Brown et al. (1995). The Journal of Experimental Medicine. doi: 10.1084/jem.182.5.1315
…addition of target cells using the following mAb: 3.168 (anti-murine CD8), Leu2b (anti-human CD8), and RL172 (anti-murine CD4). Ab was added to effector cells along with guinea pig complement (Pel- Freez Biologicals, Rogers, At O, incubated 1.5 h at 37~ and washed three times. The efficacy of cell depletion was assessed by FACS | analysis to confirm that the appropriate subpopulations were eliminated. Relative…
Pel-Freez products cited: Guinea Pig Complement (38002)
Abstract
A series of human CD8 transgenic (hCD8 Tg) mice with differential expression in the thymus and periphery were produced to investigate CD8 coreceptor regulation of repertoire selection and T cell responses. Expression of hCD8 markedly enhanced responses to both HLA class I molecules and hybrid A2/Kb molecules providing functional evidence for a second interaction site, outside of the alpha 3 domain, which is essential for optimal coreceptor function. Peripheral T cell expression of hCD8 was sufficient to augment responsiveness to HLA class I, as hCD8 Tg mice which lacked thymic expression responded as well as mice expressing hCD8 in the thymus and periphery. Both murine CD8+ and CD4+ T cells expressing hCD8 transgenes exhibited markedly enhanced responses to foreign HLA class I, revealing the ability of T cell receptor repertoires selected on either murine class I or class II to recognize human class I major histocompatibility complex (MHC). In contrast to recognition of foreign class I, thymic expression of hCD8 transgenes was absolutely required to enhance recognition of antigenic peptide restricted by self-HLA class I. Thus, our studies revealed disparate requirements for CD8 coreceptor expression in the thymus for selection of a T cell repertoire responsive to foreign MHC and to antigenic peptides bound to self-MHC, providing a novel demonstration of positive selection that is dependent on human CD8.