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.

Selective Synthesis of IFN-␥ by Ly49H ϩ Cells. We noted that, in the presence of brefeldin A, substantial proportions (i.e. Ͼ 25%) of the NK cells from MCMV-infected mice spontaneously produce intracellular IFN-␥ if allowed to incubate in the milieu of leukocytes from virus-infected tissue. For these assays to be optimal, the cells were quickly harvested and incubated without being put on ice. Usually this intracellular IFN assay is done with lymphocytes receiving an in vitro stimulus with immunogenic peptides (for T cells) or with nonspecific stimulators, such as PMA and ionomycin (24, 33) ; this may mean that something in this cellular milieu continued to stimulate the NK cells. Because of the unusual nature of this assay, several controls, in addition to the isotype controls for the anti-IFN-␥ mAb used in every assay, were run to ensure that synthesis of IFN-␥ was being observed. Using splenocytes from day 2 MCMV-infected mice as a source of IFN-␥ -producing cells, threefold molar excess of unlabeled anti-IFN-␥ mAb reduced the frequency of anti-IFN-␥-staining NK cells from 23 to 7.6%, and the mean fluorescent intensity of the remaining and very weakly staining positive cells was reduced from 638 to 120. This mAb did not block the ability of (nonNK cell) splenocytes to synthesize intracellular TNF-␣ (positive control ϭ 0.8%; anti-IFN-␥ mAbblocked ϭ 0.7%). Similarly, incubation of the anti-IFN-␥ mAb with 0.8 g/ml IFN-␥ during the IFN-␥ assay reduced the frequency of IFN-␥ staining from 20 to 7.5, and reduced the mean fluorescent intensity of the remaining positive cells from 736 to 473, without reducing the staining against TNF-␣. We conclude that this assay is indeed detecting IFN-␥ synthesis and that it is revealing a high frequency of NK cells that produce IFN-␥ when kept in the Expression of Ly49H on NK cells from different strains of mice. C57BL/6 and Balb/c splenocytes were stained with mAb DX-5 (as a pan NK cell marker that would recognize cells from each strain), anti-CD3, and mAb 1F8 (anti-Ly49 C/I/H) and mAb 5E6 (anti-Ly49 C/I). Diagrams depict the staining of gated DX-5 ϩ CD3 Ϫ lymphocytes and isotype controls for mAb 5E6 and 1F8. milieu of leukocytes from virus-infected mice. We examined IFN-␥-producing NK cells over the first 3 d of MCMV infection and found a sharp peak of production at 2 d after infection, in agreement with other publications using different types of techniques (18) .