Phenotypic characteristics of cell lines requiring interleukin 3 for growth

A number of cell lines have been derived from bone marrow cultures in the presence of WEHI-3 conditioned media (CM) that continue to require WEHI-3 CM for growth in vitro. Because the WEHI-3 cell line has been shown to constitutively produce a lymphokine (IL 3) that induces the expression of 20 alpha-hydroxysteroid dehydrogenase (20 alpha SDH) in cultures of splenic lymphocytes from athymic mice, we examined whether these cell lines were dependent upon IL 3 for growth. The results demonstrate that the factor required for growth of these cell lines copurifies with IL 3 activity on G-100, DEAE cellulose, and CM cellulose column chromatography as well as in preparative isoelectric focusing and on hydrophobic supports in reverse phase high pressure liquid chromatography. The biologic activity of peak fractions in each case was similar in both types of assays. These results strongly suggest that the WEHI-3 CM-dependent cell lines are dependent on IL 3 for growth in vitro. All the cell lines have readily detectable levels of 20 alpha SDH but have differing cell surface phenotypes. The C3HSFFV line is devoid of conventional lymphoid cell surface markers with the exception of Lyt-1, whereas the FDC-P1 expresses Thy-1, Ly-5, and H-11. Other cell lines have intermediate phenotypes.     

Functional analysis in vitro and in vivo of Mycobacterium bovis strain BCG-specific T cell clones

Several cloned T cell lines specific for PPD and BCG were obtained. All clones were able to secrete lymphokine, i.e., MAF/interferon, upon antigenic stimulation. The surface phenotype of all these different clones was Thy-1.2+, L3T4+, Lyt-2-, suggesting that these lines belonged to the helper/inducer T cell subset. The T cell clones displayed various degrees of helper activity as tested in a secondary antibody response in vitro. The capacity of these clones to elicit DTH reactions in the presence of antigen and their ability to inhibit mycobacterial growth in vivo were tested by transferring locally the different clones to normal mice. The clones which exhibited little or no helper activity were able to elicit DTH responses, whereas the clone with strong helper activity did not. Both types of functionally defined clones had the capacity to inhibit the growth of intracellular mycobacteria in vivo.     

c-myc gene expression and interleukin-2 receptor levels in cloned human CD2+,CD3+ and CD2+,CD3- lymphocytes

The levels of c-myc mRNA and interleukin-2 receptors (IL-2 Rec) were studied in human peripheral blood lymphocytes (PBL); mature CD2+,CD3+ T cell clones and CD2+,CD3- natural killer (NK) cell clones, and CD2+,CD3+ and CD2-,CD3- T lymphoma cell lines. A transient induction of the expression of c-myc and IL-2 Rec was observed in PBL after activation with phytohemagglutinin (PHA). Expression of c-myc and IL-2 Rec was also found in the CD2+,CD3+ and CD2+,CD3- clones. The CD2+,CD3+ showed higher levels of c-myc mRNA and IL-2 Rec than the CD2+,CD3- clones. In three T lymphoma cell lines constitutively high levels of c-myc mRNA but no IL-2 Rec were found. Only in JURKAT (CD2+,CD3+), c-myc mRNA levels could be further enhanced by PHA. These results suggest that in the presence of PHA, expression of c-myc and IL-2 Rec is induced via the CD3 receptor, and in the absence of PHA and/or the CD3 receptor alternative routes of induction are involved.     

Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins

A panel of antigen-specific mouse helper T cell clones was characterized according to patterns of lymphokine activity production, and two types of T cell were distinguished. Type 1 T helper cells (TH1) produced IL 2, interferon-gamma, GM-CSF, and IL 3 in response to antigen + presenting cells or to Con A, whereas type 2 helper T cells (TH2) produced IL 3, BSF1, and two other activities unique to the TH2 subset, a mast cell growth factor distinct from IL 3 and a T cell growth factor distinct from IL 2. Clones representing each type of T cell were characterized, and the pattern of lymphokine activities was consistent within each set. The secreted proteins induced by Con A were analyzed by biosynthetic labeling and SDS gel electrophoresis, and significant differences were seen between the two groups of T cell line. Both types of T cell grew in response to alternating cycles of antigen stimulation, followed by growth in IL 2-containing medium. Examples of both types of T cell were also specific for or restricted by the I region of the MHC, and the surface marker phenotype of the majority of both types was Ly-1+, Lyt-2-, L3T4+, Both types of helper T cell could provide help for B cells, but the nature of the help differed. TH1 cells were found among examples of T cell clones specific for chicken RBC and mouse alloantigens. TH2 cells were found among clones specific for mouse alloantigens, fowl gamma-globulin, and KLH. The relationship between these two types of T cells and previously described subsets of T helper cells is discussed.     

CD8 alpha+, but not CD8 alpha-, dendritic cells tolerize Th2 responses via contact-dependent and -independent mechanisms, and reverse airway hyperresponsiveness, Th2, and eosinophil responses in a mouse model of asthma

Splenic CD8alpha+ dendritic cells reportedly tolerize T cell responses by inducing Fas ligand-mediated apoptosis, suppressing IL-2 expression, or catabolizing T cell tryptophan reserves through expression of IDO. We report in this study that CD8alpha+, but not CD8alpha-, dendritic cells purified from the spleens of normal mice can tolerize the Th2 responses of cells from asthma phenotype mice through more than one mechanism. This tolerance could largely be reversed in vitro by anti-IL-10 or anti-TGFbeta Ab treatment. However, loss of direct dendritic cell-T cell contact also reduced tolerance, although to a lesser extent, as did adding the IDO inhibitor 1-methyltryptophan or an excess of free tryptophan to the cultures. Within 3 wk of reconstituting asthma phenotype mice with 1 x 10(5) OVA-pulsed CD8alpha+, but not CD8alpha-, dendritic cells, the mice experienced a reversal of airway hyperresponsiveness, eosinophilic airway responses, and pulmonary Th2 cytokine expression. This data indicates that CD8alpha+ dendritic cells can simultaneously use multiple mechanisms for tolerization of T cells and that, in vivo, they are capable of tolerizing a well-established disease complex such as allergic lung disease/asthma.     

Activation via TCR or IL-2 receptor of a CD8+ suppressor T cell clone: Effect on IL-10 production and on proliferation of the suppressor T cell

In a previous study, we established CD8⁺ suppressor T cell (Ts) clone 13G2 which produced the suppressive lymphokine, interleukin-10 (IL-10). In this study, we examined what physiological activator could induce both production of IL-10 from 13G2 and the proliferation of 13G2. Both the antigenic stimulation mimicked by the anti-CD3 antibody and the T cell growth factor interleukin-2 (IL-2) induced IL-10 production from the 13G2 clone equally well. 13G2 cells proliferated remarkably with IL-2 stimulation, while anti-CD3 only slightly induced proliferation of the clone. 13G2 cells also produced IL-10 in the presence of hydroxyurea which blocked transit of cells from G₁ to S phase. However, cycloheximide blocked the production of IL-10 from the Ts clone. The study demonstrates that both the anti-CD3 antibody and IL-2 induced IL-10 synthesis of the Ts clone equally well, and the proliferative response of Ts cells was induced more by IL-2 than by anti-CD3. IL-2 proved to be a good stimulator for Ts cells to produce suppressive lymphokine and to multiply their population.Abbreviation Ts suppressor T cell - Th helper T cell - Ag antigen - APC antigen presenting cell - IL interleukin - TCR T cell receptor - mAb monoclonal antibody     

Membrane-associated interleukin 2 epitopes on the surface of human T lymphocytes

Analysis of surface fluorescence with flow cytometry has revealed the presence of membrane-associated interleukin 2 (IL-2) epitopes on the surface of long term human T cell clones. These IL-2 epitopes could not be accounted for by soluble IL-2 binding to its specific receptor or adsorbing nonspecifically to the cells. The level of surface IL-2 antigenic determinants on the T cell clones was decreased in the presence of phorbol esters and increased in the absence of an exogenous source of IL-2. It was completely lost upon stimulation of the clones to produce the soluble lymphokine. Surface IL-2 epitopes were also detected on the Jurkat tumor cell line which secretes IL-2 upon stimulation and on another T cell tumor line MOLT 4. MLA-144 produces IL-2 constitutively; however, it did not possess membrane-associated epitopes. Tumor lines of other lineages were negative. A subpopulation of peripheral blood T lymphocytes demonstrated some membrane-bound IL-2, whereas non-T peripheral blood mononuclear cells were negative. Thus, cells with the potential of producing and secreting IL-2 upon stimulation possessed the surface epitopes of the lymphokine and cells either actively secreting IL-2 or without the potential for secretion were negative for surface expression. Membrane-associated IL-2 antigenic determinants appear to represent a T lymphocytic surface marker of potential cellular function. The relationship of this marker to the secreted lymphokine is not known. Although it is possible that the epitopes seen were present on a distinct molecule independent of secreted IL-2, the distribution on a variety of T cells and regulation via cellular activation suggest that the surface expression of IL-2 epitopes is in some way related to the soluble lymphokine.     

Distinct regulation of lymphokine production is found in fresh versus in vitro primed murine helper T cells

The kinetics of lymphokine RNA induction and secretion of biologically active lymphokine from CD4-enriched splenic T cell populations was investigated. Cells stimulated immediately after isolation from murine spleen ("fresh" T cells) and cells restimulated after 4 days of in vitro culture ("primed" T cells) were compared. Northern blot analysis and bioassays were used to analyze and quantitate production of eight lymphokines and the IL-2R. Fresh T cells produced high levels of IL-2 and low to moderate levels of IL-3, granulocyte/macrophage-CSF, and IFN-gamma. In vitro primed T cells produced IL-2, IL-3, IL-4, IL-5, IL-6, granulocyte/macrophage-CSF, IFN-gamma, and high levels of IL-2R RNA. Comparison of RNA levels and bioassays of supernatants from these populations indicated that primed T cells produced at least 10-fold more of six of the lymphokines than fresh T cells. Only IL-2 was produced in near equal amounts by fresh and primed T cells. There were also marked differences in the kinetics of lymphokine production by fresh and primed CD4+ T cells. After restimulation with Con A and PMA, primed cells produced a short burst of lymphokine RNA that peaked between 7.5 and 13 h and declined after 18 h. Fresh T cells lagged in the initial production of lymphokine RNA, with levels peaking 18 to 44 h after mitogenic stimulation. Depletion of CD4+ cells indicated that cells of helper phenotype were responsible for the majority of lymphokine production from the primed cells. Thus different subpopulations of Th cells defined by their respective ability to respond either directly (fresh T cells) or only after culture and restimulation (primed T cells) show different patterns of lymphokine gene regulation. Other studies suggest that the activity of "fresh" Th cells is due to a population with a "memory" phenotype, while the cells which require culture have a "precursor" phenotype. These distinct patterns of lymphokine gene regulation in the two populations of Th cells may account in part for differences seen in the kinetics and magnitude of the naive and memory immune responses which are regulated by Th cells.     

Lymphokine-mediated induction of antigen-presenting ability in thymic stromal cells

We have established and characterized long term thymic stromal cultures from BALB/c (H-2d) and CBA/J (H-2k) mice. All cultures contained multiple adherent cell types, whereas some also contained thymic macrophages (TM). Culture supernatants from all cultures tested contained macrophage colony-stimulating factor activity, whereas only cultures with TM had soluble or membrane-associated interleukin (IL)-1. However, a thymic epithelial cell line (3D . 1), cloned from one of these cultures, produced IL-1 bioactivity. Further analysis confirmed the production of IL-1 alpha mRNA by the epithelial cell. No IL-2 or IL-4 (formerly called B cell stimulatory factor 1) activity was detected in any of the cultures. Antigen-presenting (AP) ability was determined using the chicken ovalbumin (OVA)-specific, I-Ad-restricted T cell hybridoma 3DO-18.3. Harvested TM exhibited antigen-specific, Ia-restricted AP ability which was enhanced by IL-4 as well as interferon-gamma (IFN-gamma). In contrast, AP ability was detected in non-macrophage stromal cell cultures (NMSC) only after preincubation with IFN-gamma. AP by preinduced NMSC was also Ia-restricted and could be blocked by anti-I-Ad antibodies. Since the T cell receptor of 3DO-18.3 is known to recognize a peptide produced by CNBr degradation of OVA, these observations suggest that both TM and NMSC can process OVA to produce this peptide. Glutaraldehyde-fixation experiments confirmed that NMSC must process native OVA into antigenic peptides for successful AP. Assays using several cloned stromal cell lines of different lineages suggested that only epithelial cells could be induced with IFN-gamma to exhibit competent AP. Given the possible role for IFN-gamma in the maintenance of Ia in the thymus, we investigated whether IFN-gamma production could be ascribed to a subpopulation of thymocytes. Culture supernatants from calcium ionophore and phorbol ester-stimulated peanut agglutinin-negative, but not peanut agglutinin-positive, thymocytes induced AP ability in NMSC. Thus, some thymocytes can produce an Ia-inducing lymphokine (most likely IFN-gamma) which may play an important role in T cell ontogeny through its effects on both thymic macrophages and thymic epithelial cells.     

Phenotypic and functional characterization of T cell clones derived from the cerebrospinal fluid of multiple sclerosis patients

We describe here T cell cultures and clones established from the cerebrospinal fluid (CSF) of three patients with multiple sclerosis (MS) and one chronic meningitis patient with pleocytosis. Most of the cultures were activated with phytohemagglutinin (PHA) before growth in mitogen-free interleukin 2 (IL 2), and were never restimulated. Some of the clones obtained have been propagated for over 1 yr and are strictly IL 2-dependent. Immunofluorescence analysis performed with various monoclonal antibodies revealed that the CSF-derived lines had the characteristics of activated T cells with a stable expression of either suppressor/cytotoxic or helper/inducer surface antigens. Most of the clones established had a predominantly suppressor phenotype (OKT8+), except for the clones derived from one MS patient, which expressed only the helper phenotype (anti-Leu-3a+). Consistent with these data, the CSF-derived cultures displayed a variety of immunoregulatory functions, such as the ability to lyse nonspecific and PHA-stimulated target cells, to produce IL 2 upon mitogenic activation, and to modulate polyclonally induced Ig responses. The availability of long-term CSF T cell cultures derived from MS patients at various disease stages might provide a useful tool in investigating the factor(s) involved in the etio-pathogenesis of the disease.     

IL-4 directs the development of Th2-like helper effectors

Our studies show that the presence of IL-4 during the response of naive Th cells causes precursors to develop into a population comprised largely of "Th2-like" effectors that secrete IL-4 and IL-5, but little IL-2 or IFN-gamma. We find that the levels of IL-4 and IL-2 determine both the level of effectors developed in response to mitogen or Ag and the patterns of lymphokines they secrete when restimulated. IL-2 is required for optimum generation of effectors, and increasing levels of IL-2, augments the expansion of effectors secreting both IL-4/IL-5 and IFN-gamma. In contrast, IL-4 is required for the development of IL-4/IL-5 secreting effectors but suppresses the development of IL-2 and at higher doses IFN-gamma-secreting effectors detected after 4 days. Also dramatic are the effects of the presence or absence of IL-4 evaluated after an additional 1 to 2 wk. When cultures with or without initial IL-4 are cultured in IL-2 alone from days 4 to 11, they retain their distinct patterns of lymphokine production. Those cells that developed in cultures without IL-4 progressively secrete more IL-2 and can be maintained and expanded in IL-2. They continue to produce IFN-gamma, though the levels decrease somewhat with time, but they do not acquire the ability to produce IL-4 or IL-5. These cells thus increasingly resemble Th1 cell lines. In contrast, those cells in cultures initially exposed to IL-4, generate effectors which secrete high levels of IL-4/IL-5 (plus variable levels of IFN-gamma) at days 4 to 5, but the populations of cells developed, are not maintained well on IL-2 alone. Those cells that do survive continue to secrete IL-4 and IL-5 but not IL-2. In addition, IFN-gamma production, if present, falls off with time. Thus the cells in these cultures take on an increasingly Th2-like phenotype. It appears that the effects of low levels of IL-4 in suppressing IL-2 production by day 4 effectors appear to be transient whereas the higher levels appear to drive the development along a distinct pathway which is irreversible. These studies support the concept that different subsets of helper cells, which correspond roughly to Th1 and Th2 subsets, can develop rapidly in short term culture with respectively low vs high levels of IL-4. They support the concept that such distinct phenotypes arise from alternate pathways of differentiation that can be expected to reflect pathways available for helper T cell differentiation in the animal.     

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.     

Differentiation of NK-like cells from OKT3-, OKT11+, and OKM1+ small resting lymphocytes by culture with autologous T cell blasts and lymphokine

NK-like cells have been generated in vitro from a resting lymphocyte population of PBMC by 8 days culture with mitomycin C-treated autologous T cell blasts and lymphokine. The responder lymphocyte population was purified to the extent that it lacked classical NK cells, and lacked the precursors of MLC-derived NK-like cells and of lymphokine-activated killer cells. The NK-like cells were not generated when the responder lymphocytes were cultured with either T cell blasts or lymphokine alone. Thus, at least two signals are required for their activation. Metabolically inactive T cell blasts plus lymphokine were effective in stimulating the generation of NK-like cells, suggesting that a membrane determinant on the T cell blasts was involved in activation. The phenotype of the NK-like cells and their precursors was analyzed by monoclonal antibody and complement treatment. The phenotype of both precursor and effector cells was OKT3-, OKT11+, and OKM1+, with a distinct pattern of reactivity with OKT8 and Leu-7 for each individual donor tested. The NK-like cells were morphologically large granular lymphocytes, and they killed a variety of target cells. These studies show that signals provided by autologous T cell blasts and lymphokine are essential in triggering the differentiation of NK-like cells from appropriately purified resting lymphocytes. This mechanism of activation could occur in vivo, leading to the generation of NK cells subsequent to an antigen-specific T cell response.     

Relationships between antigen-specific helper and inducer suppressor T cell hybridomas

Ts1, or inducer suppressor T cells, share many phenotypic and functional characteristics with helper/inducer subset of T cells. In order to evaluate the relationship between these cell types, we made a series of new Ts1 hybridomas by the fusion of Ts1 cells with the functionally TCR alpha/beta-negative BW thymoma (BW 1100). Three Ts1 hybridomas (CKB-Ts1-38, CKB-Ts1-53, and CKB-Ts1-81) were established that express TCR and produce Ag-specific suppressor factors constitutively, thus making it possible to study the nature and specificity of Ag receptors, MHC restriction, and lymphokine production by the Ts1 hybridomas. Results presented in this report demonstrate that all the Ts1 hybridomas described here express CD3-associated TCR-alpha beta. These three Ts1 hybridomas recognize Ag (NP-KLH) specifically in a growth inhibition assay and this recognition is restricted by IE molecules. Two of the hybridomas also produce IL-2 or IL-2 and IL-4 upon Ag-specific activation. Thus, by these three criteria the Ts1 hybridomas appear indistinguishable from Th cells. These three Ts1 hybridomas, however, release suppressor factors (TsF1) in the supernatant that suppress both in vivo DTH and in vitro PFC responses in an Ag-specific manner. Like the TsF1 factors characterized previously, the suppression mediated by these factors are Igh restricted and lack H-2 restriction. These factors mediate suppression when given in the induction phase but not during the effector phase of the immune response. The TsF1 factors are absorbed by Ag (NP-BSA), and anti-TCR affinity columns and the suppressor activity can be recovered by elution. The data are consistent with the interpretation that Ts1 inducer-suppressor T cells are related to Th cells; the feature that distinguishes these cells is the ability to produce Ag-binding factors that specifically suppress immune responses.     

T cells in inductive and effector compartments of the intestinal mucosal immune system of nonhuman primates differ in lymphokine mRNA expression, lymphokine utilization, and regulatory function.

To define further the basis of T cell function in the inductive and effector limbs of the normal intestinal immune system, the capacity of mucosal lymphocytes to produce and use lymphokines and their effects on regulation of Ig production were determined in normal nonhuman primates. Northern blots of RNA from mitogen-activated lamina propria T cells contained more mRNA for IL-2 and IFN-gamma than did mesenteric lymph node T cells. In comparison with lymphocytes from peripheral sites, there was high expression of IL-4 and IL-5 mRNA in both mesenteric lymph node and lamina propria T cells. In studies of lymphokine utilization, T cells from lamina propria had high IL-2-induced but no IL-4-induced proliferative responses. In contrast, mesenteric lymph node T cells had high IL-4-induced and lower IL-2-induced proliferative responses compared with lamina propria T cells. Lamina propria T cells had higher helper activity in PWM-stimulated cultures and exhibited less inhibition by IL-4 than did mesenteric lymph node T cells. These data and previous studies suggest that T cells in an inductive site such as the mesenteric lymph node are a mixed population containing both "naive" cells with low potential for IFN-gamma and IL-2 production and differentiated cells with high potential for IL-4 and IL-5 production. In contrast, the data suggest that T cells in the effector compartment of the lamina propria are comprised primarily of differentiated "memory" cells that produce high levels of IL-2, IFN-gamma, IL-4, and IL-5, have high helper activity, and have a more limited ability to proliferate in response to lymphokines such as IL-4.     

Functional heterogeneity among human inducer T cell clones

Analysis of mouse CD4+ inducer T cells at the clonal level has established that a dichotomy among CD4+ T cell clones exists with regard to types of lymphokines secreted. Mouse T cell clones designated Th1 have been shown to secrete IL-2 and IFN-gamma, whereas T cell clones designated Th2 have been shown to produce IL-4 but not IL-2 or IFN-gamma. To determine if such a dichotomy in the helper inducer T cell subset occurred in man, we examined a panel of human CD4+ helper/inducer T cell clones for patterns of lymphokine secretion and for functional activity. We identified human T cell clones which secrete IL-4 but not IL-2 or IFN-gamma, and which appeared to correspond to murine Th2 clones. In marked contrast to murine IL-2 secreting Th1 clones which do not produce IL-4 or IFN-gamma, we observed that some human T cell clones secrete IL-2, and IFN-gamma as well as IL-4. Southern blot analysis indicated that these multi-lymphokine-secreting clones represented the progeny of a single T cell. IL-4 secretion did not always correlated with enhanced ability to induce Ig synthesis. Although one T cell clone which secreted IL-2, IL-4, and IFN-gamma could efficiently induce Ig synthesis, another expressed potent cytolytic and growth inhibitory activity for B cells, and was ineffective or inhibitory in inducing Ig synthesis. These results indicate that although the equivalent of murine Th2 type cells appears to be present in man, the simple division of T cells into a Th1 and Th2 dichotomy may not hold true for human T cells.     

Interleukin-2-activated murine cell lines with macrophage- and B-lymphoblast-lytic activity.

Interleukin-2 (IL-2)-activated murine killer cell lines with macrophage- and B-lymphoblastic-lytic activity were established, and their target specificity, surface markers, recognition-related structures, and requirements for optimal cell growth were characterized. Sustained growth of IL-2-activated lymphocytes was supported by the combination of IL-2 and IL-4-enriched T cell conditioned medium (CM), but was not supported by IL-2 alone or the combination of IL-2 and IL-3-containing CM in the presence of macrophages (M phi). The established line required continuous contact with M phi to maintain anti-M phi cytolytic activity. Flow cytometric analysis showed that the original line isolated by the first cloning was Thyl+, CD4-, and weakly CD8+, FcR+. The majority of these cells were CD3+ and TCR-V beta 8+. From this line, the CD3+, TCR-V beta 8+ and CD3-, TCR-V beta 8- clones were isolated by subcloning. The former clone showed Thyl+, CD3+, CD4-, CD8-, TCR-V beta 8+, FcR(+)-phenotype, and the latter clone showed Thyl+, CD3-, CD4-, CD8-, TCR-V beta 8-, FcR- phenotype. The original line and subclones showed a similar target specificity and killed resident or thioglycollate (TG)-induced peritoneal M phi and B-lymphoblasts, but did not kill T-lymphoblasts. Allogeneic M phi, M phi-like cell line P388D1, and B cell hybridoma were sensitive, whereas fresh lymphocytes, T cell lymphoma BW5147, natural killer (NK)-sensitive YAC-1, and NK-resistant P815 tumor cells were resistant to lysis by these cytotoxic lines. The addition of anti-H-2 heteroserum, anti-MHC class 1, anti-MHC class II, anti-CD3, or anti-TCR-V beta 8 monoclonal antibody (mAb) to assay cultures did not inhibit the anti-M phi cytolysis by these killer cells. In addition, the CD3- TCR-V beta 8- clone killed M phi and B lymphoblasts better than the CD3+, TCR-V beta 8+ clone. These results suggest that cytotoxic lines established in this study do not use the T cell receptor (TCR) molecules to recognize target cells and the MHC molecules are not involved in recognition. Anti-LFA-1 mAb partially inhibited anti-M phi-lysis, suggesting that the cell contact between targets and effectors is important in cytolysis. Our present data suggest that the culture condition containing IL-2, IL-4, and M phi may support the continuous growth of non-MHC-restricted killer cells with relative target specificity against M phi and B-lymphoblasts.     

Virus-induced interferon alpha/beta (IFN-alpha/beta) production by T cells and by Th1 and Th2 helper T cell clones: a study of the immunoregulatory actions of IFN-gamma versus IFN-alpha/beta on functions of different T cell populations

Spleen cells, resting T cells, activated T cells, and T cell clones characterized as type 1 (Th1) and type 2 (Th2) were investigated for their ability to produce interferon (IFN) following in vitro culture with Newcastle disease virus (NDV). All of the above cell populations, including both Th1 and Th2 T cell clones, produced high levels of IFN following in vitro culture with NDV. This IFN was characterized as a mixture of IFN-alpha and IFN-beta with IFN-alpha being the predominate species of IFN contained in the mixture. IL-2 greatly enhanced the production of IFN-alpha/beta by all cell populations in response to NDV. These different T cell populations responded very differently to the immunoregulatory actions of IFN-gamma versus IFN-alpha/beta. IFN-alpha/beta was shown to be a potent inhibitor of Con A or IL-2-induced proliferation of different T cell populations. This inhibition was not associated with a reduction in lymphokine production since spleen cells or Th1 T cell clones cultured with Con A and IFN-alpha/beta had no decrease in IL-2 or IFN-gamma production when compared to Con A-stimulated control cultures. IFN-gamma had little to no inhibitory activity on Con A-induced proliferation of spleen cells. In fact, Con A-induced proliferation was usually enhanced by IFN-gamma when nylon wool-enriched T cells were assessed. Different results were observed when IFN-gamma and IFN-alpha/beta were investigated for their ability to inhibit IL-2-induced proliferation of different T helper cell clones. IFN-gamma and IFN-alpha/beta were both capable of inhibiting IL-2-induced proliferation of T cell clones characterized as type 2 (Th2). In contrast, IFN-gamma had no effect on IL-2-induced proliferation of Th1 clones. IFN-alpha/beta, however, inhibited IL-2-induced proliferative responses of both Th1 and Th2 T cell clones. These results document the facts that (1) IFN-gamma and IFN-alpha/beta differ in their immunoregulatory actions, (2) different T cell subpopulations vary in their susceptibility to IFN-gamma regulation, and (3) virus induction of IFN-alpha/beta appears to be a ubiquitous function associated with different T cell populations.     

Interleukins 2 and 3 regulate the in vitro proliferation of two distinguishable populations of 20-alpha-hydroxysteroid dehydrogenase-positive cells

The ability of interleukin 2 (IL 2), interleukin 3 (IL 3), and granulocyte/macrophage colony-stimulating factor (GM-CSF) to induce the proliferation of cells from thymus, spleen, or bone marrow was examined and compared with their ability to induce expression of the enzyme 20-alpha-hydroxysteroid dehydrogenase (20 alpha SDH). In the thymus, the peanut agglutinin agglutinated cells (PNA+) lacked 20 alpha SDH and showed no detectable response to IL 2, IL 3, or GM-CSF in either proliferation or induction of 20 alpha SDH. In contrast, the PNA nonagglutinated (PNA-) subpopulation expressed 20 alpha SDH and proliferated in response to Con A and/or IL 2. The responding cells that could be expanded in vitro with IL 2 expressed high levels of 20 alpha SDH. Neither IL 3 nor GM-CSF in the presence or absence of Con A had a demonstrable effect on the PNA- population. In cultures of bone marrow cells, both IL 3 and GM-CSF induced proliferation, whereas IL 2 had no effect on proliferation in the presence or absence of Con A. Thy-1-depleted bone marrow cells, expanded in tissue culture with IL3, contained cells that co-expressed Thy-1 and 20 alpha SDH. In contrast, cells proliferating in vitro to GM-CSF did not expressed Thy-1 or 20 alpha SDH. In cultures of normal splenic lymphocytes, two populations of cells capable of expressing 20 alpha SDH were detected. One population could be expanded in vitro with IL 2 and Con A, whereas the second was responsive to IL 3. In spleens from athymic mice, only the latter cells were detected. These results demonstrate that IL 3 and IL 2 responsiveness distinguishes two populations of 20 alpha SDH cells. The relevance of these observations to the possible relationship of IL 3 and IL 2 in T cell differentiation is discussed.     

Murine T helper cell clones secrete granulocyte-macrophage colony-stimulating factor (GmCSF) by both interleukin-2-dependent and interleukin-2-independent pathways

Granulocyte-macrophage colony-stimulating factor (GmCSF) is a lymphokine secreted by class II major histocompatibility complex (MHC)-restricted T cells after lectin or antigen stimulation. To investigate the relationship between interleukin-2 (IL-2) and GmCSF production, we utilized long-term cultures of porcine myelin basic protein (PMBP)-specific T helper cell clones that were maintained with IL-2 in the absence of antigen or irradiated antigen-presenting cells (APC). We have found that supernatants of these T cell clones contained GmCSF activity after IL-2 stimulation. Inhibition of cell proliferation by irradiation failed to stop GmCSF production. When these clones were stimulated with PMBP and irradiated APC in the presence of anti-IL-2 receptor antibody, the T cell supernatants still contained GmCSF activity. These results indicate that (1) GmCSF production by T helper clones after IL-2 stimulation is independent of cell proliferation and (2) antigen/MHC-stimulated GmCSF production by T cell clones can occur by an IL-2-independent pathway.