Cell Responses In Autoimmune Diseases Research Articles

Glycolysis Inhibition Induces Functional and Metabolic Exhaustion of CD4+ T Cells in Type 1 Diabetes.

In Type 1 Diabetes (T1D), CD4+ T cells initiate autoimmune attack of pancreatic islet β cells. Importantly, bioenergetic programs dictate T cell function, with specific pathways required for progression through the T cell lifecycle. During activation, CD4+ T cells undergo metabolic reprogramming to the less efficient aerobic glycolysis, similarly to highly proliferative cancer cells. In an effort to limit tumor growth in cancer, use of glycolytic inhibitors have been successfully employed in preclinical and clinical studies. This strategy has also been utilized to suppress T cell responses in autoimmune diseases like Systemic Lupus Erythematosus (SLE), Multiple Sclerosis (MS), and Rheumatoid Arthritis (RA). However, modulating T cell metabolism in the context of T1D has remained an understudied therapeutic opportunity. In this study, we utilized the small molecule PFK15, a competitive inhibitor of the rate limiting glycolysis enzyme 6-phosphofructo-2-kinase/fructose-2,6- biphosphatase 3 (PFKFB3). Our results confirmed PFK15 inhibited glycolysis utilization by diabetogenic CD4+ T cells and reduced T cell responses to β cell antigen in vitro. In an adoptive transfer model of T1D, PFK15 treatment delayed diabetes onset, with 57% of animals remaining euglycemic at the end of the study period. Protection was due to induction of a hyporesponsive T cell phenotype, characterized by increased and sustained expression of the checkpoint molecules PD-1 and LAG-3 and downstream functional and metabolic exhaustion. Glycolysis inhibition terminally exhausted diabetogenic CD4+ T cells, which was irreversible through restimulation or checkpoint blockade in vitro and in vivo. In sum, our results demonstrate a novel therapeutic strategy to control aberrant T cell responses by exploiting the metabolic reprogramming of these cells during T1D. Moreover, the data presented here highlight a key role for nutrient availability in fueling T cell function and has implications in our understanding of T cell biology in chronic infection, cancer, and autoimmunity.

Anti-CD40 antibody KPL-404 inhibits T cell-mediated activation of B cells from healthy donors and autoimmune patients

BackgroundCD40-CD40L is a key co-stimulatory pathway for B cell activation. As such, its blockade can inhibit pathogenic B cell responses in autoimmune diseases, such as Sjogren’s syndrome (SjS) and systemic lupus erythematosus (SLE). In this study, we examined the in vitro effects of KPL-404, a humanized anti-CD40 monoclonal antibody (Ab), on primary human B cells derived from either healthy donors (HD) or autoimmune patients and compared them to the effects of G28-5, a partially antagonistic anti-CD40 antibody.MethodsPBMCs from HD or SjS and SLE patients were cultured in high-density cell cultures in the presence of IgG4 isotype control or anti-CD40 Abs KPL-404 or G28-5. Cells were stimulated with anti-CD3/CD28 cross-linking reagent ImmunoCult (IC) to induce CD40L-CD40-mediated B cell responses. B cell proliferation and activation, measured by dilution of proliferation tracker dye and the upregulation of CD69 and CD86, respectively, were assessed by flow cytometry. Anti-CD40 Ab cell-internalization was examined by imaging flow cytometry. Cytokine release in the PBMC cultures was quantified by bead-based multiplex assay.ResultsKPL-404 binds to CD40 expressed on different subsets of B cells without inducing cell depletion, or B cell proliferation and activation in in vitro culture. Under the same conditions, G28-5 promoted proliferation of and increased CD69 expression on otherwise unstimulated B cells. KPL-404 efficiently blocked the CD40L-CD40-mediated activation of B cells from HD at concentrations between 1 and 10 μg/ml. Treatment with KPL-404 alone did not promote cytokine production and blocked the production of IFNβ in healthy PBMC cultures. KPL-404 efficiently blocked CD40L-CD40-mediated activation of B cells from patients with SjS and SLE, without affecting their anti-IgM responses or affecting their cytokine production. Consistent with the differences of their effects on B cell responses, KPL-404 was not internalized by cells, whereas G28-5 showed partial internalization upon CD40 binding.ConclusionsAnti-CD40 mAb KPL-404 showed purely antagonistic effects on B cells and total PBMCs. KPL-404 inhibited CD40L-CD40-mediated B cell activation in PBMC cultures from both healthy controls and autoimmune patients. These data support the therapeutic potential of CD40 targeting by KPL-404 Ab for inhibiting B cell responses in SjS and SLE.

Pharmacological Targeting of GLUT1 to Control Autoreactive T Cell Responses.

An increasing body of evidence indicates that bio-energetic metabolism of T cells can be manipulated to control T cell responses. This potentially finds a field of application in the control of the T cell responses in autoimmune diseases, including in type 1 diabetes (T1D). Of the possible metabolic targets, Glut1 gained considerable interest because of its pivotal role in glucose uptake to fuel glycolysis in activated T cells, and the recent development of a novel class of small molecules that act as selective inhibitor of Glut1. We believe we can foresee a possible application of pharmacological Glut1 blockade approach to control autoreactive T cells that destroy insulin producing beta cells. However, Glut1 is expressed in a broad range of cells in the body and off-target and side effect are possible complications. Moreover, the duration of the treatment and the age of patients are critical aspects that need to be addressed to reduce toxicity. In this paper, we will review recent literature to determine whether it is possible to design a pharmacological Glut1 blocking strategy and how to apply this to autoimmunity in T1D.

The Transcriptional Coactivator Bob1 Is Associated With Pathologic B Cell Responses in Autoimmune Tissue Inflammation.

The molecular mechanisms steering abnormal B cell responses in autoimmune diseases remain poorly understood. We undertook this study to identify molecular switches controlling pathologic B cell responses in rheumatoid arthritis (RA). Candidate molecules were identified by gene expression profiling of RA synovitis and validated by quantitative polymerase chain reaction and immunohistochemistry. B cell-specific expression was confirmed by immunofluorescence, immunoblotting, and flow cytometry. The role of Bob1 in pathologic B cell responses was assessed in collagen-induced arthritis (CIA). Transcriptional profiling of RA synovitis revealed a prominent B cell signature, with the transcriptional coactivator Bob1 and its putative target BCMA being among the most up-regulated genes. Further analysis confirmed the microarray data and demonstrated elevated levels of Bob1 in B cells in RA synovium. A functional study showed that Bob1-deficient mice failed to produce pathogenic anti-type II collagen (anti-CII) antibodies and were resistant to CIA. Adoptive transfer of cells from Bob1-deficient and Bob1-sufficient mice to recombination-activating gene 1-null mice demonstrated that Bob1 deficiency exclusively in B cells abrogated germinal center (GC) B cell formation, anti-CII antibody production, and CIA development. Consistent with data from animal studies, immunophenotyping of human B cell subsets revealed increased expression of Bob1, predominantly in centrocytes and centroblasts. Correspondingly, Bob1 expression in RA synovitis was strongly correlated with CD21L, a molecular marker of GCs. In addition, similar Bob1 overexpression and correlation with CD21L expression was evidenced in parotid salivary gland tissue from patients with primary Sjögren's syndrome. These expression and functional data identify the transcriptional coactivator Bob1 as a candidate molecular switch of pathogenic B cell responses in autoimmune diseases in humans.

Experimental evidence that mutated-self peptides derived from mitochondrial DNA somatic mutations have the potential to trigger autoimmunity

Autoimmune disease is a critical health concern, whose etiology remains enigmatic. We hypothesized that immune responses to somatically mutated self proteins could have a role in the development of autoimmune disease. IFN-γ secretion by T cells stimulated with mitochondrial peptides encoded by published mitochondrial DNA was monitored to test the hypothesis. Human peripheral blood mononuclear cells (PBMCs) of healthy controls and autoimmune patients were assessed for their responses to the self peptides and mutated-self peptides differing from self by one amino acid. None of the self peptides but some of the mutated-self peptides elicited an immune response in healthy controls. In some autoimmune patients, PBMCs responded not only to some of the mutated-self peptides, but also to some of the self peptides, suggesting that there is a breach of self-tolerance in these patients. Although PBMCs from healthy controls failed to respond to self peptides when stimulated with self, the mutated-self peptide could elicit a response to the self peptide upon re-stimulation in vitro, suggesting that priming with mutated-self peptides elicits a cross-reactive response with self. The data raise the possibility that DNA somatic mutations are one of the events that trigger and/or sustain T cell responses in autoimmune diseases.

Regulation generation: the suppressive functions of human regulatory T cells.

Proper regulation of immune homeostasis is necessary to limit inflammation and prevent autoimmune and chronic inflammatory diseases. Many autoimmune diseases, such as psoriasis, are driven by vicious cycles of activated T cells that are unable to be suppressed by regulatory T cells. Effective suppression of auto-reactive T cells by regulatory T cells (Treg) is critical for the prevention of spontaneous autoimmune disease. Psoriatic Treg cells have been observed to a defect in their capacity to regulate, which clearly contributes to psoriasis pathogenesis. A challenge for translational research is the development of novel therapeutic interventions for autoimmune diseases that will result in durable remissions. Understanding the mechanism(s) of dysregulated T cell responses in autoimmune disease will allow for the development of future therapeutic strategies that may be employed to specifically target pathogenic, proinflammatory cells. Several reports have demonstrated a pathogenic role for Thl and Thl7 cells in psoriasis as well as other autoimmune diseases. Similarly, several laboratories have independently demonstrated functional defects in regulatory T cells isolated from patients with numerous divergent autoimmune diseases. One primary challenge of research in autoimmune diseases is therefore to restore the balance between chronic T cell activation and impairment of Treg suppressor mechanisms. To this end, it is critical to develop an understanding of the many suppressive mechanisms employed by Treg cells in hopes of developing more targeted therapeutic strategies for Treg-mediated autoimmune diseases.

Isolation and preservation of peripheral blood mononuclear cells for analysis of islet antigen-reactive T cell responses: position statement of the T-Cell Workshop Committee of the Immunology of Diabetes Society

Autoimmune T cell responses directed against insulin-producing β cells are central to the pathogenesis of type 1 diabetes (T1D). Detection of such responses is therefore critical to provide novel biomarkers for T1D 'immune staging' and to understand the mechanisms underlying the disease. While different T cell assays are being developed for these purposes, it is important to optimize and standardize methods for processing human blood samples for these assays. To this end, we review data relevant to critical parameters in peripheral blood mononuclear cell (PBMC) isolation, (cryo)preservation, distribution and usage for detecting antigen-specific T cell responses. Based on these data, we propose recommendations on processing blood samples for T cell assays and identify gaps in knowledge that need to be addressed. These recommendations may be relevant not only for the analysis of T cell responses in autoimmune disease, but also in cancer and infectious disease, particularly in the context of clinical trials.

Proteasomes in immune cells: more than peptide producers?

When cells are stimulated with pro-inflammatory cytokines, most of their constitutively expressed proteasomes are replaced with immunoproteasomes, which increase the production of peptides for presentation on MHC class I molecules. In addition, cortical thymic epithelial cells selectively express a type of proteasome known as the thymoproteasome that is required for the positive selection of thymocytes. Here, we discuss how these specialized types of proteasome shape the T cell receptor repertoire of cytotoxic T lymphocytes and propose that immunoproteasomes have functions, in addition to antigen processing, that influence cytokine production and T cell differentiation, survival and function. We also discuss how inhibitors of immunoproteasomes can suppress undesired T cell responses in autoimmune diseases.

Understanding B cell activation: from single molecule tracking, through Tolls, to stalking memory in malaria

B lymphocyte activation is initiated by the binding of antigens to the clonally expressed B cell receptors (BCRs) triggering signaling cascades that lead to the transcription of a variety of genes associated with B cell activation. Provided with the appropriate T cell help and the microenvironment of germinal centers antigen drives B cells to proliferate and differentiate into long-lived plasma cells and memory B cells that together constitute immunological memory. Here I describe efforts in my laboratory to gain an understanding of the cellular and molecular mechanisms that underlie three processes central to B cell biology namely, the initiation of BCR signaling, the interactions of the BCR with the innate immune system Toll-like receptors, and the generation and maintenance of B cell memory. Such knowledge is likely to aid research efforts in two areas of high public health priority, namely, the development of new therapeutics to control B cell responses in autoimmune disease and the design of effective vaccines to control infectious diseases.

Autoimmunity and molecular mimicry in the pathogenesis of post-streptococcal heart disease.

Molecular mimicry between pathogen and host has been proposed as a mechanism for the development of autoimmune diseases. Evidence suggests that microorganisms contain proteins which are similar enough to host proteins that they can stimulate existing B and T cells to respond to self proteins. The loss of immune regulation during responses against microbial antigens may explain development of pathogenic B and T cell responses in autoimmune diseases associated with infections. The study of B and T cell responses against the group A streptococcal antigens, N-acetyl-glucosamine, M protein and the autoantigen cardiac myosin has led to a better understanding of how molecular mimicry may play a role in disease. Studies of human monoclonal antibodies, T cell responses and animal models in comparison with the immunopathology in the human disease has provided information about the steps leading to inflammatory heart disease in autoimmune post-streptococcal rheumatic carditis. The new data indicate that the steps in pathogenesis of rheumatic heart disease following group A streptococcal infection include the following events. First, the development of crossreactive autoantibodies against the group A streptococcal carbohydrate antigen N-acetyl-glucosamine and cardiac myosin. Second, these antibodies react with valvular endothelium which becomes inflamed with expression of vascular cell adhesion molecule-1 (VCAM-1). After this event, T cells, CD4+ and CD8+, infiltrate through the endothelium/endocardium into the valve which is an avascular structure. Aschoff bodies or granulomatous lesions may form containing macrophages and T cells underneath the endocardium. The T cells are responsive to streptococcal M protein antigen sequences. The valve becomes scarred with eventual neovascularization and progressive, chronic disease in the valve. In the host, the mimicking antigens cardiac myosin and laminin have been involved in the myocardium and valve, respectively. As in other autoimmune diseases, both environmental and genetic factors are involved in the development of rheumatic carditis and inflammatory heart disease, a result of mimicry between the group A streptococcus and heart.

Induction of antigen-specific human CD4(+) T cell anergy by peripheral blood DC2 precursors.

Dendritic cells (DC) are antigen (Ag)-presenting cells that are essential for initiation of T cell-dependent immunity, and distinct DC subsets are known to direct different classes of immune responses. DC2 precursors (pDC2) or plasmacytoid DC were recently identified as a Th2-skewing and IFN-alpha-producing human DC subset. Here, we demonstrate that pDC2 enriched from human peripheral blood have a capacity to induce an anergic state in human Ag-specific CD4(+) T cell lines. Tetanus toxoid-specific T cell lines incubated with tetanus toxoid-pulsed autologous pDC2 failed to proliferate in secondary cultures with optimal Ag stimulation. T cell anergy induction required TCR engagement with Ag/MHC complex presented on pDC2. T cells rendered anergic lost IL-2 production but produced IFN-gamma and IL-10 upon stimulation. The pDC2-induced unresponsiveness was completely or partially reversible when a high concentration of exogenous IL-2 was added in the secondary cultures. Autoreactive CD4(+) T cell clones specific for topoisomerase I derived from a patient with scleroderma were also rendered anergic after co-culture with topoisomerase I-pulsed autologous pDC2,resulting in failure to proliferate or provide help to B cells. These results suggest that pDC2 are involved in maintenance of peripheral T cell tolerance and have potential for use in the suppression of pathogenic T cell responses in autoimmune diseases and organ transplantation.

T helper cell differentiation in multiple sclerosis and autoimmunity

Researchers from the fields of basic immunology and autoimmune disease, largely but not exclusively focused on multiple sclerosis, came together at a recent workshop 1 The International Workshop on Th1/Th2: T-Cell Differentiation in MS and Autoimmunity was held at Savannah, GA, USA, on 1–5 April 1998 1 to develop a better understanding of T helper cell responses in autoimmune disease.