Murine Cytomegalovirus Is Regulated by a Discrete Subset of Natural Killer Cells Reactive with Monoclonal Antibody to Ly49h

Daniels, Keith A.; Devora, Gene; Lai, Wayne C.; O'Donnell, Carey L.; Bennett, Michael; Welsh, Raymond M.

Preparation of mAb 1F8 to Ly49H. The amino acid sequences of Ly49 C, I, and H are very similar to each other and differ significantly from other Ly49 molecules (26) . mAb 1F8, a rat IgG2a with high reactivity to Ly49H, was prepared while attempting to generate an Ly49C-specific mAb. F334 rats were inoculated intrasplenically with 100 mg of a KLH-bound Ly49C amino acid sequence (CRPSNETLEYIKREQDRWDSKTKTVLD) that differed from Ly49I by a few amino acids. 1 mo later equal volumes of CFA and 100 mg of the peptide KLH were injected subcutaneously, followed by three additional injections at biweekly intervals. Spleen cells were removed and fused with SP2/0 myeloma cells to make mAbs. mAb specificity was screened on Bw5147 cells transfected with Ly49C or Ly49I or on RNK-16 cells transfected with Ly49H. All of the Ly49-specific mAb generated cross-reacted with Ly49 C, I, and H, but mAb 1F8 was the most potent and was useful for depletions in vivo. mAb 1F8 does not recognize Ly49 A, D, or G2. Limitations of this mAb are its cross-reactivity with Ly49 C and I, but it can be used to measure Ly49H by using it in concert with mAb 5E6, which reacts with Ly49 C and I, but not H. Another recently published mAb to Ly49H cross-reacts with Ly49I (27) . To date there is no available mAb that exclusively recognizes Ly49H.