Immunologic tolerance for immune system-mediated diseases.

Induction of long-term, antigen-specific immunologic unresponsiveness holds great promise for the treatment of many immune system-mediated diseases, including asthma, allergies, autoimmune diseases, and transplant rejection. Unlike current immunosuppressive treatments, immunologic tolerance therapies would affect only the undesired immune responses, leaving protective immunity intact. A variety of approaches to immunologic tolerance induction are being taken, reflecting the molecular and cellular complexity of immune system activation and regulation. The presentations summarized in this report represent promising strategies, some of which are being evaluated in advanced animal models and human clinical trials. Approaches presented include the following: interference with costimulatory signals in T-cell induction, T-cell receptor antagonism by altered peptides, exploitation of antigen-induced apoptosis to eliminate undesired T cells, opposition of inflammation by the induction of regulatory cytokines, induction of transplant tolerance by mixed chimerism, and deviation from deleterious allergic antibody responses by use of immunostimulatory DNA sequences. These multifaceted approaches are strongly supported by knowledge of basic immune mechanisms, which should facilitate the rational development of these therapies for controlling immune-mediated diseases.

Identification of an alternative CTLA-4 ligand costimulatory for T cell activation.

Stimulation of T cell proliferation generally requires two signals: The first signal is provided by the T cell receptor binding to antigen, and the second signal or costimulus is provided by a different receptor-ligand interaction. In mouse and human, the CD28-B7 interaction has been identified as a source of costimulatory signals. We have identified a cell surface molecule (GL1) that is distinct from B7 and abundantly expressed on activated B cells. On activated B cells GL1, rather than B7, is the predominant ligand for the T cell-activation molecule CTLA-4. GL1 provides a critical signal for T cell-dependent responses in vitro and in vivo.

Characterization of a novel cell-surface molecule expressed on subpopulations of activated T and B cells.

A mAb, GL7, is described that reacts with a 35-kDa protein on subsets of activated mouse B cells as well as activated CD4+ and CD8+ peripheral T cells. In normal mice analyzed by flow cytometry, GL7 bound at low surface density to 0 to 9% of splenic B cells and 0 to 1% of splenic T cells. In contrast, GL7 bound at high density to a subpopulation comprising approximately 20% of TCR-bright thymocytes, and to B220+ cells in the bone marrow. The activation of B cells by various stimuli resulted in high levels of expression of the surface molecule identified by GL7 on up to 70% of B cells after 48 h; the remaining B cells expressed low or undetectable levels of this molecule, despite evidence of other activation-specific changes in cell-surface phenotype. The GL7-positive population of B cells induced by IL-5 stimulation exhibited high levels of both proliferative and IgM secretory activity, whereas the GL7-negative population showed little of either activity. Activation of splenic T cells with Con A for 48 h resulted in the expression of this determinant at high density on both CD4+ and CD8+ cells. GL7 thus appears to identify a previously uncharacterized cell-surface molecule expressed selectively on subpopulations of activated B and T cells as well as on discrete subpopulations of T and B lineage cells in vivo.

Expression of variable exon A-, B-, and C-specific CD45 determinants on peripheral and thymic T cell populations.

A mAb (I/24) has been generated that is specific for a determinant on mouse CD45 molecules. Reactivity of this mAb with a panel of CD45 transfected cell lines demonstrated that the determinant recognized is dependent upon expression of one or more CD45 variable exons and that exon C is sufficient for its expression. The exon C-specific epitope detected by I/24 is expressed at high density on essentially all B lymphocytes and at an intermediate density on the vast majority of CD8+ splenic T cells. Two distinct subpopulations of CD4+ splenic T cells were detected, a minor subpopulation that expresses this exon determinant at high density and a major subpopulation that expresses it at a much lower density. This first identification of a CD45RC-specific reagent allowed a comparison of the expression of exon A-, exon B-, and exon C-specific determinants on peripheral and thymic lymphoid populations. When splenic lymphocytes were analyzed for expression of CD45RA (reactive with mAb 14.8), CD45RB (reactive with mAb 23G2 or mAb 16.A), and CD45RC (reactive with mAb I/24) determinants, it was found that each of these CD45 determinants had a distinct pattern of expression on CD4+ and CD8+ T cells and B cells. CD45RB and RC epitopes were also detected at high density on a small proportion (0.7 to 4.1%) of thymocytes. Both CD45RB and RC epitopes were found predominantly on CD4-CD8- and CD4-CD8+ thymocytes but were also found on small numbers of CD4+CD8+ and CD4+CD8- cells. The population of thymocytes that expressed CD45RB and CD45RC determinants displayed a novel TCR CD3 phenotype characterized by a level of expression that was intermediate between that seen in the larger CD3 bright and CD3 dull populations of thymocytes.

Alterations of B lymphocyte Fc gamma R II expression and ligand binding capacity induced by various activators.

Murine B lymphocytes cultured with F(ab')2 anti-mouse mu or delta lost (85%) the capacity to bind antigen-IgG antibody complexes as assessed by flow microfluorometry. Anti-mu-induced loss of binding of complexes was concentration, time, and temperature dependent, reversible, and not due to decreased expression of the receptor because binding of monoclonal anti-Fc gamma R II to B lymphocytes cultured with anti-mu was unaffected. Activation of PKC and elevation of [Ca2+]i obtained by culturing B lymphocytes with the combination of PMA and Ca2+ ionophore induced a similar loss of binding of Cx. Since stimulation of B lymphocytes with anti-mu also activates PKC and elevates [Ca2+]i, these changes may be involved in the anti-mu-induced alterations in the binding of complexes to Fc gamma R II. In contrast to the effects of other activators, LPS caused increased expression (threefold) of B lymphocyte Fc gamma R II as measured by the binding of both complexes and monoclonal anti-Fc gamma R II. Thus, different B lymphocyte activators have distinct effects on Fc gamma R II expression or ligand binding capacity and can thereby affect Fc gamma R II-generated regulatory signals.

Regulation of Fc gamma R II expression and function by B lymphocyte activators.

B lymphocytes cultured with LPS show increased expression of Fc gamma R II and increased binding of Ag-IgG complexes (both greater than 200%). In contrast, B lymphocytes cultured with either IL-4 or anti-mu show a marked loss (85-90%) of binding of Ag-IgG complexes that is specific, time and temperature dependent, and reversible. Decreased binding of complexes was not due to decreased expression of the receptor and therefore appears to be due to some form of alteration of the receptor. Based on the observation that the loss of binding of complexes requires protein synthesis, we favor the view that the loss is due to association of Fc gamma R II with another membrane molecule whose expression is induced or increased by IL-4 or anti-mu. Anti-mu induced loss of Fc gamma R II ligand binding capacity does not require cross-linking of surface IgM because the effect can be generated with F(ab') anti-mu. Anti-mu induced loss of Fc gamma R II binding of complexes was substantially prevented by IFN-gamma, whereas IFN-gamma did not reduce the anti-mu caused increase in expression of MHC class II molecules. This result shows that increased expression of the latter molecules can be dissociated from loss of Fc gamma R II ligand binding capacity. A myeloid cell line was identified that constitutively expresses Fc gamma R II binds relatively few complexes. This cell line may be useful in identifying alterations of Fc gamma R II which lead to the loss of binding of complexes. These results indicate that various B lymphocyte activators have different effects on B lymphocyte expression and function, and can thereby affect Fc gamma R II generated regulatory signals.

Detection of low numbers of lymphocyte surface membrane molecules using concentration immunofluorescence analysis (CIA).

Using viable lymphocytes in a concentration immunofluorescence assay (CIA), conditions were ascertained which allow detection of low numbers of surface membrane molecules. Utilizing 3 layer labeling [monoclonal antibody (MAb) specific for the membrane molecule, biotin-conjugated antibody specific for the MAb, and R-phycoerythrin-strepavidin], large numbers of lymphocytes in the assay wells, and the Pandex Fluorescence Concentration Analyzer, a fluorescence signal significantly above background was generated by as few as 8 x 10(7) molecules among 4 x 10(5) lymphocytes. Experiments using membrane molecules (Fc gamma R II, Ia antigens, Ly-39) which differ considerably in their level of expression indicated that comparable signals were generated by equivalent numbers of labeled molecules in a cell population irrespective of the number of molecules on an individual cell. Thus, CIA is theoretically capable of detecting membrane molecules whose expression is as low as 2 x 10(2) per cell. CIA should be useful in the assay of cytokine receptors and other lymphocyte membrane molecules expressed at low levels, and in the development of MAb specific for these molecules.

Interactions between murine B lymphocyte surface membrane molecules. Loaded but not free receptors for complement and the Fc portion of IgG co-cap independently with cross-linked surface Ig.

We have investigated the possible physical interactions between CR, receptors for the Fc gamma R and surface Ig (sIg) on the surface membrane of murine B lymphocytes. We used the rat mAb to murine CR, 8C12, and 7G6, as CR ligands, and soluble Ag-antibody complexes as FcR ligands; and F(ab')2 fragments of rabbit antibodies specific for mouse IgM and IgD as sIg ligands. We have found that: 1) sIg, CR, and Fc gamma R are not directly linked, because capping of any one did not affect the expression of the others; 2) the mAb 8C12 and 7G6 failed by themselves to cross-link CR; 3) soluble Ag-antibody complexes crosslinked some, Fc gamma R on a minority of Fc gamma R+ lymphocytes; 4) once loaded with anti-CR mAb, CR co-capped with sIg when sIg was cross-linked; 5) once loaded with Ag-antibody complexes, Fc gamma R also co-capped with sIg when sIg was sIg was cross-linked; 6) loading of Fc gamma R did not affect the co-capping of surface CR with cross-linked sIg and conversely, loading of CR did not affect the co-capping of Fc gamma R with cross-linked sIg; only loaded CR or Fc gamma R co-capped with sIg regardless of the status of the other surface molecule; 7) neither loaded nor free CR co-capped with cross-linked Fc gamma R, and neither loaded nor free Fc gamma R co-capped with cross-linked CR. These results demonstrate that both Fc gamma R and CR independently become associated with sIg when either receptor is loaded and sIg is cross-linked.

IL-4 induces loss of B lymphocyte Fc gamma R II ligand binding capacity.

Murine B lymphocytes cultured for 24 h with rIL-4 lost (mean reduction of 88%, range 81 to 96%) the capacity to bind Ag-IgG antibody complexes to B lymphocytes as assessed by flow microfluorometry. This effect was specific in that it was not seen with IL-1, IL-2, or IFN-gamma; IL-4 did not have a similar effect on other B lymphocyte membrane molecules; and the effect was completely prevented by anti-IL-4 (mAb 11B11). More than 60% inhibition of the binding of complexes was seen with as little as 1 U/ml of IL-4 although maximal inhibition was seen with greater than or equal to 30 U/ml. IL-4-induced inhibition of the binding of complexes was time dependent (the effect was first seen after 8 h and was not maximal until 24 h), temperature dependent (it did not occur at 4 degrees C), and reversible (B lymphocytes that had lost the ability to bind complexes due to IL-4 regained this capacity when re-cultured for 24 h in the absence of IL-4). The effect could be partially prevented by IFN-gamma. The inability to bind complexes appeared to be mainly due to an alteration of Fc gamma R (Fc Receptors) II rather than down-regulation of receptor expression because IL-4 induced only a moderate reduction in the binding of two Fc gamma R II specific mAb (20% for 2.4G2 and 32% for K9.361). The IL-4-induced loss of binding of complexes to B lymphocyte Fc gamma R II appears to be a novel form of receptor regulation (function rather than expression), and likely plays a role in the up-regulation of B lymphocytes by IL-4 by preventing Fc gamma R II-mediated inhibition of B lymphocyte responses.

Effects of various forms of monoclonal anti-Fc gamma R II (2.4G2) on B lymphocyte responses.

The effects of monoclonal anti-Fc gamma R II (2.4G2) in various forms on B lymphocyte proliferation and antibody (mu) secretion in vitro were evaluated. Soluble native 2.4G2 did not stimulate or inhibit the responses of B lymphocytes, either unstimulated or stimulated with rabbit F(ab')2 anti-mouse mu (anti-mu) plus lymphokines (the supernatant of Concanavalin A stimulated rat spleen cells). The failure of native 2.4G2 to affect responses was observed over a broad range of concentrations, and even when the B lymphocytes were incubated with the 2.4G2 for 24 hr prior to stimulation with anti-mu and lymphokines. Similarly, soluble chemically cross-linked 2.4G2 failed to affect B lymphocyte responses. Binding studies indicated that this failure was not due to a lack of binding and suggested that the polymerized 2.4G2 was cross-linking at least four Fc gamma R II. Larger multimers of 2.4G2 could not be evaluated due to a loss of binding activity. In contrast to the above results, 2.4G2 which was capable of extensively cross-linking Fc gamma R II (2.4G2 bound to Sepharose) or of cross-linking Fc gamma R II to surface IgM (2.4G2 hetero-cross-linked with anti-mu) specifically inhibited B lymphocyte responses to anti-mu and lymphokines. Antibody secretion was affected more than proliferation. These results provide additional evidence that Fc gamma R II regulate the responses of B lymphocytes, and suggest that cross-linking of more than four Fc gamma R II is necessary to generate the inhibitory signal. Further, the results indicate that the ligand does not have to be internalized in order to generate the regulatory signal. Finally, the results with the heterodimer suggest that it may be possible to regulate a particular antibody response using anti-Fc gamma R II cross-linked to antigen or to anti-receptor antibody (e.g. an anti-idiotype).

Interactions between B lymphocyte subpopulations. Augmentation of the responses of resting B lymphocytes by activated B lymphocytes.

Mouse B lymphocytes were fractionated from normal T lymphocyte-depleted spleen cell populations using discontinuous percoll gradients and were stimulated with rabbit F(ab')2 anti-mouse mu-specific antibodies (anti-mu) plus the supernatant of Con A-stimulated rat spleen cells (SN) as a source of lymphokines. The responses of small (mean volume 120 mu 3), dense (greater than 1.087 specific gravity), resting (least spontaneous thymidine incorporation) B lymphocytes were augmented by irradiated (4000 rad), larger (mean volume greater than 170 mu 3), less dense (less than 1.081 specific gravity), activated (greater spontaneous thymidine incorporation) B lymphocytes. Proliferation was augmented 2- to 4-fold and polyclonal antibody-forming cell responses three- to sixfold. Maximal augmentation of the responses of 5 X 10(4) resting B cells was obtained with 10(4) activated B cells. Augmenting activity was specific for activated B lymphocytes in that responses were not augmented by irradiated thymocytes, T lymphoblasts, macrophages, or additional supernatant. B lymphocytes activated in vitro by LPS or anti-mu also had augmenting activity. Augmentation of responses was maximal only when activated B lymphocytes were added simultaneously with anti-mu. The interaction between activated and resting B lymphocytes did not appear to be genetically restricted. Interestingly, the augmenting activity of activated B cells could be reconstituted by a combination of supernatant and cell membranes from these cells but not by either alone, suggesting that two components are required, one soluble and the other membrane-bound. Thus, a functional interaction has been demonstrated between B lymphocyte subpopulations which differ in their state of activation, and this interaction appears to involve a novel mechanism of action.

Independent ligand occupancy and cross-linking of surface Ig and Fc gamma receptors downregulates B-lymphocyte function. Evaluation in various B-lymphocyte populations.

When two distinct B-lymphocyte membrane receptors (Fc gamma R and sIg) are independently occupied by their respective multivalent ligands, inhibition of the antibody-forming cell response occurs but proliferation is not inhibited. This regulatory signal was examined in various B-lymphocyte populations. Unprimed B lymphocytes from immune deficient CBA/N and autoimmune MRL/l mice were responsive to this regulatory signal. In contrast, unprimed B lymphocytes from autoimmune NZB mice and antigen-primed B lymphocytes from normal DBA/2 mice were not. Together with previous results, these data suggest that resting B lymphocytes which have not encountered antigen are most susceptible to this regulatory signal. Lack of responsiveness to this downregulatory signal may contribute to the hyper-responsiveness of NZB B lymphocytes.

Cooperativity between B lymphocyte membrane molecules: independent ligand occupancy and cross-linking of antigen receptors and Fc gamma receptors down-regulates B lymphocyte function.

IgG antibody-antigen complexes bound to B lymphocyte Fc gamma receptor (Fc gamma R) but not surface immunoglobulin inhibit the antibody-forming cell response but not proliferation of these cells in response to F(ab')2 anti-mu and lymphokines. The role of B lymphocyte antigen receptors in B lymphocyte Fc gamma R-mediated inhibition was evaluated. With the use of several different antigen receptor-dependent plaque-forming cell (PFC) responses, it was found that inhibition occurred irrespective of the type of stimulatory signal used, or whether antigen receptors were bound by antibody or occupied by nominal antigen. The degree of inhibition appeared to be directly related to the extent of antigen receptor cross-linking. Maximal inhibition only occurred if both antigen receptors and Fc gamma R were occupied by their respective ligands simultaneously during the early hours of activation. In contrast, antigen receptor-independent PFC responses to the mitogen lipopolysaccharide (LPS) were not inhibited by complexes. However, the antigen-independent TNP-specific PFC response to LPS was inhibited by the combination of IgG antibody-antigen complexes and the hapten, but by neither alone. These results suggest that a down-regulatory signal is generated by functional cooperation between Fc gamma R and antigen receptors. Generation of this inhibiting signal could be mediated by the previously described physical interaction between these two receptors, and interactions between membrane receptors may be a general mechanism utilized by lymphocytes for the integration of multiple signals.

Antigen-antibody complexes bound to B-lymphocyte Fc gamma receptors regulate B-lymphocyte differentiation.

We studied the effect of soluble antigen-antibody complexes on the responses of polyclonally activated murine B lymphocytes. For this, normal B lymphocytes were stimulated with rabbit F(ab')2 anti-mu and lymphokines. IgG complexes, particularly in antigen excess, inhibited the plaque-forming cell response (55-70%), while proliferation was unaffected. Maximal inhibition was obtained with small amounts (0.2-1.0 microgram/ml) of complexes. Neither antigen or antibody alone was inhibitory. Inhibition was mediated via binding of the IgG complexes to Fc gamma receptors of B lymphocytes: (1) neither T lymphocytes or adherent accessory cells were required; (2) IgM complexes did not inhibit; and (3) inhibition was not seen when monoclonal anti-Fc gamma receptor antibodies prevented binding of the IgG complexes to these receptors. Kinetic experiments showed that B lymphocytes are susceptible to this inhibitory signal for only a short time after stimulation. We conclude that IgG complexes bound to the Fc gamma receptors of B lymphocytes regulate B-lymphocyte differentiation.

Hybridoma 2.4G2: dissociation of the B-lymphocyte-triggering activity from the monoclonal anti-Fc IgG receptor antibody.

Previously, we have reported that monoclonal antibody specific for mouse Fc IgG receptors (Fc gamma R) [purified by affinity chromatography using goat F(ab')2 anti-rat Ig linked to Sepharose from supernatants of hybridoma 2.4G2] can induce both proliferation and antibody secretion of normal B-lymphocytes in the absence of T-lymphocytes. The current studies have shown that the B-cell-triggering activity is associated with a substance produced by 2.4G2 which can be distinguished from the 2.4G2 antibody by several criteria: (1) different preparations of 2.4G2 antibody with identical binding capacity for Fc gamma R differ markedly in their ability to trigger B-lymphocytes; (2) the B-lymphocyte-triggering substance and the anti-Fc gamma R antibody can be separated using insolubilized monoclonal mouse anti-rat kappa-antibody columns; and (3) the B-lymphocyte-triggering substance has a mol. wt of less than 50,000, while the 2.4G2 antibody has a mol. wt of 160,000. The B-lymphocyte-triggering moiety produced by the 2.4G2 hybridoma may be a previously undescribed lymphokine.