Anti cd8 ab

129P2-Hvcn1^{Gt(RRN293)Byg}/Mmcd (Hvcn1-deficient; Hvcn1-KO) and WT mice were generated as previously described (20 (link)). HY-specific, TCR-transgenic Marilyn, and B6Kd-transgenic mice have been previously described (45 (link), 46 (link)).
Recipient mice were depleted of the CD8⁺ T lymphocyte subset by single i.p. injection of 300 μg of anti-CD8 Ab (BioXCell, catalog 2.43) on day –3. On day 0, skin grafting and reconstitution of the CD8⁺ T lymphocyte subset were achieved by adoptively transferring 5 × 10⁶ donor CD8⁺ T cells.
All animal protocols used in this study were approved by the Animal Use and Care Committee of Queen Mary University of London (QMUL), following the Home Office Guidance (Scientific Procedure Act 1986) and the Guide for the Care and Use of Laboratory Animals of the National Research Council (National Academies Press, 2011).

Donor tail skin was removed and cut into 1 cm² sections. Recipient mice were anesthetized using isoflurane (Halocarbon Products). When the mice reached a surgical plane of anesthesia, 0.26 μg of Metacam (Boehringer Ingelheim) was injected s.c. The mice were then stretched out to ensure easy access to the surgical site using elastic bands. The selected area was shaved, washed with 70% alcohol, and sprayed with Opsite (Smith and Nephew) spray dressing. A piece of skin was removed from the right flank to create a graft bed, any excess blood was removed, and a 1 cm² tail skin graft was placed in the graft bed. The graft was covered with Jellonet (Smith and Nephew). A plaster was then wrapped around the midriff and graft. Mice were put in a warming chamber at 32°C for the plaster to dry and for the mice to recover. Plasters were removed 7–10 days after grafting, and grafts were inspected every other day for signs of rejection. Grafts were considered rejected when less than 10% of the initial tissues remained.
In some experiments, recipient mice were depleted of CD8⁺ T cells by a single i.p. injection of 300 μg of anti-CD8 Ab (BioXCell, catalog 2.43) on day –3. On day 0 (skin grafting), mice received 5 × 10⁶ WT CD8⁺ T cells i.v.