Lipopolysaccharide from Brucella abortus behaves as a T-cell-independent type 1 carrier in murine antigen-specific antibody responses.

In order to determine the carrier nature of lipopolysaccharide from Brucella abortus (LPS-BA) in evoking humoral responses, normal and immunodeficient mice were immunized with trinitrophenyl (TNP)-conjugated LPS-BA (TNP-LPS-BA) and the responses were compared with those to known T-dependent and T-independent antigens. TNP-LPS-BA, like T-independent type 1 (TI-1) antigens such as TNP-BA and TNP-LPS from Escherichia coli (TNP-LPS-EC), generated anti-TNP responses in BALB/c, athymic BALB/c nu/nu, and CBA/N mice. In contrast, N-2,4-dinitrophenyl-beta-alanylglycylglycyl-substituted keyhole limpet hemocyanin, a typical T-dependent antigen, was not immunogenic in athymic mice, and TNP-Ficoll (T-independent type 2) was ineffective in eliciting humoral responses in CBA/N mice. These results indicate that LPS from B. abortus acts as a TI-1 carrier in generating antibody responses. In C3H/HeJ mice, TNP-LPS-BA generated higher-titer immunoglobulin G1 (IgG1), IgG2a, and IgG2b anti-TNP antibodies than TNP-LPS-EC. Compared with those from BALB/c mice, pure resting B cells isolated from C3H/HeJ mice exhibited a 30-fold lower proliferative response to LPS-EC, whereas the LPS-BA response was reduced to a lesser extent (5-fold). This suggests that the disparity observed in antibody titers was due to different abilities of LPS from B. abortus and E. coli to stimulate C3H/HeJ B cells. The ability of LPS from B. abortus to act as a carrier in generating humoral immune responses indicates that LPS-BA can be substituted for whole B. abortus organisms in vaccine development.     

Thymic-dependent anti-hapten response in congenitally athymic (nude) mice immunized with DNP-thymosin.

Immunization of congenitally athymic (nu/nu) and adult thymectomized, irradiated bone marrow, reconstituted (TxBm) mice with DNP5-thymosin (dinitrophenylated-bovine thymosin fraction 5) was found to elcit IgM and IgG anti-DNP plaque-forming cells in these animals. Further studies indicated that this response was antigen specific and not due to polyclonal activation. Since the hormonal properties of the thymosin were retained following linkage with hapten and DNP-thymosin was immunogenic in CBA/N and CBA/N female X DBA/2 male)F1 male mice, animals previously shown to have an X-linked inability to respond to thymus-independent antigens, it was concluded that DNP-thymosin functions both as a hormone and as a T-dependent antigen in eliciting an immune response in nu/nu and TxBm mice. Additional support for this conclusion was provided by the demonstration that DNP-thymosin could specifically prime for and elicit an anamnestic response in nu/nu mice. These results indicate that further investigation of the immune activities of DNP-thymosin may provide valuable insight in characterizing the maturation of helper T cells and their subsequent interaction with B cells.     

Endogenous gamma interferon mediates resistance to Brucella abortus infection.

Depletion of endogenous gamma interferon (IFN-gamma) with anti-IFN-gamma monoclonal antibody resulted in increased numbers of Brucella abortus in the spleen and liver of infected CBA mice. This increase was accompanied by a decrease in splenomegaly and a lower proportion of macrophages in the spleen. Furthermore, treatment of recipient mice with anti-IFN-gamma antibody blocked the adoptive transfer of resistance with immune T cells. Together, the results indicated that endogenous IFN-gamma plays an important role in mediating resistance to primary and secondary Brucella infection.     

Brucella abortus deficient in copper/zinc superoxide dismutase is virulent in BALB/c mice.

The gene encoding the Cu/Zn superoxide dismutase (SOD) of Brucella abortus strain 2308 was identified in a Brucella genomic library utilizing a combination of Western blotting and native gel electrophoresis. The Cu/Zn SOD gene was inactivated in vitro by ligation of a kanamycin resistance gene into the open reading frame encoding SOD. The plasmid born construct was introduced back into B. abortus by electroporation. Replacement of the wild-type Cu/Zn SOD by recombination was demonstrated by showing that both the KnR gene and the Cu/Zn SOD gene hybridized to the same band in a Southern analysis of genomic DNA. In addition, KnR strains were deficient in Cu/Zn SOD activity as assessed by lack of Cu/Zn SOD activity on a native gel and by lack of reactivity with specific serum in a Western analysis. Either strain 2308 or the Cu/Zn SOD deficient mutant injected intraperitoneally into BALB/c mice, exhibited no differences in their ability to colonize the spleen at 7 and 28 days post-inoculation. Thus, the inability to produce Cu/Zn SOD by B. abortus does not significantly impair its virulence in mice.     

Biological activities of Brucella abortus lipopolysaccharides.

Purified lipopolysaccharide (LPS) from smooth (s) and rough (R) strains of Brucella abortus and lipid A isolated from S-LPS by mild acid hydrolysis were examined in several assays of biological activity. Brucella S- and R-LPSs and Brucella lipid A activated the complement cascade. Previously reported mitogenic activation by Brucella LPSs of spleen cells from endotoxin-resistant C3H/HeJ mice was confirmed and also produced by isolated Brucella lipid A. Mitogenicity was not inhibited by polymyxin B, and amino acid analysis showed no binding of polymyxin B to Brucella LPS under conditions in which mitogenicity of phenol-water-extracted Escherichia coli LPS was inhibited. S and R Brucella LPSs and lipid A all produced equivalent polyclonal stimulation of C3H/HeJ and C3H/HeAU spleen cells. Crude and purified LPS from S but not from R B. abortus was toxic for outbred mice, with 50% lethal doses approximately six times greater than that for E. coli LPS. S- and R-LPSs were abortifacient in pregnant outbred mice. S Brucella LPS was lethal for carrageenen-pretreated C3H/HeJ and C3H/HeAU mice, whereas only C3H/HeAU mice were killed by E. coli LPS. The data are consistent with the hypothesis that the unique fatty acid composition of Brucella lipid A is responsible for its biological activity in endotoxin-resistant C3H/HeJ mice. The participation of the protein strongly bound to the lipid A cannot be excluded, but its mode of action, if any, is different from that of the lipid A-associated protein of enterobacterial LPS.     

Functional absence of a B cell subpopulation in ageing New Zealand mice

As has been found for spleen cells from ageing NZB x NZW (B/W) mice, ageing NZB mice were also found to make no antibody when stimulated in vitro with the polyclonal B cell activators (PBA) LPS and PPD. This immune defect was not due to the action of suppressor cells, since old NZB and B/W spleen cells did not suppress the PBA response of young spleen cells. Spleen cells from aged NZB mice were not able to generate antibody-forming cells when stimulated with the thymus-independent antigen, TNP-LPS, but were able to produce antibody in response to another thymus-independent antigen, TNP-AECM-Ficoll, thereby implying that there is a selective functional deletion of a B cell subpopulation in ageing New Zealand mice. The failure of B/W and NZB spleen cells to generate antibody in response to PBA is interpreted as a consequence of a continuing in vivo polyclonal B cell activation accompanying the development of autoimmune disease, leading to a scarcity of B cells available for activation by PBA and by some thymus-independent antigens in vitro.     

Mice lacking components of adaptive immunity show increased Brucella abortus virB mutant colonization

The Brucella abortus type IV secretion system (T4SS), encoded by the virB genes, is essential for survival in mononuclear phagocytes in vitro. In the mouse model, a B. abortus virB mutant was initially able to colonize the spleen at the level of the wild type for approximately 3 to 5 days, which coincided with the development of adaptive immunity. To investigate the relationship between survival in macrophages cultivated in vitro and persistence in tissues in vivo, we tested the ability of mutant mice lacking components of adaptive immunity to eliminate the virB mutant from the spleen during a mixed infection with the B. abortus wild type. Ifng(-/-) or beta(2)m(-/-) mice were able to clear the virB mutant to the same degree as control mice. However, spleens of Rag1(-/-) mice and Igh6(-/-) mice were more highly colonized by the virB mutant than control mice after 14 to 21 days, suggesting that, in these mice, there is not an absolute requirement for the T4SS to mediate persistence of B. abortus in the spleen. Macrophages isolated from Igh6(-/-) mice killed the virB mutant to the same extent as macrophages from control mice, showing that the reduced ability of these mice to clear the virB mutant from the spleen does not correlate with diminished macrophage function in vitro. These results show that in the murine model host, the T4SS is required for persistence beyond 3 to 5 days after infection and suggest that the T4SS may contribute to evasion of adaptive immune mechanisms by B. abortus.     

Vaccination with Brucella abortus rough mutant RB51 protects BALB/c mice against virulent strains of Brucella abortus, Brucella melitensis, and Brucella ovis.

Vaccination of BALB/c mice with live Brucella abortus RB51, a stable rough mutant, produced protection against challenge with virulent strains of Brucella abortus, Brucella melitensis, and Brucella ovis. Passive-transfer experiments indicated that vaccinated mice were protected against B. abortus 2308 through cell-mediated immunity, against B. ovis PA through humoral immunity, and against B. melitensis 16M through both forms of immunity. Live bacteria were required for the induction of protective cell-mediated immunity; vaccination with whole killed cells of strain RB51 failed to protect mice against B. abortus 2308 despite development of good delayed-type hypersensitivity reactions. Protective antibodies against the heterologous species were generated in vaccinated mice primarily through anamnestic responses following challenge infections. Growth of the antigenically unrelated bacterium Listeria monocytogenes in the spleens of vaccinated mice indicated that nonspecific killing by residual activated macrophages contributed minimally to protection. These results encourage the continued investigation of strain RB51 as an alternative vaccine against heterologous Brucella species. However, its usefulness against B. ovis would be limited if, as suggested here, epitopes critical for protective cell-mediated immunity are not shared between B. abortus and B. ovis.     

Interleukin-10 downregulates protective immunity to Brucella abortus.

In vivo neutralization of interleukin-10 (IL-10) with an anti-IL-10 monoclonal antibody resulted in up to 10-fold fewer bacteria in the spleens of BALB/c mice infected with the virulent Brucella abortus strain 2308. In vitro neutralization of endogenous IL-10 in brucella antigen-stimulated cultures of splenocytes from infected mice resulted in increased gamma interferon production in these cultures, whereas exogenous recombinant IL-10 inhibited the ability of peptone-elicited peritoneal macrophages to control intracellular brucellae. These data suggest that IL-10 may be downregulating the immune response to B. abortus by affecting both macrophage effector function and the production of the protective Th1 cytokine gamma interferon.     

Ontogeny of susceptibility of mouse splenic B cells to tolerance induction in vitro by TNP-D-GL.

The susceptibility of mouse spleen cells to hapten-specific tolerance induction of a primary in vitro thymus-independent antibody response was examined. Both the induction of tolerance by 2,4,6-trinitrophenyl-D-glutamic acid-D-lysine (TNP96D-GL) and of antibody formation (elicited by TNP-Brucella abortus) in neonatal spleen cell cultures were unaffected by anti-Thy-1.2 plus complement treatment. Spleen cells from neonatal mice were only slightly more sensitive to TNP96D-GL tolerance induction than were cells from adult mice. The difference in susceptibility to tolerance induction was not nearly as great as that predicted by "clonal abortion"-type theories of B cell tolerogenesis.     

Deletion of znuA virulence factor attenuates Brucella abortus and confers protection against wild-type challenge

znuA is known to be an important factor for survival and normal growth under low Zn(2+) concentrations for Escherichia coli, Haemophilus spp., Neisseria gonorrhoeae, and Pasteurella multocida. We hypothesized that the znuA gene present in Brucella melitensis 16 M would be similar to znuA in B. abortus and questioned whether it may also be an important factor for growth and virulence of Brucella abortus. Using the B. melitensis 16 M genome sequence, primers were designed to construct a B. abortus deletion mutant. A znuA knockout mutation in B. abortus 2308 (DeltaznuA) was constructed and found to be lethal in low-Zn(2+) medium. When used to infect macrophages, DeltaznuA B. abortus showed minimal growth. Further study with DeltaznuA B. abortus showed that its virulence in BALB/c mice was attenuated, and most of the bacteria were cleared from the spleen within 8 weeks. Protection studies confirmed the DeltaznuA mutant as a potential live vaccine, since protection against wild-type B. abortus 2308 challenge was as effective as that obtained with the RB51 or S19 vaccine strain.     

Variation of Brucella abortus 2308 infection in BALB/c mice induced by prior vaccination with salt-extractable periplasmic proteins from Brucella abortus 19.

The study compared the immune and protective responses induced in BALB/c mice vaccinated with six salt-extractable periplasmic protein fractions (Brucella cell surface proteins [BCSP]) of Brucella abortus 19 and later challenge exposed with B. abortus 2308. BCSP70 was precipitated with ammonium sulfate at 70% saturation, and BCSP100 was precipitated with ammonium sulfate at 100% saturation by use of supernatant fluid of BCSP70 that had been precipitated with 70% ammonium sulfate. Four subfractions were separated from BCSP100 by anion-exchange high-performance liquid chromatography (HPLC). Monophosphoryl lipid A (MPL) from Salmonella typhimurium Re mutant strain was used as a potential immune response modifier in some vaccines. Reduced or increased numbers of CFU and increased spleen size in the principal groups of mice relative to that of the nonvaccinated control group were considered protectiveness or virulence (survival) criteria. Results indicated that vaccines prepared from BCSP70 and BCSP100 were moderately protective and immunogenic. The subfractions designated BCSP100-A through BCSP100-D purified by anion-exchange HPLC were not protective when MPL was not used as an immune response modifier. However, two subfractions were associated with significant (P < 0.05) increases in CFU per spleen and splenomegaly in vaccinated mice compared with those in nonvaccinated challenge-exposed mice. MPL enhanced protection or was neutral when used with BCSP70, BCSP100, BCSP100-C, and BCSP100-D. Serologic results of an enzyme-linked immunosorbent assay indicated that MPL modulated the immunoglobulin G responses induced by BCSP70, BCSP100, and subfraction BCSP100-B vaccines only. The overall results suggest that certain proteinaceous periplasmic fractions might serve as virulence or survival factors in B. abortus infections.     

The effect of triiodothyronine on evanescent delayed hypersensitivity to sheep red blood cells and on the primary antibody response to trinitrophenylated Brucella abortus in severely undernourished weanling mice

Two experiments were conducted using weanling male CBA/J mice. Animals were fed for 14 days a nutritionally complete diet, either ad libitum, or in restricted amounts such that they lost about 30% of their weaning weight during the feeding period. Half the animals from each food intake group received 0.2 mg triiodothyronine (T3)/kg diet. Evanescent delayed hypersensitivity to sheep red blood cells (SRBC) was depressed by malnutrition but not influenced by T3 supplementation. T3 increased nucleated spleen cell number in mice immunized with trinitrophenylated Brucella abortus (TNP-BA). The number of splenic anti-TNP direct plaque-forming cells (PFCs) was decreased by malnutrition when expressed on a per spleen basis. T3 supplements increased splenic PFC number of malnourished mice when results were expressed either per spleen or per 10(6) cells, and increased PFCs per spleen in well-nourished mice. Serum anti-TNP agglutinin titres were unaffected by malnutrition and by T3. The results extend our previous observations as to the improvement by T3 of the primary anti-SRBC antibody response of malnourished weanling mice, and suggest that therapeutic hormonal treatment could be used to improve antibody responses of malnourished individuals. The mechanism of the effect of T3 is unknown, but the lack of effect on delayed hypersensitivity to SRBC implies that T3 (when administered at the dosage of the present protocol) acts on only some immune cells and does not exert a generalized adjuvant effect.     

The role of integrase/recombinase xerD and monofunctional biosynthesis peptidoglycan transglycosylase genes in the pathogenicity of Brucella abortus infection in vitro and in vivo

Brucella abortus clones identified previously using a green fluorescence protein reporter system after 4h macrophage infection provided insight regarding possible genes involved in early host-pathogen interaction. Among identified genes were an integrase/recombinase (xerD) gene involved in cell division, and a monofunctional biosynthesis peptidoglycan transglycosylase (mtgA) gene that catalyzes the final stages of the peptidoglycan membrane synthesis. Here, we evaluate the in vitro and in vivo survival of B. abortus xerD and mtgA insertional mutants. B. abortus xerD::kan and B. abortus mtgA::kan demonstrated no significant growth defects in broth culture when compared to the parental strain, S2308. Also, neither gene was required for B. abortus S2308 replication in RAW 264.7 macrophages. However, experimental evidence using interferon regulatory factor 1 knockout mice, a mouse strain highly susceptible to virulent Brucella, revealed that mice infected with B. abortus xerD::kan or B. abortus mtgA::kan survived longer than mice infected with S2308. Additionally, in immunocompetent BALB/c mice, B. abortus xerD::kan had a significantly lower level of bacterial survival when compared to S2308. Together, these results suggest that B. abortus xerD and mtgA genes play a role during the initial phase of infection in mice.     

Immune and mitogenic responses by BALB/c, C3H/HeJ, and nude mice to Brucella abortus bacterin and lipopolysaccharide.

The immunogenic and mitogenic properties of Brucella abortus 1119-3 bacterin (BA) and biologically active B. abortus lipopolysaccharide (BA-LPS) were studied using normal and athymic (nude) BALB/c and C3H/HeJ mice. Although BA stimulated 2-mercaptoethanol-sensitive (2-ME-S) primary and secondary antibody responses in all mice, nude mice, in contrast to normal BALB/c and C3H/HeJ mice, did not make substantial 2-mercaptoethanol-resistant (2-ME-R) antibody responses. Similarly, all mice injected with BA-LPS made 2-ME-S primary responses, and the secondary response of thymus-bearing mice contained a substantial 2-ME-R component. Collectively, these observations suggest that although both BA and BA-LPS can stimulate thymus-independent 2-ME-S antibody synthesis, thymus-derived cells are required for optimal immune responses containing a 2-ME-R component. The antibody responses of normal BALB/c and C3H/HeJ mice to BA and BA-LPS were qualitatively and quantitatively similar. Both BA and BA-LPS were mitogenic for spleen cells from normal and nude BALB/c and C3H/HeJ mice but not for thymus cells from normal BALB/c or C3H/HeJ mice, suggesting that both preparations are B-cell mitogens.     

Xid mouse lymphocytes respond to TI-2 antigens when co-stimulated by TI-1 antigens or lymphokines

Spleen cells from male (CBA/N x DBA/2) F1 hybrid mice do not significantly respond to in vitro stimulation by trinitrophenyl-conjugated polyacrylamide beads (TNP-PAA), whereas the same antigen elicits high PFC responses in female F1 hybrid cells. Therefore, this antigen could be classified as a T-independent type 2 (TI-2) antigen. When male spleen cells were co-stimulated by TNP-PAA and TI type 1 antigen, either LPS or Brucella abortus, they produced vigorous anti-TNP responses. A similar increase of the in vitro responsiveness of male F1 hybrid spleen cells to TNP-PAA antigen was provoked by the addition of supernatants from P 388-D1 cells stimulated by muramyl-dipeptide (MDP) mainly containing interleukin-1 (IL-1) or supernatants from phorbol 12-myristate 13-acetate (PMA)-stimulated EL-4 cells that contained T-cell factors. The PFC response to another TI-2 antigen, TNP-Ficoll, was also significantly enhanced after co-stimulation by P 388-D1 supernatants. The response to TI-2 antigens being macrophage dependent, the influence of supernatants of peritoneal macrophages from male and female F1 hybrids incubated with TNP-Ficoll on the PFC response of normal DBA/2 mouse spleen cells to sheep erythrocytes was assessed. It was found that macrophage supernatants from female hybrids regularly increased by more than two times this anti-SRBC PFC response, whereas macrophage supernatants from male F1 hybrids did not. Moreover, in a specific proliferation test measuring IL-1 activity, when macrophage supernatants from female F1 produced a 13-fold increase of thymidine incorporation, supernatants from male F1 only produced a three-fold increase. It is concluded that, in addition to the known defects of B cells from Xid mice, their macrophages are also defective.     

Protection of BALB/c Mice against Brucella abortus 544 Challenge by Vaccination with Bacterioferritin or P39 Recombinant Proteins with CpG Oligodeoxynucleotides as Adjuvant

The P39 and the bacterioferrin (BFR) antigens of Brucella melitensis 16M were previously identified as T dominant antigens able to induce both delayed-type hypersensivity in sensitized guinea pigs and in vitro gamma interferon (IFN-gamma) production by peripheral blood mononuclear cells from infected cattle. Here, we analyzed the potential for these antigens to function as a subunitary vaccine against Brucella abortus infection in BALB/c mice, and we characterized the humoral and cellular immune responses induced. Mice were injected with each of the recombinant proteins alone or adjuvanted with either CpG oligodeoxynucleotides (CpG ODN) or non-CpG ODN. Mice immunized with the recombinant antigens with CpG ODN were the only group demonstrating both significant IFN-gamma production and T-cell proliferation in response to either Brucella extract or to the respective antigen. The same conclusion holds true for the antibody response, which was only demonstrated in mice immunized with recombinant antigens mixed with CpG ODN. The antibody titers (both immunoglobulin G1 [IgG1] and IgG2a) induced by P39 immunization were higher than the titers induced by BFR (only IgG2a). Using a B. abortus 544 challenge, the level of protection was analyzed and compared to the protection conferred by one immunization with the vaccine strain B19. Immunization with P39 and CpG ODN gave a level of protection comparable to the one conferred by B19 at 4 weeks postchallenge, and the mice were still significantly protected at 8 weeks postchallenge, although to a lesser extent than the B19-vaccinated group. Intriguingly, no protection was detected after BFR vaccination. All other groups did not demonstrate any protection.     

Characterization of immunogenic properties of haptenated liposomal model membranes in mice. VII. Synergistic responses to haptenated liposomes and haptenated thymus-dependent antigens in CBA/N mice

CBA/N mice have an X-linked B-cell defect which prevents them from responding to non-mitogenic thymus-independent (TI-2) antigens such as haptenated Ficoll. The CBA/N mice do, however, respond to another group of thymus-independent (TI-1) antigens among which are haptenated liposomes. The F1 male progeny of CBA/N female mice express the same characteristics. Hapten-specific plaque-forming cell (PFC) responses to haptenated proteins (thymus-dependent, TD, antigens) by CBA/N mice and CBA/N x C3H/HeN F1 male mice can be blocked by concomitant exposure to TI-2 antigens bearing the same hapten. Simultaneous exposure to haptenated liposomes (TI-1 antigen) and TD antigens, however, results in a synergistic response to the hapten if the antigens share common epitopes. The tripeptide-enlarged hapten dinitrophenyl-beta-alanylglycylglycine (J), conjugated to phosphatidyl-ethanolamine (PE) and incorporated into liposomal model membranes, was used throughout the experiments. In F1 male mice the moderate responses to TD antigens could be restored to values equivalent to those seen in F1 female mice. This adjuvant effect of haptenated liposomes is hapten-specific, time-dependent, and restricted to certain structural forms of the liposomes.     

Induction of antigen-specific Th1-type immune responses by gamma-irradiated recombinant Brucella abortus RB51

Brucella abortus strain RB51 is an attenuated rough mutant used as the live vaccine against bovine brucellosis in the United States and other countries. We previously reported the development of strain RB51 as a bacterial vaccine vector for inducing Th1-type immune responses against heterologous proteins. Because safety concerns may preclude the use of strain RB51-based recombinant live vaccines, we explored the ability of a gamma-irradiated recombinant RB51 strain to induce heterologous antigen-specific immune responses in BALB/c mice. Exposure of strain RB51G/LacZ expressing Escherichia coli beta-galactosidase to a minimum of 300 kilorads of gamma radiation resulted in complete loss of replicative ability. These bacteria, however, remained metabolically active and continued to synthesize beta-galactosidase. A single intraperitoneal inoculation of mice with 10(9) CFU equivalents of gamma-irradiated, but not heat-killed, RB51G/LacZ induced a beta-galactosidase-specific Th1-type immune response. Though no obvious differences were detected in immune responses to B. abortus-specific antigens, mice vaccinated with gamma-irradiated, but not heat-killed, RB51G/LacZ developed significant protection against challenge with virulent B. abortus. In vitro experiments indicated that gamma-irradiated and heat-killed RB51G/LacZ induced maturation of dendritic cells; however, stimulation with gamma-irradiated bacteria resulted in more interleukin-12 secretion. These results suggest that recombinant RB51 strains exposed to an appropriate minimum dose of gamma radiation are unable to replicate but retain their ability to stimulate Th1 immune responses against the heterologous antigens and confer protection against B. abortus challenge in mice.     

Further studies on the H-2 linked dependence of the adjuvant action of Brucella abortus.

The B 19S strain of Brucella abortus is found to act as an adjuvant to the anti-sheep red blood cell (SRBC) reaction in some congenic strains of mice but not in others. If the recipient is H-2b, there is no adjuvant effect (B 19S); neither is there (thymus-dependent anti-B 19S reaction as measured by thymocyte activation and change of electrophoretic mobility. In contrast, there is a thymus-independent anti-B 19S reaction (production of haemagglutinins) as good in the H-2b mice as in the others. Experiments with T cell deprived mice show that the adjuvant action of B 19S is thymus-dependent. As the anti-SRBC reaction without adjuvant is also thymus-dependent, it is difficult to distinguish anti-SRBC and anti-B 19S reactions from the adjuvant action of B 19S on the anti-SRBC reaction. Several explanations are possible, all involving H-2 (Ir?) controlled thymus dependent mechanisms.