Anti-phosphorylcholine Antibodies Research Articles

Anti-phosphorylcholine antibodies of the T15 idiotype are optimally protective against Streptococcus pneumoniae.

In the mouse, most anti-PC antibody is found in one of the three murine anti-PC idiotype families: T15, M603, or M511. The antibodies within each of these idiotypic families have characteristic fine specificities for phosphorylcholine (PC)-analogues. In this paper we compare the ability of hybridoma IgM anti-PC antibodies of the three idiotype families to protect mice from fatal infection with S. pneumoniae. Antibody bearing the T15 idiotype was approximately 8 times as effective as antibody with the M603 idiotype and approximately 30 times as protective as antibody with the M511 idiotype. Reports by others have shown that the heavy chains of virtually all mouse anti-PC antibodies are produced by translocation of a single variable region gene and that the direct translation of this gene (in the absence of somatic mutations) results in heavy chains characteristic of the T15 idiotype. Thus, our findings suggest that the T15 germ line heavy chain variable region gene may have been selected through evolution to code for antibody binding PC-containing pathogens such as S. pneumoniae. Our observations may also explain the existence of regulatory mechanisms that result in maintenance of T15 idiotype expression in murine anti-PC immune responses.

Deposition of idiotype-anti-idiotype immune complexes in renal glomeruli after polyclonal B cell activation.

We investigated the possible role of idiotypic interactions in the pathogenesis of the glomerular lesions observed in mice undergoing polyclonal B cell activation. BALB/c mice were studied for the presence of renal deposits of T15 idiotype-anti-T15 idiotype-immune complexes (IC) after injection of bacterial lipopolysaccharides (LPS). The T15 idiotype is the major idiotype of BALB/c mice anti-phosphorylcholine (PC) antibodies, which are cross-reactive with the idiotype of the TEPC-15 myeloma protein. This model was used because T15 idiotype-anti-T15 idiotype IC have been detected in the circulation of BALB/c mice after polyclonal B cell activation. First, an idiotype-specific immunofluorescence technique allowed us to detect T15 idiotype-bearing immunoglobulins in glomeruli from day 6 to day 28 after LPS injection. Second, fluorescein isothiocyanate-conjugated TEPC-15 myeloma protein was found to localize in the glomeruli after in vivo injection 18 d after LPS administration. This renal localization was shown to be idiotype-specific and could be quantified in a trace-labeling experiment. Third, kidney-deposited immunoglobulins of mice injected with LPS were eluted, radiolabeled, and analyzed by radioimmunoassay. Both T15 idiotype-bearing immunoglobulins and anti-T15 idiotype antibodies were detected in the eluates, providing further evidence for a renal deposition of T15 idiotype-anti-T15 idiotype IC. Polyclonal B cell activation is likely to result in a simultaneous triggering of many idiotypic clones and of corresponding anti-idiotypic clones represented in the B cell repertoire. This could lead to the formation of a variety of idiotype-anti-idiotype IC that could participate in the development of glomerular lesions.

The elusive antigen. Thoughts on the structure/function relationship of homogeneous human antibodies.

The rapid progress in the elucidation of antibody structure and function had a major impact on our understanding of monoclonal immunoglobulins. It appears that monoclonicity is a basic immunologic phenomenon, closely related to specificity. The amplification of this phenomenon, observed in pathological conditions such as the various plasma cell dyscrasias, has not been fully understood due to the lack of identifiable antigens capable of triggering this response. The functional and structural data accumulated from animal experiments and the clinical investigation of anti-dextran, anti-phosphorylcholine, anti-thioflavine and homogeneous anti-arsonate antibodies as well as of cold agglutinins, point to the presence of a common structure/function relationship in these molecules. Structural and serologic data, obtained from homogeneous antibodies isolated by us from the serum of patients with cancer of the colon and of the urinary bladder, revealed significant structural similarities expressed by HVR homology and cross-idiotypic specificity.

Heterogeneity of the BALB/c IgM anti-phosphorylcholine antibody response.

Although recent advances in the study of the molecular genetics of the antibody system have greatly increased our understanding of the generation of immunological diversity1–7, the state of B-cell differentiation at which diversification and specificity commitment occur has yet to be determined. In particular, it is unclear whether the entire repertoire of antibody specificities in an individual is expressed in the absence of selective forces, such as antigenic stimulation or network regulation. It has recently been suggested that some elements of variable region diversification, particularly somatic mutations, may accompany the shift from the IgM to IgG isotype8–10. These findings support the view that much of B-cell repertoire diversity is the result of antigen-driven somatic events, because isotype shifts normally occur after antigenic stimulation11–13. As these experiments used antibodies obtained after at least one in vivo antigenic stimulation, they do not address the possibility that the selection occurring during clonal expansion and subsequent IgG synthesis may favour the use of clones expressing unique antibody specificities (clonotypes) far removed from the innate or frequently occurring germ-line sequences. To test whether B-cell diversification may precede IgM expression, we have now determined the range of diversity available in the primary IgM antibody repertoire by studying the clonal heterogeneity of monoclonal IgM antibodies expressed by BALB/c mice in response to phosphorylcholine (PC). Our results indicate that the repertoire of IgM antibodies in an inbred murine strain exceeds 107 clonotypes. This diversity is sufficiently large to preclude meaningful comparisons between the IgM and IgG repertoires and suggests that antibody diversification may occur early in B-cell differentiation.

Most B cells that have switched surface immunoglobulin isotypes generate clones of cells that do not secrete IgM.

The order of genes coding for the constant regions of immunoglobulin heavy chains is 5'-C mu, C delta, C gamma 3, C gamma 1, C gamma 2b, C gamma 2a, C epsilon, C alpha-3'. To try to determine whether switching of the expression of heavy chain genes is irreversible and occurs in a direction from 5' to 3' within a cell line, we have isolated B cells bearing particular membrane isotypes, stimulated them to generate clones, and assayed daughter cells for secreted antibody. B cell with and without surface IgM and IgD, and B cells with and without surface IgG, were isolated by fluorescence-activated cell sorting. Fractionated cells were individually stimulated with phosphorylcholine in a splenic fragment assay, and the antibody secreted by clonal progeny was assayed for several heavy chain isotypes by radioimmunoassay. The results show that 1) B cells bearing IgM and IgD can produce individual clones of cells secreting 3 isotypes of anti-phosphorylcholine antibody--IgM, IgG, and IgA; 2) most B cells expressing surface IgG generate clones that secrete IgG but not IgM; and 3) most B cells from gut-associated lymphoid tissue that do not express surface IgM or IgD generate clones of cells that secrete IgA but not IgM. Therefore, IgM- and IgD-bearing cells can give rise to progeny that secrete IgM, IgG, and IgA. Once B cells switch to a different surface isotype, they appear restricted in isotype potential, since they frequently generate clones of cells that do not secrete IgM. These findings suggest that isotype switching is generally irreversible, and isotypes are expressed in a 5'- to -3' direction that is consistent with the germline gene order.

Selected populations of alloreactive T cells contain helper T cells but lack ThId, an antigen-specific helper T cell required for dominant production of the T15 idiotype.

Isolated, alloreactive T cell populations were primed with protein carriers in vivo and tested for their ability to provide help for an anti-phosphorylcholine (PC) antibody response and for production of the T15 idiotype. It was found that alloreactive T cell populations would support anti-PC antibody response but did not selectively activate B cells capable of producing the T15 idiotype that normally dominates such responses. This failure to help for the production of the T15 idiotype was shown to be due to the absence of an antigen-specific helper T cell that is required for dominant idiotype production (ThId). These studies suggest that ThId cells have recognition structures for antigen and for self idiotype, but lack recognition structures for major histocompatibility complex-encoded antigens.

Compensation for idiotype suppression. I. Acquirement of ability to compensate for TEPC-15 idiotype suppression in mice during the early neonatal period.

Neonatal injection of BALB/c mice with antibodies specific for the idiotype of TEPC-15 myeloma protein (T15id), which is serologically identical to the major idiotype of anti-phosphorylcholine (PC) antibody, renders the recipients completely unresponsive to PC. C57BL/6, (BALB/c X C57BL/6)F1 and C.B20 mice, similarly treated with anti-T15id antibody, also displayed tolerance to PC although they were relatively more resistant (8-13%) than BALB/c mice (less than 2% control response). When anti-T15id antibody was injected into 15-day-old neonates, the resistance to the tolerance in C57BL/6 and C.B20 mice was much more apparent (up to 80% of the control response) in contrast to that in BALB/c mice, which was not significant. Adoptive transfer of spleen cells from idiotypically suppressed BALB/c mice into 20-day-old, normal C. B20 mice resulted in suppression of T15id but not in tolerance to PC, due to increased production of non-T15id-bearing anti-PC antibody. These results suggest that the ability of clonal compensation for T15id suppression is acquired during early life (2-10 days), under the influence of gene(s) associated or linked with the Igh.

Allelic forms of anti-phosphorylcholine antibodies.

Anti-idiotype antisera were prepared to 2 phosphorylcholine (PC) binding myeloma proteins, T15 of BALB/c origin and C3 of C57BL origin. These antisera were shown to recognize the respective determinants on induced anti-PC antibodies in the 2 parental strains. Genetic analysis of these V region markers in backcross progeny of (BALB/c X C57BL/6) X BALB/c demonstrated that the 2 determinants segregated as simple Mendelian alleles linked to the allotype locus. The genetic data are consistent with previously reported structural studies suggesting the allelic nature of the T15 and C3 heavy chain variable regions. The expression of the T15 and C3 antigenic determinants in Ig congenic and recombinant inbred strains derived from BALB/c-C57BL/6 crosses further supports the concept of allelism although a single exception was observed. Strain distribution analysis has indicated the potential existence of other alleles of the T15 gene in addition to C3.

Anti-phosphorylcholine antibody-producing cells in human lymphoblastoid cell lines established by transformation with Epstein-Barr virus

Human tonsillar lymphocytes infected with Epstein-Barr virus (EBV) were cultured at a cell concentration of 2 × 10 6/ml in a 0.01-ml volume in microtest plates and cell lines composed of relatively small numbers of clones (oligoclonal) were established. Culture supernatants of 1020 cell lines thus established were screened for anti-phosphorylcholine (PC) antibody production by passive hemagglutination (PHA) assay and 10 cell lines with high anti-PC PHA titers were obtained. Plaque-forming cell (PFC) assay of these cell lines revealed that in seven cell lines more than 1% of the total cells and especially in two cell lines more than 10% of the total cells were anti-PC antibody-producing cells. Free PC concentrations required to inhibit PHA (8 PHA titer) and PFC (50% inhibition) correlated well in the same cell lines, but among the different cell lines considerable differences (10- to 1000-fold) of the PC concentration were obtained, indicating that anti-PC antibody-producing cells with different binding affinities to PC were transformed in these cell lines. PHA inhibition by monospecific antisera against immunoglobulin heavy-chain classes revealed that anti-PC antibodies produced in these cell lines were of the IgM class. Cloning experiments were performed and one clone stably producing anti-PC antibodies was obtained.

Allelic forms of the immunoglobulin heavy chain variable region.

The complete variable region sequence of the heavy chain from a phosphorylcholine-binding myeloma protein of C57/BL allotype has been determined. When this sequence was compared with the germ line-coded heavy chain variable region sequence of BALB/c phosphorylcholine-binding proteins, five differences were observed. Four of the substitutions were located in the framework portion of the variable region and the fifth in the "J" or joining segment. Two of the framework substitutions were found at positions 14 and 16. Previous studies have shown that heavy chains from all anti-phosphorylcholine antibodies induced in C57/BL mice have the same amino acids at positions 14 and 16 as the C57/BL myeloma protein described in this communication. It has therefore been concluded that these residues are encoded in the C57/BL germ line in contrast to two alternatives in the BALB/c genome. This finding, in addition to the 96% homology found between the C57/BL and BALB/c sequences, suggests that these structures represent allelic forms of an entire variable region.

The lack of compensatory increases of cells producing anti-phosphorylcholine antibodies bearing other idiotypes in TEPC-15 idiotype-suppressed inbred and outbred mice.

Idiotype analyses of plaque-forming cells using anti-idiotype antibody against TEPC-15 myeloma protein (anti-T 15 id) indicated that the proportion of plaque-forming cells producing anti-phosphorylcholine (PC) antibodies with idiotypic determinants of T15 id varied depending on the strain of mice (15-97%). However, treatment of neonates of those mouse strains with the anti-T 15 id antibody rendered them unresponsive to subsequent stimulation with PC-containing antigen (less than 7% of control response). The treatment of spleen cell cultures, derived from adult mice, with anti-T 15 id antibody resulted in complete suppression of T 15 id, although the suppression of the total anti-PC response was much less pronounced as compared to that induced in vivo. These results suggest that anti-T 15 id antibodies injected in the neonatal period may chronically inhibit the differentiation of B lymphocytes specific for PC. The lack of compensatory increases in the production of anti-PC antibodies bearing other idiotypes in T 15 id-suppressed mice does not appear to be related to the clonal proportion of cells producing anti-PC with non-T 15 id. This tolerance induction by anti-idiotype antibody appears to be unique to the nature of clonal differentiation of anti-PC-producing lymphocytes.

Biological effects of anti-idiotypic antibodies on lymphocyte function: I. Analysis of the effects on B lymphocytes of combining site and framework-directed anti-T-15 idiotypic antibodies

Anti-idiotypic antibodies against TEPC-15 myeloma protein (BALB/c origin) were raised in allogeneic animals by immunization of A/J mice with the myeloma protein. The antibody activities were fractionated into two specificities by TEPC-15 immunoadsorbent affinity columns by elution with free hapten (phosphorylcholine, PC), followed by elution with acidic buffer (glycine- HCl, pH 2.3). Idiotype binding analysis indicated that the fraction eluted with hapten could be inhibited in its binding to TEPC-15 by free hapten (i.e., binding site-directed anti-idiotypic antibody), whereas the acid-eluted fraction could not (i.e., framework-directed anti-idiotypic antibody). When analyzed for their biological activities on PC-specific B lymphocytes producing T-15 idiotype-bearing antibodies, both anti-idiotypic antibody fractions had similar suppressive effects on the in vitro production of antiphosphorylcholine antibody in culture.

Thymic requirement for cyclical idiotypic and reciprocal anti-idiotypic immune responses to a T-independent antigen.

The role of the thymus in the cyclical appearance of the dominant idiotype of the myeloma protein secreted by the TEPC-15 plasmacytoma (T-15)-bearing plaque-forming cells (PFC) and anti-idiotypic cells (i.e., cells with receptors for T-15) in the spleen during a primary response to the phosphorylcholine determinant of Streptococcus pneumoniae, strain R36a (Pn) was studied using normal mice, thymus-deficient nude mice, and thymus gland-grafted nude mice (TG-nude). The nude mice and their phenotypically normal littermates (LM) were backcrossed on the BALB/c genetic background. The kinetics of the anti-Pn PFC response of BALB/c inbred mice, littermates of nude mice, and TG-nude mice were essentially the same. There was an initial peak on day 5-6 followed by a decline to near background, and then a second peak on day 12. In nude mice, the first peak of anti-Pn PFC (day 5) was comparable in magnitude to that of mice with an intact thymus; however, there was no second peak. In contrast to the cellular response measured at the level of PFC, the serum antibody response to Pn (assayed by passive hemagglutination of sheep erythrocytes coated with Pn polysaccharide) was comparable in all groups of mice and did not show a measurable oscillation. The anti-idiotypic cellular activity was determined by the ability of spleen cells to bind radiolabeled (125I) TEPC-15 myeloma protein (IgA, kappa) which carries an idiotypic determinant indistinguishable from that of most anti-phosphorylcholine antibodies in BALB/c mice. Binding of radiolabeled McPC-603 (IgA, kappa) and MOPC-315 (IgA, lambda 2) myeloma proteins (which lack the T-15 idiotypic determinant) served as controls. The changes in T-15 binding by splenic lymphocytes following the Pn immunization differed between normal and athymic mice. BALB/c, LM, and TG-nude mice showed a biphasic pattern with peaks at days 3--4 and 10--11 that was nearly reciprocal to the PFC curve. On the other hand, T-15 binding in nude mice either declined and remained depressed or was not affected by the ongoing anti-Pn response. These observations demonstrate that mature T cells are required for cyclical idiotypic and anti-idiotypic responses to immunization with a T-independent antigen and suggest that the cyclical immune response may result from an interaction between idiotypic and anti-idiotypic cell clones.

Induction of anti-phosphorylcholine antibodies of restricted heterogeneity in rabbits

Repeated injections in rabbits of pneumococcal vacxine strains R 36 A induce antibodies of restricted heterogeneity as evidences by electrophoresis and isoelectric focusing. An immunoadsorbent containing purified C-polysaccharide was used to isolate and characterize the induced antibodies. Specificity for phosphorylcholine was demonstrated by hapten elution and by inhibition of precipitation. Addition of the ligand caused an enhancement of the intrinsic fluorescence of the anti-phosphorylcholine antibodies. The association constants determined by fluorometric titration were of the order of 10 3 M −1 and were only slightly influenced by temperature.

Affinity of horse anti-phosphorylcholine antibodies for some pneumococcal polysaccharides. Contribution of the polysaccharide backbone

Antibodies to phosphorylcholine, present in the antibody pool of a horse immunized with S. pneumoniae VII, show much higher affinities with species specific pneumococcal C-teichoic acid than with phosphorylcholine, or an unrelated phosphorylcholine-containing antigen. Thus, the polysaccharide in the C-teichoic acid is also capable of binding to the combining area.

Differences in the modulations of the soluble of plasma-membrane-bound 5'-nucleotidase.

The treatment of plasma membranes by a French pressure cell in sucrose medium devoid of detergents solubilized 20% of the total protein and 95--100% of 5'-nucleotidase activity. The soluble enzyme was 40--90-fold purified by centrifugation in a sucrose gradient with a 10--20% yield with respect to the orginal lysate. The purified fraction retained the same high specificity for 5'-AMP (Km = 20 micron) as in the plasma membranes and was enriched in sphingomyelin. Whereas 5'-AMP at high concentration inhibited the membrane-bound enzyme, it had no effect on the solubilized form. The soluble enzyme was stimulated by 2 X 10(-13)-2 X 10(-15) g concanavalin A without any inhibition with higher doses of lectin. The plasma-membrane bound stimulated and inhibited 5'-nucleotidase was modulated by concanavalin A concentrations higher than 0.1 microgram. Inhibition of the activity of the soluble enzyme by antiphosphorylcholine antibodies was not observed with membranes. The regulation of 5'-nucleotidase acitvity in plasma membranes might be associated with a supramolecular organization.

Functional, Structural, and Antigenic Similarities of Guinea Pig Anti-Phosphorylcholine Antibodies

The humoral immune response to PC was measured in guinea pigs. PC-vaccine stimulated IgM and IgG2, but little IgG1, anti-PC -antibodies. No memory was induced and immunization in CFA produced tolerance. PC-KLH, on the other hand, stimulated IgM, IgG2, and IgG1 anti-PC antibodies with carrier-specific memory. Hapten inhibition of plaque formation showed uniform binding patterns with minor, but significant, differences between antiPC-vaccine and anti-PC-KLH antibodies. The antibodies were characterized by IEF and idiotypic analyses. Early after immunization with PC-vaccine, guinea pigs had restricted IEF patterns which in inbred, but not outbred animals were indistinguishable between individuals. These patterns remained restricted but more individualized with time after immunization. Anti-PC-KLH antibodies showed more heterogeneity and individuality. However, these structurally heterogeneous antibodies reacted equivalently with rabbit anti-idiotype antisera and therefore must share common structural features, regardless of isotype or the genetic background of the guinea pig.

The monoclonal anti-phosphorylcholine antibody response in several murine strains: genetic implications of a diverse repertoire.

The idiotypic identification of monoclonal antibodies has been used to define and enumerate clonotypes within the murine repertoire of B cells specific for phosphorylcholine (PC). The response in the BALB/c strain is dominated by a single antibody specificity which is identical to TEPC 15 protein; however, antibody without the TEPC 15 idiotype appears heterogeneous by idiotypic cross-reactivity and hapten inhibition of binding to antigen. Dissection of the PC-specific repertoire in the AKR, A/He, and C3H strains has indicated that some monoclonal antibodies share binding-site idiotypic determinants with TEPC 15, although these clones represent a minority of the precursor cells. In addition to providing insights into the heterogeneity and expression of the murine B-cell repertoire, these studies emphasize structural relationships between PC-specific clonotypes. Within the BALB/c strain, some antibodies share combining-site-related idiotypic specificities with TEPC 15, but differ in other variable region determinants. Among allotypically distinct strains, there exists a remarkable similarity of variable region determinants in at least a minority of antibodies.

Genetic marker in the variable region of kappa chains of mouse anti-phosphorylcholine antibodies.

A newly discovered genetic marker in the kappa light chains of mouse immunoglobulins is described. This marker, designated kappa-PC8, is located in the L chains of those anti-phosphorylcholine (PC) antibodies which show the same functional and idiotypic characteristics as a PC-binding myeloma protein, HOPC 8 (H8). Analytical isoelectric focusing of these L chains revealed two phenotypes whose strain distribution pattern suggested a genetic association with genes that determine the T lymphocyte surface antigen(s) Ly-2/Ly-3. In four strains , AKR/J, C58/J, RF/J and PL/J (AKR-type, A) the H8-like L chains have a slightly lower isoelectric point than those of C57L/J and 12 other strains (C57L-type, B). Breeding experiments showed that the kappa-PC8-A phenotype is preferentially expressed. The most probable location of the marker is the variable region since other idiotypically related kappa-chains in C57L/J and AKR/J do not show differences in their electrophoretic mobility.

Immune response to phosphorylcholine. I. Characterization of the epitope-specific antibody.

The antibodies to phosphorylcholine induced in BALB/c mice were isolated and studied with a variety of biochemical and immunochemical methods. Analysis of the idiotype and the hapten-binding specificities showed no differences to the phosphorylcholine-binding myeloma protein TEPC 15, indicating a high degree of homogeneity. However, disc electrophoresis, isoelectric focusing and amino acid composition data indicated large differences in the structure of anti-phosphorylcholine antibody and TEPC 15. By these criteria the anti-phosphorylcholine antibodies from BALB/c mice are of oligoclonal origin.