Jianya Huan

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.

Enhanced FoxP3 expression and Treg cell function in pregnant and estrogen-treated mice

Estrogen (E2) upregulates the FoxP3 gene that marks regulatory CD4+CD25+ T cells (Treg cells). However, E2 also inhibits the ability of antigen presenting cells (APC) to activate T cells. It is possible that these opposing functions might affect the degree of overt suppression during pregnancy and autoimmunity. To evaluate E2 effects on Treg cell function, we quantified FoxP3 levels and Treg suppression in CD4+CD25+ T cells from pregnant and E2-treated mice, and overt Treg suppression in E2- vs. placebo-pretreated mice with autoimmune encephalomyelitis. The data clearly demonstrate that enhanced expression of FoxP3, which occurs in pregnant mice and in mice treated exogenously with E2 pellets, results in a concomitant increase in functional suppression within the CD4+CD25(bright) Treg fraction of splenocytes. The similarities in FoxP3 expression and Treg cell function in E2-treated and pregnant mice implicate E2 as a major contributor for increasing Treg function during pregnancy. Surprisingly, suppression was not enhanced when Treg cells from E2-treated mice were activated with APC and CD4+CD25- responder T cells from the same E2-treated mice, a result consistent with impaired APC activation of Treg cells. In contrast, Treg suppression was strikingly enhanced in combined cell cultures from E2-pretreated mice that were protected from EAE induced with neuroantigen in complete Freunds adjuvant. These results suggest that E2 treatment may have opposing effects on Treg cells vs. APC that both contribute to overt suppression, but such effects are overcome and focused towards enhanced suppression in inflammatory environments produced during pregnancy and EAE.

Recombinant TCR Ligand Induces Tolerance to Myelin Oligodendrocyte Glycoprotein 35-55 Peptide and Reverses Clinical and Histological Signs of Chronic Experimental Autoimmune Encephalomyelitis in HLA-DR2 Transgenic Mice

In a previous study, we demonstrated that myelin oligodendrocyte glycoprotein (MOG)-35-55 peptide could induce severe chronic experimental autoimmune encephalomyelitis (EAE) in HLA-DR2+ transgenic mice lacking all mouse MHC class II genes. We used this model to evaluate clinical efficacy and mechanism of action of a novel recombinant TCR ligand (RTL) comprised of the α1 and β1 domains of DR2 (DRB1*1501) covalently linked to the encephalitogenic MOG-35-55 peptide (VG312). We found that the MOG/DR2 VG312 RTL could induce long-term tolerance to MOG-35-55 peptide and reverse clinical and histological signs of EAE in a dose- and peptide-dependent manner. Some mice treated with lower doses of VG312 relapsed after cessation of daily treatment, but the mice could be successfully re-treated with a higher dose of VG312. Treatment with VG312 strongly reduced secretion of Th1 cytokines (TNF-α and IFN-γ) produced in response to MOG-35-55 peptide, and to a lesser degree purified protein derivative and Con A, but had no inhibitory effect on serum Ab levels to MOG-35-55 peptide. Abs specific for both the peptide and MHC moieties of the RTLs were also present after treatment with EAE, but these Abs had only a minor enhancing effect on T cell activation in vitro. These data demonstrate the powerful tolerance-inducing therapeutic effects of VG312 on MOG peptide-induced EAE in transgenic DR2 mice and support the potential of this approach to inhibit myelin Ag-specific responses in multiple sclerosis patients.

Interferon-beta-1a treatment increases CD56bright natural killer cells and CD4+CD25+ Foxp3 expression in subjects with multiple sclerosis.

Disease modifying effects of interferon (IFN)-beta therapy in patients with multiple sclerosis (MS) may be mediated in part through enhanced immunoregulation by the CD56(bright) subpopulation of natural killer (NK) cells and by Foxp3+ (not italicized) CD4+CD25+ regulatory T cells (Treg). We found that IFN-beta-1a(IM) treatment of relapsing-remitting (RR)MS subjects over 12 months significantly increased both percentage of CD56(bright) NK cells and Foxp3 mRNA expression compared to baseline values, untreated RRMS subjects and healthy controls (HC). This striking enhancement of two prominent immunoregulatory pathways lends support to the idea that beneficial effects of IFN-beta-1a in MS include control of pernicious autoimmunity.

Serum Exosome MicroRNA as a Minimally-Invasive Early Biomarker of AML

Relapse remains the major cause of mortality for patients with Acute Myeloid Leukemia (AML). Improved tracking of minimal residual disease (MRD) holds the promise of timely treatment adjustments to preempt relapse. Current surveillance techniques detect circulating blasts that coincide with advanced disease and poorly reflect MRD during early relapse. Here, we investigate exosomes as a minimally invasive platform for a microRNA (miRNA) biomarker. We identify a set of miRNA enriched in AML exosomes and track levels of circulating exosome miRNA that distinguish leukemic xenografts from both non-engrafted and human CD34+ controls. We develop biostatistical models that reveal circulating exosomal miRNA at low marrow tumor burden and before circulating blasts can be detected. Remarkably, both leukemic blasts and marrow stroma contribute to serum exosome miRNA. We propose development of serum exosome miRNA as a platform for a novel, sensitive compartment biomarker for prospective tracking and early detection of AML recurrence.

Monomeric Recombinant TCR Ligand Reduces Relapse Rate and Severity of Experimental Autoimmune Encephalomyelitis in SJL/J Mice through Cytokine Switch

Our previous studies demonstrated that oligomeric recombinant TCR ligands (RTL) can treat clinical signs of experimental autoimmune encephalomyelitis (EAE) and induce long-term T cell tolerance against encephalitogenic peptides. In the current study, we produced a monomeric I-As/PLP 139-151 peptide construct (RTL401) suitable for use in SJL/J mice that develop relapsing disease after injection of PLP 139-151 peptide in CFA. RTL401 given i.v. or s.c. but not empty RTL400 or free PLP 139-151 peptide prevented relapses and significantly reduced clinical severity of EAE induced by PLP 139-151 peptide in SJL/J or (C57BL/6 × SJL)F1 mice, but did not inhibit EAE induced by PLP 178-191 or MBP 84-104 peptides in SJL/J mice, or MOG 35-55 peptide in (C57BL/6 × SJL/J)F1 mice. RTL treatment of EAE caused stable or enhanced T cell proliferation and secretion of IL-10 in the periphery, but reduced secretion of inflammatory cytokines and chemokines. In CNS, there was a modest reduction of inflammatory cells, reduced expression of very late activation Ag-4, lymphocyte function-associated Ag-1, and inflammatory cytokines, chemokines, and chemokine receptors, but enhanced expression of Th2-related factors, IL-10, TGF-β3, and CCR3. These results suggest that monomeric RTL therapy induces a cytokine switch that curbs the encephalitogenic potential of PLP 139-151-specific T cells without fully preventing their entry into CNS, wherein they reduce the severity of inflammation. This mechanism differs from that observed using oligomeric RTL therapy in other EAE models. These results strongly support the clinical application of this novel class of peptide/MHC class II constructs in patients with multiple sclerosis who have focused T cell responses to known encephalitogenic myelin peptides.

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 Promising Therapeutic Approach for Multiple Sclerosis: Recombinant T-Cell Receptor Ligands Modulate Experimental Autoimmune Encephalomyelitis by Reducing Interleukin-17 Production and Inhibiting Migration of Encephalitogenic Cells into the CNS

Recombinant T-cell receptor ligands (RTLs) can prevent and reverse clinical and histological signs of experimental autoimmune encephalomyelitis (EAE) in an antigen-specific manner and are currently in clinical trials for treatment of subjects with multiple sclerosis (MS). To evaluate regulatory mechanisms, we designed and tested RTL551, containing the α1 and β1 domains of the I-Ab class II molecule covalently linked to the encephalitogenic MOG-35-55 peptide in C57BL/6 mice. Treatment of active or passive EAE with RTL551 after disease onset significantly reduced clinical signs and spinal cord lesions. Moreover, RTL551 treatment strongly and selectively reduced secretion of interleukin-17 and tumor necrosis factor α by transferred green fluorescent protein-positive (GFP+) MOG-35-55-reactive T-cells and almost completely abrogated existent GFP+ cellular infiltrates in affected spinal cord sections. Reduced inflammation in spinal cords of RTL551-treated mice was accompanied by a highly significant downregulation of chemokines and their receptors and inhibition of VCAM-1 (vascular cell adhesion molecule-1) and ICAM-1 (intercellular adhesion molecule-1) expression by endothelial cells. Thus, RTL therapy cannot only inhibit systemic production of encephalitogenic cytokines by the targeted myelin oligodendrocyte glycoprotein-reactive T-cells but also impedes downstream local recruitment and retention of inflammatory cells in the CNS. These findings indicate that targeted immunotherapy of antigen-specific T-cells can result in a reversal of CNS lesion formation and lend strong support to the application of the RTL approach for therapy in MS.

Self-Enforcing Feedback Activation between BCL6 and Pre-B Cell Receptor Signaling Defines a Distinct Subtype of Acute Lymphoblastic Leukemia

Studying 830 pre-B ALL cases from four clinical trials, we found that human ALL can be divided into two fundamentally distinct subtypes based on pre-BCR function. While absent in the majority of ALL cases, tonic pre-BCR signaling was found in 112 cases (13.5%). In these cases, tonic pre-BCR signaling induced activation of BCL6, which in turn increased pre-BCR signaling output at the transcriptional level. Interestingly, inhibition of pre-BCR-related tyrosine kinases reduced constitutive BCL6 expression and selectively killed patient-derived pre-BCR(+) ALL cells. These findings identify a genetically and phenotypically distinct subset of human ALL that critically depends on tonic pre-BCR signaling. In vivo treatment studies suggested that pre-BCR tyrosine kinase inhibitors are useful for the treatment of patients with pre-BCR(+) ALL.