Anti cd4 or anti cd8 antibodies

Spleens were harvested and single cell suspensions created by filtration through 70 μm mesh in RPMI1640 culture media containing 10% fetal bovine serum and antibiotics. Splenocytes were pelleted and erythrocytes lysed using 0.84% NH₄Cl (J.T. Baker) in H₂O. For T cell repertoire profiling, splenocytes were labeled with anti-CD45, anti-CD3, and anti-CD8 antibodies (eBioscience) and MHC class I K^b tetramers provided by the NIH Tetramer Core Facility for identification of HSV-1 specific CD8⁺ T cells. Tetramer-labeled cells were analyzed using a MacsQuant flow cytometer (Miltenyi Biotec) as previously described.^{23 (link)} For adoptive transfers into Ifnar1^−/− mice, bulk preparations of CD3⁺ splenocytes were obtained by immunomagnetic isolation using anti-CD3 microbeads (Miltenyi Biotec) according to the manufacturer’s directions. Alternatively, splenocytes were labeled with anti-CD4 or anti-CD8 antibodies (eBioscience) and sorted using an S3e cell sorter (Biorad) for adoptive transfers into TCRα^−/− mice. Adoptive transfer of isolated cells was mediated by intravenous retroorbital injection. In some experiments, TCRα^−/− mice were injected i.p. with 250 μg anti-mouse CD154 (MR1 clone) or Armenian hamster IgG isotype (both from BioXcell) at days 0, 3, and 6 p.i. to evaluate CD40-dependent immune activation following adoptive transfer of CD4 T cells.