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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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Impaired T-cell cytokine production following T-cell receptor (TCR) activation in chronic spinal cord injury (SCI) mice. Isolated splenocytes (1 × 106) from uninjured (CT) or T9-SCI mice at chronic phase after injury (SCI) were stimulated ex vivo for three days with anti-CD3 + anti-CD28 + IL-2 or with IL-2 only. Brefeldin A was added six hours before cell collection. Intracellular cytokine staining and flow cytometry analysis were performed. (A) Representative dot plots show the percentage of IFN-γ+ cells and TNF-α+ cells in gated CD4+ T-cells following three-day stimulation with anti-CD3 + anti-CD28 + IL-2 or with IL-2 only. (B) Bar graph represents the mean ± SEM percentages of cytokine producing CD4+ T-cell in response to TCR activation. (C) Representative dot plots show the percentage of IFN-γ+ cells and TNF-α+ cells in gated CD8+ T-cells following three-day stimulation with anti-CD3 + anti-CD28 + IL-2 or with IL-2 only. (D) Bar graph represents the mean ± SEM percentages of cytokine producing CD8+ T-cells in response to TCR activation. n = 4 for CT, n = 5 for SCI. Twenty thousand events gated on live singlets were collected for flow cytometry analysis. n = 5 mice per group. *P < 0.05, **P < 0.01, one-tailed Student’s t-test. (E) The concentration of IFN-γ and TNF-α in the supernatant of stimulated cells was measured by ELISA.
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Figure 3: Impaired T-cell cytokine production following T-cell receptor (TCR) activation in chronic spinal cord injury (SCI) mice. Isolated splenocytes (1 × 106) from uninjured (CT) or T9-SCI mice at chronic phase after injury (SCI) were stimulated ex vivo for three days with anti-CD3 + anti-CD28 + IL-2 or with IL-2 only. Brefeldin A was added six hours before cell collection. Intracellular cytokine staining and flow cytometry analysis were performed. (A) Representative dot plots show the percentage of IFN-γ+ cells and TNF-α+ cells in gated CD4+ T-cells following three-day stimulation with anti-CD3 + anti-CD28 + IL-2 or with IL-2 only. (B) Bar graph represents the mean ± SEM percentages of cytokine producing CD4+ T-cell in response to TCR activation. (C) Representative dot plots show the percentage of IFN-γ+ cells and TNF-α+ cells in gated CD8+ T-cells following three-day stimulation with anti-CD3 + anti-CD28 + IL-2 or with IL-2 only. (D) Bar graph represents the mean ± SEM percentages of cytokine producing CD8+ T-cells in response to TCR activation. n = 4 for CT, n = 5 for SCI. Twenty thousand events gated on live singlets were collected for flow cytometry analysis. n = 5 mice per group. *P < 0.05, **P < 0.01, one-tailed Student’s t-test. (E) The concentration of IFN-γ and TNF-α in the supernatant of stimulated cells was measured by ELISA.

Mentions: To confirm the functional impairment of T-cell cytokine production isolated from chronically injured mice, we repeated the experiment using anti-CD3/anti-CD28 stimulation protocol which is more physiologically relevant. As shown in Figure 3A, B, the percentages of CD4+ T-cells producing IFN-γ or TNF-α following anti-CD3/anti-CD28 stimulation were significantly decreased in the SCI group (IFN-γ: uninjured: 2.2 ± 0.2%; chronic SCI: 1.4 ± 0.1%; P = 0.005; TNF-α: uninjured: 2.2 ± 0.2 × 106; chronic SCI: 1.4 ± 0.2 × 106; P = 0.008). CD8+ T-cells from chronic SCI mice also show a reduction in the percentages of IFN-γ and TNF-α expressing cells in response to anti-CD3/anti-CD28 stimulation (IFN-γ: uninjured: 6.2 ± 0.7%; chronic SCI: 2.6 ± 0.4%; P = 0.001; TNF-α: uninjured: 2.8 ± 0.4 × 106; chronic SCI: 1.6 ± 0.3 × 106; P = 0.03) Figure 3C, D. To further confirm the deficiency in cytokine production, we performed ELISA on the supernatant of these stimulated T-cells and found a significant reduction in the concentration of IFN-γ (uninjured: 123 ± 4 ng/ml; chronic SCI: 103 ± 5 ng/ml; P = 0.005) and TNF-α (uninjured: 0.33 ± 0.02 ng/ml; chronic SCI: 0.25 ± 0.01 ng/ml; P = 0.004) (Figure 3E).


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 following T-cell receptor (TCR) activation in chronic spinal cord injury (SCI) mice. Isolated splenocytes (1 × 106) from uninjured (CT) or T9-SCI mice at chronic phase after injury (SCI) were stimulated ex vivo for three days with anti-CD3 + anti-CD28 + IL-2 or with IL-2 only. Brefeldin A was added six hours before cell collection. Intracellular cytokine staining and flow cytometry analysis were performed. (A) Representative dot plots show the percentage of IFN-γ+ cells and TNF-α+ cells in gated CD4+ T-cells following three-day stimulation with anti-CD3 + anti-CD28 + IL-2 or with IL-2 only. (B) Bar graph represents the mean ± SEM percentages of cytokine producing CD4+ T-cell in response to TCR activation. (C) Representative dot plots show the percentage of IFN-γ+ cells and TNF-α+ cells in gated CD8+ T-cells following three-day stimulation with anti-CD3 + anti-CD28 + IL-2 or with IL-2 only. (D) Bar graph represents the mean ± SEM percentages of cytokine producing CD8+ T-cells in response to TCR activation. n = 4 for CT, n = 5 for SCI. Twenty thousand events gated on live singlets were collected for flow cytometry analysis. n = 5 mice per group. *P < 0.05, **P < 0.01, one-tailed Student’s t-test. (E) The concentration of IFN-γ and TNF-α in the supernatant of stimulated cells was measured by ELISA.
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Figure 3: Impaired T-cell cytokine production following T-cell receptor (TCR) activation in chronic spinal cord injury (SCI) mice. Isolated splenocytes (1 × 106) from uninjured (CT) or T9-SCI mice at chronic phase after injury (SCI) were stimulated ex vivo for three days with anti-CD3 + anti-CD28 + IL-2 or with IL-2 only. Brefeldin A was added six hours before cell collection. Intracellular cytokine staining and flow cytometry analysis were performed. (A) Representative dot plots show the percentage of IFN-γ+ cells and TNF-α+ cells in gated CD4+ T-cells following three-day stimulation with anti-CD3 + anti-CD28 + IL-2 or with IL-2 only. (B) Bar graph represents the mean ± SEM percentages of cytokine producing CD4+ T-cell in response to TCR activation. (C) Representative dot plots show the percentage of IFN-γ+ cells and TNF-α+ cells in gated CD8+ T-cells following three-day stimulation with anti-CD3 + anti-CD28 + IL-2 or with IL-2 only. (D) Bar graph represents the mean ± SEM percentages of cytokine producing CD8+ T-cells in response to TCR activation. n = 4 for CT, n = 5 for SCI. Twenty thousand events gated on live singlets were collected for flow cytometry analysis. n = 5 mice per group. *P < 0.05, **P < 0.01, one-tailed Student’s t-test. (E) The concentration of IFN-γ and TNF-α in the supernatant of stimulated cells was measured by ELISA.
Mentions: To confirm the functional impairment of T-cell cytokine production isolated from chronically injured mice, we repeated the experiment using anti-CD3/anti-CD28 stimulation protocol which is more physiologically relevant. As shown in Figure 3A, B, the percentages of CD4+ T-cells producing IFN-γ or TNF-α following anti-CD3/anti-CD28 stimulation were significantly decreased in the SCI group (IFN-γ: uninjured: 2.2 ± 0.2%; chronic SCI: 1.4 ± 0.1%; P = 0.005; TNF-α: uninjured: 2.2 ± 0.2 × 106; chronic SCI: 1.4 ± 0.2 × 106; P = 0.008). CD8+ T-cells from chronic SCI mice also show a reduction in the percentages of IFN-γ and TNF-α expressing cells in response to anti-CD3/anti-CD28 stimulation (IFN-γ: uninjured: 6.2 ± 0.7%; chronic SCI: 2.6 ± 0.4%; P = 0.001; TNF-α: uninjured: 2.8 ± 0.4 × 106; chronic SCI: 1.6 ± 0.3 × 106; P = 0.03) Figure 3C, D. To further confirm the deficiency in cytokine production, we performed ELISA on the supernatant of these stimulated T-cells and found a significant reduction in the concentration of IFN-γ (uninjured: 123 ± 4 ng/ml; chronic SCI: 103 ± 5 ng/ml; P = 0.005) and TNF-α (uninjured: 0.33 ± 0.02 ng/ml; chronic SCI: 0.25 ± 0.01 ng/ml; P = 0.004) (Figure 3E).

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