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.