CFSElow and CFSEhigh B cells were so injected in a ratio of 1:1 to LCMV infected BL/6 mice (11 days after infection) or na?ve BL/6 mice. Peptide-specific killing was assessed 18 hours later and revealed that more than half of LCMV-GP61 pulsed CD19+ B cells were eliminated (Fig. 6A,B). Elimination after adoptive transfer of CD4+ T cells included all B cell subtypes and, compared to na?ve BL/6 mice, IgMhighIgDlow B cells in LCMV infected mice were reduced by approximately 70% (Fig. 6C). These data indicate that LCMV-GP61 presenting IgMhighIgDlow B cells are a direct target of cytotoxic CD4+ T cells. Figure 6 Effector mechanisms of CD4+ T cell-mediated immunopathology.
To understand the mechanism of IgMhighIgDlow B cell reduction by CD4+ T cells, LCMV-immune CD4+ T cells from mice lacking important effector cytokines and cytolytic pathways such as IFN��, TNF��, perforin and FasL (gld) were generated for adoptive transfer experiments. Because virus control is different in all these knockout mice, purified CD8+ T cells (2��107 cells) of na?ve immunocompetent BL/6 mice were adoptively transferred to all mice before LCMV inoculation. Seventeen days later, LCMV was not detectable in peripheral blood by conventional in vitro focus forming assay in all mice (Fig. S2) and CD4+ T cells produced the effector cytokines IFN�� and/or TNF�� after restimulation with GP61 (Fig. S3). Splenocytes of these mice were then purified for CD4+ T cells by MACS and the number of CD4+ T cells containing 1.8��105 IFN��-producing LCMV GP61-specific CD4+ T cells from TNF��?/?, perforin?/?, FasLgld and BL/6 mice were adoptively transferred to groups of na?ve CD8-depleted BL/6 mice.
The number of CD4+ T cells from IFN��?/? mice was adjusted for TNF�� production. On day 11 after LCMV infection, B cell subgroups were analyzed as before by flow cytometry. T1, T2 and MZ B cells were clearly reduced after transfer of CD4+ T cells from TNF��?/?, IFN��?/?, perforin?/?, or FasLgld mice (Fig. 6D). There was a statistically significant elevated number of T1 B cells in CD8-depleted mice treated with CD4+ T cells lacking IFN�� or FasL in comparison to CD8-depleted mice receiving control CD4+ T cells from BL/6 mice. However, quantitatively this difference was only minimal. For T2 and MZ B cells no difference could be found between the different groups of adoptively transferred CD4+ T cells.
CD4+ T cell mediated immunopathology in the Anacetrapib liver Similar to the immune responses analyzed in the spleen, IFN�� and TNF�� production by LCMV-specific CD4+ T cells was reduced or even absent in the liver of CD8-depleted mice on day 8 and 11 after infection (Fig. 7A,B). Again, the frequency of IFN��- and TNF��-producing LCMV-specific CD4+ T cells was restored after transfer of purified LCMV-immune CD4+ T cells (Fig. 7A,B).
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