![Figure 2. Generation of LAK cells in the presence of anti-2B4 or anti-CD48, but not anti-CD2, mAbs inhibits expansion and cytotoxicity of NK cells. NK cells from B6 mice were enriched using negative depletion and cultured in 5000 U/mL/h-rIL-2 in the presence of 5 μg/mL anti-2B4, anti-CD48, or anti-CD2 mAbs for 7 days. (A) Incubation of NK cells with anti-CD48 or anti-2B4 mAbs did not inhibit the formation of IL-2–induced NK blasts. LAK cells generated in the presence of anti-CD48 or anti-2B4 mAbs showed slightly larger size in both FSC and SSC as compared with those not treated with mAbs or treated with anti-CD2 mAbs. (B) Reduced LAK-cell expansion in the presence of anti-2B4 or anti-CD48 mAbs, but not with anti-CD2 mAbs. LAK cells excluding trypan blue were counted using hemocytometer. This reduction in NK-cell expansion was not likely due to antibody-mediated toxicity, because there was no significant difference in the frequency of nonviable cells between the control and antibody-treated cells up to 3 days in culture (data not shown). Furthermore, neither isotype controls nor anti-CD2 mAbs prevented IL-2–induced NK-cell expansion. (C) 3H-thymidine incorporation into anti-2B4 and anti-CD48 mAb-treated cells was inhibited as compared with untreated or anti-CD2 mAb-treated LAK cells. Negatively enriched NK cells (15 000) were plated on the 96-well plates and cultured as described above. 3H-thymidine (1 μCi [0.037MBq]) was added at the last 16 hours of incubation. (D) LAK cells were subjected to conventional 51Cr release assay at the effector ratio indicated in each figure. Because the number of NK cells derived from anti-2B4 or anti-CD48 mAb-treated groups was reduced by approximately 50%, the cell numbers in each assay were adjusted such that equivalent numbers of viable LAK cells (CD3–NK1.1+) were contained. Target cells used were CD48–RMA/S. (E) Redirected lysis of NK cells was induced by adding 10 μg/mL anti-NK1.1 mAbs, which activates NK1.1(NKRP1-C) through Fc crosslinking via FcR present on P815 (CD48–) cells. In the absence of FcR crosslinking, LAK cells were not able to lyse P815 targets. Targets (2000) were used in each assay. Data are shown as mean ± SD and are representative of data from 5 independent experiments.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/107/8/10.1182_blood-2005-01-0185/4/zh80080694040002.jpeg?Expires=1722714833&Signature=clzIHMITAbayi6zdg8beONC71yvOu0ja0DkX4ZEVSIbLRyLwwwKE2c1RuXTHKZunx3BJ7kEVLLrqtrU6nqLKyyrHYyGqluivzw2J89TxeSmgjwv9auYvbInRoILYrB1l0KVgUjs~jB9UXjtja3~ocsqVEFR1IlwCYNdOdM6pFlMoWU7e6TnHXRVU8Ac6mg8yBiJYBfR-qzRx9BvIPotI2UipNxhYP1VI195DqJnx89xoZfewmcQ4Xsj05ziqoaGxsX9uXUF~gGvR6e7i7LxTcNdPDQU7Md2ur5QIuC03rRk6h3MxzRvrayYazwzvaRRGkTNcB4jXiafP7HeYC2KjMg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Generation of LAK cells in the presence of anti-2B4 or anti-CD48, but not anti-CD2, mAbs inhibits expansion and cytotoxicity of NK cells. NK cells from B6 mice were enriched using negative depletion and cultured in 5000 U/mL/h-rIL-2 in the presence of 5 μg/mL anti-2B4, anti-CD48, or anti-CD2 mAbs for 7 days. (A) Incubation of NK cells with anti-CD48 or anti-2B4 mAbs did not inhibit the formation of IL-2–induced NK blasts. LAK cells generated in the presence of anti-CD48 or anti-2B4 mAbs showed slightly larger size in both FSC and SSC as compared with those not treated with mAbs or treated with anti-CD2 mAbs. (B) Reduced LAK-cell expansion in the presence of anti-2B4 or anti-CD48 mAbs, but not with anti-CD2 mAbs. LAK cells excluding trypan blue were counted using hemocytometer. This reduction in NK-cell expansion was not likely due to antibody-mediated toxicity, because there was no significant difference in the frequency of nonviable cells between the control and antibody-treated cells up to 3 days in culture (data not shown). Furthermore, neither isotype controls nor anti-CD2 mAbs prevented IL-2–induced NK-cell expansion. (C) 3H-thymidine incorporation into anti-2B4 and anti-CD48 mAb-treated cells was inhibited as compared with untreated or anti-CD2 mAb-treated LAK cells. Negatively enriched NK cells (15 000) were plated on the 96-well plates and cultured as described above. 3H-thymidine (1 μCi [0.037MBq]) was added at the last 16 hours of incubation. (D) LAK cells were subjected to conventional 51Cr release assay at the effector ratio indicated in each figure. Because the number of NK cells derived from anti-2B4 or anti-CD48 mAb-treated groups was reduced by approximately 50%, the cell numbers in each assay were adjusted such that equivalent numbers of viable LAK cells (CD3–NK1.1+) were contained. Target cells used were CD48–RMA/S. (E) Redirected lysis of NK cells was induced by adding 10 μg/mL anti-NK1.1 mAbs, which activates NK1.1(NKRP1-C) through Fc crosslinking via FcR present on P815 (CD48–) cells. In the absence of FcR crosslinking, LAK cells were not able to lyse P815 targets. Targets (2000) were used in each assay. Data are shown as mean ± SD and are representative of data from 5 independent experiments.
