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2. mice lacking this CD8 subpopulation develop exaggerated immune responses to self- antigen (4). Analysis of Qa-1deficient mice has not fully clarified the contribution of Qa-1 to immunologic suppression due to the ability of the Qa-1 molecule to interact with two unique receptors. First, engagement of the T cell receptor (TCR) by Qa-1peptide complexes can lead to activation and development of antigen-specific CD8 cells. Second, engagement of the CD94/NKG2A receptor indicated by CD8 cells and natural killer (NK) cells by Qa-1/Qdm peptide ligands attenuates the activities of these cells (57). Therefore, the immune response phenotype of Qa-1-deficient mice displays these functionally unique relationships. Diminished relationships between Qa-1-restricted CD8 cells and Qa-1-deficient CD4 cells results in enhanced CD4 reactions, whereas improved NK cell lysis of triggered CD4 cells displays a loss of the inhibitory NKG2AQa-1/Qdm connection. We isolated Qa-1-dependent engagement of the TCR and NKG2A for analysis and exploited the fact that Qa-1-dependent binding to each receptor used distinct Qa-1 contact residues. We therefore generated knock-in mice that communicate a Qa-1 amino acid exchange mutation (Qa-1 D227K) that disrupts binding of Qa-1 to the CD8 coreceptor (8). Cells that communicate this Qa-1 D227K point mutation fail to present Qa-1-restricted peptides to CD8 cells but retain the ability to participate NKG2A receptors on CD8 cells and NK cells. A second Qa-1 knock-in strain (Qa-1 R72A) expresses a Qa-1 aa exchange mutation (R72A) that fails to bind to NKG2A receptors on NK cells and CD8 cells but spares Qa-1-dependent peptide presentation to the TCR (7). Manifestation of this Qa-1 aa exchange mutant by CD4 cells renders them vulnerable to NK and CD8 lysis. These two Qa-1 point mutations were backcrossed to C57BL/6 mice for analysis of CD8 T regulatory (Treg) cell activity. We statement that engagement of Qa-1 from the CD8 coreceptor is essential for Qa-1-restricted Treg activity, as indicated by findings NS13001 that Qa-1 D227K knock-in mice fail to develop regulatory CD8 activity and display enhanced proteolipid protein (PLP)-induced EAE. Analysis of Qa-1 R72A knock-in mice exposed that genetic disruption of the NKG2AQa-1 connection releases the brakes on CD8-dependent suppressive activity, permitting the development of powerful CD8+Treg activity, which results in complete resistance to the NS13001 development of EAE. == Results and Conversation == == Analysis of B6.Qa-1 D227K and R72A Knock-In Mice. == A genomic 4-Kb Qa-1 fragment comprising a D K amino acid exchange mutation at position 227 after site-directed mutagenesis (Fig. 1A) was cloned into a alternative vector and transfected into the TC1 Sera cell collection. After injection of positive homologous recombinant clones into blastocysts to produce germline chimeras (Fig. 1B), and deletion of theNeorgene after crossing to B6-EII-CRE mice, progeny were backcrossed to C57BL/6 (B6) for seven decades and intercrossed to produce homozygous B6.Qa-1 D227K mutant mice. Manifestation of cell surface Qa-1 by triggered (ConA-stimulated) CD4 cells from B6.Qa-1 D227K knock-in mice was indistinguishable NS13001 from Qa-1 WT T cells (Fig. 1C). Manifestation of the Qa-1 D227K mutation by L cells (Fig. 1D,Remaining) or activated CD4 cells (Fig. 1D,Right) failed to target these cells for lysis by Qa-1-restricted cytolytic T cells (CTLs). Qa-1/Qdm-dependent resistance of triggered B6.Qa-1 D227K CD4 Rabbit Polyclonal to ETS1 (phospho-Thr38) cells to lysis by NKG2A+NK cells was unimpairedin vitro(Fig. 1E,Remaining) andin vivo, as judged by homeostatic development inRag-2/hosts (Fig. 1E,Right). == Fig. 1. == Generation and analysis of Qa-1 D227K mutant knock-in mice. NS13001 (A) Qa-1 genomic locus and focusing on strategy. Boxes symbolize exons; exon 4 (gray box) shows the mutation site. The loxP sites are displayed by black triangles. TK, thymidine kinase gene;neor, neomycin-resistance gene. (B) Southern blot analysis of Sera cell genomic DNA. The top band (12.5 kb) corresponds to the WT allele; the lower band (7.2 kb) represents the knock-in allele.Right: PCR genotyping of knock-in mice. WT (280 bp) and knock-in (430 bp) products represent the addition of foundation pairs from the remaining loxP site and surrounding sequence (WT/WT, Qa-1 WT; DK/DK, homozygous mutant knock-in mice; WT/DK, heterozygous). (C) ConA-induced Qa-1 manifestation of WT and D227K mutant. Splenocytes from littermates (Qa-1-deficient [KO], Qa-1 WT [WT], and Qa-1 D227K knock-in [D227K]) were individually stimulated with ConA for 40 h and analyzed for surface Qa-1 manifestation by FACS analysis using anti-Qa-1 Ab (BD Bioscience)..