i. with LCMV clone 13 in the spleen (left) or after in vitro stimulation with GP33-pulsed splenocytes (right) by FACS. (F) Summary of the frequency of Ki67+ WT and PD-1 KO P14 cells for multiple mice at day 8 p.i. in the spleen. (G) Expression of CD44 versus BrdU incorporation in WT and PD-1 KO P14 cells at day 8 p.i. in the spleen after 24-h BrdU pulse for individual (left) and multiple (right) mice. Values indicate the frequency of P14 cells positive for BrdU based on FMO staining control. (H) Representative FACS histograms of protein expression of p-mTor2448 and p-S6235/236 by WT and PD-1 KO P14 cells at day 8 p.i. in the spleen after stimulation with GP33 peptide for 60 min. Values indicate MFI for phospho-proteins for individual mice (left) and multiple mice (right). All error bars indicate ±SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (paired Student’s t test) for all graphs (A–H). All data are representative of two to five independent experiments with five to eight mice per group.
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fig3: Early changes in proliferation and functionality of PD-1 KO P14 cells. For in vivo experiments, 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. P14 responses were then analyzed at the indicated time points. For early time points (days 1–4), a 1:1 ratio of mixed WT and PD-1 KO P14 cells (2.6 × 106 cells each) was adoptively transferred before infection. (A) Intracellular cytokine staining for IFNγ and TNF after stimulation with GP33 peptide (left). Values indicate the frequency of P14 cells producing IFNγ and TNF for individual (left) and multiple mice (right) at day 8 p.i. in the spleen. (B) Total number of WT and PD-1 KO P14 cells (left) and IFNγ-producing cells (right) at day 8 p.i. in the spleen. (C) Protein expression of the indicated inhibitory receptors by naive CD8+ T cells, WT P14 cells, and PD-1 KO P14 cells at day 8 p.i. in the spleens of individual mice. Values indicate MFI of expression by FACS. (D) Boolean gating analysis of the simultaneous protein expression of multiple inhibitory receptors (Lag-3, 2B4, CD160, and Tim-3) by WT and PD-1 KO P14 cells at day 8 p.i. in the spleen. Pie charts display individual populations grouped according to total number of inhibitory receptors expressed. (E) Expression of CTV as a measure of proliferation on WT and PD-1 KO P14 cells on the indicated days p.i. with LCMV clone 13 in the spleen (left) or after in vitro stimulation with GP33-pulsed splenocytes (right) by FACS. (F) Summary of the frequency of Ki67+ WT and PD-1 KO P14 cells for multiple mice at day 8 p.i. in the spleen. (G) Expression of CD44 versus BrdU incorporation in WT and PD-1 KO P14 cells at day 8 p.i. in the spleen after 24-h BrdU pulse for individual (left) and multiple (right) mice. Values indicate the frequency of P14 cells positive for BrdU based on FMO staining control. (H) Representative FACS histograms of protein expression of p-mTor2448 and p-S6235/236 by WT and PD-1 KO P14 cells at day 8 p.i. in the spleen after stimulation with GP33 peptide for 60 min. Values indicate MFI for phospho-proteins for individual mice (left) and multiple mice (right). All error bars indicate ±SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (paired Student’s t test) for all graphs (A–H). All data are representative of two to five independent experiments with five to eight mice per group.

Mentions: We next tested whether signs of exhaustion in the absence of PD-1 could be detected early during chronic infection. To interrogate this issue, the phenotype and function of WT and PD-1 KO P14 cells were analyzed at the peak of the effector response, day 8 p.i. Both WT and PD-1 KO P14 cells were highly capable of producing IFNγ at this time point (Fig. 3 A), consistent with previous work (Parry et al., 2005; Butte et al., 2007; Patsoukis et al., 2012; Yokosuka et al., 2012; Zinselmeyer et al., 2013). However, PD-1 KO P14 cells displayed a subtle, but significant, reduction in production of IFNγ and coproduction of IFNγ and TNF (Fig. 3 A and not depicted). PD-1 KO P14 cells also greatly outnumbered WT P14 cells in the spleen, blood, and other organs at day 8 p.i. (Fig. 3 B, left; and not depicted), consistent with the notion that PD-1 signals restrain CD8+ T cell responses. Therefore, despite the decrease in per-cell functionality indicated by a reduced frequency of IFNγ+ cells within the PD-1 KO P14 population, there was still a significant increase in the total number of PD-1 KO IFNγ-producing cells compared with WT (Fig. 3 B, right). PD-1 KO P14 cells also had elevated expression (Fig. 3 C) and coexpression (Fig. 3 D) of several other inhibitory receptors at this time point. Collectively, these data suggest that dysfunction of PD-1 KO P14 cells may begin early during LCMV clone 13 infection. However, the increase in total numbers of PD-1 KO P14 cells suggests that loss of PD-1 signals might improve the activation and proliferation of virus-specific CD8+ T cells, despite inducing slight defects in cytokine production and elevated inhibitory receptor expression.


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)

Early changes in proliferation and functionality of PD-1 KO P14 cells. For in vivo experiments, 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. P14 responses were then analyzed at the indicated time points. For early time points (days 1–4), a 1:1 ratio of mixed WT and PD-1 KO P14 cells (2.6 × 106 cells each) was adoptively transferred before infection. (A) Intracellular cytokine staining for IFNγ and TNF after stimulation with GP33 peptide (left). Values indicate the frequency of P14 cells producing IFNγ and TNF for individual (left) and multiple mice (right) at day 8 p.i. in the spleen. (B) Total number of WT and PD-1 KO P14 cells (left) and IFNγ-producing cells (right) at day 8 p.i. in the spleen. (C) Protein expression of the indicated inhibitory receptors by naive CD8+ T cells, WT P14 cells, and PD-1 KO P14 cells at day 8 p.i. in the spleens of individual mice. Values indicate MFI of expression by FACS. (D) Boolean gating analysis of the simultaneous protein expression of multiple inhibitory receptors (Lag-3, 2B4, CD160, and Tim-3) by WT and PD-1 KO P14 cells at day 8 p.i. in the spleen. Pie charts display individual populations grouped according to total number of inhibitory receptors expressed. (E) Expression of CTV as a measure of proliferation on WT and PD-1 KO P14 cells on the indicated days p.i. with LCMV clone 13 in the spleen (left) or after in vitro stimulation with GP33-pulsed splenocytes (right) by FACS. (F) Summary of the frequency of Ki67+ WT and PD-1 KO P14 cells for multiple mice at day 8 p.i. in the spleen. (G) Expression of CD44 versus BrdU incorporation in WT and PD-1 KO P14 cells at day 8 p.i. in the spleen after 24-h BrdU pulse for individual (left) and multiple (right) mice. Values indicate the frequency of P14 cells positive for BrdU based on FMO staining control. (H) Representative FACS histograms of protein expression of p-mTor2448 and p-S6235/236 by WT and PD-1 KO P14 cells at day 8 p.i. in the spleen after stimulation with GP33 peptide for 60 min. Values indicate MFI for phospho-proteins for individual mice (left) and multiple mice (right). All error bars indicate ±SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (paired Student’s t test) for all graphs (A–H). All data are representative of two to five independent experiments with five to eight mice per group.
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fig3: Early changes in proliferation and functionality of PD-1 KO P14 cells. For in vivo experiments, 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. P14 responses were then analyzed at the indicated time points. For early time points (days 1–4), a 1:1 ratio of mixed WT and PD-1 KO P14 cells (2.6 × 106 cells each) was adoptively transferred before infection. (A) Intracellular cytokine staining for IFNγ and TNF after stimulation with GP33 peptide (left). Values indicate the frequency of P14 cells producing IFNγ and TNF for individual (left) and multiple mice (right) at day 8 p.i. in the spleen. (B) Total number of WT and PD-1 KO P14 cells (left) and IFNγ-producing cells (right) at day 8 p.i. in the spleen. (C) Protein expression of the indicated inhibitory receptors by naive CD8+ T cells, WT P14 cells, and PD-1 KO P14 cells at day 8 p.i. in the spleens of individual mice. Values indicate MFI of expression by FACS. (D) Boolean gating analysis of the simultaneous protein expression of multiple inhibitory receptors (Lag-3, 2B4, CD160, and Tim-3) by WT and PD-1 KO P14 cells at day 8 p.i. in the spleen. Pie charts display individual populations grouped according to total number of inhibitory receptors expressed. (E) Expression of CTV as a measure of proliferation on WT and PD-1 KO P14 cells on the indicated days p.i. with LCMV clone 13 in the spleen (left) or after in vitro stimulation with GP33-pulsed splenocytes (right) by FACS. (F) Summary of the frequency of Ki67+ WT and PD-1 KO P14 cells for multiple mice at day 8 p.i. in the spleen. (G) Expression of CD44 versus BrdU incorporation in WT and PD-1 KO P14 cells at day 8 p.i. in the spleen after 24-h BrdU pulse for individual (left) and multiple (right) mice. Values indicate the frequency of P14 cells positive for BrdU based on FMO staining control. (H) Representative FACS histograms of protein expression of p-mTor2448 and p-S6235/236 by WT and PD-1 KO P14 cells at day 8 p.i. in the spleen after stimulation with GP33 peptide for 60 min. Values indicate MFI for phospho-proteins for individual mice (left) and multiple mice (right). All error bars indicate ±SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (paired Student’s t test) for all graphs (A–H). All data are representative of two to five independent experiments with five to eight mice per group.
Mentions: We next tested whether signs of exhaustion in the absence of PD-1 could be detected early during chronic infection. To interrogate this issue, the phenotype and function of WT and PD-1 KO P14 cells were analyzed at the peak of the effector response, day 8 p.i. Both WT and PD-1 KO P14 cells were highly capable of producing IFNγ at this time point (Fig. 3 A), consistent with previous work (Parry et al., 2005; Butte et al., 2007; Patsoukis et al., 2012; Yokosuka et al., 2012; Zinselmeyer et al., 2013). However, PD-1 KO P14 cells displayed a subtle, but significant, reduction in production of IFNγ and coproduction of IFNγ and TNF (Fig. 3 A and not depicted). PD-1 KO P14 cells also greatly outnumbered WT P14 cells in the spleen, blood, and other organs at day 8 p.i. (Fig. 3 B, left; and not depicted), consistent with the notion that PD-1 signals restrain CD8+ T cell responses. Therefore, despite the decrease in per-cell functionality indicated by a reduced frequency of IFNγ+ cells within the PD-1 KO P14 population, there was still a significant increase in the total number of PD-1 KO IFNγ-producing cells compared with WT (Fig. 3 B, right). PD-1 KO P14 cells also had elevated expression (Fig. 3 C) and coexpression (Fig. 3 D) of several other inhibitory receptors at this time point. Collectively, these data suggest that dysfunction of PD-1 KO P14 cells may begin early during LCMV clone 13 infection. However, the increase in total numbers of PD-1 KO P14 cells suggests that loss of PD-1 signals might improve the activation and proliferation of virus-specific CD8+ T cells, despite inducing slight defects in cytokine production and elevated inhibitory receptor expression.

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