Splenic dependence of the antibody response to thymus-independent (TI-2) antigens

The murine antibody response to T-independent (TI)-2 antigens [2,4-dinitrophenyl-Lys-Ficoll (DNP-FIC) and DNP-hydroxyethyl starch (HES)] was impaired long after splenectomy, while responses to TI-1 [trinitrophenylated lipopolysaccharide (TNP-LPS)] and thymus-dependent [TNP-keyhole limpet hemocyanin (KLH)] antigens were largely unaffected. The antibody response to these TI-2 antigens was exclusively against the conjugated epitopes [DNP-, fluorescein isothiocyanate (FITC)- or tetramethylrhodamine isothiocyanate (TRITC)-Ficoll or -HES]. Fluorescent conjugates of Ficoll and HES localize selectively to the splenic marginal zone macrophages. This localization was not affected by 750 cGy of X-irradiation, but the antibody response to the TI-2 antigens was abrogated for 14 days. Administration of spleen cells restored the antibody response to these TI-2 antigens in otherwise intact irradiated mice but not if they had been splenectomized. Our findings indicate that the antibody response to TI-2 antigens depends upon stimulation of B cells in a splenic environment. This probably involves antigen presentation by marginal zone macrophages.     

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.     

Simultaneous responses to TD, TI-1 and TI-2 antigens originate from both specific and multipotent precursors

The classification of antigens into TD, TI-1 and TI-2 varieties raises the question of whether responses to these antigens are produced by distinct or identical subpopulations of B cells. In the present study we have examined the extent of intraclonal specificity variation in the progeny of PFC appearing after stimulation with two unrelated antigens. Mouse lymphoid cells were stimulated with pairs of TD and TI antigens, PFC were individually cultured and daughter PFC examined for their specificity. In all combinations used, PFC responding to TD antigen engendered, after 48 h of culture, a high frequency of PFC daughters expressing one or the other antibody specificity, notwithstanding the specificity of parental PFC. However, PFC responding to TI antigens seemed less subject to variation in specificity, and PFC daughters engendered after a 48 h culture period were, in the majority, of the parental specificity. These results are analysed in relation to different subpopulations of B cells.     

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.     

Effects of cyclosporin A on the immune system of the mouse. I. Evidence for a direct selective effect of cyclosporin A on B cells responding to anti-immunoglobulin antibodies

Previous in vivo studies have shown that the immunsuppressive peptide cyclosporin A (CsA) selectively suppresses antibody responses to nonmitogenic T-independent (TI-2) antigens, but not those to mitogenic (TI-1) antigens. This report demonstrates that in vitro CsA suppresses the polyclonal proliferative response of B cells to anti-Ig (anti-mu) antibodies at 300-400 fold lower doses than are required to inhibit B cell proliferation induced by lipopolysaccharide. Since the proliferative response to anti-mu requires neither T cells nor macrophages, it is concluded that CsA has a direct inhibitory effect on the B cells responding to this mitogen. The data support the concept that the B cells responding to TI-2 antigens are contained within the population which is stimulated polyclonally by anti-mu, and that lipopolysaccharide stimulates a distinct B cell subpopulation. They do not, however, exclude the possibility that anti-mu and lipopolysaccharide stimulate the same B cell population via two biochemically distinct triggering mechanisms, one of which is CsA-sensitive and the other CsA-resistant.     

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.     

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.     

B cell memory to thymus-independent antigens type 1 and type 2: the role of lipopolysaccharide in B memory induction

Several studies have indicated that thymus-independent (TI) antigens, unlike their thymus-dependent (TD) counterparts, are poor at generating memory antibody responses (Immunol. Today 1981. 3:217). In contrast to this view, the present report shows that the TI type 1 (TI-1) antigen, 2,4,6-trinitrophenyl-lipopolysaccharide (TNP-LPS), elicits good secondary responses in rats. These secondary antibody responses are not only greater in magnitude than the primary responses, but display a different pattern of Ig classes with more IgG and IgA antibodies produced. In transfer experiments between congenic strains of rats which differ in their kappa light chain Ig allotype, it is shown that this memory is attributable to persistent B cell clones. The TI-2 antigen, 2,4-dinitrophenyl-hydroxyethyl starch (DNP-HES), given alone did not elicit B cell memory. However, when DNP-HES is presented to the immune system in association with LPS, the pattern of the anti-DNP response is similar to that elicited by TNP-LPS. The capacity to generate TI memory is associated with the appearance of hapten-specific B cells in the marginal zones of the spleen. Hapten-binding cells were induced in the marginal zones following immunization with TNP-LPS, but not by DNP-HES. However, concurrent immunization with DNP-HES and LPS which were not covalently linked was found to induce DNP-binding cells in the marginal zone. There is complete correlation between the appearance of hapten-binding memory B cells in the marginal zone and the capacity of these antigens to induce secondary responses.     

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.     

Cyclosporin A has differential effects on the responses of murine B cells to TI antigens and B-cell mitogens.

The effect of cyclosporin A (CyA) on the response of murine splenocytes to B-cell mitogens, TI-1 and TI-2 antigens was investigated. The proliferative response to LPS was found to be four- to five-fold less sensitive to inhibition than that to dextran sulphate. Antibody responses to a TI-2 antigen in vivo were suppressed by CyA treatment, whereas responses to the TI-1 antigen DNP-LPS were markedly enhanced. Enhanced antibody responses to DNP-LPS were also demonstrable in vitro in the presence of CyA, and the enhancement was not removed by T-cell depletion. LPS-induced antibody production in vitro was enhanced at the same CyA concentrations that inhibited proliferation by 40-50%. The implications of these findings for the mechanism of action of CyA and for our understanding of B-cell differentiation are discussed.     

Studies of the immunological activities of the outer membrane protein from Escherichia coli

The outer membrane protein (OMP) prepared from Escherichia coli was found to be a potent mitogen for murine B cells and to be capable of inducing polyclonal antibody formation as well as a proliferative response. Spleen cells from nude mice responded equally as well to OMP as those from their normal litter-mates, whereas nylon-wool-purified T cells or thymocytes failed to respond. The proliferative response was dependent on the presence of macrophages. The macrophage dependency of the polyclonal antibody response seemed to be less than that of the proliferation. OMP was mitogenic for lipopolysaccharide (LPS)-resistant C3H/HeJ spleen cells, further indicating that OMP is an unique B-cell mitogen distinct from LPS.

OMP also enhanced the specific antibody response sixty-seven-fold to an optimal dose of sheep red blood cells (SRBC) in vitro. The kinetics of the response, however, was not altered from that of cultures without OMP. The anti-SRBC response of spleen cells from C3H/HeJ mice was also enhanced by the addition of OMP, suggesting that the adjuvant effects were not due to the LPS in the preparation. Antibody responses in vitro to TI-1 antigens, trinitrophenyl-LPS (Boivin) (TNP-LPS^B) and TNP-Brucella abortus, were not enhanced in the presence of OMP. In contrast OMP enhanced the response to TI-2 antigens, TNP-LPS^W (Westphal) and dinitrophenyl-Ficoll and T cells were shown to be required for these augmented antibody responses. Enhancement was not seen in nude mouse spleen cell cultures but was seen when nylon-wool-purified T cells were added to the cultures.

     

Differences in the recruitment of virgin B cells into antibody responses to thymus-dependent and thymus-independent type-2 antigens

The bone marrow of mammals generate large numbers of B cells throughout life. Most of these have a short life span. The subject of this report is to investigate the extent to which newly formed virgin B cells can be activated by thymus-dependent (TD) and thymus-independent type 2 (TI-2) antigens carrying the hapten 2,4-dinitrophenyl. The experimental approach used chimeras made between congenic rats of different kappa immunoglobulin light chain allotype. Host (kappa la) rats were depleted of peripheral B cells by whole body irradiation but had B lymphopoietic capacity conserved by shielding the hind limbs. Their peripheral B cell pool was reconstituted by transfer of kappa lb thoracic duct lymphocytes from donors immunized previously with the TD carrier. This provides test animals where newly produced virgin B cells only express kappa la but where initially most peripheral B cells are kappa lb. The TD antigen tested was able to activated both virgin and memory B cells in the period immediately following immunization. However, long-term antibody production was attributable to repeated activation of memory B cell clones without further virgin B cell recruitment. By contrast, antibody evoked by the TI-2 antigen initially was almost exclusively due to activation of donor peripheral B cells. However, over a period following TI-2 immunization there was a progressive increase in the amount of host antibody produced with corresponding decline of the donor component of the response so that the host response was dominant by six weeks. Control experiments were conducted to show that these effects could not be explained by allotype or isotype-directed suppression. The cellular basis of these differences was investigated further by studying the rate of repopulation of different B cell compartments in these chimeras by newly formed host and mature donor B cells. The results indicate that the onset of host antibody production to the TI-2 antigen closely correlated with the appearance of host B cells in the marginal zones of the spleen, whereas good TD host anti-2,4-dinitrophenyl responses antedated the appearance of host B cells in this compartment. These results are discussed in relation to other data implicating marginal zone B cells in responses to TI-2 antigens.     

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.     

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.     

The in Vitro B-Cell Response to Pneumococcal Polysaccharides in Adults and Neonates

In order to gain insight into the relatively late appearance in ontogeny of responsiveness to T-cell independent (TI) antigens, the in vitro human B-cell response to type 4 pneumococcal polysaccharides (PS4, a human TI-2 antigen) was studied. B cells obtained from adults differentiate into anti-PS4 antibody forming cells upon culturing with PS4, but neonatal B cells obtained from cord blood fail to respond. The culture system used does, however, allow the differentiation of B cells reactive with TD antigens, for example ovalbumin. In order to evaluate the concept that in man the anti-PS4 response is derived from a particular B-cell subset we separated adult B cells on the basis of expression of the determinant recognized by the monoclonal antibody FMC7. The anti-PS4 response is found mainly, but not exclusively, in the FMC7+ subset. The selective unresponsiveness of neonatal B cells to TI-2 antigens is not, however, due to the absence of FMC7+ B cells because, unlike adult blood B cells of which about 50% are FMC7+, 100% of neonatal B cells are FMC7+.     

Sites of specific B cell activation in primary and secondary responses to T cell-dependent and T cell-independent antigens.

Techniques which identify hapten-specific B cells in tissues have been used to determine the sites of B cell activation in rat spleens in response to T cell-dependent (TD) antigens and T cell-independent type-1 (TI-1) antigens. Surface-associated hapten binding by specific memory B cells and B blasts was distinguished from the strong cytoplasmic hapten binding by specific plasma cells and plasmablasts. Blast cells in S phase were identified in tissue sections by staining cells which had been pulse labeled in vivo with 5-bromo-2'-deoxyuridine. Hapten-specific B blast cells are found in three sites: (a) around interdigitating cells in the T cell-rich zones; (b) in the follicular dendritic cell network and (c) in association with macrophages in the red pulp. Hapten-binding memory B cells, which are not in cell cycle, accumulate in the marginal zones and to a lesser extent the follicular mantles in response to TD and TI-1 antigens. The hapten-specific blast response in T zones is confined to the first few days after antigen is given and is low for primary responses to TD antigens, but massive on secondary challenge, when marginal zone memory B cells migrate to the T zones. Both the primary and secondary T zone responses to TI-1 antigens are impressive and in these responses hapten-specific B blasts are also found in the splenic red pulp. The follicular response to TD antigens starts with a small number of B blasts (fewer than five) entering each follicle. These increase in number exponentially so that by the 4th day after immunization they fill the follicle. The oligoclonality of the response is shown in simultaneous responses to two haptens where 6%-31% of the follicles on day 3 after immunization contain blasts specific for only one of the two haptens. During the 4th day classical zonal pattern of germinal centers develops. The surface immunoglobulin-positive B blasts are lost from the follicle center, while one pole of the follicular dendritic cell network fills with surface immunoglobulin-negative centroblasts. Centroblasts do not increase in numbers but divide to give rise to centrocytes, which re-express sIg and migrate into the follicular dendritic cell network. Cell kinetic studies indicate that the centrocyte population is renewed from centroblasts every 7 h. Centrocytes either leave the germinal center within this time or die in situ.(ABSTRACT TRUNCATED AT 400 WORDS)     

The Human in Vitro Anti-type 4 Pneumococcal Polysaccharide Antibody Response is Regulated by Suppressor T Cells

In order to study the regulation of the human B-cell response to T-cell independent type 2 (TI-2) antigens, we analysed the role of T cells in the in vitro antibody response to type 4 pneumococcal polysaccharide (PS4). We found that T cells can positively regulate the in vitro antibody response to PS4 when they are added into an in vitro B-cell culture system. In addition we demonstrated that T cells exert a negatively regulating activity. We found that T cells, when cultured for 24 h in the presence of high concentrations of PS4, can suppress the anti-PS4 antibody response. This down-regulation of the anti-PS4 B-cell response is shown to be antigen specific and MHC-restricted. Furthermore, PS4-specific T-cell mediated suppression appears to be radioresistant and confined to the T-cell preparations enriched for CD8+ cells. The results show that analogous to results obtained in mice, human T cells are able to exert a regulatory control of the antibody response to TI-2 antigens.     

Heterogeneous and monoclonal helper T cells induce similar anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) antibody populations in the primary adoptive response. II. Lambda light chain dominance and idiotope expression

When the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP) is presented on different carrier molecules, different anti-NP antibody responses are stimulated. On stimulation with NP-lipopolysaccharide (LPS) [T-independent type 1 (TI-1) antigen] kappa + antibodies are the major population, whereas on stimulation with NP-Ficoll [T-independent type 2 (TI-2) antigen], NP-keyhole limpet hemocyanin (KLH) or NP-chicken gamma globulin (CG) [T-dependent (TD) antigens], lambda 1+ antibodies dominate. The relative contribution of idiotopes Ac38 or Ac146 to the lambda 1+ anti-NP response was also different on comparison of TI-1 with TI-2 or TD anti-NP responses. We investigated whether light chain- or idiotype-specific T cells are responsible for these differences. Analysis of the anti-NP response of nude mice after immunization with NP-Ficoll showed lambda 1 dominance. Likewise primary adoptive transfer experiments using carrier-specific T cell lines to reconstitute the TD anti-NP response to NP-KLH or NP-CG, showed that help from carrier-specific T cells alone is capable of stimulating the characteristic lambda 1 dominant response. No significant difference could be found in the levels of Ac38 and Ac146 idiotope expression between mice reconstituted with splenic T cells and those reconstituted with T cell lines. These results suggest that light chain- or idiotype-specific T cells are required neither for the production of lambda 1 light chain dominance, nor for the appearance of idiotopes characteristic of the primary anti-NP response. The possible reasons for differences seen in both light chain and idiotope expression between primary anti-NP responses to the TI-1 antigen NP-LPS and those to TD or TI-2 antigens are discussed.     

CpG oligodeoxynucleotides overcome the unresponsiveness of neonatal B cells to stimulation with the thymus-independent stimuli anti-IgM and TNP-Ficoll.

Neonates are very vulnerable to pathogenic encapsulated bacteria due to their inability to mount an antibody response to capsular polysaccharides, which are thymus-independent type 2 (TI-2) antigens (Ag). Oligodeoxynucleotides (ODN) containing unmethylated CpG dinucleotides induced neonatal B cells to proliferate to anti-IgM, a TI-2 stimulus. CpG ODN inhibited the spontaneous and B cell receptor-mediated apoptosis of neonatal B cells and reduced the amount of the pro-apoptotic Bcl-xS, strongly correlated with anti-IgM-induced apoptosis of neonatal B cells. CpG ODN protected neonatal B cells from apoptosis by down-regulation of the Bcl-xS protein. Neonatal B cells underwent polyclonal differentiation upon stimulation with CpG ODN, but unlike in adult B cells, this was not preceded by IL-6 secretion. CpG ODN stimulated neonatal B cells to mount an Ag-specific antibody response to TNP-Ficoll, another TI-2 Ag. Thus CpG ODN could provide a novel approach to induce the immune system in neonates to respond to harmful encapsulated bacteria.     

Complement-mediated follicular localization of T-independent type-2 antigens: the role of marginal zone macrophages revisited.

In this study we demonstrate a hitherto undescribed phenomenon, namely that thymus-independent type-2 antigens (TI-2 Ag) localize in splenic follicles within 1 h after administration. The follicular localization of 2,4,6-trinitrophenyl (TNP)-Ficoll was not antibody mediated. In addition in case of high-dose administration we observed a relatively large amount of TI-2 Ag in marginal zone macrophages. However, after low-dose administration we observed a preferential localization of TNP-Ficoll in the splenic follicles. Detection of TNP-haptenated Ag in cryostat sections of murine spleens was performed with a high-affinity TNP-specific monoclonal antibody conjugated to beta-galactosidase. Within minutes after injection the TI-2 Ag localized in the marginal zone, attached to marginal zone macrophages and B cells. Twenty minutes after injection the Ag was also detected in the follicles and gradually accumulated there until 7 h after injection. Thereafter, the amount of follicular Ag gradually decreased but was still detectable up to 14 days after immunization. The follicular localization of TNP-Ficoll was complement dependent in contrast to the binding to and uptake by marginal zone macrophages. Double staining revealed that Ag was bound by macrophages, B cells and follicular dendritic cells. Haptenated thymus-dependent (TD) Ag localized exclusively in the red pulp macrophages. In vivo macrophage elimination drastically increased the amount of TNP-Ficoll in the follicles, and enhanced the humoral immune response at low doses of Ag. Moreover, complement deprivation of mice abrogated the localization of TI-2 Ag in the follicles, and led to a decreased humoral TI-2 immune response. In conclusion, we demonstrate for the first time that TI-2 Ag localize in follicles. Moreover, the presented results provide further evidence that B cells and follicular localized Ag play an important role in the induction of humoral TI-2 immune responses.