P

P. neutralizing antibody titers than those in were included in the study (Table ?(Table1),1), with the status being determined by PCR as described previously (28). For all those procedures, animals were anesthetized with ketamine HCl and managed in accordance with the guidelines of the Institutional Animal Care and Use Committees for Harvard Medical School, Dana-Farber Malignancy Institute, and Emory University or college and the (39A). Lymphocyte immunophenotyping and Mamu-A*01/SIV Gag p11C tetramer staining. EDTA-anticoagulated whole-blood specimens or lymph node lymphocytes obtained from peripheral lymph node biopsies were immunophenotyped by use of the following antibodies: anti-CD3(FN18)-allophycocyanin, anti-CD4-fluorescein isothiocyanate (19Thy5D7), anti-CD8-phycoerythrin (DK25; Dako, Carpenteria, Calif.), and anti-CD8-ECD (2ST8-5H7; Beckman Coulter, San Diego, Calif.). Specimens from deletion, as explained previously (54). Immune assays. Peripheral blood mononuclear cell Olinciguat (PBMC) specimens were cryopreserved from nine animals (five anti-CD8 MAb-treated animals and four control MAb-treated animals). Gamma interferon (IFN-)-specific enzyme-linked immunospot (ELISPOT) assays were performed on PBMC depleted of CD4+ T cells by StemSep unfavorable cell separation (Stem Cell Technologies, Vancouver, British Columbia, Canada), resulting in populations of cells with 5% residual CD4+ T cells. Serial dilutions of the PBMC (3 105 and 1 105 cells/well) in RPMI 1640-10% fetal bovine serum were plated in duplicate in 96-well Multiscreen ELISPOT IP plates (Millipore, Billerica, Mass.) coated with an anti-IFN- antibody (diaPharma, West Chester, Ohio) and then incubated with pools of SIV peptides or medium alone made up of an equivalent concentration of dimethyl sulfoxide. SIV Gag, Env, Rev, Vif, Tat, and Nef peptide pools consisted of 15-mers that overlapped by 11 residues, corresponding to either the SIVmac239 or SIVmac251 sequence, with the final concentration of each individual peptide being 2 g/ml. All peptides except the Nef pool were synthesized by Ashok Khatri of the Massachusetts General Hospital Peptide Core Facility (Boston, Mass.), using fluorenylmethoxycarbonyl chemistry; the Nef pool was obtained from the NIH AIDS Research and Olinciguat Reference Olinciguat Reagent Program. PBMC stimulated with concanavalin A (5 g/ml, 105 cells/well) served as a positive control. Plates were incubated for 12 to 18 h. After the plates were washed, IFN- spots were detected with a biotin-conjugated anti-IFN- Ab Olinciguat (diaPharma) and alkaline phosphatase-streptavidin and were developed by use of an alkaline phosphatase substrate kit (Bio-Rad, Hercules, Calif.). Spots were counted with a KS ELISPOT automated reader system (Carl Zeiss Inc., Thornwood, N.Y.) using KS ELISPOT 4.2 software (performed by Zellnet, New York, N.Y.). The results were calculated as frequencies of SIV-specific spot-forming cells (SFC) per 106 PBMC minus the frequency of SFC per 106 PBMC obtained with medium alone. The results were considered positive if there were 10 SFC per well and threefold more than the background level. CTL activity was measured as previously explained (23). Briefly, PBMC were stimulated with autologous herpesvirus papio-transformed B-cell lines (B-LCL) infected with a recombinant vaccinia computer virus (vAbt388; GHRP-6 Acetate provided by D. Panicali, Therion Biologics, Cambridge, Mass.) expressing the SIVmac251 and genes and the SIVmac239 gene in RPMI 1640-10% fetal calf serum, with 20 U/ml recombinant human interleukin-2 added after 3 days. CTL assays were performed 10 to 14 days after activation. 51Cr-loaded target cells consisted of autologous B-LCL infected with recombinant vaccinia viruses expressing either Gag derived from SIVmac251 or Env derived from SIVmac239. Chilly targets consisted of unlabeled autologous B-LCL infected with the control vaccinia computer virus strain NYCBH and were used at a cold-to-hot target ratio.