Behaviour of human immunoregulatory cells in culture. I. Variables requiring consideration for clinical studies.

The suppressor function of lymphocytes stimulated with concanavalin A (Con A) provides a potential method for examining disorders of immunoregulation. Clinical application, however, requires definition of the culture conditions that influence the expression of normal suppressor cell activity. In the present studies culture conditions were modified until a sensitive assay for non-specific suppressor cell function was reproducible utilizing the response to varying doses of phytohaemagglutinin (PHA) as an indicator system. Practical conclusions included (1) that sensitivity was not lost if the suppressor cells and responder cells were allogenic; (2) that fresh responder cells were as sensitive as precultured responder cells; (3) that a wide range of Con A concentrations could induce suppressor activity; and (4) that the sensitivity of the assay was much enhanced by using suboptimal mitogen doses of PHA. Twelve percent of normal subjects gave false negative results but these could be avoided by studying cells at more than one time point after stimulation with Con A. Cells resting in culture for 7 days could be induced to suppress after stimulation with Con A and these suppressor cells were very sensitive to pharmacological doses of dexamethasone. Studies utilizing different times of cell pre-incubation before Con A stimulation and different periods of exposure to Con A revealed fluctuation in the induction of suppression that may represent alternating periods of suppression and amplifying activity among stimulated cells in vitro. Such variations will need to be taken into account in the application of this type of assay to clinical studies seeking disordered immunoregulation.     

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.     

The regulation of T cell responses by spontaneously active suppressor cells.

In studying T cell regulation, peripheral blood mononuclear cells from normal subjects were examined for 'spontaneous', rather than mitogen-induced, suppressor cell activity. Normal blood leucocytes from 30 subjects included a subpopulation of cells capable of suppressing the response of lymphocytes to the T cell mitogen phytohaemagglutinin by 21-35%. The indicator system for these studies consisted of fresh normal lymphocytes stimulated by three concentrations of PHA in the presence or absence of normal but mitomycin C treated peripheral blood lymphocytes. To measure accurately the spontaneous suppressor cell activity, additional cultures were needed to control for the suppressive effects of crowding and metabolic competition. Allogeneic, cryopreserved 'B' cell enriched populations, supplied satisfactory control cells for this purpose. While allogeneic culture systems could induce significant suppressor cell activity after 7 days of co-culture, they could not induce this activity in the 3 days required to assay spontaneous suppressor cell effects. In developing this assay we noted that (a) crowding became a factor in the cellular response to mitogens with concentrations higher than 2 X 10(4) cells/well, (b) spontaneous suppressor cell activity decreased rapidly once cells were placed in culture and (c) both spontaneous and concanavalin A (Con A) activated suppressor cells could significantly reduce the response to PHA even when added to cultures established with mitogens 72 hr earlier. The ability to measure spontaneous suppressor cell activity in vitro will allow more physiological studies of the membrane markers and functional characteristics of these cells than is possible in conventional studies utilizing Con A. In addition, this assay allows the detection of enhanced in vivo activity of suppressor cells not easily detected in assays relying on mitogen induction of suppression. Such increased activity is thought to be an important factor in the pathogenesis of a number of human diseases.     

A suppressor cell in the response of rabbit T cells to Con A.

The response to Concanavalin A is regulated by a helper cell, described elsewhere (4), and by a suppressor cell, described in this paper. This suppressor cell is an adherent T cell with Fc receptors. Evidence for properties of the suppressor cell was obtained by two types of experiments: 1) regulatory cells gave more help if Fc-bearing subpopulations were removed from them, i.e. the suppressor cell could not be removed with Degalan beads, coated with anti-allotype antibody, but not with beads, coated with F(ab')2 fragments of the antibody; 2) help for T cells, in their response to Con A, was augmented when T cells were eliminated from the regulating adherent cell preparation. Thus, the response of spleen T cells to Con A is regulated by two adherent cells, a B helper cell and a T suppressor cell. We have previously shown that the response to phytohemagglutinin (PHA) is regulated by an adherent helper cell (4). We have found no evidence, in the present study for an adherent suppressor cell which participates in the T cell response to PHA.     

Nonspecific suppressor cell activity and specific cellular unresponsiveness in rat schistosomiasis.

Spleen cells from inbred Fischer rats infected with Schistosoma mansoni were cultured with S. mansoni antigen, phytohemagglutinin (PHA) or concanavalin A (Con A). During the first 3 weeks of infection, both a significant proliferative response and a delayed cutaneous hypersensitivity to S. mansoni antigen were observed. In contrast, during the fourth to the seventh week both responses were reduced to a nonsignificant level, and the proliferative response to PHA or Con A also decreased during this period. At the time of minimal reactivity to PHA or Con A, the depletion of cells adherent to nylon wool (but not depletion of mononuclear phagocytic cells by plating on plastic) restored a significant response to Con A. At this time, infected rat lymphocytes reduced the proliferative response of noninfected syngeneic cells induced by Con A. This inhibition was not observed when infected cells were previously passed through a nylon wool column. Inversely, nylon wool depletion of day-28 or 35-infected spleen cells did not restore lymphocyte response to S. mansoni antigen. Serum (or the dialyzable fraction thereof) from day-29-infected rats inhibited thymidine incorporation of PHA-stimulated normal lymphocytes and the response to S. mansoni of spleen cells from infected rats. This information suggests that at the end of the first month of infection in the rat, lymphocyte unresponsiveness to S. mansoni antigen could not be related to a demonstrable suppressor cell activity. Low molecular weight inhibitory factor(s) released by the parasite and acting directly on lymphocytes could partly account for this unresponsiveness in rat schistosomiasis.     

Studies on the immunobiology of rnu/rnu "nude" rats with congenital aplasia of the thymus

A detailed study of the recently described “nude” rat, which suffers from congenital aplasia of the thymus, has shown that this animal is grossly deficient in functional mature T cells. Histological investigation revealed that those areas of lymph node, spleen and Peyer's patches, which are considered to be thymus-dependent, were markedly depleted of lymphocytes. The areas of lymph node and Peyer's patches, which are normally considered to be thymus-independent, were also partially depleted of lymphocytes, whereas the thymus-independent areas of splenic white pulp showed greater activity than in control animals. Indirect immunofluorescence with a monoclonal anti-T cell antibody showed that lymph node and spleen cells were severely depleted of mature T cells, whereas staining with anti-immunoglobulin antisera revealed an increased percentage of B cells in these organs. The nature of the residual anti-T⁺ cells in rnu/rnu lymph node and spleen is not clear, but at least some of these may be precursor T cells. Spleen cells from rnu/rnu rats gave no response to phytohemagglutinin (PHA), and only occasional small responses to concanavalin A (Con A), but showed normal responses to Staphylococcus aureus bacteria. The absence of responses to PHA and Con A was not caused by suppressor cells nor by any defect in helper cell activity. In so far as PHA and Con A are considered to be T cell-dependent mitogens and S. aureus a T cell-independent mitogen, these results indicate that rnu/rnu rats lack functional T cells.     

Differential in vitro effects of etretinate and retinoic acid on the PHA and con A induced lymphocyte transformation, suppressor cell induction and leukocyte migration inhibitory factor (LMIF) production

The in vitro influence of two retinoids, etretinate and retinoic acid, on the human lymphocyte transformation, on the induction of suppressor cells and on leukocyte migration inhibitory factor (LMIF) production were investigated. Nontoxic concentration of retinoic acid increased significantly the PHA response to suboptimal mitogen concentrations, but had no effect on the Con A response; it also abolished the PHA induced LMIF production. In corresponding assays etretinate was without effect. Etretinate augmented the PHA induced suppressor cell activity, while retinoic acid was ineffective. No effect was observed on Con A induced suppressor cells by either retinoid. The findings extend the information about the immunomodulatory effects of retinoids and demonstrate that retinoic acid and etretinate have different effects on PHA and Con A induced immune responses. The mode of action of retinoids is discussed.     

Non-specific suppressor cells in murine bone marrow chimeras: their possible role in GVHD-associated immunodeficiency

Suppressor cells against several mitogen-induced responses were detected in the spleen of murine bone marrow chimeras, regardless of intravenous (i.v.) or intrasplenic (i.s.) bone marrow transplantation (BMT). According to the time-course of the suppressor activity against Con A, PHA, and PWM, they were readily detected at 11-21 days after BMT and thereafter, either gradually decreased or remained at a plateau level. In contrast, the suppressor activity against the LPS-stimulated response increased at 39-52 days as compared to 24-34 days after BMT. Characterization studies of suppressor cells early (11-21 days) after BMT revealed that those in the i.v. and i.s. chimeras were composed of host-derived plastic dish adherent and/or anti-Thy 1.2 antibody-insensitive spleen cells in general. On the contrary, those in the i.v. and i.s. chimeras that possessed severer GVHD were mainly composed of host-derived plastic dish non-adherent spleen cells. Since the suppressor activity was higher in chimeras with severe graft-versus-host disease (GVHD) than in conventional chimeras, suppressor cells against the mitogen-induced responses may be related to the immunodeficiency associated with GVHD. Particularly, plastic dish non-adherent suppressor cells may closely relate to GVHD-associated immunodeficiency as compared with plastic dish adherent suppressor cells.     

Evaluation of spleen lymphocyte responsiveness to a T-cell mitogen during early infection with larvalTaenia taeniaeformis

The effect of taeniid infection on the in vitro cellular response of the host was investigated. Infections ofTaenia taeniaeformis decreased the ability of spleen cells from susceptible C3H/He mice to respond to the T-cell mitogen concanavalin A (Con A) as early as 2 days postinfection (pi) reaching a suppression peak at day 12 pi. Similar experiments performed with spleen cells from infected BALB/c mice, resistant to the infection, revealed little or no suppression of Con A stimulation. The results suggested that susceptibility to the parasite may be due to its ability to induce a partial suppression of the host's immune system. The role of adherent splenocytes from infected C3H/He mice in the production of a deficient response to Con A during early infection was studied by coculturing experiments. These experiments demonstrated that adherent populations from infected mice did not play a direct role in the Con A-suppressor mechanisms. Concomitant with the suppressor activity an increased background proliferation was observed with nonstirnulated splenocytes from C3H/He mice infected withT. taeniaeformis. Plasma from infected mice was able to suppress the response of normal spleen cells to Con A and to stimulate a proliferative response in cultured splenocytes from noninfected animals. The results suggest the presence of factors in the plasma of infected mice which may be modulating the immune response to the parasite.     

Antigenic properties of subsets of splenic T lymphocytes responding to lectins.

The aim of the present study was to characterize the antigenic properties of spleen cells responding in vitro to various mitogenic stimuli. Mouse spleen cells were treated with alloantibodies and C and then cultured with various concentrations of lectins. The exposure of spleen cells to various dilutions of either anti-theta serum and C or anti-Ly serum and C inhibited the response to optimal concentrations of PHA to a greater extent than the response to optimal concentrations of Con A. With the exception of the optimal lectin concentration, theta-antiserum and C had a stronger inhibitory effect on the response to Con A than to PHA. The exposure of spleen cells to H-2 antiserum and C inhibited their response to Con A to a greater extent than to PHA. The inhibitory activity of H-2 antiserum and C was inversely correlated with the dose of PHA used for stimulation. The lower the concentration of PHA used for stimulation the more effective was the inhibitory effect of the antiserum. The inhibitory effect of H-2 antiserum and C on the response to Con A was highest at optimal concentrations of the mitogen, and somewhat less pronounced above or below this concentration. The present study suggests that subsets of splenic T-cells that react to various concentrations of mitogens, differ in their theta- and H-2 alloantigenicity.     

Differential effects of marijuana components on proliferation of spleen, lymph node and thymus cells in vitro

The effects of delta 9 THC and 11-OH THC on the proliferative response of murine spleen cells stimulated in vitro with the T cell mitogens Con A or PHA were compared with the effects of these drugs on the mitogen-induced proliferation of murine thymus and lymph node cells. Thymus cells were found to be suppressed at lower cannabinoid concentration than either spleen or lymph node cells. However, splenic cells were more easily suppressed than were the lymph node cells. Lymphoid cell numbers were varied from 1 X 10(6) to 8 X 10(6) cells and treated with a constant dose of either THC or 11-OH THC. When suppression was noted with spleen and lymph node cells, the smallest number of cells in the assay resulted in the greatest level of suppression of cell proliferation. No significant suppression to PHA induced proliferation was found for lymph node cells at any cell number tested. Thymus cells were always more readily suppressed than spleen or lymph node cells regardless of the number of cells in culture. Furthermore, 11-OH THC suppressed the responsiveness of the thymus cells to PHA more than to Con A under the experimental conditions used. Thus, the ability of cannabinoids to induce suppression of the proliferative response of lymphoid cells to mitogens depends on the organ source of the cells, nature of the cannabinoid (THC or 11-OH THC), dose of the cannabinoid, mitogen used (PHA or Con A), and number of cells in culture.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Effect of plant lectins on murine myeloma cells

The mitogenic response (determined by uptake of [3H]-thymidine) of two different BALB/c myeloma lines, normal BALB/c spleen cells and spleen cells from tumour-bearing mice in primary culture to PHA, Con A, PWM and Robinia pseudoaccacia (RPA) extract was determined by measuring the uptake of [3H]-thymidine over 96 h. The pattern of the response of tumour and tumour-spleen cells to the 4 different lectins was similar, and different from that of normal spleen cells. The unstimulated cultures of tumour and tumour-spleen cells displayed an initial increased uptake of [3H]-thymidine, whereas stimulated cultures displayed a low initial uptake of label. After an initial phase of inhibition, ADJ-PC5 myeloma cells were stimulated by PHA and PWM to a greater extent than were normal spleen cells. On the other hand, normal spleen cells gave a markedly better response to Con A and RPA. The mitogenic response of tumour-spleen cells to all 4 lectins was intermediate between those observed for tumour and normal spleen cells; they responded better to Con A and RPA than did tumour cells but were not as responsive as spleen cells, whereas the converse was true for their response to PWM and PHA. In agreement with other reports, the data suggest that the responsiveness of tumour-spleen cells was due to the presence of tumour cells in this tissue. These results indicate that there is no definite evidence of an impaired lectin-responsiveness in lymphoid cells of mice bearing a myeloma.     

Effect of plant lectins on murine myeloma cells.

The mitogenic response (determined by uptake of [3H]-thymidine) of two different BALB/c myeloma lines, normal BALB/c spleen cells and spleen cells from tumour-bearing mice in primary culture to PHA, Con A, PWM and Robinia pseudoaccacia (RPA) extract was determined by measuring the uptake of [3H]-thymidine over 96 h. The pattern of the response of tumour and tumour-spleen cells to the 4 different lectins was similar, and different from that of normal spleen cells. The unstimulated cultures of tumour and tumour-spleen cells displayed an initial increased uptake of [3H]-thymidine, whereas stimulated cultures displayed a low initial uptake of label. After an initial phase of inhibition, ADJ-PC5 myeloma cells were stimulated by PHA and PWM to a greater extent than were normal spleen cells. On the other hand, normal spleen cells gave a markedly better response to Con A and RPA. The mitogenic response of tumour-spleen cells to all 4 lectins was intermediate between those observed for tumour and normal spleen cells; they responded better to Con A and RPA than did tumour cells but were not as responsive as spleen cells, whereas the converse was true for their response to PWM and PHA. In agreement with other reports, the data suggest that the responsiveness of tumour-spleen cells was due to the presence of tumour cells in this tissue. These results indicate that there is no definite evidence of an impaired lectin-responsiveness in lymphoid cells of mice bearing a myeloma.     

Cutaneous tumor of northern pike, L. I. mitogenic responses of mononuclear cells from lymphoid tissues and tumor

To evaluate functional characteristics of lymphocytes from the northern pike, L., mononuclear cells were isolated by Ficoll-Isopaque gradients from lymphoid organs and cutaneous tumors of normal and tumor bearing pikes. The cells were tested in a lymphocyte proliferation assay in medium supplemented with fetal calf serum or autologous plasma using three concentrations of PHA, Con A, tuberculin PPD, and LPS. Lymphocytes of northern pike could be triggered to DNA synthesis . However, no clearcut anatomical partitioning of mitogen responses was found, since the mean optimal proliferation indices for each mitogen were similar in blood, head kidney, and spleen, while cells from head kidney, the equivalent of bone marrow, were stimulated as well with the murine T cell mitogens PHA and Con A as with the B cell mitogens PPD and LPS. Tumorous pikes seemed to have an apparently normal lymphocyte population since they responded by blastogenesis as well as normal pikes. Tumor cells exhibited a high basic metabolic rate and reactivity to mitogens was largely lacking.     

Cutaneous tumor of northern pike, Esox lucius L. I. In vitro mitogenic responses of mononuclear cells from lymphoid tissues and tumor

To evaluate functional characteristics of lymphocytes from the northern pike, Esox lucius L., mononuclear cells were isolated by Ficoll-Isopaque gradients from lymphoid organs and cutaneous tumors of normal and tumor bearing pikes. The cells were tested in a lymphocyte proliferation assay in medium supplemented with fetal calf serum or autologous plasma using three concentrations of PHA, Con A, tuberculin PPD, and LPS. Lymphocytes of northern pike could be triggered to DNA synthesis in vitro. However, no clearcut anatomical partitioning of mitogen responses was found, since the mean optimal proliferation indices for each mitogen were similar in blood, head kidney, and spleen, while cells from head kidney, the equivalent of bone marrow, were stimulated as well with the murine T cell mitogens PHA and Con A as with the B cell mitogens PPD and LPS. Tumorous pikes seemed to have an apparently normal lymphocyte population since they responded by blastogenesis as well as normal pikes. Tumor cells exhibited a high basic metabolic rate and reactivity to mitogens was largely lacking.     

犬蛔虫（Toxocaracanis）对感染鼠的细胞免疫和IL－1、IL－2免疫调节作用的影响

应用犬蛔虫（Ｔ．ｃａｒｎｉｓ）的感染性虫卵口饲感染Ｃ３Ｈ／ＨｅＮ鼠，体外培养后观察脾细胞的免疫学变化。感染第１－４周的脾细胞用ＣｏｎＡ刺激，Ｔ细胞的增殖反应，ＩＬ－２的诱生均明显地受到抑制，而且感染鼠脾细胞抑制了正常鼠脾细胞对ＣｏｎＡ的反应。感染鼠的脾细胞经ＳｅｐｈａｄｅｘＧ－１０过柱后，Ｔ细胞则不显示被抑制作用。表明影响Ｔ细胞被抑制作用的是感染鼠的巨噬细胞。相反，感染第１－４周的鼠所产生的ＩｇＧ和ＩｇＭ等抗体的Ｂ细胞活性增强；用ＬＰＳ刺激巨噬细胞诱生的白介素（ＩＬ）ＩＬ－１也增多。     

Effects of irradiation upon the response of murine spleen cells to mitogens.

The mitogenic response of murine spleen cells exposed to graded doses of radiation was evaluated. Low-dose exposures were associated with an augmented response to concanavalin A (Con A) that was most marked with 100 rads. Low-dose augmentation of phytohemagglutinin (PHA) stimulation was equivocal and most pronounced in cells exposed to 10-20 rads. Augmentation was only demonstrable when the cells were irradiated immediately prior to mitogenic stimulation. Timed exposures after stimulation with Con A or PHA showed no evidence that mitogen activation increased radioresistance, although the possibility could not be excluded that activation protects against interphase cell death. Reduced isotope incorporation was associated with all doses of radiation evaluated in cells stimulated with lipopolysaccharide (LPS) or pokeweed mitogen (PWM). On this basis it is concluded that 1) each of the mitogens tested differs in its capacity to stimulate irradiated spleen cells; 2) radiation-induced augmentation is noted with those mitogens (Con A and possibly PHA) known to activate only T cells; 3) radiation-induced augmentation may be due to the release of mitogenically active molecules by injured lymphocytes.     

Interleukin 3 activity in tumor-bearing hosts: decreased splenocyte production of and responsiveness to IL 3

A kinetic study assessing the relationship between tumor growth and the ability of BALB/c mouse splenocytes to produce Interleukin 3 (IL 3) indicated a concomitant decrease in IL 3 activity with tumor growth. Tumor-bearing host (TBH) splenocytes produced 600 pmoles/hr/10(8) cells of IL 3 activity at Day 0 but only 62 pmoles/hr/10(8) cells by Day 28 post tumor cell inoculation. Nylon wool fractionation (to remove adherent suppressor cells) did not restore IL 3 activity. Addition of purified IL 3 to mitogen proliferation assays showed that IL 3 alone was mitogenic for normal host but not TBH splenocytes. In concert with concanavalin A (Con A) and phytohemagglutinin, IL 3 augmented in vitro normal host splenocyte responsiveness but significantly further suppressed it in the TBH. An absorption assay indicated that fresh cells had acceptors to remove IL 3 from supernatants. Con A-induced normal or TBH blast cells lost their ability to absorb IL 3. Intravenous inoculation of purified IL 3 into normal and TBH resulted in further suppression of TBH splenocyte mitogen-induced blastogenesis. The exacerbation of TBH spleen cell reactivity by IL 3 may be due to a tumor-induced feedback inhibition mechanism further suppressing cellular differentiation critical to cytotoxic T lymphocyte maturation.     

Early accumulation of suppressor cell precursors in the spleen of Mycobacterium lepraemurium-infected mice and analysis of their in vitro-induced maturation.

Spleen cells harvested from mice infected intraperitoneally with M. lepraemurium 11-17 weeks prior to harvest acquired the capacity to inhibit concanavalin A (Con A) induced proliferation of normal spleen cells when precultured for up to 24 h in mitogen-free medium. The in vivo induced suppressor activity correlated with the length of the preculture period, the time post-infection and the infecting dose. These findings were interpreted as an indication that suppressor cell precursors accumulated in the spleen of infected mice during the early phase of the disease. The interaction of infection-dependent adherent suppressor cell precursors and infection-independent, non-adherent regulatory cells is necessary for the suppressor activity to develop. Both the cells which transmit the inductive signal and the precursor cells which mature into active suppressor cells are radiosensitive, whereas suppressor activity itself is a function of radioresistant adherent cells. Preculture of cells for a short period, before they were cocultured with Con A-stimulated normal spleen cells, allowed the detection of suppressor cells before they were deleterious to the infected host and also turned out to be a relevant in vitro model for characterization of suppressor cell development during M. lepraemurium infection.