A simple method for cloning hybridomas in 20-microliter hanging drops.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
M B Rittenberg
Publications and source records attributed to M B Rittenberg.
A novel in vitro assay for mouse IgE.
A simple and sensitive assay for screening mouse IgE-producing hybridomas is described. Rat basophil leukemia (RBL) cells (either live or glutaraldehyde fixed) were sensitized by mouse monoclonal IgE and then rosetted with antigen-coated sheep red blood cells. The assay was established using a mouse monoclonal IgE anti-DNP and was able to detect as little as 5 ng/ml IgE. The rosette assay is also a useful technique for studying the binding of mouse IgG monoclonal (MC) antibodies to RBL cells.
Animals↗
Immunologic memory to phosphorylcholine. VI. Heterogeneity in light chain gene expression.
BALB/c mice immunized with phosphorylcholine-conjugated keyhole limpet hemocyanin (PC-KLH) produce two types of anti-PC antibodies, designated group I and group II, which differ in their fine specificity and idiotype expression. Group II hybridomas can utilize VH genes and VL genes (in particular, V kappa 1-3) distinct from those expressed in the group I-like anti-PC myelomas. Here we have analyzed additional anti-PC hybridomas from BALB/c and (CBA/N X BALB/c)F1 male mice and also anti-PC antibodies purified from BALB/c and C57BL/6N antisera. Isoelectric focusing indicates that one of the group II hybridomas utilizes V kappa 1-3 and that related L chains are expressed in a major portion of group II serum antibodies. Other group II antibodies in antisera express different L chains, some of which are presently unidentified. However, isoelectric focusing analysis also indicates that the L chains of some group II hybridomas and serum antibodies are related to those found in the group I anti-PC myelomas and group I hybridoma and serum antibodies. In addition, one hybridoma was found to utilize lambda 2. Thus, it appears that the anti-PC antibodies with group II-like fine specificity can utilize a variety of VL genes related to, or distinct from, those expressed in group I antibodies.
Animals↗
Immunologic memory to phosphocholine. VII. Lack of T15 V1 gene utilization in Xid anti-PC hybridomas.
CBA/N mice carrying the Xid defect fail to make antibodies expressing the T15 idiotype in response to immunization with PC-KLH. Antibodies predominating in the Xid response have binding properties characteristic of group II antibodies that emerge in the memory response in BALB/c; the prototype group II antibody utilizes a VH gene product distinct from the V1 gene product expressed by T15 idiotype-positive antibodies. To examine VH gene usage in the anti-PC response of Xid B cells, hybridomas were produced from Xid mice immune to PC-KLH. Four hybridomas possessing properties typical of the predominant group II antibody response in Xid mice and two representing minor components of the response were studied. Analysis of DNA by Southern blot hybridization revealed that none of the hybridomas utilized the T15 V1 gene segment, nor did they share use of a common VDJ gene product. These results indicate that Xid group II antibodies either make use of different VH gene segments or use the same VH in combination with various D and JH segments.
Animals↗
Human thymus-independent antibody response in vitro: III. Precursor frequencies and fine specificity of human B cells stimulated by TNP-Ba in the presence and absence of Con A.
Previously, we reported that human B lymphocytes can be stimulated by trinitrophenylated Brucella abortus (TNP-Ba) in vitro to generate T-independent (TI) hapten specific plaque-forming cells (PFC). Furthermore we showed that addition of Con A on Day 3 of culture enhanced the anti-TNP response. In this report the characteristics of the anti-TNP PFC responses elicited by TNP-Ba in the presence or absence of Con A were further defined by estimating precursor frequencies and clone sizes, and by assessing the diversity of the anti-TNP-PFC response using hapten inhibition profiles. Addition of Con A to TNP-Ba-stimulated cultures was associated with a 2.3- to 3.6-fold increase in anti-TNP precursors and was also accompanied by a more vigorous clonal expansion. Fine specificity analysis of anti-TNP PFC revealed that Con A addition resulted in PFC with unique hapten inhibition profiles in that they were less inhibitable by TNP-EACA, TNP-lysine, and DNP-lysine but more inhibitable by DNP-glycine when compared to PFC generated by TNP-Ba alone. These findings suggest that at least some of the additional precursors recruited in the presence of Con A are qualitatively distinct from those activated by TNP-Ba alone since they express different V region gene products.
Antibodies, Bacterial↗
Immunologic memory to phosphocholine. IV. Hybridomas representative of Group I (T15-like) and Group II (non-T15-like) antibodies utilize distinct VH genes.
The anti-phosphocholine (PC) memory response elicited in BALB/c mice by phosphocholine-keyhole limpet hemocyanin (PC-KLH) contains two groups of antibodies distinguished by their fine specificity for PC and p-nitrophenylphosphocholine (NPPC). Group I antibodies are inhibited by both PC and NPPC, while Group II antibodies are inhibited appreciably only by NPPC; only Group I antibodies are dominated by the T15 idiotype. Anti-PC hybridomas representative of the memory response to PC-KLH were produced to examine the variable region genes expressed by memory B cells. Two IgM hybridomas were of the Group I type, because they were inhibited by both PC and NPPC and they bound to the pneumococcus R36A. However, only one of these antibodies (PCM-2) expressed a T15 idiotope, while the other (PCM-1) did not express any of three T15 idiotopes. Despite its negative T15 idiotype profile, N-terminal amino acid sequencing of PCM-1 purified heavy chain and Southern blots of the hybridoma DNA indicated that it utilizes the T15 VH and JH1 genes. Three hybridomas, IgG1, IgM, and IgE, typical of Group II antibodies, were examined; these were negative for three T15 idiotopes and displayed measurable avidity only for NPPC in a PC-protein binding inhibition assay. These three hybridoma antibodies, like serum Group II IgG1, did not measurably bind to the bacterium R36A. The heavy chain amino termini of all three of these antibodies were inaccessible for Edman degradation. Southern blots of DNA from the IgG1 hybridoma revealed the T15 VH gene to be in the germ line configuration only and unassociated with any JH segment, indicating that this Group II antibody utilizes a VH gene different from the T15 family. These results signify that, whereas some diversity of the (anti-PC) memory response may be generated by somatic diversification of variable regions important in the primary response, a significant contribution to the overall heterogeneity of memory antibodies originates in the expression of additional variable region genes.
Amino Acid Sequence↗
Immunologic memory to phosphocholine. V. Hybridomas representative of group II antibodies utilize V kappa 1-3 gene(s).
The anti-phosphocholine (PC) memory response of BALB/c mice to PC-KLH contains two groups of antibodies distinguished by fine specificity and by expression of the T15 idiotype that dominates Group I but not Group II anti-PC antibodies. The contribution of V kappa genes to this diversity was investigated by the analysis of L chains from PC-binding hybridoma proteins (PCBHP) representative of Group I and Group II. N-terminal amino acid sequence analysis was performed on the L chains of three independently derived Group II PCBHP up to residue 23 (PCG1-1) or 21 (aPC-111-1 and aPC-12-3). These three sequences differed from each other by only one or two residues, but differed by approximately 50% from the L chains of the Group I-like PC-binding myeloma proteins (PCBMP); the Group II sequences are closely related to V kappa 1-3. Isoelectric focusing analysis was also performed on the L chain of PCG1-1, as well as on L chains from PCBHP typical of Group I antibodies, and from an atypical PCBHP differing from Groups I and II in fine specificity. A Group I PCBHP and the atypical PCBHP expressed L chains related to V kappa 8 and V kappa 24, respectively. The L chains of another Group I PCBHP and of the Group II protein, PCG1-1, appeared different from those found in the PCBMP and from each other. The results indicate a more diverse expression of L chains in the memory anti-PC response than is represented by the PCBMP; both V kappa 8- and V kappa 24-derived L chains (and, presumably, somatic variants), as well as products of additional V kappa genes (V kappa 1-3), appear to be present in the anti-PC memory pool.
Amino Acid Sequence↗
Subpopulations of antibodies to phosphocholine in human serum.
We investigated the heterogeneity of anti-phosphocholine (PC) antibodies present in human serum taken from individuals before and after immunization with a multivalent pneumococcal vaccine. The fine specificity of IgM, IgG, and IgA anti-PC antibodies was determined in an ELISA by using phosphocholine or p-nitrophenyl phosphocholine (NPPC) to inhibit binding of antibody to PC-histone. We identified two populations specific for PC that differed in their binding properties. One population is inhibited by NPPC much better than by PC and is most evident in IgG antibodies. The second population has similar avidity for PC and NPPC and is consistently associated with the IgM and IgA isotypes as well as with IgG. The IgG antibodies in both populations were predominantly of the IgG2 subclass. Both populations were found in serum samples taken before immunization with pneumococcal vaccine, suggesting that they had been stimulated through prior environmental contacts with PC-containing antigens. Previously, we found populations with similar fine specificity patterns in the murine response to PC. The two murine antibody populations have been shown to derive from different immunoglobulin variable region genes. The presence of comparable antibody populations in the human suggests the possibility that these two fine specificity families have been conserved in evolution.
Animals↗
In vitro antibody synthesis in 20 microliters hanging drops: initiation of secondary responses and a simple method of cloning hybridomas.
An in vitro culture system is described in which antibody synthesis is initiated by 10(5) hapten-primed spleen cells in 20 microliters hanging drops. Plaque-forming cell (PFC) responses to thymus dependent and thymus independent antigens were obtained in drop cultures comparable to the micro Mishell--Dutton system. Antigens could be administered in soluble form or on antigen-pulsed peritoneal cells. The hanging drop method also provides a reliable yet simple procedure for cloning hybridoma cells while permitting direct visualization of the number of cells in each drop.
Animals↗
Antibodies to cell surface antigens detected by a rapid spot test using ethidium bromide.
We describe here a spot test to detect antibodies to non-erythroid cell surface antigens, using ethidium bromide staining to visualize areas of target cell lysis. This assay allows rapid screening of large numbers of samples of culture supernatants, antisera, or ascitic fluids. Antibody titers determined by this spot test are shown to correlate well with titers determined by two other methods, providing a simple method for initial estimation of the amount of antibody in positive samples.
Animals↗
Concanavalin A supernatant recruits antigen-insensitive IgG memory B lymphocyte precursors into an antigen-sensitive precursor pool.
Spleen cells from mice primed to trinitrophenyl-keyhole limpet hemocyanin (TNP-KLH) generate IgG anti-TNP memory responses when stimulated in vitro with either thymus-dependent (TD) or thymus-independent (TI) forms of the hapten. When supernatants from Con A-stimulated spleen cells (Con A Sup) were added to such secondary cultures the TI responses to DNP-dextran or TNP-T4 were augmented; the TD response to TNP-KLH was suppressed. Passage over Sephadex and addition of alpha-methyl-D-mannoside did not inhibit augmentation by Con A Sup, indicating that augmentation did not result from direct action of the lectin on the responding cells. Augmentation occurred equally well in cultures that had been depleted of T cells by treatment with anti-Thy-1.2 and complement. Limiting dilution analyses revealed that Con A Sup increased the frequency of TI-responding precursors approximately threefold while causing a concomitant decrease in TD-responding precursors. To determine the relationship of the additional TI precursors and those normally detected in the absence of Con A Sup, the TI-responding IgG precursors were first eliminated through selective suicide by using DNP-dextran plus BUdR and light treatment; subsequently no TI-responding IgG PFC could be detected to DNP-dextran unless Con A Sup was also added. The data suggest Con A Sup may augment the TI responses to DNP-dextran and TNP-T4 by recruiting additional precursors from a memory cell pool formerly insensitive to these forms of antigen.
Animals↗
In vivo allogeneic effects: shift in the isotype profile of primary TI-2 responses in mice undergoing graft-vs-host reaction.
We showed previously that primary responses to T-dependent (TD) and T-independent type 2 (TI-2) antigens were differentially affected by allogeneic effects induced in vivo during a graft-vs-host reaction (GVH). TD responses were greater than or equal to 80% suppressed, whereas the TI-2 responses were greatly enhanced, particularly the IgG component, which normally is very low. We have analyzed the IgG subclass distribution in primary responses of normal and GVH F1 mice in order to determine whether the strong T cell signals that occur during GVH reactions also induce shifts in the isotype profile. The effect of GVH on responses to TI-2 antigens was of particular interest because they are usually dominated by IgM and IgG3 classes in normal mice. We found a threefold to 10-fold increase in the PFC numbers of all four IgG subclasses in the response to TI-2 antigens, with an apparent shift from the usual IgG3 dominance to IgG1 in GVH mice. This IgG1 dominance was not found in serum antibodies where IgG3, IgG1, and IgG2b were equally expressed, although total IgG was increased greater than 20-fold. No isotype shift was found in either the TNP-KLH response, which was greater than or equal to 75% suppressed (IgG1 dominance was retained), or in the TI-1 response to TNP-Ba. The latter response was reduced (25 to 50%) in GVH mice and continued to be dominated by IgG2b/2a and IgG3. Unlike the unique isotype patterns found in primary responses, TNP-KLH primed mice challenged with TD, TI-1, or TI-2 antigens gave memory responses with identical isotype profiles that were dominated by IgG1 PFC. The role of T cells in B cell differentiation and isotype expression is discussed.
Animals↗
Lectin-induced regulation of human thymus-independent anti-TNP responses in vitro.
Recently we showed that human hapten-specific T cell-independent (TI) antibody responses could be elicited in vitro with the antigen trinitrophenylated Brucella abortus (TNP-Ba). Although by definition T cells are not required for TI responses, they have also shown capable of regulating such responses in the mouse. In this study we examine the ability of the T cell lectins Con A and PHA to modulate TI responses of human tonsil cells. Addition of the lectins to cultures on day 0 or day 3 resulted in inhibition (greater than 90%) or enhancement (greater than 150%) of the anti-TNP PFC response, respectively. The inhibition was only apparent if the lectins were added at the onset of culture but not 24 hr later, and was T cell-dependent, because T cell-depleted cultures (less than 0.5% E-rosetting cells) could not be inhibited. The enhancement observed was even greater in T cell-depleted cultures and was antigen-specific because TNP-SRBC but not PC-SRBC targets were lysed and the PFC were inhibited by soluble TNP (greater than 70%). This finding suggested that anti-TNP PFC precursors were proliferating more vigorously or that additional, previously antigen-insensitive cells were being recruited in the presence of lectin.
Antibody Formation↗
In vitro and in vivo allogeneic effects: differential modulation of B cell subpopulations.
We examined the sensitivity of primary TD and TI responses to allogeneic effects and found that primary TD responsive B cells were very sensitive to negative allogeneic effects elicited in vitro. The primary TI-2 responses, on the other hand, were not suppressed but rather enhanced twofold to threefold under the same culture conditions. Thus, primary B cell subsets resemble their IgG memory counterparts (1) in their differential sensitivity to allogeneic T cells. The ability of in vivo induced allogeneic effects of modulate antibody responses was studied using chronic graft-vs-host reaction (GVH) as a model. F1 mice immunized with TD (TNP-KLH, TNP-sheep-gamma-globulin) antigens shortly after GVH induction had reduced IgM and IgG primary responses. In contrast, the primary response to TNP-Ficoll was enhanced and included a large number of primary IgG PFC. Furthermore, mice undergoing chronic GVH were incapable of developing long-term memory after immunization with a TD antigen (TNP-KLH), as determined by secondary in vitro challenges with either TD or TI antigens. These mice retained helper T cells, indicating a failure of memory development in the B cell compartment. These studies suggest that the secondary TD- and TI-responding precursors (B2 gamma and B1 gamma, respectively) are probably both derived from primary B2 precursors (which are very sensitive to negative allogeneic effects). The maturation of the B1 memory population may thus be linked to increased resistance to allosuppression.
Animals↗
Immunologic memory to phosphorylcholine III. IgM includes a fine specificity population distinct from TEPC 15.
Group I and group II variable regions expressed in the PC-KLH memory response may originate from distinct germ-line genes or group II may arise by somatic mutation of group I (TEPC 15) germ-line genes. A current version of the somatic mutation hypothesis proposes that somatic mutation is activated by the class switch from IgM to IgG or IgA. If group II results from somatic mutation during class switching, group II IgM antibodies would not exist. This prediction was tested in the present experiments. Group I and group II antibodies were separated from whole serum by affinity chromatography on PC-Sepharose. An ELISA was used to characterize the fine specificity and idiotype of the isolated antibody populations. Group I antibodies were inhibited by both PC and NPPC haptens and were T15 idiotype-positive. Group II antibodies were inhibited appreciably only by NPPC and were negative for the T15 idiotype. The purified group II antibodies contained a significant IgM component, and high levels of group II IgM were detected during the early secondary response to PC-KLH. These observations are inconsistent with the hypothesis that group II originates by somatic mutation activated by the class switch. These results strongly suggest that one or more of the germ-line genes (V, D, or J) of groups I and II are different. Alternatively, if group II antibodies arise from group I by somatic mutation, this mutation must occur before class switching.
Animals↗
Partial reconstitution of TNP-Ficoll responses and IgG3 expression in Xid mice undergoing graft-vs-host reaction.
Explore the source record for details and available documents.
Animals↗
Immunologic memory to phosphorylcholine. II. PC-KLH induces two antibody populations that dominate different isotypes.
We examined the fine specificity of the memory response to phosphorylcholine (PC) using an enzyme-linked immunosorbent assay (ELISA) that measures the relative abilities of PC analogs to inhibit the binding of antibody to antigen. Along with the usual haptens (phosphorylcholine, L-alpha-glycerophosphorylcholine, and choline) these studies included as an inhibitor p-nitrophenyl phosphorylcholine, a compound that is homologous to the structure of the PC antigen in a protein conjugate. Evaluation of the fine specificity profiles revealed two populations of antibody elicited by PC-KLH, which were unequally dominant among the various immunoglobulin classes and IgG subclasses. Group I antibodies exhibited a similar or slightly greater avidity for PC than for NPPC and constituted the majority of IgM, IgA, and IgG3 antibodies. Group II antibodies, which were expressed primarily by IgG1, IgG2a, and IgG2b antibodies, possessed significant avidity only for NPPC. Possible mechanisms that would link IgM, IgA, and IgG3 to similar variable regions while excluding IgG1, IgG2a, and IgG2b from the same grouping are discussed.
Animals↗
Human lymphocytes can generate thymus-independent as well as thymus-dependent anti-hapten plaque-forming cell responses in vitro.
Human lymphocytes from peripheral blood, tonsils, and adenoids produced hapten-specific anti-TNP antibodies after in vitro stimulation with TNP-Ba. This antigen did not appear to behave as a polyclonal activator since TNP-Ba-stimulated cells lysed TNP-conjugated sheep erythrocytes (SRBC) but not unconjugated SRBC or DPPC-conjugated SRBC, whereas cells from PWM-stimulated cultures formed hemolytic plaques with both haptenated and unhaptenated targets. In addition, TNP-Ba generated PFC were inhibitable by soluble hapten (TNP-EACA or TNP-BSA). B cells depleted of T cells (less than 1% E-RFC cells) were able to respond to TNP-Ba but not to PWM or TNP-KLH. When varying ratios of T and B cells were added to cultures the TNP-Ba anti-TNP response was found to have a linear correlation with the number of B cells added (r = 0.987) and was maximal in the absence of added T cells. This was in contrast to the response to TNP-KLH, which required T cells at a particular T:B ratio for optimal induction of anti-TNP PFC. Thus, both T-dependent and T-independent anti-hapten responses could be elicited in human lymphoid cell cultures.
Adenoids↗