The stimulation of NK cytotoxicity by continuous CD27-CD70 interaction correlates with the reported enhanced CD8+ T-cell response of CD70-Tg mice to influenza virus infection and upon EL-4 tumour challenge. In this model continuous CD70 triggering initially enhances expansion

of the CD8+ T-cell population, combined with a higher cytotoxicity on a per cell basis 43. It is important to note that all evidenced changes for NK cells of CD70-Tg mice compared with WT mice, both phenotypical and functional, are dependent on CD27–CD70 interaction, as none of them is witnessed in CD70-Tg×CD27−/− mice. Since CD70 is up-regulated on activated B cells after antigenic stimulation, the CD70-Tg mice used in this study might provide a model for chronic CD70 expression, possibly resulting from continuous stimulation of the immune system during PD98059 molecular weight PI3K Inhibitor Library order persistent infections. Our results clearly indicate that, as previously demonstrated for the CD8+ T-cell population, continuous CD70 triggering strongly reduces the NK cell number, however inducing

higher cytotoxicity capacities on a per cell basis. CD70-Tg (eight times backcrossed to C57BL/6) 29, IFN-γ−/−×CD70-Tg and CD70-Tg×CD27−/− 29 mice were used. Because the CD70 transgene, which is under the control of the human CD19 promotor, was located on the Y chromosome, female littermates were used as WT mice. All mice were housed under specific pathogen-free conditions in our animal facility and were treated and used in agreement with the guidelines of the local ethical committee. Spleen and liver from 4- to 15-wk-old

mice were removed, PTK6 disrupted and passed through a 40 μm cell strainer (Falcon, NJ, USA). Hepatic leukocytes were prepared using two-step discontinuous Percoll gradients (GE Healthcare, IL, USA). BM cells were isolated by irrigation of femurs and tibias. Erythrocytes from spleen and BM were lysed with 0.17 M NH4Cl. For functional assays, splenocytes were enriched with DX5 Microbeads (Miltenyi Biotec, CA, USA). mAb used were anti-NK1.1 (clone PK136), anti-CD3 (clone 145-2C11), anti-CD49b (clone DX5), anti-Ly49D (clone 4E5), anti-CD314 (clone CX5), anti-CD43 (clone S7), anti-CD95 (clone Jo2), anti-CD69 (clone H1.2F3), anti-granzyme B (clone GB11), anti-CD4 (clone RM4-5), anti-CD8 (clone 53-6.7), anti-IFN-γ (clone XMG1.2), annexin-V and 7-AAD (BD Pharmingen, CA, USA). Anti-CD122 (clone TM-β1; kindly provided by Dr. T. Tanaka, Tokyo, Japan), anti-Ly49E/C (clone 4D12) 32, anti-Ly49A (clone JR9-318; kindly provided by Dr. J. Roland, Paris, France), anti-Ly49H (clone 3D10; kindly provided by Dr. W. Yokoyama, MO, USA), anti-Ly49G2 (clone 4D11; American Type Culture Collection, MD, USA), anti-CD11b (clone M1/70), anti-NKG2A/C/E (clone 3S9) 32, anti-CD27 (clone LG.7F9, eBioscience, CA, USA) and anti-CD16/CD32 (unconjugated, clone 2.4G2; kindly provided by Dr. J. Unkeless, NY, USA).