Stimulation and proliferation of CD4+ peripheral blood T lymphocytes induced by an anti-CD4 monoclonal antibody

There is experimental evidence that the CD4 molecule participates in the antigen-driven activation of T cells expressing this surface glycoprotein. Whether CD4, a member of the immunoglobulin supergene family, acts as a ligand-binding molecule and/or is directly involved in the activation pathway has yet to be established. In this study, we show that human CD4⁺ lymphocytes can be activated by exposure to the anti-CD4 monoclonal antibody (mAb) B66. Normal peripheral blood CD4⁺ cells were induced to proliferate and to synthesize interleukin 2 (IL2) by the antibody. The specificity of the antibody stimulatory activity was tested by using IL2-producing clones bearing either CD4 or CD8 on their surface. IL2 production was induced by mAb B66 in CD4+, but not CD8⁺, clones, whereas both types of clones responded to stimulation by the anti-CD3 mAb Leu-4. Despite its unique stimulatory activity, mAb B66 shared with other anti-CD4 antibodies the ability to inhibit the specific cytolytic activity of CD4⁺ effector cells. These results clearly indicate that cross-linking of surface CD4 molecules with appropriate antibodies can fully activate CD4⁺ lymphocytes. Whether the natural ligand for CD4 can trigger this activation pathway remains to be defined.     

Terminal differentiation surface antigens of myelomonocytic cells are expressed in human promyelocytic leukemia cells (HL60) treated with chemical inducers

The expression of two surface antigens present on the cell membrane of both human granulocytes and monocytes was studied during the process of myelomonocytic differentiation using two monoclonal antibodies (B9.8.1 and B13.4.1). These surface antigens are not present on immature myeloid cells nor on nonmyeloid hematopoietic cells, but can be detected when the cells are terminally differentiated. Among the bone marrow cells, B13.4.1 binds to metamyelocytes and B9.8.1 to metamyelocytes and a fraction (30%) of myelocytes. HL60 human promyelocytic leukemia cells did not react with such monoclonal antibodies. However, when such cells were induced to differentiate in vitro into mature myeloid elements by treatment with retinoic acid or dimethyl sulfoxide, 70%--90% of the differentiated cells expressed both surface antigens. Cell sorting studies on these treated HL60 cells indicated that myelocytes and metamyelocytes were the most immature cells expressing such markers. Expression of the two surface antigens was also observed when HL60 cells were induced to differentiate into monocyte/macrophage cells by treatment with the tumor promoter 12-O- tetradecanoyl-phorbol-13-acetate. Thus, human promyelocytic leukemia cells induced to differentiate in vitro by treatment with specific chemical agents express membrane antigens in the same pattern as normal bone marrow myeloid cells at the corresponding stage of differentiation.     

Induction of differentiation of human myeloid cell lines by tumor necrosis factor in cooperation with 1 alpha,25-dihydroxyvitamin D3

We analyzed the combined effect of tumor necrosis factor and 1 alpha,25-dihydroxyvitamin D3 on the differentiation of human myeloid cell lines HL-60, ML3, and U937. The two compounds synergize in inducing the morphological, phenotypic, enzymatic, and functional characteristics of cells of the monocytic lineage. Immune gamma-interferon synergizes with each compound to induce differentiation. However, recombinant tumor necrosis factor is much more effective than recombinant gamma-interferon in potentiating the effect of 1 alpha,25-dihydroxyvitamin D3 and, alone, is also more effective than recombinant gamma-interferon in inducing expression of the high-affinity Fc receptor on ML3 cells. The possible physiological or pathological relevance of the synergistic effect of tumor necrosis factor and 1 alpha,25-dihydroxyvitamin D3 on monocytic differentiation is discussed.     

Identification and characterization of a pore-forming protein of human peripheral blood natural killer cells

We show here that human peripheral blood NK cells contain a pore-forming protein (PFP) with an Mr of 70,000-72,000 that assembles structural lesions (with an average internal diameter of 150-170 A) and forms functional channels. The PFP was isolated by affinity chromatography from human NK cells, using a specific anti-C9 antiserum as the immunoadsorbent. The NK cells were isolated from PBL by positive or negative selection by indirect rosetting using a panel of monoclonal antibodies directed against different NK and T cell surface antigens. PFP was identified in NK cells freshly isolated and isolated from cultured PBL, both stimulated with interleukin 2, but not in NK cell-depleted lymphocytes. In planar bilayers, the channels formed by the NK cell-derived PFP are highly voltage resistant, with most channels persisting in the open state once they have inserted into the bilayer. The unit conductances of these channels range 0.3-1 nS in 0.1 M NaCl. The channels show poor selectivity for monovalent and divalent ions. The PFP is also released from human NK cells stimulated with the calcium ionophore A23187, suggesting that this protein, like the one produced by murine CTL lines, may be similarly secreted during cell-mediated killing. Its identification in primary human NK cell cultures indicates that this protein may play an active role in NK cell-mediated killing.     

Resistance of cytolytic lymphocytes to perforin-mediated killing. Lack of correlation with complement-associated homologous species restriction

CTL and NK cells resist self-mediated killing and lysis by their own pore-forming protein (PFP; perforin). Perforin, like C, lyses RBC. Efficient C-mediated lysis of RBC occurs when both C and RBC are from different species (homologous species restriction). A protective surface protein (C8-binding protein, homologous restriction factor) has been reported to mediate both homologous species restriction in C-dependent cytolysis and protection of some target cells against perforin-induced lysis. We show here that perforin, unlike C, lyses target cells across a variety of species, including the homologous one, while the same target cell populations resist the attack by homologous C. Perforin-containing extracts of CTL and LAK/NK cells from three species (rat, mouse, and human) and purified mouse perforin were tested against RBC from 10 different species, several nucleated target cell lines, and one primary cell population (thymocytes). While resisting lysis by homologous C, most of these cell types were lysed effectively by perforin without any homologous restriction pattern. CTL and NK cells, like other nucleated targets, are resistant to lysis by homologous but not heterologous C; however, these cell types are resistant to both homologous and heterologous perforin. Together, our results suggest that the protective mechanisms associated with C- and perforin-mediated lysis are distinct.     

Role of immune interferon in the monocytic differentiation of human promyelocytic cell lines induced by leukocyte conditioned medium

Conditioned medium (CM) from lectin-stimulated human leukocytes contains factors that induce human promyelocytic cell lines to differentiate along the monocytic pathway. In this report, we show that human promyelocytic cell lines are also induced to differentiate along this pathway by immune interferon (IFN gamma). Various preparations of IFN alpha tested did not induce this differentiation. In cultures containing IFN gamma, the cells are induced to coordinately express monocyte markers and functions such as monocyte-specific surface antigens, HLA-DR antigens, nonspecific esterase, receptors for the Fc fragment of IgG, and the ability to mediate antibody-dependent cell- mediated cytotoxicity. Our data indicate that differentiation induced by IFN gamma is not secondary to an arrest of growth of promyelocytic cell lines, but rather that a proportion of cells is induced along a programmed pathway of terminal differentiation similar to that of normal monocytes. CM contains IFN gamma, but its ability to induce differentiation is greater than expected on the basis of its content of IFN gamma. Treatments at 56 degrees C or at pH 2.0, which abolish IFN gamma activity, abrogate the differentiation ability of CM. The antiviral activity and the differentiation activity contained in the CM are coeluted from gel filtration and reverse-phase columns. Monoclonal antibodies anti-IFN gamma, which completely abrogate the differentiation ability of IFN gamma and the antiviral activity in the CM, completely suppress the induction of some monocyte markers by CM, but only reduce the expression of others. When IFN gamma is added to CM, promyelocytic cell lines are induced to differentiate to a much greater extent than that induced by either IFN gamma or IFN gamma- depleted CM alone. These results show that the differentiation activity of leukocyte CM is due to the synergistic effect of IFN gamma and other factors not yet identified.     

Monoclonal antibodies specific for differentiation antigens of human myelomonocytic cells

Several monoclonal antibodies specific for human cells of the myelomonocytic lineage at discrete stages of differentiation have been described. In this review I briefly summarize the results obtained with these reagents and discuss their possible use for the study of cell lineage and functional activities of normal peripheral blood leukocytes, for the analysis of myelomonocytic cell differentiation in vivo and in vitro and for the antigenic phenotyping of leukemia cells.     

Effects of recombinant tumor necrosis factor, lymphotoxin, and immune interferon on proliferation and differentiation of enriched hematopoietic precursor cells

We compared the effects of recombinant (r) tumor necrosis factor (TNF) and lymphotoxin (rLT) on in vitro colony formation by erythroid, multipotential and granulocyte, monocyte (CFU-GM) precursor cells. Recombinant granulocyte, macrophage-colony stimulating factor and bone marrow preparations enriched for hematopoietic precursors were used in order to examine the direct effects of the cytokines on precursors in the absence of other soluble factors or mature cells that may influence colony formation. Both rTNF and rLT inhibited colony formation by erythroid and multipotential precursor cells. rTNF and rLT did not inhibit the more mature (day-7) CFU-GM, although a synergistic inhibition was observed when the cytokines were added to cultures together with recombinant interferon gamma (rIFN gamma). However, rTNF did inhibit day-7 CFU-GM when conditioned medium from the human bladder carcinoma cell line 5637 was used as a source of colony-stimulating activity. rTNF inhibited the more immature (day-14) CFU-GM regardless of the source of colony-stimulating activity used, and was a stronger inhibitor of day-14 CFU-GM than rLT, suggesting a difference in the biological effects of the two cytokines. Noninhibitory concentrations of rTNF or rIFN increased the number of monocyte colonies formed, identified by cytochemical staining. These data suggest a mechanism for TNF and IFN gamma-induced inhibition of CFU-GM through induction of monocyte differentiation rather than through a direct toxic effect.