Engraftment of human T-cell acute lymphoblastic leukemia in immunodeficient NOD/SCID mice which have been preconditioned by injection of human cord blood.

Childhood T-cell acute lymphoblastic leukemia (T-ALL) is one of the most common childhood cancers. Study of leukemia biology, as well as preclinical testing of potential therapeutic regimens directed at T-ALL, has been impeded by the lack of an efficient in vivo model of primary leukemia. We have reported elsewhere some observations that human cord blood conditioned medium enhances leukemia colony formation in vitro and preconditioning of sublethally irradiated nonobese diabetic/ severe combined immunodeficient (NOD/SCID) mice with cord blood mononuclear cells (MNCs) facilitates the subsequent engraftment of primary T-ALL cells in these mice. Here we characterize in greater detail this in vivo xenograft model of human leukemia in NOD/SCID mice. Consistent with the thesis that cord blood facilitates engraftment, the engraftment of human leukemia can be shown to increase with increasing number of cord blood MNCs injected. In addition, we documented the expression of chemokine receptor CXCR4 by primary T-ALL from patients and found that the presence of these receptors did not result in the transmigration of T-ALL cells induced by stromal cell-derived factor-1alpha. Finally, we show that in this xenograft system T-ALL cells recovered from engrafted bone marrow are characterized by upregulated expression of interleukin 2 receptor gamma chain, suggesting that cord blood preconditioning may function in part to increase T-ALL responsiveness to growth factor(s).

Preconditioning with fetal cord blood facilitates engraftment of primary childhood T-cell acute lymphoblastic leukemia in immunodeficient mice.

Childhood T-cell acute lymphoblastic leukemia (T-ALL) is one of the most common childhood cancers. It is reported that preconditioning sublethally irradiated immunodeficient NOD/SCID (nonobese diabetic/X-linked severe combined immunodeficient) mice with human cord blood mononuclear cells facilitates the engraftment, expansion, and dissemination in these mice of primary T-ALL cells obtained from patients at the time of diagnosis. Cells recovered from mouse bone marrow or spleen resembled the original leukemia cells from patients with respect to surface lineage markers and T-cell receptor Vbeta gene rearrangements. Moreover, the pattern of leukemia dissemination in mouse tissues, resulting in universally fatal leukemia, is reminiscent of the human clinical disease. In addition, the fidelity of the model to the human disease is documented with regard to the presence of morphologically identifiable human leukemia cells in mouse bone marrow and blood and the maintenance of leukemia-initiating capacity within the leukemia-engrafted mouse. Therefore, several lines of independent approaches are used to suggest that the engrafted cells are of human leukemia origin and are not derived from cord blood. The in vivo model described here should enable the study of the growth properties of primary T-ALL cells obtained from patients and should prove useful in evaluating the potential efficacy of therapeutic strategies directed toward T-ALL.

Human cord blood conditioned medium enhances leukemia colony formation in vitro.

Childhood T-cell acute lymphoblastic leukemia (T-ALL) is one of the most common childhood cancers. Recently, we observed that pre-conditioning sub-lethally irradiated immunodeficient mice with human cord blood mononuclear cells facilitates in these mice high level engraftment of primary T-ALL cells obtained from patients. Here we report that human cord blood cells secrete a factor(s) which markedly enhances in vitro both colony number and burst size of the T-ALL clonogenic progenitors from patients. The enhancing activity does not correspond to IL-2, IL-15, nor to several other cytokines implicated in T cell proliferation/activation. Thus, it is possible cord blood may secrete an as yet unidentified factor(s) acting on leukemia clonogenic progenitors of T-ALL. Collectively, these studies should prove invaluable in addressing the growth properties of primary T-ALL cells from patients.

Functional and biochemical characterization of a novel human macrophage-derived negative regulator of haematopoiesis.

It is believed that haematopoiesis is regulated by both positive and negative signals derived from the marrow microenvironment, which includes macrophages. The identity and mechanism of action of the proteins mediating negative regulation is an area of active investigation. We report here the identification and initial characterization of a novel suppressor of early haematopoietic progenitors, designated NRH (for Negative Regulator of Haematopoiesis), isolated from the recently established human macrophage line 2MAC. The mechanism of NRH suppression appears to involve a marked decrease in the cycling of early progenitor cells. NRH activity was shown to be reversible and to correspond to an acidic, heparin-binding glycoprotein with a molecular weight of approximately 20 000 daltons ( approximately 20 kDa). By exploiting lectin specificity, hydrophobic interaction, and heparin affinity, we have developed a procedure for the rapid isolation of highly purified NRH from 2MAC-conditioned medium. By a number of functional and biochemical criteria, NRH appears to represent a novel macrophage-derived negative regulator of haematopoiesis which may have future application in certain clinical settings as a chemoprotectant of primitive haematopoietic cells.

Cytokine modulatable signalling through macrophage HLA class II. 1. IFN-gamma upregulates the efficiency of Ca2+ mobilization in response to ligation of macrophage HLA-DP.

The human macrophage line 2MAC, established recently from peripheral blood, expresses a number of lineage-specific markers as well as a broad array of intercellular adhesion molecules, including high levels of HLA class I and class II. We have presented evidence elsewhere that 2MAC can be productively applied to the study of signal transduction through macrophage HLA class II. Namely, we showed that ligation of 2MAC HLA class II, but not HLA class I, by monoclonal antibody (mAb) elicits an increase in free cytoplasmic Ca2+ concentration [Ca2+]i. Moreover, this Ca2+ flux appears to be functionally relevant: ligation of HLA-DR, but not HLA class I, by mAb results in the Ca2+ mobilization-dependent induction of tissue factor, the high-affinity cellular receptor for factor VII/VIIa. Here we show that 2MAC is uniquely valuable for addressing the efficiency of signal transduction through HLA class II. Namely, we show here that prior culture of 2MAC cells with interferon-gamma (IFN-gamma) profoundly upregulates subsequent Ca2+ mobilization in response to ligation of HLA-DP in the absence of increased cell surface HLA-DP expression. Because IFN-gamma has no effect on 2MAC HLA-DP expression, IFN-gamma must upregulate Ca2+ mobilization by increasing the efficiency of signal transduction through HLA class II (HLA-DP), by targeting some other component of the macrophage HLA class II signalling pathway.

Characterization of a new human macrophage cell line 2MAC. 1. Expression of functional macrophage CD16 (Fc gammaRIIIA/gamma) and tissue factor induction on ligation of HLA-DR.

A human macrophage-like line, designated 2MAC, has been established from peripheral blood. 2MAC expresses a number of lineage-specific markers as well as a broad array of intercellular adhesion molecules. In particular, 2MAC expresses CD16/Fc gammaRIII, the low-affinity Fc receptor for IgG, as well as high levels of HLA class I and class II. Consistent with this macrophage assignment, we present evidence that 2MAC expresses the macrophage form of CD16, namely, Fc gammaRIIIA/gamma. By several criteria also applicable to signal transducing NK CD16 and T cell CD3/TCR complexes, including modulation from the cell surface and Ca2+ mobilization in response to ligation by specific monoclonal antibody, CD16 expressed by 2MAC is functional. Ligation of 2MAC HLA class II, but not HLA class I, by specific mAb induces an increase in free cytoplasmic Ca2+ concentration ([Ca2+]i). This Ca2+ flux appears to be physiologically relevant, as ligation of HLA-DR, but not HLA class I, by mAb results in the efficient, Ca2+ mobilization-dependent induction of tissue factor by 2MAC. 2MAC, therefore, should prove useful for studying signal transduction through macrophage CD16 and HLA class II.

Phenotypic and functional characterization of a new human macrophage cell line K1m demonstrating immunophagocytic activity and signalling through HLA class II.

A human macrophage line, designated K1m, has been established from peripheral blood. K1m expresses a number of lineage-specific markers as well as a broad array of intercellular adhesion molecules. In particular, K1m expresses high levels of human leucocyte antigen (HLA) class I and class II. In response to ligation of HLA class II (HLA-DR), but not in response to ligation of HLA class 1, K1m forms tighter homotypic aggregates and develops a striking 'stellate' culture phenotype. K1m also expresses Fc receptors for immunoglobulin G (IgG) (CD64, CD32, and CD16) and can be shown to phagocytose polystyrene latex beads, as well as neuroblastoma cells in the presence of tumour-specific monoclonal antibody (mAb). The K1m cell line should therefore prove useful for studying both signalling through macrophage HLA class II and immunophagocytosis.

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.

Rearrangement and expression of T-cell antigen receptor genes in human T-lymphocyte tumor lines and normal human T-cell clones: evidence for allelic exclusion of Ti beta gene expression and preferential use of a J beta 2 gene segment.

The gene encoding the beta chain of the human T-cell receptor for antigen is composed of variable (V), diversity (D), joining (J), and constant (C) gene segments which undergo specific rearrangements during T-lymphocyte ontogeny. Southern blot analyses of seven human T-cell tumor lines and normal human T-lymphocyte clones revealed that most of these T-cell lines rearrange their Ti beta genes differently. The T-cell tumor line HPB-MLT rearranges and transcribes both of its Ti beta genes. Cloning and sequencing of the Ti beta cDNAs corresponding to these rearrangements revealed that one of the rearranged Ti beta genes is defective, while the other is functional and corresponds to the Ti beta protein expressed on the surface of these cells. Thus, this cell line displays a pattern of allelic exclusion of Ti beta gene expression. A comparison of four C beta 2-containing Ti beta cDNAs from three different cell lines revealed that three of the four utilize the same J beta 2.5 gene segment joined to different D beta and V beta genes, suggesting that there may be preferential use of this J gene during J beta 2 rearrangements. Hybridization analyses with probes for the alpha and beta genes of the T-cell receptor and the T-cell-specific T gamma gene revealed that HPB-MLT cells appear to express approximately equivalent amounts of RNA corresponding to each of the rearranged Ti alpha and Ti beta genes. However, they express a much lower level of T gamma RNA.

Isolation of complementary DNA clones encoding the human lymphocyte glycoprotein T1/Leu-1.

The T1/Leu-1/CD5 molecule, a human T-cell surface glycoprotein of relative molecular mass (Mr) 67,000, has been implicated in the proliferative response of activated T cells and in T-cell helper function. A similar involvement in T-cell proliferation has been reported for Ly-1, the murine homologue of T1. Here we report the complete amino-acid sequence of the T1 precursor molecule deduced from complementary DNA clones. The protein contains a classical signal peptide; a 347-amino-acid extracellular segment; a transmembrane region; and a 93-amino-acid intracellular segment. The extracellular segment contains many cysteine residues and is composed of two related structural domains separated by a proline/threonine-rich region. The T1 molecule has structural features characteristic of other receptor molecules.

Human T-cell gamma genes contain N segments and have marked junctional variability.

The gamma-chain genes are encoded by immunoglobulin-like gene segments in germline DNA which rearrange during the somatic development of T cells to form an active gene. The protein produced by these genes has not been identified and the diversity of the proteins that the genes can express has not been determined. We expect that the diversity of expressed gamma-chains is produced by the same three mechanisms that produce diversity of other immunoglobulin-like genes: (1) germline variable (V) and joining (J) region repertoires; (2) somatic mutation; and (3) junctional diversity. To define the contribution of each of these mechanisms to the generation of gamma-chain diversity, several gamma-chain complementary clones and rearranged gamma-chain genes have been characterized. Most of these clones seem to encode a defective gamma-chain, the variable- and constant-region portions being joined such that they would not be translated in the same reading frame. Here we report that the germline J-region diversity of the human T-cell gamma-chain is very limited and that somatic mutation does not contribute to the diversity of the gamma-chains encoded by the cloned segments. However, the junctional diversity of these gamma-chain genes is extensive. We suggest that N sequences (template-independent sequences) have been inserted enzymatically into all of the gamma-chain genes characterized.

Identification of a putative second T-cell receptor.

Framework monoclonal antibodies have identified a population of human lymphocytes that express the T3 glycoprotein but not the T-cell receptor (TCR) alpha- and beta-subunits. Chemical crosslinking experiments reveal that these lymphocytes express novel T3-associated polypeptides, one of which appears to be the product of the T gamma gene. The other polypeptide may represent a fourth TCR subunit, designated T delta.

Cloning and sequence analysis of complementary DNA encoding an aberrantly rearranged human T-cell gamma chain.

Complementary DNA (cDNA) encoding a human T-cell gamma chain has been cloned and sequenced. At the junction of the variable and joining regions, there is an apparent deletion of two nucleotides in the human cDNA sequence relative to the murine gamma-chain cDNA sequence, resulting simultaneously in the generation of an in-frame stop codon and in a translational frameshift. For this reason, the sequence presented here encodes an aberrantly rearranged human T-cell gamma chain. There are several surprising differences between the deduced human and murine gamma-chain amino acid sequences. These include poor homology in the variable region, poor homology in a discrete segment of the constant region precisely bounded by the expected junctions of exon CII, and the presence in the human sequence of five potential sites for N-linked glycosylation.

Effects of monoclonal antibodies directed against murine T lymphocyte cell surface antigens on lymphokine production by cloned T lymphocytes reactive with class I MHC or Mls alloantigens.

Monoclonal antibodies recognizing murine T lymphocyte cell surface structures implicated in T lymphocyte-mediated cytolysis, including Lyt-2, L3T4, LFA-1, and a cytolytic T lymphocyte (CTL) clonotypic determinant, were used as probes to investigate the role of these structures in lymphokine production by T cell clones induced by antigen or lectin. The clone-specific antibody 384.5 bound to and inhibited antigen-induced lymphokine production by the L3 CTL clone, but did not affect lymphokine production by other T cell clones. Antibodies against the T cell surface structures Lyt-2 or L3T4, which are expressed by mutually exclusive T cell subsets, inhibited antigen-induced lymphokine production by class I major histocompatibility complex (MHC) antigen-reactive CTL clones or an M1s-reactive helper T lymphocyte (HTL) clone, respectively. Antibody against the broadly distributed LFA-1 molecule inhibited antigen-induced lymphokine production by all of the clones tested. Lectin-induced lymphokine production by cloned T cells was not inhibited by the clonotypic antibody, anti-Lyt-2, or anti-LFA-1; slight inhibition of the HTL clone was observed with the anti-L3T4 antibody. None of these structures appear to be uniquely involved with a particular functional response. Our results suggest that each of these structures is involved with the interactions between the effector cell and the stimulating cell leading to lymphokine production.

Contributions of T cell clones to an understanding of the T cell receptor for antigen.

Cloned populations of T cells have been used to derive clone-specific monoclonal antibodies. These antibodies influence the specific immunological functions of the cloned T cells, suggesting that these antibodies react with the T cell receptor for antigen. Immunoprecipitates have been obtained with some of these antibodies, and the characteristics of the polypeptides from different cells are surprising similar. At present, little is known about this molecular complex other than that the two polypeptides are disulfide-linked and have apparent molecular weights of about 45,000 daltons and 43,000 daltons. This molecular complex, at least in human T cells, is associated with, but not covalently linked with, another molecular complex (Leu4/T3). However, modern techniques of cellular and molecular immunology should provide information very soon about the actual role of these molecules in T cell recognition of antigen. Indeed, cDNA clones that may encode genes for one of the peptide chains have been obtained.

Antigen-reactive cloned helper T cells. II. Exposure of murine cloned helper T cells to IL 2-containing supernatant induces unresponsiveness to antigenic restimulation and inhibits lymphokine production after antigenic stimulation.

Cloned murine helper T lymphocytes (HTL) reactive to alloantigen or to ovalbumin (OVA) become unresponsive to antigenic restimulation after exposure to antigen or to culture supernatant fluids (SF) containing multiple lymphokine activities. Unresponsiveness is manifest by a failure of antigen-stimulated cells to incorporate thymidine or to produce lymphokines after antigenic challenge. Antigen-unresponsive HTL, however, will incorporate thymidine when exposed to an exogenous source of interleukin 2 (IL 2). The duration of unresponsiveness to antigen is correlated with the concentration of IL 2 in SF to which the cloned HTL had been exposed. Chromatographic fractionation of IL 2-containing supernatant from EL-4 thymoma cells (EL-4 SF) yielded a pool of SF that was enriched for IL 2 activity. Exposure of HTL to lymphokines contained in this pool induced unresponsiveness to antigen that was comparable to that observed when HTL were exposed to unfractionated EL-4 SF. Unresponsiveness to antigen also developed after cloned HTL were stimulated with concanavalin A (Con A) or with OVA and syngeneic splenic filler cells. We have used monoclonal antibody (mAb) GK1.5 (anti-L3T4) to investigate the role of lymphokine production in the induction of unresponsiveness. This antibody did not inhibit IL 2-induced thymidine incorporation by cloned HTL, and did not inhibit the induction of unresponsiveness after exposure of cloned HTL to EL-4 SF. In the presence of mAb GK1.5, however, HTL that were stimulated with Con A or OVA did not become unresponsive to antigenic restimulation, an effect that was overcome by the addition of EL-4 SF. These results suggest that HTL become unresponsive to antigen after exposure to IL 2-containing SF, and that stimulation by antigen or Con A can induce the unresponsive state by virtue of stimulating lymphokine production.

Monoclonal antibody to L3T4 blocks the function of T cells specific for class 2 major histocompatibility complex antigens.

The ability of antibody to Lyt-2 and to the newly described L3T4 antigen to block the functions of helper T cells with specificity for allogeneic class 1 or class 2 MHC subregion antigens was determined. Anti-Lyt-2 blocked both allohelp delivered by unprimed T cells and IL 2 production by primed T cells when the response was directed to class 1 MHC antigens, but had no effect when the response was directed to class 2 MHC or Mls antigens. Anti-L3T4 had reciprocal activity. A control reagent, anti-LFA-1, blocked all responses tested regardless of specificity. This and other reports provide strong evidence that L3T4 is the murine equivalent of the human T cell markers Leu 3/OKT4. The ability of antibodies directed against Lyt-2 and L3T4 to block T cell function in a fashion determined by the class of MHC antigen recognized by the T cell further supports the hypotheses that Lyt-2 and L3T4 molecule in the mouse and Leu-2 and Leu-3 molecules in the human are involved in the interaction of the T cell with class-specific determinants on MHC molecules.

Characterization of the murine T cell surface molecule, designated L3T4, identified by monoclonal antibody GK1.5: similarity of L3T4 to the human Leu-3/T4 molecule.

Monoclonal antibody GK1.5 recognizes a previously undescribed murine T cell surface molecule, designated L3T4, which migrates on SDS-PAGE under reducing conditions as a single band with an apparent m.w. of 52,000. L3T4 is expressed by approximately 80% of thymocytes and by approximately 20% of spleen cells. There appears to be poor correlation between expression of L3T4 by functional T cell clones and expression of Lyt-2, expression of the cytolytic phenotype, and class I MHC antigen reactivity. On the other hand, both a class II MHC antigen-reactive HTL clone and an Lyt-1- Mls-reactive HTL clone express L3T4. Analysis of the effect of mAb GK1.5 on PFC responses in adoptive transfer suggests that L3T4 is expressed by the helper/inducer subset of murine T cells. Expression of L3T4 by murine T cells, however, may correlate primarily with class II MHC antigen reactivity rather than with functional phenotype; mAb GK1.5 profoundly blocks antigen-specific cytolysis by the cloned class II MHC antigen-reactive CTL line A15-1.17. Antigen-specific cytolysis by A15-1.17 is blocked by mAb GK1.5 at a step before the lethal hit. Collectively, the flow cytometric, functional, and biochemical data indicate that L3T4 is similar to the human Leu-3/T4 molecule.

Evidence implicating L3T4 in class II MHC antigen reactivity; monoclonal antibody GK1.5 (anti-L3T4a) blocks class II MHC antigen-specific proliferation, release of lymphokines, and binding by cloned murine helper T lymphocyte lines.

Monoclonal antibody GK1.5 recognizes a determinant, designated L3T4a, on the murine T cell surface molecule L3T4. The expression of L3T4a by functional murine T cell clones appears to correlate primarily with class II MHC antigen reactivity rather than with functional phenotype. In previous studies, antigen-specific cytolysis by a cloned class II MHC antigen(I-Ak)-reactive CTL line was found to be blocked entirely by monoclonal antibody (mAb) GK1.5, at a step before the lethal hit. In the present studies, we demonstrate that mAb GK1.5 profoundly blocks antigen-specific proliferation and release of lymphokines by cloned murine class II MHC antigen-reactive helper T lymphocyte (HTL) lines. Analysis of cloned T cell hybridomas, however, suggests that there exists clonal heterogeneity in the degree of inhibition of class II MHC antigen-specific function by mAb GK 1.5. Finally, we present evidence that mAb GK1.5 blocks class II MHC antigen-specific function by blocking class II MHC antigen-specific binding. The data presented here lend considerable support to the concept both that L3T4 and the human Leu-3/T4 molecules are similar and that L3T4 plays a role in class II MHC antigen-reactivity by murine T cells.