Amyloid-ß specific regulatory T cells attenuate Alzheimer's disease pathobiology in APP/PS1 mice.

BACKGROUND: Regulatory T cells (Tregs) maintain immune tolerance. While Treg-mediated neuroprotective activities are now well-accepted, the lack of defined antigen specificity limits their therapeutic potential. This is notable for neurodegenerative diseases where cell access to injured brain regions is required for disease-specific therapeutic targeting and improved outcomes. To address this need, amyloid-beta (Aß) antigen specificity was conferred to Treg responses by engineering the T cell receptor (TCR) specific for Aß (TCRAß). The TCRAb were developed from disease-specific T cell effector (Teff) clones. The ability of Tregs expressing a transgenic TCRAß (TCRAß -Tregs) to reduce Aß burden, transform effector to regulatory cells, and reverse disease-associated neurotoxicity proved beneficial in an animal model of Alzheimer's disease. METHODS: TCRAß -Tregs were generated by CRISPR-Cas9 knockout of endogenous TCR and consequent incorporation of the transgenic TCRAb identified from Aß reactive Teff monoclones. Antigen specificity was confirmed by MHC-Aß-tetramer staining. Adoptive transfer of TCRAß-Tregs to mice expressing a chimeric mouse-human amyloid precursor protein and a mutant human presenilin-1 followed measured behavior, immune, and immunohistochemical outcomes. RESULTS: TCRAß-Tregs expressed an Aß-specific TCR. Adoptive transfer of TCRAß-Tregs led to sustained immune suppression, reduced microglial reaction, and amyloid loads. 18F-fluorodeoxyglucose radiolabeled TCRAß-Treg homed to the brain facilitating antigen specificity. Reduction in amyloid load was associated with improved cognitive functions. CONCLUSIONS: TCRAß-Tregs reduced amyloid burden, restored brain homeostasis, and improved learning and memory, supporting the increased therapeutic benefit of antigen specific Treg immunotherapy for AD.

An open-label multiyear study of sargramostim-treated Parkinson's disease patients examining drug safety, tolerability, and immune biomarkers from limited case numbers.

BACKGROUND: The clinical utility and safety of sargramostim has previously been reported in cancer, acute radiation syndrome, autoimmune disease, inflammatory conditions, and Alzheimer's disease. The safety, tolerability, and mechanisms of action in Parkinson's disease (PD) during extended use has not been evaluated. METHODS: As a primary goal, safety and tolerability was assessed in five PD patients treated with sargramostim (Leukine®, granulocyte-macrophage colony-stimulating factor) for 33 months. Secondary goals included numbers of CD4+ T cells and monocytes and motor functions. Hematologic, metabolic, immune, and neurological evaluations were assessed during a 5-day on, 2-day off therapeutic regimen given at 3 µg/kg. After 2 years, drug use was discontinued for 3 months. This was then followed by an additional 6 months of treatment. RESULTS: Sargramostim-associated adverse events included injection-site reactions, elevated total white cell counts, and bone pain. On drug, blood analyses and metabolic panels revealed no untoward side effects linked to long-term treatment. Unified Parkinson's Disease Rating Scale scores remained stable throughout the study while regulatory T cell number and function were increased. In the initial 6 months of treatment, transcriptomic and proteomic monocyte tests demonstrated autophagy and sirtuin signaling. This finding paralleled anti-inflammatory and antioxidant activities within both the adaptive and innate immune profile arms. CONCLUSIONS: Taken together, the data affirmed long-term safety as well as immune and anti-inflammatory responses reflecting clinical stability in PD under the sargramostim treatment. Confirmation in larger patient populations is planned in a future phase II evaluation. TRIAL REGISTRATION: ClinicalTrials.gov: NCT03790670, Date of Registration: 01/02/2019, URL: https://clinicaltrials.gov/ct2/show/NCT03790670?cond=leukine+parkinson%27s&draw=2&rank=2 .

Development of an extended half-life GM-CSF fusion protein for Parkinson's disease.

Transformation of CD4+ T cell effector to regulatory (Teff to Treg) cells have been shown to attenuate disease progression by restoring immunological balance during the onset and progression of neurodegenerative diseases. In our prior studies, we defined a safe and effective pathway to restore this balance by restoring Treg numbers and function through the daily administration of the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF). These studies were conducted as a proof-of-concept testing in Parkinson's disease (PD) preclinical models and early phase I clinical investigations. In both instances, they served to ameliorate disease associated signs and symptoms. However, despite the recorded efficacy, the cytokine's short half-life, low bioavailability, and injection site reactions proved to be limitations for any broader use. To overcome these limitations, mRNA lipid nanoparticles encoding an extended half-life albumin-GM-CSF fusion protein were developed for both mouse (Msa-GM-CSF) and rat (Rsa-GM-CSF). These formulations were tested for immunomodulatory and neuroprotective efficacy using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and human wild-type alpha-synuclein (αSyn) overexpression preclinical models of PD. A single dose of the extended half-life mouse and rat mRNA lipid nanoparticles generated measurable GM-CSF plasma cytokine levels up to four days. Increased Treg frequency and function were associated with a resting microglial phenotype, nigrostriatal neuroprotection, and restoration of brain tissue immune homeostasis. These findings were substantively beyond the recorded efficacy of daily recombinant wild-type GM-CSF with a recorded half-life of six hours. Mechanistic evaluation of neuropathological transcriptional profiles performed in the disease-affected nigral brain region demonstrated an upregulation of neuroprotective CREB and synaptogenesis signaling and neurovascular coupling pathways. These findings highlight the mRNA-encoded albumin GM-CSF fusion protein modification linked to improvements in therapeutic efficacy. The improvements achieved were associated with the medicine's increased bioavailability. Taken together, the data demonstrate that mRNA LNP encoding the extended half-life albumin-GM-CSF fusion protein can serve as a benchmark for PD immune-based therapeutics. This is especially notable for improving adherence of drug regimens in a disease-affected patient population with known tremors and gait abnormalities.

CD4+ effector T cells accelerate Alzheimer's disease in mice.

BACKGROUND: Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by pathological deposition of misfolded self-protein amyloid beta (Aß) which in kind facilitates tau aggregation and neurodegeneration. Neuroinflammation is accepted as a key disease driver caused by innate microglia activation. Recently, adaptive immune alterations have been uncovered that begin early and persist throughout the disease. How these occur and whether they can be harnessed to halt disease progress is unclear. We propose that self-antigens would induct autoreactive effector T cells (Teffs) that drive pro-inflammatory and neurodestructive immunity leading to cognitive impairments. Here, we investigated the role of effector immunity and how it could affect cellular-level disease pathobiology in an AD animal model. METHODS: In this report, we developed and characterized cloned lines of amyloid beta (Aß) reactive type 1 T helper (Th1) and type 17 Th (Th17) cells to study their role in AD pathogenesis. The cellular phenotype and antigen-specificity of Aß-specific Th1 and Th17 clones were confirmed using flow cytometry, immunoblot staining and Aß T cell epitope loaded haplotype-matched major histocompatibility complex II IAb (MHCII-IAb-KLVFFAEDVGSNKGA) tetramer binding. Aß-Th1 and Aß-Th17 clones were adoptively transferred into APP/PS1 double-transgenic mice expressing chimeric mouse/human amyloid precursor protein and mutant human presenilin 1, and the mice were assessed for memory impairments. Finally, blood, spleen, lymph nodes and brain were harvested for immunological, biochemical, and histological analyses. RESULTS: The propagated Aß-Th1 and Aß-Th17 clones were confirmed stable and long-lived. Treatment of APP/PS1 mice with Aß reactive Teffs accelerated memory impairment and systemic inflammation, increased amyloid burden, elevated microglia activation, and exacerbated neuroinflammation. Both Th1 and Th17 Aß-reactive Teffs progressed AD pathology by downregulating anti-inflammatory and immunosuppressive regulatory T cells (Tregs) as recorded in the periphery and within the central nervous system. CONCLUSIONS: These results underscore an important pathological role for CD4+ Teffs in AD progression. We posit that aberrant disease-associated effector T cell immune responses can be controlled. One solution is by Aß reactive Tregs.

Safety, tolerability, and immune-biomarker profiling for year-long sargramostim treatment of Parkinson's disease.

BACKGROUND: Neuroinflammation plays a pathogenic role in Parkinson's disease (PD). Immunotherapies that restore brain homeostasis can mitigate neurodegeneration by transforming T cell phenotypes. Sargramostim has gained considerable attention as an immune transformer through laboratory bench to bedside clinical studies. However, its therapeutic use has been offset by dose-dependent adverse events. Therefore, we performed a reduced drug dose regimen to evaluate safety and to uncover novel disease-linked biomarkers during 5 days/week sargramostim treatments for one year. METHODS: Five PD subjects were enrolled in a Phase 1b, unblinded, open-label study to assess safety and tolerability of 3 µg/kg/day sargramostim. Complete blood counts and chemistry profiles, physical examinations, adverse events (AEs), immune profiling, Movement Disorder Society-Sponsored Revision of the Unified Parkinson's Disease Rating Scale (MDS-UPDRS) scores, T cell phenotypes/function, DNA methylation, and gene and protein patterns were evaluated. FINDINGS: Sargramostim administered at 3 µg/kg/day significantly reduced numbers and severity of AEs/subject/month compared to 6 µg/kg/day treatment. While MDS-UPDRS Part III score reductions were recorded, peripheral blood immunoregulatory phenotypes and function were elevated. Hypomethylation of upstream FOXP3 DNA elements was also increased. INTERPRETATION: Long-term sargramostim treatment at 3 µg/kg/day is well-tolerated and effective in restoring immune homeostasis. There were decreased numbers and severity of AEs and restored peripheral immune function coordinate with increased numbers and function of Treg. MDS-UPDRS Part III scores did not worsen. Larger patient numbers need be evaluated to assess conclusive drug efficacy (ClinicalTrials.gov NCT03790670). FUNDING: The research was supported by community funds to the University of Nebraska Foundation and federal research support from 5 R01NS034239-25.

Granulocyte-macrophage colony-stimulating factor mRNA and Neuroprotective Immunity in Parkinson's disease.

Restoring numbers and function of regulatory T cells (Tregs) is a novel therapeutic strategy for neurodegenerative disorders. Whether Treg function is boosted by adoptive cell transfer, pharmaceuticals, or immune modulators, the final result is a robust anti-inflammatory and neuronal sparing response. Herein, a newly developed lipid nanoparticle (LNP) containing mRNA encoding granulocyte-macrophage colony-stimulating factor (Gm-csf mRNA) was developed to peripherally induce Tregs and used for treatment in preclinical Parkinson's disease (PD) models. Administration of Gm-csf mRNA to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice and rats overexpressing alpha-synuclein produced dose-dependent increases in plasma GM-CSF levels and peripheral CD4+CD25+FoxP3+ Treg populations. This upregulation paralleled nigrostriatal neuroprotection, upregulated immunosuppression-associated mRNAs that led to the detection of a treatment-induced CD4+ T cell population, and decreased reactive microgliosis. The current findings strengthen prior works utilizing immune modulation by harnessing Gm-csf mRNA to augment adaptive immune function by employing a new delivery platform to treat PD and potentially other neurodegenerative disorders.

Neuroprotective Activities of Long-Acting Granulocyte-Macrophage Colony-Stimulating Factor (mPDM608) in 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine-Intoxicated Mice.

Loss of dopaminergic neurons along the nigrostriatal axis, neuroinflammation, and peripheral immune dysfunction are the pathobiological hallmarks of Parkinson's disease (PD). Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been successfully tested for PD treatment. GM-CSF is a known immune modulator that induces regulatory T cells (Tregs) and serves as a neuronal protectant in a broad range of neurodegenerative diseases. Due to its short half-life, limited biodistribution, and potential adverse effects, alternative long-acting treatment schemes are of immediate need. A long-acting mouse GM-CSF (mPDM608) was developed through Calibr, a Division of Scripps Research. Following mPDM608 treatment, complete hematologic and chemistry profiles and T-cell phenotypes and functions were determined. Neuroprotective and anti-inflammatory capacities of mPDM608 were assessed in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-intoxicated mice that included transcriptomic immune profiles. Treatment with a single dose of mPDM608 resulted in dose-dependent spleen and white blood cell increases with parallel enhancements in Treg numbers and immunosuppressive function. A shift in CD4+ T-cell gene expression towards an anti-inflammatory phenotype corresponded with decreased microgliosis and increased dopaminergic neuronal cell survival. mPDM608 elicited a neuroprotective peripheral immune transformation. The observed phenotypic shift and neuroprotective response was greater than observed with recombinant GM-CSF (rGM-CSF) suggesting human PDM608 as a candidate for PD treatment.