Transformation of human umbilical cord blood T cells by human T-cell leukemia/lymphoma virus.

Several isolates of human T-cell leukemia/lymphoma virus (HTLV) were transmitted to normal human T cells obtained from the umbilical cord blood of newborns. T cells from seven specimens were immortalized by infection with different HTLV isolates and their properties were compared with those of activated uninfected normal T cells grown in the presence of T-cell growth factor (TCGF) and with those of HTLV-positive neoplastic T-cell lines derived from patients with T-cell malignancies. The HTLV-infected cells generally belonged to a class of mature T cells (OKT4+ and Leu 3A+) and differed from the normal uninfected cells in that they could be propagated in culture indefinitely; possessed altered morphology, including convoluted nuclei and some bi- and multinucleated giant cells; formed large clumps in culture; demonstrated a diminished requirement for TCGF; had an increased density of TCGF receptors; often became completely independent of exogenous TCGF; and expressed HLA-DR determinants. These properties of the HTLV-infected cord blood T cells contrasted to those of uncultured cord blood T cells and of cord blood cells stimulated with mitogen and grown with TCGF but resembled the characteristics of T-cell lines established previously from patients with HTLV-associated T-cell malignancies. This in vitro system offers a unique opportunity to study the basic mechanism involved in abnormal growth and neoplastic transformation of a specific class of human T cells.     

Abundant transcription of a cellular gene in T cells infected with human T-cell leukemia-lymphoma virus.

Human T-cell leukemia-lymphoma virus (HTLV) is a type C retrovirus associated with a subtype of mature T-cell malignancy in humans. HTLV also infects normal human cord blood mature T lymphocytes in vitro and induces a number of phenotypic changes in these cells, including their continuous growth and partial or complete independence of T-cell growth factor (TCGF). As part of our initial study designed to analyze gene(s) specifically activated by HTLV infection, we have isolated a recombinant DNA clone by differential screening of a cDNA library made from mRNA of a human T-cell lymphoma cell line producing HTLV. This cDNA identifies a single-copy gene in all human DNAs and a single mRNA species of 2.3 kilobases expressed at several hundred copies per cell in five HTLV-positive neoplastic T-cell lines. In addition, cord blood T lymphocytes infected with HTLV, but not the uninfected counterparts, express high levels of mRNA from this gene. A survey of different human hematopoietic cell types showed that this gene is expressed at low or undetectable levels (less than 10 copies) in human T, B, myeloid, or erythroid cell lines; in moderate amounts in lymphoid precursor (immature) cell lines; and in high amounts in lectin-activated mature T-cells, comparable to those of HTLV-infected T-cell lines. The precise function of this gene has not yet been determined.     

Proliferation and differentiation of single hapten-specific B lymphocytes is promoted by T-cell factor(s) distinct from T-cell growth factor.

Hapten-specific B lymphocytes reactive to fluorescein were prepared from mouse spleen, placed singly in 10-microliters culture wells, and stimulated with fluorescein-polymerized flagellin in the presence of conditioned media (CM) from various concanavalin A-stimulated cloned T-cell tumors or hybridomas. Antigen plus appropriate CM triggered 5-9% of the B cells into both clonal proliferation and differentiation into antibody-forming cells. Antigen alone stimulated 0.5-0.8% of B cells and CM alone stimulated less than 0.1%. This bioactivity was termed B-cell growth and differentiation factor(s) (BGDF). Four CM rich in T-cell growth factor (TCGF)--namely, CM from spleen and the lines EL4, T6, and 123--contained BGDF. The lines T19.1 and WEHI-3 lacked BGDF and TCGF. Four lines of evidence suggested that BGDF and TCGF were distinct molecules. First, the BGDF/TCGF ratios in the various CM varied. Second, on gel filtration, TCGF eluted as a sharp peak corresponding to a Mr of about 35,000, whereas BGDF eluted over a range corresponding to a Mr of 25,000-60,000. Third, the activity of TCGF in EL4-CM was markedly reduced by treatment with guanidine HCl while BGDF activity was not. Fourth, BGDF showed more heterogeneity than TCGF on hydrophobic chromatography. All CM or fractions active in promoting B-cell division also promoted differentiation to antibody-forming cells. These results provide unequivocal evidence that antigen and a T-cell product can synergize to directly activate a single B lymphocyte.     

Cutaneous T-cell lymphoma: a review

Cutaneous T-cell lymphomas define a spectrum of disorders associated with T-lymphocytic proliferation with clinical manifestations occurring in the skin during the course of the disease. This review has dealt with two rather uncommon disorders, namely mycosis fungoides and Sezary syndrome which are indolent malignant lymphomas, occurring primarily in the fifth decade, and affecting males most frequently. Historically, mycosis fungoides and Sezary syndrome have been described for a relatively short time. As witnessed by Table 2, little was known concerning these disorders, other than clinical and pathologic features, until the application of immunologic, cell biologic, and cytogenetic technology which burgeoned a multitude of questions. The discovery of TCGF has allowed for both continuous growth of normal and neoplastic T cells and for the clonal expansion of some malignant clones. The establishment of these continuous cultures allowed for: (1) investigation of the mechanism of TCGF production and stimulation of T-cell growth, and (2) identification of HTLV, a retrovirus found in cell cultures from two patients with CTCL, and subsequently from patients with Japanese adult T-cell lymphoma. In addition, the HTLV has been related to a more virulent form of T-cell malignancy. The exact etiologic role of this virus in the CTCL is presently the subject of intense investigation. Through the use of immunologic methods the malignant cell of CTCL has been pheno-typically and functionally characterized as a "helper/inducer" subtype (E rosette+, anti-T-cell antisera+, T11+, T1+, T3+, 3A1-, T6-, T8-) and usually Ia-, HLADR-. Clinical manifestations of the phenotype may be clinically apparent in the serologic abnormalities present in these disorders. Utilizing these methods to investigate these disorders may provide a key to the understanding of T-cell function and cellular immunity much as myeloma provided a model for the understanding of B cells and immunity. Clinically and pathologically, these disorders behave as malignant indolent lymphomas with spread from localized cutaneous lesions to extracutaneous involvement of the blood, lymph nodes, and viscera culminating in the death of the patient from either organ dysfunction or infectious complications. At autopsy, this extracutaneous involvement is more pronounced than what was expected ante-mortem. Application of prospective staging techniques employing such special procedures as E-rosette cytology, cytogenetics, and electron microscopy in addition to usual light microscopy studies has demonstrated a greater percentage of extracutaneous involvement than otherwise expected.(ABSTRACT TRUNCATED AT 400 WORDS)     

B-lymphocytes as a source of cell surface growth-promoting factors for hematopoietic progenitors

Although B cells reside in the bone marrow, little is known concerning their functional role in hematopoiesis. We have measured the effects of surface membrane factors released from unstimulated, circulating B cells of normal donors and patients with chronic lymphocytic leukemia on human hematopoiesis in vitro. Leukemic cells augment erythroid burst formation by allogeneic blood cells (p less than 0.05). The stimulatory effect is increased in cultures containing a high B-cell seeding density, and is decreased in those with a high peripheral blood mononuclear cell seeding density. Medium conditioned by B cells (CM) from the circulation or bone marrow stimulates the formation of erythroid bursts, granulocyte-macrophage colonies, and mixed colonies containing granulocytes, erythroblasts, monocytes, and megakaryocytes (GEMM) in serum-free cultures of allogeneic and autologous marrow (p less than 0.05). This effect is localized primarily to surface membrane vesicle-rich pellets of CM. Screening of several hematopoietic and nonhematopoietic cell types reveals that membrane vesicle-associated activity is released from B cells and mitogen-stimulated, circulating T cells. In contrast, vesicles shed from freshly isolated, resting T cells, continuous T-cell leukemia cell lines, erythrocytes, and endothelial cells do not express the activity (p greater than 0.10). The stimulatory activity is augmented in cultures of marrow cells that are depleted of B4 antigen-positive lymphocytes but not of T-lymphocytes, suggesting that endogenous release of the factor(s) occurs during incubation. Furthermore, membranes partially purified from leukemic B cells also express the activity. Together with our findings that (1) the growth enhancing factor(s) is solubilized by octylglucoside, and that (2) the factor can be immunoprecipitated with BPA-neutralizing, antimembrane IgG, our results suggest that the erythropoietic activity is an integral membrane protein that may be immunologically related to BPA. The relationship of the erythroid burst stimulatory factor to other hematopoietic activities found in CM pellets is unknown.     

Comparative analysis of hematopoietic growth factors released by stromal cells from normal donors or transplanted patients.

We compared the erythroid burst-promoting activity (BPA) and colony-stimulating activity (CSA) released under serum-deprived conditions by stromal cells derived from nine normal subjects and from nine patients after bone marrow transplantation. BPA and CSA were defined according to the capacity of the conditioned media (CM) to stimulate formation of erythroid bursts and granulocyte/macrophage (GM) colonies in serum-deprived cultures of nonadherent marrow cells. Six patients (group A) failed to establish or maintain successful allografts during the study. The remaining three (group B) did not experience problems with engraftment. CM from all stromal cell cultures contained detectable levels of BPA. Preincubation of the CM with an anti-GM colony-stimulating factor (GM-CSF) monoclonal antibody (MoAb), but not with a rabbit anti-interleukin-3 (IL-3) serum, reduced BPA by an average of 94%. CM from normal and group B stromal cell cultures contained detectable CSA, and the levels correlated with the amounts of granulocyte-CSF (G-CSF) detected by a specific bioassay. G-CSF was not detectable in medium conditioned by stromal cells from transplanted patients with poor marrow function. These results indicate that CM from stromal cells from normal subjects and transplanted patients with good marrow function contain both GM-CSF and G-CSF, while CM from stromal cells from transplanted patients with poor marrow function contain detectable levels of GM-CSF only. The reduced capacity of these stromal cells to produce G-CSF is associated with a reduced capacity of the CM to sustain GM colony formation and may be associated with the inability of these patients to sustain their neutrophil counts in vivo.     

Enhancement of human erythroid progenitor cell growth by media conditioned by a human t-lymphocyte line

Recent studies have shown that soluble factors elaborated by human T lymphocytes enhance erythroid burst formation by human peripheral blood null cells. This study demonstrates that media conditioned by a long- term T lymphocyte line augmented the growth of erythroid colonies in vitro in the presence of erythropoietin (Ep). ATCC.CCl 119 (CCRF-CEM) was derived from a patient with ALL of T-lymphoblast origin. Cells from the stocks used in these experiments maintained T-cell characteristics as determined by histochemical and rosetting techniques. Increased numbers of 16 day BFU-E were seen when Ficoll-Hypaque separated peripheral blood leukocytes were cultured in the presence of a 10% (v/v) concentration of CCL 119 conditioned medium (CM). CM increased the number of BFU-E even when Ep or fetal calf serum were not growth limiting. CM also increased the number of late BFU-E observed in cultures of nonadherent bone marrow cells. When peripheral blood mononuclear cells were depleted of E-rosetting cells, only small numbers of BFU-E grew. Addition of 119 CM increased the numbers of BFU- E in E-rosette-depleted cultures. CM from B-cell, macrophage, or other T-cell lines tested did not stimulate BFU-E growth as consistently. These studies indicate that CM obtained from ATCC.CCL 119 cells contained burst-promoting activity, one of the factors required for proliferation of early erythroid progenitors.     

Human T-lymphocyte growth factor: regulation of growth and function of T lymphocytes

The discovery of T-cell growth factor (TCGF) has made it possible to now routinely grow in tissue culture normal and neoplastic human T cells for long periods and in large amounts. TCGF has been recently purified. It is a small protein released by a subset of mature T cells following lectin-antigen activation, which in turn acts upon other T- cell subsets that have developed specific receptors for TCGF after lectin-antigen stimulation. Thus, release of TCGF and development of receptors for it appear to be obligatory for the clonal expansion of all activated T cells. Unlike normal T cells, neoplastic T cells respond directly to TCGF, requiring no prior in vitro lectin-antigen activation. This has led to the development of several new cell lines from patients with T-cell leukemias and lymphomas. In some cases, these cells become independent of exogenous TCGF by producing their own growth factor, implying a role for TCGF in the continuous proliferation of these cells. These developments necessitate a reevaluation of some concepts of immunoregulation of T-cell activities in terms of production and response to TCGF. In addition, this information has clinical implications. Recent results have shown that a major defect of the athymic nude mouse is the inability to produce TCGF and that some immunosuppressive agents, such as glucocorticosteroids and cyclosporin- A, exert their effects on T cells by disrupting the TCGF-T-cell interaction. Some human immune deficiencies might be due to a failure to respond to or to produce TCGF, which in some cases might be corrected by exogenous TCGF.     

T-cell lines established from human T-lymphocytic neoplasias by direct response to T-cell growth factor.

Long-term growth of lymphoblastoid T cells from tissue samples from six of six patients with cutaneous T-cell lymphoma (CTCL) and six of six patients with acute T-lymphoblastic leukemia (ALL) has been achieved by using partially purified mitogen-free human T-cell growth factor (pp-TCGF). One cell line, CTCL-2, is now independent of added growth factor; the others continue to show absolute dependency on its presence. All lines have been in continuous culture for at least 4 months and some for > 1 year. They are erythrocyte-rosette positive and are negative for Epstein-Barr virus nuclear antigen. Most of the lines are negative for Fc and complement receptors and for surface immunoglobulin except that CTCL-1 and CTCL-2 have some cells positive for these cell surface markers. Results of histochemical studies on these cell lines are similar to the known patterns for fresh cells from their disease of origin. Cell line CTCL-3 has an abnormal karyotype, but no detectable chromosomal abnormalities were found in the other lines, consistent with the karyologic features of their clinical sources. Because T cells from normal donors do not respond to pp-TCGF unless the cells are first "activated" by a lectin mitogen such as phytohemagglutinin or an antigen, the direct response to pp-TCGF of T cells from patients with T-cell neoplasias suggests that the cell lines represent a transformed neoplastic cell population. Although some of the cell lines may be normal T cells activated by the malignant cells, the morphologic and histochemical properties of the cell lines, the abnormal karyotype of CTCL-3, and the independent growth of CTCL-2 support the conclusion that most of these cell lines are of malignant origin.     

Failure of regulation of Tac antigen/TCGF receptor on adult T-cell leukemia cells by anti-Tac monoclonal antibody

Anti-Tac monoclonal antibody, which blocks the membrane binding and action of human T-cell growth factor (TCGF), is strongly proposed to recognize TCGF receptor. We have demonstrated that anti-Tac antibody reacted with leukemic cells from patients with adult T-cell leukemia (ATL) and reacted with T-cell lines established from ATL cells. Although antigenic modulation, or down-regulation, of Tac antigen on activated normal T cells was induced by anti-Tac antibody, the expression of Tac antigen on ATL cells or T-cell lines was not affected when examined by the fluorescence-activated cell sorter (FACS) and the radioassay using 125I-staphylococcal protein A. These results indicate that regulation of Tac antigen-TCGF receptor is different between normal and malignant T cells, suggesting that failure of down- regulation of Tac antigen on leukemic cells by anti-Tac antibody may play an important role in the malignant proliferation of ATL cells.     

Production of human active BPA from TCGF independent T cell lines that do not excrete HTLV: proof of direct action of MLA-144 derived BPA using purified BFU-E

Burst promoting activity (BPA) and colony stimulating activity (CSA) were measured in the supernatants from six T-cell lines which are IL-2 independent and do not excrete human T-cell leukaemia virus. Two cell lines were shown to produce high levels of BPA, namely MLA-144 and the Jurkat cell line. MLA-144 is a gibbon T-cell line which can proliferate and produce BPA in serum free HB101 medium. The BPA from this line was shown to act directly on BFU-E by studies using highly purified peripheral blood derived BFU-E. The Jurkat CM had far less activity against purified progenitors. These cells lines will be of value in the study of erythropoietic regulation.     

Nonmalignant T cells stimulate growth of T-cell lymphoma cells in the presence of bacterial toxins

Bacterial toxins including staphylococcal enterotoxins (SEs) have been implicated in the pathogenesis of cutaneous T-cell lymphomas (CTCLs). Here, we investigate SE-mediated interactions between nonmalignant T cells and malignant T-cell lines established from skin and blood of CTCL patients. The malignant CTCL cells express MHC class II molecules that are high-affinity receptors for SE. Although treatment with SE has no direct effect on the growth of the malignant CTCL cells, the SE-treated CTCL cells induce vigorous proliferation of the SE-responsive nonmalignant T cells. In turn, the nonmalignant T cells enhance proliferation of the malignant cells in an SE- and MHC class II-dependent manner. Furthermore, SE and, in addition, alloantigen presentation by malignant CTCL cells to irradiated nonmalignant CD4(+) T-cell lines also enhance proliferation of the malignant cells. The growth-promoting effect depends on direct cell-cell contact and soluble factors such as interleukin-2. In conclusion, we demonstrate that SE triggers a bidirectional cross talk between nonmalignant T cells and malignant CTCL cells that promotes growth of the malignant cells. This represents a novel mechanism by which infections with SE-producing bacteria may contribute to pathogenesis of CTCL.     

Skin-derived interleukin-7 contributes to the proliferation of lymphocytes in cutaneous T-cell lymphoma

Cutaneous T-cell lymphomas (CTCLs) are malignancies of T cells that have a special affinity for the skin. We have previously reported that much of the T-cell receptor repertoire is altered in CTCL, and both malignant and nonmalignant clones are numerically expanded, presumably in response to T-cell trophic cytokines. We therefore examined levels of the T-cell trophic cytokines IL-2, IL-4, IL-7, IL-12, IL-13, and IL-15 in plasma in 93 CTCL patients and healthy controls. Only IL-7 levels were elevated in CTCL. We next looked at lesional skin from patients with CTCL and found elevated levels of IL-7 mRNA. Explant cultures of normal and lesional CTCL skin biopsies revealed significantly more IL-7 protein production in CTCL skin. Additionally, cultures of CTCL skin released greater numbers of T cells than normal skin; this was blocked by the addition of an IL-7 neutralizing antibody. Finally, these cultures induced proliferation of normal peripheral skin-homing T cells that were added to the cultures. These observations led us to postulate that IL-7 produced by skin cells contributes to the survival and proliferation of T cells within skin lesions and is likely the source of elevated circulating IL-7 in CTCL.     

Stimulation of human BFU(E) by products of human monocytes and lymphocytes

Regulation of normal human peripheral blood BFU(E) by lymphocytes and monocytes was investigated using plasma clot cultures. Monocyte depletion from peripheral blood mononuclear cells (MNC) resulted in 28 to 98% inhibition of BFU(E) growth in 16 of 17 experiments, but T lymphocyte depletion caused no decrease in BFU(E). When both T cells and monocytes were removed there was an 80% decrease in BFU(E) growth. Addition of as few as 10(4) monocytes alone increased BFU(E) to 88% of expected levels, but addition of 5 x 10(4) T lymphocytes resulted in an increase to only 57% of anticipated BFU(E) numbers. Conditioned media (CM) from 5 x 10(5) to 10(6) unstimulated T lymphocytes per ml stimulated BFU(E) growth from cultures of non-adherent cells, but not from cultures of T cell depleted MNC. Monocyte CM produced peak (2.2-fold) stimulation with only 10(5) monocytes/ml. CM from 5 x 10(5) to 10(6) monocytes/ml had no detectable erythroid burst promoting activity (BPA). Thus, unstimulated monocytes produce BPA, and at high cell concentrations, also produce a presumed inhibitor of BPA production or biologic activity. Although T cells may play some role in control of BFU(E), monocytes appear to be critical for optimal BFU(E) growth in vitro.     

Monoclonal antibody that defines a unique human T-cell leukemia antigen

We have generated and characterized a hybridoma monoclonal antibody, termed SN1, that defines a unique human T-cell leukemia antigen. This antibody was generated by using a human leukemia antigen preparation isolated from cell membranes of MOLT-4, a leukemia T-cell line derived from a patient with T-cell-type acute lymphoblastic leukemia (T-ALL). SN1 was characterized by a sensitive microscale radioimmunoassay using a variety of cultured and uncultured human cells. In selected cases, the cell specimens were further tested by immunoperoxidase staining and an immunofluorescence staining test. The results of the radioimmunoassay were in agreement with those of the two other tests. Among the various cultured malignant and nonmalignant cell lines, SN1 reacted only with leukemia T-cell lines derived from patients with T-ALL; it reacted with all six T-ALL cell lines tested-i.e., JM, CCRF-CEM, CCRF-H-SB2, RPMI 8402, PEER, and MOLT-4. In the case of uncultured cell specimens derived from cancer patients, SN1 reacted with four of four cases of T-ALL but did not react with specimens derived from 41 patients with other types of cancer. SN1 did not react with any normal human cell specimens tested, both cultured and uncultured. These specimens include normal lymphoblastoid cell lines, thymocytes, bone marrow cells, spleen cells, lymph node cells, peripheral blood mononuclear cells, lymphocytes containing B and T cells, purified T cells, monocytes, granulocytes, erythrocytes, and platelets. Furthermore, SN1 did not react with phytohemagglutinin-activated T cells nor with concanavalin A-activated T cells. The results show that monoclonal antibody SN1 defines a type of human leukemia antigen that is expressed on the cell surface of T-cell-type ALL cells. The results further show the usefulness of SN1 in the diagnosis of cancer patients and suggest its therapeutic potential. We designate this antigen TALLA, a T-cell ALL antigen.     

STAT3-mediated constitutive expression of SOCS-3 in cutaneous T-cell lymphoma

A characteristic feature of neoplastic transformation is the loss of external control by cytokines and extracellular matrix of cellular differentiation, migration, and mitogenesis. Because suppressors of cytokine signaling (SOCS) proteins are negative regulators of cytokine-induced signaling, it has been hypothesized that an aberrant SOCS expression plays a role in neoplastic transformation. This study reports on a constitutive SOCS-3 expression in cutaneous T-cell lymphoma (CTCL) cell lines. SOCS-3 protein is constitutively expressed in tumor cell lines (but not in nonmalignant T cells) obtained from affected skin from a patient with mycosis fungoides (MF) and from peripheral blood from a patient with Sezary syndrome (SS). In contrast, constitutive SOCS-3 expression is not found in the leukemic Jurkat T-cell line, the MOLT-4 acute lymphoblastic leukemia cell line, and the monocytic leukemic cell line U937. Expression of SOCS-3 coincides with a constitutive activation of STAT3 in CTCL tumor cells, and stable transfection of CTCL tumor cells with a dominant negative STAT3 strongly inhibits SOCS-3 expression, whereas transfection with wild-type STAT3 does not. Moreover, the reduced SOCS-3 expression in cells transfected with the dominant negative STAT3 is associated with an increased sensitivity to interferon-alpha (IFN-alpha). In conclusion, evidence is provided for a constitutive SOCS-3 expression in cancer cells obtained from patients with CTCL. Moreover, the findings indicate that the aberrant expression of SOCS-3 is mediated by a constitutive activation of STAT3 in CTCL cells and affects the IFN-alpha sensitivity of these cells. (Blood. 2001;97:1056-1062)     

Establishment and characterization of 10 cell lines derived from patients with adult T-cell leukemia.

By using human T-cell growth factor (TCGF), 10 cell lines were established from tissue samples of 10 patients with adult T-cell leukemia (ATL). Three cell lines were adapted to growth in medium lacking TCGF. The surface markers of all cell lines were characteristic of inducer/helper T cells, i.e., OKT3+, OKT4+, OKT6-, OKT8-, OKIa1+, and human Lyt2+ and Lyt3+, except that one cell line was OKT3-. The expression of the viral antigen was examined during establishment of 8 of the 10 cell lines. The viral antigen was not expressed in leukemic cells before cultivation. In 5 lines, the viral antigen was detected by immunofluorescent staining after a short period of cultivation. However, 3 cell lines, ATL-6A, ATL-9Y, and ATL-1K did not express the viral antigen during short-term culture: the ATL-6A and ATL-9Y cell lines became positive for the viral antigen after 5 and 2 months of cultivation, respectively; the ATL-1K cell line remained antigen-negative throughout a culture period of 13 months. Southern blot hybridization assay showed that all of the cell lines, including the viral antigen-negative ATL-1K cell line, contained the viral genome. Thus, the retrovirus was associated with all 10 cell lines established from ATL patients, but there was a heterogeneity in the expression time of the retroviral antigen in leukemic cells maintained in vitro. Our findings suggested that the expression of the viral antigen was not required for maintenance of the leukemic state in vivo and for growth of leukemic cells in vitro.     

Human spleen cell generation of factors stimulating human pluripotent stem cell, erythroid, and myeloid progenitor cell growth

Mitogen-stimulated murine spleen cells produce humoral substances capable of supporting murine hematopoiesis and pluripotent stem cell proliferation in vitro. Thus, we evaluated conditioned media generated by human spleen cells (SCM) in the presence or absence of mitogens for factors stimulatory for human pluripotent (CFU-GEMM), erythroid (BFU- E), and myeloid (CFU-GM) precursors. Two and one half percent to 10% SCM stimulated proliferation of all three types of precursor cells from nonadherent buoyant human marrow target cells. Mitogen-stimulated SCM augmented CFU-GM (175% to 225%), whereas CFU-GEMM and BFU-E growth was essentially unchanged. Cell separation procedures used to determine which cells provided these microenvironmental stimuli indicated that nonadherent mononuclear spleen cells provided the bulk of the CSF-GM, whereas adherent cells (95% nonspecific esterase + monocyte- macrophages) and nonadherent cells provided similar proportions of CSF- mix and erythroid burst-promoting activity (BPA). The nonadherent cells generating high levels of CSF-mix, BPA, and CSF-GM were predominantly Leu-1-negative, ie, non-T, cells. In the presence or absence of mitogens, SCM was a more potent source (1.3- to 3.8-fold) than peripheral leukocyte CM of the growth factors for the three progenitor cell types. Specific in situ cytochemical stains for analyzing morphology of myeloid colonies demonstrated that SCM stimulated the proliferation of the same types and proportions of colonies as human placental CM, suggesting that these CMs may contain similar CSF-GMs. These data show the contribution of spleen cell subsets to the generation of hematopoietic growth factors and the responsiveness of these cells to various mitogenic stimuli.     

In vitro growth of cutaneous T-cell lymphomas.

It is relatively easy to propagate EB virus-transformed normal and malignant B lymphocytes in vitro. Normal T lymphocytes divide for short periods of time after exposure to many mitogens. Exposure of normal T lymphocytes to lymphocyte-conditioned medium (LCM) permits them to divide for longer periods of time, but permanent cell lines cannot be established. Intial attempts to grow malignant T cells from patients with cutaneous T-cell lymphomas (CTCL) in unsupplemented growth medium resulted in the establishment of four EB virus-transformed B-lymphoblastoid lines. The responses of CTCL cells to mitogens and LCM varied from unresponsive to near normal. Subsequent attempts to grow CTCL cells in medium supplemented with mitogens or LCM resulted in the establishment of two cell lines lacking B-cell markers or EB virus. The cells of both lines initially had the ability to form E rosettes, although one of the lines has lost this ability with passage. The two lines can be distinguished from normal T cells by having some or all of the following properties: long-term proliferation, tumorigenecity in nude mice, gradual loss of dependence on mitogen or LCM, and convoluted nuclear morphology.     

Selective generation of erythroid burst-promoting activity by recombinant interleukin 2-stimulated human T lymphocytes and natural killer cells

Because T lymphocytes and natural killer (NK) cells produce a variety of growth factors and interleukin 2 (IL2) modulates the activity of both, we assessed the ability of IL2 to stimulate human T cells and NK cells to produce hematopoietic growth factors detectable in clonogenic marrow culture. Human recombinant interleukin 2 (rIL2) added directly to cultures of human bone marrow that had been depleted of monocytes or depleted of both monocytes and T cells caused no significant alteration of myeloid (CFU-GM) or erythroid colony formation. Conditioned media harvested from rIL2-stimulated (greater than 100 U/mL) peripheral blood mononuclear cells, T cells, Leu-2 cells, and Leu-3 cells all had erythroid burst-promoting activity (BPA) but lacked myeloid colony- stimulating factor (GM-CSF) or CFU-GM-inhibitory activity. These T cells were IL2 receptor-negative, and the addition of anti-IL2 receptor monoclonal antibody (anti-Tac) to T cell cultures did not abrogate this IL2-stimulated BPA production. In addition, Percoll gradient-enriched, large granular lymphocytes (LGL) were separated by fluorescence- activated cell sorting into Leu-11+ (NK) cells and Leu-11- (low-density Leu-4+ T) cell fractions. rIL2 stimulated LGL, Leu-11+ and Leu-11- cells to produce BPA but not detectable GM-CSF or CFU-GM-inhibitory activity. Leu-11+ (NK) cells were Tac-negative from days 0 through 14 of culture. We conclude that rIL2 at high concentrations stimulated T cells, Leu-2 and Leu-3 cell subsets, LGL, and NK cells to produce BPA but not GM-CSF and that this stimulation may be mediated by an IL2 receptor distinct from Tac or by an epitope of the IL2 receptor not recognized by the anti-Tac antibody.