Antigen-independent,IgM-induced antibody responses: requirement for “recurrent” idiotypes

Anti-idiotypic antibodies (a-Id) were produced in syngeneic mice against two monoclonal IgM antibodies of BALB/c origin, TNP.11 and SP/603. In plaque inhibition tests, using IgM-secreting hybridoma cells and anti-idiotypic antibodies, these two IgM proteins, as well as the anti-TNP myeloma protein MOPC 460 (IgA) were found to carry non-cross-reactive idiotypes. Analysis of the anti-trinitrophenyl (TNP) plaque-forming cells (PFC) in BALB/c mice, either normal or immunized with TNP-horse red blood cells, revealed that in addition to the MOPC 460 Id, also the SP/603 Id is recurrent and expressed by a fraction of the anti-TNP antibody-secreting cells in all individuals tested. In contrast, the TNP.11 Id could not be detected in any BALB/c mouse studied. TNP.11 and SP/603 antibodies were then characterized by their ability to induce an antigen-independent anti-TNP response in normal BALB/c mice. While TNP.11 was found to be inactive, the same titers of SP/603 IgM induced antigen-specific PFC all of which expressed the SPi603 Id, and increased titers of circulating IgM molecules carrying the same Id, suggesting that “recurrent” but not “nonrecurrent” Id are competent in this respect. A fraction of these SP/603-induced, SP/603-positive anti-TNP antibodies also carried MOPC 460 Id, suggesting expression on the same molecule of idiotypic determinants found on independent non-cross-reactive “recurrent” idiotypes.     

Murine anti-TNP antibodies positive for either lambda 1 or lambda 2 light chains express common idiotypic determinants

Although the lambda-bearing antibodies represent only 5% of the total mouse serum immunoglobulins, some antigens such as B1355 dextran (alpha (1-3)Dex), the 4-hydroxy-3-nitrophenyl acetyl (NP) and 2,4-dinitro or 2,4,6-trinitrophenyl (DNP/TNP) antigens can induce lambda-positive immune responses. In contrast to the lambda antibody response against alpha (1-3)Dex and NP antigens which is restricted to the lambda 1 isotype it was shown that the response to the DNP (or TNP) antigen uses lambda 1 and lambda 2 and lambda 3 isotypes. The idiotypy of the alpha (1-3)Dex and NP systems has been well characterized contrary to that of the lambda-positive anti-TNP/DNP response which has been poorly studied. In this paper, we describe two idiotopes (Id C19-3 and Id D11-2) shared by two BALB/c monoclonal anti-TNP antibodies (TNP5 and TNP9) which, respectively, use the lambda 1 and lambda 2 light chains. These idiotopes were independently expressed on other monoclonal anti-TNP/DNP antibodies and appear to require the use of a unique VH gene associated with a particular V lambda region. After TNP-Ficoll immunization, BALB/c mice recurrently express both idiotopes on lambda 1 and (lambda 2 + lambda 3) anti-TNP antibodies. In addition, all the mouse strains immunized against TNP-Ficoll give a lambda 1- and (lambda 2 + lambda 3)-positive immune response with the exception of SJL and SJA strains which present a deficit for the expression of lambda 1 light chain. The expression of Id C19-3 was restricted to the strains with the Igh-Va allotypic haplotype (including SJA) whereas the Id D11-2 was extensively expressed in the various strains.     

Early expression in (NZB X DBA/2)F1 hybrids of thymus dysfunction and abnormal antibody production inherited from the NZB parent

NZB mice, DBA/2 mice and reciprocal F1 hybrids between both strains were studied from birth to two months of age for the secretion of a serum thymic factor, thymulin (formerly named Facteur Thymique Sérique) and for spontaneous and antigen-induced immune responses: spontaneous splenic anti-2,4,6-trinitrophenyl (TNP) plaque-forming cells (PFC), spontaneous IgM serum levels, immune direct anti-sheep red blood cells (SRBC) PFC and immune serum antibody production to human gamma globulin (HGG) as well as susceptibility to tolerance induction by deaggregated HGG. An early decline of thymulin serum level was detected from two weeks of age both in NZB mice and F1 hybrids, the latter maintaining a level intermediate between that of both parental strains. Such a fall of circulating thymulin was associated to a decreased number of thymulin-secreting cells. F1 hybrids and NZB mice exhibited at two and three weeks of age spontaneous anti-TNP PFC in the spleen, and increased IgM serum levels as compared to DBA/2 mice. When immunized at birth with SRBC or at two weeks of age with HGG, NZB mice and F1 mice similarly exhibited a higher anti-SRBC antibody response, as measured 5 days later by PFC numbers, and a higher anti-HGG serum antibody production 2 weeks post-immunization, than age-matched DBA/2 mice. F1 hybrids tended to develop with age spontaneous and immune antibody responses lower than NZB mice but still much higher than DBA/2 mice. Conversely, after tolerization at birth with deaggregated HGG NZB mice but not the F1 hybrids produced higher titers of anti-HGG antibodies upon challenge than similarly tolerized DBA/2 mice. DBA/2 mothered and NZB mothered F1 hybrids did not differ for any parameter tested and no influence of the sex could be detected.     

Anti-idiotype to monoclonal anti-swine SLA antibody detects a common idiotype shared by mouse anti-SLA sera and elicits an anti-SLA activity

Heterologous anti-idiotypic reagents have been prepared against a BALB/c anti-swine MHC (SLAd) monoclonal antibody (74-11-10) in order to test for possible interspecies idiotypic cross-reactions of anti-MHC antibodies. Using these reagents to examine xenoantisera produced in BALB/c mice immunized with swine SLAdd peripheral blood lymphocytes, all animals tested were found to express detectable levels of the 74-11-10 idiotype (Id). The Id could also be detected in one out of five BALB/c mice immunized with swine SLAcc PBL. Swine anti-SLAdd alloantibodies were also tested, but failed to show detectable levels of the 74-11-10 Id. The in vivo administration of anti-idiotypic reagents to BALB/c mice induced detectable levels of 74-11-10 Id positive antibodies that bound specifically to SLAdd PBL. Similar treatment of SLAgg swine (recombinant swine expressing the class I MHC molecules of c) with anti-idiotypic antibodies failed to induce anti-SLAd antibody activity. These results thus indicate that 74-11-10 represents a shared idiotype of BALB/c anti-SLAdd antibodies. The presence of 74-11-10 Id in one mouse immunized with SLAcc PBL suggests that the failure of pigs to express the 74-11-10 Id following treatment with anti-idiotypic antibodies may be the result of tolerance rather than absence of the relevant variable region gene(s).     

Enhancement of Murine Immune Responses to Orally Administered Haptenated Streptococcus mutans

The induction of immune responses to orally administered trinitrophenyl (TNP)-haptenated Streptococcus mutans and its enhancement with muramyldipeptide (MDP), peptidoglycan (PG), and concanavalin A (Con A) were investigated in lipopolysaccharide (LPS)-non-responsive C3H/HeJ mice and the syngeneic, LPS-responsive C3H/HeN strain. Both mouse strains manifested similar immune responses, primarily of the IgM isotype, after a single gastric intubation (GI) with TNP-S. mutans. However, when groups of animals were first carrier-primed by GI with S. mutans for 2 consecutive days, followed by a single GI with TNP-S. mutans 1 week later, C3H/HeJ mice gave a significantly higher (P less than or equal to 0.01) splenic IgA anti-TNP plaque-forming cell (PFC) response than identically treated C3H/HeN mice. Furthermore, saliva, urine and serum from these C3H/HeJ mice possessed high levels of IgA anti-TNP antibodies as determined by the enzyme-linked immunosorbent assay, whereas C3H/HeN mice exhibited low antibody levels. Oral administration of Con A (either 250 micrograms or 500 micrograms/mouse) or purified PG (1 mg/mouse) at the time of TNP-S. mutans immunization resulted in significantly (P less than or equal to 0.01) enhanced splenic IgA anti-TNP PFC responses, especially in C3H/HeJ mice. On the other hand, MDP promoted IgA anti-TNP PFC responses in LPS-responsive C3H/HeN mice but did not augment responses in C3H/HeJ animals. A similar immune response pattern was seen when antibody levels were measured in serum, saliva, and urine of both mouse strains. These results demonstrate that haptenated S. mutans is a good antigen for the induction of high IgA responses in orally immunized C3H/HeJ mice and that this high response can be enhanced with the adjuvants Con A and PG. However, MDP is ineffective in C3H/HeJ mice but enhances IgA responses in normal LPS-responsive C3H/HeN animals.     

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.     

Moloney leukemia virus-induced cell surface antigen mimicry by monoclonal antibodies

We have investigated antigen-independent modulation of immune responses by monoclonal antibodies directed against both viral and nonviral antigens. BALB/c mice were immunized with monoclonal IgM (i.e. Ab1) specific for either Moloney murine leukemia virus-induced cell surface antigen (MCSA) or the hapten 2,4-dinitrophenyl (DNP). Injection with either Ab1 activated a functional idiotypic (Id) network as evidenced by production of both anti-Id (Ab2) antibodies and anti-anti-Id (Ab3) antibodies. A subset of induced Ab3 (designated Ab1), exhibited specificity for antigen (virus or DNP). In mice immunized with anti-Id antibodies (Ab2), production of Ab3 and Ab1′ was also observed. In the MCSA system, antibody-induced Ab1′ responses were effective in protecting mice from tumor development upon subsequent challenge with live virus. Furthermore, antigen-independent modulation of immunity to both viral and nonviral antigens was found to be thymus-dependent. Similar findings in other viral systems suggest that antibody-induced activation of Id networks may prove a viable alternative vaccine strategy that can elicit antigen-specific responses, and in some cases protection, in the apparent absence of exposure to antigen.     

Maternal transmission of idiotypic network interactions selecting available T cell repertoires

Up to 75% of 2,4,6-Trinitrophenyl (TNP)-self-specific helper T cells from normal BALB/c mice share a clonotypic determinant with the anti-TNP myeloma protein MOPC 460, recognized by the monoclonal antibody F6(51). Immunization of adult BALB/c mice with the MOPC 460 idiotype (Ab2 mice) leads to high titers of circulating anti-idiotypic antibodies but has no influence on the expression of the T cell clonotype. In contrast, TNP-self-specific helper cells prepared in progenies from Ab2 females, or in BALB/c mice treated as neonatals with anti-idiotypic antibodies, fail to express the corresponding T cell clonotype when immunized as adults. These results demonstrate the idiotype-dependent selection of T cell repertoires early in life and their stability in adults.     

Phosphorylcholine on isologous red blood cells induces polyclonal but not anti-phosphorylcholine plaque-forming cells in mice

It has been demonstrated in the preceding report (Bach, M. A., Beckmann, E. and Levitt, D., Eur. J. Immunol. 1984. 14: 589) that phosphorylcholine (PC) on the bacterium Streptococcus pneumoniae R36a stimulated polyclonal as well as anti-PC plaque-forming cells (PFC) in mouse spleen in vivo. In this study, red blood cells from BALB/c mice (MRBC) were either conjugated with PC, 2,4,6-trinitrophenyl (TNP) or treated with phospholipase A2 (PLA2) to expose PC on the cell membrane (determined by hemagglutination with the anti-PC myeloma HOPC8). When BALB/c mice were immunized i.v. with the conjugated or enzyme-treated MRBC, a significant polyclonal antibody response occurred (p less than 0.05) using PC-MRBC or PLA2-treated MRBC, but not with TNP-MRBC or sham-treated MRBC. No anti-PC or anti-MRBC immunoglobulin-secreting cells developed after immunization. Repeated immunization with PC-MRBC resulted in similar levels of protein A PFC after each immunization but no anti-PC, anti-MRBC or anti-PC-MRBC PFC. Thus, PC on R36a or isologous RBC stimulated increased numbers of splenic plaque-forming cells. In the case of R36a, 10-25% of these PFC produced antibodies directed towards PC. In contrast, PC-MRBC or PLA2-treated MRBC, failed to evoke any anti-PC antibody responses.     

Expression of two cross-reactive idiotypes on mouse antibodies against bromelain-treated mouse erythrocytes.

Two cross-reactive anti-idiotype (Id) antibodies were previously prepared from sera of rabbits immunized with mouse monoclonal antibodies against bromelain-treated mouse erythrocytes (BrMRBC). Most of the anti-BrMRBC plaque-forming cells (PFC) were suppressed by either of the two anti-Id antibodies. The Id profiles of anti-BrMRBC PFC were almost identical among various cell populations in a strain, but different among various mouse strains. Mouse sera contained both of the Id-bearing immunoglobulins Ig, and a significant part of the Id-bearing Ig were eliminated by absorption with BrMRBC. Nude BALB/c mice were almost equal to normal BALB/c mice in the Id patterns of anti-BrMRBC PFC and in the concentrations of the Id-bearing Ig. The injections of anti-Id antibodies into suckling mice suppressed, specifically, the development of the B cells to produce the homologous Id-bearing Ig, but the injection of Id-bearing monoclonal antibodies barely affected Id expression. It is suggested that the two Id are encoded in germ-line genes of mice, and are expressed independently of each other and Id-anti-Id regulations by T cells or B cells.     

The augmentation of tumor-specific immunity by virus help. I. Demonstration of vaccinia virus-reactive helper T cell activity involved in enhanced induction of cytotoxic T lymphocyte and antibody responses

C3H/He mice were immunized to vaccinia virus by inoculating i.p. viable virus. Their spleen cells (SC) were tested for vaccinia virus-reactive helper T cell activity capable of augmenting (a) anti-trinitrophenyl (TNP) cytotoxic T lymphocyte (CTL) response generated from unprimed C3H/He SC (responding cells) or (b) anti-TNP antibody response generated from TNP-primed C3H/He SC (responding cells) by the stimulation with syngeneic SC infected with vaccinia virus and subsequently modified with TNP (virus-self-TNP). The results demonstrate that cultures of responding cells plus 850 rds X-irradiated vaccinia virus-primed SC failed to enhance anti-TNP CTL or plaque-forming cell (PFC) responses when in vitro stimulation was provided by either virus-self or TNP-self alone. In contrast, these cultures resulted in appreciable augmentation of CTL and PFC responses when stimulated by virus-self-TNP. Such a helper activity provided by vaccinia virus-primed SC was revealed to be T cell mediated and antigen specific. These results are discussed in the context of (a) nature of virus helper antigens, (b) mechanism of help and (c) potential of virus help in augmenting CTL and antibody responses to tumor antigens.     

Inverse correlation between the utilization of an idiotype in specific immune responses and its representation in pre-immune "natural" antibodies

Previously we have characterized an idiotype (Id) that accounts for half of all specific anti-dextran B512 (Dex) antibodies in C57BL/6 mice. BALB/c mice produce the same Id in normal, pre-immune sera but fail to use it in antibody responses to Dex, although Id+ anti-Dex antibodies can be induced in this strain by anti-Id immunization. By limiting dilution analysis of B cell clonal precursors, we show here that the frequencies of Id+ B cells are comparable in both strains, but their state of activity is sharply distinct: while all Id+ B cells are small, resting lymphocytes in C57BL/6 mice, they are all large, naturally activated cells in BALB/c mice. The suggestion that naturally activated cells are poorly engaged in specific responses was supported by the delayed and lower Id+ responses obtained in BALB/c mice when they are immunized, in parallel with C57BL/6 animals, with a conjugate of anti-Id antibodies and lipopolysaccharide. Finally, C57BL/6 responder mice were found to closely reproduce the normal BALB/c situation, if analyzed 3 months after anti-Id priming: they produce low levels of serum Id and all Id+ B cells are in the large lymphocyte compartment. Upon immunization these animals develop serum Id+ responses that are undistinguishable from low-responder BALB/c mice. The relevance of these observations for the questions of physiologic self-reactivity is discussed.     

A new assay system detecting antibody production and delayed-type hypersensitivity responses to trinitrophenyl hapten in an individual mouse

A new assay system detecting antibody production and delayed-type hypersensitivity (DTH) responses to trinitrophenyl hapten in an individual mouse (AS-DAD) was established. BALB/c mice were immunized intraperitoneally with varying amounts of 2,4,6-trinitrophenylated sheep red blood cells (TNP-SRBC) on day 0. Venous blood was collected on days 2, 4, 6, 8 and 10. Levels of anti-TNP IgM and IgG in serum were assayed by enzyme-linked immunosorbent assay (ELISA). After series of bleeding the mice were challenged with 2,4,6-trinitrobenzene sulfonic acid (TNBS) solution in the footpad on day 14. Footpad swelling was measured 24 or 48 h after the challenge. Peak responses of the anti-TNP IgM and IgG production were detected 4 or 6 days after the immunization with 10⁹ TNP-SRBC. Maximum DTH response was also observed with 10⁹ TNP-SRBC 24 h after the challenge on day 14. The antibody and DTH responses were also induced in other normal inbred strains such as C3H/He and DBA/1 but not BALB/c nu/nu mice. To evaluate AS-DAD in immunopharmacological studies, various immunomodulating agents were examined in BALB/c mice by subcutaneous administration on days 0, 1, 2 and 3. Cyclosporin or cyclophosphamide at 100 mg/kg/day completely inhibited not only the anti-TNP IgM and IgG production but also the TNP-specific DTH response. Prednisolone at 0.5 mg/kg/day had no significant effect on the IgM and IgG production, whereas it inhibited the TNP-specific DTH response. Interestingly, histamine-added mouse γ-globulin at 150 mg/kg/day clearly enhanced the anti-TNP IgM and IgG production, while it showed a suppressive effect on the TNP-specific DTH response. Levamisole at 5.0 mg/kg/day showed suppressive effects on the anti-TNP IgG production without affecting the IgM production and the DTH response. These results suggest that AS-DAD is useful for evaluating the immunopharmacological action of various agents.     

Lack of age-associated auto-anti-idiotypic antibody regulation in mucosal-associated lymph nodes

It has previously been shown that the loss of immune competence in the splenic B cell population with age may be due to auto-anti-idiotypic antibody regulation (M. R. Szewczuk and R. J. Campbell, Nature 1980. 286: 164). In the present study we have investigated the appearance of auto-anti-idiotypic antibody on immune B cells from the mucosal-associated lymph nodes of old and young mice of the same strain. Various aged C57BL/6J male mice were immunized with 500 μg trinitrophenylated bovine gamma globulin (TNP-BGG) in complete Freund's adjuvant (CFA) intraperitoneally. IgM, IgG and IgA anti-TNP plaque-forming cell (PFC) responses in the spleen, mediastinal and mesenteric lymph nodes were assayed for anti-idiotype-blocked, hapten-augmentable PFC, 14 days later. It was found that 8 months or older C57BL/6J male mice produced a significantly high percentage of hapten-augmentable IgM, IgG and IgA anti-TNP PFC in the spleen. In contrast, there was a lack of hapten-augmentable anti-TNP PFC in the mesenteric and mediastinal lymph nodes with increasing age of the animal. Mice receiving antigen in the footpads and base of the tail also produced a significantly high percentage of hapten-augmentable IgG PFC in the draining peripheral lymph nodes. TNP-ε-amino-n-caproic acid (EACA) as hapten was shown to specifically augment anti-TNP PFC. Immune sera from 15-month-old mice caused a specific inhibition of anti-TNP PFC in vitro. The inhibition of plaque formation was completely reversible by addition of TNP-EACA as hapten. This PFC inhibitory activity in immune sera lacked anti-TNP antibody activity, but reacted with anti-TNP antibody of C57BL/6J origin. Immune sera from 8 month or older mice also revealed anti-(anti-TNP F(ab')₂ IgG) titer as assayed by passive hemagglutination. Thus, the results of this study indicate a division of the immune system into regulatory compartments. Auto-anti-idiotypic antibody may be involved in a down-regulation of systemic responses but with no apparent effect in the mucosal-associated lymph nodes.     

Differential induction of anti-trinitrophenyl plaque-forming cell responses to lightly and heavily conjugated trinitrophenylated heterologous and autologous erythrocytes in mice

Various anti-TNP PFC (2,4,6-trinitrophenyl plaque-forming cell) responses were obtained by injecting C3H mice with different conjugates of trinitrophenylated sheep red cells (TNP-SRC). Mice which were injected with lightly conjugated TNP-SRC (TNP_0.14SRC) produced indirect anti-TNP PFC 4 days after injection, while mice which were injected with heavily conjugated TNP-SRC (TNP₁₄SRC) produced both direct and indirect anti-TNP PFC or direct anti TNP PFC only. Heavily conjugated trinitrophenylated autologous mouse red cells elicited a low direct anti-TNP PFC response 4 days after injection. The system is another example of the capacity to modify antibody responses by modification of the antigen.     

IgA hybridomas: A method for generation in high numbers

An immunization regimen has been developed which yields a high frequency of hybridomas producing IgA isotype, antigen-specific antibody when spleen cells from immunized mice are fused with non-immunoglobulin secreting murine myeloma cells. Germfree BALB/c mice were carrier-primed with sheep erythrocytes (SRBC) by gastric intubation (GI) for 2 consecutive days followed 1 week later by GI with trinitrophenyl (TNP)-haptenated SRBC. After 7 days, spleen cells were fused with non-immunoglobulin secreting myeloma cells (X63-Ag8.653), and 2–3 weeks later, culture wells were scored for hybrid clones. Of 240 culture wells plated, 157 wells (65.4%) exhibited clones producing anti-TNP antibodies as determined by enzyme-linked immunosorbent assay. A total of 50 specific cell lines were established, of which 27 clones (54%) produced IgA isotype anti-TNP antibodies, while the anti-TNP antibodies produced by the remaining 23 clones were approximately equally distributed between the IgM and IgG isotypes. The IgA and IgM monoclonal antibodies were more effective in hemagglutinating TNP-SRBC than were IgG isotype antibodies. This study describes a method for production of a high number of antigen-specific IgA hybridomas which will allow production of IgA monoclonal antibodies to important antigens on mucosally-associated pathogens, and thus allow elucidation of functions of IgA antibody at mucosal surfaces.     

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.     

Self versus non-self carrier in primary and secondary anti-TNP B cell response: I. specificity of help

Humoral anti-hapten responses are supposed to require carrier-specific help. Yet, «TNPspecific» help can be activated with TNP coupled to syngeneic lymphocytes. To further clarify the role of hapten-specific vs. carrier-specific helper T cells (T_H) in the humoral anti-TNP response, BALB/c mice were immunized with TNP coupled to cellular or soluble self or nonself carriers, we analyzed primary and secondary B cell responses as well as the apparent specificity of help. TNP bound to syngeneic red blood cells (sRBC), syngeneic albumin (sA) and syngeneic polyclonal or monoclonal immunoglobulin (s1g/smIg) and TNP coupled to non-self carriers initiated equivalent primary anti-TNP responses. On the other hand, a secondary anti-TNP response was only obtained with heterologous carriers or with smIg. Even with heterologous carriers or with smIg, the magnitude of the secondary anti-TNP response exceeded only slightly the amplitudes of a primary anti-TNP response. Furthermore, if animals were challenged shortly after priming, they appeared un/-hyporesponsive.In vitro analysis revealed that in the primary as well as in the secondary anti-hapten response, TNP-specific T_H were involved, i.e., the primary response to s1g, sA and sRBC was exclusively due to TNP-specific help, the response after priming with TNP-smIg or TNPheterologous carrier was due to the additive effects of carrier- and TNP-specific help. Since «carrier-specific» help with smIg was independent of the antibody specificity as well as of the Ig class, we suppose that sm1g activated idiotype-specific TH, which functioned like «carrier» T_H.Two mechanisms were responsible for the low magnitude of the secondary anti-hapten response: competition for carrier-specific help (using heterologous carriers), antibody (AB)-dependent suppression.     

Anti-TNP monoclonal antibodies as reagents for enzyme immunoassay (ELISA)

The aim of this study was to produce anti-TNP monoclonal antibodies (MAbs) that could be conjugated and used for the detection of antigen-antibody reactions, in which the antigen specific-antibody had been previously bound to trinitrophenyl (TNP). For hybridoma production, SP2/0-Ag14 cells were fused with spleen cells from mice previously immunized with TNP-ovalbumin (TNP-OVA). After 10 days, enzyme-linked immunoadsorbent assay (ELISA) was used to detect anti-TNP antibodies in the supernatants, and five cultures were found to be strictly positive for TNP. Three of these were subsequently cloned by limiting dilution, and 15 clones were chosen for expansion based on the criterion of high reactivity against TNP. Anti-TNP MAbs produced by those clones were isotyped as IgG1, and purified by Sepharose-protein G affinity cromatography from ascites developed in BALB/c mice. Two purified MAbs (1B2.1B6 and 1B2.1E12) were coupled to horseradish peroxidase (HRPO). The resulting conjugates were evaluated in ELISA tests for interferon-gamma and interleukin-4 detection, in which the secondary anti-cytokine antibodies were coupled either to TNP or biotin. The performance of anti-TNP conjugates in these assays were compared with a biotin-streptavidin/peroxidase system. Both types of conjugates were similarly able to detect cytokines with r2 (linear correlation coefficient) close to unity value. Growth studies of one of those hybridomas (1B2.1B6) yielded a specific growth rate of 0.042 h(-1) and a doubling time of 16.5 h. Data discussed here show that at least two MAbs against TNP raised in this work can be used as a reagent for enzyme immunoassays.     

The induction of anti-erythrocyte antibodies in vivo by administration of lipopolysaccharide

Experiments were designed to determine whether B-cell activation of control mouse strains with lipopolysaccharide (LPS) could induce the same anti-erythrocyte antibody responses observed to occur spontaneously in the NZB strain. When BALB/c and DBA/2 mice were injected intraperitoneally with 100 micrograms of LPS, antibodies to X, HB, and HOL antigens could be detected 2 weeks later at levels comparable to those found spontaneously in NZB mice. Injection of C3H/HeJ mice, nonresponders to LPS, resulted in no detectable anti-erythrocyte antibody responses. When NZB mice were treated with LPS in this way, serum levels of anti-RBC antibodies increased. A measure of the percentage hemolysis induced by sera from these animals in the presence of an exogenous complement source revealed a higher incidence and hemolytic titer in LPS-injected BALB/c and DBA/2 strains than in PBS-injected mice. In addition, injection of LPS induced the appearance of erythrocyte-bound IgM and IgG in BALB/c, DBA/2, and NZB mice. These data suggest that LPS-induced B-cell activation results in the appearance of anti-erythrocyte antibodies.