Figure 3

TxT-induced MDSCs are immunosuppressive. (a) MDSCs were isolated by magnetic beads from spleens at 18, 24, 48 and 72 hours from sham- or TxT-treated mice and co-cultivated with CFSE-labelled spleen cells of B6.SJL mice (H-2^b, CD45.1⁺), which were activated alloantigen-specifically with irradiated spleen cells of DBA/2 mice (H-2^d, CD45.2⁺). MDSCs were added with various cell numbers to obtain different MDSC:T cell ratios. After 4 days, cells were stained for CD45.1, CD4, and CD8 and 7-AAD. Proliferation of 7-AAD⁻ CD45.1⁺ CD4⁺ and 7-AAD⁻ CD45.1⁺CD8⁺ T cells was analysed by flow cytometry and suppression of proliferation was calculated. (b) Spleen cells of 6 mice receiving TxT were pooled 24 hours after TxT and subsequently B cells were depleted. Remaining cells were stained for CD11b and Gr-1. CD11b⁺Gr-1⁺ cells were sorted according to Gr-1 expression into P1, P2 and P3. Single sorted populations were depicted as SSC/CD11b. Sorted cells were co-incubated with CFSE-labelled B6.SJL cells, which were activated with allogeneic DBA/2 spleen cells at a ratio to T cells of 1:1. After 4 days, cells were stained for CD45.1, CD3 and 7-AAD and proliferation of 7-AAD⁻ CD45.1⁺ CD3⁺ T cells was analysed by flow cytometry measuring CFSE dilution and % suppression was calculated. (c) Splenic MDSCs were isolated from sham- or TxT-treated mice after 18, 24 and 48 hours. qRT-PCRs for the expression of the immunosuppressive molecules arginase-1 and iNOS were performed and relative expression to AIP was calculated. (a,c) Data represent the mean value ± SD of triplicates from 2 experiments (18 h, 72 h) and from 3 experiments (24 h, 48 h) with spleen cells from at least 5 mice pooled. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ns = not significant. Significance was calculated by Student’s t test by comparing sham and TxT MDSCs at each time point and each MDSC: T cell ratio.