Immortalization Of Human Lymphocytes Research Articles

A Human T-cell Leukemia Virus Tax Variant Incapable of Activating NF-κB Retains Its Immortalizing Potential for Primary T-lymphocytes

The human T-cell leukemia virus type 1 (HTLV-1) transactivator (Tax) has been shown to interfere with regulated cellular proliferation. Many studies have focused on the ability of Tax to transform rodent fibroblasts; however, none has defined the molecular requirements for Tax transformation of human lymphoid cells. We show here that tax induces permanent growth of human primary T-lymphocytes by using a transformation/immortalization defective rhadinovirus vector. The cells phenotypically resemble HTLV-immortalized lymphocytes and contain episomally persisting recombinant rhadinoviral sequences, which stably express functional Tax protein. As Tax can activate major cellular signal transducing pathways including NF-kappaB and cAMP-responsive element binding protein (CREB), we asked for the relevance of these routes in the immortalization of human lymphocytes. By using Tax mutants that either activate exclusively CREB/activating transcription factor or are defective in activating this signaling pathway, we delineated that Tax can induce immortalization of primary human T-lymphocytes through a mechanism independent of NF-kappaB activation.

Immortalization of human lymphocytes by fusion with cytoplasts of transformed mouse L cells.

Fusion of mouse L929 cytoplasts with human peripheral blood lymphocytes induced lymphocyte proliferation that gave rise to lymphoid cell lines of B and T cell origin with unlimited growth potential. The immortalized cell lines were routinely grown in standard medium supplemented with fetal calf serum. Furthermore these cell lines could be propagated in chemically defined serum-free media. Each establishment of lymphoid cell lines was preceded by a proliferation phase 2 wk after cytoplast/cell fusion, which appears to be a necessary step in the immortalization process. The immortalized cells have a nearly normal human karyotype, do not form colonies in soft agar medium, and are not tumorigenic in nude mice. Cloned B cell lines produced human immunoglobulins of heavy and light chain types. No cross-reaction with DNA of herpes simplex virus, human cytomegalovirus, human T cell leukemia/lymphoma virus I and II, or polyoma virus was detected in the genome of immortalized cell lines by Southern blot hybridization. Furthermore B and T cell lines were established that appear to be free of Epstein-Barr virus genome.

The pX protein of HTLV-I is a transcriptional activator of its long terminal repeats.

Expression of the pX protein of human T-cell leukemia virus type I (HTLV-I) in animal cells demonstrates that this protein is a specific transcriptional activator of the long terminal repeats (LTR) of HTLV-I. Several other promoters are not affected by pX. No lymphocyte-specific factors are required for this activation. pX can be detected in the nucleus of transfected monkey kidney cells (line CV1) by indirect immunofluorescence. These results indicate that the pX protein is essential for the replication cycle of the virus and that it may be directly involved in the immortalization of human lymphocytes by HTLV-I.

Immortalization of human lymphocytes by co-cultivation with lethally irradiated autologous T-cell lines harbouring human T-cell leukaemia virus-I

Human adult peripheral blood lymphocytes were successfully immortalized by co-cultivation with irradiated autologous and homologous T-cell lines harbouring human T-cell leukaemia virus-I (HTLV-I). The efficiency of transformation was the same in both cases. The participation of alloantigen stimulation in co-cultivation procedures is discussed, and it is stressed that factors other than alloantigen stimulation might be required for the efficient immortalization of human T-lymphocytes in vitro by HTLV.

Lymphoma in cotton-top marmosets after inoculation with Epstein-Barr virus: tumor incidence, histologic spectrum antibody responses, demonstration of viral DNA, and characterization of viruses.

6 of 20 cotton-top tamarins (Saguinus oedipus) inoculated with Epstein-Barr virus (EBV) developed diffuse malignant lymphoma resembling reticulum cell or immunoblastic sarcoma of man. Hyperplastic lymphoreticular lesions were induced in three additional animals; in two instances the hyperplastic lesions regressed. Inapparent infection with development of antibody occured in eight animals. In two animals there was no evidence of EBV infection. One animal died in the first week after inoculation of parasitic infection. 10 animals uninoculated or mock-inoculated developed neither lymphoproliferative disease nor antibody. The malignant lymphoma appeared to arise from a cell with an uncleaved vesicular nucleus found in the center of the germinal follicle. The prominent cytologic features of this cell were extensive formation or rough endoplasmic reticulum and elaboration of the cytoplasmic membrane with microvilli. Cell lines derived from these tumors did not have receptors for complement. IgFc, or sheep erythrocytes, and the cell lines adhered to glass and plastic. EB nuclear antigen was found in imprints of two lymph nodes, one with lymphoma and one with hyperplasia. EB virus DNA was detected directly in the tumors of three animals and in cell lines from two lymphomas. Typical herpes virus particles were found in supernatant fluids from cell lines obtained from lymph nodes with tumors and hyperplasia, as well as in lines derived from blood leukocytes of marmosets with inapparent infection. These virus preparations had the biologic property characteristic of EBV, namely, stimulation of cellular DNA synthesis and immortalization of human lymphocytes. The virus derived from two cell lines was neutralized by reference human sera with EBV antibody and not by antibody-negative human sera. The virus derived from the experimental lesions is thus indistinghishable from human EBV. The marmoset has enhanced susceptibility to oncogenesis by EB virus. Among identified factors which may play a role in the heightened tumorigenicity of EB virus in this species are the increased production of virus by transformed cells and the absence of membrane receptors for complement or IgFc on transformed cells.