Regulatory functions of CD8+CD28– T cells in an autoimmune disease model

MethodsMice.Female CD28 -/-and CD8 -/-mice on a C57BL/6 background and sex-and age-matched WT controls were obtained from The Jackson Laboratories (Bar Harbor, Maine, USA).STAT4 -/-mice on a C57BL/6 background were generated as described (24).To generate CD28 -/-CD8 -/-mice, male CD28 -/-mice were first bred with the female CD8 -/-mice.Subsequently, mice from the F 1 generation, which were heterozygous in both loci, were intercrossed.CD28 -/-CD8 +/-mice were generated by breeding of CD28 -/-CD8 -/-mice with CD28 -/- mice.The mice were maintained at our facility and used at 6-8 weeks of age. Screening of CD28 -/-CD8 -/-double-knockout mice.The animals were screened by flow cytometry as well as by PCR (Figure 1).Whole blood was obtained from the retroorbital plexus of one eye, and lymphocytes were isolated after hypotonic lysis of red blood cells.The cells were stained with rat anti-mouse CD8 FITC and hamster anti-mouse CD28 phycoerythrin (PE).Appropriate isotype controls were used.All Ab's were obtained from PharMingen (San Diego, California, USA).For the PCR screening, genomic DNA was extracted from tails according to QIAGEN tail DNA protocol (QIAGEN, Valencia, California, USA).CD28 PCR was performed in a 50-µl volume using 10× reaction buffer with 15 mM Mg ++ and 10 mM dNTP mix (Boehringer Mannheim Biochemicals, Indianapolis, Indiana, USA) in an automated thermal cycler (Multiblock, Thermo Hybaid, Needham Heights, Massachusetts, USA).WT and knockout alleles were detected by multiplexing two primers specific for the WT allele with one primer for the knockout allele.This primer pairs with one of the WT primers to produce the knockout-specific band.Ten microliters of each PCR reaction was resolved on 1% agarose gels, and bands were visualized using ethidium bromide staining.Homozygous samples produced a single 740-bp band, and WT samples produced a single 600-bp band.Heterozygotes produced both bands.All PCR experiments included a no-template control and control reactions using DNA from known heterozygote and WT samples.CD8 PCR was performed as above, but using 10× reaction buffer, 25 mM Mg ++ (Perkin-Elmer Corp., Norwalk, Connecticut, USA), and primers specific for the CD8 WT and knockout alleles.Homozygous samples produced a single 343-bp band, and WT produced a single 265-bp band. EAE-induction with MOG. MOG peptide 35-55 (MOG 35-55) (M-E-V-G-W-Y-R-S-P-F-S-R-V-V-H-L-Y-R-N-G-K)corresponding to the mouse sequence was synthesized by Quality Control Biochemicals Inc. (Hopkinton, Massachusetts, USA) and purified by HPLC.Peptide purity was greater than 99% after HPLC.Mice were immunized subcutaneously in the flanks with 150-200 µg of MOG peptide in 0.1 ml PBS and 0.1 ml CFA containing 0.4 mg Mycobacterium tuberculosis (H37Ra; Difco Laboratories, Detroit, Michigan, USA) and intraperitoneally injected with 200 ng pertussis toxin (List Biological Laboratories, Campbell, California, USA) on the