Mechanisms of immunological tolerance loss versus erythrocyte self-antigens and autoimmune hemolytic anemia

Recent studies on animal and human autoimmune hemolytic anemia (AIHA) suggest that immunological tolerance loss toward red blood cells (RBC) self-antigens may be originate by different, non-mutually exclusive, mechanisms. According to now available data the identified mechanisms may be: ignorance against RBC self-antigens; molecular mimicry; polyclonal T and/or B cells activation; errors in central or peripheral tolerance; immunoregulatory disorders including cytokine network alteration. In some patients with AIHA, stimulation of PMBC by synthetic Rh peptides indicate that ignorant T and/or B cell clones may recognize cryptic RBC self-antigens. AIHA associated with bacterial or viral infections seems to be produced by polyclonal T and/or B cells activation against foreign antigens which mimic protein or carbohydrate epitopes on RBC. Polyclonal activation of host B cell clones by donor alloreactive T cells causes the AIHA in chronic GVHD. As the tolerance loss is concerned, experiments on mouse lines expressing a transgene with autoantibody activity against murine RBC have shown that non-deleted peripheral B cell clones may produce RBC autoantibodies. In humans a genetic defect of Fas/FasL autoreactive lymphocytes apoptosis may be associated to AIHA. Immunoregulatory disorders due to depletion of CD4+ CD25+ T cells or Th1/Th2 cytokines imbalance may induce autoimmune diseases. In mice AIHA may be induced or improved by cytokines or anticytokine antibodies administration. In NZB/W mice and human AIHA there is an increased production of Th2 cytokines as IL4 and IL10 but INF-gamma reduced production. In addition in human AIHA has been shown a downregulation of IL12 and therefore, an IL10/IL12 immunoregulatory circuit imbalance which might facilitate the RBC autoantibodies production.     

Immunological tolerance loss vs. erythrocyte self antigens and cytokine network disregulation in autoimmune hemolytic anaemia

Recent studies on animal and human autoimmune hemolytic anaemia (AIHA) suggest that the loss of immunological tolerance vs. erythrocyte (Er) self antigens (Ag) may be primed by different mechanisms: ignorance of Er self Ag, molecular mimicry between self and non-self Ag, polyclonal T and/or B cells activation, errors in central or peripheral tolerance, immunoregulatory disturbances including the alteration of cytokines network. In vitro stimulation by synthetic Rh peptides indicates that ignorant T and/or B cells from patients with AIHA may recognize criptic Er self Ag. The AIHA associated with bacterial or viral infections seems to be produced by polyclonal T and/or B cell activation against foreign Ag that mimics protein or carbohydrate epitopes on Er. Polyclonal activation of host B cell clones by donor T cells causes the AIHA in chronic graft-versus-host disease. Mouse lines expressing a transgene with autoantibody (autoAb) activity against murine Er have shown that non-deleted peripheral B cell clones may produce Er autoAb. In human a genetic defect of Fas/FasL autoreactive lymphocyte apoptosis may be associated with AIHA. Th1/Th2 cytokines or IL10/IL12 imbalance may induce AIHA: in NZB mice and in human AIHA there is an increased production of Th2 cytokines such as IL4 and IL10 but INF-gamma and IL12 reduced production. Particularly, IL10 seems to act as critical mediator for the Er autoAb production.     

Th1 and Th2 Cytokine Modulation by IL-10/IL-12 Imbalance in Autoimmune Haemolytic Anaemia (AIHA)

Recent studies about autoimmune diseases in animal models and in humans focused their attention on lymphocyte activation and in vitro cytokine production. The respective contribution of the Th1 and Th2 cytokines to the pathogenesis of autoimmune diseases is still a matter of debate. In this study the role of IL-2, IL-4, IFN- &#110, IL-10 and IL-12 cytokines were investigated by examining their spontaneous and mitogen-induced (OKT3 and PHA or LPS) synthesis and T-cells proliferative response by peripheral blood mononuclear cells to determine their role in the pathogenesis of AIHA. Thirteen patients affected by AIHA, idiopathic or associated with other diseases, and 13 healthy subjects, randomly selected from a group of blood donors, were investigated. This study indicated that AIHA is characterised by increased basal synthesis of IL-4 and decreased levels of IFN- &#110 compared with healthy controls ( p <0,01). These results suggest that there is a basal decrease of Th1 cytokine and an increase of the Th2 ones. Enhanced IL-2 levels in AIHA patients are likely due to the necessity of a T-cell proliferation stimulus rather than produced as Th1 prevalent stimulation. Furthermore, it has been observed a significant increase in IL-12 production in LPS stimulated cultures from healthy controls, but not in AIHA patients, that shows IL-10 increased levels, which could cause a secondary decrease in IFN- &#110 production and a stimulation of Th2 differentiation. These observations indicate that decreased production of Th1-type cytokines and prevalent Th2 ones leading to autoantibodies production in AIHA may be secondary to the imbalance between IL-10 and IL-12. These results strongly suggest that manipulation of the cytokine network, i.e. IL-10/IL-12 balance, maintained by cells of the innate immune system, can have a strong effect on the incidence of AIHA and their modulation might be useful for a therapeutic control of the disorder.     

Th1 and Th2 cytokine modulation by IL-10/IL-12 imbalance in autoimmune haemolytic anaemia (AIHA)

Recent studies about autoimmune diseases in animal models and in humans focused their attention on lymphocyte activation and in vitro cytokine production. The respective contribution of the Th1 and Th2 cytokines to the pathogenesis of autoimmune diseases is still a matter of debate. In this study the role of IL-2, IL-4, IFN-gamma, IL-10 and IL-12 cytokines were investigated by examining their spontaneous and mitogen-induced (OKT3 and PHA or LPS) synthesis and T-cells proliferative response by peripheral blood mononuclear cells to determine their role in the pathogenesis of AIHA. Thirteen patients affected by AIHA, idiopathic or associated with other diseases, and 13 healthy subjects, randomly selected from a group of blood donors, were investigated. This study indicated that AIHA is characterised by increased basal synthesis of IL-4 and decreased levels of IFN-gamma compared with healthy controls (p < 0,01). These results suggest that there is a basal decrease of Th1 cytokine and an increase of the Th2 ones. Enhanced IL-2 levels in AIHA patients are likely due to the necessity of a T-cell proliferation stimulus rather than produced as Th1 prevalent stimulation. Furthermore, it has been observed a significant increase in IL-12 production in LPS stimulated cultures from healthy controls, but not in AIHA patients, that shows IL-10 increased levels, which could cause a secondary decrease in IFN-gamma production and a stimulation of Th2 differentiation. These observations indicate that decreased production of Th1-type cytokines and prevalent Th2 ones leading to autoantibodies production in AIHA may be secondary to the imbalance between IL-10 and IL-12. These results strongly suggest that manipulation of the cytokine network, i.e. IL-10/IL-12 balance, maintained by cells of the innate immune system, can have a strong effect on the incidence of AIHA and their modulation might be useful for a therapeutic control of the disorder.     

Controlling autoimmunity--Lessons from the study of red blood cells as model antigens

The characterization of human and animal red blood cell (RBC) autoantigens in autoimmune hemolytic anemia (AIHA) has provided an opportunity study the control of specific autoimmune responses of unequivocal pathogenic relevance. The results reveal that censorship of the autoimmune helper T (Th) cell repertoire by deletion and anergy is very incomplete in healthy individuals, even for widely distributed, abundant self-antigens on RBC. There is strong evidence that autoaggression by surviving Th cells is normally held in check by other mechanisms, including failure to display the epitopes that they recognize, and active immunoregulation. AIHA is one of the first human autoimmune diseases in which regulatory T (Tr) cells that are specific for the major autoantigens have been identified. These Tr cells recognize the dominant naturally processed epitopes, and recent studies suggest that disease develops when other determinants, to which such tolerance is less secure, and which are normally inefficiently presented, are displayed at higher levels. Together, the results raise the possibility that therapy for diseases such as AIHA could be based on switching the balance of the response back towards regulation, in particular by the administration of the dominant peptides recognized by specific Tr cells.     

Change in the Th1/Th2 Phenotype of Memory T-Cell Clones from Rheumatoid Arthritis Synovium

The stability of established memory T helper (Th)1/Th2 cells in chronic inflammatory diseases is not clear, and a shift of the cytokine balance could control chronic inflammation. In order to study the regulation of the Th phenotype of memory T cells, polyclonal T-cell lines and clones with a Th1, Th0 or Th2 phenotype were developed from rheumatoid synovial tissue. Th1 [interleukin (IL)-12 + anti-IL-4] and Th2 (IL-4 + anti-IL-12) promoting environments and IL-2 were used to manipulate the cytokine profile. Polyclonal T-cell lines of predominantly Th1 type could be shifted to produce Th2 cytokines, and polyclonal Th2/Th0 lines could be shifted to produce Th1 cytokines. However, this shift was due to an amplification of CD8+ T cells with a memory phenotype and a loss of the CD4+ T cells, giving Tc2 or Tc1 profiles, respectively. Th2 clones cultured repeatedly with IL-2 switched to either a Th0 or a Th1 phenotype, while both Th1 and Th0 memory clones kept a stable phenotype. Addition of Th2-promoting conditions strongly reduced the production of both interferon-gamma and IL-17, while Th1-promoting conditions increased the production of these cytokines. These results demonstrate that RA Th2 clones readily switch, while Th1 and Th0 clones are stable. However, induction of Th2 cytokines can be obtained in polyclonal polarized memory T cells due to amplification of Tc2 cells.     

T-helper 1 dominated responses to erythrocyte Band 3 in NZB mice.

Band 3, the red blood cell (RBC) anion channel protein, is the target autoantigen for the pathogenic RBC autoantibodies and T-helper (Th) cells in New Zealand Black (NZB) mice with autoimmune haemolytic anaemia (AIHA). To determine the subpopulation of these Th cells, they were stimulated with Band 3 and the profile of the cytokines elaborated by the responding cells was measured. NZB T cells stimulated with Band 3 produced high levels of the Th1 cytokine, interferon-gamma (IFN-gamma), but little or no interleukin-4 (IL-4), IL-5 or IL-10. Similar patterns were produced by NZB T cells responding to a spectrin preparation from the RBC membrane skeleton, or to mycobacterial heat-shock protein (hsp) 65 following immunization of mice with hsp 65 in incomplete adjuvant. By contrast, T cells from CBA mice similarly immunized with hsp 65 produced high levels of IL-4 and IL-5 in response to hsp 65. Examination of the isotype of the RBC-bound immunoglobulins in NZB mice revealed that immunoglobulin G2a (IgG2a) autoantibodies were the first to be detected in most mice and that later in the disease, IgG3 autoantibodies were often prominent. It is concluded that, contrary to expectation, the development of RBC autoantibodies in NZB mice is associated with Th1 cytokine-dominated responses.     

Idiotypes and autoimmunity

The reports discussed above have increased our knowledge of idiotypes, mainly with respect to additional CRI on autoantibodies and to a relatively new aspect of 'pathogenic idiotypes'. It is obvious that much remains to be discovered about the normal role of idiotypes and how they might be involved in the pathogenesis of autoimmune disorders. The relationship of the idiotypic network to tolerance is a matter of speculation; tolerance implies the ability to distinguish between 'foreign' and self-antigens and it is important to remember that antibody V regions are also self-antigens. Several mechanisms have been proposed to explain immunological tolerance. Originally, it was envisaged that the repertoires of both T and B lymphocytes were in some way purged of potentially self-reactive clones. However, it is now evident that self-reactive lymphocytes do exist but are normally held under control. Finally, it may be that certain self-antigens are simply never exposed to immune surveillance. It seems that the control of self-reactive lymphocytes is central to the question of tolerance. In the absence of autoimmune disease, autoantibodies can be produced, for example, after many infectious diseases or vaccinations. However, this type of perturbation of the immune system is associated with the short-term production of low titres of low-affinity antibodies, generally of the IgM isotype, which are thought to represent germline gene products. Homeostasis is soon re-established, possibly by regulatory T cells interacting with autoreactive B cells through their idiotypic receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Autoreactive T cell clones isolated from normal and autoimmune-susceptible mice exhibit lymphokine secretory and functional properties of both Th1 and Th2 cells

Recent studies have suggested the existence of two mutually exclusive subpopulations of T helper (Th) cells in the murine immune system, called Th1 which produces interleukin (IL)-2 and interferon (IFN)-gamma but not IL-4 and Th2 which secretes IL-4 and IL-5 but not IL-2. Also, functionally, Th1 cells generally activate the macrophages and mediate delayed-type hypersensitivity whereas Th2 cells provide help efficiently to B cells. In the present study, we investigated the lymphokine secretory properties of two well-characterized autoreactive (self-Ia reactive) T cell clones isolated from normal DBA/2 mice and autoimmune-susceptible MRL-lpr/lpr mice. It was observed that both the autoreactive T cell clones, following activation, produced IL-2, IL-4, and IFN-gamma. They induced hyper-Ia expression and cell proliferation in syngeneic B cells as well as activated the macrophages to exhibit tumoristatic properties. Both clones could also induce T-T network interaction in which syngeneic naive CD4+ T cells responded directly to stimulation with autoreactive T cell clones. The T-T interaction was demonstrable in 1-month-old MRL-lpr/lpr mice prior to the onset of the autoimmune disease but not in 6-month-old mice having lymphadenopathy and autoimmune disease. Unlike Th1 and Th2 cells which upon antigenic stimulation respond to exogenous IL-2 and IL-4, the autoreactive T cell clones responded only to IL-2 but not to IL-4. Our data suggest the existence of a unique subset of immunoregulatory CD4+ Th cells having the lymphokine secretory and functional properties of both the murine Th1 and Th2 subsets.     

High Cytokine Serum Levels in Patients with Autoimmune Hemolytic Anemia (AIHA)

In autoimmune diseases striking abnormalities of T and B cell activation and of cytokine production are present. In 14 patients with autoimmune hemolytic anemia (AIHA), idiopathic or in the course of: lymphoma, B hepatitis, carcinoma, drug therapy (α-methyldopa), systemic lupus erythematosus (SLE), and not yet submitted to immunosuppressive therapy, the PBL proliferative response to PHA and the IL1α, IL2, IL4 and IL2R serum levels have been valued. While the stimulation index of PBL was strongly reduced in 10 cases (64±56 vs 138±45 in the control group), IL1α, IL2 and IL2R were greatly increased in all the patients, and IL4 in 5 (IL1α :199±268 pg/ml in patients vs 0.30±0.2 in controls; IL2:716±311 pg/ml vs 16±4; IL4:29±13 pg/ml vs 13±7; IL2R:1233±471 U/ml vs 256±114). Cytokine serum levels were not related with the associated disease, with the CD4+ and CD8+ cells absolute number or with PBL blastogenic in vitro response. The high serum levels of cytokines and IL2R suggest that in AIHA there exist a CD4+ lymphocyte hyperactivation (the low proliferative response of PBL might imply a temporary functional exhaustion of T lymphocytes) as in the other autoimmune diseases.     

B cells in Sjögren's syndrome: From pathophysiology to diagnosis and treatment

Primary Sj?gren's syndrome (pSS) is a chronic autoimmune systemic disease, characterized by a lymphoplasmocytic infiltration and a progressive destruction of salivary and lachrymal glands, leading to ocular and mouth dryness. T cells were originally considered to play the initiating role in the autoimmune process, while B cells were restricted to autoantibody production. However, recent years have seen growing evidence that the roles of B cells in pSS pathophysiology are multiple, and that these cells may actually play a central role in the development of the disease. B cells are over-stimulated and produce excessive amounts of immunoglobulins and various autoantibodies. Peripheral blood and salivary-gland B-cell subset distribution is altered, leading to the constitution of ectopic germinal centers where auto-reactive clones may escape tolerance checkpoints. B cells control T-cell activation by different means: B effector cells guide Th1 or Th2 differentiation, whereas regulatory B cells inhibit T-cell proliferation. Several B-cell specific cytokines, such as BAFF or Flt-3L, are instrumental in the occurrence of B-cell dysfunction. Chronic and excessive stimulation of B cells may lead to the development of lymphoma in pSS patients. Autoantibodies and blood B-cell subset analysis are major contributors of a clinical diagnosis of pSS. These considerations led to the development of B-cell depletion therapies for the management of pSS. Rituximab, a monoclonal antibody to CD20, is the best studied biologics in pSS, but other treatments hold promise, targeting for example CD22 or BAFF. Thus, during the last 20 years, the understanding of the multifaceted roles of B cells in pSS has revolutionized the management of this complex disease.     

Regulation of CD4 T Cell Reactivity to Self and Non-Self

While the thymus may be effective in inducing tolerance to lymphoid associated antigens, it is not as efficient in deleting T cells reactive to peripheral tissue specific antigens. Therefore, to maintain self tolerance to peripheral tissues, post-thymic mechanisms must be invoked. One important way to prevent autoimmune pathology mediated by autoreactive CD4 T cells is the diversion of clones to regulatory Th2 effector cells. However, many different factors contribute in vivo to the decision of stimulated CD4 T cells to develop into Th1 versus Th2 cells. For example, T cell signaling pathways may influence the types of cytokines produced by naive T cells, and studies have provided evidence for a genetic polymorphism among common mouse strains that can significantly influence the early cytokine production in stimulated naive CD4 T cells. The allele carried by the BALB/c strain promotes IL-4 production, and consequently provides resistance to autoimmune diabetes in our transgenic mouse model. In addition, antigen presenting cells can influence the development of stimulated CD4 T cells in part through the production of cytokines such as IL-12. The absorption of IL-12 in vivo can permit the expansion of Th2 type effector cells, and this phenomenon will also protect mice from autoimmunity. Finally, the relative potency of various class II positive antigen presenting cell types can influence the development of autoreactive T cells, with dendritic cells apparently being the strongest stimulator of Th1 responses. Consistent with this notion, a relB knockout mouse, which is missing dendritic cells, appears to drive Th2 development even in response to viral infection. In sum, these various influences over the Th1/Th2 decision in vivo may provide new targets for immunotherapy of autoimmune diseases.     

T cell immunity in autoimmune hepatitis

T cells play a central role in the immunopathogenesis of AIH. Until recently CD4+ T cells were thought to be critical for disease development, increasing evidence has shown that CD8+ T and gammadelta T cells also play a significant role. The predisposition of certain HLA genotypes to AIH as well as the clonal expansion of a limited number of T cell receptors suggests that the presentation of a self-antigen or a molecular mimic may be responsible for the initiation of the immune response. Given the association of AIH with viral hepatitis, it is thought that the loss of tolerance begins with an infection of hepatocytes and subsequent cytolysis by CD8+ T cells. The presentation of self-antigens or molecular mimics leads to activation and clonal expansion of T cells; this process may be increased by impaired regulatory T cells and a defect in apoptosis. Ultimately T cells initiate B cell production of autoantibodies, proinflammatory cytokines and finally hepatocyte cytotoxicity.     

Insulin-specific T cell help to TNP-specific B cells requires activation via the antigen T cell receptor

In the present study, the production of large numbers of insulin (Ins)-specific, H-2-restricted T helper (Th) cell clones is described. Among 148 clones analyzed, 121 clones had Th cell function, and these were divided into 75 Ins-specific Th cell clones and 52 autoreactive clones. The 148 clones were isolated from Ins-specific T cell lines produced by in vitro stimulation of T lymphocytes from (b X d)F1 mice immunized with Ins 7, 14, 32 or 56 days before. The following characteristics were tested with regard to the Th cell clones: restriction specificity and antigen requirement for optimal help or interleukin 2 (IL2) production. No differences in these characteristics were found among clones originating from day 7, 14, 32 or 56 T cell lines. A preference for H-2b as restriction element and an antigen concentration of about 0.01 microgram trinitrophenylated (TNP)-Ins/ml for optimal help were general traits. Optimal IL2 release is not yet obtained with 100 micrograms TNP-Ins/ml. Thus, the antigen requirement for optimal help and IL2 release differs by a factor 10(4) at least. Certain twice-cloned Th cell lines were tested for IL2 production when stimulated with anti-Thy-1 monoclonal antibodies (mAb). All clones analyzed were stimulated by mAb H155 but not with mAb H140 nor with mAb against Lyt-1, L3T4, LFA-1 or H-2K/D molecules. Therefore, we determined whether TNP-conjugated H155 mAb would mediate Th cell-B cell collaboration as well as TNP-Ins. The results with nine different Th cell clones and six different TNP-conjugated mAb used in a 10(7)-fold concentration range showed that Th cell clones have to be triggered via the T cell receptor for expression of helper function to B cells. Thus, though IL2 gene activation, synthesis or release apparently can be activated via at least two pathways: T cell receptor or Thy-1, it seems that activation of the genes responsible for synthesis and release of the helper factors, which ensure antigen-specific B cell proliferation and differentiation, needs Th cell-B contact mediated via the antigen-specific T cell receptor.     

Indoleamine 2,3 dioxygenase contributes to transferable tolerance in rat red blood cell inducible model of experimental autoimmune haemolytic anaemia

Autoimmune haemolytic anaemia (AIHA) is caused by autoantibodies against red blood cell (RBC) surface antigens that render RBC susceptible to Fc‐mediated phagocytosis and complement‐mediated lysis. Experimental AIHA can be induced by injection of rat RBC to naive mice, but a lymphocyte‐mediated regulatory mechanism eventually suppresses the production of autoantibodies specific for mouse RBC. Critically, this tolerogenic response can be transferred to naive mice by splenocytes from the rat RBC‐immunized mouse. Here we investigate whether indoleamine 2,3 dioxygenase (IDO) or the initiators of IDO cascade, including the cytotoxic T lymphocyte antigen (CTLA)‐4 receptor and its soluble isoform, contribute to this tolerogenic mechanism. Splenocytes from experimental AIHA mice were transferred adoptively to naive mice under the cover of anti‐CTLA‐4, anti‐soluble CTLA‐4 antibodies or IDO inhibitor 1‐methyl tryptophan (1‐MT). Recipient mice were immunized with rat RBC and levels of antibody against self‐RBC and rat‐RBC were monitored. Our results indicate that transfer of tolerance to naive recipients is dependent upon IDO‐mediated immunosuppression, as mice receiving previously tolerized splenocytes under the cover of 1‐MT were refractory to tolerance and developed haemolytic disease upon further challenge with rat RBC. Initiators of IDO activity, CTLA‐4 or soluble CTLA‐4 did not mediate this tolerogenic process but, on their blockade, boosted antigen‐specific effector immune responses.     

The pathogenesis of oligoarticular/polyarticular vs systemic juvenile idiopathic arthritis

Juvenile idiopathic arthritis (JIA) has had a long and difficult problem with classification. It is clearly a heterogeneous and multi-factorial autoimmune disease but all too often the distinctions among subtypes were unclear. In fact, there is now increasing evidence of a distinct pathogenesis of oligo/polyarticular JIA compared to systemic JIA. Oligo/polyarticular JIA is an antigen-driven lymphocyte-mediated autoimmune disease with abnormality in the adaptive immune system. Cartilage-derived auto-antigens activate autoreactive T cells including Th1 and Th17 cells with production of pro-inflammatory cytokines IFN-γ and IL-17. On the other hand, the inhibition of regulatory T (Treg) cells including natural Foxp3(+) Treg and self-heat shock protein-induced Treg cells with decreased anti-inflammatory cytokine IL-10 results in the loss of immune tolerance. Imbalance between autoreactive Th1/Th17 and Treg cells leads to the failure of T cell tolerance to self-antigens, which contributes to the synovial inflammation of oligo/polyarticular JIA. By contrast, systemic JIA is an autoinflammatory disease with abnormality in the innate immune system. A loss of control of the alternative secretory pathway leading to aberrant activation of phagocytes including monocytes, macrophages and neutrophils seems to be involved in the release of pro-inflammatory cytokines IL-1, IL-6, IL-18 and pro-inflammatory S100-proteins, which contribute to the multisystem inflammation of systemic JIA. Markedly distinct pathogenesis of oligo/polyarticular JIA and systemic JIA implies that they might need different treatment strategies.     

Desmoglein 3-specific T regulatory 1 cells consist of two subpopulations with differential expression of the transcription factor Foxp3

Pemphigus vulgaris (PV) is an autoimmune bullous skin disorder associated with autoantibodies against desmoglein (Dsg) 3. An imbalance of type 1 regulatory T (Tr1) cells and T helper type 2 (Th2) cells specific for Dsg3 may be critical for the loss of tolerance against Dsg3 in PV. Within the population of Dsg3-responsive, interleukin (IL)-10-secreting Tr1 cell clones, two major subpopulations were identified and sorted by fluorescence-activated cell sorting (FACS) based on their size and granularity. Upon in vitro culture, the larger subpopulation differentiated back into the two former subpopulations of the Tr1 cell clones, while the smaller subpopulation died within 2 weeks. The smaller subpopulation of the Tr1 cell clones was characterized by the expression of Foxp3, the secretion of IL-10, transforming growth factor (TGF)-beta and IL-5 upon stimulation with Dsg3, a proliferative response to IL-2 but not to Dsg3 or mitogenic stimuli, and an inhibitory effect on the proliferative response of Dsg3-responsive Th clones in a Dsg3-specific manner. In contrast, the larger subpopulation showed a Th-like phenotype, lacking Foxp3, cytotoxic T-lymphocyte antigen 4 (CTLA4) and glucocorticoid-induced tumour necrosis factor receptor (GITR) expression and IL-2 secretion, and did not mount a proliferative response to Dsg3 and mitogenic stimuli. The two Tr1 subpopulations showed expression of identical T-cell receptor (TCR) V beta chains which varied among the PV patients studied. Upon inhibition of Foxp3, the smaller Tr1 subpopulation developed a proliferate response to Dsg3 and mitogenic stimuli, no longer suppressed Dsg3-specific Th cells, lost expression of GITR and CTLA4 and secreted IL-2. Thus, our observations suggest a distinct relationship between Dsg3-specific Tr1 and Th-like cells which may be critical for the continuous generation and survival of Dsg3-specific Tr1 cells.     

The potential for induction of autoimmune disease by a randomly-mutated self-antigen

The pathology of most autoimmune diseases is well described. However, the exact event that triggers the onset of the inflammatory cascade leading to disease is less certain and most autoimmune diseases are complex idiopathic diseases with no single gene known to be causative. In many cases, a relation to an infectious disease is described, and it is thought that microbes can play a direct role in induction of autoimmunity, for instance by molecular mimicry or bystander activation of autoreactive T cells. In contrast, less attention has been given to the possibility that modified self-antigens can be immunogenic and lead to autoimmunity against wildtype self-antigens. In theory, modified self-antigens can arise by random errors and mutations during protein synthesis and would be recognized as foreign antigens by na?ve B and T lymphocytes. Here, it is postulated that the initial auto-antigen is not a germline self-antigen, but rather a mutated self-antigen. This mutated self-antigen might interfere with peripheral tolerance if presented to the immune system during an infection. The infection lead to bystander activation of na?ve T and B cells with specificity for mutated self-antigen and this can lead to epitopespreading in which T and B cells with specificity for wildtype self-antigens are activated as a result of general inflammation.     

Activation of natural killer T cells by α-carba-GalCer (RCAI-56), a novel synthetic glycolipid ligand, suppresses murine collagen-induced arthritis

Alpha-carba-GalCer (RCAI-56), a novel synthetic analogue of α-galactosylceramide (α-GalCer), stimulates invariant natural killer T (NK T) cells to produce interferon (IFN)-γ. IFN-γ exhibits immunoregulatory properties in autoimmune diseases by suppressing T helper (Th)-17 cell differentiation and inducing regulatory T cells and apoptosis of autoreactive T cells. Here, we investigated the protective effects of α-carba-GalCer on collagen-induced arthritis (CIA) in mice. First, we confirmed that α-carba-GalCer selectively induced IFN-γ in CIA-susceptible DBA/1 mice in vivo. Then, DBA/1 mice were immunized with bovine type II collagen (CII) and α-carba-GalCer. The incidence and clinical score of CIA were significantly lower in α-carba-GalCer-treated mice. Anti-IFN-γ antibodies abolished the beneficial effects of α-carba-GalCer, suggesting that α-carba-GalCer ameliorated CIA in an IFN-γ-dependent manner. Treatment with α-carba-GalCer reduced anti-CII antibody production [immunoglobulin (Ig)G and IgG2a] and CII-reactive interleukin (IL)-17 production by draining lymph node (DLN) cells, did not induce apoptosis or regulatory T cells, and significantly increased the ratio of the percentage of IFN-γ-producing T cells to IL-17-producing T cells (Th1/Th17 ratio). Moreover, the gene expression levels of IL-6 and IL-23p19, Th17-related cytokines, were reduced significantly in mice treated with α-carba-GalCer. In addition, we observed higher IFN-γ production by NK T cells in α-carba-GalCer-treated mice in the initial phase of CIA. These findings indicate that α-carba-GalCer polarizes the T cell response toward Th1 and suppresses Th17 differentiation or activation, suggesting that α-carba-GalCer, a novel NK T cell ligand, can potentially provide protection against Th17-mediated autoimmune arthritis by enhancing the Th1 response.     

Pathogenic autoantibody-inducing gamma/delta T helper cells from patients with lupus nephritis express unusual T cell receptors.

In previous work, we found that only 59 (15%) of 396 "autoreactive" T cell clones derived from five patients with lupus nephritis had the ability to selectively augment the production of pathogenic anti-DNA autoantibodies and the majority (49/59) of those autoimmune T helper (Th) clones were CD4+. Surprisingly, 7 of those Th clones were CD4-/CD8- and gamma/delta TCR+, capable of augmenting the production of pathogenic anti-DNA autoantibodies up to 125-fold. The gamma/delta Th clones responded in a MHC-nonrestricted manner to some endogenous autoantigen associated with heat shock proteins (HSP60) on the lupus B cells. The gamma/delta TCR genes expressed by 4 of these Th clones were amplified and sequenced here. Three of the 4 Th clones, each from a different lupus patient, expressed a gene from the V gamma 1 subgroup. Moreover, 2 of the Th clones expressed V delta 5, and the others V delta 1 or V delta 3. These TCRs are rarely expressed by peripheral blood gamma/delta T cells of normal adult humans. The predominant gamma/delta T cells in human peripheral blood express V gamma 2 (V gamma 9) and V delta 2 TCR genes, including HSP-responsive T cells. None of the lupus Th clones expressed this combination of TCR genes. In addition, some of these pathogenic autoantibody-inducing Th clones from the lupus patients had limited diversity and few N-nucleotide additions in their gamma/delta TCR junctional regions (CDR3), thus resembling fetal gamma/delta thymocytes early in ontogeny.