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1) Evolution of Class I MHC Receptors on Natural Killer Cells
Natural killer cells are so named because of their ability to spontaneously lyse transformed or virally infected cells. Individuals lacking NK cells or NK cell function suffer from life-threatening opportunistic viral infections including human cytomegalovirus and herpes simplex virus infections. In addition to the destruction of defective cells NK cells also secrete cytokines such as IFN-gamma, TNF-alpha, and GM-CSF to further drive the immune response and help to initiate adaptive immunity.
Control of NK cell function is absolutely vital to protect the host from autoimmunity. NK cells express a plethora of receptors that negatively and positively regulate their function. Prominent among these are the C-type lectin-like Ly49 family of class I MHC receptors. The recognition of normal MHC expression by inhibitory Ly49 on healthy cells spares them from NK cell attack. However, MHC can be downregulated after virus infection or transformation, resulting in NK cell lysis (Fig. 1).
The multigene Ly49 family is found on mouse chromosome 6 and is extremely polymorphic in terms of allelic variability and gene numbers among different Ly49 haplotypes (Fig. 2). Mapping and sequencing of bacterial artificial chromosome genome libraries of common inbred mouse strains has shown that the commonly used C57BL/6 inbred mouse has a unique haplotype shared only with C57BL/10. We have found that most common inbred mouse strains possess Ly49 haplotype represented by the BALB/c and 129 inbred mouse strains. We first sequenced 129 haplotype (650 kb; 20 genes) and is larger than the C57BL/6 cluster. On the other hand, the BALB/c Ly49 cluster is very small at 300 kb with about 8 genes. The absence of the Ly49h gene in the BALB/c cluster correlates with the lack of MCMV resistance in this mouse strain. Mouse Ly49 haplotype variability in gene number is a shared characteristic with the human functional analogues, the KIR.
Most recently, we are extending our Ly49 haplotype analysis to the non-obese diabetic (NOD) mouse strain. Initial observations include a greatly expanded repertoire of activating Ly49, a characteristic that is shared with human diabetics. Future research will involve assessing global Ly49 cluster impact on NK cell function via gene deletion approaches.
2) Class I MHC Receptors in Plasmacytoid dendritic cells.
Despite the great variability in gene number in mouse Ly49 haplotypes, all haplotypes characterized to date contain six conserved or ‘framework’ genes (Fig. 3). One of the framework genes is Ly49q. The Ly49Q protein is expressed on plasmacytoid dendritic cells (pDC), but not on NK cells. Plasmacytoid DC are a rare subset of DC that express TLR7 and TLR9 and are the most potent producers of IFN-alpha after virus infection. We are interested is identifying the function of Ly49Q on pDC.
Figure 3
Using a cell based reporter assay, we have shown that the MHC H-2Kb is a high-affinity ligand for Ly49Q. Ly49Q-BWZ reporter cells can be specifically activated by tumor cells of the H-2b haplotype or ex-vivo cells of H-2b mice (C57BL/6 or 129), but not MHC-deficient cells. The activation can be specifically inhibited with anti-Ly49Q or anti-H-2Kb mAb. Furthermore, Ly49Q-BWZ reporter cells can be specifically activated by immobilized recombinant H-2Kb/Ig but not H-2Db/Ig. To identify the specific function of Ly49Q we have produced Ly49q-deficient mice.
3) MHC-independent Regulation of Natural Killer Cells
The Nkrp1 family of NK cell receptors are structurally similar to the Ly49 and the Nkrp1 genes are located with the Ly49 in the Natural Killer Gene Complex on chromosome 6 (Fig. 4). In collaboration with Dr. James Carlyle (Sunnybrook Institute, Toronto) we have found that in direct contrast to Ly49 haplotypes, the Nkrp1 gene cluster is conserved with respect to gene numbers. Furthermore, unlike the Ly49, the ligands for the NKRP1 are not MHC, but other lectin-like proteins termed Clr/Ocil. Amazingly, Clr genes are located within the Nkrp1 cluster. Although Nkrp1/Clr gene number is conserved among BALB/c and C57BL/6 mice there are many coding sequence polymorphisms. Most prominent among these are those shared by NKRP1BBALB and NKRP1DB6, which we have proven to be highly divergent alleles. Our haplotype analysis of the BALB/c Nkrp1/Clr cluster has allowed us to identify the epitope on NKRP1CB6 giving mAb Pk136 reactivity (NK1.1) and the matching non-binding epitope in NKRP1CBALB. Interestingly, all other Nkrp1 and Clr coding regions are highly conserved.
Figure 4 |