The signal for tolerance in B cells is not transmitted through antigen-specific immunoglobulin receptors

Studies of B cell tolerance at the single-cell level require a ready source of antigen-specific B cells that are uncontaminated by T cells or accessory cells. We have isolated normal dinitrophenyl (DNP)-specific B cells from spleens of unprimed mice and propagated these cells in vitro. These B cells are uncontaminated by T cells or macrophages. Long-term cultures of these cell lines contain pre-B cells that are surface (s) IgM-, B cells with sIgM alone, and more mature B cells with sIgM, sIgD, and Ia antigens. Using the cell line lymphocytes we have shown that the early binding of the tolerogenic form of hapten to B cell receptor on mature B cells induces the same activation signal as antigen, and the negative signal induced by tolerogen occurs after B cell activation. Exposure of maturing B cells to DNP bound to murine IgG2a (MGG) for 30 days does not inhibit growth or receptor expression, but does induce tolerance that is reversible when DNP-MGG is removed. A 45-day exposure to DNP-MGG also induces a reversible tolerance.     

The role of cytoplasmic free calcium concentration in B-cell tolerance

Calcium is an important factor in the immune response. Extracellular calcium is required for antibody production by B lymphocytes. Several investigators have demonstrated that crosslinking of receptors on B lymphocytes by anti-mu antibody induces an increase in intracellular calcium. There are few data on the role of intracellular calcium mobilization or calcium influx in tolerance induction in B cells. We studied changes in free intracellular calcium concentration ([Ca+2]i) induced by exposure of dinitrophenyl (DNP)-specific B cells to the tolerance-inducing conjugate DNP-murine IgG2a (DNP-MGG). Splenic B cells enriched for DNP-specific cells and DNP-specific continuous B-cell lines were used for the studies. Exposure of B cells to the tolerogen DNP-MGG, the antigen DNP-keyhole limpet hemocyanin (DNP-KLH), or the antigen DNP-Ficoll induced an increase in free [Ca+2]i which was due to both mobilization of Ca+2 from endoplasmic reticulum (ER) and influx of extracellular Ca+2. This increase was DNP specific since no significant change was seen with carriers alone and no change was seen in cells that were not DNP specific. The DNP-MGG and DNP-Ficoll induced the same amount of Ca+2 release from ER but the release induced by DNP-KLH was higher. When B cells, which were made tolerant by in vitro incubation with DNP-MGG, were incubated with antigens, a mobilization of Ca+2 from endoplasmic reticulum occurred that was the same as that of nontolerant B cells. Since Ca+2 mobilization is associated with Ig receptor-dependent early B-cell activation, it is likely that the tolerant B cell can still receive an activation signal through the Ig receptors.     

Tolerance and immunity in antigen-specific B-lymphocyte lines: early receptor binding of either antigen or tolerogen initiates an immune response

Studies of the effect of tolerance-inducing compounds on B lymphocytes have been complicated by the fact that it is technically difficult to completely isolate the antigen-specific B cell from the effects of T cells or T-cell factors. We have used our cell lines of nonmalignant dinitrophenyl (DNP)-specific B lymphocytes derived from normal mice, which have no contaminating T cells, to study the effect of DNP-murine IgG2a (DNP-MGG), a tolerogen which is not normally immunogenic, on antigen-specific B lymphocytes. Preincubation with DNP-MGG for 48 hr, both in the presence and absence of T-cell factors from EL-4 supernatant prior to adding the antigen DNP-Ficoll, can induce tolerance in cell line B lymphocytes. The suppression is antigen-specific since preincubation with fluorescein-MGG or unconjugated MGG does not suppress the anti-DNP response. At least a 36-hr incubation is required for tolerance induction in B lymphocytes, but a 6-hr preincubation with DNP-MGG augments the immune response to DNP-Ficoll. Lymphocytes incubated for 6 or 24 hr with DNP-MGG prior to adding EL-4 supernatant and filler cells without DNP-Ficoll exhibited an immune response equal to that elicited by DNP-Ficoll and T-cell factors. A 6-hr pulse with a DNP-conjugated polymer of D-glutamic acid and D-lysine (DNP-dGL), a B-cell tolerogen which does not bind to Fc receptors, elicited the same immune response as seen with a 6-hr pulse of DNP-MGG but a 48-hr preincubation with DNP-dGL induced tolerance. Thus, it is likely that the initial binding of the tolerogen to the immunoglobulin receptor on the mature B cell elicits an activation signal similar to that seen with the antigen. The suppressive effect of the tolerogen itself appears to occur at a later stage of the process of the B-cell activation, proliferation, and differentiation.     

Membrane depolarization is induced in tolerant B lymphocytes by stimulation with antigen

Membrane depolarization is one of the earliest events in activation of cells by ligand receptor interaction. It is known that crosslinking of antigen-specific Ig receptors on B cells by antigen can induce membrane depolarization and subsequent Ia antigen expression on the cell surface. To determine whether a tolerance-inducing form of the antigen can also induce membrane depolarization after Ig receptor binding we used splenic B cells enriched for dinitrophenyl (DNP)-specific cells and determined relative membrane potential in these cells after binding of DNP-murine IgG2a (MGG) (tolerogen) or antigens (DNP-keyhole limpet hemocyanin (KLH) and DNP-Ficoll). Relative membrane potential was determined by loading the cells with the dye, 3.3-dipentyloxacarboxyanine (DiOC5(3)) after 2 hr incubation with ligand and determining relative fluorescence intensity on the fluorescence-activated cell sorter (FACS). Carriers alone did not depolarize these normal cell populations, but 100% of DNP-specific cells were depolarized by DNP-KLH and DNP-MGG while 85% were depolarized by DNP-Ficoll. To determine if tolerant B cells could be depolarized by antigen we induced tolerance in vitro or in vivo with DNP-MGG and measured the depolarization of DNP-specific B cells in response to antigens and tolerogen. DNP-specific B cells made tolerant by DNP-MGG underwent membrane depolarization when incubated with either DNP-KLH, DNP-MGG, or DNP-Ficoll but not with carriers alone. These data suggest that tolerogen induces membrane depolarization equally as well as antigen in normal cells. In addition, tolerant cells can be depolarized by Ig receptor crosslinking with either antigen or tolerogen. Thus, tolerance does not block the early membrane events induced by antigen in B cells.     

Molecular and cellular aspects of immunologic tolerance.

This review seeks to explain the most exciting recent data concerning the nature of self/non-self discrimination by the immune system in a manner accessible to a biochemical readership. The nature of recognition in the two great lymphocyte families, B cells and T cells, is described with special emphasis on the nature of the ligands recognized by each. The history of the field of immunologic tolerance is surveyed, as are the key experiments on conventional mice which provided a conceptual framework. This suggested that tolerance was essentially due to 'holes' in the recognition repertoires of both the T and B cell populations so that lymphocytes competent to react to self antigens were not part of the immunologic dictionary. There were essentially two ways to achieve this situation. On the one hand, self antigens might 'catch' developing lymphocytes early in their ontogeny and delete the cell, a process of clonal abortion. On the other hand, self antigens might signal lymphocytes (particularly immature cells) in a negative manner, reducing or abolishing their capacity for later responses, without causing death. This process is referred to as clonal anergy. Evidence for both processes exists. Special emphasis is placed on a wave of experimentation beginning in 1988 which imaginatively uses transgenic mouse technology to study tolerance. Transgenic manipulations can produce mice which synthesize foreign antigens in a constitutive and/or inducible manner, sometimes only in specific locations; mice which possess T or B lymphocytes almost all expressing a given receptor of known specificity; and mice which are an immunologic time bomb in that the antigen is present and so too are lymphocytes all endowed with receptors for that antigen. These experiments have vindicated the possibility of both clonal abortion and clonal anergy in both T and B cell populations, the choice of which phenomenon occurs depending on a number of operational circumstances. For T cell tolerance, clonal abortion occurs if the self antigenic determinant concerned is present within the thymus; if not, clonal anergy is more likely. For B cell tolerance, the strength of the negative signal and therefore the choice between abortion and anergy depends on the molar concentration of the self antigen, the capacity for multivalent presentation to a B cell, and the affinity of the B cell's receptor for the antigen in question. Some B cells with low affinity for self antigens certainly escape censorship and remain capable of secreting low affinity anti-self antibodies, which however do no harm.(ABSTRACT TRUNCATED AT 400 WORDS)     

Induction of T lymphocyte responses to a small molecular weight antigen. I. Failure to induce tolerance in azobenzenearsonate (ABA)-specific T cells in guinea pigs with an ABA conjugate of A copolymer of D-glutamic acid and D-lysine (D-GL).

2,4-Dinitrophenyl (DNP) coupled to the copolymer D-glutamic acid and D-lysine (D-GL) induces B cell tolerance but not T cell tolerance. This implies either a lack of DNP determinant recognition by T cells or a substantial difference in tolerance mechanisms for the two cell types. In the present study D-GL was conjugated with the well-defined determinant azobenzenearsonate (ABA) coupled to single amino acids shown here and previously by others to trigger effectively T lymphocytes. The experiments presented here demonstrate that these ABA conjugates of D-GL, although capable of diminishing anti-ABA antibody production, completely fail to render ABA-specific T lymphocytes tolerant thus drawing us to conclude that there are significant operational differences in the mechanisms of tolerance induction in T and B lymphocytes, respectively.     

Modification of the cytotoxic T cell repertoire in neonatal tolerance. Evidence for preferential survival of cells with low avidity for tolerogen

Clonal deletion of developing lymphocytes with potential reactivity for self is thought to play a crucial role in the establishment of self tolerance. One prediction of the clonal deletion hypothesis is that cells bearing receptors with high affinity for self are more likely than cells with low affinity receptors to be deleted from the repertoire. Experimental models of B cell tolerance have provided evidence for the preferential survival of low affinity cells with specificity for tolerogen in tolerant animals, but no comparable evidence exists for T cells. To examine this issue in T cells, cytotoxic T cell lines specific for the Kb mutant class I H-2 molecule, bm1, were generated from C57BL/6 mice rendered neonatally tolerant of bm1 and compared with anti-bm1 lines generated from normal mice. Compared with normal lines, those from tolerant mice differed in five ways: 1) they grew more slowly; 2) they were less efficient at lysing bm1 targets; 3) they showed different patterns of lysis against a panel of third party targets; 4) their cytotoxic activity against bm1 could be increased in the presence of leukoagglutinin, whereas the activity of normal lines was not increased by leukoagglutinin; and 5) their cytotoxic activity was more susceptible to inhibition by anti-Lyt-2 antibody. Taken together, these results demonstrate that the repertoire of the remaining tolerogen-specific cytotoxic T cells in neonatally tolerant mice is different from the normal C57BL/6 anti-bm1 repertoire, and the results are consistent with the idea that the remaining tolerogen-specific cells are low avidity cells that have preferentially escaped the clonal deletion process.     

Oral tolerance in experimental autoimmune encephalomyelitis. III. Evidence for clonal anergy.

We have recently reported that experimental autoimmune encephalomyelitis (EAE) can be suppressed by the oral administration of myelin basic protein (MBP). The oral introduction of 20 mg MBP together with a trypsin inhibitor results in inhibition of EAE clinical signs, decreased CNS histopathologic changes and dramatically reduced MBP-specific proliferative responses in fed and challenged Lewis rats. In the present study, we have investigated the mechanism underlying MBP-induced oral tolerance in EAE. Neither lymphoid cells (lymph node cells, spleen cells, Peyer's patch lymphocytes, thymocytes) nor humoral elements derived from tolerant donors were capable of transferring the tolerance to naive recipients. Moreover, lymphoid cells obtained from orally tolerant donors exhibited a marked decrease in their capacity to transfer EAE to naive recipient rats, even after in vitro activation with MBP or Con A. We observed that EAE could be readily transferred into orally tolerant rats using MBP-specific encephalitogenic T cell lines. In vitro cell mixing studies showed that the proliferation of lymphocytes from MBP-sensitized donors was not inhibited by the addition of lymphoid cells from tolerant donors, arguing against the role of a suppressor cell. Investigation of MBP-stimulated lymphokine production showed that both IL-2 and IFN-gamma levels were substantially decreased in spleen and lymph node cell cultures from MBP-fed rats compared to vehicle-fed control animals. Furthermore, limiting dilution analyses revealed that MBP-fed rats exhibited a profound decrease in MBP-reactive, IL-2-secreting lymphocytes relative to control animals. Thus, because lymphocytes from MBP-fed rats neither proliferate nor secrete IL-2 or IFN-gamma in response to MBP and we can find no compelling evidence for the role of suppressor cells, we propose that the oral administration of MBP results in a state of clonal anergy.     

The Florey lecture, 1986. The regulatory biology of antibody formation

The regulatory biology of antibody formation entered a new phase of study with the development of selective theories of immunity. The discovery of the 'one cell - one antibody' dogma and the demonstration that only a small minority of B cells possessed receptors specific for a given antigen were consistent with Burnet's clonal selection hypothesis, which was later formally proven by preparing antigen-specific lymphocytes and inducing clonal activation in vitro. Clonal analysis has aided precise study of immunoregulation for both B and T lymphocytes. Clonal activation of B cells in the absence of T cells is now possible with high cloning efficiency. It requires the combined action of certain antigens and growth factors, collectively termed B-cell stimulatory factors (BSFS). Single cell analysis has shown that most BSFS so far tested, in contrast to most claims in the literature, possess the capacity (in synergy with antigen) to: stimulate B cells out of the G0 phase into active cell cycle; promote sequential mitotic divisions; and induce differentiation to active secretory status. This is clearly true for IL-1, IL-2, and BSF-p2. These multiple actions resemble those of the colony-stimulating factors in haemopoiesis. Regulation of antibody production by T lymphocytes can also be profitably analysed in clonal systems. The immunoregulatory problem of tolerance can also be analysed by means of clonal techniques. Studies are summarized which indicate that T-cell-mediated suppression and functional silencing of toleragen-specific lymphocytes are both cooperatively involved in many tolerance models. For the B lymphocyte, tolerance can be induced without an actual deletion of the cell involved; rather, the tolerant cell appears to have received and stored a negative signal, rendering it unresponsive to normally immunogenic stimuli. Thus, a state termed 'clonal anergy' has been induced within the cell. Functional clonal deletion has also been noted in several models to T-lymphocyte tolerance, but here it is not known whether clonal anergy or actual death of the relevant cell is at work. Self-tolerance sufficient to be consistent with good health need not mean a total absence of cells with any degree of self-reactivity. Indeed, it is clear that some B cells capable of forming antibody with some degree of affinity for self-constituents exist in the body, and can be activated, for example by lipopolysaccharide. The requirement is to limit the amount, affinity and duration of autoantibody production. A model suggesting how this may be achieved is presented.     

Loss of carrier-determined tolerance in vitro with loss of receptor blockade.

Experiments were done to determine whether carrier-determined tolerance is reversible and whether the loss of tolerance is accompanied by the loss of receptor blockade. Spleen cells from mice made tolerant with DNP-isologous IgG remained tolerant when transferred to irradiated syngeneic mice. If these same tolerant spleen cells were incubated for 24 hr or more before transfer the tolerance was lost. Autoradiology was done on the tolerant cells with either 125I anti-DNP or 125DNP-KLH, before and after incubation in vitro. When the cells were tolerant the number of DNP ABC was decreased whereas cells having DNP on their surface were increased. When the cells lost tolerance after in vitro incubation, the hapten-bearing cells were no longer present although the number of cells free DNP receptors increased to normal. These data suggest that in carrier determined tolerance the reactivation of tolerant lymphocytes may involve reversible receptor blockade.     

Tolerance induced by TNP-derivatized syngeneic erythrocytes: evidence for cooperation between hapten-specific T and hapten-specific B lymphocytes in the immune response.

Tolerance to the DNP haptenic determinant was induced with a single i.v. injection of trinitrophenylated syngeneic red blood cells. The tolerant state lasted 1 month and was stable on transfer to irradiated thymectomized syngeneic recipients. Suppressor activity was found soon after injection of tolerogen but was lost before the termination of tolerance. The unresponsive state could be reversed by adding normal thymus cells to tolerant spleen cells but not by normal bone marrow cells. LPS when given with immunogen restored the normal immune response in tolerant mice. Thus the injection of TNP-MRBC induced partial immune unresponsiveness which was characterized by the induction of T cell suppressor activity and by a hapten-specific helper T cells tolerance. Finally, these studies suggest a cooperative interaction between DNP-specific T lymphocytes and DNP-specific B lymphocytes in the immune response to DNP-BGG.     

Macrophage-derived soluble factors mediate suppression induced by 2,4-dinitrophenyl-conjugated mouse IgG in hybridoma cells

Our laboratory has established that 2,4-dinitrophenyl-conjugated mouse IgG (DNP-MGG) can specifically suppress proliferation, antibody synthesis, and secretion in vivo in two anti-DNP secreting cell lines: hybridoma 35-12 and myeloma MOPC-315. In the present study an in vitro system was used to further analyze the mechanism of suppression of hybridoma 35-12 cells (HC) by DNP-MGG. It was found that DNP-MGG-induced suppression of HC requires macrophages (M phi) and occurs only in eclipsed HC which are mainly small, nonsecreting cells. The M phi-mediated suppression is DNP specific, requires no M phi-HC cell contact, and does not involve killing of eclipsed HC. M phi culture supernatant alone cannot mediate suppression, but supernatants obtained by culturing M phi with either HC or supernatant from HC culture can mediate suppression of eclipsed HC in the presence of DNP-MGG. DNP-MGG is not required for the generation of effective M phi factors, but it is required for suppression of HC in the presence of M phi factors. Indomethacin cannot reverse M phi-mediated suppression, suggesting prostaglandins may not be the M phi factors. These data suggest that M phi-derived factors which are not prostaglandins in nature may play a role in B-cell regulation and in B-cell suppression induced by tolerogenic forms of antigen.     

Effect of palmitate on energy coupling in lymphocyte mitochondria

In the presence of 0.1 micrograms/ml of oligomycin, DNP (40-60 microM) increases lymphocyte respiration 10-fold and more. Palmitate taken at the same concentration stimulates the respiration of isolated mitochondria (1-2 mg prot/ml) in the presence of 1 mg/ml of BSA and the respiration of lymphocytes (10(8) cells/ml). When BSA and EGTA are absent in mitochondria isolation media, the mitochondrial respiration does not increase after DNP or ADP addition. Lymphocyte preparations are mostly distinguished by mitochondrial morphology in the presence of the uncoupler; they differ less by changes in dis-C3-(5) fluorescence after addition of 5-10 microM DNP and only insignificantly by the stimulation of respiration by DNP and palmitate. These results may be explained by the increase in the uncoupler-induced permeability of mitochondria for K+ and by partial transformation of delta psi m into delta pH in some cells, which may increase the cell resistance to damaging influences.     

Antigen presentation in acquired immunological tolerance

In acquired tolerance, previous exposure to antigen under certain conditions induces specific unresponsiveness instead of specific immunological memory. It has been studied as an approach to the mechanisms of self-tolerance that operate on immunocompetent T and B lymphocytes once they leave their sites of origin in the thymus and the bone marrow. Possible mechanisms involve induction of specific suppressor cells or inactivation of antigen-specific lymphocytes (clonal anergy) as a consequence of abortive antigen presentation, in which the antigen receptor is effectively engaged but certain poorly defined accessory signals the T lymphocytes require are lacking. We propose that small, resting B lymphocytes, which lack these accessory signals, are the inactivating antigen-presenting cells in acquired tolerance to proteins and to the class II transplantation antigens. B lymphocytes, which can use their antigen receptors to gather and process antigens that are present at very low concentrations, may play a role in self-tolerance. In addition, B lymphocytes and T lymphocytes rendered anergic by encounter with self antigens could persist as self-specific suppressor cells to block an autoimmune response of autoreactive clones that had escaped deletion or anergy.     

Selection <Emphasis Type="Italic">in vitro</Emphasis> and accumulation of phytochelatins in cadmium tolerant cell line of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis>)

Cucumber (Cucumis sativus L.) cells from suspension culture were selected for their ability to grow and divide rapidly in toxic concentration of cadmium. As a result of selection a cell suspension tolerant to 100 M cadmium chloride (CdCl₂) was initiated. The selected tolerant line exhibited stable and repeatable increase in fresh and dry weight of cells in the presence of cadmium. The accumulated level of phytochelatins in cadmium sensitive (unselected) and tolerant cell line was measured by high performance liquid chromatography (HPLC) after 3, 24 h and 5 days of cadmium treatment. It was shown that in both cell lines Cd induced accumulation of phytochelatins and simultaneous glutathione depletion occurred. No distinct changes were found after 3 and 24 h of cadmium treatment whereas after 5 days of exposure to the metal, the level of phytochelatins was two times higher in the sensitive cell line as compared to the tolerant one. The accumulation of phytochelatins was correlated with cadmium concentration that increased in both cell lines during the course of cell exposure to metal. However, the level of cadmium was always lower in the tolerant cell line. The results showed no direct correlation between the tolerance of cucumber cells to Cd and the accumulated level of phytochelatins. Other mechanisms responsible for the increased tolerance of cucumber cells exposed to Cd are discussed.     

Virus-like display of a neo-self antigen reverses B cell anergy in a B cell receptor transgenic mouse model

The ability to distinguish between self and foreign Ags is a central feature of immune recognition. For B cells, however, immune tolerance is not absolute, and factors that include Ag valency, the availability of T help, and polyclonal B cell stimuli can influence the induction of autoantibody responses. Here, we evaluated whether multivalent virus-like particle (VLP)-based immunogens could induce autoantibody responses in well-characterized transgenic (Tg) mice that express a soluble form of hen egg lysozyme (HEL) and in which B cell tolerance to HEL is maintained by anergy. Immunization with multivalent VLP-arrayed HEL, but not a trivalent form of HEL, induced high-titer Ab responses against HEL in both soluble HEL Tg mice and double Tg mice that also express a monoclonal HEL-specific BCR. Induction of autoantibodies against HEL was not dependent on coadministration of strong adjuvants, such as CFA. In contrast to previous data showing the T-independent induction of Abs to foreign epitopes on VLPs, the ability of HEL-conjugated VLPs to induce anti-HEL Abs in tolerant mice was dependent on the presence of CD4(+) Th cells, and could be enhanced by the presence of pre-existing cognate T cells. In in vitro studies, VLP-conjugated HEL was more potent than trivalent HEL in up-regulating surface activation markers on purified anergic B cells. Moreover, immunization with VLP-HEL reversed B cell anergy in vivo in an adoptive transfer model. Thus, Ag multivalency and T help cooperate to reverse B cell anergy, a major mechanism of B cell tolerance.     

Induction of T cell anergy in the absence of CTLA-4/B7 interaction

Immunologic tolerance in T lymphocytes is maintained through both thymic and peripheral contributions. One peripheral tolerance mechanism is the induction of T cell anergy, a form of nonresponsiveness resulting from incomplete T cell activation, such as stimulation through the TCR in the absence of costimulation. Recent reports have suggested that engagement of the inhibitory receptor CTLA-4 by its B7 ligand is critical for the initiation of anergy. We tested the importance of CTLA-4 in anergy induction in primary T cells with an in vitro anergy system. Using both CTLA-4/B7-blocking agents and CTLA-4-deficient T cells, we found that T cell anergy can be established in the absence of CTLA-4 expression and/or function. Even in the absence of CTLA-4 signal transduction, T cells activated solely through TCR ligation lose the ability to proliferate as a result of autocrine IL-2 production upon subsequent receptor engagement. Thus, CTLA-4 signaling is not required for the development of T cell anergy.     

In vitro growth of B lymphocytes infiltrating human melanoma tissue by transformation with EBV: evidence for secretion of anti-melanoma antibodies by some transformed cells

Epstein Barr virus was used to transform the B lymphocytes infiltrating metastatic tumor tissue from seven patients with melanoma. In this way it was possible to establish continuously growing B lymphoblastoid cell lines (LCL) derived from the tumor-infiltrating B lymphocytes from each of the seven patients. Antibody production of up to 50 micrograms/ml could be achieved by such cultures, and the lymphoblastoid cells could be cloned readily by limit dilution on a feeder layer of irradiated fetal fibroblasts. Preliminary analysis of the antibodies produced by lymphoblastoid cell lines established from tumors from two of the patients indicated that most were of IgM type and bound to a panel of melanoma cell target cells, as well as to some nonmelanoma tumor cell lines. Cloned LCL were produced from the tumor-infiltrating B cells from one of the patients, and of 100 such clones tested, 9% secreted antibody that bound to autologous tumor cells, and one of these clones produced antibody that appeared to be melanoma specific.     

Photobleaching recovery studies of antigen-specific mouse lymphocyte stimulation by DNP-conjugated polymerized flagellin

We have used fluorescence photobleaching recovery (FPR) to study the lateral diffusion of antigen-receptor complexes during stimulation of DNP-specific mouse B cells by the T-independent antigens DNP-polymerized flagellin (DNP-POL). Depending on epitope density and dose, these antigens behave either as immunogens or tolerogens. Lymphocyte DNP receptors binding DNP0.5 flagellin monomer show a diffusion constant D of 2.2 X 10(-10) cm2 sec-1 and ca 50% fluorescence recovery after bleaching. For DNP-POL bound to DNP-specific lymphocytes, the observed diffusion constants decrease monotonically with increased antigen dose and epitope density. Under optimally immunogenic conditions of DNP2.3-POL at 1 micrograms/ml, D = 1.5 X 10(-11) cm2 sec-1, some 14-fold less than for a single DNP receptor. Under tolerogenic conditions lower diffusion constants approaching 0.8 X 10(-11) cm-2 sec1 are observed. The fraction of aggregates mobile on the time scale of the experiment remains constant at about 50 to 60% in all immunogenic situations, but falls abruptly to about 24 to 32% in precisely those situations where the antigen/dose combination is tolerogenic. This might support the hypotheses that there exist critical epitope densities above which antigens and receptors form rigidly cross-linked aggregates that bring about B cell tolerance. The mobility of DNP0.5 flagellin monomer bound to receptors left unoccupied after treatment with various doses and batches of DNP-POL is independent of DNP-POL presence. Receptor aggregate diffusion is unaffected by treatment with colchicine or cytochalasin B.     

Normal development in porcine thymus grafts and specific tolerance of human T cells to porcine donor MHC

The induction of T cell tolerance is likely to play an essential role in successful xenotransplantation in humans. In this study, we show that porcine thymus grafts in immunodeficient mice support normal development of polyclonal, functional human T cells. These T cells were specifically tolerant to MHC Ags of the porcine thymus donor and responded to nondonor porcine xenoantigens and alloantigens. Exogenous IL-2 did not abolish tolerance, suggesting central clonal deletion rather than anergy as the likely tolerance mechanism. Our study suggests that the thymic transplantation approach to achieving tolerance with restoration of immunocompetence may be applicable to xenotransplantation of pig tissues to humans.