Neuropathology studies of amyotrophic lateral sclerosis (ALS) and animal models of ALS reveal a strong association between aberrant protein accumulation and motor neuron damage and activated microglia and astrocytes, the resident CNS innate immune cells. While the role of neuroinflammation in the pathology of ALS is unclear imaging studies support the idea that innate immune activation occurs early disease in both humans and rodent models of ALS. In addition to innate immunity, emerging studies also reveal the presence of peripheral monocytes, macrophages, and lymphocytes in the CNS as well as at the neuromuscular junction. To better understand the association of neuroinflammation (innate and adaptive) with disease progression paraclinical studies including the use of biomarkers and imaging modalities allow monitoring of immune parameters in the disease process. Such approaches are important for patient stratification, selection, and inclusion in clinical trials, as well as to provide readouts of response to therapy. Here, we discuss the different imaging modalities e.g., MRI, MRS, PET as well as other approaches including biomarkers of inflammation in ALS, aid the understanding of the underlying immune mechanisms associated with motor neuron degeneration in ALS.
Aberrant autoantibody production is characteristic of systemic lupus erythematosus (SLE), but follicular regulatory T (TFR) cells potentially can suppress this abnormality. We investigate functional changes in TFR cells from SLE patients. Circulating TFR cells were collected from 19 SLE patients and 14 healthy controls (HC) to compare molecular expression and in vitro suppressive capacity of follicular helper T (TFH) cell proliferation. To reveal the stability of Foxp3 in TFR, pyrosequencing of conserved non-coding sequence (CNS) 2 at the Foxp3 gene locus was performed. We then tested IL-2 in SLE-TFR cells to check restoration of suppressor function. Programmed death-1 (PD-1) expression in SLE-TFR cells was positively correlated with anti-DNA antibody levels and disease activity. These cells had impaired suppressive function for TFH cells with decreased expression of suppression mediators forkhead box p3 (Foxp3), cytotoxic T-lymphocyte antigen 4 (CTLA4), and IL-2 receptor alpha (IL2Rα). Pyrosequencing identified hyper-methylation in CNS2 region of SLE-TFR cells comparing to HC. With In vitro IL-2 stimulation, PD-1 expression of TFR cells significantly decreased along with increased expression of Foxp3 and CTLA-4, especially in low-dose. Thus, SLE-TFR cells have functionally defective to TFH suppression, but low-dose IL-2 therapy might be useful to restore this ability.
Elderly individuals exhibit unbalanced bone marrow (BM) effector T cell subset differentiation, such as increased T helper (Th)-1 and T cytotoxic (Tc)-1 cell frequencies, but the underlying mechanism still unclear. Endothelial cells (ECs) , which are instructive components of the BM microenvironment, exhibit the phenotype of semi-professional antigen-presenting cells and regulate T cell recruitment and activation. Thus, we compared the frequency and function of BM ECs, especially their capacity to regulate effector T cell subsets, between young and old healthy individuals, and explored the underlying mechanism of this immunomodulatory discrepancy. Although the young and old EC percentages were comparable, young ECs showed less reactive oxygen species and better migratory and tube-forming abilities than old ECs. Notably, young ECs regulated T cells to differentiate into fewer Th1 and Tc1 cells than old ECs. Reduced T cell activation molecules and inflammatory cytokines in young BM ECs may be the possible mechanism.
Gamma-tocotrienol (γT3) is an analogue of vitamin E with beneficial effects on the immune system, including immune-modulatory properties. This study reports the immune-modulatory effects of daily supplementation of γT3 on host T-helper (Th) and T-regulatory (Treg) populations in a syngeneic mouse model of breast cancer. Female BALB/c mice were fed with either γT3 or vehicle (soy oil) for 2-weeks via oral gavage before they were inoculated with syngeneic 4T1 mouse mammary cancer cells (4T1 cells). Supplementation continued until the mice were sacrificed. Mice (n=6) were sacrificed at specified time-points for various analysis (blood leucocyte, cytokine production, and immunohistochemistry). Tumour volume was measured once every seven days. Gene expression studies were carried out on tumour-specific T-lymphocytes isolated from splenic cultures. Supplementation with γT3 increased CD4+ (p<0.05), CD8+ (p<0.05) T-cells and natural killer cells (p<0.05) but suppressed Treg cells (p<0.05) in peripheral blood when compared to animals fed with the vehicle. Higher interferon-gamma (IFN) and lower transforming growth factor-beta (TGF-) levels were noted in the T3 fed mice. Immunohistochemistry findings revealed higher infiltration of CD4+ cells, increased expression of interleukin-12 receptor-beta-2 (IL-122R), interleukin-24 (IL-24) and reduced expression of cells that express the forkhead box P3 (FoxP3) in tumours from the T3 fed animals. Gene expression studies showed the downregulation of seven prominent genes in splenic CD4+ T-cells isolated from γT3-fed mice. Supplementation with γT3 from palm oil-induced T-cell dependent cell-mediated immune responses and suppressed Treg cells in the tumour microenvironment in a syngeneic mouse model of BC.
Guillain–Barre’ syndrome (GBS) is an acute inflammatory and immune-mediated demyelinating disease of peripheral nervous system (PNS). Macrophages playing a central role in its animal model, experimental autoimmune neuritis (EAN) has been well-accepted. Additionally, NF-κB inhibitors has been used to treat cancers and showed beneficial effects. Here we investigated the therapeutic effect of M2 macrophage and NF-κB pathway is correlated with macrophages activation in experimental autoimmune neuritis (EAN) in C57BL/6 mice. We demonstrated that M2 macrophage transfusion can alleviate the clinical symptoms of EAN by reducing the proportion of M1 macrophage in the peak period, inhibiting the phosphorylation of NF-κB p65. The NF-κB inhibitor (BAY-11-7082) could alleviate the clinical symptoms of EAN and shorten the duration of symptoms by reducing the proportion of M1 macrophages and the expression of pro-inflammatory cytokines. Consequently, BAY-11-7082 exhibits strong potential as a therapeutic strategy for ameliorating EAN by influencing the balance of M1/M2 macrophages and inflammatory cytokines.
COVID-19 has been associated with both transient and persistent systemic symptoms that do not appear to be a direct consequence of viral infection. The generation of autoantibodies has been proposed as a mechanism to explain these symptoms. To understand this phenomenon in more detail, we investigated the frequency and specificity of clinically relevant autoantibodies in 84 individuals previously infected with SARS-CoV-2, suffering from COVID-19 of varying severity in both the acute and convalescent setting. These were compared with results from 32 individuals who were on ITU for non COVID reasons. We demonstrate a higher frequency of autoantibodies in the COVID-19 ITU group compared with non-COVID-19 ITU disease control patients and that autoantibodies were also found in the serum 3-5 months post COVID-19 infection. Non-COVID patients displayed a diverse pattern of autoantibodies; in contrast, the COVID-19 groups had a more restricted panel of autoantibodies including skin, skeletal muscle and cardiac antibodies. Our results demonstrate that severe COVID-19 induces a pattern of autoantibodies that may correlate with and contribute to the immune pathology associated with the long-term sequelae of infection.
Body fat has regulatory functions through producing cytokines and adipokines whose role in the pathogenesis of Systemic Sclerosis (SSc) is currently emerging. Changes in body mass, either overweight or underweight status, entail a dysregulation of the cytokines/adipokines network that may impact on SSc disease activity. We evaluated serum levels of adipokines and cytokines in SSc patients and correlated them to clinical features and body mass index (BMI) categories. The study included 89 SSc patients and 26 healthy donors (HD). Serum levels of adiponectin, leptin, resistin, visfatin, TNFα, IFNγ, IL-2, IL-10, and IL-17A were measured by Multiplex Immunoassay, and correlated to BMI, waist to hip ratio, and disease specific features. Mann-Whitney U-test or t-Student for unpaired data, Kruskal-Wallis test or ANOVA, were used for comparisons between groups. Spearman’s or Pearson’s test were used for correlation analysis. Serum levels of TNFα, IL-2, leptin, and resistin, were significantly higher in SSc than in HD. The highest levels of IL-17A, IL-2, IL-10, leptin and visfatin were detected in obese SSc patients (p <0.01). Conversely, underweight SSc patients showed the highest TNFα levels (p<0.05), which were negatively correlated with BMI (p=0.05). No correlation between adipokines/cytokines and clinical characteristics was found. Adipokines, IL-2, IL-10 and IL-17A were found to be increased in obese SSc patients, but whether they play a role in the pathogenesis of the disease remains to be investigated. Intriguingly, underweight patients had higher TNFα levels, suggesting a potential role of TNFα in inducing the cachexia observed in long-lasting disease.
Since December 2019, Coronavirus disease-19 (COVID-19) has spread rapidly across the world, leading to a global effort to develop vaccines and treatments. Despite extensive progress, there remains a need for treatments to bolster the immune responses in infected immunocompromised individuals, such as cancer patients who recently underwent a haematopoietic stem cell transplantation. Immunological protection against COVID-19 is mediated by both short-lived neutralising antibodies and long-lasting virus-reactive T cells. Therefore, we propose that T cell therapy may augment efficacy of current treatments. For the greatest efficacy with minimal adverse effects, it is important that any cellular therapy is designed to be as specific and directed as possible. Here, we identify T cells from COVID-19 patients with a potentially protective response to two major antigens of the SARS-CoV-2 virus, Spike and Nucleocapsid protein. By generating clones of highly virus-reactive CD4+ T cells, we were able to confirm a set of 9 immunodominant epitopes and characterise T cell responses against these. Accordingly, the sensitivity of T cell clones for their specific epitope, as well as the extent and focus of their cytokine response was examined. Moreover, by using an advanced T cell receptor (TCR) sequencing approach, we determined the paired TCR sequences of clones of interest. While these data on a limited population require further expansion for universal application, the results presented here form a crucial first step towards TCR-transgenic CD4+ T cell therapy of COVID-19.
Signal transducer and activator of transcription 3 (STAT3) gain-of-function (GOF) syndrome is an early-onset monogenic inborn error of immunity characterized by multi-organ autoimmune disorders, growth failure and lymphoproliferation. We describe that STAT3 GOF syndrome may be presented with hypogammaglobulinemia and recurrent severe upper and lower respiratory tract infections. The patient we present here did not have multi-organ autoimmunity and lacked early-onset autoimmune manifestations. Chest CT examinations showed mild bronchiectasis with areas of non-fibrosing alveolar-interstitial disease and maldevelopment of bilateral first ribs. By using Sanger sequencing, we revealed a novel c.508G>C, p.D170H STAT3 variant affecting the coiled coil domain (CCD) of STAT3. Functional studies confirmed that p.D170H was a GOF variant as showed by increased pSTAT3 and STAT3 transcriptional activity. Our observation suggests that STAT3 GOF syndrome can manifest in early childhood with hypogammaglobulinemia and recurrent severe respiratory tract infections and may lack autoimmune manifestations. We suggest that patients with hypogammaglobulinemia and severe, recurrent infections should be screened for STAT3 variants even if autoimmune manifestations are missing.
Summary Myelin oligodendrocyte glycoprotein antibody associated disorders (MOGAD) are rare in both children and adults, and have been recently suggested to be an autoimmune neuroinflammatory group of disorders that are different from aquaporin-4 autoantibody associated neuromyelitis optica spectrum disorder and from classic multiple sclerosis. In vivo imaging of the MOGAD patient central nervous system has shown some distinguishing features when evaluating magnetic resonance imaging of the brain, spinal cord, optic nerves, as well as retinal imaging using optical coherence tomography. In this review, we discuss key clinical and imaging characteristics of paediatric and adult MOGAD. We describe how these imaging techniques may be used to study this group of disorders and discuss how these imaging methods have led to recent insights for consideration in future studies.
Free light chains (FLC), considered a biomarker of B cell activity, are frequently elevated in different systemic inflammatory-autoimmune diseases. As systemic sclerosis (SSc) clinical course may be variable, this study aimed to assess FLCs levels in serum and urine of affected individuals, as biomarkers of disease activity. We assessed FLC levels in serum and urine of 72 SSc patients and 30 healthy controls (HC). Results were analyzed in comparison with overall clinical and laboratory findings, disease activity index (DAI) and disease severity scale (DSS). SSc patients displayed increased levels of k and λ FLC in serum, significantly higher than HC (p=0.0001) alongside with the mean levels of free k/λ ratio and of the k+λ sum (p=0.0001). In addition, SSc patients had significant higher levels in the urine of free k and of k/λ ratio than HC (p=0.0001). SSc patients with increased k+λ sum in serum showed a statistically higher levels of erythro-sedimentation rate (p=0.034), C-reactive protein (p=0.003), higher DAI (p=0.024) and DSS (p=0.015) than SSc patients with normal levels of FLC. A positive linear correlation was found between serum levels of free k and DAI (r=0.29, p=0.014). SSc patients with increased free k in urine had statistically higher DAI (p=0.048) than SSc patients with normal level of free k. The significant increased levels of FLC correlated with disease activity in SSc patients. Our results strengthen the role of FLC as useful biomarkers in clinical practice to early diagnosis and monitor disease activity with an improvement of SSc patients’ management.
Background: Typical murine models of allergic inflammation are induced by the combination of ovalbumin and aluminum hydroxide. However, accumulating evidence indicates that, in models of asthma and atopic dermatitis, allergic inflammation can be generated in the absence of aluminum hydroxide. Moreover, co-administration of S. aureus enterotoxin B with ovalbumin can enhance inflammation. Objective: The objective of this study was to establish a rapid and mast cell-dependent murine model of allergic inflammation by inducing allergic peritonitis using ovalbumin and S. aureus enterotoxin B. Methods: Allergic peritonitis was induced in C57BL/6 mice by subcutaneous sensitization and intraperitoneal challenge with ovalbumin and S. aureus enterotoxin B. Disease characteristics were assessed by flow cytometry, ELISA, Trypan Blue exclusion and colorimetric assays. Results: Time course of the allergic peritonitis revealed a peak of peritoneal inflammation 48h after challenge, as assessed by total cells and eosinophil counts. Decrease of cell numbers started 96h post challenge with complete clearance within 168h. Moreover, significantly higher levels of tryptase and increased vascular permeability were found 30 min following challenge. Allergic inflammation induction by ovalbumin and S. aureus enterotoxin B was impaired in mast cells deficient mice and partially restored by mice reconstitution with bone marrow derived mast cells, indicating the mast cell role in this model. Conclusion: We present a novel model of allergic peritonitis that is mast cell-dependent, simple and robust. Moreover, the use of S. aureus enterotoxin B better resembles human allergic inflammation, which is known to be characterized by the colonization of Staphylococcus aureus.
Arthritis is a common clinical feature of systemic lupus erythematosus (SLE) and is usually non-erosive as opposed to rheumatoid arthritis (RA). While RA synovial pathology has been extensively studied, little is known about the pathophysiology of lupus arthritis. Here, we aimed to explore the cytokine and cellular compartments in synovial fluids of SLE patients with arthritic manifestations. Acellular synovial fluid and paired serum samples from SLE patients (n=17) were analyzed with cytokine bead array for T helper associated cytokines. From two SLE patients, synovial fluid mononuclear cells (SFMC) were analyzed by multiparameter flow cytometry to dissect T cell, B cell, monocyte and dendritic cell phenotypes. SLE-derived SFMC were further stimulated in vitro to measure their capacity for producing IFN and IL-17A. All patients fulfilled the ACR 1982 classification criteria for SLE. Clinical records were reviewed to exclude the presence of comorbidities such as osteoarthritis or overlap with RA. IL-17A and IL-6 levels were high in SLE synovial fluid. A clear subset of the synovial CD4+ T cells expressed CCR6+, a marker associated with Th17 cells. IL-17-production was validated amongst CD4+CCR6+ T cells following in vitro stimulation. Furthermore, a strong IFN production was observed in both CD4+ and CD8+ cells. Our study shows high IL-17A and IL-6 levels in synovial fluids of patients with lupus arthritis. The Th17 pathway have been implicated in several aspects of SLE disease pathogenesis and our data points to Th17 involvement also for lupus arthritis.
Adoptive immunotherapy using Epstein-Barr Virus (EBV)-specific T cells is a potentially curative treatment for patients with EBV-related malignancies where other clinical options have proved ineffective. We describe improved GMP-compliant culture and analysis processes for conventional lymphoblastoid cell line (LCL)-driven EBV-specific T cell manufacture, and describe an improved phenotyping approach for analyzing T cell products. We optimized the current LCL-mediated clinical manufacture of EBV-specific T cells to establish an improved process using xenoprotein-free GMP-compliant reagents throughout, and compared resulting products with our previous banked T cell clinical therapy. We assessed effects of changes to LCL: T cell ratio in T cell expansion, and developed a robust flow cytometric marker panel covering T cell memory, activation, differentiation and intracellular cytokine release to characterize T cells more effectively. These data were analyzed using t-Stochastic Neighbour Embedding (t-SNE) algorithm. The optimized GMP-compliant process resulted in reduced cell processing time and improved retention and expansion of central memory T cells. Multi-parameter flow cytometry determined the optimal protocol for LCL stimulation and expansion of T cells and demonstrated that cytokine profiling using IL-2, TNF-α and IFN-γ was able to determine the differentiation status of T cells throughout culture and in the final product. We show that fully GMP-compliant closed-process culture of LCL-mediated EBV-specific T cells is feasible and profiling of T cells through cytokine expression gives improved characterization of start material, in-process culture conditions and final product. Visualization of the complex multi-parameter flow cytometric data can be simplified using t-SNE analysis.
Peptides that bind to and are presented on the cell surface by Human Leukocyte Antigens (HLA) molecules play a critical role in adaptive immunity. For a long time, it was believed all of the HLA bound peptides were generated through simple proteolysis of linear sequences of cellular proteins, and therefore, are templated in the genome and proteome. However, evidence for untemplated peptide ligands of HLA molecules has accumulated over the last two decades, with a recent global analysis of HLA-bound peptides suggesting that a considerable proportion of HLA bound peptides are potentially generated through splicing/fusion of discontinuous peptide segments from one or two distinct proteins. In this review, we will review recent discoveries and debates on the contribution of spliced peptides to the HLA class I immunopeptidome, consider biochemical rules for splicing, and the potential role of these spliced peptides in immune recognition.
The impact of treatment on the risk of lymphoma in patients with rheumatoid arthritis (RA) is unclear. Here, we aimed to assess if the risk of lymphoma differs according to the type of Tumor Necrosis factor inhibitor (TNFi), comparing monoclonal anti-TNF antibodies (Ab) to the soluble TNF receptor. We used BAFF-transgenic (Tg) mice as a model of autoimmunity-associated lymphoma. Six-month aged BAFF-Tg mice were treated with TNFi for 12 months. Histological examination of the spleen, assessment of the cellular composition of the spleen by flow cytometry and assessment of B cell clonality were performed at sacrifice. Crude mortality and incidence of lymphoma were significantly higher in mice treated with monoclonal anti-TNF Ab compared to both controls and mice treated with the soluble TNF receptor, even at high dose. Flow cytometry analysis revealed decreased splenic macrophage infiltration in mice treated with monoclonal anti-TNF Ab. Overall, this study demonstrates, for the first time, that a very prolonged treatment with monoclonal anti-TNF Ab increase the risk of lymphoma in B cell-driven autoimmunity. This data suggests a closer monitoring for lymphoma development in patients suffering from B cell-driven autoimmune disease with long-term exposure to monoclonal anti-TNF Ab.
Introduction The mechanisms that drives SLE remission.The aim of the present study was to measure CD4+ and CD8+ T cell exhaustion in SLE patients in prolonged remission (PR-SLE) and compared them with patients with active SLE (Act-SLE) and healthy subjects Methods We included 15 PR-SLE patients, 15 Act-SLE and 29 healthy subjects. T-cell exhaustion was determined by flow cytometry according to the expression of PD-1, Tim-3, 2B4, EOMES and T-bet in CD4+ and CD8+ T cells. Dimensionality reduction using the t-Distributed Stochastic Neighbor Embedding algorithm and Clustering Analysis was used for the identification of relevant populations. Results Percentages of CD3+, CD4+ and CD8+ T cells were similar among groups. We identified five subpopulations of CD8+ and seven of CD4+ cells. The CD4+Tbet+CD45RO+ cells identified in the unsupervised analysis were significantly increased in PR-SLE vs Act-SLE (median: 10.20, IQR: 1.74-30.50 vs. 1.68, IQR: 0.4-2.83; p<0.01). CD4+EOMES+ cells were also increased in PR-SLE vs Act-SLE (5.24, IQR: 3.38-14.70 vs. 1.39, IQR: 0.48-2.87; p<0.001). CD8+ EOMES+ cells were increased in PR-SLE vs Act-SLE (37.6, IQR: 24.9-53.2 vs 8.13, IQR: 2.33-20.5; p<0.001). Exhausted and activated T cells presented an increased frequency of PD-1, CD57 and EOMES in SLE patients vs healthy subjects. Conclusions Some subpopulations of T cells expressing markers associated with exhaustion are increased in patients in remission, supporting T-cell exhaustion as a tolerance mechanism in SLE. Exhaustion of specific populations of T cells might represent a potential therapeutic tool that will contribute to the goal of achieving sustained remission in these patients.
The factors responsible for the spectrum of COVID-19 disease severity and the genesis and nature of protective immunity against COVID-19 remain elusive. Multiple studies have investigated the immune responses to COVID 19 in various populations, including those without evidence of COVID 19 infection. Information regarding innate and adaptive immune responses to the novel SARS-CoV-2 has evolved rapidly. Here, data are accumulating defining disease phenotypes that aid in rational and informed development of new therapeutic approaches for the treatment of patients infected with SARS-CoV-2 and the development of novel vaccines. In this article, we summarize data on important innate immune responses including cytokines, specifically IL-6 and complement, and explore potential treatments. We also examine adaptive immune responses and derivative therapeutics such as monoclonal antibodies directed at spike proteins. Finally, we explore data on real-time assessments of adaptive immune responses which include CD4+/CD8+ T-cells, NKT-cells, memory B-cells, and T-follicular cells with specificities for COVID-19 peptides in infected individuals and normals. Data of two novel vaccines have been released, both showing >95% efficacy in preventing SARS-CoV-2 infection. Analysis of humoral and cellular responses to the vaccines will determine the robustness and durability of protection. In addition, long-term assessment of SARS-CoV-2 memory B and T-cell mediated immune responses in patients recovering from an infection or those with cross-reactive immunologic memory will help to define risk for future SARS-CoV infections. Finally, patients recovering from SARS-CoV-2 infection may experience prolonged immune activation likely due to T-cell exhaustion. This will be an important new frontier for study.