An antigen receptor-driven, interleukin 2-independent pathway for proliferation of murine cytolytic T lymphocyte clones.

Proliferation of T lymphocytes can be induced by IL-2, either through an autocrine pathway in which the responding cell produces its own IL-2 or through an exocrine pathway in which IL-2 secreted by Th stimulates proliferation of IL-2-dependent CTL. However, proliferation of at least some CTL clones, such as CTL L3 and CTL dB45, also can be induced by stimulation of the antigen receptor in the absence of IL-2. Stimulation of these cloned CTL with T cell-depleted allogeneic spleen cells, allogeneic tumor cells, or immobilized mAb reactive with the T cell antigen receptor (TCR) induced thymidine incorporation, entry into cell cycle, and secretion of macrophage activating factor, but these stimuli did not induce the secretion of IL-2. Several observations indicated that such proliferation of cloned CTL induced by stimulation of the TCR was independent of IL-2; IL-2 could not be detected in supernatants from stimulated CTL cells. mAbs reactive with the murine IL-2-R efficiently blocked IL-2-mediated thymidine incorporation in cloned CTL and Th, but had no inhibitory effect on TCR-driven thymidine incorporation in the CTL clones. TCR-driven thymidine incorporation in cloned Th L2 cells was profoundly inhibited by these antibodies, indicating the operation of an IL-2-mediated autocrine pathway for proliferation in this cloned Th. When antibodies to the TCR were used to stimulate cloned CTL and Th, IFN-gamma mRNA was easily shown in the cloned CTL and Th. Although IL-2 mRNA could be detected in the cloned Th, it was never observed in the cloned CTL. These findings provide evidence for the existence of a TCR-mediated, IL-2-independent pathway for induction of cellular proliferation in cloned murine CTL.

Establishment of a leukemia cell line with i(12p) from a patient with a mediastinal germ cell tumor and acute lymphoblastic leukemia.

We report the establishment of a leukemia cell line (UoC-B10) from a patient who developed leukemia several months after the diagnosis of a mediastinal yolk sac tumor. The patient's yolk sac tumor responded to combination chemotherapy, and a mature teratoma with focal areas of hematopoiesis was subsequently resected. However, 5 months after the initial diagnosis, the patient developed an acute lymphoblastic leukemia with a precursor B-cell phenotype. Cytogenetic analysis showed an i(12p) abnormality in the patient's leukemia cells and in the UoC-B10 cell line. The i(12p) was also identified retrospectively in the mediastinal tumor cells by fluorescent in situ hybridization analysis. The UoC-B10 cell line, which has been growing continuously for > 24 months in culture, was Epstein-Barr virus negative and was generally concordant with the patient's leukemia cells by analysis of immunophenotype, karyotype, and genotype. The UoC-B10 cell line possesses receptors for granulocyte-colony-stimulating factor, a cytokine which the patient received as part of his treatment protocol. This cell line may be useful in studying the relationship between i(12p) and hematological differentiation of human mediastinal germ cell tumors.

Deletion or lack of expression of CDKN2 (CDK4I/MTS1/INK4A) and MTS2 (INK4B) in acute lymphoblastic leukemia cell lines reflects the phenotype of the uncultured primary leukemia cells.

The CDKN2 gene has been recently localized to a chromosomal region found to be deleted in leukemias and solid tumors. CDKN2 encodes a 16 kDa protein product (p16INK4A), which functions as a specific inhibitor or the cyclin-dependent kinases 4 and 6. There have been many reports indicating a higher frequency of deletions of the CDKN2 gene in a variety of tumor cell lines, in comparison to primary tumors. These studies raise the possibility that deletions of CDKN2 may be a rare event in primary tumors, and in fact arise in vitro, during the establishment of permanent cell lines. To address this issue, we determined whether the CDKN2 gene deletions found in acute lymphoblastic leukemia (ALL) cell lines are also detected in the primary leukemia samples. Eleven cell lines were identified which had available frozen primary samples of their original leukemic tissue. Five out of 11 of these cell lines, as well as their primary samples had homozygous CDKN2 deletions. The remaining six cell lines and their primary samples retained at least one copy of the CDKN2 gene. Of the six CDKN2+ cell lines, five expressed CDKN2 mRNA, but only one of these expressed the p16 protein product (as did its primary sample). Our results indicate that CDKN2 deletions present in the studied ALL cell lines arose in the primary leukemic cells, and not during cell line establishment or prolonged in vitro culture.

Establishment and characterization of a megakaryoblast cell line with amplification of MLL.

A new cell line with megakaryoblastic features, designated UoC-M1, was established from the malignant cells of a 68-year-old patient with acute myeloid leukemia. The patient's leukemic cells reacted with alpha-naphthyl acetate esterase and acid phosphatase and expressed CD7, CD24, CD34, CD38, CD45, HLA-DR and CD61. Cytogenetic analysis of the patient's malignant cells (and of the UoC-M1 cells) showed a human, male hypodiploid karyotype with many chromosome rearrangements and marker chromosomes. Spectral karyotyping (SKY) analysis complemented the G-banded karyotyping and clarified several chromosomal translocations and identified the marker chromosomes. Fluorescence in situ hybridization (FISH) and SKY analysis demonstrated that one marker chromosome contained three segments of chromosome 9 interspersed with three segments of chromosome 11, as well as a portion of chromosome 19. FISH analysis with a probe for MLL revealed that the UoC-M1 cells contained four copies of the MLL gene. Southern blot analysis determined that the MLL gene had a germline profile while Northern and Western analyses showed that the MLL mRNAs and protein were of the appropriate sizes. This is the first report of amplification of the MLL gene which may be an additional mechanism of leukemogenesis or disease progression.

Growth inhibition of B-cell precursor acute lymphoblastic leukemia cell lines by monocytes: a role for prostaglandin E2.

Leukemic cell lines have proven invaluable in the molecular analysis of recurring chromosomal translocations but the optimal methods for leukemia cell line establishment are unknown. During in vitro culture, most B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cells die within 1 week at least partially mediated by inhibitors elaborated by peripheral blood mononuclear cells (PB MNCs) present within the leukemia sample. In experiments reported here, cyclooxygenase inhibitors (indomethacin and meclofenamic acid) blocked the PB MNC-mediated inhibition of BCP-ALL proliferation. Also, prostaglandin E2 (PGE2) was detected in supernatants from PB MNC cultures. When PGE2 was mixed directly with BCP-ALL cells, proliferation decreased significantly. Under the culture conditions used, PB MNCs secreted PGE2 which appears to be one of the major inhibitors of BCP-ALL growth in vitro.

Immunosuppressive effects of cyclosporin A on cloned T cells.

The effects of the immunosuppressive agent Cyclosporin A (CsA) on the immune response of T lymphocytes are not clearly understood. Much of the previous data are conflicting, possibly due to the study of bulk populations of cells. Therefore, we studied the effects of CsA on cloned murine helper T lymphocytes (HTL) and cytolytic T lymphocytes (CTL) after stimulation with Con A or with monoclonal antibodies to the cells' antigen receptors. In the HTL L2, proliferative responses and production of the lymphokines interleukin 2, interleukin 3, and interferon-gamma were blocked to background levels when CsA was added to cultures at a concentration of 0.1 to 1 microgram/ml. This inhibition of lymphokine production was found to occur at a pretranslational level, because the mRNA for these proteins was not detected when the L2 cells were stimulated in the presence of CsA. In addition, when CsA was added to cultures 3 hr after the cells had first been stimulated with the lectin Con A, levels of mRNA for the lymphokines isolated from the L2 cells 3 hr later were reduced approximately twofold. In the CTL L3, production of lymphokine (interferon-gamma) was also inhibited by CsA at a pretranslational level. However, proliferative responses to maximal stimulation with the clonotypic antibody 384.5 were not inhibited. In both HTL and CTL, the proliferative responses to recombinant IL 2 were not affected. To test whether CsA affects expression and function of the antigen receptor, we studied the effects of the drug on binding of anti-antigen receptor antibodies KJ 16-133 and 384.5 to the cell surfaces and the ability of L3 cells to lyse P815 target cells. At dosages which inhibited lymphokine production, CsA did not compete with binding of KJ 16-133 to L2 cells or 384.5 to L3 cells, as measured by flow cytometry, and the ability of L3 cells to lyse targets was unaffected. We conclude the following. CsA inhibits production of interleukin 2, interleukin 3, and interferon-gamma by L2 cells and interferon-gamma by L3 cells. This appears to occur as a result of a block in the transcription of the lymphokine genes. This pretranslational inhibition of lymphokine production can be invoked after transcription has begun. CsA does not affect expression of the T cell receptor for antigen as measured by monoclonal antibodies reactive with the receptors and by cytolytic activities of cytotoxic lymphocytes. CsA does not affect proliferative responses of HTL and CTL induced by interleukin 2.(ABSTRACT TRUNCATED AT 400 WORDS)

PMA alone induces proliferation of some murine T cell clones but not others.

The responses of cloned murine T cell lines to the phorbol ester, phorbol myristate acetate (PMA), were investigated. PMA alone was able to stimulate proliferation of some clones but not others. Two Lyt-2+, cloned cytolytic T lymphocyte (CTL) lines proliferated in response to stimulation by PMA alone, but several L3T4+, cloned helper T lymphocyte (HTL) lines did not. In contrast, all clones tested released lymphokines in response to stimulation by the combination of PMA and the calcium ionophore A23187. Moreover, all clones proliferated in response to stimulation by the combination of PMA and A23187. The proliferation of HTL in response to PMA + A23187 could be completely inhibited either by cyclosporine A (CsA) or by PC61.5, a monoclonal antibody directed against the murine IL 2 receptor; however, the proliferation of CTL in response to PMA alone was not affected either by CsA or by PC61.5. These results suggest that of the murine T cell clones tested, HTL proliferate in response to stimulation via an IL 2-dependent, autocrine pathway; in contrast, CTL, in addition to an IL 2-dependent pathway, may possess an additional IL 2-independent pathway of proliferation. CTL that proliferate in response to stimulation by PMA alone may be useful models in the study of T cell proliferation.

Interleukin-2 differentially regulates IL-2 receptors on murine cloned cytolytic and helper T cells.

The effects of interleukin-2 (IL-2) on the expression of IL-2 receptors by cloned cytolytic and helper T lymphocytes were studied using three anti-IL-2 receptor antibodies. IL-2 enhanced the expression of IL-2 receptors on all clones tested. A steep dose-response curve was observed with no measurable effect seen below 2 units/ml and maximal IL-2 receptor expression with greater than 5 units/ml. IL-2 receptor expression peaked 24-48 hr after the addition of IL-2. The subsequent decrease in IL-2 receptor expression correlated with a decrease in the levels of IL-2 remaining in the culture supernatants of cytolytic T lymphocyte cells. Removing residual IL-2 from cultures resulted in the rapid return of IL-2 receptor expression to unstimulated levels. The daily addition of low levels of IL-2 to cultures resulted in the prolonged expression of high levels of IL-2 receptors by non-IL-2-producing cloned cytolytic T cells. Cloned helper T cells which make IL-2 showed the initial increase in IL-2 receptor levels, but the daily addition of IL-2 did not prolong IL-2 receptor expression in these cells. These data suggest that IL-2 receptors on those cells which do not make IL-2 are regulated differently from receptors on cells which themselves make IL-2.

Inhibition of IL 2-driven proliferation of murine T lymphocyte clones by supraoptimal levels of immobilized anti-T cell receptor monoclonal antibody.

Both cloned murine helper T lymphocytes (HTL) and cytolytic T lymphocytes (CTL) proliferated and secreted lymphokines when stimulated with immobilized anti-T cell receptor monoclonal antibody (anti-TCR mAb). However, although proliferation of CTL increased and reached plateau levels as concentrations of anti-TCR mAb were increased, the proliferation of HTL decreased with high concentrations of anti-TCR mAb. A reduction of IL 2-dependent proliferation by CTL was observed when IL 2 was added to cultures of CTL in the presence of high concentrations of anti-TCR mAb, whereas IL 2-independent proliferation appeared to be unaffected by these concentrations of anti-TCR mAb. Inhibition of IL 2-driven proliferation caused by high concentrations of immobilized anti-TCR mAb did not seem to be mediated by soluble factors. Cells continued to express cell surface receptors for IL 2 and transferrin after treatment with immobilized anti-TCR mAb. Inhibition of IL 2-driven proliferation by high concentrations of immobilized anti-TCR mAb may represent a mechanism for regulating the proliferation of T lymphocytes. This inhibitory mechanism is initiated by stimulation of the T cell receptor, in this case by immobilized anti-TCR mAb, and is independent of other cells and factors.

Antibodies to the L3T4 and Lyt-2 molecules interfere with antigen receptor-driven activation of cloned murine T cells.

When L3T4+ cloned murine helper T lymphocytes (HTL) are stimulated with antigen or immobilized anti-T cell receptor (TCR) monoclonal antibodies (mAb) at concentrations which are optimal for proliferation, anti-L3T4 mAb inhibits activation as measured by proliferation and lymphokine production. Under similar conditions, IL 2-independent proliferation of Lyt-2+ cloned murine cytolytic T lymphocytes (CTL) stimulated by anti-TCR mAb is inhibited by anti-Lyt-2 antibodies. Proliferation of cloned HTL and CTL cells stimulated by IL 2 is not affected by the anti-L3T4 and anti-Lyt-2 mAb. The inhibition of TCR-induced activation of the T cell clones is not due to interference with the binding of the anti-TCR mAb. Stimulation of the TCR has been proposed to induce lymphokine secretion and proliferation by T cells through a pathway involving the activation of protein kinase C and the stimulation of an increase in the concentration of intracellular free calcium. However, proliferation of T cells stimulated by PMA (which activates protein kinase C) plus the calcium ionophore A23187 (which increases the concentration of intracellular free calcium) is not affected by mAb reactive with the Lyt-2 or L3T4 structures. If TCR stimulation does indeed activate T cells by activating protein kinase and increasing intracellular free calcium, then our data suggest that anti-L3T4 and anti-Lyt-2 mAb inhibit TCR-driven proliferation at some step before the activation of protein kinase C and the stimulation of a rise in intracellular free calcium concentration. Our results suggest that anti-L3T4 and anti-Lyt-2 mAb interfere with early biochemical processes induced by stimulation of the TCR. In HTL, which proliferate via an autocrine pathway, anti-L3T4 mAb appears to inhibit proliferation by interfering with signaling events involved in lymphokine production. Inhibition of IL 2-independent proliferation of Lyt-2+ cells by anti-Lyt-2 mAb appears to occur by a different mechanism. The precise molecular basis for the interference of each cell type has not yet been characterized.

Characterization of an anti-Ly-6 monoclonal antibody which defines and activates cytolytic T lymphocytes.

HK1.4 mAb was identified based on its ability to stimulate proliferation of cloned murine CTL. Within the lymphoid lineage, mAb HK1.4 bound exclusively to CTL, regardless of the expression of Lyt-2 or MHC restriction. HK1.4 mAb also bound to 40% of bone marrow cells and less than 5% of thymocytes from all mouse strains tested. Based on the tissue distribution of the determinant with which it reacted and the ability to cross-block binding of the anti-Ly-6 mAb H9/25, mAb HK1.4 appeared to react with a product of the Ly-6 locus. However, significant differences were observed between the properties of mAb HK1.4 and other, previously described anti-Ly-6 mAb. Cell proliferation and lymphokine release by cloned CTL were stimulated by culture with mAb HK1.4 alone or in the presence of non-stimulatory levels of IL-2. This proliferation and lymphokine release were not blocked by the addition of soluble anti-Lyt-2 or anti-IL-2R mAb. Activation induced by HK1.4 mAb proceeds in the absence of accessory cells, of cross-linking of the TCR, or the addition of mitogens or PMA. Stimulation of cells by anti-TCR mAb was not blocked by the addition of soluble HK1.4 mAb, and the stimulatory effects of HK1.4 and anti-TCR mAb were not additive. However, IL-2-driven proliferation of CTL clones was dramatically inhibited by the addition of HK1.4 mAb.HK1.4 mAb had no effect on Ag-specific or lectin-facilitated cytolysis. Taken together, these data indicate that mAb HK1.4 operates via an IL-2-independent pathway of activation that is also independent of the TCR.

TEL-AML1 translocations with TEL and CDKN2 inactivation in acute lymphoblastic leukemia cell lines.

The t(12;21) (p 13; q22) results in the fusion of the TEL gene located on chromosome 12 with the AML1 gene located on the derivative chromosome 21. Because this translocation is difficult to detect using standard cytogenetic techniques, 27 previously karyotyped B-lineage acute lymphoblastic leukemia (ALL) cell lines were evaluated for the presence of the TEL-AML1 fusion using the reverse transcriptase-polymerase chain reaction (RT-PCR), fluorescence in situ hybridization (FISH), and cDNA sequencing. Six cell lines expressed the TEL-AML1 chimeric transcript by RT-PCR and the t(12;21) was confirmed by FISH analysis with probes for TEL, AML1, and chromosome 12. While only one of the 6 cell lines with the t(12;21) lost the der(12)t(12;21)-encoded AML1-TEL fusion transcript, 4 cell lines lacked expression of the nontranslocated allele of TEL and 5 cell lines lacked expression of CDKN2. Moreover, in 2 patients (1 with the TEL-AML1 transcript and 1 without), TEL expression was lost with disease progression; le, TEL was expressed in the initial cell lines (established at diagnosis or first relapse) whereas TEL was not expressed in the cell lines established from these patients in late-stage disease. These data show the coexistence of multiple genetic defects in childhood B-lineage ALL Cell lines with t(12;21) will facilitate the study of TEL-AML1 and AML1-TEL fusion proteins as well as TEL and CDKN2 gene inactivation in leukemia transformation and progression.