John Cardone

Complement regulator CD46 temporally regulates cytokine production by conventional and unconventional T cells

In this study we demonstrate a new form of immunoregulation: engagement on CD4+ T cells of the complement regulator CD46 promoted the effector potential of T helper type 1 cells (TH1 cells), but as interleukin 2 (IL-2) accumulated, it switched cells toward a regulatory phenotype, attenuating IL-2 production via the transcriptional regulator ICER/CREM and upregulating IL-10 after interaction of the CD46 tail with the serine-threonine kinase SPAK. Activated CD4+ T cells produced CD46 ligands, and blocking CD46 inhibited IL-10 production. Furthermore, CD4+ T cells in rheumatoid arthritis failed to switch, consequently producing excessive interferon-γ (IFN-γ). Finally, γδ T cells, which rarely produce IL-10, expressed an alternative CD46 isoform and were unable to switch. Nonetheless, coengagement of T cell antigen receptor (TCR) γδ and CD46 suppressed effector cytokine production, establishing that CD46 uses distinct mechanisms to regulate different T cell subsets during an immune response.

The Th1 life cycle: molecular control of IFN-γ to IL-10 switching

Control of IFN-γ-secreting T helper (Th) 1 cells prevents autoimmunity and immunopathology during infection. IL-10-mediated suppression of Th1 cells is achieved not only through IL-10 produced extrinsically, but also through a negative feedback loop that induces intrinsic IL-10 expression in cells also expressing IFN-γ, during Th1 lineage differentiation. Targeting this Th1 cell IFN-γ to IL-10 switching is a tantalising prospect for developing therapeutics for Th1-mediated diseases. In this review, the molecular pathways that regulate IFN-γ versus IL-10 expression in Th1 cells are examined, with focus on the role of complement regulator and T cell co-stimulatory molecule CD46, and also discussed are challenges and controversies in the field.

Dysfunctional CD39(POS) Regulatory T Cells and Aberrant Control of T-Helper Type 17 Cells in Autoimmune Hepatitis

Autoimmune hepatitis (AIH) is an important cause of severe liver disease and is associated with both quantitative and qualitative regulatory T‐cell (Treg) impairments. Tregs express CD39, an ectonucleotidase responsible for extracellular nucleotide hydrolysis, culminating in the production of immunosuppressive adenosine. Here, we describe multiple CD39pos Treg defects that potentially contribute to the impaired immunoregulation that is characteristic of AIH. We have examined the frequency and phenotype of CD39pos Tregs by flow cytometry and measured their ectonucleotidase activity. The capacity of CD4posCD25high, CD4posCD25highCD39pos, and CD4posCD25highCD39neg subsets to suppress both proliferation of effector T cells and interleukin (IL)‐17 production was evaluated. In AIH, CD39pos Tregs are decreased in frequency, exhibit limited adenosine triphosphate/adenosine diphosphate hydrolysis activity, and fail to suppress IL‐17 production by effector CD4 T cells. Moreover, these CD39pos Tregs display a more proinflammatory profile in AIH, which is characterized by elevated CD127 positivity, and a greater propensity to produce interferon‐gamma or IL‐17 upon challenge with proinflammatory stimuli. Conclusions: In AIH, CD39pos Tregs are decreased in number, fail to adequately hydrolyze proinflammatory nucleotides and do not efficiently suppress IL‐17 production by effector CD4 T cells. CD39pos Tregs show plasticity and are unstable upon proinflammatory challenge, suggesting that defective immunoregulation in AIH might result not only from reduced Treg number and function, but also from increased conversion of Tregs into effector cells. (Hepatology 2014;59:1007–1015)

CD46 in innate and adaptive immunity: an update

CD46 was discovered in 1986 during a search for novel C3b‐binding proteins. CD46 is expressed ubiquitously and functions as a co‐factor in the factor I‐mediated proteolytic cleavage of C3b and C4b. Its vital role in preventing complement deposition on host tissue is underpinned by the fact that deficiency of CD46 is a predisposing factor for numerous disease conditions arising from complement‐mediated ‘self‐attack’. However, in the last 10 years, it has become apparent that CD46 is also heavily involved in a new and somewhat surprising functional aspect of the complement system: the down‐modulation of adaptive T helper type 1 (Th1) immune responses by regulating the production of interferon (IFN)‐γversus interleukin (IL)‐10 within these cells. Specifically, this latter function of CD46 is a tantalizing discovery – it may not only have delivered the explanation as to why so many pathogens use and abuse CD46 as cell entry receptor but clearly has important clinical implications for the better understanding of Th1‐mediated disease states and novel therapeutic approaches for their amelioration. Here, we summarize and discuss the current knowledge about CD46 and its expanding roles in the immune system.

In autoimmune hepatitis type 1 or the autoimmune hepatitis–sclerosing cholangitis variant defective regulatory T‐cell responsiveness to IL‐2 results in low IL‐10 production and impaired suppression

Defective immune regulation plays a permissive role enabling effector cells to initiate and perpetuate tissue damage, eventually resulting in autoimmune disease. Numerical and functional regulatory T‐cell (Treg) impairment has been previously reported in autoimmune liver disease (AILD; including autoimmune hepatitis and autoimmune sclerosing cholangitis ASC). However, in these early reports, Tregs were phenotypically defined as CD4+CD25+ or CD4+CD25high cells. In the current study, we reexamined phenotypic and functional properties of Tregs by adopting a more refined definition of these cells that also includes negativity or low level of expression of CD127. We studied 43 AILD patients and 22 healthy subjects (HSs) and found that CD4+CD25+CD127− Tregs were decreased in the former. This decrease was more marked in patients with active disease than in those in remission. In AILD, Treg frequencies correlated inversely with parameters of disease activity and were not affected by immunosuppressive treatment. We also document, for the first time, that, in AILD, bona‐fide Tregs produce less interleukin (IL)−10 and are impaired in their ability to suppress CD4+CD25− target cell proliferation, a feature that in HSs, but not in AILDs, is dependent, at least in part, on IL‐10 secretion. Decreased IL‐10 production by Tregs in AILD is linked to poor responsiveness to IL‐2 and phospho signal transducer and activator of transcription 5 up‐regulation. Conclusion: Tregs are numerically impaired in AILD, this impairment being more prominent during active disease. Notably, defective IL‐10 production, resulting from low Treg responsiveness to IL‐2, contributes to Treg functional impairment. (Hepatology 2015;62:863–875)

A Novel Role for CD46 in Wound Repair

The intestinal epithelium not only provides a vital physical barrier between the host and environment but is also required for uptake of nutrients and the induction of tolerance against commensals. Deregulation of any of these functions leads to several disease states including chronic infection, inflammatory bowel disease, and cancer. Here, we describe a novel role for the complement regulator CD46 in the regulation of intestinal epithelial cell (IEC) barrier function. We found that CD46 directly interacts in IECs with the cytoplasmic kinase SPAK and with transmembrane E-cadherin, both proteins necessary for epithelial cell junction and barrier formation. Further, CD46 activation on Caco-2 cells induced rapid and significant decrease in transepithelial resistance with concomitant increase in paracellular permeability. Importantly, though CD46 activation of IEC layers allowed for increased transgression of pathogenic E. coli, it also increased epithelial cell proliferation and accelerated wound repair. These data suggest a previously unappreciated role for CD46 in the maintenance of epithelial cell barrier integrity as well as barrier repair. However, this role for CD46 as “gate keeper” of the epithelium could also provide reason as to why so many pathogens bind to CD46 as such event would facilitate infection.

CD3/CD46-mediated generation of IL-10-secreting T cells is defective in rheumatoid arthritis

Background and objectives Control of effector T cell responses to foreign and self-antigens through interleukin (IL)-10-producing Th1 cells and adaptive IL-10+Tregs is vital for limiting immune pathology during infection and for preventing autoimmunity. However, the mechanisms that regulate their production remain incompletely understood. CD46 is the receptor for the C3b/C4b components of complement. The authors have previously demonstrated that activation of human CD4 T cells through the T-cell receptor and the complement regulator CD46 induces Tr1 like IL-10-producing Tregs. The authors set out to study factors that regulate the generation of IL-10 producing Tregs during normal immune homeostasis and to determine whether this pathway is defective in chronic inflammatory disease, such as rheumatoid arthritis. Materials and methods CD4 T lymphocytes were purified from PBMC of normal donors or PBMC and synovial fluid of rheumatoid arthritis (RA) and JIA patients using CD4 MicroBeads prior to activation with anti-CD3 and anti-CD46 mAb in the presence or absence of increasing concentrations of IL-2. After 36 h cytokine production was determined using the Th1/Th2 cytokine bead array. Cells were stained using the IFNγ and IL-10 cytokine secretion assay kits in combination and IFNγ+, IFNγ+/IL-10+ and IL-10+ cells isolated via cell sorting. Supernatants from cultures of these cells were then tested for their capacity to suppress proliferation of freshly purified T cells. Approval for this study was obtained from the Local Ethics Review Committee. Results IL-10-secreting T cells are derived from populations of differentiating Th1 effector T cells as they also secrete IFNγ. The authors observed that the cytokine expression profile of CD3/CD46-activated T cells is heavily influenced by the amount of IL-2 present during activation. Thus, in the presence of low IL-2, CD3/CD46-activation induces strong IFNγ-secretion and a proinflammatory effector phenotype. High IL-2 induces a ‘switch’ from IFNγ+ T cells, via an IFNγ+/IL-10+ state, to an IL-10+ phenotype. CD46-induced IFNγ+/IL-10+ and IL-10+ cells are suppressive. By contrast, analysis of T cell populations from patients with inflammatory arthritis such as RA and JIA reveals that progression to the IL-10+ state is blocked even in the presence of high exogenous IL-2. In addition, CD3/CD46-activated T cells produce 20–30 times more IFN-γ than IL-10. Conclusions CD46 activation during T cell response initiation supports IFNγ secretion to combat infection while expansion of the effector response provides high IL-2 and the signal to switch to IL-10 production and resolution of the immune response. This regulatory switch appears to be defective in chronic inflammatory disease.

Asparaginyl endopeptidase (legumain) supports human Th1 induction via cathepsin L-mediated intracellular C3 activation

Autocrine activation of the complement receptors C3aR and CD46 by complement activation components C3a and C3b produced through C3 cleavage by the protease cathepsin L (CTSL) during T cell stimulation is a requirement for IFN-γ production and Th1 induction in human CD4+ T cells. Thus, lack of autocrine CD46 activation, such as in CD46-deficient patients, is associated with defective Th1 responses and recurrent infections. We have identified LGMN [the gene coding for legumain, also known as asparaginyl endopeptidase (AEP)] as one of the key genes induced by CD46 co-stimulation during human CD4+ T cell activation. AEP processes and activates a range of proteins, among those α1-thymosin and CTSL, which both drive intrinsically Th1 activity—but has so far not been described to be functionally active in human T cells. Here we found that pharmacological inhibition of AEP during activation of human CD4+ T cells reduced CTSL activation and the CTSL-mediated generation of intracellular C3a. This translated into a specific reduction of IFN-γ production without affecting cell proliferation or survival. In line with these findings, CD4+ T cells isolated from Lgmn−/− mice also displayed a specific defect in IFN-γ secretion and Th1 induction. Furthermore, we did not observe a role for AEP-driven autocrine α1-thymosin activation in T cell-derived IFN-γ production. These data suggest that AEP is an “upstream” activator of the CTSL-C3-IFN-γ axis in human CD4+ T cells and hence an important supporter of human Th1 induction.