Anti-NeuGcGM3 antibodies, actively elicited by idiotypic vaccination in nonsmall cell lung cancer patients, induce tumor cell death by an oncosis-like mechanism.

1E10 is a murine anti-idiotypic mAb specific for an idiotypic mAb that reacts with NeuGc-containing gangliosides, sulfatides, and Ags expressed in some human tumors. In melanoma, breast, and lung cancer patients, this anti-idiotypic Ab was able to induce a specific Ab response against N-glycosylated gangliosides, attractive targets for cancer immunotherapy as these glycolipids are not naturally expressed in humans. A clinical study with nonsmall cell lung cancer patients showed encouraging clinical benefits. Immunological studies performed in 20 of these patients suggested a correlation between the induction of Abs against NeuGcGM3 and longer survival times. The induced anti-NeuGcGM3 Abs recognized and directly killed tumor cells expressing the Ag, by a mechanism independent of complement activation. In the present work, we show that this cytotoxicity differs from apoptosis because it is temperature independent, no chromatin condensation or caspase 3 induction are detected, and the DNA fragmentation induced has a different pattern than the one characteristic for apoptosis. It is a very quick process and involves cytosqeleton reorganization. The Abs induce cellular swelling and the formation of big membrane lesions that allow the leakage of cytoplasm and the loss of the cell membrane integrity. All of these characteristics resemble a process of oncotic necrosis. To our knowledge, this is the first report of the active induction in cancer patients of NeuGcGM3-specific Abs able to induce complement independent oncotic necrosis to tumor cells. These results contribute to reinforcing the therapeutic potential of anti-idiotypic vaccines and the importance of NeuGcGM3 ganglioside as antitumor target.

Primary structure of IgE monoclonal antibodies expressing an intrastrain crossreactive idiotype.

We have obtained amino acid sequences (by mRNA and amino acid sequencing) for two IgE kappa mAb that have specificity for the Ars hapten group and are related to the major idiotypic family, CRIA (crossreactive idiotype A), in the A strain of mouse. One mAb, SE20.2, fully expresses CRIA; the other, SE1.3, possesses some but not all of the characteristic idiotopes. Both IgE proteins contain VH and V kappa segments that are closely related to those associated with CRIA. The D segment of SE20.2 is also typical of CRIA+ mAb, but that of SE1.3 is one amino acid residue longer. Chain recombination experiments indicated that the L chain of SE1.3 is fully capable of supporting CRIA expression. Its deficiency with respect to idiotopes of CRIA was attributed to the extra amino acid in the D region and/or substitutions in the VH segment. A major objective was to ascertain the frequency of somatic mutations in IgE. For the VH segment (amino acids 1-98) of SE20.2, there are only three nucleotide differences and one uncertainty with respect to the nucleotide sequence of the germline gene associated with CRIA. A somewhat higher frequency of substitutions is present in the VH segment of SE1.3. The VK amino acid sequences of the IgE proteins are nearly identical to those of a prototype of the CRIA family, mAb R16.7. The results are discussed with reference to the mechanism of the IgM to IgE switch.

Natural antibodies with the T15 idiotype may act in atherosclerosis, apoptotic clearance, and protective immunity.

The immune response to oxidized LDL (OxLDL) may play an important role in atherogenesis. Working with apoE-deficient mice, we isolated a panel of OxLDL-specific B-cell lines that secrete IgM Abs that specifically bind to oxidized phospholipids such as 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphorylcholine (POVPC). These Abs block uptake of OxLDL by macrophages, recognize similar oxidation-specific epitopes on apoptotic cells, and are deposited in atherosclerotic lesions. The Abs were found to be structurally and functionally identical to classic "natural" T15 anti-PC Abs that are of B-1 cell origin and are reported to provide optimal protection from virulent pneumococcal infection. These findings suggest that there has been natural selection for B-1 cells secreting oxidation-specific/T15 antibodies, both for their role in natural immune defense and for housekeeping roles against oxidation-dependent neodeterminants in health and disease.

Idiotype-dependent repertoire generation in the network system.

The diversification of the receptors of B and T lymphocytes is determined by stepwise gene rearrangement of the V genes and the D-J segment. However, this repertoire is not yet selected within the host environment and thus is referred to as the potential repertoire. The repertoire of the peripheral mature B and T-lymphocytes is distorted and selected from the potential repertoire by various auto- and foreign antigens in the host environment and is referred to as the available repertoire. It is well documented that the peripheral T-lymphocyte repertoire is first selected in the thymus to delete or inactivate self-reactive clones. Moreover, extensive studies done during the last decade suggest that the B- and T-lymphocyte repertoires are significantly influenced by the host immunoglobulin idiotype repertoire. In this review, the role of immunoglobulin idiotypes in the selection of the B- and T-lymphocyte repertoires is discussed.

Autoantibody idiotypy and neonatal B cell repertoire.

During ontogeny, antibody variable (V) regions are subjected to selection events at the level of B-cell clones bearing on their surfaces Ig molecules useful to the developing organism. Antigenic determinants of immunoglobulin V regions (idiotypes) are believed to play an essential role in molecular recognition and immune responsiveness to exogenous and self antigens. Recent data indicate that reactivity with self-antigens is prevalent within the natural neonatal repertoire, suggesting that self-antigens are involved in the selection and shaping of the immunologic repertoire. One implication of the above considerations is that V regions having regulatory idiotypes are borne preferentially on antibodies that react with self-antigens. Here, we report on the molecular characterization of the idiotype 62 expressed by BALB/c autoantibodies to a classic self-antigen, thyroglobulin. We show that under appropriate experimental conditions, Id62 can modulate the autoantibody response through a process requiring the activation of regulatory T cells. We also demonstrate that self--reactive V regions bearing Id62 are present in newborn mice and report on the primary structure of the V-region genes encoding Id62. Lastly, we provide evidence that hybridomas derived from unstimulated splenocytes of normal neonatal BALB/c mice express Ig molecules that react prevalently with self-antigens.

Current concepts and diagnostic evaluation of autoimmune disease.

This article discusses autoimmune reactions and the numerous mechanisms by which an autoimmune response may be initiated, including genetic factors, T-cell bypass mechanisms, and idiotypes. Human autoimmune diseases are classified into three main groups, ie, organ-specific, non-organ specific, and disorders with non-organ-specific autoantibodies with lesions restricted to one or a few organs, that are examined in detail. General laboratory tests and interpretation of results in relation to state or treatment of the particular disease, age and sex of the patient, and the sensitivity of the test system used are reviewed.

Revised immune network concepts.

The idiotype network concept needs to be revised in order to be in agreement with current data on protein/protein interactions, with the phenomenon of T and B cell recognition of idiotopes, and with the failure of certain anti-idiotypes to stimulate a given immune response. It is proposed that the distinction among Ab2 alpha, beta, and gamma is abandoned, as well as the concept of an internal image idiotope which mimics the three-dimensional shape of nominal antigen. In place of these definitions, the concept of "network antigen" is introduced. Network antigens are potentially the entire repertoire of anti-idiotypes. However, their biological effectiveness is controlled and established by two factors: (i) the affinity to the idiotype Ig receptor; and (ii) the preexisting regulatory network segment that controls the outcome of immune stimulation or suppression. Screening for effective idiotype therapeutic agents has to be done with panels of anti-idiotype and idiotype antibodies in order to establish correlations between idiotope expression and disease progression. Recognizing the importance of network segments will be the first step in the direction toward a rational design of idiotype-based therapies.

The servant idiotype network.

Network Antigens are idiotopic markers which are expressed on antibodies of a given specificity. These Network Antigen Idiotopes are part of specific immune responses and are selected through the process of antigen-driven maturation. Their natural role in immunity is the regulation of the response via idiotypic network interaction. The biological power of Network Antigens can be harnessed by generating monoclonal Ab2s against polyclonal disease-derived and specific Ab1. Because network antigens play an important biological role in immunity, it becomes difficult to call the Idiotype Network selfish. We believe that the organization and selection of a small antibody repertoire, which is needed to maintain a disease free existence, is accomplished by the idiotypic network. Furthermore, if we better understand the blueprint of this network we can begin to take advantage of the primed state of clones in the network to intervene, either to stimulate beneficial responses, or to suppress harmful immunities using antibodies which recognize the key organizer and controllers in the network.

Dynamics of a class of immune networks. I. Global stability of idiotype interactions.

This paper establishes the conditions under which a class of differential equations which appear in the study of immune systems (Varela et al., 1988a, In: Theoretical Immunology Part II. New Jersey: Addison Wesley), are globally stable. This is proved by adapting a Liapunov functional originally proposed by Cohen & Grossberg (1983, IEEE Transac SMC 13, 815-826) for competitive systems. The global stability thus obtained is valid on the fast time scale where only idiotypic interactions are relevant, thus excluding both lymphocyte proliferation processes and repertoire change via recruitment from immature bone marrow B cells.

Network regulation of the immune response: modulation of suppressor lymphocytes by alternative signals including contrasuppression.

In the preceding paper we demonstrated that comparison of alternative designs for the immune network can be used to examine the functional significance of specified interactions in normal immune responses. In this paper we examine mathematically the functional significance of three interactions affecting the production of suppressor lymphocytes involved in regulation of normal immune responses. The interactions examined in detail are 1) antigenic stimulation of the production of suppressor lymphocytes, 2) idiotypic stimulation of the production of suppressor lymphocytes, and 3) antigenic inhibition of the production of suppressor lymphocytes (i.e., contrasuppression). The results of our analysis suggest that an immune system with only antigenic stimulation of suppressor production is less effective than a system with both antigenic and idiotypic stimulation of suppressor production on the basis of all of the criteria examined in this study. In turn, the latter system is less effective than a system with only idiotypic stimulation of suppressor production. Furthermore, a system with both idiotypic stimulation and antigenic inhibition of suppressor production can be equal or superior to a system with only idiotypic stimulation of suppressor production on the basis of the same criteria. Similar conclusions hold for the comparison of systems in which regulation by the suppressor lymphocytes of interest is exerted upon production of effector molecules rather than upon production of effector lymphocytes, and also for the comparison of systems in which interactions affecting the production of suppressor factors are of interest.

Pathogenic anti-DNA idiotype (16/6 Id) in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is regarded as a classical autoimmune disease. Despite this belief, no one has been able to induce the disease in naive animals, neither with DNA nor with anti-DNA antibodies. We report on the induction of SLE in BALB/c mice following immunization with a pathogenic anti-DNA idiotype (16/6 Id) or its anti-Id. We also report on a specific treatment with T suppressor cells specific for the 16/6 Id. The induction of SLE in naive mice with a pathogenic anti-DNA Id suggests an additional mechanism for the diversity of manifestations in this disease.

Recognition of idiotypic structures in the thymus.

During the passage through the thymus, T cells are selected which recognize self major histocompatibility complex (MHC) antigens with low avidity. Whether the T-cell repertoire for recognition of altered self is also built up intrathymically or in the periphery, and whether it is determined exclusively by external antigens or is shaped by the internal environment is still a matter of debate. This question was addressed by analysing the responsiveness of thymocytes during post-natal development towards a nominal antigen [trinitrophenyl (TNP)] and an anti-TNP monoclonal antibody (Sp6), which carries a recurrent idiotype. During the first weeks of life, in vitro cultures of thymocytes proliferated strongly in the absence of nominal antigen. Proliferation rates were not increased by the addition of nominal antigen [TNP-ovalbumin (OA)], but a significant increase was noted in the presence of Sp6, thymocytes recognizing the processed immunoglobulin. After in vivo stimulation with TNP conjugates, 'antigen-specific' clones could also be detected in the thymus, the frequency of clones proliferating in response to Sp6 being further augmented. With increasing age, the proliferative capacity of thymocytes from unstimulated and antigenically stimulated mice decreased significantly. Responsiveness of spleen cells (SC) differed in some respects. The response towards Sp6 decreased with age, while antigen-specific clones were detected at increasing frequencies during post-natal development. Furthermore, after antigenic stimulation, the frequency solely of antigen-specific, but not of Sp6-specific clones was increased. Thus, it appears that the T-cell repertoire is shaped already during the intrathymic passage, being influenced primarily by the B-cell repertoire and modulated further by external antigen.

Regulation of antibody secretion by hybridoma cells. II. Mechanism of idiotype-induced suppression of antibody secretion by hybridoma cells.

We have previously reported that antibody secretion by B.22 hybridoma cells can be suppressed in an MHC-restricted manner, by idiotype-specific T cells. It was shown that T cells of both helper and suppressor phenotypes are involved, and that the suppression is mediated by soluble factors. In the present paper, we have characterized the effects of T-cell-mediated suppression at the level of B.22 antibody mRNA expression and stability. Nuclear run-on analysis comparing suppressed and control B.22 cells indicates no change in the transcription rates of heavy and light chains. Northern blot analysis demonstrates that steady-state levels of heavy and light chain mRNAs are also unchanged. Thus, the suppression of antibody secretion by B.22 cells probably occurs at the levels of translation or secretion.

Inhibitory and stimulatory signaling via immunoglobulin receptors: dichotomous responses elicited in clonal B cell populations.

The paradox that cross-linking of IgM antigen receptors on B cells by native or surrogate antigen can inhibit, as well as stimulate, B cell functions has previously been attributed to changes during maturation and activation, programming either a negative or a positive response at defined developmental stages. In contrast to this concept, we show here that some B cells possess the potential for both types of response at the same stage. In three clonal malignant human B cell populations, bivalent soluble monoclonal antibodies to IgM or idiotype, but not IgD completely inhibited spontaneous DNA synthesis, but significantly induced [3H]thymidine uptake when coupled to insoluble compounds. In co-incubation experiments mitogenic stimuli were dominant over inhibitory ones and were still effective after prolonged pretreatment of the B cells with inhibitory reagents. Ionomycin, known to increase intracellular calcium levels, and low doses of phorbol ester, described to activate protein kinase C, also suppressed DNA synthesis. High doses of phorbol ester alone or in combination with ionomycin, however, induced DNA synthesis in two of the lymphomas. We conclude that some B cells may respond to cross-linking of surface IgM in a dose-dependent manner so that all signals increase DNA synthesis once a threshold has been reached. These dose-dependent effects may in part involve signaling via breakdown of membrane inositol phosphates.

A new strain-specific cross-reactive idiotope with possible regulatory function expressed on BALB/c anti-alpha (1-3) dextran antibodies.

We obtained a mAb, B5, defining a major crossreactive Id, the B5 Id, having an in vivo regulatory function. BALB/c mice immunized against the bacterial dextran Ag B1355S produce lambda 1-bearing anti-alpha (1-3) glucosidic linkage of dextran B1355S (DEX) antibodies. Of these antibodies 30% expressed the B5 Id determinant. As was observed for the IdX-Id, B5 idiotope expression was also linked to the BALB/c H chain allotype. The antibody family expressing the B5 Id included IdX+ as well as IdX- defined monoclonal anti-alpha (1-3) DEX antibodies. Injection into BALB/c mice of mAb B5 conjugated to keyhole limpet hemocyanin led to enhancement of serum anti-alpha (1-3) DEX antibody synthesis, and to a strong increase in B5+ Ig lacking dextran-binding activity. IdX+ serum Ig were also increased in these mice. Injection of this mAb into allotype congenic nonresponder C.B20 mice induced a significant serum lambda 1-bearing anti-alpha (1-3) dextran antibody response (5 to 50 micrograms/ml). However, in A/J nonresponder mice, a similar treatment induced only the dextran-nonbinding component, which reached high levels (approximately 7 mg/ml) in the sera of these mice. In the majority of these A/J mice, we detected a specific anti-(4-hydroxy-5-iodo-3-nitrophenyl)acetyl antibody response characterized by B5 Id-positive antibodies. This observation can be explained by an in vivo regulatory idiotope-mediated cross-regulation.

The role of the 16/6 idiotype network in the induction and manifestations of systemic lupus erythematosus.

Experimental systemic lupus erythematosus (SLE) has been induced in mice by immunization with either a human anti-DNA mAb bearing a common idiotype (Id) designated 16/6 Id (antibody 1, Ab1) or with a murine anti-16/6 Id mAb (Ab2). In the present study a murine mAb (5G12-4, Ab3) that bears the 16/6 Id and binds to DNA was produced and was found to bind rabbit anti-16/6 Id sera and murine anti-16/6 Id mAb similarly to the human mAb 16/6 Id (Ab1). Moreover, mAb 5G12-4 was shown to share T cell epitopes with the human 16/6 Id mAb, since lymph node cells of mice immunized with the mAb 5G12-4 proliferated significantly to the human 16/6 mAb and vice versa. Following immunization of mice with the murine mAb bearing the 16/6 Id, antibodies to dsDNA, ssDNA, 16/6 Id, anti-16/6 Id, and to HeLa nuclear extract proteins were detected, similarly to those observed previously upon immunization with Ab1 or Ab2. Six months following the immunization, the mice exhibited leukopenia, increased erythrocyte sedimentation rates, and proteinuria. Examination of the kidneys of the mice disclosed immune complex deposits, thickening of the Bowman's capsule and glomerular necrosis. These results show the importance of the 16/6 Id network in the induction and progression of SLE in mice.

Detection of a regulatory idiotype on a spontaneous neonatal self-reactive hybridoma antibody.

To investigate the physiologic relevance of an idiotype-driven regulation of the immune system, we began a search for spontaneous self-reactive hybridomas in neonatal BALB/c mice. We sought hybridoma antibodies reactive with thyroglobulin (Tg) and expressing Id62, a recurrent idiotype with regulatory properties borne on induced adult autoantibodies to Tg. We describe herein such a neonatal Tg binding/Id62-positive monoclonal antibody (mAb) B10H2, and compare it with prototype mAb 62, an adult Tg-binding/Id62-positive mAb. Both mAb react with the same Tg epitope, as demonstrated by their abilities to totally inhibit the binding of the other to Tg. Moreover, as assessed by a double-reciprocal plot of their binding to Tg, their relative affinities for Tg are comparable. Likewise, mAb B10H2 appears idiotypically similar to mAb 62 because it totally inhibits the binding of mAb 62 to homologous anti-idiotype. Finally, as previously shown for mAb 62, mAb B10H2 expresses Id62 independently on both heavy (H) and light (L) chains, as evidenced by the immunoblot binding of a specific anti-Id62 probe to separated H and L chains. By molecular genetic analysis both antibodies appear to have made use of a member of the same VH 7183 gene family. Altogether, these findings suggest that neonatal mAb B10H2 and adult mAb 62 are very similar if not identical, and regulatory idiotype Id62 may be germline encoded. Furthermore this observation supports, in general, the concept of an idiotype-driven regulation for autoimmune responses.