Idiotype variants emerging after anti-idiotype monoclonal antibody therapy of a murine B cell lymphoma

One of the difficulties encountered with the treatment of human B cell malignancies with anti-Id antibodies is the emergence of Id variants. The current study was designed to investigate this phenomenon further by using the murine B cell lymphoma model 38C13. Tumors were harvested that developed despite treatment with the anti-Id antibody S1C5 in mice inoculated with 38C13 cells and evaluated by immunofluorescence. Various phenotypes were found among escaping tumor cells. Some cells continued to react with S1C5 whereas others lost S1C5 reactivity. Among these latter cells, some continued to express surface IgM kappa, whereas others no longer expressed surface mu or kappa. After Id variant cell lines were established, immunofluorescence and ELISA of cell lysates from the surface IgM kappa- lines revealed persistent intracellular mu H chain but no detectable kappa. Surface IgM kappa+ lines were fused with myeloma cells and the Ig proteins secreted by the resultant hybridomas analyzed. The apparent m.w. of the mu-chains of these rescued Ig was the same as wild-type 38C13, whereas the kappa-chains were either the same or different in m.w. from the wild type. The IgM kappa of the variant line, T3C, weakly reacted with S1C5 and did not react with other anti-Id antibodies. The IgM kappa of the other variants were nonreactive with all the antibodies. Immunofluorescence of these surface Ig+ variants confirmed this finding. Some of the surface Ig+ and Ig- variant lines grew identically to wild-type tumor in vivo, but only the weakly S1C5-reactive variant T3C was inhibited in its growth by S1C5. Moreover, T3C was the only one of these lines capable of being lysed in vitro with S1C5 by antibody-dependent cellular cytotoxicity. Further studies revealed that surface Ig+ and Ig- variants emerge in escaping tumors with similar frequency and that these variants represent a major mode of tumor escape from anti-Id treatment in this model.     

Idiotype shifts caused by neonatal tolerance to phosphorylcholine

The injection of as little as 0.5 microgram phosphorylcholine-(PC) conjugated mouse immunoglobulin into BALB/c neonates within 48 hr of birth results in complete unresponsiveness to PC for 3 to 4 wk. Thereafter, anti-PC responses can be detected in tolerized animals, but these responses differ significantly from those of normal BALB/c mice. First, the magnitude of responsiveness does not approach normal levels even 9 mo after birth. Second, although the initial responses as tolerance is broken can be T15+, idiotypic dominance is not established; instead, a heterogeneous T15- population eventually emerges, which includes clones with higher and with lower avidity than T15. Unirradiated unresponsive mice will help transplanted normal B cells to produce T15+ responses to thymus-dependent PC antigens. The responses of animals recovered from tolerance are stable upon adoptive transfer. We have, moreover, found no evidence of either loss of idiotype-specific T cell help or generation of suppression. Therefore, neonatal exposure to PC tolerogen can effect profound, permanent changes in the antigen-specific B cell compartment independent of any influence on conventional T cell regulatory mechanisms.     

Induction of autoimmunity in a transgenic model of B cell receptor peripheral tolerance: changes in coreceptors and B cell receptor-induced tyrosine-phosphoproteins.

Abrogation of peripheral tolerance in transgenic mice that express a uniform B-cell receptor may create a powerful tool to examine the molecular mechanisms that underlie the autoimmune response in B cells. Here we report that processes that induce a systemic lupus erythematosus-like syndrome in normal mice, namely chronic graft vs host reaction, trigger systemic autoimmunity in a well-established transgenic mice model of B cell receptor peripheral tolerance. The induction of graft vs host reaction in mice that carry both a rearranged B cell Ag receptors specific for hen egg lysozyme and expressing chronically circulating hen egg lysozyme Ag resulted in induction of high and sustained levels of circulating anti-hen egg lysozyme autoantibodies and glomerulonephritis with proteinuria. This was associated with marked changes in expression of cell-surface proteins, such as CD23 and complement receptor 2. B cells from the graft vs host-induced mice could proliferate in vitro in response to self-Ag, and upon stimulation with anti-IgD demonstrated rapid phosphotyrosine phosphorylation of specific proteins, which could not be induced in the anergic double transgenic B cells. Conversely, loss of tolerance was not associated with a higher induction in the level of Syk kinase phosphorylation following stimulation with anti-IgD. Taken collectively, these data establish that 1) processes that induce a systemic lupus erythematosus-like syndrome in normal mice can abrogate peripheral tolerance in transgenic mice expressing self-tolerized B cells, and that 2) loss of tolerance in this model is associated with marked changes in surface expression of B cell coreceptors as well as with selective changes in IgD-induced signaling by discrete tyrosine-phosphoproteins, but not Syk kinase.     

An intra-Peyer's patch gene transfer model for studying mucosal tolerance: distinct roles of B7 and IL-12 in mucosal T cell tolerance

Development of mucosal immunity and tolerance requires coordinated expression of a number of genes within the mucosa-associated lymphoid tissue (MALT). To study the roles of these genes in the MALT, we have established a MALT-specific gene transfer model using replication-defective adenovirus as vector. In this model, the target gene of interest is directly delivered into the Peyer's patch by intra-Peyer's patch injection of the recombinant virus. Using this gene transfer model, we investigated the roles of B7-1 and IL-12 in the development of mucosal tolerance. We found that intra-Peyer's patch injection of OVA induced Ag-specific T cell hyporesponsiveness, as manifested by decreased T cell proliferation and IL-2/IFN-gamma production upon subsequent immune challenge. Intra-Peyer's patch B7-1 gene transfer at the time of OVA administration partially reversed the inhibition of T cell proliferation and IL-2 secretion, but had no effect on IFN-gamma production. By contrast, intra-Peyer's patch IL-12 gene transfer completely restored T cell proliferation and IFN-gamma secretion and partially reversed IL-2 inhibition. Using an adoptive TCR transgenic model, we further demonstrated that B7 and IL-12 played distinct roles during the inductive phase of mucosal tolerance. B7 selectively increased T cell proliferation and IL-2 secretion without affecting IFN-gamma production, whereas IL-12 increased both IL-2 and IFN-gamma production. These results indicate that B7 alone may not be sufficient to abrogate mucosal tolerance, and that cytokines such as IL-12 may also be required. Based on these findings, we propose a new model to explain the paradoxical roles of B7 in mucosal immunity and tolerance.     

Natural suppressor cell inhibiting T killer responses against retroviruses: a model for self tolerance

We previously reported the characterization of a spontaneous suppressor T cell population (NSC) present in naive mice and able to suppress the cytotoxic response (CTL) against tumor cells induced only by endogenous Gross virus (GLV). In this study we demonstrate the existence of such NSC inhibiting the CTL activity against tumor cells induced by the normally exogenous Moloney virus (M-MLV) in mice of the Mov-13 (V+) strain in which the M-MLV has been artificially endogenized and which express the virus during the embryonal life. These NSC are not found in other Mov strains in which the endogenized M-MLV is not expressed during fetal life. The implication of these data in the mechanism of self tolerance is discussed.     

Idiotype vaccination against murine B cell lymphoma. Inhibition of tumor immunity by free idiotype protein

A murine B cell lymphoma (38C13) was used as a model to study the induction of idiotype (Id)-specific tumor immunity. Immunization of syngeneic mice with Id protein derived from the tumor resulted in the production of anti-Id antibodies by the host and in the induction of a state of resistance to tumor growth. Tumor immunity could be established only if the Id protein was conjugated to a strongly immunogenic carrier protein such as keyhole limpet hemocyanin or thyroglobulin, and if the conjugate was administered at least 1 week prior to tumor challenge. Free Id protein, such as that present in tumor bearing animals, was found to inhibit tumor immunity in a dose-dependent manner. Although tumor immunity could be induced in animals with pre-existent serum Id protein, the expression of the immune state was inhibited by the presence of the soluble protein.     

Spatial regulation of exocytosis and cell polarity: yeast as a model for animal cells

Exocytosis is the major mechanism by which new membrane components are delivered to the cell surface. In most, if not all, eukaryotic cells this is also a highly spatially regulated process that is tightly coordinated with the overall polarity of a cell. The Rho/Cdc42 family of GTPases and the lethal giant larvae/Sro7 family are two highly conserved families of proteins which appear to have dual functions both in cell polarity and exocytosis. Analysis of their functions has begun to unravel the coordination between these processes and propose a model for polarized vesicle docking and fusion at the site of asymmetric cell growth.     

The Xenopus oocyte: a model for studying the metabolic regulation of cancer cell death

Abnormal metabolism and the evasion of apoptosis are both considered hallmarks of cancer. A remarkable biochemical model system, the Xenopus laevis oocyte, exhibits altered metabolism coupled to its apoptotic machinery in a similar fashion to cancer cells. This review considers the theory that these two hallmarks of cancer are coupled in tumor cells and provides strong proof that the Xenopus laevis oocyte system is an appropriate model in which to dissect the biochemical events underlying the connection between the two hallmarks. By further elucidating the mechanisms through which metabolism suppresses apoptotic machinery, we may gain a better understanding about how normal cells transform into cancer cells.     

Idiotype vaccination against murine B cell lymphoma. Humoral and cellular requirements for the full expression of antitumor immunity

The roles of humoral and cellular antitumor immune responses induced by immunization with tumor-derived idiotypic IgM were studied in a syngeneic, transplantable B cell lymphoma (38C13) of C3H mice. Id vaccination with keyhole limpet hemocyanin-conjugated Id induced protection against a subsequent lethal tumor challenge. Such immunizations elicited anti-idiotypic antibodies that were cytotoxic in in vitro antibody-dependent cellular cytotoxicity assays as well as in vivo passive transfer experiments. L3T4+ T cells, which proliferated in vitro in response to the specific Id protein, were also induced. However, cells mediating direct cytotoxicity, either in vitro or in vivo, were not observed in the lymph nodes, spleens, or peritoneal cavity of immune mice or at the site of tumor regression as demonstrated by using a tumor sponge implantation model. In addition, in vitro sensitization of immune lymphocytes against 38C13 tumor cells failed to induce cytotoxicity. Immunization with lipid conjugated Id also elicited a T cell proliferative response but failed to induce anti-idiotypic antibodies and did not confer resistance to tumor growth. These results suggest that anti-idiotypic antibodies play the major role in the destruction of 38C13 tumor cells. However, in vivo depletion of L3T4+ or Lyt-2+ cells from 38C-Id-keyhole limpet hemocyanin-immunized mice resulted in diminished protection against a tumor challenge. Thus, although humoral responses appear to play the predominant part in tumor destruction, cellular responses are also required for the full expression of antitumor immunity in this system.     

Idiotypic regulation of B cell differentiation

We study the equilibrium properties of idiotypically interacting B cell clones in the case where only the differentiation of B cells is affected by idiotypic interactions. Furthermore, we assume that clones may recognize and be stimulated by self antigen in the same fashion as by antiantibodies. For idiotypically interacting pairs of non-autoreactive clones we observe three qualitatively different dynamical regimes. In the first regime, at small antibody production an antibody-free fixed point, the virgin state, is the only attractor of the system. For intermediate antibody production, a symmetric activated state replaces the virgin state as the only attractor of the system. For large antibody production, finally, the symmetric activated state gives way to two asymmetric activated states where one clone suppresses the other clone. If one or both clones in the pair are autoreactive there is no virgin state. However, we still observe the switch from an almost symmetric activated state to two asymmetric activated states. The two asymmetric activated states at high antibody production have profoundly different implications for a self antigen which is recognized by one of the clones of the pair. In the attractor characterized by high autoantibody concentration the self antigen is attacked vigorously by the immune system while in the opposite steady state the tiny amount of autoantibody hardly affects the self antigen. Accordingly, we call the first state the autoimmune state and the second the tolerant state. In the tolerant state the autoreactive clone is down-regulated by its anti-idiotype providing an efficient mechanism to prevent an autoimmune reaction. However, the antibody production required to achieve this anti-idiotypic control of autoantibodies is rather large.     

B cell tolerance and positive selection in lupus

Systemic lupus erythematosus is considered a prototype of systemic autoimmune diseases; however, despite considerable advances in recent years in the understanding of basic mechanisms in immunology, little progress has been made in elucidating the etiology and pathogenesis of this disease. This even holds for inbred mice, such as the lupus-prone New Zealand Black/New Zealand White F(1) mice, which are all genetically programmed to develop lupus at a predetermined age. This frustrating state of affairs calls for a fundamental change in our scientific thinking and the opening of new directions in lupus research. In this study, we suggest that intrinsic B cell tolerance mechanisms are not grossly impaired in lupus-prone mice, but that an unusually strong positive selection event recruits a small number of autoreactive B cells to the germinal centers. This event could be facilitated by nucleic acid-protein complexes that are created by somatic changes in the susceptible animal.     

A model for regulation of the cell cycle incorporating cyclin A, cyclin B and their complexes

Abstract. t. A mathematical model for the cell cycle is proposed that incorporates the known biochemical reactions involving both cyclin A and cyclin B, the interactions of these cyclins with cdc2 and cdk2, and the controlling effects of cdc25 and weel. The model also postulates the existence of an as yet unknown phosphatase involved in the formation of maturation promoting factor. The model produces solutions that agree qualitatively with a wide variety of experimentally observed cell-cycle behavior. Conditions under which the model could explain the initial rapid divisions of embryonic cells and the transition to the slower somatic cell cycle are also discussed.     

Role of B7 in T cell tolerance

The induction of effective immune responses requires costimulation by B7 molecules, and Ag recognition without B7 is thought to result in no response or tolerance. We compared T cell responses in vivo to the same Ag presented either by mature dendritic cells (DCs) or as self, in the presence or absence of B7. We show that Ag presentation by mature B7-1/2-deficient DCs fails to elicit an effector T cell response but does not induce tolerance. In contrast, using a newly developed adoptive transfer system, we show that naive OVA-specific DO11 CD4+ T cells become anergic upon encounter with a soluble form of OVA, in the presence or absence of B7. However, tolerance in DO11 cells transferred into soluble OVA transgenic recipients can be broken by immunization with Ag-pulsed DCs only in B7-deficient mice and not in wild-type mice, suggesting a role of B7 in maintaining tolerance in the presence of strong immunogenic signals. Comparing two double-transgenic models--expressing either a soluble or a tissue Ag--we further show that B7 is not only essential for the active induction of regulatory T cells in the thymus, but also for their maintenance in the periphery. Thus, the obligatory role of B7 molecules paradoxically is to promote effective T cell priming and contain effector responses when self-Ags are presented as foreign.     

Regulation of B cell tolerance by murine gangliosides

The potential role of gangliosides as modulators of the triggering of neonatal primary B lymphocytes at the single precursor cell level was evaluated. Tolerance was induced in splenic fragment cultures containing an excess of carrier-primed T cells. Gangliosides at low concentrations (20 ng/culture) abrogated the tolerogenic effect of haptens presented on carriers not recognized by environmental T cells. The permanent arrest of immature B-cell responsiveness resulting from tolerogen treatment was eliminated by the presence of gangliosides during tolerogen treatment. The active moiety in the glycolipid preparation which protected B cells during tolerogen treatment was separated by ion-exchange chromatography and demonstrated to be in the disialoganglioside fraction.     

A macrophage cell model for pH and volume regulation

A whole-cell model of a macrophage (mphi) is developed to simulate pH and volume regulation during a NH4Cl prepulse challenge. The cell is assumed spherical, with a plasma membrane that separates the cytosolic and extracellular bathing media. The membrane contains background currents for Na+, K+ and Cl-, a Na(+)-K+ pump, a V-type H(+)-extruder (V-ATPase), and a leak pathway for NH4+. Cell volume is controlled by instantaneous osmotic balance between cytosolic and extracellular osmolytes. Simulations reveal that the mphi model can mimic alterations in measured pH(i) and cell volume (Vol(i)) data during and after delivery of an ammonia prepulse, which induces an acid load within the cell. Our analysis indicates that there are substantial problems in quantifying transporter-mediated H+ efflux solely from experimental observations of pH(i) recovery, as is commonly done in practice. Problems stemming from the separation of effects arise, since there is residual NH4+ dissociation to H+ inside the mphi during pH(i) recovery, as well as, proton extrusion via the V-ATPase. The core assumption of conventional measurement techniques used to estimate the H+ extrusion current (I(H)) is that the recovery phase is solely dependent on transporter-mediated H+ extrusion. However, our model predictions suggest that there are major problems in using this approach, due to the complex interactions between I(H), NH3/NH4+ buffering and NH3/NH4+ efflux during the active acid extrusion phase. That is, the conventional buffer capacity-based I(H) estimation must also take into account the perturbation that a prepulse challenge brings to the cytoplasmic acid buffer itself. The importance of this whole-cell model of mphipH(i) and volume regulation lies in its potential for extension to the characterization of several other types of non-excitable cells, such as the microglia (brain macrophage) and the T-lymphocyte.     

Establishment of a leukemic cell model for studying human pre-B to B cell differentiation

Reproducible models for examining early stages of human B cell differentiation are poorly developed. We now describe the establishment and characterization of a novel human leukemic cell line that recapitulates the pre-B to B cell stage of differentiation. This cell line, designated BLIN-1, was initially established in tissue culture medium containing low m.w. B cell growth factor, and consistently shows a dependency on this cytokine for optimal growth at low density. BLIN-1 cells have a 9p chromosomal abnormality, identical to the abnormality present in the leukemic blasts from the patient's original bone marrow aspirate. The immunologic phenotype of BLIN-1 is consistent with a cell arrested at the pre-B cell stage of development. Analysis of Ig gene rearrangement and Ig expression in a series of BLIN-1 subclones show that the cells spontaneously rearrange kappa light chain genes, leading to the differentiation of surface kappa-negative pre-B cells into surface kappa-positive B cells. The BLIN-1 cell line is, to our knowledge, the first defined human model for examining this critical developmental stage in human B cell ontogeny. As such, it offers a unique resource for examining variables influencing onset of kappa L chain gene rearrangement and expression.