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Objective: To assess safety, tolerability, and efficacy of the antigen-specific immunotherapy ATX-MS-1467 in participants with relapsing multiple sclerosis using different treatment protocols to induce tolerance., Methods: Two open-label trials in adult participants with relapsing multiple sclerosis were conducted. Study 1 was a multicenter, phase 1b safety evaluation comparing intradermal (i.d.) (cohort 1) with subcutaneous (cohort 2) administration in 43 participants. Both cohorts received ATX-MS-1467 dosed at 25, 50, 100, 400, and 800 μg at 14-day intervals over 8 weeks, followed by 8 weeks with 4 additional 800-μg doses at 14-day intervals and 32 weeks off study medication. Study 2 was a phase 2a, multicenter, single-arm trial enrolling 37 participants. ATX-MS-1467 was titrated from 50 μg i.d. on day 1 to 200 μg on day 15 and 800 μg on day 29 followed by biweekly administration of 800 μg for 16 weeks and 16 weeks off study medication. Efficacy was evaluated on MRI parameters and clinical variables. Safety endpoints included treatment-emergent adverse events and injection-site reactions., Results: In study 1, there was a significant decrease in new/persisting T1 gadolinium-enhanced (GdE) lesions in cohort 1 from baseline to week 16, returning to baseline values at week 48. In study 2, the number of T1 GdE lesions were significantly reduced on treatment and remained reduced at study completion. Safety results were unremarkable in both studies., Conclusion: Relatively slow ATX-MS-1467 titration and a longer full-dose i.d. treatment period is associated with reduction in GdE lesions and a sustained effect post treatment. Further trials of ATX-MS-1467 are warranted., Classification of Evidence: This work provides Class IV evidence that for patients with relapsing multiple sclerosis, slow ATX-MS-1467 titration and a longer full-dose i.d. treatment period is associated with reduction in GdE lesions., (© 2018 American Academy of Neurology.)

Administration of peptides i.n. induces peripheral tolerance in Tg4 myelin basic protein-specific TCR-Tg mice. This is characterized by the generation of anergic, IL-10-secreting CD4+ T cells with regulatory function (IL-10 Treg). Myelin basic protein Ac1-9 peptide analogs, displaying a hierarchy of affinities for H-2 A(u) (Ac1-9[4K]<<[4A]<[4Y]), were used to investigate the mechanisms of tolerance induction, focusing on IL-10 Treg generation. Repeated i.n. administration of the highest affinity peptide, Ac1-9[4Y], provided complete protection against EAE, while i.n. Ac1-9[4A] and Ac1-9[4K] treatment resulted in only partial protection. Ac1-9[4Y] was also the most potent stimulus for IL-10 Treg generation. Although i.n. treatment with Ac1-9[4A] gave rise to IL-10-secreting CD4+ T cells, the population as a whole was also capable of secreting IFN-gamma after an in vitro recall response to Ac1-9[4A] or [4Y]. In addition to IL-10 production, other facets of tolerance, namely, anergy and suppression (both in vitro and in vivo), were affinity dependent, with i.n. Ac1-9[4Y]-, [4A]- or [4K]-treated CD4+ T cells being the most, intermediate and least anergic/suppressive, respectively. These findings demonstrate that the generation of IL-10 Treg in vivo is driven by high signal strength.

Most autoantigens implicated in multiple sclerosis (MS) are expressed not only in the central nervous system (CNS) but also in the thymus and the periphery. Nevertheless, these autoantigens might induce a strong autoimmune response leading to severe destruction within the CNS. To investigate the influence of a dominantly presented autoantigen on experimental autoimmune encephalomyelitis (EAE), we generated transgenic mice expressing the autoantigenic peptide MBP 1-10 covalently bound to the MHC class II molecule I-Au. These mice were crossed either with B10.PL or with TCR-transgenic Tg4 mice, specific for the transgenic peptide-MHC combination. In double transgenic mice we found strong thymic deletion and residual peripheral T cells were refractory to antigen stimulation in vitro. Residual peripheral CD4+ T cells expressed activation markers and a high proportion was CD25 positive. Transfer of both CD25-negative and CD25-positive CD4+ T cells from double transgenic animals into B10.PL mice strongly inhibited the progression of EAE. Despite this thorough tolerance induction, some double transgenic mice developed severe signs of EAE after an extended period of time. Our data show that in the circumstances where autoantigenic priming persists, and where the number of antigen-specific T cells is high enough, autoimmunity may prevail over very potent tolerance-inducing mechanisms.

Altered peptide ligands (APL) with increased MHC-binding properties are highly effective at inducing T cell tolerance after systemic administration in soluble form, preventing experimental autoimmune encephalomyelitis (EAE) induced with the myelin basic protein (MBP) Ac1-9 peptide. We have previously described a diverse Ac1-9-reactive T cell repertoire with differing TCR affinities. A remaining question is what proportion of this repertoire is silenced by peptide therapy? Here, we show that the sensitivity of a T cell to peptide-induced tolerance is related to its avidity for native Ac1-9. These data provide new evidence that self-reactive T cells bearing low-affinity TCRs are able to escape therapeutic induction of tolerance.

Regulatory CD4(+) T cells were induced in the Tg4 TCR transgenic mouse specific for the N-terminal peptide (Ac1-9) of myelin basic protein by intranasal administration of a high-affinity MHC-binding analog (Ac1-9[4Y]). Peptide-induced tolerant cells (PItol) were anergic, failed to produce IL-2, but responded to Ag by secretion of IL-10. PItol cells were predominantly CD25(-) and CTLA-4(+) and their anergic state was reversed by addition of IL-2 in vitro. PItol cells suppressed the response of naive Tg4 cells both in vitro and in vivo. The in vitro suppression mediated by these cells was not reversed by cytokine neutralization and was cell-cell contact-dependent. However, suppression of proliferation and IL-2 production by PItol cells in vivo was abrogated by neutralization of IL-10. These results emphasize an important role for IL-10 in the function of peptide-induced regulatory T cells in vivo and highlight the caution required in extrapolating mechanisms of T regulatory cell function from in vitro studies.

The peripheral T-cell population is educated to recognize a maximum of pathogen-derived epitopes while ignoring self-antigens. As the total number of T-cell clones is limited, each T-cell receptor (TCR) needs to be cross-reactive in order to achieve a wide repertoire. This opens the possibility for T cells to diverge from their defending role and induce auto-aggression by mistake. The factors involved in the initiation of such autoimmune responses remain to be fully understood. In an attempt to assess the role of antigen presenting cells (APC) in the triggering of autoimmunity, we studied the cross-reactivity of TCR transgenic Tg4 T cells, reactive to the Ac1-9 peptide of myelin basic protein (MBP). Using different APC populations and a range of peptide analogues of Ac1-9, we found that the activation of APC enhanced the cross-reactivity of Tg4 cells, and that this effect could be mimicked by resting APC supplemented with exogenous co-stimulation. Further, we observed that the inhibitory effect of an antagonist peptide of the Tg4 TCR was greatly reduced when activated APC were used. However, when co-stimulation was blocked, TCR antagonism was restored to its normal level. Our results show for the first time that the activation of naturally occurring APC, namely dendritic cells, B cells and macrophages, can modulate the reactivity of T cells, both in terms of cross-reactivity and TCR antagonism, and that this effect is most likely due to enhanced levels of co-stimulation.

The rules governing which T cells are inactivated during peptide-induced tolerance are unclear. Here we show that MBP(89-101) contains three overlapping but distinct T cell epitopes that are restricted by a single major histocompatibility complex (MHC) class II molecule. The dominant epitope is not processed from MBP and is not relevant to the induction of autoimmunity. Pathogenic T cells recognize two minor epitopes that are processed from MBP but are presented only poorly after exposure to MBP(89-101). Induction of immunological tolerance by MBP(89-101) therefore inactivates T cells that recognize the dominant epitope and disease-relevant T cells escape tolerance. The topology of the three epitopes implicates asparagine endopeptidase as the enzyme that controls recognition of this region of MBP. Our results highlight the need to use peptides that mimic the binding of processed antigen fragments to MHC molecules for successful modulation of disease-relevant T cells.

Little is known about the processing of putative human autoantigens and why tolerance is established to some T cell epitopes but not others. Here we show that a principal human HLA-DR2-restricted epitope--amino acids 85-99 of myelin basic protein, MBP(85-99)--contains a processing site for the cysteine protease asparagine endopeptidase (AEP). Presentation of this epitope by human antigen-presenting cells is inversely proportional to the amount of cellular AEP activity: inhibition of AEP in living cells greatly enhances presentation of the MBP(85-99) epitope, whereas overexpression of AEP diminishes presentation. These results indicate that central tolerance to this encephalitogenic MBP epitope may not be established because destructive processing limits its display in the thymus. Consistent with this hypothesis, AEP is expressed abundantly in thymic antigen-presenting cells.

Thymic selection depends on positive and negative selective mechanisms based on the avidity of T cell interaction with antigen-major histocompatibility complex complexes. However, peripheral mechanisms for the recruitment and clonal expansion of the responding T cell repertoire remain obscure. Here we provide evidence for an avidity-based model of peripheral T cell clonal expansion in response to antigenic challenge. We have used the encephalitogenic, H-2 A(u)-restricted, acetylated NH(2)-terminal nonameric peptide (Ac1-9) epitope from myelin basic protein as our model antigen. Peptide analogues were generated that varied in antigenic strength (as assessed by in vitro assay) based on differences in their binding affinity for A(u). In vivo, these analogues elicited distinct repertoires of T cells that displayed marked differences in antigen sensitivity. Immunization with the weakest (wild-type) antigen expanded the high affinity T cells required to induce encephalomyelitis. In contrast, immunization with strongly antigenic analogues led to the elimination of T cells bearing high affinity T cell receptors by apoptosis, thereby preventing disease development. Moreover, the T cell repertoire was consistently tuned to respond to the immunizing antigen with the same activation threshold. This tuning mechanism provides a peripheral control against the expansion of autoreactive T cells and has implications for immunotherapy and vaccine design.

T regulatory (Treg) cells expressing the transcription factor FoxP3 play a key role in protection against autoimmune disease. GFP-FoxP3 reporter mice have been used widely to study the induction, function and stability of both thymically- and peripherally-induced Treg cells. The N-terminal modification of FoxP3, however, affects its interaction with transcriptional co-factors; this can alter Treg cell development and function in certain self-antigen specific animal models. Interestingly, Treg cell function can be negatively or positively affected, depending on the nature of the model. In this study, we focused on the effect of the GFP-FoxP3 reporter on Treg cell development and function in the Tg4 mouse model. In this model, T cells express a transgenic T cell receptor (TCR) specific for the Myelin Basic Protein (MBP) peptide Ac1-9, making the animals susceptible to experimental autoimmune encephalomyelitis (EAE), a disease akin to multiple sclerosis in humans. Unlike diabetes-susceptible mice, Tg4 FoxP3gfp mice did not develop spontaneous autoimmune disease and did not demonstrate augmented susceptibility to induced disease. Concurrently, thymic generation of natural Treg cells was not negatively affected. The induction of FoxP3 expression in naive peripheral T cells was, however, significantly impaired as a result of the transgene. This study shows that the requirements for the interaction of FoxP3 with co-factors, which governs its regulatory ability, differ not only between natural and inducible Treg cells but also between animal models of diseases such as diabetes and EAE. [ABSTRACT FROM AUTHOR]

The N-terminal peptide (Ac1-9) of myelin basic protein (MBP) is the immunodominant encephalitogenic epitope in H-2 mice. Previous studies have defined the role of amino acid residue 4 in binding to I-Au. Accordingly, substitutions at this residue have generated peptides spanning a wide range of affinities for the MHC. In the present study, we have tested the toterogenlcity of three of these peptides, Ad-9, Ac1-9[4A] and Ac1-9[4Y], by administering these to mice i.p. in the absence of adjuvant Significantly, mice treated with the high affinity analogues Ac1-9[4A] and Ac1-9[4Y] prior to immunization became less susceptible to Ad1-9-induced experimental autoimmune encephalomyelitis (EAE), whereas those given the low affinity peptide Ac1-9 were only moderately protected. T cell priming, as assessed by proliferate and lymphokine assays, demonstrated a direct correlation between the level of disease inhibition and T cell unresponsiveness. In treatment studies, Ac1-9 and Ac1-9[4Y] were also shown to be effective when given on the first day of disease onset. Priming of T cells, when measured by proliferation , however, became more resistant to inactivation when soluble peptides were administered close to the day of assay. Kinetic studies revealed that tolerance could be achieved in primed mice but that this takes time to develop. Two conclusions can be drawn from this study: (I) administration of peptide in solution is effective in prevention of EAE both before and after autoreactive T cell activation, and (II) high affinity analogues of self-peptides inactivate their cognate T cells readily whereas lower affinity peptides, being less efficient in tolerance induction, may allow potentially autoreactive T cells to persist in healthy individuals. [ABSTRACT FROM PUBLISHER]

The peptide rAc1–11 represents the dominant T cell epitope of rat myelin basic protein (MBP) in mice of the H-2 haplotype. Residue 4 has been shown previously to govern binding of the peptide to the class II molecule, I-A. We have constructed peptide analogues bearing amino acid substitutions at position 4 and have assessed their ability to stimulate an antigen-specific T cell hybridoma when presented by viable antigen presenting cells (APC). Complexes between I-A and one such analogue, rAc1–11[4A], were rapidly lost from the surface of live APC displaying a half-life (½) of ˜10 min. Neither shedding of intact complexes from the cell surface, nor their Internallzatlon and recycling through an acidic intracellular compartment were found to account for their loss. The possible dissociation of rAc1–11[4A] from the peptide binding cleft was therefore addressed by comparing the t½, of complexes between I-A and peptide analogues of higher affinity. The tyroslne-substltuted analogue, rAc1–11[4Y], remained stably bound to I-A for at least 4 h, thereby displaying a ½, far in excess of that evident for rAc1–11[4A]. Significantly, the wild type peptide, rAc1–11 , bound so transiently that functional complexes could not be detected on the surface of peptlde-pulsed APC. The physiological relevance of these findings was confirmed by extending our studies to an analysis of the homologous epitope of murine MBP; evidence that this epitope likewise displays minimal affinity for I-A suggests a novel strategy for the escape from tolerance induction by encephalitogenlc T cells. [ABSTRACT FROM PUBLISHER]

Peripheral immune regulation is critical for the maintenance of self-tolerance. Here we have investigated signaling processes that distinguish T cells with regulatory capability from effector T cells. The murine Tg4 T cell receptor recognizes a peptide derived from the self-antigen myelin basic protein. T cells from Tg4 T cell receptor transgenic mice can be used to generate effector T cells and three types of T cells with regulatory capability, inducible regulatory T cells, T cells tolerized by repeated in vivo antigenic peptide exposure or T cells treated with the tolerogenic drug UCB9608 (a phosphatidylinositol 4 kinase IIIβ inhibitor). We comparatively studied signaling in all of these T cells by activating them with the same antigen presenting cells presenting the same myelin basic protein peptide. Supramolecular signaling structures, as efficiently detected by large-scale live cell imaging, are critical mediators of T cell activation. The formation of a supramolecular signaling complex anchored by the adaptor protein linker for activation of T cells (LAT) was consistently terminated more rapidly in Tg4 T cells with regulatory capability. Such termination could be partially reversed by blocking the inhibitory receptors CTLA-4 and PD-1. Our work suggests that attenuation of proximal signaling may favor regulatory over effector function in T cells. [ABSTRACT FROM AUTHOR]

Tregs are important mediators of immune tolerance to self antigens, and it has been suggested that Treg inactivation may cause autoimmune disease. Therefore, immunotherapy approaches that aim to restore or expand autoantigen-specific Treg activity might be beneficial for the treatment of autoimmune disease. Here we report that Treg-mediated suppression of autoimmune disease can be achieved in vivo by taking advantage of the ability of the liver to promote immune tolerance. Expression of the neural autoantigen myelin basic protein (MBP) in the liver was accomplished stably in liver-specific MBP transgenic mice and transiently using gene transfer to liver cells in vivo. Such ectopic MBP expression induced protection from autoimmune neuroinflammation in a mouse model of multiple sclerosis. Protection from autoimmunity was mediated by MBP-specific CD4+CD25+Foxp3+ Tregs, as demonstrated by the ability of these cells to prevent disease when adoptively transferred into nontransgenic mice and to suppress conventional CD4+CD25- T cell proliferation after antigen-specific stimulation with MBP in vitro. The generation of MBP-specific CD4+CD25+Foxp3+ Tregs in vivo depended on expression of MBP in the liver, but not in skin, and occurred by TGF-beta-dependent peripheral conversion from conventional non-Tregs. Our findings indicate that autoantigen expression in the liver may generate autoantigen-specific Tregs. Thus, targeting of autoantigens to hepatocytes may be a novel approach to prevention or treatment of autoimmune diseases. [ABSTRACT FROM AUTHOR]