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.     

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.     

Cell-mediated immune functions and immunoregulatory cells in Beh?et's syndrome.

Lymphocyte responsiveness to mitogens (phytohaemagglutinin (PHA), concanavalin A (Con A), and pokeweed mitogen (PWM)), T-cell subsets (T mu and T gamma) and short-lived suppressor cell activity were investigated in the peripheral blood of seven patients with Behçet's syndrome and compared to normal individuals and patients with systemic lupus erythematosus (SLE). Amongst patients with Behçet's syndrome, responses to mitogens PHA and Con A were normal or slightly reduced; numbers of circulating T gamma cells were unaltered whereas T mu cells were reduced (P less than 0 . 05) compared with normal individuals. This was in contrast to SLE where a marked reduction in responses to PHA and Con A was found with reduced T gamma cell but normal T mu cell numbers. Although the mean suppressor cell activity in the Behçet's group was significantly reduced, all patients had values within the normal range, in contrast with SLE where the reduction was much more marked and most patients had values below the normal range. In conclusion, the pattern of alteration in T mu and T gamma cells in Behçet's syndrome is distinct from that in SLE, and the reduction of short-lived suppressor cell activity is only mild in Behçet's syndrome but marked in SLE.     

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.     

Lymphocyte subpopulations and reactivity to mitogens in patients with scleroderma.

T lymphocyte subpopulations were studied in 40 patients with scleroderma (PSS), 26 of whom were studied simultaneously for lymphoproliferative responses to phytohaemagglutinin (PHA), concanavalin A (Con A) and pokeweed mitogen (PWM). PSS patients exhibited a reduction relative to 42 age- and sex-matched controls in the absolute number and percentage of early E rosettes, late E rosettes and E rosettes formed with aminoethylisothiouronium bromide (AET) treated sheep red blood cells. There was no difference between patients and controls in the proportions of B lymphocytes. PSS patients exhibited normal lymphocyte transformation responses to PHA and ConA and an augmented response to PWM. The mitogen responses did not correlate with the absolute number or percentage of lymphocytes or T and B lymphocyte subpopulations. No correlation was observed between any immunological variable studied and the extent of skin or organ involvement, disease duration or therapy.     

Alteration of lymphocyte subpopulations with cytomegalovirus infection in infancy.

The distribution of immunoglobulin bearing (Ig+), T, and null lymphocyte subpopulations and the lymphocyte response to phytohaemagglutinin (PHA) and pokeweed mitogen (PWM) were determined in three infants with cytomegalovirus (CMV) infection. These infants had significantly decreased percentages of T cells (13%, 29% and 40%) compared to age-matched controls (61 +/- 2%). Compensatory increases in the percentages of Ig+ and null cells occurred. Decreased lymphocyte reactivity to PHA and PWM occurred in two patients. Purified T cells from these patients had normal reactivity indicating a disproportion of T cells in the peripheral blood. These abnormalities may result from CMV infection of lymphocytes and could be responsible for prolonged CMV viruria.     

Inhibition of mitogen-induced human lymphocyte responsiveness by polymixin antibiotics

Polymixin antibiotics, polymixin B and polymixin E (colistin) inhibited the mitogen-induced lymphoproliferative response of human lymphocytes. Inhibition of the lymphocyte response to PHA, PWM and Con A was evident at a low concentration of 1 U/ml of antibiotics. Lymphocytes in which the signals for proliferation had occurred were similarly prevented from proliferating. The effects were not due to cell death (toxicity). Since polymixin concentrations at which inhibition of lymphocyte proliferation was observed are employed in tissue culture medium and are also attained in plasma of patients, the results suggest that the use of the antibiotics in lymphocyte cultures limits lymphocyte responsiveness and that patients receiving polymixin antibiotics may experience a state of immunosuppression.     

Ontogeny of surface markers on functionally distinct T cell subsets in the chicken

Three subsets of chicken peripheral T cells (T1, T2 and T3) have been identified in peripheral blood of adult chickens on the basis of fluorescence intensity after staining with certain xenogeneic anti-thymus cell sera (from turkeys and rabbits). They differentiate between 3-10 weeks of age in parallel with development of responsiveness to the mitogens concanavalin A (Con A), phytohemagglutinin (PHA) and pokeweed mitogen (PWM). Functional tests on the T subsets, sorted with a fluorescence-activated cell sorter, have shown that T2, 3 cells respond to Con A, PHA and PWM and are capable of eliciting a graft-vs.-host reaction (GvHR). In contrast, although T1 cells respond to Con A, they respond poorly to PHA and not at all to PWM or in GvHR. There was some indication of cooperation between T1 and T2,3 cells for the PHA response. Parallels between these chicken subsets and helper and suppressor/cytotoxic subsets in mammalian systems are discussed.     

Inhibition of antigen- and lectin-induced proliferation of rat spleen cells by a Taenia taeniaeformis proteinase inhibitor

Rat splenic lymphocytes, cultured in vitro for 3 days in the presence of a larval cestode proteinase inhibitor, exhibited a marked suppression of proliferation when stimulated with Con A, PHA, PWM and ovalbumin. Reduced responsiveness was observed over a full range of concentrations of Con A (16-fold), PHA (50-fold), PWM (four-fold) and ovalbumin (16-fold). These results indicated that the inhibitory action could not be overcome by increasing the mitogen or antigen doses beyond optimal levels. This suppressive effect disappeared when the Taenia taeniaeformis proteinase inhibitor was added 20 h after the initiation of culture, suggesting that the inhibitor affects lymphocyte blastogenesis during the early stages of lymphocyte activation.     

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.     

L-canavanine acts on suppressor-inducer T cells to regulate antibody synthesis: lymphocytes of systemic lupus erythematosus patients are specifically unresponsive to L-canavanine

L-Canavanine (LC) is an amino acid contained in alfalfa seeds that provokes a disease state similar to systemic lupus erythematosus (SLE) in primates. In vitro experiments showed that LC stimulated proliferation of human phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC) and T cells of healthy donors but not of pokeweed mitogen (PWM)-stimulated PBMC. LC inhibited spontaneous generation of immunoglobulin-secreting cells (ISC) of PBMC, while it enhanced ISC generation of CD8(-) cells. LC inhibited PWM-induced ISC generation of CD8(-) cells but not of CD4(-) cells, indicating that LC stimulates CD8(-) cells more strongly than CD4(-) cells. The stimulation index of lymphocyte proliferation (PHA + LC/PHA) was greater in CD8(-)Leu8(+) cells than CD8(-)Leu8(-) cells. The stimulation index was also higher in PBMC than in PBMC plus CD8(-)Leu8(-) cells, the former population containing relatively increased CD8(-)Leu8(+) cells. These findings suggest that LC acts mainly on CD8(-)Leu8(+) cells. That LC acts on CD8(-)Leu8(+) cells was confirmed by the finding that LC inhibited ISC generation of non-T plus CD4(+)Leu8(+), but not of non-T plus CD8(-)Leu8(-) cells. In addition, we found that PBMC of SLE patients were specifically unresponsive to LC stimulation. The stimulation index of lymphocyte proliferation (PHA + LC/PHA) in SLE patients (n = 16) was 0.97 +/- 0.19, whereas that in age-matched healthy control (n = 17) was 1.45 +/- 0.40 (P less than 0.001). Patients with active disease were especially unresponsive to LC. Its responsiveness did not correlate with the dose of prednisolone administered. These findings suggest that the lymphocyte response to LC depends primarily on the existence of functional CD8(-)Leu8(+) cells. Moreover, it appears that suppressor-inducer T cells, responsive to LC, are especially deficient in SLE.     

Regulation of immune functions by human surfactant

Human peripheral blood mononuclear cells (MNC) were incubated in vitro with highly purified human surfactant to examine its effect on various T cell functions. Surfactant inhibited DNA synthesis by lymphocytes in response to concanavalin A (Con A), phytohemagglutinin (PHA), and in the autologous mixed lymphocyte reaction (AMLR). In contrast, surfactant had no effect on pokeweed-mitogen (PWM, T cell-dependent B lymphocyte mitogen)-induced DNA synthesis or on interleukin-2 (IL-2) receptor expression on T cells activated with PHA, Con A or PWM. Furthermore, surfactant had either no effect or enhanced (depending upon the concentration of IL-2 used) the response of exogenous recombinant IL-2 on IL-2-dependent T cell line, In vitro addition of recombinant IL-2 corrected the suppressive effect of surfactant on the AMLR. These data show immunosuppressive effect of surfactant on T lymphocyte functions.     

Carrageenan-induced suppression of chicken lymphocyte proliferation

Mitogenic responsiveness of peripheral blood lymphocytes (PBL) of chickens was suppressed by either pretreatment with or addition to the culture medium of various concentrations of carrageenan (CGN). Pretreatment for 1 hr significantly suppressed response to Concanavalin A (Con A) and Pokeweed mitogen (PWM) but did not affect Phytohemagglutinin (PHA) induced stimulation. Extension of the pretreatment period to 4 hrs suppressed response induced by all three mitogens. On the other hand, addition of carrageenan to the culture medium caused a dose-dependent suppression of PHA and Con A mediated response, but the effect on stimulation due to PWM was equivocal. In addition, low concentrations of CGN were weakly mitogenic to PBL and splenic lymphocytes.     

Specificity of immediate skin reaction with streptokinase-streptodornase (SK-SD) in humans.

Five patients with no detectable serum IgA (less than 20 mug/ml) and one patient with low serum IgA were compared to normal subjects. The number of circulating E-RFC was normal as was the lymphocyte DNA synthesis induced by PHA, Con A, and streptokinase-streptodornase. The patients had normal numbers of IgA-bearing lymphocytes and normal or increased numbers of B cells. Purified anti-immunoglobulin antibodies specific for IgG, IgA and IgM induced a normal lymphocyte DNA synthesis as did PWM. The patients' lymphocytes were able in vitro to transform into actively secreting IgA plasmocytes. This transformation was determined by counting the IgA and immunoglobulin-containing cells and then measuring the IgA and IgG secretion in the cultures. In some patients PWM was selectively suppressive in IgA B-cell transformation into IgA secreting cells; in the other patients PWM had no effect on the IgA B-cell differentiation. PWM enhanced the IgG secretion in the patients' cultures as well as IgA and IgG secretion in the normal controls.     

Bovine con A-induced suppressor cells: generation, macrophage requirements and possible mechanisms of regulatory action.

Bovine Concanavalin A-induced suppressor cells were generated from lymphocytes which were non-adherent to anti-immunoglobulin coated dishes and cells possessing receptors for peanut agglutinin. Bovine lymphocytes, preincubated with 25 microgram/ml of Con A for 40-45 hr, could suppress the responses of autologous cells to the mitogens Con A, PHA and PWM as much as 90% when they were cultured together at a ratio of 1:1 (suppressor cell to responder cell) or higher. Suppressor cells were not necessary at the initiation of the mitogenic assay as they could regulate responding cells if added at 48 hr in a 72 hr assay. Allogeneic responder cells could be suppressed at the same level as autologous cells indicating a lack of genetic restriction. Macrophages were not required for suppressor cell generation because peripheral blood lymphocytes (PBL) depleted of macrophages by Sepharose G-10 columns, and subsequently incubated with Con A, could suppress autologous cells to a similar degree as unseparated PBl's. Responder cells depleted of macrophages had normal mitogen responsiveness and were suppressed indicating macrophages were not required in transmission of suppressor signals. Cell to cell contact was not required for suppression connoting a soluble factor(s) as the modulator of suppression.     

Purification of human T and B cells by a discontinuous density gradient of percoll.

A simple technique for isolating human peripheral B and T cells is described. This method is based on the different mobility of cells in a discontinuous density gradient of Percoll. A population with a mean of 72.8% of surface stable Ig (B cells) is found in the top layer (fraction I) whereas T cells with less than 3% of Ig are distributed in the two lower layers (fractions II and III). B cells obtained by this method were highly viable and reacted strongly with anti-DRw specific sera, whereas T cells did not respond. The clear-cut difference between positive and negative reactions makes this technique very suitable for DRw typing. The proliferative response to PHA is decreased in fractions I and III while greatly increased in fraction II. The different mitogenic responses in the two fractions containing T cells probably represent two functionally distinct T cell subsets. Cells from all fractions are less activated than unseparated cells when Con A and PWM are used as mitogen. The technique is very rapid, avoids the interaction of cells with their specific receptors and gives both T and B cells in one step, all advantages over other methods commonly used.     

Regulatory Influences on the Response of Rabbit T Cells to Concanavalin A and Phytohaemagglutinin

The proliferative response, induced in rabbit spleen cells by concanavalin A (Con A) and phytohaemagglutinin (PHA), is abolished when T cells are killed with antibody against rabbit thymus lymphocyte antigen (RTLA) in the presence of complement. The response was examined with purified spleen T cells, to which various helper cell fractions were added; it could be shown that B cells help the responding T cells. The helper effect in the response to PHA is abolished and the response to Con A is reduced by any manoeuvre which destroys or removes B cells. Help by B cells is given when helper cells have lost proliferative capacity as a consequence of mitomycin-C treatment. Spleen cells adhering to the walls of culture tubes help suspended T spleen cells in their response to Con A. This help could be abolished by complement mediated cell kill with antibody to rabbit bursal equivalent lymphocyte antigen (RABELA). On the other hand, the helper effect in Con A response was increased when T cells were removed. Thus the response of T cells to Con A is regulated by helper B cells and suppressor T cells.     

Effect of mitogens on the changes in intracellular cyclic AMP concentration in the lymphocytes of healthy persons and patients with lymphoblastic leukemia

In the lymphocytes of 20 healthy subjects and 17 patients with chronic lymphatic leukaemia (CLL) the cAMP level was determined before and at different time periods during cell incubation with mitogens (phytohaemagglutinin--PHA, concanavalin A--Con A, and pokeweed mitogen PWM). The experiments demonstrated that PHA-induced lymphocyte proliferation was associated in healthy subjects during the first 10--20 minutes of incubation with a rise in the intracellular cAMP concentration, while the level of cAMP decreased systematically during 30 minutes of incubation with Con A and PWM. Incubation of lymphocytes of the patients with CLL during 30 minutes with PHA, Con A and PWM caused no changes in the low cAMP level in these cells. The obtained results suggest that PHA activation of lymphocytes in healthy subjects is connected with changes in the intracellular concentration of cAMP.     

In Vitro studies on human B and T cell purified populations. Stimulation by mitogens and allogeneic cells, and quantitative binding of phytomitogens.

The property of T cells to form rosettes with sheep red blood cells has been used to separate peripheral blood lymphocytes into purified T- and B-cell suspensions after density gradient centrifugation. A study of lymphocyte markers has shown that 2-6 per cent of E rosettes only were recovered in the B cell-enriched population. Lymphocyte stimulation in vitro was obtained with PHA, con A and PWM in unseparated and T-cell populations, but never in B-cell suspensions. Experiments of recombination between the two purified fractions have demonstrated that 10% of T cells added to B cells were able to induce a response to PHA and PWM. Otherwise, only T cells responded to allogenic stimulation. Lastly, B and T cells seemed to bind qualitatively and quantitatively the same mitogens on their membranes.