Mechanisms of checkpoint inhibition-induced adverse events.

Immune checkpoint inhibition has revolutionized the treatment of several solid cancers, most notably melanoma and non-small-cell lung cancer (NSCLC). Drugs targeting cytotoxic T lymphocyte antigen (CTLA)-4 and programmed cell death 1 (PD-1) have made their way into routine clinical use; however, this has not been without difficulties. Stimulation of the immune system to target cancer has been found to result in a reduction of self-tolerance, leading to the development of adverse effects that resemble autoimmunity. These adverse effects are erratic in their onset and severity and can theoretically affect any organ type. Several mechanisms for immune-related toxicity have been investigated over recent years; however, no consensus on the cause or prediction of toxicity has been reached. This review seeks to examine reported evidence for possible mechanisms of toxicity, methods for prediction of those at risk and a discussion of future prospects within the field.

Neuroimmune interactions: how the nervous and immune systems influence each other.

In recent years, researchers worldwide have expanded our understanding of how, and the degree to which, the immune system interacts with the nervous system, and vice versa. In this issue of Clinical & Experimental Immunology, we are pleased to present our new Review Series: 'Neuroimmune interactions: how the nervous and immune systems influence each other', a collection of four Review articles commissioned by Leonie S. Taams from leading researchers in this exciting interdisciplinary field. The collection covers key technical, experimental and clinical findings in the fast-developing field of neuroimmunology.

Comparative analysis of immune cell subsets in peripheral blood from patients with periodontal disease and healthy controls.

Periodontitis is a chronic inflammatory disease caused by the colonization of teeth by the bacterial plaque biofilm and the resultant host immune responses in adjacent periodontal tissues. Disease severity can vary dramatically between patients with periodontitis, with some subjects displaying inflammation without bony destruction (gingivitis), while others experience chronic progressive or rapidly aggressive gingival connective tissue damage and bone loss. To determine whether peripheral immune dysregulation is associated with periodontitis, we performed extensive analysis of immune cell subsets in peripheral blood from patients with chronic or aggressive periodontitis versus periodontally healthy control subjects. Peripheral blood mononuclear cells (PBMC) from patients with chronic periodontitis or aggressive periodontitis and from periodontally healthy controls were analysed by 8-10-colour flow cytometry for the frequencies of various lymphocyte subsets, including interleukin (IL)-17-, interferon (IFN)-γ-, tumour necrosis factor (TNF)-α- and IL-10-producing cells, and the frequencies and phenotype of monocytes. Cytokine levels in serum from the different groups were determined by Luminex assay. We found no significant differences in the frequencies of major immune cell populations [CD4+ T cells, CD8+ T cells, γδ T cells, CD4+ CD45RO+ CD25+ CD127low regulatory T cells (Tregs ), CD19+ B cells, CD14+ monocytes] or of cytokine-producing T cells, or in the phenotype of CD14+ monocytes in peripheral blood from these patient cohorts. Additionally, no significant differences were observed in serum levels of prototypical inflammatory cytokines. These results suggest that the local gingival inflammatory response is not reflected by obvious changes in major blood immune cell subset frequencies.

A randomized controlled trial protocol assessing the effectiveness, safety and cost-effectiveness of methotrexate vs. ciclosporin in the treatment of severe atopic eczema in children: the TREatment of severe Atopic eczema Trial (TREAT).

BACKGROUND: Oral systemic immunomodulatory medication is regularly used off-licence in children with severe atopic eczema. However, there is no firm evidence regarding the effectiveness, safety, cost-effectiveness and impact on quality of life from an adequately powered randomized controlled trial (RCT) using systemic medication in children. OBJECTIVES: To assess whether there is a difference in the speed of onset, effectiveness, side-effect profile and reduction in flares post-treatment between ciclosporin (CyA) and methotrexate (MTX), and also the cost-effectiveness of the drugs. Treatment impact on quality of life will also be examined in addition to whether FLG genotype influences treatment response. In addition, the trial studies the immune-metabolic effects of CyA and MTX. METHODS: Multicentre, parallel group, assessor-blind, pragmatic RCT of 36 weeks' duration with a 24-week follow-up period. In total, 102 children aged 2-16 years with moderate-to-severe atopic eczema, unresponsive to topical treatment will be randomized (1 : 1) to receive MTX (0·4 mg kg-1 per week) or CyA (4 mg kg-1 per day). RESULTS: The trial has two primary outcomes: change from baseline to 12 weeks in Objective Severity Scoring of Atopic Dermatitis (o-SCORAD) and time to first significant flare following treatment cessation. CONCLUSIONS: This trial addresses important therapeutic questions, highlighted in systematic reviews and treatment guidelines for atopic eczema. The trial design is pragmatic to reflect current clinical practice.

Inflammatory cytokines compromise programmed cell death-1 (PD-1)-mediated T cell suppression in inflammatory arthritis through up-regulation of soluble PD-1.

The programmed cell death 1 (PD-1) receptor plays a major role in regulating T cell activation. Our aim was to determine how inflammation influences PD-1-mediated T cell suppression. Flow cytometry analysis of rheumatoid arthritis (RA) and psoriatic arthritis (PsA) synovial fluid (SF) mononuclear cells showed an increase in the percentage of PD-1+ cells within the CD4+ and CD8+ T cell compartment compared to paired peripheral blood (PB). Upon in-vitro T cell receptor (TCR) stimulation of healthy control (HC) CD4+ T cells in the presence of plate-bound PD-L1fc chimera, significantly decreased proliferation and interferon (IFN)-γ secretion was observed. In contrast, CD4+ T cells from RA and PsA PB and SF appeared resistant to such PD-1-mediated inhibition. Addition of the proinflammatory cytokines tumour necrosis factor (TNF)α, interleukin (IL)-6 and IL-1ß, which were increased in RA and PsA SF compared to osteoarthritis (OA) SF, consistently abrogated PD-1-mediated suppression in HC CD4+ T cell cultures. This effect was reversed by inhibitors of these cytokines. Soluble PD-1 (sPD-1) levels were increased in cell culture supernatants from TNFα and IL-6-stimulated cultures compared to untreated controls, and also in RA and PsA, but not in OA, serum and SF. Functionally, addition of sPD-1fc counteracted PD-1-mediated suppression of HC CD4+ T cells, and increased T cell proliferation in HC CD4+ T cell/monocyte co-cultures. These in-vitro findings indicate that CD4+ T cells from patients with RA and PsA show increased resistance to PD-1-mediated suppression, which may be explained in part by the presence of soluble PD-1 in the inflammatory environment.

Contribution of the IL-17 Pathway to Psoriasis and Psoriatic Arthritis.

Investigators have accrued compelling evidence that the IL-17 pathway is central to the pathogenesis of psoriasis and psoriatic arthritis. The evidence comprises genome-wide association studies (GWAS), data from experimental murine models and findings from in vitro studies on patients' cells or tissue biopsies. More recently, the success of drugs blocking the IL-17 pathway in treating both psoriasis (PsO) and psoriatic arthritis (PsA) confirms that IL-17 is a clinically relevant therapeutic target. However, there remain many unanswered questions: is PsA simply an extension of PsO from the skin to the synovial tissue or are there differences in the underlying pathogenesis of these diseases? Which cell type represents the primary source of IL-17 in PsO and PsA? And how are these cells regulated? This review outlines the IL-17 pathway, summarises the evidence supporting its role in PsO and PsA and discusses recent data that may help to address these yet unresolved questions.

Enhanced and persistent levels of interleukin (IL)-17⁺ CD4⁺ T cells and serum IL-17 in patients with early inflammatory arthritis.

Prognosis of patients with early inflammatory arthritis (EIA) is highly variable. The aim of this study was to compare, longitudinally and cross-sectionally, the levels of cytokine-expressing cells in peripheral blood (PB) from patients with EIA to those in established rheumatoid arthritis (RA) and healthy controls (HC). PB mononuclear cells from HC (n = 30), patients with EIA (n = 20) or RA (n = 38) were stimulated with phorbol myristate acetate (PMA)/ionomycin for 3 h, and stained for cell markers and cytokines. Serum cytokines and chemokines were measured by Luminex. Patients with EIA were reassessed at 6 and 12 months. The percentage of interleukin (IL)-17⁺ interferon (IFN)-γ⁻ CD4⁺ T cells [T helper type 17 (Th17)] was increased in RA and EIA versus HC. Serum IL-1ß, IL-2, IL-4 IL-17 and macrophage inflammatory protein (MIP)-1α were increased in RA and EIA versus HC. IL-1Ra, IL-15 and IFN-α were increased in EIA versus HC. IL-6 and tumour necrosis factor (TNF)-α was increased in RA but not EIA versus HC. Disease activity scores in EIA patients improved over 12 months' treatment. Th17 percentage at baseline was correlated with both rheumatoid factor (RF) titre and functional deficit at 12 months. Baseline levels of serum granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-6 and IL-8 were correlated with Larsen score at 12 months. There were no significant changes in cytokine-expressing CD4⁺ T cells over time, although the percentage of IL-6⁺monocytes increased. IL-17⁺ CD4⁺ T cells and serum IL-17 levels are increased in EIA. IL-6-expressing monocytes increase during the first year of disease, irrespective of disease-modifying anti-rheumatic drug (DMARD) therapy. We observed incomplete clinical responses, suggesting EIA patients need more intensive early therapy.

Harnessing engineered antibodies of the IgE class to combat malignancy: initial assessment of FcɛRI-mediated basophil activation by a tumour-specific IgE antibody to evaluate the risk of type I hypersensitivity.

BACKGROUND: IgE antibodies, sequestered into tissues and retained locally by the high-affinity IgE receptor, FcɛRI, on powerful effector cells such as mast cells, macrophages and eosinophils, may offer improvements in the therapy of solid tumours. The chimeric antibody, MOv18 IgE, against the human ovarian carcinoma antigen, folate receptor α (FRα), is more effective than its IgG1 counterpart in xenograft models of ovarian cancer. Although MOv18 IgE binds to a single epitope on FRα and cannot cross-link IgE receptors on basophils, there remains a risk that components in the circulation of ovarian cancer patients might cross-link FRα-MOv18-IgE-receptor-FcɛRI complexes on basophils to cause type I hypersensitivity. OBJECTIVE: To assess the propensity for MOv18 used in a therapeutic setting to cause FcɛRI-mediated type I hypersensitivity. METHODS: As validated readouts of the potential for MOv18 to cause FcɛRI-mediated type I hypersensitivity we measured release of a granule-stored mediator from a rat basophilic leukaemia cell line RBL SX-38 stably transfected with human tetrameric (αßγ2) FcɛRI, and induction of CD63 on blood basophils from patients with ovarian carcinoma and healthy controls ex vivo. RESULTS: Serum FRα levels were increased in ovarian cancer patients compared with healthy controls. MOv18 IgE alone, or in the presence of its antigen recombinant human FRα, or of healthy volunteer (n=14) or ovarian carcinoma patient (n=32) sera, did not induce RBL SX-38 cell degranulation. Exposure to FRα-expressing ovarian tumour cells at target-to-effector ratios expected within tumours induced degranulation. MOv18 IgE did not induce expression of CD63 in blood basophils from either healthy volunteers (n=6), or cancer patients, despite detectable levels of circulating FRα (n=5). CONCLUSION AND CLINICAL RELEVANCE: These encouraging data are compatible with the hypothesis that, when ovarian carcinoma patients are treated with MOv18, FcɛRI-mediated activation of effector cells occurs within the tumour mass but not in the circulation mandating, with due caution, further pre-clinical studies.

Translational mini-review series on Th17 cells: CD4 T helper cells: functional plasticity and differential sensitivity to regulatory T cell-mediated regulation.

CD4(+) T cells display considerable flexibility in their effector functions, allowing them to tackle most effectively the range of pathogenic infections with which we are challenged. The classical T helper (Th) 1 and Th2 subsets have been joined recently by the Th17 lineage. If not controlled, the potent effector functions (chiefly cytokine production) of which these different cells are capable can lead to (sometimes fatal) autoimmune and allergic inflammation. The primary cell population tasked with providing this control appears to be CD4(+) regulatory T (T(reg)) cells expressing the forkhead box P3 (FoxP3) transcription factor. Here we consider the comparative capacity of FoxP3(+) T(regs) to influence the polarization, expansion and effector function of Th1, Th2 and Th17 cells in vitro and in vivo as well as in relation to human disease. This remains a particularly challenging series of interactions to understand, especially given our evolving understanding of T(reg) and T effector interrelationships, as well as recent insights into functional plasticity that cast doubt upon the wisdom of a strict categorization of T effector cells based on cytokine production.

Peripheral generation and function of CD4+CD25+ regulatory T cells.

The balance between immunity and tolerance is important to maintain immune homeostasis. Several mechanisms are in place to ensure that the immune response is controlled, such as T cell anergy, apoptosis and immune ignorance. A fourth mechanism of peripheral tolerance is the active suppression by regulatory or suppressor T cells. The existence of suppressor T cells was first described in the early 1970s, but these cells became discredited in the 1980s. The work of Shimon Sakaguchi and others, however, has brought these cells back into the limelight and nowadays research into regulatory/suppressor T cells is a very active field of immunology. Different types of regulatory T cells have been described, including CD4+CD25+ T cells that constitutively express CTLA-4, GITR and Foxp3, TGF-beta producing Th3 cells, IL-10 producing Tr1 cells, and CD8+CD28- T cells. This review will focus on the generation and function of CD4+CD25+ regulatory T cells. CD4+CD25+ regulatory cells were originally described as thymus-derived anergic/suppressive T cells. Recent papers, however, indicate that these cells might also be generated in the periphery. CD4+CD25+ regulatory T cells can be activated by self-antigens and non-self-antigens, and once activated can suppress T cells in an antigen nonspecific manner. Interestingly, the suppressive effects of these cells are not restricted to the adaptive immune system (T and B cells) but can also affect the activation and function of innate immune cells (monocytes, macrophages, dendritic cells). These features make the CD4+CD25+ regulatory T cell subset an interesting target for immunotherapy of chronic inflammatory or autoimmune diseases.

Anergic T cells as active regulators of the immune response.

T cell anergy is one of the mechanisms leading to the establishment and maintenance of peripheral tolerance. Recent data from our and other laboratories indicate that anergic T cells are not functionally inert but in fact are capable of regulating the immune response in an active manner. In this review, we describe our viewpoint on how anergic self-reactive T cells could contribute to regulation of the immune response.

Antigen presentation by T cells versus professional antigen-presenting cells (APC): differential consequences for T cell activation and subsequent T cell-APC interactions.

We compared the effects of antigen (Ag) presentation by T cells and professional antigen-presenting cells (APC) on T cell proliferation, cytokine production and surface molecule expression. Ag presentation by T cells (T-T presentation) induced an initial T cell activation phase as measured by proliferation and IL-2 production. These activated T cells became anergic upon antigenic restimulation by professional APC, as shown by a failure to proliferate or produce IL-2 or IFN-gamma. Interestingly, such T cells were not intrinsically defective in their signal transduction pathways since they did proliferate and produce cytokines upon restimulation with mitogenic stimuli. Flow cytometric analysis revealed a more profound TCR and CD3 down-regulation during T-T presentation than during APC-T presentation. However, no up-regulation of CD80, CD86, CD45RC and OX40 (CD134) was observed on T cells during T-T presentation or subsequent antigenic restimulation of anergic T cells in the presence of professional APC, whereas increased expression of these molecules was observed during professional APC-T presentation of non-anergic T cells. The impaired expression of co-stimulatory and activation molecules on T cells after T-T presentation of Ag might lead to altered interactions between T cells and professional APC upon antigenic restimulation. We propose that T cell anergy is a functional consequence of these altered T cell-APC interactions.

Dose-dependent induction of distinct anergic phenotypes: multiple levels of T cell anergy.

T cell anergy has been proposed as one of the mechanisms underlying peripheral T cell tolerance. In recent years, the functional relevance of T cell anergy has been studied extensively in vitro and in vivo, using different species, cell systems, and ways to induce anergy. Although these studies concurred about the induction of unresponsiveness, conflicting findings were obtained with respect to the function of anergic T cells and to the persistence of T cell anergy. In the present study, T cell anergy was induced through T-T presentation of the specific Ag by rat MHC class II+ T cells in the absence of professional APC. We show that, depending on the Ag dose with which T cells were incubated, distinct anergic phenotypes were induced. Incubation of T cell clones with a low (suboptimal) Ag dose induced hyporesponsiveness. Incubation with a higher (optimal) Ag dose induced an anergic state capable of exerting immunoregulatory effects. Incubation with a high (supraoptimal) Ag dose led to an anergic suppressive phenotype that was persistent and was not reversed by APC, Ag, and rIL-2. These findings demonstrate that T cell anergy is not confined to a single state of functional inactivation. Instead, multiple levels of T cell anergy exist. Thus, anergic T cells can contribute to the regulation of the immune response either in a persistent and active manner or in a passive manner, depending on their level of T cell anergy.

Phenotypic analysis of IL-10-treated macrophages using the monoclonal antibodies RFD1 and RFD7.

Suppressive or tolerogenic antigen-presenting cells (APC) might play an important role in the control of auto/hyperreactivity and the resolution of the immune response. Recent studies have provided evidence that tolerogenic APC can be induced by anergic T cells or interleukin-10 (IL-10). The aim of this study is to investigate how anergic T cells and IL-10 induce the suppressive APC phenotype and how this affects the immune response. Previously, two monoclonal antibodies (RFD1 and RFD7) were described by our lab which distinguish inductive (RFD1+RFD7-), phagocytic (RFD1-RFD7+) and suppressive (RFD1+RFD7+) macrophages. RFD1 recognizes an MHC class II-associated epitope which has restricted expression, and RFD7 recognizes a predominantly cytoplasmic antigen. Macrophages were derived from the adherent fraction of peripheral blood mononuclear cells from healthy donors. At day 5, IL-10 or IFNgamma (a cytokine which should lead to the inductive APC phenotype) was added to the cultures. At day 7, the macrophages were harvested and their phenotypes were assessed by immunohistochemical staining and FACS analysis. Upon culture of macrophages with IL-10 RFD1 staining and HLA class II expression were reduced, whereas RFD7 staining was increased. Incubation of APC with IFNgamma led to upregulation of RFD1 and HLA class II, without affecting RFD7 staining. This suggests that IL-10 induced the suppressive RFD1+RFD7+ APC population, whereas IFNgamma treatment led to the inductive RFD1+RFD7- APC subset. Thus the use of IL-10 and/or IFNgamma, and the discrimination offered by mAbs RFD7 and RFD1 represent a model whereby APC function in terms of T cell stimulation or T cell anergy can be assessed.

'Anergic' T cells modulate the T-cell activating capacity of antigen-presenting cells.

Nowadays there is compelling evidence for immunoregulation by T cells. Recently, we showed that so-called 'anergic' T cells are not functionally inert but can act as regulatory cells by actively suppressing other T cell responses. We now show that 'anergic' T cells mediate this suppressive effect via modulation of the T-cell activating capacity of the antigen-presenting cell (APC). Upon removal of the 'anergic' T cells, the suppressive APC phenotype persisted, indicating that 'anergic' T cells conditioned the APC to become a mediator of T cell suppression. The inhibitory signal delivered by 'anergic' T cells depended on the presence of the cognate ligand for the 'anergic' T cell, and appeared to be dominant since previously activated APC were rendered inhibitory as well. These findings imply that APC upon cross-talk with T cells can adopt distinct functional phenotypes ranging from T-cell stimulatory to T-cell suppressive. The contribution of 'anergic' T cells to the functional tuning of APC offers an explanation for the maintenance of 'anergic' T cells in the repertoire, and for their role in immunoregulation.

A self-hsp60 peptide acts as a partial agonist inducing expression of B7-2 on mycobacterial hsp60-specific T cells: a possible mechanism for inhibitory T cell regulation of adjuvant arthritis?

We previously reported that resistance to the induction of adjuvant arthritis after preimmunization with mycobacterial hsp60 was mediated by T cells recognizing a conserved epitope (M256-270) of mycobacterial hsp60. These T cells were cross-reactive with the homologous rat hsp60 peptide sequence and the natural self-epitope on stressed antigen-presenting cells. Recognition of peptide M256-265, the conserved core of peptide M256-270, was shown to be essential for the generation of self-reactive T cells. The rat homologue of peptide M256-265, peptide R256-265, differs with three conservative amino acid substitutions from the mycobacterial core peptide. Thus peptide R256-265 could act as an altered peptide ligand with the potential of inducing a different functional phenotype in M256-270-specific T cells. We now show that peptide R256-265 was recognized by M256-270-specific T cells as a partial agonist, inducing TCR down-regulation and up-regulation of activation/adhesion molecules in the absence of proliferative responses. Peptide R256-265 did not induce anergy but induced B7-2 (but not B7-1) expression on M256-270-specific T cells, as opposed to the mycobacterial peptide, which preferentially induced B7-1. These effects were more pronounced at low peptide concentrations. Therefore also in vivo at the more relevant low physiological level of expression, the self-hsp could induce such phenotype. It is discussed how this selective up-regulation of B7-2 expression on (self-hsp60) autoreactive T cells might be a way by which destructive autoimmune responses are controlled.

Competition for antigenic sites during T cell proliferation: a mathematical interpretation of in vitro data.

By fitting different mathematical T cell proliferation functions to in vitro T cell proliferation data, we studied T cell competition for stimulatory signals. In our lymphocyte proliferation assays both the antigen (Ag) availability and the concentration of T cells were varied. We show that proliferation functions involving T cell competition describe the data significantly better than classical proliferation functions without competition, thus providing direct evidence for T cell competition in vitro. Our mathematical approach allowed us to study the nature of T cell competition by comparing different proliferation functions involving (i) direct inhibitory T-T interactions, (ii) Ag-specific resource competition, or (iii) resource competition for nonspecific factors such as growth factors, and access to the surface of Ag-presenting cells (APCs). We show that resource competition is an essential ingredient of T cell proliferation. To discriminate between Ag-specific and nonspecific resource competition, the Ag availability was varied in two manners. In a first approach we varied the concentration of APCs, displaying equal ligand densities; in a second approach we varied the Ag density on the surface of the APCs, while keeping the APC concentration constant. We found that both resource competition functions described the data equally well when the Ag availability was increased by adding APCs. When the APC concentration was kept constant, the nonspecific resource competition function yielded the best description of the data. Our interpretation is that T cells were competing for "antigenic sites" on the APCs.