Autoreactive T-cell Proliferation Research Articles

Obesity and the risk of Multiple Sclerosis. The role of Leptin

ObjectiveTo investigate the effects of leptin on different T‐cell populations, in order to gain more insight into the link between leptin and obesity.MethodsThree hundred and nine RRMS patients and 322 controls participated in a cross‐sectional survey, to confirm whether excess weight/obesity in adolescence or early adulthood increased the risk of MS. Serum leptin levels were determined by ELISA. MBP83–102, and MOG63–87 peptide‐specific T cells lines were expanded from peripheral blood mononuclear cells. Leptin receptor expression was measured by RT‐PCR and flow cytometry. Bcl‐2, p‐STAT3, pERK1/2, and p27kip1 expression were assayed using ELISA, and apoptosis induction was determined by Annexin V detection. Cytokines were assessed by ELISPOT and ELISA, and regulatory T cells (Tregs) by flow cytometry.ResultsLogistic regression analysis, showed excess weight at age 15, and obesity at 20 years of age increased MS risk (OR = 2.16, P = 0.01 and OR = 3.9, P = 0.01). Leptin levels correlated with BMI in both groups. The addition of Leptin increased autoreactive T‐cell proliferation, reduced apoptosis induction, and promoted proinflammatory cytokine secretion. Obese patients produced more proinflammatory cytokines compared to overweight/normal/underweight subjects. Inverse correlation was found between leptin levels and circulating Treg cells (r = −0.97, P < 0.0001). Leptin inhibited Treg proliferation. Effects of leptin on CD4+CD25− effector T cells were mediated by increased STAT3 and ERK1/2 phosphorylation, and down modulation of the cell cycle inhibitor P27kip1. In contrast, leptin effects on Tregs resulted from decreased phosphorylation of ERK1/2 and upregulation of p27kip1.InterpretationLeptin promotes autoreactive T‐cell proliferation and proinflammatory cytokine secretion, but inhibits Treg‐cell proliferation.

Autoantigen presentation by splenic dendritic cells is required for RBC-specific autoimmunity.

Failure of humoral tolerance to red blood cell (RBC) antigens may lead to autoimmune hemolytic anemia (AIHA), a severe and sometimes fatal disease. Previous studies have shown that although tolerance is robust in HOD mice, autoantibodies are generated upon adoptive transfer of OTII CD4+ T cells, which are specific for an epitope contained within the HOD antigen. These data imply that antigen-presenting cells (APCs) are presenting RBC-derived autoantigen(s) and are capable of driving T-cell activation. Given that multiple APCs participate in erythrophagocytosis, we used a transgenic approach to determine which cellular subsets were required for autoantigen presentation and subsequent autoreactive T-cell activation. HOD mice, which express an RBC-specific antigen consisting of hen egg lysozyme, ovalbumin, and human blood group molecule Duffy, were bred with IAbfl/fl and Cre-expressing transgenic animals to generate mice that lack I-Ab expression on particular cell subsets. OTII CD4+ T cell proliferation was assessed in vivo in HOD+ I-Abfl/fl xCre+ mice and in vitro upon coculture with sorted APCs. Analysis of HOD+ I-Abfl/fl xCre+ mice demonstrated that splenic conventional dendritic cells (DCs), but not macrophages or monocytes, were required for autoantigen presentation to OTII CD4+ T cells. Subsequent in vitro coculture experiments revealed that both CD8+ and CD8- DC subsets participate in erythrophagocytosis, present RBC-derived autoantigen and stimulate autoreactive T-cell proliferation. These data suggest that if erythrocyte T-cell tolerance fails, DCs are capable of initiating autoimmune responses. As such, targeting DCs may be a fruitful strategy for AIHA therapies.

Cepharanthine blocks TSH receptor peptide presentation by HLA-DR3: Therapeutic implications to Graves’ disease

We have previously identified a signature HLA-DR3 pocket variant, designated HLA-DRβ1-Arg74 that confers a high risk for Graves' Disease (GD). In view of the key role of HLA-DRβ1-Arg74 in triggering GD we hypothesized that thyroid-stimulating hormone receptor (TSHR) peptides that bind to the HLA-DRβ1-Arg74 pocket with high affinity represent key pathogenic TSHR peptides triggering GD, and that blocking their presentation to CD4+ T-cells can be used as a novel therapeutic approach in GD. There were several previous attempts to identify the major pathogenic TSHR peptide utilizing different methodologies, however the results were inconsistent and inconclusive. Therefore, the aim of our study was to use TSHR peptide binding affinity to HLA-DRβ1-Arg74 as a method to identify the key pathogenic TSHR peptides that trigger GD. Using virtual screening and ELISA and cellular binding assays we identified 2 TSHR peptides that bound with high affinity to HLA-DRβ1-Arg74 - TSHR.132 and TSHR.197. Peptide immunization studies in humanized DR3 mice showed that only TSHR.132, but not TSHR.197, induced autoreactive T-cell proliferation and cytokine responses. Next, we induced experimental autoimmune Graves’ disease (EAGD) in a novel BALB/c-DR3 humanized mouse model we created and confirmed TSHR.132 as a major DRβ1-Arg74 binding peptide triggering GD in our mouse model. Furthermore, we demonstrated that Cepharanthine, a compound we have previously identified as DRβ1-Arg74 blocker, could block the presentation and T-cell responses to TSHR.132 in the EAGD model.

Delayed type hypersensitivity‐induced myeloid‐derived suppressor cells regulate autoreactive T cells

Mild but efficient treatments of autoimmune diseases are urgently required. One such therapy, long-term maintenance of chronic delayed type hypersensitivity, has been described for alopecia areata (AA), a hair follicle-affecting autoimmune disease. The molecular mechanisms underlying the therapeutic efficacy are unknown, but may involve myeloid-derived suppressor cells (MDSCs). AA-affected mice were treated with squaric acid dibutyl ester (SADBE). The immunoreactivity of SADBE-treated AA lymphocytes and of AA lymphocytes co-cultured with SADBE-induced MDSCs was analyzed. The curative effect of SADBE was abolished by all-transretinoic acid, which drives MDSCs into differentiation, confirming a central role for MDSCs in therapeutic SADBE treatment. SADBE and SADBE-induced MDSCs strongly interfered with sustained autoreactive T-cell proliferation in response to AA skin lysate (autoantigen), which was accompanied by weak ζ-chain down-regulation and strongly impaired Lck activation. In contrast, activation of the mitochondrial apoptosis pathway and blockade of the anti-apoptotic PI3K/Akt pathway by SADBE-induced MDSCs did not require T-cell receptor engagement. Apoptosis induction correlated with high TNF-α expression in SADBE-induced MDSCs and elevated TNFRI levels in AA lymphocytes. SADBE-induced MDSCs interfere with persisting autoreactive T-cell proliferation and promote apoptosis of these T cells, which qualifies MDSCs induced and maintained by chronic delayed type hypersensitivity reactions as promising therapeutics in organ-related autoimmune diseases.

Congenic Mesenchymal Stem Cell Therapy Reverses Hyperglycemia in Experimental Type 1 Diabetes

OBJECTIVEA number of clinical trials are underway to test whether mesenchymal stem cells (MSCs) are effective in treating various diseases, including type 1 diabetes. Although this cell therapy holds great promise, the optimal source of MSCs has yet to be determined with respect to major histocompatibility complex matching. Here, we examine this question by testing the ability of congenic MSCs, obtained from the NOR mouse strain, to reverse recent-onset type 1 diabetes in NOD mice, as well as determine the immunomodulatory effects of NOR MSCs in vivo.RESEARCH DESIGN AND METHODSNOR MSCs were evaluated with regard to their in vitro immunomodulatory function in the context of autoreactive T-cell proliferation and dendritic cell (DC) generation. The in vivo effect of NOR MSC therapy on reversal of recent-onset hyperglycemia and on immunogenic cell subsets in NOD mice was also examined.RESULTSNOR MSCs were shown to suppress diabetogenic T-cell proliferation via PD-L1 and to suppress generation of myeloid/inflammatory DCs predominantly through an IL-6-dependent mechanism. NOR MSC treatment of experimental type 1 diabetes resulted in long-term reversal of hyperglycemia, and therapy was shown to alter diabetogenic cytokine profile, to diminish T-cell effector frequency in the pancreatic lymph nodes, to alter antigen-presenting cell frequencies, and to augment the frequency of the plasmacytoid subset of DCs.CONCLUSIONSThese studies demonstrate the inimitable benefit of congenic MSC therapy in reversing experimental type 1 diabetes. These data should benefit future clinical trials using MSCs as treatment for type 1 diabetes.

Antigen‐specific Treg impair CD8+ T‐cell priming by blocking early T‐cell expansion

Foxp3(+) Treg are crucial for the maintenance of self-tolerance and have been shown to control CD8(+) T-cell effector functions. In addition, Treg are thought to control the priming of CD8(+) T cells, which recognize the same antigens as Treg. Taking advantage of our model of peripheral tolerance induction to influenza hemagglutinin (HA) after HA gene transfer, we found that HA-specific Treg suppress antigen-linked CTL responses through early blockade of CD8(+) T-cell expansion. Confronted with their cognate antigen, Treg expand more rapidly than CD8(+) T cells and are highly suppressive only during the initial stages of immune priming. They nullify HA-specific CD8(+) T-cell responses, local inflammatory responses and rejection of HA transduced cells. When HA gene transfer is performed with extensive tissue inflammation, HA-specific Treg are less effective but still reduce the frequency of newly primed HA-specific CD8(+) T cells and the ensuing frequency of memory CD8(+) T cells. Our results demonstrate that Treg control CTL priming in an antigen-specific manner at the level of T-cell expansion, highlighting how self-reactive Treg could prevent the induction of autoimmune responses through selective blockade of autoreactive T-cell proliferation.

Novel immunomodulatory properties of cirsilineol through selective inhibition of IFN-γ signaling in a murine model of inflammatory bowel disease

Regulation of signal transducer and activator of transcription (STAT) 1 signaling is being explored as a new approach to the treatment of inflammatory bowel diseases. However, few chemicals have been reported to inhibit IFN-γ/STAT1 signaling for Crohn's disease therapy. In the present study, we found that cirsilineol, a small natural compound isolated from Artemisia vestita, significantly ameliorated trinitro-benzene sulfonic acid (TNBS)-induced T-cell-mediated experimental colitis in mice, which was closely associated with reduced autoreactive T-cell proliferation and activation. Moreover, the regulatory action of pro-inflammatory and anti-inflammatory cytokine by cirsilineol treatment was found to decrease the activity of effector Th1 cells but increase the activity of regulatory T cells as characterized by down-regulation of IFN-γ and corresponding up-regulation of IL-10 and TGF-β. The therapeutic effect of cirsilineol was attributable to a novel regulatory mechanism with selective inhibiting IFN-γ signaling in colonic lamina propria CD4+ T cells, which was mediated through down-regulating STAT1 activation and T-bet expression. Furthermore, cirsilineol was found to down-regulate the activation of JAK2, a critical kinase for IFN-γ/STAT1 signaling, and abrogate the expression of T-bet, resulting in markedly decreased proliferation and activation of T cells in vitro. Importantly, the inhibition of IFN-γ/STAT1 signaling by cirsilineol was reversible in the presence of high level of IFN-γ. These results strongly suggest that cirsilineol might be potentially useful for treating T-cell-mediated human inflammatory bowel diseases.

Modulation of autoimmunity to beta-cell antigens by proteases.

Proteases are used in therapy for autoimmune diseases yet the mechanism of their action remains to be determined. We studied the immunological basis of protease therapy in the context of Type I (insulin-dependent) diabetes mellitus. We studied the effects of proteases (trypsin, papain, chymotrypsin, bromelain) on immune reactivity of a series of autoreactive T-cell clones from prediabetic subjects and patients with a recent onset of Type I diabetes and specific to the autoantigens GAD65, IA-2 and insulin-secretory granule protein. Cell surface expression of adhesion, co-stimulatory and homing molecules on both antigen-presenting cells and T cells was changed after protease treatment. Cytokine analyses showed a selective inhibition of proinflammatory (Th-1) but not Th-2 cytokine production. Autoreactive T-cell proliferation was inhibited at pharmacological serum concentrations, whereas non-specific proliferation to phytohaemagglutinin was not affected at these concentrations. Preincubation experiments on T cells and antigen-presenting cells separately showed that this effect was mediated by APCs, but not T-cells. Proteases have pleiotropic immunological effects supporting an immunomodulatory potential for the intervention of chronic inflammatory diseases and Th-1 mediated oedema formation.

Soluble forms of intercellular adhesion molecule-1 in insulin-dependent diabetes mellitus

Summary Soluble adhesion molecules are detectable at low levels in healthy people but are increased in various disorders. However, their physiological role is unknown. Circulating intercellular adhesion molecule-1 (cICAM-1) may modulate inflammation or arise as a consequence of inflammation. We have described elevated concentrations of cICAM-1 in subjects at risk of developing insulin-dependent diabetes mellitus (IDDM), compared with recent-onset IDDM patients and healthy controls. Here we tested the ability of a monomeric soluble recombinant form of ICAM-1 (rICAM-1), to prevent the proliferation of T cells to islet-cell and other antigens. We also tested the ability of two multivalent ICAM-1-immunoglobulin (ICAM-1-Ig) fusion proteins to stop proliferation of T cells in vitro. Autoreactive T-cell proliferation was suppressed by monoclonal antibodies directed against ICAM-1 or lymphocyte-function antigen-1 (LFA-1). Furthermore, 100 μmol rICAM-1 blocked T-cell proliferation in response to an islet-cell autoantigen, and multivalent ICAM-1-Ig fusion proteins were approximately 1000-fold more effective. The usual interleukin-2-induced proliferation of T cells was unaffected by ICAM or ICAM-Ig. In addition, rICAM-1 blocked primary T-cell responses from peripheral blood mononuclear cells of newly diagnosed IDDM patients in concentrations similar to elevated cICAM-1 concentrations in individuals at risk for the disease. Thus, naturally circulating ICAM-1 may downregulate inflammation in subjects at risk of developing IDDM. Ig-ICAM-1 fusion proteins may thus provide novel means to intervene in the pathogenesis of autoimmune diseases.

Tumour Growth Causes Suppression of Autoreactive T‐Cell Proliferation by Disrupting Macrophage Responsiveness to Interferon‐γ

Normal immune homeostasis is regulated partly by a small population of CD4+ T cells that react to autologous major histocompatibility complex class-II molecules on self-cells. Decreased autoreactive T-cell responses are associated with cancer. Tumour growth causes syngeneic macrophages (M phi) to suppress autoreactive T-cell proliferation by decreasing M phi class-II expression and increasing M phi production of the suppressor molecule prostaglandin E2 (PGE2). Because interferon-gamma (IFN-gamma) is a potent M phi activation molecule which regulates both M phi PGE2 and class-II expression, the effects of IFN-gamma on tumour-induced suppression of autoreactive T-cell proliferation were investigated. Exogenous IFN-gamma increased normal host (NH) CD4+ autoreactive T-cell proliferation stimulated by syngeneic NH M phi but decreased proliferation stimulated by tumour-bearing host (TBH) M phi. Antibody (Ab) neutralization of endogenous IFN-gamma activity reduced TBH M phi-mediated suppression. Kinetic studies showed that endogenous IFN-gamma suppressor activity was not exclusive during T-cell activation. Indomethacin treatment blocked IFN-gamma-induced suppression in TBH M phi-T cell cultures. TBH M phi-T cell cultures contained significantly more PGE2 than those containing NH M phi. Exogenous IFN-gamma increased early PGE2 production in TBH M phi cultures but decreased production in NH M phi cultures. The Ab-mediated neutralization of endogenous transforming growth factor-beta or tumour necrosis factor-alpha reduced TBH M phi-mediated suppression and blocked IFN-gamma-induced suppression. Short-term treatment of M phi with IFN-gamma before their addition to T cells caused TBH M phi to stimulate T-cell proliferation, which suggests that early suppressor molecule production by TBH M phi inhibits synthesis or activity of IFN-gamma-induced stimulatory monokines. These results show that tumour growth causes M phi to suppress autoreactive T-cell responses by allowing IFN-gamma to induce M phi suppressor molecules, which block production or activity of stimulatory monokines.

Serial cell-mediated immunological changes in terminal uremic patients on continuous ambulatory peritoneal dialysis therapy.

To investigate the mechanism of immunologic defects in uremia, we examined the cell-mediated immunity in 20 uremic patients with well balanced dietary assessments before continuous ambulatory peritoneal dialysis (CAPD) therapy and the serial changes in cell-mediated immunity 3, 6, 9, and 12 months after therapy. Absolute lymphocyte count, active T, total T, OKT4, OKT8, and B-cell levels were significantly lower in uremic patients than in controls, but progressively increased after CAPD treatment. The lymphoproliferative responses to mitogens decreased and were more evidently suppressed in the presence of autologous plasma. The suppressive effect of autologous plasma could be abolished by indomethacin treatment. The plasma prostaglandin E2 level, which was increased in uremic patients, decreased after CAPD treatment. These results suggest that prostaglandin E2 may play an important role in the suppression of T-cell function. The T-cell response to the stimulation of autologous non-T cells (autologous mixed-lymphocyte reaction; AMLR) in uremic patients was also low in patients before CAPD treatment. The patients' autoreactive T cells could suppress AMLR, and so did the supernatant from AMLR and isolated T-cell culture of uremic patients. These results suggest the existence of suppressor autoreactive T cells which can release suppressor factor(s). However, to our surprise, the autoreactive T-cell proliferation increased, and the suppressive effect of the supernatant was abolished after CAPD therapy. The mechanism for this peculiar phenomenon is not known and deserves further investigation.(ABSTRACT TRUNCATED AT 250 WORDS)

Murine neonatal spleen contains natural T and non-T suppressor cells capable of inhibiting adult alloreactive and newborn autoreactive T-cell proliferation

The spleen of neonatal mice is known to be a rich source of cells capable of suppressing a variety of immune functions of adult lymphocytes in vitro. From such observations has emerged the concept that the gradual development in ability to express immune functions after birth is due in part to the parallel normal physiological decay of naturally occurring regulatory suppressor cells. There is, however, some confusion in the literature as to the exact nature of the newborn inhibitory cell type(s). In contrast to most previous reports which detect only a single type of neonatal suppressor cell, usually a T cell, we show here that newborn spleen harbors both T and non-T inhibitory cells. Both types of suppressor cells could be shown to suppress the proliferative response of adult spleen to alloantigens as well as newborn T cells reacting against self-Ia antigen in the autologous mixed lymphocyte reaction (AMLR). Newborn suppressor T cells were characterized as being non-adherent to Ig-anti-Ig affinity columns, soybean agglutinin receptor negative (SBA −), and susceptible to lysis by anti-T-cell specific antiserum plus complement. Non-T suppressor cells were identified as non-phagocytic, SBA receptor positive (SBA +), and resistant to cytotoxic treatment with anti-T-cell antibodies and complement. The apparent controversy surrounding previous reports as to the T versus non-T nature of newborn suppressor cells can be reconciled by the present observation that both types of inhibitory cells coexist in the spleen. Furthermore, the, demonstration that newborn suppressor cells can effectively regulate T-cell proliferative activity mediated by other newborn cells provides more direct support for the contention that such inhibitory cells play a physiological role in controlling immune responsiveness during early ontogeny.