Lifeless cells were excluded by staining with DAPI (Sigma-Aldrich, St. an antigen-specific manner. However, the infection-induced growth of LCMV-specific T cells, viral clearance, and the humoral immune response were seriously impaired in CD4Cre/R-DTA mice as compared to control mice. Transfer of polyclonal na?ve CD4 T cells from wild-type mice but not anti-PD-L1 blockade restored the expansion Mutant IDH1-IN-2 and function of endogenous CD8 TML cells in CD4Cre/R-DTA mice. Materials and Methods Mice and Illness Homozygous CD4Cre mice (23) were crossed to R-DTA mice (22) to generate lymphopenic CD4Cre+R-DTA+ mice (CD4Cre/R-DTA) and CD4Cre+R-DTA? control mice (CD4Cre). B6_CD45.1 congenic mice (B6.SJL-Ptprca Pepcb/BoyJ) were originally obtained from The Jackson Laboratory and crossed to normal C57BL/6J mice to generate heterozygous CD45.1+CD45.2+ congenic mice. C57BL/6J mice were purchased from Charles River Laboratories (Sulzfeld, Germany). Mice were maintained in the Franz-Penzoldt-Zentrum in Erlangen under specific pathogen-free conditions. Mice were infected with 200?pfu of LCMV-WE intravenously under biosafety level 2 and analyzed at indicated points in time. All experiments were performed in accordance with German animal protection law and European Union guidelines 86/809 and were approved by the Federal Government of Lower Franconia. Flow Cytometry Single-cell suspensions of spleens were generated under biosafety level 2 by mechanical disruption and erythrocytes were lysed with ACK-buffer (0.15?M NH4Cl, 1?mM KHO3, 0.1?mM Na2EDTA). Cells were preincubated with anti-CD16/CD32 mAb (clone 2.4G2; BioXcell, West Lebanon, NH, USA) and stained with respective antibodies. The following antibodies were used for surface staining: PerCP-Cy5.5- or APCe780-labeled anti-CD4 (clone RM4-5), FITC-, PE-, or APC-labeled anti-CD8 (clone 53-6.7), PE-Cy7-labeled anti-CD62L (clone MEL-14), eFluor660-labeled anti-GL-7 (clone GL-7), FITC- or eFluor450-labeled anti-CD45R (clone RA3-6B2), FITC-labeled anti-CD44 (clone IM7), e450-labeled anti-KLRG1 (clone 2F1), PerCP-labeled anti-CD45.2 (clone 104), and PE- or e450-labeled anti-CD45.1 (clone A20) were purchased from eBioscience (San Diego, CA, USA). PE-Cy7-labeled anti-CD38 (clone 90), PE-Cy7-labeled anti-CD4 (clone RM4-5), and PE-Cy7-labeled or biotinylated anti-PD-1 (clone RMP1-30) were purchased from BioLegend (San Diego, CA, USA). Vioblue- or APC-labeled anti-CD44 (clone IM7.8.1) was purchased from Miltenyi Biotec (Bergisch Gladbach, Germany), PE-labeled anti-CXCR5 (clone 2G8) and V500-labeled Streptavidin were from BD Biosciences (San Jose, CA, USA). For dextramer stainings (gp33_H2-Db coupled to APC; Immudex, Copenhagen, Denmark), cells were washed with PBS made up of 5% FCS, incubated with 5?l dextramer per IKK-gamma antibody sample for 10?min at room temperature and then the antibody mixture for surface staining was added for an additional Mutant IDH1-IN-2 20?min at 4C. Tetramer staining (gp66_I-Ab coupled to PE, NIH tetramer core facility) was performed in RPMI1640 Mutant IDH1-IN-2 (PAN-Biotech, Aidenbach, Germany) made up of 10% FCS. Cells were incubated with 0.3?ng tetramer for 2?h at 37C, washed, and stained with respective antibodies. FITC-labeled anti-mouse IFN- (clone XMG1.2; BioLegend) and PE-labeled anti-mouse TNF- (clone MP6-XT22; eBioscience) were used for intracellular staining after cells had been fixed with 4% paraformaldehyde and permeabilized with the Intracellular Staining Perm Wash Buffer (BioLegend) according to the manufacturers protocol. Dead cells were excluded by staining with DAPI (Sigma-Aldrich, St. Louis, MO, USA), fixable viability dye APC-eFluor780, or fixable viability dye APC-eFluor506 (both from eBioscience). Samples were acquired with FACS Canto II (BD Bioscience) and MACSQuant (Miltenyi Biotec). Data were analyzed using FlowJo software (TreeStar, Ashland, OR, USA). Restimulation of T Cells Single-cell suspensions were either restimulated with 1?g/ml gp33- (KAVYNFATM) or gp61- (GLKGPDIYKGVYQFKSVEFD) peptide (JPT, Berlin, Germany) for 4?h. After 2?h, 10?g/ml Brefeldin A (Sigma-Aldrich, St. Louis, MO, USA) was added. IFN- and TNF- production was measured by intracellular staining. Quantitative RT-PCR RNA was prepared from the indicated organs with the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturers protocol. To transcribe the viral RNA genome, 1?g of total RNA was reversely transcribed into cDNA with the LCMV-gp-specific reverse primer. Quantitative PCR was performed with SYBR Select Grasp Mix (Thermo Fisher Scientific, Waltham, Mutant IDH1-IN-2 MA, USA), and the following primer sequences: LCMV-gp forward primer 5-CATTCACCTGGACTTTGACAGACTC-3 and LCMV-gp reverse primer 5-GCAACTGCTGTGTTCCCGAAAC-3 under the following conditions: 95C 30?s, 60C 20?s, 72C 20?s; 40 cycles. Copy numbers of LCMV genomes per gram organ were determined using a plasmid made up of 115?bp of LCMV gp gene. Cytotoxicity Assay Splenocytes from na?ve B6 mice were stained with 5?M CellTraceViolet (Thermo Fisher Scientific), loaded with 1?M gp33 peptide (JPT), and mixed with splenocytes from CD45.1 congenic mice loaded with 1?M m45 peptide of MCMV (JPT). The 4.5??106 cells per group Mutant IDH1-IN-2 were mixed and transferred into LCMV-infected or na?ve mice at day 8 after infection. Analysis took place 90?min after cell transfer. Adoptive T Cell Transfers CD4 or CD8 T cells from spleens of CD45.1.