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Purpose FOXP3 deficiency results in severe multisystem autoimmunity in both mice and humans, driven by the absence of functional regulatory T cells. Patients typically present with early and severe autoimmune polyendocrinopathy, dermatitis, and severe inflammation of the gut, leading to villous atrophy and ultimately malabsorption, wasting, and failure to thrive. In the absence of successful treatment, FOXP3-deficient patients usually die within the first 2 years of life. Hematopoietic stem cell transplantation provides a curative option but first requires adequate control over the inflammatory condition. Due to the rarity of the condition, no clinical trials have been conducted, with widely unstandardized therapeutic approaches. We sought to compare the efficacy of lead therapeutic candidates rapamycin, anti-CD4 antibody, and CTLA4-Ig in controlling the physiological and immunological manifestations of Foxp3 deficiency in mice. Method We generated Foxp3-deficient mice and an appropriate clinical scoring system to enable direct comparison of lead therapeutic candidates rapamycin, nondepleting anti-CD4 antibody, and CTLA4-Ig. Results We found distinct immunosuppressive profiles induced by each treatment, leading to unique protective combinations over distinct clinical manifestations. CTLA4-Ig provided superior breadth of protective outcomes, including highly efficient protection during the transplantation process. Conclusion These results highlight the mechanistic diversity of pathogenic pathways initiated by regulatory T cell loss and suggest CTLA4-Ig as a potentially superior therapeutic option for FOXP3-deficient patients.

FOXP3-expressing regulatory T cells (T

The ability of immune-modulating biologics to prevent and reverse pathology has transformed recent clinical practice. Full utility in the neuroinflammation space, however, requires identification of both effective targets for local immune modulation and a delivery system capable of crossing the blood-brain barrier. The recent identification and characterization of a small population of regulatory T (Treg) cells resident in the brain presents one such potential therapeutic target. Here, we identified brain interleukin 2 (IL-2) levels as a limiting factor for brain-resident Treg cells. We developed a gene-delivery approach for astrocytes, with a small-molecule on-switch to allow temporal control, and enhanced production in reactive astrocytes to spatially direct delivery to inflammatory sites. Mice with brain-specific IL-2 delivery were protected in traumatic brain injury, stroke and multiple sclerosis models, without impacting the peripheral immune system. These results validate brain-specific IL-2 gene delivery as effective protection against neuroinflammation, and provide a versatile platform for delivery of diverse biologics to neuroinflammatory patients. The work was supported by the VIB, an ERC Consolidator Grant TissueTreg (to A.L.), an ERC Proof of Concept Grant TreatBrainDamage (to A.L.), an ERC Starting Grant AstroFunc (to M.G.H.), an ERC Proof of Concept Grant AD-VIP (to M.G.H.), FWO Research Grant 1513616N (to M.G.H.), Thierry Latran Foundation Grant SOD-VIP (to M.G.H.), an ERNAET Chair (H2020-WIDESPREAD-2018-2020-6; NCBio: 951923; to M.G.H.), FWO Research Grants 1503420N (to E.P.) and 1513020N (to J.W.), an SAO-FRA pilot grant (20190032, to E.P.), and the Biotechnology and Biological Sciences Research Council through Institute Strategic Program Grant funding BBS/E/B/000C0427 and BBS/E/B/000C0428, and the Biotechnology and Biological Sciences Research Council Core Capability Grant to the Babraham Institute. E.P., V.L., M.M., P.G., J.W. and A.d.B. were supported by fellowships from the FWO. R.L. is a senior clinical investigator of FWO Flanders. P.B., O.A. and C.P.F. were supported by an ERA-NET-NEURON grant EJTC 2016 to C.P.F. and by the Netherlands Organization for Scientific research (NWO). The authors acknowledge the important contributions of J. Haughton (VIB) for mouse husbandry, M. Rincon (VIB) for advice on AAV design and production, K. Vennekens for technical support, P. -A. Penttila and the KUL FACS Core, J. Wouters and the KUL Molecular Small Animal Imaging Center (MoSAIC), S. Walker and the Babraham Institute Imaging Core, the VIB Bio-Imaging Core, the VIB Single Cell Sequencing Core, and R. Breedijk, M. Hink and the Leeuwenhoek Center for Advanced Microscopy at the University of Amsterdam.

Single domain antibodies (VHHs) are potentially disruptive therapeutics, with important biological value for treatment of several diseases, including neurological disorders. However, VHHs have not been widely used in the central nervous system (CNS), largely because of their restricted blood-brain barrier (BBB) penetration. Here, we propose a gene transfer strategy based on BBB-crossing adeno-associated virus (AAV)-based vectors to deliver VHH directly into the CNS. As a proof-of-concept, we explored the potential of AAV-delivered VHH to inhibit BACE1, a well-characterized target in Alzheimer's disease. First, we generated a panel of VHHs targeting BACE1, one of which, VHH-B9, shows high selectivity for BACE1 and efficacy in lowering BACE1 activity in vitro. We further demonstrate that a single systemic dose of AAV-VHH-B9 produces positive long-term (12 months plus) effects on amyloid load, neuroinflammation, synaptic function, and cognitive performance, in the AppNL-G-F Alzheimer's mouse model. These results constitute a novel therapeutic approach for neurodegenerative diseases, which is applicable to a range of CNS disease targets. We want to thank the collaborators of the VIB Nanobody Core and VIB Protein Core for their valuable contribution to the research presented in this paper. We thank Joost Schymkowitz, Frederic Rousseau, and Roosmarijn Vandenbroucke for discussions and advice. We thank Jeason Haughton for expert support with mouse husbandry. MM was supported by a predoctoral fellowship from the Fonds Wetenschappelijk Onderzoek (FWO) (SB/ 1S48018N). MYR received a postdoctoral fellowship from the FWO (133722/ 1204517N) and acknowledges the continuous support of the Fundacion Cardiovascular de Colombia. SID was supported by The Foundation for Alzheimer Research (SAO-FRA) (P#14006). MGH was supported by the Thierry Latran Foundation (SOD-VIP), FWO (Grant 1513616N), and European Research Council (ERC) (Starting Grant 281961—AstroFunc; Proof of Concept Grant 713755—AD-VIP). He is currently the ERANet Chair (NCBio) at i3S Porto funded by the European Commission (H2020-WIDESPREAD-2018- 2020-6; NCBio; 951923). Work in the BDS Lab is supported by the Opening the Future campaign of the KU Leuven, SAO-FRA (P#16017), FWO, KU Leuven, VIB, a Methusalem grant from KU Leuven and the Flemish Government, the Flanders Network for Dementia Research (VIND, Strategic Basic Research Grant 135043) and the Alzheimer’s Association. BDS is supported by the Geneeskundige Stichting Koningin Elisabeth and the Bax-Vanluffelen Chair for Alzheimer’s disease. The synopsis image was produced by David Pennington (@PenningtonArt.co.uk).

Background: Alzheimer’s Disease is a neurodegenerative disease with a neuroinflammatory component. Due to the multifunctional capacity of regulatory T cells to prevent and reverse inflammation, regulatory T cells have been proposed as a potential therapeutic in Alzheimer’s Disease, either as a direct cell therapy or through the use of IL2 as a biologic to expand the endogenous population. Methods: Here we characterize the longitudinal immunological changes occurring in T cells in the AppNL-G-F mouse model of Alzheimer’s disease. Results: Age-dependent immunological changes, in both the brain and periphery, occurred in parallel in both AppNL-G-F mice and control AppNL mice. As the endogenous IL2 axis was disturbed with age, we sought to determine the effect of IL2 supplementation on disease progression. Using a genetic model of IL2 provision in the periphery or in the brain, we found that expanding regulatory T cells in either location was unable to alter the progression of key pathological events or behavioral changes. Conclusions: These results suggest that either the AppNL-G-F mouse model does not recapitulate key regulatory T cell-dependent process of Alzheimer’s disease, or that regulatory T cell therapy is not a promising candidate for APP-mutation-driven Alzheimer’s disease.

BackgroundAlzheimer’s Disease is a neurodegenerative disease with a neuroinflammatory component. Due to the multifunctional capacity of regulatory T cells to prevent and reverse inflammation, regulatory T cells have been proposed as a potential therapeutic in Alzheimer’s Disease, either as a direct cell therapy or through the use of IL2 as a biologic to expand the endogenous population.MethodsHere we characterize the longitudinal immunological changes occurring in T cells in the AppNL-G-F mouse model of Alzheimer’s disease.ResultsAge-dependent immunological changes, in both the brain and periphery, occurred in parallel in both AppNL-G-F mice and control AppNL mice. As the endogenous IL2 axis was disturbed with age, we sought to determine the effect of IL2 supplementation on disease progression. Using a genetic model of IL2 provision in the periphery or in the brain, we found that expanding regulatory T cells in either location was unable to alter the progression of key pathological events or behavioral changes.ConclusionThese results suggest that either the AppNL-G-F mouse model does not recapitulate key regulatory T cell-dependent process of Alzheimer’s disease, or that regulatory T cell therapy is not a promising candidate for APP-mutation-driven Alzheimer’s disease.

Cognitive decline is a common pathological outcome during aging, with an ill-defined cellular or molecular basis. Among the cellular changes observed with age are alterations to neuronal plasticity, changes in the glial compartment and the decline of the neurogenic niche. In the recent years, the concept of inflammaging, defined as a low-grade inflammation increasing with age, has emerged as a nexus for age-related diseases. This increase of basal inflammation is also observed in the central nervous system. While not classically considered a neurological cell type, infiltrating T cells increase in the brain with age, and may be responsible for amplification of inflammatory cascades and disruptions to the neurogenic niche. Recently, a small resident population of regulatory T cells has been identified in the brain, and the capacity of IL2-mediated expansion of this population to counter neuroinflammatory disease has been demonstrated. Here we test a brain-specific IL2 delivery system for the prevention of neurological decline in aging mice. We identify the molecular hallmarks of aging in the brain glial compartments, and identify partial restoration of this signature through IL2 treatment. At a behavioral level, brain IL2 delivery prevented the age-induced defect in spatial learning, without improving the general decline in motor skill or arousal. These results identify immune modulation as a potential path to preserving cognitive function for healthy ageing.

Regulatory T cells (Tregs) control inflammation and maintain immune homeostasis. The well-characterised circulatory population of CD4+Foxp3+ Tregs is effective at preventing autoimmunity and constraining the immune response, through direct and indirect restraint of conventional T cell activation. Recent advances in Treg cell biology have identified tissue-resident Tregs, with tissue-specific functions that contribute to the maintenance of tissue homeostasis and repair. A population of brain-resident Tregs, characterised as CD69+, has recently been identified in the healthy brain of mice and humans, with rapid population expansion observed under a number of neuroinflammatory conditions. During neuroinflammation, brain-resident Tregs have been proposed to control astrogliosis through the production of amphiregulin, polarize microglia into neuroprotective states, and restrain inflammatory responses by releasing IL-10. While protective effects for Tregs have been demonstrated in a number of neuroinflammatory pathologies, a clear demarcation between the role of circulatory and brain-resident Tregs has been difficult to achieve. Here we review the state-of-the-art for brain-resident Treg population, and describe their potential utilization as a therapeutic target across different neuroinflammatory conditions. ispartof: IMMUNOLOGY LETTERS vol:248 pages:26-30 ispartof: location:Netherlands status: published

To improve the anti-tumor efficacy of immune checkpoint inhibitors, numerous combination therapies are under clinical evaluation, including with IL-12 gene therapy. The current study evaluated the simultaneous delivery of the cytokine and checkpoint-inhibiting antibodies by intratumoral DNA electroporation in mice. In the MC38 tumor model, combined administration of plasmids encoding IL-12 and an anti-PD-1 antibody induced significant anti-tumor responses, yet similar to the monotherapies. When treatment was expanded with a DNA-based anti-CTLA-4 antibody, this triple combination significantly delayed tumor growth compared to IL-12 alone and the combination of anti-PD-1 and anti-CTLA-4 antibodies. Despite low drug plasma concentrations, the triple combination enabled significant abscopal effects in contralateral tumors, which was not the case for the other treatments. The DNA-based immunotherapies increased T cell infiltration in electroporated tumors, especially of CD8+ T cells, and upregulated the expression of CD8+ effector markers. No general immune activation was detected in spleens following either intratumoral treatment. In B16F10 tumors, evaluation of the triple combination was hampered by a high sensitivity to control plasmids. In conclusion, intratumoral gene electrotransfer allowed effective combined delivery of multiple immunotherapeutics. This approach induced responses in treated and contralateral tumors, while limiting systemic drug exposure and potentially detrimental systemic immunological effects. ispartof: CANCER GENE THERAPY vol:29 issue:7 pages:984-992 ispartof: location:England status: published

To improve the anti-tumor efficacy of immune checkpoint inhibitors, numerous combination therapies are under clinical evaluation, including with IL-12 gene therapy. The current study evaluated the simultaneous delivery of the cytokine and checkpoint-inhibiting antibodies by intratumoral DNA electroporation in mice. In the MC38 tumor model, combined administration of plasmids encoding IL-12 and an anti-PD-1 antibody induced significant anti-tumor responses, yet similar to the monotherapies. When treatment was expanded with a DNA-based anti-CTLA-4 antibody, this triple combination significantly delayed tumor growth compared to IL-12 alone and the combination of anti-PD-1 and anti-CTLA-4 antibodies. Despite low drug plasma concentrations, the triple combination enabled significant abscopal effects in contralateral tumors, which was not the case for the other treatments. The DNA-based immunotherapies increased T cell infiltration in electroporated tumors, especially of CD8

Paraneoplastic cerebellar degeneration (PCD) is a rare immune-mediated disease that develops mostly in the setting of neoplasia and offers a unique prospect to explore the interplay between tumor immunity and autoimmunity. In PCD, the deleterious adaptive immune response targets self-antigens aberrantly expressed by tumor cells, mostly gynecological cancers, and physiologically expressed by the Purkinje neurons of the cerebellum. Highly specific anti-neuronal antibodies in the serum and cerebrospinal fluid represent key diagnostic biomarkers of PCD. Some anti-neuronal antibodies such as anti-Yo autoantibodies (recognizing the CDR2/CDR2L proteins) are only associated with PCD. Other anti-neuronal antibodies, such as anti-Hu, anti-Ri, and anti-Ma2, are detected in patients with PCD or other types of paraneoplastic neurological manifestations. Importantly, these autoantibodies cannot transfer disease and evidence for a pathogenic role of autoreactive T cells is accumulating. However, the precise mechanisms responsible for disruption of self-tolerance to neuronal self-antigens in the cancer setting and the pathways involved in pathogenesis within the cerebellum remain to be fully deciphered. Although the occurrence of PCD is rare, the risk for such severe complication may increase with wider use of cancer immunotherapy, notably immune checkpoint blockade. Here, we review recent literature pertaining to the pathophysiology of PCD and propose an immune scheme underlying this disabling disease. Additionally, based on observations from patients' samples and on the pre-clinical model we recently developed, we discuss potential therapeutic strategies that could blunt this cerebellum-specific autoimmune disease.

The brain is a site of relative immune privilege. Although CD4 T cells have been reported in the central nervous system, their presence in the healthy brain remains controversial, and their function remains largely unknown. We used a combination of imaging, single cell, and surgical approaches to identify a CD69+ CD4 T cell population in both the mouse and human brain, distinct from circulating CD4 T cells. The brain-resident population was derived through in situ differentiation from activated circulatory cells and was shaped by self-antigen and the peripheral microbiome. Single-cell sequencing revealed that in the absence of murine CD4 T cells, resident microglia remained suspended between the fetal and adult states. This maturation defect resulted in excess immature neuronal synapses and behavioral abnormalities. These results illuminate a role for CD4 T cells in brain development and a potential interconnected dynamic between the evolution of the immunological and neurological systems. VIDEO ABSTRACT. ispartof: CELL vol:182 issue:3 pages:625-+ ispartof: location:United States status: published

Neuroinflammation is often associated with blood-brain-barrier dysfunction, which contributes to neurological tissue damage. Here, we reveal the pathophysiology of Susac syndrome (SuS), an enigmatic neuroinflammatory disease with central nervous system (CNS) endotheliopathy. By investigating immune cells from the blood, cerebrospinal fluid, and CNS of SuS patients, we demonstrate oligoclonal expansion of terminally differentiated activated cytotoxic CD8+ T cells (CTLs). Neuropathological data derived from both SuS patients and a newly-developed transgenic mouse model recapitulating the disease indicate that CTLs adhere to CNS microvessels in distinct areas and polarize granzyme B, which most likely results in the observed endothelial cell injury and microhemorrhages. Blocking T-cell adhesion by anti-α4 integrin-intervention ameliorates the disease in the preclinical model. Similarly, disease severity decreases in four SuS patients treated with natalizumab along with other therapy. Our study identifies CD8+ T-cell-mediated endotheliopathy as a key disease mechanism in SuS and highlights therapeutic opportunities., Susac syndrome is an inflammatory pathology of the brain endothelium. Here the authors show that the pathology is driven by CD8 T cells attacking the endothelium, and that blocking T cell-endothelial adhesion ameliorates the disease in a mouse model, and associates with improved clinical score in 4 patients.

Central nervous system (CNS) inflammation occurs in a large number of neurological diseases. The type and magnitude of CNS inflammation, as well as the T-cell contribution, vary depending on the disease. Different animal models of neurological diseases have shown that T cells play an important role in CNS inflammation. Furthermore, recent studies of human neurological disorders have indicated a significant role for T cells in disease pathology. Nevertheless, how individual T-cell subsets affect neuronal survival, damage and/or loss remains largely unclear. In this review we discuss the processes by which T cells mediate either beneficial or deleterious effects within the CNS, with emphasis on the direct interaction between T cells and neurons, as occurs in multiple sclerosis, paraneoplastic cerebellar degeneration, and viral encephalitis. The therapeutic approaches targeting T cells and their mediators as treatment for neurological diseases are also described here.