Stable growth transformation of human T-lymphocytes by Herpesvirus saimiri (H. saimiri) subgroup C

#The invention concerns the transformation of human T-lymphocytes to stable growth by infecting said T-cells with strains of H. saimiri subgroup NonA NonB, preferably subgroup C. The transformed cells grow independently of antigen stimulation and can have a phenotype of activated mature T-lymphocytes, expressing cell surface markers characteristic for the distinct differentiation status of the T-cells. Infection with strains of H. saimiri belonging to the subgroups above can thus be used to generate human T-cell lines for a variety of immunological and biochemical applications as well as for therapeutic use.

#Studies of normal human lymphocyte function are largely dependent on clones of stimulated cells since tumor lines usually express aberrant phenotypes and have modified functional properties. Expansion of human B-lymphocyte cultures by infection with the Epstein-Barr virus, a gamma.sub.1 herpesvirus, has provided continuously growing human B cells for the study of B-cell differentiation and immunoglobulin synthesis. The distantly related gamma.sub.2 prototype, H. saimiri, is indigenous to and apathogenic in squirrel monkeys (Fleckenstein, B. & Desrosiers, R. C. (1982) in The Herpesviruses, ed. Roizman, B. (Plenum, New York), Vol. 1, pp. 253-331) but causes fulminant T-cell lymphomas and acute lymphocytic leukemias in other New World primates (Fleckenstein, B. & Desrosiers, R. C. (1982) loc. cit.) and rabbits.

#It also immortalizes cultured peripheral blood lymphocytes of cotton top marmosets (Saguinus oedipus) (Schirm, S. et al. (1984) J. Virol. 49, 938-946) and common marmosets (Callithrix jacchus) (Desrosiers, R. C. et al. (1986) J. Virol. 57, 701-705). Genes required for lymphocyte growth transformation and oncogenicity were localized to the left terminal region of the 112-kilobase-pair (kbp) L-DNA in the H. saimiri genome (Desrosiers, R. C. et al. (1985) Science 228, 184-187). Based on extreme sequence variations at the same left terminus of L-DNA, the known H. saimiri strains have been divided into subgroups (A, B, and C as well as NonA NonB) (Desrosiers, R. C. & Falk, L. A. (1982) J. Virol. 43, 352-356; Medveczky, P. et al. (1984) J. Virol. 52, 938-944) that differ in their oncogenic capacity (Medveczky et al. (1989) J. Virol. 63, 3601-3611).

#Cultures of human mononuclear cells from peripheral blood, cord blood, or thymus infected with e.g. strains 484-77 and 488-77 of H. saimiri subgroup C (Desrosiers, R. C. et al., loc. cit.; Medveczky, P. et al., loc. cit.) yielded continuously growing lymphoblastoid cell lines, whereas all cells infected with strains 11 and OMI (group A) or SMHI (group B) and uninfected cells ceased to proliferate within approx. 4 weeks. Numerous lymphoid cell lines were consistently established by infection with the subgroup C strain 488, of which six lines were selected for further characterization (Table 1). All lines have been observed for at least 9 months. When total cellular DNA on Southern blots was hybridized with a 1.7-kbp AccI fragment from the left end of L-DNA, covering the transformation-associated gene (FIG. 1), genomic H. saimiri DNA sequences were found in all cell lines, except line PB-W, at 30-60 genome equivalents per diploid cellular genome. The lymphoid cell lines contained the multicopy viral genomes as episomes, as has been observed in lymphoma-derived monkey cells (Kaschka-Dierich, C. et al. (1982) J. Virol. 44, 295-310) and in persistently infected human tumor cell lines (Grassmann, R. & Fleckenstein, B. (1989) J. Virol. 63, 1818-1821; Alt, M. et al. (1991) Gene 102, 265-269). Estimates based on results of a polymerase chain reaction (PCR) indicated that the peripheral blood-derived line PB-W probably carries 1 genome copy per cell. The immortalized human lymphoblastoid lines did not secrete detectable amounts of infectious virus into the culture supernatant. Transcription of the putative oncogene STP-C488/ORF-2 (ORF=open reading frame) (Jung, J. U. et al., loc. cit.) was demonstrated in all cell lines by hybridization of RNA on Northern blots with ORF-2-specific primers (FIG. 1).

#All cell lines tested expressed mature T-cell phenotypes (CD2.sup.+, CD3.sup.+, CD5.sup.+, CD7.sup.+, and TCR1.sup.+) but did not have the markers CD1 and TCR2. All lines, except one (Lucas), were either CD4.sup.+ CD8.sup.- or CD4.sup.- CD8.sup.+ (Table 1). The thymus-derived cell line Lucas had a mixed phenotype.

#Double-fluorescence analysis indicated that the Lucas cell line contains CD4.sup.+ and CD8.sup.+ single-positive cells and smaller subpopulations of CD4/8 double-positive and -negative cells. All cell lines were negative for the nonspecific NK cell marker CD57 (HNK-1). A second marker for lymphocytes with Natural Killer (NK) activity, CD56 (NKH-1), was found on all lines with the exception of PB-W. Strong cytotoxic activity on K562 cells was observed for the CD8.sup.+ lines (Table 1). The CD4.sup.+ CDS.sup.- lines showed no (PB-W) or low (CB-15) NK activity. All cell lines appeared to represent activated T-cells expressing HLA-class II molecules and were positive for the T-cell activation marker CD30. They all expressed the interleukin 2 receptor .beta. chain. No reactivity was observed with the pan-B-cell marker CD19.