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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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The number of splenocytes and splenic T-cells are not changed during chronic spinal cord injury (SCI). (A) Bar graph represents the mean ± SEM spleen weights of uninjured mice (CT) and T9-SCI mice at chronic phase after injury (SCI). n = 12 mice/group. Data are pooled across three independent experiments. (B) Bar graph represents the mean ± SEM of total splenocyte numbers for CT and SCI mice. n = 17 for CT, n = 19 for SCI. Data were pooled across four independent experiments. (C) Representative dot plots show the percentage of T-cells (CD45+CD3+) in live splenocytes (upper panels), as well as the percentages of CD4+ T-cells (CD4+CD8−, bottom right quadrant) and CD8+ T-cells (CD4−CD8+, upper left quadrant) in gated T-cells (bottom panels). (D) Bar graph represents the mean ± SEM number of splenic T-cells in CT and SCI mice. (E) Bar graph show the mean ± SEM numbers of splenic CD4+ T-cells and CD8+ T-cells in CT and SCI mice. Ten thousand events gated on live singlets were collected. n = 12 mice/group. Data are pooled across three independent experiments. No statistical difference was detected between the two groups. P > 0.05, two-tailed Student’s t-test.
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Figure 1: The number of splenocytes and splenic T-cells are not changed during chronic spinal cord injury (SCI). (A) Bar graph represents the mean ± SEM spleen weights of uninjured mice (CT) and T9-SCI mice at chronic phase after injury (SCI). n = 12 mice/group. Data are pooled across three independent experiments. (B) Bar graph represents the mean ± SEM of total splenocyte numbers for CT and SCI mice. n = 17 for CT, n = 19 for SCI. Data were pooled across four independent experiments. (C) Representative dot plots show the percentage of T-cells (CD45+CD3+) in live splenocytes (upper panels), as well as the percentages of CD4+ T-cells (CD4+CD8−, bottom right quadrant) and CD8+ T-cells (CD4−CD8+, upper left quadrant) in gated T-cells (bottom panels). (D) Bar graph represents the mean ± SEM number of splenic T-cells in CT and SCI mice. (E) Bar graph show the mean ± SEM numbers of splenic CD4+ T-cells and CD8+ T-cells in CT and SCI mice. Ten thousand events gated on live singlets were collected. n = 12 mice/group. Data are pooled across three independent experiments. No statistical difference was detected between the two groups. P > 0.05, two-tailed Student’s t-test.

Mentions: Several studies have shown that following acute SCI (< seven days post-injury) there was a dramatic reduction in the number of splenic T-cells [30-32]. To evaluate the impact of spinal cord injury on spleen function at a chronic time point, namely five to seven weeks post-SCI, we first assessed the cell numbers with a focus on T-cells. As shown in Figure 1A, the spleen weight was not significantly different between the uninjured and chronic SCI groups (uninjured: 77.1 ± 2.1 mg; chronic SCI: 70.6 ± 5.3 mg; P = 0.27) nor was the total spleen cell number (uninjured: 86.5 ± 8.4 × 106; chronic SCI: 89.4 ± 9.2 × 106; P = 0.82) (Figure 1B). Furthermore, the spleens of uninjured and chronic SCI mice contained similar numbers of T-cells (uninjured: 27.0 ± 1.9 × 106; chronic SCI: 22.6 ± 2.4 × 106; P = 0.16) (Figure 1C, D) with no significant differences in the numbers of CD4+ T-cells (uninjured: 12.4 ± 1.0 × 106; chronic SCI: 10.0 ± 0.7 × 106; P = 0.06) or CD8+ T-cells (uninjured: 11.5 ± 0.8 × 106; chronic SCI: 9.3 ± 1.2 × 106; P = 0.14) (Figure 1C, E) between groups.


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)

The number of splenocytes and splenic T-cells are not changed during chronic spinal cord injury (SCI). (A) Bar graph represents the mean ± SEM spleen weights of uninjured mice (CT) and T9-SCI mice at chronic phase after injury (SCI). n = 12 mice/group. Data are pooled across three independent experiments. (B) Bar graph represents the mean ± SEM of total splenocyte numbers for CT and SCI mice. n = 17 for CT, n = 19 for SCI. Data were pooled across four independent experiments. (C) Representative dot plots show the percentage of T-cells (CD45+CD3+) in live splenocytes (upper panels), as well as the percentages of CD4+ T-cells (CD4+CD8−, bottom right quadrant) and CD8+ T-cells (CD4−CD8+, upper left quadrant) in gated T-cells (bottom panels). (D) Bar graph represents the mean ± SEM number of splenic T-cells in CT and SCI mice. (E) Bar graph show the mean ± SEM numbers of splenic CD4+ T-cells and CD8+ T-cells in CT and SCI mice. Ten thousand events gated on live singlets were collected. n = 12 mice/group. Data are pooled across three independent experiments. No statistical difference was detected between the two groups. P > 0.05, two-tailed Student’s t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 1: The number of splenocytes and splenic T-cells are not changed during chronic spinal cord injury (SCI). (A) Bar graph represents the mean ± SEM spleen weights of uninjured mice (CT) and T9-SCI mice at chronic phase after injury (SCI). n = 12 mice/group. Data are pooled across three independent experiments. (B) Bar graph represents the mean ± SEM of total splenocyte numbers for CT and SCI mice. n = 17 for CT, n = 19 for SCI. Data were pooled across four independent experiments. (C) Representative dot plots show the percentage of T-cells (CD45+CD3+) in live splenocytes (upper panels), as well as the percentages of CD4+ T-cells (CD4+CD8−, bottom right quadrant) and CD8+ T-cells (CD4−CD8+, upper left quadrant) in gated T-cells (bottom panels). (D) Bar graph represents the mean ± SEM number of splenic T-cells in CT and SCI mice. (E) Bar graph show the mean ± SEM numbers of splenic CD4+ T-cells and CD8+ T-cells in CT and SCI mice. Ten thousand events gated on live singlets were collected. n = 12 mice/group. Data are pooled across three independent experiments. No statistical difference was detected between the two groups. P > 0.05, two-tailed Student’s t-test.
Mentions: Several studies have shown that following acute SCI (< seven days post-injury) there was a dramatic reduction in the number of splenic T-cells [30-32]. To evaluate the impact of spinal cord injury on spleen function at a chronic time point, namely five to seven weeks post-SCI, we first assessed the cell numbers with a focus on T-cells. As shown in Figure 1A, the spleen weight was not significantly different between the uninjured and chronic SCI groups (uninjured: 77.1 ± 2.1 mg; chronic SCI: 70.6 ± 5.3 mg; P = 0.27) nor was the total spleen cell number (uninjured: 86.5 ± 8.4 × 106; chronic SCI: 89.4 ± 9.2 × 106; P = 0.82) (Figure 1B). Furthermore, the spleens of uninjured and chronic SCI mice contained similar numbers of T-cells (uninjured: 27.0 ± 1.9 × 106; chronic SCI: 22.6 ± 2.4 × 106; P = 0.16) (Figure 1C, D) with no significant differences in the numbers of CD4+ T-cells (uninjured: 12.4 ± 1.0 × 106; chronic SCI: 10.0 ± 0.7 × 106; P = 0.06) or CD8+ T-cells (uninjured: 11.5 ± 0.8 × 106; chronic SCI: 9.3 ± 1.2 × 106; P = 0.14) (Figure 1C, E) between groups.

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