Early monocyte depletion prevents IFNγ producing T cell infiltration into the brain but does not affect peripheral PbTg-specific CTL responses.

<p>C57BL/6 mice were infected with 5*10e4 PbTg iRBC i.v.. Simultaneously, groups of mice received either anti-Gr1, anti-Ly6G or anti-CCR2 mAb. Six days later, T cell subsets in the brain and spleen were analysed and cellular immune responses were evaluated. (A) Frequencies of CD8⁺ cells and (B) CD4⁺ cells among brain infiltrates were determined by flow cytometry. Each group contained 4–5 mice. (C) Leukocyte preparations from the brains of individual mice were re-stimulated <i>ex vivo</i> and intracellular stained to determine the frequency of IFNγ producing CD8⁺ T cells. Representative images of brain derived IFNγ⁺CD8⁺ T cells by flow cytometry are shown. Calculated frequencies of CD8⁺ IFNγ⁺ T cells and CD8⁺ IFNγ⁺ T cells in the brains as analysed from (A) are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124080#pone.0124080.t001" target="_blank">Table 1</a>. (D) Fold increase of IFNγ mRNA levels relative to GAPDH in the brains of PbTg-infected mice ± d0 depletion on day 6 p.i. n = 6–8 per group, Kruskal Wallis test with Dunn’s Post test was performed. (E) Total cell count of splenocytes from all d0 depletion groups and controls at day 6 p.i. (F) Frequency of CD3⁺CD8⁺ splenocytes in percent from all d0 mAb depletion groups and controls at day 6 p.i. (G) Calculated total amount of CD8⁺ splenocytes according to data from F and G. (H) <i>In vivo</i> cytotoxicity assay analysing PbTg-specific T cells. Recipient PbTg-infected mice, that were or were not additionally treated with depletion antibodies, received an adoptive transfer of CSFE-labelled T cells, that were loaded with ovalbumin-derived MHC class I peptide SIINFEKL and unloaded control cells i.v. (1x10⁷/mouse) on day 5 p.i.. 18 hours later the level of lytic activity was measured by flow cytometry in the spleen. (I) In addition, splenocytes from the same animals as in (H) were re-stimulated with SIINFEKL <i>ex vivo</i> for 24 hours and IFNγ production was quantified by sandwich ELISA. (A-I) Bars show mean ± SEM from n = 4–5 mice per group from 1 out of 3 independent depletion-infection experiments. Statistical analysis was performed using Kruskal-Wallis test and Dunn’s Post test and significant differences are indicated by the stars in brackets between the groups (* p<0.05, ** p<0.01).