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Lymphoid cells infiltrating metastatic melanomas were grown directly from cell suspensions of tumour tissue by the addition of T cell growth factor. Lymphoid cells grew' out at the expense of tumour cells in six of seven freshly excised tumours. and cells from two cultures were expanded for in vitro testing of cytolytic function against different target cells. Early in culture the tumour derived lymphocytes killed fresh autologous melanoma cells and, particularly later in culture, were highly arid non-specifically cytolytic For cultured melanoma and non-melanoma cells, Cultured peripheral blood lymphocytes from patients with melanoma, and from normal subjects. were cytolytic to the same degree as tumour derived lymphocytes, and also resembled cells grown from tumour tissue in possessing acid phosphatase activity which was resistant to tartrate. Cultured lymphoblasts from both tumour and peripheral blood had a T cell phenotype when analysed with monoclonal antibodies. An in vitro co-culture system was employed to study the kinetics and the precursors of these non-specific killer cells among blood mononuclear cells. Blood mononuclear cells cultured with irradiated B lymphoblasts led to the generation of non-specific cytolytic cells, referred to as activated lymphocyte killer (ALK) cells, after 7 10 days of culture and the progenitors of these ALK cells were demonstrated to be distinct from those ol specific cytolytic T cells. [ABSTRACT FROM AUTHOR]

Mouse-rosette formation was investigated in a variety of leukaemias and lymphomas, and it was found that this phenomenon was restricted to the B lymphocytes of chronic lymphocytic leukaemia and hairy-cell leukaemia. Eighteen of nineteen cases of chronic lymphocytic leukaemia expressed the mouse receptor, but only eight of fifteen cases of hairy-cell leukaemia showed significant mouse rosette formation by peripheral blood cells. A much higher percentage of splenic hairy cells, compared with peripheral blood cells, formed mouse rosettes, but receptor expression was found to be dependent on the surface immunoglobulin (SIg) phenotype. Cases forming mouse rosettes had multiple heavy chain isotypes including IgG on the surface, but the ontogenically more mature SIgG-only cells from other eases failed to form rosettes, whether from peripheral blood or spleen. Cultured hairy cells lost the ability to form mouse rosettes, and receptor expression by peripheral blood hairy cells fluctuated widely in the days following splenectomy. The results suggest that the mouse erythrocyte receptor is an early marker in B cell differentiation and that receptor expression by individual cells may depend on the age of the cell. [ABSTRACT FROM AUTHOR]

A new case of hairy-cell leukaemia (HCL) in which the hairy cells (HCs) formed spontaneous sheep erythrocyte (E) rosettes is described, and this E-rosette formation is shown to be an active process independent of surface immunoglobulin (SIg). Sequential studies with a panel of markers of this and of a case of HCL with T-cell features previously described from this laboratory show that both these cases, at certain times of study, possessed combined T- and B-cell features. Extensive follow-up study revealed wide fluctuations in the surface phenotype of peripheral blood (PB) mononuclear cells, while the total leucocyte count remained stable. At various times, the PB mononuclear cells showed the following phenotypes: E+SIg+yFc+C3-E-SIgyFcC3-, E-SIg-yFC+yFc+C3-, E+SIg-yFc+/-C3- and the significance of these phenotypic changes is discussed. The importance of sequential studies with a panel of markers in the characterization of lymphoproliferative disorders is emphasized. [ABSTRACT FROM AUTHOR]

Immunological surface marker analysis of the mature lymphoid and plasma cells of a range of human lymphoproliferative diseases is used to show that the surface immunoglobulin (sIg) phenotype of plasma cells depends upon the isotype being secreted. Using a sensitive rosette assay, the plasma cells of all IgG (eight), IgA (six) and IgE (one) myelomas studied and of one of two cases of IgD myeloma were negative for all classes of sig. The plasma cells from the other case of IgD myeloma expressed only sIgD, while a proportion of plasma cells from two cases of IgM myeloma and one of BJ myeloma had both sTgM and sTgD at the cell surface. The mature lymphoid and plasma cells from the two patients with mixed Iymphuid/plasmacytoid proliferations ot IgA type (2) carried slgA alone, while the mature bone marrow cells from a case of Waldenstr&ouml'm's macroglobulinaemia had only sIgM. In all the various types of immunoproliferative disease studied, a greater proportion of lymphoid cells than plasma cells possessed γFc receptors, and plasma cells were negative for receptors for EAC and μFc, and lacked the la-like p29, 34 antigen. These results are used to provide a scheme illustrating the terminal stages of B cell development. Examination of the peripheral blood from nineteen cases without morphological evidence of blood involvement demonstrated a monoclonal B cell proliferation in 40% of these cases. Immunological analysis of lymphocyte subpopulations showed that both E-rosette-forming cells and sIgM+ and slgD+ normal B cells were depressed in most cases and that there was a corresponding increase in Fc+slg- and/or null (Fc-slg-) cells. [ABSTRACT FROM AUTHOR]

It has been demonstrated that in two patients with T-cell Sézary syndrome the pathological cells express a receptor for EA(IgM) when cultured in a medium with foetal calf or autologous serum. A study of the time-course of this rosette formation revealed that a large proportion, but not all, of E-rosetting cells progressively expressed this receptor. In agreement with previous reports by others on the normal T-cell population bearing the IgM receptor, incubation at 37 °C in the presence of serum was shown to be a necessary condition. The implications of this pathological subpopulation of T-cells are discussed. [ABSTRACT FROM AUTHOR]

Immunological marker studies using direct immunofluorescence and rosette methods were performed on the hone marrow and peripheral blood of an IgM myeloma, and on the peripheral blood of two cases of IgA myeloma and one IgG myeloma. These studies confirmed the peripheral blood involvement in plasma cell dyscrasias. In the IgA myeloma, the membrane and cytoplasmic immunoglobulin was restricted to IgA, and the IgG myeloma did not express immunoglobulin at the surface of the cells shown to contain cytoplasmic IgG. The IgM myeloma cells, on the other hand, expressed membrane immunoglobulin, including IgD, of a single light chain type. The majority of plasma cells secreting IgA or IgG did not express the IgG Fc receptor, but most of the cells from the IgM myeloma, including plasma cells, did express IgG Fc receptors. [ABSTRACT FROM AUTHOR]

By the use of a rosette test employing yeast particles coated with C3 activated by the alternative pathway, we have demonstrated that the hairy cells of leukaemic reticuloendotheliosis (LRE) do not possess a receptor for C3. It is also shown that hairy cells display a consistently lower percentage of rosette formation with OXEAC than with ox EA IgM and this is attributed to varying degrees of steric hindrance of IgM or incomplete coating by complement. Blocking with monomeric IgM confirmed this interpretation. These results are used to reconcile previously conflicting reports concerning the presence or absence of a receptor for C3 on the hairy cells of LRE, anti to suggest that the disease is accompanied by a severe reduction of normal B lymphocytes. [ABSTRACT FROM AUTHOR]

Human thymocytes differed from peripheral blood I cells, in that they did not proliferate in response to mouse monoclonal antibodies specific for the T3 antigen complex (UCHT1 and OKT3), even in the presence of 1-cell growth factor (IL-2) or with monocytes plus IL-2. The failure to respond was not the result of inhibition by cortical thymocytes, since OKT6-negative cells also did not respond when tested under conditions of limiting dilution. Of more importance, these antibodies rendered human thymocytes unresponsive to the lectin phytohaemagglutinin when added before, during, or immediately after addition of the lectin, an effect which was much more profound than the decrease in mitogenesis caused with blood mononuclear cells. These results illustrate a clear difference between medullary thymocytes and peripheral T cells in their ability to respond to signals transduced through the 13 antigen complex. [ABSTRACT FROM AUTHOR]

The presence of a receptor for the Fc of IgM (μFcR) was demonstrated on the pathological B cells of all of sixteen patients with hairy-cell leukaemia and most, but not all, of twenty-four cases of chronic lymphocytic leukaemia, by a rosette method employing ox erythrocytes sensitized with purified IgM (EAM). This μFcR was also demonstrated on a small population of normal human mononuclear cells from peripheral blood. Pathological B cells with this receptor (BM) simultaneously expressed a different and distinct receptor for the Fc of IgG, and were detectable without pre-incubation in medium containing foetal calf serum (FCS). The μFcR on B cells was blocked by Fc5μ and IgM, but not by F(ab')2μ fragments, or by IgG, whether monomeric or aggregated. Monomeric IgM and IgM bound to its antigen blocked much more effectively than pentameric IgM. B cells also possessed surface immunoglobulin and the Ia-like P29,34 antigen, and an antiserum to this antigen blocked the μFcR. The μFcR on B cells differs in a number of ways from the μFcR reported on T cells, and these differential characteristics are discussed in some detail. The μFcR was rapidly shed and resynthesized when washed BM cells were maintained in medium not containing FCS and the general importance of this phenomenon in any study of μFcR is considered. It is suggested that BM cells are memory cells and that the μFcR plays a part in the immune response. [ABSTRACT FROM AUTHOR]

Mixed lymphocyte cultures were set up between blood mononuclear cells and irradiated autologous or allogeneic B lymphoblasts infected with Epstein Barr virus. The resulting cytotoxic effector cells were cloned and tested for activity against the stimulating B lymphoblast, K562 and melanoma targets. Specific clones which killed only the stimulating B lymphoblasts (cytotoxic T lymphocytes: CTL) were re-cloned and the subclones tested for cytolysis of B lymphoblasts and melanoma cells. Of seven primary CTL clones generated in allogeneic culture, 308 subclones developed the ability to kill melanoma cells and none retained specific CTL function. In the autologous system, 180 subclones were derived from three specific primary clones: of these. 13 (7%) retained specific function 29(16%) were able to kill both B lymphoblasts and melanoma cells, and 93 (52%) killed only the melanoma target. Testing of random clones demonstrated that whereas both B lymphoblast killing (CTL function) and melanoma cell killing (anomalous killer; AK function) were blocked by a monoclonal antibody to LFA-1, only CTL function was blocked by anti-T3 or anti-T8 antibodies. The factor(s) causing the progression of CTL to AK cells are discussed. These data thus demonstrate that the majority of CTL are capable of mediating AK cell function and are thus potentially suitable for passive immunotherapy. [ABSTRACT FROM AUTHOR]

The LIM 1863 colon carcinoma cell line grows in suspension as morphologically and functionally organized organoids in serum-containing medium. Addition of TGF-β caused the organoids to adhere and inhibited DNA synthesis. A 20 min incubation with TGF-β was sufficient to induce adherence and this could be inhibited by cycloheximide. The adhesion and DNA synthesis inhibition were demonstrated to be separate events. We were not able to detect any changes in matrix or cell membrane antigens. Similarly there were no changes in synthesized proteins (by two-dimensional gel electrophoresis), and no upregulation of proteoglycan. When adhered organoids were lysed from the tissue culture plastic surface, untreated organoids adhered to this surface. This 'conditioned' surface was destroyed by trypsin but not collagenase or medium from normal LIM 1863 cultures. However, the adherent phenotype was prevented when organoids were treated with transforming growth factor-β (TGF-β) in the presence of medium conditioned by normal LIM 1863 cultures rather than in fresh medium. The adhesion process was inhibited by an antibody (QE2E5) against β1 integrin although no quantitative changes in integrins were observed (by immunoprecipitation or RNA analysis). A second anti-β1 integrin antibody (61.2C4) inhibited LIM 1863 adhesion to collagen but not TGF-β induced adhesion, implying that TGF-β induced a specific conformational change or interaction of a β1 integrin, In this morphologically structured system TGF-β induced a number of subtle effects including formation of new extracellular matrix and conformational change of a β1 integrin, rather than the major quantitative changes in cell/matrix molecules reported previously. [ABSTRACT FROM AUTHOR]

Blood from patients with hairy cell leukaemia (HCL) was used to investigate subpopulations of the non-specific activated lymphocyte killer (ALK) cells generated in mixed lymphocyte culture (MLC). Mononuclear cells from four of live patients with HCL had decreased natural killer (NK) cell activity and., of these, all showed a decrease in ALK cell activity against K-562 target cells (NK like cells), but three had normal levels of ALK cells able to kill melanoma cells. One patient who had received splenectomy had normal NK cell activity, and in MLC his cells generated normal levels of both subpopulations of ALK cells. The results support the hypothesis of separate subsets of ALK cells: NK like cells with NK cell precursors and which preferentially lyse the K-562 target cell and a second separate population which arises independent of NK cell precursors and which has strong cytolytic activity against an NK insensitive melanoma cell line. [ABSTRACT FROM AUTHOR]

A sensitive reverse haemolytic plaque assay was used on freshly isolated blood cells from normal human subjects to show that T lymphocytes and monocytes were both necessary for immunoglobulin production by unstimulated B cells cultured only for the time necessary to form plaques. When lymphocyte preparations were fully depleted of T cells by E-rosette formation overnight on ice followed by Ficoll-Hypaque centrifugation, the number of plaque-forming cells was reduced by up to 94% this reduction was reversed by the replacement of T cells, although excess T cells suppressed plaque formation. Moreover, when T-cell function was blocked by 10–1000 ng of two monoclonal anti-T-cell antibodies, OKT3 or UCHT1. this significantly reduced or abolished spontaneous IgG plaque, and higher concentrations of either OKT3 or UCHT1 reduced the numbers of IgA and IgM plaques formed by B cells. The role of monocytes in spontaneous plaque formation was investigated. The removal of plastic-adherent cells from mononuclear cell preparations did not consistently result in a reduction in the numbers of plaques, but complement-mediated lysis of monocytes with either of two monoclonal antibodies with specificity for rnonocytes, OKM1 and FMC1T, reduced by 50% the number of IgG, IgA and IgM plaques. This effect was reversed by addition of as few as 1% plastic-adherent cells. Decreased plaque formation by B cells, resulting from either blocking of T-cell function with monoclonal antibody or complement-mediated lysis of monocytes, or both, was fully reversed by soluble factors present in cell-free conditioned medium from lectin-activated T cells. Thus spontaneous plaque formation by human peripheral blood B cells requires T cells and a small number of monocytes, and the major function of these cells is to help B cells by the production of soluble factors. [ABSTRACT FROM AUTHOR]

Acid phosphatase and esterase cytochemistry performed on purified normal human T-cell populations showed that both methods produced distinctive localized dot patterns of reactivity in 60–70% of cells. By examination of rosette preparations formed with ox erythrocytes coated with IgM (EAM), with IgG (EAG), or anti-human κ and λ light chains, it was shown that this pattern of reactivity was largely restricted to small T lymphocytes possessing receptors for the Fc of IgM (Tμ cells). In addition, both B lymphocytes and T cells with receptors for the Fc of IgG (Tγ cells) were larger lymphocytes with more abundant cytoplasm and usually displayed scattered granular acid phosphatase activity; in esterase preparations both cell types were either negative or possessed similar scattered granular positivity. As compared with Tμ cells, Tγ cells were seen to form loose spontaneous rosettes with sheep erythrocytes. Combined esterase and acid phosphatase staining showed that both enzyme activities in the Tμ cells are localized in the same area, and ultrastructural acid phosphatase cytochemistry established that this was in distinctive lysosomal structures. Tμ staining by both esterase and acid phosphatase cytochemistry was greatly reduced after rosetting with EAG, but not after rosette formation with EAM or sheep erythrocytes. [ABSTRACT FROM AUTHOR]

The level of expression of surface antigens was studied on neutrophils from paired samples of blood and synovial fluid of patients with rheumatoid arthritis (RA). By measuring the immunofluorescence of labelled monoclonal antibodies it was shown that on joint neutrophils there was an increase in expression of complement receptor 3 (CR3) and granulocyte functional antigens 1 and 2 compared with blood neutrophils, whereas the expression of GpIIb-IIIa was reduced. The pattern of expression was the same as that seen with in vitro activation and strongly suggest that activation is occurring within the joint. [ABSTRACT FROM PUBLISHER]