Influence of recombinant interleukin-2 and alpha- and gamma-interferons on the induction of novel class I antigens

Previous results obtained in our laboratory showed that novel class I antigens, closely related to HLA-A (TM antigen related to HLA-A9 and GO antigen related to HLA-A24), were expressed on activated HLA-A9 or HLA-A24 peripheral blood lymphocytes (PBL), whatever activation factor was used [mitogenic stimulation (PHA, PWM, Con A), EBV transformation or alloactivation], but not on resting T and B lymphocytes. These antigens were also expressed on HLA-A9 or HLA-A24 common acute lymphoblastic leukaemias (cALL) and some 'immature' chronic lymphocytic leukaemias (B-CLL), but not in hairy cell leukaemias (HCL), most of the B-CLL, acute myeloblastic leukaemias (AML) and acute myelomonoblastic leukaemias (AMoL). In order to investigate the regulation mechanisms of these novel class I antigens, PBL and different leukaemic cell types were treated in vitro with recombinant IL-2 (rIL-2) and alpha and gamma-interferon (rIFN). Our results showed that without previous activation, but after culture with rIL-2, TM and GO antigens could be induced in HLA-A9 or HLA-A24 PBL and enhanced in HLA-A9 or HLA-A24 B-CLL cases, whereas no expression was found in HLA-A9 or HLA-A24 HCL, T-ALL, AML or AMoL. After culture with rINFs, the expression of TM and GO antigens could be induced on HLA-A9 PBL and all HLA-A9 leukaemic cell varieties. Our results support the hypothesis that the expression of class I antigens is induced at an early stage of the cell cycle.     

Normal Human Sera Cytotoxic to Cells of Human Acute Leukemia

Eight human sera from healthy individuals with no history of immunization with human transplantation antigens have demonstrated complement dependent cytotoxicity to cells of some patients with active acute leukemia. The antigen(s) detected by these sera are absent from normal and remission lymphocytes and appear most often in ALL patients with a high peripheral lymphoblast count. Some B and T lymphoblastoid cell lines carry the antigen(s) as evidenced by their ability to react with and absorb the antileukemia activity. The data support the existence of at least two overlapping specificities detected by these antileukemia sera. The leukemia antigen(s) show no strong correlation with any known HLA antigen. These observations provide evidence for a human leukemia blast associated antigen or set of antigens which may be immunogenic in man. Their relationship to normal HLA antigens of loci other than A, B or C is unknown.     

Expression of HLA-DR and common acute lymphoblastic leukemia antigens on glioma cells

The expression on several established human glioma cell lines of two well-defined differentiation antigens, HLA-DR and the common acute lymphoblastic leukemia antigen (CALLA) has been demonstrated. Rabbit anti-CALLA antiserum and monoclonal anti-la antibodies specifically lysed glioma cells in the presence of complement. Absorption of anti-CALLA antiserum and anti-Ia antibodies by glioma cells abolished their cytotoxicity against blasts isolated from a common acute lymphoblastic leukemia. Immunoprecipitation of solubilized glioma cells by monoclonal anti-Ia antibodies revealed two polypeptide chains of 28 and 33 kDa, whereas the anti-CALLA antiserum precipitated a single polypeptide chain of 100 kDa.     

Immunophenotypic and immunogenotypic analyses of acute leukemia and lymphoblastic lymphoma expressing myeloid and lymphoid antigens]

By using monoclonal antibodies against lymphoid and myeloid differentiation antigens, surface marker analysis was performed on the tumor cells from 42 patients with acute leukemia and lymphoblastic lymphoma. Nine (21%) of 42 cases were diagnosed biphenotypic leukemia. Two (17%) of the 12 patients with acute myeloid leukemia, four (18%) of 22 with acute lymphocytic leukemia and three (38%) of 8 with lymphoblastic lymphoma expressed both lymphoid and myeloid antigens. Tumor cells from six patients expressed both T-cell and myeloid antigens, and those from three other expressed both B-cell and myeloid antigens. Southern blot analysis was performed on the DNA from four patients with biphenotypic leukemia cells expressing T-cell and myeloid antigens. DNA from one patient showed clonal rearrangement of the immunoglobulin heavy chain (IgH) gene, and that from one other showed clonal rearrangement of both IgH gene and T-cell receptor beta-chain gene. DNA from two other patients showed a germline configuration of both genes. These results indicate that biphenotypic leukemia, especially T-cell and myeloid phenotype, is not so rare in acute leukemia and lymphoblastic lymphoma. The results of immunogenotypic analysis were not consistent with those of immunophenotypic analysis in biphenotypic leukemia.     

Identification of murine leukemia viral antigens detected on mouse cells by H-2 alloantisera.

H-2 alloantisera have been previously reported to contain antibodies against murine leukemia viral antigens, but the nature of the viral antigens on mouse cells which interact with these antibodies has not been established. We have found that H-2 alloantisera recognize components of molecular weight 70 000-80 000 mouse lymphocytes and leukemia cells. These components were also detected by a goat antiserum against the murine leukemia virus (MuLV) glycoprotein (gp 70) and are therefore closely related to or identical with that viral protein. Although most H-2 alloantisera detected gp 70-like molecules on lymphocytes and leukemia cells from a great variety of mouse strains, only one H-2 alloantiserum was found to interact with a gp 70 component on cells from C57BL/10 and C57BL/6 mice. Animals such as C57BL/10 mice that lacked the component reacting with most H-2 alloantisera showed increased serum levels of anti-MuLV antibodies after injection of B10.A spleen cells having a gp 70 component detectable by other H-2 alloantisera. In contrast, strains with cells reactive to antiviral antibodies in the H-2 alloantisera had low responses to MuLV antigens after a similar immunization procedure. Serum levels of anti-MuLV antibodies in both groups of mice, however, were increased after injection of Freund's adjuvant. These observations suggest that anti-MuLV antibodies in mouse alloantisera may arise from a response to viral antigens on the immunizing cells and general stimulation of the immune system.     

Human monocyte-histiocyte differentiation antigens identified by monoclonal antibodies

Two distinct differentiation antigens of human myelomonocytic cells are defined using murine monoclonal antibodies. The antigens recognized by antibodies 20.2 and 20.3 are expressed by all cells of the monocyte lineage in both peripheral blood and bone marrow. Cell-sorting experiments demonstrated that histiocytes and immature bone marrow cells with detectable alpha-naphthyl butyrate esterase activity also express both antigens. Within cells of other lineages, the antigens had distinct patterns of expression. Immature myeloid cells were 20.2 negative, but 20.3 positive; whereas mature myeloid cells were 20.2 positive, but 20.3 negative. Nucleated erythroid cells and platelets expressed only the 20.3 antigen. These results indicate that myeloid and monocytic cells share common differentiation antigens with cells of the erythroid and megakaryocytic lineages. The 20.2 and 20.3 antibodies reacted with the leukemic cells from some patients with acute nonlymphocytic leukemia (FAB, M1-M5) and with some cell lines derived from patients with nonlymphocytic leukemia, but not with blast cells from patients with lymphoid leukemia or with lymphoid leukemic cell lines. These antibodies may prove useful in studying the differentiation of bone marrow stem cells, in defining the cellular origins and classification of leukemias, and in the identification of distinct prognostic subgroups of acute nonlymphocytic leukemia.     

Characterization of a monoclonal antibody (A12) that defines a human acute lymphoblastic leukemia-associated differentiation antigen

A human leukemia-associated differentiation antigen has been identified by a monoclonal antibody (A12) raised to the lymphoblastoid cell line NALM-1. The A12 antigen was expressed on the surface of leukemic cells from patients with common acute lymphoblastic leukemia (c-ALL) as well as on cells of the hematopoietic cell lines NALM-1, Reh-6, Raji, Daudi, CEM, and 8402 as determined by an antibody-binding radioimmunoassay, as well as by indirect immunofluorescence and FACS analysis. This antigen was not detected on normal blood lymphocytes, normal bone-marrow cells or leukemic cells from patients with acute myeloid leukemia (AML). The A12 antigen had an apparent molecular weight of 100 kD as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and appeared to be related to if not identical with the acute lymphoblastic leukemia antigen CALLA described by others. Cross-blocking experiments indicated that preincubation of NALM-1 cells with antibody A12 or J5 (anti-CALLA) could block subsequent binding of 125I-labeled A12 and J5 antibody. These results suggest that the two monoclonal antibodies recognize identical or closely located antigenic sites. The surface membrane expression of A12 antigen in NALM-1 cells was modulated when the cells were cultured in the presence of A12 antibody. Under these conditions, the expression of Ia antigens was unaffected. Re-expression of A12 antigen occurred within 24 h after transfer of the modulated cells into medium devoid of monoclonal antibody.     

AFTR: a fifth human B-cell-specific surface antigen

We report the identification and characterization of a fifth B-cell-specific surface antigen (AFTR) detected by three murine monoclonal antibodies. AFTR is present on all malignant human B-cell lines tested except those of plasma cells and on malignant B cells from 49/49 patients, including those with pre-B-cell acute lymphoblastic leukemia. Similarly, AFTR is found on all CD19+ B cells from normal bone marrow, peripheral blood, and tonsil, although it is only weakly expressed on Epstein-Barr virus-transformed normal B cells. AFTR is not expressed on T lymphocytes, thymocytes, myelocytes, monocytes, granulocytes, or erythrocytes, or on endothelial or epithelial cells. Isolation of AFTR from surface-iodinated cells by immune precipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals bands at 38 and 42 kd in the acute lymphoblastic leukemia cell lines Laz 221 and NALM-6. Molecular weights of these two bands vary slightly between different B-cell populations but change little under reducing and nonreducing conditions. In contrast, relative intensities of these bands vary considerably between different B-cell populations. Like the B-cell-specific surface immunoglobulin and CD19 antigens, but unlike CD20, expression of AFTR is specifically reduced or modulated when cells are incubated with monoclonal antibodies at 37 degrees C. The molecular weight, behavior, and cellular distribution of AFTR are distinct from those of known B-cell surface antigens. One of these MoAbs (J3-109) is a prototype reagent for the CD72 antigen cluster.     

Occurrence of Specific Phytohaemagglutinin- Reactive Immunoglobulins in the Sera of Certain Individuals

While investigating sera for possible antibodies to TL-like determinants, multiparous sera were selected that had specific cytotoxic reactivity against phytohaemagglutinin (PHA)-activated lymphocytes of certain individuals and were negative against unstimulated T or B cells from the same donor. The reactivity patterns were not correlated to any HLA specificities. However, in an indirect immunofluorescence assay with flow cytometry, these same sera had strong specific monomorphic reactivity to PHA-activated lymphocytes from all the individuals in the panel. Unstimulated cells remained negative. In contrast, other human sera lacked reactivity with PHA-stimulated lymphocytes. The addition of PHA to fresh lymphocytes followed by incubation at 4 degrees C for 30 min resulted in the same pattern of reactive and non-reactive sera. When incubated with PHA, different cell types, including 0- erythrocytes and a murine lymphoid cell line, reacted similarly with the sera. Using plates coated directly with PHA-E, L, and P in a cell-free ELISA system. PHA-specific sera reacted specifically with PHA-coated wells. The anti-PHA activity was removed by absorption with 0- erythrocytes coated with PHA without affecting the titre of the anti-HLA antibody in the same serum. These findings suggested that IgG molecules in certain sera react directly with residual PHA bound to the cell surface and not necessarily with molecules of cellular origin induced by exposure to PHA, complicating the search for antibodies specific for T-cell activation antigens.     

Monoclonal antibodies against human erythrocyte membrane antigens and their reactivities with hematopoietic cells

Four monoclonal antibodies against human erythrocyte membrane antigens were established. The antigenic determinants of KOR-E1, E3, E6 were Pr1h antigen, Wrb antigen, and the trypsin sensitive portion of glycophorin A (EnaTS) respectively. The antigen recognized by KOR-E4 could not be determined. The reactivities of these antibodies with normal hematopoietic cells, malignant hematopoietic cell lines (N = 31), and fresh leukemic cells obtained from 128 patients with various types of leukemias were studied. All antibodies reacted only with erythrocytes among peripheral blood cells, and also KOR-E6 reacted only with erythroid cells among bone marrow cells. KOR-E3 had no reactivity with any cell lines examined, and KOR-E1 and KOR-E4 were reactive with some lymphoid cell lines. However, KOR-E6 had specific reactivities with erythroid (HEL, K562), megakaryocytic (CMK-1), multiphenotypic (KOPM-28), and basophilic (KU-812) cell lines. The antigen (glycophorin A) recognized by KOR-E6 was expressed on a small population of mononuclear cells separated from acute lymphoblastic leukemia (3/70), acute myelogenous leukemia (2/12), monosomy 7-myeloproliferative disorder (1/1), juvenile CML (1/1), and transient myeloproliferative disorder with Down's syndrome (4/12), although it could not be determined whether these cells were leukemic cells or not. KOR-E6 was reactive with a large population of leukemic blasts in erythroleukemia (2/2) and acute megakaryoblastic leukemia (3/6). Thus, KOR-E6 appears to be an erythroid marker of leukemic cells.     

Induction of non-specific human suppressor cells in vitro by defined Onchocerca volvulus antigens

In the present study the activity of Onchocerca volvulus total antigens (OVA) on the proliferative response of human lymphocytes from healthy donors was investigated. Normal human lymphocytes were cultured for 72 h with polyclonal activators, phytohaemagglutinin (PHA) or pokeweed mitogen (PWM), together with OVA, and thymidine uptake was measured. The proliferation of normal lymphocytes was markedly inhibited by the parasite antigens. The inhibition was not attributable to a cytotoxic effect of OVA, since at least 80% viable cells were recovered at the end of cell cultures. The inhibition was not abrogated by removal of the adherent cell population. The passage of OVA through immunosorbent column containing human antibodies to O. volvulus significantly reduced the suppressive activity of OVA. The in vitro response to mitogens (PHA, PWM) of normal human lymphocytes was suppressed by co-culture with allogeneic or syngeneic lymphocytes, which had previously been exposed for 72 h to OVA. The suppression was not abrogated by the irradiation of mononuclear cells before the exposure to OVA. A significant reduction of the suppression was however observed when OVA pre-treated cells were T cell depleted by centrifugation of E rosettes. Thus, parasite antigens, which are recognized by antibodies in infected human sera may participate in the modulation of the cellular immune response during O. volvulus infection by inducing suppressor cells. This suppression could in addition contribute to the survival of the parasite in its host.     

Cell surface antigens and function of monocytes and a monocyte-like cell line before and after infection with HIV

The human immunodeficiency virus (HIV-1) preferentially infects cells that express the CD4 molecule, including monocytes and cells of the monocyte lineage. The monocyte-like cell line U937 and monocytes isolated from peripheral blood lymphocytes (PBL) were infected with HIV-1. Cell surface antigen expression was determined in infected and noninfected cells as was the ability to stimulate in mixed lymphocyte reaction. The CD4 antigen decreased in infected cells U937 and PBL monocytes. MHC class II antigens HLA-DR, HLA-DQ, and HLA-DP increased in HIV-1 infected U937 cells. In infected PBL-derived monocytes, HLA-DR increased, HLA-DQ decreased, and HLA-DP was unchanged. Infected U937 and PBL monocytes were capable of stimulating allogeneic lymphocytes, thus demonstrating retention of the alloantigen presentation function of HIV-1-infected monocytes.     

Frequent expression of myeloid antigen (CD13) on immature T cells in culture

Leukemic cells from 12 patients with lymphoblastic lymphoma (LBL) and T cell acute lymphoblastic leukemia (T-ALL) were studied to determine the inducibility of myeloid antigens in culture in the presence and absence of 12-O-tetradecanoylphorbol-13-acetate (TPA) in association with discrete phenotypic and genotypic analyses on these cells. The investigation revealed that leukemic cells corresponding to common or mature thymocytes were never induced to express any myeloid antigens, and showed rearrangements of T cell antigen receptor (TcR) beta and gamma chain genes. Concomitant examination on leukemic cells from mature T cell malignancies, including adult T cell leukemia (ATL), T cell chronic lymphocytic leukemia (T-CLL) and T cell non-Hodgkin's lymphoma (T-NHL), also failed to express myeloid antigens in culture. By contrast, one of the panmyeloid antigens, CD13 (MCS-2) antigen was induced on leukemic cells corresponding to early thymocytes in 5 out of 7 cases in TPA-added culture and in 3 cases even in TPA-free culture. All of these CD13 antigen inducible cases exhibited the germ line configurations of TcR beta and gamma chain genes except for one case of T-ALL with sole TcR gamma chain gene rearrangement. These findings suggest that primitive T cells, still not undergoing TcR gene rearrangements, retain the characteristics of multipotent progenitor cells to possess different lineage markers and are able to express myeloid antigen not exceptionally. Both phenotypically and genotypically immature thymocytes are considered to be less restricted in the differentiation pathway of hematopoietic cells committed to T cell lineage.     

Histocompatibility antigens acting as helper determinants for tumor-associated antigens of murine lymphosarcoma

The mechanism of effectiveness of allogeneic versus syngeneic immunization in mounting a humoral specific immune response against tumor-associated antigens of murine urethan-induced thymic lymphosarcomas was studied. C57BL mice immunized with untreated or blocked C3Hf lymphoma cells yielded sera with complement-dependent cytotoxicity for C57BL lymphosarcomas, whereas they did not respond to repeated injections of blocked syngeniec lymphoma cells, indicating a helper activity of alien alloantigens in the reflex of tumor antigens. Thymectomized, lethally-irradiated and T, but not B-reconstituted mice produced active sera, suggesting a T-dependent response. The anti-tumor activity of the serum ran parallel with the anti-histocompatibility one. The tumor-associated and the alien histocompatibility determinants had to be present on the same cell membrane. In addition, active sera were obtained when immunizing syngeneic lymphoma cell inocula were followed by allogeneic normal immunocompetent cells. It is suggested that either a helper mechanism with B and T cells cooperating in recognizing haptenic tumor-associated and carrier histocompatibility antigens or a graft-versus-host reaction determining an abnormal induction of the immune system is needed in order to detect the weak tumor-associated antigens of urethan-induced lymphomas.     

Cellular antigens in nephroblastoma: identification with monoclonal antibodies which recognize hemopoietic cells

The correlation between expression of differentiation antigens and morphogenesis was examined in 11 human nephroblastomas using monoclonal antibodies which recognize both hemopoietic and renal cells. Analysis of frozen tissue sections by indirect immunofluorescence revealed that blastema cells and some tubules were recognized by BA-1 and OKB2, which identify unstimulated B cells and granulocytes. Stroma, some tubules, and focal blastema were recognized by BA-2, which identifies a 24-kDa antigen on leukemic cells and platelets. Mature epithelium in glomerular bodies was identified by C3bR and J5, which recognize CR1 and the common acute lymphoblastic leukemia antigen, respectively. Tissue sections from a clear cell sarcoma and a mesoblastic nephroma were notably reactive only with BA-2. These observations demonstrate that the relationship between antigen expression and morphology in nephroblastomas is similar to that observed in fetal kidney and suggest that expression of these cell surface antigens may have a role in morphogenesis.     

The response of cultured human kidney capillary endothelium to immunologic stimuli

We have developed in culture for the first time one of the actual target cells in kidney rejection by establishing long-term cultures of human kidney capillary endothelium and have begun to explore their response to immunologic stimuli. Microvascular endothelial cells were obtained from the human kidney cortex by mechanical separation, enzyme digestion, and density gradient centrifugation. The cells were exposed to 1% PHA, a PHA-stimulated PBL supernatant, human and mouse IL-1 and human gamma-interferon. They were stained with FITC-conjugated monoclonal antibodies specific for beta 2-microglobulin, HLA-DR, and DQ and analyzed by flow cytometry (FACS-II). HKCE cells express HLA-ABC antigens during growth and confluent phases but do not express DR or DQ antigens. Only the PHA-stimulated PBL supernatant and gamma-interferon induced class II antigen expression. A change in cellular morphology was noted after 72 hr of incubation in the presence of the lectin-stimulated PBL supernatant. Our findings show that microvascular endothelial cells express selected Ia antigens only after immunologic induction. This is in agreement with studies using umbilical and aortic endothelial cells. These data suggest that a dynamic interaction takes place between activated lymphocytes whose products induce expression of class II antigens and human kidney endothelial cells.     

Detection of B-lymphocyte (B-cell)-associated antigens on human leukemic lymphocytes. Masking of membrane antigens.

An indirect immunofluorescent technic has been used for rabbit antisera (anti-EP) that demonstrated complement-dependent cytotoxicity against human leukemic lymphocytes but not against normal blood lymphocytes. With the immunofluorescent technic, the antisera were found to react with 2--23% of normal blood lymphocytes. Simultaneous staining of normal cells with anti-EP and for surface immunoglobulins (SIg) on bone-marrow-derived B-cells showed that the proportions of stained cells were similar to percentages of cells stained by anti-EP alone or for SIg alone. The percentage of anti-EP reactive cells also approximated the percentages of cells reactive to a known antiserum to human B-cell associated, or Ia-like, antigens. The anti-EP reacted with lymphoblastoid cells from two B-cell lines lacking the Epstein-Barr viral genome. The antigens detected by anti-EP probably are B-cell-associated. The anti-EP intensely stained neoplastic cells of acute or chronic lymphocytic leukemia and lymphosarcoma-cell leukemia. Cells from two patients with chronic lymphocytic leukemia and from two patients with acute lymphocytic leukemia showed increased intensity of anti-EP staining and/or increased proportions of stained cells following overnight incubation in culture medium, compared with the preincubation samples. This observation suggests the presence of a "blocking component(s)" on cell surfaces, which interfered with anti-EP reactivity. After overnight incubation, the component might have been removed from the antigenic sites on cell surfaces. Further studies of leukemia lymphocytes using anti-EP for the cell-bound "blocking component" may reveal important pathogenetic mechanisms.     

Production of human--human hybridomas secreting antibody to sheep erythrocytes after in vitro immunization of peripheral blood lymphocytes

This report describes the formation of human hybridomas after in vitro immunization of peripheral blood lymphocytes (PBL) with an antigen and fusion of the stimulated lymphocytes with a HAT-sensitive human myeloma cell line, RPMI 8226. PBL were stimulated in vitro with sheep erythrocytes (SRBC) plus fresh human serum. PBL of some donors produced anti-SRBC antibody when they were cultured at 2 X 10(6) cells per well in a 24-well plate with the antigen plus fresh human serum for 7 days. Although lymphocytes of some donors were "low-responders" under the above conditions, they responded to SRBC when they were cultured with not only the antigen plus fresh human serum but also with the culture supernatant obtained after phytohemagglutinin (PHA) stimulation of a mixture of PBL from two donors (MLC-PHA sup). The cells sensitized by this procedure were fused with RPMI 8226 cells. Hybrids secreting IgM or IgG anti-SRBC antibodies were obtained. Additionally the ratio of total IgG-producing hybridomas to IgM-producing ones was higher when the MLC-PHA sup was used at the time of the in vitro immunization.     

Chromosome mapping of cell membrane antigens expressed on activated B cells

Hybrids formed by fusion of either human acute lymphoblastic or chronic lymphocytic leukemia cells and the mouse myeloma P3.X63.Ag8/653 have been used to show that the expression of two cell surface antigens, Bp37 and p76, associated with B cell activation and detected by the monoclonal antibodies BB1 and BB2, respectively, segregate with human chromosomes 12 and 19, respectively. Another antigen expressed on activated B cells (p24) also maps to chromosome 12 (Katz et al., Eur. J. Immunol. 1984. 13: 1008) which is of interest in the light of the frequent involvement of this chromosome in certain B cell leukemias and lymphomas.     

All monocyte antigens are not expressed on renal endothelium

Recently a tissue restricted antigen system, which is expressed on endothelial cells and monocytes (E-M antigens) but not lymphocytes, has been associated with kidney graft rejection. In screening sera from recipients of kidney, bone marrow or skin grafts for possible reactivity with endothelial cell antigens, we have found that all (13 of 13) endothelial reactive sera also reacted with monocytes, but that many (21 of 34) monocyte reactive sera did not react with endothelial cells. Additionally, one well-defined monoclonal antibody (M1/70), which was cytotoxic for human monocytes, neither stained renal endothelium nor was absorbed by renal endothelium when perfused through a human kidney. Thus, not all monocyte antigens appear to be expressed in high concentrations on renal vascular endothelium. This may explain why monocyte reactive antibodies do not always correlate with kidney graft rejection.