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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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Related in: MedlinePlus

Impaired T-cell cytokine production in response to PMA/ionomycin stimulation after chronic spinal cord injury (SCI). Isolated splenocytes (1 × 106) from uninjured (CT) or T9-SCI mice at chronic phase after injury (SCI) were stimulated ex vivo with PMA/ionomycin in the presence of brefeldin A for four hours and then processed for flow cytometry analysis. The unstimulated controls were incubated only with brefeldin A. (A) Representative dot plots show the percentage of IFN-γ+ and TNF-α+ cells in gated CD4+ T-cells following PMA/ionomycin stimulation compared to unstimulated or isotype control. (B) Bar graph represents the mean ± SEM percentages and numbers of cytokine producing CD4+ T-cell in response to PMA/ionomycin stimulation. (C) Representative dot plots show the percentage of IFN-γ+ and TNF-α+ cells in gated CD8+ T-cells following PMA/ionomycin stimulation compared to unstimulated or isotype control. (D) Bar graph represents the mean ± SEM percentages and numbers of cytokine producing CD8+ T-cells in response to PMA/ionomycin stimulation. Ten thousand events gated on live singlets were collected. n = 14 for CT, n = 12 for SCI. Data were pooled across four independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, one-tailed Student’s t-test.
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Figure 2: Impaired T-cell cytokine production in response to PMA/ionomycin stimulation after chronic spinal cord injury (SCI). Isolated splenocytes (1 × 106) from uninjured (CT) or T9-SCI mice at chronic phase after injury (SCI) were stimulated ex vivo with PMA/ionomycin in the presence of brefeldin A for four hours and then processed for flow cytometry analysis. The unstimulated controls were incubated only with brefeldin A. (A) Representative dot plots show the percentage of IFN-γ+ and TNF-α+ cells in gated CD4+ T-cells following PMA/ionomycin stimulation compared to unstimulated or isotype control. (B) Bar graph represents the mean ± SEM percentages and numbers of cytokine producing CD4+ T-cell in response to PMA/ionomycin stimulation. (C) Representative dot plots show the percentage of IFN-γ+ and TNF-α+ cells in gated CD8+ T-cells following PMA/ionomycin stimulation compared to unstimulated or isotype control. (D) Bar graph represents the mean ± SEM percentages and numbers of cytokine producing CD8+ T-cells in response to PMA/ionomycin stimulation. Ten thousand events gated on live singlets were collected. n = 14 for CT, n = 12 for SCI. Data were pooled across four independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, one-tailed Student’s t-test.

Mentions: Effective T-cell response against pathogen requires the production of cytokines such as IFN-γ and TNF-α [33-36]. To evaluate the function of these T-cells, we isolated splenocytes from the spleens of uninjured and chronic SCI mice and stimulated them ex vivo with PMA/ionomycin. As shown in Figure 2A and 2B, a significantly smaller percentage of CD4+ T-cells produced IFN-γ following stimulation (uninjured: 8.3 ± 0.5%; chronic SCI: 5.8 ± 0.4%; P = 0.0005) also correlating with a significantly reduced number of IFN-γ+CD4+ T-cells in chronically injured mice compared to controls (uninjured: 1.0 ± 0.1 × 106; chronic SCI: 0.7 ± 0.1 × 106; P = 0.02). The percentage or number of CD4+ T-cell expressing TNF-α (Figure 2A, B) following stimulation was not significantly different between chronic SCI and control (percentage: uninjured: 9.5 ± 0.6%; chronic SCI: 9.1 ± 0.8%; P = 0.37; cell number: uninjured: 1.3 ± 0.1 × 106; chronic SCI: 1.4 ± 0.2 × 106; P = 0.38). However, the percentage and number of CD8+ T-cells expressing TNF-α was significantly reduced following chronic SCI (percentage: uninjured: 7.7 ± 0.7%; chronic SCI: 6.0 ± 0.6%; P = 0.04; cell number: uninjured: 0.80 ± 0.06 × 106; chronic SCI: 0.60 ± 0.05 × 106; P = 0.007) (Figure 2C, D), when neither the percentage nor the number of IFN-γ+CD8+ T-cells was changed by chronic SCI (percentage: uninjured: 20.1 ± 1.1%; chronic SCI: 18.5 ± 0.9%; P = 0.14; cell number: uninjured: 2.2 ± 0.2 × 106; chronic SCI: 1.9 ± 0.2 × 106; P = 0.17) (Figure 2C, D).


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)

Impaired T-cell cytokine production in response to PMA/ionomycin stimulation after chronic spinal cord injury (SCI). Isolated splenocytes (1 × 106) from uninjured (CT) or T9-SCI mice at chronic phase after injury (SCI) were stimulated ex vivo with PMA/ionomycin in the presence of brefeldin A for four hours and then processed for flow cytometry analysis. The unstimulated controls were incubated only with brefeldin A. (A) Representative dot plots show the percentage of IFN-γ+ and TNF-α+ cells in gated CD4+ T-cells following PMA/ionomycin stimulation compared to unstimulated or isotype control. (B) Bar graph represents the mean ± SEM percentages and numbers of cytokine producing CD4+ T-cell in response to PMA/ionomycin stimulation. (C) Representative dot plots show the percentage of IFN-γ+ and TNF-α+ cells in gated CD8+ T-cells following PMA/ionomycin stimulation compared to unstimulated or isotype control. (D) Bar graph represents the mean ± SEM percentages and numbers of cytokine producing CD8+ T-cells in response to PMA/ionomycin stimulation. Ten thousand events gated on live singlets were collected. n = 14 for CT, n = 12 for SCI. Data were pooled across four independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, one-tailed Student’s t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4230802&req=5

Figure 2: Impaired T-cell cytokine production in response to PMA/ionomycin stimulation after chronic spinal cord injury (SCI). Isolated splenocytes (1 × 106) from uninjured (CT) or T9-SCI mice at chronic phase after injury (SCI) were stimulated ex vivo with PMA/ionomycin in the presence of brefeldin A for four hours and then processed for flow cytometry analysis. The unstimulated controls were incubated only with brefeldin A. (A) Representative dot plots show the percentage of IFN-γ+ and TNF-α+ cells in gated CD4+ T-cells following PMA/ionomycin stimulation compared to unstimulated or isotype control. (B) Bar graph represents the mean ± SEM percentages and numbers of cytokine producing CD4+ T-cell in response to PMA/ionomycin stimulation. (C) Representative dot plots show the percentage of IFN-γ+ and TNF-α+ cells in gated CD8+ T-cells following PMA/ionomycin stimulation compared to unstimulated or isotype control. (D) Bar graph represents the mean ± SEM percentages and numbers of cytokine producing CD8+ T-cells in response to PMA/ionomycin stimulation. Ten thousand events gated on live singlets were collected. n = 14 for CT, n = 12 for SCI. Data were pooled across four independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, one-tailed Student’s t-test.
Mentions: Effective T-cell response against pathogen requires the production of cytokines such as IFN-γ and TNF-α [33-36]. To evaluate the function of these T-cells, we isolated splenocytes from the spleens of uninjured and chronic SCI mice and stimulated them ex vivo with PMA/ionomycin. As shown in Figure 2A and 2B, a significantly smaller percentage of CD4+ T-cells produced IFN-γ following stimulation (uninjured: 8.3 ± 0.5%; chronic SCI: 5.8 ± 0.4%; P = 0.0005) also correlating with a significantly reduced number of IFN-γ+CD4+ T-cells in chronically injured mice compared to controls (uninjured: 1.0 ± 0.1 × 106; chronic SCI: 0.7 ± 0.1 × 106; P = 0.02). The percentage or number of CD4+ T-cell expressing TNF-α (Figure 2A, B) following stimulation was not significantly different between chronic SCI and control (percentage: uninjured: 9.5 ± 0.6%; chronic SCI: 9.1 ± 0.8%; P = 0.37; cell number: uninjured: 1.3 ± 0.1 × 106; chronic SCI: 1.4 ± 0.2 × 106; P = 0.38). However, the percentage and number of CD8+ T-cells expressing TNF-α was significantly reduced following chronic SCI (percentage: uninjured: 7.7 ± 0.7%; chronic SCI: 6.0 ± 0.6%; P = 0.04; cell number: uninjured: 0.80 ± 0.06 × 106; chronic SCI: 0.60 ± 0.05 × 106; P = 0.007) (Figure 2C, D), when neither the percentage nor the number of IFN-γ+CD8+ T-cells was changed by chronic SCI (percentage: uninjured: 20.1 ± 1.1%; chronic SCI: 18.5 ± 0.9%; P = 0.14; cell number: uninjured: 2.2 ± 0.2 × 106; chronic SCI: 1.9 ± 0.2 × 106; P = 0.17) (Figure 2C, D).

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