Cytoplasmic as opposed to surface Ia antigens expressed on human peripheral blood lymphocytes and monocytes.

Peripheral blood leucocytes were examined for the presence of HLA-Dr or Ia-like antigen on the cell surface and in the cytoplasm. Surface staining of viable cell suspensions, using monoclonal anti-Ia antibody in both immunofluorescence and immunoperoxidase methods gave similar results. Using a newly developed immunoperoxidase double staining procedure, which stains the cells when either viable or fixed, both the surface and cytoplasm of the individual cells were labelled and examined. Twenty to thirty percent of the mononuclear cells were positive for Ia staining on the cell surface and in the cytoplasm. Morphologically these positive cells were identified as both lymphocytes and monocytes. A small percentage (5-9%) of these monocytes and lymphocytes consistently labelled only cytoplasmic Ia determinants. In contrast, only surface binding was observed with monoclonal antibodies recognizing T cell subpopulations. The ability to detect intracellular Ia antigenic determinants is of value in distinguishing stages of lymphocyte and monocyte development.

The immunological classification of leukaemia based on a rapid microcytotoxicity test.

We describe a rapid, accurate, and reproducible cytotoxic antibody test for the immunological classification of leukaemia. Well characterized heteroantisera and monoclonal antibodies were distributed in a microcytotoxicity tray referred to as a leukaemia screening tray (LST). Leukaemia cells were tested for the presence of Ia-like, 'blast', thymocyte, common acute lymphoblastic leukaemia (cALL), and acute myeloblastic leukaemia (AML) antigens. Utilizing this technique, T ALL could be distinguished from non-T ALL, ALL and AML could be differentiated, the myeloid blast crisis of chronic myelogenous leukaemia (CML) could be distinguished from the lymphoid blast crisis, and the T lymphocyte and B lymphocyte lymphoid leukaemias were readily identified. It has been shown that subclassification of leukaemia according to surface markers, as described here, offers considerable improvement in the diagnosis and treatment of leukaemia over the classical morphological methodologies. Because the test can be completed in 2 hr and unlimited amounts of monoclonal antibodies are available, the LST or similar tests should become universally available in the future to supplement morphological data and replace other lengthy enzyme and rosetting tests.

Immunologic classification of lymphocytic leukemias based on monoclonal antibody-defined cell surface antigens.

A panel of monoclonal antibodies reactive with normal lymphocyte subsets was used to classify cases of lymphocytic leukemia on the basis of cell surface antigen expression. The antibodies employed were commercially available and included a common framework HLA-DR antibody, two pan-T antibodies (Leu-1 and OKT-3), and antibodies defining cytotoxic/suppressor (Leu-2 and OKT-8) and helper/inducer (Leu-3 and OKT-4) subpopulations of normal T lymphocytes. Cases of ALL could be subgrouped into non-T non-B, pre-T and T-ALL on the basis of reactivity with HLA-DR, Leu-1, and OKT-3 antibodies. Leukemic cells from patients with T-cell CLL could be divided into Leu-2/OKT-8 reactive and Leu-3/OKT-4 reactive subpopulations, as well as a subgroup in which the majority of cells were unreactive with either of these antibodies. With the exception of one individual, all Sézary cell leukemias expressed a phenotypic pattern similar to that of the Leu-3 subgroup of T-CLL. Malignancies of B-cell lineage (B-CLL, prolymphocytic leukemia, and lymphosarcoma) that were examined were reactive with both the HLA-DR and Leu-1 antibodies. On the contrary, normal B lymphocytes and lymphoid cell lines of B-cell origin did not express surface antigens recognized by the Leu-1 antibody.

Monoclonal antibodies reactive with acute myelogenous leukemia cells.

Two cytotoxic monoclonal antibodies (D5D6 and C10H5) were raised to acute myelogenous leukemia (AML) cells. One of them, D5D6, reacted with 100% of the cells from 44/50 AML patients. The second, C10H5, reacted with 100% of the cells from 13/39 AML patients. D5D6 and C10H5 were cytotoxic to 100% of the monocytes from all healthy donors tested. Apart from monocytes, these antibodies did not react with any other peripheral blood cells from normal donors or with cancerous cells either from cell culture lines or from patients with chronic myelogenous leukemia (CML), lymphoid leukemias, or solid tumors. These antibodies inhibited the growth of the myeloid-monocytic stem cell, the CFU-C, and reacted with a minor population of normal bone marrow cells by immunofluorescence. Both D5D6 and CH5 have potential utility for the differential diagnosis of acute leukemia, for the detection of early leukemic relapse, and for enrichment of human stem cells.

An antigen expressed by cells of the myelo-monocytic lineage.

We describe an antigen(s) characterized by a heteroantiserum raised in rabbits against mature human granulocytes. This antigen was found on neutrophils, monocytes, platelets, acute and chronic myelocytic leukemia cells and on granulocyte-macrophage progenitor cells grown in agar. It was not found on lymphocytes, eosinophils, erythrocytes, or erythroid progenitor cells. On the basis of tissue distribution and absorption studies, the antigen (tentatively designated the "myelo-monocytic" antigen) is distinct from antigens previously identified on human neutrophils. Restriction of the "myelo-monocytic" antigen to normal and malignant cells of the myelo-monocytic series suggests that it may represent a normal differentiation antigen of the myelo-monocytic lineage.

Immunologic classification of childhood acute lymphocytic leukemia.

Immunologic approaches to the classification of acute lymphocytic leukemia (ALL) have led to a new awareness of the heterogeneity of this disease. Surface membrane markers including surface membrane immunoglobulin, complement receptors, and sheep erythrocyte (E) receptors have demonstrated at least three subtypes of ALL, which include non-B, non-T, ALL, T-ALL, and B-ALL. In addition, hetero-antisera to Ia-like antigens and ALL-associated antigens have been used to positively identify non-B, non-T ALL, which was previously a diagnosis of exclusion. This paper reports 17 cases of childhood ALL whose lymphoblasts were studied for surface membrane immunoglobulin, sheep erythrocyte receptors, and the presence of four antigens detected by well-characterized heteroantisera. Every non-B, non-T lymphoblast was positively identified by the Ia-like antiserum and/or the ALL antiserum. One T lymphoblast was identified by E receptors and the T antiserum, whereas two did not have E receptors but did react to the T antiserum. None of these three T lymphoblasts reacted with the Ia-like antiserum.

Immunologic classification of acute lymphoblastic leukemia. Implications for normal lymphoid differentiation.

Acute lymphoblastic leukemia (ALL) is a heterogeneous disease as defined by clinical characteristics and immunologic techniques. The standard cell surface markers are sheep erythrocyte receptors for T lymphocytes and surface membrane immunoglobulin for B lymphocytes. Utilizing these markers, three subtypes of ALL designated T-ALL, B-ALL and non-B, non-T or null ALL have been defined. We have studied 70 patients with ALL utilizing these standard cell surface markers. In addition, we have further subclassified these patients by testing each cell for an ALL-associated antigen, the la-like antigen, and thymocyte antigen(s) all defined by well-characterized antisera. We can define 12 subgroups of ALL by their surface antigenic characteristics. These subgroups may have relevance to the clinical expression of disease and may define identifiable stages of normal lymphocyte development.

Characterization of three different human T cell membrane antigens, two being present on T lymphocyte subpopulations.

Heteroantisera were raised in rabbits to thymocytes, HSB2 cells, and Sezary cells. Following absorption with Ia-positive leukemia cells, these sera appeared to be specific for different T cell antigens. Both the anti-HSB2 and the anti-Sezary sera reacted with approximately 50% and the antithymocyte serum with 100% of normal peripheral blood T lymphocytes. None of the sera reacted with B cells. The apparent molecular weights of the antigens being detected were determined by immunoprecipitation followed by SDS polyacrylamide gel electrophoresis. A dimer of 170,000 daltons consisting of two similar 85,000-dalton polypeptide chains was immunoprecipitated by the anti-HSB2 serum whereas single polypeptides of 53,000 and 64,000 daltons were immunoprecipitated by the anti-Sezary and antithymocyte sera, respectively.

Dual B and T markers in acute and chronic lymphocytic leukemia.

Leukemic cells from 20 patients with chronic lymphocytic leukemia (CLL) and 60 patients with acute lymphocytic leukemia (ALL) were studied for T- and B-lymphocyte cell surface membrane markers. B-cell markers included surface membrane immunoglobulin, erythrocyte-antibody complement rosette formation, and B-cell (a-like or HLA-DR) antigens detected by a B-cell antiserum. T-cell markers included spontaneous sheep red blood cell rosette formation and a cytotoxic reaction to a specific T-cell antiserum. Seven patients with CLL and two with ALL had dual B and T markers. We propose that dual B- and T-cell markers are more common in CLL and ALL patients than previously reported. With newer and more sensitive tests for identification of B and T cells, this observation may be recognized more frequently.

B lymphocyte antigens in the differential diagnosis of human neoplasia.

Human B lymphocyte antigens (HBLA) were detected with fluorescent-labeled antibodies on malignant cells of 102 patients with Hodgkin disease and other lymphomas, plasma cell myeloma, and nonlymphoreticular neoplasms including carcinomas of the breast, lung, and ovary, soft tissue sarcomas, and neuroblastoma. HBLA were present in Hodgkin disease and other lymphomas of B cell or histiocyte derivation. They were absent in plasma cell myeloma and nonlymphoreticular neoplasms. Absorption studies revealed that malignant T cells had smaller amounts of HBLA, usually not detected by immunofluorescence. Expression of HBLA was dependent on both cell differentiation and origin. Detection of HBLA enabled immunologic distinction of Reed-Sternberg and other lymphoma cells from morphologically similar cells of nonlymphoreticular origin. The rapidity, reproducibility, and economy of the immunofluorescence test make this a useful clinical tool for the differential diagnosis of lymphoma from other malignant disorders in man.

Immunofluorescent method for positive identification of null-cell type acute lymphocytic leukemias: use of heterologous antiserum.

Acute lymphoblastic leukemia (ALL) patients could be subclassified into two groups depending on whether or not their leukemia cells expressed a B-lymphocyte antigen. The antigen was detected by an indirect immunofluorescence test using rabbit antisera. In the positive group, consisting of 26 of 32 patients, the leukemia cells were of the "null"-cell type, i.e., they did not appear to express currently recognized T- and -b-cell markers. Absroption studies indicated that the positive null-cell group expressed a common antigen which was not expressed on the negative group. Of the 6 negative cases,, 5 expressed complement receptors and 3 expressed T-cell markers. The negative group was also characterized by high white cell counts and the pressence of a mediastinal mass.

Soluble HLA antigens present in normal human serum.

HLA antigens have been detected in normal human serum by inhibition of monospecific HLA antisera in the complement-dependent cytotoxicity test. The antigens present in serum were the same as those found on the donor's lymphocytes. HLA-A9 was unique in that it was present in serum at inhibitory titers of 1:8 to 1:32 whereas the majority of A-and B-locus antigens were present at titers of 1:4 or less. Serum HLA-A9 antigens were found in the high density lipoprotein fraction with a molecular weight greater than 200,000. In the presence of detergent they have an apparent molecular weight of 86,000 which is reduced to 46,000 when treated with papain. These values are similar to those obtained for detergent-solubilized cellular HLA antigens.

Group-specific human granulocyte antigens on a chronic myelogenous leukemia cell line with a Philadelphia chromosome marker.

Group-specific human granulocyte antigens are serologically detectable with granulocytotoxic-positive human alloantisera on a cell line, K562, of chronic myelogenous leukemia origin which bears a Philadelphia chromosomal marker. The same cell line lacks serologically detectable HLA, B2 microglobulin, and B-lymphocyte antigens. Granulocyte antigens are important cell markers for cell lines of suspected myeloid lineage.

Blocking of HLA and B lymphocyte alloantigens with F (ab')2 fragments of rabbit antibodies.

F(ab')2 fragments of rabbit antibodies specific for human B lymphocytes will block human anti-B lymphocyte alloantibodies. F(ab')2 of anti-beta2m will block HLA alloantibodies. The F(ab')2 fragments can be used to improve the typing of B cell lines and certain leukemia cells which express both HLA and B lymphocyte alloantigens. Pretreatment of these cells with anti-beta2m F(ab')2 preparations allows the observation of B cell alloantigen reactions independently of HLA reactions. Similar use of rabbit anti-B cell F (ab')2 allows HLA reactions to be observed free from the "extra reactions" caused by B alloantibodies that are present in many HLA typing sera.

Isolation and characterization of human B cell alloantigens.

Human B lymphocyte alloantigens were solubilized from malignant spleen cell membranes from lymphoma patients by detergent treatment or papain digestion. Antigenic activity was detected by inhibition of cytotoxicity of rabbit and human anti-B cell antisera. Extracts from one patient (AC) specifically inhibited only B alloantisera of B groups 1 and 3; sera from B groups 4 and 5 were not inhibited. Inhibitory titers were greater than 1:400 against positive sera and less than 1:2 against negative sera. The papain extracts were purified by QAE-A50 Sephadex and Concanavalin A Sepharose 4B chromatography, and preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis (PAGE) under nondenaturing conditions. By testing gel slices for antigenic activity the m.w. of the papain-extracted B antigen was 58,000 whereas the detergent-extracted antigen was 65,000. Antigenic activity detected by rabbit and human antisera copurify together, suggesting that these sera were reacting with the same molecules. Immunoprecipitation of sodium deoxycholate-solubilized extracts of a cultured human B lymphoid cell line 8392 with the rabbit anti-B cell antisera revealed two B cell polypeptides of apparent m.w. 27,000 and 35,000 that were not found on the paired T line 8402. It is suggested that these polypeptides might be subunits of the 65,000 dalton native B lymphocyte alloantigen. The polypeptide subunits do not appear to be linked covalently by disulfide bonds.