Tumors are considered to be possible targets of immunotherapy using stimulated and expanded cytot... more Tumors are considered to be possible targets of immunotherapy using stimulated and expanded cytotoxic Tlymphocytes (CTL). It is important to consider the drug-induced effects when chemotherapeutic regimens and CTL-mediated immunotherapy is planned to be used in parallel. In this study, we characterized the effect of 29 frequently used chemotherapeutic agents on the cytotoxic activity of autologous and allogeneic CTLs. We found that treatment of CTLs with the following drugs: docetaxel, vincristine, chlorambucil, mitomycin C, oxaliplatin, doxorubicin, and bleomycin effectively inhibited CTL-mediated killing, without affecting their viability. On the other hand, the following drugs enhanced or permitted efficient CTL-mediated killing in vitro at concentrations comparable with the maximally achieved therapeutic concentration in vivo in humans: daunorubicin, prednisolone, vinorelbine, cisplatin, methotrexate, hydroxyurea, cytarabine, cyclophosphamide, topotecan, epirubicin, fluorouracil, carboplatin, asparaginase, 6-mercaptopurine, and bortezomib. Our results could poten-tially be used in the future to design new CTL-based adjuvant immunotherapy protocols. Financial Disclosure: The authors have declared there are no financial conflicts of interest related to this work. Henriette Skribek and Michael Uhlin contributed equally to this work. Authors' contributions: The project was conceived and designed by Laszlo Szekely and Gyorgy Stuber. The experiments were mainly carried out and/or coordinated by Laszlo Markasz. Michael Uhlin created and cultures all CTL lines and LCLs used in the experiments. Gyorgy Stuber, Laszlo Markasz, and Rita Otvos took part in preparation of the microtiter plates for in vitro drug sensitivity assays. Laszlo Markasz and Rita Otvos were responsible for measuring the plates using the automated laser confocal fluorescent microscope. Laszlo Szekely and Emilie Flaberg wrote the computer programs QuantCapture 4.0 and CytotoxCount 3.0. Laszlo Markasz and Henriette Skribek analyzed and interpreted the data. Staffan Eksborg made comparable the in vitro results with the in vivo data. Eva Olah, Henriette Skribek together with the other authors have been involved in the planning of the experimental details, and the drafting, and critical reading of the manuscript. All authors read and approved the final manuscript. Reprints: Laszlo Szekely,
The EBV carrying lines MEC1 and MEC2 were established earlier from explants of blood derived cell... more The EBV carrying lines MEC1 and MEC2 were established earlier from explants of blood derived cells of a chronic lymphocytic leukemia (CLL) patient at different stages of progression to prolymphocytoid transformation (PLL). This pair of lines is unique in several respects. Their common clonal origin was proven by the rearrangement of the immunoglobulin genes. The cells were driven to proliferation in vitro by the same indigenous EBV strain. They are phenotypically different and represent subsequent subclones emerging in the CLL population. Furthermore they reflect the clinical progression of the disease. We emphasize that the support for the expression of the EBV encoded growth program is an important differentiation marker of the CLL cells of origin that was shared by the two subclones. It can be surmised that proliferation of EBV carrying cells in vitro, but not in vivo, reflects the efficient surveillance that functions even in the severe leukemic condition. The MEC1 line arose before the aggressive clinical stage from an EBV carrying cell within the subclone that was in the early prolymphocytic transformation stage while the MEC2 line originated one year later, from the subsequent subclone with overt PLL characteristics. At this time the disease was disseminated and the blood lymphocyte count was considerably elevated. The EBV induced proliferation of the MEC cells belonging to the subclones with markers of PLL agrees with earlier reports in which cells of PLL disease were infected in vitro and immortalized to LCL. They prove also that the expression of EBV encoded set of proteins can be determined at the event of infection. This pair of lines is particularly important as they provide in vitro cells that represent the subclonal evolution of the CLL disease. Furthermore, the phenotype of the MEC1 cells shares several characteristics of ex vivo CLL cells.
We studied the expression of the 20-kDa homologous restriction factor (CD59/HRF20), a complement ... more We studied the expression of the 20-kDa homologous restriction factor (CD59/HRF20), a complement regulatory protein, on two subsets of blood derived B cells and on Burkitt's lymphoma lines. Both low-density (activated) and high-density (resting) B cell populations expressed high levels of CD59. CD59 was detectable, however, only on a minority of cells or not at all on three Epstein-Barr virus (EBV)-negative BL lines (BL41, BL28 and DG75) and on clones of an EBV-positive BL line (Mutu) that phenotypically resembled resting B lymphocytes. On the other hand, CD59 was detected at high or medium levels on Mutu cells which had a lymphoblastoid cell-like phenotype. Expression of CD59 was upregulated by 5-azacytidine, a drug inhibiting cytosine methylation, on CD59-negative cell lines. Induction was accompanied by a partial hypomethylation in the 5' region of CD59 coding sequences.
Most humans carry the potentially life-endangering Epstein-Barr virus (EBV). The immediate danger... more Most humans carry the potentially life-endangering Epstein-Barr virus (EBV). The immediate danger after infection is imposed by proliferation of the B cells that carry the viral genome. Although a number of different cell types can be infected with EBV, B lymphocytes are exceptionally sensitive; they express a set of virus-encoded proteins, which collaborate with host proteins to induce proliferation. This phenomenon can be demonstrated in vitro with experimentally infected B cells. These viral genes are expressed only in B lymphocytes and are restricted to a defined differentiation stage. This limitation is of high importance for the maintenance of the controlled EBV-carrier state of humans. The emergence of EBV-induced B-cell malignancies is counteracted by highly efficient immunologic mechanisms. Recognition of EBV-transformed immunoblasts in an MHC class I-restricted manner by cytotoxic CD8 T cells and, to a lesser extent, by CD4 T cells, is thought to play the major role. The i...
To understand the mechanism for the refractoriness of B-chronic lymphocytic leukemia (B-CLL) cell... more To understand the mechanism for the refractoriness of B-chronic lymphocytic leukemia (B-CLL) cells for Epstein-Barr virus (EBV)-induced immortalization. Cultures were initiated with EBV-infected tonsillar B and B-CLL cells. Expression of EBNA-2 and some of the key players regulating G1/S phase transition such as c-myc expression, phosphorylation of Rb protein, expression of G1 cyclins, and the cyclin-dependent kinase inhibitor p27 were followed. In line with earlier studies, EBV infection induced c-myc expression, pRb phosphorylation, D2 and D3 expression, and disappearance of p27 in normal B cells. In contrast, EBV-infected B-CLL cells remained resting and they did not express c-myc; cyclin D2, ppRb and cyclin D3 were seen only in occasional cells. Importantly, p27 expression was maintained. In B-CLL cells, the expression of the EBV-encoded nuclear proteins EBNAs is not followed by entrance to the cell cycle. Thus, the difference in the interaction of EBV-normal B cells and EBV-B-C...
Using reverse transcription of whole cellular RNA and nested PCR, we have performed experiments m... more Using reverse transcription of whole cellular RNA and nested PCR, we have performed experiments mixing different proportions of Epstein-Barr virus (EBV)-carrying and EBV-negative cells. Based on the results, a method that detects viral transcripts for EBNA-1, EBNA-2, LMP1, and LMP2a from less than one positive cell among 10(5) negative cells was developed. With this method we have shown that the EBV DNA positive cells among small, high-density peripheral blood B-lymphocytes of normal healthy persons express EBNA-1-mRNA but not EBNA-2 or LMP1. A similar EBV expression pattern is found in type I Burkitt lymphoma cells. We suggest that the expression pattern in the lymphoma cells reflects the viral strategy in normal resting B cells and meets the requirements of latent persistence.
Three categories of tumor promoters and chemically related but inactive substances were tested fo... more Three categories of tumor promoters and chemically related but inactive substances were tested for their effect on the cytotoxic activity of human blood lymphocytes against K562 and Daudi targets. Lymphocytes incubated overnight in the presence of phorbol esters 12-O-tetradecanoylphorbol-13-acetate and phorbol-12,13-dibutyrate [P(Bu)2] had enhanced function. Incubation with 4-alpha-phorbol-12,13-didecanoate was without effect. Enhancing activity was also exerted by the indole alkaloids, teleocidin and lyngbyatoxin A, and the polyacetates, aplysiatoxin and debromoaplysiatoxin, but not by dihydroteleocidin. Only the tumor-promoting compounds activated the cytotoxic potential. The substances acted in a dose-dependent manner with optimal activity at characteristic concentrations. Overnight incubation of lymphocytes at 4 degrees did not change their spontaneous cytotoxicity but abolished the enhancing effect of P(Bu)2. Thus, P(Bu)2-induced activation occurred only on metabolically active...
The mechanisms by which CD4+ T cells are eliminated during HIV infection are poorly understood. W... more The mechanisms by which CD4+ T cells are eliminated during HIV infection are poorly understood. We have previously shown that HIV infected cell lines activate and fix C3 via the alternative complement pathway (ACP). In the present study we examined the ability of blood lymphocytes from 40 HIV+ individuals to fix C3. A large fraction of the CD4+ T cells reacted with anti-gp120 antibodies. These cells also carried C3 fragments in vivo and could further fix C3 if exposed to human serum in vitro. C3 activation occurred via the ACP. In some cases exposure of the lymphocytes to human serum under conditions allowing ACP activation resulted in partial elimination of CD4+ T cells. The results suggest that complement activation and fixation by CD4+ T cells opsonized with HIV particles or gp120 may contribute to their selective destruction.
Mouse somatic cell hybrids between a high NK-sensitive lymphoma line (YACIR) and various sarcoma ... more Mouse somatic cell hybrids between a high NK-sensitive lymphoma line (YACIR) and various sarcoma or L-cell-derived cell lines (all weakly sensitive) all exhibited low NK sensitivity. This was not due to a general resistance to cell-mediated lysis, since these mouse hybrids could be killed by in vitro-sensitized specific T cells. Alloantigens are usually codominantly expressed in somatic cell hybrids, whereas differentiationrelated markers are usually suppressed. Because NK sensitivity was found to be suppressed, in contrast to the expression of alloantigens and tumorassociated antigens, we suggest that the target structure may be either a differentiation-related antigen or a differentiation-related "'membrane property. "'
During the past decades, anticancer immunotherapy has evolved from a promising therapeutic option... more During the past decades, anticancer immunotherapy has evolved from a promising therapeutic option to a robust clinical reality. Many immunotherapeutic regimens are now approved by the US Food and Drug Administration and the European Medicines Agency for use in cancer patients, and many others are being investigated as standalone therapeutic interventions or combined with conventional treatments in clinical studies. Immunotherapies may be subdivided into "passive" and…
Barr virus; FISH, fluorescence in situ hybridization; IG, immunoglobulin; IGHV, IG heavy chain va... more Barr virus; FISH, fluorescence in situ hybridization; IG, immunoglobulin; IGHV, IG heavy chain variable; IGL/KV, IG lambda/kappa light chain variable; LCL, lymphoblastoid cell line; LMP-1, EBV-encoded latent membrane protein 1; MDA-LDL, oxidized low density lipoprotein of the malonedialdehyde type; miR, micro-RNA; nLDL, native low density lipoprotein; oxLDL, oxidized low density lipoprotein; SNP, single nucleotide polymorphism; RQ-PCR, real-time quantitative polymerase chain reaction Chronic lymphocytic leukemia (CLL) cells express the receptor for Epstein-Barr virus (EBV) and can be infected in vitro. Infected cells do not express the growth-promoting set of EBV-encoded genes and therefore they do not yield LCLs, in most experiments. With exceptional clones, lines were obtained however. We describe a new line, HG3, established by in vitro EBV-infection from an IGHV1-2 unmutated CLL patient clone. All cells expressed EBNA-2 and LMP-1, the EBV-encoded genes pivotal for transformation. The karyotype, FISH cytogenetics and SNP-array profile of the line and the patient's ex vivo clone showed biallelic 13q14 deletions with genomic loss of DLEU7, miR15a/miR16-1, the two micro-RNAs that are deleted in 50% of CLL cases. Further features of CLL cells were: expression of CD5/CD20/CD27/CD43 and release of IgM natural antibodies reacting with oxLDL-like epitopes on apoptotic cells (cf. stereotyped subset-1). Comparison with two LCLs established from normal B cells showed 32 genes expressed at higher levels (. 2-fold). Among these were LHX2 and LILRA. These genes may play a role in the development of the disease. LHX2 expression was shown in self-renewing multipotent hematopoietic stem cells, and LILRA4 codes for a receptor for bone marrow stromal cell antigen-2 that contributes to B cell development. Twenty-four genes were expressed at lower levels, among these PARD3 that is essential for asymmetric cell division. These genes may contribute to establish precursors of CLL clones by regulation of cellular phenotype in the hematopoietic compartment. Expression of CD5/CD20/CD27/CD43 and spontaneous production of natural antibodies may identify the CLL cell as a self-renewing B1 lymphocyte.
The EBV carrying lines MEC1 and MEC2 were established earlier from explants of blood derived cell... more The EBV carrying lines MEC1 and MEC2 were established earlier from explants of blood derived cells of a chronic lymphocytic leukemia (CLL) patient at different stages of progression to prolymphocytoid transformation (PLL). This pair of lines is unique in several respects. Their common clonal origin was proven by the rearrangement of the immunoglobulin genes. The cells were driven to proliferation in vitro by the same indigenous EBV strain. They are phenotypically different and represent subsequent subclones emerging in the CLL population. Furthermore they reflect the clinical progression of the disease. We emphasize that the support for the expression of the EBV encoded growth program is an important differentiation marker of the CLL cells of origin that was shared by the two subclones. It can be surmised that proliferation of EBV carrying cells in vitro, but not in vivo, reflects the efficient surveillance that functions even in the severe leukemic condition. The MEC1 line arose before the aggressive clinical stage from an EBV carrying cell within the subclone that was in the early prolymphocytic transformation stage while the MEC2 line originated one year later, from the subsequent subclone with overt PLL characteristics. At this time the disease was disseminated and the blood lymphocyte count was considerably elevated. The EBV induced proliferation of the MEC cells belonging to the subclones with markers of PLL agrees with earlier reports in which cells of PLL disease were infected in vitro and immortalized to LCL. They prove also that the expression of EBV encoded set of proteins can be determined at the event of infection. This pair of lines is particularly important as they provide in vitro cells that represent the subclonal evolution of the CLL disease. Furthermore, the phenotype of the MEC1 cells shares several characteristics of ex vivo CLL cells.
Tumors are considered to be possible targets of immunotherapy using stimulated and expanded cytot... more Tumors are considered to be possible targets of immunotherapy using stimulated and expanded cytotoxic Tlymphocytes (CTL). It is important to consider the drug-induced effects when chemotherapeutic regimens and CTL-mediated immunotherapy is planned to be used in parallel. In this study, we characterized the effect of 29 frequently used chemotherapeutic agents on the cytotoxic activity of autologous and allogeneic CTLs. We found that treatment of CTLs with the following drugs: docetaxel, vincristine, chlorambucil, mitomycin C, oxaliplatin, doxorubicin, and bleomycin effectively inhibited CTL-mediated killing, without affecting their viability. On the other hand, the following drugs enhanced or permitted efficient CTL-mediated killing in vitro at concentrations comparable with the maximally achieved therapeutic concentration in vivo in humans: daunorubicin, prednisolone, vinorelbine, cisplatin, methotrexate, hydroxyurea, cytarabine, cyclophosphamide, topotecan, epirubicin, fluorouracil, carboplatin, asparaginase, 6-mercaptopurine, and bortezomib. Our results could poten-tially be used in the future to design new CTL-based adjuvant immunotherapy protocols. Financial Disclosure: The authors have declared there are no financial conflicts of interest related to this work. Henriette Skribek and Michael Uhlin contributed equally to this work. Authors' contributions: The project was conceived and designed by Laszlo Szekely and Gyorgy Stuber. The experiments were mainly carried out and/or coordinated by Laszlo Markasz. Michael Uhlin created and cultures all CTL lines and LCLs used in the experiments. Gyorgy Stuber, Laszlo Markasz, and Rita Otvos took part in preparation of the microtiter plates for in vitro drug sensitivity assays. Laszlo Markasz and Rita Otvos were responsible for measuring the plates using the automated laser confocal fluorescent microscope. Laszlo Szekely and Emilie Flaberg wrote the computer programs QuantCapture 4.0 and CytotoxCount 3.0. Laszlo Markasz and Henriette Skribek analyzed and interpreted the data. Staffan Eksborg made comparable the in vitro results with the in vivo data. Eva Olah, Henriette Skribek together with the other authors have been involved in the planning of the experimental details, and the drafting, and critical reading of the manuscript. All authors read and approved the final manuscript. Reprints: Laszlo Szekely,
The EBV carrying lines MEC1 and MEC2 were established earlier from explants of blood derived cell... more The EBV carrying lines MEC1 and MEC2 were established earlier from explants of blood derived cells of a chronic lymphocytic leukemia (CLL) patient at different stages of progression to prolymphocytoid transformation (PLL). This pair of lines is unique in several respects. Their common clonal origin was proven by the rearrangement of the immunoglobulin genes. The cells were driven to proliferation in vitro by the same indigenous EBV strain. They are phenotypically different and represent subsequent subclones emerging in the CLL population. Furthermore they reflect the clinical progression of the disease. We emphasize that the support for the expression of the EBV encoded growth program is an important differentiation marker of the CLL cells of origin that was shared by the two subclones. It can be surmised that proliferation of EBV carrying cells in vitro, but not in vivo, reflects the efficient surveillance that functions even in the severe leukemic condition. The MEC1 line arose before the aggressive clinical stage from an EBV carrying cell within the subclone that was in the early prolymphocytic transformation stage while the MEC2 line originated one year later, from the subsequent subclone with overt PLL characteristics. At this time the disease was disseminated and the blood lymphocyte count was considerably elevated. The EBV induced proliferation of the MEC cells belonging to the subclones with markers of PLL agrees with earlier reports in which cells of PLL disease were infected in vitro and immortalized to LCL. They prove also that the expression of EBV encoded set of proteins can be determined at the event of infection. This pair of lines is particularly important as they provide in vitro cells that represent the subclonal evolution of the CLL disease. Furthermore, the phenotype of the MEC1 cells shares several characteristics of ex vivo CLL cells.
We studied the expression of the 20-kDa homologous restriction factor (CD59/HRF20), a complement ... more We studied the expression of the 20-kDa homologous restriction factor (CD59/HRF20), a complement regulatory protein, on two subsets of blood derived B cells and on Burkitt's lymphoma lines. Both low-density (activated) and high-density (resting) B cell populations expressed high levels of CD59. CD59 was detectable, however, only on a minority of cells or not at all on three Epstein-Barr virus (EBV)-negative BL lines (BL41, BL28 and DG75) and on clones of an EBV-positive BL line (Mutu) that phenotypically resembled resting B lymphocytes. On the other hand, CD59 was detected at high or medium levels on Mutu cells which had a lymphoblastoid cell-like phenotype. Expression of CD59 was upregulated by 5-azacytidine, a drug inhibiting cytosine methylation, on CD59-negative cell lines. Induction was accompanied by a partial hypomethylation in the 5' region of CD59 coding sequences.
Most humans carry the potentially life-endangering Epstein-Barr virus (EBV). The immediate danger... more Most humans carry the potentially life-endangering Epstein-Barr virus (EBV). The immediate danger after infection is imposed by proliferation of the B cells that carry the viral genome. Although a number of different cell types can be infected with EBV, B lymphocytes are exceptionally sensitive; they express a set of virus-encoded proteins, which collaborate with host proteins to induce proliferation. This phenomenon can be demonstrated in vitro with experimentally infected B cells. These viral genes are expressed only in B lymphocytes and are restricted to a defined differentiation stage. This limitation is of high importance for the maintenance of the controlled EBV-carrier state of humans. The emergence of EBV-induced B-cell malignancies is counteracted by highly efficient immunologic mechanisms. Recognition of EBV-transformed immunoblasts in an MHC class I-restricted manner by cytotoxic CD8 T cells and, to a lesser extent, by CD4 T cells, is thought to play the major role. The i...
To understand the mechanism for the refractoriness of B-chronic lymphocytic leukemia (B-CLL) cell... more To understand the mechanism for the refractoriness of B-chronic lymphocytic leukemia (B-CLL) cells for Epstein-Barr virus (EBV)-induced immortalization. Cultures were initiated with EBV-infected tonsillar B and B-CLL cells. Expression of EBNA-2 and some of the key players regulating G1/S phase transition such as c-myc expression, phosphorylation of Rb protein, expression of G1 cyclins, and the cyclin-dependent kinase inhibitor p27 were followed. In line with earlier studies, EBV infection induced c-myc expression, pRb phosphorylation, D2 and D3 expression, and disappearance of p27 in normal B cells. In contrast, EBV-infected B-CLL cells remained resting and they did not express c-myc; cyclin D2, ppRb and cyclin D3 were seen only in occasional cells. Importantly, p27 expression was maintained. In B-CLL cells, the expression of the EBV-encoded nuclear proteins EBNAs is not followed by entrance to the cell cycle. Thus, the difference in the interaction of EBV-normal B cells and EBV-B-C...
Using reverse transcription of whole cellular RNA and nested PCR, we have performed experiments m... more Using reverse transcription of whole cellular RNA and nested PCR, we have performed experiments mixing different proportions of Epstein-Barr virus (EBV)-carrying and EBV-negative cells. Based on the results, a method that detects viral transcripts for EBNA-1, EBNA-2, LMP1, and LMP2a from less than one positive cell among 10(5) negative cells was developed. With this method we have shown that the EBV DNA positive cells among small, high-density peripheral blood B-lymphocytes of normal healthy persons express EBNA-1-mRNA but not EBNA-2 or LMP1. A similar EBV expression pattern is found in type I Burkitt lymphoma cells. We suggest that the expression pattern in the lymphoma cells reflects the viral strategy in normal resting B cells and meets the requirements of latent persistence.
Three categories of tumor promoters and chemically related but inactive substances were tested fo... more Three categories of tumor promoters and chemically related but inactive substances were tested for their effect on the cytotoxic activity of human blood lymphocytes against K562 and Daudi targets. Lymphocytes incubated overnight in the presence of phorbol esters 12-O-tetradecanoylphorbol-13-acetate and phorbol-12,13-dibutyrate [P(Bu)2] had enhanced function. Incubation with 4-alpha-phorbol-12,13-didecanoate was without effect. Enhancing activity was also exerted by the indole alkaloids, teleocidin and lyngbyatoxin A, and the polyacetates, aplysiatoxin and debromoaplysiatoxin, but not by dihydroteleocidin. Only the tumor-promoting compounds activated the cytotoxic potential. The substances acted in a dose-dependent manner with optimal activity at characteristic concentrations. Overnight incubation of lymphocytes at 4 degrees did not change their spontaneous cytotoxicity but abolished the enhancing effect of P(Bu)2. Thus, P(Bu)2-induced activation occurred only on metabolically active...
The mechanisms by which CD4+ T cells are eliminated during HIV infection are poorly understood. W... more The mechanisms by which CD4+ T cells are eliminated during HIV infection are poorly understood. We have previously shown that HIV infected cell lines activate and fix C3 via the alternative complement pathway (ACP). In the present study we examined the ability of blood lymphocytes from 40 HIV+ individuals to fix C3. A large fraction of the CD4+ T cells reacted with anti-gp120 antibodies. These cells also carried C3 fragments in vivo and could further fix C3 if exposed to human serum in vitro. C3 activation occurred via the ACP. In some cases exposure of the lymphocytes to human serum under conditions allowing ACP activation resulted in partial elimination of CD4+ T cells. The results suggest that complement activation and fixation by CD4+ T cells opsonized with HIV particles or gp120 may contribute to their selective destruction.
Mouse somatic cell hybrids between a high NK-sensitive lymphoma line (YACIR) and various sarcoma ... more Mouse somatic cell hybrids between a high NK-sensitive lymphoma line (YACIR) and various sarcoma or L-cell-derived cell lines (all weakly sensitive) all exhibited low NK sensitivity. This was not due to a general resistance to cell-mediated lysis, since these mouse hybrids could be killed by in vitro-sensitized specific T cells. Alloantigens are usually codominantly expressed in somatic cell hybrids, whereas differentiationrelated markers are usually suppressed. Because NK sensitivity was found to be suppressed, in contrast to the expression of alloantigens and tumorassociated antigens, we suggest that the target structure may be either a differentiation-related antigen or a differentiation-related "'membrane property. "'
During the past decades, anticancer immunotherapy has evolved from a promising therapeutic option... more During the past decades, anticancer immunotherapy has evolved from a promising therapeutic option to a robust clinical reality. Many immunotherapeutic regimens are now approved by the US Food and Drug Administration and the European Medicines Agency for use in cancer patients, and many others are being investigated as standalone therapeutic interventions or combined with conventional treatments in clinical studies. Immunotherapies may be subdivided into "passive" and…
Barr virus; FISH, fluorescence in situ hybridization; IG, immunoglobulin; IGHV, IG heavy chain va... more Barr virus; FISH, fluorescence in situ hybridization; IG, immunoglobulin; IGHV, IG heavy chain variable; IGL/KV, IG lambda/kappa light chain variable; LCL, lymphoblastoid cell line; LMP-1, EBV-encoded latent membrane protein 1; MDA-LDL, oxidized low density lipoprotein of the malonedialdehyde type; miR, micro-RNA; nLDL, native low density lipoprotein; oxLDL, oxidized low density lipoprotein; SNP, single nucleotide polymorphism; RQ-PCR, real-time quantitative polymerase chain reaction Chronic lymphocytic leukemia (CLL) cells express the receptor for Epstein-Barr virus (EBV) and can be infected in vitro. Infected cells do not express the growth-promoting set of EBV-encoded genes and therefore they do not yield LCLs, in most experiments. With exceptional clones, lines were obtained however. We describe a new line, HG3, established by in vitro EBV-infection from an IGHV1-2 unmutated CLL patient clone. All cells expressed EBNA-2 and LMP-1, the EBV-encoded genes pivotal for transformation. The karyotype, FISH cytogenetics and SNP-array profile of the line and the patient's ex vivo clone showed biallelic 13q14 deletions with genomic loss of DLEU7, miR15a/miR16-1, the two micro-RNAs that are deleted in 50% of CLL cases. Further features of CLL cells were: expression of CD5/CD20/CD27/CD43 and release of IgM natural antibodies reacting with oxLDL-like epitopes on apoptotic cells (cf. stereotyped subset-1). Comparison with two LCLs established from normal B cells showed 32 genes expressed at higher levels (. 2-fold). Among these were LHX2 and LILRA. These genes may play a role in the development of the disease. LHX2 expression was shown in self-renewing multipotent hematopoietic stem cells, and LILRA4 codes for a receptor for bone marrow stromal cell antigen-2 that contributes to B cell development. Twenty-four genes were expressed at lower levels, among these PARD3 that is essential for asymmetric cell division. These genes may contribute to establish precursors of CLL clones by regulation of cellular phenotype in the hematopoietic compartment. Expression of CD5/CD20/CD27/CD43 and spontaneous production of natural antibodies may identify the CLL cell as a self-renewing B1 lymphocyte.
The EBV carrying lines MEC1 and MEC2 were established earlier from explants of blood derived cell... more The EBV carrying lines MEC1 and MEC2 were established earlier from explants of blood derived cells of a chronic lymphocytic leukemia (CLL) patient at different stages of progression to prolymphocytoid transformation (PLL). This pair of lines is unique in several respects. Their common clonal origin was proven by the rearrangement of the immunoglobulin genes. The cells were driven to proliferation in vitro by the same indigenous EBV strain. They are phenotypically different and represent subsequent subclones emerging in the CLL population. Furthermore they reflect the clinical progression of the disease. We emphasize that the support for the expression of the EBV encoded growth program is an important differentiation marker of the CLL cells of origin that was shared by the two subclones. It can be surmised that proliferation of EBV carrying cells in vitro, but not in vivo, reflects the efficient surveillance that functions even in the severe leukemic condition. The MEC1 line arose before the aggressive clinical stage from an EBV carrying cell within the subclone that was in the early prolymphocytic transformation stage while the MEC2 line originated one year later, from the subsequent subclone with overt PLL characteristics. At this time the disease was disseminated and the blood lymphocyte count was considerably elevated. The EBV induced proliferation of the MEC cells belonging to the subclones with markers of PLL agrees with earlier reports in which cells of PLL disease were infected in vitro and immortalized to LCL. They prove also that the expression of EBV encoded set of proteins can be determined at the event of infection. This pair of lines is particularly important as they provide in vitro cells that represent the subclonal evolution of the CLL disease. Furthermore, the phenotype of the MEC1 cells shares several characteristics of ex vivo CLL cells.
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