Effects of lymphocytes from Marek's disease-infected chickens on mitogen responses of syngeneic normal chicken spleen cells.

PHA responses have been measured in lymphoid cell cultures prepared by mixing normal chicken spleen cells with spleen or thymus cells from syngeneic chickens infected with the oncogenic herpesvirus MDV. Results of these studies may be summarized as follows: 1) spleen cells from MDV-infected chickens with visceral lymphomas inhibit the PHA response of normal spleen cells possibly by release of soluble inhibitory factors in response to the mitogen; 2) lymphoid cells from asymptomatic MDV-infected chickens, although hyporeactive themselves to PHA, can have a stimulatory effect on PHA responses of normal spleen cells in mixed cultures; 3) spleen cells from MDV-infected chickens, effectively protected from viral oncogenesis by HVT vaccination, show normal reactivity to PHA in spearate cultures and may react in mixed cultures like normal lymphocytes, with neither a pronounced stimulatory nor inhibitory effect on the PHA response of normal 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.     

Splenic suppressor macrophages induced in mice by injection of Corynebacterium parvum.

Spleen cells from C57BL/6N mice injected with killed Corynebacterium parvum (CP) had a marked growth inhibitory effect on the in vitro proliferation of RBL-5 murine lymphoma cells. It was most marked 12 to 14 days after injection and was usually no longer detectable later than 21 days. It could be demonstrated at effector cell to target ratios between 20:1 and 5:1 at which normal spleen cells had a growth-promoting effect. Addition of CP to an in vitro mixture of spleen cells and tumor cells augmented the inhibitory effect of spleen cells from CP-injected mice although it conferred no inhibitory potential on normal spleen cells. Growth inhibiton by CP spleen cells was not mediated by T cells and various depletion experiments suggested that the effector cells of the phenomenon were macrophages. Spleen cells of CP-injected mice also showed strongly depressed responses to the T cell mitogens PHA and Con A and suppressed the mitogen responses of syngeneic normal spleen cells. The characteristics of the suppressor cells mediating this effect appeared to be very similar to those inhibiting lymphoma cell growth. The responses to LPS were also strongly suppressed in mice injected with 2.1 mg of CP. However, after injection of one-tenth of the dose a relative sparing of the LPS response was noted, whereas the PHA response was still suppressed.     

In vitro generation of suppressor cell activity: suppression of in vitro induction if cell-mediated cytotoxicity.

It was observed that when normal mouse spleen cells were cultured alone in vitro (precultured) for 3 to 7 days, these cells lost the ability to generate cell-mediated cytotoxicity (CML) during subsequent in vitro sensitization with allogeneic spleen cells, trinitrophenyl (TNP)-modified syngeneic spleen cells, or syngeneic tumor cells. These precultured cells, which were themselves unable to generate CML, were also shown in mixing experiments to suppress, actively, the generation of CML by freshly explanted spleen cells. Suppression occurred at the sensitization phase of CML, and not at the effector level; supernatants from suppressive precultured cells were not suppressive. Suppression was totally abrogated by the treatment of spleen cells with a T cell-specific rabbit anti-mouse brain serum and complement (RalphaMB+C) either before or after preculturing, suggesting that a T cell eas essential both to the generation of suppressor activity and to its expression. Suppressor activity was entirely absent in precultured nylon wool column-nonadherent spleen cells, a T cell-enriched population containing most of the RalphaMB+C-sensitive cells in the spleen. Precultured nylon column-adherent cells (T cell-depleted) did have suppressive activity, and a mixture of nylon-adherent and nylon-non-adherent cells was a suppressive after preculture as the precultured unseparated spleen. Moreover, the ability of nylon-adherent spleen cells to generate suppressive activity during preculturing was abrogated by treatment with RalphaMB+C. Thus, the "spontaneous" generation of CML-suppressive activity was dependent upon a limited subpopulation of splenic T cells isolated in the nylon column-adherent fraction. The relationship of these data to a previously described synergy between subpopulations of normal spleen in the generation of CML is discussed, and the findings related to other suppressor systems described in the literature.     

Immunologic tolerance to HGG in mice. I. Suppression of the HGG response in normal mice with spleen cells or a spleen cell lysate from tolerant mice.

Adoptive transfer of spleen cells or spleen cell lysates from mice tolerant to human-gamma-globulin (HGG) specifically suppressed the response of normal syngeneic recipients to HGG. The suppressive activity could be transferred for over 100 days after tolerance induction. The suppression induced by both spleen cells and spleen cell lysate was found to be specific as evidenced by a normal response to a challenge with turkey-gamma-globulin or goat erythrocytes. The activity of the suppressive lysate could be removed by passing the material through an HGG immunoadsorbent column but not by passing it through an anti-HGG column or a BSA column. These results indicated that the factor had antigen specificity and was probably not antigen-antibody complexes. That this suppression was not due to a shifting of the kinetics of the antibody response has also been demonstrated. The antigen-specific suppressor factor in the tolerant spleen cell lysates was a protein with a m.w. of approximately 45,000 daltons. The kinetics of the appearance of both suppressor cells and suppressor factor were consistent with a mechanism of active suppression functioning in the maintenance of tolerance to HGG.     

Immunoregulation in experimental schistosomiasis: in vitro induction and assay of spleen cell suppressor activity

Spleen cells from normal CBA/J mice or mice infected with Schistosoma mansoni were exposed for 48 to 72 hr to either concanavalin A (Con A), soluble egg antigen (SEA), or soluble worm antigenic preparation (SWAP), treated with mitomycin C to prevent further DNA synthesis, and admixed with either normal or sensitized syngeneic spleen cells exposed to a concentration gradient of phytohemagglutinin (PHA) or SEA, respectively. Both nonspecific (by Con A) and "antigen-specific" (by SEA and SWAP in infected mice only) induction of suppression was observed when using PHA-induced blastogenesis as the final assay. The number of mice with inducible splenic suppressive activity and the degree of PHA suppression induced by exposure to SEA appeared to decline between 8 and 20 weeks of infection. In contrast, when the response of spleen cells from mice infected for 8 weeks to SEA served as the final assay, strong suppressive activity was induced from the spleen cells of all chronically infected mice (20 weeks of infection). This model permits parallel analysis of the induction of suppressor activity by nonspecific and schistosome antigen-specific signals during the course of this chronic, immunoregulated condition, schistosomiasis mansoni.     

Generation of cytolytic T lymphocytes in vitro. X. Induction of primary and secondary CTL responses by the mitogen sodium periodate.

Short-term (15 min) sodium periodate (NaIO4) treatment of mouse spleen cells previously primed in vivo or in vitro against alloantigens induced the formation of secondary (2 degree) cytolytic T lymphocytes (CTL) specific for the priming antigens. CTL formation was readily demonstrable within 24 hr after treatment.This early CTL response occurred equally well in the presence or absence of cytosine arabinoside (Ara C), indicating that NaIO4 could induce CTL independently of DNA synthesis. Forty-eight hours after periodate treatment, the lytic activity was similar to that observed in parallel cultures stimulated with irradiated allogeneic spleen cells, although the peak activity was reached earlier (day 4) and was somewhat lower than that induced by alloantigen. The addition of irradiated NaIO4-treated unprimed syngeneic spleen cells to cultures of untreated alloimmune spleen cells also led to CTL formation, which suggests an indirect mechanism of activation. In contrast to alloimmune spleen cells, normal spleen cells treated with NaIO4 developed only very low levels of cytotoxicity after 4 days of incubation. However, in the presence of PHA, such cells were capable of lysing syngeneic and allogeneic target cells.     

Effect of rubomycin and carminomycin on suppressor cells participating in the regulation of the immune response

The effect of rubomycin and carminomycin on induction of suppressor cells in immunization of mice with high doses (2 . 10(9) cells) of sheep red blood cells (SRBC) or with the allogeneic spleen cells was studied. When the spleen cells of syngeneic animals hyperimmunized with SRBC were administered to intact cells, a marked specific suppression of both the antibody formation and the reaction of the delayed type hypersensitivity were observed. A decrease in the suppressor activity or its complete elimination as a result of exposure of the spleen cell donors to the antibiotics was indicative of a relatively selective effect of these drugs on the suppressor cells. This effect of the antibiotics increased after treatment of the spleen immune cells with the anti-T-serum and complement. However, the blocking effect of the antibiotics on the suppressor cells was not strictly specific, since they were detected in the syngeneic and allogenic systems with the cell transfer.     

Abrogation of the capacity of delayed-type hypersensitivity responses to alloantigens by intravenous injection of neuraminidase-treated allogeneic cells

BALB/c or C3H/He mice were inoculated i.v. with allogeneic spleen cells untreated or treated with neuraminidase. Appreciable or potent anti-allo-delayed-type hypersensitivity (DTH) responses were observed when mice were inoculated i.v. with untreated allogeneic cells or inoculated i.v. with those cells followed by s.c. immunization with untreated allogeneic cells. In contrast, i.v. inoculation of neuraminidase-treated allogeneic cells (presensitization) not only failed to induce any significant anti-allo-DTH responses but also abolished the capability of the animals to develop DTH responses after s.c. immunization, indicating the tolerance induction. This tolerance was alloantigen-specific, and rapidly inducible and long lasting. The induction of suppressor cell activity was demonstrated in tolerant mice. However, this activity was associated only with the tolerant state around 4 to 7 days after the i.v. presensitization, but was no longer detected in mice more than 14 days after the presensitization, although these mice exhibited complete tolerant state. When spleen cells from such tolerant mice were transferred i.v. into 600 R x-irradiated syngeneic recipient mice alone or together with normal syngeneic spleen cells, these tolerant spleen cells themselves failed to induce DTH responses but did not exhibit suppressive effect on the generation of DTH responses induced by normal spleen cells co-transferred. These results indicate that i.v. administration of neuraminidase-treated allogeneic cells results in the induction of alloantigen-specific tolerance which is not always associated with the induction of suppressor cell activity but rather with the elimination or functional impairment of alloantigen-specific clones.     

Mechanism of histamine-induced inhibition of lymphocyte response to mitogens in mice

Histamine induced, in mice, an inhibition of lymphocyte response to PHA and LPS, at molar concentrations ranging from 10^?3 to 10^?9M. This inhibition occurs as a specific interaction between histamine and T lymphocytes bearing H2-type receptors for this hormone (H + cells) and Ly 2 membrane antigens. Two features of the suppressive activity of this T-cell subpopulation were observed: (i) when histamine is added at the beginning of the culture period with PHA or LPS, it activates the suppressor activity of H + cells which act on the lymphocyte population responding to PHA and LPS; (ii) preincubation of spleen lymphocytes with histamine for 24 hr induces suppressor cells which inhibit the response to PHA, but not to LPS, of syngeneic lymphocytes in a coculture system, and which are radiosensitive. The role of PHA as a second stimulus of histamine-induced suppressor cells, and the relation between these cells and PHA or Con A-induced suppressor cells, are discussed.     

Stimulation of antibody production to the hapten, 2,4-dinitrobenzene by affinity-labeled murine lymphoid cells. II. Suppressive activity of an excess of thymocytes.

The suppressive effects of excess numbers of normal thymocytes on the transfer of immunity to 2,4-dinitrophenyl (DNP) into irradiated syngeneic recipients with metanitrobenzenediazonium fluoborate (m-NBDF)-labeled spleen cells and normal thymocytes (NT) was investigated. The significant immune response to DNP obtained with the optimal dose of 10 × 10⁷ NT was completely ablated when 20 × 10⁷ NT were used. The suppression was not the result of supro-optimal helper activity. T cell interference of m-NBDF-labeled spleen cell homing, or nonspecific suppression due to excessive numbers of cells. The suppressor activity was T cell dependent and was present in both the cortisone-resistant and cortisone-sensitive thymocyte populations. The suppression was obliterated by 1000 R of irradiation. Thus, the suppressor activity could be the result of either a radiosensitive property of normal thymocytes when they are in excess or a subpopulation of radiosensitive, suppressor thymocytes.     

Differential in vitro modulation of suppressor and antitumor functions of mouse macrophages by lymphokines and/or endotoxin

Peritoneal macrophages of normal mice exhibited natural suppressor activity, as indicated by their ability to inhibit the proliferation of spleen cells in response to stimulation with phytohemagglutinin (PHA) or concanavalin A (Con A). Their suppressor function could be modulated in vitro with a variety of treatment regimens. High-dose lipopolysaccharide (LPS) (LPSH; 10 micrograms/ml) or lymphokines (supernatant from Con A-stimulated spleen cells) plus low-dose LPS (LPSL; 10 ng/ml) caused a reduction in the suppressor activity of adherent peritoneal macrophages. In contrast, these same treatments induced the macrophages to become tumoricidal and cytostatic for tumor cells, indicating a major dissociation between the regulation of suppressor and cytotoxic activities of macrophages. The lack of correlation between these activities was further demonstrated by macrophages that had been activated in vitro by Corynebacterium parvum: these cells expressed high tumoricidal and cytostatic activities, and also strong suppressor activity. The suppressor function could be selectively downregulated by in vitro pretreatment with LPSH.     

Immunoregulation in disseminated histoplasmosis: Characterization of splenic suppressor cell populations

Potent immunosuppressor cell activity was induced during the course of disseminated histoplasmosis in C3H/Anf mice. Spleen cells from infected mice severely suppressed the primary antibody response in vitro of normal syngeneic spleen cells to both a T-dependent antigen (sheep red blood cells) and a T-independent antigen (trinitrophenyl-lipopolysaccharide) at Weeks 1 and 3 of infection, respectively. Likewise, marked suppressor cell activity was present within lymph nodes. In a kinetic study, suppressor activity was detected first on Day 2 and increased to the maximum level on Day 4 after inoculation of Histoplasma capsulatum. Two populations of spleen cells express suppressor function in this model. One population, identified as T cells, was nonadherent to nylon wool columns; its suppressor capacity was abolished by anti-Thy 1 and reduced greatly by low-dosage X-irradiation (500 R). Cells of the second suppressor population had macrophage-like properties; although poorly adherent to plastic surfaces, they adhered to nylon wool columns and could be removed from spleen cell suspensions by carbonyl iron treatment; high-dosage X-irradiation (3000 R) and mitomycin C treatment failed to abrogate suppression by these cells.     

Experimental cutaneous leishmaniasis. I. Nonspecific immunodepression in BALB/c mice infected with Leishmania tropica

Leishmania tropica in BALB/c mice causes a fatal infection accompanied by the development of multiple metastatic lesions. Spleen cells from these mice were shown to have depressed proliferative responses to concanavalin A (Con A), phytohemagglutinin (PHA), and lipopolysaccharide (LPS). Coinciding with this immunodepression was the development of a cell population capable of suppressing normal spleen cell responses to Con A. This suppressor cell activity was first observed at 6 wk and was present throughout the remainder of the infection. At 12 wk the suppressor cells could be removed by Sephadex G-10 passage or carbonyl iron treatment; however, Sephadex G-10 passage could not reverse the suppression at 18 wk. Indomethacin, a prostaglandin synthetase inhibitor, was found to abrogate the activity of the adherent suppressor cell, suggesting that prostaglandin production may be involved in the immunosuppression seen in these mice. In addition, Sephadex G-10 passage and indomethacin were found to markedly augment spleen cell responses to leishmanial antigen, indicating that the adherent suppressor cell is capable of regulating specific immunologic responses.     

Induction of suppressor cells by a tumor-derived suppressor factor

Murine fibrosarcomas produce a factor that activates suppressor cells to inhibit expression of delayed-type hypersensitivity (DTH) responses to dinitrochlorobenzene (DNCB). This tumor-derived suppressor factor (TDSF) was partially purified by preparative isoelectric focusing of spent medium and 3 M KCl extracts of cultured methylcholanthrene-induced and spontaneous fibrosarcomas of C3H/He mice. Incubation of 1 micrograms/ml of a fraction, isoelectric pH less than 2.9, with normal syngeneic spleen cells for 1-6 hr at 37 degrees C induced suppressor cells that inhibited the primary DTH response to DNCB upon intraperitoneal transfer to normal C3H/HeJ mice. TDSF was not present in extracts of either syngeneic embryonic fibroblasts or normal spleen cells or in medium conditioned by normal peritoneal exudate cells but was present in 3 M KCl extracts of and the spent medium from four different cultured murine fibrosarcomas. TDSF activity was not restricted at the major histocompatibility complex. The suppressor cells inhibited the efferent limb of the DTH response because (1) hyporesponsive recipients of TDSF-treated spleen cells had splenic effector T cells capable of transferring DTH to DNCB into naive secondary recipients and (2) the ability of Lyt 1+,2- effector Tdth cells to transfer a secondary DTH response to DNCB was inhibited by co-incubation with macrophages or Lyt 1-,2+ T cells treated with TDSF. Preliminary biochemical analysis suggested that TDSF was an RNA- protein complex. Thus, several murine fibrosarcomas produced a soluble factor that activated splenic suppressor cells to depress the immune response to nonneoplastic antigens. These suppressor factors represent a novel group of regulatory molecules which may be ribonucleoprotein complexes.     

Induction of allospecific suppressor T cells in vitro.

Incubation of mouse thymic lymphocytes with irradiated allogeneic spleen cells gave rise to suppressor cells. The suppressor activity was assayed by adding the incubated cell mixture to a mixed lymphocyte culture (MLC) in which the responder cells were syngeneic with the sensitized thymocytes and the stimulator cells were syngeneic with the sensitizing spleen cells. Such addition suppressed significantly thymidine incorporation in the mixed lymphocyte reaction (MLR). The suppressor cells were found to carry the θ antigen and to function allospecifically, as shown by cross-testing in three allogeneic combinations. Our data suggest that these cells may originate from immature cortisone-sensitive thymic lymphocytes and also provide some preliminary information concerning their mode of action.     

Abnormal activation and loss of suppressor T cells in the spontaneously hypertensive rat.

Suppressor T cell function in the spontaneously hypertensive rat (SHR) and normotensive Wistar Kyoto (WKY) rats was analyzed using syngeneic mixed lymphocyte reaction (SMLR) and concanavalin A (Con A) activation. A depressed SMLR was found in adult SHR but not in adult WKY. IL-2 synthesized by SHR was 40-fold lower than that of WKY, and the suppressor T cells generated in the SMLR were incapable of suppressing IgG synthesis. Precursors of cells that can be activated by Con A to become functional suppressor cells are reduced in adult SHR. Supernatant fluids derived from Con A-activated spleen cells from adult SHR failed to significantly inhibit IgG synthesis by cultures of syngeneic spleen cells compared to supernatant fluids from young SHR or WKY Con A-activated spleen cells. However, spleen cells from both adult SHR and WKY proliferated strongly and released equivalent amounts of IL-2 in response to Con A. Addition of exogenous IL-2 to the SMLR cultures in vitro restored the ability of SHR T cells to respond in the SMLR, with generation of cells capable of suppressing IgG synthesis. Administration of SHR with IL-2 in vivo also restored the suppressor T cell function in the SMLR. These results suggest a defective suppressor T cell activation and loss of suppressor T cell activity as the SHR age.     

Studies of the T-lymphocytes resident in the spleens of thymectomized, irradiated, bone marrow-reconstituted (TXB) mice.

The T-lymphocytes resident in the spleens of thymectomized, lethally irradiated mice that had been reconstituted with syngeneic bone marrow (TXB) were characterized. Both recently reconstituted N-TXB, (approximately 3 weeks after bone marrow injection) and aged (>6 months after reconstitution) A-TXB animals were studied. The T-lymphocytes from spleens of recently reconstituted N-TXB donors did not respond to PHA but did react significantly to Concanavalin A (Con A). The lack of PHA sensitivity was not due to dilution of reactive cells by other cell types. Removal of adherent cells, likewise, did not restore N-TXB spleen cell PHA responsiveness. N-TXB splenic T-cells were cortisone resistant. N-TXB spleen cells by themselves did not cause a graft vs host response. However, N-TXB spleen cells amplified the graft vs host response of normal lymph node cells but not N-TXB lymph node cells. Addition of cyclic GMP enhanced [³H]thymidine uptake of N-TXB spleen cells caused by Con A. N-TXB spleen cells were exclusively spleen seeking. The Con A reactive cell within N-TXB spleens was demonstrated to be of donor origin. Fetal liver as well as syngeneic bone marrow contained cells capable of reconstituting the Con A response. Spleen cells from aged. (>6 months) A-TXB were found to be PHA sensitive. Competitive inhibition assays measuring θ expression in A-TXB spleen cells indicate a significant increase in the θ positive lymphocyte population occurred with time. The data indicate that considerable reconstitution of θ positive cells had occurred in A-TXB donors. The results also suggest that the T-lymphocyte population of the TXB spleen may be a unique subpopulation of T-lymphocytes that resides exclusively in spleen and bone marrow.     

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.     

Suppressor cells in the spleens of tumor-bearing mice: enrichment by centrifugation on hypaque-ficoll and characterization of the suppressor population.

Spleen cells from mice bearing methylcholanthrene-induced sarcomas or a mammary adenocarcinoma suppressed the mitogen responses of normal spleen and lymph node cells. Lymph node cells from tumor bearers had no suppressive effects. Centrifugation of spleen cells layered on Hypaque-Ficoll (specific gravity of 1.08) produced a dense fraction which pelleted and a light fraction which was retained at the Hypaque-Ficoll-medium interface. Suppressive activity was not found in either fraction of normal spleen cells. In tumor-bearer spleen cells suppressor activity was greatly enriched in the light fraction. Treatment of the suppressor fraction with anti-theta or anti-Ig serum and complement did not remove suppressor activity. However, the suppressor cells were removed by passage through nylon wool or by carbonyl iron treatment. Also, the population which adhered to plastic Petri dishes contained the suppressor cell activity.