CMV drives the expansion of highly functional memory T cells expressing NK‐cell receptors in renal transplant recipients

Cytomegalovirus (CMV) is a common opportunistic infection encountered in renal transplant recipients (RTRs) and may be reactivated without symptoms at any time post‐transplant. We describe how active and latent CMV affect T‐cell subsets in RTRs who are stable on maintenance therapy. T‐cell responses to CMV were assessed in RTRs (n = 54) >2 years post‐transplant, and healthy controls (n = 38). Seven RTRs had CMV DNA detectable in plasma. CMV antibody and DNA aligned with increased proportions of CD8⁺ T cells and reduced CD4/CD8 ratios. This paralleled an expansion of effector memory T‐cell (T_EM), terminally differentiated T‐cell (T_EMRA) and CD57⁺ T_EMRA cell populations. Expression of NK‐cell receptors, LIR‐1 and KLRG1 on CD4⁺ and CD8⁺ CD57⁺ T_EM and T_EMRA cells correlated with elevated interferon‐γ and cytotoxic responses to anti‐CD3 and increased cytotoxic responses to CMV phosphoprotein (pp) 65 in RTRs who carried CMV DNA. CD8⁺ T cells from all CMV seropositive RTRs responded efficiently to CMV immediate early (IE) ‐1 peptides. The data show that latent and active CMV infection can alter T‐cell subsets in RTRs many years after transplantation, and up‐regulate T‐cell expression of NK‐cell receptors. This may enhance effector responses of CD4⁺ and CD8⁺ T cells against CMV.     

Tyrosine kinase 2 is not limiting human antiviral type III interferon responses

Tyrosine kinase 2 (TYK2) associates with interferon (IFN) alpha receptor, IL‐10 receptor (IL‐10R) beta and other cytokine receptor subunits for signal transduction, in response to various cytokines, including type‐I and type‐III IFNs, IL‐6, IL‐10, IL‐12 and IL‐23. Data on TYK2 dependence on cytokine responses and in vivo consequences of TYK2 deficiency are inconsistent. We investigated a TYK2 deficient patient, presenting with eczema, skin abscesses, respiratory infections and IgE levels >1000 U/mL, without viral or mycobacterial infections and a corresponding cellular model to analyze the role of TYK2 in type‐III IFN mediated responses and NK‐cell function. We established a novel simple diagnostic monocyte assay to show that the mutation completely abolishes the IFN‐α mediated antiviral response. It also partly reduces IL‐10 but not IL‐6 mediated signaling associated with reduced IL‐10Rβ expression. However, we found almost normal type‐III IFN signaling associated with minimal impairment of virus control in a TYK2 deficient human cell line. Contrary to observations in TYK2 deficient mice, NK‐cell phenotype and function, including IL‐12/IL‐18 mediated responses, were normal in the patient. Thus, preserved type‐III IFN responses and normal NK‐cell function may contribute to antiviral protection in TYK2 deficiency leading to a surprisingly mild human phenotype.     

Role of the MHC restriction during maturation of antigen‐specific human T cells in the thymus

In the thymus, a T‐cell repertoire able to confer protection against infectious and noninfectious agents in a peptide‐dependent, self‐MHC‐restricted manner is selected. Direct detection of Ag‐specific thymocytes, and analysis of the impact of the expression of the MHC‐restricting allele on their frequency or function has never been studied in humans because of the extremely low precursor frequency. Here, we used a tetramer‐based enrichment protocol to analyze the ex vivo frequency and activation‐phenotype of human thymocytes specific for self, viral and tumor‐antigens presented by HLA‐A*0201 (A2) in individuals expressing or not this allele. Ag‐specific thymocytes were quantified within both CD4CD8 double or single‐positive compartments in every donor. Our data indicate that the maturation efficiency of Ag‐specific thymocytes is poorly affected by HLA‐A2 expression, in terms of frequencies. Nevertheless, A2‐restricted T‐cell lines from A2⁺ donors reacted to A2⁺ cell lines in a highly peptide‐specific fashion, whereas their alloreactive counterparts showed off‐target activity. This first ex vivo analysis of human antigen‐specific thymocytes at different stages of human T‐cell development should open new perspectives in the understanding of the human thymic selection process.     

An in vitro platform supports generation of human innate lymphoid cells from CD34+ hematopoietic progenitors that recapitulate ex vivo identity

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Activation‐dependent cell death of human monocytes is a novel mechanism of fine‐tuning inflammation and autoimmunity

In patients with juvenile idiopathic arthritis (JIA), increased release of IFN‐γ and GM‐CSF in cells infiltrating synovial tissue can be a potent driver of monocyte activation. Given the fundamental role of monocyte activation in remodeling the early phases of inflammatory responses, here we analyze the GM‐CSF/IFN‐γ induced activity of human monocytes in such a situation in vitro and in vivo. Monocytes from healthy donors were isolated and stimulated with GM‐CSF ± IFN‐γ. Monocyte activation and death were analyzed by flow cytometry, immunofluorescence microscopy, ELISA, and qPCR. T‐cell GM‐CSF/IFN‐γ expression and monocyte function were determined in synovial fluid and peripheral blood from 15 patients with active JIA and 21 healthy controls. Simultaneous treatment with GM‐CSF and IFN‐γ induces cell death of monocytes. This cell death is partly cathepsin B‐associated and has morphological characteristics of necrosis. Monocytes responding to costimulation with strong proinflammatory activities are consequently eliminated. Monocytes surviving this form of hyperactivation retain normal cytokine production. Cathepsin B activity is increased in monocytes isolated from synovial fluid from patients with active arthritis. Our data suggest GM‐CSF/IFN‐γ induced cell death of monocytes as a novel mechanism to eliminate overactivated monocytes, thereby potentially balancing inflammation and autoimmunity in JIA.     

Interplay of innate lymphoid cells and the microbiota

Innate lymphoid cells (ILC) are a recently identified group of innate lymphocytes that are preferentially located at barrier surfaces. Barrier surfaces are in direct contact with complex microbial ecosystems, collectively referred to as the microbiota. It is now believed that the interplay of the microbiota with host components (i.e. epithelial cells and immune cells) promotes host fitness by regulating organ homeostasis, metabolism, and host defense against pathogens. In this review, we will give an overview of this multifaceted interplay between ILC and components of the microbiota.     

Respiratory syncytial virus infection augments NOD2 signaling in an IFN‐β‐dependent manner in human primary cells

Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infections in infants, with remarkable variability in disease severity. An exaggerated proinflammatory response and influx of leukocytes is part of the pathogenesis of severe RSV disease. Here, we show an increase in proinflammatory cytokine production by human immune cells after stimulation with RSV and muramyl dipeptide (MDP), which is recognized by nucleotide‐binding oligomerization domain containing 2 (NOD2). PBMCs from Crohn's disease patients homozygous for the 3020insC mutation in the NOD2 gene did not show a synergistic response to stimulation with RSV and MDP, suggesting that NOD2 is essential for the observed synergy. Further experiments aimed at identifying the viral ligand indicated that viral RNA plays an essential role in the recognition of RSV. Stimulation with RSV or Poly(I:C) induced IFN‐β expression, which resulted in an increased expression of the viral receptors TLR3 and RIG‐I, as well as an increased NOD2 expression. Our data indicate that IFN‐β induction by viral RNA is an essential first step in the increased proinflammatory response to MDP. We hypothesize that the enhanced proinflammatory response to MDP following RSV infection may be an important factor in determining the outcome of the severity of disease.     

Bone marrow type 2 innate lymphoid cells: a local source of interleukin‐5 in interleukin‐33‐driven eosinophilia

T helper type 2 (Th2) cells, type 2 innate lymphoid cells (ILC2s) and eosinophil progenitors have previously been described to produce interleukin‐5 (IL‐5) in the airways upon allergen provocation or by direct administration of IL‐33. Eosinophilic airway inflammation is known to be associated with IL‐5‐dependent eosinophil development in the bone marrow, however, the source of IL‐5 remains unclear. T helper cells, ILC2s and CD34⁺ progenitors have been proposed to be involved in this process, therefore, we investigated whether these cells are taking part in eosinophilopoiesis by producing IL‐5 locally in the bone marrow in IL‐33‐driven inflammation. Airway exposure with IL‐33 led to eosinophil infiltration in airways and elevated eotaxin‐2/CCL24. Importantly, IL‐5 production as well as expression of the IL‐33 receptor increased in ILC2s in the bone marrow under this treatment. A small but significant induction of IL‐5 was also found in CD34⁺ progenitors but not in T helper cells. Similar results were obtained by in vitro stimulation with IL‐33 where ILC2s rapidly produced large amounts of IL‐5, which coincided with the induction of eosinophil hematopoiesis. IL‐33‐mediated eosinophil production was indeed dependent on IL‐5 as both airway and bone marrow eosinophils decreased in mice treated with anti‐IL‐5 in combination with IL‐33. Interestingly, the responsiveness of ILC2s to IL‐33 as well as IL‐33‐induced eotaxin‐2/CCL24 were independent of the levels of IL‐5. In summary, we demonstrate for the first time that IL‐33 acts directly on bone marrow ILC2s, making them an early source of IL‐5 and part of a process that is central in IL‐33‐driven eosinophilia.     

Semaphorin 7A modulates cytokine‐induced memory‐like responses by human natural killer cells

Cytokine‐induced memory‐like (CIML) NK cells are endowed with the capacity to mediate enhanced effector functions upon cytokine or activating receptor restimulation for several weeks following short‐term preactivation with IL‐12, IL‐15, and IL‐18. Promising results from a first‐in‐human clinical trial highlighted the clinical potential of CIML NK cells as adoptive immunotherapy for patients with hematologic malignancies. However, the mechanisms underlying CIML NK cell differentiation and increased functionality remain incompletely understood. Semaphorin 7A (SEMA7A) is a potent immunomodulator expressed in activated lymphocytes and myeloid cells. In this study, we show that SEMA7A is substantially upregulated on NK cells stimulated with cytokines, and specifically marks activated NK cells with a strong potential to release IFN‐γ. In particular, preactivation of NK cells with IL‐12+IL‐15+IL‐18 resulted in greater than tenfold upregulation of SEMA7A and enhanced expression of the ligand for SEMA7A, integrin‐β1, on CIML NK cells. Strikingly, preactivation in the presence of antibodies targeting SEMA7A lead to significantly decreased IFN‐γ production following restimulation. These results imply a novel mechanism by which cytokine‐enhanced SEMA7A/integrin‐β1 interaction promotes CIML NK cell differentiation and maintenance of increased functionality. Our data suggest that targeting SEMA7A/integrin‐β1 signaling might provide a novel immunotherapeutic approach to potentiate antitumor activity of CIML NK cells.     

TAP‐mediated processing of exoerythrocytic antigens is essential for protection induced with radiation‐attenuated Plasmodium sporozoites

MHC class I dependent CD8⁺ T cells are essential for protection induced by radiation‐attenuated Plasmodium sporozoites (RAS) in murine malaria models. Apart from the mechanism of activation of CD8⁺ T cells specific for the circumsporozoite protein, the major sporozoite antigen (Ag), CD8⁺ T cells specific for other exoerythrocytic Ags that have been shown to mediate protection have not been thoroughly investigated. Specifically, mechanisms of processing and presentation of exoerythrocytic Ags, which includes liver stage (LS) Ags, remain poorly understood. We hypothesize that as exogenous proteins, LS Ags are processed by mechanisms involving either the TAP‐dependent phagosomal‐to‐cytosol or TAP‐independent vacuolar pathway of cross‐presentation. We used TAP‐deficient mice to investigate whether LS Ag mediated induction of naïve CD8⁺ T cells and their recall during sporozoite challenge occur by the TAP‐dependent or TAP‐independent pathways. On the basis of functional attributes, CD8⁺ T cells were activated via the TAP‐independent pathway during immunizations with Plasmodium berghei RAS; however, IFN‐γ⁺CD8⁺ T cells previously induced by P. berghei RAS in TAP‐deficient mice failed to be recalled against sporozoite challenge and the mice became parasitemic. On the basis of these observations, we propose that TAP‐associated Ag processing is indispensable for sterile protection induced with P. berghei RAS.     

Oncostatin M overexpression induces skin inflammation but is not required in the mouse model of imiquimod‐induced psoriasis‐like inflammation

Oncostatin M (OSM) has been reported to be overexpressed in psoriasis skin lesions and to exert proinflammatory effects in vitro on human keratinocytes. Here, we report the proinflammatory role of OSM in vivo in a mouse model of skin inflammation induced by intradermal injection of murine OSM‐encoding adenovirus (AdOSM) and compare with that induced by IL‐6 injection. Here, we show that OSM potently regulates the expression of genes involved in skin inflammation and epidermal differentiation in murine primary keratinocytes. In vivo, intradermal injection of AdOSM in mouse ears provoked robust skin inflammation with epidermal thickening and keratinocyte proliferation, while minimal effect was observed after AdIL‐6 injection. OSM overexpression in the skin increased the expression of the S100A8/9 antimicrobial peptides, CXCL3, CCL2, CCL5, CCL20, and Th1/Th2 cytokines, in correlation with neutrophil and macrophage infiltration. In contrast, OSM downregulated the expression of epidermal differentiation genes, such as cytokeratin‐10 or filaggrin. Collectively, these results support the proinflammatory role of OSM when it is overexpressed in the skin. However, OSM expression was not required in the murine model of psoriasis induced by topical application of imiquimod, as demonstrated by the inflammatory phenotype of OSM‐deficient mice or wild‐type mice treated with anti‐OSM antibodies.     

Monocyte‐derived dendritic cells enhance protection against secondary influenza challenge by controlling the switch in CD8+ T‐cell immunodominance

Influenza virus infection triggers an increase in the number of monocyte‐derived dendritic cells (moDCs) in the respiratory tract, but the role of these cells during antiviral immunity is still unclear. Here we show that during influenza infection, moDCs dominate the late activation of CD8⁺ T cells and trigger the switch in immunodominance of the CD8⁺ T‐cell response from acidic polymerase specificity to nucleoprotein specificity. Abrogation of monocyte recruitment or depletion of moDCs strongly compromised host resistance to secondary influenza challenge. These findings underscore a novel function of moDCs in the antiviral response to influenza virus, and have important implications for vaccine design.     

Antigen presentation by B cells guides programing of memory CD4+ T‐cell responses to a TLR4‐agonist containing vaccine in mice

The contribution of B cells to immunity against many infectious diseases is unquestionably important and well characterized. Here, we sought to determine the role of B cells in the induction of T‐helper 1 (T_H1) CD4⁺ T cells upon vaccination with a tuberculosis (TB) antigen combined with a TLR4 agonist. We used B‐cell deficient mice (μMT^?/?), tetramer‐positive CD4⁺ T cells, markers of memory “precursor” effector cells (MPECs), and T‐cell adoptive transfers and demonstrated that the early antigen‐specific cytokine‐producing T_H1 responses are unaffected in the absence of B cells, however MPEC induction is strongly impaired resulting in a deficiency of the memory T_H1 response in μMT^?/? mice. We further show that antigen‐presentation by B cells is necessary for their role in MPEC generation using B‐cell adoptive transfers from wt or MHC class II knock‐out mice into μMT^?/? mice. Our study challenges the view that B‐cell deficiency exclusively alters the T_H1 response at memory time‐points. Collectively, our results provide new insights on the multifaceted roles of B cells that will have a high impact on vaccine development against several pathogens including those requiring T_H1 cell‐mediated immunity.     

Donor bone marrow cells are essential for iNKT cell‐mediated Foxp3+ Treg cell expansion in a murine model of transplantation tolerance

Mixed chimerism induction is the most reliable method for establishing transplantation tolerance. We previously described a novel treatment using a suboptimal dose of anti‐CD40 ligand (anti‐CD40L) and liposomal formulation of a ligand for invariant natural killer T cells administered to sub‐lethally irradiated recipient mice after donor bone marrow cell (BMC) transfer. Recipient mice treated with this regimen showed expansion of a Foxp3‐positive regulatory T(Treg) cell phenotype, and formation of mixed chimera. However, the mechanism of expansion and bioactivity of Treg cells remains unclear. Here, we examine the role of donor BMCs in the expansion of bioactive Treg cells. The mouse model was transplanted with a heart allograft the day after treatment. The results showed that transfer of spleen cells in place of BMCs failed to deplete host interferon (IFN)‐γ‐producing CD8⁺T cells, expand host Ki67⁺CD4⁺CD25⁺Foxp3⁺ Treg cells, and prolong graft survival. Severe combined immunodeficiency mice who received Treg cells obtained from BMC‐recipients accepted skin grafts in an allo‐specific manner. Myeloid‐derived suppressor cells, which were a copious cell subset in BMCs, enhanced the Ki67 expression of Treg cells. This suggests that donor BMCs are indispensable for the expansion of host bioactive Treg cells in our novel treatment for transplant tolerance induction.     

Hierarchy of immunosuppressive strength among myeloid‐derived suppressor cell subsets is determined by GM‐CSF

CD11b⁺/Gr‐1⁺ myeloid‐derived suppressor cells (MDSC) contribute to tumor immune evasion by restraining the activity of CD8⁺ T‐cells. Two major MDSC subsets were recently shown to play an equal role in MDSC‐induced immune dysfunctions: monocytic‐ and granulocytic‐like. We isolated three fractions of MDSC, i.e. CD11b⁺/Gr‐1^high, CD11b⁺/Gr‐1^int, and CD11b⁺/Gr‐1^low populations that were characterized morphologically, phenotypically and functionally in different tumor models. In vitro assays showed that CD11b⁺/Gr‐1^int cell subset, mainly comprising monocytes and myeloid precursors, was always capable to suppress CD8⁺ T‐cell activation, while CD11b⁺/Gr‐1^high cells, mostly granulocytes, exerted appreciable suppression only in some tumor models and when present in high numbers. The CD11b⁺/Gr‐1^int but not CD11b⁺/Gr‐1^high cells were also immunosuppressive in vivo following adoptive transfer. CD11b⁺/Gr‐1^low cells retained the immunosuppressive potential in most tumor models. Gene silencing experiments indicated that GM‐CSF was necessary to induce preferential expansion of both CD11b⁺/Gr‐1^int and CD11b⁺/Gr‐1^low subsets in the spleen of tumor‐bearing mice and mediate tumor‐induced tolerance whereas G‐CSF, which preferentially expanded CD11b⁺/Gr‐1^high cells, did not create such immunosuppressive environment. GM‐CSF also acted on granulocyte–macrophage progenitors in the bone marrow inducing local expansion of CD11b⁺/Gr‐1^low cells. These data unveil a hierarchy of immunoregulatory activity among MDSC subsets that is controlled by tumor‐released GM‐CSF.