The 4E5 monoclonal antibody specifically recognizes Ly-49D, which is expressed on subsets of natural killer (NK) cells in C57BL/6, C3H/He (at a very low frequency), and SJL, but not DBA/2, AKR, CBA/J, or BALB/c mice. Unlike Ly-49D antigen has not been betected on NK-1.1+ (or DX5+) T cells. In 129/J mice, the 4E5 antibody cross-reacts with Ly-49O, Ly-49R, and Ly-49V. The Ly-49 family of NK-cell receptors, members of the C-type lectin superfamily, are disulfide-linked type-II transmembrane protein homodimers with extracellular carbohydrate-recognition domains, which 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. Ly-49D weakly binds to MHC class I antigens of the k halpotype, and Ly-49D+ IL-2-activated NK cells lyse target cells expressing H-2[a], H-2[b], H-2[d], H-2[k], H-2[p], H-2[q], and H-2[s] and the CHO (Chinese hamster ovary) cell line. Ly-49D+ cells mediate allogenic resistance to H-2d bone marrow transplantation. In vitro studies suggest that the Ly-49D receptor mediates activation of NK-cell cytolytic activity via tyrosine phosphorylation of their ITIMs (Immunoreceptor Tyrosine-based Inhibitory Motifs). Molecular differences between the Ly-49D stimulatory receptor and the inhibitory members of the Ly-49 family include the absence of an ITIM in Ly-49D, the lack of phosphorylation of Ly-49D in activated NK cells, and the association of a novel tyrosine-phosphorylated protein (pp16) with Ly-49D in activated NK cells. Ly-49O and Ly-49V are closely related to Ly-49A[B6] and, like Ly-49A, have ITIM domains. Ly-49O- and Ly-49V-transfected 293T (human kidney epithelial) cells bind tetramers of H-2D[b], D[b], D[k], and L[d]. In addition, the Ly-49V-transfected cells also bind K[b], K[d], and K[k]. Ly-49R is closely related to Ly-49D[B6] and is putative activating receptor due to its lack of an ITIM domain. Ly-49R-transfected 293T cells bind soluble tetramers of H-2D[b], D[d], D[k], and L[d].
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.