Autoantigen-specific CD4 Research Articles

Autoantigen-specific CD4+ T cells acquire an exhausted phenotype and persist in human antigen-specific autoimmune diseases

Pro-inflammatory autoantigen-specific CD4+ T helper (auto-Th) cells are central orchestrators of autoimmune diseases (AIDs). We aimed to characterize these cells in human AIDs with defined autoantigens by combining human leukocyte antigen (HLA)-tetramer-based and activation-based multidimensional exvivo analyses. In aquaporin4-antibody-positive neuromyelitis optica spectrum disorder (AQP4-NMOSD) patients, auto-Th cells expressed CD154, but proliferative capacity and pro-inflammatory cytokines were strongly reduced. Instead, exhaustion-associated co-inhibitory receptors were expressed together with FOXP3, the canonical regulatory Tcell (Treg) transcription factor. Auto-Th cells responded invitro to checkpoint inhibition and provided potent B cell help. Cells with the same exhaustion-like (ThEx) phenotype were identified in soluble liver antigen (SLA)-antibody-autoimmune hepatitis and BP180-antibody-positive bullous pemphigoid, AIDs of the liver and skin, respectively. While originally described in cancer and chronic infection, our data point to Tcell exhaustion as a common mechanism of adaptation to chronic (self-)stimulation across AID types and link exhausted CD4+ Tcells to humoral autoimmune responses, with implications for therapeutic targeting.

Type XVII Collagen–Specific CD4+ T Cells Induce Bullous Pemphigoid by Producing IL-5

Bullous pemphigoid (BP) is an autoimmune subepidermal blistering disease caused by anti-type XVII collagen (COL17) antibodies. BP has some immunological features such as eosinophilic infiltration and the deposition of IgE autoantibodies in the skin; however, the mechanism behind such features remains largely unclear. We focused on the autoantigen-specific CD4+ T cells, which are considered to regulate immune response. We established COL17-specific CD4+ T cell lines in vitro. Wild-type mice were immunized with synthesized peptides that include a pathogenic epitope of COL17, and lymphocytes were subjected to a limiting dilution assay. We established 5 T cell lines and examined the pathogenicity by transferring them with COL17-primed B cells into Rag-2-/-/COL17-humanized mice that express human COL17 but not mouse COL17 in the skin. Notably, 3 lines induced BP-like skin changes associated with subepidermal separation and eosinophilic infiltration histologically, and the production of anti-COL17 antibodies. The other 2 lines did not induce such phenotypes. RNA-sequencing analysis revealed that Th2 cytokines, particularly IL-5, were highly expressed in the pathogenic T cell lines. Anti-IL-5 antibody administration significantly reduced the skin changes and attenuated the production of autoantibodies. Thus, the production of IL-5 is critical for COL17-specific CD4+ T cells to induce BP phenotypes in vivo.

Regulatory T cells inhibit autoantigen-specific CD4+ T cell responses in lupus-prone NZB/W F1 mice.

Progressive loss of regulatory T cell (Treg)-mediated control over autoreactive effector T cells contributes to the development of systemic lupus erythematosus (SLE). Accordingly, we hypothesized that Treg may also have the capacity to suppress the activation of autoreactive CD4+ T cells that are considered to drive autoimmunity. To investigate whether Treg are involved in the control of autoreactive CD4+ T cells, we depleted CD25+ Treg cells either in vivo or in vitro, or combined both approaches before antigen-specific stimulation with the SLE-associated autoantigen SmD1(83-119) in the NZB/W F1 mouse model either after immunization against SmD1(83-119) or during spontaneous disease development. Frequencies of autoantigen-specific CD4+ T cells were determined by flow cytometry using the activation marker CD154. Both in vitro and in vivo depletion of CD25+ Treg, respectively, increased the frequencies of detectable autoantigen-specific CD4+ T cells by approximately 50%. Notably, the combined in vivo and in vitro depletion of CD25+ Treg led almost to a doubling in their frequencies. Frequencies of autoantigen-specific CD4+ T cells were found to be lower in immunized haploidentical non-autoimmune strains and increased frequencies were detectable in unmanipulated NZB/W F1 mice with active disease. In vitro re-addition of CD25+ Treg after Treg depletion restored suppression of autoantigen-specific CD4+ T cell activation. These results suggest that the activation and expansion of autoantigen-specific CD4+ T cells are partly controlled by Treg in murine lupus. Depletion of Treg therefore can be a useful approach to increase the detectability of autoantigen-specific CD4+ T cells allowing their detailed characterization including lineage determination and epitope mapping and their sufficient ex vivo isolation for cell culture.

Sepsis impedes EAE disease development and diminishes autoantigen-specific naive CD4 T cells.

Evaluation of sepsis-induced immunoparalysis has highlighted how decreased lymphocyte number/function contribute to worsened infection/cancer. Yet, an interesting contrast exists with autoimmune disease development, wherein diminishing pathogenic effectors may benefit the post-septic host. Within this framework, the impact of cecal ligation and puncture (CLP)-induced sepsis on the development of experimental autoimmune encephalomyelitis (EAE) was explored. Notably, CLP mice have delayed onset and reduced disease severity, relative to sham mice. Reduction in disease severity was associated with reduced number, but not function, of autoantigen (MOG)-specific pathogenic CD4 T cells in the CNS during disease and draining lymph node during priming. Numerical deficits of CD4 T cell effectors are associated with the loss of MOG-specific naive precursors. Critically, transfer of MOG-TCR transgenic (2D2) CD4 T cells after, but not before, CLP led to EAE disease equivalent to sham mice. Thus, broad impairment of antigenic responses, including autoantigens, is a hallmark of sepsis-induced immunoparalysis.

Nuclear antigen–reactive CD4+ T cells expand in active systemic lupus erythematosus, produce effector cytokines, and invade the kidneys

Systemic lupus erythematosus is a systemic and chronic autoimmune disease characterized by loss of tolerance towards nuclear antigens with autoreactive CD4+ T cells implicated in disease pathogenesis. However, very little is known about their receptor specificity since the detection of human autoantigen specific CD4+ T cells has been extremely challenging. Here we present an analysis of CD4+ T cells reactive to nuclear antigens using two complementary methods: T cell libraries and antigen-reactive T cell enrichment. The frequencies of nuclear antigen specific CD4+ T cells correlated with disease severity. These autoreactive T cells produce effector cytokines such as interferon-γ, interleukin-17, and interleukin-10. Compared to blood, these cells were enriched in the urine of patients with active lupus nephritis, suggesting an infiltration of the inflamed kidneys. Thus, these previously unrecognized characteristics support a role for nuclear antigen-specific CD4+ T cells in systemic lupus erythematosus.

T cell receptor transduction onto regulatory T cells to treat autoimmune disease

Background & Aim Autoimmune disease is caused by failure of self-tolerance mechanisms. Central to self-tolerance is the regulatory T cell (Treg) subset, which actively suppresses pro-inflammatory self-reactivity. Tregs, like all T cells, are activated when their T cell receptor (TCR) recognises its cognate antigen in the context of peptide-MHC complexes. Using the model autoimmune disease, Goodpasture's, we have shown that a reduction in Tregs specific for the Goodpasture's autoantigen potentiates disease. Here, we show that lentiviral transduction of TCRs specific for the GP autoantigen is a feasible way of developing autoantigen-specific Treg therapy. Methods, Results & Conclusion Autoantigen-specific CD4+ T cells were isolated from Goodpasture's disease patient PBMCs by single cell flow cytometric sorting using tetramers. The single cells then underwent T cell receptor sequencing to determine the TCR alpha and TCR beta chain CDR3 sequence and Variable, Diverse and Joining gene usage. After analysing dozens of paired alpha-beta TCR sequences, a clonal population expressing the same TCR was identified in each patient (GP1-TCR and GP2-TCR). Lentiviral vectors coding for these alpha-beta TCR genes plus GFP reporter were constructed and used to transduce Jurkat cells, a human T cell line lacking endogenous TCR expression. The Jurkat cells were shown to express both GFP and GP1-TCR or GP2-TCR by flow cytometry. Co-culture of the transduced Jurkat cells with a DR15-specific B-Lymphoblastiod Cell Line (as antigen presenting cells) in the presence or absence of GP peptide resulted in an increase of Jurkat cell activation only in the group with GP peptide. This was measured by an upregulation of CD69 cell surface expression by flow cytometry and increased IL2 secretion by ELISA. This suggests GP1-TCR and GP2-TCR are specific for GP peptide. Using custom designed lentiviral constructs, GP1TCR was successfully lentivirally transduced onto primary human Tregs. Therefore, the use of single cell sequencing and enhanced lentiviral transduction of TCRs onto Tregs allow for the development of potent autoantigen specific Tregs that can be used to treat autoimmune disease.

Altered homeostasis and development of regulatory T cell subsets represent an IL-2R-dependent risk for diabetes in NOD mice.

The cytokine interleukin-2 (IL-2) is critical for the functions of regulatory T cells (Tregs). The contribution of polymorphisms in the gene encoding the IL-2 receptor α subunit (IL2RA), which are associated with type 1 diabetes, is difficult to determine because autoimmunity depends on variations in multiple genes, where the contribution of any one gene product is small. We investigated the mechanisms whereby a modest reduction in IL-2R signaling selectively in T lymphocytes influenced the development of diabetes in the NOD mouse model. The sensitivity of IL-2R signaling was reduced by about two- to threefold in Tregs from mice that coexpressed wild-type IL-2Rβ and a mutant subunit (IL-2RβY3) with reduced signaling (designated NOD-Y3). Male and female NOD-Y3 mice exhibited accelerated diabetes onset due to intrinsic effects on multiple activities in Tregs Bone marrow chimera and adoptive transfer experiments demonstrated that IL-2RβY3 Tregs resulted in impaired homeostasis of lymphoid-residing central Tregs and inefficient development of highly activated effector Tregs and that they were less suppressive. Pancreatic IL-2RβY3 Tregs showed impaired development into IL-10-secreting effector Tregs The pancreatic lymph nodes and pancreases of NOD-Y3 mice had increased numbers of antigen-experienced CD4+ effector T cells, which was largely due to impaired Tregs, because adoptively transferred pancreatic autoantigen-specific CD4+ Foxp3- T cells from NOD-Y3 mice did not accelerate diabetes in NOD.SCID recipients. Our study indicates that the primary defect associated with chronic, mildly reduced IL-2R signaling is due to impaired Tregs that cannot effectively produce and maintain highly functional tissue-seeking effector Treg subsets.

Tolerogenic Ag-PLG nanoparticles induce tregs to suppress activated diabetogenic CD4 and CD8 T cells

Type 1 diabetes (T1D) is mediated by destruction of pancreatic β cells by autoantigen-specific CD4+ and CD8+ T cells, thus the ideal solution for T1D is the restoration of immune tolerance to β cell antigens. We demonstrate the ability of carboxylated 500 nm biodegradable poly(lactide-co-glycolide) (PLG) nanoparticles PLG nanoparticles (either surface coupled with or encapsulating the cognate diabetogenic peptides) to rapidly and efficiently restore tolerance in NOD.SCID recipients of both activated diabetogenic CD4+ BDC2.5 chromagranin A-specific and CD8+ NY8.3 islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)-specific TCR transgenic T cells in an antigen-specific manner. Further, initiation and maintenance of Ag-PLG tolerance operates via several overlapping, but independent, pathways including regulation via negative-co-stimulatory molecules (CTLA-4 and PD-1) and the systemic induction of peptide-specific Tregs which were critical for long-term maintenance of tolerance by controlling both trafficking of effector T cells to, and their release of pro-inflammatory cytokines within the pancreas, concomitant with selective retention of effector cells in the spleens of recipient mice.

T cell receptor \u03b2-chains display abnormal shortening and repertoire sharing in type 1 diabetes

Defects in T cell receptor (TCR) repertoire are proposed to predispose to autoimmunity. Here we show, by analyzing >2 × 108TCRB sequences of circulating naive, central memory, regulatory and stem cell-like memory CD4+ T cell subsets from patients with type 1 diabetes and healthy donors, that patients have shorter TCRB complementarity-determining region 3s (CDR3), in all cell subsets, introduced by increased deletions/reduced insertions during VDJ rearrangement. High frequency of short CDR3s is also observed in unproductive TCRB sequences, which are not subjected to thymic culling, suggesting that the shorter CDR3s arise independently of positive/negative selection. Moreover, TCRB CDR3 clonotypes expressed by autoantigen-specific CD4+ T cells are shorter compared with anti-viral T cells, and with those from healthy donors. Thus, early events in thymic T cell development and repertoire generation are abnormal in type 1 diabetes, which suggest that short CDR3s increase the potential for self-recognition, conferring heightened risk of autoimmune disease.

Tregs restrain dendritic cell autophagy to ameliorate autoimmunity.

Design of efficacious Treg-based therapies and establishment of clinical tolerance in autoimmune diseases have proven to be challenging. The clinical implementation of Treg immunotherapy has been hampered by various impediments related to the stability and isolation procedures of Tregs as well as the specific in vivo targets of Treg modalities. Herein, we have demonstrated that Foxp3+ Tregs potently suppress autoimmune responses in vivo through inhibition of the autophagic machinery in DCs in a cytotoxic T-lymphocyte-associated protein 4-dependent (CTLA4-dependent) manner. Autophagy-deficient DCs exhibited reduced immunogenic potential and failed to prime autoantigen-specific CD4+ T cells to mediate autoimmunity. Mechanistically, CTLA4 binding promoted activation of the PI3K/Akt/mTOR axis and FoxO1 nuclear exclusion in DCs, leading to decreased transcription of the autophagy component microtubule-associated protein 1 light chain 3β (Lc3b). Human DCs treated with CTLA4-Ig, a fusion protein composed of the Fc region of IgG1 and the extracellular domain of CTLA4 (also known as abatacept, marketed as Orencia), demonstrated reduced levels of autophagosome formation, while DCs from CTLA4-Ig-treated rheumatoid arthritis patients displayed diminished LC3B transcripts. Collectively, our data identify the canonical autophagy pathway in DCs as a molecular target of Foxp3+ Treg-mediated suppression that leads to amelioration of autoimmune responses. These findings may pave the way for the development of therapeutic protocols that exploit Tregs for the treatment of autoimmunity as well as diseases in which disturbed tolerance is a common denominator.

Inhibition of microRNA-155 ameliorates experimental autoimmune myocarditis by modulating Th17/Treg immune response.

Experimental autoimmune myocarditis (EAM) is an inflammatory cardiac disease driven by autoantigen-specific CD4+ T cells. Th17 and Treg cells are crucial participants in immune response. A wide variety of immune disorders are associated with Th17/Treg imbalance. MicroRNA-155 (miR-155) is a pivotal regulator of the immune system. However, the modulatory effect of miR-155 on Th17/Treg immune response during EAM is unknown. Our study aims to investigate the potential role of miR-155 on the development of autoimmune myocarditis. In this study, we revealed that miR-155 expression was highly elevated in heart tissue and CD4+ T cells during EAM. Also, we identified a proliferative and functional imbalance of Th17/Treg in EAM, which is due to a more active development of Th17 cells and an increased resistance of Th17 cells to Treg-mediated suppression. MiR-155 inhibition in EAM resulted in attenuated severity of disease and cardiac injury, reduced Th17 immune response, and decreased dendritic cell (DC) function of secreting Th17-polarizing cytokines. Furthermore, CD4+ T cells from miR-155-inhibited EAM mice exhibited reduced proliferation and IL-17A secretion in response to autoantigen. Finally, we confirmed an indispensable positive role of miR-155 on the differentiation of Th17 cells and the DC function of secreting Th17-polarizing cytokines through in vitro studies. These findings demonstrated that miR-155 adversely promotes EAM by driving a Th17/Treg imbalance in favor of Th17 cells, and anti-miR-155 treatment can significantly reduce the autoimmune response thus to ameliorate EAM, suggesting that miR-155 inhibition could be a promising therapeutic strategy for the treatment of autoimmune myocarditis. MiR-155 expression was highly elevated in EAM mice. An imbalance of Th17/Treg existed in EAM mice. MiR-155 inhibition in EAM attenuated disease severity and cardiac injury. MiR-155 inhibition suppressed Th17 immune response in EAM. MiR-155 inhibition reduced DC function of secreting Th17-polarizing cytokines in EAM.

Interleukin-7 is required for CD4+ T cell activation and autoimmune neuroinflammation

IL-7 is known to be vital for T cell homeostasis but has previously been presumed to be dispensable for TCR-induced activation. Here, we show that IL-7 is critical for the initial activation of CD4+ T cells in that it provides some of the necessary early signaling components, such as activated STAT5 and Akt. Accordingly, short-term in vivo IL-7Rα blockade inhibited the activation and expansion of autoantigen-specific CD4+ T cells and, when used to treat experimental autoimmune encephalomyelitis (EAE), prevented and ameliorated disease. Our studies demonstrate that IL-7 signaling is a prerequisite for optimal CD4+ T cell activation and that IL-7R antagonism may be effective in treating CD4+ T cell-mediated neuroinflammation and other autoimmune inflammatory conditions.

Abstract LB-157: VISTA deficiency synergizes with a non-redundant immune checkpoint pathway and leads to enhanced immune activation

Abstract V-domain Ig suppressor of T cell activation (VISTA) is a novel negative checkpoint ligand that suppresses T-cell mediated immune responses. Previous studies using VISTA-neutralizing monoclonal antibody show that VISTA-blockade enhances T cell-activation in an inflammatory disease model EAE, as well as in murine tumor models. Current study describes a comprehensive characterization of VISTA knockout (KO) mice. We show that despite the apparent normal hematopoietic development in young ko mice, VISTA genetic deficiency leads to a pro-inflammatory phenotype in aged animals, as well as enhanced T-cell activation in response to acute antigen immunization. In addition, we show that VISTA deficiency significantly enhanced disease development in a spontaneous model of autoimmune disease, which is correlated with the spontaneous activation of auto-antigen specific CD4+ T cells. Lastly, when combined with the genetic deficiency of another checkpoint molecule, synergistic or additive immune activation was observed. The implication of such enhanced immunity in cancer development and treatment will be discussed. Citation Format: Li Wang, Isabelle LeMercier, Arief Suriawinata, Wenna Chen, Randolph J. Noelle, Jiannan Li. VISTA deficiency synergizes with a non-redundant immune checkpoint pathway and leads to enhanced immune activation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-157. doi:10.1158/1538-7445.AM2014-LB-157

VISTA deficiency synergizes with a non-redundant immune checkpoint pathway and leads to enhanced immune activation (IRM10P.744)

Abstract V-domain Ig suppressor of T cell activation (VISTA) is a novel negative checkpoint ligand that suppresses T-cell mediated immune responses. Previous studies using VISTA-neutralizing monoclonal antibody show that VISTA-blockade enhances T cell-activation in an inflammatory disease model EAE, as well as in murine tumor models. Current study describes a comprehensive characterization of VISTA knockout (KO) mice. We show that despite the apparent normal hematopoietic development in young ko mice, VISTA genetic deficiency leads to a pro-inflammatory phenotype in aged animals, as well as enhanced T-cell activation in response to acute antigen immunization. In addition, we show that VISTA deficiency significantly enhanced disease development in a spontaneous model of autoimmune disease, which is correlated with the spontaneous activation of auto-antigen specific CD4+ T cells. Lastly, when combined with the genetic deficiency of another checkpoint molecule, synergistic or additive immune activation was observed.

The type 1 diabetes resistance locus B10 Idd9.3 impairs B cell development and implicates microRNA-34a as a regulator of T1D pathogenesis (BA14P.200)

Abstract Type 1 diabetes (T1D) is an autoimmune disease resulting from the T cell-mediated destruction of insulin-producing pancreatic beta cells. Multiple insulin-dependent diabetes (Idd) loci confer T1D susceptibility or resistance in both humans and nonobese diabetic (NOD) mice, but the genes and mechanisms by which they control disease development are largely unknown. NOD.B10 Idd9.3 mice contain the Idd9.3 locus from T1D-resistant C57BL/10 mice that encodes 19 genes, including microRNA (miR)-34a, and confers partial T1D protection. B cells have been shown to play a critical role in priming autoantigen-specific CD4+ T cells in NOD mice. We show that early B cell development is impaired in NOD.B10 Idd9.3 mice, resulting in significant reduction of splenic B cells compared to NOD mice. Molecular analysis revealed that miR-34a expression was significantly elevated in B cell progenitors and marginal zone B cells of NOD.B10 Idd9.3 mice compared to NOD mice. Furthermore, miR-34a expression inversely correlated with levels of Foxp1, an essential regulator of B cell lymphopoiesis, which is directly repressed by miR-34a. Importantly, peripheral B cell paucity in NOD.B10 Idd9.3 mice was associated with impaired proliferation of islet-antigen-specific CD4+ T cells in response to endogenous autoantigen in pancreatic lymph nodes. These data suggest that miR-34a negatively regulates B cell development in NOD.B10 Idd9.3 mice and thereby may contribute to their T1D protection.

The type 1 diabetes resistance locus B10 Idd9.3 mediates impaired B‐cell lymphopoiesis and implicates microRNA‐34a in diabetes protection

NOD.B10 Idd9.3 mice are congenic for the insulin-dependent diabetes (Idd) Idd9.3 locus, which confers significant type 1 diabetes (T1D) protection and encodes 19 genes, including microRNA (miR)-34a, from T1D-resistant C57BL/10 mice. B cells have been shown to play a critical role in the priming of autoantigen-specific CD4(+) T cells in T1D pathogenesis in non-obese diabetic (NOD) mice. We show that early B-cell development is impaired in NOD.B10 Idd9.3 mice, resulting in the profound reduction of transitional and mature splenic B cells as compared with NOD mice. Molecular analysis revealed that miR-34a expression was significantly higher in B-cell progenitors and marginal zone B cells from NOD.B10 Idd9.3 mice than in NOD mice. Furthermore, miR-34a expression in these cell populations inversely correlated with levels of Foxp1, an essential regulator of B-cell lymphopoiesis, which is directly repressed by miR-34a. In addition, we show that islet-specific CD4(+) T cells proliferated inefficiently when primed by NOD.B10 Idd9.3 B cells in vitro or in response to endogenous autoantigen in NOD.B10 Idd9.3 mice. Thus, Idd9.3-encoded miR-34a is a likely candidate in negatively regulating B-cell lymphopoiesis, which may contribute to inefficient expansion of islet-specific CD4(+) T cells and to T1D protection in NOD.B10 Idd9.3 mice.

Autoantigen-specific memory CD4+ T cells are prevalent early in progression to Type 1 diabetes

Autoreactive CD4+ T cells contribute to the destruction of insulin producing beta cells in Type 1 diabetes (T1D). Using MHC class II tetramers, we have analyzed the frequency of GAD65- (274–286; 555–567) and insulin- (A1–15; A6–21) specific CD4+ T cells in 31 children with T1D, 65 multiple autoantibody-positive children and 93 HLA- and age-matched controls. In a smaller group of children T-cell responses of memory origin to the same autoantigens were investigated. We observed a higher response to GAD65 555–567 in the autoantibody-positive children than in the controls (P=0.017). Memory T-cell responses to GAD65 555–567 were more frequent among T1D patients (P=0.025) and autoantibody-positive (P=0.054), while all controls were negative (n=28). In summary, the presence of antigen experienced GAD65-specific T cells in the subjects with diabetes-associated autoimmunity is encouraging for further directions in the prediction of T1D.

Unmasking of autoreactive CD4 T cells by depletion of CD25 regulatory T cells in systemic lupus erythematosus

ObjectiveAutoreactive CD4 T cells specific for nuclear peptide antigens play an important role in tolerance breakdown during the course of systemic lupus erythematosus (SLE). However, reliable detection of these cells...

Generation and expansion of regulatory human CD4 + T-cell clones specific for pancreatic islet autoantigens

Autoantigen-specific regulatory T cells (Treg) are a potential cell therapy for human autoimmune disease, provided they could be generated in adequate numbers and with stable function. To this end, we determined the feasibility of cloning and expanding human CD4 + Treg specific for the type 1 diabetes autoantigens, GAD65 and proinsulin. Blood CD4 + cells stimulated to divide in response to GAD65 (in three healthy individuals) or proinsulin (in one type 1 diabetic) were flow sorted into single cells and cultured on feeder cells in the presence of anti-CD3 monoclonal antibody, IL-2 and IL-4. Clones were expanded over 4–6 weeks and screened for autoantigen-dependent suppression of tetanus toxoid-specific T-cell proliferation. Suppression by Treg clones was then confirmed against autoantigen-specific non-Treg clones. Of a total of 447 clones generated, 98 (21.9%) had autoantigen-dependent suppressor function. Treg clones were anergic but proliferated to autoantigen after addition of IL-2 or in co-culture with stimulated bulk T cells, without loss of suppressor function. Treg clones were stored over liquid N 2, thawed and further expanded over 12 days, whereupon they exhibited decreased suppressor function. Expansion of Treg clones overall was in the order 10 7–10 8-fold. Treg clones were not distinguished by markers of conventional CD4 +CD25 + Treg and suppressed independently of cell–cell contact but not via known soluble suppressor factors. This study demonstrates that autoantigen-specific CD4 + Treg clones with potential application as a cell therapy for autoimmune disease can be generated and expanded from human blood.

Narcolepsy: autoimmunity, effector T cell activation due to infection, or T cell independent, major histocompatibility complex class II induced neuronal loss?

Human narcolepsy with cataplexy is a neurological disorder, which develops due to a deficiency in hypocretin producing neurons in the hypothalamus. There is a strong association with human leucocyte antigens HLA-DR2 and HLA-DQB1*0602. The disease typically starts in adolescence. Recent developments in narcolepsy research support the hypothesis of narcolepsy being an immune-mediated disease. Narcolepsy is associated with polymorphisms of the genes encoding T cell receptor alpha chain, tumour necrosis factor alpha and tumour necrosis factor receptor II. Moreover the rate of streptococcal infection is increased at onset of narcolepsy. The hallmarks of anti-self reactions in the tissue--namely upregulation of major histocompatibility antigens and lymphocyte infiltrates--are missing in the hypothalamus. These findings are questionable because they were obtained by analyses performed many years after onset of disease. In some patients with narcolepsy autoantibodies to Tribbles homolog 2, which is expressed by hypocretin neurons, have been detected recently. Immune-mediated destruction of hypocretin producing neurons may be mediated by microglia/macrophages that become activated either by autoantigen specific CD4(+) T cells or superantigen stimulated CD8(+) T cells, or independent of T cells by activation of DQB1*0602 signalling. Activation of microglia and macrophages may lead to the release of neurotoxic molecules such as quinolinic acid, which has been shown to cause selective destruction of hypocretin neurons in the hypothalamus.