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D W Metzger

Publications and source records attributed to D W Metzger.

At least 19 recordsLinked to original sources
PubMed1998

Single-chain Fv of anti-idiotype 11-1G10 antibody interacts with antibody NC41 single-chain Fv with a higher affinity than the affinity for the interaction of the parent Fab fragments.

A single-chain Fv (scFv) fragment of anti-idiotype antibody 11-1G10, which recognizes an idiotope of anti-neuraminidase antibody NC41, was constructed by joining VH and VL domains with a (Gly4Ser)3 linker, with a pelB leader sequence, and two C-terminal FLAG tag sequences, and expressed in E. coli (10 mg/L). The 11-1G10 scFv was isolated by affinity chromatography on an anti-FLAG M2 antibody column as a 2:1 mixture of monomer and dimer forms which were separated by Superdex 75 chromatography; monomer (at 100 microg/ml) was stable for 7 days at 21 degrees C and 30 days at 4 degrees C, whereas the dimer slowly dissociated to monomer to yield a 2:1 monomerdimer equilibrium mixture after 30 days at 4 degrees C. The dimer was bivalent, with each combining site binding an NC41 Fab to yield a stable complex of Mr approximately 156,000. Binding affinities, determined in solution using a BIAcore biosensor, showed that the affinity for the interaction of 11-IG10 scFv monomer with NC41 scFv monomer was five- to six-fold higher than the interaction of the parent Fab pair. This is the first example of an scFv derived from a monoclonal antibody with a higher affinity than its parent Fab.

Amino Acid Sequence↗
PubMed1997

Interleukin-12 acts as an adjuvant for humoral immunity through interferon-gamma-dependent and -independent mechanisms.

Interleukin-12 (IL-12) is a pivotal cytokine that has dramatic effects on cell-mediated immunity. It is now becoming increasingly recognized that IL-12 also strongly controls humoral immunity. We have investigated the mechanism by which IL-12 induces alterations in antibody isotype expression by determining the influence of IL-12 on in vitro immunoglobulin (Ig) production in polyclonally activated murine spleen cell cultures. Cells exposed to IL-12 plus lipopolysaccharide or anti-CD40 monoclonal antibody showed dramatically elevated IgG2a and suppressed IgG1 production compared to cells cultured in the absence of IL-12. IL-12 treatment of spleen cell cultures induced expression of gamma2a germ-line transcripts, consistent with initiation of switch recombination to IgG2a. In addition, exposure of limiting dilution cultures to IL-12 increased IgG2a+ cell precursor frequency. All of the above results were dependent on interferon-gamma (IFN-gamma). However, in the absence of IFN-gamma, IL-12 still had significant effects on Ig secretion. Specifically, IL-12 enhanced IgG1 and IgG2b anti-DNP antibody levels in mice containing specific disruptions in the IFN-gamma gene. Our results suggest that IL-12 induces T helper type 1 and natural killer cells to secrete large amounts of IFN-gamma which then causes B cells to switch to IgG2a and IgG3 production. In addition, IL-12 has direct or indirect effects on B cells that are independent of IFN-gamma. The IFN-gamma-independent effects may include enhancement of Ig expression by post-switched cells.

Adjuvants, Immunologic↗
PubMed1997

Primary human immune response to Neisseria meningitidis serogroup C in interleukin-12-treated severe combined immunodeficient mice engrafted with human peripheral blood lymphocytes.

Lack of primary immune response in severe combined immunodeficient (SCID) mice engrafted with human peripheral blood lymphocytes (hu-PBL) has limited the applicability of this model. Use of human cytokines, in particular interleukin (IL)-12, was studied in the hu-PBL-SCID model. SCID mice were treated with IL-12 and reconstituted with hu-PBL in T replacement factor. The hu-PBL-SCID mice were immunized with serogroup C meningococcal polysaccharide (MCPS). The MCPS-specific antibody response was determined by ELISA. Thirteen of the 15 immunized, IL-12-treated hu-PBL-SCID mice demonstrated a primary human antibody response to MCPS ranging from 0.25 to 3.3 microg/mL, while no MCPS-specific antibody response was detectable in the 18 controls. Expression of cross-reactive idiotypic markers found on human anti-MCPS antibodies in the immunized hu-PBL-SCID mice was similar to that observed in immunized volunteers.

Adult↗
PubMed1997

Single-chain Fv fragments of anti-neuraminidase antibody NC10 containing five- and ten-residue linkers form dimers and with zero-residue linker a trimer.

Single-chain variable fragments (scFvs) of anti-neuraminidase antibody NC10 were constructed by joining the VH and VL domains with 10-residue (Gly4Ser)2 and five-residue (Gly4Ser) linkers; a zero-residue linker scFv was constructed by joining the C-terminal residue of the VH domain to the N-terminus of the VL domain. The scFv with the 10- and five-residue linkers exclusively formed dimeric antibody fragments (M(r) 52000). These were shown to be bivalent and were able to cross-link two neuraminidase tetramers to form a 'sandwich' type complex; each antigen combining site could also bind an anti-idiotype Fab'. The zero-residue linker scFv (M(r) 70000) was shown to form a trimer with three active antigen combining sites, each binding an anti-idiotype Fab' to yield a complex of M(r) 212000. The orientation of the combining sites in the zero-residue linker scFv, however, was such that it could not cross-link tetramers of neuraminidase. BIAcore biosensor experiments showed that the affinity of each individual antigen combining site in both the 10- and five-residue linker scFv dimers and zero-residue linker scFv trimer was essentially the same when the scFvs were immobilized onto the sensor surface. However, when the scFvs were used as the analyte, the dimeric and trimeric scFvs showed an apparent increase in binding affinity due to the avidity of binding the multivalent scFvs.

Amino Acid Sequence↗
PubMed1996

Enhancement of humoral immunity by interleukin-12.

We have found that IL-12 treatment of mice leads to long-lasting enhancement in production of most antibody isotypes in conventional B-cell responses. Initial recruitment of new B-cell clones into the response is mediated by IFN-gamma, but subsequent enhancement of Ig secretion appears to be IFN-gamma-independent. We have further found that activated B cells can directly bind IL-12. Taken together, our results suggest a two-step model for the role of IL-12 in enhancement of humoral immunity. Initially, IL-12 induces production of IFN-gamma from Th1 and NK cells. Enough cytokine can be produced from either cell type to then mediate gamma 2a heavy chain isotype switching as well as temporary suppression of IgG1 production. IL-12 further stimulates post-switched cells, including cells producing IgG1, to secrete greatly increased amounts of antibody. This step is not mediated by IFN-gamma but might be due to direct IL-12 binding to activated B lymphocytes. Depletion of B1 cells by IL-12 may further enhance antibody responsiveness since B1 cells are known to competitively inhibit Ig secretion by conventional B cells. The end result is that IL-12 causes a generalized upregulation in production of all antibodies and therefore acts as a strong adjuvant for humoral as well as cellular immunity.

Adjuvants, Immunologic↗
PubMed1996

Inhibition of murine B1 lymphocytes by interleukin-12.

B1 cells are a subset of B lymphocytes found in many species and are implicated in the development of autoimmunity. B1 cells have previously been shown to be suppressed by the T helper (Th)1 cytokine interferon (IFN)-gamma, and to be stimulated by the Th2 cytokines interleukin (IL)-2, IL-4, IL-5 and IL-10. To examine further the interactions of B1 cells and Th1 cells, we have now tested the effects of the Th1 cell-inducing cytokine IL-12 on murine B1 cells. BALB/c mice were immunized with phosphorylcholine conjugated to keyhole limpet hemocyanin (PC-KLH) and simultaneously treated with 1 microgram recombinant murine IL-12 for 3 consecutive days. In addition to altering the isotype and idiotype distribution of anti-PC antibodies, IL-12 treatment was found to cause a loss of peritoneal, but not splenic B lymphocytes in immunized mice. B cell depletion required exposure to IL-12 plus antigenic stimulation. Levels of peritoneal B lymphocytes were fully restored by day 45, but the majority of these cells belonged to the B2 subset. Additionally, proliferation of B1 cells in vitro induced by IL-5 was substantially inhibited by IL-12. IL-12 itself had no effect on viable cell recovery of peritoneal cells (PeC) cultured in vitro, but viable cell recovery was significantly decreased in PeC cultured with IL-5 plus IL-12. These results show that IL-12 causes the loss of murine peritoneal B1 cells and suggest that treatment with this cytokine may be useful for disease conditions that involve B1 cell dysfunction.

Animals↗
PubMed1996

Direct binding of IL-12 to human and murine B lymphocytes.

IL-12 has been shown to play a central role in cell-mediated inflammatory reactions through direct activation of T cells and NK cells. IL-12 also strongly influences humoral immunity but these effects have been thought to be indirect and caused by intermediary cytokines. Using flow cytometry, we now show that IL-12 directly interacts with B cells. Freshly isolated murine peritoneal B-1 and conventional B lymphocytes bound IL-12, but splenic B cells failed to react unless first stimulated with lipopolysaccharide. All murine B cell sources were found to express IL-12R beta 1 subunit transcripts as detected by PCR and RNase protection assays. IL-12 binding was also detected on phytohemagglutinin-stimulated human T cell blasts and Staphylococcus aureusl IL-2-stimulated B cell blasts but not on freshly isolated peripheral blood lymphocytes. Similarly, IL-12 directly bound to the human SKW6.4 Burkitt's B cell lymphoma line. In all cases positive staining was ablated by omitting IL-12 from the procedure, showing that it was not due to detection of endogenous IL-12. These findings indicate that B cells represent another major target for IL-12 in addition to T and NK cells, and that IL-12 can directly affect humoral immunity.

Animals↗
PubMed1995

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Amino Acid Sequence↗
PubMed1995

Antibody response of murine B1 cells to hen eggwhite lysozyme.

Cell transfer studies were performed to investigate the ability of murine peritoneal B1 cells to produce specific IgG antibody against the T-dependent protein antigen, hen eggwhite lysozyme (HEL). Peritoneal cells (PeC) from normal BALB/c mice were transferred into newborn, allotype-congenic, C.B-17 severe combined immunodeficient (SCID) mice alone or together with splenic T cells from HEL-primed C.B-17 mice. After immunization with HEL, only those mice that received both PeC and primed T cells produced HEL-specific IgG of the PeC donor allotype. To identify the B cell subset responsible for antibody production, PeC were sorted before transfer into B1 and conventional B (B2) cell populations. It was found that transfer of purified B1 cells plus primed T cells resulted in high levels of IgG1 anti-HEL in approximately half of the SCID recipients, while mice receiving B2 cells produced little detectable antibody. The responses consisted primarily of IgG1 kappa anti-HEL, with no significant IgM or lambda-bearing components. Seventeen HEL-specific hybridomas of BALB/c origin, i.e., derived from the B1 cell donor, were obtained from reconstituted SCID mice after various periods of immunization and analyzed for fine specificity using a panel of avian lysozymes. All but one of the B1 cell-derived mAbs recognized an HEL epitope not present on the closely related bobwhite quail lysozyme (differing from HEL at only 4 of 129 amino acid positions). While IEF analyses demonstrated the presence of extensive clonotypic diversity, the epitope specificity pattern, which is rare among B2-cell-derived antibodies, suggests that the B1 cell recognition repertoire for HEL is severely limited.

Amino Acid Sequence↗
PubMed1995

The rabbit B cell antigen receptor is non-covalently associated with unique heteromeric protein complexes: possible insights into the membrane IgM/IgD coexpression paradox.

We describe several proteins that are components of the rabbit B cell receptor complex. Two proteins (37 kDa and 42 kDa) were found in non-covalent association with IgM expressed on B cells from peripheral blood. These proteins were also immunoprecipitated by anti-B29 (Ig-beta) and anti-mb1 (Ig-alpha) monoclonal antibodies. As in the mouse and human, the IgM associated molecules were found as heteromeric structures with non-reduced apparent molecular weights of approximately 70-75 kDa. On rabbit B cells we also found these proteins in a 100-135 kDa complex which may represent trimeric or tetrameric structures. By Western blot, the 37 kDa protein was identified as rabbit Ig-beta (B29), suggesting that the 42 kDa protein is rabbit Ig-alpha. These data suggest that rabbit IgM is associated with both Ig-alpha/beta and Ig-(alpha beta)2 or alpha beta gamma complexes. When similar immunoprecipitation studies were performed on lysates made from B cells isolated from appendix follicles, we found two additional IgM associated protein complexes containing 34 kDa and 36 kDa proteins.

Animals↗
PubMed1995

Protection of mice from group A streptococcal skin infection by interleukin-12.

It has been shown that interleukin (IL)-12 induces cell-mediated immunity and provides significant protection against intracellular organisms. The ability of this cytokine to enhance immunity in a mouse model of group A streptococcal skin infection was studied. Outbred CD1 mice were injected for 3 consecutive days with 0.1 microgram of recombinant murine IL-12 before or after challenge with strain 64/14 group A streptococci. In both cases, in vivo IL-12 treatment significantly decreased the rate of death after infection and increased survival over the period of experimental observation. Thus, IL-12 may be useful for treatment of gram-positive bacterial infections. The time course of the experiments suggests that IL-12 is acting in this model system to enhance natural, rather than acquired, immunity.

Animals↗
PubMed1995

Interleukin 12 alters the isotype-restricted antibody response of mice to hen eggwhite lysozyme.

Protein antigens elicit humoral responses in mice that consist predominantly of IgG1 antibodies. We have now investigated the ability of IL-12, a cytokine reported to augment IgG2a anti-hapten responses through activation of Th1 cells, to alter antibody responses to hen eggwhite lysozyme (HEL). The normal response of BALB/c mice to HEL is highly restricted to IgG1 expression and therefore provides an excellent system for determining effects of cytokines on expression of other isotypes. Seven days after immunization, IL-12 treated mice demonstrated greatly elevated HEL-specific IgG2a antibody levels and suppressed IgG1 production, while PBS-treated control mice showed a typical IgG1-restricted response. On day 28, IL-12-treated mice showed heightened serum antibody levels of both isotypes. Delaying cytokine treatment until after the typical IgG1 anti-HEL response had already been established also led to significant elevation of serum IgG2a antibody levels. These effects correlated with increased IFN-gamma production; however, administration of IL-12 plus anti-IFN-gamma had little influence on IgG2a enhancement, although it did relieve the early IgG1 suppression. Furthermore, the differential effects of Il-12 on isotype expression did not correlate with time; in fact, IgG2a enhancement correlated with loss of IgG1 suppression. Our findings indicate that (i) IL-12 reproducibly induces large amounts of IgG2a HEL-specific antibodies in vivo; (ii) it can alter isotype profiles of both primary and secondary responses; and (iii) its effects on humoral immunity are not completely explained by induction of Th1 cell derived IFN-gamma.

Animals↗
PubMed1993

Enhanced skin allograft survival after photodynamic therapy. Association with lymphocyte inactivation and macrophage stimulation.

It has been found previously that peritoneal exposure to hematoporphyrin derivative (HpD) photodynamic therapy (PDT) can induce systemic immunosuppression of contact hypersensitivity. We have now found that HpD-PDT also significantly prolongs survival of murine skin allografts. Normal A/J mice transplanted with BALB/c skin rejected the grafts within 10 +/- 0.9 days. Recipient mice treated 24 hr previously with HpD-PDT rejected skin allografts at 16 +/- 1.2 days. HpD alone or irradiation alone had no effect on skin graft survival, nor did HpD-PDT administered shortly after grafting. Flow cytometric analyses showed a nearly complete depletion of peritoneal lymphocytes 3 days after HpD-PDT. Lymphocyte levels were normal in the spleen, an organ not directly targeted by the PDT treatment, but the cells were totally unresponsive to Con A and LPS mitogens. Conversely, peritoneal HpD-PDT caused a striking enhancement in macrophage function as measured by phagocytosis of antibody-coated sheep erythrocytes. Humoral immunity to hen egg-white lysozyme was not significantly changed by HpD-PDT. These results demonstrate that HpD-PDT causes systemic immunosuppression of cellular immunity which, in turn, allows prolonged survival of allografts. Humoral immunity appears to remain largely unaffected by HpD-PDT and macrophages become activated, suggesting that this therapy might be more effective in specifically targeting T cell-mediated immunity than current immunosuppressive treatments.

Animals↗
PubMed1990

Identification of rabbit genomic Ig-VH pseudogenes that could serve as donor sequences for latent allotype expression.

Synthetic DNA oligomers specific for the VHa allotypes of rabbit Ig genes have been used to identify latent allotypic sequences in homozygous a1 and a2 rabbits. Two Ig VH pseudogenes containing latent a3 regions have been cloned from the genome of a homozygous a2 rabbit. Analysis of the regions associated with allotype expression indicates that these two pseudogenes contain VHa- sequences in framework region 1 (FR1) and VHa3 sequences in FR3. One gene has undergone an unusual rearrangement with a third VH gene, deleting their intervening sequences and recombining in FR3 with sequences 5' to the leader exon. Our results demonstrate the presence of latent VH sequences in the genomic DNA of normal rabbits and suggest that a mechanism such as gene conversion is responsible for expression of genetically-unexpected Ig VH genes.

Amino Acid Sequence↗
PubMed1988

Do antibodies recognize amino acid side chains of protein antigens independently of the carbon backbone?

In an effort to understand the structural basis for antigen mimicry by internal image antibodies, we determined the variable (V) region sequences of two mouse mAbs that mimic the rabbit Ig a1 allotype. The results showed that while the mAb light chains did not contain any allotype-related residues, both heavy chain V regions contained within complementarity-determining region 2 an unusual sequence homologous to the nominal antigen but in opposite orientation with respect to the carbon backbone. The ability of the internal image reversed sequence to express an a1-like determinant was tested directly by producing synthetic peptides that corresponded to the presumed antigenic regions of rabbit Ig and the mAb internal images, respectively. Although the two peptides presented the homologous residues in opposite orientations, they both completely inhibited at similar concentrations the binding of rabbit Ig to anti-a1 antibody. Conservative substitutions in the peptide sequence identified a paired Thr and Glu as being critical for expression of the a1 epitope. These findings indicate that antibodies can recognize the molecular environments created by amino acid side chains independently from the orientation of the protein carbon backbone.

Amino Acid Sequence↗
PubMed1988

In vivo activation of quiescent B cells by anti-immunoglobulin. II. Production of lysozyme-specific monoclonal antibodies of desired isotype.

We wished to determine whether injection of mice with anti-isotype antibody would be a means to regulate in vivo isotype expression and to obtain hybridomas secreting monoclonal antibodies (mAbs) of desired antigen specificity and isotype. Treatment with a rat mAb (7D2) reactive with both the IgG2a and IgG2b isotypes of mouse Ig resulted in large increases in the serum concentrations of mouse IgG2(a + b). Moreover, injection of antigen-7D2 conjugates had a profound effect on the isotype distribution of hybridomas subsequently obtained from these animals. Thus, while greater than 95% of anti-hen eggwhite lysozyme (HEL) mAbs prepared from mice immunized with HEL alone were of the IgG1 isotype, 12/15 (80%) of the mAbs from mice injected with HEL-7D2 conjugates were of the IgG2a or IgG2b isotype. When tested for effector functions using HEL-coated erythrocytes, the mAbs showed the expected activities, i.e., the IgG2, but not IgG1 anti-HEL mAbs were able to fix complement, bind protein A, and mediate antibody-dependent cytotoxicity and phagocytosis. These results indicate that in vivo immunization with anti-isotype-antigen conjugates can be used to produce hybridomas of predetermined antigen and isotype specificities.

Animals↗