Anti-hapten Antibody Research Articles

Epitope specificity of bee venom phospholipase A2-specific suppressor T cells which produce antigen-binding glycosylation inhibiting factor.

From the spleen cells of BALB/c mice primed with bee venom phospholipase A2 (PLA2), we established seven T cell hybridomas which constitutively secreted glycosylation inhibiting factor (GIF), expressed both CD3 and TCR alpha beta, and responded to antigen-pulsed antigen presenting cells (APC) for the formation of IgE-binding factor. Upon stimulation with antigen-pulsed APC, four of the seven hybridomas produced GIF having affinity for native PLA2. The antigen-binding GIF could suppress the anti-hapten antibody response of BALB/c mice to dinitrophenyl (DNP)-PLA2 conjugates in a carrier-specific manner and bound to immunosorbents coupled with either the mAb 14-12 or anti-TCR alpha chain, H28-710. Analysis of the epitope specificity of the TCR on the GIF-producing T hybridomas indicated that all of the hybridomas which could produce antigen-binding GIF upon antigenic stimulation recognized the synthetic peptide representing amino acid residues 19-34 in PLA2 molecules in the context of the product of the I-Ad subregion and the antigen-binding GIF formed by the cells had affinity for the peptide. The 3-D structure of bee venom PLA2 indicates that the sequence of amino acid 14-24 forms a loop in the PLA2 molecule and represents an external structure of the antigen, while peptide 25-37 forms an alpha helix. Evidence was obtained which suggests that the sequence of 25-34 contains amino acid residues interacting with Ia molecules, while peptide 19-24 contains residues involved in the interaction of p19-34-Ia complexes with TCR on the hybridomas. It was also found that not only the synthetic peptide 19-34, but also the peptides 13-28 and 19-30 inhibited the binding of antigen-binding GIF to PLA2-coupled Sepharose, while peptide 25-40 failed to do so. The results collectively indicate that the antigen-binding GIF and TCR on the cell source of the factor interact with a common epitope which is exposed on the surface of a nominal antigen.

Affinity maturation of anti-hapten antibodies in a single mouse analyzed by two-dimensional affinity electrophoresis.

Affinity maturation of anti-hapten antibodies in an inbred mouse (BALB/c) was analyzed by two-dimensional affinity electrophoresis (2D-AEP) in which the antibodies specific to a hapten of dinitrophenyl (DNP) group were separated into a large number of IgG families according to the differences in their isoelectric points (pI) and in their affinities for the ligand of DNP immobilized in the gel matrices. Each of the IgG families consisted of several spots showing an identical affinity for the ligand but different pI, and the spots were specifically stained by immunostaining with an anti-murine IgG subclass antibody. These results lead us to the conclusion that each of the IgG families is derived from a single clone of antibody-producing cells specific to DNP. The mass and affinity of the IgG families varied in the course of immunization with DNP-conjugated chicken serum albumin (DNP-CSA). The first injection of DNP-CSA induced a small amount of IgG families showing a wide variety in their affinity for DNP. The second injection induced a large amount of IgG families, especially of families having a medium affinity for DNP. Additional injections, however, did not change the mass or affinity of the IgG families significantly. This suggests that an antigen-induced somatic mutation of the immunoglobulin gene does not occur frequently to mature the affinity of antibodies by the additional injections after the second injection. 2D-AEP enables us to analyze the affinity maturation of antibodies in vivo in a single mouse and to document the subclass switch of the antibody in the course of immunization.

Nucleotide sequence of messenger RNA encoding VHDJH and VKJK of a highly conserved idiotype-defined primary response anti-hapten antibody.

The primary humoral immune response of mice to the hapten phthalate (Xmp) is focused upon two adjacent immunodominant negatively charged carboxyl groups on a benzene ring that are in positions meta and para to the azolinkage (i.e., Xmp) to the protein carrier keyhole limpet hemocyanin. A significant fraction of the anti-Xmp antibodies raised in several different inbred mouse strains (BALB/c, DBA/2, A/HeHa; C3H, and SM/J), and many wild mouse populations express a cross-reactive Id, CRIXmp-1. This CRIXmp-1 is conspicuously absent in C57BL/6 mice. In order to obtain a better understanding of the events and parameters that influence the selection and regulation of the primary response B cell repertoire, and to explore the structural basis of Ag binding, we have determined the nucleotide sequence of the entire V region gene complexes, which encode the H and L chains of these highly conserved and dominant CRIXmp-1+ antibodies. Our data establish that the H chain gene complex consists of a single VH germ-line gene that is identical to VH Oxazolone-1, encoding the H chain of another highly conserved and dominant cross-reactive Id family associated with the primary response to Oxazolone. In CRIXmp-1+ Xmp-specific hybridomas this gene is joined to a limited set of D region sequences that express a conserved amino acid motif-GLR. At least three of the five D regions examined are coded for by DFL16.2. This VHD complex can be utilized with one of three different JH region genes (JH1, JH2, and JH4) without any significant effect upon antibody fine specificity or Id. In spite of this lack of JH fidelity all of the CRIXmp-1+ hybridomas have precisely maintained the same length in the H chain CDR3 and FRW4 by altering either the length of the D segment or the length of JH. Nucleotide sequence analysis of the VL gene complex of CRIXmp-1+ anti-Xmp antibodies indicates that the L chain V region is also encoded by a single germ-line gene. The amino acid sequence predicted from the nucleotide sequence of the VKJK from Xmp-specific CRIXmp-1+ hybridomas is identical to the sequence of the anti-arsonate antibody 1210.7, which is the prototype of another Id family (CRI) that is conserved and dominant in BALB/c mice.

Germ line variable regions that match hypermutated sequences in genes encoding murine anti-hapten antibodies.

We asked whether there are germ line immunoglobulin variable (V) segments that match sites of hypermutation in V regions encoding murine antibodies. Murine germ line DNA was probed with a panel of short deoxyoligonucleotides identical in sequence to segments of hypermutated V regions from hybridomas generated in the BALB/c response to the hapten 2-phenyloxazolone (Ox). Germ line sequences that match mutations in both heavy and kappa light chain V regions were identified, and clones of some of these germ line V segments were obtained. Comparison of these clones with hypermutated V regions revealed regions of identity ranging in size from 7 to over 50 nucleotides. In an effort to separate the effects of antigen selection from the mutagenic process, we also searched for matches to a panel of silent mutations in VH regions from germinal center B cells. Fourteen silent mutations occur among a collection of 36 hypermutated VH regions from two separate germinal centers of C57BL/6 mice stimulated with the hapten 4-hydroxy-3-nitrophenyl. Matches to nine of these silent mutations can be found among published sequences of C57BL/6 VH regions of the J558 family. Taken together, these data are consistent with the possibility that a template-dependent mutational process, like gene conversion, may contribute to somatic hypermutation.

Susceptibility and resistance to autoimmunity following neonatal injection of semi-allogeneic spleen cells in rats

A model of neonatal allotolerance was developed in rats. Brown-Norway (BN) neonates injected with semi-allogeneic (BN × Lewis) F1 hybrid spleen cells express a long-lasting chimerism and exhibit polyclonal B cell activation demonstrated by hyperimmunoglobulinemia affecting mainly IgE and IgG1, anti-laminin and anti-DNA autoantibodies as well as glomerulonephritis and anti-hapten antibodies. These abnormalities are autoregulated although the chimerism persists. In contrast, Lewis (LEW) neonates injected with semi-allogeneic (BN × LEW) F1 hybrid spleen cells exhibit a very short-lasting chimerism and transient activation of B cells, as reflected by increased allo-class II antigen expression, but do not develop an autoimmune disease. The autoimmune syndrome observed in BN rats is similar to that reported in mice during host-versus-graft reaction. Similarities between the drug-induced models of autoimmunity and allogeneic reactions in BN rats are also striking. The susceptibility of BN rats and the resistance of LEW rats to these autoimmune diseases might respectively reflect the involvement of TH2-like or of TH1-like subsets.

Separation of monoclonal antibodies from antihapten antisera by two-dimensional affinity electrophoresis

A high-resolution two-dimensional affinity electrophoresis (2D-AEP) method was developed, using capillary polyacrylamide gel (PAG) isoelectric focusing in the first and slab PAG affinity electrophoresis in the second direction. Using this method, anti-hapten antibodies were separated into a number of monoclonal antibody [immunoglobulin G (IgG)] families, each of which is composed of several IgG spots having an identical affinity to the hapten but different isoelectric points. 2D-AEP may offer a powerful tool for solving fundamental problems in immunochemistry such as antibody heterogeneity, its hapten binding specificity and antigen-dependent somatic mutation.

Characterization of anti-hapten antibodies generated in vitro by channel catfish peripheral blood lymphocytes

Secondary in vitro stimulation of channel catfish peripheral blood lymphocytes with haptenated T-dependent antigen (TNP-KLH) elicited large numbers of hapten-specific Ab-producing cells and relatively high levels (10–96 μg/mL) of TNP-specific Ab in the culture medium. These in vitro generated Abs were compared to in vivo generated Abs from the serum of the same fish with respect to covalent structure, affinity, and isotypic composition of heavy and light chains. SDS-PAGE analysis under both reducing and nonreducing conditions revealed that the in vitro Abs were structurally similar to the serum Abs. Similarly, in vitro pulse-labeled Abs also exhibited the eight band profile characteristic of channel catfish serum Abs when run under nonreducing denaturing conditions. Scatchard analysis of equilibrium dialysis data revealed that the affinities of the culture- and serum-derived Abs were quite similar, that is, exhibited association constants of ∼2.0 × 10 6 M −1. However, it was routinely observed that the in vitro generated Abs exhibited somewhat fewer binding sites per molecule than those derived from serum. The use of murine monoclonal Abs specifically for different isotypes of channel catfish heavy and light chains demonstrated that the isotypic composition of the culture- and serum-derived fish anti-TNP Abs were similar; exceptions occurred with cultures producing lower levels of Abs. These results strongly suggest that channel catfish in vitro Ab responses closely reflect what normally occurs in vivo.

Divergent immune responses to respiratory and contact chemical allergens: antibody elicited by phthalic anhydride and oxazolone

In previous studies we observed that exposure of mice to the contact allergen 2,4-dinitrochlorobenzene (DNCB) and the respiratory allergen trimellitic anhydride (TMA) resulted in qualitatively different immune responses characteristic of selective Th1- and Th2-type T helper cell activation respectively. The purpose of the present investigation was to determine whether the effects recorded with DNCB and TMA are characteristic of immune responses to contact and respiratory chemical allergens in general. Experiments have been performed with phthalic anhydride, a known human respiratory sensitizer, and with oxazolone, a potent contact allergen. Under conditions of exposure where both chemicals elicited an IgG anti-hapten antibody response, only phthalic anhydride caused an increase in the serum concentration of IgE. Furthermore, like TMA, phthalic anhydride preferentially induced IgG2b rather than IgG2a antibody. In contrast, oxazolone, like DNCB, induced a markedly stronger IgG2a than IgG2b antibody response. These data provide confirmatory evidence that chemical respiratory allergens and chemical contact allergens elicit qualitatively different immune responses which reflect their clinical effects in man.

Characterization of murine immune responses to allergenic diisocyanates

Chemicals may cause contact allergy. Some allergens may, in addition, cause respiratory sensitization. In previous investigations we have found that contact and respiratory sensitizers induce differential immune responses in mice characteristic of T H1 and T H2 T helper cell activation, respectively. In the present study we have examined immune responses in mice following topical exposure to three allergenic diisocyanates; diphenylmethane-4,4′-diisocyanate (MDI), dicyclohexylmethane-4,4′-diisocyanate (HMDI), and isophorone diisocyanate (IPDI). All three chemicals are contact allergens. MDI is in addition a known human respiratory allergen. HMDI and IPDI appear not to induce respiratory sensitization or at least do so very rarely. Exposure of mice to all chemicals resulted in a vigorous lymphocyte proliferative response in lymph nodes draining the site of application, and each caused contact sensitization. In common with other respiratory allergens, MDI induced an increase in the serum concentration of IgE and provoked considerably more IgG2b than IgG2a anti-hapten antibody; responses consistent with a preferential activation of T H2 cells. In contrast, under conditions where both caused lymph node cell proliferation and contact sensitization, neither HMDI nor IPDI induced a measurable antibody response of any class. These data provide additional evidence that different classes of chemical allergen cause divergent immune responses in mice. The possibility that these characteristics may facilitate not only the identification, but also classification, of chemical allergens is discussed.

Effect of anti-DNP IgG1- and IgG2a-secreting hybridomas in vivo on the development of an anti-DNP IgE antibody response in mice.

We reported previously that CBA mice pretreated with dinitrophenyl-Bordetella pertussis (DNP-BP) conjugates exhibited sharply decreased anti-DNP IgE, and increased IgG2a antibodies following immunization with DNP-ovalbumin (DNP-OA) in alum. The objective of the present experiments was to determine whether the decrease in anti-DNP IgE was attributed to a regulatory effect exerted by IgG2a antibodies. Anti-DNP monoclonal antibodies (Mab) of the IgG1 or IgG2a isotype were passively transferred to mice, 24 h before a primary immunization with DNP-OA in alum. Anti-DNP IgE production was drastically suppressed in recipients of IgG1 but not of IgG2a Mab. Similar results were obtained when the Mab were endogenously produced by intraperitoneal implantation of anti-DNP-secreting hybridomas into (BALB/cxCBA)F1 (BCF1) mice. However, neither IgG1 nor IgG2a isotypes suppressed IgE antibody production if the hybridoma implantation took place 10 days after hapten priming. These results are, to our knowledge, the first to show a clear dissociation between the effect of either passively transferred or endogenously secreted IgG1 and IgG2a antibodies in their ability to inhibit a primary anti-hapten IgE antibody response.

Requirement of certain epitope specificities of glycosylation inhibiting factor for the suppression of in vivo IgE and IgG antibody responses.

The ovalbumin (OVA)-specific T cell hybridoma 71B1, which constitutively secretes glycosylation inhibiting factor (GIF) and is specific for the immunogenic epitope represented by amino acids 323-339 in the OVA molecules, failed to form GIF having affinity for nominal antigen upon stimulation with OVA-pulsed antigen-presenting cells (APC). However, the GIF produced by the antigen-stimulated 71B1 cells bound to the mAb 14-12, which is specific for the antigen-binding chain of effector type suppressor T cell factor (TseF), and to mAb specific for TCR. The GIF constitutively released from unstimulated 71B1 cells failed to bind to any of these antibodies. Gel filtration of GIF preparations showed that the 14-12+ GIF from the antigen-stimulated 71B1 cells are composed of 80-100 and 25-35 kDa species, while the GIF from unstimulated cells was 12-15 kDa. Reduction and alkylation treatment of the GIF from the antigen-stimulated cells resulted in the disappearance of the 80-100 and 25-35 kDa GIF, which was accompanied by the formation of the 12-15 kDa GIF. Thus, the GIF from the antigen-stimulated 71B1 cells was similar to the previously described OVA-binding GIF from the 231F1 cells with respect to their antigenic structures and molecular size, and both factors appear to be composed of the 14-12+ polypeptide chain and 12-15 kDa non-specific GIF. However, the GIF from the antigen-stimulated 71B1 cells lacked affinity for the native OVA or synthetic peptide 323-339, and failed to suppress the in vivo antibody response to dinitrophenyl (DNP)-OVA. In contrast, the OVA-binding GIF has affinity for native OVA and the peptide 307-317, to which the cell source of the factor is specific, and suppressed the in vivo anti-hapten antibody response to DNP-OVA. The results suggest that formation of antigen-specific TsF is confined to T cells with certain epitope specificities. It was also found that the OVA-binding GIF failed to suppress the in vivo anti-hapten antibody response to DNP-conjugates of urea-denatured OVA (UD-OVA), which does not bind OVA-binding GIF. However, APC pulsed with UD-OVA appear to express the epitope 307-317 for which the OVA-binding GIF has affinity. The results collectively suggest that the affinity of GIF for an immunizing antigen, rather than processed antigen, is required for immunosuppression.

Detection of One Attomole of [Arg8]-Vasopressin by Novel Noncompetitive Enzyme Immunoassay (Hetero-Two-Site Complex Transfer Enzyme Immunoassay)1

One attomole of [Arg8]-vasopressin (AVP) was detected by a novel noncompetitive enzyme immunoassay (hetero-two-site complex transfer enzyme immunoassay). AVP was indirectly biotinylated using N-hydroxysuccinimidobiotin and trapped onto an anti-AVP IgG-coated polystyrene ball. After washing, biotinylated AVP was eluted from the polystyrene ball with HCl and was reacted with 2,4-dinitrophenyl-fluorescein disulfide-bovine serum albumin-rabbit anti-AVP IgG conjugate. The complex formed was trapped on [anti-2,4-dinitrophenyl group] IgG-coated polystyrene balls and, after washing, reacted with avidin-beta-D-galactosidase conjugate. The polystyrene balls were washed, and the complex of the three components was eluted with 2,4-dinitrophenyl-L-lysine and transferred to anti-fluorescein IgG-coated polystyrene balls. After washing, the complex was released from the polystyrene balls by reduction with 2-mercaptoethylamine and transferred to [anti-rabbit IgG] IgG-coated polystyrene balls. beta-D-Galactosidase activity bound to the last polystyrene balls was assayed by fluorometry. The detection limit of AVP was 1.1 fg (1 amol)/tube. Interference by proteins in biological fluids was eliminated by separation of peptides from proteins using a molecular sieve. The principle of the present method may be applicable to the measurement of haptens, including peptides, that can be derivatized so as to be bound simultaneously by both anti-hapten antibody and avidin molecules.

Heavy and light chain variable region sequences and antibody properties of anti-phosphotyrosine antibodies reveal both common and distinct features.

Phosphotyrosine and similar analogs have been used to elicit antibodies that have found widespread use in the study of cellular tyrosine phosphorylation. In order to better understand the anti-phosphotyrosine immune response and to elucidate the details of the specific association between a tyrosine phosphate and an antibody combining site, we have undertaken a detailed comparison of antibody stability, specificity, apparent affinity, and primary structure for eight different anti-phosphotyrosine antibodies derived from immunizations with three different antigens. Two of these, 2G8 and 1G2, were derived from an immunization using azobenzylphosphonate conjugated to carrier, and five others, Py2, Py20, Py42, Py54, and Py69, were the products of an immunization with phosphotyrosine conjugated to carrier. Each of these anti-hapten antibodies was an IgG. One antibody, 129, an IgM, was the result of an immunization with a mixture of tyrosine-phosphorylated proteins which had been purified from growth factor treated cells. We found that antibody binding was significantly inhibited by millimolar levels of divalent cations or high concentrations of monovalent salt, with the exception of the antibody 129 where binding was significantly enhanced by both. Under optimal conditions, the highest apparent affinities for phosphotyrosine were observed for antibodies Py69 and Py20 (10(-6)-10(-7) M) and the lowest for 129 and 1G2 (10(-3)-10(-4). The heavy and light chain variable regions of seven of these antibodies were cloned and sequenced and a predominant anti-phosphotyrosine response was observed. The light chains of these antibodies could be assigned to one of two major VK groups, VK10 and VK19, with sequence identity between the different light chains of each class ranging from 65 to 100% at the amino acid level. Similar sequence identity was found among the heavy chain sequences (89-98% identity at the amino acid level) with the exception of one antibody, 2G8, which was only distantly related to the others (61-64% amino acid identity). These heavy chains belong to the same heavy chain family, J558. Two of the antibodies, Py20 and Py69, were clearly derived from the same progenitor cell since both share a highly unusual apparent V-D-D-JH organization. However, a significant level of somatic mutation has occurred between the two antibodies resulting in subtle changes in their apparent affinity and specificity.

Inhalation Exposure of Mice to Trimellitic Anhydride Induces Both IgG and IgE Anti-Hapten Antibody

The development of antibody responses resulting from inhalation exposure to chemical allergens has been studied in mice. Inhalation exposure of BALB/c mice to atmospheres containing approximately 5 mg/m3 of the respiratory allergen trimellitic anhydride (TMA) resulted in the appearance of both serum IgG and IgE anti-hapten antibody. IgE anti-TMA was first detectable 2-3 weeks following the initiation of exposure and was still present at 6 weeks. Under the same conditions of exposure, 2,4-dinitrochlorobenzene (DNCB), a contact allergen which apparently lacks the capacity for respiratory sensitization, failed to elicit detectable amounts of anti-dinitrophenol (DNP) antibody. Exposure to increased concentrations of atmospheric DNCB (15 mg/m3) did, however, result in an IgG anti-DNP response but not in IgE antibody. These data demonstrate firstly, that atmospheres containing low molecular weight respiratory allergens can initiate specific IgE responses in mice, and secondly, that inhaled chemicals may differ in their ability to induce IgE antibody.

Antigen targetting with monoclonal antibodies as vectors—II. Further evidence that conjugation of antigen to specific monoclonal antibodies enhances uptake by antigen presenting cells

Immunization of rats with haptenized monoclonal antibodies (mAbs) against accessory cells enhances anti-hapten antibody responses. To see whether the mAb-conjugates really targetted the antigen (hapten) to the antigen presenting cells, we have investigated the lymph node distribution of locally injected radiolabelled conjugates. Compared with control conjugates, i.e. haptenized non-binding mAbs, a much larger proportion of the specific conjugates were retained in the draining lymph nodes. Whereas control conjugates were rapidly phagocytosed and degraded by macrophages, the specific conjugates were associated with the targetted accessory cells, which were radiolabelled for extended periods. Haptenated MRC OX6 (anti-MHC class II) gave strong labelling of interdigitating cells (IDC) in the paracortex with 70% of IDC still labelled by 4 days and 15% by 16 days following injection. By Western blots intact OX6 conjugates were still detected in the draining lymph node as long as 3 days after injections, whereas control conjugates were hardly detectable even by 24 h. The findings substantiate the idea that mAbs can be exploited for vectorial transport of antigens to accessory cells.

Selective Impairment of T Lymphocyte Activation following Contact Sensitization with Oxazolone

We have previously reported that topical exposure of mice to oxazolone results in the appearance of regulatory mechanisms which markedly depress lymph node cell (LNC) proliferative responses to subsequent challenge with the same chemical. In the present study, we have sought to identify the cellular targets for such immunoregulation. Autoradiographic analyses revealed that although pre-exposure to oxazolone caused a substantial reduction of paracortical hyperplasia following challenge, the frequency of proliferating cells in lymphoid follicles was slightly increased. That B lymphocyte responses are unaffected by oxazolone-induced immunoregulation was confirmed by investigation of anti-hapten antibody formation by draining LNC. Challenge with oxazolone resulted in an accelerated antibody response in mice previously exposed to the same chemical. These data reveal that the active immunoregulation induced following sensitization with oxazolone is selective for T lymphocytes. Evidence is presented that CD4+ and CD8+ T lymphocytes possess equivalent sensitivity to these mechanisms.

Antibody caging of a nuclear-targeting signal.

We have developed a technique for reversibly masking a peptide-targeting signal. A fluoresceinated derivative of the simian virus 40 large tumor antigen nuclear-targeting signal was synthesized and cross-linked to bovine serum albumin. The conjugated protein was efficiently transported into rat liver nuclei unless the peptide-targeting signal was sterically hindered by binding of an anti-fluorescein antibody. Addition of free 5-aminofluorescein competed for antibody binding and rapidly restored nuclear accumulation of the derivatized bovine serum albumin. General use of hapten derivatization and anti-hapten antibodies for caging portions of macromolecular surfaces can be extended to a variety of proteins, including antibodies themselves.

Novel and sensitive noncompetitive (two-site) immunoassay for haptens with emphasis on peptides

A novel and sensitive noncompetitive (two-site) enzyme immunoassay is described to measure attomole amounts of haptens, especially small peptides, which cannot be bound simultaneously by two different antibodies directed to the corresponding two different epitopes on the hapten molecules; consequently, they cannot be measured by the conventional two-site immunoassays. The principle of the assay is to label haptens to be measured with an appropriate substance, so that the hapten molecule can be bound simultaneously by both a binding substance molecule for the label, and anti-hapten antibody molecule, which allows two-site assay. Feasibility of the principle was demonstrated by using biotin as a label and angiotensin I, a 10 amino acid single chain peptide, as a model hapten. Angiotensin I was biotinylated and, after removal of unreacted biotin and other biotinylated substances, was measured using anti-angiotensin I Fab'-horseradish peroxidase conjugate and streptavidin-coated polystyrene balls. The detection limit of angiotensin I was 10 amol (13 fg); this was 100-fold lower than that by competitive radioimmunoassay using the same antibody and 125I-angiotensin. I. This principle was successfully applied to the measurement of arginine vasopressin, a nine amino acid single chain peptide with an intramolecular disulfide bridge. Possible modifications, advantages, and disadvantages of the assay method are discussed.

Polarity of immunogens: implications for vaccine design

Peptide constructs have been engineered consisting of amino acid sequence determinant recognized by T cells (TD) co-linearly linked to haptenic peptides. It was found that high anti-hapten antibody titers were induced after immunization with those constructs which had the TD sequence in the N-terminal position with respect to the hapten. Low or zero titers were elicited when the TD was in C-terminal position. Also, a high anti-hapten antibody titer corresponded to a low or zero anti-TD antibody titer and vice versa. These results suggest that immunogens are polar and stress the relevance of searching the more adequate position of the TD within a peptide construct when designing immunogens or synthetic peptide vaccines.

Characterization of the interleukin 5‐reactive splenic B cell population

The characteristics of the interleukin (IL) 5-reactive splenic B cell population of C57BL/6 nu/nu mice, with respect to IL 5/IL2 reactivity, cell surface phenotype, VH gene family usage, autoreactivity and the structure of the IL5 receptor (IL5R), were analyzed. It was found that 2%-4% of splenic B cells express relatively high levels of IL 5R as determined by the binding of the anti-IL 5R monoclonal antibody R52.120. Over 90% of the splenic B cells that mature to IgM secretion upon activation with IL5 are comprised in this small subpopulation of B cells. Moreover, the vast majority of splenic B cells that mature to IgM-secreting cells when activated by IL2 also reside in this IL5R+B cell population. The cell surface phenotype of the IL5R+ splenic B cells is IgM+, B220+, Ly-1- and IL2R p55-. Upon activation with IL5 this cell surface phenotype changes, in that a vast majority of the B cells then express the p55 chain of the IL2R, whereas the level of IL5R decreases. VH gene family usage in the IL5-activated splenic B cells was analyzed by in situ hybridization. VH gene family usage was found to be random and not different from the VH genes expressed in LPS-activated B cells. Hybridoma collections from IL5-activated splenic B cells and LPS-activated B cells were screened and compared for the production of autoantibodies and antibodies directed against the haptens (4-hydroxy-3-iodo-5-nitrophenyl)acetyl (NIP) and 2,4,6-trinitrophenyl (TNP). In both collections high, but not significantly different frequencies of autoantibody-(32% IL5, 31.4% LPS) and of anti-hapten antibody (27.8% IL5, 18.6% LPS)-producing hybridomas were found. The structure of the IL5R on IL5-activated B cells was analyzed by 125I-labeled IL5 binding and cross-linking. About 100 high-affinity (10(-11) M) and 1000 low-affinity (10(-9) M) IL5-binding sites are present on IL5-activated splenic B cells, and both high- and low-affinity IL5R are similar to those expressed on the IL5-dependent B13 cell line. Cross-linking of 125I-labeled IL5 to the receptors on IL5-activated B cells revealed one major IL5-binding protein of 45-50 kDa molecular mass and another minor binding protein of 130-140 kDa. The same IL5-binding proteins are present on the IL5-dependent B13 cell line.(ABSTRACT TRUNCATED AT 400 WORDS)