In the periphery, Th1 cells can promote macrophage proinflammatory responses through the ligation of CD154 expressed by Th1 cells and CD40 expressed by macrophages [21]. Previously, we have reported the critical role of microglial CD40 in the development of L5Tx-induced mechanical hypersensitivity [13]. To further identify the infiltrating CD4+ T lymphocyte-mediated downstream responses that contribute to the development of sustained mechanical hypersensitivity following L5Tx, we investigated the potential involvement of the interaction between microglial CD40 and CD154 expressed by spinal cord-infiltrating CD4+ T lymphocytes. First, we confirmed that L5Txinduced spinal cord infiltrating CD4+ T lymphocytes express CD154 via IHC. Serial sections of L5 spinal cord segments from two WT BALB/c mice 7 days post-L5Tx were stained for CD4 and CD154. Consistent with what we have shown previously, the majority of CD4+ T lymphocytes were observed in the ipsilateral side of the dorsal horn [4]. CD154+CD4+ T lymphocytes were detected in the ipsilateral side of the dorsal horn region (see representative images in Figure 4A). Due to the limited numbers of infiltrating CD4+ T lymphocytes, we could not accurately quantify the CD154+CD4+ T lymphocytes via IHC. To further investigate the CD40–CD154 pathway, an in vivo study with CD154 KO mice was conducted. We tested the mechanical sensitivity of BALB/c CD154 KO mice and WT BALB/c mice following either L5Tx or sham surgery with von Frey filaments. No basal differences in mechanical sensitivity were observed between the CD154 KO mice and WT mice. Interestingly, L5Tx induced significant mechanical hypersensitivity starting at day 1 and up to 21 days post-surgery in both CD154 KO and WT mice compared to the corresponding sham groups, while no significant differences in mechanical sensitivity between CD154 KO and WT mice were detected (Figure 4B; Two-way repeated ANOVA, ptime<0.001, pgroup<0.001, and ptime × group<0.001). Thus, it is unlikely that Th1 CD4+ T lymphocytes mediate L5Tx-induced behavioral hypersensitivity via the CD40–CD154 pathway.