Blood was collected from the tail vein pre-infection (d0) or on days 1, 2, 3, and 4 post-infection in BD Microtainer tubes (catalog no. binding c-FMS inhibitor of fluorochrome-conjugated peptide-MHC class-I (SCLEFWQRV-H-2Kb) multimer. Shown are color-coded density plots with red and blue representing highest and lowest density, respectively. c-FMS inhibitor (FL) fluorescence channel and fluorescence intensity. Percentages of main interest are indicated for gated areas.(TIF) ppat.1004100.s002.tif (257K) GUID:?477E92E6-B91E-4D94-B055-D538F0188EE1 Physique S3: MC deficiency has no influence around the viral epitope hierarchy of the CD8 T-cell response in the lungs nor on recognition of infected cells. MC-sufficient WT C57BL/6 (black bars) and MC-deficient (open bars) mice were infected intravenously. On day 6 post-infection, immunomagnetically-purified CD8 T lymphocytes derived from the lungs of 6 mice per group were used as responder cells in IFN-based ELISpot assays. (A) Hierarchy of viral epitopes recognized by pulmonary CD8 T cells. EL-4 stimulator cells were exogenously loaded with synthetic peptides representing the indicated MHC-I-presented epitopes. (?) stimulator cells with no peptide added. (B) Recognition of infected cells (MEF) presenting naturally-processed viral peptides despite the expression of viral immune evasion genes. (n.i.) uninfected MEF. Throughout, frequencies of responding, IFN-secreting cells were determined by intercept-free linear regression analysis. Bars represent most probable numbers, and error bars indicate 95% confidence intervals.(TIF) ppat.1004100.s003.tif (150K) GUID:?7FD81D76-4208-4FB9-8B08-8D1532C673A6 Physique S4: MC deficiency does not notably alter the activation phenotype of pulmonary CD8 T cells. MC-sufficient WT C57BL/6 (left panels) and MC-deficient (right panels) mice were infected intravenously. Multi-color cytofluorometric analyses were performed on day 6 post-infection for lung infiltrate cells pooled from 6 mice per group and pre-gated on CD8+ cells (for the gating strategy, recall Fig. 1B). Shown are color-coded density plots with red and blue representing highest and lowest density, respectively. The analyzed cell surface markers are indicated. Viral epitope-specific CD8 T cells were identified with TCR-specific peptide-MHC class-I multimers, specifically with M45-Db and M57-Kb Dextramers. (FL) fluorescence channel and fluorescence intensity. Percentages of main interest are indicated for gated areas and quadrants.(TIF) ppat.1004100.s004.tif (972K) GUID:?347AD2C6-3A18-4BF3-B203-222B694DB381 Physique S5: MC deficiency does not alter the mice were derived from the experiment described in Physique S3, so that the different effector functions Rabbit polyclonal to Caspase 2 can be directly compared. Here, the CD8 T cells were analyzed for their cytolytic effector function with no preceding expansion in cell culture. Viral epitope-specific cytolysis was assayed at graded effector-to-target (ET) cell ratios with EL-4 lymphoma cells as target cells pulsed with saturating concentrations of synthetic peptides corresponding to the viral epitopes indicated.(TIF) ppat.1004100.s005.tif c-FMS inhibitor (111K) GUID:?19C6EC3B-479C-4EC9-8B28-AB0894D198B2 Abstract The lungs are a noted predilection site of acute, latent, and reactivated cytomegalovirus (CMV) infections. Interstitial pneumonia is the most dreaded manifestation of CMV disease in the immunocompromised host, whereas in the immunocompetent host lung-infiltrating CD8 T cells confine the infection in nodular inflammatory foci and prevent viral pathology. By using murine CMV contamination as a model, we provide evidence for a critical role of mast cells (MC) in the recruitment of protective CD8 T cells to the lungs. Systemic contamination brought on degranulation selectively in infected MC. The viral activation of MC was associated with a wave of CC chemokine ligand 5 (CCL5) in the serum of C57BL/6 mice that was MC-derived as verified by contamination of MC-deficient sash mutants. In these mutants, CD8 T cells were recruited less efficiently to the lungs, correlating with enhanced viral replication and delayed virus clearance. A causative role for MC was verified by MC reconstitution of c-FMS inhibitor sash mice restoring both, efficient CD8 T-cell recruitment and contamination control. These results reveal a novel crosstalk axis between innate and adaptive immune defense against CMV, and identify MC as a hitherto unconsidered player in the immune surveillance at a relevant site of CMV disease. Author Summary Being strategically located beneath endothelial and epithelial surfaces, mast cells (MC) serve as sentinels for invading pathogens at host-environment boundaries as part of the innate defense against contamination. Host genetic resistance against c-FMS inhibitor cytomegaloviruses (CMV) is largely determined by the innate immune response, but an implication of MC in the adaptive immune defense against CMV has not been considered so far and is almost impossible to.