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Phenotypic and functional characteristics of AIDS-related lymphoma cell lines
324
Malignancies of the immune system
subgmups and none of the samples eXpreSSed the VH2 or VK4 subgroup. The panel of the paraproteins rarely expressed the probed VH or &associated CRI. These data suggest that the IgV genes may not be randomly expressed in the malignant cells of Iranian MM patients and these genes might be highly mutated, as evidenced by the lack of expression of the probed CRI.
P.5.12.03
V region genes encoding an Immunocytoma-secreted antl-phosphollpid autoantibody
M. Blay, C. Benito, J.C. Reverter I, E. Campo 2, F. Bosch 3, T. Gallart. ’ Services of Immunology: Hemorhempy and Hemostasis, Hospital Clinic Universitari, Barcelona, Spain, 2Services of Pathology, Hospkal Clinic Universitari, Barcelona, Spain, 3Services of Clinical Hematology; _. Hospital Clinic Universitari, Sarcelona, Spain Introductfon: The study of B cell malignancies secreting circulating autoantibodies may provide information about ihe possibility that normal Eicells producing natural IgM autoantibodies with unmutated VH and VL lg region genes, could be prone to be involved in autoimmune responses of pathogenic IgG autoantibodies and/or to undergo malignant transformation. Therefore studies were performed in a patient with spleen immunocytoma associated with serum monoclonal IgMA and circulating anti-phospholipid (AP) antibody. Materiel and Methods: Fusion of patient’s splenccytes with F3B6 heteromyeloma cells was done to obtain heterohybridomas secreting AP antibodies. ELISA and phospholipid-dependent in vitro coagulation tests were used to detect AP antibodies. To analyze rearranm VH and VL Ig region genes in malignant spleen B cells and in splenocyte-derived heterohybridomas, a PCR-based approach was employed. Isoelectrofocusing, flow cytometry and immunohistochemistry were also performed. Resutts: Circulating anti-cardiolipin (AC) antibody and lupus anticoagulant (LA) were found: and both antibody activities resided in the serum monoclonal IgMA. Splenocyte-derived heterohybrfdoma clones producing IgMA with both AC and LA activities were obtained. PCR analysis, “fingetptinting”. and sequencing of rearranged VH and VL Ig region genes in the DNA from splenocytes and cDNA from heterohybrfdoma clones demonstrated the clonal identity between heterohybridoma clones and spleen tumor B cells. Moreover, the isolectric mobility of separated Ig chains of both serum and heterohybtidoma-secreted IgMA was identical. The AC and LA activities of both serum and heterohybrtdomasecreted IgMA did not reauire Bn-alvcourotein I co-factor. The VH and VL aene -__ segments-were homologbus (97%) to ihe reported germ-line gene segments, VH3-30 (1.Qlll, DP-49) and VA3.1 (IGLV3S1, DPL16), respectivelv. Conclurlon: Data bemonstrate.that serum IgMA &th AC/LA antibody activities was produced by spleen tumor B cell clone. The VH and VA gene segments encoding this autoantibody have been obsetved frequentty in other autoantibodies. In contrast to what is usually thought, data clearly show that AC and LA antibody activities can reside in the same lg molecule and can be cofactor-independent. Cofactor-independent AC andlor IA antibodies such as those found in infections are thought to lack autoimmune pathogenic properties, a notion consistent with the absence of AP-related clinical manifestations in the patient.
24 June 1997 - Poster presentations
increase of IL10 production in HBL2 (from 150 to 3440 pg/ml), HBLl (from 550 to 2137 pa/ml) and PA662 (from 460 to 2550 psy’ml). Conclueions: Our data suggest that membrane CD40 in AIDS SNCCL may be a key element in the regulation of their pathophysidogy by regulating membrane antigen expression and cytoklne production. BCBL cell lines have their own phenotype, characterized by absence of lineage specific B cell antigens usually involved in the transmission of stimulatory signals, but constitutlvely express an “activated” B cell phenotype. Which molecules drive their proliferation and cytokine production remains to be established.
P.5.12.05
CM+ B cells from chronic lymphocytlc leukemla lnhlblt IgG production by autologous bone marrow plasma cells
A. Sampalo, G. Navas. J.A. Brfeva. Servicio de Inmunologia, Hospital Universitatfo Puerta de/ Mar, Cadiz, Spain Introduction: B-Chronic Lymphocytic Leukemia (CLL) is defined as a clonal proliferation of CD5+ B lymphocytes. Hv~ammaalobulinemia is a common feature in CLL, but its p&h&en& mechanism remains uncertain. The aim of this study was to investigate the possible effect of CD5+ B cells on IgG secretion bv autoloaous bone marrow olasma cells. Material and Methods: i5 B-CLL patients were included in the study. CD5+ B calls were purified from peripheral blood (CD5+ 8). and this fraction was removed from bone mar& samples (CDS-‘BM) by using suited rnAbs and magnetic beads. CD5+ B fraction consisted of more than 95% CDlQ+CD5+ cells. CD5- BM contained less than 5% CDlQ+CD5+ cells. CD5+ B cells were stimulated or not with PMA (10 @ml for 16 h), and cocultured with autologous CD5- MO cells. After 7 davs of culture. the IaG oroduction was measured by ELISA. Results are expressed as percentagesof ihe IgG secretion observed in the corresponding CD5- BM cell culture alone (control value). Reautts: Unsttmulated CD5+ B cells reduced autoloaous CD5- BM IgG production to a 66.6% f 21 (mean f SD). This inhibitory effect was increased by PMA-preincubation (52.6% f 15). The inhibition was dependent on cellular contact, since the separation of CD5+ B and CD5- MO fractions in a transwell system reversed the effect (IgG secretion returned to 92% f 11). The molecules involved in this cellular interaction were studied by adding certain blocking mAb. rnAb against CD5, CD72 and CD95 reversed the present inhibition by PMA-CD5+ B cells (97% f 6. 76% f 13 and 62% f 12. of control value, respectively). Moreoier. CD95 was expressed on plasma ceils from B-CLL BM cells. mAb against other adhesion molecules (CD54, CD56, CD22, CDlla, CDllc and CD60) did not modii the described inhibitory effect. Conclusion: CD5+ B lymphocytes from B-CLL inhibit in vitro IgG production by autologous BM cells. This effect needs cellular contact, and CD5, CD72 and CD95 molecules seem to be involved in this mechanism. These findings could contribute to explain the hypogammaglobulinemia of B-CLL.
1P.5.12.06 ) Expresslon of lymphocyte activation antigens In human leukemia8 and lymphomas L.N.Shlapatskaya ‘, A.G. Berdova ‘, S.V. Mikhalap’, O.V. Yurchenko l,
1P.5.12.04
1 Phenotyplc and functional characterlstlcs of AIDS-related lymphoma cell lines
P. De Paoli ‘, M. Cozzi2, R. Tedeschi ‘, A. Gloghini 2, A.M. Cilia2, G. Gaidano 3, A. Carbane 2. 1Microbio/o~ Immunolcgy, l&o/logy. Centro Msrfmento Oncologico, Aviano, Ifal,! 2Pathology, Centro Rit%mento Oncolqico, A&no, ltak 3Medical Sciences, University of Torino, My
Introduction:Cell lines derived from AIDS-related non-Hodgkin Lymphomas, including small non-cleaved cell lymphoma (SNCCL) and body cavity-based lymphoma (BCBL), have been used to characterize the membrane expression of antigens involved in B cell proliferation (CD40) and in B cell differentiation (CD45 isoforms, CD30. CDlO, CDQ5);we also wanted to establish the functional role of some of these antigens in the production by these cell lines of cytokines, like interleukin 10, that have been suggested to have a role in AIDS-NHL pathogenesis. MaterlaIr and Methods: 7 SNCCL cell lines (HBLl, HBL2, BRGIgA, AS263A. LamC3+. PA662. Eslll) and 1 BCBL cell line fHBL61 were studied by quaniitative flow cytometry with clustered MAb CD&, Cl&RA and RO, CD30, CDlO. CD95 and with MAb from VI HLDA. Cell lines were also stimulated in vitro by CD4OL transfected L cells and cytokine (ILB. ILIO) production in supematants tested by ELISA. Results: The 7 SNCCL cell lines were positive for CD40, but quantitative measurements display different levels of reactivity, from 11,000 (HBL2) to 162.000 molecules/cell (HBLl); high CD40 density cell lines display a selected phenotype having reactivity with CD45RA, CD45RO and CDQ5. The BCBL cell line was negative for CD40 and with lineage specific B cell MAb, but reacted with antibodies from VI HLDA B cell activation panel (821, 30,50, 92, 93). The incubation of SNCCL with CD4OL L cells induced after 24 h a sharp
D.F. Gluzman ‘, E.A. Clark2, S.P. Sidorenko ‘. ’ Kavetsky institute of Experimental Pathokg~ Oncology and Radiobiology Ukrainian Academy of Science, Kim Ukraine, 2Department of lmmunol~ University of Washington, Seattle, USA Introductton: The aim of the work was to study the expression of the activation antigens CDQ5, CDwl50 and CD25 on leukemia and lymphomas for the potential regulation of tumor cell fate. Materials and Methods: Monoclonal antibodies IPO-3 (CDwl50) and IPC-4 (CDQ5) were generated after immunization with B lymphoblastoid cell line RPMI-1766. The hybridoma producing mAb IPO-51 (CD25) wasdeveloped following immunization with spleen cells from patient with hairy cell leukemia. The expression of Ags was examined by indirect immunofluorescence and immunocytochemistty on cells from peripheral blood and lymph nodes of 165 patients with leukemia and lymphoma, cell lines and by flow cytometry on mononuclear cells isolated from peripheral blood and tonsils of practically healthy persons. Rerulte: CD25, CD95 and CDwl50 were found on T and B cell lines with highest level of expression on lymphoblastoid cell lines. CDwl50 was detected on CD25+ CD45RO+ suboooulation of T and B cells. and also on dendritic and endothelial cells. CD25, cd95 and CDwl50 were ;p-regulated on tonsillar B cells after activation via CD40 and T-cells after stimulation via CD3 atone or together with CD26. Activation of resting B cells via mlgM induce rapid upregulation of CD25 and CDwl50, however did not affect the level of CD95 expression. In tonsil and lymph nodes CDQ5+and CDwl50* cells localized in germinal centers of B cell follicules. Mab IPO-3, IPO-4 and IPO-51 reacted with neoplasms of B-cell origin. including CLL, HCL and NHL. Epithelial cells in Lennert’s lymphoma had high level of surface CDQS.CDwl50 was detected on lyrnphoblasts and immunoblasts in Hodgkin’s desease, however Reed-Stemberg cells were negative. These mAbs did not label cells at myelomas, CALL,
Malignancies of the immune system
subgmups and none of the samples eXpreSSed the VH2 or VK4 subgroup. The panel of the paraproteins rarely expressed the probed VH or &associated CRI. These data suggest that the IgV genes may not be randomly expressed in the malignant cells of Iranian MM patients and these genes might be highly mutated, as evidenced by the lack of expression of the probed CRI.
P.5.12.03
V region genes encoding an Immunocytoma-secreted antl-phosphollpid autoantibody
M. Blay, C. Benito, J.C. Reverter I, E. Campo 2, F. Bosch 3, T. Gallart. ’ Services of Immunology: Hemorhempy and Hemostasis, Hospital Clinic Universitari, Barcelona, Spain, 2Services of Pathology, Hospkal Clinic Universitari, Barcelona, Spain, 3Services of Clinical Hematology; _. Hospital Clinic Universitari, Sarcelona, Spain Introductfon: The study of B cell malignancies secreting circulating autoantibodies may provide information about ihe possibility that normal Eicells producing natural IgM autoantibodies with unmutated VH and VL lg region genes, could be prone to be involved in autoimmune responses of pathogenic IgG autoantibodies and/or to undergo malignant transformation. Therefore studies were performed in a patient with spleen immunocytoma associated with serum monoclonal IgMA and circulating anti-phospholipid (AP) antibody. Materiel and Methods: Fusion of patient’s splenccytes with F3B6 heteromyeloma cells was done to obtain heterohybridomas secreting AP antibodies. ELISA and phospholipid-dependent in vitro coagulation tests were used to detect AP antibodies. To analyze rearranm VH and VL Ig region genes in malignant spleen B cells and in splenocyte-derived heterohybridomas, a PCR-based approach was employed. Isoelectrofocusing, flow cytometry and immunohistochemistry were also performed. Resutts: Circulating anti-cardiolipin (AC) antibody and lupus anticoagulant (LA) were found: and both antibody activities resided in the serum monoclonal IgMA. Splenocyte-derived heterohybrfdoma clones producing IgMA with both AC and LA activities were obtained. PCR analysis, “fingetptinting”. and sequencing of rearranged VH and VL Ig region genes in the DNA from splenocytes and cDNA from heterohybrfdoma clones demonstrated the clonal identity between heterohybridoma clones and spleen tumor B cells. Moreover, the isolectric mobility of separated Ig chains of both serum and heterohybtidoma-secreted IgMA was identical. The AC and LA activities of both serum and heterohybrtdomasecreted IgMA did not reauire Bn-alvcourotein I co-factor. The VH and VL aene -__ segments-were homologbus (97%) to ihe reported germ-line gene segments, VH3-30 (1.Qlll, DP-49) and VA3.1 (IGLV3S1, DPL16), respectivelv. Conclurlon: Data bemonstrate.that serum IgMA &th AC/LA antibody activities was produced by spleen tumor B cell clone. The VH and VA gene segments encoding this autoantibody have been obsetved frequentty in other autoantibodies. In contrast to what is usually thought, data clearly show that AC and LA antibody activities can reside in the same lg molecule and can be cofactor-independent. Cofactor-independent AC andlor IA antibodies such as those found in infections are thought to lack autoimmune pathogenic properties, a notion consistent with the absence of AP-related clinical manifestations in the patient.
24 June 1997 - Poster presentations
increase of IL10 production in HBL2 (from 150 to 3440 pg/ml), HBLl (from 550 to 2137 pa/ml) and PA662 (from 460 to 2550 psy’ml). Conclueions: Our data suggest that membrane CD40 in AIDS SNCCL may be a key element in the regulation of their pathophysidogy by regulating membrane antigen expression and cytoklne production. BCBL cell lines have their own phenotype, characterized by absence of lineage specific B cell antigens usually involved in the transmission of stimulatory signals, but constitutlvely express an “activated” B cell phenotype. Which molecules drive their proliferation and cytokine production remains to be established.
P.5.12.05
CM+ B cells from chronic lymphocytlc leukemla lnhlblt IgG production by autologous bone marrow plasma cells
A. Sampalo, G. Navas. J.A. Brfeva. Servicio de Inmunologia, Hospital Universitatfo Puerta de/ Mar, Cadiz, Spain Introduction: B-Chronic Lymphocytic Leukemia (CLL) is defined as a clonal proliferation of CD5+ B lymphocytes. Hv~ammaalobulinemia is a common feature in CLL, but its p&h&en& mechanism remains uncertain. The aim of this study was to investigate the possible effect of CD5+ B cells on IgG secretion bv autoloaous bone marrow olasma cells. Material and Methods: i5 B-CLL patients were included in the study. CD5+ B calls were purified from peripheral blood (CD5+ 8). and this fraction was removed from bone mar& samples (CDS-‘BM) by using suited rnAbs and magnetic beads. CD5+ B fraction consisted of more than 95% CDlQ+CD5+ cells. CD5- BM contained less than 5% CDlQ+CD5+ cells. CD5+ B cells were stimulated or not with PMA (10 @ml for 16 h), and cocultured with autologous CD5- MO cells. After 7 davs of culture. the IaG oroduction was measured by ELISA. Results are expressed as percentagesof ihe IgG secretion observed in the corresponding CD5- BM cell culture alone (control value). Reautts: Unsttmulated CD5+ B cells reduced autoloaous CD5- BM IgG production to a 66.6% f 21 (mean f SD). This inhibitory effect was increased by PMA-preincubation (52.6% f 15). The inhibition was dependent on cellular contact, since the separation of CD5+ B and CD5- MO fractions in a transwell system reversed the effect (IgG secretion returned to 92% f 11). The molecules involved in this cellular interaction were studied by adding certain blocking mAb. rnAb against CD5, CD72 and CD95 reversed the present inhibition by PMA-CD5+ B cells (97% f 6. 76% f 13 and 62% f 12. of control value, respectively). Moreoier. CD95 was expressed on plasma ceils from B-CLL BM cells. mAb against other adhesion molecules (CD54, CD56, CD22, CDlla, CDllc and CD60) did not modii the described inhibitory effect. Conclusion: CD5+ B lymphocytes from B-CLL inhibit in vitro IgG production by autologous BM cells. This effect needs cellular contact, and CD5, CD72 and CD95 molecules seem to be involved in this mechanism. These findings could contribute to explain the hypogammaglobulinemia of B-CLL.
1P.5.12.06 ) Expresslon of lymphocyte activation antigens In human leukemia8 and lymphomas L.N.Shlapatskaya ‘, A.G. Berdova ‘, S.V. Mikhalap’, O.V. Yurchenko l,
1P.5.12.04
1 Phenotyplc and functional characterlstlcs of AIDS-related lymphoma cell lines
P. De Paoli ‘, M. Cozzi2, R. Tedeschi ‘, A. Gloghini 2, A.M. Cilia2, G. Gaidano 3, A. Carbane 2. 1Microbio/o~ Immunolcgy, l&o/logy. Centro Msrfmento Oncologico, Aviano, Ifal,! 2Pathology, Centro Rit%mento Oncolqico, A&no, ltak 3Medical Sciences, University of Torino, My
Introduction:Cell lines derived from AIDS-related non-Hodgkin Lymphomas, including small non-cleaved cell lymphoma (SNCCL) and body cavity-based lymphoma (BCBL), have been used to characterize the membrane expression of antigens involved in B cell proliferation (CD40) and in B cell differentiation (CD45 isoforms, CD30. CDlO, CDQ5);we also wanted to establish the functional role of some of these antigens in the production by these cell lines of cytokines, like interleukin 10, that have been suggested to have a role in AIDS-NHL pathogenesis. MaterlaIr and Methods: 7 SNCCL cell lines (HBLl, HBL2, BRGIgA, AS263A. LamC3+. PA662. Eslll) and 1 BCBL cell line fHBL61 were studied by quaniitative flow cytometry with clustered MAb CD&, Cl&RA and RO, CD30, CDlO. CD95 and with MAb from VI HLDA. Cell lines were also stimulated in vitro by CD4OL transfected L cells and cytokine (ILB. ILIO) production in supematants tested by ELISA. Results: The 7 SNCCL cell lines were positive for CD40, but quantitative measurements display different levels of reactivity, from 11,000 (HBL2) to 162.000 molecules/cell (HBLl); high CD40 density cell lines display a selected phenotype having reactivity with CD45RA, CD45RO and CDQ5. The BCBL cell line was negative for CD40 and with lineage specific B cell MAb, but reacted with antibodies from VI HLDA B cell activation panel (821, 30,50, 92, 93). The incubation of SNCCL with CD4OL L cells induced after 24 h a sharp
D.F. Gluzman ‘, E.A. Clark2, S.P. Sidorenko ‘. ’ Kavetsky institute of Experimental Pathokg~ Oncology and Radiobiology Ukrainian Academy of Science, Kim Ukraine, 2Department of lmmunol~ University of Washington, Seattle, USA Introductton: The aim of the work was to study the expression of the activation antigens CDQ5, CDwl50 and CD25 on leukemia and lymphomas for the potential regulation of tumor cell fate. Materials and Methods: Monoclonal antibodies IPO-3 (CDwl50) and IPC-4 (CDQ5) were generated after immunization with B lymphoblastoid cell line RPMI-1766. The hybridoma producing mAb IPO-51 (CD25) wasdeveloped following immunization with spleen cells from patient with hairy cell leukemia. The expression of Ags was examined by indirect immunofluorescence and immunocytochemistty on cells from peripheral blood and lymph nodes of 165 patients with leukemia and lymphoma, cell lines and by flow cytometry on mononuclear cells isolated from peripheral blood and tonsils of practically healthy persons. Rerulte: CD25, CD95 and CDwl50 were found on T and B cell lines with highest level of expression on lymphoblastoid cell lines. CDwl50 was detected on CD25+ CD45RO+ suboooulation of T and B cells. and also on dendritic and endothelial cells. CD25, cd95 and CDwl50 were ;p-regulated on tonsillar B cells after activation via CD40 and T-cells after stimulation via CD3 atone or together with CD26. Activation of resting B cells via mlgM induce rapid upregulation of CD25 and CDwl50, however did not affect the level of CD95 expression. In tonsil and lymph nodes CDQ5+and CDwl50* cells localized in germinal centers of B cell follicules. Mab IPO-3, IPO-4 and IPO-51 reacted with neoplasms of B-cell origin. including CLL, HCL and NHL. Epithelial cells in Lennert’s lymphoma had high level of surface CDQS.CDwl50 was detected on lyrnphoblasts and immunoblasts in Hodgkin’s desease, however Reed-Stemberg cells were negative. These mAbs did not label cells at myelomas, CALL,