Idiotype vaccine production using hybridoma technology.

Non-Hodgkin's lymphoma (NHL) is the most common hematological malignancy both in Europe and in the United States. Follicular lymphoma (FL), a tumor comprised of mature B cells, represents one fourth of all NHL and, despite good response rates to standard treatments, tends to frequently relapse to such an extent that it is still considered incurable. Among several alternative therapeutic options actively being pursued, immunotherapy by idiotypic vaccination is in the forefront of clinical experimental medicine. The idiotype vaccine consists of the tumor-specific immunoglobulin conjugated with keyhole limpet hemocyanin (KLH) and administered together with an adjuvant. Over the last 20 years, researchers have proven that this vaccine can induce specific immune responses. Too, those patients with such responses experience a disease-free survival longer than normally achievable, although these latter results require further confirmation in large clinical trials. Traditionally, idiotype vaccines have been produced through hybridoma technology. In this chapter this technology is described.

Expanding the Ig superfamily code for laminar specificity in retina: expression and role of contactins.

Bipolar, amacrine, and retinal ganglion cells elaborate arbors and form synapses within the inner plexiform layer (IPL) of the vertebrate retina. Specific subsets of these neuronal types synapse in one or a few of the ≥10 sublaminae of the IPL. Four closely related Ig superfamily transmembrane adhesion molecules--Sidekick1 (Sdk1), Sdk2, Dscam, and DscamL--are expressed by non-overlapping subsets of chick retinal neurons and promote their lamina-specific arborization (Yamagata and Sanes, 2008). Here, we asked whether contactins (Cntns), six homologs of Sdks and Dscams, are expressed by and play roles in other subsets. In situ hybridization showed that cntn1-5 were differentially expressed by subsets of amacrine cells. Immunohistochemistry showed that each Cntn protein was concentrated in a subset of IPL sublaminae. To assess roles of Cntns in retinal development, we focused on Cntn2. Depletion of Cntn2 by RNA interference markedly reduced the ability of Cntn2-positive cells to restrict their arbors to appropriate sublaminae. Conversely, ectopic expression of cntn2 redirected neurites of transduced neurons to the Cntn2-positive sublaminae. Thus, both loss- and gain-of-function strategies implicate Cntn2 in lamina-specific neurite targeting. Studies in heterologous cells showed that Cntn2 mediates homophilic adhesion, but does not bind detectably to Sdks, Dscams, or other Cntns. Overexpression analysis showed that Cntns1 and 3 can also redirect neurites to appropriate sublaminae. We propose that Cntns, Sdks, and Dscams comprise an Ig superfamily code that uses homophilic interactions to promote lamina-specific targeting of retinal dendrites in IPL.

Differential idiotype utilization for the in vivo type 14 capsular polysaccharide-specific Ig responses to intact Streptococcus pneumoniae versus a pneumococcal conjugate vaccine.

Murine IgG responses specific for the capsular polysaccharide (pneumococcal capsular polysaccharide serotype 14; PPS14) of Streptococcus pneumoniae type 14 (Pn14), induced in response to intact Pn14 or a PPS14-protein conjugate, are both dependent on CD4(+) T cell help but appear to use marginal zone versus follicular B cells, respectively. In this study, we identify an idiotype (44.1-Id) that dominates the PPS14-specific IgG, but not IgM, responses to intact Pn14, isolated PPS14, and Group B Streptococcus (strain COH1-11) expressing capsular polysaccharide structurally identical to PPS14. The 44.1-Id, however, is not expressed in the repertoire of natural PPS14-specific Abs. In distinct contrast, PPS14-specific IgG responses to a soluble PPS14-protein conjugate exhibit minimal usage of the 44.1-Id, although significant 44.1-Id expression is elicited in response to conjugate attached to particles. The 44.1-Id elicited in response to intact Pn14 was expressed in similar proportions among all four IgG subclasses during both the primary and secondary responses. The 44.1-Id usage was linked to the Igh(a), but not Igh(b), allotype and was associated with induction of relatively high total PPS14-specific IgG responses. In contrast to PPS14-protein conjugate, avidity maturation of the 44.1-Id-dominant PPS14-specific IgG responses was limited, even during the highly boosted T cell-dependent PPS14-specific secondary responses to COH1-11. These results indicate that different antigenic forms of the same capsular polysaccharide can recruit distinct B cell clones expressing characteristic idiotypes under genetic control and suggest that the 44.1-Id is derived from marginal zone B cells.

Persistence of a large population of exhausted monoclonal B cells in mixed cryoglobuliemia after the eradication of hepatitis C virus infection.

PURPOSE: Functionally exhausted and mostly autoreactive B-cells with a peculiar CD21(low)CD11c(+) phenotype accumulate in several human immunological disorders including common variable immunodeficiency, HIV infection and rheumatoid arthritis. In HCV-associated mixed cryoglobulinemia (MC) there is accumulation of exhausted clonal B cells expressing a V(H)1-69-encoded cross-reactive idiotype; these cells are phenotypically heterogeneous, displaying either a CD21(low)CD11c(+) or a marginal zone (MZ)-like (IgM(+)CD27(+)CD21(+)CD11c(-)) phenotype. Irrespective of their phenotype, V(H)1-69(+) B-cells are unresponsive to the stimulation of Toll-like receptor 9 (TLR9). We investigated the fate of these cells after the eradication of HCV. METHODS: Fourteen MC patients were studied before and after antiviral therapy. V(H)1-69(+) B-cells were identified using the G6 monoclonal antibody and their phenotype and responsiveness to the stimulation of TLR9 were investigated. RESULTS: In seven virological non-responders, cryoglobulin levels and the number and phenotype of V(H)1-69(+) B cells remained substantially unchanged. By contrast, in sustained viral responders cryoglobulinemia subsided and the number of V(H)1-69(+) B cells declined. However, high proportions of MZ-like V(H)1-69(+) B cells retaining unresponsiveness to TLR9 stimulation persisted for several months in these patients. CONCLUSIONS: Clonal expansion of CD21(low) V(H)1-69(+) B cells may depend on continual stimulation by HCV, whereas their MZ-like counterparts may persist for years after the eradication of infection. Prolonged survival of exhausted MZ-like B cells after withdrawal of the initial inciting stimulus may contribute to the accumulation of autoreactive B cells in immunological disorders.

The endoplasmic reticulum stress-inducible protein, Herp, is a potential triggering antigen for anti-DNA response.

Anti-dsDNA Abs are highly specific indicators of systemic lupus erythematosus (SLE) and play a pathogenic role in lupus nephritis. Human anti-dsDNA Abs are most likely generated by an Ag-driven mechanism. However, the Ag responsible for triggering anti-dsDNA Ab production has not been identified. To search for proteins that are cross-reactive with anti-dsDNA Abs, we screened a cDNA library from a patient with SLE with single-chain Fv of O-81 human anti-ss/dsDNA mAb by using a two-hybrid system. Homocysteine-induced ER protein (Herp), an endoplasmic reticulum (ER) stress-inducible ER membrane protein, was identified and shown to bind to original O-81 Ab and human lupus anti-dsDNA Abs. Some IgG purified from patients with active SLE by Herp-immobilized affinity chromatography bound to dsDNA. BALB/c mice immunized with Herp showed IgG anti-dsDNA Abs, IgG anti-nucleosome Abs, and glomerular IgG deposition. Herp reactivity was strongly positive in a proportion of PBLs from patients with active SLE, but undetectable in those from healthy controls. Moreover, activation of caspases was observed in the Herp-positive cells, implying that ER stress-induced apoptosis likely occurs in patients with active SLE. Herp is exposed on blebs of ER stress-induced apoptotic cells, suggesting that Herp can be recognized by immune cells. These results indicate that Herp mimics structural determinants of DNA immunologically and can be immunogenic in vivo. Thus, Herp represents a candidate autoantigen for anti-DNA Abs. This study may help explain how common environmental factors induce the production of anti-DNA Abs and contribute the development of SLE.

Cell-free production of Gaussia princeps luciferase--antibody fragment bioconjugates for ex vivo detection of tumor cells.

Antibody fragments (scFvs) fused to luciferase reporter proteins have been used as highly sensitive optical imaging probes. Gaussia princeps luciferase (GLuc) is an attractive choice for a reporter protein because it is small and bright and does not require ATP to stimulate bioluminescence-producing reactions. Both GLuc and scFv proteins contain multiple disulfide bonds, and consequently the production of active and properly folded GLuc-scFv fusions is challenging. We therefore produced both proteins individually in active form, followed by covalent coupling to produce the intended conjugate. We used an Escherichia coli-based cell-free protein synthesis (CFPS) platform to produce GLuc and scFv proteins containing non-natural amino acids (nnAAs) for subsequent conjugation by azide-alkyne click chemistry. GLuc mutants with exposed alkyne reactive groups were produced by global replacement of methionine residues in CFPS. Antibody fragment scFvs contained a single exposed azide group using a scheme for site-specific incorporation of tyrosine analogs. Incorporation of tyrosine analogs at specific sites in proteins was performed using an engineered orthogonal tRNA-tRNA synthetase pair from an archaebacterium. The unique azide and alkyne side chains in GLuc and the antibody fragment scFv facilitated conjugation by click chemistry. GLuc-scFv conjugates were shown to differentiate between cells expressing a surface target of the scFv and cells that did not carry this marker.

Construction, expression and in vitro biological effects of idiotype Ig Fab fragment of B-chronic lymphocytic leukemia.

The purpose of this study was to construct expression vectors of idiotype (Id) SmIg in patients with B-chronic lymphocytic leukemia and to express them in E.coli to obtain recombinant Id, and to investigate the effect of the protein on the proliferation and secretion of IL-2 and IFN-gamma of stimulated peripheral blood mononuclear cells (PBMC) in vitro. Light chain gene and Fd fragment of heavy chain gene were inserted into fd-tet-DOG2 vector to construct fd-tet-DOG2-Fab. Fab gene was further cloned into expression vector pHEN2 to construct the soluble expression vector pHEN2-Fab. After induction by IPTG, Fab protein was purified by Ni-NTA-chromatography. MTT was used to determine the effects of purified protein on the proliferation of stimulated PBMC in vitro and the concentrations of IL-2 and IFN-gamma in the culture supernatants were detected by ELISA. The results showed that recombinant pHEN2-Fab expression vector was constructed successfully. Fab protein was expressed in positive clone after induced by IPTG and two specific bands at 24-25 kD position were observed by SDS-PAGE electrophoresis. Proliferation of PBMC could be induced by purified Fab and the concentrations of IL-2 and IFN-gamma in culture supernatants were increased significantly after induction. It was suggested that the expression vector of SmIg Fab fragment was constructed successfully, and expressed and secreted from E. coli. The Fab protein could induce proliferation of PBMC and promote secretion of IL-2 and IFN-gamma.

Growth inhibition of myeloma cells by anti-idiotype antibodies in the absence of membrane-bound immunoglobulin.

Immunoglobulins are expressed as membrane-bound or secreted forms. Plasma cells produce little or no membrane immunoglobulin but secrete immunoglobulin molecules in large amounts. Immunoglobulin idiotypes of malignant B cells are tumor-specific antigens that may be targeted for immunotherapy. Thus, idiotype vaccination is being evaluated in clinical trials to control residual disease in multiple myeloma and non-Hodgkin's lymphoma. It is traditionally considered that anti-idiotype antibodies are not effective against plasma cell tumors, because the large amounts of immunoglobulin molecules secreted by the tumors block anti-idiotype antibodies, and because the absence of membrane immunoglobulin on the surface of these tumor cells renders them resistant to the effect of anti-idiotype antibodies. While the obstacle of abundant circulating idiotype may be obviated by reducing tumor burden to minimal residual disease, the absence of membrane immunoglobulin has been considered as a limiting factor that prevents tumor eradication by anti-idiotype antibodies. We demonstrate here that murine plasmacytoma cells can produce small amounts of membrane immunoglobulin M (IgM) heavy chains. However, the latter are precursor molecules that do not reach the cell surface. Although membrane-bound IgM is absent, the cells stain positively for surface IgM, reflecting molecules of the secreted form in the process of secretion. In spite of the relatively low levels of secreted immunoglobulin on the cell surface, anti-idiotype antibodies are effective in retardation of tumor growth in vivo. Thus, while there is no doubt that idiotype-specific cell-mediated responses are very important, myeloma patients in complete remission may additionally benefit from idiotype-specific humoral responses.

Cell-free production of scFv fusion proteins: an efficient approach for personalized lymphoma vaccines.

The unique immunoglobulin (Ig) idiotype on the surface of each B-cell lymphoma represents an ideal tumor-specific antigen for use as a therapeutic vaccine. We have used an Escherichia coli-based, cell-free protein-expression system to produce a vaccine within hours of cloning the Ig genes from a B-cell tumor. We demonstrated that a fusion protein consisting of an idiotypic single chain Fv antibody fragment (scFv) linked to a cytokine (GM-CSF) or to an immunostimulatory peptide was an effective lymphoma vaccine. These vaccines elicited humoral immune responses against the native Ig protein displayed on the surface of a tumor and protected mice against tumor challenge with efficacy equal to that of the conventional Ig produced in a mammalian cell and chemically coupled to keyhole limpet hemocyanin. The cell-free E coli system offers a platform for rapidly generating individualized vaccines, thereby allowing much more efficient application in the clinic.

Expression of idiotypic antibodies-1 and -2 and breastfeeding in relation to antibody levels against Haemophilus influenzae type B in children.

The aim of the study was to determine the concentrations of serum antibodies against Haemophilus influenzae type b in preschool children in relation to the distribution of idiotypic antibodies 1 and 2 (Id-1 and Id-2) and the exposure to breastfeeding in infancy. Sera were obtained from 74 control children recruited in an earlier case-control study before the introduction of general Hib vaccination. Duration of breastfeeding was monitored, and prevalence of noninvasive infections was registered. Concentrations of IgG1 and IgG2 anti-Hib, as well as of total Id-1 and Id-2, were determined in ELISA. The expression of Id-1 antibodies increased with age in contrast to the Id-2 antibodies that were found only in children up to 24 months of age. Expression of Id-1 antibodies was positively correlated with higher anti-Hib levels of both the IgG1 and IgG2 isotype. Children expressing Id-2 antibodies showed higher IgG2 anti-Hib concentrations than those who did not have Id-2 (P = 0.001). The concentrations of neither Id-1 nor Id-2 antibodies were related to the duration of breastfeeding. Duration of breastfeeding was related to increased anti-Hib IgG2 in healthy children above 18 months of age. These study shows that the expression of idiotype-1 and idiotype-2 antibodies was associated with higher IgG2 anti-Hib concentration and that breastfeeding could enhance the anti-Hib IgG2 production in children.

Systemic autoimmune disease caused by autoreactive B cells that receive chronic help from Ig V region-specific T cells.

B cells present BCR V region-derived Id-peptides on their MHC class II molecules to Id-specific CD4+ T cells. Prolonged Id-driven T-B collaboration could cause autoimmune disease, but this possibility is difficult to test in normal individuals. We have investigated whether mice doubly transgenic for an Id+ Ig L chain and an Id-specific TCR develop autoimmune disease. Surprisingly, T cell tolerance was not complete in these mice because a low frequency of weakly Id-reactive CD4+ T cells accumulated with age. These escapee Id-specific T cells provided chronic help for Id+ B cells, resulting in a lethal systemic autoimmune disease including germinal center reactions, hypergammaglobulinemia, IgG autoantibodies, glomerulonephritis, arthritis, skin affection, and inflammatory bowel disease. Inflamed tissues contained foci of Id-driven T-B collaboration, with deposition of IgG and complement. The disease could be transferred with B and T cells. The results demonstrate a novel mechanism for development of autoimmune disease in which self-reactive Id+ B cells receive prolonged help from Id-specific T cells, thus bypassing the need for help from T cells recognizing conventional Ag.

Association of B-1 B cells with follicular dendritic cells in spleen.

Although CD5(+) B-1 B cells have been recognized as an infrequent B cell subset in mice for many years, attempts to identify their histologic location in normal mouse spleen have proven difficult due to both their paucity and low level expression of CD5. In this study we have studied V(H)11/D(H)/J(H) gene-targeted mice, V(H)11t, that develop elevated numbers of CD5(+) V(H)11/V(k)9 B cells with an anti-phosphatidylcholine (anti-PtC) autoreactive specificity, allowing B-1 B cell detection by anti-PtC Id-specific Abs in spleen section staining. Using this approach we found that anti-PtC B-1 cells first appear within the white pulp in neonates, expand in association with follicular dendritic cells (FDC), and localize more centrally than other (non-B-1) IgD(high) follicular B cells in adults. Among neonatal B cells, CD5(+) B-1 cells in both normal and V(H)11t mouse spleen and peritoneal cavity express the highest levels of CXCR5, which is important for FDC development. Injection of purified spleen or peritoneal B-1 cells into RAG knockout mice resulted in B-1 cell follicle formation in spleen, inducing FDC development and plasma cell generation. These results indicate that B-1 B cells are the first B cells to express fully mature levels of CXCR5, thereby promoting the development of FDC.

A unique population of extrathymically derived alpha beta TCR+CD4-CD8- T cells with regulatory functions dominates the mouse female genital tract.

A better understanding of the regulatory role of genital tract T cells is much needed. In this study, we have analyzed the phenotype, distribution, and function of T lymphocytes in the female genital tract of naive, pregnant, or Chlamydia trachomatis-infected C57BL/6 mice. Unexpectedly, we found that the dominant lymphocyte population (70-90%) in the genital tract was that of CD3(+)alphabetaTCR(int)CD4(-)CD8(-) T cells. Moreover, these cells were CD90(low) but negative for the classical T cell markers CD2 and CD5. The CD3(+)B220(low) cells were NK1.1 negative and found in nude mice as well as in mice deficient for MHC class II, beta(2)-microglobulin, and CD1, indicating extrathymic origin. They dominated the KJ126(+)Vbeta8.2(+) population in the genital tract of DO11.10 OVA TCR-transgenic mice, further supporting the idea that the CD3(+)B220(low) cells are truly T cells. The function of these T cells appeared not to be associated with immune protection, because only CD4(+) and CD8(+) T cells increased in the genital tract following chlamydial infection. Notwithstanding this, the infected, as well as the uninfected and the pregnant, uterus was dominated by a high level of the CD3(+)CD4(-)CD8(-)B220(low) cells. Following in vitro Ag or polyclonal stimulation of the CD3(+)CD4(-)CD8(-)B220(low) cells, poor proliferative responses were observed. However, these cells strongly impaired splenic T cell proliferation in a cell density-dependent manner. A large fraction of the cells expressed CD25 and produced IFN-gamma upon anti-CD3 plus anti-CD28 stimulation, arguing for a strong regulatory role of this novel T cell population in the mouse female genital tract.

Restoration of Ig secretion: mutation of germline-encoded residues in T15L chains leads to secretion of free light chains and assembled antibody complexes bearing secretion-impaired heavy chains.

We previously described T15H chain mutants that were impaired in assembly with L chain and in ability to be secreted from the cell. The unmutated T15L chain is unusual in that it is secretion-impaired in the absence of assembly with H chain. The T15L chain preferentially pairs with T15H in vivo, suggesting that if we introduced mutations that would allow secretion of free T15L chain, they might also lead to the secretion of the complex with the defective H chain. We mutated four positions in the germline T15L that had amino acids infrequently found in other kappa-chains. Mutation to the most frequently occurring amino acid at three of the four positions allowed secretion of free L chain, while the combination of two secretion-restoring mutations was synergistic. Coexpression of secretion-restored mutant L chains with the secretion-defective mutant H chains rescued secretion of the assembled H(2)L(2) complex, suggesting that during somatic hypermutation in vivo, deleterious mutations at the H chain may be compensated by mutations on the L chain. To our knowledge, this is the first example of mutations in IgL chains that are able to restore secretion-defective H chains to secretion competence in mammalian cells.

Evidence for involvement of a hydrophobic patch in framework region 1 of human V4-34-encoded Igs in recognition of the red blood cell I antigen.

The monoclonal IgM cold agglutinins that bind to the I/i carbohydrate Ags on the surface of RBCs all have Ig H chains encoded by the V4-34 gene segment. This mandatory use indicates that distinctive amino acid sequences may be involved in recognition. Critical amino acids exist in framework region 1 (FR1) of V4-34-encoded Ig, and these generate a specific Id determinant which apparently lies close to the I binding site. However, I binding by Id-expressing Ig can be modulated by sequences in complementarity-determining region (CDR)(H)3. Examination of the crystal structure of an anti-I cold agglutinin has revealed a hydrophobic patch in FR1 involving residue W7 on beta-strand A and the AVY motif (residues 23-25) on beta-strand B. In this study we used mutagenesis to show that each of the strand components of the hydrophobic patch is required for binding the I carbohydrate Ag. In addition, the crystal structure reveals that amino acids in the carboxyl-terminal region of CDR(H)3 form a surface region adjacent to the hydrophobic patch. We propose that the I carbohydrate Ag interacts simultaneously with the entire hydrophobic patch in FR1 and with the outside surface of CDR(H)3. This interaction could leave most of the conventional binding site available for binding other Ags.

Regulating the isotypic and idiotypic profile of an anti-PC antibody response: lessons from peptide mimics.

Protection against microbial invasion depends not only on the host's ability to mount an immune response, but on its ability to mount the correct immune response. Whether an antibody response is protective or not depends on both the fine antigenic specificity, that may be associated with particular idiotypes and epitope binding characteristics, and the isotype, determining antibody effector function. Thus, both the variable and the constant region of the antibodies induced by a peptide mimotope must be considered when assessing the success of any immunization. Phosphorylcholine (PC), an epitope present on the cell-wall C-polysaccharide of all pneumococcal serotypes, is capable of eliciting a protective antibody response to pneumococcal infection in mice and provides an attractive model system for understanding the immune response generated by peptide mimics. In this system, both the idiotype and isotype of protective antibodies have been determined and the characteristics of the in vivo response are well described and highly reproducible. We describe here the immune response generated by two peptide mimics of PC. Mice immunized with the peptides developed antibodies binding PC and C-polysaccharide. The idiotypic profile of the response differed depending on the peptide, but never included canonical T15(+) antibodies. The isotype of the response to peptide mimics differed depending on a combination of peptide and adjuvant, and included both IgG2a and IgG2b antibodies which are not typically seen in the response to PC. Thus, peptide mimotopes may elicit anti-polysaccharide responses, but fail to elicit the idiotypes and isotypes observed in the protective response to the microbial antigen.

Aging-dependent exclusion of antigen-inexperienced cells from the peripheral B cell repertoire.

Aging is accompanied by greatly reduced B cell production in the bone marrow, yet peripheral B cell numbers do not decline. We hypothesize that this may reflect filling of the peripheral pool with B cells that are long-lived as a consequence of specificity for, and chronic stimulation by, environmental Ags. To begin to explore this possibility, we analyzed the effects of aging on B cell population dynamics in the anti-H2(k/b) 3-83 mu-delta Ig-transgenic mouse. We predicted that, because they presumably do not bind environmental Ags, B cells bearing the transgenic receptor may be lost in aged animals. As seen in nontransgenic animals, total splenic B cell numbers remained constant with age in the Ig-transgenic animals despite reduced B cell production. Importantly, although the few newly produced B cells in the bone marrow of aged mice are 3-83 positive, the peripheral compartment of these mice is dominated by B cells that express endogenous Ig genes rather than the transgenes. This population includes large numbers of marginal zone-like and CD21(low/-)CD23(low/-)IgM(low) B cells, as well as elevated numbers of CD5+ B cells. Many of these cells express only non-B220 CD45 isoforms, suggesting that they may be memory cells. A significant proportion of aged transgenic animals produce autoantibodies that are reactive with ssDNA, dsDNA, or histones. Results support the hypothesis that, in the face of severely reduced production with age, B cells are selected based on reactivity to environmental Ags, accumulate, and display activated phenotypes. Cells bearing 3-83-transgenic receptors are excluded from this population due to their specificity. Beyond their importance in aging, these findings define a novel form of receptor revision in which B cells are selected rather than deleted based on Ag reactivity.

Immunization with a recombinant adenovirus encoding a lymphoma idiotype: induction of tumor-protective immunity and identification of an idiotype-specific T cell epitope.

The Ig Id of a B cell lymphoma is a tumor-specific Ag, although as a self-Ag it is likely to be a weak immunogen. Provision of a foreign gene may enhance the immunogenicity of the idiotype. Viral vectors allow highly efficient transfer of genetic material and are themselves innately immunogenic. We have investigated the ability of recombinant adenoviral vectors, encoding the idiotypic gene with or without fusion to the human Fc region, to produce anti-idiotypic Ab- and T cell-mediated responses in a syngeneic BALB/c A20 murine lymphoma model. The idiotypic V(H) and V(L) sequences were assembled as a single chain variable fragment (scFv) and adenoviral vectors encoding the A20 scFv (Ad.A20) and A20 scFv linked to the Fc fragment of human IgG1 (Ad.A20hFc) were constructed. A single immunization of BALB/c mice with Ad.A20hFc but not Ad.A20 induced a specific anti-idiotypic Ab response. T cell lines generated from mice vaccinated with either vector displayed specific cytotoxicity, proliferation, and IFN-gamma release against a syngeneic dendritic cell line transduced using a retroviral vector to express the A20 scFv idiotype (XS52.A1.A20). Importantly, both T cell lines lysed the A20 lymphoma cells. An immunodominant H-2K(d)-restricted CD8(+) T cell peptide, DYWGQGTEL (A20[106-114]), was identified as a naturally occurring A20 scFv epitope. A single immunization with Ad.A20hFc but not Ad.A20 provided protection in >40% of animals challenged with a lethal dose of the A20 tumor line and was more effective, in this model, than a previously optimized plasmid vaccine.

T15-idiotype-negative B cells dominate the phosphocholine binding cells in the preimmune repertoire of T15i knockin mice.

T15i knockin (KI) mice express a H chain that is encoded by a rearranged T15 VDJ transgene which has been inserted into the J(H) region of chromosome 12. This T15H chain combines with a kappa22-33 L chain to produce a T15-Id+ Ab having specificity for phosphocholine (PC). Inasmuch as T15-Id+ Abs dominate the primary immune response to PC in normal mice, it was surprising to find that 80% of the PC-dextran-binding B cells in unimmunized homozygous T15i KI mice were T15-Id-. Analysis of L chains expressed in these T15-Id-, PC-specific B cells revealed that two L chains, kappa8-28 and kappa19-15, were expressed in this population. The V(kappa) region of these L chains was recombined to J(kappa)5, which is typical of L chains present in PC-specific Abs. When T15i KI mice were immunized with PC Ag, T15-Id+ B cells expanded 6-fold and differentiated into Ab-secreting cells. There was no indication that the T15-Id- B cells either proliferated or differentiated into Ab-secreting cells following immunization. Thus, T15-Id- B cells dominate the PC-binding population, but they fail to compete with T15-Id+ B cells during a functional immune response. Structural analysis of T15H:kappa8-28L and T15H:kappa19-15L Abs revealed L chain differences from the kappa22-33 L chain which could account for the lower affinity and/or avidity of these Abs for PC or PC carrier compared with the T15-Id+ T15H:kappa22-33L Ab.

Insulin binds to glucagon forming a complex that is hyper-antigenic and inducing complementary antibodies having an idiotype-antiidiotype relationship.

We demonstrate using physico-chemical techniques that insulin binds to glucagon with a Kd of 0.89 micromolar. While such binding is of little significance physiologically, it has important immunological consequences. Hormone binding is mirrored by specific binding between insulin antibody and glucagon antibody to form idiotype-antiidiotype complexes observable by Ouchterlony immunodiffusion and ELISA. These complexes may provide new insights into the formation of circulating immune complexes in diabetes. The insulin-glucagon complex is hyper-antigenic, inducing antibody production at concentrations that do not elicit immune responses from the individual hormones. The resulting immune response is not primarily against the individual hormones, but against the complex. In fact, all so-called insulin antibodies tested (rabbit, guinea pig, mouse and human) show substantially higher affinity for insulin-glucagon complex than for insulin alone, suggesting that this complex is the primary antigen in most, if not all, cases. These results lead to several testable predictions, including the possibility that glucagon antibody will bind to insulin receptors to cause type 2 (antibody mediated) insulin resistance.