The adjuvant activity of synthetic N-acetylmuramyl-dipeptide: evidence of initial target cells for the adjuvant activity.

MurNAc-l-Ala-d-isoGln (N-acetylmuramyl-l-alanyl-d-isoglutamine, MDP), a synthetic compound, acts as an adjuvant on the humoral immune response and on the T cell-mediated immune response. In this report, we attempted to directly demonstrate the initial target cells of MDP for its adjuvant activity in vitro by using cell separation procedures.It was demonstrated that MDP enhanced the immune response following direct interaction with antigen-stimulated T and B lymphocytes, but nonstimulated lymphocytes, shortly after triggering by antigen, and that there was no macrophage requirement for MDP to elicite the adjuvant action in the primary anti-SRBC PFC response in vitro. It has also been demonstrated that the adjuvant activity of MDP is due to an enhancing effect which is different from the possible mitogenic activity to spleen cells and MDP replaces neither a function of macrophages, which is substituted by 2-mercaptoethanol nor a helper function of T cells.     

Inhibitory and stimulatory effects of a synthetic glycopeptide (MDP) on the in vitro PFC response: factors affecting the response.

A synthetic adjuvant active glycopeptide, N-acetyl-muramyl-l-alanyl-d-isoglutamine (MDP), has been previously shown to enhance the in vitro immune response of mouse spleen cells to T-dependent or independent antigens. Data presented here show that the net activity of MDP on the in vitro immune response is closely related to the cell culture conditions: Two distinct patterns of MDP activities could actually be detected. Marked stimulation of the PFC response was observed at “low density” cell cultures. In contrast, suppression could be seen at “high density” cell cultures. Moreover, the culture conditions which permitted characterization of either the enhancement or suppression of the immune response by MDP were strongly dependent on the strain of mouse used. However, these activities were not dependent on antigen concentration, on kinetics of responses, or on cytotoxic effects.     

In vitro spleen cell responsiveness to various analogs of MDP (N-acetylmuramyl-L-alanyl-D-isoglutamine), a synthetic immunoadjuvant, in MDP high-responder mice

N-Acetylmuramyl-l-alanyl-d-isoglutamine (MDP), a synthetic immunoadjuvant, was incubated with spleen cells of DBA/2 or Balb/c mice and optimal responses were obtained after 4 or 5 days of culture in a serum-free medium supplemented with 2-mercaptoethanol. In contrast, lymphocytes of (C57B1/6 × AKR)F₁ hybrids responded weakly under the same conditions. The results reported here show that like in the case of DBA/2 and Balb/c strains, spleen cells of Swiss mice and of inbred AKR and CBA mice could be stimulated in vitro whereas C57B1/6 and LPS-refractory C3H/He mice did not respond. Fourteen synthetic MDP analogs (eight known to be adjuvant active and six devoid of activity) were tested in DBA/2 high-responder mice. A good correlation was observed between in vitro stimulation and the presence or absence of adjuvant activity in vivo of these compounds.     

Target cells for the activity of a synthetic adjuvant: muramyl dipeptide.

Muramyl dipeptide (MDP), a synthetic adjuvant, increased the primary response of CBA mice to sheep red blood cells (SRBC). In reconstituted irradiated recipients, cooperation between T and B lymphocytes was required for the expression of adjuvant activity and MDP increased the efficiency of SRBC-educated T cells. The role of T-derived lymphocytes in mediating the MDP adjuvant activity was also demonstrated in irradiated mice and in mice reconstituted with various splenic cellular types of donors which had received SRBC and MDP 24 hr earlier. In our experiments, the macrophage did not seem to be involved, since MDP did not increase the phagocytic capacity of peritoneal exudate cells and MDP- and SRBC-pretreated macrophages had no increased ability to induce an anti-SRBC immune response. These results demonstrate the importance of T lymphocytes as mediators of the adjuvant activity of MDP.     

The immunoregulatory effects of edeine analogues in mice

The edeines analogs were tested in several in vitro and in vivo assays using the mouse model, with edeine B (peptide W1) and cyclosporine A as reference compounds. The peptides displayed moderate, stimulatory effects on concanavalin A-induced (ConA-induced) splenocyte proliferation, whereas their effects on pokeweed mitogen-induced (PWM-induced) splenocyte proliferation were inhibitory. The peptides inhibited lipopolysacharide-induced (LPS-induced) tumor necrosis factor alpha production but had little effect on interleukin 6 production. In the model of the humoral immune response in vitro to sheep red blood cells, peptide 1 was distinctly stimulatory in the investigated concentrations (1-100 μg/ml), whereas peptides 3 and 4 only stimulated the number of antibody-forming cells at the highest concentration (100 μg/ml). In the model of the delayed type hypersensitivity in vivo to ovalbumin, the peptides were moderately suppressive (3 being the most active). The reference peptide W1 stimulated ConA-induced cell proliferation at 1–10 μg/ml but was inhibitory at 100 μg/ml. It also inhibited PWM-induced cell proliferation in a dose-dependent manner. This peptide had no effect on the humoral immune response in vitro or on cytokine production, but inhibited DTH reaction in vivo. The relationship between structure and activity, and a possible mode of action of the peptides, is discussed in this paper.     

Suppressor T-cell activity induced as a result of thermal injury.

Many thermally injured patients survive their initial trauma only to succumb to infection at 2 to 4 weeks after the burn. Both clinical and experimental data have suggested that acute thermal insult compromises immune function. In this report we have sequentially examined the ability of thermally injured mice to generate a specific in vitro primary antibody-forming cell (AFC) response to sheep red blood cells (SRBC) at various times after thermal injury. Thermally injured mice appear to lose the ability to generate de novo antibody-forming cells in vitro after thermal injury. The defect was dissected as to the involvement of macrophage (φ), thymus-derived cell (T-cell), or bursal equivalent (B-cell) defects. Murine B cells from burned animals exhibited normal immunological function in the in vitro AFC system. T cells from burned mice were demonstrated as not only dysfunctional in the generation of immune AFC, but also as able to suppress generation of an AFC response by syngeneic normal cells.     

Antibody formation by one or both lymphoid populations present in chimeric marmosets

Development of an isoimmune serum capable of identifying a specific leukocyte antigen in the marmoset, Saguinus fuscicollis illigeri, permitted detection of lymphoid cell chimerism in this species by the cytotoxic test. This reagent was then used to identify the cell population in the chimera responsible for antibody production against a test antigen, sheep red blood cells. Primary in vitro antibody formation as measured by plaque-forming cells with blood leukocytes or splenic lymphocytes of six animals chimeric for the leukocyte antigen, MLA-1, revealed an immune response by both cell types of the chimeric population from three animals and a response by only one cell type in the other three.     

Structural specificity of synthetic peptide adjuvant for induction of experimental allergic encephalomyelitis

The peptide N-acetylmuramyl-l-alanyl-d-isoglutamine (MDP), which has adjuvant activities, and 17 of its derivatives and analogs were synthesized and assayed to elucidate the structure necessary for adjuvant activity in induction of experimental allergic encephalomyelitis (EAE) in guinea pigs. The results revealed the importance of the d configuration and the α-carboxamide group of the isoglutaminyl residue of MDP for adjuvant activity. Replacement of the l-alanyl residue of MDP by d-alanine, but not by l-serine or glycine, resulted in a marked decrease in the activity. The β-methyl glycoside of MDP was found to be more active than the α-methyl derivative. 6-O-Stearoyl-N-acetylmuramyl-l-alanyl-d-isoglutamme showed activity.     

In vitro studies of the rabbit immune system: I. Hemolytic plaque-forming response of dissociated spleen cells from normal and primed rabbits

The conditions for the in vitro generation of primary and secondary immune responses by rabbit spleen cells to sheep red blood cell (SRBC) antigen have been examined. Spleen cells from many normal and all previously immunized rabbits are capable of producing in vitro plaque-forming cell (PFC) responses when cultured as dissociated cell suspensions in the presence of antigen. Primed spleen cells generate approximately 100 times the number of PFCs obtained in normal cultures with a shorter lag period. Both types of cultures demonstrate a period of exponential increase in PFCs during which the doubling time is 12–14 hr. This increase occurs after 1 day of culture of spleen cells from primed rabbits and after 4 days of culture of spleen cells from unprimed rabbits. The PFCs which arise in cultures of primed cells appear not to be the progeny of those generated in vivo but to be derived from an increased number of PFC precursors. Repeated immunization of the spleen cell donor is required to produce significant numbers of indirect (IgG) PFC or indirect precursors; most of the PFC found after a single immunization in vivo or in vitro are direct (IgM). There is no evidence for conversion of IgM to IgG PFC in vitro. This system should provide a means for further identification of the cellular interactions involved in the immune response of the rabbit.     

Inhibition of in vitro immune response by extracts from long-term cultured lymphoid cells.

Ultrasonic cell extracts prepared from long term calf lymphoid cell cultures (LTE) inhibit the primary in vitro response to sheep red blood cells, either of young calf lymphocytes or of mouse spleen cells. LTE do not affect lymphocyte stimulation by Con A or LPS. The activity disappears after treating LTE with protease. The molecular weight of the active factors estimated by Diaflo ultrafiltration is 10,000 to 15,000 D.     

Augmentation of mouse natural killer cell activity by muramyl dipeptide and its analogs

The ability of muramyl dipeptide (MDP) and its structural analogs (des-MDP, abu-MDP, and des-abu-MDP) to influence mouse natural killer (NK) cells in two different strains of mice was examined. In CBA/J mice, administration of MDP by both intraperitoneal (ip) and intravenous (iv) routes enhanced splenic NK cell activity. Maximum augmentation of NK cell activity was observed 3 days after MDP treatment. NK cell activity was also stimulated upon in vitro culture of CBA/J mouse spleen cells with MDP. Only iv inoculation of MDP to C57BL/6 mice 7 days previously enhanced NK cell activity of spleen cells. Peritoneal NK cell activity was not affected in either strain of mice, regardless of the route of inoculation of MDP. Two structural analogs of MDP, abu-MDP and des-abu-MDP, enhanced peritoneal NK cell activity, whereas des-MDP had no effect when tested 3 days after ip treatment of CBA/J mice with these compounds. Peritoneal NK cell activity of C57BL/6 mice was not modulated by des-MDP, abu-MDP, or des-abu-MDP. A synergistic effect on peritoneal NK cell activity was observed in both CBA/J and C57BL/6 mice treated first with MDP and then with lipopolysaccharide (LPS) or Bacillus Calmette-Guerin (BCG).     

In vivo regulation of humoral and cellular immune responses of mice by a synthetic adjuvant, N-acetyl-muramyl-L-alanyl-D-isoglutamine, muramyl dipeptide for MDP.

In vitro immunizations by T-dependent or T-independent antigens can be modulated by muramyl dipeptide (MDP). Enhancement or suppression of the antibody responses was observed according to the spleen-cell concentrations. Data presented here show that MDP can also suppress the immune response in vivo if used at relatively high dosage and injected before the antigen (SRBC). In vitro generation of cytotoxic T-lymphocyte recovered from mice which had been treated by MDP under the same experimental conditions was also decreased whereas macrophage cytostatic activity was not affected. By MDP pretreatment, a significant increase of antibody-dependent cell-mediated cytotoxicity was observed.     

Cellular immunity in the mouse. V. Further studies on leukemia virus activation in allogeneic reactions of mice: stimulatory parameters.

The relationship between activation of thymic (T)-derived lymphocytes and mouse leukemia virus (MuLV) induction were studied in vivo and in vitro. The results indicate that there is no simple relationship between the severity of GVH, assayed by splenomegaly, alteration of T-cell reactivity in vitro, the activation of mouse leukemia viruses, and the subsequent development of lymphoma. Allogeneic stimulation either in vivo or in vitro is a potent activator of MuLV, as is the drug iododeoxyuridine. However, nonspecific T-cell mitogens such as PHA or Con-A, the drug cyclophosphamide, or specific antigenic stimulus such as sheep red blood cells after in vivo sensitization are not effective virus activators. The source of the cell supporting MuLV replication in vitro appears to be a theta-positive (T) lymphoblast.     

Time and dosage dependence of immunoenhancement by murine type II interferon preparations.

Preparations of Type II immune induced Interferon enhanced the plaque forming cell response of mice to sheep red blood cells both in vivo and in vitro. The enhancement of the antibody response was dependent on the dosage of interferon used and the time of administration of interferon. The expression of the antiviral and immuno-enhancing activities of Type II interferon preparations shared several physical-chemical properties, including pH 2 lability and heat stability. The plaque forming cell response to lipopolysaccharide, a T-independent antigen, could not be enhanced by treatment with Type II interferon. In addition, treatment of spleen cell cultures of nude thymic deficient mice with Type II interferon could not cause an enhancement of the plaque forming cell response to lipopolysaccharide. These data suggest that the immunoenhancing effect of Type II interferon on antibody responses is produced by an effect on T lymphocytes in contrast with the immunosuppressive effect which appears to be mediated through an effect on B lymphocytes.     

Immune response regulation by lymphocyte products. Potentiator and suppressor factors detected during responses to concanavalin A, E. coli lipopolysaccharide and dinitrophenylated bovine serum albumin.

The efferent lymph from immunostimulated popliteal lymph nodes of sheep was analysed for modulatory effects on the in vitro response of peripheral blood lymphocytes to phytohemagglutinin. Both efferent lymph and the supernates prepared from lymph cells exhibited potentiating activity when collected during the first 6 hours of responses to Concanavalin A and dinitrophenylated bovine serum albumin.² Cells collected on the third day of responses to these antigens and to E. coli lipopolysaccharide produced supernates that almost totally suppressed the in vitro response. The possible contribution made by these cells and cell products to the regulation of the immune response in vivo is discussed.     

Immune cytolysis of human tumor cells mediated by xenogeneic "immune" RNA

Normal, nonimmune, human peripheral blood lymphocytes, when incubated with RNA extracted from lymphoid organs of guinea pigs or sheep immunized with human tumor cells, mediated the immune cytolysis of those tumor cells in vitro. Lymphocytes incubated without RNA, or with control RNA preparations, failed to evidence cytotoxic activity. Treatment of the active RNA preparations with ribonuclease abrogated the cytotoxic activity, but treatment with deoxyribonuclease or pronase did not effect activity.     

Formulation,High Throughput In Vitro Screening and In Vivo Functional Characterization of Nanoemulsion-Based Intranasal Vaccine Adjuvants

Vaccine adjuvants have been reported to induce both mucosal and systemic immunity when applied to mucosal surfaces and this dual response appears important for protection against certain pathogens. Despite the potential advantages, however, no mucosal adjuvants are currently approved for human use. Evaluating compounds as mucosal adjuvants is a slow and costly process due to the need for lengthy animal immunogenicity studies. We have constructed a library of 112 intranasal adjuvant candidate formulations consisting of oil-in-water nanoemulsions that contain various cationic and nonionic surfactants. To facilitate adjuvant development we first evaluated this library in a series of high-throughput, in vitro assays for activities associated with innate and adaptive immune activation in vivo. These in vitro assays screened for the ability of the adjuvant to bind to mucin, induce cytotoxicity, facilitate antigen uptake in epithelial and dendritic cells, and activate cellular pathways. We then sought to determine how these parameters related to adjuvant activity in vivo. While the in vitro assays alone were not enough to predict the in vivo adjuvant activity completely, several interesting relationships were found with immune responses in mice. Furthermore, by varying the physicochemical properties of the surfactant components (charge, surfactant polar head size and hydrophobicity) and the surfactant blend ratio of the formulations, the strength and type of the immune response generated (T_H1, T_H2, T_H17) could be modulated. These findings suggest the possibility of using high-throughput screens to aid in the design of custom adjuvants with unique immunological profiles to match specific mucosal vaccine applications.     

Nonspecific activation of murine spleen cells in vitro by a synthetic immunoadjuvant (N-acetyl-muramyl-L-alanyl-D-isoglutamine)

We reported previously that synthetic N-acetyl-muramyl-l-alanyl-d-isoglutamine (MDP) displayed marked adjuvant activity but was devoid of mitogenicity in vitro. The data reported here establish that, under different cultural conditions, thymidine uptake and blast cells can be increased by MDP in spleen cells of DBA/2 and Balb/c mouse strains. Optimal responses were obtained on culture in a serum-free medium supplemented with 2-mercaptoethanol for 4 or 5 days. This effect was also obtained with spleen cells of Balb/c nude mice. When the synthetic MDP was compared to a natural water-soluble adjuvant (neo-WSA), extracted from Mycobacterium smegmatis cells, both were found to stimulate [³H] thymidine incorporation by mouse spleen cells. However, with the neo-WSA, the effect peaked on Day 2 and was weak or absent on Days 4 and 5. When the cells were cultured in a medium containing fetal calf serum, neo-WSA activation was completely abolished, while MDP-mediated stimulation was decreased.     

Immunologic significance of nonspecific suppressor cells in spleens of tumor-bearing mice.

Spleen cells from mice bearing a progressively growing syngeneic tumor failed to respond to stimulation with mitogens in vitro. This lack of reactivity was due to the presence of nylon wool-adherent cells in the population that could inhibit the mitogen response of normal lymphocytes. Paradoxically, at times when strong suppressor cell activity could be detected in tumor-bearing mice, the animals responded normally to in vivo immunization with sheep erythrocytes and allogeneic tumors, and to in vitro sensitization with allogeneic tumor cells. Regression of a highly antigenic syngeneic tumor also was unaffected by the presence of these suppressor cells. Thus, the occurrence of nonspecific suppressor cells in the spleens of tumor-bearing mice did not influence the overall immunologic competence of these animals.     

Differential effects of concanavalin A and phytohemagglutinin on murine immunity. Suppression and enhancement of humoral immunity.

The abilities of concanavalin A (Con A) and phytohemagglutinin P (PHA) to selectively induce different T-cell activities affecting humoral immunity were evaluated. The mitogens were intravenously injected before, with, or after injection of sheep red blood cells (SRBC) into mice, and the 3 to 6-day plaque-forming cell (PFC) responses were assessed. Mitogenic treatment differentially influenced the resultant in vivo PFC responses to SRBC. The in vivo suppressive effects induced by Con A were shown to be temporary; only the Day 4 PFC response was inhibited. Con A given 3 hr before, with, or after the antigenic challenge enhanced the PFC response. In contrast, PHA given at all intervals inhibited both the 4- and 5-day PFC response. Neither mitogen appeared to affect the kinetics of the in vivo PFC response to SRBC. Both mitogens enhanced in vivo DNA synthesis by the splenic cells, and Con A appeared biphasic in its stimulation. Con A-induced effects on the humoral immune response were short-lived and transient, while PHA induced a longer-lasting effect on humoral immunity.