Yuan K. Chou

Decreased FOXP3 Levels in Multiple Sclerosis Patients

Autoimmune diseases such as multiple sclerosis (MS) may result from the failure of tolerance mechanisms to prevent expansion of pathogenic T cells. Our study is the first to establish that MS patients have abnormalities in FOXP3 message and protein expression levels in peripheral CD4+CD25+ T cells (Tregs) that are quantitatively related to a reduction in functional suppression induced during suboptimal T‐cell receptor (TCR) ligation. Of importance, this observation links a defect in functional peripheral immunoregulation to an established genetic marker that has been unequivocally shown to be involved in maintaining immune tolerance and preventing autoimmune diseases. Diminished FOXP3 levels thus indicate impaired immunoregulation by Tregs that may contribute to MS. Future studies will evaluate the effects of therapies known to influence Treg cell function and FOXP3 expression, including TCR peptide vaccination and supplemental estrogen.

Treatment of multiple sclerosis with T–cell receptor peptides: Results of a double–blind pilot trial

A T–cell receptor (TCR) peptide vaccine from the Vβ5.2 sequence expressed in multiple sclerosis (MS) plaques and on myelin basic protein (MBP)–specific T cells boosted peptide–reactive T cells in patients with progressive MS. Vaccine responders had a reduced MBP response and remained clinically stable without side effects during one year of therapy, whereas nonresponders had an increased MBP response and progressed clinically. Peptide–specific T helper 2 cells directly inhibited MBP–specific T helper 1 cells in vitro through the release of interleukin–10, implicating a bystander suppression mechanism that holds promise for treatment of MS and other autoimmune diseases.

Functional assay for human CD4+CD25+ Treg cells reveals an age-dependent loss of suppressive activity.

CD4+CD25+ regulatory T cells (Treg cells) prevent T cell‐mediated autoimmune diseases in rodents. To develop a functional Treg assay for human blood cells, we used FACS‐ or bead‐sorted CD4+CD25+ T cells from healthy donors to inhibit anti‐CD3/CD28 activation of CD4+CD25− indicator T cells. The data clearly demonstrated classical Treg suppression of CD4+CD25− indicator cells by both CD4+CD25+high and CD4+CD25+low T cells obtained by FACS or magnetic bead sorting. Suppressive activity was found in either CD45RO− (naive) or CD45RO+ (memory) subpopulations, was independent of the TCR signal strength, required cell–cell contact, and was reversible by interleukin‐2 (IL‐2). Of general interest is that a wider sampling of 27 healthy donors revealed an age‐ but not gender‐dependent loss of suppressive activity in the CD4+CD25+ population. The presence or absence of suppressive activity in CD4+CD25+ T cells from a given donor could be demonstrated consistently over time, and lack of suppression was not due to method of sorting, strength of signal, or sensitivity of indicator cells. Phenotypic markers did not differ on CD4+CD25+ T cells tested ex vivo from suppressive vs. nonsuppressive donors, although, upon activation in vitro, suppressive CD4+CD25+ T cells had significantly higher expression of both CTLA‐4 and GITR than CD4+CD25− T cells from the same donors. Moreover, antibody neutralization of CTLA‐4, GITR, IL‐10, or IL‐17 completely reversed Treg‐induced suppression. Our results are highly consistent with those reported for murine Treg cells and are the first to demonstrate that suppressive activity of human CD4+CD25+ T cells declines with age. Published 2003 Wiley‐Liss, Inc.

Frequency of T cells specific for myelin basic protein and myelin proteolipid protein in blood and cerebrospinal fluid in multiple sclerosis

T cell sensitization to two myelin components, myelin basic protein (MBP) and myelin proteolipid protein (PLP), may be important to the pathogenesis of multiple sclerosis (MS). Using the limiting dilution assay, we demonstrated that the blood of MS patients had an increased frequency of MBP-reactive T cells compared with normal subjects and patients with other neurological diseases (OND) and rheumatoid arthritis. There was no difference in T cell frequency to a synthetic peptide, PLP139-151, or Herpes simplex virus. Within cerebrospinal fluid (CSF), 37% of IL-2/IL-4-reactive T cell isolates from MS patients responded either to MBP or PLP139-151 while only 5% of similar isolates from OND patients responded to these myelin antigens. The mean relative frequency of MBP-reactive T cells within CSF from MS patients was significantly higher than that of OND patients (22 x 10(-5) cells versus 1 x 10(-5) cells) and was similar to that of MBP reactive T cells within the central nervous system of rats with experimental autoimmune encephalomyelitis. These results lend new support to the hypothesis that myelin-reactive T cells mediate disease in MS.

Rudimentary TCR Signaling Triggers Default IL-10 Secretion by Human Th1 Cells

Understanding the process of inducing T cell activation has been hampered by the complex interactions between APC and inflammatory Th1 cells. To dissociate Ag-specific signaling through the TCR from costimulatory signaling, rTCR ligands (RTL) containing the α1 and β1 domains of HLA-DR2b (DRA*0101:DRB1*1501) covalently linked with either the myelin basic protein peptide 85–99 (RTL303) or CABL-b3a2 (RTL311) peptides were constructed to provide a minimal ligand for peptide-specific TCRs. When incubated with peptide-specific Th1 cell clones in the absence of APC or costimulatory molecules, only the cognate RTL induced partial activation through the TCR. This partial activation included rapid TCR ζ-chain phosphorylation, calcium mobilization, and reduced extracellular signal-related kinase activity, as well as IL-10 production, but not proliferation or other obvious phenotypic changes. On restimulation with APC/peptide, the RTL-pretreated Th1 clones had reduced proliferation and secreted less IFN-γ; IL-10 production persisted. These findings reveal for the first time the rudimentary signaling pattern delivered by initial engagement of the external TCR interface, which is further supplemented by coactivation molecules. Activation with RTLs provides a novel strategy for generating autoantigen-specific bystander suppression useful for treatment of complex autoimmune diseases.

Recombinant TCR ligand induces early TCR signaling and a unique pattern of downstream activation

Recombinant TCR ligands (RTLs) consisting of covalently linked α1 and β1 domains of MHC class II molecules tethered to specific antigenic peptides represent minimal TCR ligands. In a previous study we reported that the rat RTL201 construct, containing RT1.B MHC class II domains covalently coupled to the encephalitogenic guinea pig myelin basic protein (Gp-MBP72–89) peptide, could prevent and treat actively and passively induced experimental autoimmune encephalomyelitis in vivo by selectively inhibiting Gp-MBP72–89 peptide-specific CD4+ T cells. To evaluate the inhibitory signaling pathway, we tested the effects of immobilized RTL201 on T cell activation of the Gp-MBP72–89-specific A1 T cell hybridoma. Activation was exquisitely Ag-specific and could not be induced by RTL200 containing the rat MBP72–89 peptide that differed by a threonine for serine substitution at position 80. Partial activation by RTL201 included a CD3ζ p23/p21 ratio shift, ZAP-70 phosphorylation, calcium mobilization, NFAT activation, and transient IL-2 production. In comparison, anti-CD3ε treatment produced stronger activation of these cellular events with additional activation of NF-κB and extracellular signal-regulated kinases as well as long term increased IL-2 production. These results demonstrate that RTLs can bind directly to the TCR and modify T cell behavior through a partial activation mechanism, triggering specific downstream signaling events that deplete intracellular calcium stores without fully activating T cells. The resulting Ag-specific activation of the transcription factor NFAT uncoupled from the activation of NF-κB or extracellular signal-regulated kinases constitutes a unique downstream activation pattern that accounts for the inhibitory effects of RTL on encephalitogenic CD4+ T cells.

Therapeutic vaccination with a trivalent T‐cell receptor (TCR) peptide vaccine restores deficient FoxP3 expression and TCR recognition in subjects with multiple sclerosis

Therapeutic vaccination using T‐cell receptor (TCR) peptides from V genes commonly expressed by potentially pathogenic T cells remains an approach of interest for treatment of multiple sclerosis (MS) and other autoimmune diseases. We developed a trivalent TCR vaccine containing complementarity determining region (CDR) 2 peptides from BV5S2, BV6S5 and BV13S1 emulsified in incomplete Freunds adjuvant that reliably induced high frequencies of TCR‐specific T cells. To evaluate induction of regulatory T‐cell subtypes, immunological and clinical parameters were followed in 23 treatment‐naïve subjects with relapsing‐remitting or progressive MS who received 12 monthly injections of the trivalent peptide vaccine over 1 year in an open‐label study design. Prior to vaccination, subjects had reduced expression of forkhead box (Fox) P3 message and protein, and reduced recognition of the expressed TCR repertoire by TCR‐reactive cells compared with healthy control donors. After three or four injections, most vaccinated MS subjects developed high frequencies of circulating interleukin (IL)‐10‐secreting T cells specific for the injected TCR peptides and significantly enhanced expression of FoxP3 by regulatory T cells present in both ‘native’ CD4+ CD25+ and ‘inducible’ CD4+ CD25− peripheral blood mononuclear cells (PBMC). At the end of the trial, PBMC from vaccinated MS subjects retained or further increased FoxP3 expression levels, exhibited significantly enhanced recognition of the TCR V gene repertoire apparently generated by perturbation of the TCR network, and significantly suppressed neuroantigen but not recall antigen responses. These findings demonstrate that therapeutic vaccination using only three commonly expressed BV gene determinants can induce an expanded immunoregulatory network in vivo that may optimally control complex autoreactive responses that characterize the inflammatory phase of MS.

A novel regulatory pathway for autoimmune disease: Binding of partial MHC class II constructs to monocytes reduces CD74 expression and induces both specific and bystander T-cell tolerance

Treatment with partial (p)MHC class II-β1α1 constructs (also referred to as recombinant T-cell receptor ligands - RTL) linked to antigenic peptides can induce T-cell tolerance, inhibit recruitment of inflammatory cells and reverse autoimmune diseases. Here we demonstrate a novel regulatory pathway that involves RTL binding to CD11b(+) mononuclear cells through a receptor comprised of MHC class II invariant chain (CD74), cell-surface histones and MHC class II itself for treatment of experimental autoimmune encephalomyelitis (EAE). Binding of RTL constructs with CD74 involved a previously unrecognized MHC class II-α1/CD74 interaction that inhibited CD74 expression, blocked activity of its ligand, macrophage migration inhibitory factor, and reduced EAE severity. These findings implicate binding of RTL constructs to CD74 as a key step in both antigen-driven and bystander T-cell tolerance important in treatment of inflammatory diseases.

Specificity of regulatory CD4+CD25+ T cells for self-T cell receptor determinants.

Although the phenotypic and regulatory properties of the CD4+CD25+ T cell lineage (Treg cells) have been well described, the specificities remain largely unknown. We demonstrate here that the CD4+CD25+ Treg population includes the recognition of a broad spectrum of human TCR CDR2 determinants found in the germline V gene repertoire as well as that of a clonotypic nongermline‐encoded CDR3β sequence present in a recombinant soluble T cell receptor (TCR) protein. Regulatory activity was demonstrated in T cell lines responsive to TCR but not in T cell lines responsive to control antigens. Inhibitory activity of TCR‐reactive T cells required cell–cell contact and involved CTLA‐4, GITR, IL‐10, and IL‐17. Thus, the T–T regulatory network includes Treg cells with specificity directed toward self‐TCR determinants.

Recombinant HLA-DP2 Binds Beryllium and Tolerizes Beryllium-Specific Pathogenic CD4+ T Cells

Chronic beryllium disease is a lung disorder caused by beryllium exposure in the workplace and is characterized by granulomatous inflammation and the accumulation of beryllium-specific, HLA-DP2-restricted CD4+ T lymphocytes in the lung that proliferate and secrete Th1-type cytokines. To characterize the interaction among HLA-DP2, beryllium, and CD4+ T cells, we constructed rHLA-DP2 and rHLA-DP4 molecules consisting of the α-1 and β-1 domains of the HLA-DP molecules genetically linked into single polypeptide chains. Peptide binding to rHLA-DP2 and rHLA-DP4 was consistent with previously published peptide-binding motifs for these MHC class II molecules, with peptide binding dominated by aromatic residues in the P1 pocket. 9Be nuclear magnetic resonance spectroscopy showed that beryllium binds to the HLA-DP2-derived molecule, with no binding to the HLA-DP4 molecule that differs from DP2 by four amino acid residues. Using beryllium-specific CD4+ T cell lines derived from the lungs of chronic beryllium disease patients, beryllium presentation to those cells was independent of Ag processing because fixed APCs were capable of presenting BeSO4 and inducing T cell proliferation. Exposure of beryllium-specific CD4+ T cells to BeSO4-pulsed, plate-bound rHLA-DP2 molecules induced IFN-γ secretion. In addition, pretreatment of beryllium-specific CD4+ T cells with BeSO4-pulsed, plate-bound HLA-DP2 blocked proliferation and IL-2 secretion upon re-exposure to beryllium presented by APCs. Thus, the rHLA-DP2 molecules described herein provide a template for engineering variants that retain the ability to tolerize pathogenic CD4+ T cells, but do so in the absence of the beryllium Ag.