CD8+ lymphocyte-mediated injury of dorsal root ganglion neurons during lentivirus infection: CD154-dependent cell contact neurotoxicity

Abstract

Neuronal damage in dorsal root ganglia (DRGs) with accompanying axonal injury is a key feature of human immunodeficiency virus (HIV)-related distal sensory polyneuropathy (DSP). In a model of HIV-related DSP, we observed numerous CD3+ T lymphocytes (p < 0.05) in DRGs from feline immunodeficiency virus (FIV)-infected animals, which also exhibited low CD4+ and high CD8+ lymphocyte levels in blood accompanied by a selective loss of small-diameter sural nerve axons (p < 0.05). FIV-infected lymphocytes cocultured with syngeneic DRGs caused neuronal damage, indicated by neurite retraction, neuronal soma atrophy, and loss (p < 0.05). In contrast, supernatants from FIV-infected or uninfected lymphocytes were minimally neurotoxic, despite high FIV virion levels. Among lymphocyte subsets cocultured with DRG cultures, CD8+ T cells from both FIV-infected and uninfected lymphocytes selectively caused DRG neuronal injury (p < 0.05). FIV-infected CD8+ T cells showed markedly increased CD154 expression (p < 0.05), whereas neurons were the predominant cells expressing CD40 in DRGs. Blocking CD154 on activated CD8+ T cells protected DRG neurons (p < 0.05). These findings indicated that CD8+ T cells were principal effectors of DRG neuronal injury after FIV infection through a CD40-CD154 interaction in a cell contact-dependent manner.

Figure 1.

Blood lymphocyte subsets and CD3+ T cell infiltration in DRGs of FIV-infected animals. A, Mean CD4 cell counts in blood were decreased in FIV-infected animals at 12 weeks after infection. B, Mean CD8 cell counts in blood were increased in FIV-infected animals at 8 weeks (8wk) and 12 weeks (12wk) after infection compared with controls. Few CD3+ T cells were observed in DRGs of uninfected animals (C), whereas CD3+ T cells were increased in DRGs of FIV-infected animals (D), often proximal to neurons (Student's t test; *p < 0.05).

Figure 2.

Interactions of peripheral blood lymphocytes with neurons and neurotoxicity. DIC imaging revealed T cells (arrows) interacted with neurons (A; arrowhead), which were defined by CD3 (green) and NF200 (red) immunostaining (B), respectively. C, Neurite length was diminished in FIV-infected and uninfected T cell-treated DRG cultures compared with untreated controls (185.7 ± 17 μm) (mean ± SEM), and FIV-infected T cells with PMA stimulation were more cytotoxic in terms of neurite length than uninfected T cells. D, Neuronal soma area was reduced with FIV-infected and uninfected T cell application, but only those T cells with 25 ng/ml PMA stimulation, regardless of FIV infection status, caused a significant reduction in soma size compared with untreated controls (2220.8 ± 105 μm²) (mean ± SEM). E, Neuronal loss caused by FIV-infected T cells was increased compared with uninfected T cells and untreated controls (162 ± 16/well; mean ± SEM; Bonferroni's multiple comparison test; *p < 0.05).

Figure 3.

Application of PBL supernatants to DRG cultures. A, Supernatants from either FIV-infected or uninfected T cell cultures had no effects on neurite length (untreated control DRG cultures, 212 ± 20 μm; ±SEM). B, Neuronal soma area was reduced by supernatant application from PMA-stimulated T cells compared with untreated controls, although supernatants from FIV-infected T cell cultures induced soma atrophy (untreated control DRG cultures, 2355 ± 95 μm²; mean ± SEM). C, Neuronal survival was not affected by supernatants from FIV-infected and uninfected T cell cultures (neuron number in untreated control DRG cultures, 162.5 ± 12.5; mean ± SEM). D, FIV RNA quantity in PBL culture supernatants displayed no differences between PMA-stimulated or unstimulated PBLs (Bonferroni's multiple comparison test; *p < 0.05).

Figure 4.

Effects of lymphocyte subsets on DRG neurons. A, FACS analyses revealed differential proportions of CD4+, CD8+, and CD22+ lymphocyte populations in total cultured lymphocytes applied to DRG cultures. B, Proviral load was detected in FIV-infected CD4 and CD8 T cells, showing CD4+ T cells had higher levels of FIV provirus and that PMA stimulation enhanced the virus infection. CD4+ T cell or CD22+ B cell treatments did not cause retraction of neurite length (C, E) (untreated control DRG cultures, 198 ± 27 μm; mean ± SEM) or soma size atrophy (D, F) (untreated control DRG cultures, 2043 ± 164 μm²; mean ± SEM). G, FIV-infected and uninfected CD8+ T cells induced DRG neuronal injury terms of retraction of neurite length regardless of PMA stimulation. H, Neuronal soma atrophy was caused by PMA-stimulated CD8+ T cells, regardless of FIV infection status (Bonferroni's multiple comparison test; *p < 0.05).

Figure 5.

Expression of CD40 and CD154 in DRGs from FIV-infected animals. A, FIV infection increased CD154 transcript abundance in cultured PBLs compared with uninfected PBLs. Conversely, PMA decreased CD154 expression in uninfected PBLs. B, CD40 immunoreactive cells (green, arrows) were colocalized with NF200 immunoreactive neurons (C, yellow, arrow) in DRGs from FIV-infected animals. D, Infiltrating CD3+ T cells (green) were expressed CD154 (yellow) (Student's t test; *p < 0.05).

Figure 6.

Inhibition of DRG neuronal injury by CD154 antibody. Pretreatment with CD154 antibody prevented neurite retraction (A) (neurite length in untreated control DRG cultures, 209.1 ± 26 μm; mean ± SEM) and neuronal soma atrophy (B) (untreated control DRG cultures, 1836 ± 78 μm²; mean ± SEM) caused by CD8+ T cells. In contrast, CD154 antibody or recombinant CD154 peptide treatment of DRG cultures had no effects on neuronal viability (Bonferroni's multiple comparison test; *p < 0.05).