Suppression of antibody responses in allogeneic mice by products of lymphoid tissue. I. Allogeneic suppressive factor (ASF) from spleens repopulated with thymus cells.

A cellfree extract prepared from the spleen cells of C3H mice is capable of suppressing antibody responses to SRBC when extract material is exposed to alloantigens. The observed immunosuppression was attributed to a soluble factor in the extract. This allogeneic suppressive factor (ASF) was detected in extracts prepared from the spleen cells of unirradiated mice as well as those of irradiated mice repopulated with thymocytes, provided that mice were previously immunized with SRBC. Donors of actively suppressive ASF preparations did not need to be previously exposed to alloantigens. Extracts from thymus and marrow cells of unirradiated mice and the spleen cells of irradiated mice repopulated with marrow cells (or no cells) did not contain ASF. C3H thymocytes stimulated with SRBC generated more ASF activity in spleens of C3BF1 hosts than in those of C3H hosts, indicating that alloantigenic stimulation enhances the production or activity of ASF. Once produced, C3H ASF was able to suppress antibody responses in cell transfer experiments only if exposed to C3BF alloantigens of either donor lymphoid cells or irradiated hosts. Once exposed to alloantigens, ASF appears to be capable of suppressing antibody responses of syngeneic C3H or semi-allogeneic C3BF cells. When both donor lymphoid cells and hosts were syngeneic with the donor of the ASF, there was enhancement of antibody formation in cell transfer experiments. C3H ASF did not interfere with education of C3BF thymocytes to SRBC or with the generation of precursors of anti-SRBC antibody-forming cells by C3BF1 marrow cells. ASF may interfere with cellular cooperative events necessary for humoral immune responses or with terminal differentiation of B cells. Production of ASF could partially account for the suppression of antibody responses observed during graft-vs-host reactions.     

Morphine inhibits mucosal antibody responses and TGF-beta mRNA in gut-associated lymphoid tissue following oral cholera toxin in mice

In this study, we investigated the effect of morphine on the mucosal immune system using fragment cultures of ileal segments, Peyer's patches (PPs), and mesenteric lymph nodes. Mice were implanted s.c. with a morphine slow release pellet. Control groups received a naltrexone slow release pellet, a placebo pellet, or both a morphine and a naltrexone pellet. After 48 h, mice were orally immunized with cholera toxin (CT) and were boosted orally 1 wk later. Animals were sacrificed 1 wk after the booster immunization, and PPs, mesenteric lymph nodes, and ileal segments were cultured in 24-well plates for 12 days. Morphine resulted in a highly significant inhibition of CT-specific IgA and IgG production in fragment culture supernatants of all three tissues compared with placebo. Naltrexone blocked the reduction in Ab levels induced by morphine, indicating that the effect is opioid receptor mediated. Morphine did not significantly alter total IgA levels in any of the tissue culture supernatants. Morphine also inhibited CT-specific IgA and IgG levels in serum. By flow cytometry, morphine did not alter the lymphoid cell composition in PPs compared with placebo. The effect of morphine on TGF-beta, IL-5, and IL-6 mRNA expression in PPs and ileal segments was determined following oral immunization with CT. Morphine significantly decreased TGF-beta mRNA compared with that in the placebo group, and naltrexone blocked this effect. These results indicate that morphine inhibits Ag-specific IgA responses in gut-associated lymphoid tissue at least partially through the inhibition of TGF-beta, a putative IgA switch factor, in the gastrointestinal tract.     

Macrophage-mediated suppression of immune responses in Toxoplasma-infected mice. III. Suppression of antibody responses to parasite itself

In acute Toxoplasma infection, anti-sheep erythrocytes (SRBC) antibody responses were strongly suppressed in the infected C57BL/6 mice, and the mice produced low titers of only 2-mercaptoethanol (2-ME)-sensitive antibodies but not 2-ME-resistant antibodies. By contrast, the infected BALB/c mice produced much higher titers of both 2-ME-sensitive and -resistant anti-SRBC antibodies than the infected C57BL/6 mice. In anti-Toxoplasma antibody responses, the 2-ME-resistant antibody titers were significantly lower in the infected C57BL/6 mice than in the BALB/c mice in the early phase of infection, suggesting that the suppressive effect of Toxoplasma infection affects antibody responses to Toxoplasma itself as well as to the unrelated antigen, SRBC. A histological study revealed that in the infected C57BL/6 mice, a large number of acid phosphatase-positive, macrophage-like cells infiltrated into the follicles of their spleens, and an involution of follicles occurred in the acute phase of infection. This histological change was not observed in the infected BALB/c mice. The infected C57BL/6 mice, which had the suppressed anti-Toxoplasma antibody responses, made five times as many as cysts in their brains as compared with the BALB/c mice at the fifth week of infection.     

Suppression of in vitro antibody responses by Listeria primed spleen cells.

Spleen cells from mice primed with virulent Listeria monocytogenes do not develop an anti-SRBC plaque forming cell response to SRBC in culture. Furthermore, when Listeria primed spleen cells are co-cultured with normal spleen cells and SRBC, the anti-SRBC response of the normal cells is suppressed. Listeria primed spleen cells from T cell depleted donors are equally effective at immunosuppression. The immunosuppressive effect does not appear to be due to the presence of the bacterium or its products per se in the cultures. Furthermore, the effect cannot be transferred across a 0.45 μm pore membrane. Kinetic studies show that the immunosuppressive effect develops by 2 days post-Listeria inoculation and peaks by Day 6. Low doses of Listeria are not immunosuppressive and produce some enhancement effect. From these results, it is suggested that a population of non-T cell dependent cells develop in Listeria primed hosts that nonspecifically suppress the response of B cells to an unrelated antigen in culture.     

Characterization of suppressive immunoglobulin-binding factor (IBF). II. Purification and molecular weight determination of IBF produced by L-5178-Y theta-positive lymphoma.

L-5178-Y thymoma cells were used to produce radioactive immunoglobulin-binding factor (IBF). For this purpose, the cells were internally labeled by incubation with radioactive amino acids and/or fucose. The supernatants contained radioactive material that bound to IgG-sensitized erythrocytes and suppressed the in vitro antibody response to sheep red blood cells. Upon filtration on Sephadex G-200 both the IgG-binding activity and the suppressive activity eluted at peaks of 140,000 and above 300,000 d. However, on SDS polyacrylamide gels, after precipitation with antigen-IgG-antibody complexes. IBF was found in a single peak of 80,000 d. This molecule could be dissociated in the presence of mercaptoethanol into a major unit of 40,000 d and a minor unit of 20,000 d. These data suggest that IBF is a molecule of 80,000 d, which contains chains of 40,000 d and probably 20,00 d linked by disulfide bridges. In cell supernatants, however, the factor exists in polymeric forms of 140,000 d and more than 300,000 d.     

Suppression of B-cell and T-cell responses by the prostaglandin-induced T-cell-derived suppressor (PITS). I. Analysis of the PITS beta factor

Within populations of mitogenically (PWM) stimulated normal human lymphocytes, the proliferation of B lymphocytes is terminated by T cells. In contrast, T cells limit their own proliferation. T cells thus apparently measure and terminate the proliferation of B cells as well as themselves, suggesting an important role for them in limiting amplification during immune response. Under the culture conditions employed, PWM-induced B- and T-cell proliferation was uncoupled from B-cell differentiation into plasmacytes. Termination of B-cell proliferation in this in vitro model of humoral immune response is independent of B-cell differentiation.     

Characterization of suppressive immunoglobulin-binding factor (IBF). I. Production of IBF by a theta-positive lymphoma (L-5178-Y).

L-5178-Y, a theta-positive, Fc receptor-bearing mouse thymoma cell line spontaneously releases immunoglobulin-binding factor (IBF) upon short-term incubation in vitro. IBF produced by L-5178-Y cells is identical in its biologic activity with IBF produced by Fc receptor positive alloantigen-activated T cells. It suppresses the in vitro plaque response of mouse spleen cells to sheep erythrocytes by interfering mainly with the late phase of the generation of antibody-forming cells. Therefore, L-5178-Y thymoma affords a homogeneous source of IBF in sufficient quantities for the study of its biochemical nature and the mechanism by which it interferes with cells participating in antibody synthesis.     

Suppression of B-cell and T-cell responses by the prostaglandin-induced T-cell-derived suppressor (PITS). II. Resolution of multiple PITS beta factors

It has been shown that T cells cultured with prostaglandin E2 are induced to release at least two peptide-containing lymphokines (PITS). Both the high-molecular-weight (35,000; PITS alpha) and the low-molecular-weight (2000 to 5000; PITS beta) factors were shown to be potent inhibitors of both T-cell- and B-cell-dependent mitogen responses. Data are reported here which show that the PITS beta factor may be reproducibly resolved into seven components by high-pressure liquid chromatography. Although each of these components is capable of suppressing the phytohemagglutinin-induced blastogenic response, not all of these factors will suppress the in vitro antibody response to sheep erythrocytes, or a mixed lymphocyte reaction. Nevertheless, the broad-range suppressive effects previously reported for PITS beta now seem to be mediated by multiple low-molecular-weight PITS factors.     

Studies of immune responses in mice prone to autoimmune disorders. II. Decreased down-regulation by auto-anti-idiotype antibody in autoimmune-prone mice

Three lines of evidence are presented which suggest that autoimmune-prone mice are deficient in the production of auto-anti-idiotype antibody during their immune response to trinitrophenylated Ficoll (TNP-F). NZB, MRL lpr/lpr and older BXSB male mice have no hapten-augmentable plaque-forming cells (PFC). Hapten-augmentable PFC have been previously shown to be cells whose secretion of antibody has been inhibited by the binding of auto-anti-idiotype antibody to cell surface idiotype. Sera from TNP-F immunized NZB mice lack PFC inhibiting activity (anti-idiotype antibody). Spleen cells from TNP-F immune NZB mice fail to transfer anti-idiotype antibody-mediated suppression to naive mice as do spleen cells from immune non-autoimmune-prone mice. Taken together these data suggest that autoimmune-prone mice are deficient in auto-anti-idiotype antibody-mediated downward regulation of their immune responses. It was further shown that the immune response of NZB mice to TNP-F shows a slower decline in splenic PFC and a greater heterogeneity of PFC affinity than do the responses of non-autoimmune-prone strains. Since athymic (nude) mice, which were previously shown to be defective in the production of auto-anti-idiotype antibody, also show a slower decline in splenic PFC and an increased heterogeneity of PFC affinity, it is suggested that these peculiarities of the immune responses of autoimmune-prone and athymic mice are also the consequences of the lack of auto-anti-idiotype antibody-mediated down-regulation.     

Influenza virus-specific human cytotoxic T cell clones: Heterogeneity in antigenic specificity and restriction by class II MHC products

Human cytotoxic T lymphocytes specific for A/JAP/57 (H2N2) influenza virus were cloned from in vitro stimulations of peripheral blood lymphocytes. Analysis of the viral specificity in cytotoxic function revealed one clone that killed all type A influenza-infected targets, another clone that was specific for the hemagglutinin subtype of the immunizing influenza virus, and the third clone that demonstrated cytotoxicity restricted to the hemagglutinin of A/JAP/57 and A/JAP/62 (H2N2) and not other type A influenza strains with the H2N2 subtypes. The phenotype of these three clones was Leu 2^?, Leu 3⁺, Leu 4⁺; MHC restriction of their cytotoxic function was mapped to HLA-DR by a panel of target cells as well as by inhibition of cytotoxicity with monoclonal antibodies. Proliferation of these clones, examined in a tritiated thymidine incorporation assay, was found to be driven by antigen in the absence of exogenous lymphokines. For all three clones antigen-dependent production and secretion of lymphokines with IL-2 activity was demonstrated. The antigen specificity of proliferation and factor production was shown to be identical to the pattern that each clone revealed in its cytotoxic function.     

The B-cell response in lymphoid tissue of mice immunized with various antigenic forms of the influenza virus hemagglutinin.

Protection of BALB/c (H-2d) mice against secondary challenge with influenza A viruses is primarily dependent on appropriate recognition of the hemagglutinin (HA) molecule by effectors of humoral immunity, the B lymphocytes and their product the immunoglobulin molecules. The influence of the antigenic form of the HA in eliciting protective antibodies is not clearly defined. We directly monitored the kinetics, character, localization, and helper T-cell dependence of the primary antibody-forming cell (AFC) response and the development of B-cell memory in lymphoid tissues associated with the upper and lower respiratory tracts, and in the spleen and bone marrow, to three forms of HA with various degrees of antigenic organization. Our results show that the antigenic organization of HA substantially influences B-cell immunity, namely, the capacity to generate both primary AFCs and memory B cells responsive to lethal challenge. Immunization by infection is the most efficient means of generating protective memory B cells, in contrast to subunit vaccine. The data also indicate that memory AFCs are predominantly localized to the regional lymphoid tissue where challenge HA is found, unlike primary AFCs, which are restricted to the priming site and which require in vivo CD4+ T-cell help.     

Suppression of the in vitro secondary antibody response of rabbit lymphoid cells by Concanavalin A.

The effect of various concentrations of concanavalin A (Con A) on the in vitro secondary antibody response of rabbit lymph node and spleen cells to sheep red blood cells (SRBC) was studied. Complete suppression of the IgM plaque-forming cell (PFC) response of both lymph node and spleen cultures was observed when 10 mug/ml of Con A was added at the time of initiation of the cultures whereas only partial suppression was observed when 1 mug/ml of Con A was added. Moreover, marked suppression of the immune responses of both spleen and lymph node cultures was observed when 10 mug/ml of Con A was added at 24 hr after antigenic challenge and to a lesser extent when added at 48 hr. Suppression of the IgM PFC response was also detected when spleen cultures were exposed to 10 mug/ml of Con A for as little as 2 hr after antigenic challenge. However, substantial increases in DNA synthesis were observed only in those cultures which were in contact with Con A for at least 24 hr. Finally evidence is presented that the Con A-induced suppression is mediated by a soluble substance(s).     

Suppression of in vitro antibody response by ribosome-associated factor(s) from interferon-treated cells.

Initiation factor preparation (eIF-IF) from mouse L cells treated with virus-type interferon suppressed the in vitro plaque-forming cell (PFC) response to sheep red blood cells. The eIF-IF preparation had previously been shown to block formation of the ternary complex Met-tRNA_f-eIF-GTP. The formation of this complex is a necessary step in initiation of protein synthesis. Initiation factor preparation (eIF) from untreated L cells affected neither the PFC response nor the participation of eIF in the formation of ternary complex. The induced factor was shown not to be Interferon by antibody neutralization experiments with anti-interferon. The factor must be present in the PFC cultures during the early stages of antigen induction in order to suppress the immune response. Speculatively, eIF-IF may act at the level of the macrophage, perhaps entering the cell by pinocytosis. This may account for its inability to inhibit virus replication in L cells. The production of the inhibitory factor is blocked or partially blocked by actinomycin D. It is possible that this factor is a mediator of the immunosuppressive effects of virus-type interferon. This is the first report of biological activity on cells, which is associated with a ribosome-associated factor induced by interferon.     

Suppression of cell-mediated immune responses after total lymphoid irradiation (TLI). I. Characterization of suppressor cells of the mixed lymphocyte reaction

Total lymphoid irradiation (TLI) was administered to (BALB/c X C57BL/6)F1 mice in eight daily doses of 200 rad (total 1600 rad). Spleen cells isolated from mice after treatment with TLI do not respond to alloantigens in vitro in a one-way mixed lymphocyte reaction (MLR), but normal reactivity recovers after approximately 2 mo. Radioresistant, antigen-nonspecific suppressor cells are documented in the spleens of TLI-treated mice immediately after radiotherapy, but suppressive capacity gradually disappears within 30 days. After TLI, the spleen is repopulated with large cells, the proportion of which is greatest at a time when theta-bearing cells are still depleted. Radioresistant suppression is mediated predominantly by the large cell subset and is thymus independent. Suppressor function can be abolished by lethal physicochemical procedures including formaldehyde fixation, multiple freeze-thawing, and heating to 56 degrees C, and it cannot be conferred by supernatants of TLI-suppressed MLR suspensions. Suppression cannot be overcome by adding various cell factors including T cell growth factor (TCGF) and lymphocyte-activating factor (LAF), nor is it affected by a prostaglandin inhibitor. Equally potent radioresistant suppressive activity is documented by co-culturing cells derived from other sources enriched in large, immature hematopoietic cells, including fetal liver cells and bone marrow cells obtained from normal and congenitally athymic mice. The presence of a large cell population and MLR suppressor function is also documented in the spleens of mice treated with single dose or fractionated doses of lethal whole body irradiation, followed by reconstitution with bone marrow cells obtained from normal mice. The data suggest that MLR suppressor cells, which are large, immature and predominantly radioresistant, can be induced after a short and well-tolerated TLI regimen.     

Effects of deoxycoformycin in mice. I. Suppression and enhancement of in vivo antibody responses to thymus-dependent and -independent antigens

The effect of 2'-deoxycoformycin (DCF) on the PFC responses of AKR mice to SE, TNP-Ficoll, and TNP-B. abortus was examined. Subcutaneous injection of DCF 4 days before antigen caused suppression of all three responses by 70 to 78%. In contrast, injection of DCF 1 day after antigen caused enhancement of both the anti-SE and the anti-TNP-Ficoll responses. Although a single high dose of cortisone acetate injected 4 days before antigen caused a similar suppression, the effect of DCF was not mediated via a steroid release, inasmuch as DCF also suppressed the immune response in adrenalectomized mice. The response of BALB/c mice to TNP-Ficoll was also inhibited by DCF pretreatment and enhanced by injection of DCF after antigen. In contrast, in athymic mice DCF caused suppression of the anti-TNP-Ficoll PFC response, whether injected before or after antigen. These results are interpreted as suggesting that DCF causes suppression primarily via an effect on B cells. The enhancement seen in normal but not in athymic mice may possibly be ascribed to an effect on suppressor T cells. Apparently the enhancement of both TD and TI responses caused by DCF injected 1 day after antigen in normal mice is the net result of these two opposing effects. The results imply that helper T cells are resistant to DCF.