In vivo immune response suppression by the supernatant from concanavalin A-activated spleen cells.

Supernatants from concanavalin A- (Con A) activated murine spleen cells have been shown to suppress the in vitro plaque-forming cell (PFC) response to sheep red blood cells (SRBC). The present study examined the effect of such Con A-activated spleen cell supernatants (herein termed CONS) on the in vivo immune response to SRBC in C57BL/6, BALB/c and CDF1 mice. CONS derived from BALB/c spleen cells suppressed direct PFC 4 and 8 days after immunization with 2 X 10(8) SRBC. CONS also suppressed indirect PFC 8 days after immunization, as well as serum hemagglutinins to SRBC. The PFC response of C57BL/6 (H-2b) mice was suppressed as much as that of BALB/c (H-2d) by CONS derived from BALB/c mice, indicating a lack of H-2 specificity of the CONS. In addition to suppression of the antibody response to SRBC, in vivo CONS administration resulted in reduction in spleen cell number. This reduction was not sufficient to explain the decreased PFC response. When the CONS was separated into less than 10,000 m.w. and greater than or equal to 10,000 m.w. fractions, the immunosuppressive activity was found in the less than 10,000 m.w. fraction. This observation suggests that intact interferon, SIRS, and MIF were not responsible for the results obtained.     

Epitopic suppression in synthetic vaccine models: analysis of the effector mechanisms

The induction of an immune response against synthetic peptides usually requires the use of an immunogenic carrier. The use of tetanus toxoid (TT) has been proposed for this purpose as it is highly immunogenic and has been used extensively in humans. Previous studies have demonstrated that an epitope-specific suppression of IgG antibody responses occurs when mice previously primed with TT are subsequently immunized with SODP, a haptenic epitope linked to TT. In the present investigation, we characterized the effector populations which regulate anti-SODP antibody responses in TT/TT-SODP immunized mice. In vitro studies showed that epitopic suppression did not arise due to nonspecific suppressor phenomena. Coculture experiments demonstrated that epitopic suppression was partially mediated by suppressor T cells which specifically inhibited the anti-hapten but not the anti-carrier antibody response. The majority of these T cells were shown to possess the Lyt-2+ phenotype. Apart from the T suppressor population we demonstrated a deficiency at the B-cell level which contributed to the total suppressive effect. Epitopic suppression, therefore, resulted from the effects of dual specific suppressor mechanisms.     

Selective impairment of B cell function by Neisseria meningitidis

Spleen cells from CBA/J mice infected with Neisseria meningitidis displayed depressed in vitro plaque-forming cell (PFC) responses to T-dependent (sheep red blood cell; SRBC) and T-independent (TNP-LPS, TNP-Ficoll) antigens. The inhibition was observed over a wide range of antigen concentrations. The decreased responsiveness of splenocytes from infected mice was due to a selective impairment of B-cell function since helper-T-cell activity was intact in infected mice as shown by the ability of T-enriched lymphocytes to cooperate with normal B-enriched lymphocytes in the generation of an anti-SRBC response, accessory macrophage function was preserved since adherent spleen cells from bacteria-injected mice were shown to produce normal or increased levels of IL-1 and were able to cooperate with normal non-adherent spleen cells in the generation of PFC against SRBC. Addition of peritoneal cells from normal animals or extraneous IL-1 both failed to restore normal PFC responses in cultures of splenocytes from infected mice. Finally, B-enriched lymphocytes from infected mice produced poor anti-SRBC responses when cultured with either Con A supernatant or T-enriched lymphocytes from normal or infected mice. Cell-mixing experiments failed to detect the presence of suppressor cells in cultures of unfractionated spleen cells or B-enriched lymphocytes from infected mice. Therefore, the immunological unresponsiveness associated with a Neisseria meningitidis infection was attributed to a meningococcus-induced defect(s) in B-cell function. In vivo polyclonal B-cell activation leading to clonal exhaustion did not play a major role in the depression of humoral responses since meningococcal infection induced little or no polyclonal Ig secretion.     

Roles of I-E molecule and CD28 costimulation in induction of suppression by staphylococcal enterotoxin B in vivo.

Exposure to bacterial superantigens leads to the induction of a subsequent state of immune hyporesponsiveness. Using a transwell coculture system, a previous report demonstrated that splenocytes from staphylococcal enterotoxin B (SEB)-injected BALB/c mice secreted soluble mediators to suppress the proliferative response of naive syngeneic splenocytes to SEB stimulation. We show in the present study that, in contrast to the suppressive effect induced by SEB in BALB/c (H-2(d) haplotype), MRL(+/+), and MRL-lpr/lpr (H-2(k)) mice, SEB-primed splenocytes from I-E(-) strains such as B6, B10, A.BY (H-2(b)), and A.SW (H-2(s)) mice failed to inhibit the CD25 expression and the proliferative activity of their syngeneic naive responder splenocytes. Further results revealed that the SEB-primed cells from BALB/c, but not B6, mice inhibited the CD25 expression and proliferation of naive responder cells from either BALB/c or B6 mice, indicating the critical regulatory role of the effector cells. Unlike SEB, staphylococcal enterotoxin A induced profound suppression in both BALB/c and B6 mice. Moreover, the suppressive competence of SEB-primed splenocytes was diminished in CD28-deficient BALB/c mice. Taken together, our results indicate that when SEB is used as a stimulator in vivo, both the I-E molecule and CD28 costimulation are required for the induction of regulatory cells bearing suppressive activity.     

Immunoregulatory pathways in adult responder mice. II. Regulation of delayed-type hypersensitivity responses by GAT-specific suppressor factors present in GAT-tolerant adult responder mice

We studied the effects of T cell extracts from adult responder BALB/c mice tolerized with poly(Glu60Ala30Tyr10) (GAT)-coupled syngeneic spleen cells (GAT-SP) on delayed-type hypersensitivity (DTH), T cell-proliferative (Tprlf), and plaque-forming cell (PFC) responses. Adult responder mice injected i.v. with GAT-SP develop Lyt-1-2+ suppressor T cells (Ts), which suppress the induction of GAT-specific DTH and PFC, but not Tprlf responses. Sonicates from these Ts contain an afferent-acting, soluble factor(s) (GAT-TsFdh) that specifically suppresses the same responses as the intact Ts (i.e., DTH and PFC, but not Tprlf). Immunosorbent chromatography studies were employed to determine the molecular nature of the suppressive material active on both cellular and humoral responses. In both assay systems, GAT-TsFdh was found to bear determinants encoded by the I subregion of the H-2 complex and a receptor(s) for GAT. BALB/c-derived GAT-TsFdh suppressed the induction of GAT DTH in syngeneic BALB/c and H-2-compatible B10.D2, but not in allogeneic C57BL/6 or CBA/Cum, suggesting a possible H-2 restriction in the suppression. It was also shown that one target of functional regulation by GAT-TsFdh is the T helper cell for DTH responses (DTH-Th). The results suggest that similar Ts and TsF regulate humoral and cell-mediated responses, perhaps by affecting a target common to both pathways (e.g., the T helper cell). The resistance of Tprlf responses to suppression by GAT-TsFdh indicates that the effector DTH-Th target is not a major component of the proliferative response. These data are discussed with respect to GAT-specific TsF-regulating PFC responses, which have been identified in nonresponders and in responders tolerized as neonates with GAT.     

Oral administration of chitin down-regulates serum IgE levels and lung eosinophilia in the allergic mouse

Previous studies showed that local macrophages phagocytose nonantigenic chitin particles (1-10 micrometer polymers of N-acetyl-d -glucosamine) through mannose receptors and produce IL-12, IL-18, and TNF-alpha. These cytokines lead to the production of IFN-gamma by NK cells. To determine whether chitin could down-regulate Th2 responses, chitin was given orally (8 mg/day for 3 days before and 13 days during ragweed allergen immunization) in BALB/c and C57BL/6 mice. These ragweed-immunized mice were given ragweed intratracheally on day 11. Three days after the challenge, the immunized mice with saline (controls) showed increases in serum IgE levels and lung eosinophil numbers. The chitin treatment resulted in decreases of these events in both strains. To dissect the inhibitory mechanisms of Th2 responses, spleen cells (4 x 106 cells/ml) isolated from the ragweed-immunized mice (controls) were cultured in the presence of ragweed and/or chitin for 3 days (recall responses). Ragweed alone stimulated the production of IL-4 (0.6 ng/ml), IL-5 (20 U/ml), and IL-10 (3.2 ng/ml), but not IFN-gamma. Ragweed/chitin stimulation resulted in significant decreases of IL-4, IL-5, and IL-10 levels and the production of IFN-gamma (48 U/ml). Moreover, spleen cells isolated from the chitin-treated mice showed ragweed-stimulated IFN-gamma production (15 U/ml) and significantly lower levels of the Th2 cytokines, suggesting that the immune responses were redirected toward a Th1 response. Collectively, these results indicate that chitin-induced innate immune responses down-regulate Th2-facilitated IgE production and lung eosinophilia in the allergic mouse.     

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.     

Regulation of IgG1 and IgG2 subclass expression by adjuvant-activated splenic suppressor T cells

Spleen cells from mice immunized with different adjuvants are able to suppress secondary in vitro IgG plaque-forming cell (PFC) responses. The suppressive effect is mediated by Lyt-2-positive T cells. IgG subclasses are affected differentially depending on the number of T suppressor (Ts) cells added in the assays. At low Ts cell concentration IgG2a and IgG2b PFC responses are selectively inhibited. At higher Ts cell concentration IgG1 responses could also be completely inhibited, but IgA and IgM responses are not affected. Suppressor cells responsible for IgG1, IgG2a, and IgG2b suppression are never found in the draining lymph nodes of adjuvant-immunized animals.     

<Emphasis Type="Italic">Brucella abortus</Emphasis> phosphoglyceromutase and dihydrodipicolinate reductase induce Th1 and Th2-related immune responses

Brucellae are intracellular bacterial pathogens that cause Brucellosis, bringing great economic burdens to developing countries. The pathogenic mechanisms of Brucella are still poorly understood. Earlier immune response plays an important role in the Brucella infection. Phosphoglyceromutase (PGM) and dihydrodipicolinate reductase (DapB) were cloned, expressed, purified, and their immunocompetence was analyzed. Cytokines were detected by murine macrophages (RAW 264.7) and splenocytes that stimulated with the two recombinant proteins. The immune responses were analyzed by ELISA from mice with the two recombinant proteins immunized. TNF-α, IL-6 and IL-8 were produced in stimulated RAW 264.7 cells and splenocytes. Th1-type cytokines, IFN-γ and IL-2, induced in RAW 264.7 cells and splenocytes were higher then Th2-type cytokines, IL-4 and IL-5. Th2-related immune response was induced in splenocytes obtained 35 days after mice immunized with the two proteins. The production of IgG1 was higher than IgG2a in immunized mice. Taken together, our results demonstrated that the two proteins could induce Th1 and Th2-type immune responses in vivo and in vitro.     

Modulation of mouse anti-SRBC antibody response by placental extracts

Mouse placental extracts (PE) and corresponding Sephadex G-200 fractions were administered to isogeneic CBA mice along with an optimal immunizing dose of SRBC. Spleen cells were harvested 8 days later and transferred to CBA recipients, subsequently immunized with SRBC. The immunoregulatory activity of spleen cells from PE-treated donors was compared to cells from liver extract (LE)-treated controls or from mice immunized with SRBC only, using Cunningham's PFC direct and indirect tests. Within the dose range used, selective modulatory activities were obtained with cells from PE, but not from LE, treated mice, the latter being comparable to cell transfer effects from donors immunized with SRBC only. Spleen cells from animals injected with low doses of PE (0.25 to 4 mg per mouse) added to immunizing SRBC had a suppressive effect on the primary IgM response of recipients immunized against SRBC. In contrast, when SRBC were given to donor animals with higher doses of PE (8 to 13 mg), transferred spleen cells potentiated the IgM response of the recipients. These opposite suppressive and potentiating activities were found in distinct Sephadex G-200 fractions of 40 and 60 kDa, respectively. When the effect of PE treatment was tested within the same animal, the indirect secondary PFC response following a challenge with SRBC was significantly modified. We observed an overall suppression of the different isotypes after treatment with lower doses of PE or with its 40-kDa fraction. PE doses of 0.5 to 2 mg resulted in a stronger inhibition of IgM than IgG₁ production. This phenomenon was also obtained with the 40 KDa fraction. IgG₂ responses were significantly reduced by all doses of this fraction. In contrast, all doses of the 60-kDa fraction gave a strong stimulation of IgG₂ and IgM responses and a constant suppression of the IgG₁ response. This shows a clear dissociation between IgG₁ and C'-fixing (IgM, IgG₂) antibody classes as far as the influence of placental substances is concerned in their regulation. These data emphasize the relevance of isogeneic placental products as a useful physiological material capable of modulating xenogeneic immune responses (as well as allogeneic systems).     

In vitro immunization. Effect of growth and differentiation factors on antigen-specific B cell activation and production of monoclonal antibodies to autologous antigens and weak immunogens

The antigen-specific activation of murine nonimmunized B lymphocytes subsequently used in hybridization experiments has been investigated by using phylogenetically conserved antigens or autologous immunogens. This in vitro immunization was supported by B cell growth and differentiation factors derived from phorbol myristate acetate-stimulated EL-4 thymoma cells and mixed lymphocyte cultures (MLC). A filter immuno-plaque assay was used to evaluate the effect of different activation procedures on the number of antigen-specific plaque-forming cells (PFC). We first determined the requirement for MLC-derived lymphokines in the in vitro immunization. An optimal number of antigen-specific PFC was obtained when using 33 to 50% of the supernatant from a 48-hr MLC to support the activation. B cell growth and differentiation factors derived from EL-4 cultures were then tested for their abilities to potentiate the number of PFC by using both unseparated spleen cells and highly purified Ig-positive B cells as target cells. The combination of lymphokines found in supernatants from 25% EL-4 thymoma culture and 33% MLC yielded the highest number of PFC when used to support an in vitro immunization. This optimal factor preparation was used to determine the kinetics (4 to 7 days) and the dose response (0.01 to 10 micrograms antigen/ml) of antigen-specific B cell activation before using the immunized splenocytes as parental cells in cell fusion experiments. Mouse albumin and hemoglobin, actin (25 micrograms/ml), RNA polymerase II (5 micrograms/ml), as well as syngeneic mouse serum were used to immunize BALB/c spleen cells in vitro. We obtained antigen-specific PFC by using all of the different immunogens, including syngeneic mouse serum, and the in vitro immunized cells were then used in hybridization experiments. The specific efficiencies of each fusion that made use of cells immunized with mouse albumin, hemoglobin, syngeneic mouse serum, actin, or RNA polymerase II were 12, 31, 33, 52, and 22%, respectively, which illustrated the apparent lack of immune tolerance found when the immunization was performed in culture.     

Soluble factors produced during an immune response regulate Ia antigen expression by murine adenocarcinoma and fibrosarcoma cells

LT-85 is an alveologenic adenocarcinoma of C3Hf/HeN mice. Comparisons of the in vitro and in vivo surface properties of these cells revealed that under normal conditions, they expressed I-A and I-E antigens iv vivo only. By using clonally derived cells, it was established that this phenomenon was not due to the selection of an Ia antigen-positive tumor cell subpopulation, but resulted from phenotypic conversion of Ia antigen-negative tumor cells. These tumor cells and 1053 cells (a fibrosarcoma of C3H/HeN MTV- mice) could, however, be induced to express I-A, I-E, and much higher levels of H-2 antigens in vitro by co-culturing them with spleen cells from LT-85 tumor-bearing C3H/HeN MTV- mice. In vitro induction of Ia and H-2 antigens did not result from contaminating splenocytes or from antigen transfer, because splenocytes from BALB/c (H-2d) mice immunized with A/J (H-2k/d) cells were able to induce the expression of Iak antigens by both tumor cell lines. It was found that this phenomenon was neither H-2-restricted nor antigen-specific. The results clearly indicated, however, that an immune response was required to generate phenotypic conversion of the tumor cells, both in vivo and in vitro. It was further found that soluble, rather than cellular, factors produced during an immune response induced the expression of Ia antigens by LT-85 and 1053 tumor cells. In contrast to what has been reported about the induction of Ia antigens on macrophages and normal epithelial and endothelial cells, the induction of Ia antigens on LT-85 and 1053 cells did not appear to require T cells, and did not involve gamma-interferon. These findings demonstrate that some tumor cells are capable of altering their MHC antigen phenotype in response to factors produced during an immune response in vivo or in vitro. Because of the involvement of Ia antigens in several aspects of immune phenomena, the ability of tumor cells to differentially express Ia antigens in response to environmental factors may have profound effects on host-tumor interactions. Furthermore, the differences seen in the phenotypes of tumor cells grown in vitro and in vivo suggest that in vitro methodologies of tumor cell characterization may not present a complete picture of the natural state of the tumor cell surface.     

Prostaglandin-mediated suppression of delayed-type hypersensitivity to infected erythrocytes during Babesia microti infection in mice

The mechanism of suppression of delayed-type hypersensitivity (DTH) to intraerythrocytic Babesia microti which occurs during infection in mice was examined. The suppression was not specific for anti-parasite DTH; infected mice immunized and challenged with sheep red blood cells had a similar depression of anti-sheep red blood cell DTH. Sublethal or lethal irradiation did not significantly alter the suppression of the DTH response, and cyclophosphamide pretreatment of infected mice also had no effect on suppression. Multiple passive transfer experiments using serum or regional lymph node cells from immunized or infected and immunized (suppressed) donor animals failed to demonstrate any ability to transfer suppression of DTH. Adherent cells from the spleens or peritoneal exudates of suppressed mice, however, did significantly depress the ability of immunized mice to express a DTH response. The cells responsible for this suppression were Thy 1- and nonspecific esterase+. Treatment of suppressive cell populations with 10 micrograms/ml indomethacin for 24 hr in vitro abrogated their suppressive ability, and in vivo administration of indomethacin to suppressed mice also restored DTH to normal levels. By examining levels of prostaglandin E2 (PGE2) in supernates of cultured peritoneal exudate cells from immune or suppressed mice, it was shown that infected mice had peritoneal exudate cells which produced significantly more PGE2 than similar cells from immune mice. These data suggest that B. microti infection elicits synthesis of PGE2 by macrophage-like cells which results in suppression of DTH to parasite as well as heterologous antigens.     

Multiple suppressive effects of transforming growth factor beta 1 on the immune response in chickens

The immunosuppressive effect of human recombinant TGF-beta 1 on chicken immune responses in vitro was evaluated. TGF-beta 1 at 1-10 ng/ml reduced T cell proliferation in response to concanavalin A by 50-80% and B cell proliferation in response to LPS by greater than 90%. In contrast, when added to immune spleen cells, it reduced the secondary PFC response to sheep erythrocytes by less than 50%, particularly when added at the same time as antigen on Day 2 of incubation. When TGF-beta 1 was added during a 2-day incubation to nylon wool-nonadherent immune or normal spleen cells, it caused the maintenance and/or appearance of suppressor cells. These suppressor cells, in coculture with immune spleen cells, inhibited the secondary PFC response in vitro without any further exposure to TGF-beta 1. The phenotype of the cells giving rise to suppressor cells under the influence of TGF-beta 1 was CT8+, TCR2+(alpha,beta), CT4-, TCR1-(gamma,delta) cells. The results suggest that, in addition to direct suppressive effects on the proliferation of B cells and of some T cells, TGF-beta 1 may suppress immune responses by maintaining or by promoting the development of suppressor T cells.     

Suppressor cells in tumor-bearing mice capable of nonspecific blocking of in vitro immunization against transplant antigens.

Spleen cells from mice with progressively-growing methyl-cholanthrene-induced tumors, when immunized in vitro against transplant alloantigens, developed less cytotoxic activity against these antigens as measured by a short-term chromium-release assay than did spleen cells from normal mice. The hyporesponsiveness of spleen cells from the tumor-bearing mice seemed to be due to the presence of suppressor cells which could be removed by nylon-column passage but not by anti-theta treatment and which, in mixture experiments, could inhibit the response of normal spleen cells. The suppression appeared to occur at the sensitization stage and not at the effector stage of the in vitro tests. No evidence was found for mediation of the suppression by soluble factors. These observations emphasize the growing importance of suppressive mechanisms in tumor immune systems.     

Immune suppression induced by protoscoleces of Echinococcus multilocularis in mice. Evidence for the presence of CD8dull suppressor cells in spleens of mice intraperitoneally infected with E. multilocularis.

Immunoregulatory states induced by i.p. inoculation with the metazoan parasite Echinococcus multilocularis in the murine system were investigated. Proliferative responses and IL-2 production induced by Con A in spleen cells from BALB/c mice were significantly depressed at an early stage after infection with E. multilocularis protoscoleces (PSC). Addition of plastic-adherent cells from normal syngeneic mice to the nonadherent spleen cells from infected mice did not restore the depressed Con A responsiveness. On the other hand, exogenous IL-2 reconstituted completely the proliferative responses to Con A. Flow cytometry analysis revealed that CD4- CD8+ cells with a low density of CD8 Ag (CD8dull cells) increased in spleens from infected mice 2 weeks after inoculation. Addition of the spleen cell subpopulation containing the CD8dull cells, but not that depleted of the CD8dull cells, to normal spleen cells resulted in marked suppression of the Con A responses. These findings suggest that the CD8dull cells detected in spleens of mice inoculated with E. multilocularis PSC may play a key role in the suppressive regulation of immune responses. The relevance of the immune suppression seen in the early stages of experimental infection with E. multilocularis PSC to the eventual establishment of a host-parasite relationship is discussed.     

Functional differentiation in the genetic control of murine T lymphocyte responses to human fibrinopeptide B

The ability to generate proliferative and helper T lymphocyte responses in mice was compared by using the 14 amino acid peptide, human fibrinopeptide B (hFPB). Lymph node or peritoneal exudate T cells from mice immunized with hFPB were assessed for in vitro proliferation to soluble hFPB as determined by the uptake of 3H-thymidine. The T cell proliferative response to hFPB was found to be under MHC-linked Ir gene control; mice possessing the H-2a,k haplotypes were responders, whereas H-2b,d,q,s mice were nonresponders. The influence of non-H-2 genes on these responses was not investigated, so exclusive regulation by H-2 is provisional. The absence of a detectable lymph node and peritoneal exudate T cell proliferative response persisted in H-2b,d,q,s mice after immunization and boosting with several doses of hFPB. In addition, the capacity to produce a T cell proliferative response was inherited in an autosomal dominant manner and gene(s) controlling responsiveness to hFPB mapped to the I-A subregion of the H-2 complex. To measure peptide-specific helper T cell activity, an in vitro microculture assay in which hFPB-primed lymph node T cells and normal spleen B cells and macrophages were used was developed measuring anti-fluorescein isothiocyanate (FITC) IgM and IgG plaque-forming cell (PFC) responses after culture with FITC-conjugated peptide. Immunization of B10.BR, C57BL/10, B10.D2, and B6AF mice with hFPB primed for significant helper T cell activity as assessed by the ability to augment a primary in vitro IgM response to FITC. The normal B cell IgM responses were completely dependent on hFPB-primed T cells and required that hapten (FITC) and carrier (peptide) be linked. In addition, immunization with FITC-conjugated peptide elicited positive in vivo PFC responses to FITC in B10.BR and C57BL/10 mice, indicating similar genetic control of helper activity in both the intact animals and the in vitro microcultures. Thus, B10.BR mice show both T help and T proliferative responses to hFPB, whereas C57BL/10 mice show only T help and no T proliferative responses. In contrast to B10.BR mice, C3H and CBA mice immunized with hFPB were completely unresponsive when assayed for helper T cell activity in vitro despite their ability to generate positive lymph node T cell proliferative responses. These results indicate responsiveness to hFPB by T helper and proliferating cells is different and is under separate genetic control.     

H-2 I alloantigens and recall of memory cytotoxic responses

AQR mice were immunized with H-2K and H-2 I encoded alloantigens presented by (Ax6R)F₁ splenocytes. Spleen cells from these alloimmune mice were subsequently restimulated in vitro with B10.A lymphocytes and/or B10.T(6R) lymphocytes, thus presenting them with the immunizing H-2K and H-2 I alloantigens independently. When stimulated with B10.A lymphocytes, alloimmune lymphocytes develop significant cytotoxicity against the immunizing H-2K target antigens. When stimulated with a similar number of B10.T(6R) spleen cells, alloimmune lymphocytes undergo a prominant proliferative response, but develop little, if any, cytotoxicity against the immunizing H-2 K target antigens. The most efficient restimulation of cytotoxicity occurs when the alloimmune spleen cells are simultaneously restimulated by B10.A and B10.T(6R) lymphocytes. Stimulation with the immunizing H-2 I alloantigens alone is not sufficient for regeneration of detectable cytotoxic responses from alloimmune spleen populations. Stimulation with the immunizing H-2K alloantigens alone appears to be both necessary and sufficient to stimulate alloimmune cytotoxic responses. Although the immunizing H-2 I alloantigens are apparently not required to generate alloimmune cytotoxic responses, they markedly potentiate the cytotoxic responses induced by the immunizing H-2K alloantigens.     

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).     

Immunomodulating effects in vitro of interleukin-2 and interferon-gamma on human blood and bone marrow mononuclear cells

Mononuclear cells (MNC) derived from peripheral blood (PBMNC) of 23 normal donors and 4 AIDS patients, and from bone marrow (BMMNC) of 15 normal donors were incubated at 37 degrees C in culture medium alone or in the presence of either natural or recombinant human interleukin-2 (IL-2) or recombinant human interferon-gamma (IFN-gamma; 1-1,000 U/ml). The cultured cells were washed on days 1, 4 or 7 and tested for various immune functions in vitro and for cell surface phenotype. IL-2, but not IFN-gamma, was found mitogenic for both PBMNC and BMMNC. The natural killer (NK) activity of both PBMNC and BMMNC was the only function tested that was markedly augmented (over 100-fold compared to medium control) by both lymphokines. Pretreatment of PBMNC with IL-2 at greater than or equal to 10 U/ml profoundly suppressed (up to 90%) various functions, such as mitogenic responses (phytohemmagglutinin, concanavalin A, pokeweed mitogen), allogeneic mixed leukocyte reaction, antibody production and T cell colony formation in agar. In contrast, some BMMNC functions were elevated at low doses of IL-2 and IFN-gamma, and significant suppression of BMMNC was seen only with high doses of IL-2 (greater than or equal to 100 U/ml) and IFN-gamma (1,000 U/ml). IL-2 was by far more effective than IFN-gamma in both the amplification of NK activity and the suppression of most of the other functions. IL-2, but not IFN-gamma, was found to activate/induce suppressor cells and increased the proportion of Leu-2+ (CD8) cells in PBMNC; the suppressive effect was time- and dose-dependent. The IL-2-induced suppression could be diminished by inclusion of anti-IL-2 antibody during the pretreatment phase. Similar suppressive effects were noted in PBMNC from AIDS patients. These findings suggest that: (a) high-dose IL-2 may elicit immunosuppression which can be mediated by nondiscriminative highly cytotoxic cells (i.e. lymphokine-activated killer cells) and/or by noncytotoxic, nonspecific suppressor cells, and (b) that PBMNC respond differently to the lymphokines than do BMMNC.