Leukemic cells arise from cloned cytotoxic lymphocytes during cell culture

Abstract

In spite of many promising attempts to apply T cell clones to questions of in vitro and in vivo function of T cells it is still unclear to what extent continuous propagation of T lymphocytes in vitro effects their original properties. This study describes the appearance of malignant cells from long-term cultured C57BL/6 (B6) cytotoxic T lymphocytes (CTL). Four out of five T cell lines (CTLL.1,3,4,5) representing distinct stages of development of T effector cells in vitro were repeatedly cloned and all five CTLL were tested for various cellular parameters. It is shown that transformation of H-Y-specific CTLL into malignant cells in vitro was accompanied by alterations in growth characteristics, successive loss of specificity and cytolytic function and by quantitative changes in the expression of cell surface markers. Whereas growth of the H-Y-specific CTLL (CTLL.l) was dependent on antigen and concanavalin A (Con A) supernatant (Con ASN) the CTLL variants could be either maintained in Con ASN alone (CTLL.3) or in the absence of both antigen and lymphokine sources (CTLL.4,5). CTLL.l was cytolytic for male B6 target cells and lysed P815 tumor targets in the presence but not the absence of lectin. In contrast, CTLL.3 lost its original specificity but lysed P815 cells in the absence or presence of lectin. CTLL.2 representing an intermediary stage showed cytolytic activity on both male B6 and P815 target cells. In contrast, CTLL.4 and CTLL.5 lost the ability to lyse any of the indicated target cells. Although all CTLL expressed the surface markers Thy-1, Lyt-2, K^b, D^b and interleukin 2 receptor (IL 2R), Thy-1 and Lyt-2 markers were drastically reduced and K^b/ D^b and IL 2 R structures significantly increased on CTLL.4 and CTLL.5 compared to CTLL.1,2,3. In addition, multiple karyotypic alterations including the appearance of metacentric chromosomes were observed in long-term cultured CTLL. Investigations on the expression of theα-, β-, and γ-chains of the T cell antigen receptor in CTLL. 1-5 indicate that all three chains were expressed as mRNA irrespective of whether the lymphocytes expressed their original specificity and/or function. However, distinct β variable chain genes were used by H-Y-specific CTLL and its longterm culture variants CTLL.2 and CTLL.3 suggesting that the expression of the new specificity was accompanied by the rearrangement of a new β-chain gene in T effector cells. The new β-chain gene was also expressed as mRNA in CTLL.4 and CTLL.5. When injected i.p. into B6 male and female mice CTLL.4 but not CTLL.1,2,3 showed progressive growth in ascites or grew as solid tumor. CTLL-derived metastases were found in central organs (liver, lung, spleen, uterus) as well as in the central nervous system (brain). The fact that specificity, functional and malignant variants can be isolated from the same H-Y-specific CTLL in vitro may allow the study of mechanisms which alter antigen specificity, function and growth control of T effector cells. In addition, this model system may be suitable for studying the parameters responsible for the conversion of normal diploid CTLL to cancer cells.