Effect of HIV on antigen presentation by dendritic cells and macrophages

The antigen-presenting function of dendritic cells (DC) and macrophages (MO) following infection with HIV in vitro was examined. Using non-infected cells, DC, but not MO, stimulated primary proliferative responses in allogeneic lymphocytes in the mixed leukocyte reaction. Both DC and MO stimulated secondary responses to influenza virus and to tetanus toxoid in autologous T lymphocytes. After exposure of DC and MO to HIV1 in vitro for 2 days, 27 % of DC but < 1 % MO became infected as assessed by in situ hybridization. DC were blocked in their capacity to stimulate responses to alloantigens or to the recall antigens. By contrast, MO retained the ability to stimulate responses to the recall antigens. Similar effects during in vivo infection would allow activated T-cell clones to respond to antigens presented by MO early in infection. However, any loss of activated T cells might prove cumulative and damaging in the absence of an effective DC recruitment mechanism for resting T cells.     

Selective T cell killing of human lymphocytes by ultraviolet radiation

The effects of ultraviolet radiation (uv) on human B and T lymphocytes were studied. In vitro studies showed that T lymphocytes were more sensitive to uv than B lymphocytes as assessed by eosin-dye exclusion. Following uv exposure, the viable lymphocytes responded to mitogens (PHA, PWM), and functional B lymphocytes were present at a time when no viable T cells were detected. Varying doses of uv were required to abrogate different in vitro responses (proliferative response to antigen or allogeneic cells, MIF production, and cell-mediated lympholysis). In vivo, uv was able to diminish an established cutaneous delayed hypersensitivity response. In vitro uv treatment of parental mouse spleen cells eliminated a graft-versus-host reaction in F₁ recipients as determined by the spleen index. The basis for the differential effect of uv on B and T lymphocyte viability and functional responses is unknown.     

Phylogeny of lymphocyte heterogeneity. II. Differential effects of temperature on fish T-like and B-like cells.

Lymphocytes from the bluegill, a freshwater fish, were observed to undergo in vitro mitogenic responses to a variety of “classical” mitogens. Using cell fractionation approaches based upon surface markers and in vitro mitogenesis, bluegill lymphocytes could be divided into two populations. One population responded to PHA and Con A but not to LPS, contained surface antigens in common with bluegill brain, and did not form spontaneous rosettes with rabbit erythrocytes. The other population responded to LPS but not to PHA or Con A, did not appear to contain surface antigens in common with brain, and did form rosettes with rabbit erythrocytes. The former population responded to mitogenic stimulation very well at 32 °C, whereas the latter population responded better at 22 than at 32 °C. The pattern of mitogenic responses and brain antigen distribution coupled with the observation that mixed lymphocyte responses were obtained at 32 but not at 22 °C makes it likely that the 32 °C responsive population represents the fish equivalent of T cells. The other population may be B cells. These data suggest that the immunosuppressive effects of low temperatures on cold-blooded animals may be effects on the generation of functional T cells and not on B cells.     

Immunosuppressive alpha globulin from bovine thymus. I. Preparation and assay

DEAE chromatography at pH 5.0 of the saline-soluble proteins from bovine thymus glands yields a protein fraction similar in activity to the immunosuppressive alpha-2 globulins previously described from bovine and human serum. Of 32 preparations 16 had consistent and reproducible suppressive activity to DNA synthesis in phytohemagglutinin (PHA)—stimulated lymphocytes in concentrations ranging from 50–600 μg/ml in vitro. In vivo immunosuppression, not directly related to the degree of in vitrolymphocyte suppression, occurred in doses of 4–5 mg per mouse, and was assessed by the hemagglutinin response to sheep red blood cells. A variety of other protein and nucleic acid fractions were not suppressive in these assays; in particular, fractions A and B, which precede the immunosuppressive Fraction C in the elution from DEAE-cellulose, are neither suppressive, nor do they significantly alter the effects of Fraction C.     

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.     

Studies on the specificity of in vitro-induced lymphocytotoxicity to methylcholanthrene-induced fibrosarcoma cell lines

Cytotoxic effector lymphocytes were induced by in vitro immunization of lymph node and spleen cells from CS7B16(H2^b) and Balb/c(H2^d) mice to syngeneic or allogeneic methylcholanthrene-induced fibrosarcoma (MCAF) cell lines. The T cell-dependent cytotoxicity was specific to target cell lines to which the lymphocytes were immunized in vitro. Normal fibroblasts as stimulator cells did not induce lymphocytotoxicity to syngeneic MCAF cells or to normal syngeneic fibroblasts. The results indicate that the in vitro-immunized lymphocytes recognize individual specific tumor-associated antigens of the MCAF cells. In experiments in which the lymphocytes were immunized in vitro to allogeneic MCAF cells, cytotoxic reactions to alloantigens, but not to tumor-associated antigens, were detected. Incubation with phytohemagglutinin (PHA) during the sensitization period modified the specificity of the cell-mediated lysis of MCAF cells: Allogeneic as well as syngeneic target cells were destroyed by these effector cells. PHA induced a nonspecific cytotoxic effect which increased the specific lysis of target cells. The cytotoxicity of the in vitro-immunized lymphocytes was inhibited by incubation with membrane protein preparations from the syngeneic MCAF cell lines. In contrast to the specificity of the cytotoxic effect to the different syngeneic cell lines, the membrane extract of one individual syngeneic MCAF cell line was able to inhibit the lymphocytotoxicity to all other syngeneic cell lines. Membrane protein preparations from allogeneic MCAF cells or from normal syngeneic fibroblasts were not inhibitory. The in vitro-immunized cytotoxic lymphocytes did not impair the tumor growth in vivo as could be demonstrated by passive transfer of the lymphocytes in a Winn assay.     

Mitogen Synergism in low-responding CBA/CaJ mice.

Although neither phytohemagglutinin (PHA) nor concanavalin A (Con A) stimulated blood cultures in vitro from low-responding CBA/CaJ mice effectively, a mixture of PHA and Con A over a range of concentrations stimulated a response from CBA/CaJ mouse blood that was greater than the sum of the responses produced by using PHA or Con A individually. This synergistic effect was expressed as the percentage by which the responses to the PHA and Con A mixture exceeded the sum of the responses to PHA alone and Con A alone. When the mitogen concentrations that gave maximum responses individually were used, the synergistic effect averaged 319% in cultures of blood from low-responding CBA/CaJ mice. Apparently simultaneous exposure to PHA and Con A stimulates DNA synthesis in white blood cells of CBA/CaJ mice that fail to respond to either mitogen alone.     

Dipetalonema viteae: in vitro blastogenesis of hamster spleen and lymph node cells to phytohemagglutinin and filarial antigens

Hamsters of the randomly bred LAKZ and inbred LSH strains were infected with Dipetalonema viteae, and the in vitro responses of lymph node and spleen lymphocytes to male and female worm antigens and phytohemagglutinin (PHA) were measured by a [³H]-thymidine-uptake assay at various times after infection. The PHA response remained unchanged at the level of controls in infected LAKZ hamsters while LSH hamsters showed a depressed response to the mitogen during late infection. Stimulation of lymph node cells by filarial antigens was maximal in both strains of hamsters at Week 4 postinfection, almost reaching values obtained in PHA stimulated cultures. A similar high lymphocyte transformation reaction was measured after the injection of dead third stage larvae. During transient microfilaremia, when antibody titers reached a maximal level, the lymphocyte reactivity to filarial antigens decreased drastically and only occasionally was demonstrated in hamsters 20 and 30 weeks after infection. No correlation between lymphocyte reactivity and parasitological findings (worm load or intensity and duration of microfilaremia) could be demonstrated. The cellular unresponsiveness to filarial antigens was further analyzed in chronically infected LAKZ hamsters. No suppressor cells could be found in lymphocyte suspensions of nonresponding hamsters. A challenge infection did not restore lymphocyte reactivity. Serum of chronically infected hamsters caused marked inhibition when added to filarial antigen-sensitive lymphocytes. Lymphocytes from hamsters with a mixed D. viteae and Schistosoma mansoni infection responded as well to soluble schistosomal egg antigens at Week 30 of a D. viteae infection as lymphocytes from hamsters infected with S. mansoni alone. The humoral immune response to schistosomal antigens, however, was significantly lower in animals with a mixed infection.     

Stage-related capacity for limb chondrogenesis in cell culture.

Cells from wing buds of varying-stage chick embryos were dissociated and grown in culture to test their capacity for cartilage differentiation. Micro-mass cultures were initiated with a cell layer greater than confluency, which occupied a restricted area of the culture dish surface (10–13 mm²). Cells from stage 24 chick embryo wing buds (prior to the appearance of cartilage in vivo) undergo cartilage differentiation in such cultures. Typically, during the first 1–2 days of culture, cells form aggregates (clusters of cells with a density 1.5 times greater than that of the surrounding nonaggregate area). By Day 3, virtually all aggregates differentiate into cartilage nodules which are easily recognized by their Alcian blue staining (pH 1.0) extracellular matrix. Subsequently, nodules increase in size, and adjacent nodules begin to coalesce. Micro-mass cultures were used to test the chondrogenic capacity of wing bud cells from chick embryos representing the different stages of limb development up to the appearance of cartilage in vivo (stages 17–25). Cells from embryo stages 21–24 form aggregates which differentiate into cartilage nodules in vitro with equal capacity (scored as number of nodules per culture). In contrast, cells from embryo stages 17–19 form aggregates in similar numbers, but these aggregates never differentiate into nodules under routine conditions. However, aggregates which form in cultures of stage 19 wing bud cells do differentiate into cartilage nodules if exposed to dibutyryl cyclic AMP and theophylline. Cells from stage 20 embryos manifest a varying capacity to form cartilage nodules; apparently, this is a transition stage. Cells from stage 25 embryos produce cartilage in vitro without forming either aggregates or nodules. Based on the results presented in this paper, the authors propose a model for cartilage differentiation from embryonic mesoderm cells involving: (1) aggregation, (2) acquisition of the ability to respond to the environment in the aggregate, (3) elevated intracellular cyclic AMP levels, and (4) stabilization and expression of cartilage phenotype.     

Linear density gradient separation of human lymphocyte subsets. II. Characterization of cells responding in secondary MLC and CML.

Lymphocytes were separated on linear density gradients (LDG) after they had been sensitized in vitro against allogeneic cells and had reverted to small cells. Cells from individual density fractions were restimulated with autologous, specific, or third-party cells and assayed 48 hr later for their response in secondary mixed leukocyte culture (MLC) and cell-mediated lympholysis (CML). Memory cells capable of responding in secondary MLC were broadly distributed and found in both heavy and light fractions. The various density classes of memory cells differed with respect to the degree of their specificity for the restimulating cells. In secondary MLC the greatest specificity for the originally sensitizing cells and the least cross-reactivity for third-party cells were primarily features of light- and medium-density cells. Memory killer cells for CML were fairly homogeneously grouped. Following restimulation, killers were enriched in light to medium fractions also, as was previously seen at the peak of the response on Day 6.     

Modulation of lymphocyte functions by group A streptococcal membrane

Protoplast membranes isolated from group A streptococci suppress functions of mouse B cells in vivo and in vitro. Intraperitoneal injection 24 or 72 hr (but not 12 hr) before collection of lymphoid cells results in a selective decrease in the mitogenic response of bone marrow cells to dextran sulfate (DS). The response of bone marrow cells to lipopolysaccharide (LPS), and spleen cells to both DS and LPS, is unaltered. In vitro exposure of lymphocytes to membranes concomitantly with mitogen reduces the response to both DS and LPS, however, the DS response is more susceptible to low doses of membrane. Suppression of the response to DS in vitro is not mediated by cells bearing Thy 1.2 antigen. Neither the phytohemagglutinin (PHA)-responsive cells nor the adherent cells participate in suppression of the LPS response in vitro. In contrast to the suppression of B-cell functions neither the PHA nor concanavalin A (Con A) response of mouse bone marrow, spleen, or thymus cells is altered by streptococcal protoplast membranes injected 24 hr before collection of cells. In vitro exposure of spleen cells to a limited range of concentrations of membrane results in an enhanced proliferative response of spleen cells stimulated by suboptimal doses of PHA. This synergism is not mediated by the adherent cells. Addition of membranes to spleen cell cultures in vitro has no effect upon the response of spleen cells to suboptimal doses of Con A or to optimal doses of either Con A or PHA. Higher concentrations of membranes reduce the proliferative response of both control and mitogen-stimulated cells. This nonselective suppression by high doses of membranes is not due to toxicity. Delayed hypersensitivity to sheep erythrocytes is potentiated by injection of membranes. These studies suggest that streptococcal membranes preferentially suppress the immature B cells and enhance certain T-cell functions.     

Depressed T-cell reactivity and suppressor activity of lymphoid cells from cyclophosphamide-treated mice

A study was made of the in vitro proliferative activity of thymus-derived lymphoid cells from cyclophosphamide-treated mice (Cy-mice) and the relationship between this and some in vivo immunological responses. The proliferative response to phytohaemagglutinin (PHA) and allogeneic cells was depressed for up to 3 weeks after drug treatment in spleen and lymph node cells, responsiveness recovering more rapidly in lymph node cells. Cell concentration in culture was shown to be important in such measurements as cells from some Cy-mice were able to inhibit their own proliferation and that of normal lymph node cells. No stable soluble factor responsible for this effect could be isolated. It was shown that in vitro proliferative activity is not a good indicator of in vivo T-cell capability as indicated by the very rapid recovery of ability to reject skin grafts and the fairly rapid recovery of ability to produce cytotoxic cells compared to the slower recovery of in vitro T-cell activities.     

Suppression of bovine T- and B-lymphocyte responses by fetuin, a bovine glycoprotein

Fetuin, a bovine glycoprotein present in milligram amounts in fetal calf serum, suppressed lymphocyte responses to phytohemagglutinin (PHA) by 70–90%. T-lymphocyte-enriched populations were suppressed by fetuin in their response to PHA indicating direct T-cell regulation and suggesting that macrophages were not required for fetuin activity. Lymphocyte responses to the lipopolysaccharides (LPS) of Escherichia coli and Brucella abortus in the presence of fetuin were markedly reduced compared to cultures without fetuin. Fetuin also suppressed alloantigen recognition in 85% of the mixed-leukocyte responses, and primary in vitro antibody responses induced by the LPS of B. abortus. No toxicity to responding cells or inhibition of early cell division could be demonstrated. A mechanism of regulation appeared to be via suppressor cells. Lymphocytes maintained in fetuin for 48 hr, but not beyond 72 hr, exhibited suppressor cells which could modulate normal adult lymphocyte responses to PHA.     

Ontogeny of murine B-cell responses to thymus-independent trinitrophenyl antigens.

The ontogeny of B-cell responsiveness to three thymus-independent trinitrophenyl (TNP) antigens has been examined in BALB/c mice in vivo and in vitro. When in vivo splenic plaque-forming cell (PFC) responses to TNP-conjugated lipopolysaccharide (TNP-LPS), Ficoll (TNP-Ficoll), and Brucella abortus (TNP-Brucella) were measured in neonatal and adult mice, a defined sequence of responsiveness was observed. Newborn mice responded well to TNP-LPS, but not to TNP-Ficoll or TNP-Brucella. Neonates injected at 1 day of age responded to TNP-LPS and TNP-Ficoll and mice 5 to 14 days of age responded to TNP-LPS, TNP-Ficoll, and TNP-Brucella. Furthermore, the antigen-reactive populations increased at different rates for the three antigens in the first 2 weeks of life. In vitro experiments confirmed the results obtained in vivo although slightly earlier responsiveness to TNP-Brucella was observed in vitro. PFC inhibition assays with free TNP hapten were performed so that avidity profiles could be examined in neonatal and adult anti-TNP PFC responses. The results clearly demonstrate that once a response becomes detectable in neonatal mice immunized with any of the three TI TNP antigens, fully heterogeneous or “adult-like” responses are found. In addition, experiments comparing avidity profiles in athymic (nu/nu) BALB/c mice and their normal (nu/+) littermates demonstrate that T cells are not required for the generation of fully heterogeneous anti-TNP PFC responses. These results indicate that B cells responsive to different TI TNP antigens mature at different times and at different rates during ontogeny. Late maturation events of such B cells do not include the acquisition of additional V-region specificities as detected in a PFC inhibition assay.     

Kinetics of oligodendrocyte-like cells in primary culture of mouse embryonic brain

An oligodendrocyte-like cell population was identified and its kinetic features were studied in primary cultures of mouse brain. Cells from telencephalon and diencephalon of 14-day-old embryos were dissociated before seeding on poly-l-lysine or collagen-coated glass coverslips. After some divisions and migrations, cells differentiated throughout the cultures; round and small overlying cells appeared after the 9th day in vitro. Their morphology, based on light microscopy, after May-Grunwald-Giemsa staining, scanning electron microscopy, and transmission electron microscopy, looked like that of oligodendrocytes. These cells are referred to here as oligodendrocyte-like cells. Their filiation, kinetics, and proliferation were studied by quantitative radioautography. Their density increased from the 9th to the 16th day in vitro. Two groups were found: large and clearly labeled oligodendrocyte-like cells and small dark unlabeled ones which undoubtedly derived from the first. Analogies between our observations in vitro and those carried out in vivo by several experimenters, suggest that in vitro these cells are probably oligodendrocytes.     

Modulation of lymphocyte mitogen responses by cocultured fibroblasts

Immunological reactions in vivo occur in an environment rich in fibroblasts (FB) and other connective tissue cells. The possibility that FB might affect mononuclear cell proliferative responses to mitogens in vitro was examined in a microculture system. Human peripheral blood mononuclear cells (MC) were cocultured with mitomycin C-treated FB in the presence of phytohemagglutinin (PHA) or pokeweed mitogen (PWM). Cocultured FB (at a 1:100 to 1:10 FB:MC ratio) suppressed the response of MC to PHA (by as much as 35%) but did not significantly affect PWM responses. Cocultures of FB and MC were characterized by 10- to 30-fold increases in prostaglandin E₂ concentrations compared to MC or FB cultured alone. Inhibition of prostaglandin synthesis with indomethacin or mefenamic acid significantly reversed the FB-mediated suppression of lymphocyte PHA responses. Prostaglandin-dependent FB suppression of lymphocyte PHA responses was seen only when FB:MC coculture was initiated at the onset of exposure of MC to PHA. When FB were added to MC after 24 hr of culture with PHA, no effect was seen. Addition at 48 or 66 hr resulted in prostaglandin-independent enhancement of lymphocyte proliterative responses to PHA. Thus in cocultures of FB and MC, MC reactivity to PHA may be influenced in part via alterations in FB prostaglandin metabolism. The interaction between FB and MC may be important in the modulation of immune responses in inflammatory lesions.     

Bone Marrow-Derived Mesenchymal Stem Cells Drive Lymphangiogenesis

It is now well accepted that multipotent Bone-Marrow Mesenchymal Stem Cells (BM-MSC) contribute to cancer progression through several mechanisms including angiogenesis. However, their involvement during the lymphangiogenic process is poorly described. Using BM-MSC isolated from mice of two different backgrounds, we demonstrate a paracrine lymphangiogenic action of BM-MSC both in vivo and in vitro. Co-injection of BM-MSC and tumor cells in mice increased the in vivo tumor growth and intratumoral lymphatic vessel density. In addition, BM-MSC or their conditioned medium stimulated the recruitment of lymphatic vessels in vivo in an ear sponge assay, and ex vivo in the lymphatic ring assay (LRA). In vitro, MSC conditioned medium also increased the proliferation rate and the migration of both primary lymphatic endothelial cells (LEC) and an immortalized lymphatic endothelial cell line. Mechanistically, these pro-lymphangiogenic effects relied on the secretion of Vascular Endothelial Growth Factor (VEGF)-A by BM-MSC that activates VEGF Receptor (VEGFR)-2 pathway on LEC. Indeed, the trapping of VEGF-A in MSC conditioned medium by soluble VEGF Receptors (sVEGFR)-1, -2 or the inhibition of VEGFR-2 activity by a specific inhibitor (ZM 323881) both decreased LEC proliferation, migration and the phosphorylation of their main downstream target ERK1/2. This study provides direct unprecedented evidence for a paracrine lymphangiogenic action of BM-MSC via the production of VEGF-A which acts on LEC VEGFR-2.     

Expression of IgG memory response in vitro to thymus-dependent and thymus-independent antigens

A model is described in which expression of IgG secondary antihapten responses of large magnitude can be initiated in vitro without resorting to in vivo boosting prior to culture. The number of IgG plaque-forming cells (PFC) is frequently as much as 100-fold greater than that of IgM PFC. Spleen cells from mice primed with trinitrophenylated keyhole limpet hemocyanin (TNP-KLH) several months earlier are stimulated in vitro to produce an anti-TNP plaque-forming cell response 7–10 days later. The in vitro IgG response can be elicited with either a thymus-dependent antigen (TNP-KLH) or thymus-independent antigens (TNP-T₄ bacteriophage or DNP-dextran). The kinetics of the responses to these two forms of antigen differ in that the thymus-independent response peaks two days earlier. The IgG response to both forms of antigen requires the presence of 2-mercaptoethanol (2-ME) even though macrophages are not depleted prior to culture. In the absence of the reducing agent both thymus-dependent and thymus-independent IgG responses were diminished ≥90%. The magnitude of the response to thymus-independent antigens emphasizes the ability of these materials to elicit IgG expression in memory B cells provided optimal conditions for memory development and in vitro expression exist.     

In vitro and in vivo immunomodulatory activity of sulfated polysaccharides from Enteromorpha prolifera

Water-soluble sulfated polysaccharides extracted from Enteromorpha prolifera and fractionated using ion-exchange chromatography (crude, F₁, F₂ and F₃ fractions) were investigated to determine their in vitro and in vivo immunomodulatory activities. The sulfated polysaccharides, especially the F₁ and F₂ fractions, stimulated a macrophage cell line, Raw 264.7, inducing considerable nitric oxide (NO) and various cytokine production via up-regulated mRNA expression. The in vivo experiment results show that the sulfated polysaccharides (the crude and F₂ fractions) significantly increased Con A-induced splenocyte proliferation, revealing their potential comitogenic activity. In addition, IFN-γ and IL-2 secretions were considerably increased by the F₂ fraction without altering the release of IL-4 and IL-5. This implies that the F₂ fraction can activate T cells by up-regulating Th-1 response and that Th-1 cells might be the main target cells of the F₂ fraction. These in vitro and in vivo results suggest that the sulfated polysaccharides are strong immunostimulators.     

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.