Showing papers on "Immune tolerance published in 1993"

Spontaneous loss of T-cell tolerance to glutamic acid decarboxylase in murine insulin-dependent diabetes.

Abstract: INSULIN-DEPENDENT diabetes mellitus (IDDM) in non-obese diabetic (NOD) mice results from the T-lymphocyte-mediated destruction of the insulin-producing pancreatic β-cells and serves as a model for human IDDM1. Whereas a number of autoantibodies are associated with IDDM2, it is unclear when and to what β-cell antigens pathogenic T cells become activated during the disease process. We report here that a T-helper-1 (Thl) response to glutamate decarboxylase develops in NOD mice at the same time as the onset of insulitis. This response is initially limited to a confined region of glutamate decarboxylase, but later spreads intramolecularly to additional determinants. Subsequently, T-cell reactivity arises to other β-cell antigens, consistent with intermolecular diversification of the response. Prevention of the spontaneous anti-glutamate decarboxylase response, by tolerization of glutamate decarboxylase-reactive T cells, blocks the development of T-cell autoimmunity to other β-cell antigens, as well as insulitis and diabetes. Our data suggest that (1) glutamate decarboxylase is a key target antigen in the induction of murine IDDM; (2) autoimmunity to glutamate decarboxylase triggers T-cell responses to other β-cell antigens, and (3) spontaneous autoimmune disease can be prevented by tolerization to the initiating target antigen.

Receptor editing: an approach by autoreactive B cells to escape tolerance.

Abstract: To determine the fate of anti-DNA antibody-bearing B cells in normal mice, we generated transgenic mice bearing the heavy (H) and light (L) chain genes of a well-characterized anti-double-stranded DNA antibody. This antibody was originally isolated from a diseased MRL/lpr mouse and has characteristics common to spontaneously arising anti-DNA antibodies. Results show that the H/L transgene (tg) immunoglobulin receptor is not expressed by animals bearing both tgs, although single tg animals (H or L) express their transgenes. Young H/L tg animals express few B cells, whereas adult H/L tg animals maintain almost normal B cell numbers. Analysis of the immunoglobulin receptors used by adult B cells shows that all contain the tg H chain in association with endogenous L chains. These B cells transcribe the L tg as well as the rearranged endogenous L chain gene, and loss of endogenous L chain gene transcription results in resurrection of the 3H9 H/L tg product. Examination of the endogenous L chains used by these cells shows that they represent a highly restricted subset of V genes. Taken together, these data suggest that autoreactive transgenic B cells can rearrange endogenous L chain genes to alter surface receptors. Those L chains that compete successfully with the L tg for H chain binding, and that create a nonautoreactive receptor, allow the B cell to escape deletion. We suggest that this receptor editing is a mechanism used by immature autoreactive B cells to escape tolerance.

"Infectious" transplantation tolerance

Abstract: The maintenance of transplantation tolerance induced in adult mice after short-term treatment with nonlytic monoclonal antibodies to CD4 and CD8 was investigated. CD4+ T cells from tolerant mice disabled naive lymphocytes so that they too could not reject the graft. The naive lymphocytes that had been so disabled also became tolerant and, in turn, developed the capacity to specifically disable other naive lymphocytes. This process of "infectious" tolerance explains why no further immunosuppression was needed to maintain long-term transplantation tolerance.

The influence of antigen organization on B cell responsiveness

Abstract: The influence of antigen epitope density and order on B cell induction and antibody production was assessed with the glycoprotein of vesicular stomatitis virus serotype Indiana [VSV-G (IND)]. VSV-G (IND) can be found in a highly repetitive form the envelope of VSV-IND and in a poorly organized form on the surface of infected cells. In VSV-G (IND) transgenic mice, B cells were unresponsive to the poorly organized VSV-G (IND) present as self antigen but responded promptly to the same antigen presented in the highly organized form. Thus, antigen organization influences B cell tolerance.

Human T-cell clonal anergy is induced by antigen presentation in the absence of B7 costimulation.

Abstract: The maximal T-cell response to its antigen requires presentation of the antigen by a major histocompatibility complex class II molecule as well as the delivery of one or more costimulatory signals provided by the antigen-presenting cell (APC) Although a number of candidate molecules have been identified that are capable of delivering a costimulatory signal, increasing evidence suggests that one such critical pathway involves the interaction of the T-cell surface antigen CD28 with its ligand B7, expressed on APCs In view of the number of potential costimulatory molecules that might be expressed on the cell surface of APCs, artificial APCs were constructed by stable transfection of NIH 3T3 cells with HLA-DR7, B7, or both Here, we show that in a human antigen-specific model system, when tetanus toxoid peptide antigen is presented by cells cotransfected with HLA-DR7 and B7, optimal T-cell proliferation and interleukin 2 production result In contrast, antigen presentation, in the absence of B7 costimulation, results in T-cell clonal anergy These results demonstrate that it is possible to induce antigen-specific clonal tolerance in human T cells that have been previously sensitized to antigen The artificial antigen-presenting system provides a useful model for the investigation of the biochemical events involved in the generation of tolerance and for the study of signals necessary to overcome tolerance

Inhibition of Langerhans cell antigen-presenting function by IL-10. A role for IL-10 in induction of tolerance.

Abstract: IL-10 is a product of activated keratinocytes and is released during the induction phase of contact sensitivity. As IL-10 effects have been described as being mediated by APC, we investigated effects of IL-10 on epidermal Langerhans cells (LC), the resident APC in the epidermis. Initial studies failed to demonstrate effects of IL-10 on MHC class II Ag expression by LC or anti-CD3 mAb- or alloantigen-induced LC-dependent T cell proliferation. However, production of IFN-gamma and IL-2, (but not IL-6) was markedly reduced in these assays. When the soluble-protein Ag specific T cell clones AE7 (Th1) and D10.G4 (Th2) were substituted for unprimed T cells, differential effects of IL-10 on T-cell proliferation were observed. Whereas IL-10-pretreated and untreated LC supported Th2 cell proliferation equally well, IL-10-pretreated LC were essentially unable to induce Th1 cell proliferation in response to native protein or peptide Ag. The inhibitory influence of IL-10 on Th1 cells was observed when fresh or 1 day cultured LC were used; 2- or 3-day cultured LC were affected to a much lesser extent by IL-10 pretreatment. Further, coculture experiments using IL-10-pretreated or untreated LC of a different haplotype suggest that IL-10 negatively regulates a costimulatory signal required for induction of Th1 cell proliferation. To assess whether T cells incubated with Ag and IL-10-pretreated LC were responsive to further stimulation, T cells were rescued after 1 day of coculture with IL-10-pretreated LC and restimulated, either immediately or after 1 to 5 days of rest, with untreated LC in the presence of Ag. T cells incubated with IL-10-pretreated LC were found to be anergic, whereas T cells incubated with untreated LC proliferated normally after further stimulation. However, anergic T cells responded vigorously to IL-2. These data indicate that although IL-10-pretreated LC are effective APC for Th2 cells, they fail to induce Th1 cell proliferation and rather induce clonal anergy in these cells.

Cytokine control of parasite-specific anergy in human lymphatic filariasis Preferential induction of a regulatory T helper type 2 lymphocyte subset

Abstract: The immunological mechanisms involved in maintenance of an asymptomatic microfilaremic state (MF) in patients with lymphatic filariasis remain undefined. MF patients have impaired filarial antigen (Ag)-specific lymphocyte proliferation and decreased frequencies (Fo) of Ag-specific T cells, and yet elevated serum IgE and antifilarial IgG4. To investigate the mechanism of Ag-specific anergy in MF patients in contrast to amicrofilaremic individuals with chronic lymphatic obstruction (CP), the Fo of Ag-specific lymphocytes from peripheral blood mononuclear cells secreting either IL-4 or IFN-gamma were assessed by filter spot enzyme-linked immunosorbent assay, and IL-10 and transforming growth factor-beta (TGF-beta) mRNA transcript levels were assessed by a semiquantitative reverse transcriptase polymerase chain reaction technique. The Fo of filaria-specific IL-4-secreting lymphocytes were equivalent in both MF (geometric mean [GM] = 1:11,700) and CP (GM = 1:29,300 P = 0.08), whereas the Fo of IFN-gamma-secreting lymphocytes were lower in MF (GM = 1:39,300) than in CP (GM = 1:4,200, P < 0.01). When the ratio of IL-4/IFN-gamma (T helper type 2 [Th2]/Th1)-secreting cells was examined, MF subjects showed a predominant Th2 response (8:1) compared with a Th1 response in CP individuals (1:4). mRNA transcript levels of IL-10 were also significantly elevated in MF compared with CP individuals (P < 0.01). Further, IL-10 and TGF-beta were shown to have a role in modulating the Ag-specific anergy among MF subjects, in that neutralizing anti-IL-10 or anti-TGF-beta significantly enhanced lymphocyte proliferation response (by 220-1,300%) to filarial Ags in MF individuals. These findings demonstrate that MF subjects respond to parasite antigen by producing a set of suppressive cytokines that may facilitate persistence of the parasite within humans while producing little clinical disease.

T cell immunity after a viral infection versus T cell tolerance induced by soluble viral peptides.

Abstract: The fate of in vivo activated CD8+ cytotoxic T cells was studied in transgenic mice expressing a T cell receptor (TCR) specific for the lymphocytic choriomeningitis virus (LCMV) glycoprotein peptide 33-41 presented by major histocompatibility complex (MHC) class I molecules. LCMV infection of TCR transgenic mice induced LCMV-specific effector and memory T cells whereas injection of soluble LCMV glycoprotein peptide 33-41 resulted in tolerance by peripheral deletion and anergy of LCMV-specific T cells after an initial expansion phase. Similarly, LCMV peptide 33-41-specific tolerance could be achieved in normal C57BL/6 mice and was not abrogated by an LCMV infection. These results obtained with a classically MHC-restricted peptide antigen parallel previous findings with retroviral or bacterial superantigens and indicate a possibility to modulate specifically mature peripheral cytotoxic T lymphocytes in vivo.

Suppression of B lymphopoiesis during normal pregnancy.

Abstract: We describe a dramatic reduction in numbers and activity of committed B lymphocyte precursors in the bone marrow of normal pregnant mice. Changes in cells responsive to IL-7 were evident as early as 6.5 d of pregnancy and values were < 10% of normal at parturition. B lineage precursors, identified by display of CD45R and absence of surface IgM, were also substantially depressed, and subpopulations representing different stages in the B lineage were assessed by three-color flow cytometry. Early pro-B cells are medium to large in size and have been previously characterized by low expression of the heat-stable antigen (HSA). This category of cells was not reduced, and in fact may have been slightly elevated, during pregnancy. In contrast, all subsequent populations of B lineage precursors, defined by patterns of expression of heat-stable and CD43 antigens, were substantially depressed. The immediate precursors of B cells (small pre-B cells) were identified by small size, expression of CD45R, absence of CD43, and lack of surface IgM. These were the most reduced of any phenotypically defined population in bone marrow. Numbers of newly formed B cells, characterized by the presence of sIgM, but not sIgD, were also diminished. However, B cells with a mature phenotype (sIgM+, sIgD+) were present in normal to somewhat elevated numbers. Mitogen-responsive B cells clonable in a semisolid agar assay were not significantly affected. A bromodeoxyuridine (BrdU) labeling technique was used to evaluate mitotic activity, which revealed an increased proportion of long-lived lymphocytes in the bone marrow of pregnant mice. These observations indicate that B lymphopoiesis is markedly downregulated during pregnancy and that all precursor populations beyond the early pro-B cell stage are affected. The pregnancy-related changes in bone marrow were selective for B lineage precursors, as cells expressing myeloid and erythroid markers were not reduced. In spleen, evidence was obtained for partial depletion of one subset of B cells. These cells, which have been reported to be recent immigrants from marrow, are characterized as having high levels of sIgM and HSA. Changes in other major B lymphocyte subsets in the spleen were less remarkable. When considered with results from the BrdU labeling procedure, the findings indicate that both production and export of lymphocytes from marrow may be substantially decreased. Numbers of B cell precursors were higher in postpartum animals whose litters were removed at birth, suggesting that lactation may prolong regeneration of lymphocyte production.(ABSTRACT TRUNCATED AT 400 WORDS)

B7 but not intercellular adhesion molecule-1 costimulation prevents the induction of human alloantigen-specific tolerance.

Abstract: Presentation of antigen by the major histocompatibility complex to T lymphocytes without the requisite costimulatory signals does not induce an immune response but rather results in a state of antigen-specific unresponsiveness, termed anergy. To determine which costimulatory signals are critical for the T cell commitment to activation or anergy, we developed an in vitro model system that isolated the contributions of alloantigen and each candidate costimulatory molecule. Here, we show that transfectants expressing HLA-DR7 and either B7 or intercellular adhesion molecule 1 (ICAM-1) deliver independent costimulatory signals resulting in alloantigen-induced proliferation of CD4-positive T lymphocytes. Although equivalent in their ability to costimulate maximal proliferation of alloreactive T cells, B7 but not ICAM-1 induced detectable interleukin 2 secretion and prevented the induction of alloantigen-specific anergy. These results are consistent with the hypothesis that blockade of the ICAM-1:lymphocyte function-associated 1 pathway results in immunosuppression, whereas blockade of the B7:CD28/CTLA4 pathway results in alloantigen-specific anergy. This approach, using this model system, should facilitate the identification of critical costimulatory pathways which must be inhibited in order to induce alloantigen-specific tolerance before human organ transplantation.

Direct evidence for anergy in T lymphocytes tolerized by oral administration of ovalbumin.

Abstract: The present study investigated bystander suppression, specific suppression and anergy as mechanisms for oral tolerance. Oral tolerance was induced in mice by a single gastric intubation of 20 mg ovalbumin (OVA) and was evaluated in vitro by the absence of T lymphocyte proliferative responses to OVA after priming by OVA-complete Freund's adjuvant (CFA). T lymphocyte unresponsiveness was antigen specific, systemic and was not affected by the vehicle used for immunization. T lymphocytes derived from tolerant popliteal lymph nodes (PLN) responded to an acetone precipitate (AP) of mycobacteria present in CFA; this response was not suppressed by co-culture with OVA, thereby arguing against a mechanism of bystander suppression in our system. Responses of PLN T lymphocytes derived from OVA-CFA primed, non-tolerant mice, or those of an OVA-specific T lymphocyte line, were not suppressed by PLN or spleen cells derived from OVA tolerant mice. These results excluded the possibility that oral tolerance was induced and maintained by a mechanism of specific suppression. At the cellular level, we found that OVA-tolerant T lymphocytes did not produce interleukin-2 (IL-2) nor express IL-2 receptor in response to OVA stimulation in vitro; both observations are indicative of a state of anergy. Incubation of OVA-tolerant PLN T lymphocytes together with murine recombinant IL-2 for 5 days, released anergic T lymphocytes and a concomitant OVA-specific proliferative response of CD4+ T cells was detected. Taken together, our experimental system excludes the involvement of bystander or specific suppression in the induction of oral tolerance to OVA, and provides direct evidence to show that oral tolerance results from specific T lymphocyte anergy.

The Fas protein is expressed at high levels on CD4+CD8+ thymocytes and activated mature lymphocytes in normal mice but not in the lupus-prone strain, MRL lpr/lpr.

Abstract: The lymphoproliferation (lpr) mutation in the MRL strain of mice is caused by the insertion of the early transposable element ETn in the Fas gene. The insertion causes a striking decrease in Fas mRNA expression and is associated clinically with marked acceleration of the lupus-like disease. To further explore the role of the Fas protein in T-cell selection in the thymus and tolerance in the peripheral immune system, we produced a monospecific polyclonal anti-murine Fas antibody that binds to a polymorphic region of the protein. Fas protein expression was detected on approximately 90% of BALB/c and MRL +/+ thymocytes, and the expression was highest on CD4+CD8+ thymocytes, the stage at which most thymocytes die by apoptosis. In contrast to the high level of expression of Fas on thymocytes, Fas was detected on < 10% of normal splenic T cells. After activation of splenic T cells with Con A or anti-CD3 and interleukin 2, Fas expression increased approximately 10-fold. Fas expression on splenic B cells was also markedly up-regulated after activation with lipopolysaccharide or anti-mu antibodies. The Fas protein was not detected on resting or activated lymphocytes obtained from MRL lpr/lpr mice. Together, these findings suggest that Fas plays a role in both thymic selection and T-cell survival in the periphery and that the accelerated autoimmunity in MRL lpr/lpr mice results from a defect in both of these pathways.

Evolution and pathophysiology of the human natural anti-alpha-galactosyl IgG (anti-Gal) antibody.

Abstract: Anti-Gal is a human natural antibody which interacts specifically with the mammalian carbohydrate structure Galα1-3Galβ1-4GlcNAc-R, termed, the α-galactosyl epitope. This antibody constitutes approximately 1% of circulating IgG in human serum and is produced, upon stimulation, by 1% of circulating B lymphocytes. Anti-Gal is also present as IgA antibodies in body secretions such as saliva, milk and colostrum. The antigenic source for the constant production of anti-Gal seems to be the α-galactosyl-like epitopes found on many bacteria of the gastrointestinal flora. Whereas anti-Gal is abundant in humans, apes and Old World monkeys, it is absent from New World monkeys, prosimians and nonprimate mammals. The latter group of species produces, however, large amounts of α-galactosyl epitopes (> 106 epitopes per cell). It is estimated that anti-Gal appeared in ancestral Old World primates less than 28 million years ago, possibly as a result of an evolutionary event which exerted a selective pressure for the suppression of α-galactosyl epitopes expression by inactivation of the gene for the enzyme α1,3 galactosyltransferase. This also resulted in the loss of immune tolerance to the α-galactosyl epitope and the production of anti-Gal. The physiologic role of this antibody is not clear as yet. It may participate in the protection against gastrointestinal bacteria. In addition it seems to contribute to the removal of normal and pathologically senescent red cells by interacting with the few hundred cryptic α-galactosyl epitopes which are exposed de novo in the course of red cell aging, thereby opsonizing these cells for phagocytosis by reticuloendothelial macrophages. The α-galactosyl epitope has been found to be aberrantly expressed on human cells and the interaction of anti-Gal with such epitopes may result in autoimmune disease. Preliminary data suggest such a mechanism in Graves' disease. Anti-Gal has been found to interact with therapeutic recombinant proteins expressing α-galactosyl epitopes, but so far there is no indication that it affects the half-life in the circulation and the biologic activity. Detection of anti-Gal in the seminal fluid and in the cerebrospinal fluid may serve as a simple means for assessment of damage to the blood-genital tract barrier or the blood-brain barrier. Studies on the interaction of anti-Gal with aberrantly expressed α-galactosyl epitopes on human cells may elucidate the possible role of anti-Gal in human autoimmune diseases.

Specificity of HLA class I antigen recognition by human NK clones: evidence for clonal heterogeneity, protection by self and non-self alleles, and influence of the target cell type.

Abstract: Prior studies using polyclonal populations of natural killer (NK) cells have revealed that expression of certain major histocompatibility complex (MHC) class I molecules on the membrane of normal and transformed hematopoietic target cells can prevent NK cell-mediated cytotoxicity. However, the extent of clonal heterogeneity within the NK cell population and the effect of self versus non-self MHC alleles has not been clearly established. In the present study, we have generated more than 200 independently derived human NK cell clones from four individuals of known human histocompatibility leukocyte antigens (HLA) type. NK clones were analyzed for cytolytic activity against MHC class I-deficient Epstein Barr virus (EBV) transformed B lymphoblastoid cell lines (B-LCL) stably transfected with several HLA-A, -B, or -C genes representing either self or non-self alleles. All NK clones killed the prototypic HLA-negative erythroleukemia K562 and most lysed the MHC class I-deficient C1R and 721.221 B-LCL. Analysis of the panel of HLA-A, -B, and -C transfectants supported the following general conclusions. (a) Whereas recent studies have suggested that HLA-C antigens may be preferentially recognized by NK cells, our findings indicate that 70% or more of all NK clones are able to recognize certain HLA-B alleles and many also recognize HLA-A alleles. Moreover, a single NK clone has the potential to recognize multiple alleles of HLA-A, HLA-B, and HLA-C antigens. Thus, HLA-C is not unique in conferring protection against NK lysis. (b) No simple patterns of HLA specificity emerged. Examination of a large number of NK clones from a single donor revealed overlapping, yet distinct, patterns of reactivity when a sufficiently broad panel of HLA transfectants was examined. (c) Both autologous and allogeneic HLA antigens were recognized by NK clones. There was neither evidence for deletion of NK clones reactive with self alleles nor any indication for an increased frequency of NK clones recognizing self alleles. (d) With only a few exceptions, protection conferred by transfection of HLA alleles into B-LCL was usually not absolute. Rather a continuum from essentially no protection for certain alleles (HLA-A*0201) to very striking protection for other alleles (HLA-B*5801), with a wide range of intermediate effects, was observed. (e) Whereas most NK clones retained a relatively stable HLA specificity, some NK clones demonstrated variable and heterogeneous activity over time. (f) NK cell recognition and specificity cannot be explained entirely by the presence or absence of HLA class I antigens on the target cell.(ABSTRACT TRUNCATED AT 400 WORDS)

Clonal anergy blocks in vivo growth of mature T cells and can be reversed in the absence of antigen.

Abstract: Experiments in various models have indicated that immunological tolerance can result from the physical elimination (deletion) of reactive lymphocytes as well as from anergy. We have previously reported that mature CD4-CD8+ T cells when confronted with their antigen can proliferate extensively but are finally eliminated or become intrinsically anergic such that remaining cells are refractory to stimulation by any T cell receptor ligands, even in the presence of exogenous interleukin 2. Here we show that in vivo the anergy can be reversed in the absence of antigen, such that the cells are then able to proliferate extensively in vivo to a new challenge with the antigen in question.

Oral tolerance in experimental autoimmune uveoretinitis. Distinct mechanisms of resistance are induced by low dose vs high dose feeding protocols.

Abstract: Studies of oral tolerance in LEW rat models of autoimmune diseases including S-antigen (S-Ag)-mediated experimental autoimmune uveoretinitis (EAU), and myelin basic protein-induced experimental autoimmune encephalomyelitis have produced conflicting evidence for the roles of clonal anergy and suppression. Using subpeptides from a region of S-Ag known to induce oral tolerance a protective site was localized to a nonamer of residues 347-355. This site was also uveitogenic, providing the basis for testable hypotheses for tolerance to be due to clonal anergy in pathogenic T cells specific for that site, or to suppression. Evidence for suppression was strongly supported by several observations. 1) Induction of oral tolerance with low dose feedings (250 micrograms/feeding) of peptide 343-362 conferred resistance to EAU induction by intact S-Ag, which should not be possible if only T cells specific for epitopes in 343-362 were rendered unresponsive, since there are several other pathogenic sites in S-Ag. 2) Low dose feeding induced resistance to EAU induction by a distinct, spatially separate peptide, residues 270-289, of S-Ag. 3) The requirement for linked recognition was shown by the inability of tolerance induced by feeding 343-362 to protect from EAU induction by a peptide, residues 521-540, derived from interphotoreceptor retinoid binding protein, a different uveitogenic retinal protein. 4) Resistance could be adoptively transferred. Conversely, induction of tolerance with high doses of peptide (5 mg/feeding) resulted in loss of resistance to EAU induced by S-Ag, although disease induction by the fed peptide was inhibited; observations that are consistent with clonal anergy. The apparent lack of suppression after high dose feeding could mean that suppressor T cells can also be rendered unresponsive or that induction of T suppressor cells is dependent on CD4+ Th cells, which were rendered anergic, leading to lack of T suppressor development. We suggest that oral tolerance operates by at least two distinct mechanisms that depend on the feeding dose; low doses induce suppression, whereas high doses induce unresponsiveness.

Epitopes of myelin basic protein that trigger TGF-beta release after oral tolerization are distinct from encephalitogenic epitopes and mediate epitope-driven bystander suppression.

Abstract: We have been studying the suppression of experimental autoimmune encephalomyelitis in the Lewis rat after oral administration of myelin basic protein (MBP). Suppression is mediated by CD8+ T cells that adoptively transfer protection and suppress immune responses in vitro. This suppression is mediated by secretion of TGF-beta following triggering by the fed antigen. In the present study, we tested the ability of overlapping 20 amino acid peptides from MBP to trigger suppression mediated by spleen cells from Lewis rats orally tolerized to MBP. Using a transwell system, we found that spleen cells from MBP orally tolerized animals stimulated by residues 21-40, 51-70 and 101-120 of MBP suppress proliferative responses of an ovalbumin specific cell line. This suppression correlated with secretion of TGF-beta by cells stimulated with the peptide. In addition, T cells from animals fed the tolerogenic peptide 21-40 alone secreted TGF-beta whereas no TGF-beta release or in vitro suppression was observed in animals fed the MBP encephalitogenic determinant 71-90. The 71-90 peptide triggered proliferation of MBP primed cells from animals immunized with MBP/CFA whereas the suppressor epitopes identified above did not. Furthermore, oral administration of peptide 21-40 suppressed disease induced by peptide 71-90. DTH responses to 71-90 were not affected by oral administration of peptide 21-40 whereas DTH responses to whole MBP were suppressed. These results demonstrate that distinct suppressor determinants exist on MBP which are separate from encephalitogenic determinants, and that epitope-driven bystander suppression plays an important role in down-regulation of tissue specific autoimmune processes following oral tolerization. These findings have important implications for the design of tissue specific targeted immunotherapy by oral tolerization in humans.

An autoimmune-disease with multiple molecular targets abrogated by the transgenic expression of a single autoantigen in the thymus

Abstract: Many autoimmune diseases are characterized by autoantibody reactivities to multiple cellular antigens. Autoantigens are commonly defined as targets of the autoimmune B cell response, but the role, if any, of these autoantigens in T cell-mediated autoimmune diseases is generally unknown. Murine experimental autoimmune gastritis is a CD4+ T cell-mediated organ-specific autoimmune disease induced by neonatal thymectomy of BALB/c mice. The murine disease is similar to human autoimmune gastritis and pernicious anemia, and is characterized by parietal and chief cell loss, submucosal mononuclear cell infiltrates, and autoantibodies to the alpha and beta subunits of the gastric H/K ATPase. However, the specificity of T cells that cause the disease is not known. To examine the role of the H/K ATPase in this T cell-mediated disease, transgenic mice were generated that express the beta subunit of the H/K ATPase under the control of the major histocompatibility complex class II I-Ek alpha promoter. We show that transgenic expression of the gastric H/K ATPase beta subunit specifically prevents the onset of autoimmune gastritis after neonatal thymectomy. In addition, thymocyte transfer experiments suggest that tolerance of pathogenic autoreactive T cells is induced within the thymus of the transgenic mice. We conclude that the beta subunit of the gastric H/K ATPase is a major T cell target in autoimmune gastritis and that thymic expression of a single autoantigen can abrogate an autoimmune response to multiple autoantigens.

Induction of long-term specific tolerance to allografts in rats by therapy with an anti-CD3-like monoclonal antibody.

Abstract: Monoclonal antibodies to CD3 have been shown to activate T cells in vivo and in vitro but have also been shown to render T cells anergic in vitro. In this study G4.18, a mouse IgG3 mAb, was produced that appeared to recognize CD3 by its binding to all peripheral T cells, including a population not recognized by mAb to TCR-alpha/beta that was presumed to be TCR-gamma/delta cells. It precipitated molecules in the 24-26 kd region consistent with the CD3 complex as well as molecules approximately 45 and approximately 49 kd that corresponded to TCR alpha and beta chains and a 92-kd complex. Incubating T cells for 24 hr with saturating concentrations of G4.18 caused modulation of the TCR complex. In vitro, it activated T cells but only if prebound to plastic. In solution it inhibited MLC and CML, but not PHA or Con A activation. In vivo, G4.18 was not toxic even in high doses, and this was thought to be due to the inability of this mAb to activate T cells in vitro because the rat lacks Fc receptors for mouse IgG3. Therapy with G4.18 resulted in transient modulation of TCR/CD3 on T cells and depletion of these cells from blood. G4.18 had no depleting effects by lymph node or spleen cells but caused marked, transient thymic involution. Therapy with G4.18 also induced indefinite survival (> 100 days) of PVG (RTIc) heart grafts but not skin grafts in DA (RTIa) hosts. These hosts with long-surviving cardiac transplants, when grafted from PVG skin, accepted these grafts but rejected third-party skin in first-set. Thus G4.18 was shown to induce long-term specific tolerance to an organ allograft.

Immunosuppressive factors in human cancer.

Abstract: Publisher Summary This chapter reviews the factors on immunosuppressive molecules that are elaborated by tumor cells or found in sera and effusions. Immunosuppressive factors can be found in normal sera, but their level is increased in cancer sera. These factors may contribute to the defective cellular responses of patients. Some well-characterized immunosuppressive molecules are transforming growth factor β (TGF-β), lymphocyte blastogenesis inhibitory factor (LBIF), p15E, and suppressive E-receptor (SER). TGF-β is produced by most cells and has an extraordinarily wide range of biological activities. p15E protein possesses a remarkable range of immunosuppressive activities. The SER is isolated from malignant effusions derived from patients with several types of cancer and inhibited several cellular immune responses. The exact mechanism of the immunosuppression is still uncertain, but a multiplicity of factors contributes to its occurrence: suppressor T cells and suppressor macrophages, immune complexes, acute phase proteins, tumor-derived suppressor molecules, suppressor substances in sera and effusion fluids of patients, and chemotherapy and irradiation. The colony-stimulating factors, acute-phase proteins, and miscellaneous molecules are some of the immunosuppressive factors in human cancer.

Autoimmune gld mutation uncouples suicide and cytokine/proliferation pathways in activated, mature T cells.

Abstract: Antigen receptor-directed suicide plays an important role in the elimination of potentially autoaggressive immature T cells during thymic differentiation. Here we demonstrated evidence for a second pathway of receptor-directed suicide in mature T cells that is missing in a mutant strain (gld) of mice with an "autoimmune" lymphoproliferative syndrome. The defect is evident within the gld activated T cell and does not require the presence of an antigen-presenting cell for its expression. Receptor-driven suicide is intact in immature T cells of animals with this mutation. These results support the significance of receptor-directed suicide in the mature T cell compartment and suggest that the immune system may use three independent pathways for regulating programmed cell death in shaping and controlling the immune response.

Thymic recognition of class II major histocompatibility complex allopeptides induces donor-specific unresponsiveness to renal allografts.

Abstract: Recent data show that intrathymic injection of allogeneic cells induces donor-specific unresponsiveness to allografts. There is also evidence to suggest that, in addition to recognizing intact MHC molecules, T cells can recognize processed MHC peptides, although the role of this indirect mode of allo-recognition in allograft rejection is unknown. We report that a single intrathymic injection of 100 micrograms of a mixture of eight 25-mer synthetic polymorphic class II MHC allopeptides, representing the full-length sequence of RT1.Bu beta and RT1.Du beta (WF) into incompatible (RT1l) LEW recipients, induced a state of long-term unresponsiveness to subsequent engraftment 2 days later of WF, but not third party (RT1n) BN renal allografts. Intrathymic injection of 100 micrograms of either RT1.Bu beta or RT1.Du beta peptide mixtures alone were insufficient to prolong renal allograft survival. Intravenous or intrasplenic injection of the allopeptide mixture did not affect renal allograft survival, establishing the role of thymic recognition of class II MHC allopeptides in inducing systemic unresponsiveness. The induction of intrathymic donor-specific unresponsiveness was abrogated if thymectomy was performed on the day of renal transplantation or 5 days later. PBLs from long-term surviving animals exhibited marked reduction of proliferation to WF, but not third party BN stimulator lymphocytes in the standard mixed lymphocyte response assay in vitro. These observations emphasize the role of recognition of processed MHC molecules in vascularized allograft rejection and confirm the role of the thymus in acquired systemic tolerance to alloantigens.

A rheumatoid factor transgenic mouse model of autoantibody regulation

Abstract: To address whether B cells expressing a disease-associated autospecificity are regulated in normal mice, we have established a rheumatoid factor (RF) transgenic model of autoimmunity, using V genes derived from an IgA anti-IgG2a RF isolated from an autoimmune MRL/lpr mouse. As we wished to study induction of tolerance during B cell development, we cloned the VH gene into an IgM expression vector. The RF we chose binds only IgG2a of the 'a' allotype (IgG2a) but not IgG2ab allowing us to produce transgenic animals on IgHa and IgHb backgrounds, which either express or lack the self-antigen. Two transgenic lines were studied. Using mice which lack the self-antigen, we show by fluorescence activated cell sorting and hybridoma analysis that the H and L transgenes are expressed to the exclusion of endogenous genes in most splenic B cells. In spite of good allelic exclusion, transgenic mice which are genetically capable of expressing IgG2aa have reduced but significant (approximately 50 micrograms/ml) serum levels. Nonetheless, the frequency and numbers of transgene-expressing B cells in peripheral lymphoid organs of such mice which have the self-antigen are similar to those which lack it (IgHb mice). Thus, B cells expressing an anti-self IgG2a surface receptor can develop in this system. Whether such B cells are anergic or otherwise regulated in autoantigen-expressing mice is discussed.

The inability to process a self-peptide allows autoreactive T cells to escape tolerance.

Abstract: It is now clear that antigen presenting cells (APCs) do not present all the possible peptides of self-proteins to the immune system. When then, is the fate of T cells specific for those self-peptides that escape processing? In this study, the COOH-terminal peptide (residues 81-104) of self cytochrome c (cyt c) elicited strong autoimmune T cells, as well as autoantibodies specific for this immunogen. These T cells did not respond to stimulation with the whole self cyt c molecule, demonstrating that APCs cannot process and present the self 81-104 peptide. Whereas mice were unresponsive to immunization with the whole mouse cyt c molecule, the mouse 81-104 fragment together with the whole self-molecule induced and amplified the autoimmune T cell response to sites within the 1-80 peptide. T cells that never contact the relevant self-peptide are functionally ignorant. They do not become tolerized or deleted, nor do they normally participate in immune responses to the native whole self-protein, since APCs cannot present the 81-104 peptide.

Mature CD4+ T lymphocytes from MRL/lpr mice are resistant to receptor-mediated tolerance and apoptosis.

Abstract: The goal of this study was to examine the functional responses and tolerance susceptibility of T lymphocytes from mice of the autoimmune strain, MRL/lpr. A population of autoreactive CD4+ T cells can be readily expanded from the lymphoid tissues of young lpr mice. Lines of IL-2-producing autoreactive and alloreactive lpr and alloreactive +/+ T cells were developed to study their responses to tolerance-inducing stimuli. Culture of +/+ T cells with high concentrations of immobilized anti-CD3 antibody induces both functional anergy and apoptosis. By contrast, lpr-derived T cell lines are relatively resistant to anergy and apoptosis. The implications of these findings for the development of autoimmunity, and the possible role of the Fas Ag in determining resistance or susceptibility to tolerance, are discussed.

In vivo role of interleukin 4 in T cell tolerance induced by aqueous protein antigen.

Abstract: High doses of aqueous protein antigens induce a form of immunological tolerance in which interleukin 2 (IL-2)- and interferon gamma (IFN-gamma)-secreting T helper type 1 (Th1) cells are inhibited, but IL-4-secreting (Th2) cells are not. This is manifested by reduced proliferation of antigen-specific T cells upon in vitro restimulation, and marked suppression of specific antibody responses of the immunoglobulin (Ig)G2a, IgG2b, and IgG3 isotypes, but not of IgG1 and IgE. The role of the immunomodulatory cytokine IL-4 in this model of unresponsiveness to protein antigens has been examined. Administration of tolerizing antigen itself primes splenic CD4+ T cells for secretion of lymphokines, both IL-2 and IL-4. Neutralization of IL-4 in vivo with the anti-IL-4 antibody 11B11 during tolerance induction augments IFN-gamma production by T cells of tolerant mice, and reverses the suppression of IgG2a, IgG2b, and IgG3. This blockade of IL-4 function does not, however, restore the proliferative responses of T cells, suggesting that reduced T cell proliferation is due to direct T cell inactivation or anergy. Inhibiting the activity of IL-4 in vivo also inhibits the expansion of antigen-specific Th2-like cells, which are resistant to the induction of unresponsiveness. Thus, the immunologic consequences of high-dose tolerance are due to a combination of clonal T cell anergy and IL-4-mediated immune regulation.

Self-Ignorance in the Peripheral T-Cell Pool

Abstract: The induction of immunological tolerance is a most fundamental property of the immune system that prevents responsiveness to self constituents while allowing reactivity to foreign invaders. It may be expected that in a system as complex and vitally important as that imposing and maintaining self tolerance, diverse mechanisms must operate to neutralize auto-aggressive lymphocytes. Indeed, various toleragenic mechanisms have been suggested at one time or another. They may be classified under two main groups. In one, the tolerant state is imposed as a result of the physical or functional silencing of potentially reactive lymphocytes following antigen encounter. This may involve clonal deletion, clonal abortion or clonal anergy (Nossal 1986). In the second, the immune system's own regulatory mechanisms hold reactive lymphocytes in check, through various cellular interactions yet to be defined precisely (Fowell et al. 1991). In many studies investigating the processes involved in the induction of immunological tolerance, the immune system has been confronted, not with self constituents, but with exogenous antigens introduced systemically. Of the several reasons why this may not mimic the natural situation, one is that an immune response does not result from the simple encounter of antigen and lymphocyte, but from a complex interaction of diverse cell types and molecules. The activation of such a cellular network must be subjected to appropriate control mechanisms. Hence, unresponsiveness following the introduction of exogenous antigen may reflect some normal control processes which limit the immune response, rather than events actually occurring during the induction of tolerance to 5 /̂/\" constituents. This should be borne in mind when considering self tolerance mechanisms

Studies of T cell deletion and T cell anergy following in vivo administration of SEB to normal and lupus-prone mice.

Abstract: This study examines the responses of lupus-prone NZB, (NZB x NZW) F1, BXSB, MRL-lpr/lpr and control mice (H-2 and Mls matched) to in vivo administration of the superantigen staphylococcal enterotoxin B (SEB). Two weeks after i.v. administration of 500 micrograms SEB, CD4+V beta 8+ lymph node T cells were deleted equivalently by lupus-prone and control mice. However, IE+ strains deleted a greater proportion (47% to 77%) of their CD4+V beta 8+ cells than did IE- strains (24% to 27%). CD8+V beta 8+ cells were deleted less than CD4+V beta 8+ cells by injection of 500 micrograms SEB. IE- strains failed to delete CD8+V beta 8+ cells, whereas six of seven IE+ strains deleted > 25% of their CD8+V beta 8+ cells. IE+ MRL-lpr/lpr mice showed some impairment in deletion: they failed to delete CD8+V beta 8+ cells at all doses of SEB and had reduced deletion of CD4+V beta 8+ cells at low doses of in vivo SEB (10 and 50 micrograms). Peripheral expansion of the intrathymically deleted V beta 7 TCR family was not observed in lupus-prone mice 2 wk after 500 micrograms in vivo SEB. In vitro restimulation with SEB of mice previously injected with 500 micrograms SEB demonstrated anergy in T cells from all strains, including the IE- and MRL-lpr/lpr. This result contrasts with previous reports of tolerance defects in lupus-prone strains using B cell read-out assays as measures of tolerance. The present study demonstrates that there is no global defect in peripheral T cell deletion or anergy in lupus-prone mice to the superantigen SEB. Although additional Ag would need to be studied, these experiments raise the possibility that some reported tolerance defects in lupus-prone strains may reflect excessive B cell responses to relatively normal T cell signals.