Lymphoid Lines Research Articles

ATF-2-binding regulatory element is responsible for the Ly49A expression in murine T lymphoid line, EL-4

To understand the mechanism of Ly49A-expression and its significance in T-cell differentiation, we analyzed the 5′-flanking region of the Ly49A gene in a search for the Ly49A-regulatory element. Since very few known regulatory elements have been found in this region, presumably a novel regulatory sequence(s) could exist. Accordingly, we defined the 13-bp regulatory element, 5′-ATGACGAGGAGGA-3′, restricted to Ly49A-expression in EL-4 cells in comparison with two other representative cell lines tested. This element, designated as EL13, proved to be previously undiscovered by homology search and is highly homologous with several virus DNAs. Using EL13 as a probe we have cloned a cDNA encoding a binding protein to EL13. Its deduced nucleotide sequence revealed that EL13-binding protein is almost identical with rat ATF-2. Although ATF-2 is known to bind to cyclic AMP responsive element (CRE), EL13 shares five out of eight nucleotides with this consensus sequence. Our results suggested that ATF-2 may play an important role via binding to EL13 for the expression of Ly49A. These data will provide useful information for understanding T-cell and NK-cell differentiation in murine immune system.

IL-6 Rescues Enterocytes from Hemorrhage Induced Apoptosisin Vivoandin Vitroby abcl-2Mediated Mechanism

Following a hemorrhagic event, damage to the highly metabolic intestinal tissue induces loss of barrier function leading to bacterial escape and LPS contamination of the host. Orally administered IL-6 restores intestinal barrier function following hemorrhage in both rat and mouse models. IL-6 prevents apoptosis in a variety of lymphoid cells and lines, through the activation of the proto-oncogenebcl-2.This communication elucidates the role of the IL-6–bcl-2interaction in intestinal apoptosis following hemorrhagic shock. Terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling (TUNEL) and p53 immunohistochemical staining were used to examine intestines from mice hemorrhaged and fed saline or IL-6 and enterocytes (IEC-6) exposed to hypoxia and LPS alone or LPS and IL-6in vitro. In situhybridization forbcl-2expression was performed on intestines or enterocytes. Intestinal sections from mice hemorrhaged and fed IL-6 showed reduction in apoptosis and increases inbcl-2gene expression relative to sections taken from mice hemorrhaged and fed saline. IEC-6 cells exposed to hypoxia and LPS had high numbers of TUNEL staining cells. Subsequent exposure to IL-6 after hypoxia and LPS reduced apoptotic cell numbers and increasedbcl-2gene expression. The data show that exposure of intestinal epithelial cells to IL-6 either by oral administration in hemorrhaged mice or by coculture following hypoxia and LPS treatment results in increasedbcl-2gene expression and reduced damage from apoptosis.

Retroviral Transfer of Antisense Sequences Results in Reduction of C-Abl and Induction of Apoptosis in Hemopoietic Cells

The c-abl proto-oncogene is ubiquitously expressed during mammalian development. Activated forms of c-Abl proteins are oncogenic and have been shown to suppress apoptosis. The biological role of normal c-Abl protein is unknown. In this study, we have introduced c-abl antisense sequences into various hemopoietic cells by retroviral gene transfer. Introduction and expression of the antisense sequence effectively reduced the amount of c-Abl protein in a number of transduced hemopoietic cells, that consequently underwent apoptosis. When factor-dependent cell lines were examined, we observed that the addition of sufficient amounts of growth factors could suppress apoptosis in myeloid but not in lymphoid lines. The ability of myeloid cells to be rescued by growth factors correlated with upregulation of mRNA level of IL-3 receptor subunits. Our data suggest that c-Abl provides an anti-apoptotic signal during mammalian cell growth, and that myeloid and lymphoid cells are different in their resistance to apoptosis.

Retroviral transfer of antisense sequences results in reduction of C-Abl and induction of apoptosis in hemopoietic cells.

The c-abl proto-oncogene is ubiquitously expressed during mammalian development. Activated forms of c-Abl proteins are oncogenic and have been shown to suppress apoptosis. The biological role of normal c-Abl protein is unknown. In this study, we have introduced c-abl antisense sequences into various hemopoietic cells by retroviral gene transfer. Introduction and expression of the antisense sequence effectively reduced the amount of c-Abl protein in a number of transduced hemopoietic cells, that consequently underwent apoptosis. When factor-dependent cell lines were examined, we observed that the addition of sufficient amounts of growth factors could suppress apoptosis in myeloid but not in lymphoid lines. The ability of myeloid cells to be rescued by growth factors correlated with upregulation of mRNA level of IL-3 receptor subunits. Our data suggest that c-Abl provides an anti-apoptotic signal during mammalian cell growth, and that myeloid and lymphoid cells are different in their resistance to apoptosis.

A point mutation within CD45 exon A is the cause of variant CD45RA splicing in humans.

The leukocyte common antigen (CD45) is alternatively spliced, generating various isoforms expressed on hemopoietic cells. The splicing pattern of CD45 in T cells is altered in some individuals who show abnormal expression of high molecular weight isoforms containing exon A. The variant splicing pattern was shown to be associated with heterozygosity for a silent point mutation within CD45 exon A. This C to G transition is located 77 nucleotides downstream of the splice acceptor junction of exon A (198 bp total length). Here we report that this mutation is the cause of abnormal splicing. To isolate the mutant gene, somatic cell hybrids of lymphocytes with a CD45 splicing defect and a mouse lymphoid line were produced and clones expressing different isoforms of CD45 were isolated. Expression of the high molecular weight isoform containing exon A was associated with the mutation within exon A. All hybrids expressing the low molecular weight isoforms lacking exon A contained the normal allele of CD45 only. In addition, minigenes including this mutation were constructed and transfected into various cell lines (COS-7, HeLa, CHO). Semi-quantitative reverse transcription polymerase chain reaction showed an increase of more than tenfold in splicing to CD45RA (concomitant with a decrease in splicing to CD45RO) when compared with the normal minigene. Taken together, these results demonstrate a causal relationship between the mutation in CD45 exon A and the variant splicing pattern observed. The involvement of trans-acting splicing factors that interact with this region of CD45 pre-mRNA is currently under investigation.

Mouse Lymphoma Cells Destined to Undergo Apoptosis in Response to Thapsigargin Treatment Fail to Generate a Calcium-mediated grp78/grp94 Stress Response

grp78/grp94 induction is critical for maintaining the viability of epithelial cells and fibroblasts following treatment with thapsigargin (TG), an inhibitor of Ca2+ uptake into the endoplasmic reticulum. In contrast to these cell types, WEHI7.2 mouse lymphoma cells undergo apoptosis when treated with TG, prompting us to examine the grp78/grp94 stress response in WEHI7.2 cells. TG treatment failed to induce grp78/grp94 transcription in WEHI7.2 cells, measured by Northern hybridization and nuclear run-on assays, even if the cells were protected from apoptosis by overexpressing bcl-2. However, grp78/grp94 transcription was induced by the glycosylation inhibitor tunicamycin, suggesting that there are at least two grp78/grp94 signaling pathways, one in response to TG-induced endoplasmic reticulum Ca2+ pool depletion, which is inoperable in WEHI7.2 cells, and one in response to glycosylation inhibition, which is operable in WEHI7.2 cells. Studies of additional lymphoid lines, as well as several nonlymphoid lines, suggested a correlation between grp78/grp94 induction and resistance to apoptosis following TG treatment. In conclusion, the vulnerability of TG-treated WEHI7.2 cells to apoptosis may be due to failure to signal a grp78/grp94 stress response.

Accumulation of major stress protein 70kDa protects myeloid and lymphoid cells from death by apoptosis.

The major heat shock protein, hsp70, is known to contribute to the mechanisms of cell protection against a variety of stress and cytotoxic factors, providing an increase of cell survival. Whether hsp70 could be implicated in the rescue of cells from stress-induced death proceeding on apoptotic pathway is not well established. Here we report that susceptibility of myeloid and lymphoid cell lines to apoptosis induced by heat shock or ethanol coincides with hsp70 content and can be modulated by changes in expression of this protein. Cells of lymphoid and myeloid lines differing in basal and inducible level of the protein were tested. The cells containing higher amounts of hsp70 (U937, Jurkat, Molt4) were more resistant to the apoptosis-inducing stimuli then cells which accumu-late lower amounts of the protein (HL60) and especially those lacking the protein (NSO). Inhibition of hsp70 accumulation by quercetin made cells more susceptible to the same apoptotic inducer. Enhancement of hsp70 expression by previous heating or by liposomal delivery of the exogenic protein to the cells lacking hsp70 made them more resistant to apoptosis. The possible mechanisms of the hsp70 protective effect in apoptosis are discussed.

L1 adhesion molecule on human lymphocytes and monocytes: expression and involvement in binding to alpha v beta 3 integrin.

The L1 adhesion molecule is a member of the immunoglobulin (Ig) superfamily initially identified in the nervous system which contains six Ig-like domains. Besides the known L1-L1 homotypic interaction, L1 was recently shown to bind to very late antigen (VLA)-5 in the mouse and alpha v beta 3 in the human. The sixth Ig domain is critical for this function. We now demonstrate that human CD4+ peripheral blood T lymphocytes, monocytes and B lymphocytes, but not CD8+ T lymphocytes, express L1. When compared to the expression of CD31, another ligand for alpha v beta 3 on T lymphocytes, only a small proportion of cells were CD31+L1+ double positive. L1 was also detected on the surface of human monocytic and lymphoid tumor lines and was shown to have a molecular mass of approximately 220 kDa, similar to the molecule present on neuroblastoma cells. The function of the sixth Ig domain of human L1 as an integrin ligand was also investigated. Using an RGD-containing peptide derived from the sixth Ig domain as well as a fusion protein of the sixth Ig domain of L1 and the Fc portion of human IgG1 (6.L1-Fc), we demonstrated the binding of human MED-B1 (alpha v beta 3hi, alpha 5 beta 1lo) tumor cells and this binding was blocked by alpha v-specific mAb. In contrast, human Nalm-6 cells (alpha v beta 3lo, alpha 5 beta 1hi) did not bind to the 6.L1-Fc fusion protein. MED-B1 cells could also be stained with the 6.L1-Fc fusion protein. Our results suggest that human L1 binds predominantly to alpha v beta 3 and that its presence on leukocytes could be important for adhesion and migration.

Dominance of tissue-restricted cytotoxic T lymphocytes in the response to allogeneic renal epithelial cell lines.

Our current knowledge of cytotoxic T lymphocytes (CTL) is largely derived from studies of effector populations generated in allogeneic mixed leukocyte cultures (MLC) and assayed for lytic activity to lymphoid cell (LC) targets. We herein report that the CTL response to allogeneic renal epithelial cell lines (REC) is dominated by effectors that efficiently lyse REC targets but show little cross-reactivity with LC targets. In contrast, CTL generated against allogeneic spleen cell stimulators (ie., in MLC) lysed REC and LC targets at comparable levels. Lytic activity in both types of cultures was mediated by CD8+TCRalpha/beta+ cells directed to classical H2 class I alloantigens. Anti-REC effectors cross-reacted with fibroblast and macrophage targets but not with targets commonly used to detect alloreactive CTL, such as lipopolysaccharide- or Con A-stimulated lymphoblasts or lymphoid tumor lines, whereas MLC-elicited effectors efficiently lysed all targets. CTL clones propagated from anti-REC cultures exhibited the same allospecificity and tissue specificity as bulk anti-REC effectors. Individual CTL clones were highly heterogeneous in their capacity to recognize the same class I alloantigen expressed on cells derived from different tissues. These data demonstrate that the cellular environment in which CD8 precursors encounter class I alloantigens can have a profound effect on the cell-type specificity of CTL populations. An important implication of these data is that conventional assays of CTL lytic activity may fail to reveal a significant component of the host response to allogeneic tissues.

Lymphoid cell resistance to glucocorticoids in HIV infection

In humans infected with the HIV-1 virus there may be a disproportionate severity of signs and symptoms of illness compared to the fraction of CD4 + infected T-lymphoid cells. In part, this may be due to altered intercellular signalling systems and intracellular signal transduction. Glucocorticoids are well known for their effects on the vitality and function of lymphoid cells. Patients with HIV infections often show elevated circulating levels of cortisol, suggesting some misfunction in the regulatory systems that maintain the levels of this critical hormone. At the cellular level, it is known that both acute HIV infection and glucocorticoids can cause apoptotic cell death in thymic lymphocytes. However, chronically HIV-infected cells appear to be resistant to glucocorticoid-evoked cell death. Glucocorticoid receptor-ligand binding studies on patients' cells have shown reduced affinity between the receptor binding sites and test steroids. In vitro, chronically HIV-infected cells of the lymphoid CEM line displayed resistance to glucocorticoid-induced apoptosis. These cells showed reduced numbers of binding sites with little alteration in apparent affinity between ligand and receptor. Thus it appears that there may often be malfunction of the normal glucocorticoid response in HIV-infected cells probably due to altered interactions between the glucocorticoid receptor and its hormone. Such alterations may have clinical consequences, including the possibility of a relatively longer life span of infected CD4 + T-lymphocytes, as well as systemic effects of chronically elevated cortisol levels.

Characterization of Lymphocyte Fibronectin

In vitrocultured “activated” peripheral blood lymphocytes and T-cell lines synthesized a high-molecular-weight gelatin binding molecule (MW 500 kDa), whereas resting lymphocytes showed poor or negligible synthesis of the same component. Concanavalin A-mediated anchorage of the lymphocytes to a substratum potentiated synthesis of the high-molecular-weight molecule. Western blotting of the gelatin-binding lymphocyte molecule demonstrated reactivity with antibodies specific for human fibronectin. Furthermore, immunocytochemistry showed reactivity of anti-fibronectin antibodies with T-lymphocytes at the single-cell level. The lymphocyte-derived fibronectin was preferentially cell associated and relatively small amounts were present in the culture medium. RT-PCR of total RNA from CD4+T-cells and the lymphoid T-cell line MOLT-4 showed that the most abundant species of fibronectin mRNA lacked the entire III CS exon encoding the α4β1binding region LDV. Amplification of the III CS region from other T-cell lines revealed that these cells expressed several fibronectin mRNA isoforms most of which were lacking the LDV coding sequence. In conclusion, synthesis of fibronectin is demonstrated to occur in T-lymphocytes and to be regulated by signals which activate the cells.

Integration of an Epstein-Barr virus episome 3′ into the gene encoding immunoglobulin heavy-chain α1 in a lymphoblastoid cell line

For the first time we have characterized an unoccupied site of Epstein-Barr (EBV) virus integration in a lymphoblastoid cell line, RGN1. The site of integration is about 1.5 kb downstream from the gene encoding the heavy chain constant αl, specifying immunoglobulin A (IgA). Sequence and Southern analysis allowed us to hypothesize that integration occurred via a double exchange involving the viral latent origin of DNA replication ( oriP) and the human DNA. The region involved in the integration is transcribed into poly(A)+RNA in all the tested lymphoid lines, but not in the RGN1 line. We suggest a mechanism of integration primed by interactions between oriP and cell ori and its potential role in the establishment and/or evolution of EBV-carrying lines.

Expression of 18.6/CD23 antigen on human lymphoid progenitor cell lines and phorbol 12-myristate 13-acetate (PMA)-induced microglia-shaped cells.

The nature of lymphoid progenitors and factor(s) determining commitment to either the T- or B-lymphocyte pathway are poorly understood in the human system. In this study, we generated a monoclonal antibody (MoAb), 18.6, that recognizes a cell surface antigen on a human lymphoid progenitor cell line (FL4.4). MoAb 18.6 reacted with lymphoid progenitor lines, B lymphoid cell lines, and myelomonocytic cell lines. It did not react with any T cell or erythroid leukemic cell lines. Two color FACS analyses of normal lymphoid tissues showed that MoAb 18.6 reacted with a majority of CD20+ mature B cells and a minority of CD64+ monocytes. Molecules of 3 different sizes with MW of 34, 45, and 68 Kd were precipitated with MoAb 18.6 from the lymphoid progenitor cell line. The 18.6 antigen was not expressed on a fetal liver-derived lymphoid progenitor-like cell line, FL1.4, which has the capacity to differentiate into microglia-shaped cells upon PMA-stimulation. Stimulation of FL1.4 cells with PMA induced expression of the 18.6 antigen within 24 hr and the microglia-shaped cells stained positively with MoAb 18.6. Finally, cloning of a cDNA that encoded the 18.6 antigen revealed that the 18.6 antigen is identical to the CD23 antigen. Taken together, these data suggest that the 18.6/CD23 antigen is expressed on lymphoid precursors at a very early stage of differentiation.

Constitutive activation of different Jak tyrosine kinases in human T cell leukemia virus type 1 (HTLV-1) tax protein or virus-transformed cells.

HTLV-1 infection causes an adult T cell leukemia in humans. The viral encoded protein tax, is thought to play an important role in oncogenesis. Our previous data obtained from a tax transgenic mouse model revealed that tax transforms mouse fibroblasts but not thymocytes, despite comparable levels of tax expression in both tissues. Constitutive tyrosine phosphorylation of a 130-kD protein(s) was observed in the tax transformed fibroblast B line and in HTLV-1 transformed human lymphoid lines, but not in thymocytes from Thy-tax transgenic mice. Phosphotyrosine immunoprecipitation followed by Western blot analysis with a set of Jak kinase specific antibodies, identified p130 as Jak2 in the tax transformed mouse fibroblastic cell line and Jak3 in HTLV-1 transformed human T cell lines. Phosphorylation of Jak2 in tax transformed cells resulted from high expression of IL-6. Tyrosine phosphorylation of this protein could also be induced in Balb/c3T3 cells using a supernatant from the B line, which was associated with induction of cell proliferation. Both phosphorylation and proliferation were inhibited by IL-6 neutralizing antibodies. Constitutive phosphorylation of Jak kinases may facilitate tumor growth in both HTLV-1 infected human T cells and the transgenic mouse model.

FAS-induced apoptosis is mediated via a ceramide-initiated RAS signaling pathway

Fas receptor-induced apoptosis plays critical roles in immune homeostasis. However, most of the signal transduction events distal to Fas ligatlon have not been elucidated. Here, we show that Ras is activated following ligation of Fas on lymphoid lines. The activation of Ras is a critical component of this apoptotic pathway, since Inhibition of Ras by neutralizing antibody or a dominant-negative Ras mutant interfered with Fas-induced apoptosis. Furthermore, ligatlon of Fas also resulted in stimulation of the sphingomyelin signaling pathway to produce ceramides, which, in turn, are capable of inducing both Ras activation and apoptosis. This suggests that ceramides acts as second messengers in Fas signaling via Ras. Thus, ligation of the Fas molecule on lymphocyte lines induces activation of Ras via the action of ceramide, and this activation is necessary, but not sufficient, for subsequent apoptosis.

Characterization of surface lymphotoxin forms. Use of specific monoclonal antibodies and soluble receptors.

Lymphotoxin (LT) is a cytokine related to TNF, found in human systems in both secreted and membrane bound forms. The well characterized secreted form is a trimer of a single protein, LT-alpha, whereas the surface form is composed of a complex between two related molecules, LT-alpha and LT-beta. Because there is a distinct receptor for the complex, the membrane form is believed to signal via events different from those elicited by TNF and secreted LT-alpha. By using a battery of anti-LT-alpha and LT-beta mAbs, it is clear that two LT surface forms exist on the surface of PMA-activated II-23 cells, a human T cell hybridoma. Assuming that these surface forms are trimers, a minor form appears early after induction having an apparent stoichiometry of LT-alpha 2/beta 1 and is recognized by one group of anti-LT-alpha mAbs and the p55-TNF receptor. The second and predominant form has an apparent LT-alpha 1/beta 2 composition and is recognized by a second group of pantrophic anti-LT-alpha mAbs and the LT-beta receptor. Neither of the heteromeric forms nor a putative LT-beta homotrimeric form were found to be secreted. The properties of surface LT on the II-23 cell system were similar to those of the surface LT forms on Chinese hamster ovary cells transfected with both LT-alpha and LT-beta genes and a number of lymphoid tumor lines. These experiments point toward the LT-alpha 1/beta 2 complex as the predominant membrane form of LT on the lymphocyte surface, and this complex is the primary ligand for the LT-beta receptor.

Age-related changes in the secretory-dendritic cells of the pigeon bursa of Fabricius: An immunohistochemical and ultrastructural study

The present study was undertaken to determine, by means of immunohistochemical techniques, image analysis and ultrastructural methods, whether the secretory-dendritic cells (SDC) of the pigeon bursa of Fabricius undergo changes from hatching to the involutive stage (120 days) of the organ. A monoclonal antibody against vimentin (VIM) was used to label SDC. VIM-like immunoreactivity (VIM-L IR) was observed labelling dendritic cell profiles in all age groups. These cells are primarily localized within the medulla and at the cortico-medullary border of the lymphoid follicles. At hatching VIM-L IR was present mainly in the cell bodies, whereas during post-hatching bursal growth (7 to 75 days) it was also present in the cell processes. Conversely, the involutive period examined (90-120 days) was characterized by a progressive decrease of VIM-L IR in the SDC processes. Quantitative studies confirmed the immunohistochemical findings. At the ultrastructural level, there was a progressive increase from 0 to 90 days of age in both the number and size of secretory granules and break-down bodies, as well as in the length of the SDC processes. The involutive stage showed the reverse phenomena. The present results demonstrate that the SDC of the pigeon bursa of Fabricius undergo age-related changes parallel with that of the organ. The possible involvement of SDC in the maintenance of the bursal microenvironment and their role in the maturation of lymphoid line cells is discussed.

Segregation of APO-1/Fas antigen- and tumor necrosis factor receptor-mediated apoptosis.

The cytokine tumor necrosis factor (TNF) and the APO-1/Fas ligand represent typical inducers of apoptosis, i.e. programmed cell death. A limited sequence homology between the TNF receptor TR60 (p55) and the APO-1/Fas (CD95) antigen in their intracellular domains suggests overlapping signaling pathways. The TNF-sensitive cell line KYM-1, which expresses high numbers of both TNF receptors and the APO-1/Fas antigen, is highly sensitive towards triggering of apoptosis by each of the TNF receptors, but resistant to APO-1/Fas stimulation. The opposite response pattern was obtained using the B lymphoid line SKW 6.4, which also co-expresses all three cell surface molecules. Furthermore, no co-modulation of APO-1/Fas by down-regulation of cell surface expressed TR60 and/or TR80 receptors, and vice versa, was found. These data argue against a physical interaction of TNF receptors with APO-1/Fas and, in addition, demonstrate cell specific control of induction of apoptosis and usage of distinct signaling pathways by these receptor molecules.

Allogeneic T-Cell Clones Able to Selectively Destroy Philadelphia Chromosome-Bearing (Ph1+) Human Leukemia Lines Can Also Recognize Ph1- Cells From the Same Patient

Immunocompetent cells in bone marrow allografts have been associated with a graft-versus-leukemia (GVL) effect. To further characterize effector mechanisms that may be involved in this GVL phenomenon, we have previously established an in vitro model to identify allogeneic T-cell clones that selectively mediate cytotoxicity against a patient's leukemic cells, but not against nonleukemic lymphocytes from the same patient. We have modified this in vitro model to test whether the Ph1 chromosome and the P210 fusion protein it controls have a detectable role in leukemia-specific recognition by allogeneic T-cell clones. In this report, T-cell lines reactive with allogeneic Ph1 chromosome-bearing (Ph1+) chronic myeloid leukemia (CML) cell lines were derived and selected to be minimally reactive with Ph1 negative (Ph1-) lymphoid lines from the same patient. However, after prolonged culture, these same T-cell lines also mediated significant destruction of the Ph1- target cells from the same patients. These T-cell lines specifically recognized cells from the allogeneic CML patient to which they were sensitized, and were not contaminated by an outgrowth of natural killer cells. Furthermore, subclones could be derived from these T-cell lines, and some of these subclones again showed selective killing of the allogeneic Ph1+ leukemia cell lines, and not of the Ph1- cell line from the same patient. Analyses of T-cell receptor (TCR) genes showed the alloreactive T-cell lines and the Ph1+ selective subclones derived from them to be of the same clonal origin. This suggests that the same T cells reacting with antigens expressed on the nonleukemic Ph1- targets can at times selectively and preferentially kill the allogeneic Ph1+ cells. As the same TCR that recognizes Ph1+ cells also can recognize the Ph1- targets, it appears that the Ph1+ chromosome does not play a detectable role in recognition by these allogeneic T-cell clones. This in vitro observation may provide a model for evaluating the relationship between GVL and graft-versus-host disease effects.

Allogeneic T-cell clones able to selectively destroy Philadelphia chromosome-bearing (Ph1+) human leukemia lines can also recognize Ph1- cells from the same patient

Immunocompetent cells in bone marrow allografts have been associated with a graft-versus-leukemia (GVL) effect. To further characterize effector mechanisms that may be involved in this GVL phenomenon, we have previously established an in vitro model to identify allogeneic T- cell clones that selectively mediate cytotoxicity against a patient's leukemic cells, but not against nonleukemic lymphocytes from the same patient. We have modified this in vitro model to test whether the Ph1 chromosome and the P210 fusion protein it controls have a detectable role in leukemia-specific recognition by allogeneic T-cell clones. In this report, T-cell lines reactive with allogeneic Ph1 chromosome- bearing (Ph1+) chronic myeloid leukemia (CML) cell lines were derived and selected to be minimally reactive with Ph1 negative (Ph1-) lymphoid lines from the same patient. However, after prolonged culture, these same T-cell lines also mediated significant destruction of the Ph1- target cells from the same patients. These T-cell lines specifically recognized cells from the allogeneic CML patient to which they were sensitized, and were not contaminated by an outgrowth of natural killer cells. Furthermore, subclones could be derived from these T-cell lines, and some of these subclones again showed selective killing of the allogeneic Ph1+ leukemia cell lines, and not of the Ph1- cell line from the same patient. Analyses of T-cell receptor (TCR) genes showed the alloreactive T-cell lines and the Ph1+ selective subclones derived from them to be of the same clonal origin. This suggests that the same T cells reacting with antigens expressed on the nonleukemic Ph1- targets can at times selectively and preferentially kill the allogeneic Ph1+ cells. As the same TCR that recognizes Ph1+ cells also can recognize the Ph1- targets, it appears that the Ph1+ chromosome does not play a detectable role in recognition by these allogeneic T-cell clones. This in vitro observation may provide a model for evaluating the relationship between GVL and graft-versus-host disease effects.