SummaryHuman lymphocytes with natural killer (NK) activity, including most activated 3'/8 + T lymphocytes, recognize and lyse tumor target cells without requiring recognition of major histocompatibility complex antigen. However, unlike 3'/8 + T lymphocytes, NK cells do not express CD3/T cell receptor (TCR) molecules, and the receptors involved in ceU-mediated cytotoxicity are unknown. To further delineate circulating NK cells, we developed monoclonal antibodies (mAbs) against the human NK leukemia YT2C2. We report the isolation of a mAb termed BY55, recognizing at the cell surface a novel 80-kD protein with restricted expression, In addition to the immunizing cell line, this mAb binds to circulating NK cells, 3'/8* cells, and a minor subset of ot/B + T lymphocytes. Expression of the BY55 mAb-reactive epitope/ molecule is regulated by activation, as short-term culture of peripheral blood lymphocytes (PBL) with phorbol ester induced its downmodulation. Furthermore, BY55 mAb reactivity was found neither with the NK nor with the TCR a/B + and 3"/8 + clones tested. Biochemical studies as well as phenotypic analysis revealed that this structure is different from all previously identified molecules on the lymphocyte cell surface. Interestingly, we found that BY55 + cells exert most NK activity obtained with fresh circulating lymphocytes. We report that within fresh E rosette-positive PBL only a subset of the CD16 +, CD56 +, and CD57 + cells coexpressed BY55 molecule, indicating that BY55 mAb defines a unique subset mediating NK activity of circulating PBL.N 'K cells are circulating lymphocytes found within the large granular lymphocyte population (1, 2) and constitute, with CTL (3), the major cytotoxic effector lymphoid elements of the immune system. The characteristic surface markers expressed by NK cells are CD56 (4-6), CD57 (4, 5), CD16 (7-10), CD11b (11), and CDllc (11, 12). Most circulating cells with NK activity express the CD2 molecule but are distinct from the T lineage, as they do not express CD3 and do not rearrange any of the TCR genes (13, 14). In addition, NK cells mediate cytotoxicity without requiring recognition of MHC molecules on target cells. However, most activated TCR 3'/8 lymphocytes (15-18), and under certain circumstances some TCR o~/B cloned lymphocytes (19), can also mediate MHC-unrestricted cytotoxicity. The NK cell receptor(s) participating in target recognition remain(s) largely unknown despite extensive studies (20)(21)(22).In an attempt to define NK cell-specific surface structures, we characterized a mAb obtained by repeated immunization with YT2C2, a human cell line with NK functional characteristics. This cell line was selected for intermediate affinity binding of 12sI-IL-2 with the IL-2R p75 subunit (23). In the present study, we report the isolation ofa mAb, termed BY55, reacting at the cell surface with a novel 80-kD protein structure. BY55 mAb reactivity is observed with 15-25% of circulating lymphocytes. Two-color immunofluorescence staining of fresh E rosette-positive (E +) PBL o...

We had previously reported, using BY55 monoclonal antibody, a cell surface 80-kDa protein restricted to human functional peripheral blood cytotoxic lymphocytes with either natural killer CD3-or cytotoxic T lymphocyte CD3+CD8+ phenotype. In the present report, we studied the cytotoxic lymphocytes in adult bone marrow and newborn cord blood as these organs are commonly used as sources of hematological stem cells for allogeneic transplantation. Our results showed that BYS mAb labeled only 5-10% of the bone marrow lymphocytes, which included a major proportion of CD3+CD8+ cytotoxic T lymphocytes. Interestingly, within cord blood cells, BY55+ lymphocytes represented 20-35% of the lymphocytes corresponding exclusively to a CD3-cell subset. Furthermore, we detected in cord blood no cytotoxic T lymphocyte activity but we demonstrated that the CD3-BY55+ cell subset contained the whole natural killer activity.Natural killer (NK) cells are circulating lymphocytes found within the large granular lymphocyte population (1, 2). The characteristic surface markers expressed by circulating NK cells are CD56 (3, 4), CD16 (5-7), CD11b (8, 9), and CD122 (interleukin 2 receptor p75) (10, 11). To further delineate circulating cells with cytotoxic activity, we developed a monoclonal antibody (mAb) obtained by repeated immunization with YT2C2, a human cell line with NK functional characteristics (12). This mAb, termed BY55, reacts with a 80-kDa protein structure expressed exclusively by circulating cytotoxic lymphocytes (13,14). In normal human peripheral blood, cytotoxic lymphocytes labeled with BY55 mAb corresponded to 20-25% of cells, including mostly CD3 negative lymphocytes, the majority of T-cell receptor (TCR) yS positive T lymphocytes, and a minor subset of CD8+afB+ with cytotoxic T-lymphocyte (CTL) activity.Bone marrow transplantation for hematopoietic reconstitution is the unique treatment for some inherited diseases or for various bone marrow malignancies. However, graftversus-host disease (GVHD), which can be lethal and involves cytotoxic lymphocytes from the donor, is the major limitation to the frequent usage ofthis therapeutic procedure. We had recently reported that umbilical cord blood appeared to be a source of transplantable stem cells without causing severe GVHD (15, 16). Therefore, it was important to analyze the cytotoxic lymphocyte subsets within bone marrow and cord blood. Furthermore, conflicting results were reported concerning the cytotoxic function of the lymphocytes isolated from cord blood (17)(18)(19). In the present study, we used BY55 mAb to delineate the cytotoxic lymphocyte population within newborn cord blood and adult bone mar- Sorted populations were collected as described (20), washed twice, and tested as effector cells in cytotoxic assays. Trypan blue exclusion was performed on the unsorted population and sorted populations, and the viability was always >90%.mAb. BY55 mAb was obtained as described elsewhere (13). Other mAb such as CD3, CD4, CD8, CD11b, and TCRa,3 were produced locally. The...