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.     

Opposite modulation of lymph node cell and spleen cell responses to mitogens by muramyl dipeptide and its desmethyl analog

N-acetylmuramyl-l-alanyl-d-isoglutamine (MDP), the minimal structure necessary for adjuvant activity of mycobacterial cell wall preparations, was evaluated as an immunostimulant in mice. MDP treatment, which increased carbon clearance and nonspecific resistance to lethal Klebsiella challenge, induced lymph node cellular hyperplasia (4-fold). In contrast, spleen and resident peritoneal cell recovery was comparable to controls. Lymph node cells (LNC) from MDP-treated mice had enhanced [³H]thymidine uptake in unstimulated (4-fold) and lipopolysaccharide (LPS) (5-fold)-, concanavalin A (Con A) (2-fold)-, and phytohemagglutinin (PHA) (1.5-fold)-stimulated cultures. In contrast, spleen cells exhibited depressed responses when stimulated with LPS (2-fold), Con A (2- to 5-fold), and PHA (3-fold). Depressed responses of spleen cells to mitogens were demonstrated over a range of mitogen concentrations. The desmethyl analog produced similar effects, although spleen cells were not as hyporeactive. Opposing modulations of the immune system by MDP resembles that reported after BCG infection, and correlates with increased nonspecific host resistance to microbial challenge.     

Specific antigens inhibit plasmacytoma cells bearing phosphorylcholine-binding myeloma proteins.

The influence of a synthetic adjuvant active glycopeptide, N-acetylmuramyl-l-alanyl-d-isoglutamine (MDP), and of some of its analogs on the in vitro immune response to sheep red blood cells was studied using Mishell and Dutton in vitro stimulation system. When MDP and adjuvant active analogs were incubated with normal spleen cells, increased cell recovery was observed after 3 or 4 days of culture, showing a good correlation between the adjuvant activity in vivo and the enhancement of cell viability in vitro. The analogs which were found to have an adjuvant activity in vivo were equally effective in stimulating in vitro both the background hemolytic PFC and the immune response to sheep red blood cells. However, those which were inactive in vivo were effective in vitro but only at high concentration levels.     

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.     

Adenovirus fiber protein (FP) functions as a mitogen and an adjuvant

Fiber protein (FP) from adenovirus serotype 12 and 2 was shown to be mitogenic for lymphocytes of normal BALB/c mice. Maximum increase in [³H]thymidine incorporation was observed with 50–75 μg/ml of adenovirus 12 FP after 48 hr of culture. Also, FP induced blast cell transformation of mouse lymphocytes. The mitogenic activity was abolished with corresponding antiserum. Enriched T cells were not activated by FP, while B cells from athymic nude mice were stimulated to levels of approximately those of whole spleen cells. The stimulatory activity of FP was amplified by the presence of an adherent cell population (probably macrophages). Furthermore, FP served as an adjuvant in vivo, increasing IgM synethesis to SRBCs in mice immunized with FP along with SRBC. The implications of these findings are discussed.     

Transplantation antigen expression on murine trophoblast detection by induction of specific alloimmunity

Cell-mediated immune responses to murine embryonic trophoblast cells were investigated using lymphocyte trophoblast cultures (LTC) and cell-mediated lympholysis (CML). Spleen cells from CBA (H-2^k) or C57BL/6 (H-2^b) mice hyperimmunized with 3.5-day-old Balb/c (H-2^d) blastocysts did not undergo DNA synthesis after in vitro exposure to Balb/c blastocyst outgrowths nor were cytotoxic lymphocytes (CTL) generated against H-2^d alloantigens. Splenocytes from Balb/c mice presensitized with semiallogeneic (Balb/c female × C57BL/6 male) trophoblast cells derived from 17- to 20-day placental tissue expressed a weak proliferative response in the presence of semiallogeneic placental trophoblast and produced a moderate number of CTL against H-2^b (paternal strain) alloantigens when compared to mixed lymphocyte cultures (MLC) between Balb/c responder and semiallogeneic (stimulator) spleen cells. CTL were also generated in vitro after splenocytes from Balb/c mice hyperimmunized with semiallogeneic spleen cells were restimulated in vitro with placental trophoblast cells. These studies showing that early-stage trophoblast cells fail to evoke transplantation immunity and placental trophoblast is capable of generating alloimmunity only after combined in vivo hyperimmunization with in vitro restimulation suggest that these trophoblast cells are poorly immunogenic due in part to the relatively weak functional expression of major transplantation antigens.     

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.     

Polyclonal B Cell Activation by Cell Wall Preparations of Gram-Positive Bacteria

The effects of polyclonal B cell activation (PBA) of cell walls and their cell wall fractions obtained from several kinds of gram-positive bacteria were studied using the anti-sheep red blood cell (SRBC) or anti-trinitrophenylated (TNP) SRBC plaque forming cell (PFC) responses of cultured spleen cells from Balb/c, athymic nu/nu, their littermates (nu/+), C3H/He (LPS-responder), C3H/HeJ (LPS-non-responder), (CBA/N × Balb/c) F1 male with an X-linked defect in B cell function and the F1 female mice. The cell walls of Staphylococcus epidermidis (ATCC 155), Lactobacillus plantarum (ATCC 8014), Micrococcus lysodeikticus (NCTC 2665), Mycobacterium rhodochrous (ATCC 184), Streptomyces gardneri (ATCC 23911) and Nocardia corynebacteriodes (ATCC 14898) had the ability to induce polyclonal B cell responses in the spleen cells of Balb/c, nu/nu, nu/+, C3H/He and C3H/HeJ mice. The cell wall fractions prepared by enzymatic digestion from the cell walls of S. epidermidis, S. gardneri or N. corynebacteriodes were also capable of inducing polyclonal B cell responses. The responses of spleen cells from (CBA/N × Balb/c) F1 male mice to these active preparations, except the cell walls of M. rhodochrous, were much lower than those of the F1 female mice. These findings indicate that the majority of the cell wall preparations lacks PBA ability for spleen cells with the CBA/N defect, except for the cell walls of M. rhodochrous which possess this ability. The PBA-ability of synthetic peptidoglycan, muramyl dipeptide (N-acetylmuramyl-L -alanyl-D -isoglutamine, MDP), was also examined, and a similar activity was observed in MDP.     

Distinctive adjuvanticity of synthetic analogs of mycobacterial water-soluble components.

Synthetic N-acetyl-muramyl-l-alanyl-d-isoglutamine represents the minimal structure capable of duplicating the activity of mycobacteria in Freund's complete adjuvant. In contrast to mycobacterial adjuvant preparations, that function only in the form of water-in-oil emulsions, this compound and a second synthetic analog (N-acetyl-muramyl-l-alanyl-d-isoglutamic acid) augment the humoral immune responses of mice equally as well as aqueous solutions. Whereas N-acetyl-muramyl-l-alanyl-d-isoglutamic acid administered to guinea pigs in water-in-oil emulsion has no effect, N-acetyl-muramyl-l-alanyl-d-isoglutamine induces delayed hypersensitivity to ovalbumin and azobenezene-arsonate N-acetyl-l-tyrosine and increases the level of antibody against ovalbumin. Under these conditions, challenge with the synthetic adjuvants themselves evokes no skin responses. Moreover, Freund's complete adjuvant sensitizes guinea pigs to tuberculin and to native mycobacterial water-soluble adjuvant but not to the synthetic analogs.     

Stimulation of splenic T cells on syngeneic monolayers of epidermal basal cells (EBC) in mice. Regulation by Lyt 1+ and Lyt 2+ cell subsets

Syngeneic proliferative response of splenic T cells against monolayers of epidermal basal cells (EBC) was obtained with C57BL/6 and DBA/2 mice. Optimal response, as assessed by [³H]thymidine uptake, occurred on the 6th day of coculture. The level of [^3H]thymidine uptake by unseparated spleen cells was lower than by fractionated T cells from C57BL/6 mice, and null for DBA/2 mice. It was not significantly different when lymphocytes were cocultured with syngeneic or allogeneic epidermal cells. Ia antigens did not appear to be involved in the syngeneic response, since it was not prevented by pretreating stimulator monolayers with monoclonal anti-Ia^k antibody or by adding this antibody directly to the cultures. When the proliferative responses of separated Lyt 1⁺ and Lyt 2⁺ cell subsets were compared, the prominent role of Lyt 1⁺ cells was demonstrated. Enhancement of the T-cell reactivity by eliminating Lyt 2⁺ cells and suppression of the response of a constant number of Lyt 1⁺ cells by adding Lyt 2⁺ cells suggested that Lyt 2⁺ cells could suppress and modulate the Lyt 1⁺ cell proliferation.     

Lymphokines secreted from sodium periodate-treated lymphocytes

This study was designed to determine if sodium metaperiodate (NaIO₄)-treated lymphocytes secrete lymphokines and if these lymphokines are similar to those obtained from mitogen- or antigen-stimulated lymphocytes. A brief exposure of CBA spleen cells to NaIO₄ induced the secretion of significant amounts of migration inhibitory factor (MIF). This MIF had a molecular weight range between 30,000 and 58,000, and was stable when heated at 56 °C for 30 min, but unstable at 80 °C. These characteristics are similar to those previously reported for mitogen- and antigen-induced MIF. In addition, NaIO₄ induced the secretion of lymphotoxin (LT) from CBA and Balb/c spleen cells, as well as from guinea pig lymph node cells. NaIO₄ was compared to the other inducers in regard to the quantity of LT secreted. Supernatant derived from NaIO₄-treated mouse spleen cells contained less LT than supernatants derived from concanavalin A- or phytohemagglutinin-treated cells, but contained more activity than those supernatants derived from lipopolysaccharide-treated cells. CBA spleen cells secreted significantly more LT than Balb/c spleen cells after NaIO₄ stimulation. NaIO₄-stimulated CBA spleen cells secreted LT in cultures with or without serum, but stimulated Balb/c spleen cells secreted LT only in serum-containing cultures. The advantages of NaIO₄ as an inducer of lymphokines, as opposed to other mitogens or antigens, is the brief exposure of this agent to the cells after which the NaIO₄ is removed, and the lymphokines can be obtained free from the inducer.     

Inhibition by specific monosaccharides of interleukin 2-induced thymocyte proliferation

When rat thymocytes are cultured for 3 days in serum-free medium and are stimulated to divide by interleukin 2 (IL 2), concanavalin A, or sodium periodate oxidation, addition to the medium of 10–25 mMd-ribose, 2-deoxy-d-ribose, or N-acetyl-d-galactosamine inhibits by 40% or more the incorporation of [³H]thymidine. d-ribose and lectin-free IL 2 generated from sodium periodate oxidation of rat spleen cells were used to study the characteristics of this inhibition and to test possible mechanisms of inhibition. Viability of thymocytes cultured with d-ribose is similar to that of cells cultured without this sugar. In order to be inhibitory, d-ribose has to be added to the cultures within the first 24 hr, and the inhibition can be prevented if the sugar is removed 18–24 hr after the start of culture. d-Ribose does not block the absorption of IL 2 by unstimulated rat thymocytes or by concanavalin A-generated thymic or splenic blast cells. When thymocytes are cultured with d-ribose for 24 hr, inactivated with mitomycin C, and then cultured for 3 days with fresh mitogenically stimulated cells, [³H]thymidine incorporation into the latter is not altered. This suggests that the sugar does not generate suppressor cells or suppressor supernates. d-Ribose does not appear to be a general metabolic inhibitor since [³H]leucine incorporation into thymocyte proteins and the release of [³H]leucine into medium after a 2-hr. [³H]leucine pulse are not altered by d-ribose. Trivial or artifactual effects (nonspecific cytotoxicity, changes in thymidine transport, or changes in isotonicity of the culture medium) cannot explain the inhibition. A hypothetical mechanism of inhibition is discussed.     

Enhancement of carrier-specific helper T cell function by the synthetic adjuvant, N-acetyl muramyl-L-alanyl-D-isoglutamine (MDP).

The adjuvant effect of a synthetic peptidoglycan, muramyl dipeptide (N-acetyl muramyl-L-alanyl-D-isoglutamine, MDP), was studied by using the anti-Tnp PFC and hemagglutinin responses of BALB/c mice to hapten-carrier conjugates. Administration of Tnp-OVA and MDP in saline to mice, followed 2 weeks later by a boost of Tnp-OVA in saline, led to significantly higher IgM and IgG anti-Tnp PFC and total anti-Tnp-hemagglutinin responses than those obtained in mice not treated with MDP in the initial immunization. A similar adjuvant effect by MDP on anti-hapten PFC responses was seen if mice were primed with KLH together with MDP and challenged with Tnp-KLH 2 weeks later. This apparent effect on carrier priming for helper function was confirmed and quantitated by double adoptive transfer experiments with graded numbers of spleen cells from KLH +/- MDP-primed mice and a fixed number of hapten-primed spleen cells from syngeneic Tnp-OVA immunized animals. These data suggest that at least one mode of action of the synthetic adjuvant MDP is via the enhanced stimulation of the helper T cell function.     

Primary in vitro antibody response of rabbit lymphoid cells and T-B cell collaboration in the absence of detectable mitogens

To investigate whether the antibody response and T-B-cell collaboration in vitro can be obtained in the absence of mitogens, a method of obtaining an in vitro primary anti-sheep red blood cell antibody response by rabbit spleen and lymph node cells was developed. We used Marbrook culture vessels and a specially prepared medium containing 10% autologous serum and maintained at pH 7.4–7.6. The system was shown to be devoid of any polyclonal mitogens as assessed by [³H]thymidine incorporation and by direct examination for blast cells in stained smears. The primary response increased continuously over the 5-day cultivation period and only IgM but not IgG plaque-forming cells (PFC) were detected. In over 20 experiments, the response ranged from 357 ± 17 to 4425 ± 110 PFC/10⁷ cultured cells with a median stimulation index of 52. The spleen cells required less antigen than the lymph node cells and 2-mercaptoethanol inhibited the response of the spleen cells but not that of the lymph node cells. Lymphocytes were separated into highly pure T- and B-cell populations by negative selection using antibody-coated human erythrocytes to rosette either T or B cells and Ficoll-Hypaque centrifugation to remove rosetted cells. Upon cultivation, B cells alone gave a low IgM response, whereas B cells reconstituted with T cells gave a response similar to that obtained with unseparated lymphoid cells. We concluded that: (a) optimal conditions for obtaining primary in vitro antibody responses using rabbit spleen and lymph node cells were established, (b) T-B-cell collaboration was demonstrated in the rabbit primary antibody response to sheep erythrocytes, and (c) the primary antibody response in vitro and T-B-cell collaboration may occur in the absence of detectable polyclonal mitogens.     

Random recirculation of small T lymphocytes from thoracic duct lymph in the mouse

The migration of ⁵¹Cr-labeled nylon-wool separated mouse thoracic duct T cells has been followed in order to determine whether there is a circulation of small (nondividing) T cells through the small intestine. Approximately 6% of the injected dose of T-TDL localized in the small intestine (minus Peyer's patches). Experiments revealed that this gut-localizing cell population consisted almost entirely, if not exclusively, of lymphoblasts present in mouse T-TDL. When lymphoblasts and small lymphocytes from mouse T-TDL were separated by velocity sedimentation, and the migration of separated fractions was studied, we found large cells (66% blasts) migrated well to the gut but poorly to the lymph nodes, whereas small cells (2% blasts) showed minimal migration to the gut but localized randomly in lymph nodes and spleen. The in vivo distribution of small cells from T-TDL was similar to that of T-PLN. Furthermore, the recirculatory patterns of both ⁵¹Cr-labeled T-TDL and T-PLN were found to be identical as accessed by their rate of recovery in the thoracic duct lymph of recipient mice. These results support the notion that the vast majority of T-TDL and T-PLN are part of a common pool of recirculating T cells which recirculate randomly through lymph nodes and spleen and not the small intestine.     

Components of mycobacteria and muramyl dipeptide with adjuvant activity induce lymphocyte activating factor

Several components of mycobacteria including a water-soluble extract (WSA) and an interphase material (IPM) as well as the synthetic cell wall analog muramyl dipeptide (MDP) all stimulated human mononuclear cells (MNL) to produce a factor which was mitogenic for murine thymocytes. The mediator induced by MDP is probably lymphocyte-activating factor (LAF) because it was produced by adherent but not nonadherent MNL and yields two characteristic peaks of activity in the 16,000–22,000 and 60,000–70,000 molecular weight range when eluted from Bio-Gel P-100 columns. The 6-O-stearoyl derivative of MDP was an active inducer of MNL LAF production, whereas, the d-alanine analog of MDP was somewhat less potent. Unfractionated as well as adherent, but not nonadherent, mouse peritoneal cells also produced LAF in response to WSA, IPM, and MDP. P388D₁ cell line macrophages, which are completely devoid of lymphocytes, could be stimulated by WSA, IPM, and MDP to produce LAF after prolonged incubation. These adjuvants did not stimulate nonadherent Balb/C or human blood cells to produce a mitogenic factor. However, when the P388D₁ macrophages were stimulated with these adjuvants in the presence of nonadherent murine or human peripheral blood cells, a mitogenic activity was produced in a shorter period of incubation suggesting that activated lymphocytes can facilitate the production of LAF by macrophages.     

Identification and characterization of a soluble suppressor factor(s) in the serum of AKR mice bearing lymphocytic leukemia

Leukemia in AKR mice was found to be associated with the presence of a serum factor(s) termed AKR leukemic suppressor factor (AKR-LSF). Suppression was quantitated by measuring the inhibition of PHA-stimulated [³H]thymidine incorporation by normal AKR spleen cells at various dilutions of leukemic mouse serum (LMS). AKR-LSF activity was expressed as units per milliliter, which is the reciprocal of the LMS dilution that inhibited [³H]thymidine uptake by 50% with respect to fetal calf serum control cultures. The amount of activity in the serum directly correlated to the rate of tumor cell growth. Mice receiving 10⁷ BW5147 transplanted leukemia cells had 130 ± 12 units of AKR-LSF activity/ml of serum compared to 40 ± 8 units/ ml for mice with spontaneous leukemia. Normal mouse serum contained 33 ± 11 units/ml. The leukemic serum exhibited no strain specificity in either phytohemagglutinin or lipopolysaccharide assays, but was found to be twofold more inhibitory against mouse spleen cells than that against rat spleen cells. Human lymphocyte blastogenesis was not inhibited by the leukemic serum. LMS did not inhibit the growth of L929 fibroblasts or murine tumor cells in vitro. Further work is necessary to determine what role the suppressor factor may play in the regulation of antitumor cell immunity.     

Deficiency in the incorporation of labeled thymidine and inhibition in the biosynthesis of interleukin-2 in lymphocytes obtained from Histoplasma capsulatum infected mice

The incorporation of (³H) thymidine and the biosynthesis of interleukin-2(IL-2) were investigated in Concanavalin A (ConA) and histoplasmin stimulated lymphocytes from spleen of infected Balb/c mice with the yeast phases of Histoplasma capsulatum. The ability to incorporate (³H) thymidine of Con A stimulated lymphocytes in culture from spleen of Histoplasma capsulatum infected mice, as well as the IL-2 content present in the supernatants of that cultures, were depressed along the first three weeks of the experiments, but starting week five, normal values were restored or even discretly increased. Incorporation of (³H) thymidine in histoplasmin stimulated lymphocytes remained inhibited along the seven weeks the experiment lasted. Results showed that inoculation of H. capsulatum yeast in mice provoked a temporary immunosuppression on cell mediated immunity, that can be explained by means of the inability of T cells to produce enough IL-2 necessary for the proliferation of T cells in culture.     

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).