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B cells from leukemic patients are relatively resistant to in-vitro EBV transformation
Leukemia Research Vol. 10, No. 8, pp. 1059-1060, 1986. Printed in Great Britain.
0145-2126 $3.00 + .00 Pergamon Journals Ltd.
BRIEF COMMUNICATION B CELLS FROM LEUKEMIC PATIENTS ARE RELATIVELY RESISTANT TO IN-VITRO EBV TRANSFORMATION ANNE-FRAN~OISE TILKIN, MARTINE BAGOT, FRAN~OISE PICARD, FRANt~OISE DREYFUS, JEAN PAUL VERNANT and JEAN PAUL L~VY Laboratoire d'Immunologie et de Virologie des Tumeurs, INSERM U 152, H6pital Cochin, 27 rue du Faubourg Saint Jacques, 75654 Paris Cedex 14, France
(Received 3 February 1986. Revision accepted 18 February 1986) Abstract---Samples of peripheral blood mononuclear cells (PBMC) from normal donors or from leukemic patients were used to obtain Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCL). Whereas the rate of transformation was around 85% with normal donors, it was only around 20% with leukemic patients. However no explanation for this resistance of B cells from leukemic patients to in-vitro EBV transformation could be found. Indeed no correlation exists between this EBV resistance and the age, sex, diagnosis or chemotherapeutic regimen. Furthermore no correlation is apparent between the percentage of B2+ cells, which should have EBV receptors, and the successful EBV transformation.
Key words: EBV-induced lymphoblastoid cell line, B2 antigen in intermediate mature B cells, gp 140, the C3d receptor of human B lymphocytes, which is also the EBV receptor.
THE PATI'ERN of lymphoid subsets in leukemic patients treated by chemotherapy remains poorly documented especially for B cells. Therefore, we think that it would be useful to report data which have been observed fortuitously in the course of a study of alloreactive and autoreactive T cells in allogeneic bone marrow graft patients. Samples of peripheral blood mononuclear cells (PBMC) have been taken from these patients just before graft, from their bone marrow donor and in most cases from their parents and the other available siblings. From these PBMC, Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCL) were established according to classical procedures using the B958E Cell line as a source of infectious EBV [1]. Fourteen families have been studied. We were surprised to observe that it was very difficult to obtain EBV-transformed LCL from the recipient while such LCL were
Abbreviations: PBMC, peripheral blood mononuclear cells; EBV, Epstein-Barr virus; LCL, EBV-induced-lymphoblastoid cell line; PBL, peripheral blood lymphocyte; HLA, human leucocyte antigen; CML, chronic myeloid leukemia; AML, acute myeloid leukemia; ALL, acute lymphoid leukemia. Correspondence to: Dr Tilkin, INSERM U 152, H6pital Cochin, 27 rue du Faubourg Saint Jacques, 75654 Paris Cedex 14, France.
easily obtained in most cases from the donor and the other members of the family. After repeated attempts, an EBV transformation was finally obtained with some recipients' PBLs while it was usually found at the first attempt with the other subjects. Nevertheless, only 4/14 (28.5%) of the bone marrow recipients provided an EBV-transformed LCL in contrast to 12/14 (85.7%) of the H L A identical donors. In our laboratory, using the same method the rate of transformation obtained with a panel of more than 60 normal blood donors was around 85%, while only 2/15 (13%) EBV transformations were obtained with an additional panel of 15 leukemic patients not selected from marrow graft recipients. Therefore, overall only 6/29 (20.6%) of the leukemic patients were found sensitive to in-vitro EBV transformation. However, we were unable to establish any correlation between the possibility of obtaining an EBV-transformed LCL in leukemia patients and their age, sex or diagnosis (CML, A M L and ALL). Furthermore, the chemotherapy r e # m e n was apparently not important in this respect since neither PBL from CML patients with classical treatments nor PBL from the heavily treated A M L or LCL patients could be EBV-transformed. Delays between the last chemotherapy treatment and the moment of the test varied from 10 days to 2 months without apparently altering the efficiency of transformation. The whole lymphocyte numbers, which varied from 2 x 10 l°cells/1 to
1059
1060
A-F. TILKIN et al.
6.10 x 10Jlcells/1 was also irrelevant. Similarly, the number of circulating B cells among PBMC, when tested varied from 120/ram 3 to 360/ram 3 as indicated by the expression of cell surface immunoglobulins. Thus, this excludes a decrease in total B-cell number as an explanation for the i n - v i t r o EBV resistance. However, since the EBV receptor is a recently identified glycoprotein (gp140) [2] apparently related to the B2 antigen [3] which is expressed selectively on "intermediate mature B cells" [4,5] it was more interesting to study the B2+ subset of B lymphocytes. Nadler et al. had shown that 6-+ 1% PBMC from normal donors are B2+. Among our patients the percentage of B2+ cells which has been been measured in all patients varied from 0 to 10% with a median value Of 3.4%, but here again no clear correlation appeared between this percentage and successful EBV transformation. Further studies using different markers specific for B-cell subpopulations would be necessary to understand the resistance of B cells from leukemia patients to EBV transformation. Furthermore kinetics experiments should be done to test whether the proliferative capacity of the leukemic patient's B lymphocytes is either intact, or totally or partially impaired. Nevertheless this observation deserves to be reported for two main reasons: (1) it emphasizes the probable abnormality of B-cell subsets in these patients, since it is highly probable from our observations that normal EBV-sensitive lymphocytes are quantitatively or qualitatively altered in resistant patients; (2) this property of B cells from leukemic patients hampers immunological studies with these
patients involving EBV-transformed LCL as target cells. A c k n o w l e d g e m e n t s - - W e thank Dr R. Frade for very helpful discussions and Mich61eHeslan and Mich61e Kaybanda for the excellent technical assistance.
REFERENCES 1. Nilsson K., Klein G., Henke W. & Heule G. (1971) The establishment of lymphoblastoid cell lines from adult and fetal human lymphoid tissue and its dependence on EBV. Int. J. Cancer 8, 443. 2. Frade R., Barel M., Ehlin-Henriksson B. & Klein G. (1985) gp140, the C3d receptor of human B lymphocytes, is also the Epstein-Barr virus receptor. Proc. natn. Acad. Sci. U.S.A. 82, 1490. 3. Frade R., Myones B.L., Barel M., Krikorian L., Charriant C. & Ross G. D. (1985) gp140, EC3b binding membrane component of lymphocyte is the B cell C3dg/C3d receptor (CR2) and is distinct from the neutrophil C3dg receptor (CR4). Eur. J. Immun. 15, 1192. 4. Nadler L. M., Stashenko P., Hardy R., van Agthoven A., Terhorst C. & Schlossman S. F. (1981) Characterization of human B cell-specific antigen (B2) distinct from B1. J. lmmun. 126, 1941. 5. Barel M., Charriant C. & Frade R. (1981) Isolation and characterization of a C3b receptor-like molecule from membranes of a human B lymphoblastoid cell line (Raji). F E B S Lett. 136, 111.
0145-2126 $3.00 + .00 Pergamon Journals Ltd.
BRIEF COMMUNICATION B CELLS FROM LEUKEMIC PATIENTS ARE RELATIVELY RESISTANT TO IN-VITRO EBV TRANSFORMATION ANNE-FRAN~OISE TILKIN, MARTINE BAGOT, FRAN~OISE PICARD, FRANt~OISE DREYFUS, JEAN PAUL VERNANT and JEAN PAUL L~VY Laboratoire d'Immunologie et de Virologie des Tumeurs, INSERM U 152, H6pital Cochin, 27 rue du Faubourg Saint Jacques, 75654 Paris Cedex 14, France
(Received 3 February 1986. Revision accepted 18 February 1986) Abstract---Samples of peripheral blood mononuclear cells (PBMC) from normal donors or from leukemic patients were used to obtain Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCL). Whereas the rate of transformation was around 85% with normal donors, it was only around 20% with leukemic patients. However no explanation for this resistance of B cells from leukemic patients to in-vitro EBV transformation could be found. Indeed no correlation exists between this EBV resistance and the age, sex, diagnosis or chemotherapeutic regimen. Furthermore no correlation is apparent between the percentage of B2+ cells, which should have EBV receptors, and the successful EBV transformation.
Key words: EBV-induced lymphoblastoid cell line, B2 antigen in intermediate mature B cells, gp 140, the C3d receptor of human B lymphocytes, which is also the EBV receptor.
THE PATI'ERN of lymphoid subsets in leukemic patients treated by chemotherapy remains poorly documented especially for B cells. Therefore, we think that it would be useful to report data which have been observed fortuitously in the course of a study of alloreactive and autoreactive T cells in allogeneic bone marrow graft patients. Samples of peripheral blood mononuclear cells (PBMC) have been taken from these patients just before graft, from their bone marrow donor and in most cases from their parents and the other available siblings. From these PBMC, Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCL) were established according to classical procedures using the B958E Cell line as a source of infectious EBV [1]. Fourteen families have been studied. We were surprised to observe that it was very difficult to obtain EBV-transformed LCL from the recipient while such LCL were
Abbreviations: PBMC, peripheral blood mononuclear cells; EBV, Epstein-Barr virus; LCL, EBV-induced-lymphoblastoid cell line; PBL, peripheral blood lymphocyte; HLA, human leucocyte antigen; CML, chronic myeloid leukemia; AML, acute myeloid leukemia; ALL, acute lymphoid leukemia. Correspondence to: Dr Tilkin, INSERM U 152, H6pital Cochin, 27 rue du Faubourg Saint Jacques, 75654 Paris Cedex 14, France.
easily obtained in most cases from the donor and the other members of the family. After repeated attempts, an EBV transformation was finally obtained with some recipients' PBLs while it was usually found at the first attempt with the other subjects. Nevertheless, only 4/14 (28.5%) of the bone marrow recipients provided an EBV-transformed LCL in contrast to 12/14 (85.7%) of the H L A identical donors. In our laboratory, using the same method the rate of transformation obtained with a panel of more than 60 normal blood donors was around 85%, while only 2/15 (13%) EBV transformations were obtained with an additional panel of 15 leukemic patients not selected from marrow graft recipients. Therefore, overall only 6/29 (20.6%) of the leukemic patients were found sensitive to in-vitro EBV transformation. However, we were unable to establish any correlation between the possibility of obtaining an EBV-transformed LCL in leukemia patients and their age, sex or diagnosis (CML, A M L and ALL). Furthermore, the chemotherapy r e # m e n was apparently not important in this respect since neither PBL from CML patients with classical treatments nor PBL from the heavily treated A M L or LCL patients could be EBV-transformed. Delays between the last chemotherapy treatment and the moment of the test varied from 10 days to 2 months without apparently altering the efficiency of transformation. The whole lymphocyte numbers, which varied from 2 x 10 l°cells/1 to
1059
1060
A-F. TILKIN et al.
6.10 x 10Jlcells/1 was also irrelevant. Similarly, the number of circulating B cells among PBMC, when tested varied from 120/ram 3 to 360/ram 3 as indicated by the expression of cell surface immunoglobulins. Thus, this excludes a decrease in total B-cell number as an explanation for the i n - v i t r o EBV resistance. However, since the EBV receptor is a recently identified glycoprotein (gp140) [2] apparently related to the B2 antigen [3] which is expressed selectively on "intermediate mature B cells" [4,5] it was more interesting to study the B2+ subset of B lymphocytes. Nadler et al. had shown that 6-+ 1% PBMC from normal donors are B2+. Among our patients the percentage of B2+ cells which has been been measured in all patients varied from 0 to 10% with a median value Of 3.4%, but here again no clear correlation appeared between this percentage and successful EBV transformation. Further studies using different markers specific for B-cell subpopulations would be necessary to understand the resistance of B cells from leukemia patients to EBV transformation. Furthermore kinetics experiments should be done to test whether the proliferative capacity of the leukemic patient's B lymphocytes is either intact, or totally or partially impaired. Nevertheless this observation deserves to be reported for two main reasons: (1) it emphasizes the probable abnormality of B-cell subsets in these patients, since it is highly probable from our observations that normal EBV-sensitive lymphocytes are quantitatively or qualitatively altered in resistant patients; (2) this property of B cells from leukemic patients hampers immunological studies with these
patients involving EBV-transformed LCL as target cells. A c k n o w l e d g e m e n t s - - W e thank Dr R. Frade for very helpful discussions and Mich61eHeslan and Mich61e Kaybanda for the excellent technical assistance.
REFERENCES 1. Nilsson K., Klein G., Henke W. & Heule G. (1971) The establishment of lymphoblastoid cell lines from adult and fetal human lymphoid tissue and its dependence on EBV. Int. J. Cancer 8, 443. 2. Frade R., Barel M., Ehlin-Henriksson B. & Klein G. (1985) gp140, the C3d receptor of human B lymphocytes, is also the Epstein-Barr virus receptor. Proc. natn. Acad. Sci. U.S.A. 82, 1490. 3. Frade R., Myones B.L., Barel M., Krikorian L., Charriant C. & Ross G. D. (1985) gp140, EC3b binding membrane component of lymphocyte is the B cell C3dg/C3d receptor (CR2) and is distinct from the neutrophil C3dg receptor (CR4). Eur. J. Immun. 15, 1192. 4. Nadler L. M., Stashenko P., Hardy R., van Agthoven A., Terhorst C. & Schlossman S. F. (1981) Characterization of human B cell-specific antigen (B2) distinct from B1. J. lmmun. 126, 1941. 5. Barel M., Charriant C. & Frade R. (1981) Isolation and characterization of a C3b receptor-like molecule from membranes of a human B lymphoblastoid cell line (Raji). F E B S Lett. 136, 111.