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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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Adoptive transfer of WT and PD-1 KO P14 cells as a model to study T cell exhaustion in the absence of PD-1. In brief, CD8+ T cells were isolated from peripheral blood of naive WT or PD-1 KO P14 mice. 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. (A) Representative FACS plots of gating scheme for WT and PD-1 KO P14 cells. P14 cells were gated from total CD8+ T cells (far left) by expression of H-2Db GP33 tetramer, congenic markers, and PD-1, as indicated (shaded gray: antibody isotype control). (B) Longitudinal analysis of viral load in serum of mice that received the indicated numbers of WT and PD-1 KO P14 cells followed by infection with LCMV clone 13 (±SEM). *, P < 0.05 (unpaired Student’s t test). (C) Survival curve of mice that received the indicated numbers of WT and PD-1 KO P14 cells after LCMV clone 13 infection. “Adjusted Survival” indicates loss of >20% of total body weight and subsequent euthanasia. (D) Representative FACS plots of endogenous GP33 and WT P14 responses in naive mice at day 9 posttransfer after inoculation with serum from a naive mouse or from a mouse that received co-adoptive transfer of WT and PD-1 KO P14 cells followed by LCMV clone 13 infection (day 39 p.i.). Values indicate frequency of endogenous GP33 and P14 responses as a percentage of CD8+ T cells. (E) Summary of the number of naive WT mice with (intact) or without (mutated) GP33-specific T cell responses after inoculation with serum from mice with WT and PD-1 KO P14 cells. All data are representative of three independent experiments with at least four mice per group (A–E).
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fig1: Adoptive transfer of WT and PD-1 KO P14 cells as a model to study T cell exhaustion in the absence of PD-1. In brief, CD8+ T cells were isolated from peripheral blood of naive WT or PD-1 KO P14 mice. 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. (A) Representative FACS plots of gating scheme for WT and PD-1 KO P14 cells. P14 cells were gated from total CD8+ T cells (far left) by expression of H-2Db GP33 tetramer, congenic markers, and PD-1, as indicated (shaded gray: antibody isotype control). (B) Longitudinal analysis of viral load in serum of mice that received the indicated numbers of WT and PD-1 KO P14 cells followed by infection with LCMV clone 13 (±SEM). *, P < 0.05 (unpaired Student’s t test). (C) Survival curve of mice that received the indicated numbers of WT and PD-1 KO P14 cells after LCMV clone 13 infection. “Adjusted Survival” indicates loss of >20% of total body weight and subsequent euthanasia. (D) Representative FACS plots of endogenous GP33 and WT P14 responses in naive mice at day 9 posttransfer after inoculation with serum from a naive mouse or from a mouse that received co-adoptive transfer of WT and PD-1 KO P14 cells followed by LCMV clone 13 infection (day 39 p.i.). Values indicate frequency of endogenous GP33 and P14 responses as a percentage of CD8+ T cells. (E) Summary of the number of naive WT mice with (intact) or without (mutated) GP33-specific T cell responses after inoculation with serum from mice with WT and PD-1 KO P14 cells. All data are representative of three independent experiments with at least four mice per group (A–E).

Mentions: Direct infection of PD-1 or PD-L1 KO mice with LCMV clone 13 causes lethal immunopathology (Barber et al., 2006; Frebel et al., 2012). Previous experiments using adoptive transfer of LCMV-specific TCR-transgenic CD8+ T cells (P14 cells) also resulted in immunopathology when 104 PD-1 KO P14 cells were transferred (Frebel et al., 2012). We hypothesized that using lower numbers of P14 cells would avoid immune-mediated damage, allowing for analysis of PD-1 KO P14 cell exhaustion during chronic infection. Thus, a co-adoptive transfer strategy was used in which equal numbers of WT and PD-1 KO P14 cells were transferred into congenically distinct WT mice, followed by infection with LCMV clone 13 (Fig. 1 A). This strategy allowed for direct comparison of WT and PD-1 KO P14 cell responses in the same recipient mice during chronic viral infection while controlling for precursor frequency, TCR repertoire, viral load, inflammatory environment, and immunopathology. To determine whether this co-adoptive transfer strategy mitigated alterations in viral load and/or morbidity that occur as a result of transfer of high numbers of P14 cells (Blattman et al., 2009; Frebel et al., 2012), viremia and pathogenesis were assessed in mice that received varying numbers of WT and PD-1 KO P14 cells. Transfer of >1,000 total P14 cells (500 WT + 500 PD-1 KO) resulted in reduced viral load (Fig. 1 B) and significant morbidity (Fig. 1 C) compared with no P14 transfer, consistent with previous studies (Blattman et al., 2009; Frebel et al., 2012). However, transfer of 500 total P14 cells (250 WT + 250 PD-1 KO) did not significantly alter viremia or morbidity during chronic LCMV infection compared with mice that did not receive P14 cells (Fig. 1, B and C). One concern is that the presence of a monoclonal P14 T cell population might drive epitope escape (Pircher et al., 1989; Blattman et al., 2009). However, under the conditions used here, virus derived from recipients of WT and PD-1 KO P14 cells was capable of inducing a robust GP33-specific CD8+ T cell response when inoculated into new naive mice, indicating an intact GP33 sequence even in the presence of PD-1 KO P14 cells (Fig. 1, D and E). These results demonstrate that a co-adoptive transfer approach with small numbers of WT and PD-1 KO P14 cells can be used to address the development of CD8+ T cell exhaustion in the absence of cell-intrinsic PD-1 signals.


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)

Adoptive transfer of WT and PD-1 KO P14 cells as a model to study T cell exhaustion in the absence of PD-1. In brief, CD8+ T cells were isolated from peripheral blood of naive WT or PD-1 KO P14 mice. 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. (A) Representative FACS plots of gating scheme for WT and PD-1 KO P14 cells. P14 cells were gated from total CD8+ T cells (far left) by expression of H-2Db GP33 tetramer, congenic markers, and PD-1, as indicated (shaded gray: antibody isotype control). (B) Longitudinal analysis of viral load in serum of mice that received the indicated numbers of WT and PD-1 KO P14 cells followed by infection with LCMV clone 13 (±SEM). *, P < 0.05 (unpaired Student’s t test). (C) Survival curve of mice that received the indicated numbers of WT and PD-1 KO P14 cells after LCMV clone 13 infection. “Adjusted Survival” indicates loss of >20% of total body weight and subsequent euthanasia. (D) Representative FACS plots of endogenous GP33 and WT P14 responses in naive mice at day 9 posttransfer after inoculation with serum from a naive mouse or from a mouse that received co-adoptive transfer of WT and PD-1 KO P14 cells followed by LCMV clone 13 infection (day 39 p.i.). Values indicate frequency of endogenous GP33 and P14 responses as a percentage of CD8+ T cells. (E) Summary of the number of naive WT mice with (intact) or without (mutated) GP33-specific T cell responses after inoculation with serum from mice with WT and PD-1 KO P14 cells. All data are representative of three independent experiments with at least four mice per group (A–E).
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fig1: Adoptive transfer of WT and PD-1 KO P14 cells as a model to study T cell exhaustion in the absence of PD-1. In brief, CD8+ T cells were isolated from peripheral blood of naive WT or PD-1 KO P14 mice. 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. (A) Representative FACS plots of gating scheme for WT and PD-1 KO P14 cells. P14 cells were gated from total CD8+ T cells (far left) by expression of H-2Db GP33 tetramer, congenic markers, and PD-1, as indicated (shaded gray: antibody isotype control). (B) Longitudinal analysis of viral load in serum of mice that received the indicated numbers of WT and PD-1 KO P14 cells followed by infection with LCMV clone 13 (±SEM). *, P < 0.05 (unpaired Student’s t test). (C) Survival curve of mice that received the indicated numbers of WT and PD-1 KO P14 cells after LCMV clone 13 infection. “Adjusted Survival” indicates loss of >20% of total body weight and subsequent euthanasia. (D) Representative FACS plots of endogenous GP33 and WT P14 responses in naive mice at day 9 posttransfer after inoculation with serum from a naive mouse or from a mouse that received co-adoptive transfer of WT and PD-1 KO P14 cells followed by LCMV clone 13 infection (day 39 p.i.). Values indicate frequency of endogenous GP33 and P14 responses as a percentage of CD8+ T cells. (E) Summary of the number of naive WT mice with (intact) or without (mutated) GP33-specific T cell responses after inoculation with serum from mice with WT and PD-1 KO P14 cells. All data are representative of three independent experiments with at least four mice per group (A–E).
Mentions: Direct infection of PD-1 or PD-L1 KO mice with LCMV clone 13 causes lethal immunopathology (Barber et al., 2006; Frebel et al., 2012). Previous experiments using adoptive transfer of LCMV-specific TCR-transgenic CD8+ T cells (P14 cells) also resulted in immunopathology when 104 PD-1 KO P14 cells were transferred (Frebel et al., 2012). We hypothesized that using lower numbers of P14 cells would avoid immune-mediated damage, allowing for analysis of PD-1 KO P14 cell exhaustion during chronic infection. Thus, a co-adoptive transfer strategy was used in which equal numbers of WT and PD-1 KO P14 cells were transferred into congenically distinct WT mice, followed by infection with LCMV clone 13 (Fig. 1 A). This strategy allowed for direct comparison of WT and PD-1 KO P14 cell responses in the same recipient mice during chronic viral infection while controlling for precursor frequency, TCR repertoire, viral load, inflammatory environment, and immunopathology. To determine whether this co-adoptive transfer strategy mitigated alterations in viral load and/or morbidity that occur as a result of transfer of high numbers of P14 cells (Blattman et al., 2009; Frebel et al., 2012), viremia and pathogenesis were assessed in mice that received varying numbers of WT and PD-1 KO P14 cells. Transfer of >1,000 total P14 cells (500 WT + 500 PD-1 KO) resulted in reduced viral load (Fig. 1 B) and significant morbidity (Fig. 1 C) compared with no P14 transfer, consistent with previous studies (Blattman et al., 2009; Frebel et al., 2012). However, transfer of 500 total P14 cells (250 WT + 250 PD-1 KO) did not significantly alter viremia or morbidity during chronic LCMV infection compared with mice that did not receive P14 cells (Fig. 1, B and C). One concern is that the presence of a monoclonal P14 T cell population might drive epitope escape (Pircher et al., 1989; Blattman et al., 2009). However, under the conditions used here, virus derived from recipients of WT and PD-1 KO P14 cells was capable of inducing a robust GP33-specific CD8+ T cell response when inoculated into new naive mice, indicating an intact GP33 sequence even in the presence of PD-1 KO P14 cells (Fig. 1, D and E). These results demonstrate that a co-adoptive transfer approach with small numbers of WT and PD-1 KO P14 cells can be used to address the development of CD8+ T cell exhaustion in the absence of cell-intrinsic PD-1 signals.

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