B cell responses to a peptide epitope. X. Epitope selection in a primary response is thermodynamically regulated

We examine the etiological basis of hierarchical immunodominance of B cell epitopes on a multideterminant Ag. A model T-dependent immunogen, containing a single immunodominant B cell epitope, was used. The primary IgM response to this peptide included Abs directed against diverse determinants presented by the peptide. Interestingly, affinity of individual monomeric IgM Abs segregated around epitope recognized and was independent of their clonal origins. Furthermore, affinity of Abs directed against the immunodominant epitope were markedly higher than that of the alternate specificities. These studies suggested that the affinity of an epitope-specific primary response, and variations therein, may be determined by the chemical composition of epitope. This inference was supported by thermodynamic analyses of monomer IgM binding to Ag, which revealed that this interaction occurs at the expense of unfavorable entropy changes. Permissible binding required compensation by net enthalpic changes. Finally, the correlation between chemical composition of an epitope, the resultant affinity of the early primary humoral response, and its eventual influence on relative immunogenicity could be experimentally verified. This was achieved by examining the effect of various amino-terminal substitutions on immunogenicity of a, hitherto cryptic, amino-terminal determinant. Such experiments permitted delineation of a hierarchy of individual amino acid residues based on their influence; which correlated well with calculated Gibbs-free energy changes that individual residue side chains were expected to contribute in a binding interaction. Thus, maturation of a T-dependent humoral response is initiated by a step that is under thermodynamic control.     

Structural basis of a conserved idiotope expressed by an autoreactive human B cell lymphoma. Evidence that a VH CDR3 mutation alters idiotypy and specificity

Our laboratory has previously investigated the relationship of autoimmune disease and B cell neoplasia in a patient with a diffuse, well differentiated splenic B cell lymphoma and associated autoimmune hemolysis due to an anti-Pr2 antibody. EBV-immortalized B cell clones, established from this lymphoma, were shown to secrete the same pathologic anti-Pr2 antibody. The antiidiotypic mAb, RI.1, defined a private Id (IdRI.1) of the anti-Pr2 antibody that was related to the Ag-binding site and was expressed by both the lymphoma and derived cell lines. This unique Id was expressed by the majority of splenic tumor B cells and also was conserved over a period of 4 yr. In this report, the structural basis of IdRI.1 expression was investigated by analysis of Id- variants isolated by flow microfluorimetry using RI.1. Six Id- cell lines that secrete IgM kappa but lack Pr2 specificity were generated from an Id+ cell line, LS2. These lines were shown to be related to LS2 and the lymphoma by karyotype and by restriction fragment analysis of Ig gene rearrangements. Shared and unshared nucleotide substitutions in the VH and VL regions of the six independent clones were used to construct a genealogic tree relating the Id- clonal members to a common Id+ precursor. The tree illustrates that the base changes occurred sequentially, suggesting that they were introduced by somatic point mutation. Only one VH CDR3 bp difference from the LS2 nucleic acid sequence is common to all Id- sequences, resulting in an amino acid substitution of cysteine 108 to tyrosine. Taken together, these findings suggest that both the expression of IdRI.1 and Ag binding are affected by a single mutation localized to the D region of the anti-Pr2 antibody.     

Evaluations of Alkyl hydroperoxide reductase B cell antigen epitope as a potential epitope vaccine against Campylobacter jejuni

ObjectiveThe present study aimed to screen and find alkyl hydroperoxide reductase (AhpC) B cell dominant epitope of Campylobacter jejuni (C. jejuni).Materials and methodsBio-informatic algorithms were used to predict B cell epitopes of AhpC. The AhpC protein and chemically synthesized antigenic epitopes of C. jejuni were considered as antigens, and the AhpC antibody was used as the primary antibody, ELISA and dot blot were used to analyze and screen the dominant epitope. The specific IgG of mice serum and IL-4 in splenocyte culture supernatant were detected by ELISA. The protective efficacy was evaluated by animal disease index and tissue histopathological staining of the jejunum.ResultsSeven epitopes of AhpC were predicted, one epitope (AhpC_4–16) was found to recognize the antibodies of AhpC and had strong antigenicity by ELISA and dot blot analysis. In epitope AhpC_4–16 immunized mice, specific IgG of serum and IL-4 in splenocyte culture supernatant were significantly higher. The illness index decreased significantly, the protective rate was 66.67%. Histopathology displayed that the jejunum morphology was better than the control group.ConclusionsThese findings suggested that epitope AhpC_4–16 showed effective protective role against C. jejuni and is a candidate epitope of vaccine against this pathogen.     

Characterization of a soluble suppressor of human B cell function produced by a continuous human suppressor T cell line. II. Evidence for suppression through a direct action of CTC-SISS-B on human B cells

CTC-SISS-B is an antigen-nonspecific suppressive lymphokine elaborated by an interleukin 2-dependent suppressor T cell line that produces noncytotoxic inhibition of human B cell but not T cell function. Like SISS-B, a soluble suppressive lymphokine present in the supernatants of Con A-activated peripheral blood T cell cultures, CTC-SISS-B is of 60,000 to 90,000 m.w., and its action is blocked by the simple sugar L-rhamnose. CTC-SISS-B inhibits human B cell Ig production and proliferation through a direct interaction with human B cells rather than through indirect effects on immunoregulatory T cells or monocytes. CTC-SISS-B suppression occurs through inhibition of an early event(s) in B cell activation since proliferation and Ig production by established human B cell lines are not inhibited by this lymphokine. Despite sharing many biochemical and biologic properties, CTC-SISS-B and gamma-interferon appear to be distinct mediators.     

Unusually diverse T cell response to a repeating tripeptide epitope.

The immune system utilizes a diverse T cell repertoire for the recognition of foreign antigens in the context of self MHC gene products. We have examined the potential diversity of the T cell response directed to a immunodominant repeating tripeptide epitope (EYA)5. This peptide represents one of the two T cell epitopes on the synthetic alpha-helical polypeptide antigen Poly 18, Poly EYK(EYA)5 in H-2d mice and does not require antigen processing prior to presentation to Poly 18-specific T cell hybridomas. The T cell response directed to the repeating tripeptide epitope (EYA)5 is extremely heterogenous even though the epitope has a relatively simple amino acid sequence. We have analyzed the fine specificity of 21 randomly chosen Poly 18-reactive, (EYA)5-specific and H-2d-restricted T cell hybridomas derived from H-2d, H-2bxd, and H-2b----H-2bxd Poly 18-responding mice to determine the number of unique antigen reactivity patterns represented by this T cell population. We used alanine- and/or lysine-substituted (EYA)5 peptides and a panel of haplotype-varied splenocytes and observed a great deal of microheterogeneity in response. We find that 13 of the 21 hybridomas have a distinct fine antigen specificity and T cell receptors. The binding of (EYA)5 to the antigen-binding groove of I-Ad appears to generate a highly diversified T cell response. Therefore, (EYA)5-I-Ad complex allows the activation of unrelated T cell clonotypes with the same overall antigen specificity and MHC restriction, but with distinct microheterogeneity in response and receptor usage.     

Selective deficiency of IgG and IgA as a first stage defect in B cell differentiation.

A case of selective deficiency of IgG and IgA, in a 13-year old girl, is described. Immunologic investigations, showing an almost complete absence of IgG- and IgA-bearing lymphocytes and significant amounts of IgD and IgM positive cells, suggest the possibility of a block in the shift from IgM to IgG synthesis at the B lymphocyte level.     

Insulin-dependent phosphorylation of DPP IV in liver. Evidence for a role of compartmentalized c-Src

Dipeptidyl peptidase IV (DPP IV, CD26, EC 3.4.14.5) serves as a model aimed at elucidating protein sorting signals. We identify here, by MS, several tyrosine-phosphorylated proteins in a rat liver Golgi/endosome (G/E) fraction including DPP IV. We show that a pool of DPP IV is tyrosine-phosphorylated. Maximal phosphorylation was observed after 2 min following intravenous insulin injection. DPP IV coimmunoprecipitated with the cellular tyrosine kinase Src (c-Src) with maximal association also observed after 2 min following insulin injection. DPP IV was found phosphorylated after incubation of nonsolubilized G/E membranes with [gamma-32P]ATP. The c-Src inhibitor PP2 inhibited DPP IV phosphorylation. Oriented proteolysis experiments indicate that a large pool of c-Src is protected in G/E fractions. Following injection of the protein-tyrosine phosphatase inhibitor bpV(phen), DPP IV levels markedly decreased by 40% both in plasma membrane and G/E fractions. In the fraction designated Lh, DPP IV levels decreased by 50% 15 min following insulin injection. Therefore, a pool of DPP IV is tyrosine-phosphorylated in an insulin-dependent manner. The results suggest the presence of a yet to be characterized signalling mechanism whereby DPP IV has access to c-Src-containing signalling platforms.     

A newly characterized HLA-DP beta-chain allele. Evidence for DP beta heterogeneity within the DPw4 specificity

cDNA clones corresponding to the DPw4 alpha- and DPw4 beta-chains were isolated from a cDNA library prepared from a DPw4 homozygous cell line, their nucleotide sequences were determined, and the corresponding amino acid sequences were deduced. This DPw4 alpha-chain is identical to the conserved DP alpha-chains from DPw4 and DPw2 haplotypes, although the DPw4 beta-chain (referred to as DPw4b beta) differs from all reported DP beta-chain sequences. The DPw4b beta-chain differs from the reported DPw4 beta sequence (referred to as DPw4a beta) at three amino acid positions in the first domain (36, 55, and 56). The DPw4b beta-chain sequence differs from the DPw2 beta-chain sequence only at position 69 in the first domain, suggesting that the lysine at position 69 in DPw4b beta and the glutamic acid at position 69 in DPw2 beta contribute to the epitopes that define "DPw4-ness" and "DPw2-ness," respectively. In addition, the patterns of sequence identities and differences among the DPw4b beta-, DPw4a beta-, DPw2 beta-, and DPw3 beta-chains suggest that the DPw4b beta sequence arose via a gene conversion event or a point mutation. The I-LR1 mAb, which was previously found to bind only to DPw2, DPw3, and DR5 molecules, binds to an L cell transfectant expressing the DPw4 alpha:DPw4b beta molecule. The DPw4b beta sequence provides the first evidence for structural heterogeneity within the DPw4 specificity.     

Induction of IgG secretion in a human B cell clone with recombinant IL 2

A human B cell clone (SGB3) responsive to IL 2 was established. Both recombinant IL 2 and B cell differentiation factor (BCDF) induced IgG secretion in SGB3 cells in a dose-dependent manner, but IL 2 did not affect the proliferation of SGB3 cells. FACS analysis showed that SGB3 cells expressed Tac antigen and anti-Tac antibody inhibited IL 2-induced IgG secretion without any inhibitory effect on BCDF-induced IgG secretion. These results showed that IL 2 could directly act on B cells and provide a differentiation signal through IL 2 receptors distinct from BCDF receptors. Physiologic relevance of IL 2 in the antibody response was discussed.     

Proteins of BrdU-dependent hamster cell lines as characterized by two-dimensional gel electrophoresis.

Cell lines which exhibit the BrdU-dependent phenotype (B4 and HAB) were studied with respect to BrdU-induced alterations in genetic expression by two-dimensional gel electrophoresis. A comparison of the proteins from the HAB cells, in which the DNA is 100% substituted by BrdU, to those of the unsubstituted parent line (3460) showed 55 protein alterations; the synthesis of 15 increased while that of the other 40 decreased. When 3460 cells were grown in BrdU such that their DNA was greater than 50% substituted, 27 protein changes could be detected; of these, the synthesis of 10 increased while that of 17 decreased. A comparison of all these changes in the various cell lines showed six which were common to the BrdU-substituted cell lines. The proteins from another Syrian hamster cell line, BHK-21 (C-13) and those of HAB cells grown in thymidine or BrdC were also examined on two-dimensional gels. Although BrdU has a dramatic effect on many cellular functions, relatively few changes in the pattern of protein synthesis could be detected in these cell lines, perhaps reflecting the specialized action of this analogue on particular cellular functions.     

B cell responses to a peptide epitope. VIII. Immune complex-mediated regulation of memory B cell generation within germinal centers.

Using an in vivo reconstitution assay, we examine here the role of immune complexes in both formation of germinal centers (GC) and processes that occur subsequently within. The presence of Ag, as immune complexes, was found not to constitute a limiting requirement for the initiation of GC formation. No detrimental effect either on numbers or sizes of the resulting GC was observed when Ag-containing immune complexes were omitted during reconstitution. Thus, both recruitment and proliferation of Ag-activated B cells within GC appear not to be limited by Ag concentrations. In contrast, the presence of immune complexes was observed to be obligatory for the generation of Ag-specific memory B cells. This optimally required immune complexes to be constituted by IgG-class Abs with epitope specificities that were homologous to those of the GC B cells. The GC reaction was also found to be characterized by an enhancement of Ab specificity for the homologous epitope. Although some improvement in specificity was noted in recall responses from immune complex-deficient GC, the presence of appropriate immune complexes served to further optimize the outcome. Here again, isotype and epitope-specificity of the Ab constituent in immune complexes proved to be important.     

Suppression of B cell function by methotrexate and trimetrexate. Evidence for inhibition of purine biosynthesis as a major mechanism of action

Methotrexate (MTX) is a widely used drug in the treatment of a variety of human neoplasms. Trimetrexate (TMQ) is a lipid-soluble quinazoline derivate of MTX that, unlike MTX, is not dependent upon membrane folate transport for cellular entry. A number of studies have demonstrated that MTX and, more recently, TMQ possess potent immunosuppressive properties. To examine the cellular events associated with the immunomodulatory effects of anti-folates on humoral immunity, a murine B cell maturation model was used. In vitro, MTX and TMQ reduced the number of antibody-forming cells to SRBC, as well as IgM production. B cells stimulated with anti-Ig demonstrated a dose-related suppression in [3H]UdR incorporation after addition of either drug, suggestive of a decrease in de novo DNA synthesis. B cell activation events preceding S phase were also suppressed by both anti-folates, as evidenced by inhibition of RNA synthesis. However, neither drug affected surface expression of Ia Ag nor inositol phosphate accumulation. Addition of TdR caused a slight non-significant increase in the antibody-forming cell response in the presence of 10(-7) M MTX. However, addition of hypoxanthine or adenine, but not guanine, resulted in complete restoration. Timed addition revealed that the ability of MTX to suppress antibody responses was diminished if added after 48 h of culture, similar to the reversal of this suppression mediated by hypoxanthine. Cell cycle analysis of LPS-stimulated B lymphocytes demonstrated that both drugs modulated events preceding, as well as during, the S phase. The present studies suggest that although drug-induced impairments in dTMP biosynthesis may be responsible for deficient lymphoid proliferation, anti-folate-induced impairment in purine biosynthesis is a major mechanism in anti-folate-induced suppression of humoral immunity.     

Fc fragment from human IgG induces PGE2 secretion. II. Negative regulation in B cell differentiation

In humans, in vitro, Fc fragment of IgG at a low concentration induces plasma cell generation. However, Fc fragment at a high concentration induces PGE2 release of monocyte activation capable of inhibiting this differentiation. The levels of PGE2 in the supernatant culture from mononuclear cells from normal donors were examined as a function of culture duration and concentration of Fc, Fab fragments and IgG. Plasma cells containing intracytoplasmic Ig were demonstrated by immunofluorescence with a polyvalent antiserum to human immunoglobulin(s). PGE2 levels, from mononuclear cell cultures, were analyzed by the RIA test. The results indicated that the Fc fragments are able to induce PGE2 secretion. The maximal release of PGE2 occurs after 24 hr of culture; this level is proportionate to the quantity of Fc fragments introduced. The addition of indomethacin in the cell culture stimulated by a high concentration of Fc fragments reestablishes the percentage of plasma cells. These results suggest the regulatory role of Fc fragment by PGE2 secretion in B cell differentiation.     

Identification of an immunodominant B cell epitope on the hepatitis C virus nonstructural region defined by human monoclonal antibodies.

Several EBV-transformed B cell lines (BCL) were obtained from two patients with chronic hepatitis C virus (HCV) infection that secreted IgG class antibodies to the HCV nonstructural Ag c100-3. Two cloned BCL, derived from the same parental line, generated stable cloned lines that secreted up to 20 mg/liter of specific IgG1(kappa). Supernatants from oligoclonal and cloned BCL were also analyzed by immunoblot and all strongly reacted with recombinant polypeptides derived from the putative NS4 region of HCV, c100-3 and 5-1-1 (a 42-amino acid fragment of c100-3), whereas no reaction with the viral nucleoprotein, the NS3 nonstructural protein or the superoxide dismutase moiety of the c100-3 fusion protein could be documented. The fine specificity of these antibodies was also evaluated using overlapping synthetic peptides (20-mers) covering the 5-1-1 sequence. All oligoclonal and clonal IgG displayed high affinity binding to peptides covering residues 120-137 of Chiron's c100-3 sequence at the aminoterminus of 5-1-1. In addition, a minimal B cell epitope, N-VLYREF-C, was defined by human oligoclonal and monoclonal antibodies corresponding to residues 132-137. Interestingly, predominant recognition of the N-terminus of 5-1-1 was also observed in more than 80% of sera from patients with HCV infection. In conclusion, we have successfully produced human B cell cloned lines that secrete abundant quantities of IgG1(kappa)-specific for a polypeptide encoded by the NS4 region of HCV. Such antibodies recognize an immunodominant epitope, relative to this region, located at the N-terminus of the 5-1-1 fragment.     

T cell regulation of immunoglobulin class expression in the B cell response to TNP-Ficoll: characterization of the T cell responsible for preferential enhancement of the IgG2a response

Syngeneic T cells injected into athymic nu/nu mice cause a preferential enhancement in the amount of IgG2a anti-TNP Ab produced by these mice to TNP-Ficoll. This enhancement appears to be caused by T cell effects on the IgG switching pathway. Through the use of F1----parent chimeras, the helper T cells were shown to affect TNP-Ficoll-responsive B cells in an H-2-unrestricted manner. The ability of T cells to mediate this IgG2a enhancement did not appear to be unique to any particular murine genetic background, because it was observed with T cells and nu/nu mice of C57BL/10, BALB/c, CBA/Ca, and B10.D2 strains. Priming of T cell donors with Ficoll or TNP-Ficoll did not increase the ability of splenic T cells, on a per cell basis, to enhance the IgG2a Ab response to TNP-Ficoll. The T cell population responsible for modulating the isotypic response was found to be sensitive to C-mediated cytotoxicity with both anti-Lyt-2 and anti-Lyt-1 hybridoma Ab. Although T cells from both the thymus and the spleen expressed enhancing activity, splenic T cells were more effective, on a per cell basis, than were thymocytes. The observations suggest that T cells that appear to enhance the switch to IgG2a in TNP-Ficoll-responsive B cells are not effectively primed by the antigen and interact with TNP-Ficoll-activated B cells through an H-2-unrestricted mechanism.     

Human B cell differentiation by Fc fragment. III. Effect of IL-1 and IL-2 on differentiation of human B lymphocytes induced by Fc fragments of human IgG

The human Fc fragment of IgG, when added to blood mononuclear cells in vitro, induces B cell differentiation after 6 days of culture. This activity requires the presence of T cells and monocytes. This work explores the roles of interleukin 1 (IL-1) and interleukin 2 (IL-2) in B cell differentiation induced by Fc fragments. Peripheral blood mononuclear cells (PBMC) from normal donors were examined for plasma cell differentiation following stimulation with Fc fragment (15 and 30 micrograms/ml) with or without IL-1 (6 U/ml) or IL-2 (2 U/ml). Results indicate that both IL-1 and IL-2 accelerated B cell differentiation by the Fc fragment to 3 days of culture, compared to 6 days required with the Fc fragment alone. The time required for differentiation was not further shortened when both IL-1 and IL-2 were present in culture; both IL-1 and IL-2 were able to partially induce B differentiation alone at 6 days of culture. The importance of IL-2 in B cell differentiation was further supported by the finding that antibodies specific for the IL-2 receptor blocked B cell differentiation induced by Fc fragments, with or without additional IL-1 or IL-2. The depletion of monocytes also blocked B cell differentiation and the requirement for monocytes could not be replaced by exogenous IL-1; however, Fc fragments were shown to induce monocytes to secrete IL-1 beta after 24 hr in culture. These results suggest that accelerated differentiation of B cells into plasma cells requires a double signal provided by Fc fragments and IL-1 or IL-2. Monocytes are necessary for Fc fragment-induced differentiation and cannot be replaced by either IL-1 or IL-2.     

The human B cell response to IL-13 is dependent on cellular phenotype as well as mode of activation.

Normal mature quiescent human B lymphocytes, isolated as a function of buoyant density, require activation for up-regulation of IL-13R constituents. Cell activation through a combination of surface Ig and CD40 receptor ligation leads to the most substantial message production for IL-13Ralpha1. Functional consequences of this receptor variation, in initially quiescent cells, includes demonstrable effects on cellular proliferation in response to ligand exposure. Variations in the method of surface activation, with particular emphasis on the CD40 receptor, reveals that immobilized CD40 ligand may be sufficient, in and of itself, to up-regulate IL-13Ralpha1, which may bear significance for B-lymphocyte bystander proliferation. Regulation of the IL-13Ralpha1 protein and message also differs as a function of cellular phenotype. Although values are greater in memory than naive B cells, as they are initially isolated from extirpated tonsils, variations in the magnitude of message and protein, as a function of surface stimulation, are more substantial in the naive subset. The magnitude of variation in message production in naive cells is associated with a more vigorous proliferative response to IL-13 than seen in memory lymphocytes. The cellular response to IL-13, as a function of activation and phenotype, is the converse of that demonstrated for IL-2. Evaluation of proliferation, receptor message, ligand binding protein production, and the response to putatively synergistic cytokines reveals that IL-2 is the predominant lymphokine utilized by memory cells. This is in contradistinction to IL-13, which along with IL-4, are the predominant moieties for naive lymphocytes.