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PubMed1985

Cloning of mitogen- and antigen-reactive B lymphocytes on filter paper discs. I. A description of the technique and of methods for the analysis of colonies.

A novel technique for establishing short term clones of antigen- or mitogen-activated splenic B lymphocytes is described. Spleen cells are plated onto the surface of filter paper discs and subsequently stimulated by antigen or mitogen in situ; activated B cells proliferate and differentiate into pure colonies of cells analogous to bacterial colonies growing on agar. These colonies of lymphocytes may be characterized in a series of replica hemolytic-plaque, autoradiographic, or immunoenzyme assays making possible a full characterization of the frequency of secreted idiotopes and paratopes and of the cells that produce them. Colony induction by either antigen or mitogen occurs under identical conditions, thus a rigorous comparison between the mitogen-selected and antigen-selected antibody repertoires may be made.

Animals↗
PubMed1986

The influence of antibody affinity on the radioallergosorbent test (RAST) and in vitro histamine release. Studies with hapten-specific monoclonal IgE antibodies.

Four murine monoclonal IgE antibodies specific for the hapten, 4-hydroxy-3-nitrophenylacetyl (NP), had been previously found to be heteroclitic in nature in that they bound the crossreacting hapten, 4-hydroxy-3-iodo-5-nitrophenylacetyl (NIP), with greater affinity than NP. The influence of antibody affinity on the results of two commonly used assays for IgE, namely the radioallergosorbent test (RAST) and histamine release from rat peritoneal mast cells, was studied using these antibodies. In general, in agreement with previous reports, it was found that affinity influences both RAST and histamine release; however, the affinity constants deduced from equilibrium dialysis measurements for the reactions with monovalent haptens were not directly related to the activities of the antibodies as reflected in assays using multivalent hapten protein conjugates.

Animals↗
PubMed1978

Immune reactivity during aging. I. T-helper dependent and independent antibody responses to different antigens, in vivo and in vitro.

The immunological status during aging was assessed by measuring the antibody response of the long-lived (C3H/eb X C57Bl/6J)F1 mice to various antigens in vivo and in vitro. In vivo, a decrease in antibody production to DNP and NIP haptenic determinants coupled on to BGG, as well as the response to SRBC, was observed. The decline was more pronounced in the IgG as compared to IgM antibodies. The results were recorded when various parameters such as antigen dose and kinetics of the response, were considered. Reduction of the antibody response was also noted when PVP was employed as immunogen. Similar results were noted when the responses to SRBC and DNP--polylysine were induced and followed in spleen organ cultures. In all of these experimental systems, the peak response was observed in mice 6--12 months old. From then on a gradual decrease was manifested, mice 30--36 months old producing significantly low responses. The results demonstrate that decrease in antibody production is expressed in the isolated spleen tissue in the same manner as in the intact animal. Furthermore, they were interpreted as indicating that the lesion may be at the T helper and the B cell compartments.

Aging↗
PubMed1983

Mouse IgG antibodies have subclass associated affinity differences.

Subclasses of IgG were separated from pools of mouse sera by letting immunoglobulins absorb on protein A-Sepharose and by eluting with buffers of decreasing pH. Most donor mice were immunized with a conjugate of a hapten (NIP) and chicken gamma globulin 20 days previously. The results indicate that concentrations of IgG varied from 5.1 to 8.6 mg/ml in the pools of immune sera and was 3.0 mg/ml in one normal serum tested. One half of this was IgG1, ca. 20% of IgG2a and IgG2b each, and 10% IgG3 in the pools of BALB/c sera. IgG2a and IgG3 could not be separated from C57BL sera (due to allotype b), but their combined share of IgG appears to be higher than in BALB/c. Immune sera contained 0.5-1.6 mg/ml of anti-NIP antibodies. Of this 90-98% was IgG1 and the remainder was split between the other subclasses. Up to one half of the protein in the IgG1 fraction was anti-NIP antibody. This surprising finding was confirmed by demonstrating that nearly 50% of the u.v.-light absorption was specifically removed by a NIP-immunosorbent. Subclass-associated affinity-differences were observed. IgG1 anti-NIP had a greater average affinity than IgG2a anti-NIP antibodies. The difference was ca. 1.5-fold when the equilibrium dialysis was focusing on the high-affinity bracket of the total population (concentration of free hapten 16-200 nM). At higher hapten concentrations the trend was the same but the data are fewer. Antibodies in subclasses IgG2b and IgG3 appear to share the lower affinity of IgG2a.

Animals↗
PubMed1984

Photoaffinity-labeled hapten-binding T-cell receptor on a suppressor T-cell hybridoma.

A T-cell hydridoma, 7C3-13-Ag6, which produces a (4-hydroxy-3-nitrophenyl)acetyl (NP)-specific suppressor T-cell factor associated with an I-J determinant, was utilized to study the hapten-binding receptor of T-cells. This hybridoma had been shown to express NP-binding receptor molecules on the cell surface with heteroclitic fine specificity for a cross-reactive hapten, (4-hydroxy-5-iodo-3-nitrophenyl) acetyl (NIP). The stoichiometric analysis of the hapten binding by 7C3-13-Ag6 cells was performed by the measurement of direct binding of highly radioactive haptens to the cell surface. The affinity constant (Ka) of the receptor for N125IP-epsilon-aminocaproic acid (N125IP-cap), as calculated from a Hill plot, was 5.75 X 10(7) M-1 [Hill coefficient (a) = 0.86; expression of receptor sites per cell = approximately 1 X 10(3) on average]. The receptor molecule was specifically affinity labeled with photoreactive nitroaryl azide derivatives of N125IP (510-570 Ci/mmole). The specificity of photoaffinity labeling was demonstrated both by competitive inhibition of labeling with NIP- or NP-cap and by differential photoaffinity labeling based on the reversibility of hapten-receptor interaction. The gel electrophoretic analysis of the photoaffinity-labeled molecule indicated that the hapten-binding receptor of 7C3-13-Ag6 has a mol. wt of 28,000 +/- 3000 and an isoelectric point of 5.6-5.7. No immunoglobulin determinants were detected on the molecule. A comparative immunoprecipitation analysis of the membrane lysate of 7C3-13-Ag6 with monoclonal anti-I-J reagents identified a separate I-J molecule of 25,000 +/- 1000 mol. wt that is distinct from the photoaffinity-labeled hapten-binding molecule.

Affinity Labels↗
PubMed1987

Regulation of the response to alpha(1-3) dextran: an anti-dextran associated idiotope of BALB/c mice is also expressed on A/J anti-NIP antibodies.

The functional properties of a mAb reactive to the B5 idiotope were analyzed in BALB/c, A/J and C.B20 mice. the B5 idiotope was specifically associated with the alpha(1-3) DEX response in a strain-specific manner in BALB/c mice (responder strain). It was serologically distinct from the previously described IdX idiotype and overlapped on part of the IdX+ and IdX- BALB/c anti-alpha(1-3) DEX response. While treatment of adult BALB/c mice with mAb B5 enhanced the serum anti-alpha(1-3) DEX antibody synthesis and induced de novo antibody synthesis in non-responder C.B20 mice, A/J non-responder mice remained unresponsive, despite elevated levels of serum B5+ dextran non-binding Ab3 immunoglobulins. However, in some of these A/J mice, we detected an enhancement of anti-NIP antibody synthesis. This anti-NIP antibody component was (1) specifically induced by mAb B5, (2) found in A/J, but not in BALB/c or C.B20 mice, and (3) specific towards the NIP hapten. These A/J anti-NIP antibodies bore either the lambda 1 or the kappa light chain and some of them also expressed the B5 idiotope. The B5 id thus appears as a typical id determinant shared by antibodies having different binding specificities, and it may have a role in the regulation of these responses.

Animals↗
PubMedDate not supplied

LPS greatly enhances the antibody response to hapten-polysaccharide conjugates, but not to hapten-protein conjugates.

In confirmation of earlier findings, we observed that an injection of bacterial lipopolysaccharide (LPS) into mice caused a considerable increase in the serum concentrations of IgM and IgG (total Ig rose three- to four-fold in 7 days), and a corresponding increase in the concentrations of "natural" anti-(3-iodo-4-hydroxy-5-nitrophenyl) acetyl (NIP) and anti-trinitrophenol (TNP) antibodies. Our main purpose was to determine what effect LPS had on antigen-dependent responses. Hapten conjugates of a polysaccharide and of proteins were used as antigens. Hapten-protein conjugates induced a strong anti-hapten antibody response (up to 1 mg/ml of anti-hapten antibodies on day 7). Hapten-polysaccharide conjugates induced only a meagre increase in anti-hapten antibodies from the pre-immunization level (maximal concentration 65 micrograms/ml on day 7). LPS, when injected with the antigen, greatly enhanced the antibody response to the hapten-polysaccharide conjugates (up to 2.6 mg/ml of anti-hapten antibodies on day 7). It had little effect on antibody responses to hapten-protein conjugates. The combination treatment had the same effect on immunoglobulin concentrations as LPS alone.

Animals↗
PubMed2001

'Troy-bodies': antibodies as vector proteins for T cell epitopes.

A major objective in vaccine development is the design of reagents that give a strong, specific T cell response. Targeting of antigens to antigen presenting cells (APC) results in enhanced antigen presentation and T cell activation. In this paper, we describe a novel targeting reagent denoted 'Troy-bodies', namely recombinant antibodies with APC-specificity and with T cell epitopes integrated in their C regions. We have made such antibodies with V regions specific for either IgD or MHC class II, and five different T cell epitopes have been tested. All epitopes could be introduced into loops of C domains without disrupting immunoglobulin (Ig) folding. Four have been tested in T cell activation studies, and all could be released and presented by APC. Furthermore, whether IgD- or MHC-specific, the molecules tested enhanced T cell stimulation compared to non-specific control antibodies in vitro as well as in vivo. Using this technology, specific reagents can be designed that target selected antigenic peptides to an APC of choice. Troy-bodies may therefore be useful for manipulation of immune responses, and in particular for vaccination purposes.

Animals↗
PubMedDate not supplied

Drastic change in idiotypic but not antigen-binding specificity of an antibody by a single amino-acid substitution.

In proliferating B lymphocytes, somatic mutation of rearranged antibody variable (V)-region genes occurs at high frequency and may have a key role in the selection of these cells. It is of interest in this context to learn in which way single mutations can affect antigen binding and/or idiotypic specificity of an antibody. Previous investigations have analysed spontaneous mutants of myeloma and hybridoma cells in which the mutation affected the antigen-binding specificity of the antibody. Here we describe an antibody mutant that has fully retained antigen-binding specificity but has lost or drastically changed all V-region antigenic determinants (idiotopes) of the wild type as defined by monoclonal anti-idiotope antibodies. The mutant phenotype is generated by a glycine to arginine exchange in the middle of the diversity (D) element, at position 103 of the heavy chain.

Amino Acid Sequence↗
PubMed1993

Activation of complement by an IgG molecule without a genetic hinge.

The hinge region links the two Fab arms to the Fc portion of the IgG molecule. It mediates flexibility to the molecule and serves as a connecting structure between the two heavy chains. In addition it provides space between the Fab and Fc parts. All three properties have been proposed to be important for the ability of IgG to initiate complement activation leading to complement-mediated cell lysis (CML). Here we report the construction of a hinge-deleted mouse-human chimaeric IgG3 molecule with specificity for the hapten NIP (3-iodo-4-hydroxy-5-nitrophenacetyl), HM-1. HM-1 lacks the genetic hinge, but has an introduced cysteine between Ala 231 (EU numbering) and Pro 232 in the lower hinge encoded by the CH2 exon. The introduced cysteine forms a disulphide bond between the two heavy chains of the molecule. In CML, HM-1 shows a greater activity than IgG3 wild type. This is the first time an IgG molecule without a genetic hinge has been found to be active in CML. We conclude that the hinge functioning as a spacer is not a prerequisite for complement activation. Rather, its major role seems to be to connect the heavy chains to each other in the amino-terminal part of CH2. Because HM-1 is expected to have low Fab-Fc flexibility, this molecular feature is probably of no importance for complement activation.

Animals↗
PubMed2000

Pharmacologic suppression of target cell recognition by engineered T cells expressing chimeric T-cell receptors.

Adoptive therapy with autologous T cells expressing chimeric T-cell receptors (chTCRs) is of potential interest for the treatment of malignancy. To limit possible T-cell-mediated damage to normal tissues that weakly express the targeted tumor antigen (Ag), we have tested a strategy for the suppression of target cell recognition by engineered T cells. Jurkat T cells were transduced with an anti-hapten chTCR tinder the control of a tetracycline-suppressible promoter and were shown to respond to Ag-positive (hapten-coated) but not to Ag-negative target cells. The engineered T cells were then reacted with hapten-coated target cells at different effector to target cell ratios before and after exposure to tetracycline. When the engineered T cells were treated with tetracycline, expression of the chTCR was greatly decreased and recognition of the hapten-coated target cells was completely suppressed. Tetracycline-mediated suppression of target cell recognition by engineered T cells may be a useful strategy to limit the toxicity of the approach to cancer gene therapy.

Flow Cytometry↗
PubMed1999

Antigen-specific membrane fusion mediated by the haemagglutinin protein of influenza A virus: separation of attachment and fusion functions on different molecules.

Using genetic engineering techniques, two strategies for changing the receptor-binding specificity of the influenza A virus haemagglutinin (HA) protein whilst preserving its membrane fusion function, have been explored. The aim was to investigate whether the HA protein could be developed as an attachment/entry protein for targeting enveloped virus gene therapy vectors to specific cell populations. In the first strategy, a single chain antibody Fv region (scFv) specific for the hapten NIP was inserted between HA1 codons 139 and 145, to create a scFv-HA chimaeric protein. This protein was shown to possess anti-NIP binding activity, but membrane fusion activity could not be demonstrated. The possibility that linking the scFv domain directly to HA may have inhibited the HA fusion function led to the development of the second strategy. This involved separating the receptor-binding and membrane fusion functions of HA on to two different molecules. The feasibility of this strategy was tested by looking for fusion between NIP-conjugated red blood cells which lacked sialic acid (the HA protein's natural receptor) and Chinese hamster ovary cells that expressed both the above anti-NIP scFv-HA chimaeric protein (as a non-fusigenic, receptor-binding molecule) and wild-type HA protein (as a fusigenic, non-binding molecule) on their surface. Cell-to-cell fusion was observed in this system, indicating that the receptor-binding function of HA can be transferred to an adjacent molecule, and also changed in its specificity, without compromising its membrane fusion activity. This finding strongly suggests that the development of a two-molecule attachment and entry system for retargeting enveloped virus gene therapy vectors, based on HA, is a viable proposition.

Animals↗
PubMed1992

Metal-binding chimeric antibodies expressed in Escherichia coli.

Metallothionein, a well-characterized biological chelator of metals, has been genetically fused to the binding domain of an antibody and expressed in the periplasm of Escherichia coli. Specific delivery of 109Cd to immobilized hapten or to haptenated cells was demonstrated directly in periplasmic extracts. This approach is potentially useful for targeted radiotherapy and diagnostic imaging. We find six to seven atoms of metal per active antigen-combining site. Absence of the Fc portion of the immunoglobulin along with low immunogenicity of metallothionein-metal complexes should reduce immunologic reactions.

Amino Acid Sequence↗
PubMed1976

Cell-mediated lympholysis of N-(3-nitro-4-hydroxy-5-iodophenylacetyl)-beta-anaylglycylglycyl-modified autologous lymphocytes. Effector cell specificity to modified cell surface components controlled by the H-2K and H-2D serological regions of the murine major histocompatibility complex.

Splenic lymphocytes from four C57BL/10 congenic mouse strains were sensitized in vitro to N(-3-nitro-4-hydroxy-5-iodophenylacetyl)-beta-alanylglycylglycyl-(N) modified autologous lymphocytes. The effector cells generated after 5 days of culture were assayed on a series of either N-modified phytohemagglutinin-stimulated spleen cells or N-modified tumor cells. The results indicated in all cases that both N modification of the targets and H-2 homology between the modified stimulating and target cells are required for lysis to occur. In each case the effector cells were found to lyse N-modified target cells only when there was homology at either or both ends of the major histocompatibility complex (MHC) between the stimulator and target cells. B10.BR lysed targets sharing alleles at K (or K plus I-A) and/or at D. B10.A effector cell specificity was mapped to K (or K plus I-A) and/or the D half of the MHC (D or D plus I-C and/or S). The two regions of specificity determined for B10.D2 effector cells were D (or D plus S plus I-C) and a region not including D of the MHC. C57BL/10 effector cells lysed N-modified targets only if there was target cell H-2 homology at K, I-A, and I-B or at the D serological region. As in the trinitrophenyl (TNP) system (6) B10.BR and B10.A effector cells lysed targets sharing K end H-2 serological regions greater than target cells sharing D-end serological regions. The C57BL/10 effector cells were shown to react to the K end greater than the D end, which differed from the equal reactivity seen in the TNP system for this strain. The data are consistent with the hypothesis that the antigen recognized by the effector cell includes an altered H-2 serological cell surface product. That the reaction is not "hapten specific" and the H-2 homology is required only for effector:target cell interaction was excluded by the use of two F1 combinations in which lysis of only N-modified target cells sharing the H-2 haplotype with the stimulating parental strain was obtained. Finally, it was demonstrated that N and TNP modification create distinct new antigenic determinants, since an effector cell sensitized to one modifying agent will lyse only H-2 matched target modified with that same modifying agent.

Animals↗
PubMed1976

Antigen-induced aggregation and modulation of receptors on hapten-specific B lymphocytes.

Mouse spleen cells were subjected to a fractionation procedure designed to enrich for 4-hydroxy-3-iodo-5-nitro-phenylacetyl (NIP)- or DNP-specific B lymphocytes, which depended on adherence of specific cells to a layer of hapten-gelatin at 4 degrees C, recovery of bound cells by melting, and digestion of adherent antigen by collagenase. A population of cells resulted which contained 90% typical B cells and 37% of cells capable of binding a fluorescent, haptenated polymeric protein. Fractionated cells were reacted in vitro with fluorescent conjugates of the specific haptens with polymerized flagellin [NIP-polymerized flagellin (POL)-tetramethylrhodamine isothiocyanate conjugate or DNP-POL-fluorescein isothiocyanate conjugate] under a variety of conditions, with the aim of investigating the behavior of Ig receptors on B lymphocytes after exposure to antigen; Experiments were performed with immunogenic and tolerogenic concentrations of antigen. Furthermore, four experimental designs were used, namely: (a) brief labeling with fluorescent antigen followed by culture without antigen (pulse design); (b) culture in the continuous presence of fluorescent antigen (continuous-labeling design); (c) culture in the continuous presence of nonlabeled antigen followed by labeling of unoccupied receptors by fluorescent antigen (receptor status design); and (d) culture with nonlabeled antigen for 2 h followed by incubation without further antigen for 20 h and labeling with fluorescent antigen (modulation design). Further insight into receptor occupancy and distribution was gained by the use of fluorescent antihapten and antiglobulin reagents. It was found that both immunogenic and tolerogenic antigen concentrations caused rapid patching and capping of the receptors to which they attached, followed by endocytosis and probably some shedding of Ig receptors. However, a proportion of cells continued to bear some cell surface antigen for 24 h. The immunogenic antigen concentration failed to completely remove the receptor coat from the cell surface. At all stages of immunogenesis, plentiful unoccupied receptors could be demonstrated. The tolerogenic concentration nearly saturated available receptors, and in its continuous presence, only few unoccupied or antigen-occupied surface receptors could be detected after 24 h of culture. Experiments of the modulation design showed that brief incubation with the tolerogenic concentration appeared to suppress receptor resynthesis, as few new receptors could be demonstrated after 20 h of further culture without antigen. Experiments were performed to determine whether fractionated cells prepared from spleens of 8-day-old mice showed an unusual tendency for modulation, even with immunogenic antigen concentrations. They were found to behave essentially like adult fractionated cells. The results are discussed in the framework of current theories of B-lymphocyte activation and tolerization.

Animals↗
PubMed1976

The specificity of cellular immune responses II. The structure of antigenic determinants leading to T-lymphocyte stimulation.

T cells from guinea pigs immunized with the hapten 2,4-dinitrophenyl (DNP)-coupled directly to mycobacteria are of interest since they recognize and respond to DNP conjugated to many but not all carriers. The experiments reported here further analyze the structure of the complex, chemically defined antigenic determinants recognized by such T cells. These antigenic determinants can have DNP coupled either to the xi-amino group of lysyl residues or to the hydroxyl group of tyrosyl residues. Furthermore, essential contributions to the determinant recognized by such T cells are made by amino acid residues to which the hapten is not attached. Such residues are thought to be close to the hapten group itself, since introducing a small spacer between hapten and carrier prevents recognition. The hapten itself is also recognized and discriminated from other haptens with great precision by these T lymphocytes. The strain of guinea pig immunized affects the precise specificity characteristics of the responding T cells, in a way that may reflect the activity of histocompatibility-linked immune response genes. Finally, the characteristics of the immunogen have been studied. It is thought that the lipid content of the mycobacteria may be critical in inducing the hapten-reactive T cells, and this is supported by finding similar responses in T cells from guinea pigs immunized with DNP protein to which lipid has been covalently attached. Thus, the T-cell population being studied, while recognizing haptens with great precision, appears to require a larger determinant for activation than do hapten-specific B lymphocytes.

Animals↗