Haptenic Determinants Research Articles

Splenic macrophages: antigen presenting cells for T1–2 antigens

Thymus-independent antigens type 2 (T1–2) such as DNP-Ficoll or DNP-hydroxyethyl starch are selectively retained in mice and rats by a distinct population of macrophages in the marginal zone of the white pulp of the spleen and in the marginal sinus and septa and medulla of lymph nodes. There is suggestive evidence that the antibody response to the haptenic determinants, which is rapid in onset and persists for many weeks, is largely made by a sessile subpopulation of B cells in the spleen marginal zone characterized by the presence of surface IgM but lacking IgD. Splenectomy in adult mice, rats and humans greatly diminishes and delays the antibody response to DNP-Ficoll, but has no such effect on the response to thymus-dependent, or (in mice) to T1-1 immunogens. In humans, if DNP-Ficoll had been administered prior to splenectomy, a normal antibody response was made when the antigen was readministered after splenectomy, indicating that once the responding B cells had been stimulated they were no longer confined to the spleen. Experiments in mice showed that after elimination of B cells by irradiation, responsiveness to T1–2 antigens could be restored by spleen cells isolated as single-cell suspensions provided that the architecture of the spleen remained, but could not be restored if the mice had been splenectomized. This implies that both the spleen B cells and the marginal zone macrophages are required. Since administration of cyclophosphamide, which selectively depletes the marginal zone B cell subpopulation, abolished the antibody response to T1–2 antigens in mice and rats until this subpopulation recovered, the B cells which initiate the antibody response are presumably contained therein. The capacity to initiate a thymus-independent rapid IgM antibody response to multi-epitope polymeric bacterial capsular polysaccharides depends largely upon the spleen. If splenectomy is contemplated, primary immunization with such antigens should be undertaken before rather than after the spleen is removed.

Hapten-modified basophils: a model of human immediate hypersensitivity that can be elicited by IgG antibody.

Trimellitic anhydride (TMA) was incubated with human leukocytes to produce leukocytes with trimellityl (TM) haptenic determinants. The basophils in these TM leukocytes released histamine when challenged with human serum containing IgG but no IgE antibodies against TM determinants. Controls of TMA-treated cells exposed to normal human sera or untreated cells exposed to anti-TM sera released no histamine. The most relevant site of TMA modification of the basophil was receptor-bound IgE. This was demonstrated by the marked reduction in histamine release if IgE was removed from the leukocytes before TMA treatment and challenge and also by activation of release from untreated cells by passive sensitization with TM-IgE before challenge. Affinity-purified IgG from an anti-TM serum released much more histamine from TMA-treated cells than did the IgG-poor effluent at similar protein concentrations. These observations suggest a new model of hypersensitivity resulting from human exposure to chemicals capable of combining with autologous proteins. In some individuals this could result in both an immune response to the new determinants without the production of significant quantities of IgE antibodies and hapten modification of receptor-bound IgE on mast cells and basophils. After sufficient antibody was produced, reexposure to the chemical with hapten modification of mast cells or basophils or both could result in mediator release and clinical symptoms. This mechanism may be relevant to immediate-type chemical hypersensitivity reactions in which IgE antibody has not been demonstrated.

Langerhans Cells: Antigen Presenting Cells of the Epidermis

While epidermis in the skin provides an excellent barrier to the environment, it is an incomplete one. Some antigenic material can penetrate through the stratum corneum (or be introduced pathologically) where strategically placed epidermal Langerhans cells reside. In this review, we have assembled relevant data concerning the antigen presenting potential of epidermal Langerhans cells. Strong circumstantial evidence derived from in vitro studies of epidermal cell suspensions enriched for Langerhans cells indicates that Langerhans cells possess this capability. In vivo studies with intact skin indicate that critical numbers of functioning Langerhans cells are essential for successful induction of contact hypersensitivity by epicutaneously applied haptens. And within the past several months, experiments with purified preparations of epidermal Langerhans cells have proven that these cells, and perhaps they alone among epidermal cells, possess the capacity of processing and presenting haptenic determinants to the immune system. The challenge for the future is to determine the extent to which this unique property of Langerhans cells affords physiologic protection to the skin and under what pathologic circumstances altered Langerhans cell function leads to disease.

Antisera specific for the oligoribonucleotide sequences AAU and A 2U 2 and their recognition of oligonucleotide conformation

Antisera specific for two synthetic oligoribonucleotide sequences, AAU and A 2U 2, were elicited in rabbits. The oligonucleotides were synthesized using polynucleotide phosphorylase under high salt conditions. Each oligomer was isolated by ion exchange chromatography, and was conjugated to bovine serum albumin, and injected into rabbits as an emulsion with complete Freund's adjuvant. The specificities of the resulting sera were analyzed using a modified Farr-type radioimmunoassay employing homologous oligonucleotide-protein conjugates radiolabeled with [ 3H]acetic anhydride and unlabeled free oligonucleotides as inhibitors. The antiserum elicited by AAU-BSA reacted well with AAU-RSA but a major fraction of the antibodies was directed to determinants of the conjugate that were not present on the free hapten. With respect to the haptenic determinants, AAU was a better inhibitor than any of the constituent mono- or dinucleotides, implying that features of the entire trinucleotide were being recognized. The other members of the A 2U n family reacted to about the same extent as AAU, while other trinucleotides required an up to 21-fold higher concn in order to achieve similar inhibition. The most striking aspect of this antiserum was its failure to bind free ApA, although it could bind the ApA-containing oligonucleotides A 3, AAG, AAC and A 2U n . It seems likely that the ApA sequence in solution does not contain a significant proportion of a conformation present to a great extent in the ApA-containing oligomers. The antiserum elicited by A 2U 2-BSA was like anti-AAU-BSA in that some of the antibodies were directed against determinants not present on the free hapten. The most striking result of the inhibition experiments was the specificity of the antiserum for members of the A 2U n series. When the A 2U n series was compared with AA, AMP or any member of the U n series, approximately four orders of magnitude separated the inhibition curves. The poor binding of component mono- and dinucleotides implies that the conformation recognized by the antibody is present only to a significant extent in the trimeric sequence; the equality of binding of AAU with A 2U 2, A 2U 3 and A 2U 4 suggests that this conformation of the triplet is preserved in the longer sequences. These studies demonstrate the utility of immunochemical procedures for the study of oligonucleotide conformation in solution.

Immunogenicity and effector functions of glutaraldehyde-treated rabbit and mouse immunoglobulin G

Rabbit and mouse IgG treated with glutaraldehyde (GA) were immunogenic in homologous species. Glutaraldehyde treatment induced in the IgG molecule two types of antigenic determinants. One of them was found on the monomeric fraction of GA-treated rabbit IgG (haptenic determinant) and the other on the polymeric fraction (structural determinant). The haptenic determinants were found also on monoaldehyde-treated rabbit IgG and GA-treated Fab and Fc fragments. It was demonstrated that rabbit and mouse antibodies are specific for GA-treated IgG and have species specificity. While GA treatment did not alter the antigen binding capacity of rabbit IgG antibody, its effector functions (except protein A binding) were much affected. Thus it was found that GA treatment enhances IgG ability to react with rheumatoid factor, reduces drastically its capacity to activate the complement system, abolishes the cytophilic properties of IgG and accelerates its catabolic rate. The possible blocking effect of GA on the amino acid residues (mainly Lys) situated in or very close to the effector sites of the IgG molecule is suggested.

Evidence for low-molecular weight antibodies in the serum of a urodele amphibian, Ambystoma mexicanum

Adult axolotls ( A. mexicanum) were hyperimmunized with the haptenic determinant, azobenzene-arsonate (ARS). Specific antibodies were isolated from their serum by immune-affinity chromatography on immobilized ARS columns. Analysis of the specific ARS-binding molecules by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) indicated that these animals were capable of producing both high molecular-weight (presumably IgM) and low molecular-weight antibodies to the ARS hapten. The low molecular-weight anti-ARS antibody produced by one inbred colony of axolotls did not show any restricted heterogeneity, as assessed by isoelectric focusing. Our results suggest that regulatory events, not the absence of genetic information, may be responsible for the apparent lack of Ig isotype diversity in this species.

Immunological activities of purified preparations of enterobacterial common antigen.

The immunological activities of three purified preparations of enterobacterial common antigen (ECA) obtained by different procedures were studied. ECA-Ma (method of A. Marx) was from Salmonella typhimurium TV149 (Ra mutant), ECA-My (method of H. Mayer) was from S. montevideo, and ECA-Ro (method of E. Romanowska) was from Shigella sonnei phase I. These preparations, on a weight basis, neutralized similar amounts of ECA antibodies, indicating that the serological activities were comparable. Neither ECA-My nor ECA-Ro elicited specific delayed-type hypersensitivity skin reactions at 24 or 48 h in immunized guinea pigs. ECA-Ma, as well as the nonpurified preparations of the antigens used for immunization, elicited reactions at 24 h but not at 48 h. Thus, ECA-specific delayed-type hypersensitivity was not detected in immunized guinea pigs. Striking differences were noted in the immunogenicity of these antigens, ECA-Ma being highly immunogenic in the rabbit in contrast to ECA-My and ECA-Ro. ECA-Ma was a potent mitogen for guinea pig spleen cells, stimulating high levels of DNA synthesis; ECA-My was only slightly active. The three antigens were mitogenic to spleen cells from both CBA/J and C3H/HeJ mice, although not to the same degree, indicating that this effect is not due to contaminating lipopolysaccharide, since the latter strain of mice is resistant to endotoxin. Since an ECA-Ma extract made from an ECA-negative mutant proved to be mitogenic to murine spleen cells, the mitogenicity is not due to the ECA haptenic determinant. The mitogenic effect is polyclonal in nature, ECA-Ma producing a maximum response on day 3. Thus, the ECA preparations are both B-cell mitogens and polyclonal activators in murine spleen cells. From these studies it is evident that the biological and immunological activities of these purified antigens depend not only on the haptenic determinant but also on associated or bound components of the preparations.

The effect of Pseudomonas aeruginosa on the immunogenicity of enterobacterial common antigen.

Pseudomonas aeruginosa produces a factor (PF) which alters the enterobacterial common antigen (ECA). Its effect on the immunogenicity of two types of immunogenic ECA, namely, the ethanol-soluble preparation freed of lipopolysaccharide and the LPS-coupled form from the R-mutant E. coli 014 was investigated. The antibody response following intravenous immunization was determined by means of the hemagglutination test. It is shown that PF abolishes the immunogenicity of the former but not of the latter. PF obtained from a strain of P. maltophilia yielded the same results. Antiserum against Pseudomonas aeruginosa of types 1 and 6 neutralizes PF produced by either type. These results suggest that PF alters the lipid part and not the haptenic determinant of ECA and that this activity is neutralized by P. aeruginosa antiserum of either type 1 or type 6. This interpretation is compatible with the identification of PF as a lipase.

Radioallergosorbent test with conjugates specific for 'minor' haptenic determinants in the diagnosis of IgE-mediated penicillin allergy in man.

Penicillamine-polylysine, benzylpenicillanyl-human serum albumin and ampicillin-polymers were coupled to CNBr-activated paper discs and used in addition to penicilloyl G and penicilloyl V discs in RAST investigations. Sera from sixty patients with case histories of penicillin allergy and with positive or negative skin tests to different penicillin determinants and from seventeen subjects with atopic diseases caused by allergens other than penicillin were tested. The penicilloyl-('major' determinant) specific RAST (Phadebas RAST Penicilloyl G and V) was positive in twenty out of twenty-four patients with positive skin tests to penicilloyl-polylysine, in eight out of twelve patients with sole skin reactivity to 'minor' determinants, and in five out of twenty-four patients with negative skin tests to all antigens used. The penicillamine-specific RAST was the only positive in vitro test in four patients with negative skin tests (two) or sole positive skin reactivity to 'minor' determinants (two), whereas benzylpenicillanyl and ampicillin-polymer discs added no more information than the penicilloyl structures in all patients tested. Antibodies specific to 'minor' determinants apparently show strong in vitro cross-reactivity with the penicilloyl determinant. It is, therefore, concluded that 'minor' determinant specific conjugates are of marginal importance for in vitro diagnosis of penicillin-allergic patients.

T LYMPHOCYTE CLONES HAVING DEFINED IMMUNOLOGICAL FUNCTIONS

T cell clones are proving to be useful for analysis of various immunological phenomena. Although some clones grow well in IL 2-containing CM alone, the combination of antigen, "filler" cells, and CM improves cloning efficiency and may favor the retention of stable clones expressing a "normal" phenotype. The antigen recognition repertoire of T cells appears to be much larger than was predicted using conventional approaches. CTLs can react with at least 23 different determinants on the H-2Kb molecule of the MHC. CTLs demonstrate MHC-restricted recognition of viral and haptenic determinants, but the restriction is not strict for all cloned cells, and a variety of reactivity patterns are found. Antigen-responsive cloned T cells appear to recognize antigens in association with alloantigens encoded by the I-A region of the MHC. However, the relevant structures formed in hybrid animals include all possible combinations of peptides including those formed by trans-complementation of I-A and I-E region gene products. Culture supernatants from antigen-responsive cloned T cells contain a variety of biological activities including IL 2, B cell-stimulating factor, colony-stimulating factor, migration inhibition factor, interferon, and factors that modulate expression of Ia antigens on macrophages and influence production of complement components. These factors may be important in mediating interactions between T lymphocytes and other cells. Cloned T cells have proved to be useful for studying the importance of particular phenotypic characteristics associated with given immunological functions. Although expression of Lyt-2 generally correlates with cytolytic activity and Lyt-1 with helper activity, important exceptions have been found with T cell clones. Cloned T cells promise to be useful for further characterization of the molecular basis of immune responses.

Allorestriction and xenorestriction phenomena in the induction of delayed-type hypersensitivity (DTH) responses in the guinea pig against azobenzenearsonate- (ABA) modified peritoneal exudate cells.

The role of MHC products in the induction of DTH responses Ia antigens play a crucial role in these cells in the recognition of ABA determinants by lymphocytes. Second, JY-1 guinea pigs were employed as responder animals. A clear allorestriction phenomenon was observed again when weakly modified ABA-PEC were used. On the other hand, the allorestriction phenomenon was apparently abrogated when heavily modified ABA-PEC were employed: allogeneic as well as syngeneic ABA-PEC induced strong and comparable DTH responses. Even in this condition, xenogeneic (mouse) ABA-PEC were barely immunogeneic, On the basis of these observations, the cross-reactivity of Ia antigens in the conjunctive recognition of haptenic determinants by T cells is discussed.

Immune response of BALB/c mice to phthalate: characterization of a new and useful model for studying immune regulation.

The humoral immune response to a unique haptenic determinant has been characterized at the single cell level. We report here that the hapten, phthalate, when conjugated to keyhole limpet hemocyanin (4-azophthalate-KLH) is highly immunogenic in adult BALB/c mice, and that the immunogenicity of the hapten is largely restricted to the two charged carboxylate groups on phthalate. Based upon plaque-forming cell (PFC) inhibition with increasing concentrations of free phthalate, relative affinity distribution profiles of antibody-forming cells have been established for individual mice. The antibody-forming cells in the spleens of these mice consist of five or six individual relative affinity sets. PFC inhibition studies with hybridomas secreting phthalate specific monoclonal antibodies reveal, as expected, a more restricted inhibition profile. The majority of PFC produced by the hybridomas could be assigned to one of the relative affinity sets. PFC inhibition studies employing a panel of cross-reactive phthalate...

Ontogeny of B lymphocyte function. X. Strain differences in maturation of the capacity of the B lymphocyte population to produce a high-affinity antibody response.

AbstractThe ontogeny of the B lymphocyte population to be capable of producing a high‐affinity plaque‐forming cell (PFC) response to a haptenic determinant was studied by cell‐transfer techniques in five different mouse strains. Lethally irradiated adult mice were reconstituted with liver from fetal or neonatal donors or spleen from young donors as a source of B lymphocytes. In addition, all animals received 1 × 108 adult thymus cells. Recipients were immunized with dinitrophenylated bovine gamma‐globulin (DNP‐BGG) one day after cell transfer. The heterogeneity of affinity of their anti‐DNP PFC response was assayed by hapten inhibition of plaque formation. It was established by use of congenic mice differing at the Igh locus that only donor cells responded under these conditions. By use of this transfer system, it was shown that the B cell populations of A/HeJ, AKR and C57L/J mice aquire the capacity to produce a high‐affinity PFC response between day 15 and 18 of fetal life. The B cell populations of LAF1 and DBA/1 J mice mature between 7 and 10 days after birth. The B cell population of C57BL/6 J mice matures between 21 and 28 days of age. In LAF1 and AKR mice, the B cell population acquires the capacity to generate a high‐affinity indirect PFC response at approximately the same time as the capacity to generate a high‐affinity direct PFC response. The time of maturation of the capacity to generate indirect PFC was found to differ in different strains of mice. Maturation of the capacity to produce indirect PFC appears to represent a dinstinctly different differentiation event from the acquisition of the capacity to produce a high‐affinity PFC response. The data suggest that the maturation of the B cell population to be capable of producing high‐affinity PFC is a precisely regulated differentiation event, the timing of which is strain‐related. Preliminary data suggest that this regulation is not linked to either the H‐2 haplotype or to the Igh allotype.

Carrier-priming leads to hapten-specific suppression.

Studies over the past two decades have established1,2 that ‘helper’ T lymphocytes primed to a ‘carrier’ protein increase B-lymphocyte antibody responses to ‘haptenic’ determinants on the carrier protein. T cells from donors primed with one commonly used carrier protein, keyhole limpet haemocyanin (KLH), have been shown by many workers to augment B-cell IgG anti-dinitrophenyl (DNP) responses in adoptive recipients stimulated with DNP-KLH. Therefore, we were surprised recently to find that the KLH-primed mice we normally use as donors for carrier-specific helper T cells in adoptive transfers3 show reduced rather than augmented IgG in situ anti-DNP antibody production when stimulated with DNP-KLH. Here we show that hapten-specific regulation of antibody production is responsible for this response failure. That is, that KLH-priming before DNP-KLH exposure reduces the production of IgG anti-DNP antibody without interfering with the anti-KLH response or with the development of anti-DNP memory B cells, and that IgG anti-DNP production remains low after further stimulation with DNP on KLH or an unrelated carrier. Thus initial stimulation with a carrier-protein creates an oddly compromised animal in which IgG antibody production to a ‘new’ haptenic determinant on the carrier is kept minimal despite the presence of normal anti-hapten memory and carrier-specific help capable of supporting other responses to the hapten-carrier conjugate.

Cell-type restriction in tolerance induced by hapten-modified cells

Tolerance to the TNP haptenic determinant was induced by a single intravenous injection of trinitrophenylated syngeneic cells. Syngeneic spleen or thymus cells were capable of acting as carriers for tolerance induction while syngeneic bone marrow cells were not. Syngeneic spleen cells depleted of θ-positive and adherent cells were also suitable carriers for tolerance induction. Sonicated haptenated spleen cells, but not sonicated haptenated bone marrow cells induced tolerance. The ability of haptenated cells to induce tolerance was not correlated with their localization in lymphoid organs. Furthermore, cells recovered from the spleens of lethally irradiated animals reconstituted with bone marrow cells 1 week previously were incapable of inducing tolerance after hapten-modification. However, after 3 weeks, spleen cells from bone marrow-reconstituted mice had acquired the ability to induce tolerance. These results suggest that only certain types of syngeneic cells have the ability to act as carriers for tolerance induction; merely being syngeneic, and therefore presumably nonimmunogenic, is not sufficient to permit the cell to act as a carrier for tolerance induction.

Recognition of Hapten-Modified Cells In Vitro by Human T Lymphocytes

Clearer definition of the recognitive structures of human T lymphocytes for antigens will be required to elucidate the molecular basis of diseases and immunological responses induced or regulated by normal or abnormal T-cell function. For this purpose we have investigated the cellular requirements for immune responses in vitro to trinitrophenyl-conjugated peripheral blood mononuclear cells. The responding cell was characterized as a T cell on the basis of rosetting with sheep erythrocytes. T-cell recognition of hapten in proliferative responses depended upon presentation of antigen in an appropriate stimulator-cell context. Neither autologous hapten-modified erythrocytes nor T cells restimulated responses of in vitro-primed lymphocytes. Moreover, hapten-conjugated non-T cells were more effective than modified unfractionated cells in restimulating proliferative responses. Both macrophages and non-T lymphocytes effectively restimulated hapten-conjugate responses.Cell-mixing experiments indicated that the failure of haptenated T cells to stimulate proliferative responses was not because of a lack of fresh macrophages; these experiments suggested instead that T cells do not express appropriate structures necessary to present haptenic determinants in an immunogenic form. Hapten-modified T cells, however, were capable of inducing primed lymphocytes to become efficient cytotoxic effector cells, indicating that T-cell recognitive units for stimulation of proliferative and cytotoxic responses are different. These data support the concept that for induction of proliferative responses, human T cells recognize conventional antigens in association with HLA-D-region-encoded Ia-like molecules.

Immunogenicity of glutaraldehyde treated homologous monomeric albumin in rabbits

Previous studies (Onicaˇ et al., 1978) have shown that glutaraldehyde polymerized homologous albumin is immunogenic in rabbits. The reaction mixture consisted of high proportion of polymeric and oligomeric molecules, but a small quantity of monomeric ones. The present investigation shows that the monomeric fraction isolated from the reaction mixture is altered in comparison with untreated monomeric albumin. The modification produced in the homologous monomeric albumin by glutaraldehyde conferred to the molecule immunogenic properties, due to the presence of new antigenic sites (haptenic determinants). Glutaraldehyde treatment induced similar haptenic determinants on albumins of different origins as shown by the cross-reactivity between glutaraldehyde treated human and rabbit albumin, which precipitate both with human and rabbit antibodies. Rabbit antibodies were shown to be specific both for the glutaraldehyde modified monomeric albumin and the “aged” fraction which exist in albumin stored either in dry state (commercial albumin) or in solution at 37°C. The same antibodies failed to react with freshly prepared albumin. Our data strongly suggest that the glutaraldehyde treatment alter the monomeric albumin in a similar way as in vitro “ageing” does.

Recognition of idiotypes in lymphocyte interactions. I. Idiotypic selectivity in the cooperation between T and B lymphocytes.

Idiotypic restrictions are demonstrated in vitro for the cooperation between T and B lymphocytes with specificity for Group A streptococcal carbohydrate. T helper cells which have been primed in vivo with anti-idiotypic antibodies to the A5A idiotype and which are therefore essentially A5A idiotype-positive, cooperate only with A5A idiotype-positive B cells, even when mixtures of A5A idiotype-positive and A5A idiotype-negative B cells are present. Essentially A5A idiotype-negative T helper cells that have been primed in vivo with Group A streptococcal vaccine after in vivo suppression with anti-A5A idiotypic antibody are unable to cooperate with B cells which have been primed with anti-A5A idiotype antibody and which are therefore essentially A5A idiotype-positive. Mixtures of A5A idiotype-negative and A5A idiotype-positive T cells cooperate with both A5A idiotype-negative and A5A idiotype-positive B cells. Idiotypic restrictions could not be demonstrated for T and B cells recognizing carrier and hapten determinants, respectively, in experiments in which the cooperation of genetically VH-identical T and B cells was compared to the cooperation of genetically VH-different T and B cells. The data are discussed with respect to various models for the communication between T and B cells. It is proposed that for successful T-B cooperation, ordinarily two types of T helper cells are required, one recognizing the antigen and the other recognizing the idiotype of the B cell.

Inhibition of Human Urinary Kallikrein (Urokallikrein) by Anti-Enzyme Fab

Abstract Monospecific rabbit IgG and Fab antibodies against human urokallikrein inhibit the proteolytic and esterolytic functions of the enzyme in a substrate dose-dependent fashion, indicating an antibody specificity for antigenic determinants at the active site of the enzyme. The specific anti-urokallikrein Fab fragments exhibited charge heterogeneity on isoelectric focusing but were oligoclonal in comparison with the distribution of the entire Fab pool. The inhibition of the kinin-generating activity of urokallikrein by the specific Fab fragments was complete at 30 sec, the earliest time interval measured. The average inhibition constant for the specific monovalent Fab fragments is the same as for the specific IgG, namely, 2.2 × 10-8 M, a finding compatible with the observations of others employing haptenic determinants. Since the molar concentrations required for comparable inhibition by anti-urokallikrein Fab were greater than the molar concentrations of anti-urokallikrein IgG, the inhibition of enzyme function by anti-urokallikrein IgG reflects both active site neutralization and additional nonspecific effects including precipitation. Analysis of the inhibition of urokallikrein kiningenerating activity by anti-urokallikrein Fab by the method of Dixon indicated the mechanism to be competitive. The additional finding that anti-urokallikrein Fab gave a dose-related inhibition of the action of urokallikrein on incremental concentrations of the synthetic substrate, α-N-p-tosyl-l-arginine methyl ester, indicated action at the active site. Analysis of these data by Dixon plots revealed competitive inhibition at the lower concentrations of Fab with an average inhibitory constant of 2.5 × 10-7 M and mixed inhibition at higher concentrations, thereby suggesting the presence of some specificities for antigenic determinants other than those at the active site. These studies demonstrate that anti-urokallikrein IgG yields a population of anti-urokallikrein Fab directed so as to neutralize proteolytic and esterolytic functions of the enzyme by a competitive mechanism.

The diversity of the influenza-specific primary B-cell repertoire in BALB/c mice.

The primary immune response of BALB/c mice to influenza (PR8) hemagglutinin (HA), a complex protein antigen, has been examined by the splenic focus assay, and the resulting monoclonal anti-HA antibodies have been characterized by their reactivity with heterologous viruses. The analysis of the primary B-cell response to HA revealed marked differences from responses previously defined for haptenic determinants. There were following differences: (a) the frequency of HA-specific B cells in both conventional and germ-free BALB/c mice was 1 in 1.0-1.5 X 10(5) splenic B cells, which is substantially lower than the frequency of B cells responsive to various simple haptenic determinants; (b) monoclonal anti-HA antibodies were predominantly of the IgA or IgM isotypes instead of IgG, which dominates antihapten responses; and (c) after immunization, the frequency of anti-HA-specific B cells increases by 10- to 50-fold, which is much greater increase than that observed after immunization with haptenic determinants. Fine specificity analysis of primary monoclonal HA-specific antibodies revealed extensive diversity and a considerable overlap with the specificities obtained from immune mice. Given the low overall frequency of HA-specific B cells, it could be calculated that the representation of most HA-specific clonotypes within the B-cell repertoire could not exceed 1 in 10(7) B cells. These findings indicate that the primary B-cell clonotype repertoire is extremely diverse and largely antigen independent in its generation.