Fc-signalling in the modulation of immune responses by passive antibody.

Passive antibody can both suppress and augment immune responses. Until recently, there was virtual unanimity on the importance of the interaction of the Fc portion of modulating antibody with Fc-receptors (Fc-signalling), especially in experiments involving the suppression by antibody. Experiments reported in the last few years, that do not demonstrate the range of Fc-portion/Fc-receptor influences on the suppression of immune responses by passive antibody, have introduced new uncertainty into this field. The purpose of this paper is to review how the initial controversy on the influence of Fc-signalling in inhibition by passive antibody was resolved. Old and new approaches are suggested that may help in resolving the current uncertainty engendered by recent experimental results that were interpreted to mean that passive suppressive antibody does not utilize the inhibitory FcgammaRIIB receptor. An understanding of the factors that influence negative Fc-signalling is needed in order to optimize clinical therapies whose action depends on the suppressive property of antibody.

Immunoreceptor tyrosine-based inhibitory motifs on activating molecules.

Immunoreceptor tyrosine-based inhibitory motifs (ITIMs) have the restricted consensus sequence V/I/xYxxL/V, but may be more broadly defined by the sequence V/I/L/SxYxxL/V/I/S. If one includes the ITIM of CTLA-4, then the sequence becomes psixYxxpsi, where psi represents amino acids with nonpolar side chains. Aside from their presence in various inhibitory molecules, ITIMs are also found on many activating receptors and pathways. ITIMs with the restricted consensus sequence occur on IL-4Ralpha, IL-3Rbeta type II, gp130 cytokineR, OB-R (leptinR), LIF-Rbeta TNF-RI, G-CSF-R, PDGF-R, Blk, Ctk/Ntk, Lsk, Zap-70, PKB/RACalpha, PKC-alpha, PKC-beta, PKC-gamma, PKC-delta, PKC-zeta, PKC-epsilon, PKC-eta, PKC-phi, PKC-mu, calmodulin-dependent kinase IIdelta, SLP-76-associated protein, FYN-binding protein, Shc binding protein, RasGRF2, CDC25 homologue, Jak2, Jak3, PLCbeta1, and PLCbeta3. If ITIMs are defined by a broader consensus sequence, the list of ITIMs on activating molecules becomes even larger. In some instances, these ITIMs have been shown to associate with inhibitory phosphatases. Whether these ITIMs on activating receptors/pathways are necessary and sufficient for negative control of activating events and for immunologic tolerance is not yet known. In some instances, ITIMs on coinhibitory receptors are also required for appropriate negative regulation. By studying events leading to negative control during activation and to immunologic tolerance, it should be possible to discern the balance between antigen receptor-based negative events and coinhibition.

Why so many coinhibitory receptors?

Receptors that display negative signalling functions on lymphocytes and other cells of the reticuloendothelial system now number about 30. These negative receptors are transmembrane glycoproteins activated by phosphorylation of a tyrosine residue in immunoreceptor tyrosine-based inhibitory motifs that bind various phosphatases to induce dominant negative signals. Since these receptors are armed by the action of activating receptors and inhibit signalling by activating receptors, we have termed them coinhibitory receptors and the negative outcome is coinhibition. Coinhibitory receptors and some inhibitory mediators include FcgammaRIIB, CTLA-4, CD5, CD22, p58/70/140 KIR, gp49B1/gp91, PIRB1-5, LAIR-1, NKB1, Ly49 A/C/E/F/G, NKG2-A/B APC-R, CD66, CD72, PD-1, SHPS-1, SIRP-alpha1, ILT1-5, MIR7, 10, hMIR(HM18), hMIR(HM9), LIR1-3,5,8, Fas (CD95), TGFbeta-R, TNF-R1, IFNgamma-R (alpha and beta chains), mast cell function Ag, H2-M, HLA-DM, CD1, CD1-d, CD46, c-cbl, Pyk2/FADK2, P130 Ca rel prot, PGDF-R, LIF, LIF-R, CIS, SOCS13 and 5, and others are being defined regularly. This long list suggests that coinhibitors are needed not only for self-nonself discrimination, but also for control of ongoing responses to foreign antigens so that infectious agents are ideally dealt with by an appropriate level of immune responses to nonself and an appropriate amount of immunopathology and sickness behaviour.

FcR-mediated inhibition of cell activation and other forms of coinhibition.

The tripartite inactivation model proposed that coaggregation of the B cell antigen receptor (BCR) with the Fc receptor (FcR) by antigen and specific IgG antibody complexes explained the Fc-dependent inhibition of immune responses by antibody. This model has since been substantiated by many observations and its impact on studies of immune regulation has been threefold: (1) IgG antibody, via Fc gamma RIIB, mediates inhibition of cell activation in many cell types, demonstrating the general importance of this mechanism in immune regulation; (2) Fc gamma RIIB was the first receptor described that regulates immune responses by coinhibition, that is, regulation as a result of interaction between activating receptors (BCR, TCR, Fc epsilon RI, Fc gamma RIII, Fc gamma RIIA) and inhibitory receptors (Fc gamma RIIB, CTLA4, CD5, CD22, p58/70/140 KIR, gp49B1/gp91, Ly49A/C/E/F/G, NKG2-A/B, APCR, Fas (CD95), TGF beta-R, TNF-R, IFN gamma-R, and others). The list of coinhibitors is expanding, just as the list of costimulators has grown. Tolerance through multiple coinhibitors implies that Signal 1 alone is not tolerogenic; and (3) Studies of Fc gamma RIIB coinhibitory mechanisms have pointed the way to potential general inhibitory signaling pathways used by many receptors, involving the competing effects of various kinases and phosphatases, and other competitive events. Investigations of Fc gamma RIIB physiologic function and of other coinhibitory receptors, together with recent biochemical analyses, give an initial understanding of the biology of these inhibitory receptory receptors. Paradoxes within and between theoretical constructs, functional observations, and mechanistic studies point to critical questions for future study.

Effect of major histocompatibility complex expression on murine intestinal graft survival.

BACKGROUND: Clinical intestinal transplantation has been plagued by frequent and severe graft rejection. It has been proposed that the major histocompatibility complex (MHC) antigens might play a critical role in this process owing to their extensive expression on enterocytes and mucosa-associated immune cells. METHODS: The present study examined the role of MHC antigens in intestinal graft rejection using MHC class I-deficient and MHC class II-deficient donors. RESULTS: Grafts with normal MHC expression were rejected by 9 days, whereas survival was prolonged to 14 days in the MHC class II-deficient grafts (P=NS) and to 20 days in the MHC I-deficient grafts (P<0.002). In all groups, early rejection was characterized by (1) increased crypt cell apoptosis, as detected by the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) technique of in situ labeling; and (2) the increased expression of perforin and a CD8 phenotype in the graft-infiltrating cells. CONCLUSIONS: These data suggest that MHC antigens, CD8-positive T cells, and perforin-expressing cells contribute to intestinal graft rejection. Apoptosis of the progenitor epithelial crypt cells during early intestinal rejection may impair the gut's ability to regenerate and repair mucosal damage.

Hypogammaglobulinaemia occurs in Fas-deficient MRL-lpr mice following deletion of MHC class II molecules.

Fas (CD95)-mediated apoptosis in B and T cells is deficient in both human autoimmune lymphoproliferative syndrome and in MRL-lpr mice, a model for systemic lupus erythematosis (SLE). Autoimmune disease in these mice is associated with polyclonal B cell activation, increased serum immunoglobulin and autoantibodies. In non-autoimmune mice MHC class II is not required for normal serum immunoglobulin expression, and previously we have shown using MHC class II-deficient MRL-lpr mice (MRL-lpr Ab-/-) that generation of specific antibodies to DNA requires MHC class II-directed T cell help. In contrast, in the present study we demonstrate that MRL-lpr Ab-/- mice also have a profound reduction of total serum immunoglobulin levels, suggesting abnormal polyclonal regulation of B cells by MHC class II-directed T cells occurs in the autoimmune MRL-lpr strain. This abrogation of immunoglobulin production does not occur in MHC class II-deficient non-obese diabetic (NOD) mice, nor in MHC class I-deficient NOD or MRL-lpr mice. Reduced immunoglobulin levels in MRL-lpr Ab-/- mice were not due to a lack of B cells or to an increased loss of circulating immunoglobulin, but were associated with reduced numbers of surface IgG-positive B cells. These results define a general abnormal regulation of B cells in MRL-lpr mice through a process requiring MHC class II, and suggest that Fas deficiency may allow expansion of totally T-dependent B cells.

Co-stimulation and co-inhibition: equal partners in regulation.

Specific immune responses are controlled by two counterbalancing mechanisms-co-stimulation and co-inhibition. Antigen receptors determine specificity, activate co-stimulation and/or co-inhibition, and interact with these co-stimulatory/co-inhibitory mechanisms to dictate the direction of the immune response, either positive or negative. Co-stimulatory or co-inhibitory ligands interact with their specific receptors and may indicate the context in which antigen is perceived by lymphocytes. Ligation of antigen receptors may activate only co-stimulatory or co-inhibitory mechanisms, and thus may influence secondarily the direction of the immune response. Furthermore, the activity of a given co-stimulator or co-inhibitory receptor is modified depending on signalling via the antigen receptor. If neither co-stimulators nor co-inhibitors are present, lymphocytes, activated in response to antigen receptor signalling, produce low levels of effector elements and then revert to inactivity. Co-inhibitors are defective in autoimmune disease.

Defective antigen-receptor-mediated regulation of immunoglobulin production in B cells from autoimmune strains of mice.

B cells are stimulated by antigens or by polyclonal activators such as bacterial lipopolysaccharide (LPS) to produce antibody. In nonautoimmune strains of mice, LPS-stimulated antibody responses are inhibited by crosslinking the B cell antigen-receptor (BCR), while antigen-driven responses are shut down by co-crosslinking the BCR and the receptor for the Fc portion of IgG (Fc gamma R). BCR signals are poor at shutting off LPS-induced antibody production, including anti-ssDNA antibody production, in B cells from NZB, NZB/WF1, and BXSB lupus-prone mice but not MRL/lpr or NZW mice. In the current studies, the defect in NZB B cells was shown to be independent of T cells and macrophages. The inheritance pattern of resistance to BCR ligation of LPS-induced Ig production in BXSB mice could not be assigned to either founding strain. In New Zealand mixed (NZM) recombinant inbred mice, slightly but significantly more resistance was found in a line (NZM2410) that demonstrates a greater degree of clinical autoimmunity than another line (NZM64) with fewer autoimmune problems. The autoimmune defect is specific to BCR signals because inhibition of LPS activation by ligation of MHC class II occurs normally in NZB B cells. Bypassing the BCR by direct stimulation of second messengers with phorbol esters or ionomycin did not overcome the defect, suggesting that defects in downstream signaling events, rather than in the BCR mechanism itself, are responsible for the reduced ability to inhibit the LPS response in NZB B cells. The inability of the BCR signaling pathway to control LPS-induced Ig production in NZB mice was apparent at the level of H mu-chain mRNA for secreted IgM. These results suggest that autoimmunity-associated B cell defects in BCR signaling and subsequent regulation of LPS-driven antibody responses have a number of inheritance patterns and involve downstream events in signaling pathways in B cells. The defect can result in aberrant regulation of H mu-chain mRNA levels for secreted IgM production, and may be a predisposing factor in murine systemic autoimmune disease.

The trouble with transgenic mice.

Observations on many antigen-receptor transgenic models with anti-self specificities have been interpreted as proof for clonal deletion or for mechanisms involved in clonal deletion. At the same time, there is increasing evidence that many lymphocytes that recognize self exist, are activated and produce end products, even in individuals without clinical autoimmunity. Except perhaps for the amount of anti-self activity, there is little agreement on what distinguishes immune products normally recognizing self from those associated with clinical autoimmunity. To resolve this paradox, the tendency in immunology is to accept conclusions from transgenic models as normal, while judging those from the unmanipulated state as suspect. However, transgenics have a major weakness. Transgenes encoding antigen-receptors are derived from highly selected mature lymphocytes and are expressed in developing lymphocytes that normally do not display the antigen-receptors of mature lymphocytes. Such precocious expression of antigen-receptors could have profound abnormal effects on lymphocyte development. Other transgenic models suggest that processes in lymphocyte differentiation not involving antigen-receptor binding specificity exert powerful influences on lymphocyte development; therefore, mechanisms other than classical positive and negative selection are important.

Do lymphocytes require calibration?

Complexity in the activation/regulatory apparatus and the variable nature of the antigen-binding site dictate that B and T cells establish and select, during their development, appropriate activation and control mechanisms beyond simple antigen-binding specificity. These mechanisms are established partly by fixed interactions dictated by genetically defined structures, but they are also attained by calibration during ontogeny. This calibration depends on the ordered expression of early components (each of which is invariant), on their interaction with specific ligands, and on the receipt of invariant signals for calibration. Lymphocytes calibrate themselves by expressing various cell surface components, such as restricted heavy chain D-regions and pseudo-light chains. These are expressed in association with elements that will make up the antigen-receptor complex of mature lymphocytes. Calibration by invariant signals results in the establishment, selection and active maintenance of cellular activities which serve to control lymphocyte function. Since these cellular activities are one of a number of possible conditions, they are referred to as variant controls. Effectively calibrated basic cellular functions, specialized responses and cellular interactions allow lymphocytes to attain self-nonself discrimination. If calibration fails, lymphocytes will develop abnormalities, such as immunodeficiency and autoimmunity.

Lipopolysaccharide-induced IgM production is not suppressed by antigen receptor ligation in B cells from some autoimmune strains of mice.

Ligation of surface immunoglobulin on resting or activated nonautoimmune B cells inhibited lipopolysaccharide (LPS)-induced total IgM production. B cells from NZB, (NZB x NZW)F1, and BXSB mice were relatively resistant, but B cells from NZW or MRL/lpr mice were inhibited. The resistance occurs in B cells from young and old NZB mice, and in both resting and activated splenic NZB B cells. Anti-ssDNA responses induced by lipopolysaccharide occur in the presence of antigen-receptor-ligating antibody in NZB, but not in DBA/2, B cells. Antagonism in signaling between the antigen and LPS receptor is not a general B cell hyporesponsiveness, but defects in antagonism specifically between antigen and LPS signaling may be a predisposing factor to autoimmune disease in some autoimmune strains of mice.

Mutual antagonism between antigen- and lipopolysaccharide-induced antibody production.

B cells are induced to antibody production by antigens or by mitogens, such as lipopolysaccharide (LPS). We observed a mutually antagonistic relationship between activation through the antigen-receptor (AgR) and LPS-receptor (LPSR) in vitro. Prior exposure of B cells to AgR-ligating antibody prevented antibody forming cell (AFC) production induced by LPS, but not that induced by specific antigen (SRBC, TNP-Ficoll, or TNP-LPS). AFC production induced by antigen could be abrogated by concomitant exposure to LPS; the shutdown of the antigen-driven response was apparent when LPS-induced AFC were prevented by pre-exposure to antibody against the AgR. The ability of signaling through the AgR to inhibit antibody production stimulated by LPS was seen in DBA/2 and BALB/c mouse strains, and not in the New Zealand Black (NZB) strain. The results suggest that mutual antagonism is distinct from other forms of immune hyporesponsiveness, and that defects in antagonism may be a factor in the development of autoimmune disease.

Blockade of immunoregulatory Fc-signalling by HIV peptides: oligopeptides from HIV gp120 and gp41 bind the Fc portion of IgG and increase the in vitro anti-ssDNA response.

Concomitant ligation of antigen receptors with Fc-receptors negatively signals B cells. Antibodies to the Fc portion of IgG prevent this negative Fc-signalling, provided that these antibodies do not emit Fc signals. Prevention of Fc signals leads to augmented antibody responses to self and foreign antigens, and reduces the requirement for T cells by 10- to 100-fold in T cell-dependent antibody responses. In ELISA assays, peptides from conserved portions of the glycoproteins, HIV-1 gp120 or gp41 from HIV-1 and HIV-2 bind to the Fc portion of IgG, but do not bind the F(ab')2 portion of IgG. HIV-derived peptides, which bind to the Fc portion of IgG, augment the antibody-forming cell response to single-stranded (ss)DNA. The spontaneous response to ssDNA using spleen cells from young mice, and the response in the presence of exogenous DNA using spleen cells from old mice, are augmented to the greatest extent. These results demonstrate that HIV peptides bind to the Fc portion of IgG and augment immune responses to DNA; they suggest the possibility that blockade of the Fc portion of IgG antibodies is associated with a reduction in Fc-mediated regulation of anti-self responses. Blockade of regulatory Fc-signalling may account for increased circulating immunoglobulins and autoantibodies in clinical AIDS.

Tentativeness and fervor in cell biology require negative and positive feedforward control.

End-product feedback regulates early steps in metabolic pathways, affecting activation and the rate of end-product synthesis. Early formation of end-product also modifies later steps in the synthesis of end-product. We designate this form of regulation feedforward. Negative feedback/feedforward by end-products may result in homeostasis, but also in physiologic tentativeness. Positive feedback/feedforward by end-product gives rise to fervid events. Tentativeness and fervor, due to negative and positive feedforward rather than feedback, explain otherwise puzzling aspects of immunology and endocrinology.

Immunoregulatory characteristics of the in vitro anti-ssDNA response.

Continuous blockade of B-cell antigen receptors (BCRs) with Fab alpha sIg prevents the anti-ssDNA response of high, but not low, density B cells. Signaling via the BCRs, by prior exposure to crosslinking F(ab')2 alpha sIg, had no effect on the spontaneous anti-DNA response, but prevented a lipopolysaccharide-induced anti-DNA response. Pretreatment with intact alpha sIg, which provides exogenously derived Fc signals, reduced the response. An Fc-signal-blocking agent, F(ab')2 anti-IgG-Fc antibody, increased the number of anti-DNA antibody-forming cells produced in the absence of exogenous IgG anti-ssDNA antibody. Thus, activation is dependent on the availability of the BCRs, prior BCR crosslinking does not interfere with activation, and endogenous IgG anti-ssDNA antibody limits the activation of anti-ssDNA-specific B cells most of which are T-cell independent. These results indicate that the anti-ssDNA response is driven through the BCR.

Low-dose steroid therapy in cyclosporine-treated renal transplant recipients with well-functioning grafts. The Canadian Multicentre Transplant Study Group.

OBJECTIVE: Low-dose prednisone given on alternate days as a steroid adjunct to cyclosporine therapy was investigated primarily for its influence on kidney graft and patient survival and, secondarily, on renal function and complications. DESIGN: Multicentre randomized double-blind clinical trial. SETTING: Fourteen Canadian transplant centres. PATIENTS: A total of 523 patients with well-functioning renal transplants (cadaveric grafts or grafts from living related donors) and without active graft rejection reactions who were entered into the trial from 1982 to 1985. INTERVENTION: Patients were randomly assigned 90 days after transplantation to receive either placebo (260 patients) or low-dose prednisone (263 patients). MAIN OUTCOME MEASURES: Graft and patient survival. MAIN RESULTS: After at least 5 years of follow-up 50 patients assigned placebo had lost their graft and 17 had died; the corresponding figures for those assigned prednisone were 38 and 16. After an average interval of 1.4 years 143 patients in the placebo group and 123 patients in the prednisone group had stopped therapy with the test drug or had had their treatment group decoded or both. Patients were withdrawn from the study 2 years after stopping the test therapy. The actuarial 5-year graft survival rates were 73% and 85% in the placebo and prednisone groups respectively (p = 0.03), and the actuarial 5-year patient survival rates were 92% and 94% respectively (p = 0.6). This analysis included 43 and 29 graft losses and 14 and 12 deaths in the placebo and prednisone groups respectively. Weibull parametric modelling of graft survival identified the following variables as risk factors for graft loss: histocompatibility leukocyte antigen B (HLA-B) mismatching (p = 0.007), donor death from cerebrovascular accident (p = 0.01), increased donor age (p = 0.02) and being a male recipient (p = 0.05). When these factors were included in the Cox proportional hazards model, the influence of assigned treatment on graft survival was reduced to p = 0.1. Donor death from cerebrovascular accident (p = 0.002), diabetes mellitus in the recipient (p = 0.02) and increased recipient age (p = 0.05) were risk factors for patient death. Renal function and incidence of complications were similar in the treatment groups. CONCLUSIONS: Continued administration of low-dose prednisone on alternate days is advisable, particularly in patients with cadaveric grafts and those with previously failed transplants.