Ontogeny of T-cell mitogen response in Lewis rats. III. Juvenile adherent suppressor cells block adult mitogen responses

Spleen cells from suckling female Lewis rats (4 to 20 days old) were able to suppress mitogenic responses to concanavalin A (Con A) and phytohemagglutinin (PHA) of spleen or thymus cells from adult female Lewis rats and thymus cells from suckling Lewis rats. Thymus cells from suckling rats were unable to suppress adult spleen cell mitogenic responses to Con A. Removal of carbonyl iron (cFe)-, plastic-, or nylon-wool-adherent cells removed the suppressive action of juvenile spleen cells, but irradiation did not. Separated plastic-adherent spleen cells from suckling animals suppressed adult mitogenic responses to Con A. at optimal Con A doses 2-mercaptoethanol (2-ME, 2 X 10(-5) M) abolished the suppressive effect of juvenile cells, however, at the hyperoptimal dose of Con A (125 micrograms/ml) even higher doses of 2-ME did not relieve suppression by juvenile cells. These suppressor cells in suckling pups were affected by early weaning which decreased suppression, resulting in enhanced mitogenic responses of juvenile cells and removal of the ability to suppress adult mitogenic response.     

BCG-induced suppressor cells. I. Demonstration of a macrophage-like suppressor cell that inhibits cytotoxic T cell generation in vitro.

Spleen cells obtained from mice 5 to 40 days after infection with viable BCG organisms (BCG-spleens) were found to be unresponsive in vitro to both mitogenic and alloantigenic stimuli. Moreover, suppressor cells could be demonstrated in the spleens from these infected animals. When spleen cells from BCG-infected mice were added to either syngeneic or allogeneic normal spleen cells, the mixtures neither proliferated nor developed cytotoxic activity when cultured with alloantigen or with concanavalin A (Con A). The development of unresponsiveness post-infection paralleled the onset of suppressive activity. Spleen cells obtained from mice given heat-killed BCG were neither suppressive nor unresponsive. The suppressive activity of BCG-spleen cells was associated with an adherent, phagocytic cell that lacked membrane-associated Thy-1 antigen. Removal of this cell by passage through nylon wool columns resulted in a cell population that was no longer capable of suppression and that responded normally to alloantigen and to Con A. It would thus appear that BCG infection results in the development of a "suppressor" macrophage-like cell population within the spleen. The role of this cell type in regulation of the immune response in BCG-infected animals is as yet undefined.     

Effects of environmental temperature on the population of T-cell subsets, the blastogenic responses of lymphocytes in blood and spleen and splenic NK cell activity in mice

The population of T-cell subsets, the blastogenic responses of lymphocytes in blood and spleen and splenic NK cell activity were examined in mice transferred from 22 degrees C to 12 degrees C or 32 degrees C environments. The percentage of Thy-1.2 positive cells and Lyt-1.2 positive cells in the spleen decreased after the transfer. However the percentage of Lyt-2.2 positive cells in the spleen was not affected. Thy-1.2 and Lyt-1.2 positive cells in the blood also decreased. The percentage of Lyt-2.2 positive cells in the blood was not affected in mice exposed to 12 degrees C. However, Lyt-2.2 positive cells in the blood decreased on day 1 but increased on day 3 in mice exposed to 32 degrees C. Blastogenic responses of spleen lymphocytes to concanavalin A (Con A) and pokeweed mitogen (PWM) were suppressed in transferred mice, but responses to lipopolysaccharide (LPS) and phytohemagglutinin-P (PHA-P) were not affected in any group. Blastogenic responses of blood lymphocytes to Con A, PHA-P, and PWM tended to be weaker in transferred mice than in mice kept in the 22 degrees C environment. In particular the response to PWM in mice exposed to 12 degrees C was less than 8% of that in the 22 degrees C mice. Splenic NK cell activity decreased in transferred mice, but was not suppressed as much as in mice administered 5mg of cortisone acetate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Generation of suppressor cells by concanavalin A: a new perspective

Significantly lower mitogenic responses of fresh cells co-cultured with Con A-stimulated cells were found when compared with the responses of fresh cells co-cultured with preincubated control cells. We do not agree with the interpretation that this effect represents the generation of suppressor cells by Con A, since the responses of fresh cells cultured alone were also significantly less than when co-cultured with control cells and the same as when co-cultured with the Con A-stimulated cells. Treatment with mitomycin C was sufficient to prevent the preincubated cells from contributing to the mitogenic response of the fresh cells. The increased responses of fresh cells when co-cultured with preincubated cells seems analagous to the increased mitogenic responses of cells aged in vitro by preincubation without mitogen. This effect seems to be transferable to fresh cells in the absence of cell division. Although preincubation in the presence of Con A abrogates this effect, we do not interpret this as the generation of suppressor cells.     

Suppression of the polyclonal B-cell responses by concanavalin A-treated bone marrow B cells in vitro

A suppressor cell that inhibits the development of a polyclonal antibody response of splenic B cells to lipopolysaccharide is generated in the bone marrow cell culture in response to a mitotic dose (10 micrograms/ml) of concanavalin A (Con A). The Con A-responding suppressor cell is radioresistant and found in a bone marrow B (BM-B) cell population of normal as well as athymic mice. The suppressor activity of Con A-treated BM-B cells was consistently higher (P less than 0.01-0.0001) than those of untreated BM-B and fresh BM cells. The BM-B cell population recovered from short-term (3-day) cultures with Con A contained about 65% surface immunoglobulin (Ig)-positive cells, about 6% T cells, and less than 0.5% plastic-adherent cells, the latter two of which did not contribute to the suppressive activity. Thus, cytolytic treatment with various anti-T-cell antibodies could not eliminate the suppressive activity of the Con A-treated BM-B cells, and the Con A-treated macrophage population provided no significant suppression. The Con A-treated BM-B cells adherent to anti-Ig or anti-Con A dishes exhibited highly enriched suppressive activity. It was therefore concluded that an immature B-cell population of bone marrow could develop in response to stimulation with Con A into surface Ig-positive suppressor cells, contributing to the regulation of nonspecific B-cell responses.     

Studies on the mechanisms of action of BCG

Summary Bacillus Calmette-Guérin was given intravenously (i.v.) or subcutaneously (s.c.), 14 days before antigenic or mitogenic stimulation.When given i.v., BCG increased plaque-forming cell production against thymus-dependent antigens (sheep red blood cells and a hapten coupled to hemocyanin) and against a thymus-independent antigen (the same hapten coupled to polymerized flagellin). BCG given s.c. was generally ineffective in potentiating humoral responses.In contrast, a marked depression of spleen cell reactivity in vitro to a T-cell mitogen, concanavalin A (Con A) but an increased response to a B-cell mitogen, dextran sulfate, were observed after BCG i.v., and to a lesser extent after BCG s.c. This inhibitory effect was reversed after removal of cells adherent to nylon or plastic. Normal spleen cell reactivity to Con A was strongly depressed when the cells were cultivated in presence of unfractionated or plastic-adherent BCG-treated spleen cells, suggesting that BCG induces suppressor cells which inhibit T-cell responses in vitro. Similar impairment of T-cell functions in vivo was not observed since BCG given i.v. significantly increased the intensity of delayed-type hypersensitivity to picryl chloride.BCG also induced macrophage activation, measured by macrophage tumoricidal activity in vitro, more strongly and more rapidly when given i.v. than s.c.Modifications of the distribution of lymphocytes from the circulation to the organs were observed. An early increase of lymphocyte migration to the spleen and a long-lasting depression of cell migration to mesenteric lymph nodes and bone marrow were seen after BCG given i.v. When given s.c., BCG induced a progressive increase of cell migration to the draining lymph nodes concomitant with a depression of migration to mesenteric nodes.These results demonstrate the complexity of the mode of action of BCG, but many of its effects on immune responses may result from a primary action on macrophages.     

Mitogenic activation of EL-4 cells does not require surface Thy-1 expression

The ability of monoclonal anti-Thy-1 antibodies to stimulate IL-2 production and T-cell proliferation has raised the possibility that Thy-1 may play an important role in T-cell activation. To examine this postulated role we have produced Thy-1-negative variants of the murine T lymphoma EL-4 by mutagenesis with ethyl methanesulfonate (EMS) and subsequent negative selection with anti-Thy-1 monoclonal antibodies (mAbs) and complement. Although the parental EL-4 cell line produced interleukin-2 (IL-2) in response to concanavalin A (Con A), phytohemagglutinin, anti-Thy-1 mAbs, and an anti-T3 mAb, as well as after exposure to phorbol myristate acetate (PMA), only PMA was capable of inducing IL-2 production by several Thy-1-negative cell lines. The loss of responsiveness to cell surface stimulatory ligands appeared to be correlated with loss of Thy-1 expression because mutagenized cells selected for high levels of Thy-1 expression all responded normally to Con A. However, when Thy-1 expression was reconstituted in the "nonresponder" (Thy-1-negative) cell lines either by transfection of a Thy-1.2 gene or by 5-azadeoxycytidine treatment, the revertant cell lines were still unable to produce IL-2 when stimulated with Con A, anti-Thy-1, or anti-T3. Furthermore, several other independently derived Thy-1-negative EL-4 cell lines responded normally to mitogens and mitogenic mAbs. Taken together, these results suggest that Thy-1 expression is not required for the T-cell activation process and that the EMS mutagenesis procedure resulted in an additional mutation(s) responsible for the inability of certain Thy-1-negative cell lines to be triggered by mitogens and mitogenic mAbs. These cell lines may prove to be valuable tools for further biochemical and molecular studies of the sequence of events associated with T-cell activation.     

Concanavalin A-inducible suppressor cells in pleural effusions and peripheral blood of cancer patients

Summary Carcinomatous pleural effusions of 18 of 20 patients with lung cancer contained suppressor cell precursors that could be activated by concanavalin A (Con A) to suppress the proliferative responses of autologous and allogeneic lymphocytes to phytohemagglutinin and Con A. However, pleural effusion cells showed no suppressor function without prior activation by Con A. In contrast, the peripheral blood of the cancer patients exhibiting impaired mitogenic response contained nonspecific spontaneous suppressor cells capable of inhibiting the lymphoproliferative response to mitogens without prior activation by Con A, but these cells were not able to show further suppressor function even after activation by Con A. The maximum suppression was observed after 48-h treatment of lymphocytes with optimally mitogenic doses of Con A. The Con A-inducible suppressor cells of the pleural effusion and spontaneous suppressor cells of the peripheral blood of cancer patients had the same characteristics with regard to the capacity to suppress the mitogenic responses of autologous and allogeneic lymphocytes, belonging to the group of nylon wool-nonadherent T cells and being sensitive to in vitro culture and resistant to treatment with mitomycin C.     

Properties and applications of monoclonal antibodies directed against determinants of they Thy-1 locus.

Fusion of cells of the mouse myeloma line, P3/X63-Ag8 with spleen cells from AKR/J mice immunized against C3H thymocytes or from (BALB/c x BALB.K)F1 mice immunized against AKR/J thymocytes gave rise to hybrid cell lines that continuously secrete antibodies specific for the Thy-1.2 and Thy-1.1 antigens, respectively. Monoclonal antibodies from four such cell lines were analyzed in detail. All were 19S IgM, and, in the presence of complement (C), had high lytic titers on T cells of the appropriate antigenicity. Their specificity was shown by lysis of thymocytes from Thy-1 congenic mouse strains, A/J(Thy-1.2) and A. Thy 1.1. Furthermore, they lyse only 60 to 70% of lymph node cells, suggesting cytotoxicity for mature T cells and not B cells. Treatment of peripheral lymphocyte populations with monoclonal antibody plus C eliminated effector cytotoxic T lymphocytes, their precursors, and the mitogenic response to Con A, but did not affect the response to LPS. Purified, fluorescein-labeled monoclonal anti-Thy-1 antibody could be used to distinguish T and B cells. Purified antibody coupled to Sepharose 6MB was used to separate viable T and B cells. Two independently isolated anti-Thy-1.2 hybridomas are indistinguishable and bind the same determinant whereas a third is unique and may bind a separate site.     

Subpopulation of T Cells Sensitive to Natural Thymocytotoxic Autoantibody (NTA) of New Zealand Mice: I. Distinct Cytotoxic Sensitivity of Functional T Cell Subsets to NTA and Anti-Thy-1 Antibodies

NZB mice produce a natural thymocytotoxic autoantibody (NTA) capable of specifically injuring thymocytes and T cells. NTA-reactive antigen (NTA-A) shows a different density distribution among T cells, and partial killing with NTA and complement can eliminate T cells bearing NTA-A in high density. Thy-1 antigen is similar to NTA-A in this respect. To determine the effects of NTA and anti-Thy-1 on distinct functional subsets of T cells, Con A-induced suppressor T cell (Con A-Ts) activity against the allogeneic mixed lymphocyte reaction (MLR), responding T cell (T_MLR) activity in the allogeneic MLR, and Con A-induced cytotoxic T cell (Con A-Tc) activity were examined simultaneously in BALB/c spleen cells before and after partial elimination of NTA- and anti-Thy-1-sensitive T cells. Treatment with NTA and complement resulted in a marked reduction in Con A-Ts activity, a significant increase in T_MLR activity and a slight and inconstant decrease in Con A-Tc activity. Since Con A-generated Ts were much less sensitive to NTA, the NTA-sensitive T cells involved in Con A-Ts activity appear to be precursors or promoters of the Con A-Ts. In contrast, the precursors of Con A-Tc seem to be relatively resistant to NTA. The increase in T_MLR activity caused by NTA suggests the possibility that NTA is less cytotoxic for T_MLR and cytotoxic for some suppressor T cells in allogeneic MLR. The monoclonal anti-Thy-1 antibody showed no such preferential cytotoxic effects on the three T cell functions. The NTA-sensitive T cells, in contrast to anti-Thy-1-sensitive T cells, were reduced gradually during Con A stimulation. All these findings indicate that NTA-A not only differs from Thy-1 antigen but that it appears to be a unique T cell antigen.     

Cloned T cells with "co-helper" activity. I. Biologic activities of the clone

Clones of sheep erythrocyte-(SRBC) specific helper T cells with the surface phenotype Thy-1+, Ly-1+, Ly-2- have been derived that grow in vitro in the absence of exogenous antigen or added growth factors. The IL 2-independent clone, 101.6 has been shown to produce a supernatant factor that augments the primary anti-SRBC but not anti-burro RBC responses of whole spleen cells or Ly-1 T plus B cell cultures. The supernatant does not help B cells directly. This augmenting activity is terminated "co-helper" because the enhancement requires the presence of normal Ly-1 T cells. The supernatant of 101.6 was not shown to contain IL 2; co-helper activity was distinguishable from IL 2 activity by absorption with SRBC but not with Con A blasts, and we observed that co-helper activity does not act on spleen cells that differ at the major histocompatibility complex.     

Lymphocyte function in experimental African trypanosomiasis. VIII. Loss of suppressor T cell function in lymph nodes

The immunosuppression that occurs in mice experimentally infected with African trypanosomiasis has been examined further. In the present study we have examined lymph node cells from Trypanosoma rhodesiense-infected C57Bl/6J mice for the ability to produce mitogen induced antigen-nonspecific suppressor T cells (Ts). Inguinal, mesenteric, and brachial lymph node cells were harvested from uninfected control mice and from mice at different periods of infection. These cells were cultured with or without concanavalin A (Con A) for 48 hr to induce Ts activity. After stimulation, the control and infected lymph node cells were passed over Sephadex G-10 columns to remove suppressor macrophages that arise during the infection from Con A-induced Ts. The column passed cells were then added to normal mouse responder spleen cells in a primary in vitro antibody response culture system with sheep erythrocytes (SRBC) as antigen. The resultant plaque-forming cell responses to SRBC indicated that Ts function was not induced in infected lymph node cell populations. However, early in the infection, a stimulatory signal was provided by both the untreated and Con A-treated infected lymph node cells, which was lost in the terminal stage. Determinations of T cell subpopulations revealed that the infected Lyt 2.2-bearing subpopulation was not significantly altered from normal controls. We conclude that T. rhodesense infected mice fail to mount normal lymph node cell antigen nonspecific Ts responses and that this loss of activity may be due to an intrinsic dysfunction in the suppressor T cell population.     

Reversal of Con A-induced suppression by a platelet-derived factor which binds to activated suppressor T cells

A platelet-derived factor found in serum as well as in platelet releasate prepared either with calcium ionophore or with thrombin was shown to reverse Con A-induced suppression of the plaque forming cell (PFC) response to sheep erythrocytes (SRBC) in vivo in (CB6)F1 mice. In addition, as shown previously, lymphoma cell-induced suppression in SJL mice was similarly reversed. The factor could be injected prior to Con A on the day before SRBC injection, or on the same day as antigen with comparable results. It also enhanced PFC responses in the absence of Con A. Suppressor cell induction by Con A in vivo, as demonstrated by assay on PFC responses of normal spleen cells in vitro, was abrogated by simultaneous injection of the platelet factor. Cells from mouse spleen and lymph node, but not from thymus could absorb the factor from human serum at 4 degrees C. The phenotype of the relevant spleen cells was L3T4-, Ly1-, Ly2+, Thy1+, Ly22+, Qa1+, Qa4+, Qa5+, and Ly6.IE+. These results suggest that this factor binds to activated peripheral T cells of the suppressor cell phenotype.     

Cellular transfer of experimental allergic encephalomyelitis in Lewis rats: Effects of different sensitization regimens on in vitro reactivity of donor spleen cells to concanavalin A

Splenocytes from Lewis rats sensitized to guinea pig spinal cord (GPSC) and complete Freund's adjuvant (CFA) or to myelin basic protein (MBP)-CFA plus pertussis vaccine were less effective than spleen cells from MBP-CFA sensitized donors in transferring EAE to syngeneic recipients following culture with concanavalin A (Con A). Moreover, splenocytes from rats sensitized to GPSC-CFA plus pertussis vaccine showed no EAE transfer activity following culture with Con A. Diminished EAE transfer activity occurred in parallel with decreased proliferative responses of primed splenocytes to Con A. These effects were due, at least in part, to the use of pertussis vaccine and to Con A activation of a suppressive adherent cell subpopulation in sensitized donor spleens. Proliferative responses and EAE transfer activity were restored upon removal of plastic-adherent cells from splenocytes of rats sensitized to MBP-CFA plus pertussis vaccine prior to Con A activation of the non adherent lymphoid cells. Deletion of plastic-adherent cells from splenocytes of donors sensitized to GPSC-CFA plus pertussis vaccine prior to activation with Con A, however, had no effect on proliferative responses or EAE transfer activity. Furthermore, EAE transfer activity of Con A-activated splenocytes from MBP-CFA-sensitized donors was lost when such cells were cultured with splenocytes from donors sensitized to GPSC-CFA plus pertussis vaccine.     

Mechanism for the suppression of gamma-interferon responsiveness in mice after thermal injury

As reported previously, gamma-interferon production was decreased after the administration of inducers to thermally injured mice as compared with noninjured controls. Similarly, spleen cells from injured mice had decreased ability to produce interferon in vitro after stimulation with inducers. The present study demonstrated that the decrease in interferon production was associated with the presence of suppressor cells in the spleen of burned mice that were capable of inhibiting interferon production by normal splenic lymphocytes in vitro. Passive transfer of spleen cells containing suppressor cell activity derived from injured mice induced suppression in normal mice, and the time of the appearance of suppressor cell activity in injured mouse spleens closely approximated the time of the appearance of the suppression of interferon production observed in mice after thermal injury. The suppressor cells were characterized as a population of macrophages by the following: they adhered to plastic surface and could be removed from spleen cells by carbonyl-iron treatment; treatment of plastic-adherent cells with anti-Thy-1.2 and anti-mouse immunoglobulin antisera followed by complement failed to abrogate the suppression produced by these cells.     

Thy-1+ epidermal cells proliferate in response to concanavalin A and interleukin 2

Mouse epidermal cells (EC) are composed of at least two phenotypically discrete populations of cells that in epidermal sheets have a dendritic morphology: Ia+ Langerhans cells (LC) and dendritic, bone marrow-derived, Ia- cells that express Thy-1 antigen (Thy-1+ dEC). Thy-1+ dEC lack other typical T cell markers such as L3T4, Lyt-1, and Lyt-2; however they do express Ly-5 and asialo GM1 in common with NK cells and certain other leukocytes. To investigate the functional capabilities of Thy-1+ dEC in vitro, cell suspensions prepared from trypsin-disaggregated sheets of mouse body wall epidermis were first enriched to 8 to 20% Ia+ and 20 to 40% Thy-1+ cells by centrifugation over Isolymph and then were cultured for 2 to 10 days with Concanavalin A (Con A) and/or partially purified rat IL 2. Con A-induced proliferation of EC was readily seen, with the maximal response occurring at a Con A concentration of 2.5 micrograms/ml on day 5 of culture. Con A responses were significantly enhanced by the continuous presence of 1 microgram/ml indomethacin. Responses both in the presence and absence of Con A were significantly enhanced by the addition of 5 to 10 U/ml of partially purified rat IL 2; proliferation in cultures stimulated by both Con A and IL 2 continued to increase throughout the 10-day culture period. Culture of fluorescence-activated cell sorter (FACS)-separated EC suspensions revealed that Thy-1-depleted EC and irradiated Thy-1+ EC failed to proliferate in response to Con A and IL 2, whereas unirradiated purified Thy-1+ EC gave enhanced Con A- and IL 2-induced responses compared with the unseparated population. Finally, to distinguish between the proliferation of small numbers of mature peripheral T cells and that of Thy-1+ dEC, antibody and complement-depletion studies were conducted with an unusual monoclonal anti-Thy-1 reagent, 20-10-5S, and with the anti-T cell reagents, anti-L3T4 and anti-Lyt-2. Thy-1+ dEC, but not LC, express the 20-10-5S determinant; furthermore, in CBA (Thy-1.2) mice 20-10-5S reacts with Thy-1+ dEC, thymocytes, and peripheral T cells, whereas in AKR/J (Thy-1.1) mice, it reacts only with Thy-1+ dEC and thymocytes and not with peripheral T cells. Pretreatment of AKR/J EC with 20-10-5S and complement abolished the capacity of such cells to respond to Con A and to IL 2.(ABSTRACT TRUNCATED AT 400 WORDS)     

Dual effects of OK-432 on mitogenic response of splenocytes to concanavalin A

OK-432, a streptococcal preparation, was studied for its effect on the concanavalin A (Con A)-induced mitogenesis of the host spleen cells. When mice were given a single intraperitoneal injection of OK-432, there was a substantial increase in the mitogenic response of splenocytes, whereas multiple injections conversely resulted in a marked reduction of the mitogenic response, when the spleen cells were cultured at high cell densities of over than 5 X 10(5) cells/well. The reduced Con A-responsiveness in the latter was not restored by mixing spleen cells from mice given multiple OK-432 injections with those from normal mice. Moreover, splenic macrophages from OK-432-injected mice exhibited marked inhibitory activity against Con A-mitogenesis of normal splenocytes, while normal splenic macrophages failed to show such an effect. Splenic T cells from OK-432-injected mice also showed an inhibitory activity against Con A-mitogenesis of normal splenocytes and similar activity was also noted in normal splenic T cells. Therefore, the OK-432-spleen cells contain two types of suppressor cells; one is a newly elicited suppressor macrophage and the other is a suppressor T cell supposedly resident also in normal spleen cells. In the OK-432-injected spleen cells, accessory cell function for T cell Con A-mitogenesis was markedly reduced. On the other hand, it was noted that the interleukin 2-producing ability of the OK-432-splenocytes was augmented more than that of normal splenocytes, indicating that multiple OK-432 injections also cause an increase in the helper T cell activity of the host spleen cells.     

Suppression of Con A mitogen-induced proliferation of normal spleen cells by macrophages from chickens with hereditary muscular dystrophy

Spleen cells from chickens with hereditary muscular dystrophy (MD) give low blastogenic responses to the T cell mitogen concanavalin A (Con A) while exhibiting normal mitogen stimulated blastogenic responses to the T cell mitogen phytohemagglutinin (PHA). The addition of MD spleen cells to normal spleen cells caused a marked suppression of the Con A response of the normal cells while not affecting the PHA response of the normal cells. The suppressive activity by the MD spleen cells requires viable cells and is contact mediated. The suppressive activity is attributed to the presence in MD spleens of a population of suppressor cells with characteristics typical of macrophages. The suppressor cell activity was not removable by complement-mediated lysis using anti-T or anti-B sera, but it was reversible by treatment with carrageenan or carbonyl iron magnet, by passage through a Sephadex G-10 column, and by adherence to plastic petri dishes or glass beads. MD spleen cells depleted of the suppressor cell population remained unable to respond to Con A.     

In vitro studies of the rabbit immune system. IV. Differential mitogen responses of isolated T and B cells.

The mitogenic responses of separated rabbit lymphocyte populations functionally analogous to mouse T and B cells have been tested in vitro. Purified T cells were prepared by passage over nylon wool (NW) and purified B cells prepared by treatment with antithymocyte serum and complement (ATS + C). ATS + C kills 70% of peripheral blood lymphocytes (PBL's) and 50% of the spleen cells while passage over NW yields 40% of the applied PBL's and 5–23% of the applied spleen cells. NW-purified T cells from the spleen or PBL's respond fully to concanavalin A (Con A) but have a reduced response to phytohemaglutinin (PHA) and little or no response to goat anti-rabbit immunoglobulin (anti-Ig). PBL's that survive ATS + C (B cells) are stimulated by anti-Ig but not by Con A or PHA. B cells purified from spleen do not respond to Con A or PHA but will respond to anti-Ig under appropriate conditions. A full spleen B-cell response to anti-Ig required removal of Ig produced by the cultures that blocked anti-Ig stimulation. It is concluded that, for rabbit lymphocytes, Con A and PHA are primarily T-cell mitogens and that anti-Ig is primarily a B-cell mitogen. However, the mitogen response of unfractionated PBL or spleen cell populations indicates an overlap in reactivity. This could be due to cells sharing T and B properties, alteration of cell populations by the fractionation procedures used, or recruitment of one population in the presence of a mitogenic response of the other population.     

Mechanisms of corticosteroid action on lymphocyte subpopulations. IV. Effects of in vitro hydrocortisone on naturally occuring and mitogen-induced suppressor cells in man.

The effects of in vitro hydrocortisone (OHC) on human peripheral blood (PB) suppressor cell function were investigated. Two types of suppressor cells were studied: (i) the naturally occurring PB suppressor cell seen in 10% of normal people whose lymphocytes do not respond to in vitro PWM stimulation with direct anti-SRBC PFC responses, and (ii) Con A-generated suppressor cells. The addition of OHC to PWM-stimulated cultures from nonresponders reconstituted the PFC response in two of three individuals. The addition of OHC to allogenic cocultures of nonresponder and responder lymphocytes completely inhibited the ability of the naturally occurring suppressor cell of the nonresponder cultures to inhibit the PFC responses of normal responders. Preincubating the nonresponder cultures in 10^?5M OHC for 30 min followed by washing did not inhibit suppressor function, whereas readdition of OHC to cocultures did inhibit nonresponder suppressor cell function. The addition of up to 10^?4M OHC to previously generated Con A-activated suppressor cell-fresh cell cocultures in vitro did not prevent or inhibit mitogen-activated suppressor cell function. However, preincubation of PB cells for 6 hr prior to the addition of Con A prevented the generation of suppressor cells and in two of eight experiments generated a population of cells which were in and of themselves mitogenic for autologous fresh PB. Thus, the function of naturally occurring suppressor cells as well as the induction but not the function of Con A-activated suppressor cells is sensitive to pharmacologic levels of OHC. The effect of OHC on naturally occurring suppressor cell function or on the generation of suppressor cells by Con A did not involve cell lysis, but rather was a reversible phenomenon requiring the continued presence of OHC in culture.