Furthermore, CD8 CTL expansion was not seen in animals infected with LLOSer92 or LLOSer92 30 minutes or 48 hours after transfer

Furthermore, CD8 CTL expansion was not seen in animals infected with LLOSer92 or LLOSer92 30 minutes or 48 hours after transfer. lysis of erythrocytes with ammonium chloride. Splenocytes were resuspended in RP10+, consisting of RPMI 1640 (GIBCO BRL; Life Technologies Chloroquine Phosphate Inc. Gaithersburg, Maryland, USA) supplemented with 10% FCS, L-glutamine, HEPES (pH 7.5), -mercaptoethanol, penicillin (100 U/ml), streptomycin (100 g/ml) and gentamycin (50 g/ml). We incubated 40 106 responder splenocytes in the presence of 30 106 irradiated, syngeneic splenocytes that were peptide-pulsed for 1 hour at 37C with 10C9 M LLO91-99 peptide. Cell lines were cultured in 10 ml RP10+ medium supplemented with 0.5 ng/ml IL-2 and 4 ng/ml IL-7 (R&D Systems Inc., Minneapolis, Minnesota, USA). After 7 days, responder T cells were restimulated as described above. One to two weeks after the second in vitro restimulation, CD8 CTLs were washed with PBS and resuspended at a concentration of 6 106 epitope-specific CD8 CTLs per 250 l PBS for injection into syngeneic mice. Tetrameric H2-KdCpeptide complexes. MHC-peptide tetramers for staining of epitope-specific T cells were generated as previously described (27). Staining and flow cytometric analysis. One hundred thousand CD8 CTLs from T cell cultures or 2 106 Chloroquine Phosphate splenocytes were added to wells of a 96-well plate. After incubation at 4C for 20 minutes with unconjugated streptavidin (0.5 mg/ml; Molecular Probes Inc., Eugene, Oregon, USA) and Fc Block (BD PharMingen, San Diego, California, USA) in FACS staining buffer (SB: PBS [pH 7.45] 0.5% BSA, and 0.02% sodium azide), cells were stained with FITC-conjugated Chloroquine Phosphate anti-CD62L (BD PharMingen), phycoerythrin-conjugated H2-Kd LLO91-99 tetramers (0.25C0.5 mg/ml), peridinin chlorophyll proteinCconjugated anti-Thy1.1 (BD PharMingen), and allophycocyaninCconjugated anti-CD8 (BD PharMingen) in SB for 60 minutes at 4C. Cells were also stained with either FITC- or Chloroquine Phosphate phycoerythrin-conjugated anti-CD25 or anti-CD44 and FITC-conjugated antiCT cell receptor V (antiCTCR V) segments (TCR V2, 3, 4, 5, 6, 7, 8.1, 8.2, 8.1C8.3, 9, 10, 11, 12, 13, 14, 17). Subsequently, cells were washed three times in SB and then fixed with 1% paraformaldehyde/PBS (pH 7.4). Four-color flow cytometry was performed using a FACSCalibur or LSR flow cytometer, and data were analyzed with CellQuest software (Becton Dickinson Immunocytometry Systems Mountain View, California, USA). Intracellular cytokine staining. One to two weeks after the second in vitro stimulation, 5 106 CD8 CTLs per milliliter RPMI+ were incubated for 5 hours at 37C and 5% CO2 with 1 g/ml brefeldin A (BD PharMingen) in a 24-well flat-bottom plate coated with anti-CD3 mAb (10 g/ml) (BD PharMingen). Cells Chloroquine Phosphate were fixed by incubation with Cytofix/Cytoperm (BD PharMingen) at 4C for 20 minutes. Thereafter, cells were washed twice Rabbit Polyclonal to Amyloid beta A4 (phospho-Thr743/668) with 1 Perm/Wash buffer (BD PharMingen) and stained at 4C for 30 minutes with FITC-conjugated antiCIFN-, FITC-conjugated anti-TNF, or FITC-conjugated IgG1 isotype control. Finally, cells were again washed with 1 Perm/Wash Buffer and resuspended in FACS SB for flow cytometric analysis. CTL assays. Standard Cr-release assays using 51Cr-labeled P815 target cells were performed as previously described (28). For peptide titrations, the percentage specific lysis was decided over a range of different peptide concentrations at a constant effector-to-target ratio of approximately 20:1. Carboxyfluorescein diacetate succinimidyl ester labeling. CD8 CTLs were washed with PBS and resuspended at 5 107 per milliliter in PBS made up of 1 M carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes Inc.). The cell suspension was incubated at 37C, 5% CO2 for 10 minutes and immediately washed with cold RPMI 1640/10% FCS before transfer into mice. Contamination of mice with L. monocytogenes, harvesting of spleen, and bacterial quantification. Thirty minutes, 48 hours, or 7 days after transfer of 6 106 LLO91-99Cspecific Thy1.1 CD8 CTLs, Thy1.2 recipient mice were infected with 5 103 10403S or LLOSer92 via lateral tail vein injection. Seventy-two hours after contamination, spleens and livers were removed and viable bacterial counts in these organs were assessed by homogenizing the tissues through a wire mesh into PBS made up of 0.05% Triton X-100. Subsequently, aliquots were plated onto brain-heart infusion agar plates (Life Technologies Inc.) and CFUs were counted after 24C48 hours of incubation. In vivo administration of mAbs. Anti-CD40 mAb was purified from the FGK-45 hybridoma, and 100 g was injected intraperitoneally into recipient mice at the indicated time points. Results Characterization of in vitroCgenerated epitope-specific CD8 CTL line. To characterize the ability of adoptively transferred CD8 T cells to confer protective immunity, we generated LLO91-99Cspecific CD8 T cell lines by in vitro peptide stimulation. This epitope induces an immunodominant T cell response with a complex TCR repertoire (29). Previous work indicated that this activation status could influence the capacity of CD8 T cells to confer protective.