Tregs Attenuate Peripheral Oxidative Stress and Acute Phase Proteins in ALS.

Oxidative stress (OS) induces inflammation, which in turn exacerbates OS and the expression of acute phase proteins (APPs). Regulatory T lymphocyte (Treg) therapy was assessed for suppression of OS and APP responses in longitudinal serum samples from subjects with amyotrophic lateral sclerosis (ALS) enrolled in a phase I clinical trial. The first round of Treg therapy suppressed levels of oxidized low-density lipoprotein (ox-LDL). During a 6-month washout period, ox-LDL levels increased. A second round of therapy again suppressed ox-LDL levels and then rose following the cessation of treatment. Serum levels of APPs, soluble CD14, lipopolysaccharide binding protein, and C-reactive protein, were stabilized during Treg administrations, but rose during the washout period and again after therapy was discontinued. Treg therapy potentially suppresses peripheral OS and the accompanying circulating pro-inflammatory induced APPs, both of which may serve as peripheral candidates for monitoring efficacies of immunomodulating therapies. ANN NEUROL 2022;92:195-200.

Amyotrophic lateral sclerosis is a systemic disease: peripheral contributions to inflammation-mediated neurodegeneration.

PURPOSE OF REVIEW: Neuroinflammation is an important mediator of the pathogenesis of disease in amyotrophic lateral sclerosis (ALS). Genetic mutations such as C9orf72 have begun to define the numerous cell autonomous pathways that initiate motor neuron injury. Yet, it is the signalling to surrounding glia and peripherally derived immune cells that initiates the noncell autonomous inflammatory process and promotes self-propagating motor neuron cell death. The purpose of this review is to explore the systemic immune/inflammatory contributions to the pathogenesis of ALS: what are the peripheral pro-inflammatory signatures, what initiates their presence and do they represent potential therapeutic targets. RECENT FINDINGS: In ALS, motor neuron cell death is initiated by multiple cell autonomous pathways leading to misfolded proteins, oxidative stress, altered mitochondria, impaired autophagy and altered RNA metabolism, which collectively promote noncell autonomous inflammatory reactivity. The resulting disease is characterized by activated microglia and astrocytes as well as peripherally derived pro-inflammatory innate and adaptive immune cells. In this unrelenting disorder, circulating blood monocytes and natural killer cells are pro-inflammatory. Furthermore, regulatory T lymphocytes are dysfunctional, and pro-inflammatory cytokines and acute phase proteins are elevated. SUMMARY: The collective dysregulation of cells and cytokines in patients with ALS accurately reflect increased disease burdens, more rapid progression rates and reduced survival times, reinforcing the concept of ALS as a disorder with extensive systemic pro-inflammatory responses. These increased systemic pro-inflammatory immune constituents provide potentially meaningful therapeutic targets.

A robust, good manufacturing practice-compliant, clinical-scale procedure to generate regulatory T cells from patients with amyotrophic lateral sclerosis for adoptive cell therapy.

Regulatory T cells (Tregs) play a fundamental role in the maintenance of self-tolerance and immune homeostasis. Defects in Treg function and/or frequencies have been reported in multiple disease models. Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder affecting upper and lower motor neurons. Compelling evidence supports a neuroprotective role for Tregs in this disease. Indeed, rapid progression in ALS patients is associated with decreased FoxP3 expression and Treg frequencies. Thus, we propose that strategies to restore Treg number and function may slow disease progression in ALS. In this study, we developed a robust, Good Manufacturing Practice (GMP)-compliant procedure to enrich and expand Tregs from ALS patients. Tregs isolated from these patients were phenotypically similar to those from healthy individuals but were impaired in their ability to suppress T-cell effector function. In vitro expansion of Tregs for 4 weeks in the presence of GMP-grade anti-CD3/CD28 beads, interleukin (IL)-2 and rapamcyin resulted in a 25- to 200-fold increase in their number and restored their immunoregulatory activity. Collectively, our data facilitate and support the implementation of clinical trials of adoptive therapy with ex vivo expanded and highly suppressive Tregs in patients with ALS.

A phase 1 proof-of-concept study evaluating safety, tolerability, and biological marker responses with combination therapy of CTLA4-Ig and interleukin-2 in amyotrophic lateral sclerosis.

Objective: To determine whether a combination therapy with abatacept (CTLA4-Ig) and interleukin-2 (IL-2) is safe and suppresses markers of oxidative stress, inflammation, and degeneration in ALS. Methods: In this open-label study, four participants with ALS received subcutaneous injections of low dose IL-2 (1 × 106 IU/injection/day) for 5 consecutive days every 2 weeks and one subcutaneous injection of CTLA4-Ig (125 mg/mL/injection) every 2 weeks coinciding with the first IL-2 injection of each treatment cycle. Participants received a total of 24 treatment cycles during the first 48 weeks in this 56-week study. They were closely monitored for treatment-emergent adverse events (TEAEs) and disease progression with the ALSFRS-R. Phenotypic changes within T cell populations and serum biological markers of oxidative stress [4-hydroxynonenal (4-HNE) and oxidized-LDL (ox-LDL)], inflammation (IL-18), and structural neuronal degeneration [neurofilament light chain (Nf-L)] were assessed longitudinally. Results: CTLA4-Ig/IL-2 therapy was safe and well-tolerated in all four participants over the 56-week study. During the first 24 weeks, the average rate of change in the ALSFRS-R was +0.04 points/month. Over the 48-week treatment period, the average rate of change was -0.13 points/month with one participant improving by 0.9 points/month while the other three participants experienced an average decrease of -0.47 points/month, which is slower than the average - 1.1 points/month prior to initiation of therapy. Treg suppressive function and numbers increased during treatment. Responses in the biological markers during the first 16 weeks coincided with minimal clinical progression. Mean levels of 4-HNE decreased by 30%, ox-LDL decreased by 19%, IL-18 decreased by 23%, and Nf-L remained the same, on average, in all four participants. Oxidized-LDL levels decreased in all four participants, 4-HNE and IL-18 levels decreased in three out of four participants, and Nf-L decreased in two out of four participants. Conclusion: The combination therapy of CTLA4-Ig and IL-2 in ALS is safe and well-tolerated with promising results of clinical efficacy and suppression of biomarkers of oxidative stress, neuroinflammation and neuronal degeneration. In this open-label study, the efficacy as measured by the ALSFRS-R and corresponding biomarkers suggests the therapeutic potential of this treatment and warrants further study in a phase 2 double-blind, placebo-controlled trial. Clinical trial registration: ClinicalTrials.gov, NCT06307301.

T cell-microglial dialogue in Parkinson's disease and amyotrophic lateral sclerosis: are we listening?

Neuroinflammation is a pathological hallmark in Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS), and is characterized by activated microglia and infiltrating T cells at sites of neuronal injury. In PD and ALS, neurons do not die alone; neuronal injury is non-cell-autonomous and depends on a well-orchestrated dialogue in which neuronally secreted misfolded proteins activate microglia and initiate a self-propagating cycle of neurotoxicity. Diverse populations and phenotypes of CD4(+) T cells crosstalk with microglia, and depending on their activation status, influence this dialogue and promote neuroprotection or neurotoxicity. A greater understanding of the T cell population that mediates these effects, as well as the molecular signals involved should provide targets for neuroprotective immunomodulation to treat these devastating neurodegenerative diseases.

A phase 1 open-label pilot study of low-dose interleukine-2 immunotherapy in patients with Alzheimer's disease.

TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT05821153, Registered April 20 2023, Retrospectively registered, https://classic. CLINICALTRIALS: gov/ct2/show/NCT05821153.

Regulatory T lymphocytes from ALS mice suppress microglia and effector T lymphocytes through different cytokine-mediated mechanisms.

Activated microglia and infiltrating lymphocytes are neuropathological hallmarks of amyotrophic lateral sclerosis (ALS), a fatal motoneuron disease. Although both cell types play pivotal roles in the ALS pathogenic process, the interactions between microglia and lymphocytes, specifically regulatory CD4+CD25High T lymphocytes (Tregs) and cytotoxic CD4+CD25- T lymphocytes (Teffs), have not been addressed. When co-cultured with mSOD1 adult microglia, mSOD1 Tregs suppressed the cytotoxic microglial factors NOX2 and iNOS through an IL-4-mediated mechanism, whereas Teffs were only minimally effective; IL-4 inhibitory antibodies blocked the suppressive function of mSOD1 Tregs, and conditioned media from mSOD1 Tregs or the addition of IL-4 reduced microglial NOX2 expression. During the stable disease phase, the total number of Tregs, specifically the numbers of CD4+CD25HighIL-4+, CD4+CD25HighIL-10+ and CD4+CD25HighTGF-ß+ Tregs, were increased in ALS mice compared with WT mice; Tregs isolated during this phase reduced Teff proliferation. In contrast, during the rapidly progressing phase, the number of mSOD1 Tregs decreased while the proliferation of mSOD1 Teffs increased. The combination of IL-4, IL-10, and TGF-ß was required to inhibit the proliferation of mSOD1 Teffs by mSOD1 Tregs that were isolated during the slow phase, while inhibition of mSOD1 Teffs by mSOD1 Tregs during the rapid phase, as well as WT Teffs, was not dependent on these factors. Thus, mSOD1 Tregs at the slow phase suppressed microglial toxicity and SOD1 Teff proliferation through different mechanisms; microglial activation was suppressed through IL-4 whereas mSOD1 Teffs were suppressed by IL-4, IL-10 and TGF-ß. These data suggest that mSOD1 Tregs contribute to the slowly progressing phase in ALS mice and may offer a novel therapeutic option for ALS patients.

Endogenous regulatory T lymphocytes ameliorate amyotrophic lateral sclerosis in mice and correlate with disease progression in patients with amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis is a relentless and devastating adult-onset neurodegenerative disease with no known cure. In mice with amyotrophic lateral sclerosis, CD4+ T lymphocytes and wild-type microglia potentiate protective inflammatory responses and play a principal role in disease pathoprogression. Using this model, we demonstrate that endogenous T lymphocytes, and more specifically regulatory T lymphocytes, are increased at early slowly progressing stages, augmenting interleukin-4 expression and protective M2 microglia, and are decreased when the disease rapidly accelerates, possibly through the loss of FoxP3 expression in the regulatory T lymphocytes. Without ex vivo activation, the passive transfer of wild-type CD4+ T lymphocytes into amyotrophic lateral sclerosis mice lacking functional T lymphocytes lengthened disease duration and prolonged survival. The passive transfer of endogenous regulatory T lymphocytes from early disease stage mutant Cu2+/Zn2+ superoxide dismutase mice into these amyotrophic lateral sclerosis mice, again without ex vivo activation, were substantially more immunotherapeutic sustaining interleukin-4 levels and M2 microglia, and resulting in lengthened disease duration and prolonged survival; the stable disease phase was extended by 88% using mutant Cu2+/Zn2+ superoxide dismutase regulatory T lymphocytes. A potential mechanism for this enhanced life expectancy may be mediated by the augmented secretion of interleukin-4 from mutant Cu2+/Zn2+ superoxide dismutase regulatory T lymphocytes that directly suppressed the toxic properties of microglia; flow cytometric analyses determined that CD4+/CD25+/FoxP3+ T lymphocytes co-expressed interleukin-4 in the same cell. These observations were extended into the amyotrophic lateral sclerosis patient population where patients with more rapidly progressing disease had decreased numbers of regulatory T lymphocytes; the numbers of regulatory T lymphocytes were inversely correlated with disease progression rates. These data suggest a cellular mechanism whereby endogenous regulatory T lymphocytes are immunocompetent and actively contribute to neuroprotection through their interactions with microglia. Furthermore, these data suggest that immunotherapeutic interventions must begin early in the pathogenic process since immune dysfunction occurs at later stages. Thus, the cumulative mouse and human amyotrophic lateral sclerosis data suggest that increasing the levels of regulatory T lymphocytes in patients with amyotrophic lateral sclerosis at early stages in the disease process may be of therapeutic value, and slow the rate of disease progression and stabilize patients for longer periods of time.

Extracellular Vesicles Derived From Ex Vivo Expanded Regulatory T Cells Modulate In Vitro and In Vivo Inflammation.

Extracellular vehicles (EVs) are efficient biomarkers of disease and participate in disease pathogenesis; however, their use as clinical therapies to modify disease outcomes remains to be determined. Cell-based immune therapies, including regulatory T cells (Tregs), are currently being clinically evaluated for their usefulness in suppressing pro-inflammatory processes. The present study demonstrates that ex vivo expanded Tregs generate a large pool of EVs that express Treg-associated markers and suppress pro-inflammatory responses in vitro and in vivo. Intravenous injection of Treg EVs into an LPS-induced mouse model of inflammation reduced peripheral pro-inflammatory transcripts and increased anti-inflammatory transcripts in myeloid cells as well as Tregs. Intranasal administration of enriched Treg EVs in this model also reduced pro-inflammatory transcripts and the associated neuroinflammatory responses. In a mouse model of amyotrophic lateral sclerosis, intranasal administration of enriched Treg EVs slowed disease progression, increased survival, and modulated inflammation within the diseased spinal cord. These findings support the therapeutic potential of expanded Treg EVs to suppress pro-inflammatory responses in human disease.

Combined Regulatory T-Lymphocyte and IL-2 Treatment Is Safe, Tolerable, and Biologically Active for 1 Year in Persons With Amyotrophic Lateral Sclerosis.

BACKGROUND AND OBJECTIVES: In a phase 1 amyotrophic lateral sclerosis (ALS) study, autologous infusions of expanded regulatory T-lymphocytes (Tregs) combined with subcutaneous interleukin (IL)-2 were safe and well tolerated. Treg suppressive function increased and disease progression stabilized during the study. The present study was conducted to confirm the reliability of these results. METHODS: Participants with ALS underwent leukapheresis, and their Tregs were isolated and expanded in a current Good Manufacturing Practice facility. Seven participants were randomly assigned in a 1:1 ratio to receive Treg infusions (1 × 106 cells/kg) IV every 4 weeks and IL-2 (2 × 105 IU/m2) injections 3 times/wk or matching placebo in a 24-week randomized controlled trial (RCT). Six participants proceeded into a 24-week dose-escalation open-label extension (OLE). Two additional participants entered directly into the OLE. The OLE included dose escalation of Treg infusions to 2 × 106 cells/kg and 3 × 106 cells/kg at 4-week intervals. RESULTS: The Treg/IL-2 treatments were safe and well tolerated, and Treg suppressive function was higher in the active group of the RCT. A meaningful evaluation of progression rates in the RCT between the placebo and active groups was not possible due to the limited number of enrolled participants aggravated by the COVID-19 pandemic. In the 24-week OLE, the Treg/IL-2 treatments were also safe and well tolerated in 8 participants who completed the escalating doses. Treg suppressive function and numbers were increased compared with baseline. Six of 8 participants changed by an average of -2.7 points per the ALS Functional Rating Scale-Revised, whereas the other 2 changed by an average of -10.5 points. Elevated levels of 2 markers of peripheral inflammation (IL-17C and IL-17F) and 2 markers of oxidative stress (oxidized low-density lipoprotein receptor 1 and oxidized LDL) were present in the 2 rapidly progressing participants but not in the slower progressing group. DISCUSSION: Treg/IL-2 treatments were safe and well tolerated in the RCT and OLE with higher Treg suppressive function. During the OLE, 6 of 8 participants showed slow to no progression. The 2 of 8 rapid progressors had elevated markers of oxidative stress and inflammation, which may help delineate responsiveness to therapy. Whether Treg/IL-2 treatments can slow disease progression requires a larger clinical study (ClinicalTrials.gov number, NCT04055623). CLASSIFICATION OF EVIDENCE: This study provides Class IV evidence that Treg infusions and IL-2 injections are safe and effective for patients with ALS.

Ex vivo expanded human regulatory T cells modify neuroinflammation in a preclinical model of Alzheimer's disease.

BACKGROUND: Regulatory T cells (Tregs) play a neuroprotective role by suppressing microglia and macrophage-mediated inflammation and modulating adaptive immune reactions. We previously documented that Treg immunomodulatory mechanisms are compromised in Alzheimer's disease (AD). Ex vivo expansion of Tregs restores and amplifies their immunosuppressive functions in vitro. A key question is whether adoptive transfer of ex vivo expanded human Tregs can suppress neuroinflammation and amyloid pathology in a preclinical mouse model. METHODS: An immunodeficient mouse model of AD was generated by backcrossing the 5xFAD onto Rag2 knockout mice (5xFAD-Rag2KO). Human Tregs were expanded ex vivo for 24 days and administered to 5xFAD-Rag2KO. Changes in amyloid burden, microglia characteristics and reactive astrocytes were evaluated using ELISA and confocal microscopy. NanoString Mouse AD multiplex gene expression analysis was applied to explore the impact of ex vivo expanded Tregs on the neuroinflammation transcriptome. RESULTS: Elimination of mature B and T lymphocytes and natural killer cells in 5xFAD-Rag2KO mice was associated with upregulation of 95 inflammation genes and amplified number of reactive microglia within the dentate gyrus. Administration of ex vivo expanded Tregs reduced amyloid burden and reactive glial cells in the dentate gyrus and frontal cortex of 5xFAD-Rag2KO mice. Interrogation of inflammation gene expression documented down-regulation of pro-inflammatory cytokines (IL1A&B, IL6), complement cascade (C1qa, C1qb, C1qc, C4a/b), toll-like receptors (Tlr3, Tlr4 and Tlr7) and microglial activations markers (CD14, Tyrobp,Trem2) following Treg administration. CONCLUSIONS: Ex vivo expanded Tregs with amplified immunomodulatory function, suppressed neuroinflammation and alleviated AD pathology in vivo. Our results provide preclinical evidences for Treg cell therapy as a potential treatment strategy in AD.

Neuroinflammation modulates distinct regional and temporal clinical responses in ALS mice.

An inflammatory response is a pathological hallmark of amyotrophic lateral sclerosis (ALS), a relentless and devastating degenerative disease of motoneurons. This response is not simply a late consequence of motoneuron degeneration, but actively contributes to the balance between neuroprotection and neurotoxicity; initially infiltrating lymphocytes and microglia slow disease progression, while later, they contribute to the acceleration of disease. Since motor weakness begins in the hindlimbs of ALS mice and only later involves the forelimbs, we determined whether differential protective versus injurious inflammatory responses in the cervical and lumbar spinal cords explained the temporally distinct clinical disease courses between the limbs of these mice. Densitometric evaluation of immunohistochemical sections and quantitative RT-PCR (qRT-PCR) demonstrated that CD68 and CD11c were differentially increased in their spinals cords. qRT-PCR revealed that protective and anti-inflammatory factors, including BDNF, GDNF, and IL-4, were increased in the cervical region compared with the lumbar region. In contrast, the toxic markers TNF-α, IL-1ß and NOX2 were not different between ALS mice cervical and lumbar regions. T lymphocytes were observed infiltrating lumbar spinal cords of ALS mice prior to the cervical region; mRNA levels of the transcription factor gata-3 (Th2 response) were differentially elevated in the cervical cord of ALS mice whereas t-bet (Th1 response) was increased in the lumbar cord. These results reinforce the important balance between specific protective/injurious inflammatory immune responses in modulating clinical outcomes and suggest that the delayed forelimb motor weakness in ALS mice is partially explained by augmented protective responses in the cervical spinal cords.

CD4+ T cells support glial neuroprotection, slow disease progression, and modify glial morphology in an animal model of inherited ALS.

Neuroinflammation, marked by gliosis and infiltrating T cells, is a prominent pathological feature in diverse models of dominantly inherited neurodegenerative diseases. Recent evidence derived from transgenic mice ubiquitously overexpressing mutant Cu(2+)/Zn(2+) superoxide dismutase (mSOD1), a chronic neurodegenerative model of inherited amyotrophic lateral sclerosis (ALS), indicates that glia with either a lack of or reduction in mSOD1 expression enhance motoneuron protection and slow disease progression. However, the contribution of T cells that are present at sites of motoneuron injury in mSOD1 transgenic mice is not known. Here we show that when mSOD1 mice were bred with mice lacking functional T cells or CD4+ T cells, motoneuron disease was accelerated, accompanied by unexpected attenuated morphological markers of gliosis, increased mRNA levels for proinflammatory cytokines and NOX2, and decreased levels of trophic factors and glial glutamate transporters. Bone marrow transplants reconstituted mice with T cells, prolonged survival, suppressed cytotoxicity, and restored glial activation. These results demonstrate for the first time in a model of chronic neurodegeneration that morphological activation of microglia and astroglia does not predict glial function, and that the presence of CD4+ T cells provides supportive neuroprotection by modulating the trophic/cytotoxic balance of glia. These glial/T-cell interactions establish a novel target for therapeutic intervention in ALS and possibly other neurodegenerative diseases.

Serum programmed cell death proteins in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a multifactorial, multisystem pro-inflammatory neuromuscular disorder. Activation of programmed cell death-1 (PD-1), and its ligands, programmed cell death-ligand 1 and 2 (PD-L1/L2), leads to immune suppression. Serum soluble forms of these proteins, sPD-1/sPD-L1/sPD-L2, inhibit this suppression and promote pro-inflammatory responses. The purpose of this study was to determine if sPD-1, sPD-L1, and sPD-L2 were increased in sera of patients with ALS. sPD-1 and sPD-L2 were elevated in sera of patients and accurately reflected patients' disease burdens. Increased sera levels of programmed cell death proteins reinforce the concept that peripheral pro-inflammatory responses contribute to systemic inflammation in patients with ALS.

Ex vivo expansion of dysfunctional regulatory T lymphocytes restores suppressive function in Parkinson's disease.

Inflammation is a pathological hallmark of Parkinson's disease (PD). Chronic pro-inflammatory responses contribute to the loss of neurons in the neurodegenerative process. The present study was undertaken to define the peripheral innate and adaptive immune contributions to inflammation in patients with PD. Immunophenotyping revealed a shift of peripheral myeloid and lymphoid cells towards a pro-inflammatory phenotype. Regulatory T cells (Tregs) were reduced in number, and their suppression of T responder proliferation decreased. The PD Tregs did not suppress activated pro-inflammatory myeloid cells. Ex vivo expansion of Tregs from patients with PD restored and enhanced their suppressive functions while expanded Tregs displayed increased expression of foxp3, il2ra (CD25), nt5e (CD73), il10, il13, ctla4, pdcd1 (PD1), and gzmb. Collectively, these findings documented a shift towards a pro-inflammatory peripheral immune response in patients with PD; the loss of Treg suppressive functions may contribute significantly to this response, supporting PD as a disorder with extensive systemic pro-inflammatory responses. The restoration and enhancement of Treg suppressive functions following ex vivo expansion may provide a potential cell therapeutic approach for patients with PD.

Extracellular mutant SOD1 induces microglial-mediated motoneuron injury.

Through undefined mechanisms, dominant mutations in (Cu/Zn) superoxide dismutase-1 (mSOD1) cause the non-cell-autonomous death of motoneurons in inherited amyotrophic lateral sclerosis (ALS). Microgliosis at sites of motoneuron injury is a neuropathological hallmark of ALS. Extracellular mutant SOD1 (mSOD1) causes motoneuron injury and triggers microgliosis in spinal cord cultures, but it is unclear whether the injury results from extracellular mSOD1 directly interacting with motoneurons or is mediated through mSOD1-activated microglia. To dissociate these potential mSOD1-mediated neurotoxic mechanisms, the effects of extracellular human mSOD1(G93A) or mSOD1(G85R) were assayed using primary cultures of motoneurons and microglia. The data demonstrate that exogenous mSOD1(G93A) did not cause detectable direct killing of motoneurons. In contrast, mSOD1(G93A) or mSOD1(G85R) did induce the morphological and functional activation of microglia, increasing their release of pro-inflammatory cytokines and free radicals. Furthermore, only when microglia was co-cultured with motoneurons did extracellular mSOD1(G93A) injure motoneurons. The microglial activation mediated by mSOD1(G93A) was attenuated using toll-like receptors (TLR) 2, TLR4 and CD14 blocking antibodies, or when microglia lacked CD14 expression. These data suggest that extracellular mSOD1(G93A) is not directly toxic to motoneurons but requires microglial activation for toxicity, utilizing CD14 and TLR pathways. This link between mSOD1 and innate immunity may offer novel therapeutic targets in ALS.

Restoring regulatory T-cell dysfunction in Alzheimer's disease through ex vivo expansion.

Inflammation is a significant component of Alzheimer's disease pathology. While neuroprotective microglia are important for containment/clearance of Amyloid plaques and maintaining neuronal survival, Alzheimer inflammatory microglia may play a detrimental role by eliciting tau pathogenesis and accelerating neurotoxicity. Regulatory T cells have been shown to suppress microglia-mediated inflammation. However, the role of regulatory T cells in ameliorating the proinflammatory immune response in Alzheimer's disease requires further investigation. Forty-six patients with Alzheimer disease, 42 with mild cognitive impairment and 41 healthy controls were studied. The phenotypes of peripheral regulatory T cells were assessed with multicolour flow cytometry. Regulatory T cells were co-cultured with responder T cells and proliferation was determined by 3H-thymidine incorporation. In separate experiments, regulatory T cells were added to induced pluripotent stem cell-derived pro-inflammatory macrophages and changes in interleukin-6/tumour necrosis-alpha transcripts and protein levels were measured. Freshly isolated regulatory T cells were expanded ex vivo in the presence of CD3/CD28 expander beads, interleukin-2 and rapamycin to promote their suppressive function. We found that the suppressive function of regulatory T cells on responder T-cell proliferation was compromised at the Alzheimer disease stage, compared with mild cognitive impairment and healthy controls. CD25 mean fluorescence intensity in regulatory T-cell population was also reduced in Alzheimer dementia patients. Regulatory T cells did not suppress pro-inflammatory macrophages at baseline. Following ex vivo expansion, regulatory T-cell suppression of responder T-cell proliferation and pro-inflammatory macrophage activation increased in both patients and controls. Expanded regulatory T cells exerted their immunoregulatory function on pro-inflammatory macrophages through a contact-mediated mechanism. In conclusion, regulatory T-cell immunophenotype and function are compromised in Alzheimer's disease. Following ex vivo expansion, the immunomodulatory function of regulatory T cells is enhanced even at advanced stages of Alzheimer's disease. Restoration of regulatory T-cell function could be explored as a means to modulate the inflammatory status of Alzheimer's disease.

Elevated acute phase proteins reflect peripheral inflammation and disease severity in patients with amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a multifactorial, multisystem pro-inflammatory neuromuscular disorder compromising muscle function resulting in death. Neuroinflammation is known to accelerate disease progression and accentuate disease severity, but peripheral inflammatory processes are not well documented. Acute phase proteins (APPs), plasma proteins synthesized in the liver, are increased in response to inflammation. The objective of this study was to provide evidence for peripheral inflammation by examining levels of APPs, and their contribution to disease burden and progression rates. Levels of APPs, including soluble CD14 (sCD14), lipopolysaccharide binding protein (LBP), and C-reactive protein (CRP), were elevated in sera, and correlated positively with increased disease burden and faster progression. sCD14 was also elevated in patients' CSF and urine. After a 3 year follow-up, 72% of the patients with sCD14 levels above the receiver operating characteristics cutoff were deceased whereas only 28% below the cutoff were deceased. Furthermore, disease onset sites were associated with disease progression rates and APP levels. These APPs were not elevated in sera of patients with Alzheimer's Disease, frontotemporal dementia, or Parkinson's Disease. These collective APPs accurately reflect disease burden, progression rates, and survival times, reinforcing the concept of ALS as a disorder with extensive systemic pro-inflammatory responses.

Immunosuppressive Functions of M2 Macrophages Derived from iPSCs of Patients with ALS and Healthy Controls.

Amyotrophic lateral sclerosis (ALS) is a disorder with immune alterations that augment disease severity. M2 macrophages benefit diabetic and nephrotic mice by suppressing the pro-inflammatory state. However, neither have M2 cells been investigated in ALS nor have human induced pluripotent stem cell (iPSC)-derived M2 cells been thoroughly studied for immunosuppressive potentials. Here, iPSCs of C9orf72 mutated or sporadic ALS patients were differentiated into M2 macrophages, which suppressed activation of pro-inflammatory M1 macrophages as well as proliferation of ALS CD4+CD25- Tc (Teffs). M2 cells converted ALS Teffs into CD4+CD25+Foxp3+ regulatory T cells (Tregs) and rescued Tregs of ALS patients from losing CD25 and Foxp3. Furthermore, Tregs induced or rescued by iPSC-derived M2 had strong suppressive functions. ALS iPSC-derived M2 cells including those with C9orf72 mutation had similar immunomodulatory activity as control iPSC-derived M2 cells. This study demonstrates that M2 cells differentiated from iPSCs of ALS patients are immunosuppressive, boost ALS Tregs, and may serve as a candidate for immune-cell-based therapy to mitigate inflammation in ALS.

Immune dysregulation in amyotrophic lateral sclerosis: mechanisms and emerging therapies.

Neuroinflammation is a common pathological feature of many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), and is characterised by activated CNS microglia and astroglia, proinflammatory peripheral lymphocytes, and macrophages. Data from clinical studies show that multiple genetic mutations linked to ALS (eg, mutations in SOD1, TARDBP, and C9orf72) enhance this neuroinflammation, which provides compelling evidence for immune dysregulation in the pathogenesis of ALS. Transgenic rodent models expressing these mutations induce an ALS-like disease with accompanying inflammatory responses, confirming the immune system's involvement in disease progression. Even in the absence of known genetic alterations, immune dysregulation has been shown to lead to dysfunctional regulatory T lymphocytes and increased proinflammatory macrophages in clinical studies. Therefore, an improved understanding of the biological processes that induce this immune dysregulation will help to identify therapeutic strategies that circumvent or ameliorate the pathogenesis of ALS. Emerging cell-based therapies hold the promise of accomplishing this goal and, therefore, improving quality of life and extending survival in patients with ALS.