Supplementary MaterialsSupplementary data cs1280825ntsadd. pathogen-free conditions and had access to standard rodent food and water and T-cell activation, we made use of the adoptive transfer of T-cells from DO11.10 mice that are transgenic for an OVA peptide (pOVA)-specific T-cell receptor. This T-cell transfer increases the frequency of OVA-specific T-cells in wt mice to levels that allow their visualization T-cell assays, spleen cells from naive mice were depleted from CD3+ cells by using mouse anti-CD3 antibodies (clone 17A2; BD Biosciences) and sheep anti-rat IgG Dynabeads at a ratio of two beads per cell (Invitrogen) in combination with Dynal MPC-1 Magnetic Particle Concentrator (Dynal A.S.) and were used as APCs with a purity of more than 95% CD3? cells. Next, 3105 APCs that had been irradiated with 30 Gy were co-cultured with 2105 Rabbit Polyclonal to CDCA7 CFSE-labelled T-cells from naive DO11.10 mice, along with CD3+ T-cells from pLNs of sham-treated or injured mice at various ratios. All cultures were set up in triplicate (96-well plates) in the presence or absence of Cefiderocol 0.1?g/ml pOVA. After 3?days, supernatants were harvested and analysed by IFN- ELISA. In both T-cell assays, the dilution of CFSE during the proliferation of OVA-specific T-cells was assessed by flow cytometry. Flow cytometry We stained pLN cells with fluorochrome-labelled antibodies against the T-cell receptor specific for pOVA (clone KJ1-26; Caltag) in combination with anti-CD4 (clone RM4-5) or in combination with anti-CD69 (clone H1.2F3) and anti-CD25 (clone PC61.5). For tracking OVACFITC, pLN cells were stained with anti-CD11c (clone N418) and anti-CD11b (clone M1/70). Antibodies against CD11c, CD40 (clone HM40-3) and CD86 (clone GL1) were used to analyse DC. For characterization of NK cells, pLN cells were stained against CD3 clone 145-2C11), CD11b and CD49b (clone DX5). All antibodies were obtained from BD Biosciences. Intracellular staining of FoxP3 was performed with the FoxP3/Transcription Factor Staining Buffer Set from eBioscience according to the manufacturer’s instructions. For all specific antibodies, appropriate isotype antibodies served as negative control. Flow cytometry was performed with a FACSCalibur flow cytometer (BD Biosciences) and CellQuest Pro software (BD Biosciences). Statistical analyses Data are expressed as means S.D. of triplicate cultures, individual mice or multiple experiments. Statistically significant differences between two or more groups were detected with Student’s (Figure 3C). However, after the application of OVA-loaded BMDCs, pLN cells from injured mice released larger amounts of IFN- than did cells from sham-treated mice (Figure 3C). Open in a separate window Figure 3 Inverse capability of exogenously loaded DCs and endogenous APCs to Th1-cell priming were injected into the hind footpads 4?days after injury or sham treatment and after transfer of CFSE-labelled OVA-specific Th-cells. After 3?days, pLN cells were pooled per group. (A) Overview of the experimental design. (B) Percentage of CD25+ and CD69+ cells in gated OVA-specific Th-cells. (C) Content of IFN- in the supernatants after restimulation of pLN cells with pOVA. Data show mean S.D. of triplicate cultures and are representative of three independent experiments with (Supplementary Figure S2D). Thus, after skeletal muscle injury, the priming of antigen-specific Th-cells by resident APCs in the pLNs is not restricted and the Th1 differentiation ability of transferred OVA-specific T-cells is not generally impaired. Endogenous T-cells mediate the suppression of OVA-specific Th-cells after injury Tregs are frequently involved in Th-cell suppression. Cefiderocol We investigated whether endogenous T-cells acquire regulatory activity after injury and suppress the priming of subsequently transferred OVA-specific Th-cells. Therefore, we isolated CD3+ T-cells Cefiderocol from pLNs 24?h after injury or sham treatment and transferred them into naive mice, along with naive CFSE-labelled OVA-specific T-cells. One day later, we injected OVA s.c. into the footpads and 3?days later we measured the proliferation, activation and cytokine secretion of OVA-specific T-cells from pLN cells (for experimental approach see Figure 4A). Irrespective of whether the T-cells injected into Cefiderocol the naive mice came from sham-treated or injured mice, the co-injected OVA-specific T-cells proliferated to the same extent and reached the same percentages.