The idiotypic network in the regulation of autoimmunity: Theoretical and experimental studies.

The regulation of autoimmunity is a key issue in fundamental immunology. Despite outstanding achievements on this front, we currently have more questions than answers. The idea of an immune network as a regulatory mechanism is quite attractive, since it enables us to explain the selectivity (specificity), and moreover the clonality, of the regulation. Nevertheless it remains unclear how this mysterious network of immune cells is organized, how it operates, and how it exerts control over autoimmunity. This article presents an attempt to understand how the immune network functions and how it controls autoreactivity. We present a mathematical model of the immune network that is based on principles of immune network organization and function that we arrived at from a survey of the available literature. To test the principles on which the mathematical model is based, we studied the model and compared the different responses to antigen that it generated with the results obtained from experimental studies of immune response. The modeled kinetics of idiotype and anti-idiotype in response to the administration of antigen are in good agreement with the experimental kinetics of idiotypic and anti-idiotypic antibodies. To obtain evidence of the existence of idiotypic mechanisms for regulating autoimmunity, we studied a mathematical model containing autoclones and compared the model results with data from experimental studies in a model of autoimmune hemolytic anemia in mice. Because the results from the theoretical and the experimental studies coincide, there is justification to conclude that autoreactive lymphocytes are normal components of the immune network within which they are regulated. We discuss a possible molecular/cellular mechanism for negative control of autoreactive cells as affected by anti-idiotypic antibodies.

Cloning variable region genes of clonal lymphoma immunoglobulin for generating patient-specific idiotype DNA vaccine.

Available therapies for lymphoplasmacytic lymphoma (LPL) provide no survival advantage if started before signs or symptoms of end-organ damage develop; hence, current recommendations are to follow a program of observation while patients are in the asymptomatic phase of disease. We hypothesize that using idiotypic determinants of a B-cell lymphoma's surface immunoglobulin as a tumor-specific marker, we can develop patient-specific chemokine-idiotype fusion DNA vaccines that induce an immune response against LPL. By activating the host immune system against the tumor antigen, we postulate that disease control of asymptomatic phase lymphoplasmacytic lymphoma can be maintained. These chemokine-idiotype fusion DNA vaccines provide protection in a lymphoma mouse model and have recently entered clinical trials. Herein, we describe procedures for the generation of therapeutic vaccines, particularly "second-generation" recombinant vaccines. Specifically, in the Methods section we describe how to identify lymphoma-associated immunoglobulin V (IgV) genes from patient biopsy and how to assemble these genes as single-chain variable gene fragment (scFv) in-frame with MIP-3α to generate novel DNA fusion vaccines.

Phosphocholine-binding antibody activities are hierarchically encoded in the sequence of the heavy-chain variable region: dominance of self-association activity in the T15 idiotype.

A methodology based on the representation of each amino acid of a protein sequence by the electron-ion interaction potential and subsequent analysis by signal processing was used to determine the characteristic or common frequency (in Hz) that reflects the biological activity shared among phosphocholine (PC)-binding antibodies. The common frequency for the variable portion of the heavy chain (VH) of the PC-specific antibodies is found to be at f = 0.37 Hz. The VH sequences of the PC-binding antibodies exhibit three subsites for the PC moiety where hypervariable region 2 (CDR2) plays a role in the interaction with the phosphate group. Mutations in this VH region have an impact on the ability of mutant variants to bind PC and its carrier molecule, as well as on the characteristic frequency shift toward f = 0.12 Hz for mutants failing to bind both hapten and carrier. The VH sequence of mutants that retain the ability to bind PC still shows f = 0.37 Hz, suggesting that this frequency determines PC binding. However, this statement was not confirmed as mutation in another PC subsite impairs PC binding but retains both the phosphate-group recognition and the frequency at f = 0.37 Hz. Herein, this finding is discussed to promote the idea that the VH sequence of the PC-binding antibodies encodes the subsite for phosphate-group binding as a dominant functional activity and that only CDR2 of the T15-idiotype antibodies together with FR3 region form an autonomous self-association function represented by the T15VH50-73 peptide with f = 0.37±0.05 Hz. Thus, these data confirmed that T15VH50-73 peptide might be used in superantibody technology.

Translational development of vaccination strategies in follicular NHL.

Follicular lymphoma expresses a unique immunoglobulin molecule termed idiotype that has been used as a tumor-specific antigen for vaccine development. Early stage clinical studies revealed that vaccination consisting of keyhole limpet hemocyanin-conjugated lymphoma idiotype protein in combination with granulocyte-macrophage colony-stimulating factor induced tumor-specific immune responses and molecular remission in patients with follicular lymphoma. Three double-blind, randomized, Phase III trials were conducted to further determine the clinical benefit of this vaccine therapy. Compared to the placebo, prolonged disease-free survival in vaccinated patients was concluded only in one study where all the patients enrolled in the trial already had complete remission from induction chemotherapy. Next generation idiotype vaccines are being developed with the focus on simplifying vaccine formulation and potentiating tumor-specific immunity. This category includes genetically modified idiotype single-chain DNA vaccine, liposome-encapsulated idiotype vaccine and dendritic cell vaccine. Although preclinical data supported the immunogenicity and therapeutic advantage of these new vaccines, their clinical benefits remain to be tested. Optimizing new generation idiotype vaccines may require combination with immune adjuvants that potentiate vaccine-induced antitumor immunity, have direct effects against tumor or block immune regulatory checkpoints. Moreover, identification of a universal follicular lymphoma antigen is important for future development of vaccine therapy against this disease.

Plant-produced idiotype vaccines for the treatment of non-Hodgkin's lymphoma: safety and immunogenicity in a phase I clinical study.

Plant-made vaccines have been the subject of intense interest because they can be produced economically in large scale without the use of animal-derived components. Plant-made therapeutic vaccines against challenging chronic diseases, such as cancer, have received little research attention, and no previous human clinical trials have been conducted in this vaccine category. We document the feasibility of using a plant viral expression system to produce personalized (patient-specific) recombinant idiotype vaccines against follicular B cell lymphoma and the results of administering these vaccines to lymphoma patients in a phase I safety and immunogenicity clinical trial. The system allowed rapid production and recovery of idiotypic single-chain antibodies (scFv) derived from each patient's tumor and immunization of patients with their own individual therapeutic antigen. Both low and high doses of vaccines, administered alone or co-administered with the adjuvant GM-CSF, were well tolerated with no serious adverse events. A majority (>70%) of the patients developed cellular or humoral immune responses, and 47% of the patients developed antigen-specific responses. Because 15 of 16 vaccines were glycosylated in plants, this study also shows that variation in patterns of antigen glycosylation do not impair the immunogenicity or affect the safety of the vaccines. Collectively, these findings support the conclusion that plant-produced idiotype vaccines are feasible to produce, safe to administer, and a viable option for idiotype-specific immune therapy in follicular lymphoma patients.

A common idiotype in IgE and its relation to recognition of the grass pollen allergen Phl p 2.

The variable regions of allergen-specific IgE, the isotype mediating allergic responses, are poorly defined to date. In this study we define the character of human antibody binding sites recognizing Phl p 2, a major allergen from timothy grass pollen. Independently raised specificities developed by phage display technology tended to have common sequence motifs (idiotypes), such as IGHV4-31 germline gene origin and heavy chain complementarity-determining region (CDR) 3 length and sequence. They also combined with highly related light chain sequences. Such heavy chain variable domain-encoding transcripts have also been found in the IgE-encoding transcriptome of yet other grass pollen allergic subjects. Altogether these data argue that a common idiotype is used to establish specific antibodies with a potential to mediate allergic responses to Phl p 2. Such a restriction may contribute to the limited molecular diversity observed in some IgE populations.

Idiotype protein vaccination in combination with adjuvant cytokines in patients with multiple myeloma--evaluation of T-cell responses by different read-out systems.

Anti-idiotypic T cells were analyzed in myeloma patients (n=18) vaccinated with idiotypic protein together with the adjuvant cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF) and/or interleukin-12 (IL-12). In the group given IL-12/GM-CSF, 78% developed idiotype specific T cells as compared to 22% in the group given only IL-12 (proliferation/ELISPOT assays) (p<0.05). The percentage of immune-responding patients increased when quantitative real time polymerase chain reaction assays for cytokines were included. A predominance of a Th1 (IFN-gamma/TNF-alpha) immune response was noted in the IL-12 group while a Th2 (IL-5) response prevailed in the IL-12/GM-CSF group (p=0.053). Application of multiple read-out systems improved the characterization of the immune response.

Production of myeloid dendritic cells (DC) pulsed with tumor-specific idiotype protein for vaccination of patients with multiple myeloma.

BACKGROUND: Immunotherapy of cancer with DC vaccines has produced encouraging results in clinical trials. Antigen (Ag)-pulsed DC have elicited CD4+ and CD8+ T-cell immunity and tumor regression in humans. However, there is no standard method of DC production. The DC phenotype, number and Ag-loading process used in these studies have varied, making comparisons between trials difficult. METHODS: In the present report a reproducible method was developed for the production of a DC-based vaccine. Monocytes were enriched by adhesion from healthy donor apheresis products and cultured with growth factors for maturation into DC. The cells were loaded with the tumor Ag idiotype proteins from patients with multiple myeloma. DC culture and Ag loading were performed in an automated and closed system. The DC product was characterized for phenotype by flow cytometry and for function in Ag uptake and Ag presentation. RESULTS: These monocyte-derived DC expressed high levels of costimulatory molecules (CD80/86). Ag-pulsed DC functioned to induce allogeneic proliferative lymphocyte responses and Ag-specific cytotoxic T lymphocyte (CTL) responses. The DC viability, phenotype and function were well preserved following prolonged frozen storage. Aliquots from the product of a single DC preparation could be used for sequential vaccinations without batch to batch variability. DISCUSSION: Ag-pulsed DC can be reproducibly generated for clinical use. These standardized methods are now being employed for a clinical trial to evaluate idiotype-pulsed DC vaccine therapy following non-myeloablative transplant for the treatment of multiple myeloma.

Variable-region-identical antibodies differing in isotype demonstrate differences in fine specificity and idiotype.

A central tenet of the current understanding of the relationship between Ab structure and function is that the variable region domain is solely responsible for Ag specificity. However, this view was recently challenged by the observation that families of mouse-human chimeric Abs with identical V regions demonstrate differences in fine specificity and by reports of changes in Ab Id structure with isotype switching. Here we revisited this question by evaluating the reactivity of two families of murine IgG switch variants that differed in V region usage for Cryptococcus neoformans glucuronoxylomannan, glucuronoxylomannan peptide mimetics, and anti-Id mAbs. The results reveal isotype-related differences in fine specificities and Id for two mAb isotype switched families, thus establishing the validity of this observation with sets of homologous Abs. The results suggest that the C region affects V region protein conformation, leading to differences in fine specificity and Id. The finding that isotype can affect fine specificity has major implications for current concepts of the generation of secondary responses, idiotypic network regulation, and isotype function. Given that isotype class switching and Ig gene somatic hypermutation share molecular mechanisms, these observations unify these processes in the sense that both can alter specificity and affinity.

Adoptive transfer of anti-idiotypic T cells cure mice of disseminated B cell lymphoma.

There is extensive interest in idiotypic vaccination as a treatment of lymphoma. An alternative approach is the adoptive transfer of in vitro generated T cells. This strategy has been used to treat posttransplantation EBV-related diseases. The ability to generate in vitro T cells to peptides derived from immunoglobulin idiotypes raises the possibility of directly using such cells as a treatment of lymphoma. Investigating the adoptive transfer of specific T cells to idiotype derived peptides in a murine lymphoma model is therefore an important part of the clinical translation of this alternative approach. We have generated an idiotype-specific T cell line, able to recognise a defined, naturally processed idiotype-derived epitope. This line has been used to successfully treat mice with disseminated lymphoma supporting the clinical use of idiotype specific T cells.

Hypotension with intravenous immunoglobulin therapy: importance of pH and dimer formation.

Therapy with intravenous immunoglobulin preparations has been used effectively in a wide range of conditions. Although generally well tolerated, intravenous immunoglobulin preparations may be associated with transient hypotension in some patients. This study examined the role of different immunoglobulin G fractions in the development of intravenous immunoglobulin-induced hypotension in an anaesthetized rat model and assessed the effects of a new liquid immunoglobulin prepared at a low pH on both the formation of immunoglobulin G dimers and the development of hypotension. The effects of this new preparation in an experimental autoimmune encephalomyelitis model were also evaluated. Results from the haemodynamic studies indicated that immunoglobulin G dimers in polyclonal immunoglobulin G are responsible for the hypotensive events associated with some immunoglobulin preparations. They also showed that adjustment to an acidic pH results in the rapid dissociation of immunoglobulin G dimers and prevents the development of hypotension. Additional experiments demonstrated that only immunoglobulin G dimers with a functional Fc fragment can bind to Fcgamma receptors on macrophages to induce the release of blood pressure-lowering mediators. Moreover, essentially monomeric Fc fragments can block the blood pressure-lowering effects of immunoglobulin G dimers. Preparation of a new liquid intravenous immunoglobulin with the pH adjusted to 4.3 prevents the formation of immunoglobulin G dimers even over long-term storage and does not significantly affect blood pressure in a rat model. This preparation is as effective as other intravenous immunoglobulin preparations in ameliorating symptoms of experimental autoimmune encephalomyelitis. These results, like those from previous studies, indicate that preparation of intravenous immunoglobulin at a low pH substantially reduces immunoglobulin G dimerization; this effect significantly decreases the potential for intravenous immunoglobulin to induce hypotension without reducing its clinically relevant biological activity.

Rational design of peptide vaccines for autoimmune disease: harnessing molecular recognition to fix a broken network.

Autoreactive T-cells and antibodies are found at low levels in normal individuals and are thought to be kept at bay by regulatory T-cells and a network of idiotypic and anti-idiotype-bearing antigen receptors on lymphocytes as well as idiotypic anti-idiotypic antibodies. Disruption of this network by genetic, environmental and unknown factors is thought to result in autoimmune diseases. An obvious, ideal and specific therapy for such disorders would be to harness this regulatory network to re-establish immunologic homeostasis. In practice, however, this is not an easy task as most autoimmune diseases involve polyclonal responses to self antigen. Thus, we are faced with the conundrum of not knowing which autoreactive idiotype-bearing antibody or antigen receptor(s) to target in order to restore or induce network regulatory function. The thesis of this review is that understanding a fundamental property governing peptide/protein shape can be used in part to circumvent the problems of self reactivity and polyclonality in autoimmune disorders. More specifically, an algorithm has been developed to design peptide vaccines with shapes that are thought to be complementary in contour to self epitopes which seem to be the focus of autoimmunity. In theory, such complementary shapes should be engendered in certain autoreactive antigen receptors--these complementary constructs consequently represent receptor mimetics. By targeting an immune response against such mimetics, one generates a polyclonal anti-idiotype response that matches the complexity of the autoimmune response itself. This article will describe the algorithm for vaccine design, summarize the in vitro and in vivo evidence for its efficacy and discuss possible therapeutic utility in human autoimmune diseases.

Induction of cellular immunity by anti-idiotypic antibodies mimicking GD2 ganglioside.

Gangliosides are potentially useful targets for tumor destruction by antibodies. However, the role of gangliosides in T cell-mediated immunity to tumors is unclear. We produced three murine monoclonal anti-idiotypic antibodies (Ab2) against a monoclonal antibody (Ab1) that binds strongly to melanoma-associated GD2 ganglioside and weakly to GD3 ganglioside. All three Ab2 induced anti-anti-idiotypic antibodies (Ab3) with Ab1-like binding specificity to tumor cells and antigen in rabbits. The Ab3 specifically bound to GD2(+) tumor cells and isolated GD2, and shared idiotopes with the Ab1. Two of the three Ab2 induced GD2-specific delayed-type hypersensitivity responses in BALB/c and C57BL/6 mice, but not in C57BL/6/CD4(-/-) mice. Peripheral blood mononuclear cells (PBMC) from a melanoma patient proliferated specifically in response to in vitro stimulation with Ab2. Proliferation was accompanied by Th1-type cytokine production. Our studies demonstrate the induction of ganglioside-specific T cell-dependent immunity by Ab2 in mice. These T cells showed specific reactivity to ganglioside expressed by tumor cells.

Production and characterization of an anti-idiotypic single chain Fv that recognizes an anti-DNA antibody.

A well-characterized recombinant anti-idiotype to an anti-DNA antibody can be useful for studies of the regulation of anti-DNA-producing B cells. Using a hybridoma technique, a monoclonal anti-idiotypic antibody, designated O2F3, was obtained, and its scFv gene was constructed. O2F3 single chain Fv (scFv) was produced against an idiotope of a monoclonal anti-DNA antibody, 3D8, that was obtained from an autoimmune-prone mouse, MRL-lpr/lpr. Here we describe the production and in vitro characterization of the O2F3 scFv, and compare it with its parent monoclonal antibody, O2F3 IgM. To characterize O2F3 scFv and O2F3 IgM, we generated recombinant 3D8 fragments, including 3D8 scFv, 3D8 VH, and 3D8 VL, that were used as antigens in several assays. ELISA and Western blot analysis showed that both O2F3 scFv and O2F3 IgM recognized a conformational determinant formed by the association of the variable region heavy and light chains of the 3D8 antibody, suggesting that O2F3 scFv retained a similar binding pattern to its parent O2F3 antibody. The idiotope recognized by O2F3 was shown by competitive ELISA to be outside of the DNA binding site of the 3D8 antibody. This characterized O2F3 scFv could be applied for the regulation of anti-DNA antibody production and the manipulation of recombinant antibody-based proteins to which toxins, enzymes, and chemical agents can be connected.

Induction of cytotoxic T-cell responses against immunoglobulin V region-derived peptides modified at human leukocyte antigen-A2 binding residues.

Cytotoxic T-lymphocyte (CTL) responses can be generated against peptides derived from the immunoglobulin (Ig) V region in some but not all patients. The main reason for this appears to be the low peptide-binding affinity of Ig-derived peptides to major histocompatibility complex (MHC) class I molecules and their resulting low immunogenicity. This might be improved by conservative amino acid modifications at the MHC-binding residues of the peptides (heteroclitic peptides). In this study, it was found that in 18 Ig-derived peptides, that heteroclitic peptides from the Ig gene with improved binding to human leukocyte antigen (HLA)-A*0201 can be used to improve CTL responses. Amino acid substitution substantially increased predicted binding affinity, and there was a strong correlation between predicted and actual binding to HLA-A*0201. CTLs generated against the heteroclitic peptide had not only enhanced cytotoxicity against the heteroclitic peptide but also increased killing of antigen-presenting cells pulsed with the native peptide. Surprisingly, no difference was observed in the frequency of T cells detected by MHC class I peptide tetramers after stimulation with the heteroclitic peptide compared with the native peptide. CTLs generated against heteroclitic peptides could kill patients' tumor cells, showing that Ig-derived peptides can be presented by the tumor cell and that the failure to mount an immune response (among other reasons) likely results from the low immunogenicity of the native Ig-derived peptide. These results suggest that heteroclitic Ig-derived peptides can enhance immunogenicity, thereby eliciting immune responses, and that they might be useful tools for enhancing immunotherapy approaches to treating B-cell malignant diseases.

Architecture of idiotypic networks: percolation and scaling behavior.

We investigate a model where idiotypes (characterizing B lymphocytes and antibodies of an immune system) and anti-idiotypes are represented by complementary bit strings of a given length d allowing for a number of mismatches (matching rules). In this model, the vertices of the hypercube in dimension d represent the potential repertoire of idiotypes. A random set of (with probability p) occupied vertices corresponds to the expressed repertoire of idiotypes at a given moment. Vertices of this set linked by the above matching rules build random clusters. We give a structural and statistical characterization of these clusters, or in other words of the architecture of the idiotypic network. Increasing the probability p one finds at a critical p a percolation transition where for the first time a large connected graph occurs with probability 1. Increasing p further, there is a second transition above which the repertoire is complete in the sense that any newly introduced idiotype finds a complementary anti-idiotype. We introduce structural characteristics such as the mass distribution and the fragmentation rate for random clusters, and determine the scaling behavior of the cluster size distribution near the percolation transition, including finite size corrections. We find that slightly above the percolation transition the large connected cluster (the central part of the idiotypic network) consists typically of one highly connected part and a number of weakly connected constituents and coexists with a number of small, isolated clusters. This is in accordance with the picture of a central and a peripheral part of the idiotypic network and gives some support to idealized architectures of the central part used in recent dynamical mean field models.

Structural determinants of the human idiotype HibId-1.

The human antibody response to the capsular polysaccharide of Haemophilus influenzae type b is predominated by antibodies expressing a light-chain-associated idiotype designated HibId-1. HibId-1 is expressed by kappa light chains encoded by either the A2 or A18 variable region genes. In this report we use site-directed mutagenesis and molecular modeling to show that HibId-1 expression is determined by residues in the first and second complimentarity determining regions that are widely separated in the primary sequence, but closely juxtaposed by the tertiary folding of the mature light chain molecule. Of the known human light chains, only alleles of A2 and A18 encode these residues at these positions in their germline configuration. VIG10, a mouse monoclonal antibody of unknown specificity that expresses HibId-1, and 23F.2, an A2-utilizing Streptococcus pneumoniae 23F polysaccharide-specific human Fab fragment that lacks HibId-1, provide examples of the HibId-1 determinant both arising and being lost by somatic mutation. In addition, we show that the residues responsible for HibId-1 expression can be disassociated from those required for antigen binding.

Enhanced antitumor immunity by fusion of CTLA-4 to a self tumor antigen.

The idiotypic determinant (Id) of the immunoglobulin expressed by a B-cell malignancy can serve as an effective tumor-specific antigen but is only weakly immunogenic. This study demonstrates that the immunogenicity of the tumor Id protein can be dramatically increased by directing it to antigen-presenting cells (APCs). Cytotoxic T-lymphocyte antigen 4 (CTLA-4) present on activated T cells has a strong binding affinity to both B7-1 and B7-2 molecules, which are primarily expressed on APCs. After construction of a fusion protein consisting of Id and CTLA-4 (Id-CTLA4), mice immunized with the fusion protein induced high titers of Id-specific antibody and T-cell proliferative responses without adjuvants and were protected from lethal tumor challenge. The Id-CTLA4 fusion protein was so potent that even low doses (down to 0.1 microg) of the immunogen were able to elicit strong antibody responses. By using an Id-CTLA4 mutant protein, the ability to bind B7 molecules on APCs was shown to be required for the enhanced immunogenicity of Id-CTLA4. These findings demonstrate that fusing CTLA-4 to a potential tumor antigen represents an effective approach to prime antitumor immunities in vivo and may be applicable to the design of vaccines for a variety of other diseases. (Blood. 2000;96:3663-3670)

Secondary V(D)J rearrangements and B cell receptor-mediated down-regulation of recombination activating gene-2 expression in a murine B cell line.

It has recently become clear that recombination of Ig genes is not restricted to B cell precursors but that secondary rearrangements can also occur under certain conditions in phenotypically immature bone marrow and peripheral B cells. However, the nature of these cells and the regulation of secondary V(D)J recombination in response to B cell receptor (BCR) stimulation remain controversial. In the present study, we have analyzed secondary light chain gene rearrangements and recombination activating gene (RAG) expression in the surface IgM+, IgD- murine B cell line, 38C-13, which has previously been found to undergo kappa light chain replacement. We find that 38C-13 cells undergo spontaneous secondary Vkappa-Jkappa and RS rearrangements in culture, with recombination occurring on both productive and nonproductive alleles. Both 38C-13 cells and the Id-negative variants express the RAG genes, indicating that the presence of RAG does not depend on activation via the 38C-13 BCR. Moreover, BCR cross-linking in 38C-13 cells leads to a rapid and reversible down-regulation of RAG2 mRNA. Therefore, 38C-13 cells resemble peripheral IgM+, IgD- B cells undergoing light chain gene rearrangement and provide a possible in vitro model for studying peripheral V(D)J recombination.