Volume 31, Number 3, March 2009 S isolate antigens stimulate APC derived IL-10 and IL-6 Figure 2 Bone marrow macrophages (BMMΦ) stimulated with S isolate excretory/secretory antigen (E/S) secrete higher levels of IL-10 and IL-6 compared to BMMΦ stimulated with E or J isolate E/S. Bone marrow was isolated and pooled from 3–5 naïve C57BL/6 mice and cultured into BMMΦ. The BMMΦ were incubated for 48 h, in vitro with medium alone – unstimulated (U), 50 μg/mL E isolate E/S (E), 50 μg/mL J isolate E/S (J), 50 μg/mL S isolate E/S (S) or 100 ng Lipopolysaccharide (LPS). Levels of IL-10, IL-12p70, TGF-β, IL-6, TNF-α and MIP-1α were measured by CBA; all cytokine data are pg/mL. Values represent the mean ± SD for four wells per treated group. Signiﬁcant difference between the BMMΦ stimulated with the S isolate E/S antigen and BMMΦ stimulated with the E or J isolate E/S antigens. **P < 0·01 and ***P < 0·001. Experiments were performed four times and data shown are a representative experiment. expression were measured using the geomean. Geomeans for relevant isotypes were also obtained and used to create a fold-change value of geomean sample/geomean isotype control. Examples of CD40 expression of cells cultured in medium alone with the relevant isotype control for CD40 (Figure 3d), cells cultured with LPS and stained with anti-CD40 and relevant isotype control (Figure 3e) and levels of CD40 expression on unstimulated BMDCs compared to cells exposed to LPS (Figure 3f) are shown. In two out of four experiments the S isolate E/S increased the levels of CD80 and CD86 expression on CD11c+CD11b+ 7AAD− cells (Figure 3g). In all four experiments, BMDCs stimulated with either the E, J or S isolate E/S consistently increased levels of CD40 expression compared to medium alone samples (Figure 3g). There was however no consistent dif- ference between the isolates. LPS up-regulated the levels of expression of CD80, CD86, MHC class II and CD40 on BMDCs compared to unstimulated levels in all four experiments. Levels of IL-10 and IL-6 secreted by BMDCs were signiﬁcantly increased following stimulation with S isolate E/S compared to the E or J isolate E/S BMDCs cultured with isolate-speciﬁc E/S, LPS or medium alone (unstimulated) were analysed for a panel of cytokines. As seen for BMMΦ, levels of IL-10 and IL-6 secreted by BMDCs were signiﬁcantly elevated following S isolate stimulation compared to stimulation with E/S from the other two isolates (Figure 4a,d, respectively). Levels of TNF-α, IL-12p70 and MIP-1α did not vary between isolates, but in all cases, were elevated from unstim- ulated levels and were equivalent to levels from cells exposed to LPS (Figure 4b,e,f ). Unpublished observations from our laboratory have also shown no differences in BMDC- derived TGF-β levels after exposure to the three isolate antigens. In accordance with data presented for BMMΦs, levels of Th2 associated cytokines IL-4, IL-5, IL-9 and IL- 13 were analysed and found to be undetectable (data not shown). DISCUSSION In this study we explore the hypothesis that the ability of C57BL/6 mice to mount a protective Th2 immune response to the J and E isolates of T. muris but a nonprotective Th1 immune response to the S isolate is due to isolate-speciﬁc E/S antigens interacting differently with key APCs. Little is known about the responses of macrophages or DCs to T. muris antigens although it has been reported that BMMΦ from C57BL/6 wild type mice and mannose recep- tor KO mice, secrete increased levels of IL-6, IL-10, CCL3 (MIP1-α) and CCL9 following stimulation with the E isolate of T. muris E/S antigen (26). Here we culture BMMΦ and BMDCs from naïve C57BL/ 6 mice with the E/S from the E, J and S isolates of T. muris to identify differences in BMMΦ and/or BMDC responses (at the level of cytokine secretion or cell surface marker expression) which may underlie the subsequent documented © 2009 The Authors Journal compilation © 2009 Blackwell Publishing Ltd, Parasite Immunology, 31, 123–131 127