Establishment of a bank of blood donor derived epstein barr virus specific T cell lines for treatment of post-transplant lymphoproliferative disease

ABSTRACT Epstein-Barr virus (EBV)-specific T-cell lines generated by repeated stimulation with EBV-immortalized lymphoblastoid B-cell lines (LCL) have been successfully used to treat EBV-associated posttransplant lymphoproliferative disease (PTLD) in hematopoietic stem cell transplant recipients. However, PTLD in solid-organ transplant recipients and other EBV-associated malignancies respond less efficiently to this adoptive T-cell therapy. LCL-stimulated T-cell preparations are polyclonal and contain CD4+ and CD8+ T cells, but the composition varies greatly between lines. Because T-cell lines with higher CD4+ T-cell proportions show improved clinical efficacy, we assessed which factors might compromise the expansion of this T-cell population. Here we show that spontaneous virus production by LCL and, hence, the presentation of viral antigens varies intra- and interindividually and is further impaired by acyclovir treatment of LCL. Moreover, the stimulation of T cells with LCL grown in medium supplemented with fetal calf serum (FCS) caused the expansion of FCS-reactive CD4+ T cells, whereas human serum from EBV-seropositive donors diminished viral antigen presentation. To overcome these limitations, we used peripheral blood mononuclear cells pulsed with nontransforming virus-like particles as antigen-presenting cells. This strategy facilitated the specific and rapid expansion of EBV-specific CD4+ T cells and, thus, might contribute to the development of standardized protocols for the generation of T-cell lines with improved clinical efficacy.

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A Novel Epstein-Barr Virus-Like Virus, HVMNE, in aMacaca Nemestrina With Mycosis Fungoides

Blood ◽

10.1182/blood.v94.6.2090.418k28_2090_2101 ◽

1999 ◽

Vol 94 (6) ◽

pp. 2090-2101

Author(s):

E.D. Rivadeneira ◽

M.G. Ferrari ◽

R.F. Jarrett ◽

A.A. Armstrong ◽

P. Markham ◽

...

Keyword(s):

T Cell ◽

Cell Lines ◽

Epstein Barr Virus ◽

Cd8 T Cell ◽

Macaca Nemestrina ◽

Type I ◽

Polymerase Gene ◽

Barr Virus ◽

Epstein Barr ◽

T Cell Lines

Epstein-Barr virus (EBV) infection of humans has been associated with the development of lymphoid malignancies mainly of B-cell lineage, although occasionally T-cell lymphomas have been reported. We describe here the characterization of a novel EBV-like virus (HVMNE) isolated from a simian T-cell lymphotropic virus type I/II (STLV-I/II) seronegative pigtailed macaque (Macaca nemestrina) with a cutaneous T-cell lymphoma. Immunohistochemistry studies on the skin lesions demonstrated that the infiltrating cells were of the CD3+/CD8+ phenotype. Two primary transformed CD8+ T-cell lines were obtained from cultures of peripheral blood mononuclear cells (PBMC) and skin, and, with time, both cell lines became interleukin-2–independent and acquired the constitutive activation of STAT proteins. Polymerase chain reaction analysis of the DNA from the cell lines and tissues from the lymphomatous animal demonstrated the presence of a 536-bp DNA fragment that was 90% identical to EBV polymerase gene sequences, whereas the same DNA was consistently negative for STLV-I/II sequences. Electron microscopy performed on both cell lines, after sodium butyrate treatment, showed the presence of a herpes-like virus that was designated HVMNE according to the existing nomenclature. In situ hybridization studies using EBV Epstein-Barr viral-encoded RNA probes showed viral RNA expression in both CD8+ T-cell lines as well as in the infiltrating CD8+ T cells of skin-tissue biopsies. Phylogenetic analysis of a 465-bp fragment from the polymerase gene of HVMNE placed this virus within theLymphocryptovirus genus and demonstrated that HVMNEis a distinct virus, clearly related to human EBV and other EBV-like herpesviruses found in nonhuman primates.

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Epstein-Barr virus (EBV)-carrying and -expressing T-cell lines established from severe chronic active EBV infection

Blood ◽

10.1182/blood.v87.4.1446.bloodjournal8741446 ◽

1996 ◽

Vol 87 (4) ◽

pp. 1446-1457 ◽

Cited By ~ 76

Author(s):

S Imai ◽

M Sugiura ◽

O Oikawa ◽

S Koizumi ◽

M Hirao ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Lines ◽

Epstein Barr Virus ◽

Gene Rearrangement ◽

Barr Virus ◽

Ebv Infection ◽

Epstein Barr ◽

T Cell Lines

Four novel Epstein-Barr virus (EBV)-carrying T-cell lines, designated SIS, AIK-T8, AIK-T4, and SKN, were established from peripheral blood lymphocytes (PBL) of patients with severe chronic active EBV infection, in the presence of interleukin-2 and 4-deoxyphorbol ester. AIK-T8 and - T4 were derived from a single patient. Cell marker and genotype analyses showed that SIS, AIK-T8, and AIK-T4 had mature T-cell phenotypes with clonally rearranged T-cell receptor (TCR) genes, whereas SKN had an immature T-cell phenotype without TCR gene rearrangement. None of the cell lines expressed B, natural killer, or myeloid antigens or had Ig gene rearrangement. All lines carried EBV genomes in a single episomal form. SIS, AIK-T8, and SKN showed the same phenotype, TCR gene configuration, and/or EBV clonotype as their source or biopsied materials; therefore, they represented EBV-infected T cells proliferating in the patients. TCR gene and EBV episomal structures similar to those of AIK-T4 were not found in its source PBL, probably due to the few parental clones in vivo. All lines expressed EBV-encoded small RNA (EBER) 1, nuclear antigen (EBNA) 1, and latent membrane protein (LMP) 1, -2A, and -2B, but not other EBNAs that could be recognized by EBV-specific immune T cells. EBV replicative antigens were rarely expressed or induced. Such EBV latency reflects the in vivo situation, in which the T cells may evade immune surveillance and be insensitive to antiherpesvirus drugs. Collectively, the data suggest that EBV can target and latently infect T cells at any stage of differentiation in vivo, thus potentially causing uncontrolled T-cell proliferation. These cell lines will facilitate further analyses of possible EBV-induced oncogenicity in T cells.

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EPSTEIN-BARR VIRUS-NEGATIVE HUMAN MALIGNANT T-CELL LINES

Journal of Experimental Medicine ◽

10.1084/jem.139.5.1070 ◽

1974 ◽

Vol 139 (5) ◽

pp. 1070-1076 ◽

Cited By ~ 61

Author(s):

Joseph Kaplan ◽

Thomas C. Shope ◽

Ward D. Peterson

Keyword(s):

T Cell ◽

Cell Lines ◽

Epstein Barr Virus ◽

Malignant Cells ◽

Barr Virus ◽

Virus Antigens ◽

Epstein Barr ◽

T Cell Lines

Two lymphoblastoid lines, CCRF-CEM and HSB-2, with properties of malignant cells, derived from children with leukemia secondary to lymphosarcoma, have T-cell properties and lack Epstein-Barr virus antigens.

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An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection

Journal of Visualized Experiments ◽

10.3791/56515 ◽

2018 ◽

Author(s):

Chongfeng Xu ◽

Junhong Ai ◽

Qingxun Zhang ◽

Ting Li ◽

Xiaorong Wu ◽

...

Keyword(s):

T Cell ◽

Virus Infection ◽

Cell Lines ◽

Epstein Barr Virus ◽

Epstein Barr Virus Infection ◽

Simple Method ◽

Barr Virus ◽

Epstein Barr ◽

Barr Virus Infection ◽

T Cell Lines

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Isolation of Epstein-Barr Virus (EBV)-Specific Cytotoxic T Lymphocytes That Lyse Reed-Sternberg Cells: Implications for Immune-Mediated Therapy of EBV+ Hodgkin's Disease

Blood ◽

10.1182/blood.v89.6.1978 ◽

1997 ◽

Vol 89 (6) ◽

pp. 1978-1986 ◽

Cited By ~ 85

Author(s):

Amy P. Sing ◽

Richard F. Ambinder ◽

Doley J. Hong ◽

Michael Jensen ◽

Wendy Batten ◽

...

Keyword(s):

T Cell ◽

Cell Lines ◽

Hodgkin's Disease ◽

Epstein Barr Virus ◽

Hodgkin’S Disease ◽

Adoptive T Cell Therapy ◽

T Cell Therapy ◽

Barr Virus ◽

Epstein Barr ◽

T Cell Lines

Abstract A subset of Hodgkin's disease (HD) patients have detectable Epstein-Barr virus (EBV) genomes in the malignant Reed-Sternberg (R-S) cells. R-S cells express only a limited set of latent EBV proteins, but only LMP1 and LMP2 can potentially elicit a CD8+ cytotoxic T-lymphocyte (CTL) response. We have evaluated if either of these proteins could be used as targets for specific adoptive T-cell therapy for EBV-positive (EBV+) HD. The success of this strategy requires that R-S cells are susceptible to lysis by CD8+ CTL, and that CTL specific for LMP1 and LMP2 can be detected and potentially amplified in HD patients. Antigen presentation and CTL sensitivity was evaluated with an in vitro maintained, phenotypically representative R-S cell line, HDLM-2. The R-S cells were able to process and present viral proteins, and to be efficiently lysed by specific CTL in a Class I–restricted manner. Since CTL responses to LMP1 and LMP2 do not represent the dominant responses to EBV, we examined if CTL clones specific for these proteins could be isolated despite the presence of weak or nondetectable responses in polyclonal T-cell lines. LMP-specific clones were generated from individuals either by cloning from the polyclonal EBV-reactive T-cell lines or by direct stimulation of peripheral blood mononuclear cells (PBMC) with cells expressing LMP1 or LMP2 as the only EBV protein. Our ability to isolate CTL specific for LMP proteins from individuals with HD and the sensitivity of R-S cells for CTL-mediated lysis suggest that the pursuit of specific adoptive immunotherapy represents a viable strategy for the subset of HD patients with EBV+ tumors.

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A Novel Epstein-Barr Virus-Like Virus, HVMNE, in aMacaca Nemestrina With Mycosis Fungoides

Blood ◽

10.1182/blood.v94.6.2090 ◽

1999 ◽

Vol 94 (6) ◽

pp. 2090-2101 ◽

Cited By ~ 15

Author(s):

E.D. Rivadeneira ◽

M.G. Ferrari ◽

R.F. Jarrett ◽

A.A. Armstrong ◽

P. Markham ◽

...

Keyword(s):

T Cell ◽

Cell Lines ◽

Epstein Barr Virus ◽

Cd8 T Cell ◽

Macaca Nemestrina ◽

Type I ◽

Polymerase Gene ◽

Barr Virus ◽

Epstein Barr ◽

T Cell Lines

Abstract Epstein-Barr virus (EBV) infection of humans has been associated with the development of lymphoid malignancies mainly of B-cell lineage, although occasionally T-cell lymphomas have been reported. We describe here the characterization of a novel EBV-like virus (HVMNE) isolated from a simian T-cell lymphotropic virus type I/II (STLV-I/II) seronegative pigtailed macaque (Macaca nemestrina) with a cutaneous T-cell lymphoma. Immunohistochemistry studies on the skin lesions demonstrated that the infiltrating cells were of the CD3+/CD8+ phenotype. Two primary transformed CD8+ T-cell lines were obtained from cultures of peripheral blood mononuclear cells (PBMC) and skin, and, with time, both cell lines became interleukin-2–independent and acquired the constitutive activation of STAT proteins. Polymerase chain reaction analysis of the DNA from the cell lines and tissues from the lymphomatous animal demonstrated the presence of a 536-bp DNA fragment that was 90% identical to EBV polymerase gene sequences, whereas the same DNA was consistently negative for STLV-I/II sequences. Electron microscopy performed on both cell lines, after sodium butyrate treatment, showed the presence of a herpes-like virus that was designated HVMNE according to the existing nomenclature. In situ hybridization studies using EBV Epstein-Barr viral-encoded RNA probes showed viral RNA expression in both CD8+ T-cell lines as well as in the infiltrating CD8+ T cells of skin-tissue biopsies. Phylogenetic analysis of a 465-bp fragment from the polymerase gene of HVMNE placed this virus within theLymphocryptovirus genus and demonstrated that HVMNEis a distinct virus, clearly related to human EBV and other EBV-like herpesviruses found in nonhuman primates.

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