Induction of immune and adjuvant immunoglobulin G responses in mice by Brucella lipopolysaccharide

The immunogenic and adjuvant properties of Brucella abortus and Escherichia coli lipopolysaccharides (LPSs) were studied in endotoxin-responsive, athymic, and euthymic BALB/c mice and in responsive C3H/HeAu mice and congenic nonresponsive C3H/HeJ mice. Consistent with previous reports, E. coli LPS did not stimulate significant primary or secondary antibody responses in C3H/HeJ mice and induced the production of immunoglobulin M (IgM) and low levels of IgG in C3H/HeAu mice. In contrast, B. abortus smooth and rough LPS stimulated primary and secondary antibody responses and induced the production of IgM and high levels of IgG in both responsive and nonresponsive strains of C3H/He mice and in nude mice. When used as adjuvant, B. abortus LPS augmented the IgG plaque-forming-cell response of C3H/HeAu and BALB/c euthymic mice to the T-dependent antigen sheep erythrocytes. E. coli LPS augmented only the IgM plaque-forming-cell response in the same mouse strains. Neither B. abortus nor E. coli LPS was adjuvant for C3H/HeJ or nude mice. The dichotomy between the antibody and adjuvant responses of both C3H/HeJ mice and athymic mice to B. abortus LPS may be a function of the true thymus independence and dependence of these responses. In addition, the refractiveness of C3H/HeJ and nude mice to B. abortus LPS as adjuvant, but not as mitogen or polyclonal B cell activator, clearly dissociates these phenomena.     

Influence of endotoxin-protein in immunoglobulin G isotype responses of mice to Brucella abortus lipopolysaccharide

Brucella abortus endotoxin preparations, containing approximately 5 to 6% protein, induce strong immune and adjuvant immunoglobulin G (IgG) responses as compared with Escherichia coli endotoxin preparations, with equivalent amounts of protein, which induce responses in which IgM antibody predominates. Using an enzyme-linked immunoassay with isotype-specific conjugates, we found that antibody of all four subclasses of IgG were evoked during the course of the immune responses of C3H/HeAu mice to B. abortus endotoxin. Secondary responses of endotoxin-hyporesponsive C3H/HeJ mice were similar to those seen in C3H/HeAu mice, although lower levels of antibody were produced during their primary responses. The primary responses of BALB/c athymic mice consisted almost entirely of IgG3, and IgG1 appeared following a second injection. The effects of lipopolysaccharide (LPS)-associated protein on the immunogenic properties of B. abortus endotoxin were examined by comparing responses to endotoxin with those to a purified B. abortus LPS containing less than 1% protein. The endotoxin evoked strong primary and secondary responses in which antibody directed to LPS determinants consisted mainly of IgG3 and those to the protein determinants were largely IgG1 antibody. Primary and secondary responses to purified LPS consisted mainly of IgG3 antibody. The potential mechanism of the contribution of protein to the immunogenic properties of the endotoxin as well as possible immune mechanisms involved in these responses are discussed.     

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.     

Immune response against the T-independent antigen alpha (1 leads to 3) dextran. I. Demonstration of an unexpected IgG response of athymic and germ-free-raised euthymic BALB/c mice

The primary antibody response in BALB/c mice to the T-independent bacterial antigen dextran B1355S [alpha(1 leads to 3)dextran] (Dex) was studied by means of isoelectric focusing, hemagglutination and immunodiffusion techniques. In response to a single immunization with 10 micrograms Dex all mice produce specific IgM antibodies. In addition, about 30% of conventionally raised BALB/c and BALB/c nu/ + mice, but 95% of germ-free (GF)-raised normal BALB/c and 100% of athymic BALB/c nu/nu mice produce specific IgG class anti-Dex antibodies. These antibodies include all IgG subclasses, carry predominantly the lambda light chain and the cross-reactive J558 idiotype and are specific for the alpha(1 leads to 3)glucosidic linkage. As compared to athymic and GF-raised mice, conventionally raised mice exhibit only a weak IgG response. The pronounced IgG production of GF-raised mice was not altered when adult mice were removed from their GF environment and housed under conventional conditions for several weeks prior to immunization with Dex. Reconstitution with isolated splenic T cells from conventionally raised, unprimed BALB/c mice reduces the remarkable capacity of BALB/c nu/nu mice to produce IgG anti-Dex antibodies. These findings suggest that the reduced capacity of conventionally raised BALB/c mice to mount an IgG response to the T-independent antigen Dex is due to a T cell-mediated suppressive mechanism which is neonatally induced by contact with environmental, i.e. bacterial, antigens.     

Immunomodulation of the antibody response to lipopolysaccharide in C3H/HeJ mice by complexing with heterologous ribosomes.

We show that formaldehyde fixation of Salmonella typhimurium lipopolysaccharide (LPS) to ribosomes purified from Brucella abortus induced a primary immunoglobulin M (IgM) response to LPS in C3H/HeJ mice and upon revaccination resulted in elevated titers of IgM and induction of IgG antibody to the O antigens of LPS, as measured by an enzyme-linked immunosorbent assay. A similar LPS-Aspergillus fumigatus ribosomal complex yielded IgM and IgG antibody to LPS only after secondary stimulation. These results demonstrate that the hyporesponsiveness of C3H/HeJ mice with respect to antibody formation to LPS can be overcome by complexing this molecule to ribosomal particles and provide a theoretical mechanism for the action of some "ribosomal" vaccines. The results are compatible with the hypothesis that LPS in complex with the ribosomes is converted to a T-dependent form of the antigen to which the C3H/HeJ mice can respond.     

The induction of B cells refractory to antibody-specific immunoregulation

Subsequent to primary immunization with a hapten-carrier conjugate, and concomitant with an initial antibody response to that antigen, a regulatory mechanism is induced that specifically limits the stimulation of hapten-specific primary B cell responses through the recognition of B cell antibody. Nonimmune B cells are sensitive to this regulation, while secondary B cells are refractory to its suppressive effects. Experiments were conducted to determine the conditions under which refractory B cell populations are generated. B cells from BALBlc and athymic BALB/c nu/nu mice were examined following immunization with the T-dependent antigen dinitropheny-lated (DNP) hemocyanin and the T-independent antigen DNP-Ficoll to assess the T cell dependence of the generation of refractory B cells. Evidence is presented that this process is not dependent on the presence or participation of T cells during in vivo immunization, since both T-dependent and T-independent antigens have the potential to induce a refractory B cell population. However, under certain circumstances, T cells can regulate the induction of refractory B cells during in vivo immunization. In addition, it was determined that the immunoregulatory phenomenon can be induced following immunization with both T-dependent and T-independent antigens.     

Hapten-specific B cell blockade of the immune response to a thymus-independent-1 antigen produced by concomitant administration of a thymus-independent-2 antigen.

CBA/N mice harbour an X-linked B cell defect which is transmitted by CBA/N female mice to their hybrid male progeny. These mice mount normal responses to thymus-dependent (TD) and some thymus-independent (TI-1) antigens, while the response to TI-2 antigens is absent. Hapten-specific plaque-forming cell (PFC) responses to TD antigens can be blockaded by concomitant exposure of these mice to TI-2 antigens bearing the same hapten. This paper investigates in defective mice the blockade of their response to TNP3-LPS (trinitrophenylated lipopolysaccharide, a TI-1 antigen), imposed by DNP59-Ficoll (dinitrophenylated Ficoll, a TI-2 antigen). The effectiveness of the blocking agent, DNP59-Ficoll, differed in various inbred mouse strains: CBA/N X C3H/HeN F1 male greater than CBA/N female greater than CBA/N X C3H/HeN F1 female. The role of T cells in the observed hapten-specific blockade phenomenon was investigated using athymic CBA/N nude mice and a B cell tolerogen. Our findings indicate that T cell participation is not essential for the blockade of CBA/N PFC responses and they suggest that direct blockade of TI- and TD-responsive B cell populations is likely to occur.     

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.     

T-cell-dependent and T-cell-independent mechanisms of tolerance to glucuronoxylomannan of Cryptococcus neoformans serotype A.

Glucuronoxylomannan (GXM), a type 2 T-independent antigen, is the major component of the capsular polysaccharide (CnCAP) of Cryptococcus neoformans. Previous studies have described the tolerogenic effects of high doses of CnCAP on the specific humoral response. In this investigation, evidence for both high-dose and low-dose tolerance to GXM is presented. BALB/cBy female mice, primed with either 5 ng or 50 micrograms of GXM, then coimmunized 3 days later with immunogenic doses of both GXM and type 3 pneumococcal polysaccharide (SSS-III), showed an antigen-specific inhibition in their splenic plaque-forming cell (PFC) responses to GXM compared with control groups primed with normal saline. SSS-III PFCs remained unchanged between GXM-primed and normal saline-primed groups. Low-dose tolerance appeared to be T dependent, whereas high-dose tolerance appeared to be T independent. Low-dose tolerance to GXM could not be induced in athymic BALB/c nu/nu mice, whereas high-dose tolerance in the same mice could be induced. Furthermore, low-dose tolerance was adoptively transferred with B-cell-depleted splenocytes to naive BALB/c mice, while high-dose tolerance was not. Complement-mediated depletion of CD4+ but not CD8+ splenocytes from low-dose-primed mice abrogated the transfer of low-dose tolerance. These findings indicate T-dependent and T-independent mechanisms of antigen-specific B-cell tolerance to GXM in BALB/c mice at low and high antigen doses, respectively.     

Adjuvant effects of CpG oligodeoxynucleotides on responses against T-independent type 2 antigens

Oligodeoxynucleotides containing CpG motifs (CpG-ODN) are potent in vitro B-cell activators and they have been successfully used to increase in vivo antibody responses to T-dependent peptide and protein antigens. In contrast, the use of CpG-ODN to enhance in vivo antibody responses to various T-independent type 2 (TI-2) antigens has recently generated contradictory results. In this study, we compared the CpG-ODN stimulatory effect on antibody responses of adult and young BALB/c mice to trinitrophenylaminoethyl-carboxymethyl (TNP) -Ficoll and to polysaccharides (PS) from several distinct serotypes of Streptococcus pneumoniae (SPn). CpG-ODN co-administration significantly enhanced antigen-specific immunoglobulin M (IgM), IgG, IgG1 and IgG2a titres to TNP-Ficoll. The depletion of CD4+ cells by monoclonal antibodies (GK1.5) identified their essential role in CpG-ODN-mediated enhancement of antibody responses. In contrast to TNP-Ficoll, CpG-ODN failed to enhance IgM and IgG responses to any of the 18 SPnPS serotypes tested. Providing T-cell epitopes by the conjugation of SPnPS to the carrier protein tetanus toxoid again allowed CpG-ODN to mediate enhancement of IgG, IgG2a and IgG3 responses to most SPnPS serotypes. Thus, antigen-presenting cell/T-cell interaction appears to largely mediate the in vivo influence of CpG-ODN on antibody responses to TI-2 antigens. In early life, additional factors limit CpG-ODN modulation of antibody responses to TI-2 antigens.     

Plasmodium falciparum: natural and experimental transmission-blocking immunity

Fc-dependent regulation of humoral immune responses was investigated by immunization of BALB/c mice with immune complexes. These complexes were composed of DNP- and PC-conjugated KLH or Ficoll, and monoclonal T15 idiotype-positive anti-PC antibodies of different isotypes but indistinguishable V-region properties. Since the response to DNP was analyzed, effects due to masking of antigenic determinants by anti-PC antibodies are excluded. The responses to free and complexed antigens showed significant differences in the proportions of DNP-specific IgM, IgG1, IgG2 and IgG3 antibodies. Complexes containing the T-dependent carrier KLH elicited serum antibodies to DNP with significantly decreased IgM levels, irrespective of the isotype in the complex. Under these conditions, however, DNP-specific IgG classes were augmented to various extents, depending on the isotype in the complex. In the case of the T-independent carrier Ficoll, only complexes with IgM or IgG3 suppressed the IgM response. Moreover, immunization with complexes composed of IgM or IgG2a led to a significant decrease of DNP-specific IgG1. In contrast to changes induced by antibodies bound to the T-dependent antigen, immunization with T-independent complexes did not enhance the production of any of the immunoglobulin isotypes.     

Comparison between Nu/Nu and normal BALB/c T-helper activity in anti-hapten and anti-carrier responses

Nu/nu BALB/c mount a primary in vivo anti-TNP response to T-dependent TNP-antigens in the range of normal BALB/c mice. However, the response against the carrier (horse red blood cells, HRBC) was in the magnitude of about 5% as compared to normal BALB/c mice. Neither against the hapten, nor against the carrier a secondary response was observed. It could be shown by in vitro experiments that nu/nu contain a small population of TNP specific as well as HRBC specific T-helper (THTNP, THHRBC) cells. But even within the small nu/nu T-cell population, TH cells are less frequent than in T-cell populations of normal mice. Furthermore, the difference in help between nu/nu and normal BALB/c was more pronounced with respect to the anti-HRBC than the anti-TNP response. These observations could explain the lack of a secondary in vivo response being due to the low number of TH cells and the apparent in vivo unresponsiveness against HRBC as a consequence of the low frequency of THHRBC.     

Role of different lymphocyte subpopulations in the formation of non-specific immunoglobulins induced by antigen injection

The formation of antibody and non-specific immunoglobulin under the influence of T-dependent (TD) and type 2 T-independent (TI-2) antigens in mice of two congenic strains CBA (Lyb5-, Lyb5+) and CBA/N (Lyb5-) was studied. TD antigens induced in mice of both strains not only the appearance of antibody-forming cells (AFC), but also a great increase in the number of cells producing non-specific immunoglobulins (nIFC). TI-2 antigens induced the AFC and antigen-dependent nIFC formation in CBA mice only. It is concluded that during immune response to TI-2 antigens not only the AFC appearance but the increase in nIFC formation (polyclonal activation) is due mainly to the mature Lyb5+ B cells.     

Mechanism of Enhanced Humoral Response in Rodents by a Synthetic Polymer

We previously reported that administration of a low molecular weight (MW=800) synthetic polymer, NED 137, significantly increases humoral and cellular immune responses in the rat. The effect of NED 137 on the murine humoral response to T-dependent (TD) and T-independent (TI) antigens was studied in C57 BL/6, CBA/J and Balb/c mice. The TD antigens (SRBC, DNP-DA with adjuvant) or TI antigen (DNP-Ficoll) were administered simultaneously with NED 137. The polymer significantly increased the direct PFC response to all antigens tested in normal mice. However, it could not restore the PFC response to SRBC in athymic (nu/nu) mice. The effect of NED 137 on accessory cells was studied by the assessment of the in vitro response to SRBC in normal and macrophage-depleted rat spleen cultures. The polymer stimulated both, the primary and secondary IgM response and its immunopotentiating activity was the greatest in macrophage-depleted spleen cell preparations. The lack of effect of NED 137 in systems devoid of functional T cells, dependency on and specificity for a sensitizing antigen and its ability to stimulate a secondary response suggest that this polymer does not act as a “conventional” B-cell polyclonal activator.     

Mechanism of Enhanced Humoral Response in Rodents by a Synthetic Polymer

Abstract

We previously reported that administration of a low molecular weight (MW=800) synthetic polymer, NED 137, significantly increases humoral and cellular immune responses in the rat. The effect of NED 137 on the murine humoral response to T-dependent (TD) and T-independent (TI) antigens was studied in C57 BL/6, CBA/J and Balb/c mice. The TD antigens (SRBC, DNP-DA with adjuvant) or TI antigen (DNP-Ficoll) were administered simultaneously with NED 137. The polymer significantly increased the direct PFC response to all antigens tested in normal mice. However, it could not restore the PFC response to SRBC in athymic (nu/nu) mice. The effect of NED 137 on accessory cells was studied by the assessment of the in vitro response to SRBC in normal and macrophage-depleted rat spleen cultures. The polymer stimulated both, the primary and secondary IgM response and its immunopotentiating activity was the greatest in macrophage-depleted spleen cell preparations. The lack of effect of NED 137 in systems devoid of functional T cells, dependency on and specificity for a sensitizing antigen and its ability to stimulate a secondary response suggest that this polymer does not act as a “conventional” B-cell polyclonal activator.     

Influence of lipopolysaccharide on graft versus host reactivity of lipopolysaccharide-unresponsive C3H/HeJ mice.

It was initially reported that lipopolysaccharide (LPS)-unresponsive C3H/HeJ mice are refractory to LPS at the B-lymphocyte level, but more recently it has been shown that other cells are similarly unaffected. The current study was undertaken to study an in vivo LPS-modulated disease process involving macrophage-T cell interactions. Adult CBA/J and C3H/HeJ mice were used as spleen donors, and graft versus host reactions were induced in BALB/c neonates. Prior LPS treatment of CBA/J adults decreased the ability of their spleen cells to cause fatal graft versus host disease in BALB/c neonates, whereas no difference was found between injection of spleen cells from normal or LPS-treated C3H/HeJ mice. Similar results were obtained with these cell types when the mouse spleen mixed leukocyte culture system was used. In a carbon clearance assay for stimulation of the reticuloendothelial system with LPS, it was found that the rate of phagocytosis was significantly increased in BALB/c and CBA/J mice 72 h after inoculation of LPS. No stimulation was seen in rate of carbon uptake in the C3H/HeJ animals after treatment with phenol-extracted LPS or with butanol-extracted LPS. An LPS-induced protective serum factor was produced only in the LPS-responsive CBA/J mice and was specific for the syngeneic cells.     

Rheumatoid Synovial Fluid Reconstitutes the B-Cell Defect in CBA/N Mice

Synovial fluid from patients with rheumatoid arthritis (RA-SF) contains a biological activity which can replace T cells for activation of antibody secretion in human blood lymphoid cells and which can also induce the selective differentiation of IgG2b-secreting cells in lipopolysaccharide (LPS)-pre-activated mouse spleen cells. The B-cell activity of this factor was studied in CBA/N mice which have an X-linked B-cell immunodeficiency which manifests itself as a defective humoral response to certain thymus-independent antigens (TI-2). RA-SF has now been shown to reconstitute partly the B-cell deficiency in CBA/N splenic B cells in vitro. Addition of RA-SF to LPS-pretreated cell cultures results in IgG2b secretion in CBA/N spleen cells as well. In contrast to cells from normal CBA mice, cells from CBA/N mice cannot respond to interleukin 4 (IL-4) after addition of LPS with production of IgG1 antibodies in vitro. However, the addition of RA-SF completely restores a normal IL-4-induced IgG1 response. No other biologically active factors have been shown to allow the production of IgG antibody producing cells in CBA/N splenic B cells. It is postulated that the xid immunodeficiency could be the result of a deficient production of a biological activity which is abundant in RA-SF.     

Primary antibody responses to thymus-independent antigens in the lungs and hilar lymph nodes of mice.

B lymphocytes from the pulmonary lymphoid tissues were stimulated with a variety of thymus-independent (TI) antigens by intratracheal (i.t.) immunization. Immune responses in the lungs and hilar lymph nodes (HLN), which are part of the localized lymphoid tissue, as well as in the spleen, the systemic lymphoid organ, were studied. Thus, primary i.t. immunization of mice with the TI-1 antigen trinitrophenyl-lipopolysaccharide (TNP-LPS) elicited both antigen-specific and polyclonal plaque-forming cell responses from HLN, lung, and splenic B lymphocytes. These responses appeared as early as 3 days after immunization and declined by day 7. Similar immunization with another TI-1 antigen, TNP-Brucella abortus, resulted in anti-TNP responses in both pulmonary and systemic lymphoid tissues, although the kinetics of the antibody response were different than those to TNP-LPS. Interestingly an i.t. immunization with a TI-2 antigen, TNP-Ficoll, failed to induce an anti-TNP PFC response from HLN and lung B cells, although there was good antibody formation from splenic B cells. Antibody response to TNP-Ficoll was restored in pulmonary tissues when mice were immunized with TNP-Ficoll mixed with unconjugated B. abortus. In conclusion, our results indicate that TI-1 and TI-2 antigens differ in their ability to induce antibody responses in the pulmonary lymphoid tissues. The inability of TNP-Ficoll to elicit an antibody response in pulmonary lymphoid tissues has significance in the development of vaccines containing bacterial polysaccharides.     

Adjuvant actions of linear mannan-possessing lipopolysaccharide (LPS) in LPS-resistant C3H/HeJ mice.

Immunopotentiation has been demonstrated when Klebsiella O3 lipopolysaccharide (KO3 LPS), which possesses a linear mannan as the O-specific side chain, was injected subcutaneously into endotoxin resistant C3H/HeJ mice together with soluble protein antigens. The LPS exhibited significantly positive adjuvant effects on antibody responses in vivo after secondary antigen challenge and on delayed-type hypersensitivity (DTH) reactions against protein antigens. However, KO3 LPS was not a polyclonal B cell activator (PBA) in C3H/HeJ mice nor mitogenic in cultures of spleen cells of C3H/HeJ. Thus, the activity of the LPS in C3H/HeJ mice is confined to the potentiation of T-dependent immune responses. The contribution of the mannan O side chain to the adjuvant action of KO3 LPS was suggested.     

The glucuronoxylomannan of Cryptococcus neoformans serotype A is a type 2 T-cell-independent antigen.

The humoral immune response of inbred mice to immunization with the glucuronoxylomannan (GXM) of Cryptococcus neoformans was investigated both serologically and in plaque-forming cells (PFCs). The T-helper-cell-independent quality of the GXM was demonstrated by using BALB/c nu/nu mice. Primary and secondary dose responses to three antigenic forms of GXM, (i) the native antigen, (ii) a GXM-bovine serum albumin protein conjugate, and (iii) a cryptococcal whole-cell vaccine, revealed a lack of isotype class switching and anamnestic responses. Both the levels of complement-fixing anti-GXM antibody in serum and the PFC responses in the athymic mice showed no significant differences from those in the wild-type controls. However, T cells are involved in the suppression of the primary response to GXM. When BALB/cBy mice were given rabbit anti-mouse thymocyte serum along with 0.5 microgram of GXM, both antibody levels in serum and PFC responses were significantly increased over those of control mice that received GXM and normal rabbit serum. In addition, T cells were also shown to enhance the primary immune response to GXM. BALB/cBy mice were given GXM and anti-mouse thymocyte serum on day 1. On day 2, the experimental group was given anti-mouse thymocyte serum and the control group was given saline. On day 5, comparison of the PFC responses and anti-GXM antibody titers of the two groups revealed a significant increase in the immune response of the control over the experimental group. The type 2 T-cell-independent quality of GXM was also demonstrated in CBA/cHN xid mice. These mice lack the Lyb+ subset of B cells and are unable to respond to type 2 T-independent antigens but respond normally to type 1 T-independent antigens. Type III pneumococcal polysaccharide, a type 2 T-independent antigen, was used as a negative control, and trinitrophenyl-lipopolysaccharide, a type 1 T-independent antigen, was used as a positive control. The CBA/cHN xid mice failed to respond to either type III pneumococcal polysaccharide or GXM but did not respond to immunization with trinitrophenyl-lipopolysaccharide. BALB/cBy mice responded normally to all three antigens.