Cytokine-Neuroantigen Fusion Proteins as a New Class of Tolerogenic, Therapeutic Vaccines for Treatment of Inflammatory Demyelinating Disease in Rodent Models of Multiple Sclerosis

Myelin-specific induction of tolerance represents a promising means to modify the course of autoimmune inflammatory demyelinating diseases such as multiple sclerosis (MS). Our laboratory has focused on a novel preclinical strategy for the induction of tolerance to the major encephalitogenic epitopes of myelin that cause experimental autoimmune encephalomyelitis (EAE) in rats and mice. This novel approach is based on the use of cytokine-NAg (neuroantigen) fusion proteins comprised of the native cytokine fused either with or without a linker to a NAg domain. Several single-chain cytokine-NAg fusion proteins were tested including GMCSF-NAg, IFNbeta-NAg, NAgIL16, and IL2-NAg. These cytokine-NAg vaccines were tolerogenic, therapeutic vaccines that had tolerogenic activity when given as pre-treatments before encephalitogenic immunization and also were effective as therapeutic interventions during the effector phase of EAE. The rank order of inhibitory activity was as follows: GMCSF-NAg, IFNbeta-NAg > NAgIL16 > IL2-NAg > MCSF-NAg, IL4-NAg, IL-13-NAg, IL1RA-NAg, and NAg. Several cytokine-NAg fusion proteins exhibited antigen-targeting activity. High affinity binding of the cytokine domain to specific cytokine receptors on particular subsets of APC resulted in the concentrated uptake of the NAg domain by those APC which in turn facilitated the enhanced processing and presentation of the NAg domain on cell surface MHC class II glycoproteins. For most cytokine-NAg vaccines, the covalent linkage of the cytokine domain and NAg domain was required for inhibition of EAE, thereby indicating that antigenic targeting of the NAg domain to APC was also required in vivo for tolerogenic activity. Overall, these studies introduced a new concept of cytokine-NAg fusion proteins as a means to induce tolerance and to inhibit the effector phase of autoimmune disease. The approach has broad application for suppressive vaccination as a therapy for autoimmune diseases such as MS

Subset-specific co-stimulatory signals are required for IL-2 production but not growth inhibition responses by T cell hybrids specific for myelin basic protein

Two distinct types of T cell hybridomas (designated THYB-1 and THYB-2) were derived by fusing BW5147 thymoma cells with encephalitogenic T helper cells from Lewis rats. Both subsets required MHC-restricted presentation of determinants within the 72-86 peptide sequence of myelin basic protein (MBP) as a requisite signal for IL-2 production. Unlike THYB-1 hybrids, however, THYB-2 hybrids required additional accessory cell activities that were mediated by radiosensitive nonadherent (RS-NAdh) splenocytes (SPL). In this study, we describe two observations indicating that RS-NAdh SPL enable MBP-specific responses of THYB-2 hybrids by providing subset-specific co-stimulatory signals that act independently of antigen recognition pathways. First, RS-NAdh SPL were required by THYB-2 hybrids for MBP-stimulated IL-2 production but were not needed when MBP-specific inhibition of hybrid growth was used as an alternative measure of cellular activation. Second, PMA and ionomycin induced optimal IL-2 production by both tHYB-1 hybrids and BW5147 thymoma cells but only stimulated low or marginal levels of IL-2 production by THYB-2 hybrids. Together, these observations indicate that RS-NAdh SPL were required for the specific response of IL-2 production regardless of whether the response was stimulated by antigen or by mitogens that bypass initial antigen recognition events. This study thereby provides additional evidence that distinct stimulus-response relationships define two T-helper cell lineages in experimental autoimmune encephalomyelitis.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/30179/1/0000564.pd

The Extracellular Domain of Myelin Oligodendrocyte Glycoprotein Elicits Atypical Experimental Autoimmune Encephalomyelitis in Rat and Species

Atypical models of experimental autoimmune encephalomyelitis (EAE) are advantageous in that the heterogeneity of clinical signs appears more reflective of those in multiple sclerosis (MS). Conversely, models of classical EAE feature stereotypic progression of an ascending flaccid paralysis that is not a characteristic of MS. The study of atypical EAE however has been limited due to the relative lack of suitable models that feature reliable disease incidence and severity, excepting mice deficient in gamma-interferon signaling pathways. In this study, atypical EAE was induced in Lewis rats, and a related approach was effective for induction of an unusual neurologic syndrome in a cynomolgus macaque. Lewis rats were immunized with the rat immunoglobulin variable (IgV)-related extracellular domain of myelin oligodendrocyte glycoprotein (IgV-MOG) in complete Freund’s adjuvant (CFA) followed by one or more injections of rat IgV-MOG in incomplete Freund’s adjuvant (IFA). The resulting disease was marked by torticollis, unilateral rigid paralysis, forelimb weakness, and high titers of anti-MOG antibody against conformational epitopes of MOG, as well as other signs of atypical EAE. A similar strategy elicited a distinct atypical form of EAE in a cynomolgus macaque. By day 36 in the monkey, titers of IgG against conformational epitopes of extracellular MOG were evident, and on day 201, the macaque had an abrupt onset of an unusual form of EAE that included a pronounced arousal-dependent, transient myotonia. The disease persisted for 6–7 weeks and was marked by a gradual, consistent improvement and an eventual full recovery without recurrence. These data indicate that one or more boosters of IgV-MOG in IFA represent a key variable for induction of atypical or unusual forms of EAE in rat and Macaca species. These studies also reveal a close correlation between humoral immunity against conformational epitopes of MOG, extended confluent demyelinating plaques in spinal cord and brainstem, and atypical disease induction

fMRI scanner noise interaction with affective neural processes

The purpose of the present study was the investigation of interaction effects between functional MRI scanner noise and affective neural processes. Stimuli comprised of psychoacoustically balanced musical pieces, expressing three different emotions (fear, neutral, joy). Participants (N=34, 19 female) were split into two groups, one subjected to continuous scanning and another subjected to sparse temporal scanning that features decreased scanner noise. Tests for interaction effects between scanning group (sparse/quieter vs continuous/noisier) and emotion (fear, neutral, joy) were performed. Results revealed interactions between the affective expression of stimuli and scanning group localized in bilateral auditory cortex, insula and visual cortex (calcarine sulcus). Post-hoc comparisons revealed that during sparse scanning, but not during continuous scanning, BOLD signals were significantly stronger for joy than for fear, as well as stronger for fear than for neutral in bilateral auditory cortex. During continuous scanning, but not during sparse scanning, BOLD signals were significantly stronger for joy than for neutral in the left auditory cortex and for joy than for fear in the calcarine sulcus. To the authors' knowledge, this is the first study to show a statistical interaction effect between scanner noise and affective processes and extends evidence suggesting scanner noise to be an important factor in functional MRI research that can affect and distort affective brain processes

Use of surface plasmon resonance for the measurement of low affinity binding interactions between HSP72 and measles virus nucleocapsid protein

The 72 kDa heat shock protein (HSP72) is a molecular chaperone that binds native protein with low affinity. These interactions can alter function of the substrate, a property known as HSP-mediated activity control. In the present work, BIAcore instrumentation was used to monitor binding reactions between HSP72 and naturally occurring sequence variants of the measles virus (MV) nucleocapsid protein (N), a structural protein regulating transcription/replication of the viral genome. Binding reactions employed synthetic peptides mimicking a putative HSP72 binding motif of N. Sequences were identified that bound HSP72 with affinities comparable to well-characterized activity control reactions. These sequences, but not those binding with lesser affinity, supported HSP72 activity control of MV transcription/replication. BIAcore instrumentation thus provides an effective way to measure biologically relevant low affinity interactions with structural variants of viral proteins

In Search of HPA Axis Dysregulation in Child and Adolescent Depression

Dysregulation of the hypothalamic–pituitary–adrenal (HPA) axis in adults with major depressive disorder is among the most consistent and robust biological findings in psychiatry. Given the importance of the adolescent transition to the development and recurrence of depressive phenomena over the lifespan, it is important to have an integrative perspective on research investigating the various components of HPA axis functioning among depressed young people. The present narrative review synthesizes evidence from the following five categories of studies conducted with children and adolescents: (1) those examining the HPA system’s response to the dexamethasone suppression test (DST); (2) those assessing basal HPA axis functioning; (3) those administering corticotropin-releasing hormone (CRH) challenge; (4) those incorporating psychological probes of the HPA axis; and (5) those examining HPA axis functioning in children of depressed mothers. Evidence is generally consistent with models of developmental psychopathology that hypothesize that atypical HPA axis functioning precedes the emergence of clinical levels of depression and that the HPA axis becomes increasingly dysregulated from child to adult manifestations of depression. Multidisciplinary approaches and longitudinal research designs that extend across development are needed to more clearly and usefully elucidate the role of the HPA axis in depression

Multidimensional profiling of human T cells reveals high CD38 expression, marking recent thymic emigrants and age-related naive T cell remodeling

Thymic involution is a key factor in human immune aging, leading to reduced thymic output and a decline in recent thymic emigrant (RTE) naive T cells in circulation. Currently, the precise definition of human RTEs and their corresponding cell surface markers lacks clarity. Analysis of single-cell RNA-seq/ATAC-seq data distinguished RTEs by the expression of SOX4, IKZF2, and TOX and CD38 protein, whereby surface CD38 hi expression universally identified CD8 + and CD4 + RTEs. We further determined the dynamics of RTEs and mature cells in a cohort of 158 individuals, including age-associated transcriptional reprogramming and shifts in cytokine production. Spectral cytometry profiling revealed two axes of aging common to naive CD8 + and CD4 + T cells: (1) a decrease in CD38 ++ cells (RTEs) and (2) an increase in CXCR3 hi cells. Identification of RTEs enables direct assessment of thymic health. Furthermore, resolving the dynamics of naive T cell remodeling yields insight into vaccination and infection responsiveness throughout aging

A GMCSF-neuroantigen fusion protein is a potent tolerogen in experimental autoimmune encephalomyelitis (EAE) that is associated with efficient targeting of neuroantigen to APC

Cytokine-NAg fusion proteins represent an emerging platform for specific targeting of self-antigen to particular APC subsets as a means to achieve antigen-specific immunological tolerance. This study focused on cytokine-NAg fusion proteins that targeted NAg to myeloid APC. Fusion proteins contained GM-CSF or the soluble extracellular domain of M-CSF as the N-terminal domain and the encephalitogenic 69–87 peptide of MBP as the C-terminal domain. GMCSF-NAg and MCSF-NAg fusion proteins were ∼1000-fold and 32-fold more potent than NAg in stimulating antigenic proliferation of MBP-specific T cells, respectively. The potentiated antigenic responses required cytokine-NAg covalent linkage and receptor-mediated uptake. That is, the respective cytokines did not potentiate antigenic responses when cytokine and NAg were added as separate molecules, and the potentiated responses were inhibited specifically by the respective free cytokine. Cytokine-dependent targeting of NAg was specific for particular subsets of APC. GMCSF-NAg and MCSF-NAg targeted NAg to DC and macrophages; conversely, IL4-NAg and IL2-NAg fusion proteins, respectively, induced an ∼1000-fold enhancement in NAg reactivity in the presence of B cell and T cell APC. GMCSF-NAg significantly attenuated severity of EAE when treatment was completed before encephalitogenic challenge or alternatively, when treatment was initiated after onset of EAE. MCSF-NAg also had significant tolerogenic activity, but GMCSF-NAg was substantially more efficacious as a tolerogen. Covalent GMCSF-NAg linkage was required for prevention and treatment of EAE. In conclusion, GMCSF-NAg was highly effective for targeting NAg to myeloid APC and was a potent, antigen-specific tolerogen in EAE

Autologous rat myelin basic protein is a partial agonist that is converted into a full antagonist upon blockade of CD4. Evidence for the integration of efficacious and nonefficacious signals during T cell antigen recognition.

Abstract A central question of TCR function is based on the observation that some MHC ligands may bind TCR without stimulating biologic activity. To address the role of CD4 in this mechanism, we studied the interactions of the anti-CD4 mAb W3/25 with an encephalitogenic line of T helper cells. Proliferative responses to guinea pig (GP) myelin basic protein (GPMBP) were mediated by distinct W3/25-sensitive and W3/25-insensitive mechanisms whereas responses to autologous rat (R) MBP (RMBP) were exclusively mediated by W3/25-sensitive pathways. In assays of IL-2 production, RMBP was a partial agonist that stimulated an intermediate level of IL-2 production but antagonized high levels of GPMBP-stimulated IL-2 production to that intermediate level. In the presence of W3/25, RMBP lacked stimulatory activity but instead exhibited inhibitory activity that completely blocked GPMBP-stimulated proliferative responses. The inhibitory mechanism did not involve antigenic competition for MHC glycoproteins, a blockade of mitogenic signaling, or induction of high zone tolerance. Rather, the mechanism involved specific occupancy of TCR with antagonistic MHC ligands derived from the 72-86 region of RMBP. In proliferative assays, GPMBP was approximately 10-fold more active than RMBP. In the presence of W3/25 however, GPMBP-induced agonism and RMBP-induced antagonism exhibited overlapping dose-response curves. RMBP and W3/25 not only fully inhibited GPMBP-stimulated proliferation, this synergistic combination also elicited an extended phase of T cell energy. In conclusion, RMBP-derived MHC ligands occupy TCR to exhibit full efficacy for CD4-dependent signaling pathways while simultaneously lacking efficacy for W3/25-resistant signaling pathways. These data support an "integrative" model of T cell Ag recognition whereby MHC glycoproteins actively guide specific clustering of MHC-ligated TCR to enable quantitative comparisons of efficacious (nonself) and nonefficacious (self) signals by T cells.</jats:p