Pathogenesis of NOD diabetes is initiated by reactivity to the insulin B chain 9-23 epitope and involves functional epitope spreading.

Type 1 diabetes (T1D) is mediated by destruction of pancreatic ß-cells by CD4 and CD8 T cells specific for epitopes on numerous diabetogenic autoantigens resulting in loss of glucose homeostasis. Employing antigen-specific tolerance induced by i.v. administration of syngeneic splenocytes ECDI cross-linked to various diabetogenic antigens/epitopes (Ag-SP), we show that epitope spreading plays a functional role in the pathogenesis of T1D in NOD mice. Specifically, Ag-SP coupled with intact insulin, Ins B(9-23) or Ins B(15-23), but not GAD65(509-528), GAD65(524-543) or IGRP(206-214), protected 4-6 week old NOD mice from the eventual development of clinical disease; infiltration of immune cells to the pancreatic islets; and blocked the induction of DTH responses in a Treg-dependent, antigen-specific manner. However, tolerance induction in 19-21 week old NOD mice was effectively accomplished only by Ins-SP, suggesting Ins B(9-23) is a dominant initiating epitope, but autoimmune responses to insulin epitope(s) distinct from Ins B(9-23) emerge during disease progression.

CCL22 regulates experimental autoimmune encephalomyelitis by controlling inflammatory macrophage accumulation and effector function.

EAE is a demyelinating disease of the CNS and serves as a mouse model of MS. Expression of CCL22 in the draining LNs and spinal cord correlated with the onset of clinical EAE development and remained elevated. Administration of anti-CCL22 at the time of autoantigen immunization delayed the initiation of clinical disease and dampened the severity of peak initial disease and relapses. Reduced EAE severity correlated with the reduction of pathology and leukocytes in the CNS, particularly, activated CD11b+Ly6C(hi) macrophages. There were no differences in effector T cell-proliferative responses or effector T cell IFN-γ or IL-17 responses. However, treatment at the onset of disease did not reduce disease progression. Treatment of adoptive T cell transfer recipient mice with anti-CCL22 resulted in decreased clinical disease development accompanied by a decrease in CNS accumulation of CD11b+Ly6C(hi) macrophages. Neutralization of CCL22 resulted in a macrophage population whose effector cytokine expression consisted of decreased TNF and increased IL-10, a phenotype more consistent with M2 macrophages. This was corroborated by in vitro cultures of macrophages with CCL22. These results suggest that CCL22 functions to regulate development of EAE through macrophage chemoattraction and effector function.

Endoplasmic reticulum stress response as a potential therapeutic target in multiple sclerosis.

Multiple Sclerosis (MS) is an inflammatory demyelinating disorder of the central nervous system (CNS). Since current treatments are aimed at nonspecifically down-regulating inflammation and natural mechanisms of repair and remyelination within the CNS are inadequate for recovery of function, MS patients presently only have available treatments that delay symptom progression. The complex nature of this disease means that only multifaceted treatments hold the promise of a cure. Recent studies indicate that the ER stress response, a cellular pathway that allows a cell to survive and recover from a stressful event, could be elicited to help the myelin-generating and myelin-support cells of the CNS survive inflammatory insult.

Anti-thymocyte globulin (ATG) prevents autoimmune encephalomyelitis by expanding myelin antigen-specific Foxp3+ regulatory T cells.

The T cell-depleting polyclonal antibody, anti-thymocyte globulin (ATG) has long been used in organ transplantation to treat acute rejection episodes. More recently, it is also being used as part of an induction regimen to protect allografts. It has been proposed that ATG might deplete effector T cells (T-effs) while sparing regulatory T cells (T-regs). In order to test whether ATG is effective in autoimmune disease, we used Foxp3gfp 'knock-in' mice in combination with a myelin oligodendrocyte glycoprotein (MOG)(35-55)/IA(b) tetramer to study more closely the effect of ATG treatment on antigen-specific T cell responses in vivo during MOG-induced experimental autoimmune encephalomyelitis (EAE), an animal model for Multiple Sclerosis. ATG treatment enhanced the expansion of MOG-specific T-regs (CD4(+)Foxp3(+)) in MOG-immunized mice. T-effs were depleted, but on a single-cell basis, the effector function of residual T-effs was not compromised by ATG. Thus, ATG tipped the balance of T-effs and T-regs and skewed an auto-antigen-specific immune reaction from a pathogenic T cell response to a potentially protective T-reg response. In both acute and relapsing remitting disease models, ATG treatment resulted in the attenuation from EAE, both in a preventive and early therapeutic setting. We conclude that ATG treatment enforces the development of a dominant immunoregulatory environment which may be advantageous for the treatment of T cell-driven autoimmune diseases.

CD4+ T cell expressed CD80 regulates central nervous system effector function and survival during experimental autoimmune encephalomyelitis.

CD80 expressed on the surface of APCs provides a positive costimulatory signal to naive CD4+ T cells during activation. Therefore, it was hypothesized that treatment of SJL mice with various forms of anti-CD80 mAb during remission from the acute phase of relapsing experimental autoimmune encephalomyelitis (R-EAE) would ameliorate disease progression. We previously reported that treatment of SJL mice with anti-CD80 Fab during R-EAE remission blocked activation of T cells specific for endogenous myelin epitopes, inhibiting epitope spreading and clinical disease progression; however, treatment with the native form of the same anti-CD80 mAb exacerbated disease progression. The current data show that intact anti-CD80 mAb binds both CNS-infiltrating CD4+ T cells and CD11c+ dendritic cells and that exacerbation of R-EAE directly correlates with increased survival and activity of myelin-specific CD4+ T cells, while the percentage of CD11c+ dendritic cells in the CNS and their APC activity was not altered. In vitro data show that cross-linking CD80 on the surface of CD4+ T cells activated in the presence of Th1-promoting cytokines increases the level of T cell activation, effector function, and survival by directly up-regulating the expression levels of transcripts for T-bet, IFN-gamma, and Bcl-xL. These findings indicate a novel regulatory role for CD80-mediated intracellular signals in CD4+ T cells and have important implications for using anti-costimulatory molecule mAb therapy in established autoimmune disease.

Cutting Edge: Anti-CD25 monoclonal antibody injection results in the functional inactivation, not depletion, of CD4+CD25+ T regulatory cells.

CD4+CD25+ T regulatory (T(R)) cells are an important regulatory component of the adaptive immune system that limit autoreactive T cell responses in various models of autoimmunity. This knowledge was generated by previous studies from our lab and others using T(R) cell supplementation and depletion. Contrary to dogma, we report here that injection of anti-CD25 mAb results in the functional inactivation, not depletion, of T(R) cells, resulting in exacerbated autoimmune disease. Supporting this, mice receiving anti-CD25 mAb treatment display significantly lower numbers of CD4+CD25+ T cells but no change in the number of CD4+FoxP3+ T(R) cells. In addition, anti-CD25 mAb treatment fails to both reduce the number of Thy1.1+ congenic CD4+CD25+ T(R) cells or alter levels of CD25 mRNA expression in treatment recipients. Taken together, these findings have far-reaching implications for the interpretation of all previous studies forming conclusions about CD4+CD25+ T(R) cell depletion in vivo.

Targeting the TCR: T-cell receptor and peptide-specific tolerance-based strategies for restoring self-tolerance in CNS autoimmune disease.

A principal theme in autoimmunity is the breakdown of central tolerance resulting in the persistence and eventual activation of autoreactive T cells. Because CD4(+) T cells are key contributors to the underlying pathogenic mechanisms responsible for the onset and progression of most autoimmune diseases, they are a logical target for therapeutic interventions. One technique for restoring self-tolerance is to exploit the endogenous regulatory mechanisms that govern CD4(+) T-cell activation. In this review, we discuss promising techniques with the common goal of inducing antigen (Ag)-specific tolerance. Emphasis is given to the use of non-mitogenic anti-CD3 and peptide-specific tolerance strategies that specifically target the T-cell receptor (TCR) in the absence of costimulatory signals. These approaches produce a TCR signal of insufficient strength to cause CD4(+) T-cell activation and instead induce functional T-cell anergy or deletion while avoiding generalized long-term immunosuppression.

CD28 regulates glucocorticoid-induced TNF receptor family-related gene expression on CD4+ T cells via IL-2-dependent mechanisms.

The glucocorticoid-induced TNF-related gene receptor (GITR) is the newest member of the costimulatory molecule family and is expressed on both resting CD4+CD25+ regulatory T (T(R)) cells and activated CD4+ T cells. We investigated the endogenous mechanisms that regulate GITR expression on both T(R) and CD4+ T cells, as well as the functional interaction between GITR and other costimulatory molecules. CD28 stimulation increased GITR expression on both T(R) and CD4+ T cells via IL-2-dependent mechanisms. In addition, ligation of GITR and/or CD28 increased the level of CD4+ T cell proliferation and effector function under both APC-dependent and -independent conditions, suggesting that these costimulatory molecules cooperate to regulate CD4+ T cell activation and function by directly signaling to the CD4+ T cell. Thus, GITR may serve opposing functional roles on CD4+ T(R) and effector cells and alterations in GITR expression and/or function may tip the balance between immune tolerance and effector function.

Therapeutic blockade of TCR signal transduction and co-stimulation in autoimmune disease.

Autoimmune diseases are initiated and maintained by presentation of self antigen through complex interactions between different cells of the immune system. In most autoimmune disorders, autoantigen-specific responses are induced by the activation of specific T cells with self peptides displayed on activated antigen presenting cells (APCs). These T cells may then activate and drive B cell responses that either initiate or contribute to chronic disease pathogenesis. In order to activate the T cell, two signals are required: T cell receptor (TCR) engagement by autoantigen presented in the context of self MHC class II and costimulation (CD28-CD80/CD86 interactions). Feedback must also be provided to the APC through MHC class II engagement by the TCR and through costimulatory events controlling T cell differentiation and effector function (CD154-CD40 interactions, among others). With this in mind, numerous strategies have been developed to block the engagement and activation of self-reactive cells. We review and discuss recent progress in understanding the efficacy and underlying molecular mechanisms of three separate immunotherapeutic strategies targeting the TCR and costimulatory molecules: i) blocking TCR signaling (using non-mitogenic anti-CD3 monoclonal antibody); ii) blocking CD28 costimulation (anti-B7 monoclonal antibody blockade); and iii) blocking CD40 engagement on APCs (anti-CD154 monoclonal antibody blockade).

Treatment with nonmitogenic anti-CD3 monoclonal antibody induces CD4+ T cell unresponsiveness and functional reversal of established experimental autoimmune encephalomyelitis.

In vivo administration of anti-CD3 Ab induces both immune tolerance and undesirable side-effects resulting from nonspecific proinflammatory cytokine production. In the current study, we investigated the therapeutic potential of two structurally altered forms of the anti-CD3 Ab in ameliorating established experimental autoimmune encephalomyelitis. Administration of either a chimeric (NM-IgG3) or digestion product (NM-F(ab')2) form of the anti-CD3 Ab during established experimental autoimmune encephalomyelitis conferred significant protection from clinical disease progression and was associated with decreased Ag-specific T cell proliferation, cytokine production, and CNS inflammation. Interestingly, while this protection correlated with an increase in the frequency of CD4(+)CD25(+) regulatory T cells, neither prior depletion of regulatory T cells nor anti-TGF-beta treatment abrogated the treatment's efficacy. Importantly, both treatments induced normal levels of intracellular Ca(2+)-flux, but significantly diminished levels of TCR signaling. Consequent to this decreased level of TCR-mediated signaling were alterations in the level of apoptosis and CD4+ T cell trafficking resulting in a profound lymphopenia. Collectively, these results indicate that nonmitogenic anti-CD3 directly induces a state of immune unresponsiveness in primed pathogenic autoreactive effector cells via mechanisms that may involve the induction of T cell tolerance, apoptosis, and/or alterations in cell trafficking.

Cutting edge: ligation of the glucocorticoid-induced TNF receptor enhances autoreactive CD4+ T cell activation and experimental autoimmune encephalomyelitis.

The glucocorticoid-induced TNFR (GITR) is expressed at high levels on resting CD4(+)CD25(+) T regulatory (T(R)) cells and regulates their suppressive phenotype. Accordingly, we show that anti-GITR mAb treatment of SJL mice with proteolipid protein 139-151-induced experimental autoimmune encephalomyelitis significantly exacerbated clinical disease severity and CNS inflammation, and induced elevated levels of Ag-specific T cell proliferation and cytokine production. Interestingly, prior depletion of T(R) cells failed to result in exacerbated experimental autoimmune encephalomyelitis suggesting alternative targets for the anti-GITR mAb treatment. Importantly, naive CD4(+)CD25(-) T cells up-regulated GITR expression in an activation-dependent manner and anti-GITR mAb treatment enhanced the level of CD4(+) T cell activation, proliferation, and cytokine production in the absence of T(R) cells both in vivo and in vitro. Taken together, these findings suggest a dual functional role for GITR as GITR cross-linking both inactivates T(R) cells and increases CD4(+)CD25(-) T cell effector function, thus enhancing T cell immunity.

Regulation of experimental autoimmune encephalomyelitis (EAE) by CD4+CD25+ regulatory T cells.

Multiple endogenous mechanisms exist to inhibit thymic development of functional autoreactive T cells. In spite of this, autoreactive CD4+ T cell populations persist in normal individuals and retain the capacity to initiate autoimmune disease. Thus, additional regulatory mechanisms operative in the peripheral immune system are required to protect against both the generation of self-directed immune responses and the initiation of autoimmune diseases. One such mechanism involves the active inhibition of T cell responses by CD4+CD25+ regulatory T (T(reg)) cells. In this study, we investigated the protective role of T(reg) cells during experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis (MS). Our findings indicate that T(reg) cells confer significant protection from the development of MOG(35-55)-induced EAE that may result from the promotion of a protective Th2 response and decreased homing of autoreactive cells to the CNS. Importantly, T(reg) cells differentially expressed elevated levels of ICAM1 and P selectin, molecules which may facilitate T-T cell interactions and contribute to the mechanism by which T(reg) cells inhibit CD4+ T cell responses. Collectively, these findings support a role for T(reg) cells as an active regulatory mechanism that may protect individuals from the onset of MS, as well as other autoimmune diseases.

Role of ICAM-1 and P-selectin expression in the development and effector function of CD4+CD25+regulatory T cells.

Dynamic regulatory mechanisms prevent autoreactive T cell activation. Upon T cell receptor crosslinking, CD4+CD25+ T regulatory (T(R)) cells block both the proliferation and cytokine production of CD4+CD25- effector cells in an apparent antigen non-specific manner. Within the T(R)population, L-selectin (CD62L)(hi)T(R)cells have been described as more efficient suppressors of T cell proliferation than CD62L(low)T(R)cells. We have previously reported that CD4+CD25+CD62L(hi)T(R)cells express elevated levels of two additional adhesion molecules, ICAM-1 (CD54) and P-selectin (CD62P) in comparison to non-T(R)cells. In the current study, we investigated the functional contribution of CD54 and CD62P expression to the suppressive phenotype of T(R)cells both in vitro and in vivo. While the CD4+CD25+ T(R)cell population was demonstrated to be significantly larger in CD62P-/- mice than in wild-type C57BL/6 mice, CD62P-/- T(R)cell function was deficient in vitro, but not in vivo. Interestingly, we detected no deficiencies in T(R)cell numbers or effector function in CD54-/- mice suggesting that T(R)cells may differ from effector CD4+ T cells in the requirement for CD54 expression within the immunological synapse. Collectively, these findings indicate that CD62P may influence T(R)cell differentiation/development and that T(R)cell activation occurs independently of CD54 expression.

Adaptive immunity in mice lacking the beta(2)-adrenergic receptor.

The beta-2-adrenergic receptor (beta(2)AR) is expressed by most lymphocyte populations and binds the sympathetic neurotransmitter norepinephrine (NE). Stimulation of the beta(2)AR is reported to be the primary mechanism by which signals from the sympathetic nervous system influence both cell-mediated and humoral immunity. We report here that body/organ weights, lymphoid organ cell number/phenotype/histology, the contact sensitivity response, and the amount, avidity, and isotype of antibody resulting from a T cell-dependent antibody response in beta(2)AR deficient mice (beta(2)AR-/- mice) were all similar to measures made in beta(2)AR+/+ mice. Other members of the adrenergic receptor family did not appear to compensate for the absence in beta(2)AR expression. In contrast, beta(2)AR-/- B cells cultured in vitro were unable to respond to NE in a manner similar to beta(2)AR+/+ B cells. Thus, mice in which expression of the beta(2)AR gene is defective from early development to adulthood may no longer require that NE stimulate the beta(2)AR to maintain immune homeostasis, and this may be due to a non-adrenergic mechanism that provides compensation in vivo.

Mimicking the way to autoimmunity: an evolving theory of sequence and structural homology.

Although the etiology of autoimmune diseases remains largely unknown, a prevailing theory concerns the infection-induced activation of self-reactive lymphocytes via the process of molecular mimicry. Here, we discuss the theory of molecular mimicry and its continued evolution from the initial basic considerations of sequence similarity to the current theories of structural homology. Such findings serve to further our understanding of T-cell receptor degeneracy and might one day provide a direct link between infection and autoimmune disease.

Cutting edge: CD4+CD25+ regulatory T cells suppress antigen-specific autoreactive immune responses and central nervous system inflammation during active experimental autoimmune encephalomyelitis.

Autoreactive CD4(+) T cells exist in normal individuals and retain the capacity to initiate autoimmune disease. The current study investigates the role of CD4(+)CD25(+) T-regulatory (T(R)) cells during autoimmune disease using the CD4(+) T cell-dependent myelin oligodendrocyte glycoprotein (MOG)-specific experimental autoimmune encephalomyelitis model of multiple sclerosis. In vitro, T(R) cells effectively inhibited both the proliferation of and cytokine production by MOG(35-55)-specific Th1 cells. In vivo, adoptive transfer of T(R) cells conferred significant protection from clinical experimental autoimmune encephalomyelitis which was associated with normal activation of autoreactive Th1 cells, but an increased frequency of MOG(35-55)-specific Th2 cells and decreased CNS infiltration. Lastly, transferred T(R) cells displayed an enhanced ability to traffic to the peripheral lymph nodes and expressed increased levels of the adhesion molecules ICAM-1 and P-selectin that may promote functional interactions with target T cells. Collectively, these findings suggest that T(R) cells contribute notably to the endogenous mechanisms that regulate actively induced autoimmune disease.

B cell receptor- and beta 2-adrenergic receptor-induced regulation of B7-2 (CD86) expression in B cells.

The costimulatory molecule B7-2 (CD86) is expressed on the surface of APCs, including B cells. Considering the importance of B7-2 in regulating both T and B cell function, it may be important to understand the regulatory mechanisms governing its expression. We report in this study that stimulation of the B cell receptor (BCR) and/or a neurotransmitter receptor, the beta(2)-adrenergic receptor (beta(2)AR), may cooperate to regulate B cell-associated B7-2 expression in vitro and in vivo. beta(2)AR stimulation further enhanced the level of BCR-induced B7-2 expression in B cells potentially via protein tyrosine kinase-, protein kinase A-, protein kinase C-, and mitogen-activated protein kinase-dependent mechanisms. Importantly, BCR and/or beta(2)AR stimulation, but not histone hyperacetylation and DNA hypomethylation alone, increased B cell-associated B7-2 expression by increasing B7-2 mRNA stability, NF-kappa B nuclear binding, and NF-kappa B-dependent gene transcription. Thus, this study provides additional insight into the signaling intermediates and molecular mechanisms by which stimulation of the BCR and beta(2)AR may regulate B cell-associated B7-2 expression.