The 5E6 (also known as clone SW5E6) antibody reacts with Ly-49C[BALB], Ly-49C[B6], Ly-49C[NZB], and Ly-49I[B6], inhibitory receptors which are expressed on subsets of natural killer (NK) cells and NK-1.1+ (or DX5+) T lymphocytes (NK-T cells) in all strains tested except C57BR and RIII, on a population of memory CD8+ T lymphocytes and NK1.1+ γδ T cells in C57BL/6 mice, and on a distinct subset of B-1 cells of BALB/c and C57BL/6 mice. The proportion of NK T cells expressing Ly-49C/I is higher (2-5 fold) in thymus than in liver (immature and mature NK T cells, respectively), and there is evidence that the down-regulation of Ly-49 receptor expression is necessary for normal NK T-cell development. Most NK cells express a single allele of Ly-49C, although occasionally they may express more than one allele. The Ly-49 family of NK-cell receptors are disulfide-linked type-II transmembrane protein homodimers with extracellular carbohydrate-recognition domains (CRD) that bind to MHC class I alloantigens. The Ly-49 family members are expressed independently, such that an individual NK or T cell may display more than one class of Ly-49 receptor homodimers. The 5E6 antibody is specific for the Ly-49C CRD. The Ly-49C[BALB] and Ly-49C[B6] alloantigens bind to MHC class I antigens of the b, d, k, and s haplotypes, and the 5E6 antibody blocks this binding. Binding of Ly-49C[BALB]- and Ly-49C[B6]- expressing transfectants to lymphoblasts of H-2[f], H-2[q], H-2[r], and H-2[v] strains has also been detected. Ly-49I[B6] transfectants bind H-2[r] lymphoblasts and bind much more weakly to the b, d, k, q, s, and v haplotypes. The levels of the Ly-49 inhibitory receptors are down-regulated by their ligands in vivo, and the various levels of expression of an Ly-49 inhibitory receptor may affect the specificity of NK cells. Ly-49C is specifically downregulated in the presence of H-2K[b] class I molecules (one of the Ly-49C ligands). Ly-49C[+] and/or Ly-49I[+] cells mediate allogeneic and hybrid resistance to H-2d bone marrow transplantation. In vitro and in vivo studies suggest that the Ly-49C and/or Ly-49I receptors mediate negative regulation of NK-cell cytolytic activity via tyrosine phosphorylation of their ITIMs (Immunoreceptor Tyrosine-based Inhibitory Motifs).
The epitope recognized by this antibody on Ly49C may be masked on freshly isolated primary NK cells due to cis interactions with MHC class I molecules. This observation has been reported for other Ly49C monoclonal antibodies that bind to the same structural region.
The antibody was conjugated to BD Horizon™ BUV661 which is part of the BD Horizon Brilliant™ Ultraviolet family of dyes. This dye is a tandem fluorochrome of BD Horizon BUV395 with an Ex Max of 348-nm and an acceptor dye with an Em Max at 661-nm. BD Horizon Brilliant BUV661 can be excited by the ultraviolet laser (355 nm) and detected with a 670/25 filter and a 630 nm LP. Due to cross laser excitation of this dye, there may be significant spillover into channels detecting APC-like emissions (eg, 670/25-nm filter).
Due to spectral differences between labeled cells and beads, using BD™ CompBeads can result in incorrect spillover values when used with BD Horizon BUV661 reagents. Therefore, the use of BD CompBeads or BD CompBeads Plus to determine spillover values for these reagents is not recommended. Different BUV661 reagents (eg, CD4 vs. CD45) can have slightly different fluorescence spillover therefore, it may also be necessary to use clone-specific compensation controls when using these reagents.