Human blood lymphocyte subsets separated on the basis of nylon adherence, SRBC and EA rosetting: Natural cytotoxicity and characterization with monoclonal reagents (original) (raw)
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Journal of Immunological Methods, 1983
Lymphocyte subsets from human blood obtained by different procedures were analyzed for cytotoxic potential and phenotypic characteristics. Nylon wool column passed lymphocytes were fractionated on the basis of: (1) E and Fcy receptor expression, (2) cell density and Fc'y receptor expression, (3) Fc3, receptor expression. The cytotoxic subsets obtained by separation on the basis of E and EA rosetting differed in their phenotypic composition from those separated on the basis of density or on immune complex monolayers. The E-Fcy-population contained few LGL and OKM1 positive cells. The E-Fc-r ,+ population was made up almost entirely of LGL and OKMI positive cells. The low density population was highly enriched in LGLs; among these the Fcy-cells were OKT3 positive. In contrast to the E-population the low dense Fcy + cells were mainly LGLs and were OKMI positive. Fc3, + subsets had less killer activity against Daudi cells.
Scandinavian Journal of Immunology, 1976
In this article we present methods for the purification and fractionation of human blood lymphocytes, which have been used in our laboratory to characterize antibody-dependent cytotoxic effector cells (K cells). The assay system consists of highly purified lymphocytes, 51Cr-labelled chicken erythrocytes (Ec) and IgG rabbit anti-Ec in high dilutions. Various ways of comparing K-cell potentials of different lymphocyte preparations in this system are discussed. When purified lymphocytes are partially depleted (60-85% depletion) of cells forming rosettes with sheep erythrocytes (E+ cells), the K-cell activity of the depleted fraction is increased, indicating the the majority of the E+ cells are inactive in this assay. Depletion of EAC-rosette-forming cells shows that most or all K cells have complement receptors. For depletion of B cells, the lymphocytes may be passed through glass bead columns, charg ed with F(ab')2 fragments of human IgG and F(ab')2 fragments of rabbit antibodies to the F(ab')2 part of human IgG. These columns give high yields of B-cell depleted fractions. These preparations are rich in E+ cells and contain approximately 80% of the Fc-receptor lymphocytes which form rosettes with bovine erythrocytes, coated with IgG antibodies. Their K-cell activity is unchanged or slightly elevated, indicating the mature B cells, i.e. SIg+ cells, have little or no K-cell activity. In contrast, passage of the lymphocytes through immune complex columns (ovalbumin/anti-ovalbumin) leads to approximately 70% depletion of Fc receptor-bearing cells, while most of the B cells (SIg+ cells) pass through the columns. The relative frequency of E+ cells in the passed fraction frequently shows a slight reduction. These preaparations have a very low K-cell activity, indicating that K cells are lymphocytes with Fc receptors of relatively strong avidity.
Natural killer activity of human blood lymphocytes
Molecular Immunology, 1982
Natural killer (NK) activity is an operational designation. It implies the iit vitro cytotoxicities registered in short-term tests exerted by lymphocytes derived from donors with no known immunization history against the particular target. The strength of the effect exerted by unmanipulated blood lymphocytes shows an individual variation. Short-term in ritro treatment with interferon elevates the lytic potential of lymphocytes. Owing to the heterogeneity of the cytotoxic blood lymphocytes with regard of cell surface properties it is not possible to separate all active cells and all inactive cells in clean populations. A considerable enrichment of active cells can be achieved if nylon wool non-adherent, large, granular Fc gamma receptor positive SRBC receptor negative-or low-avidity SRBC receptor positive-OKMI-reactive cells are separated. Negative cells are concentrated in the Fc receptor and OKMI-negative high-avidity SRBC receptor positive high cell density subset. The activity of lymphocytes in the former category is potentiated by interferon and the latter acquire the lytic function if PHA is added to the assay system. Freshly separated, non-cultured tumor cells are not or weaklv sensitive to the effect of unmanipulated lymphocytes. However, when the lymphocytes are treated with interferon prior to the assay a lytic potential can be induced even against these in allogeneic effector target combinations. Cytotoxic cells which acquired the function after in I;ico and,'or in tritro immunization are designated as 'cytotoxic T-lymphocytes' (CTL), and were shown to act on the basis of antigen recognition. The expression of known T-markers on at least a fraction of the active cells and the recognition of alloantigens in NK systems suggest that the distinction between CTL and NK cells is not as sharp as initially suggested.
Comparison of NK Activity in Mouse Spleen and Peripheral Blood Lymphocytes
Immunobiology, 1988
Natural killer (NK) cells originating in mouse peripheral blood were studied with regard to their lytic activity against YAC-1 target cells and to their expression of asialo-GM1 marker on their surface. In Balb/c, CBA/LAK and A/J mice, PBL were found to be approximately twice as effective as splenocytes. Splenic and peripheral NK cells were shown by flow cytometry to have similar lytic potential per cell; the difference in NK activity found in the spleen and in PBL was solely due to the differences in the size of the NK cell population found in the two sites. Strain distribution of NK activity in PBL followed the same pattern observed in splenocytes. The difference in NK activity between CBA and Balb/c mice was shown to be due to the fact that the lytic potential per NK cell was approximately twice as high in the former.
Cellular Immunology, 1983
Recent investigations have indicated that the OKM I hybtidoma monoclonal antibody reactive with cells of the myelomonocytic series identifies a subpopulation of human peripheral blood mononuclear cells (PBMNC) which mediate natural and antibody-dependent cellular cytotoxicity (ADCC). However, it was not clear whether this OKMl+ group was heterogeneous with regard to cytotoxic function or the presence of receptors for sheep erythrocytes. Thus, the purpose of the present study was to further define the phenotype of the ADCC effector cell and natural killer (NK) cell using a combination of reactivity with hybridoma antibodies and separation of subsets by sheep erythrocyte rosette (E+) formation. Furthermore, the phenotypes of the NK population were assessed directly by performing two-color immunofluorescent staining on tumor cell conjugates. These studies led to the following conclusions: (1) that NK activity is mediated by both E+ OKMI+ and E-OKMl+ cells; the E+ OKT3+ cell posses& essentially no ADCC or NK activity; (2) that E+ OKMl+ cells mediated more NK activity on a per cell basis than E-OKM I+ cells; this was verified by separating OKMl+ cells on a cell sorter into E+ and Ewith the OKTll monoclonal antibody (anti-E-receptor antibody); (3) that E+ OKMl+ cells mediated both ADCC and NK activity; (4) that the phenotypes of PBMNC forming tumor cell conjugates were (a) OKM 1 + (both E-receptor positive and negative) and (b) OKM 1-E-receptor positive.
Scandinavian Journal of Immunology, 1974
Normal human peripheral blood lymphocytes were sensitized to autologous or allogeneic lymphoblastoid cells in vitro. Purified T lymphocytes were found to be able to respond both by performing DNA synthesis and by functioning as killer cells. Surface marker analysis of blast-transformed lymphocytes in tbe in vitro cultures showed a bigh proportion (around 50%) of blasts lacking any surface marker attributable to B or T blasts; sucb 'null' blasts have previously not been found in conventional mixed leukocyte culture or after phytobemagglutinin or concanavalin A activation. Since the 'null' blasts could be shown to be produced in a higb percentage from originally almost 'pure' sheep erythrocyte (SRBC)binding lymphocytes and displayed a similar killing capacity per unit cell number as SRBC-binding lymphoblasts, we consider the 'null' blast to be of T origin.