Similar to the regulation of vasodilation, the balance between NO and superoxide (O2-) regulates expansion of activated T cells in mice. Reduction of suppressive NO levels by O2- is essential for T cell expansion and development of autoimmunity. In mice primed with heat-killed Mycobacterium, a splenocyte population positive for Gr-1 (Ly-6G/C) is the exclusive source of both immunoregulatory free radicals. Distinct Gr-1+ cell subpopulations were separated according to Ly-6G expression. In culture with activated T cells, predominantly monocytic Ly-6G- Gr-1+ cells produced T cell-inhibitory NO but no O2-. However, mostly granulocytic Ly-6G+ cells produced O2- simultaneously but had no measurable effect on proliferation. Recombination of the two purified Gr-1+ subpopulations restored controlled regulation of T cell proliferation through NO and O2- interaction. Coculture of p47phox-/- and inducible NO synthase-/- Gr-1+ cells confirmed this intercellular interaction. These data suggest that bacterial products induce development of distinct Gr-1+ myeloid lineages, which upon stimulation by activated T cells, interact via their respective free radical products to modulate T cell expansion.

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NO, which suppresses T cell proliferation, may be inactivated by superoxide (O2−) due to their strong mutual affinity. To examine this possibility, preactivated Th clones were cocultured with stimulated macrophages. PMA neutralized the inhibitory activity of NO, which was dependent on extracellular O2− production. In contrast, macrophages from p47phox −/− (pKO) mice, which lack functional NADPH oxidase, retained their NO-dependent inhibition of T cell proliferation upon stimulation with PMA, indicating that NADPH oxidase is the major source of NO-inactivating O2− in this system. To examine the NO-O2− interaction in vivo, the role of NADPH oxidase in experimental autoimmune encephalomyelitis was studied in pKO mice. No clinical or histological signs were observed in the pKO mice. Neither a bias in Th subsets nor a reduced intensity of T cell responses could account for the disease resistance. Although spleen cells from pKO mice proliferated poorly in response to the immunogen, inhibition of NO synthase uncovered a normal proliferative response. These results indicate that NO activity may play a critical role in T cell responses in pKO mice and that in normal spleens inhibition of T cell proliferation by NO may be prevented by simultaneous NADPH oxidase activity.

Acute infection of the central nervous system by the neurotropic JHM strain of mouse hepatitis virus (JHMV) induces nucleocapsid protein specific cytotoxic T lymphocytes (CTL) not found in the periphery (S. Stohlman, S. Kyuwa, J. Polo, D. Brady, M. Lai, and C. Bergmann, J. Virol. 67:7050-7059, 1993). Peripheral induction of CTL specific for the nucleocapsid protein of JHMV by vaccination with recombinant vaccinia viruses was unable to provide significant protection to a subsequent lethal virus challenge. By contrast, the transfer of nucleoprotein-specific CTL protected mice from a subsequent lethal challenge by reducing virus replication within the central nervous system, demonstrating the importance of the CTL response to this epitope in JHMV infection. Transfer of these CTL directly into the central nervous system was at least 10-fold more effective than peripheral transfer. Histological analysis indicated that the CTL reduced virus replication in ependymal cells, astrocytes, and microglia. Although the CTL were relatively ineffective at reducing virus replication in oligodendroglia, survivors showed minimal evidence of virus persistence within the central nervous system and no evidence of chronic ongoing demyelination.

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