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Chronic thoracic spinal cord injury impairs CD8+ T-cell function by up-regulating programmed cell death-1 expression.

Zha J, Smith A, Andreansky S, Bracchi-Ricard V, Bethea JR - J Neuroinflammation (2014)

Bottom Line: Chronic SCI impaired both CD4+ and CD8+ T-cell cytokine production.The observed T-cell dysfunction correlated with increased expression of programmed cell death 1 (PD-1) exhaustion marker on these cells.Blocking PD-1 signaling in vitro restored the CD8+ T-cell functional defect.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Miami Project to Cure Paralysis, Department of Neurosurgery, Miller School of Medicine, University of Miami, Miami, FL 33136, USA. VBracchi@med.miami.edu.

ABSTRACT

Background: Chronic spinal cord injury (SCI) induces immune depression in patients, which contributes to their higher risk of developing infections. While defects in humoral immunity have been reported, complications in T-cell immunity during the chronic phase of SCI have not yet been explored.

Methods: To assess the impact of chronic SCI on peripheral T-cell number and function we used a mouse model of severe spinal cord contusion at thoracic level T9 and performed flow cytometry analysis on the spleen for T-cell markers along with intracellular cytokine staining. Furthermore we identified alterations in sympathetic activity in the spleen of chronic SCI mice by measuring splenic levels of tyrosine hydroxylase (TH) and norepinephrine (NE). To gain insight into the neurogenic mechanism leading to T-cell dysfunction we performed in vitro NE stimulation of T-cells followed by flow cytometry analysis for T-cell exhaustion marker.

Results: Chronic SCI impaired both CD4+ and CD8+ T-cell cytokine production. The observed T-cell dysfunction correlated with increased expression of programmed cell death 1 (PD-1) exhaustion marker on these cells. Blocking PD-1 signaling in vitro restored the CD8+ T-cell functional defect. In addition, we showed that chronic SCI mice had higher levels of splenic NE, which contributed to the T-cell exhaustion phenotype, as PD-1 expression on both CD4+ and CD8+ T-cells was up-regulated following sustained exposure to NE in vitro.

Conclusions: These studies indicate that alteration of sympathetic activity following chronic SCI induces CD8+ T-cell exhaustion, which in turn impairs T-cell function and contributes to immune depression. Inhibition of the exhaustion pathway should be considered as a new therapeutic strategy for chronic SCI-induced immune depression.

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Blocking PD-1 restores the TNF-α production by CD8+ T-cells from chronic spinal cord injury (SCI) mice. Splenocytes (1 × 106) isolated from uninjured mice were stimulated ex vivo with PMA/ionomycin in the presence of brefeldin A for four hours (group: CT). Splenocytes (1 × 106) isolated from chronic SCI mice were also stimulated ex vivo with the same condition as the CT group, except that 10 μg/mL anti-PD-1 blocking antibody (group: SCI + αPD-1) or 10 μg/mL rat IgG2a, κ isotype (group: SCI + Isotype) were added to the culture. (A) Representative dot plots show the percentage of IFN-γ+ cells in gated CD4+ T-cells. (B) Bar graph represents the mean ± SEM percentage of IFN-γ+ cells in CD4+ T-cells. (C) Representative dot plots show the percentage of TNF-α+ cells in gated CD8+ T-cells. (D) Bar graph represents the mean ± SEM percentage of TNF-α+ cells in CD8+ T-cells. Ten thousand events gated on live singlets were collected. n = 9 for CT, n = 11 for SCI + Isotype, n = 11 for SCI + αPD-1. Data are pooled across three independent experiments. *P < 0.05, **P < 0.01, n.s. no significant difference was detected, P > 0.05, one-tailed Student’s t-test.
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Figure 7: Blocking PD-1 restores the TNF-α production by CD8+ T-cells from chronic spinal cord injury (SCI) mice. Splenocytes (1 × 106) isolated from uninjured mice were stimulated ex vivo with PMA/ionomycin in the presence of brefeldin A for four hours (group: CT). Splenocytes (1 × 106) isolated from chronic SCI mice were also stimulated ex vivo with the same condition as the CT group, except that 10 μg/mL anti-PD-1 blocking antibody (group: SCI + αPD-1) or 10 μg/mL rat IgG2a, κ isotype (group: SCI + Isotype) were added to the culture. (A) Representative dot plots show the percentage of IFN-γ+ cells in gated CD4+ T-cells. (B) Bar graph represents the mean ± SEM percentage of IFN-γ+ cells in CD4+ T-cells. (C) Representative dot plots show the percentage of TNF-α+ cells in gated CD8+ T-cells. (D) Bar graph represents the mean ± SEM percentage of TNF-α+ cells in CD8+ T-cells. Ten thousand events gated on live singlets were collected. n = 9 for CT, n = 11 for SCI + Isotype, n = 11 for SCI + αPD-1. Data are pooled across three independent experiments. *P < 0.05, **P < 0.01, n.s. no significant difference was detected, P > 0.05, one-tailed Student’s t-test.

Mentions: We next asked whether inhibiting PD-1 signaling will restore the functional defects in cytokine production observed in the splenic T-cells isolated from chronic SCI mice. Splenocytes from uninjured mice and chronic SCI mice were stimulated with PMA/ionomycin in the presence of either anti-PD-1 or an isotype control antibody. Compared with splenocytes from uninjured mice, the splenocytes from chronic SCI mice showed a significant reduction in the percentage of IFN-γ-expressing CD4+ T-cells (uninjured: 7.0 ± 0.8%; chronic SCI + Isotype: 5.1 ± 0.6%; P = 0.04) (Figure 7A, B) and TNF-α-expressing CD8+ T-cells (uninjured: 6.9 ± 0.9%; chronic SCI + Isotype: 4.2 ± 0.6%; P = 0.009) (Figure 7C, D) after PMA/ionomycin restimulation in presence of isotype control antibodies. Blocking PD-1 restored the percentage of CD8+ T-cells expressing TNF-α (chronic SCI + anti-PD-1: 8.6 ± 1.6%; P = 0.01) (Figure 7C, D). However, IFN-γ production by CD4+ T-cells from chronic SCI mice was not restored (chronic SCI + anti-PD-1: 5.4 ± 1.0%; P = 0.43) (Figure 7A, B).


Chronic thoracic spinal cord injury impairs CD8+ T-cell function by up-regulating programmed cell death-1 expression.

Zha J, Smith A, Andreansky S, Bracchi-Ricard V, Bethea JR - J Neuroinflammation (2014)

Blocking PD-1 restores the TNF-α production by CD8+ T-cells from chronic spinal cord injury (SCI) mice. Splenocytes (1 × 106) isolated from uninjured mice were stimulated ex vivo with PMA/ionomycin in the presence of brefeldin A for four hours (group: CT). Splenocytes (1 × 106) isolated from chronic SCI mice were also stimulated ex vivo with the same condition as the CT group, except that 10 μg/mL anti-PD-1 blocking antibody (group: SCI + αPD-1) or 10 μg/mL rat IgG2a, κ isotype (group: SCI + Isotype) were added to the culture. (A) Representative dot plots show the percentage of IFN-γ+ cells in gated CD4+ T-cells. (B) Bar graph represents the mean ± SEM percentage of IFN-γ+ cells in CD4+ T-cells. (C) Representative dot plots show the percentage of TNF-α+ cells in gated CD8+ T-cells. (D) Bar graph represents the mean ± SEM percentage of TNF-α+ cells in CD8+ T-cells. Ten thousand events gated on live singlets were collected. n = 9 for CT, n = 11 for SCI + Isotype, n = 11 for SCI + αPD-1. Data are pooled across three independent experiments. *P < 0.05, **P < 0.01, n.s. no significant difference was detected, P > 0.05, one-tailed Student’s t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 7: Blocking PD-1 restores the TNF-α production by CD8+ T-cells from chronic spinal cord injury (SCI) mice. Splenocytes (1 × 106) isolated from uninjured mice were stimulated ex vivo with PMA/ionomycin in the presence of brefeldin A for four hours (group: CT). Splenocytes (1 × 106) isolated from chronic SCI mice were also stimulated ex vivo with the same condition as the CT group, except that 10 μg/mL anti-PD-1 blocking antibody (group: SCI + αPD-1) or 10 μg/mL rat IgG2a, κ isotype (group: SCI + Isotype) were added to the culture. (A) Representative dot plots show the percentage of IFN-γ+ cells in gated CD4+ T-cells. (B) Bar graph represents the mean ± SEM percentage of IFN-γ+ cells in CD4+ T-cells. (C) Representative dot plots show the percentage of TNF-α+ cells in gated CD8+ T-cells. (D) Bar graph represents the mean ± SEM percentage of TNF-α+ cells in CD8+ T-cells. Ten thousand events gated on live singlets were collected. n = 9 for CT, n = 11 for SCI + Isotype, n = 11 for SCI + αPD-1. Data are pooled across three independent experiments. *P < 0.05, **P < 0.01, n.s. no significant difference was detected, P > 0.05, one-tailed Student’s t-test.
Mentions: We next asked whether inhibiting PD-1 signaling will restore the functional defects in cytokine production observed in the splenic T-cells isolated from chronic SCI mice. Splenocytes from uninjured mice and chronic SCI mice were stimulated with PMA/ionomycin in the presence of either anti-PD-1 or an isotype control antibody. Compared with splenocytes from uninjured mice, the splenocytes from chronic SCI mice showed a significant reduction in the percentage of IFN-γ-expressing CD4+ T-cells (uninjured: 7.0 ± 0.8%; chronic SCI + Isotype: 5.1 ± 0.6%; P = 0.04) (Figure 7A, B) and TNF-α-expressing CD8+ T-cells (uninjured: 6.9 ± 0.9%; chronic SCI + Isotype: 4.2 ± 0.6%; P = 0.009) (Figure 7C, D) after PMA/ionomycin restimulation in presence of isotype control antibodies. Blocking PD-1 restored the percentage of CD8+ T-cells expressing TNF-α (chronic SCI + anti-PD-1: 8.6 ± 1.6%; P = 0.01) (Figure 7C, D). However, IFN-γ production by CD4+ T-cells from chronic SCI mice was not restored (chronic SCI + anti-PD-1: 5.4 ± 1.0%; P = 0.43) (Figure 7A, B).

Bottom Line: Chronic SCI impaired both CD4+ and CD8+ T-cell cytokine production.The observed T-cell dysfunction correlated with increased expression of programmed cell death 1 (PD-1) exhaustion marker on these cells.Blocking PD-1 signaling in vitro restored the CD8+ T-cell functional defect.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Miami Project to Cure Paralysis, Department of Neurosurgery, Miller School of Medicine, University of Miami, Miami, FL 33136, USA. VBracchi@med.miami.edu.

ABSTRACT

Background: Chronic spinal cord injury (SCI) induces immune depression in patients, which contributes to their higher risk of developing infections. While defects in humoral immunity have been reported, complications in T-cell immunity during the chronic phase of SCI have not yet been explored.

Methods: To assess the impact of chronic SCI on peripheral T-cell number and function we used a mouse model of severe spinal cord contusion at thoracic level T9 and performed flow cytometry analysis on the spleen for T-cell markers along with intracellular cytokine staining. Furthermore we identified alterations in sympathetic activity in the spleen of chronic SCI mice by measuring splenic levels of tyrosine hydroxylase (TH) and norepinephrine (NE). To gain insight into the neurogenic mechanism leading to T-cell dysfunction we performed in vitro NE stimulation of T-cells followed by flow cytometry analysis for T-cell exhaustion marker.

Results: Chronic SCI impaired both CD4+ and CD8+ T-cell cytokine production. The observed T-cell dysfunction correlated with increased expression of programmed cell death 1 (PD-1) exhaustion marker on these cells. Blocking PD-1 signaling in vitro restored the CD8+ T-cell functional defect. In addition, we showed that chronic SCI mice had higher levels of splenic NE, which contributed to the T-cell exhaustion phenotype, as PD-1 expression on both CD4+ and CD8+ T-cells was up-regulated following sustained exposure to NE in vitro.

Conclusions: These studies indicate that alteration of sympathetic activity following chronic SCI induces CD8+ T-cell exhaustion, which in turn impairs T-cell function and contributes to immune depression. Inhibition of the exhaustion pathway should be considered as a new therapeutic strategy for chronic SCI-induced immune depression.

Show MeSH
Related in: MedlinePlus