Nicole Culbertson

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.

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.

Functional Suppression by FoxP3+CD4+CD25high Regulatory T Cells during Acute Hepatitis C Virus Infection

BACKGROUND Infection with hepatitis C virus (HCV) is characterized by impairment of viral effector T cell responses and a high propensity for viral persistence. Previous studies have demonstrated that chronic HCV infection is associated with an increased frequency of regulatory T (T(reg)) cells, compared with that in persons whose infection resolved and in healthy persons. However, all patients in prior analyses had exposures in the distant past, precluding the ability to determine whether T(reg) cells play a causal role in establishing persistence during the earliest stages of infection or whether they are expanded because of viral persistence. METHODS For the first time, we longitudinally analyzed T(reg) cells in patients with acute HCV infection (n = 27). We used a multiparameter approach, including fluorescence-activated cell sorting analysis of cell-surface and intracellular antigens, coculture experiments with highly purified CD4(+)CD25(high) regulatory and CD4(+)CD25(-) responder cell populations, and multiplex analysis of secreted cytokines. RESULTS Forkhead transcription factor 3 (FoxP3) expression and T(reg) cell suppression were greater in patients with acute HCV infection than in healthy control subjects but were not different at the first time point among patients who subsequently developed persistence or resolved HCV infection spontaneously; however, 6 months later, the resolution of disease was associated with a relative loss of functional suppression. CONCLUSIONS Collectively, these data indicate that patients with acute HCV infection who develop chronicity versus spontaneous resolution exhibit temporal changes in T(reg) cell function. It is possible that repetitive viral antigenic stimulation alters the function of T(reg) cells over time.

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.

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 highly immunogenic trivalent T cell receptor peptide vaccine for multiple sclerosis

Background: T cell receptor (TCR) peptide vaccination is a novel approach to treating multiple sclerosis (MS). The low immunogenicity of previous vaccines has hindered the development of TCR peptide vaccination for MS. Objective: To compare the immunogenicity of intramuscular injections of TCR BV5S2, BV6S5 and BV13S1 CDR2 peptides in incomplete Freund’s adjuvant (IFA) with intradermal injections of the same peptides without IFA. Methods: MS subjects were randomized to receive TCR peptides/IFA, TCR peptides/saline or IFA alone. Subjects were on study for 24 weeks. Results: The TCR peptides/IFA vaccine induced vigorous T cell responses in 100% of subjects completing the 24-week study (9/9) compared with only 20% (2/10) of those receiving the TCR peptides/saline vaccine (P =0.001). IFA alone induced a weak response in only one of five subjects. Aside from injection site reactions, there were no significant adverse events attributable to the treatment. Conclusions: The trivalent TCR peptide in IFA vaccine represents a significant improvement in immunogenicity over previous TCR peptide vaccines and warrants investigation of its ability to treat MS.

Vβ CDR3 motifs associated with BP recognition are enriched in OX-40+ spinal cord T cells of Lewis rats with EAE

Spinal cord (SC) T cells were isolated at the onset of actively induced experimental autoimmune encephalomyelitis (EAE) and sorted for the presence of the OX‐40 activation marker. Previously, we reported an enhanced bias in Vβ8.2 expression as well as enhanced proliferative responses to basic protein antigens among the OX‐40+ SC T cells. Here we demonstrate that CDR3 motifs associated with EAE are present at a significantly higher frequency in Vβ8.2 sequences of OX‐40+ SC T cells (16/17) compared with those of OX‐40− SC T cells (5/17). Thus, the OX‐40 antigen may be useful as a marker to isolate and characterize autoantigen‐specific T cells from the site of inflammation in T‐cell‐mediated autoimmune 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.

T‐cell hybridoma specific for myelin oligodendrocyte glycoprotein‐35–55 peptide produced from HLA‐DRB1*1501‐transgenic mice

The goal of this study was to establish an unlimited and standardized source of humanized myelin peptide‐specific T cells for in vitro testing of biological function. Thus, we perpetuated myelin oligodendrocyte glycoprotein (MOG)‐35–55 peptide‐specific T cells obtained from immunized HLA‐DRB1*1501‐transgenic (Tg) mice by somatic fusions with BW5147 thymoma cells or BW5147 T‐cell receptor (TCR) α−β− variant (BW5147 variant) cells. The resulting T‐cell hybridomas responded strongly to both mouse MOG‐35–55 (42S) and human MOG‐35–55 peptide (42P), regardless of which peptide was used for initial immunization, and were DRB1*1501 restricted. The MOG‐35–55‐reactive T‐cell hybridomas were CD3+CD4+CD8− and expressed intracellular Th1 cytokines upon concanavalin A stimulation. Clones from either human MOG‐35–55‐ or mouse MOG‐35–55‐selected hybridomas uniquely expressed the TCR BV8 gene in combination with AV17 and AV11 genes. V gene analyses confirmed the expression of TCR AV1, AV11, AV16, BV1, and BV5 gene segments in the widely used fusion partner BW5147 and demonstrated deletion of TCR AV1, AV11, and BV1 in the BW5147 variant. T‐cell hybridomas were positively stained with anti‐TCR β‐chain antibody on the cell surface, whereas neither BW5147 nor its variant had positive TCR surface expression. For functional application, we found that a monomeric form of the human HLA‐DR2‐derived recombinant T‐cell receptor ligand (RTL) covalently linked to human MOG‐35–55 peptide specifically inhibited proliferation of a hybridoma clone selected with human MOG‐35–55 but not a different hybridoma clone selected with myelin basic protein. The RTL‐induced inhibition in vitro of the human MOG‐35–55 peptide‐specific hybridoma reflected the ability of the RTL to inhibit experimental autoimmune encephalomyelitis induced by human MOG‐35–55 peptide in HLA‐DR2 transgenic mice. Thus, the MOG‐35–55 peptide‐specific T‐cell hybridoma from DR2‐Tg mice represents a novel humanized T‐cell reagent useful for standardized biological screening of both DR2‐restricted stimulation and RTL‐dependent inhibition of response to human MOG‐35–55 peptide.

Diminished frequency of interleukin-10-secreting, T-cell receptor peptide-reactive T cells in multiple sclerosis patients might allow expansion of activated memory T cells bearing the cognate BV gene †

T cells responsive to T‐cell receptor (TCR) determinants may regulate pathogenic Th1 responses in patients with multiple sclerosis (MS) through interleukin (IL)‐10‐dependent bystander suppression. In this study, innate IL‐10‐ and interferon (IFN)‐γ‐secreting T cells responsive to TCR peptides were quantified in peripheral blood mononuclear cells of MS patients and healthy controls (HC) using the ELISPOT assay. Most HC had vigorous IL‐10 but low IFN‐γ frequencies to BV5S2 and BV6S1 peptides. In contrast, MS patients had significantly lower IL‐10 frequency responses to the TCR peptides but normal responses to concanavalin A. Patients undergoing TCR‐peptide vaccination had moderate responses that fluctuated in concert with vaccination. In an MS patient and HC, expression of BV6S1 by activated memory T cells was inversely associated with the presence of IL‐10‐secreting BV6S1‐reactive T cells. These results suggest that MS patients have diminished frequencies of innate TCR‐reactive T cells that may allow oligoclonal expansion of activated autoreactive Th1 effector cells expressing cognate V genes. J. Neurosci. Res. 66:171–176, 2001. Published 2001 Wiley‐Liss, Inc.