Regulation of T-cell function by antibodies: enhancement of the response of human T-cell clones to hepatitis B surface antigen by antigen-specific monoclonal antibodies.

Eight mouse monoclonal antibodies specific for hepatitis B surface antigen (HBsAg) were examined for their effects on the antigen-induced proliferative response and lymphokine production of human HBsAg-specific T-cell clones in vitro. While all specifically enhanced the T-cell proliferative response, antibodies of the IgG class were generally more effective than those of the IgM class. Both the divalent F(ab')2 and the monovalent Fab fragments of an IgG monoclonal antibody had no effects, indicating that the Fc portion of the antibody molecules was required. Since antigen-presenting cells bear surface receptors for the Fc of IgGs and fewer or none for that of IgMs, the above results also suggest that antibodies enhance the capture of antigens by antigen-presenting cells as a result of the binding of antigen-antibody complexes to the Fc receptors on these cells. In addition to potentiating the proliferation of the T-cell clones, antibodies also increased the antigen-induced production of interferon-gamma by these cells. The present in vitro studies suggest that antibodies may regulate immune responses and do so by enhancing antigen presentation and thus augmenting antigen-induced activation and clonal expansion of T cells.     

Characterization of monoclonal antibodies to human monocytes

A set of monoclonal antibodies reacting with human peripheral blood monocytes is characterized. Two of these antibodies also bind to a fraction of granulocytes (BL-M/G-1) respectively to all of them (BL-M/G-2). On the other hand the BL-M-3 appears to be specific for monocytes only. All three monoclonals do not react with enriched B and T lymphocytes, platelets, erythrocytes and several lymphoid or erythroid permanent cell lines. The antigens recognized by all three monoclonals are expressed by monoblastic cell line U-937, whereas the myeloid line HL-60 and the cell line KG-1 express only antigen recognized by monoclonal antibody BL-M/G-2.     

Staining of Langerhans cells with monoclonal antibodies to macrophages and lymphoid cells.

Langerhans cells are Ia-bearing antigen-presenting cells in the epidermis that share many functions with macrophages. We have used monoclonal antibodies to the macrophage antigens, Mac-2 and-3, Ia antigen, Fc fragment receptor and the common leukocyte antigen CLA to compare the cell surface antigens of these cells with those of interdigitating and follicular dendritic cells and of macrophages in lymphoid tissues. Immunoperoxidase staining was carried out with epidermal sheets from BALB/c mice and epidermal cell suspensions enriched for Langerhans cells by Fc rosetting. Langerhans cells stained for all of these antigens. Comparison with the staining properties of other dendritic cells and macrophages, in combination with previous observations, indicates a close relationship of Langerhans cells to the interdigitating cells of lymphoid tissues.     

Monoclonal antibody to human cytotoxic-suppressor T-lymphocytes cross-reacts with canine lymphocytes and inhibits cell-mediated lympholysis of canine cells

Of 19 murine antihuman Tp32 (T8) monoclonal antibodies tested, only antibody T811 showed cross-reactivity with canine lymphoid cells. It recognized an antigen expressed on 19%-27% of peripheral blood mononuclear cells (PBMC), 20%-30% of peripheral Thy 1+ T-lymphocytes, 40% of puppy thymocytes, 35%-45% of alloantigen-activated peripheral lymphocytes, and 100% of PBMC from a dog with acute T-cell leukemia. The antigen was not expressed on peripheral Thy 1- cells, bronchoalveolar cells, or null-type acute leukemia cells. The antibody inhibited cell-mediated lympholysis in the absence of complement, while antibody F3-20-7 directed at the canine Thy 1 antigen did not. It was not possible to precipitate a corresponding cell surface antigen on canine cells using standard 125I radioimmunoprecipitation techniques nor did the antibody cause antigenic modulation. Our data suggest that monoclonal antibody T811 reacts with a functional subset of canine T-lymphocytes similar to that of human cells; however, significant differences may exist between the antigens recognized on these cells in the two species.     

Characterization of B-cell type chronic lymphocytic leukemia cells by surface markers and a monoclonal antibody

This study was carried out to determine the reactivity of the Leu-1 mouse monoclonal antibody with B-chronic lymphocytic leukemia and other B-cell leukemias. This antibody has been previously reported to recognize a surface antigen expressed by almost all human thymocytes and peripheral T cells. It was also detected on surface immunoglobulin-bearing cells of most patients with chronic lymphocytic leukemia but was not detectable in normal B-cells and B-cell lines. In the present series, the neoplastic lymphocytes in 33 cases of B-chronlc lymphocytic leukemia expressed surface immunoglobulin, la antigen, and receptors for Fc, C3, and mouse erythrocytes. All but one case expressed the Leu-1 antigen as detected by indirect immunofluorescence using flow cytometry. In three other cases, the leukemic cells in the peripheral blood reacted with anti-Leu-1, whereas the bone marrow lymphocytes did not. Moreover, quantitative differences in the surface density of Leu-1 were apparent by flow cytometry. The peripheral blood and bone marrow lymphocytes in other B-cell leukemias, including 15 cases of leukemic B-cell lymphomas and three cases of hairy-cell leukemia, failed to stain positively with the anti-Leu-1 antibody. The recognition of the Leu-1 antigen adds to the phenotypic characterization of B-chronic lymphocytic leukemia and may contribute to a better understanding of the pathogenesis of the disease and its expression in different tissues.     

Immunologic classification of leukemia and lymphoma

Important insights into leukocyte differentiation and the cellular origins of leukemia and lymphoma have been gained through the use of monoclonal antibodies that define cell surface antigens and molecular probes that identify immunoglobulin and T cell receptor genes. Results of these studies have been combined with markers such as surface membrane and cytoplasmic immunoglobulin on B lymphocytes, sheep erythrocyte receptors on T lymphocytes, and cytochemical stains. Using all of the above markers, it is now clear that acute lymphoblastic leukemia (ALL) is heterogeneous. Furthermore, monoclonal antibodies that identify B cells, such as the anti-B1 and anti-B4 antibodies in combination with studies of immunoglobulin gene rearrangement, have demonstrated that virtually all cases of non-T-ALL are malignancies of B cell origin. At least six distinct subgroups of non-T-ALL can now be identified. T-ALL is subdivided by the anti-Leu-9, anti-Leu-1, and antibodies that separate T lymphocyte subsets into three primary subgroups. Monoclonal antibodies are also useful in the subclassification of non-Hodgkin's lymphoma, and certain distinct markers can be correlated with morphologic classification. The cellular origin of the malignant Reed-Sternberg cell in Hodgkin's disease remains uncertain. A substantial number of investigators favor a myelocyte/macrophage origin based on cytochemical staining; however, consistent reactivity with antimonocyte reagents has not been demonstrated. Although monoclonal antibodies are useful in distinguishing acute myeloid from acute lymphoid leukemias, they have less certain utility in the subclassification of acute myelogenous leukemia (AML). Attempts to subclassify AML by differentiation-associated antigens rather than by the French-American-British (FAB) classification are underway in order to document the potential prognostic utility of surface markers. Therapeutic trials using monoclonal antibodies in leukemia and lymphoma have been reported. Intravenous (IV) infusion of unlabeled antibodies is the most widely used method; transient responses have been demonstrated. Antibodies conjugated to radionuclides have been quite successful in localizing tumors of less than 1 cm in some studies. Therapy trials with antibodies conjugated to isotopes, toxins, and drugs are currently planned. Purging of autologous bone marrow with monoclonal antibodies and complement in vitro has been used in ALL and non-Hodgkin's lymphoma; preliminary data suggest that this approach may be an effective therapy and may circumvent many of the obstacles and toxicities associated with in vivo monoclonal antibody infusion.     

Immunologic classification of lymphoma and lymphoid leukemia

Important insights into lymphocyte differentiation and the cellular origins of lymphoma and lymphoid leukemia have been gained through the use of monoclonal antibodies that define cell surface antigens and molecular probes that identify immunoglobulin and T cell receptor genes. Results of these studies have been combined with markers such as surface membrane and cytoplasmic immunoglobulin on B lymphocytes, sheep erythrocyte receptors on T lymphocytes, and cytochemical stains. Utilising all of the above markers, it is now clear that acute lymphoblastic leukemia (ALL) is heterogeneous. Furthermore, monoclonal antibodies that identify B cells such as the anti-B1 and anti-B4 antibodies in combination with studies of immunoglobulin gene rearrangement have demonstrated that virtually all cases of non-T-ALL involve B lymphocytes. At least six distinct subgroups of non-T-ALL can now be identified. T-ALL is subdivided by the anti-Leu-9, anti-Leu-1, and additional antibodies that separate T lymphocyte subsets into three primary subgroups. Monoclonal antibodies are also useful in the subclassification of non-Hodgkin's lymphoma, and certain distinct markers can be correlated with morphologic classification.     

Expression of two natural killer cell antigens, H-25 and H-366, by human immature myeloid cells and by erythroid and granulocytic/monocytic colony-forming units

Two monoclonal antibodies (MoAbs), H-25 and H-366, shown previously to react with human peripheral blood large granular lymphocytes with natural killer (NK) cell activity and some peripheral blood monocytes, have now been shown to also react with a significant proportion of the myeloid and erythroid precursor cells in human bone marrow and peripheral blood. In FACS IV cell sorting and immune rosetting of bone marrow cells, the antigens recognized by H-25 and H-366 were found to be expressed on most blasts and promyelocytes but sequentially fewer of the more mature cells of the myeloid lineage. Both antigens were also found on most monocytes but only a minor proportion of lymphoid and nucleated red cells in the bone marrow. In vitro assays detecting hematopoietic colony-forming units revealed that these antigens are expressed by virtually all mature erythroid colony-forming units (day-7 CFU-E), and the majority of the more primitive erythroid burst forming units (day-14 BFU-E). H-25 but not H-366 was also found on a variable proportion of the day-7 and day-14 granulocytic/monocytic colony- forming units (CFU-GM) in the bone marrow. The same type of precursor cells are also found in the H-25 and H-366 positive cell populations isolated from peripheral blood. In preliminary testing of cells from acute leukemic patients, FACS analysis showed that both antigens are also expressed on leukemic cells from patients with T cell acute lymphocytic leukemia and with myeloid leukemias. These studies demonstrate that the H-25 and H-366 positive NK cells in the peripheral blood retain some of the cell surface properties of early hematopoietic precursor cells, thus providing further evidence supporting the bone marrow origin of NK cells.     

Heterogeneity in a lymphoid tumor: coexpression of T and B surface markers

Heterogeneity of leukemic cells was defined in a case of lymphoma. Four phenotypically distinct subpopulations of leukemic cells were identified. One subpopulation was observed to simultaneously express B- and T-cell characteristics. B-cell characteristics included monoclonal IgM (lambda) surface immunoglobulin, HLA-DR antigens, and expression of the B-cell antigen identified by the BA-1 monoclonal antibody. T-cell characteristics included E-rosette formation, expression of the pan-T- associated antigens recognized by the Leu-1 and OKT-11 monoclonal antibodies, and expression of the suppressor cytotoxic T-cell- associated antigen recognized by the Leu-2 and OKT-8 monoclonal antibodies. In addition to this subpopulation, three other phenotypically distinct subpopulations were identified, two of which expressed monoclonal IgM (lambda) surface immunoglobulin. The results of this investigation indicates that three phenotypically distinct lymphoid subpopulations bearing B-cell characteristics, and probably a fourth T-cell subgroup, were derived from a common lineage. These findings suggest that the malignancy involved a lymphoid progenitor cell that may possess diverse maturational capacity.     

Changes of adult T cell leukemia cell surface antigens at relapse or at exacerbation phase after chemotherapy defined by use of monoclonal antibodies

Surface phenotypes of leukemic cells from six patients with adult T cell leukemia (ATL) were analyzed by the use of monoclonal antibodies, both at the time of initial diagnosis and at either relapse or exacerbation phase after chemotherapy. Changes of cell surface antigens were observed in four of the six cases. The majority of the leukemic cells of these patients were reactive with anti-Leu-1 and anti-Leu-3a, but unreactive with anti-Leu-2a and MAS 036c monoclonal antibodies at the time of initial diagnosis, indicating that ATL cells are of peripheral inducer/helper T cell origin. In three cases, the Leu-1 antigen disappeared at relapse or at exacerbation phase, and in one of these cases, a small percentage of ATL cells became reactive with MAS 036c, which identifies cortical thymocyte antigen. ATL cells of one other case did not have Leu-1 antigen from the start, but gained Leu-2a antigen at exacerbation phase and became double-labeled cells (Leu-2a+, 3a+), which is known to be a feature of thymocytes. Thus, it appeared that ATL cells sometimes change their surface phenotype to that of an earlier stage of T cell differentiation at relapse or at exacerbation phase. Chronic myelocytic leukemia (CML) cells also usually change to immature cells at blastic crisis involving morphological change. However, this morphological change was not so prominent in the ATL cases studied, except one, in which typical ATL cells with nuclear indentation changed to large immature cells with basophilic cytoplasm at relapse.     

The murine monoclonal antibody, 14A2.H1, identifies a novel platelet surface antigen

A murine monoclonal antibody 14A2.H1, raised against acute myeloid leukaemia cells, identifies a previously undescribed 27 kDa platelet surface glycoprotein which is expressed at low copy number (10(3)/platelet). MAb 14A2.H1 caused aggregation of platelets which was dependent on Fc gamma RII. Binding of the antibody to platelets was not altered by activation by thrombin or phorbol ester. In haemopoietic cell populations the antibody bound to megakaryocytes, monocytes (weakly), several myeloid leukaemic cell lines and fresh myeloid leukaemic blasts from some patients. Lymphocytes, lymphoid cell lines, neutrophils and haemopoietic progenitor cells were negative. Expression of the antigen was not restricted to haemopoietic cells as epithelial cells in tonsillar crypts and endothelial cells were positive.     

Analysis of antigenic determinants on human monocytes and macrophages

Mo1, 2, 3, and 4, and Plt-1 are a series of five distinct antigens detected on the surface of human peripheral blood monocytes by mouse monoclonal antibodies. Mo2 and 3 are restricted to the monocyte- macrophage series, while Mo1, as previously reported, is also expressed by human granulocytes and null cells. Mo3, as distinguished from Mo1 and Mo2, is weakly expressed by virgin peripheral blood monocytes but becomes well expressed if monocytes are cultured overnight at 37 degrees C. Mo4 is coexpressed by monocytes and platelets, while Plt-1 appears to be a platelet-specific antigen whose detection on monocytes reflects adherence of platelets to monocyte membranes. That Mo2-4 are true monocyte antigens is demonstrated by their resynthesis following protease treatment of monocytes (Mol expression is resistant to proteolytic digestion). During myeloid-monocyte differentiation, the Mo antigens are infrequently expressed by immature myeloid cells but are found at higher frequency on leukemic monocytic forms. Macrophages from cultured peripheral blood monocytes and HL-60 cells exposed to lymphokines or phorbol diester express Mo1-4, but noncirculating peritoneal macrophages lack Mo3. The Mo antigens are differentiation markers whose expression reflects membrane heterogeneity during myeloid- monocyte-macrophage maturation.     

Phenotypic analysis of acute lymphoblastic leukemia (ALL) cells which are classified as non-T non-B and negative for common ALL antigen

When phenotypic marker analysis of acute lymphoblastic leukemia (ALL) cells (102 cases) was performed, a group of ALL cells (15 cases) classified as non-T non-B, and negative for common-ALL antigen (CALLA) was characterized in a focused manner. "Non-T non-B" was defined as negative for T cell properties such as E-rosetting or reactivity with anti-human T-cell monoclonal antibodies (T101, WT1), and absence of any B-cell characteristics (cell surface and/or cytoplasmic immunoglobulin and reactivity with B1 monoclonal antibody). Despite their marked heterogeneity, CALLA(-) non-T non-B ALL cells revealed three different phenotypic patterns in terms of presence of terminal deoxynucleotidyl transferase (TdT) and of reactivity with antimyeloid (MCS1) or myelomonocyte (MCS2 and OKM1) monoclonal antibodies. Four of 15 cases reacted with some myeloid-specific antibodies, but were negative for TdT. Six cases had both MCS2 antigen and TdT. The remaining five cases expressed no myeloid antigens, but were positive for TdT with some exceptions. These findings showed that acute leukemias with myeloid antigens might be involved in CALLA(-) non-T non-B ALL having no relationship to the presence of TdT, and, furthermore, that the blasts with simultaneous expression of TdT and myeloid-specific antigen (MCS2) might represent an immature stage in hematopoietic differentiation closely corresponding with the bifurcation of the lymphocyte/myeloid pathway. Alternatively, only five cases remained "unclassified leukemia." We therefore think that the detailed examination of CALLA(-) non-T non-B ALL cells using myeloid specific antibodies is helpful in clarifying the characteristics of myeloid precursors and the common bipotential stem cell of lymphoid and myeloid progenitors.     

Immunocytometric characteristics of a monoclonal antibody (Bra55) recognizing the leukocyte common antigen (LCA)

The immunofluorescence cytofluorometric reactivity pattern of monoclonal antibody Bra55 (IgG1) elicited with a non-T, non-B ALL cell line (REH), with a panel of human neoplastic hemopoietic cell lines (including non-T, non-B, T and myeloid leukemia cell lines) and with isolated peripheral blood lymphocytes, monocytes and granulocytes from healthy donors corresponded to the previously described microscopic immunofluorescence, ELISA, immunoprecipitation and immunocytochemic data indicating that this monoclonal antibody recognizes a 170-220 kDa cell surface glycoprotein (leukocyte common antigen) expressed selectively on hemopoietic cells. The purified, FITC-conjugated Bra55 monoclonal antibody was effectively inhibited in its binding to the surface of LCA-positive cells by reference anti-LCA monoclonal antibodies; no inhibition of this activity by LCA-unrelated monoclonal antibodies (such as anti-MHC class I and class II antibodies) was observed. These data confirm the previously reported hemopoietic cell specificity (anti-LCA, CD45) of the Bra55 monoclonal antibody.     

Murine monoclonal antibody therapy in two patients with chronic lymphocytic leukemia

We infused the murine monoclonal antibody T101 into two patients with advanced refractory chronic lymphocytic leukemia (CLL) after confirming its reactivity with their CLL cells. One patient received doses of 1, 3, and 12 mg; the second patient received 10 mg. Antibody was delivered over 10--15 min. The major observations were: (1) T101 murine monoclonal antibody did bind to cells with T65 surface antigen and saturated these cells in vivo; (2) cells that bound T101 disappeared from the circulation by 2 hr after treatment, as evidenced by a marked drop in lymphocyte counts; (3) T101 serotherapy resulted in some intravascular cell injury associated with sequestration and probably destruction in the liver and lung; (4) free serum T101 was demonstrable, but disappeared by 2--4 hr after infusion; (5) rapid infusion of T101 did not induce significant modulation of T65; (6) rapid infusion of greater than 10 mg of T101 was associated with significant systemic reactions. Monoclonal antibodies may someday have an application in leukemia therapy, but additional experimental trials are clearly indicated.     

Lymphokine inducing "terminal differentiation" of the human monoblast leukemia line U937: a role for gamma interferon

The human monoblast leukemia line, U937, is growth-inhibited and induced to develop markers of mature monocytes by lymphokine preparations. Lymphokine is cytostatic and induces expression of Fc receptors in U937 and in myelomonocytic leukemic lines RC-2A and KG-1, but does not have these effects on T- and B-lymphocytic lines. In addition to previously described properties, including complement receptors, phagocytosis, and antibody-dependent cellular cytotoxicity (ADCC), Mac-1 and Mac-3 surface antigens defined by monoclonal antibodies are induced on U937 cells by lymphokine and phorbol ester. The Mac-1 surface component appears to have a regulatory role in differentiation of the monocyte lineage line, since antibodies to this antigen block the induction of Mac-3 antigen. The lymphokine activity was concentrated by salt precipitation and characterized by ion- exchange and size chromatography. Fractions of about 40,000 daltons were responsible for growth inhibition and induction of Fc receptors and Mac-1 antigen in U937 cells. However, ADCC was not induced in U937 by individual fractions of lymphokine, suggesting that this cytotoxic capacity may be regulated by a lymphokine of a different size, which is only effective after initial maturation steps. Since gamma-interferon is present on the 40K size range of lymphokine, the possibility that interferon is a differentiation modulator for the monoblast cells was investigated. Highly purified gamma-interferon (10(7) U/mg protein) at 10-300 U/ml inhibited growth and induced Fc receptors in U937 similar to the effect of lymphokine. The Fc-receptor-inducing activity of lymphokine was inhibited by a neutralizing monoclonal antibody to gamma- interferon, suggesting that this differentiation factor in lymphokine is gamma-interferon.     

Fc gamma receptor II (CD32) on malignant B cells influences modulation induced by anti-CD19 monoclonal antibody

Antigenic modulation is one of many factors determining the effectiveness of monoclonal antibody (MoAb)-mediated therapy. To select the isotype of a CD19 MoAb most suitable for radioimmunotherapy of patients with B-cell malignancies, we studied the influence of MoAb isotype on modulation, after binding of the MoAb to different cell-line cells. The CD19-IgG1 MoAb was found to induce modulation of CD19 antigens on Daudi cell line cells more rapidly than did its IgG2a switch variant. We provide evidence that this difference in modulation rate is caused by the expression of Fc gamma receptor II (Fc gamma RII) on these cells. Experiments aimed at elucidating the mechanism of Fc gamma RII involvement in modulation induction by CD19-IgG1 showed that Fc gamma RII did not comodulate with CD19 MoAbs. However, cocrosslinking of CD19 and Fc gamma RII with CD19-IgG1 MoAb resulted in enhanced calcium mobilization in Daudi cells. This increased signal induction accompanies the enhanced capping and subsequent modulation of CD19 antigens. Because Fc gamma RII is expressed in varying densities on malignant B cells in all differentiation stages, our results have implications for the MoAb isotype most suitable for use in MoAb-based therapy of patients with B-cell malignancies.     

Lymphocytes bearing Fc gamma receptors in rheumatoid arthritis. II. Phenotypic characterisation of mononuclear cells forming Facb rosettes in RA.

We have previously reported an increased proportion of Facb-rosette forming cells in the peripheral blood of patients with rheumatoid arthritis in comparison with healthy controls. The present study investigates the surface phenotype of these cells by means of monoclonal antibodies and a variety of rosetting and lymphocyte fractionation techniques. Facb-R+ cells were found to lack surface markers characteristic of T and B lymphocytes. Studies with monoclonal reagents showed a positive reaction with OKIa1, OKM1, and another monocyte-specific antibody. Fac-R+ cells were recognised by anti-HLA-DR reagents but did not bind the monoclonal antibody 17.15 that recognises a determinant on HLA-DR antigens expressed by lymphocytes but not monocytes. These results show that Facb-R+ cells share certain surface characteristics with monocytes, though they are not phagocytic. These observations are consistent with an accessory role for Facb-R+ cells in the immune response.     

In situ immunologic characterization of cellular constituents in lymph nodes and spleens involved by Hodgkin's disease

The cellular constituents in lymph nodes and spleens of patients with Hodgkin's disease were studied with a series of monoclonal antibodies directed against human thymocyte, peripheral T-cell, and la antigens. Utilizing both an immunoperoxidase technique on frozen tissue sections and indirect immunofluorescence on cell suspensions, wer found that a majority of lymphocytes were T cells, since they stained with anti-T1 and anti-T3 antibodies, which react with all peripheral T cells. In addition, most of these cells were reactive with anti-T4 antibody, which defines the helper/inducer T-cell population, whereas only a minority of cells stained with anti-T5 and anti-T8 antibodies, which are reactive with suppressor/cytotoxic T cells. Moreover, a large proportion of T cells expressed T10 antigen, which is found on activated T cells. A minority of the T cells also expressed la antigen(s), again suggesting that some of the T cells are activated. In contrast, the Reed-sternberg cells did not react with any of these anti- T-cell antibodies or with anti-IgM antiserum, but displayed strong membrane and cytoplasmic staining with anti-la antibody. Taken together, these findings suggest that Reed-Sternberg cells are not of T- cell lineage but may be derived from antigen-presenting reticulum cells in the thymus-dependent areas of lymphoid tissues; these cells are normally associated with T4+ cells.