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-122R), 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.

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.

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.

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.

When Thetis dipped her son Achilles into the River Styx to make him immortal, she held him by the Heel, which was not submerged, and thus created a weak spot that proved deadly for Achilles. Millennia later, Achilles Heel is part of today’s lexicon meaning an area of weakness or a vulnerable spot that causes failure. Also implied is that an Achilles Heel is often missed, forgotten or underappreciated, until it is under attack, and then failure is fatal. Paris killed Achilles with an arrow ‘guided by the Gods’. At the International Congress of the Immunology of Diabetes Society, 2018, five leading experts were asked to present the case for a particular cell/element that could represent the Achilles Heel of T1D. Their arguments are summarized here, to make this case.

Autoimmune neutropenia (AIN) in childhood is characterized by chronic neutropenia and positivity for antineutrophil antibodies, resulting in the excessive destruction of neutrophils. In this study, we investigated the involvement of regulatory T cells (Tregs) in the pathogenesis of AIN in childhood. Tregs have been classified into three subpopulations based on the expressions of CD45RA and FOXP3: resting Tregs, activated Tregs, and non-suppressive Tregs. The frequency of activated Tregs (CD4+CD25+FOXP3highCD45RA− T cells) as well as that of total Tregs (CD4+CD25+FOXP3+ T cells) in peripheral blood was significantly decreased in patients with AIN. Analysis of the T cell receptor (TCR)-Vβ repertoire of CD4+ T cells revealed skewed usages in patients with AIN compared with that observed in age-matched control subjects. Regarding T cell subsets, the use of four of 24 TCR-Vβ families in Tregs and one in conventional T cells were increased in patients with AIN. The number of patients with AIN who showed skewed usages of TCR-Vβ family in conventional and Tregs was significantly higher than that reported in control subjects. When the preference between Tregs and conventional T cells in each TCR-Vβ family was individually compared, different use was prominently observed in the TCR-Vβ 9 family in patients with AIN. These results suggest that the quantitative abnormalities of Tregs and the skew of the TCR-Vβ repertoire in CD4+ T cells, including Tregs and conventional T cells, may be related to autoantibody production through a human neutrophil antigen-reactive T cell clone.

Tuberculosis kills more people than any other single infectious disease globally. Despite decades of research, there is no vaccine to prevent TB transmission. Bacille Calmette-Guerin (BCG) vaccine developed a century ago has little effect on pulmonary TB and does not control transmission. Lack of an effective vaccine emanates from lack of knowledge on correlates of protective immunity on which to base vaccine design and development. However, some household contacts who are extensively exposed to Mtb infection remain persistently negative to tuberculin skin test and interferon-gamma assay. These individuals called “resisters” clear Mtb infection early before the development of acquired immunity. The immunological basis of early Mtb clearance is yet to be established, however, innate lymphocytes such as monocytes/macrophages, dendritic cells, neutrophils and natural killer cells, and innate like T cells such as mucosal associated invariant T cells, invariant natural killer T cells and gamma-delta (γδ) T cells have been implicated in this early protection. One of the cells that has attracted increasing attention in recent years, in protection against Mtb is the natural killer cell. Emerging data from animal and epidemiological studies indicate that NK cells may play a significant role in the fight against Mtb. NK cells express various surface markers to recognize and kill both Mtb and Mtb-infected cells. In this review, recent advances in our understanding of NK cells in the fight against Mtb early during infection, with emphasis on cohort studies, will be presented.

Tissue damage associated with trauma might release a sufficient autoantigen substrate to break immune tolerance. In a previous study, we showed that the leukopenia observed following severe inflammation is related to adenosine A1-receptor (A1R) desensitization and A2AR upregulation. We hypothesized that, under destructive pathological conditions this mechanism is beneficial in reducing prevalence of autoimmunity. In this study, we aim to evaluate the protective role of A1R and A2AR in prevention of autoimmune diseases. We used two murine models of autoimmune diseases: type 1 diabetes (T1D) induced by low-dose streptozotocin and pristane-induced lupus (PIL) and on neutrophils we studied NETosis regulation by adenosine. In both the T1D and PIL models, A1R-KO mice were predisposed to the development of autoimmunity. In the PIL model, in WT mice, parallel to the decline of A1R mRNA levels, lymphocytes number dropped (-85%) 6h after pristane injection. WT mice remained without any sign of disease at 36 weeks. In contrast, following pristane 43% of A1R-KO mice suffered from lupus-like disease. Compared to A1R-KO, in WT mice at 10 days A2AR mRNA levels were significantly higher. Similar to PIL, in T1D model the presence of A1R and A2AR was protective. In addition, we found that A1R increases and A2AR suppresses NETosis. We suggest that adenosine-dependent immune suppression and reduction in neutrophil extracellular traps (NETs) limits the reactive T-cells and development of anti-double strand DNA (dsDNA) antibodies that promote autoimmunity.

Follicular helper T cells (Tfh) cells are generally considered critical in secondary lymphoid tissues; however, they are also identified in the circulation and in tertiary lymphoid structures in chronic inflammation. Gingival tissues with periodontitis reflect chronic inflammation so genomic footprints of Tfh cells should occur in these tissues and may differ related to aging effects. Methods: Macaca mulatta monkeys were used in a model of ligature-induced periodontitis [adult group (12-23 years of age); young group (3-7 years)]. Gingival tissue and subgingival microbiome samples were obtained at matched healthy sites, sites during ligature-induced disease, and in samples after clinical resolution. Microarray analysis examined Tfh genes (n=40) and the microbiome samples were examined using 16S MiSeq. Results: An apparent increase in the major transcription factor of Tfh cells, BCL6, was found with disease in both adult and young animals, while the master transcription markers of other T cell subsets were either decreased or showed minimal change. A number of the Tfh related genes, including surface receptors, secreted products and transcription factors were also significantly increased during disease. Unique microbial complexes showed patterns of interactions with Tfh genes that differed in health and disease. Conclusions: An increase in Tfh cell responsiveness occurred later in the progression of periodontitis, affected by age and strongly related to specific microbial complexes. The capacity of gingival Tfh cells to contribute to localized B cell activation and active antibody responses, including affinity maturation may be critical for controlling periodontal lesions and contributing to limiting and/or resolving the lesions.

Rheumatoid arthritis (RA) is a systemic inflammatory autoimmune disease that leads to joint destruction and disability. Despite a significant progress in administration of biological agents for RA patients, there is still a need for improved therapy. Intravenous-immunoglobulins (IVIG) , a pooled polyspecific immunoglobulin-G (IgG) extracted from 20,000 healthy subjects, showed beneficial therapeutic effect in patients with immune-deficiency, sepsis, and autoimmune diseases. The current study aim to investigate the beneficial effect of treatment with IVIG in established collagen induced arthritis in DBA/1j mice. Murine arthritis was induced in DBA/1j mice. The treatment with IVIG started when the disease was established. The clinical score was followed twice a week until day 48. The mice were bled for plasma, the paws were H&E stained. Cytokine profile in the plasma was analyzed by Luminex technology, titers of circulating anti-collagen antibodies in the plasma was tested by ELISA. Our results show that treatment with IVIG in murine, significantly rreduced the clinical arthritis score (P<0.001). Moreover, mode of action show that IVIG significantly reduced circulating level of inflammatory cytokines (IFN, IL-1β, IL-17, IL-6, TNFα) (P<0.001), inhibit anti-collagen antibodies (P < 0.001) in the plasma of CIA mice. Importantly, histopathological examination revealed that IVIG treatment prevented the migration of inflammatory immune cells into the cartilage and synovium, reduced the extent of joint damage and preserved joint architecture. Our results proved for the first time the valuable anti-inflammatory treatment of IVIG in experimental RA. We propose IVIG therapy for a subgroup of patients with as rheumatological-related diseases.

The skin is a unique immune organ that constitutes a complex network of physical, chemical, and microbiological barriers against external insults. Keratinocytes are the most abundant cell type in the epidermis. These cells form the physical skin barrier and represent the first line of the host defense system by sensing pathogens via innate immune receptors, initiating antimicrobial responses and producing various cytokines, chemokines and antimicrobial peptides, which are important events in immunity. A damaged epidermal barrier in atopic dermatitis allows the penetration of potential allergens and pathogens to activate keratinocytes. Among the dysregulation of immune responses in atopic dermatitis, activated keratinocytes play a role in several biological processes that contribute to the pathogenesis of atopic dermatitis. In this review, we summarize the current understanding of the innate immune functions of keratinocytes in the pathogenesis of atopic dermatitis, with a special emphasis on skin-derived antimicrobial peptides and atopic dermatitis-related cytokines and chemokines in keratinocytes. An improved understanding of the innate immunity mediated by keratinocytes can provide helpful insight into the pathophysiological processes of atopic dermatitis and support new therapeutic efforts.