Functional analysis of human T cell subsets defined by monoclonal antibodies. III. Regulation of helper factor production by T cell subsets

In the present report we extended our previous studies demonstrating that obligatory T-T interactions are important in regulating human immune responses in vitro. Functionally distinct human T cell subsets were isolated by complement-mediated lysis using the monoclonal antibodies OKT4 and OKT8. Evidence was obtained that during allogeneic interactions, OKT4+, but not OKT8+, responder T cells are required to generate helper factor(s) capable of polyclonally activating human B cells independent of additional T cell help. Importantly, the alloantigen-induced helper factor(s) production and/or release was found to be suppressed by addition of graded numbers of radiosensitive OKT8+ cells. On the other hand, no evidence was obtained that supernatant derived from alloactivated OKT8+ cells could counterbalance the helper activity generated in the presence of supernatant from alloactivated OKT4+ cells. Furthermore, OKT8+ cells, known to suppress PWM-driven B cell differentiation in the presence of OKT4+ cells, do not suppress B cell differentiation induced by preformed helper factor even in the presence of OKT4+ cells. These data further underscore the importance of functional T-T interactions in immunoregulation in vitro and support the idea that the target of suppression of B cell differentiation, induced either by alloantigen-triggered helper factor or PWM, are OKT4+ cells and not B cells themselves.     

Helper cell function of Leukemic Leu-2a+, histamine receptor+, T gamma lymphocytes

Leukemic cells from a patient with an 11-yr history of chronic lymphocytic leukemia (CLL) were found to have the surface phenotype Leu-1+, Leu-2a+, Leu-3a-, sheep erythrocyte rosette+, IgGFc receptor+. The cells also bore a receptor for histamine inhibitable by cimetidine (H-2). The clonal nature of the proliferation was documented by the presence of a consistent marker chromosome (22-trisomy) in metaphases elicited by culture with T cell growth factors. Although the surface phenotype suggested that these cells might function as suppressor lymphocytes, they had an enhancing effect on the pokeweed- mitogen- (PWM) driven generation of plasma cells and reverse hemolytic plaque-forming cells in vitro. This helper activity was modified neither by irradiation of the leukemic cells nor by removal of a minor population of Leu-3a+ cells, suggesting that the effects were attributable to the CLL cells themselves. In addition to these functions, the CLL cells were active in antibody-dependent cellular cytotoxicity (ADCC) assays in association with expression of Fc receptors for IgG. The ADCC was diminished when a transient loss of the Fc receptor expression was observed. No activity in natural killer cell assays employing K-562 cells or herpes simplex virus- (HSV) infected cells as targets could be attributed to the leukemic clone. These studies indicate that the cell surface phenotype, as defined by monoclonal antibodies, may not always predict the functional state of a particular cell, and suggest that within the Leu-2a+ (TH-2+) population of human lymphocytes, some helper as well as suppressor/cytotoxic cells are to be found.     

A defect in the suppressor circuits among OKT4+ cell populations in patients with systemic lupus erythematosus occurs independently of a defect in the OKT8+ suppressor T cell function

The autologous mixed lymphocyte reaction (MLR) is thought to be part of a regulatory role of T cells on B cell function. OKT4+, but not OKT8+, cells can proliferate in response to autologous non-T cells. Moreover, the OKT4+ cell population activated early in the course of autologous MLR functioned as inducer cells for the differentiation of B cells, whereas later in the response, the activated OKT4+ cells were particularly enriched in suppressor cells. A part of the autologous MLR appears to be an important pathway for the activation of feedback suppression mechanisms among cells contained within the OKT4+ populations. Patients with systemic lupus erythematosus (SLE) were studied with regard to the following OKT4+ cell functions in vitro after activation in the autologous MLR: a) proliferative response, and b) helper and suppressor activities for differentiation of B cells. A marked reduction in the proliferative response of OKT4+ cells was observed in SLE patients. SLE OKT4+ cells activated in the autologous MLR could function as helper cells but could not exert any suppressor activity. This OKT4+ cell abnormality was present regardless of the disease activity, and occurred in the absence of autoantibodies including anti-T cell antibodies. Instead, SLE anti-T cell antibodies could preferentially eliminate cells bearing the OKT8+ phenotype characteristic of suppressor cells in populations of normal T cells. These results suggest that the defect in the suppressor circuits among OKT4+ cell populations is intrinsic to SLE lymphocytes and that the OKT8+ suppressor T cell defect is caused by antibodies produced by the B cells of SLE patients.     

Morphological and immunophenotypic features of chronic lymphocytic leukemia

In this review, we summarize the morphological features and immunophenotypic profile of chronic lymphocytic leukemia (CLL) cells, discuss the value of these investigations as front line diagnostic tests, and emphasize their correlation with the clinical features, disease progression, molecular genetics and pathogenesis of CLL. In CLL, the morphology of the circulating cells is characteristic and typical in the majority of cases. However, 15% of patients, either at diagnosis or during the course of the disease, show atypical morphology reflected by either (1) an increased (> 10%) number of circulating prolymphocytes, designated CLL/PL, or (2) an increased (> 15%) number of circulating lymphoplasmacytic and cleaved cells, designated 'atypical' CLL. There is strong evidence of a close association between atypical morphology (CLL/PL) and atypical (CLL) and clinical features, e.g. disease progression, advanced stage and survival, molecular genetics, particularly trisomy 12, but also the rare cases with t(11;14) or t(14;19), p53 abnormalities, unmutated immunoglobulin (Ig) VH genes and origin of the cell (naive, pregerminal center cell). CLL cells have a distinct immunological repertoire different from that of other lymphoproliferative disorders. The typical CLL phenotype is CD5+, CD23+, FMC7-, weak expression of surface Ig (sIg) and weak or absent expression of membrane CD22 and CD79b. The latter marker identifies an extracellular epitope of the B-cell receptor (BCR) beta chain and its weak or absent expression in CLL may derive from the expression of a truncated form. This, together with the low expression of CD22, might explain the abnormal signal transduction of CLL cells similar to that of anergic B lymphocytes. Because no single marker is specific for CLL, a composite phenotype considering this set of 5 or 6 markers compounded into a scoring system helps to distinguish CLL from the other B-cell malignancies. Immunophenotypic analysis has also been shown to be useful for minimal residual disease detection and adds valuable prognostic information because the expression of certain markers, such as FMC7 or CD38, seems to be associated with a poor outcome. In addition, CLL cells express a variety of Bcl-2 family proteins with a profile that favors inhibition of apoptosis which, together with the interaction with microenvironmental (e.g. stromal) cells and the release of cytokines, explains the long life span and subsequent accumulation of CLL cells in various organs. Despite controversies relating to the expression of adhesion molecules (selectins and integrins) in CLL cells, it appears that some of these molecules do play a role in the pathogenesis, biology and clinical patterns of the disease. In conclusion, morphology and immunophenotype are the two essential investigations, which must be carried out in all cases of CLL. Both provide relevant information in terms of diagnosis, course of the disease, prognosis and pathogenesis.     

A DC-specific cytolytic T lymphocyte line is OKT8+1

A human cytotoxic T lymphocyte (CTL) line, A9, was generated by limiting dilution and was selected because of its apparent DC specificity. A9 is 100% OKT3+, 90% OKT4+, and 10% OKT8+, but by negative selection the CTL present are entirely OKT8+. These OKT8+ CTL are totally inhibitable by Genox 3.53, an anti-DC1 monoclonal antibody (mAb), and Leu-10, an anti-DC subgroup mAb, but are not inhibitable by a panel of anti-HLA-DR mAb. These CTL are also inhibitable by anti-OKT3 and anti-LFA-2 but not by OKT4 or OKT8 mAb. These findings extend previous studies that showed that OKT8+ CTL recognize HLA-A,B antigens, whereas OKT4+ CTL recognize HLA-DR and SB antigens. It is possible that an as yet undefined T cell surface molecule is involved in DC recognition.     

Isolation of myelin basic protein-reactive T-cell lines from normal human blood

T-Cell lines which responded by proliferation to the autoantigen, myelin basic protein (MBP), were isolated from the blood of six of nine normal humans. These T-cell lines could be maintained in in vitro culture for up to 2 months through the use of Interleukin 2 and repeated MBP stimulation. Optimal antigen-induced proliferation required both antigen and antigen-presenting cells found in the adherent cell population of autologous peripheral blood mononuclear cells (PBM). The T-cell lines were predominantly of the helper phenotype (OKT3+, OKT4+, OKT8-) and responded to both human and guinea pig myelin basic protein.     

Antigen-specific human T lymphocyte clones: induction, antigen specificity, and MHC restriction of influenza virus-immune clones

Human peripheral blood lymphocytes from an HLA-Dw1,3 individual were primed in vitro with influenza A virus (A/Texas/1-77/x-49) and subsequently cloned by limiting dilution in TCGF. Of the 96 TLCs originally obtained, nine were characterized in detail. TLCs were antigen specific, responding to influenza A virus, not to influenza B, TGAL, GAT, tetanus toxoid, or KLH, and only when antigen was presented by cells unable to form rosettes with AET-treated SRBC. Presentation of antigen by unseparated PBL often resulted in significant "back stimulation," probably via production of growth factors. The MHC requirements for the induction of TLC proliferation were analyzed. Of four representative clones analyzed, three required Dw1;DR1 compatibility for successful presentation of viral antigens by a panel of antigen-presenting cells. In contrast, one TLC showed an unusual pattern of response that could not be correlated to a particular HLA haplotype. Monoclonal anti-T cell antibody analysis of the surface phenotype of two TLCs maintained in continuous culture for 5 mo indicated that they were OKT3+, 4+, and 8-, consistent with an inducer/helper phenotype. To confirm the clonal nature of TLCs, data on the functional properties of TLC subclones are also presented.     

Sézary syndrome with hyposplenism

Phenotypic, functional and clinical analysis of two patients with Sezary Syndrome are presented. Both patients had an elevated lymphocyte count with the helper/inducer cell phenotype by analysis with monoclonal antibodies (OKT3+, T4+, T6-, T8-, M1-), had the characteristic cerebriform nucleus of Sezary cells, and were Fc-IgG receptor negative. The functional tests revealed no proliferative, cytotoxic or immunoregulatory activity of patient E.P.'s leukemic cells, while the lymphocytes of patient A.N. responded to mitogen stimulation and had helper cell capacity in pokeweed mitogen driven B cell differentiation and maturation. Both patients presented skin involvement, pruritus, hepatomegaly and patient E.P. showed generalized lymphadenopathy. The spleen size of patient A.N. was below the normal range with an estimated spleen weight of approximately 160 g (normal 180-229 g). Patient E.P. had an extremely small spleen size with an estimated weight of approximately 20 g as shown by abdominal sonography and spleen scintigraphy and had Howell-Jolly bodies within the erythrocytes. The size of the spleen in various other diseases with T cell proliferations is discussed with respect to the possible proliferative centers of the various T-cell subpopulations.     

Characterization of antigen-specific T cells in multiple sclerosis twins with elevated proliferative responses to measles virus

The proliferative response to measles virus in normal individuals is low compared with the response to mumps virus. This is probably due to a low precursor frequency of OKT4+, IL 2-secreting helper cells. The presence of a measles high-responder state has previously been identified in some twin individuals with multiple sclerosis. Further characterization of the measles response in these high-responder individuals has demonstrated that the enhanced measles responses are due to a greater response by OKT4+ cells, which secrete higher levels of IL 2; this contrasting with the low levels of IL 2 secretion and OKT4+ cell proliferation seen in the unaffected twins. No evidence for suppression by either accessory or T cells, which would account for the quantitative differences between the high responders with multiple sclerosis and their unaffected low-responder twin siblings, was detected. The results indicate that a clonally expanded population of measles-specific responder cells is responsible for the high-responder state in these twins with multiple sclerosis. The mechanism producing this state may have relevance to possible immunoregulatory abnormalities producing autoimmunity in multiple sclerosis.     

T lymphocyte subsets in patients with malignant melanoma

Summary T lymphocyte subset profiles were determined by monoclonal antibodies on cryopreserved peripheral blood lymphocytes from 57 patients with malignant melanoma and 19 healthy controls. Quantitation of percentages of total T cells (OKT3.PAN), helper (OKT4.IND) or suppressor (OKT8.SUP) cells, and the ratio of helper/suppressor subsets revealed no correlation of these markers with stage of disease or clinical outcome. A sequential study of these markers on peripheral blood lymphocytes from three stage I melanoma patients with subsequent recurrent disease showed no fluctuations that could be correlated to tumor progression. This study indicates that there is no systemic imbalance in T cell subsets in malignant melanoma and that quantitation of these subsets cannot predict the clinical course of this disease.     

Systemic administration of autologous,alloactivated helper-enriched lymphocytes to patients with metastatic melanoma of the lung

Summary A phase I study was carried out to test the feasibility and toxicity of infusing large numbers of autologous, alloactivated helper lymphocytes into patients with metastatic melanoma. Patient peripheral blood lymphocytes (Pt-PBL) obtained by lymphopheresis and expressing the helper phenotype BT5/9 were separated and stimulated for 48 or 72 h with a pool of PBL from four to six healthy donors. Patients were then infused with such activated lymphocytes over a 2–3 h period. A total of 4 phereses and infusions (2/week for 2 weeks) were carried out for each cycle in each patient. Of the five patients treated, two received a second round of infusions. Infusion of autologous PBL stimulated in vitro for 48 h caused chills, fever, headache, and increased blood pressure. All symptoms disappeared in 2–3 h and were easily controlled by appropriate therapy. When lymphocytes were given after 72 h of allostimulation, no or very mild toxicity was observed. Serum chemistry, coagulation, autoimmunity, and urine analysis showed no gross abnormalities during therapy or follow-up of the patients. Immunological parameters (OKT4/OKT8 ratio, NK activity and cytotoxic T cell activity to autologous melanoma) were evaluated before starting the therapy, during its course and during the 3 to 6 months follow-up. The OKT4/OKT8 ratio increased significantly but transiently soon after the first course of infusions in one of the two patients tested. NK activity increased after 75–100 days in the three patients tested and in one of them it was high even after 180 days. No correlation between NK activity and prognosis was apparent. Cytotoxicity to autologous tumor was assessed in two patients, only one of whom exhibited an increased activity from 75 to 180 days, which was associated with a prognosis better than that of the negative patient. Five patients were treated: two had progressive disease, two had stable disease for 5 and 6 months, respectively. In the first of these patients, a new cycle of lymphocyte infusions was carried out which caused a measurable reduction of lung tumor nodules whose growth, however, resumed 4 months later. This patient died 14 months after the onset of therapy. The fifth patient had a partial regression of pulmonary and intracranial metastases after therapy, but eventually died 3 months later. These results indicate that infusion of a high numbers of autologous, allostimulated helper PBL is a feasible and safe procedure, which could therefore be used in future studies of adoptive immunotherapy of cancer.     

Human T cell clones exerting multiple cytolytic activities show heterogeneity in susceptibility to inhibition by monoclonal antibodies

Human cytotoxic T cell clones (CTL) were obtained by limiting dilution after in vitro priming against an allogeneic Epstein Barr virus (EBV)-transformed B cell line (B-LCL) BSM. Three OKT3+, OKT8+ E rosette-forming (RFC) but EA gamma-RFC- clones with cytotoxic activity against the stimulator cell and one "non-cytolytic" clone were expanded for over 50 generations and further characterized. Clone G9 showed allospecific lysis of Cw3+ lymphocytes and B cell lines. Three cytolytic clones (G9, D11, and A3) showed cytotoxicity to the stimulator B-LCL, to the human plasma cell leukemia-derived line LICR-LON-HMY2 and to short-term cultured melanoma cells (O-mel). Four other EBV-transformed B-LCL unrelated to the stimulator B-LCL were not lysed. These clones also exerted cytotoxic activity against NK-sensitive target cells (TC), e.g., the erythroleukemia cell line K562. Other NK-sensitive TC, e.g., lymphoma-derived Daudi cells, were killed provided they were pretreated with phytohemagglutinin (PHA). Cytolytic activity against the B-LCL cell LICR-LON and O-mel, but not against K562 or PHA-treated target cells, was inhibited by monoclonal anti-HLA ABC antibodies (MCA). The cytolytic activities of OKT3+,8+ clones G9 and A3 but not that of OKT3+,8+ clone D11 were inhibited by OKT8. Another MCA, 13.3, directed against the murine glycoprotein T-200, inhibited the cytolytic activity of clone D11 against K562 but not against the stimulator cells. Clone G9 was not inhibited by MCA 13.3. The four clones, including the OKT4+ "non-cytotoxic" clone K12, exerted lytic activity against TC that are normally resistant to lysis provided these TC were pretreated with PHA. The TC specificity range of the clones was confirmed by cold target inhibition experiments. A correlation between blocking of lytic activity by cold TC and the percentage of conjugate formation with the particular cold TC was observed. Because these clones also show differential susceptibility to inhibition of lysis by various MCA, it is concluded that human cytotoxic T cell clones can exert multiple lytic activities, i.e., the operationally defined lytic mechanisms differ at least at certain stages of the lytic cycle.     

Functional analysis of human T cell subsets defined by monoclonal antibodies. V. Suppressor cells within the activated OKT4+ population belong to a distinct subset

In the present report, we characterize a monoclonal antibody directed at a surface differentiation antigen on human T cells. The monoclonal antibody, OKT17, recognizes a cell surface antigen present on the majority of resting normal peripheral T cells. In contrast, OKT17 is unreactive with normal B cells, B cell lines, T cell lines, or SIg+ CLL. Interestingly, after activation, the antigen recognized by OKT17 is lost from a subset of OKT4+ cells. We took advantage of this finding to explore further the functional heterogeneity within activated OKT4+ cells. Evidence was obtained that the PWM-activated OKT4+ subset remaining after depletion of OKT17-reactive T cells (OKT4+ 17-) contains radiosensitive helperr cells but is devoid of suppressor cells. In contrast, the activated OKT4+ 17+ population contains potent radiosensitive suppressor cells as well as radioresistant helpe cells. Taken together, these studies suggest that the OKT17 monoclonal antibody can differentiate two functionally mature, activated OKT4+ human T cells: OKT4+ OKT17+ radiosensitive suppressor cells and OKT4+ 17- radiosensitive helper cells.     

Release of hemopoietic factors by normal human T cell lines with either suppressor or helper activity

We analyzed the release of activities capable of stimulating the in vitro growth of human hemopoietic progenitor cells by long-term cultured T cell growth factor (TCGF)-dependent human T lymphocytes. Seven cell lines tested produced colony-stimulating activity (CSA) as well as burst-promoting activity (BPA). The CSA stimulated primarily the growth of the cells forming colonies after 14 days of incubation. In addition the supernatants from these seven T-cell lines showed the ability to induce the in vitro growth of mixed granulocyte, erythroid, megakaryocyte, macrophage colonies (CFU-GEMM). The release of hemopoietic factors did not depend on the presence of accessory cells or phytohemagglutinin or serum during the incubation for factor production. In six of the T cell lines the majority of the cells were reactive to the OKT 8 monoclonal antibody (MoAb), whereas one cell line contained mostly OKT 4+ cells. Suppressor activity was detected in three tested OKT 8+ cell lines, while the one OKT 4+ displayed helper activity. All cell lines produced hemopoietic factors with equal efficiency. These results indicate that factors affecting human hematopoiesis are produced by normal T lymphocytes in long-term culture and this property is not related to the helper or suppressor activity of the cultured cells.     

Clonal analysis of murine graft-vs-host disease. I. Phenotypic and functional analysis of T lymphocyte clones

Acute and chronic graft-vs-host disease (GVHD) due to non-MHC histocompatibility differences differ histopathologically. Acute GVHD is characterized by the cytotoxic destruction of recipient tissues, whereas chronic GVHD is characterized by increased collagen deposition. In an attempt to determine if acute and chronic GVHD represent two phases of the same pathophysiologic process or two distinct processes, the T lymphocytes from the C57BL/6 (B6) recipients of LP spleen cells (non-H-2 GVHD) have been cloned and compared to clones from immune mice (LP anti-B6). Acute GVHD (G) clones were established on day 10-14 posttransplant and chronic GVHD (CG) clones on day 50 from animals with clinical chronic GVHD. Immune (I) clones were established 10 to 14 days after immunization. All I clones exhibited B6-specific blastogenesis and cytotoxicity and had a Thy-1.2+, Lyt-2.2+, L3T4- phenotype. All CG clones were noncytotoxic, had I-Ab-specific blastogenesis, and had a Thy-1.2+, Lyt-2.2-, L3T4+ phenotype. The acute GVHD (G) clones were heterogeneous. Fourteen of 23 clones exhibited B6-specific blastogenesis and had a Lyt-2.2+, L3T4- phenotype (B6-G clones). Seven of 9 B6-G clones were cytotoxic for B6 targets. Nine of 23 G clones exhibited I-Ab-specific blastogenesis, and all but one clone had a Lyt-2.2-, L3T4+ phenotype as the did CG clones. Thus, the principal clonogenic T lymphocytes from mice with acute and chronic GVHD differ in terms of 1) their antigenic specificity, 2) their cytotoxic capacity, and 3) their surface phenotype. The presence of I-Ab-specific T lymphocytes with a phenotype identical to CG clones early after transplantation suggests that the immunologic events that result in chronic GVHD begin soon after transplantation. These results indicate that acute GVHD is due primarily to recipient-specific cytotoxic donor T lymphocytes, whereas chronic GVHD is due to autoreactive helper T lymphocytes.     

Interleukin 2-dependent natural killer (NK) cell lines from patients with primary T cell immunodeficiencies

Bulk cultured cell lines with natural killer (NK) activity were derived by in vitro culture with interleukin 2-containing conditioned medium (IL 2-CM) of peripheral blood mononuclear leukocytes (PBL) from patients with primary T cell deficiencies. Lines were developed from three patients with severe combined immunodeficiency (SCID) and one patient with Nezelof's syndrome and contained several populations of cells with distinct phenotypes. All lines contained a cell population expressing the Leu-5 (50K) (sheep red blood cell receptor), 3A1 (40K), and OKT10 antigens, but lacking the pan T cell antigens Leu-1 (67K) and Leu-4 (19K) as well as the markers of T cell subsets Leu-2a (32K) and Leu-3a (56K). These cells failed to express the Leu-7 antigen and only weakly expressed OKM1. In addition, one line contained a population of Leu-5+, 3A1+, OKT10+, Leu-2a+, Leu 1-, and Leu 4- cells. Three of the lines also contained populations with classic T cell (Leu-1 and-Leu 4+) phenotypes. The lines were enriched in NK activity compared with the PBL from which they were derived. Their growth was strictly dependent on IL 2-CM. Highly purified IL 2, lacking any other detectable protein contaminants or lymphokine activities, was capable of supporting the growth of the Leu-5+, 3A1+ "null" cell populations from these lines without alteration in their functional activity or phenotype. Thus, studies of in vitro expanded cell lines from patients with severe disorders of T cell function and thymic involution indicate that this "null" cell population does not require thymic maturation to develop its effector function. This "null" cell population can be maintained in vitro in the presence of IL 2. This finding is analogous to the data obtained from study of NK cells in athymic (nude) mice.     

Lysis of fresh human solid tumor cells by autologous lymphocytes activated in vitro by allosensitization

Summary We have previously demonstrated that cancer patients' peripheral blood lymphocytes (PBL) allosensitized against single or pool normal donor PBL are capable of lysing fresh autologous tumor cells in a 4-h ⁵¹Cr-release assay. In this report, we present further investigations into this phenomenon. These alloactivated killer cells (A-AK cells) lysed autologous and allogeneic tumors and allogeneic but not autologous PBL. Furthermore, cold target inhibition studies demonstrated that autologous and allogeneic tumors were lysed by the same effector cells. Multiple metastases from the same patient were equivalently lysed by these A-AK cells. The presence of adherent cells and proliferation of the precursors were necessary to generate A-AK cells, although the effector cell itself was radioresistant and nonadherent. The effects of allosensitization were enhanced by the addition of lectin-free interleukin-2 preparations to the in vitro culture by partial depletion of adherent cells prior to sensitization. The A-AK effector cell was OKT3+, OKT8+, OKT4–, OKM1– and could be generated by just 3 days of allosensitization. The precursors for A-AK cells could be separated from NK cells on percoll gradients and lysis could be generated from thoracic duct lymphocytes, a population devoid of NK cells. The phenotype of the majority of the precursor cells was OKT3+, OKT4–. These allocatived PBL could be expanded in crude or lectin-free interleukin-2 without loss of cytotoxicity for fresh autologous tumor cells. Activated T cells represent a population of non-NK cells with broad lytic specificity for fresh tumor cells. Such cells may be of value in the adoptive immunotherapy of human solid tumors and may play a role in immune surveillance.     

Identification of the T cell subset that produces human gamma interferon

Positive and negative selection procedures combined with cytofluorographic analysis and lysis with monoclonal antibodies were utilized to identify the T lymphocyte subset that produces human gamma interferon (gamma-IFN) (formerly referred to as "immune" or "type II" interferon) in response to mitogen stimulation. Lymphocytes were separated on the basis of their Fc receptors for IgG or IgM, their nonreactivity with IgM or IgG antibodies, and their reactivity with the monoclonal antibodies OKT4, OKT8, OKT11a, and OKM1. Isolated T cell subsets were incubated with the gamma-IFN inducer, phytohemagglutinin. Three days after induction, the cell supernatants were harvested and assayed for interferon. The T cell subset that produces gamma-IFN was identified as E rosette positive with the phenotype: T gamma, T non-micro, OKM1+, OKT4-, OKT8- and OKT11a+. gamma-IFN production by cells was resistant to doses of x-irradiation that abrogate mitogen-induced T suppressor function but was highly sensitive to low doses of 4-hydroperoxycyclophosphamide. These data demonstrate that gamma-IFN is produced by the T gamma, OKM1+ lymphocyte subset, but these cells may also require the presence of accessory monocytes for elaboration of gamma-IFN. The anti-proliferative activity of gamma-IFN may be responsible for the previously described suppressor function of this subset, and gamma-IFN production by T gamma cells may distinguish this subset from the suppressor/cytotoxic functions of the OKT8+ subset or the mitogen-induced OKT4+ suppressor.     

Homologous human macrophage hybridomas that produce a novel cytotoxic factor in their culture supernatants

Homologous human macrophage hybridoma cell lines were obtained by somatic cell fusion between peripheral blood monocyte-derived macrophages and a subclone of the myelomonocytic cell line, U937-F9. The hybridoma cell lines grown in vitro for more than a year were confirmed by manifestations of phagocytosis, adherence, nonspecific esterase, acid phosphatase, chromosome numbers and other cell surface antigens. Cell surface antigens on hybridomas were detected by flow cytometry analysis with monoclonal antibodies. With interclonal differences, a typical phenotype of hybridoma cells was CDw14+, OKM5+, Mac-1+ (equivalent to OKM1 and Mol), OKT9+, HLA-DR- and CD20+. After stimulation with lipopolysaccharide and calcium ionophore A23187, culture supernatants of clones c18A and c29A showed cytotoxic activity against human melanoma A375 Met-Mix and other cell lines which were resistant to the tumor necrosis factor, lymphotoxin and interleukin 1. This cytotoxic factor was found to be distinct from the tumor necrosis factor, lymphotoxin and interleukin 1 using the anti-tumor necrosis factor, anti-lymphotoxin and anti-interleukin 1 antisera.     

Lymphokine production by human T cell hybridomas

Human T cell hybridomas were generated by hybridization of SH9 cells, the 6-thioguanine-resistant variant of human T lymphoma Hut102-B2, with concanavalin A-stimulated human peripheral blood lymphocytes. The hybrid nature of the established cell lines was documented by the difference in D14S1 restriction fragment length polymorphism between SH9 and the hybridoma cell DNA, and by the expression of OKT11 antigen on hybrid cells. T cell growth factor, macrophage growth factor (MGF), and interferon (IFN) activities have been demonstrated in the supernatants of different hybrid cultures, but not in SH9 cell cultures. Substantial quantities of MGF were secreted by several hybrids including the L23 line. MGF activity was dose-dependent, heat-labile, and synergistic with indomethacin. High titers of IFN activity were found in the cultures of hybridoma L415 and its subclones. Neutralization with specific antisera showed the IFN synthesized by L415 clones was immune interferon (IFN-gamma). Like the parental SH9 line, all of the hybridomas producing these lymphokines exhibited a cell surface phenotype typical for helper T cells. The hybridoma system therefore shows potential for the study of various lymphokines produced by human helper T lymphocytes.