Molecular structures that influence the immunomodulatory properties of the lipid A and inner core region oligosaccharides of bacterial lipopolysaccharides.

The relationship between chain length as well as the position of fatty acyl groups to the ability of lipid A to abolish the expression of suppressor T-cell (Ts) activity was examined. Fatty acyl chain lengths of C12 to C14, as in the lipid A of Escherichia coli and Salmonella minnesota, appear to be optimal for this bioactivity, since lipid A preparations with fatty acyl groups of relatively short chain length (C10 to C12 for Pseudomonas aeruginosa and Chromobacterium violaceum) or predominantly long chain length (C18 for Helicobacter pylori) are without effect. The presence of an acyloxyacyl group of appropriate chain length at the 3' position of the glucosamine disaccharide backbone of lipid A also plays a decisive role. By contrast, the lipid A proximal inner core region oligosaccharides of some bacterial lipopolysaccharides increase the expression of Ts activity; this is due mainly to the capacity of such oligosaccharides, which are relatively conserved in structure among gram-negative bacteria, to enlarge or expand upon the population of CD8+ Ts generated during the course of a normal antibody response to unrelated microbial antigens. The minimal structure required for the expression of the added immunosuppression observed appears to be a hexasaccharide containing one 2-keto-3-deoxyoctonate residue, two glucose residues, and three heptose residues to which are attached two pyrophosphorylethanolamine groups. The relevance of these findings to virulence and to the pathogenesis of gram-negative infections is discussed.

The influence of monophosphoryl lipid A (MPL) on erythrocyte autoantibody formation.

The onset and the amount of erythrocyte autoantibodies induced by the injection of C57BL/6N mice with rat red blood cells (RRBC) were hastened and increased, respectively, after the administration of monophosphoryl lipid A (MPL); this was not the case for similarly treated BALB/cAnN mice, which make a lower autoantibody response after immunization with RRBC. The transfer of spleen cells from donor C57BL/6N mice immunized with RRBC suppressed autoantibody formation in recipient mice subsequently immunized with RRBC; however, treatment with MPL prevented neither the induction nor the expression of such suppression. This suggests that the increased autoantibody response in RRBC-immunized C57BL/6N mice treated with MPL is not due to the inactivation of suppressor cell activity which, in other studies, was found to be extremely sensitive to MPL.

Immunosuppressive effects induced by the polysaccharide moiety of some bacterial lipopolysaccharides.

The immunomodulatory properties of several lipopolysaccharides (LPS) derived from clinical isolates of Pseudomonas aeruginosa, Branhamella catarrhalis, and Bordetella pertussis were evaluated for their capacity to influence the magnitude of the antibody response to type III pneumococcal polysaccharide (SSS-III), which is known to be regulated by suppressor and amplifier T cells (Ts and Ta, respectively). The administration of LPS, two days after immunization resulted in a significant increase in the antibody response. Such enhancement may be due mainly to the ability of the lipid A moiety of LPS to abolish the negative effects of activated Ts, thereby enabling Ta function to be more fully expressed; however, B cell mitogenicity of the LPS molecule also may be involved. By contrast, treatment with LPS at the time of immunization with SSS-III induces significant suppression of the SSS-III-specific antibody response; such suppression is not induced by LPS or lipid A derived from Escherichia coli and Salmonella minnesota, and is independent of the capacity of LPS to activate B cells polyclonally, an activity generally attributed to the lipid A fraction of LPS. Studies conducted with the LPS of P. aeruginosa indicated that the suppression induced is T cell dependent and mediated by the polysaccharide (PS) fraction of LPS; it appears to be due-at least in part-to the capacity of PS to expand or increase the size of the precursor pool of Ts, activated in response to SSS-III. The significance of these findings to the pathogenesis of certain gram-negative infections is discussed.

CD4+ T cells regulate the magnitude of the antibody response to several helper T cell independent antigens.

The effect of prior depletion of CD4+ T cells on the magnitude of the primary antibody response to several antigens considered to be helper T cell independent (TI) was examined. Treatment of mice with the monoclonal antibody GK1.5 to remove CD4+ T cells had little effect on the magnitude of the specific antibody response to the lipopolysaccharide (LPS) of Escherichia coli 0113; however, such treatment reduced the magnitude of the splenic IgM antibody response to E. coli 055 LPS, Serratia marcescens LPS, TNP-Ficoll, and Type III pneumococcal polysaccharide (SSS-III), compared to control untreated or rat IgG treated mice. Thus, CD4+ T cells positively influence of magnitude of the antibody response to the majority of helper T cell independent antigens tested.

Differential effects of monophosphoryl lipid A on expression of suppressor T cell activity in lipopolysaccharide-responsive and lipopolysaccharide-defective strains of C3H mice.

Lipopolysaccharide (LPS)-responsive and LPS-defective strains of C3H mice did not differ in the capacity to make an antibody response to type III pneumococcal polysaccharide or in the degree of thymus-derived suppressor cell (Ts) activity generated following exposure to type III pneumococcal polysaccharide. However, treatment with monophosphoryl lipid A (MPL) abolished the expression of Ts function in LPS-responsive but not LPS-defective mice. Since this effect was elicited by different preparations of MPL, it appears to be a general property of MPL mediated by direct action of MPL on activated Ts.

Antigen-specific suppressor T cells respond to recombinant interleukin-2 and other lymphokines.

Previous studies have shown that transfer of whole spleen cell populations obtained from primed donors or transfer of purified T cells enriched for suppressor activity (Ts) to recipient mice decreased the antibody response to pneumococcal polysaccharide type III (SSS-III) when the animals were simultaneously immunized with SSS-III. In the present studies, such suppression of the antibody response was transferred with 10- to 100-fold fewer primed spleen cells when the cells were treated in vitro with recombinant interleukin-2 (rIL-2) before transfer; spleen cells from naive mice or mice primed with an unrelated antigen (dextran) and then treated with rIL-2 did not cause suppression of the antibody response to SSS-III, thereby eliminating the possibility of nonspecific carryover effects induced by rIL-2. In vivo administration of rIL-2 at the time of immunization with an optimally immunogenic dose of SSS-III resulted in significant (P less than 0.05) suppression of the antibody response relative to that of control animals, suggesting that IL-2 augments the clonal expansion of Ts cells in vivo. Further, the ability of passively administered anti-IL-2 receptor antibody to inhibit generation of Ts cells in vivo is consistent with such a view. Spleen cells from primed animals treated with rIL-4, rIL-5, or gamma interferon--but not those from primed animals treated with rIL-6--likewise were able to transfer suppression of the antibody response with fewer cells than those required when primed cells not treated with lymphokines were used. Thus, these studies indicate that Ts cell activity is greatly influenced by lymphokines produced by helper T cells. The studies also suggest that these lymphokines are required during activation and/or clonal expansion of Ts cells.

T cells regulate the IgM antibody response of BALB/c mice to dextran B1355.

The IgM antibody response of BALB/c mice to bacterial (Leuconostoc) dextran B1355 is influenced in a positive and negative manner by regulatory CD4+ and CD8+ T cells, respectively. Treatment with concanavalin A (ConA) at the time of immunization or 2 days later caused suppression and enhancement of the antibody response, respectively. Priming of mice with a sub-immunogenic dose of dextran resulted in profound suppression upon subsequent immunization 3 days later. None of these effects were demonstrable in athymic mice. Transfer of T cells from mice primed 18 h previously with a subimmunogenic dose of dextran suppressed the antibody response in immunized recipients; such suppression was abolished by the treatment of transferred cells with anti Thy 1.2 or anti Lyt 2.2 (CD8) antibody in the presence of complement. By contrast, the transfer of T cells from mice, which had been given an immunogenic dose of dextran 4 days previously, increased the antibody response in immunized recipients; such enhancement was abolished by treating transferred cells with anti Thy 1.2 or anti L3T4 (CD4) antibody in the presence of complement. These findings indicate that the immune response to dextran B1355 is regulated by CD4+ T-amplifier cells (Ta cells) and by CD8+ T-suppressor cells (Ts cells) which are activated during the course of a normal antibody response.

Inactivation of suppressor T cell activity by the nontoxic lipopolysaccharide of Rhodopseudomonas sphaeroides.

Antibody responses of mice immunized with type III pneumococcal polysaccharide were examined with and without treatment with nontoxic lipopolysaccharide from Rhodopseudomonas sphaeroides (Rs-LPS). The results obtained were similar to those described previously for mice treated with monophosphoryl lipid A (MPL) except that lower amounts of Rs-LPS were needed. Both were without effect when given at the time of immunization with type III pneumococcal polysaccharide but elicited significant enhancement when given 2 to 3 days later. Such enhancement was T cell dependent and not due to polyclonal activation of immunoglobulin M synthesis by B cells. Treatment with either Rs-LPS or MPL abolished the expression but not induction of low-dose paralysis, a form of immunological unresponsiveness known to be mediated by suppressor T cells (Ts). The in vitro treatment of cell suspensions containing Ts with extremely small amounts of Rs-LPS or MPI completely eliminated the capacity of such cells to transfer suppression to other mice. These findings indicate that the immunomodulatory effects of both MPL and Rs-LPS are mainly the result of eliminating the inhibitors effects of Ts; this permits the positive effects of amplifier T cells to be more fully expressed, thereby resulting in an increased antibody response. The significance of these and other findings to the use of Rs-LPS as a pharmacotherapeutic agent for gram-negative bacterial sepsis is discussed.

Analysis of the optimal conditions for the adsorption of type III pneumococcal polysaccharide to plastic for use in solid-phase ELISA.

An economical, sensitive enzyme-linked immunosorbent assay (ELISA) method for measuring all isotypes of immunoglobulin specific for type III pneumococcal polysaccharide (SSS-III) is described, using 96-well polystyrene microtiter plates coated directly with antigen. To achieve substantial binding of SSS-III to plastic plates, the polysaccharide had to be dissolved in a buffer (0.1 M Hepes) of pH 4.0 or less. Optimal conditions for adsorption of SSS-III to plates were found to be pH 3.5 and a concentration of 1.0 microgram SSS-III/ml (0.1 microgram/well). Under these conditions, murine anti-SSS-III polyclonal or monoclonal antibodies could be detected to a limit of about 5-10 ng/ml. The implications of these findings for assays that use mixtures of polysaccharides adsorbed to plastic plates are discussed.

Characterization of the immunodeficiency of RIIIS/J mice: immune response to polysaccharide antigens.

RIIIS/J mice lack an autosomal dominant gene(s) that influences the magnitude of the antibody response to several polysaccharide antigens of bacterial origin. Low responsiveness is demonstrable whether polysaccharide is administered as a T-helper-cell-independent or -dependent antigen conjugated to an immunogenic carrier; however, RIIIS/J mice make good anti-hapten antibody responses to haptenated polysaccharides. The low antibody responses of RIIIS/J mice to type III pneumococcal polysaccharide do not appear to be the results of an imbalance in the activity of regulatory T lymphocytes. Compared with other strains of mice, RIIIS/J mice elicit low antibody responses to lipopolysaccharide (LPS). They do not develop a cyclic primary or secondary antibody response to Escherichia coli O113 LPS; the latter is not due to a lack of mitogenic response to E. coli O113 LPS. They also produce auto-anti-idiotypic antibody after being immunized with trinitrophenyl-Ficoll.

Metabolic activity is necessary for activation of T suppressor cells by B cells.

Ag-primed B cells must express cell-surface IgM, but not IgD or Ia Ag, and must remain metabolically active, in order to activate suppressor T cells (Ts) specific for type III pneumococcal polysaccharide. Ag-primed B cells that were gamma-irradiated with 1000r, or less, retained the ability to activate Ts; however, Ag-primed B cells exposed to UV light were not able to do so. gamma-Irradiated and UV-treated Ag-primed B cells both expressed comparable levels of cell-surface IgM, and both localized to the spleen after in vivo transfer; neither could proliferate in vitro in response to mitogens. By contrast, gamma-irradiated primed B cells were still able to synthesize proteins, whereas UV-treated primed B cells could not. These findings suggest that in order for Ag-primed B cells to activate Ts, they must a) express cell-associated IgM (sIgM) antibody bearing the idiotypic determinants of antibody specific for type III pneumococcal polysaccharide, and b) be able to synthesize protein for either the continued expression of sIgM after cell transfer, or for the elaboration of another protein molecule that is also required for the activation of Ts; this molecule does not appear to be Ia Ag.

Enrichment of suppressor T cells by means of binding to monophosphoryl lipid A.

The binding and elution of spleen cells from plastic dishes coated with monophosphoryl lipid A (MPL) resulted in a greater than 1,000-fold enrichment of antigen-specific suppressor T-cell (TS) activity when spleen cells from mice 18 to 24 h after exposure to a low dose of type III pneumonococcal polysaccharide (SSS-III) were used. The removal of MPL-adherent TS cells resulted in an increase in the degree of amplifier T-cell (TA) activity present in the remaining MPL-nonadherent cell fraction; however, both TS and TA activities were found in the MPL-adherent cell fraction when spleen cells from mice 4 days after immunization with an optimal dose of SSS-III were examined. These findings, as well as others, suggest that both TS and TA, once activated, acquire a cell surface receptor that enables them to bind to MPL. Because of differences in the kinetics for the activation of TS and TA during the course of the antibody response and the fact that TS, but not TA, activity appears as early as 18 to 24 h after exposure to SSS-III, it is possible to use this experimental approach to obtain cell suspensions greatly enriched in TS activity.

Specific immunological unresponsiveness to bacterial lipopolysaccharides develops in a cyclic manner.

Prior exposure (priming) of BALB/cByJ mice to a low dose of lipopolysaccharide derived from Escherichia coli 055 or Serratia marcescens, followed by immunization with an optimally immunogenic dose of the same lipopolysaccharide 2 to 30 days later, results in the expression of substantially reduced antibody responses. Such unresponsiveness, which is antigen specific, occurs in a cyclic manner with time after priming.

Immunomodulatory activity of monophosphoryl lipid A in C3H/HeJ and C3H/HeSnJ mice.

Treatment with nontoxic monophosphoryl lipid A (MPL) derived from a polysaccharide-deficient, heptoseless Re mutant of either Salmonella typhimurium or Salmonella minnesota R595 enhanced the immunoglobulin M (IgM) anti-type III pneumococcal polysaccharide (SSS-III) antibody response of C3H/HeSnJ mice. Such an adjuvant effect was not observed in lipopolysaccharide-nonresponder C3H/HeJ mice. Nevertheless, C3H/HeJ spleen cells produced a weak mitogenic response to both preparations of MPL in vitro, and C3H/HeJ mice showed a significant increase in serum IgM levels without an increase in numbers of splenic IgM-secreting plaque-forming cells after in vivo treatment with MPL. A significant increase in serum IgG3 levels was accompanied by a transient decrease in serum IgG1 levels in C3H/HeSnJ mice given MPL; such non-antigen-specific polyclonal effects were not observed in C3H/HeJ or in athymic nu/nu mice. Since the enhanced antibody response to SSS-III has been attributed to the inactivation of suppressor T cells by MPL and since suppressor-T-cell activity is demonstrable in both C3H/HeSnJ and C3H/HeJ mice, these findings imply that (i) the suppressor T cells of C3H/HeJ mice are refractory to inactivation by MPL and (ii) some of the polyclonal and mitogenic effects produced in C3H/HeJ mice are due to the direct action of MPL on B lymphocytes.

Adjuvant effects of trehalose dimycolate on the antibody response to type III pneumococcal polysaccharide.

Treatment with trehalose dimycolate (TDM) increases the magnitude of the immunoglobulin M (IgM) antibody response of mice to type III pneumococcal polysaccharide (SSS-III). Such enhancement is demonstrable over a wide range of immunizing doses and does not require thymus-derived (T) cells to be elicited. Although young adult mice immunized with SSS-III do not usually make anti-SSS-III antibodies of the IgG1 and IgG3 classes, antibodies of one or both isotypes were produced after immunization and treatment with TDM and/or monophosphoryl lipid A (MPL); the additive nature of the effect produced by both TDM and MPL suggests that the two immunomodulators act by different mechanisms. TDM and MPL have different effects on the induction and expression of low-dose immunological paralysis, a form of unresponsiveness known to be mediated by suppressor T cells. The relevance of these findings to the modes of action of TDM and MPL is discussed.

Characterization of the immunodeficiency of RIIS/J [corrected] mice. I. Association with the CD5 (LY-1) [corrected] B cell lineage.

RIIIS/J mice produce low antibody responses to several polysaccharide Ag of bacterial origin. They have low levels of serum IgM and IgG3 and high levels of serum IgG2a and IgG2b. Low serum IgM and IgG3 have been attributed to a low frequency of CD5 (Ly-1) B cells, which play an important role in the production of natural antibodies. Indeed, RIIIS/J mice have a low frequency of CD5 (Ly-1)+, IgM bright+, Ly-5 (B220)dull+ (i.e., CD5 (Ly-1) B) cells in their peritoneum. RIIIS/J mice treated with LPS produce a low anti-bromelain-treated mouse RBC splenic plaque-forming cell response and a normal anti-mouse transferrin splenic PFC response. Those data are compatible with the fact that CD5 (Ly-1) B cells contain the precursors of B lymphocytes secreting anti-bromelain-treated mouse RBC antibody. However, they have a higher frequency of IgM bright+, Mac-1+ cells in their peritoneum. These cells represent the CD5 (Ly-1) "sister population" of CD5 (Ly-1) B cells described by others. This suggests that characteristics usually associated with the CD5 (Ly-1) lineage are applicable only to the CD5 (Ly-1)+ Mac-1+ IgM+ population, but not the related CD5 (Ly-1)- Mac-1+ IgM+ population. RIIIS/J mice should thus prove a valuable model to study the CD5 (Ly-1) B cell lineage.

Ability of monophosphoryl lipid A to augment the antibody response of young mice.

Treatment with nontoxic monophosphoryl lipid A increased the magnitude of the immunoglobulin M (IgM) antibody response to type III pneumococcal polysaccharide in young (2- to 4-week-old) mice. This was accompanied by the appearance of significant numbers of IgG1- and IgG3- secreting antibody-forming cells in 4-week-old mice. These findings indicate that monophosphoryl lipid A can be used as an adjuvant to improve the immunogenicity of poorly immunogenic antigens in young, immunologically immature animals.

Prior exposure to subimmunogenic amounts of some bacterial lipopolysaccharides induces specific immunological unresponsiveness.

Pretreatment (priming) of BALB/c mice with a low (subimmunogenic) dose of Escherichia coli O113 lipopolysaccharide (LPS) generates immunological memory 7 to 30 days later; the direct (immunoglobulin M) plaque-forming cell (PFC) responses produced after subsequent immunization with an optimal dose are 4 to 20 times greater than those of unprimed mice. By contrast, priming with a low dose of E. coli O55 LPS, followed by immunization with an optimally immunogenic dose 2 to 30 days later, resulted in a significantly reduced antibody response. Similar results were obtained with Serratia marcescens LPS. Dose-response studies indicated that such unresponsiveness is antigen specific and could be induced with subimmunogenic amounts of LPS. Priming reduced the magnitude of the PFC response to all immunizing doses of LPS tested. Unresponsiveness is not due to (i) an alteration in the time course of the PFC response or to (ii) a change in the isotype of the anti-LPS antibody produced after priming and immunization.

Mechanisms of specific immunological unresponsiveness to bacterial lipopolysaccharides.

Low-dose priming of mice with Escherichia coli O113 lipopolysaccharide (LPS) results in the development of immunological memory, whereas low-dose priming with E. coli O55 LPS or Serratia marcescens LPS induces significant antigen-specific unresponsiveness. All three preparations of LPS induced proliferation of mouse splenocytes with similar time course and [3H]thymidine uptake. There was no correlation between the small amounts of serum antibody detected by enzyme-linked immunosorbent assay after low-dose priming and the subsequent generation of either memory or unresponsiveness. Further, the passive transfer of small amounts of LPS-specific antibody had no significant effect on the magnitude of the plaque-forming cell (PFC) response elicited after subsequent immunization. Reduction of the PFC response to E. coli O55 LPS occurred after low-dose priming of nu/nu (as well as nu/+) mice; however, unresponsiveness could not be generated in nu/nu mice by low-dose priming with S. marcescens LPS. Thus, although the development of low-dose unresponsiveness to S. marcescens LPS appears to involve T cells, the response of E. coli O55 LPS does not. Enhancement of the primary PFC response to S. marcescens LPS could be transferred with low-dose primed spleen cells depleted of Lyt-2+ T cells; this suggests that the magnitude of the PFC response to this preparation of LPS is negatively influenced by Lyt-2+ T cells and positively influenced by Lyt-2- spleen cells (i.e., L3T4+ T cells). These findings indicate that T cells appear to be involved in regulating the magnitude of the antibody response to some types of bacterial LPS.

Enhancement of non-Candida antibody responses by Candida albicans cell wall glycoprotein.

Two cell wall glycoprotein extracts from Candida albicans (glycoprotein [GP] and peptidoglucomannan [PGM]) were tested for their influence on antibody responses to type III pneumococcal polysaccharide and sheep erythrocytes. GP was isolated from lipid-extracted cell walls with ethylenediamine, whereas PGM was extracted with dilute sodium hydroxide. Both glycoproteins increased the number of antibody-producing plaque-forming cells in the spleens of mice immunized with type III polysaccharide or sheep erythrocytes, although PGM appeared to be about 10 times more effective. PGM could be administered up to 3 days prior to immunization with sheep erythrocytes to elicit enhancement; it did not have to be administered by the same route as the immunogen to cause significant enhancement. Enhancement did not appear to be the result of a direct mitogenic effect of GP and PGM on lymphocytes, nor did these glycoproteins appear to stimulate the production of B-cell growth factors or interleukin 2.