Cytotoxic T Lymphocyte-associated Antigen Research Articles

T-cell immunosuppression in sepsis is augmented by sciatic denervation-induced skeletal muscle atrophy.

Skeletal muscle atrophy is a known risk factor for immunosuppressive conditions and for a poor prognosis in sepsis. However, its immunopathology has not been experimentally elucidated. This study investigated the effects of skeletal muscle atrophy on the immunopathology of sepsis. Male C57BL/6J mice were subjected to sciatic denervation (DN) and caecal ligation and puncture (CLP) to induce muscle atrophy or sepsis. The macrophages, myeloid-derived suppressor cells (MDSC), and T-cells in peritoneal and spleen were analysed using flow cytometry. DN-induced muscle atrophy did not affect macrophage and MDSC populations. In contrast, the percentage of cytotoxic T-lymphocyte-associated antigen (CTLA)-4+ CD4+ T-cells, programmed death (PD)-1+ CD8+ T-cells, regulatory T-cells, and the CTLA-4+ regulatory T-cells was statistically significantly higher in DN-CLP mice than in sham-CLP mice. Skeletal muscle atrophy before sepsis triggers excessive Tcell immunosuppression, which may contribute to the exacerbation of sepsis under skeletal muscle atrophy.

Immune-checkpoint inhibitor-mediated myocarditis: CTLA4, PD1 and LAG3 in the heart.

Immune-checkpoint inhibitors (ICIs) have revolutionized oncology, with nearly 50% of all patients with cancer eligible for treatment with ICIs. However, patients on ICI therapy are at risk for immune-related toxicities that can affect any organ. Inflammation of the heart muscle, known as myocarditis, resulting from ICI targeting cytotoxic Tlymphocyte-associated antigen 4 (CTLA4), programmed cell death protein 1 (PD1) and PD1 ligand 1 (PDL1) is an infrequent but potentially fatal complication. ICI-mediated myocarditis (ICI-myocarditis) is a growing clinical entity given the widespread use of ICIs, its increased clinical recognition and growing use of combination ICI treatment, a well-documented risk factor for ICI-myocarditis. In this Review, we approach ICI-myocarditis from a basic and mechanistic perspective, synthesizing the recent data from both preclinical models and patient samples. We posit that mechanistic understanding of the fundamental biology of immune-checkpoint molecules may yield new insights into disease processes, which will enable improvement in diagnostic and therapeutic approaches. The syndrome of ICI-myocarditis is novel, and our understanding of immune checkpoints in the heart is in its nascency. Yet, investigations into the pathophysiology will inform better patient risk stratification, improved diagnostics and precision-based therapies for patients.

The Role of Biologics in Rheumatoid Arthritis: A Narrative Review.

Rheumatoid arthritis (RA) is a chronic inflammatory joint disease that can cause cartilage and bone damage as well as a disability. Various cytokines play an essential role in disease formation such as tumor necrosis factor (TNF)-alpha, interleukin (IL)-1, IL-6, IL-17, and macrophages; osteoclast is also activated by the cytokines, which cause bone degradation. Early diagnosis is key to optimal therapeutic success, particularly in patients with well-characterized risk factors for poor outcomes such as high disease activity, presence of autoantibodies, and early joint damage. Treatment algorithms involve measuring disease activity with composite indices, applying a treatment-to-target strategy, and using conventional, biological, and new non-biological disease-modifying antirheumatic drugs. After the treatment target of stringent remission (or at least low disease activity) is maintained, dose reduction should be attempted. Although the prospects for most patients are now favorable, many still do not respond to current therapies. The biologics have changed the disease progression over the past few decades, such as TNF-alpha inhibitors (infliximab, etanercept, adalimumab, golimumab, certolizumab), IL-1 inhibitors (anakinra), IL-6 inhibitors (tocilizumab), CD20 inhibitors (rituximab), and cytotoxic T-lymphocyte associated antigen (CTLA)-4 inhibitors (abatacept). In treatment with biologics, only little is known if "biologic-free" remission is possible in patients with sustained remission following intensive biological therapy. Infliximab and etanercept, in the long run, develop the drug antibody. This article has reviewed the action of the cytokine on joints and biological drug's action in blocking the cytokine degradation effect, benefits of biologics, and adverse effects in the long and short term. They are also effective alone or in combination with other drugs.

Utility of Antibodies in the Diagnoses of Thyroid Diseases: A Review Article.

Thyroid problems are among the most widespread endocrine illnesses, affectingindividuals in India andthe global population. A thyroid function test is used to diagnose, screen, and monitor patients. Hyperthyroidism is a clinical condition due to excessive circulation of thyroid hormone; in contrast, hypothyroidism is due to a deficiency of thyroid hormone. Graves' disease (GD) is a form of hyperthyroidism due to thyroid-stimulating hormonereceptor autoantibodies (TRAb), and anti-thyroid peroxidase antibodies (anti-TPO antibodies). The most common reason for hypothyroidism is Hashimoto's thyroiditis (HT), in which patients have thyroid receptor antibodies (TRAb), antibodies to thyroid peroxidase (TPO), and thyroglobulin antibodies.Many essential genes, including the thyroid-specific genes thyroglobulin (TSGT), TSH-receptor gene, human leukocyte antigen (HLA) genes, cytotoxic T lymphocyte-associated antigen (CTLA)genes, thyroglobulin gene, vitamin D receptor gene, and many immune-regulatory geneswere associated with autoimmune thyroid diseases' (AITDs') etiology. This review paper aims to determine if antibodies are beneficial in detecting autoimmune thyroid disease or not. We have also discussed the etiology of autoimmune thyroid illness, serum antibodies in autoimmune thyroid disease, pathophysiology, and TSH receptor features.

Mechanisms underlying immune-related adverse events during checkpoint immunotherapy

Immune checkpoint (IC) proteins are some of the most important factors that tumor cells hijack to escape immune surveillance, and inhibiting ICs to enhance or relieve antitumor immunity has been proven efficient in tumor treatment. Immune checkpoint blockade (ICB) agents such as antibodies blocking programmed death (PD) 1, PD-1 ligand (PD-L) 1, and cytotoxic T lymphocyte-associated antigen (CTLA)-4 have been approved by the U.S. Food and Drug Administration (FDA) to treat several types of cancers. Although ICB agents have shown outstanding clinical success, and their application has continued to expand to additional tumor types in the past decade, immune-related adverse events (irAEs) have been observed in a wide range of patients who receive ICB treatment. Numerous studies have focused on the clinical manifestations and pathology of ICB-related irAEs, but the detailed mechanisms underlying irAEs remain largely unknown. Owing to the wide expression of IC molecules on distinct immune cell subpopulations and the fact that ICB agents generally affect IC-expressing cells, the influences of ICB agents on immune cells in irAEs need to be determined. Here, we discuss the expression and functions of IC proteins on distinct immune cells and the potential mechanism(s) related to ICB-targeted immune cell subsets in irAEs.

Immune Checkpoint Blockade Enhances Mutated Calreticulin-Induced T Cell Immunity in Myeloproliferative Neoplasms

Abstract Somatic mutations in the calreticulin (CALR) gene are key drivers of cellular transformation in myeloproliferative neoplasms (MPN) and are the second most common driver mutations in essential thrombocythemia (ET) and myelofibrosis (MF) patients. Each CALR mutation leads to a shift in the reading frame resulting in the formation of an altered protein with an identical 36-amino acid sequence in the C-terminus. We hypothesized that the recurrence and uniformity of the novel sequence would provide the mutated CALR an attractive candidate as a MPN-specific tumor antigen that might elicit anti-tumor immune responses. Therefore, we investigated the immunogenicity of the altered CALR by employing in silico peptide binding prediction algorithms to interrogate the binding affinities of mutated CALR peptides to human leukocyte antigens (HLA). These analyses indicated that the mutated epitopes can bind to class I and II HLA alleles with high affinity supporting the hypothesis that mutant CALR could be immunogenic in vivo . We then tested the immunogenicity of the overlapping peptides spanning the mutated region in vitro by utilizing intracellular staining (ICS) and Enzyme-Linked ImmunoSPOT (ELISPOT) assays. Naive T cells derived from the peripheral blood mononuclear cells (PBMNCs) isolated from healthy donors displayed effector functions after priming with the mutated peptides but not with the corresponding wild type peptides . Incubation withthe mutated CALR peptides induced T cells proliferation, upregulation of CD137 (4-1BB) and production of interferon (IFN)-γ and tumor necrosis factor (TNF)-α. Moreover, as suggested by in silico peptide-HLA binding predictions, both CD8+ and CD4+ T cell precursors displayed increased IFN-γ production in response to stimulation with mutated CALR peptides. There was an overall 4±3-fold increase in IFN-γ production by CD4+ T cells and a 2.5±0.5-fold increase by CD8+ T cells after stimulation with mutated peptides as compared to control self-antigens (n = 14 healthy donors out of which 11 showed CD4+ and 5 showed CD8+ T cell responses). These results demonstrate that mutated CALR elicits T cell responses in vitro supporting its potential immunogenic functions in vivo . We next investigated the presence of mutant CALR-specific T cell immunity in MPN patients with CALR mutations. PBMNCs from a total of 18 CALR-mutated MPN patients with ET (7), MF (5) or MF arising from ET (6) were evaluated. MPN T cells were then stimulated with the pooled mutant CALR peptides and T cell responses were assessed by ELISPOT. Out of 18 patients, increased IFN-γ production upon stimulation was observed in only 2 patients (one with ET and one with ET-MF). The less than robust MPN T cell responses suggested that immune suppressive mechanisms might be operating. To this end, we evaluated the expression of cell surface inhibitory receptors, programmed cell death (PD)-1 and the cytotoxic T-lymphocyte-associated antigen (CTLA)-4, by T cells from MPN patients. Both CD8+ and CD4+ T cells from MPN patients exhibited increased levels of PD-1 and CTLA-4 when compared to the healthy controls (HC). For instance, 9.3±8.2% of MPN patients CD4+ T cells expressed PD-1 (n = 12) as compared to 2.5±1.7% of HC CD4+ T cells (n=10) (p value = 0.0482). Similarly, there were twice as many PD-1 expressing CD8+ T cells in MPN patients as compared to HC. We next assessed MPN T cell responses against mutated peptides in the presence of neutralizing PD-1 or CTLA-4 antibodies. PD-1 inhibition restored T cell immunity in cultures derived from 3 MPN patients and CTLA-4 inhibition restored response in one patient (n=16). Of note, following the PD-1 or CTLA-4 inhibition, the number of IFN-γ producing cells and the amount of IFN-γ produced on a per-cell-basis increased upon stimulation with mutant-CALR peptides as compared to control peptides. These results indicate that the limited MPN T cell responses to CALR mutated peptides were due to T cell exhaustion caused, at least in part, by activation of the PD-1/CTLA-4 pathway. In summary, our studies establish mutated CALR as a MPN-specific tumor antigen thereby providing a rationale for the development of immunotherapies targeting mutated CALR in patients. Moreover, our data indicate that immune checkpoint signaling might be responsible for T cell exhaustion in some MPN patients thereby supporting the use of checkpoint blockade inhibitors for treatment of these patients. Disclosures Mascarenhas: Incyte: Other: Clinical Trial Steering Committee , Research Funding; CTI Biopharma: Research Funding; Janssen: Research Funding; Merck: Research Funding; Novartis: Other: DSMB member , Research Funding; Promedior: Research Funding. Bhardwaj: Merck & Co., Inc.: Membership on an entity's Board of Directors or advisory committees; Dendreon Corporation: Membership on an entity's Board of Directors or advisory committees; Dynavax Technologies Corporation: Consultancy; Crucell: Patents & Royalties; Neostem, Inc.: Patents & Royalties.

Circulating Biomarkers of Response and Toxicity of Immunotherapy in Advanced Non-Small Cell Lung Cancer (NSCLC): A Comprehensive Review.

Simple SummaryAlthough immunotherapy has dramatically revolutionized non-small cell lung cancer (NSCLC) treatment, not all the patients will benefit from this innovative therapy. The identification of potential biomarkers able to predict efficacy and toxicity of immunotherapy represents an urgent need for tailored treatment regimens. Liquid biopsy is a minimally invasive and economical tool that could provide important information about patients’ selection and treatment monitoring. Currently, several blood biomarkers are under investigation (circulating immune and tumor cells, soluble immunological mediators, peripheral blood cells). Prospective clinical trials are needed to validate their use in clinical practice.Immune checkpoint inhibitors (ICIs) targeting the programmed cell death (PD)-1 protein and its ligand, PD-L1, and cytotoxic T-lymphocyte-associated antigen (CTLA)-4, have revolutionized the management of patients with advanced non-small cell lung cancer (NSCLC). Unfortunately, only a small portion of NSCLC patients respond to these agents. Furthermore, although immunotherapy is usually well tolerated, some patients experience severe immune-related adverse events (irAEs). Liquid biopsy is a non-invasive diagnostic procedure involving the isolation of circulating biomarkers, such as circulating tumor cells (CTC), cell-free DNA (cfDNA), and microRNAs (miRNAs). Thanks to recent advances in technologies, such as next-generation sequencing (NGS) and digital polymerase chain reaction (dPCR), liquid biopsy has become a useful tool to provide baseline information on the tumor, and to monitor response to treatments. This review highlights the potential role of liquid biomarkers in the selection of NSCLC patients who could respond to immunotherapy, and in the identification of patients who are most likely to experience irAEs, in order to guide improvements in care.

Uncoupling Therapeutic Efficacy from Immune-Related Adverse Events in Immune Checkpoint Blockade.

SummaryImmunotherapy with monoclonal antibodies targeting immune checkpoint molecules, including programmed death-1 (PD-1), PD ligand-1 (PD-L1), and cytotoxic T-lymphocyte-associated antigen (CTLA)-4, has become prominent in the treatment of many types of cancer. However, a significant number of patients treated with immune checkpoint inhibitors (ICIs) develop immune-related adverse events (irAEs). irAEs can affect any organ system, and although most are clinically manageable, irAEs can result in mortality or long-term morbidity. Factors that can predict irAEs remain elusive. Understanding the etiology of ICI-induced irAEs and ways to limit these adverse events are needed. In this review, we provide basic science and clinical insights on the mechanisms responsible for ICI efficacy and ICI-induced irAEs. We further provide insights into approaches that may uncouple irAEs from the ability of ICIs to kill tumor cells.

Abstract B072: Immune checkpoint blockade enhances mutated calreticulin-induced T-cell immunity in myeloproliferative neoplasms

Abstract Somatic mutations in the CALR gene are key drivers of cellular transformation in myeloproliferative neoplasms (MPN) and are the second most common driver mutations in essential thrombocythemia (ET) and myelofibrosis (MF) patients. CALR mutations are 1-bp frameshift mutations that result in the formation of a novel C-terminus with an identical 36-amino acid (aa) sequence that is shared by all CALR+ MPN patients. We hypothesized that the recurrence and uniformity of the novel C-terminus mark the mutated (mut)-CALR an attractive candidate as an MPN-specific tumor neoantigen that might elicit anti-tumor immune responses across patients who express this mutation. To assess the immunogenicity of mut-CALR neopeptide, we interrogated the binding affinity of epitopes from the altered C-terminus using in silico peptide binding prediction algorithms. The analysis showed that multiple epitopes could bind to class I and II human leukocyte antigens (HLA). We investigated whether we could elicit T-cell responses against mut-CALR neopeptide from CALR+ MPN patients. Peripheral blood mononuclear cells (PBMCs) from 18 MPN patients with ET (7), MF (4) or MF arising from ET (7) were stimulated in vitro with pooled overlapping long peptides (OLPs; 14-15 aa) spanning the mutated or wild type (WT) CALR C-terminus and T-cell responses were assessed by enzyme-linked immunospot assay (ELISPOT). There was a significant increase in interferon (IFN)-γ production upon stimulation with mut-CALR OLPs, whereas the corresponding WT sequence did not induce a T-cell response. Next, we evaluated whether mut-CALR-specific T-cells were undergoing exhaustion. Examination of the expression of checkpoint receptors on T-cells ex vivo determined that T-cells from peripheral blood of CALR+ MPN patients exhibited increased expression of multiple cell surface inhibitory molecules compared to healthy donor T-cells, including programmed cell death (PD)-1 and cytotoxic T lymphocyte-associated antigen (CTLA)-4. In order to address whether PD-1 and CTLA-4 overexpression suppressed mut-CALR-specific T-cell responses in CALR+ MPN patients, PD-1 or CTLA-4 signaling on T-cells was blocked through addition of monoclonal blocking antibodies. T-cell responses against mut-CALR peptides were recovered in 3 CALR+ MPN patients that were previously nonresponsive to mut-CALR stimulation. To gain further insights into checkpoint receptor-mediated T-cell suppression in CALR+ MPN patients, we investigated mut-CALR-specific T-cell responses in a CALR+ MPN patient receiving PD-1 inhibitor pembrolizumab, in our ongoing clinical trial (NCT03065400). We monitored the changes in the frequencies of peripheral blood T-cells in this patient before and after pembrolizumab treatment by both flow cytometry and T-cell receptor (TCR) sequencing. Prior to therapy, this patient had low proportion of T-cells (4.33% of PBMCs) and 32.3% of the T-cells expressed PD-1. After 2 cycles of pembrolizumab, the T-cell proportion greatly increased (26% of PBMCs) and remained high when measured after 6 cycles (18% of PBMCs). Sequencing of TCR Vβ chains of peripheral blood T-cells revealed that T-cells exhibited greater clonality after pembrolizumab treatment. Furthermore, we evaluated changes in mut-CALR-specific T-cell responses in this CALR+ MPN patient prior to and during pembrolizumab administration. At baseline, peripheral blood T-cells from this patient did not respond to stimulation with mut-CALR peptides. After pembrolizumab treatment, however, mut-CALR-specific T-cell responses were evident, as shown by increased IFN-γ production. Together, these results establish mut-CALR as a novel MPN-specific tumor shared-neoantigen and provide a strong rationale to study its immunogenic properties to inform the design of novel immunotherapies targeting mut-CALR in MPN patients carrying the mutation. Citation Format: Cansu Cimen Bozkus, Vladimir Roudko, John P. Finnigan, John Mascarenhas, Ronald Hoffman, Camelia Iancu-Rubin, Nina Bhardwaj. Immune checkpoint blockade enhances mutated calreticulin-induced T-cell immunity in myeloproliferative neoplasms [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B072.

Interleukin-37 Enhances the Suppressive Activity of Naturally Occurring CD4+CD25+ Regulatory T Cells.

Naturally occurring CD4+CD25+ regulatory T cells (Tregs) are essential for the suppression of autoimmunity and can control the immune-mediated pathology during the early phase of sepsis. Our previous data showed that silencing interleukin-37 (IL-37) in human CD4+CD25+ Tregs obviously reduced the suppressive activity of CD4+CD25+ Tregs. Here, we found that rhIL-37 stimulation markedly enhanced the suppressive activity of CD4+CD25+ Tregs isolated from naive C57BL/6 J mice in the absence or presence of lipopolysaccharide (LPS). Treatment with rhIL-37 could significantly upregulate the expression of cytotoxic T-lymphocyte-associated antigen (CTLA)-4 and forkhead/winged helix transcription factor p3 (Foxp3) on CD4+CD25+ Tregs. Also, rhIL-37 stimulation promoted the production of transforming growth factor-β1 (TGF-β1) but not IL-10 in the supernatants of cultured CD4+CD25+ Tregs. Pretreated CD4+CD25+ Tregs with rhIL-37 in the presence or absence of LPS were cocultured with CD4+CD25− T cells, ratio of IL-4/interferon-γ in the supernatants obviously increased in IL-37-stimulated groups. In addition, early administration of IL-37 significantly improved the survival rate of septic mice induced by cecal ligation and puncture. Taken together, we concluded that rhIL-37 enhances the suppressive activity of CD4+CD25+ Tregs and might be a potential immunomodulator for the treatment of septic complications.

Cutaneous adverse reactions to molecular targeted antitumor drugs and their management

There are three kinds of molecular targeted antitumor drugs: inhibitors of membrane-associated therapeutic targets, inhibitors of intracellular signaling pathways, and immunomodulators. Inhibitors of membrane-associated therapeutic targets include epidermal growth factor receptor inhibitors (EGFRIs), KIT and BCR-ABL inhibitors, antiangiogenic agents and multikinase inhibitors. Inhibitors of intracellular signals include inhibitors of the RAS-RAF-MEK-ERK pathway, PI3K-AKT-mTOR pathway and Hedgehog signaling pathway. Inhibitors of cytotoxic T lymphocyte associated-antigen (CTLA)and programmed death 1 (PD-1)belong to immunomodulatory agents. Cutaneous adverse effects of different molecular targeted antitumor drugs share some common features, but also differ from each other. Most of the side effects are dose-dependent and reversible. Management strategies should be adjusted according to the severity of skin eruptions. Dose tapering and even discontinuation of antitumor drugs are necessary for very severe cases, but for mild ones, symptomatic treatment might be enough. This article reviews cutaneous adverse reactions to molecular targeted therapy as well as their prevention and management. Key words: Molecular targeted therapy; Antineoplastic agents; Drug toxicity; Skin manifestations

Tuftsin-derived T-peptide prevents cellular immunosuppression and improves survival rate in septic mice

The primary mechanisms of sepsis induced cellular immunesuppression involve immune dysfunction of T lymphocytes and negative immunoregulation of regulatory T cells (Tregs). It has been found that tuftsin is an immune modulating peptide derived from IgG in spleen. T-peptide is one of tuftsin analogs. Herein, we examined the effect of T-peptide on cell-mediated immunity in the presence of lipopolysaccharide (LPS) and the survival rate in septic mice. T-peptide regulated the proliferative ability of CD4+CD25− T cells in dual responses. Meanwhile, 10 and 100 μg/ml T-peptides were able to enhance the apoptotic rate of CD4+CD25− T cells compared with 1 μg/ml T-peptide, but markedly lowered interleukin (IL)-2 levels. When CD4+CD25+ Tregs were treated with T-peptide for 24 hours, and co-cultured with normal CD4+CD25− T cells, the suppressive ability of CD4+CD25+ Tregs on CD4+CD25− T cells was significantly lowered, along with decreased expression in forkhead/winged helix transcription factor p-3 (Foxp-3) as well as cytotoxic T lymphocyte-associated antigen (CTLA)-4, and secretion of transforming growth factor (TGF)-β. Moreover, T-peptide has the ability to improve outcome of septic mice in a dose- and time- dependent manner, and associated with improvement in the microenvironment of cellular immunosuppression in septic mice.

Expression of IL-37 contributes to the immunosuppressive property of human CD4+CD25+ regulatory T cells.

Interleukin-37 (IL-37) possesses the function of down-regulate systemic and local inflammation. It is unknown whether IL-37 is expressed in human regulatory T cells (Tregs) and its role in modulating the immune response of Tregs. In the present study, cell surface molecules and secretory cytokines were analyzed in order to determine the function of IL-37 in regulating inhibitory effect of human CD4+CD25+Tregs. Meanwhile, the effects of IL-37 on T cell differentiation and proliferation as co-culture of CD4+CD25+Treg/CD4+CD25−T cell were also investigated. It was showed that IL-37 was expressed in cytoplasm of CD4+CD25+Tregs, and the levels of IL-37 were gradually elevated with the enhanced activity of CD4+CD25+Tregs. Secretory cytokines such as transforming growth factor (TGF)-β and interleukin (IL)-10, and expressions of cell surface molecules, including forkhead/winged helix transcription factor p3 (FOXP3) and cytotoxic T-lymphocyte associated antigen (CTLA)-4, were significantly decreased when IL-37 gene was silenced by siRNA. Furthermore, down-regulation of IL-37 expression in human CD4+CD25+Tregs obviously promoted proliferation of co-cultured T cell and differentiation, together with observably enhancement of IL-2 formation. These results demonstrated that IL-37 might manifest as a critical protein involving in immunosuppression of human CD4+CD25+Tregs.

IL-1R1 is expressed on both Helios(+) and Helios(-) FoxP3(+) CD4(+) T cells in the rheumatic joint.

Synovial fluid from rheumatic joints displays a well-documented enrichment of forkhead box protein 3 (FoxP3)(+) regulatory T cells (tissue Tregs ). However, we have previously demonstrated that the mere frequency of FoxP3 expressing cells cannot predict suppressive function. Instead, extrinsic factors and the functional heterogeneity of FoxP3(+) Tregs complicate the picture. Here, we investigated FoxP3(+) Tregs from blood and synovial fluid of patients with rheumatic disease in relation to Helios expression by assessing phenotypes, proliferative potential and cytokine production by flow cytometry. Our aim was to investigate the discriminatory potential of Helios when studying FoxP3(+) Tregs in an inflammatory setting. We demonstrate that the majority of the synovial FoxP3(+) CD4(+) T cells in patients with inflammatory arthritis expressed Helios. Helios(+) FoxP3(+) Tregs displayed a classical Treg phenotype with regard to CD25 and cytotoxic T lymphocyte-associated antigen (CTLA)-4 expression and a demethylated Treg -specific demethylated region (TSDR). Furthermore, Helios(+) FoxP3(+) T cells were poor producers of the effector cytokines interferon (IFN)-γ and tumour necrosis factor (TNF), as well as of the anti-inflammatory cytokine interleukin (IL)-10. The less abundant Helios(-) FoxP3(+) T cell subset was also enriched significantly in the joint, displayed a overlapping phenotype to the double-positive Treg cells with regard to CTLA-4 expression, but differed by their ability to secrete IL-10, IFN-γ and TNF upon T cell receptor (TCR) cross-linking. We also demonstrate a striking enrichment of IL-1R1 expression in synovial CD4(+) T cells that was restricted to the CD25-expressing FoxP3 population, but independent of Helios. IL-1R1 expression appears to define a tissue Treg cell phenotype together with the expression of CD25, glucocorticoid-induced TNF receptor family-related gene (GITR) and CTLA-4.

Regulatory T-cell deficiency and immune dysregulation, polyendocrinopathy, enteropathy, X-linked–like disorder caused by loss-of-function mutations in LRBA

A number of heritable immune dysregulatory diseases result from defects affecting regulatory T (Treg) cell development, function, or both. They include immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome, which is caused by mutations in forkhead box P3 (FOXP3), and IPEX-like disorders caused by mutations in IL-2 receptor α (IL2RA), signal transducer and activator of transcription 5b (STAT5b), and signal transducer and activator of transcription 1 (STAT1). However, the genetic defects underlying many cases of IPEX-like disorders remain unknown. We sought to identify the genetic abnormalities in patients with idiopathic IPEX-like disorders. We performed whole-exome and targeted gene sequencing and phenotypic and functional analyses of Treg cells. A child who presented with an IPEX-like syndrome and severe Treg cell deficiency was found to harbor a nonsense mutation in the gene encoding LPS-responsive beige-like anchor (LRBA), which was previously implicated as a cause of common variable immunodeficiency with autoimmunity. Analysis of subjects with LRBA deficiency revealed marked Treg cell depletion; profoundly decreased expression of canonical Treg cell markers, including FOXP3, CD25, Helios, and cytotoxic Tlymphocyte-associated antigen 4; and impaired Treg cell-mediated suppression. There was skewing in favor of memory Tcells and intense autoantibody production, with marked expansion of T follicular helper and contraction of T follicular regulatory cells. Whereas the frequency of recent thymic emigrants and the differentiation of induced Treg cells were normal, LRBA-deficient T cells exhibited increased apoptosis and reduced activities of the metabolic sensors mammalian target of rapamycin complexes 1 and 2. LRBA deficiency is a novel cause of IPEX-like syndrome and Treg cell deficiency associated with metabolic dysfunction and increased apoptosis of Treg cells.

Immunologic checkpoints in cancer therapy: focus on the programmed death-1 (PD-1) receptor pathway.

T-lymphocytes have the potential to recognize cancer antigens as foreign and therefore eliminate them. However, immune checkpoints such as cytotoxic T-lymphocyte-associated antigen (CTLA)-4 and programmed cell death (PD)-1 receptor and its ligands (PD-L1, PD-L2) suppress the activity of T-lymphocytes. Advances in the understanding of immunology and its role in cancer have led to the development of immune checkpoint inhibitors that block CTLA-4 and PD-1 and result in durable responses in patients with a wide range of cancers. PD-1 and PD-L1 inhibitors are currently in many stages of clinical investigation, and the anti-PD-1 antibody, pembrolizumab, was recently approved by the US Food and Drug Administration. Many questions remain to be answered, such as the optimal administration schedule, biomarkers that associate with benefit, and potential for use of PD-1 agents in combination approaches. Nonetheless, immunotherapy with PD-1 blocking antibodies is now becoming an integral part in the management of cancer.

Imaging manifestations of unusual side effects of new anticancer medications

Aim To illustrate imaging manifestations of unusual side effects of some new anti-cancer medications. Many of the new anti-cancer medications target specific biologic pathways and are expected to cause less adverse effects on healthy tissues. Some of these agents are associated with unusual side effects, recognition of which is clinically important. Ipilimumab is a monoclonal antibody against cytotoxic T lymphocyte-associated antigen (CTLA)-4 used for the treatment of metastatic melanoma. Though it has been shown to increase overall survival rates, it also results in various immune-related adverse effects, one of which is hypophysitis. Crizotinib is an anaplastic lymphoma kinase (ALK) and c-ros oncogene 1 inhibitor, approved for treatment of non-small cell lung carcinoma. Its side effects include the unusual formation of complex renal cysts which mimic renal abscesses or malignancy. In this presentation, we illustrate the imaging manifestations of Ipilimumab associated hypophysitis, early recognition of which is important as cessation of the drug in affected patients leads to resolution of hypophysitis, and also demonstrate complex renal cysts associated with the use of Crizotinib, knowledge of which is important to avoid misdiagnosis of renal abscess/ malignancy and consequent inappropriate treatment or stoppage of Crizotinib.

Ipilimumab induces simultaneous regression of melanocytic naevi and melanoma metastases

Ipilimumab blocks cytotoxic T-lymphocyte-associated antigen (CTLA)-4, potentiating the antimelanoma T-cell host response. Ipilimumab has been shown to improve overall survival in patients with previously treated metastatic melanoma. CTLA-4 antibodies generate immune responses to the melanoma-associated antigens Melan-A, NY-ESO-1 and glycoprotein (gp)100 in metastatic melanoma. Digital epiluminescence microscopy (DELM) is a noninvasive method permitting the monitoring of the morphology of melanocytic lesions over time. A 50-year-old man with metastatic melanoma received four ipilimumab injections after failure of dacarbazine chemotherapy. Positron emission tomography revealed regression of pulmonary metastases, and simultaneously, DELM showed regression of several melanocytic naevi. On histological examination of the regressing naevi, prominent CD8+, CD4+ and CD45R0 lichenoid lymphohistiocytic infiltrates were seen, whereas nonregressing naevi were almost free of inflammatory infiltrate. Expression of melanoma-associated antigens in benign melanocytic naevi may explain the induction of naevus regression by ipilimumab. DELM could represent a valuable noninvasive method to monitor ipilimumab efficacy.

Regulatory effect of tumor necrosis factor-α induced protein 8 like-2 expression on immunosuppressive activity of CD4 + CD25 + regulatory T cells in mice

Objective To observe the expression of tumor necrosis factor-α induced protein 8 like-2 (TIPE2) in CD4 + CD25 + regulatory T cells ( CD4 + CD25 + Tregs) and analyze its potential effect on immunosuppressive activity of CD4 + CD25 + Tregs. Methods CD4 + CD25 + Tregs were purified from spleen of the BALB/c mice by using magnetic cell sorting system.The expressions of TIPE2 mRNA and protein in CD4 + CD25 + Tregs were detected by using RT-PCR and Western blot.CD4 + CD25 +Tregs were further infected with the recombinant lentiviruses that carried small interference RNA(siRNA)to knock down the TIPE2 expression.Based on the expressions of cell surface molecules including cytotoxic T-lymphocyte-associated antigen (CTLA)-4 and forkhead/winged helix transcription factor p3 (Foxp3) detected by flow cytometry and the levels of cytokines including interleukin (IL)-10 and transforming growth factor (TGF)-3 examined by ELISA in CD4 + CD25 + Tregs,the functional role of TIPE2 in controlling suppressive activity of CD4 + CD25 + Tregs was analyzed.In the meantime,the proliferation activities of T effector cells were assayed by MTT test. Results A 147 bp TTPE2 gene band and a clear TIPE2 band with a molecular mass of approximately 21 000 in CD4 + CD25 + Tregs were detected by using Westem blot.Cell surface molecule as well as cytokine expressions were significantly down-regulated when the CD4 + CD25 + Treg stimulated and activated by anti-CD3/CD28 was cultured for 24 hours after the siRNA silenced CD4 + CD25 + Treg cells ( P ＜ 0.01 ).Meanwhile,the suppression role of CD4 +CD25 + Tregs on the proliferation activity of T effector cells was weakened obviously ( P ＜ 0.05 ). Conclusions As an important immunoregulatory molecule,TIPE2 not only expresses in the CD4 + CD25 +Tregs,but also affects the immunosuppressive function of CD4 + CD25 + Tregs. Key words: Immunity, cellular; Tumor necrosis factor alpha; Mice; Protein 8 like-2

Expression of tumor necrosis factor-α induced protein 8 like-2 contributes to the immunosuppressive property of CD4+CD25+ regulatory T cells in mice

Tumor necrosis factor-α induced protein 8 like-2 (TNFAIP8L2, TIPE2), a lately discovered negative regulator of innate immunity and cellular immunity, shares considerable sequence homology with members of the tumor necrosis factor-α induced protein 8 (TNFAIP8) family. It is preferentially expressed in lymphoid-derived and marrow-derived cells. However, it is unclear whether TIPE2 is expressed in the regulatory T cells (Tregs) to contribute to its negative regulatory property in immune response. The present study was designed to examine whether naturally occurring CD4(+)CD25(+) Tregs isolated from murine spleens expressed TIPE2 by the use of Western blot and reverse transcription-polymerase chain reaction (RT-PCR). Based on the expression of cell surface molecules, including cytotoxic T-lymphocyte-associated antigen (CTLA)-4 and forkhead/winged helix transcription factor p3 (Foxp3) on CD4(+)CD25(+) Tregs, and cytokines including interleukin (IL)-10 as well as transforming growth factor (TGF)-β were analyzed to investigate the functional role of TIPE2 in controlling suppressive activity of CD4(+)CD25(+) Tregs. Meanwhile, IL-2, the ratio of interferon (IFN)-γ/IL-4 in CD4(+)CD25(+)Treg/CD4(+)CD25(-) T cell coculture supernatant and nuclear factor of activated T cells (NF-AT) activation in T lymphocytes were determined to examine the effects of TIPE2 on the T-cell proliferation and differentiation induced by CD4(+)CD25(+) Tregs. It was found that TIPE2 was a cytoplasmic protein expressed in CD4(+)CD25(+) Tregs, and cell surface molecules as well as cytokines (IL-10, TGF-β) expressions were significantly down-regulated when TIPE2 gene silenced by siRNA. On the other hand, CD4(+)CD25(+) Tregs treated with TIPE2 knock-down promoted T-cell proliferation as well as differentiation, and markedly upregulated IL-2 expression and intranuclear NF-AT activation. The results suggested that TIPE2 appeared to be a critical immunoregulatory molecule involved in immunosuppressive function of CD4(+)CD25(+) Tregs.