Monoclonal antibody (B27M2) subdividing HLA-B27.
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Antibodies, Monoclonal↗
Biomedical subjects
E G Engleman
Publications and source records attributed to E G Engleman.
Production of functional human T-T hybridomas in selection medium lacking aminopterin and thymidine.
The production of hybridomas between immunologically activated T cells and malignant T-cell lines offers a potentially unlimited source of soluble T-cell-derived products. Recently, human T-T hybrids have been described; however, their use has been hampered by slow growth and chromosomal instability due at least in part to the presence of thymidine in the traditional hypoxanthine/aminopterin/thymidine (HAT) selection medium. In this report, we describe the development of a rapidly growing hypoxanthine phosphoribosyltransferase-deficient human T-cell line designated J3R7, the use of azaserine/hypoxanthine (AH) medium as an alternative selection medium to HAT medium, and the production of functional T-T hybrids by using the J3R7 line and the AH selection technique. Hybrids selected in AH medium were 4-fold greater in number and 3-fold faster in growth rate than hybrids grown in HAT medium. No stable clones were obtained from HAT cultures whereas AH-derived hybrids could be readily cloned by the method of limiting dilution. Evidence for hybridization included (i) the presence of approximately twice the number of chromosomes in hybrids than in J3R7 cells; (ii) the presence on hybrid cells of the Leu-3a surface antigen, present on normal helper T cells but not on J3R7 cells; (iii) the expression of HLA antigens of both the normal T-cell partner and the J3R7 line; and (iv) the constitutive secretion of interleukin 2 from multiple hybrid clones but not from the J3R7 cell line. Thus far, these clones have maintained their rapid growth, chromosome number, surface phenotype, and constitutive secretion of interleukin 2 for 4 months.
Aminopterin↗
Cultured human epidermal cells do not synthesize HLA-DR.
All nucleated cells express HLA-A, B, and C antigens. However, only a few cells, including epidermal cells, demonstrate HLA-DR antigens which are potent transplantation immunogens in man. The current study was undertaken to determine if epidermal cell continue to synthesize and/or express HLA-DR antigens after prolonged in vitro culture. Epidermal cells cultured for 7 days or more no longer stimulated allogeneic lymphocytes in the epidermal cell-lymphocyte reaction. Indirect immunofluorescence light microscopy of cultured cells using mouse monoclonal antibody to HLA-DR antigen confirmed that these cells do not express HLA-DR antigens whereas they retain beta 2-microglobulin. Detergent extracts of 12-day cultured epidermal cells biosynthetically labeled with 35 S-methionine were immunoprecipitated with monoclonal anti-DR antibody and analyzed by the method of two-dimensional polyacrylamide gel electrophoresis. No radiolabeled proteins were found on these gels in the regions where HLA-DR molecules are known to migrate. These data indicate that HLA-DR antigen is absent from cultured epidermal cells. Finally, we describe a technique for growing epidermal cells on a gelatin membrane which allows subsequent removal of intact cell monolayers from the culture dish. Such monolayers may be useful for purposes of transplantation.
Cells, Cultured↗
Use of the fluorescence-activated cell sorter to quantitate and enrich for subpopulations of human skin cells.
A variety of immunologic staining techniques were compared in a quantitative study of antigen expression by human epidermal cells. Virtually all nucleated epidermal cells express beta 2-microglobulin, which is associated with HLA-A, -B, and -C antigens, whereas only about 4% expressed T6, an antigen expressed by Langerhans cells but not other cells in the skin. With the fluorescence-activated cell sorter (FACS), epidermal cell suspensions were selectively enriched 10- to 15-fold for T6-positive Langerhans cells. An average of 6.5% of cells were specifically stained by anti-HLA-DR antibody. When dispersed cells stained with anti-DR plus peroxidase were examined with the technique of immunoelectron microscopy, only mononuclear leukocytes (probably Langerhans cells) were stained. After separating HLA-DR positive skin cells with the FACS, the DR-positive population but not the DR-negative population stimulated proliferation of allogeneic responder lymphocytes, indicating that sorted cells are metabolically active. We conclude that HLA-DR antigen is not expressed by keratinocytes in normal human skin cell suspensions and that the FACS can be used to selectively enrich or deplete skin cell suspensions of antigenically distinct subpopulations such as Langerhans cells.
Antibodies, Monoclonal↗
Human skin cells synthesize HLA-DR molecules of the same charge and molecular weight as those synthesized by autologous lymphocytes.
Between 5 and 14% of human skin cells, separated by trypsinization, express HLA-DR antigen as detected by indirect immunofluorescence with monoclonal anti-DR antibody. To determine the source and structure of these DR molecules, skin cell suspensions were biosynthetically labeled with 35S-methionine and the radiolabeled DR molecules were analyzed by the method of two-dimensional polyacrylamide gel electrophoresis. The results indicated the presence of heavy, intermediate, and light chains indistinguishable from those of B lymphocyte DR. Gels in which DR immunoprecipitates from skin cells and autologous lymphocytes were run simultaneously confirmed this finding. Thus, skin cells and lymphocytes synthesize HLA-DR antigen of identical charge and molecular weight.
B-Lymphocytes↗
Separation of human skin cells by velocity sedimentation into functionally distinct fractions.
The epidermis consists of a heterogeneous population of cells including Langerhans cells, Merkel cells, melanocytes, and keratinocytes in various stages of differentiation. The current study was undertaken to determine if skin cell suspensions can be separated into morphologically and/or functionally distinct fractions. Skin cells were suspended by trypsinization and separated into multiple fractions by velocity sedimentation. Certain fractions reproducibly stimulated proliferation of allogeneic lymphocytes in the skin cell lymphocyte reaction, whereas other fractions, containing larger cells, supported growth of keratinocyte colonies in cell cultures. These results indicate that stimulation in the skin cell lymphocyte reaction and growth of keratinocyte colonies are mediated by distinct cells, separable by velocity sedimentation.
Cell Division↗
Dissection of immunoregulatory subpopulations of T lymphocytes within the helper and suppressor sublineages in man.
The helper/inducer (Leu-3) and suppressor/cytotoxic (Leu-2) sublineages of human peripheral blood T cells can both the subdivided into functionally distinct subsets with anti-Leu-8, a new monoclonal antibody that identifies 75 +/- 10% of the Leu-3 cells and 60 +/- 10% of the Leu-2 cells. Using the autologous MLR as a T cell-dependent stimulus for immunoglobulin synthesis, we have shown that the major helper effect for antibody formation lies within the numerically minor Leu-3,8- subset. In addition, neither Leu-2,8- cells alone suppress Leu-3-induced immunoglobulin synthesis, but in combination these subsets are markedly inhibitory. These results indicate that at least two phenotypically distinct cell types of suppressor lineage interact to produce suppression of an immune response in man.
Animals↗
Immunoregulation in the synovium of rheumatoid arthritis.
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Arthritis, Rheumatoid↗
Treatment of intractable rheumatoid arthritis with total lymphoid irradiation.
Eleven patients with intractable rheumatoid arthritis were treated with total lymphoid irradiation (total dose, 2000 rad) in an uncontrolled feasibility study, as an alternative to long-term therapy with cytotoxic drugs such as cyclophosphamide and azathioprine. During a follow-up period of five to 18 months after total lymphoid irradiation, there was a profound and sustained suppression of the absolute lymphocyte count and in vitro lymphocyte function, as well as an increase in the ratio of Leu-2 (suppressor/cytotoxic) to Leu-3 (helper) T cells in the blood. Persistent circulating suppressor cells of the mixed leukocyte response and of pokeweek mitogen-induced immunoglobulin secretion developed in most patients. In nine of the 11 patients, these changes in immune status were associated with relief of joint tenderness and swelling and with improvement in function scores. Maximum improvement occurred approximately six months after irradiation and continued for the remainder of the observation period. Few severe or chronic side effects were associated with the radiotherapy.
Antibodies, Monoclonal↗
Monoclonal anti-HLA-B27 antibody (B27M1): production and lack of detectable typing difference between patients with ankylosing spondylitis, Reiter's syndrome, and normal controls.
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Antibodies, Monoclonal↗
Antibodies to membrane structures that distinguish suppressor/cytotoxic and helper T lymphocyte subpopulations block the mixed leukocyte reaction in man.
Two major subsets of human T lymphocytes that are functionally analogous to the mouse Lyt-2+ and Lyt-2- subsets have been defined by their expression of two thymus-dependent membrane antigens, Leu-2 and Leu-3. Leu-2+,3- cells have suppressor/cytotoxic functions and Leu-2-,3+ cells have helper functions. These studies were designed to determine the effects of adding IgG1 monoclonal anti-Leu-2 and anti-Leu-3 antibodies to the mixed leukocyte reaction (MLR). At high concentrations, each antibody partially inhibited the proliferative response of unseparated T cells and abolished the response of the isolated subset having the appropriate phenotype. An IgG1 monoclonal antibody to HLA-A2 and an IgG2a antibody to Leu-1, a pan-T antigen, failed to inhibited the MLR. These results suggest that the Leu-2 and Leu-3 antigens may have a direct role in the mechanism whereby T cells recognize and respond to alloantigen.
Animals↗
Treatment of NZB/NZW F1 hybrid mice with Mycobacterium bovis strain BCG or type II interferon preparations accelerates autoimmune disease.
NZB/NZW F1 hybrid mice develop a spontaneous autoimmune disease characterized by the appearance of antinuclear antibodies and premature death due to immune complex glomerulonephritis. To investigate the possible effects of cellular immune stimulation on this disorder, groups of female NZB/NZW mice, aged 2, 5, and 7 months, were treated either with the nonspecific immunostimulatory agent Mycobacterium bovis strain BCG or with saline. Mice treated with BCG at ages 5 and 7 months died sooner than age-matched controls, and death was associated with severe glomerulonephritis, suggesting that BCG may have accelerated autoimmunity in these mice. Since BCG is known to stimulate the production of type II (or gamma) interferon, a substance with potent immunoregulatory effects, a second study was carried out to assess the effects of type II interferon on NZB/NZW disease. A greater number of type II interferon-treated mice died by 9 months of age when compared to controls, and the increased death rate was associated with a more rapid development of antinuclear antibodies and histologically confirmed glomerulonephritis. These data, together with a recent report of increases in the level of serum type II interferon in patients with active systemic lupus erythematosus, suggest that type II interferon may play a role in the pathogenesis of autoimmune disease.
Animals↗
Studies of a human T lymphocyte antigen recognized by a monoclonal antibody.
A monoclonal antibody (designated L17F12) detects an antigen present on 95-100% of human peripheral T lymphocytes, the majority of thymocytes, and acute lymphocytic leukemia T cells but not B cells, B-cell lines, or monocytes. Examination of frozen tissue sections by the immunoperoxidase method revealed that the cells expressing this antigen were found predominantly in the medulla of thymus and in T-cell zones of lymph node and spleen. The antigen recognized by L17F12 was associated with a cell-surface glycoprotein of 67,000 daltons. L17F12 was used to isolate this molecule from human thymocytes, normal peripheral T cells, leukemic T cells, and T-cell lines. Expression of this antigen on normal T cells was not diminished by prolonged exposure in vitro to various T-cell stimuli. In the absence of complement, L17F12 bound to T cells without altering proliferative functions, thus enabling rapid purification of functionally intact T cells. In the presence of complement, L17F12 was cytolytic for T cells, providing the basis for depletion of T cells from heterogeneous populations. These data suggest that the monoclonal antibody L17F12 recognizes a specific T-cell differentiation protein. This antibody will be useful in studies of the human immune system.
Animals↗
Induction of immunoglobulin-secreting cells in the allogeneic mixed leukocyte reaction: regulation by helper and suppressor lymphocyte subsets in man.
The existence of functionally distinct T lymphocyte subsets in man, initially demonstrated with heteroantisera, has been confirmed with monoclonal reagents. Two major subsets have been defined: Leu-2 cells, which effect cell mediated lymphocytotoxicity (CML), and Leu-3 cells, which amplify CML and other T cell functions. This study is an effort to determine the effects of these subsets on the immunoglobulin secretion induced in the human mixed leukocyte reaction (MLR). T cells were separated from non-T cells by rosetting with sheep erythrocytes and were fractionated into Leu-2 and Leu-3 subsets by solid phase immunoabsorption with monoclonal antibodies. T cell subsets were cultured with autologous non-T cells and irradiated allogeneic stimulator non-T cells. Secretion of IgM and IgG was measured by a reverse hemolytic plaque assay. MLR-induced antibody secretion was specifically dependent on the Leu-3 T lymphocyte subset. The Leu-2 subset was incapable of generating large numbers of IgM- or IgG-secreting cells, and, in fact, suppressed the Leu-3-induced response. Exposure of Leu-3 cells to a dose of irradiation sufficient to prevent their proliferation in MLR did not reduce induced immunoglobulin secretion. Leu-2-mediated suppression, however, was sensitive to low dose irradiation. Thus Ig secretion in human MLR is regulated by a balance of helper activity from the Leu-3 subset and suppressor activity from the Leu-2 subset.
Antibody-Producing Cells↗
Activation of human T lymphocyte subsets: helper and suppressor/cytotoxic T cells recognize and respond to distinct histocompatibility antigens.
This study was directed at determining the major histocompatibility complex (MHC) antigens recognized by helper (Leu-3) and suppressor-cytotoxic (Leu-2) T lymphocyte subsets in man. These 2 subsets were isolated from peripheral blood with monoclonal antibodies and challenged in vitro with various stimuli. Only Leu-3 cells proliferated in response to autologous nonrosetting cells and soluble antigens, suggesting that helper but not suppressor-cytotoxic T cells recognize autologous HLA-DR antigen. Furthermore, only Leu-3 T cells responded to allogeneic DR antigen; this was shown in reactions between 2 siblings who were HLA-identical except for a single disparity at HLA-DR caused by a crossover event. Leu-3 cells activated in primary allogeneic mixed leukocyte reactions (MLR) responded equally in secondary allogeneic MLR to the priming cells and to cells that were identical at HLA-DR but discordant at HLA-A, B, and C to the priming cell. The antigens responsible for stimulating Leu-2 cells in allogeneic MLR were not identified, although the results are compatible with a role for HLA-A and B antigens and exclude a dominant role for HLA-DR. These data indicate that the helper and suppressor-cytotoxic T cell subsets in man respond differentially to MHC antigens in a manner analogous to the murine Lyt-2- 3- and Lyt-2+ 3+ populations.
Antibodies, Monoclonal↗
Induction of immunoglobulin secreting cells in the human autologous mixed leukocyte reaction: regulation by helper and suppressor lymphocyte subsets defined with monoclonal antibodies.
The generation of IgM- and IgG-secreting cells in the human autologous MLR has been confirmed by using a microwell culture system and a reverse hemolytic plaque assay. The regulation of this response by T cell subsets was examined by using monoclonal antibodies that have previously allowed the definition of a Leu-3 helper/inducer subpopulation and a Leu-2 suppressor/cytotoxic subpopulation. These subsets were isolated by "panning" with monoclonal antibodies and were then cultured with autologous non-T cells, which serve as both stimulators of the reaction and a B cell source. The induction of antibody-forming cells was found to be dependent upon the Leu-3 subset whereas the Leu-2 subset not only failed to support C cell maturation, it actively suppressed the Leu-3 induced response. The Leu-3 response was found to be partially sensitive to radiation doses of 1000 rad or greater, whereas Leu-2 mediated suppression, even at high doses of cells, was totally ablated by 1500 rad. Thus, the generation of antibody-forming cells in the human autologous MLR is regulated by a balance of Leu-3 cell help and Leu-2 cell suppression. Such a finding may prove to be of relevance to immunoregulation in both health and disease.
Antibodies, Monoclonal↗
Ia antigen on peripheral blood mononuclear leukocytes in man. II. Functional studies of HLA-DR-positive T cells activated in mixed lymphocyte reactions.
Blast transformation of T cells in response to allogeneic lymphocytes is followed by expression of HLA-DR antigen on up to 60% of the T cells. Murine monoclonal antibody to HLA-DR antigen was used to separate alloactivated T cells into those T cells that express high quantities of DR antigen (DR+) and those that express little or no DR antigen (DR-), and each population was tested in a variety of assays. DR+, but not DR-, T cells stimulated fresh allogeneic and autologous T cells to proliferate and supported proliferation by fresh autologous T cells to soluble antigens. Alloactivated T cells were suppressive of fresh mixed lymphocyte reactions (MLR) and suppression by irradiated DR+ T cells was specific for the DR antigens of the initial stimulator cell. Suppression of the MLR by DR+ T cells was not a result of altered kinetics or cell-mediated cytotoxicity. DR+ T cells released soluble factors that suppressed fresh allogeneic responses. These data indicate that alloactivated DR+ T cells may provide antigen-specific feedback inhibition of the MLR.
Animals↗
Ia antigen on peripheral blood mononuclear leukocytes in man. I. Expression, biosynthesis, and function of HLA-DR antigen non-T cells.
Murine monoclonal antibodies to monomorphic components of HLA-DR antigen were used to analyze the distribution and function of DR molecules on non-T mononuclear leukocytes from peripheral blood. On the basis of indirect immunofluorescence and complement-mediated cytolysis. DR antigen was detected on approximately 70% of non-T cells (DR+) and could not be detected on approximately 30% of non-T cells (DR-). A fluorescence-activated cell sorter was used to separate non-T cells into DR+ and DR- populations, and each population was studied. At least one-third of DR- cells were monocytes, and the remainder were surface-immunoglobulin-negative lymphocytes. Analysis of [35S]methionine-labeled proteins by the method of two-dimensional polyacrylamide gel electrophoresis indicated that DR+, but not DR-, cells biosynthesize DR molecules DR+ cells stimulated strongly in the autologous and allogeneic mixed lymphocyte reactions (MLR) and supported T cell proliferation to soluble antigens, whereas DR- cells stimulated in the allogeneic MLR but failed to stimulate in the autologous MLR or to support T cell proliferation to soluble antigens. When present continuously in culture, one monoclonal anti-DR antibody (antibody 2.06) modestly inhibited T cell proliferative responses. Another antibody (antibody 1.35) markedly enhanced the autologous MLR and the proliferative response to soluble antigens, but had no effect on the allogeneic MLR. These data suggest that DR+ and DR- non-T populations are functionally distinct, and that DR antigen may be required for presentation of soluble antigen and stimulation in the autologous MLR. Antigens in addition to DR may stimulate allogeneic T cell proliferation.
Animals↗