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.     

Distribution of antigens defined by OKB monoclonal antibodies on benign and malignant lymphoid cells and on nonlymphoid tissues

Monoclonal antibodies OKB1, OKB2, OKB4 and OKB7 have been previously shown to detect distinctive antigens displayed on B, but not on T, lymphocytes. Benign and malignant lymphoid cells were investigated for their reactivity with these antibodies in cell suspension by indirect immunofluorescence and in cryostat tissue sections by the avidin-biotin complex immunoperoxidase technique. Fetal liver pre-B cells and pre-B and common type acute lymphoblastic leukemia cells isolated from 15 patients were OKB1-OKB2+OKB4-OKB7-. All mature lymphoid tissue B cells and the neoplastic cell surface immunoglobulin-positive (SIg+) B cells isolated from each of 47 B cell neoplasms were OKB2+. OKB1 and OKB7 were expressed by interfollicular, follicular center, and many, but not all, mantle zone B cells. OKB4 was expressed by follicular center cells, but not by mantle zone or interfollicular B cells. The neoplastic SIg+ B cells isolated from 45 of 47 B cell malignancies were OKB1+OKB4+, and those isolated from 45 of 46 B cell malignancies were OKB7+. The neoplastic B cells of one mantle zone lymphoma were OKB1-, of one small lymphocytic cell lymphoma were OKB7-, of one large cell lymphoma were OKB4-, and of one small lymphocytic cell lymphoma with a monoclonal gammopathy were OKB1-OKB4-. Normal and myeloma plasma cells were OKB-. The malignant T cells isolated from 12 T cell neoplasms were OKB2-OKB4-, but were OKB1+ and/or OKB7+ in 3 cases. Thus, the OKB antibodies appear to detect distinctive antigens that may be expressed at different stages of B cell differentiation. In addition, OKB4 reacted with selected renal and respiratory epithelium, and OKB2 reacted with a wide range of epithelial tissues. The OKB antibodies should prove useful in the investigation of B cell differentiation and may aid in the identification and characterization of lymphoproliferative malignancies with significant therapeutic and prognostic differences not identifiable by conventional histopathologic and immunologic methods.     

Detection of cells of megakaryocyte lineage in haematological malignancies by immuno-alkaline phosphatase labelling cell smears with a panel of monoclonal antibodies

Immuno-alkaline phosphatase staining (by the APAAP technique) has been used to identify promegakaryoblasts in cell smears from 10 cases of leukaemia (three acute leukaemia, seven blast transformations). In all cases promegakaryoblasts were labelled by at least two anti-platelet glycoprotein (gp) antibodies, the highest percentages being obtained with anti-gp IIIa (antibody C17). HLA-DR was expressed by a variable percentage of neoplastic cells in all cases, the T11 (CD2) antigen (sheep red cell receptor) in four of seven cases tested and the p150,95 antigen in three of the six cases tested. In some cases of acute myeloid leukaemia APAAP staining of blood smears revealed circulating promegakaryoblasts and micromegakaryocytes (which superficially resemble small lymphoid cells). It is concluded that immuno-alkaline phosphatase staining of cell smears offers a convenient means of diagnosing acute megakaryoblastic leukaemia in the routine laboratory.     

Treatment of patients with malignant lymphomas with monoclonal antibodies

Malignant lymphomas represent a heterogenous group of B and T cell-derived malignancies. Most lymphomas are sensitive to chemo- and radiotherapy, however many patients will eventually relapse. Immunotherapeutic approaches including monoclonal antibodies, cytokines or vaccination approaches may offer an alternative treatment of chemotherapy-resistant residual cells especially in cases with low tumor burden or residual disease following chemo- or radiotherapy. Monoclonal antibodies have been successfully applied in their native form, or coupled with radioisotopes or toxins to selectively destroy lymphoma cells and promising results in early clinical trials have been obtained. Alternatively, bispecific antibodies and idiotypic vaccination strategies are used to target autologous T cells to eliminate lymphoma cells. A humanized anti-CD20 antibody showed excellent results in chemotherapy refractory lymphomas and has recently been approved for clinical application in CD20 positive B cell lymphomas.     

Identification of neutrophil subpopulations with monoclonal antibodies

Two monoclonal antibodies have been produced by the hybridoma technique that recognize subpopulations of human neutrophils. The antibodies, termed 1B5 and 4D1, react with a mean percentage of 57% and 51% of peripheral blood granulocytes, respectively. The antigens recognized appear to be neutrophil specific in that these antibodies do not react with eosinophils, platelets, erythrocytes, monocytes, or nonadherent peripheral blood mononuclear cells. Although the neutrophil subpopulations recognized by these antibodies are nearly identical (coinclusive), the antigenic determinants recognized appear to be different. These monoclonal antibodies to neutrophil subpopulations may prove useful to studying functional heterogeneity among neutrophils as well as for investigations of normal and abnormal myeloid differentiation.     

A single monoclonal antibody identifies T-cell lineage of childhood lymphoid malignancies

Immunophenotyping studies with monoclonal antibodies have revealed the heterogeneity of childhood acute lymphoblastic leukemia (ALL) and non- Hodgkin's lymphoma (NHL). The lymphoid malignancies of T-cell lineage are particularly heterogeneous and, until now, no single monoclonal antibody has been found to identify all cases of T-ALL and T-NHL. A monoclonal antibody, 4H9, recognizes an antigen of 40,000 molecular weight on normal and malignant T cells. Thirty-six cases of childhood T- ALL and T-NHL were tested, and in all cases, the malignant blast cells were reactive with 4H9, whereas malignant cells from 61 cases of non-T ALL and NHL were not reactive with 4H9. Monoclonal antibody 4H9 is a sensitive and specific reagent for the identification of childhood T- cell ALL and NHL and should be extremely useful in immunophenotyping studies of lymphoid malignancies.     

Normal equivalent cells of B cell malignancies: analysis with monoclonal antibodies

Immunoglobulin heavy chain expression and reactivity of monoclonal antibodies RFA-1, -2, -3, -4 and OKT10 discriminate between majority B lymphocyte populations in the bone marrow and in peripheral lymphoid organs. In this study normal tissues and various B cell malignancies were studied in cell suspensions and tissue sections. Pre-B acute lymphoblastic leukaemia and multiple myeloma apparently reflect the phenotypes on normal B lineage cells of the bone marrow, while centroblastic-centrocytic lymphoma, B chronic lymphoid leukaemia (CLL) and prolymphocytic leukaemia (PLL) show the characteristics of distinct peripheral B lymphoid subsets found at different sites in the lymphoid tissue. These 'normal equivalent' cells are centroblasts and centrocytes in the germinal centre. CLL-like cells at the edge of the germinal centre (a minority population) and strongly Ig positive cells in the lymphocyte corona. Malignant cells in macroglobulinaemia are apparently more closely related to PLL and the corresponding normal peripheral B cells (in the corona) than to myeloma cells or the equivalent normal plasma cells in the bone marrow.     

磁激活细胞分选术联合混合抗体提高模拟恶性腹水中游离癌细胞检出效率的分析

背景磁激活细胞分选术(magnetic activated cell sorting,MACS)是一种新的免疫磁性分离技术.其原理是基于抗体对抗原的特异性识别,将50 nm磁性微珠直接或者间接耦联在抗体上,与有相应抗原表达的细胞相连,在高强度、高梯度磁场中达到细胞磁性分离的目的.该法具有分离纯度和回收率均较高的优势,也能分离出体液中存在的少量肿瘤细胞.目的评价MACS联合一组肿瘤细胞标志物的方法对提高模拟恶性腹水中游离癌细胞检出效率的作用.方法 选择5种与恶性腹水病因有关的肿瘤细胞株作为研究对象,采用免疫荧光反应和流式细胞仪(FCM)检测上皮相关抗原(epithlial-related antigen,EpCAM)、CA125、癌胚抗原和TAG-72共4种单克隆抗体及其混合抗体在各肿瘤细胞的表达.将肿瘤细胞以不同比例掺入单个核细胞中模拟恶性腹水细胞成分,与联合单个抗体进行分选对比,观察MACS术联合混合抗体分选肿瘤细胞的效率.结果 FCM结果表明,混合抗体在5种肿瘤细胞的阳性表达率均高于4种抗体单独反应的阳性率.MACS术联合混合抗体检出模拟恶性腹水中肿瘤细胞的得率比联合单个抗体的得率要高,其中以2种胃癌细胞和结肠癌细胞的平均得率提高较多(分别为69.18%±20.84%比45.23%±11.54%、78.75%±15.42%比59.73%±16.64%和85.63%±12.30%比76.88%±8.65%),卵巢癌细胞次之(32.49%±3.58%比31.79%±4.82%),肝癌细胞最少(11.78%±0.43%比7.16%±0.46%).结论 与联合单个抗体进行分选相比,应用MACS术联合一组混合抗体的方法可有效提高恶性腹水中游离癌细胞的检出效率,尤其对胃肠道肿瘤所致的恶性腹水有潜在的临床应用价值.     

The phenotype of the neoplastic cells of hairy cell leukemia studied with monoclonal antibodies

Eighteen cases of hairy cell leukemia were studied with a battery of polyclonal anti-Ig and nine monoclonal antibodies to determine the lineage of the hairy cells (HC) and the stage of their maturation arrest. Hairy cells tend to nonspecifically bind many antisera and precautions had to be taken to avoid nonspecific fluorescence of the cells. All but one case was reactive with one light chain type and one or more heavy chain isotype antisera as reported before. All cases studied were positive for monomorphic HLA-DR determinants, using monoclonal antibody 7.2. Most cases tested (6/7) were positive with the B-lineage related antibody PI 153/3. While most cases (15/18) were nonreactive with the B-lineage related antibody BA-1, they became positive (5/5) following in vitro culture. Seven out of nine cases were reactive with OKM1. Common acute lymphoblastic leukemia antigen (CALLA) was absent in all (15) cases tested and the ALL associated structure p24/BA-2 was absent from 16 of 18 cases. HC from none of the cases were clearly positive with the T-cell antibodies 9.6., or TA-1, whereas in only 1/18 the cells reacted with T101. The results of this study support the B cell lineage of most HC, and show the presence of multiple phenotypes. In combination with the surface Ib present on the cells, a hierarchy of phenotypes is postulated, with SIg-ormu delta, BA- 1+, PI 153/3+, HLA-DR+ being the most immature, and SIg(alpha) gamma, BA-1-, PI 153/3-, HLA-DR+ the most mature.     

Lipopolysaccharide responsiveness of malignant lymphoid cells in a patient with hairy cell leukemia.

The malignant cells in a patient with hairy cell leukemia responded most evidently to lipopolysaccharide (LPS) in in vitro culture for 3 1/2 days when the conventional tritiated thymidine uptake method was used. Since the malignant cells from patients with several other forms of leukemia and the peripheral blood mononuclear cells from healthy individuals did not show a comparable degree of responsiveness to LPS, we could exclude the possibility that this response was due to effects on contaminating normal mononuclear cells or to the nonspecific conditioning effect through LPS-affected contaminating normal monocytes. Morphological changes were observed with photo- and electronmicroscopy. It is likely that the hairy cells from the patient did respond to LPS, and whether or not this phenomenon may be confined to this type of lymphoid leukemia is not being investigated.     

Cytoskeletal binding of monoclonal anti-DNA antibodies derived from tonsillar lymphoid cells of a normal subject

The ability of monoclonal IgM anti-DNA autoantibodies derived from normal human lymphoid cells to bind to cellular constituents of human epithelial cells (HEp-2) was examined by immunofluorescence. Hybridoma supernatants from 10 different clones were studied. Four of them gave a strong fibrillar cytoplasmic staining that resembled cytoskeletal staining, 1 showed strong nuclear staining only, 3 showed weak nucleolar staining only, and 2 showed no staining. The hybridoma supernatants that reacted with HEp-2 cytoskeleton were either polyspecific for various nucleic acid antigens, such as single-stranded DNA, DNA, poly(dA:dT), poly(dG).poly(dC), RNA, and cardiolipin, or were restricted to cardiolipin. Cytoskeletal staining identical to the hybridoma supernatant staining was also seen with mouse monoclonal anti-vimentin antibody B11.5.1. Inhibition of the cytoskeletal staining was accomplished in 3 of the 4 hybridoma supernatants by preabsorption of these hybridoma supernatants with cardiolipin and/or single-stranded DNA, or by preincubation of the HEp-2 cells with the mouse monoclonal anti-vimentin antibody.     

Double fluorochrome analysis of human bone marrow lymphoid cells: studies with terminal transferase and anti-T-cell monoclonal antibodies

Mature thymus-derived (T) lymphocytes are generally believed to be derived from a bone marrow progenitor cell. Data from studies with animals suggest that the enzyme terminal deoxynucleotidyl transferase (Tdt) is expressed in many T-cell progenitors in bone marrow. In this study we attempted to identify Tdt+ bone marrow cells in man that may be committed to T lineage based on coexpression of Tdt and antigens that have previously been useful in characterization of thymocytes or peripheral-blood T cells. We used a panel of ten monoclonal antibodies against such antigens to analyze Tdt+ bone marrow cells using two-color immunofluorescence. We found that T-cell-associated antigens were not expressed on Tdt+ bone marrow cells and that T cells in bone marrow have a phenotype similar if not identical to peripheral-blood T cells. These results support the hypothesis that many postthymic immunocompetent T cells are found in human marrow. Our results also suggest that if Tdt+ bone marrow cells are committed to T lineage, then the acquisition of mature T-cell-associated antigens is an intrathymic event.     

Identification of mutant monoclonal antibodies with increased antigen binding.

Sib selection and an ELISA have been used to isolate hybridoma subclones producing mutant antibodies that bind antigen better than the parental monoclonal antibody. Such mutants arise spontaneously in culture at frequencies of 2.5-5 X 10(-5). The sequences of the heavy and light chain variable regions of the mutant antibodies are identical to that of the parent and the Ka values of the mutants and the parent are the same. The increase in binding is associated with abnormalities of the constant region polypeptide and probably reflect changes in avidity of these antibodies.     

Studies on B lymphoid tumors treated with monoclonal anti-idiotype antibodies: correlation with clinical responses

Monoclonal anti-idiotype antibodies can be made which are exquisitely specific for B lymphocytic malignancies. We have conducted a clinical trial in which some patients' tumors regressed after infusion of such antibodies. Here, we evaluated characteristics of the antibodies, the tumors, and the patients to determine which features best correlated with the clinical response. Neither the isotype of the murine antibodies, nor their avidity were predictive of clinical outcome. The specific epitope to which the antibodies bound was characterized by immunochemical techniques. Reactivity with a heavy-light chain combinatorial determinant correlated somewhat with clinical effect. Variations in the characteristics of the individual tumors such as antigen sites per cell and ability to modulate the surface immunoglobulin were not predictive of response. In one patient with prolymphocytic leukemia the anti-idiotype antibody had a direct antiproliferative effect on tumor cells in vitro. This patient's tumor response was explainable by such a direct mechanism. In the other patients, who had lymphomas, therapeutic outcome correlated with the number of host nontumor cells infiltrating the tumor. The vast majority of these nontumor cells were mature T lymphocytes of the Leu 4, Leu 3 (T3, T4) phenotype. Thus, a preexistent host-tumor interaction seems to be important in the in vivo effect of anti-idiotype antibodies in B cell tumors.     

Ferritins in malignant and non-malignant lymphoid cells

Lymphoid cells from peripheral blood, thymus, malignant and non-malignant lymph nodes were analysed for ferritin content using radioimmunoassays specific for the 'acidic' H-subunit-rich and for 'basic' L-subunit-rich isoferritins, and the data were compared with the immunological characteristics of the cells. All tissues with high proportion of T or 'null' cells contained the lowest concentration of L-subunit-rich isoferritins, while the H-subunit-rich forms increased from low levels in the quiescent peripheral blood lymphocytes (PBL), to higher values in the immature and proliferating thymocytes and lymphoblasts, malignant or not. B-cell lymphomas contained concentrations of both ferritin types higher than those found in PBL. No significant difference was found in the isoferritin concentrations between non-malignant lymph nodes and tissues involved in Hodgkin's disease. These findings indicate that maturation stage, proliferative status and anatomical localization affect isoferritin expression in lymphoid cells.     

Do some malignant melanoma cells share antigens with the myeloid monocyte lineage?

Melanoma cells freshly isolated from 63 advanced primary lesions and 103 metastases were analyzed by staining with monoclonal antibodies MEM 28 directed against a 200 kDa antigen present on all leukocytes and tissue macrophages (CD 45), MEM 18 directed against a monocyte antigen of 53 kDa, anti CD 14--Immunotech, Marseille and 3.9 directed against a 150 kDa antigen expressed on monocytes and to even greater degree on most tissue macrophages (CD 11 c). All antibodies showed variable reactivity with melanoma cells, percentage of positive tumor cells ranged from 0 to 70.