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Early studies indicate that rats may have a repertoire of MHC class Ib-reactive Ly49 stimulatory receptors capable of mounting memory-like NK cell alloresponses. In this article, we provide molecular and functional evidence for this assumption. Pairs of Ly49 receptors with sequence similarities in the lectin-like domains, but with opposing signaling functions, showed specificity for ligands with class Ia-like structural features encoded from the first telomeric MHC class Ib gene cluster, RT1-CE , which is syntenic with the H2-D/H2-L/H2-Q cluster in mice. The activating Ly49s4 receptor and its inhibitory counterparts, Ly49i4 and Ly49i3, reacted with all allelic variants of RT1-U, whereas Ly49s5 and Ly49i5 were specific for RT1-E u NK cell cytolytic responses were predictably activated and inhibited, and potent in vivo NK alloresponses were induced by repeated MHC class Ib alloimmunizations. Additional Ly49-class Ib interactions, including RT1-C l with the Ly49s4/Ly49i4/Ly49i3 group of receptors, were characterized using overexpressed receptor/ligand pairs, in vitro functional assays, and limited mutational analyses. Obvious, as well as subtle, Ly49-class Ib interactions led to ligand-induced receptor calibration and NK subset expansions in vivo. Together, these studies suggest that in vivo NK alloresponses are controlled by pleomorphic Ly49-class Ib interactions, some of which may not be easily detectable in vitro., (Copyright © 2018 by The American Association of Immunologists, Inc.)

Natural killer cells are able to recognize and kill target cells according to differences in MHC class I expression. In rodents, the Ly49 receptors are primarily responsible for this MHC differentiation. We previously described the cloning of a novel C-type lectin-like receptor, KLRH1, encoded in the NK complex adjacent to the Ly49 genes and expressed by subsets of NK and NKT cells. MHC influence on selection of KLRH1(+) NK cells in congenic strains suggested that KLRH1 may have an MHC ligand, although we were unable to identify any such ligand. In this study, we have used a sensitive reporter system and Fc fusion protein to demonstrate that KLRH1 binds specifically to the classical MHC class I molecule RT1-A2 of the RT1(n) haplotype. Cytolytic activity of KLRH1-transfected RNK-16 cells was also inhibited by target cells expressing RT1-A2(n). Thus, KLRH1 represents a novel family of MHC allele-specific inhibitory receptors expressed by NK cells.

The Ly49 family of lectin-like receptors in rodents includes both stimulatory and inhibitory members. Although NK alloreactivity in mice is regulated primarily by inhibitory Ly49 receptors, in rats activating Ly49 receptors are equally important. Previous studies have suggested that activating rat Ly49 receptors are triggered by polymorphic ligands encoded within the nonclassical class Ib region of the rat MHC, RT1-CE/N/M, while inhibitory Ly49 receptors bind to widely expressed classical class Ia molecules encoded from the RT1-A region. To further investigate rat Ly49-mediated regulation of NK alloreactivity, we report in this study the identification and characterization of two novel paired Ly49 receptors that we have termed Ly49 inhibitory receptor 5 (Ly49i5) and Ly49 stimulatory receptor 5 (Ly49s5). Using a new mAb (mAb Fly5), we showed that Ly49i5 is an inhibitory receptor that recognizes ligands encoded within the class Ib region of the u and l haplotypes, while the structurally related Ly49s5 is an activating receptor that recognizes class Ib ligands of the u haplotype. Ly49s5 is functionally expressed in the high NK-alloresponder PVG strain, but not in the low alloresponder BN strain, in which it is a pseudogene. Ly49s5 is hence not responsible for the striking anti-u NK alloresponse previously described in BN rats (haplotype n), which results from repeated alloimmunizations with u haplotype cells. The present studies support the notion of a complex regulation of rat NK alloreactivity by activating and inhibitory Ly49 members, which may be highly homologous in the extracellular region and bind similar class Ib-encoded target ligands.