Antigen and Unspecific Mitogen Stimulation of Lymphocytes Eluted from Rheumatoid Inflammatory Tissue

Lymphocytes were eluted from synovial tissues of 17 patients with classical rheumatoid arthritis, using a procedure previously reported. Stimulation was obtained wit h the unspecific mitogens phytohemaggalutinin (PHA), pokeweed mitogen (PWM), and concanavalin A (Con A) as well as with purified protein derivative of tuberculin (PPD) and mitomycin-C-treated allogeneic lymphocytes, whereas Candida antigen usually gave a low response. The pattern of reactivity o t unspecific mitogens was similar to that obtained with lymphocytes from peripheral blood of rheumatoid arthritis patients. Two different PPD preparations usually gave transformation of the same magnitude as seen with PHA. This was in contrast to the reactivity of the peripheral blood lymphocytes. It could be demonstrated that the elution procedure initiated some degree of lymphocyte transformation, mainly potentiating the responses to PHA and Con A.     

Lymphocyte transformation in human plasma without addition of synthetic medium. A study of immune function in patients with chronic uraemia or diabetes mellitus.

The in vitro response of lymphocytes obtained from normal subjects, uraemic patients on haemodialysis and diabetic patients was studied using cultures containing either medium plus plasma (medium cultures) or plasma alone (plasma cultures). The study demonstrated that plasma alone can adequately support lymphocyte transformation induced by nonspecific mitogens (PHA and PWM) and allogeneic lymphocytes in mixed lymphocyte culture (MLC) reaction. This investigation further confirms our previously reported findings that uraemic patients undergoing haemodialysis have a normal lymphocyte response in MLC and to PHA and PWM. Plasma cultures give results similar to conventional medium cultures in subjects where lymphocyte transformation is normal. The lymphocyte hyporactivity observed in diabetics is, however, better shown in the plasma cultures. The suppressed response of the diabetic patient's lymphocytes to PHA and PWM both in the presence of autologous and normal AB plasma suggests intrinsic lymphocyte dysfunction as the explanation for impaired immune function. Plasma cultures may provide a better in vitro system for the evaluation of immune function in certain groups of patients where it is desirable to distinguish between intrinsic abnormalities of lymphocyte function and the effect of humoral immunosuppressive factors.     

T-cell growth factor-mediated proliferation of lymphocytes from a T-chronic lymphocytic leukaemia patient lacking mitogen and alloantigen responsiveness

Peripheral blood lymphocytes from a patient with chronic lymphocytic leukaemia of T cell origin (T-CLL PBL), were found to lack PHA and Con A responsiveness and to have a very poor responder activity in mixed lymphocyte cultures. In spite of the presence of Ia-like antigens on 36% of the T-CLL PBL, negligible stimulator capacity in MLC was observed. Proliferation of the T-CLL PBL could be induced and maintained both by exogenous T cell growth factor (TCGF) containing PHA, and mitogen-free TCGF. In addition, restoration of the responder and stimulator capacity in MLC was obtained in the presence of mitogen-free TCGF. These results indicate that the lack of response to mitogens and alloantigens of the T-CLL PBL has to be attributed to the failure of these cells to produce TCGF upon activation.     

Lymphocyte stimulation and plasma inhibition in patients with malignant neoplasms. A comparative study with three mitogens.

The blastogenic response of lymphocytes from patients with malignant neoplasms was evaluated by stimulation with three phytomitogens (PHA, PWM, and Con A). The response of patient lymphocytes to all three mitogens was significantly lower than that of control lymphocytes, and most patients with abnormal PHA responses also responded abnormally to PWM and Con A. However, a few patients with normal PHA responses were abnormal to Con A, suggesting the suppression of a Con A-sensitive population. The observation that PWM responses were abnormal in patients with lowered PHA lymphocyte stimulation indicates that both T and B lymphocyte mitogen responses were suppressed in these patients. Plasma from patients was capable of either inhibiting or enhancing lymphocyte mitogen stimulation. However, inhibitory plasmas were generally from patients with abnormal mitogen responses.     

Suppression of human lymphocyte responses by Trypanosoma cruzi.

Virtually nothing is known about the basis for the immunosuppression associated with human T. cruzi infection. We have used an in vitro system to explore this effect. Incubation of human peripheral blood mononuclear cells (PBMC) with blood forms of T. cruzi abrogated their responses to suboptimal, optimal and supraoptimal doses of Con A, PHA or PWM, whether or not monocytes were depleted. Killed parasites were not suppressive. Maximal suppression (74%) occurred when the parasites were present during the entire culture period (96 hr), although significant suppression (33%) was seen when the organisms were added 24, 48 or 72 hr after initiation, suggesting that the early stages of lymphocyte activation had been impaired and that a second generation of cells was also affected. The 4-day supernatant medium of a T. cruzi suspension supported PBMC responses to Con A as well as medium incubated alone, indicating that suppression did not result from parasite removal of essential nutrients. Furthermore, 96 hr after mitogenic stimulation, the proportions of viable PBMC in cultures containing or lacking the parasites were comparable. Although T. cruzi binds Con A and PHA, this absorption was not the cause of reduced responsiveness since optimal concentrations of Con A and PHA remained in solution under our conditions. Levels of IL-2 in PHA-stimulated PBMC cultures were markedly reduced in the presence of T. cruzi. However, exogenous IL-2 failed to restore lymphocyte responsiveness. T. cruzi neither absorbed nor inactivated IL-2. Thus, the noted suppression appeared to involve at least deficient production and utilization of IL-2.     

Immunoregulation in experimental filariasis. IV. Induction of non-specific suppression following in vitro exposure of spleen cells from infected jirds to Brugia pahangi antigen.

Studies with inbred jirds chronically infected (greater than 5 months) with Brugia pahangi have demonstrated splenic suppressor cells which modulate in vitro responsiveness to mitogens and parasite antigens. The stimuli which induce suppression were characterized by analysing the effect of activated cells from inbred normal or B. pahangi-infected jirds on the PHA and PWM responsiveness of cultures on normal cells. Regulatory cells were stimulated in vitro with concanavalin A (Con A; 5 micrograms/ml) or an extract of adult B. pahangi (20 micrograms/ml) for 72 hr and irradiated (1500 rads) prior to cocultivation with normal cells. Addition of Con A-activated normal spleen cells to normal cells produced moderate suppression of PHA and enhancement of PWM responsiveness. However, Con A-stimulated spleen cells from infected animals consistently suppressed both the PHA and PWM responsiveness of normal cells by 80-90%. Spleen cells from chronically infected jirds were also induced by B. pahangi antigen to suppress both the PHA and PWM responsiveness of normal lymphocytes. In contrast, spleen cells from animals 3-15 weeks after infection and lymph node cells from all time points were capable of suppressing only PWM responses when stimulated by antigen. Normal spleen cells were not induced by B. pahangi antigens to exhibit immunoregulatory activity. The suppression mediated by antigen-induced spleen cells from chronically infected jirds was partially or totally alleviated by removal of non-specific suppressor cells which are plastic adherent and cyclophosphamide-sensitive, or by removal of antigen-specific suppressor cells which bear receptors for histamine. the results suggest the involvement of regulatory cell circuits in experimental filarial infections.     

Poliovirus-induced suppression of lymphocyte stimulation: a macrophage-mediated effect.

Poliovirus was shown to suppress the in vitro response of human mononuclear blood cells stimulated with phytohaemagglutinin (PHA), pokeweed mitogen (PWM), tuberculin purified protein derivative (PPD) or allogeneic cells. The suppression required infectious virus and the presence of macrophages. Experiments with combined cultures of human lymphocytes stimulated with PHA, PWM, PPD or allogeneic cells, and different numbers of autologous macrophages indicated that both lymphocyte stimulation and the inhibitory effect of poliovirus increased with increasing ratio of macrophages to lymphocytes. The response of human lymphocytes to PHA also was enhanced when autologous macrophages were replaced with murine macrophages. Mouse hepatitis virus inhibited this enhancing effect whereas poliovirus failed to do so. The findings confirm our previous suggestion that poliovirus inhibits stimulation of lymphocytes by suppressing the enhancing effect of macrophages.     

Altered regulation of mitogen responsiveness by suppressor cells in multiple sclerosis.

Suppression of the lymphocyte response to concanavalin A (Con A), phytohaemagglutinin (PHA) and protein A from Staphylococcus aureus (SpA) by Con A-induced suppressor cells was measured in twenty-four patients with recently active-recovering multiple sclerosis (MS), twelve with inactive MS and twenty-three healthy controls. Patients with recently active disease displayed significantly greater suppression of the response to Con A. Suppression of the responses to PHA and SpA did not differ among the groups. Lymphocyte stimulation in cultures not showing suppression was similar in all three types of subjects. These results suggest a disturbance of lymphocyte regulation in patients with recently active-recovering MS and illustrate the potential usefulness of measuring the suppression of responsiveness to several mitogens.     

Mitogen-induced lymphocyte-mediated cytotoxicity in vitro: effect of mitogens selectively activating T or B cells

Concanavalin A (Con A) and phytohemagglutinin (PHA) are selective T cell mitogens, whereas lipopolysaccharide (LPS) and purified protein derivative of tuberculin (PPD) are selective B cell mitogens. Con A and PHA induced mouse lymphocyte-mediated cytotoxicity in vitro, which was equally expressed on autologous, allogeneic and heterologous target fibroblasts, as measured by thymidine release from prelabeled targets. The dose response curves of Con A-induced lymphocyte-mediated cytotoxicity and DNA synthesis were parallel, 5 μg/ml being optimal, both higher and lower concentrations being less effective. Con A pretreated mouse lymphocytes failed to express cytotoxicity even though they were irreversibly activated with regard to DNA synthesis and were morphologically transformed. However, when Con A pretreated lymphocytes were added to target fibroblasts in the presence of soluble Con A, the cytotoxic effect markedly exceeded that induced by Con A in non-pretreated lymphocytes. It is suggested that cytotoxicity not only requires close contact between target and aggressor lymphocyte, but actual binding of the lymphocyte to the target, in this case effectuated by Con A. Con A and PHA failed to induce lymphocyte-mediated cytotoxicity in B lymphocytes. LPS and PPD did not induce cytotoxicity in normal or in B lymphccytes. Even LPS or PPD pretreated B lymphocytes failed to exert cytotoxicity when aggregated to the targets by Con A. It is suggested that the lymphocytes responsive to LPS and PPD are not the non-T cells responsible for cell-mediated cytotoxicity on antibody-coated target cells. Con A and PHA, but not LPS and PPD, induced lymphocyte-mediated cytotoxicity in human lymphocytes on allogeneic and heterologous target fibroblasts. The dose response curves for induction of cytotoxicity and DNA synthesis by Con A were not always identical in this case, cytotoxicity persisting at low Con A concentrations, which failed to activate DNA synthesis.     

An in vitro study of immunomodulatory effects of some saponins

The in vitro immunomodulatory activities of a number of saponins (crude Quillaja saponin, Quillayanin, Quil-A and glycyrrhizic acid) are described. Addition of these saponin preparations to mouse spleen cell cultures resulted in significant cell proliferation. B-cells were induced to proliferate in the presence of the crude saponin, and T-cells in the presence of Quil-A. On the other hand, Quillayanin and glycyrrhizic acid stimulated both T- and B-lymphocytes equally. The selective proliferation of subtypes of lymphocytes correlated with restimulation responses by polyclonal mitogens. Pretreatment by lymphocytes with crude saponins induced significant T-cell responses to PHA and Con A, and to T-independent B-cell stimulation by LPS. Pulse exposure of spleen cells to Quil-A resulted in enhanced cell proliferation when restimulated with PHA, Con A and PWM. In comparison, similar exposure of lymphocytes to Quillayanin or glycyrrhizic acid produced markedly increased responses to PHA, Con A, PWM and LPS. Incubation of lymphocytes in the presence of Quillaja saponins and Quillayanin caused effector cell generation as determined in a one-way mixed lymphocyte reaction. In the case of lymphocytes cultured in the presence of crude saponins or glycyrrhizic acid, the supernatants contained active soluble factors. This was demonstrated by the observation that the addition of supernatants to spleen cell cultures induced spontaneous cell proliferation, and also amplified their responses to a suboptimal dose of PHA. The experimental data suggest that different components in the Quillaja saponin preparations may have selective effects on various subtypes of cell populations. Glycyrrhizic acid has the most profound immunomodulatory activity in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human peripheral Null lymphocytes I. Isolation,immunological and functional characterization

A Null lymphocyte-enriched population was isolated from human peripheral blood of healthy donors using a combination of Ficoll density gradient centrifugation followed by elimination of mononuclear phagocytes, passage through Ig-anti-Ig columns and sedimentation of E rosettes. After each separation step lymphocyte fractions were examined for morphology, cell surface markers, mitogen responsiveness and effector functions in antibody-dependent (ADCC) and spontaneous cellular cytotoxicity (SCMC) reactions against an allogeneic melanoma cell line. The final Null lymphocyte preparation was recovered at a rate of 1 to 3 % from the population passed through Ig-anti-Ig columns (fraction FFF-C). The marker analysis revealed over 99 %of surface Ig-negative lymphoid cells; 50 to 60 % of these cells were ‘real Null’ cells lacking immunological cell surface markers, 7 % formed EA, 13 % EAC and 24 %E rosettes. Regarding the mitogen responses, passage through Ig-anti-Ig columns drastically reduced concanavalin A (Con A), pokeweed mitogen (PWM)and tuberculin (PPD) responses, whereas the phytohemagglutinin (PHA) response was reduced in absolute counts but not in the stimulation index. Compared to the T cell-enriched lymphocyte fraction, the Null cells showed significantly diminished proliferative responses to PHA and Con A and slightly increased reactivity to PWM and PPD. Although depleted of high affinity Fc receptor lymphocytes, the Null cell fractions exhibited good ADCC and SCMC activities being about 4 to 6 times higher than in the T cell fraction.     

Impaired mitogen-induced lymphocyte responses and the hypothalamic-pituitary-adrenal axis in depressive disorders

The lymphocyte stimulation responses to the mitogens phytohemagglutinin (PHA), concanavalin A (Con A) and pokeweed (PWM) were investigated in 30 hospitalized depressed women undergoing a dexamethasone suppression test (DST). Patients were classified according to DSM-III as having major depression with melancholia, without melancholia, and minor depression. The Hamilton Depression Rating Scale (HDRS) and the State-Trait Anxiety Inventory (STAI) were measured. Patients with major depression showed significantly decreased lymphocyte stimulation induced by PHA, Con A, and PWM as compared to those with minor depression. These differences could not be attributed to age, body weight, weight loss, total number of leukocytes, menopausal status, sleep disturbances, concomitant use of low-dosage benzodiazepines or length of drug-free period before testing. The group mean differences in lymphocyte stimulation counts were not affected by the severity of illness or the severity of state and trait anxiety. There were no significant differences in the lymphocyte responses to PHA, Con A, and PWM between DST non-suppressors and DST suppressors. No significant correlations were established between baseline and post dexamethasone cortisol values and the lymphocyte stimulation counts.     

Human spleen as a source of T cell growth factor.

Human spleen cells were tested for the ability to produce T cell growth factor (TCGF) upon stimulation with PHA. Quantitative analysis of the amounts of TCGF produced under optimal conditions indicated that supernatants obtained from spleen cell cultures were approximately five times more active than those derived from peripheral blood leucocytes (PBL). Moreover, in contrast to PBL, there was no significant difference in TCGF production between individual spleen cell populations. Among splenic T cells, TG-depleted cell fractions were superior to TG-enriched cell fractions in producing TCGF upon PHA stimulation. These supernatants induced intense proliferation of blast cell populations isolated from mixed leucocyte-tumour cell cultures (MLTC) established with PBL and irradiated allogeneic myelogenous leukaemic cells. Within 7 days of culture in TCGF, the number of MLTC blast cells increased approximately 300-fold. Concomitantly, the lytic activity (on a per-cell basis) of these populations against the corresponding myelogenous leukaemic cell targets increased approximately 80-fold.     

5-bromodeoxyuridine-light inactivation of human lymphocytes stimulated mitogens and allogeneic cells: evidence for distinct T-lymphocyte subsets.

The study of human peripheral blood currently permits enumeration of circulating B and T lymphocytes as well as the analysis of functional responses in vitro following stimulation with mitogens, antigens or allogeneic cells. In the present experiments, subsets of these major lymphocyte populations were analyzed by dissecting in vitro responses using an ablative technique. After an initial culture period of lymphocytes with a mitogen, the proliferating cells were inactivated with 5-bromodeoxyuridine and light, then the capacity of the remaining lymphocytes to respond to the same or a different mitogenic influence was tested. Responsiveness to a different stimulant in the presence of no further response to the first stimulant was taken as evidence for a different responding cell population. A large subset of peripheral blood lymphocytes was responsive to both phytohemagglutinin (PHA) and concanavalin A (Con A); ablation of the cells responsive to one left little or no cells responsive to the other. Pokeweed mitogen (PWM) stimulated a portion of the PHA-Con A-responsive subset and an approximately equal subset unresponsive to PHA or Con A. Other evidence indicates that with each of these mitogens (especially with PHA and Con A in a soluble form), most of the proliferative response of peripheral blood B lymphocytes is indirectly triggered and is dependent on T cell stimulation. The population of PHA-Con A-responsive cells is, therefore, interpreted to represent a major T cell subset plus recruited cells; the PWM-responsive population would include a T cell subset having also PHA and Con A responsiveness, and another subset of T (or perhaps B) cells. The mitogen-sensitive population showed no overlap with cells responsive to allogeneic stimulation in mixed leukocyte culture. Ablation of the mitogen-responsive cells potentiated the mixed leukocyte reaction, suggesting that a suppressive influence was removed with the inactivation of the mitogen-responsive cells. It appears, therefore, that distinct subsets of T lymphocytes differentially responsive to PHA-Con A, to PWM and to allogeneic stimulation are present in the human peripheral blood.     

Effects of Thymosin and Evidence of Monocyte Suppression of Both T- and B-Cell Functions in Two Cases of 'Common Variable Immunodeficiency'

We have studied two patients with common variable immunodeficiency (CVID) impaired cell-mediated immunity. and high percentages of monocytes in their peripheral blood. Removal of monocytes from cultures of peripheral blood mononuclear cells from both patients increased the in vilro responses to phytohaemagglutinin (PHA) and concanavalin A (Con A) but not to purified protein derivative (PPD), as measured by [³H]thymidine uptake. Similarly, supernatants of monocyte cultures from both patients. unlike supernatants of normal monocytes, suppressed the in vitro responses to PHA and Con A but enhanced the response to PPD by cultured mononuclear cells from the patients and from normal donors. Addition of unfractionaicd mononuclear cells from both patients 10 normal mononuclear cells suppressed both pokeweed mitogen (PWM) stimulation and IgG production: this effect was abrogated by removal of monocytes from the patients' mono-nuclear cell populations. The effect of thymosin on both patients' mononuclear cells was assayed in vitro. Thymosin was ineffective in vitro with cells from the first patient: for the other patient. [³H]thymidine uptake by mononuclear cells stimulated with PPD increased. whereas uptake by Con A-stimulated cells decreased, as did the percentage of E rosette-forming cells, providing further evidence of heterogeneity of the CVID syndrome. The effects of thymosin were also dependent on monocytes.     

Activation of lymphocyte subpopulations in patients with chronic lymphocytic leukemia.

T lymphocytes from the peripheral blood of normal donors and patients with CLL were isolated by nylon wool filtration or E-rosette separation and tested for functional activities. Unseparated CLL lymphocytes showed a poor response to phytomitogens and to allogeneic cells. Subpopulations enriched in E-RFC showed an increased PHA- and a normal MLC-reactivity; the Con A responses, however, were markedly reduced in all experiments. Subpopulations which were T cell depleted showed no reactivity to all mitogens. Purified T cells from normal donors showed a decreased reactivity to PHA, Con A and PWM. The Con A responses were completely abolished in almost all experiments. These diminished responses could be restored by unseparated cells or by small numbers of syngeneic, mitomycin-treated monocytes. The data suggest that mitogen-induced T lymphocyte stimulation in vitro in general depends on the presence of monocytes.     

The use of supernatants from neuraminidase and galactose oxidase (NAGO)-treated lymphocytes as a source of T cell growth factor

Human peripheral blood or tonsil lymphocytes produce T cell growth factor (TCGF), when activated with neuraminidase (NA) and galactose oxidase (GO). Partial purification of NAGO-TCGF on Sepharose G-100 columns gave a TCGF-active fraction within the same molecular weight range as the conventional lectin-induced TCGF (approximately 15,000 Da). Human T cells, activated in mixed lymphocyte culture (MLC) with irradiated allogeneic EB-virus transformed B-cells (LCL) could be maintained in continuous culture for several months with retained functional activities. The cells showed similar growth patterns when cultured in the presence of either NAGO-TCGF or PHA-TCGF. The growing cells were characterized by means of monoclonal antibodies. After 4 weeks of culture 98% of these were OKT3+ and 87% were also OKT8+. The cytolytic activities of the cultures were tested in cell-mediated lympholysis (CML) against allogeneic LCL as target cells, in natural cytotoxicity (NK) against K562 cells and in antibody dependent cytotoxicity (ADCC) against bovine erythrocytes. Cultures displaying one or several of these functions were obtained. The results indicate, that TCGF obtained from supernatants of NAGO-activated lymphocytes is as potent as the T cell growth promoting factor obtained by lectin stimulation. One major advantage of using NAGO-generated TCGF is that contamination with lectin is avoided.     

Defective gamma-interferon production in peripheral blood leukocytes of patients with acute tuberculosis

Production of interferon (IFN)-gamma by peripheral blood leukocytes (PBL) was examined in cultures of unseparated fresh whole blood exposed to phytohemagglutinin (PHA), concanavalin A (Con A), or pokeweed mitogen (PWM). The yield of IFN-gamma was measured by a newly developed immunoradiometric assay. Nine of 14 patients with acute pulmonary tuberculosis (TB) showed a depressed IFN-gamma response to Con A and/or PWM. Only four of these TB patients also showed a depressed IFN-gamma response to PHA. Stimulation of the patients' PBL cultures with PHA in the presence of exogenous interleukin 2 (IL 2) produced normal IFN-gamma yields in all but the most severely depressed patients. PBL cultures of TB patients with defective IFN-gamma production in response to mitogenic lectins also produced less IFN-gamma after stimulation with tuberculin PPD. Although some patients showed a moderate degree of lymphopenia, their OKT4/T8 lymphocyte ratios were mostly normal or close to normal, with the notable exception of one TB patient who has been diagnosed to have the acquired immune deficiency syndrome (AIDS).     

Functional properties of continuously cultured human T lymphocytes.

Human T lymphocytes (CCTC) have been maintained in continuous culture by a new method. The ability of CCTC to produce three types of T cell response has been determined and compared to the published responses of T cells grown in 'T cell growth factor' (TCGF) medium. Unlike TCGF cells, CCTC will not give proliferative responses when stimulated with PHA, Con A, or allogeneic lymphocytes even when supplemented with autologous PBL as a source of accessory cells. Instead, CCTC are potent inhibitors of the proliferative response of normal cells in MLC. This suppressive activity is not specific for histocompatible cells. Finally, CCTC express cytotoxic activity to a number of human lymphoid cell lines and this activity appears to be different from the polyclonal cytotoxicity reported for TCGF-maintained cells. Our method of long-term T cell culture therefore appears to grow cells with different properties from TCGF cells.     

Peripheral Blood Mononuclear Cell Sonicates as an Alternative to Irradiated Allogeneic Cells to Stimulate a Mixed Lymphocyte Reaction and to Enumerate CD69 Alloreactive T Cells

ABSTRACT: An alloreactive reaction similar to that occurring during GvHD can be generated in a mixed lymphocyte culture. The presence of both stimulator and responder cells in these cultures makes the identification and enumeration of alloreactive cells difficult and unreliable. We describe the use of PBMC sonicates as an alternative to the standard MLC method to stimulate an allogeneic reaction. Using combinations of autologous or allogeneic PBMC sonicates, we showed that the lymphocyte proliferative response to cell sonicates was comparable to the response using irradiated cells. The proliferative response was concentration dependent and reached maximum levels at day 6. Both irradiated cells and PBMC sonicates induced significantly lower responses when the stimulating cells were partially HLA-DR matched rather than completely mismatched. Alloreactive T cells stimulated with sonicates were enumerated by the flow cytometric detection of CD69 or CD25. In HLA-mismatched cultures, approximately 7% of CD3+ T cells were CD69+ or CD25+, suggesting alloreactivity. Although there was a significant correlation between the expression of these activation markers and lymphocyte proliferative responses, significant individual variations in the results of these two assays were observed. The results in this study demonstrate the potential of using PBMC sonicates instead of irradiated lymphocytes for the study and identification of alloreactive cells at the cellular and molecular level.