CD4 T-cell aging exacerbates neuroinflammation in a late-onset mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is an adult-onset progressive neurodegenerative disorder characterized by the loss of upper and lower motor neurons in the brain and spinal cord. Accumulating evidence suggests that ALS is not solely a neuronal cell- or brain tissue-autonomous disease and that neuroinflammation plays a key role in disease progression. Furthermore, whereas both CD4 and CD8 T cells were observed in spinal cords of ALS patients and in mouse models of the disease, their role in the neuroinflammatory process, especially considering their functional changes with age, is not fully explored. In this study, we revealed the structure of the CD4 T-cell compartment during disease progression of early-onset SOD1G93A and late-onset SOD1G37R mouse models of ALS. We show age-related changes in the CD4 T-cell subset organization between these mutant SOD1 mouse models towards increased frequency of effector T cells in spleens of SOD1G37R mice and robust infiltration of CD4 T cells expressing activation markers and the checkpoint molecule PD1 into the spinal cord. The frequency of infiltrating CD4 T cells correlated with the frequency of infiltrating CD8 T cells which displayed a more exhausted phenotype. Moreover, RNA-Seq and immunohistochemistry analyses of spinal cords from SOD1G37R mice with early clinical symptoms demonstrated immunological trajectories reminiscent of a neurotoxic inflammatory response which involved proinflammatory T cells and antigen presentation related pathways. Overall, our findings suggest that age-related changes of the CD4 T cell landscape is indicative of a chronic inflammatory response, which aggravates the disease process and can be therapeutically targeted.

Applying causal discovery to single-cell analyses using CausalCell.

Correlation between objects is prone to occur coincidentally, and exploring correlation or association in most situations does not answer scientific questions rich in causality. Causal discovery (also called causal inference) infers causal interactions between objects from observational data. Reported causal discovery methods and single-cell datasets make applying causal discovery to single cells a promising direction. However, evaluating and choosing causal discovery methods and developing and performing proper workflow remain challenges. We report the workflow and platform CausalCell (http://www.gaemons.net/causalcell/causalDiscovery/) for performing single-cell causal discovery. The workflow/platform is developed upon benchmarking four kinds of causal discovery methods and is examined by analyzing multiple single-cell RNA-sequencing (scRNA-seq) datasets. Our results suggest that different situations need different methods and the constraint-based PC algorithm with kernel-based conditional independence tests work best in most situations. Related issues are discussed and tips for best practices are given. Inferred causal interactions in single cells provide valuable clues for investigating molecular interactions and gene regulations, identifying critical diagnostic and therapeutic targets, and designing experimental and clinical interventions.

Randomised clinical trial: Psychological intervention improves work productivity and daily activity by reducing abdominal pain and fatigue in Crohn's disease.

BACKGROUND: Chronic abdominal pain and fatigue are characteristics of Crohn's disease (CD) and contribute to functional impairments. AIMS: To examine whether CD-tailored cognitive-behavioural and mindfulness intervention (COBMINDEX) is effective in reducing abdominal pain and fatigue in patients with CD and whether changes in abdominal pain and fatigue mediate any beneficial effects of COBMINDEX on impairments in work productivity and daily activities. METHODS: This is a secondary analysis of a parallel-group multicentre randomised controlled trial. Patients with mild-to-moderate CD (n = 142) were randomised into either intervention group receiving COBMINDEX, or control group receiving treatment-as-usual for 3 months followed by COBMINDEX. Complete data were collected from 120 patients (34.0 ± 10.7 years, 62.5% female, intervention = 60, control = 60). Analysis of covariance assessed group differences in 3-month follow-up scores, controlling for baseline scores. Multiple parallel mediation analysis assessed the proposed mechanisms for the entire sample. RESULTS: The intervention group demonstrated significantly lower levels of abdominal pain (F = 17.46, p < 0.001, η2 p  = 0.13), fatigue (F = 7.26, p = 0.008, η2 p  = 0.06) and impairments at work (F = 4.82, p = 0.032, η2 p  = 0.07) and daily activities (F = 6.26, p = 0.014, η2 p  = 0.05), compared with treatment-as-usual. Moreover, changes in abdominal pain and fatigue significantly mediated the beneficial effects of COBMINDEX on patients' work productivity (b = -9.90, SE = 2.86, 95% CI: -16.11 to -4.94) and daily activities (b = -9.65, SE = 1.91, 95% CI: -13.77 to 6.35), independent of changes in disease activity. CONCLUSIONS: COBMINDEX is effective at reducing abdominal pain and fatigue in patients with CD, which in turn leads to improvement in functioning. Clinicians should incorporate screening for severe abdominal pain and fatigue and consider offering cognitive-behavioural and mindfulness training. CLINICALTRIALS: gov, Number: NCT05085925. Ministry of Health in Israel (https://my.health.gov.il/CliniTrials/Pages/MOH_2020-02-24_008721.aspx).

Targeting the overexpressed mitochondrial protein VDAC1 in a mouse model of Alzheimer's disease protects against mitochondrial dysfunction and mitigates brain pathology.

BACKGROUND: Alzheimer's disease (AD) exhibits mitochondrial dysfunctions associated with dysregulated metabolism, brain inflammation, synaptic loss, and neuronal cell death. As a key protein serving as the mitochondrial gatekeeper, the voltage-dependent anion channel-1 (VDAC1) that controls metabolism and Ca2+ homeostasis is positioned at a convergence point for various cell survival and death signals. Here, we targeted VDAC1 with VBIT-4, a newly developed inhibitor of VDAC1 that prevents its pro-apoptotic activity, and mitochondria dysfunction. METHODS: To address the multiple pathways involved in AD, neuronal cultures and a 5 × FAD mouse model of AD were treated with VBIT-4. We addressed multiple topics related to the disease and its molecular mechanisms using immunoblotting, immunofluorescence, q-RT-PCR, 3-D structural analysis and several behavioral tests. RESULTS: In neuronal cultures, amyloid-beta (Aß)-induced VDAC1 and p53 overexpression and apoptotic cell death were prevented by VBIT-4. Using an AD-like 5 × FAD mouse model, we showed that VDAC1 was overexpressed in neurons surrounding Aß plaques, but not in astrocytes and microglia, and this was associated with neuronal cell death. VBIT-4 prevented the associated pathophysiological changes including neuronal cell death, neuroinflammation, and neuro-metabolic dysfunctions. VBIT-4 also switched astrocytes and microglia from being pro-inflammatory/neurotoxic to neuroprotective phenotype. Moreover, VBIT-4 prevented cognitive decline in the 5 × FAD mice as evaluated using several behavioral assessments of cognitive function. Interestingly, VBIT-4 protected against AD pathology, with no significant change in phosphorylated Tau and only a slight decrease in Aß-plaque load. CONCLUSIONS: The study suggests that mitochondrial dysfunction with its gatekeeper VDAC1 is a promising target for AD therapeutic intervention, and VBIT-4 is a promising drug candidate for AD treatment.

Immune aging in multiple sclerosis is characterized by abnormal CD4 T cell activation and increased frequencies of cytotoxic CD4 T cells with advancing age.

BACKGROUND: Immunosenescence (ISC) describes age-related changes in immune-system composition and function. Multiple sclerosis (MS) is a lifelong inflammatory condition involving effector and regulatory T-cell imbalance, yet little is known about T-cell ISC in MS. We examined age-associated changes in circulating T cells in MS compared to normal controls (NC). METHODS: Forty untreated MS (Mean Age 43·3, Range 18-72) and 49 NC (Mean Age 48·6, Range 20-84) without inflammatory conditions were included in cross-sectional design. T-cell subsets were phenotypically and functionally characterized using validated multiparametric flow cytometry. Their aging trajectories, and differences between MS and NC, were determined using linear mixed-effects models. FINDINGS: MS patients demonstrated early and persistent redistribution of naïve and memory CD4 T-cell compartments. While most CD4 and CD8 T-cell aging trajectories were similar between groups, MS patients exhibited abnormal age-associated increases of activated (HLA-DR+CD38+; (P = 0·013) and cytotoxic CD4 T cells, particularly in patients >60 (EOMES: P < 0·001). Aging MS patients also failed to upregulate CTLA-4 expression on both CD4 (P = 0·014) and CD8 (P = 0·009) T cells, coupled with abnormal age-associated increases in frequencies of B cells expressing costimulatory molecules. INTERPRETATION: While many aspects of T-cell aging in MS are conserved, the older MS patients harbour abnormally increased frequencies of CD4 T cells with activated and cytotoxic effector profiles. Age-related decreased expression of T-cell co-inhibitory receptor CTLA-4, and increased B-cell costimulatory molecule expression, may provide a mechanism that drives aberrant activation of effector CD4 T cells that have been implicated in progressive disease. FUNDING: Stated in Acknowledgements section of manuscript.

Brain-immune axis regulation is responsive to cognitive behavioral therapy and mindfulness intervention: Observations from a randomized controlled trial in patients with Crohn's disease.

BACKGROUND AND AIMS: Crohn's disease (CD) is a chronic inflammatory bowel disease associated with psychological stress that is regulated primarily by the hypothalamus-pituitary-adrenal (HPA) axis. Here, we determined whether the psychological characteristics of CD patients associate with their inflammatory state, and whether a 3-month trial of cognitive-behavioral and mindfulness-based stress reduction (COBMINDEX) impacts their inflammatory process. METHODS: Circulating inflammatory markers and a wide range of psychological parameters related to stress and well-being were measured in CD patients before and after COBMINDEX. Inflammatory markers in CD patients were also compared to age- and sex-matched healthy controls (HCs). RESULTS: CD patients exhibited increased peripheral low-grade inflammation compared with HCs, demonstrated by interconnected inflammatory modules represented by IL-6, TNFα, IL-17, MCP-1 and IL-18. Notably, higher IL-18 levels correlated with higher score of stress and a lower score of wellbeing in CD patients. COBMINDEX was accompanied by changes in inflammatory markers that coincided with changes in cortisol: changes in serum levels of cortisol correlated positively with those of IL-10 and IFNα and negatively with those of MCP-1. Furthermore, inflammatory markers of CD patients at baseline predicted COBMINDEX efficacy, as higher levels of distinct cytokines and cortisol at baseline, correlated negatively with changes in disease activity (by Harvey-Bradshaw Index) and psychological distress (global severity index measure) following COBMINDEX. CONCLUSION: CD patients have a characteristic immunological profile that correlates with psychological stress, and disease severity. We suggest that COBMINDEX induces stress resilience in CD patients, which impacts their well-being, and their disease-associated inflammatory process.

Randomized Controlled Trial of Cognitive-Behavioral and Mindfulness-Based Stress Reduction on the Quality of Life of Patients With Crohn Disease.

BACKGROUND: Patients with Crohn disease have debilitating psychological symptoms, mental fatigue, and poor quality of life. Psychological intervention may improve these symptoms. METHODS: We performed a randomized parallel-group physician-blinded trial of cognitive-behavioral and mindfulness-based stress reduction (COBMINDEX) on quality of life and psychological symptoms in adults with mild-moderate Crohn disease. COBMINDEX was taught by social workers in one-on-one video conferences over 3 months; quotidian home practice was mandated. RESULTS: Fifty-five COBMINDEX and 61 waitlist control patients completed the study; mean age was 33 years and 65% of participants were women. At 3 months, COBMINDEX patients had significantly reduced disease activity (per Harvey-Bradshaw Index score, C-reactive protein level, and calprotectin level), increased quality of life (Short Inflammatory Bowel Disease Questionnaire [SIBDQ] score increased from baseline 41 to 50; P < 0.001), decreased psychological symptoms (Global Severity Index [GSI], 0.98-0.70; P < 0.001), reduced fatigue (Functional Assessment of Chronic Illness Therapy-Fatigue, 26-33; P < 0.001), and increased mindfulness disposition (Freiburg Mindfulness Inventory, 33-38; P < 0.001). Waitlist patients had a significant but small change in Harvey-Bradshaw Index, SIBDQ, and GSI scores, without improvement in fatigue or mindfulness. There were significant correlations (0.02 > P < 0.002) in COBMINDEX patients between baseline SIBDQ, GSI, Freiburg Mindfulness Inventory, and Functional Assessment of Chronic Illness Therapy-Fatigue scores with a relative change (baseline to 3 months) of the SIBDQ score, but none among waitlist patients. Predictors of relative change of the SIBDQ score in COBMINDEX patients included the GSI score (90% quantile; coefficient 0.52; P < 0.001), somatization (90%; 0.20; P = 0.001), depression (75%; 0.16; P = 0.03), and phobic anxiety (75%; 0.31; P = 0.008). CONCLUSIONS: COBMINDEX was effective in increasing patients' quality of life and reducing psychological symptoms and fatigue. Patients with severe baseline psychological symptoms benefited the most from COBMINDEX.

An optimized protocol for the retroviral transduction of mouse CD4 T cells.

Transduction of primary T cells has become prominent with the introduction of chimeric antigen receptor T-cell therapy. Although there are many protocols for the transduction of human T cells, it remains a challenge to transduce murine T cells. We present an optimized protocol for the retroviral transduction of murine CD4 T cells, which overcomes major challenges including large-scale production and long-term culturing of transduced cells. The optimized protocol combines high transduction efficiency with a low rate of cell death. For complete details on the use and execution of this protocol, please refer to Eremenko et al., 2019.

An optimized intracerebroventricular injection of CD4+ T cells into mice.

The blood-brain barrier acts as a major barrier for the entrance of most therapeutics into the brain, impeding treatment for neurological disorders. Intracerebroventricular (ICV) injection of T cells is a useful tool for cell therapy of neurological disorders including neurodegenerative and neuropsychiatric diseases and brain tumors. Here, we present an optimized ICV injection of T cells with improved injection efficiency at pathological sites within the brain parenchyma. We describe details of the surgical procedure and verification of injection via immunohistochemistry. For complete details on the use and execution of this protocol, please refer to Fisher et al. (2014); Strominger et al., (2018); Mittal et al. (2019); Eremenko et al. (2019).

Protein kinase C eta is activated in reactive astrocytes of an Alzheimer's disease mouse model: Evidence for its immunoregulatory function in primary astrocytes.

Alzheimer's disease (AD) is the primary cause of age-related dementia. Pathologically, AD is characterized by synaptic loss, the accumulation of ß-amyloid peptides and neurofibrillary tangles, glial activation, and neuroinflammation. Whereas extensive studies focused on neurons and activation of microglia in AD, the role of astrocytes has not been well-characterized. Protein kinase C (PKC) was also implicated in AD; however, its role in astrocyte activation was not elucidated. Using the 5XFAD mouse model of AD, we show that PKC-eta (PKCη), an astrocyte-specific stress-activated and anti-apoptotic kinase, plays a role in reactive astrocytes. We demonstrate that PKCη staining is highly enriched in cortical astrocytes in a disease-dependent manner and in the vicinity of amyloid-ß peptides plaques. Moreover, activation of PKCη, as indicated by its increased phosphorylation levels, is exhibited mainly in cortical astrocytes derived from adult 5XFAD mice. PKCη activation was associated with elevated levels of reactive astrocytic markers and upregulation of the pro-inflammatory cytokine interleukin 6 (IL-6) compared to littermate controls. Notably, inhibiting the kinase activity of PKCη in 5XFAD astrocyte cultures markedly increased the levels of secreted IL-6-a phenomenon that was also observed in wild-type astrocytes stimulated by inflammatory cytokines (e.g., TNFα, IL-1). Similar increase in the release of IL-6 was also observed upon inhibition of either the mammalian target of rapamycin (mTOR) or the protein phosphatase 2A (PP2A). Our findings suggest that the mTOR-PKCη-PP2A signaling cascade functions as a negative feedback loop of NF-κB-induced IL-6 release in astrocytes. Thus, we identify PKCη as a regulator of neuroinflammation in AD.

Thymus involution sets the clock of the aging T-cell landscape: Implications for declined immunity and tissue repair.

Aging is generally characterized as a gradual increase in tissue damage, which is associated with senescence and chronic systemic inflammation and is evident in a variety of age-related diseases. The extent to which such tissue damage is a result of a gradual decline in immune regulation, which consequently compromises the capacity of the body to repair damages, has not been fully explored. Whereas CD4 T lymphocytes play a critical role in the orchestration of immunity, thymus involution initiates gradual changes in the CD4 T-cell landscape, which may significantly compromise tissue repair. In this review, we describe the lifespan accumulation of specific dysregulated CD4 T-cell subsets and their coevolution with systemic inflammation in the process of declined immunity and tissue repair capacity with age. Then, we discuss the process of thymus involution-which appears to be most pronounced around puberty-as a possible driver of the aging T-cell landscape. Finally, we identify individualized T cell-based early diagnostic biomarkers and therapeutic strategies for age-related diseases.

A TRAIL-TL1A Paracrine Network Involving Adipocytes, Macrophages, and Lymphocytes Induces Adipose Tissue Dysfunction Downstream of E2F1 in Human Obesity.

Elevated expression of E2F1 in adipocyte fraction of human visceral adipose tissue (hVAT) associates with a poor cardiometabolic profile. We hypothesized that beyond directly activating autophagy and MAP3K5 (ASK)-MAP kinase signaling, E2F1 governs a distinct transcriptome that contributes to adipose tissue and metabolic dysfunction in obesity. We performed RNA sequencing of hVAT samples from age-, sex-, and BMI-matched patients, all obese, whose visceral E2F1 protein expression was either high (E2F1high) or low (E2F1low). Tumor necrosis factor superfamily (TNFSF) members, including TRAIL (TNFSF10), TL1A (TNFSF15), and their receptors, were enriched in E2F1high While TRAIL was equally expressed in adipocytes and stromal vascular fraction (SVF), TL1A was mainly expressed in SVF, and TRAIL-induced TL1A was attributed to CD4+ and CD8+ subclasses of hVAT T cells. In human adipocytes, TL1A enhanced basal and impaired insulin-inhibitable lipolysis and altered adipokine secretion, and in human macrophages it induced foam cell biogenesis and M1 polarization. Two independent human cohorts confirmed associations between TL1A and TRAIL expression in hVAT and higher leptin and IL6 serum concentrations, diabetes status, and hVAT-macrophage lipid content. Jointly, we propose an intra-adipose tissue E2F1-associated TNFSF paracrine loop engaging lymphocytes, macrophages, and adipocytes, ultimately contributing to adipose tissue dysfunction in obesity.

Aging promotes reorganization of the CD4 T cell landscape toward extreme regulatory and effector phenotypes.

Age-associated changes in CD4 T-cell functionality have been linked to chronic inflammation and decreased immunity. However, a detailed characterization of CD4 T cell phenotypes that could explain these dysregulated functional properties is lacking. We used single-cell RNA sequencing and multidimensional protein analyses to profile thousands of CD4 T cells obtained from young and old mice. We found that the landscape of CD4 T cell subsets differs markedly between young and old mice, such that three cell subsets-exhausted, cytotoxic, and activated regulatory T cells (aTregs)-appear rarely in young mice but gradually accumulate with age. Most unexpected were the extreme pro- and anti-inflammatory phenotypes of cytotoxic CD4 T cells and aTregs, respectively. These findings provide a comprehensive view of the dynamic reorganization of the CD4 T cell milieu with age and illuminate dominant subsets associated with chronic inflammation and immunity decline, suggesting new therapeutic avenues for age-related diseases.

CD4 T Cells Induce A Subset of MHCII-Expressing Microglia that Attenuates Alzheimer Pathology.

Microglia play a key role in innate immunity in Alzheimer disease (AD), but their role as antigen-presenting cells is as yet unclear. Here we found that amyloid ß peptide (Aß)-specific T helper 1 (Aß-Th1 cells) T cells polarized to secrete interferon-γ and intracerebroventricularly (ICV) injected to the 5XFAD mouse model of AD induced the differentiation of major histocompatibility complex class II (MHCII)+ microglia with distinct morphology and enhanced plaque clearance capacity than MHCII- microglia. Notably, 5XFAD mice lacking MHCII exhibited an enhanced amyloid pathology in the brain along with exacerbated innate inflammation and reduced phagocytic capacity. Using a bone marrow chimera mouse model, we showed that infiltrating macrophages did not differentiate to MHCII+ cells following ICV injection of Aß-Th1 cells and did not support T cell-mediated amyloid clearance. Overall, we demonstrate that CD4 T cells induce a P2ry12+ MHCII+ subset of microglia, which play a key role in T cell-mediated effector functions that abrogate AD-like pathology.

BDNF-producing, amyloid ß-specific CD4 T cells as targeted drug-delivery vehicles in Alzheimer's disease.

BACKGROUND: The delivery of therapeutic proteins to selected sites within the central nervous system (CNS) parenchyma is a major challenge in the treatment of various neurodegenerative disorders. As brain-derived neurotrophic factor (BDNF) is reduced in the brain of people with Alzheimer's disease (AD) and its administration has shown promising therapeutic effects in mouse model of the disease, we generated a novel platform for T cell-based BDNF delivery into the brain parenchyma. METHODS: We generated amyloid beta-protein (Aß)-specific CD4 T cells (Aß-T cells), genetically engineered to express BDNF, and injected them intracerebroventricularly into the 5XFAD mouse model of AD. FINDINGS: The BDNF-secreting Aß-T cells migrated efficiently to amyloid plaques, where they significantly increased the levels of BDNF, its receptor TrkB, and various synaptic proteins known to be reduced in AD. Furthermore, the injected mice demonstrated reduced levels of beta-secretase 1 (BACE1)-a protease essential in the cleavage process of the amyloid precursor protein-and ameliorated amyloid pathology and inflammation within the brain parenchyma. INTERPRETATION: A T cell-based delivery of proteins into the brain can serve as a platform to modulate neurotoxic inflammation and to promote neuronal repair in neurodegenerative diseases.

Co-assembled Ca2+ Alginate-Sulfate Nanoparticles for Intracellular Plasmid DNA Delivery.

Successful gene therapy requires the development of suitable carriers for the selective and efficient delivery of genes to specific target cells, with minimal toxicity. In this work, we present a non-viral vector for gene delivery composed of biocompatible materials, CaCl2, plasmid DNA and the semi-synthetic anionic biopolymer alginate sulfate (AlgS), which spontaneously co-assembled to form nanoparticles (NPs). The NPs were characterized with a slightly anionic surface charge (Zeta potential [ζ] = -14 mV), an average size of 270 nm, and their suspension was stable for several days with no aggregation. X-ray photoelectron spectroscopy (XPS) validated their ternary composition, and it elucidated the molecular interactions among Ca2+, the plasmid DNA, and the AlgS. Efficient cellular uptake (>80%), associated with potent GFP gene expression (22%-35%), was observed across multiple cell types: primary rat neonatal cardiac fibroblasts, human breast cancer cell line, and human hepatocellular carcinoma cells. The uptake mechanism of the NPs was studied using imaging flow cytometry and shown to be via active, clathrin-mediated endocytosis, as chemical inhibition of this pathway significantly reduced EGFP expression. The NPs were cytocompatible and did not activate the T lymphocytes in human peripheral blood mononuclear cells. Proof of concept for the efficacy of these NPs as a carrier in cancer gene therapy was demonstrated for Diphtheria Toxin Fragment A (DT-A), resulting in abrogation of protein synthesis and cell death in the human breast cancer cell line. Collectively, our results show that the developed AlgS-Ca2+-plasmid DNA (pDNA) NPs may be used as an effective non-viral carrier for pDNA.

The Choroid Plexus Functions as a Niche for T-Cell Stimulation Within the Central Nervous System.

The choroid plexus (CP) compartment in the ventricles of the brain comprises fenestrated vasculature and, therefore, it is permeable to blood-borne mediators of inflammation. Here, we explored whether T-cell activation in the CP plays a role in regulating central nervous system (CNS) inflammation. We show that CD4 T cells injected into the lateral ventricles adhere to the CP, transmigrate across its epithelium, and undergo antigen-specific activation and proliferation. This process is enhanced following peripheral immune stimulation and significantly impacts the immune signaling induced by the CP. Ex vivo studies demonstrate that T-cell harboring the CP through its apical surface is a chemokine- and adhesion molecule-dependent process. We suggest that, within the CNS, the CP serves an immunological niche, which rapidly responds to peripheral inflammation and, thereby, promotes two-way T-cell trafficking that impact adaptive immunity in the CNS.

Old Mice Accumulate Activated Effector CD4 T Cells Refractory to Regulatory T Cell-Induced Immunosuppression.

Chronic low-grade inflammation and reduced lymphocyte potency are implicated in the pathogenesis of major illnesses associated with aging. Whether this immune phenotype results from a loss of cell-mediated regulation or intrinsic dysregulated function of effector T cells (Teffs) requires further research. Here, we report that, as compared with young C57BL6 mice, old mice show an increased frequency of CD4+CD62L- Teffs with a dysregulated activated phenotype and markedly increased effector functions. Analysis of the frequency and suppressive function of CD4+FoxP3+ regulatory T cells (Tregs) indicates an increase in the frequency of FoxP3+ T cells with aging which, however, occurs within the CD4+CD25- T cells. Furthermore, whereas Tregs from young and old mice similarly suppress Teffs from young mice, both have a compromised suppressive capacity of Teffs from old mice, a phenomenon which is partially recovered in the presence of IL-2-producing CD4+CD62L+ non-Teffs. Finally, we observed that Teff subsets from old mice are enriched with IL-17A-producing T cells and exhibit intrinsically dysregulated expression of genes encoding cell-surface molecules and transcription factors, which play a key role in T-cell activation and regulation. We, thus, demonstrate an age-related impairment in the regulation of effector CD4 T cells, which may underlie the higher risk for destructive inflammation associated with aging.

TGF-ß affinity-bound to a macroporous alginate scaffold generates local and peripheral immunotolerant responses and improves allocell transplantation.

Enhancing vascularization of cell-transplantation devices is necessary for maintaining cell viability and integration within the host, but it also increases the risk of allograft rejection. Here, we investigated the feasibility of generating an immunoregulatory environment in a highly vascularized macroporous alginate scaffold by affinity-binding of the transforming growth factor-ß (TGF-ß) in a manner mimicking its binding to heparan sulfate. Using this device to transplant allofibroblasts under the kidney capsule resulted in the induction of local and peripheral TGF-ß-dependent immunotolerance, characterized by higher frequency of immature dendritic cells and regulatory T cells within the device and by markedly reduced allofibroblast-specific T-cell response in the spleen, thereby increasing the viability of the transplanted cells. Culturing whole splenocytes in the TGF-ß-bound scaffold indicated that the regulatory function of TGF-ß is IL-10-dependent. We thus demonstrate a novel platform for transplantation devices, designed to promote an immunoregulatory microenvironment suitable for cell transplantation and autoimmune regulation. STATEMENT OF SIGNIFICANCE: Allogeneic cell graft transplantation is a potentially optimal treatment for many clinical deficiencies. It is yet challenging to overcome chronic rejection without compromising host immunity to pathogens. We present the features and function of a cell transplantation device designed based on the principle of affinity binding of angiogenic and immunoregulatory factors to extracellular matrix in aim to achieve sustained release of these factors. We show that presentation of these factors in such manner generates the infrastructure for device vascularization and induces profound local allocell-specific tolerance, which then evokes peripheral T-cell tolerance. The tolerance is antigen specific, does not cause immune deficits and may thus serve to improve allocell survival as well as a platform to mitigate pathogenic autoimmunity.