We then examined the phenotype of the lumbar spinal cord-infiltrating CD4+ T lymphocytes in the naive, L5Tx, and sham groups. At day 7 post-surgery, lumbar spinal cord mononuclear cells were harvested and analyzed as described above via intracellular flow cytometric analysis (Figure 2). First, we examined the expression of the transcription factors T-bet and GATA-3 within the lumbar spinal cord-infiltrating CD4+ T lymphocytes. Within each set of experiments, the number of T-bet+CD4+ T lymphocytes in the L5Tx group was consistently higher than that of the naive and sham groups. As a result, there was a significant increase in the number of T-bet+CD4+ T lymphocytes in the L5Tx group compared to both the naive and sham groups. A slight, but not significant, increase in T-bet+CD4+ T lymphocytes was also observed in sham animals compared to naive mice (Figure 2B, one-way ANOVA, p<0.05). On the other hand, GATA-3+ CD4+ T lymphocytes were below the level of detection. Thus, these data indicated that the L5Tx-induced lumbar spinal cord infiltrating CD4+ T lymphocytes were predominantly Th1 cells. We further examined the cytokine expression of the infiltrating CD4+ T lymphocytes by measuring the number of IFN-γ+, IL-4+, TNF-α+, and GM-CSF+ CD4+ T lymphocytes. TNF-α and GM-CSF are two other cytokines predominantly associated with Th1 rather than Th2 cells. There were L5Tx-induced significant increases in the number of IFN-γ+ and GM-CSF+ lumbar spinal cord infiltrating CD4+ T lymphocytes (Figure 2C and E, one-way ANOVA, p<0.05). L5Tx induced similar, but not significant, changes in the number of TNF-α+ CD4+ T lymphocytes (Figure 2D, one-way ANOVA, p>0.05; p=0.051, L5Tx vs. sham), while no significant changes in the number of IL-4+ CD4+ T lymphocytes were detected (data not shown). These data further support lumbar spinal cord-infiltrating Th1 CD4+ T lymphocytes mediating L5Txinduced maintenance of mechanical hypersensitivity, and also suggest that this mediation might involve multiple Th1 cytokines.