1INSERM Unit 395, BP 3028, F-31024 Touluse Cedex, France.
²Biovector Therapeutics, BP 169, F-31676 Cedex, France.

Jorge Carneiro

The class differentiation and cross-regulation of Th lymphocytes, for which the Th1/Th2 dichotomy is the main paradigm, is believed to play a major role in determining the behavior of the immune system, and in the fate of the immune response in particular. It might provide the cellular basis for dominant or infectious tolerance, and in this way it may represent a target for autoimmune therapy or the control of organ transplantation. Although some critical components of the mechanisms of class regulation have been identified by thorough molecular analysis, the organization of those mechanisms, and how they are reflected in the global function of the immune system remains elusive. The present work tries to identify what regulatory mechanism operate at the cell population level, trying to bridge the gap between the information at the molecular level and the observations at higher levels. Empirical evidence has suggested the type of APC-B lymphocyte, macrophage or dendritic cells (DC)-influences and may determine which class of Th cells dominates an immune response. This simplistic view, however, was recently proven to be ellusive by two independent reports indicating that Th1 or Th2 cell lines can be generated in vitro under different doses of antigen [Hosken, et al. J Exp Med 1995;182:1579; Constant, et al. J Exp Med 1995;182:1591]. We previously proposed [Carneiro Int Immunol 1995;7:1265] a model for Th1/Th2 population dynamics that predicted this dependence on antigen dose. This model called into action, together with other direct lymphocyte-lymphocyte interactions, the influence of cytokines produced by Th lymphocytes on the selectivity and function of their APCs. In the present work, we study what type of behavior can be expected solely on the basis of such indirect regulation via modulation of APC, neglecting any other cross-regulatory influences.

We propose a minimal model for the population dynamics of two classes of differentiated Th lymphocytes, which are stimulated by a single population of APCs. The APCs can be in two functional states which are interconvertible. The conversion of the APC from one state to another is determined by its interactions with Th cells. The APC, depending on its functional state, will interact differently with (selectivity) and stimulate (function) the cells from either class of Th cells. The model is implemented as an explicit set of coupled ODEs. The parameters under which the model can account for the impact of the antigen dose on the generation of Th1 and Th2 cells are identified and discussed. These and other theoretical results are compared with empirical data sets available in the literature, in different domains of application of the Th1/Th2 paradigm.

The general significance of the main postulate that immune regulation can be mediated by factors produced by T lymphocytes that tune the functional state of their APCs is discussed. Special attention is payed to the phenomena known as dominant tolerance.

Theoretical Biology. Institute Gulbenkian of Science. Oeiras. Portugal. Theoretical Biology and Bioinformatics. University of Utrecth. The Netherlands.

DA Chow, H Wang, ZY Zhang

Extensive evidence, including passive transfer studies supports a role for polyclonal serum natural antibody (NAb) acting as a mediator of natural resistance against tumors in mice However, little is known about their mechanisms of action or about the phenotype of susceptible cells. The expression of NAb binding structures appears to be elevated by constitutive increases in the basal activation of PKC in both a ras-transformed and a preneoplastic rat PKC-B1 overexpressing model in 10Ti C3fI fibroblasts. This, coupled with corresponding decreases in expression of membrane PKC-a and NAb binding in confluent IOTi cells raised the possibility that in general, cells activated through PKC are NAb sensitive. Together with the increased in vivo elimination of the high NAb binding PKC-B1 overerexpressing cells, the data extend the support of a role for NAb in tumor surveillance to defend against preneoplasia and argued for NAb contributing to the homeostasis of the organism. Tumor promoter generation of variants and flow cytometry selection for high Nab binding L5178Y-F9 cells was correlated with selection for reduced tumorigenicity and increased the expression of CD4SRA and higher molecular weight isoforms of CD45. Considering the transient increased expression of CO45RA soon after T-cell activation, the most NAb-sensitive cells may be those passing through the early stages of activation

Department of Immunology, University of Manitoba, Winnipeg, MB, Canada, R3F OW3.

Cindy Wong, Jean-Pierre Mach, Ricarda Finnern

Efforts are being made to develop anti-cancer agents with the ability to distinguish between normal and tumor cells. The mucin MUC-1 is overexpressed in adenocarcinoma e.g. breast and ovarian, and due to aberrant glycosylation of the core protein new epitopes are being exposed, making this a suitable target for tumor therapy. So far, only murine antibodies have been described for the targeting of MUC-1 expressing tumors, but they do have limitations for their use in human immunotargeting and immunotherapy. Targeting of tumors with human single chain Fv (scFv) antibody fragments may overcome some of the limitations of murine antibodies. We isolated and characterised human scFv which bind to the MUC-1 core protein using the phage display technology. The described human scFv have been selected directly on a MUC-1 expressing breast adenocarcinoma cell line for binding to the core protein of MUC-1.

University of Laussane, Department of Biochemistry, Laussane, Switzerland.

AM Vázquez, R Pérez, A Pérez, AM Hernández, M Alfonso, G Bombino, I Beausoleil

Five mouse anti-idiotypic (Ab2) MAbs were generated against a mouse anti-NeuGc containing-ganglioside MAbs (P3 MAb). These IgG1 Ab2 MAbs were able to block the binding of the Ab1 MAb to the NeuGcGM3 ganglioside and to a breast human carcinoma cell line recognized by P3 MAb. Most of them produced strong anti-anti-idiotypic antibody (Ab3) response in syngeneic mice and three of these Ab2 MAbs induced antibodies bearing P3 MAb idiotopes, but they failed to elicit Ab3 antibodies with the same specificity as P3 MAb. These results demonstrate that these Ab2 MAbs are not internal image antibodies and may be classified as g-type Ab2 MAbs. The capacity of one of these Ab2 (1E10 MAb) to induce an Ab3 response in monkeys was tested and the administration of this Ab2 developed a predominant anti-idiotypic response, indicating the immunodominance of the idiotypic determinants of 1E10 MAb. Preliminary results showed that 1E10 MAb was able to inhibit pulmonary metastases in murine models.

Hybridoma Group. Department of Antibody Engineering. Center of Molecular Immunology, Havana, Cuba.

C Jancin, E Chuluyan, A Morelli, A Larregina, M Saracco, M Barboza, L Fainborn

Dendritic cells (DC) are professional antigen presenting cells that can prime naive T helper lymphocytes and elicit cytotoxic T lymphocyte responses to soluble antigens. In vitro antigen-pulsed DC can induce antigen-specific T cell responses, supporting the potential use in clinical immunotherapy. Indeed, tumor regression has been observed after vaccination with DC pulsed with lymphoma specific idiotypes.

Based on their critical role in antigen presentation and their low number in peripheral blood (1% of mononuclear cells) there have been several efforts to establish in vitro cultures of DC to be used in clinical trials, Currently, it is possible to generate DC from bone marrow, cord blood or adult peripheral blood by applying appropriate combinations of growth factors: GM-CSF, IL-4, TNF a, Fit3 ligand and IL-13. DC obtained under different protocols have been thoroughly phenotypically and functionally studied. Most functional studies have examined the ability of monodentritic cells (moDC) to stimulate mixed reaction lymphocyte (MRL). However, none of these studies have tested the capacity of these cells to interact with the extracellular matrix or endothelial cells. The aim of this study was to further compare the phenotypic characteristic of the skin derived DC (sDC), with DC derived from monocytes cultured with IL-4 and GM-CSF and to evaluate their adhesion to fibronectin (FN) and endothelial cells. Since these cells are intended to be used for immunotherapy, we also compared the phenotype and functional behavior of moDC cultured with 10% heat inactivated human serum (HS) or fetal bovine serum (FBS). We examined the phenotypic characteristic of skin dendritic cells sDC and moDC generated under different culture conditions and their interactions with fibronectin FN and endothelial cells. The moDC were obtained after culturing monocytes with GM-CSF (800U/mL) and IL-4 (500U/mL), with either 10% FBS or 10% allogeneic HS. Regardless the serum used, most moDC expressed HLA-DR, and CD86. On day 5 of incubation, 20-67% of the moDC cultured in the presence of HS (HS moDC) showed the presence of CD1a, b and c vs 94-97% when cultured in presence of FBS (FBS moDC). DC showed a differential gradient of adhesion to FN; FBS-moDC>sDC-monocyte. Both FBS and HS-moDC were strongly positive for CD49e (a4 integrin), a MAb against CD 49e blocked the adhesion of both types of moDC.

Laboratory of Immunogenetics, Hospital de Clínicas, School of Medicine, University of Buenos Aires, Argentina.

A Macías, S Arce, R Bolaños, J León, L Suárez, R Pérez, A Lage

The manipulation of idiotypical interaction has shown to be of real value, as suggested by the success reported for the treatment of various autoimmune diseases with a human IgG pool, containing an important fraction of idiotipically connected natural autoantibodies.

We have obtained highly connected anti-idiotypic MAbs by immunizing Balb/c mice with an antiGM2 specific IgM antibody. The anti-idiotypic MAbs B7 and 34B7 belong to the IgG2a and IgG1 subclass. Both were classified as "alfa" anti-idiotypes. However, coupled to KLH and injected in Balb/c mice they induced an antigen-independent B-cell secreting serological antibody response (AB1') against the gangliosides, but not against other antigens, suggesting the activation of ganglioside multi-specific natural autoantibodies.

These anti-idiotypes showed other striking properties, such as heterogeneous binding to the variable region of murine and human immunoglobulins, and some of the in vitro effects reported for the human immunoglobulin pool.

Preliminary results showed that intravenous injection of high doses of these MAbs induced reduction in the number of lung metastases in C57Bl/6 mice inoculated with melanoma B-16 and Lewis lung carcinoma tumor cells.

These results suggest the assess of the possible use of these multi-specific anti-idiotypes as an immunoglobulin pool surrogate.

Center of Molecular Immunology, Havana, Cuba.

Ernesto Moreno

Several approaches have been developed to dock relatively small ligands to protein receptors. Few of them include some degree of protein flexibility. In this study, a new method was developed to screen a large amount of receptor conformations for ligand binding. These conformations were generated via molecular dynamics applied on a large number of residues, including backbone atoms. The DOCK 3.5 program, which deals with a rigid receptor, was taken as a basis to develop a program called DOCKdyna, where this method was implemented. Important modifications were made in certain essential parts of the DOCK algorithm and new features were added. In particular, a fast and totally automated method was developed to represent the geometry and physico-chemical properties of the receptor binding site. Ligand flexibility was also included.

The program was tested on three model systems taken from the protein data bank: the GGBP-glucose (2GBP) complex, the MC603 antibody complexed with phosphocholine (2MCP) and the BR96 antibody complexed with Lewis Y (1CLY). More than 50 residues (~800 atoms) in the binding site region were allowed to move during the dynamic simulations and were subsequently included in the calculations.

For each system, one thousand receptor coordinate sets were collected and screened for ligand binding in about 24 h, using an average workstation. In the three cases, most of the best scoring solutions given by the program had very nearly a crystal geometry and a post-docking energy relaxation of these solutions further improved the results. Thus, in the screening process the ligand picked out those receptor conformations that were more appropriate for binding, implicitly taking into account the induced fit that should occur in nature. The speed of the method makes it very promising for structure prediction via docking.

Center of Molecular Immunology, Havana, Cuba.
Institute of Medical Biochemistry, University of Gothenburg, Medicinaregatan 9A, S-41390, Gothenburg, Sweden.

Robert Lechler

In many strain combinations, passenger cell-depleted rodent organ allografts are accepted in the absence of any immunosuppression, while allografts containing bone marrow-derived antigen-presenting cells are promptly rejected. These observations highlighted the crucia1 distinction between antigenicity and immunogenicity. Furthermore, the prolonged residence of an accepted allograft induces a state of profound, donor-specific tolerance in many of these experimental models. We have recently observed the same phenomenon in clinical transplant recipients, in that the frequency of anti-donor, IL-2-secreting Tcells was markedly lower than that against equally mismatched third party cells in 40% of the transplanted patients. The invitro analysis provide a mechanistic explanation for these findings, in that alloantigen presentation by primary cultures of gamma-interferon-treated epithelial cells from the thyroid or the kidney induced allospecific Tcell unresponsiveness. This appeared to result from Fas-mediated deletion in naive, and the induction of anergy in memory T-cells.

The other aspect of the tolerant state induced by the prolonged residence of an allograft is that it is actively maintained, as judged by the fact that it can be transferred to a naive animal by CD4+ Tcells. The mechanisms of transferrable tolerance remain a matter of debate. At least two mechanisms may contribute; first, cytokines such as TGFb and IL-10 may regulate potentially inflammatory cells. This mechanism of regulation is well supported in several models of autoimmune diseases, however it fails to provide a satisfactory explanation for transplantation tolerance in that there is hardly evidence to suggest that Th2 bias occurs in tolerant recipient animals. An alternative mechanism invokes regulation by anergic Tcells; this may simply reflect their capacity to compete with responsive Tcells for the APC surface, and for locally produced growth factors. or it may reflect a more active mechanism dependent upon cell-cell contact.

These mechanisms leading to the induction and the maintenance of Tcell tolerance may play important physiologica1 roles in the regulation of autoreactive Tcells. Equally these mechanisms may interfere with attempts to induce anti-tumor immune responses. Means of circumventing these obstacles merit discussion in the design of strategies to target Tcell effector mechanisms against tumors.

Department of Immunology, Division of Medicine, Imperial College School of Medicine, London.

E Montero, F Quintana, A Rojas, I Beausoleil, E Renjifo, JF Amador, R Pérez, A Lage

The autoimmune recognition of tumor antigens is emerging as a paradigm with implications to induce tumor immunity as autoimmunity. From this view, the main question is essentially how to break the state of immune tolerance or ignorance in order to induce tumor rejection.

Analysis relying on a recessive model of natural tolerance limits the approach to try to activate peripheral lymphocytes increasing co-stimulatory signals or using modified self antigens for immunization. Recently, non-deletional or dominant mechanisms have been receiving experimental support. It has been shown that healthy adult individuals contain effectors and regulatory autoreactive cells protecting autologous tissues from immune aggression by pathogenic autoreactive cells.

Derived from this analysis, the question was addressed whether the immune system can actively respond to normal tissue autoantigens expressed by cancer cells and to induce a specific autoimmunity against tumor cells and their growth arrest following the removal of regulatory Tcells that suppress available immunocompetent autoreactive cells capable of destroying tumor cells. In this regard, the effects of immunosuppression with Cyclosporin A and MAbs anti-CD4 and anti-CD8 on syngeneic Balb/c Ehrlich ascites tumor cell growth were evaluated in Balb/c or Balb xid mice. Tumor growth arrest as well as both survival and life span interval increases were obtained in treated animals compared to the control.

The issue was evaluated regardless of whether the process of peripheral expansion of lymphocytes is driven by antigens and whether it is possible to fulfill the "lymphoid space" skewed towards selected specificities. In this regard a selected immunosuppression was induced with MAbs combined with human epidermal growth factor (hEGF) vaccination to learn the effects on the regenerated repertoire. High antibody titers on hEGF were obtained in mice immunosuppressed with anti-CD4 and lower with anti-CD8. Titers of antibodies to murine (self) EGF were also found.

These studies fit into the dominant model of natural tolerance and support a model of effective manipulation of natural autoreactivity against dominant self-antigens on tumor cells as well as mechanisms able to directionally generate specific immune responses against selected antigens.

Center of Molecular Immunology, Havana, Cuba.

Pedro J Ruíz,¹ Roland Wolkowicz,² Ari Waisman,² David L Hirschberg,¹ Pnina Carmi,² Netta Erez,² Hideki Garren,¹ Johannes Herkel,² Marcela Karpuj,² Lawrence Steinman,¹ Varda Rotter,² Irun R Cohen²

1Department of Neurology and Neurological Sciences, Stanford University Medical Center, Stanford, California, 94305, USA.
²Departments of Cellular and Molecular Biology, and Immunology, Weizmann Institute of Science, Rehovot, 76100, Israel.

L Fernández, A Carr, G Marquina, Z Mazorra, F Estévez, A Mullet, O Valiente, R Pérez

Although thousands of patients have received a variety of cancer vaccines over the last 25 years, significant tumor responses are only occasionally observed. Many tumor antigens have been described at molecular level in these years but the main problem with human cancer antigens is their poor immunogenicity. This is particularly true for most carbohydrate tumor antigens, which are both Tcell independent and "self" antigens.

The mechanisms by how "self" carbohydrates are ignored by the immune system remains unknown and usually the attempts to reproduce the T dependent transposition successfully applied to bacterial polysaccharides fails with own gangliosides and other saccharides.

N-glycosylated gangliosides are absent from human tissues, hence, they were considered as potential tumor "specific" antigens provided they were present in neoplastic cells. We recently found that in invasive ductal carcinomas of the breast the average expression of the NGcGM3 ganglioside is about 107 molecules/cell, and then designed, for the first time, a heterophile ganglioside cancer vaccine project for human cancer therapy.

NGcGM3 was hydrophobically incorporated to the outer membrane protein complex (OMPC) of Neisseria meningitidis and a soluble complex vaccine was obtained. Inmunization of chickens (in which NGcGM3 is also a heterophile antigen) with OMPC/NGcGM3 vaccine + Montanide ISA 51, induced unusually high titers of IgG specific antibodies. Moreover, the chicken antisera showed a powerful cytocidal effect against the murine myeloma X63 cell line that depended not only on the complement.

Additionally, the murine monoclonal antibody 14F7 (IgG1 vs NgcGM3), generated with our immunization strategy, also showed a powerful cytocidal effect against the murine myeloma X63 cell line in vitro and antitumor activity in vivo.

These results suggest that the combination of an appropriate strategy of xenogenization (hydrophobic rather than covalent conjugation) with the heterophilic character (non-self) of NGcGM3 could provide a highly efficient way to fight against circulating breast tumor cells and micrometastases.

Center of Molecular Immunology, Havana, Cuba.

PCL Beverley, H Drakesmith, D O'Neil, S Schneider, E Sercarz, B Chain

Identification of the target antigens of cytotoxic T lymphocytes in murine and human tumours, has shown that many are unaltered tissue specific differentiation antigens. The majority of Tcells with high affinity for self should be deleted in the thymus so that it might be difficult to generate a powerful immune response against epitopes of these molecules normally produced by antigen processing. However, here we show that under experimental conditions responses to cryptic epitopes can be generated.

Dendritic cells (DC) can be grown from mouse bone marrow using GM-CSF and these cells are capable of processing the model antigen hen egg lysozyme (HEL), for its presentation to MHC class II-restricted Tcell hybridomas. We have found that a pre-treatment of the DC overnight with interleukin- 6 (IL-6) prior to pulsing with HEL, alters the specificity of antigen processing so that responses to normally dominant epitopes may be lost and those to cryptic epitopes revealed. We also show that when mice are injected with IL-6 treated DC pulsed with HEL or with antigen plus IL-6- in incomplete Freund's adjuvant, responding Tcells from the draining node or spleen show altered specificity consistent with the in vitro results.

These results demonstrate that manipulation of the specificity of antigen processing in the class II pathway is possible and can reveal responses to cryptic epitopes. We suggest that tolerance to cryptic self epitopes may be incomplete and the manipulation of the specificity of responses to tumor antigens might be useful in tumour immunotherapy. Responses to cryptic epitopes may also play a role in autoimmunity.

The Edward Jenner Institute for Vaccine Research, Compton, Berkshire RG20 7NN, UK.

Kalet León, Jorge Carneiro, Enrique Montero, Rolando Pérez, Agustín Lage

Here we summarize our results in the study of immunological tolerance throughout the development of a mathematical model of the immune system. Our general approach is to conceive tolerance as an active dominant phenomena related, at least partially, to the intrinsic non-linear dynamics of the immune system, which is a consequence of complex specific interactions involving variable region molecules.

This work was based on a mathematical model developed by Carneiro, et al. in 1996, in which special attention is devoted to the interface of co-operation between T and B lymphocytes. Although this model allowed to address some important questions related to the organisation of the immune system, it had certain limitations in addressing dominant tolerance.

In a first attempt to tackle some of these limitations, two major modifications were introduced: the average connectivity is now an explicit function of time, based on empirical evidence; and the antigen-driven activation of T lymphocytes is described by a log-bell shaped dose response curve. The modified model provides a new interpretation for tolerance induction during the neonatal period, and for the adult tolerance induction by low or high doses of antigen.

Further development is obtained by modifying the treatment of Tcell behavior in the model. The existence of several Tcells for each antigen is allowed, APCs are considered a limiting resource for Tcell activation, T-B cell co-operation process also stimulate Tcell proliferation and three different reversible functional states for Tcells are introduced. The new modified model suggests: 1) the need of a diverse self recognition in order to tolerate self antigens; 2) low levels of immune suppression of helper, but not effector cells, can induce adult tolerance and 3) tolerance transference can be mediated either by helper or effector cells.

Our studies propose that dominant tolerance can be explained by the intrinsic and complex dynamics of the immune system, based on a strong competition between its different components.

Center of Molecular Immunology, PO Box 16040, Havana 11600, Cuba.

Normando Iznaga-Escobar, Tania Crombet, Juan Félix Amador, Rolando Pérez

The use of antibodies as targeting agents for the delivery of radioisotopes to tumors is an appealing concept that has been receiving widespread attention since the advent of monoclonal antibody technology. Cell directed radiation therapy (CDRT or Radioimmunotherapy) has the potential of killing cells within a tumor whether or not they express the antigen to which the antibody binds. However, characteristics and distribution of target antigens remain crucial considerations in the development of monoclonal antibody based therapies.

In the present work the epidermal growth factor receptor (EGF-R) and the o-lynked glycoprotein carbohydrate chain tumor associated antigen (TAA ior C2) have been evaluated as potential targets for CDRT. The EGF-R and the o-lynked glycoprotein carbohydrate chain TAA ior C2 are highly expressed on the surface of various tumor types (e.g breast, lung, bladder and brain tumors for the EGF-R and colon and rectum tumors for the TAA ior C2), but are present to a much lesser degree on normal human tissues. Therefore, monoclonal antibodies to murine EGF-R ior egf/r3 and humanized h-R3 and to TAA ior C2 murine ior C5 are increasingly observed as important biological reagents that once coupled with beta radioisotopes will complement the group of existing cytotoxic drugs for cancer treatments.

This study provides information on pharmacokinetics, normal organ biodistribution, absorbed radiation doses to normal organs and tumors and toxicity of anti-EGF-receptor and anti-TAA ior C2 antibodies labeled with 188Re. A review on TAA as a pharmacodymanic concept has been carried out.

Center of Molecular Immunology, Havana, Cuba.

G González,¹ B Sánchez,¹ I Beausoleil,¹ OL Pardo,² JL García,² T Crombet,¹ M Catal,³ JC Hernández,³ V Mirabal,³ Y González,¹ P Marinello,¹ A Domarco,¹ G Guillén,² R Pérez,¹ A Lage¹

1Center of Molecular Immunology, Havana, Cuba.
²Center for Genetic Engineering and Biotechnology, Havana, Cuba.
³Centro de Investigaciones Médico-Quirúrgicas, Havana, Cuba.

Copyright 1998 Elfos Scientiae