IFNγ+ Treg in-vivo and in-vitro represent both activated nTreg and peripherally induced aTreg and remain phenotypically stable in-vitro after removal of the stimulus

Background: IFNγ-producing CD4+CD25+Foxp3+CD127- Treg represent the first line of Treg during an immune response. In the present study we determined whether IFNγ+ Treg in-vivo and in-vitro are Helios-positive representing activated natural (nTreg) or Helios-negative representing adaptive Treg (aTreg) and whether they originate from CD4+CD25+ and/or CD4+CD25- PBL. Furtheron, we investigated whether they are inducible by recombinant IFNγ (rIFNγ) as a single stimulus, decrease in-vitro after elimination of the stimulus, and have a demethylated Foxp3 Treg-specific demethylated region (TSDR) which is associated with stable Foxp3 expression. Method: Subsets of IFNγ+ Treg were determined in peripheral blood of healthy controls using eight-color flow cytometry and were further investigated in-vitro. Foxp3 TSDR methylation status was determined using bisulphite polymerase chain reaction (PCR) and high resolution melt (HRM) analysis. Results: Nearly all Treg in the peripheral blood were Helios+IFNγ- (1.9 ± 1.1/μl) and only few were Helios+IFNγ+ or Helios-IFNγ+ Treg (both 0.1 ± 0.1/μl). Enriched IFNγ+ Treg subsets showed in part strong Foxp3 TSDR demethylation. In-vitro, rIFNγ was unable to induce Treg. CD4+CD25+ enriched PBL stimulated with PMA/Ionomycin in the presence of rIFNγ were rather resistant to the effect of rIFNγ, in contrast to CD4+CD25- enriched PBL which showed increasing total Treg with Helios+ Treg switching from IFNγ- to IFNγ+ and increasing Helios-IFNγ+ Treg. The data indicate that rIFNγ, in combination with a polyclonal stimulus, activates nTreg and induces aTreg. When phorbol 12-myristate 13-acetate (PMA)/Ionomycin was washed out from the cell culture after 6 h stimulation, Treg induction continued for at least 96 h of cell culture, contradicting the hypothesis that removal of the stimulus results in significant decrease of IFNγ- and IFNγ+ CD4+CD25+Foxp3+CD127- Treg due to loss of Foxp3 expression. Conclusions: IFNγ+Helios- aTreg as well as IFNγ+Helios+ nTreg are detectable in the blood of healthy individuals, show in part strong Foxp3 TSDR demethylation and are inducible in-vitro. The present data provide further insight concerning the in-vivo and in-vitro characteristics of IFNγ+ Treg and help to understand their role in immunoregulation. Alloantigen-specific demethylated IFNγ+Helios+ nTreg might represent a suitable marker for monitoring graft-specific immunosuppression in renal transplant recipients

Cytokine expression during early and late phase of acute Puumala hantavirus infection

<p>Abstract</p> <p>Background</p> <p>Hantaviruses of the family <it>Bunyaviridae </it>are emerging zoonotic pathogens which cause hemorrhagic fever with renal syndrome (HFRS) in the Old World and hantavirus pulmonary syndrome (HPS) in the New World. An immune-mediated pathogenesis is discussed for both syndromes. The aim of our study was to investigate cytokine expression during the course of acute <it>Puumala </it>hantavirus infection.</p> <p>Results</p> <p>We retrospectively studied 64 patients hospitalised with acute <it>Puumala </it>hantavirus infection in 2010 during a hantavirus epidemic in Germany. Hantavirus infection was confirmed by positive anti-hantavirus IgG/IgM. Cytokine expression of IL-2, IL-5, IL-6, IL-8, IL-10, IFN-γ, TNF-α and TGF-β1 was analysed by ELISA during the early and late phase of acute hantavirus infection (average 6 and 12 days after onset of symptoms, respectively). A detailed description of the demographic and clinical presentation of severe hantavirus infection requiring hospitalization during the 2010 hantavirus epidemic in Germany is given. Acute hantavirus infection was characterized by significantly elevated levels of IL-2, IL-6, IL-8, TGF-β1 and TNF-α in both early and late phase compared to healthy controls. From early to late phase of disease, IL-6, IL-10 and TNF-α significantly decreased whereas TGF-β1 levels increased. Disease severity characterized by elevated creatinine and low platelet counts was correlated with high pro-inflammatory IL-6 and TNF-α but low immunosuppressive TGF-β1 levels and <it>vice versa </it>.</p> <p>Conclusion</p> <p>High expression of cytokines activating T-lymphocytes, monocytes and macrophages in the early phase of disease supports the hypothesis of an immune-mediated pathogenesis. In the late phase of disease, immunosuppressive TGF-β1 level increase significantly. We suggest that delayed induction of a protective immune mechanism to downregulate a massive early pro-inflammatory immune response might contribute to the pathologies characteristic of human hantavirus infection.</p

Inhibition of Allogeneic T Cell Proliferation by Indoleamine 2,3-Dioxygenase–expressing Dendritic Cells: Mediation of Suppression by Tryptophan Metabolites

Indoleamine 2,3-dioxygenase (IDO), an enzyme involved in the catabolism of tryptophan, is expressed in certain cells and tissues, particularly in antigen-presenting cells of lymphoid organs and in the placenta. It was shown that IDO prevents rejection of the fetus during pregnancy, probably by inhibiting alloreactive T cells, and it was suggested that IDO-expression in antigen-presenting cells may control autoreactive immune responses. Degradation of tryptophan, an essential amino acid required for cell proliferation, was reported to be the mechanism of IDO-induced T cell suppression. Because we wanted to study the action of IDO-expressing dendritic cells (DCs) on allogeneic T cells, the human IDO gene was inserted into an adenoviral vector and expressed in DCs. Transgenic DCs decreased the concentration of tryptophan, increased the concentration of kynurenine, the main tryptophan metabolite, and suppressed allogeneic T cell proliferation in vitro. Kynurenine, 3-hydroxykynurenine, and 3-hydroxyanthranilic acid, but no other IDO-induced tryptophan metabolites, suppressed the T cell response, the suppressive effects being additive. T cells, once stopped in their proliferation, could not be restimulated. Inhibition of proliferation was likely due to T cell death because suppressive tryptophan catabolites exerted a cytotoxic action on CD3+ cells. This action preferentially affected activated T cells and increased gradually with exposure time. In addition to T cells, B and natural killer (NK) cells were also killed, whereas DCs were not affected. Our findings shed light on suppressive mechanisms mediated by DCs and provide an explanation for important biological processes in which IDO activity apparently is increased, such as protection of the fetus from rejection during pregnancy and possibly T cell death in HIV-infected patients

Association of low serum TGF-β level in hantavirus infected patients with severe disease

Background: Hantaviruses are emerging zoonotic pathogens which cause hemorrhagic fever with renal syndrome, an immune-mediated pathogenesis is discussed. The aim of the present study was to investigate the role of TGF-β expression in acute hantavirus infection. Results: We retrospectively studied 77 patients hospitalised with acute Puumala infection during a hantavirus epidemic in Germany in 2012. Hantavirus infection was confirmed by positive anti-Puumala hantavirus IgG and IgM. Plasma levels of transforming growth factor (TGF)-β1 and TGF-β2 were analysed. Based on glomerular filtration rate on admission, patients were divided in mild and severe course of disease. Puumala virus RNA was detected by PCR amplification of the viral L segment gene. Out of 77 Puumala virus infected patients, 52 (68%) were male. A seasonal distribution was detected in our cohort with a peak in summer 2012, the highest incidence was observed in the age group of 30–39 years. Puumala virus RNA was detectable in 4/77 cases. Patients with severe disease had a significant longer hospital stay than patients with mild disease (6.2 vs 3.6 days). Thrombocyte count (186 vs 225 per nl), serum TGF-β1 (74 vs 118 ng/l) and TGF-β2 (479 vs 586 pg/l) were significantly lower in severe compared to mild disease. However, C-reactive protein (CRP) was significantly higher in patients with severe disease (62 vs 40 mg/l). TGF-β1/Cr was the most sensitive and specific marker associated with renal dysfunction. Conclusion: High serum CRP and low serum TGF-β in the early phase of hantavirus infection is associated with a severe course of disease. Our results support the hypothesis of an immune-mediated pathogenesis in hantavirus infection

IL-23 plasma level is strongly associated with CMV status and reactivation of CMV in renal transplant recipients

Background: Cytomegalovirus seropositivity is an independent risk factor for atherosclerosis in patients with ESRD. Donor CMV seropositivity is associated with higher graft loss. Dendritic cells, macrophages and Th17 lymphocytes are defined as producers of IL-23. IL-23 is thought to be involved in the promotion of Th17 cell polarization. Latent CMV-induced Th17 might be involved in the pathogenesis of CMV infection in patients with ESRD. We aimed to evaluate associations of Th17-dependent cytokines with ESRD, CMV status and post-transplant outcome in kidney transplantation. Results: IL-21 plasma levels were similar in patients and healthy controls (p = 0.47), whereas IL-9 (p = 0.02) and IL-23 (p < 0.0001) levels were significantly higher in ESRD patients. CMV-seronegative (p = 0.002) and –seropositive (p < 0.001) patients had significantly higher IL-23 plasma levels than controls. CMV-seropositive patients showed excessively higher IL-23 (p < 0.001) plasma levels than CMV-seronegative patients. Patients with post-transplant CMV reactivation had higher IL-23 plasma levels than patients without CMV reactivation (p = 0.025). Conclusions: Our results indicate that latent CMV induces IL-23. IL-23 might be an inflammatory mediator of latent CMV infection in patients with ESRD and predisposes patients for post-transplant CMV reactivation

Autoantigen-specific immunosuppression with tolerogenic peripheral blood cells prevents relapses in a mouse model of relapsing-remitting multiple sclerosis

Background: Dendritic cells (DCs) rendered suppressive by treatment with mitomycin C and loaded with the autoantigen myelin basic protein demonstrated earlier their ability to prevent experimental autoimmune encephalomyelitis (EAE), the animal model for multiple sclerosis (MS). This provides an approach for prophylactic vaccination against autoimmune diseases. For clinical application such DCs are difficult to generate and autoantigens hold the risk of exacerbating the disease. Methods: We replaced DCs by peripheral mononuclear cells and myelin autoantigens by glatiramer acetate (Copaxone®), a drug approved for the treatment of MS. Spleen cells were loaded with Copaxone®, incubated with mitomycin C (MICCop) and injected into mice after the first bout of relapsing-remitting EAE. Immunosuppression mediated by MICCop was investigated in vivo by daily assessment of clinical signs of paralysis and in in vitro restimulation assays of peripheral immune cells. Cytokine profiling was performed by enzyme-linked immunosorbent assay (ELISA). Migration of MICCop cells after injection was examined by biodistribution analysis of 111Indium-labelled MICCop. The number and inhibitory activity of CD4+CD25+FoxP3+ regulatory T cells were analysed by histology, flow cytometry and in vitro mixed lymphocyte cultures. In order to assess the specificity of MICCop-induced suppression, treated EAE mice were challenged with the control protein ovalbumin. Humoral and cellular immune responses were then determined by ELISA and in vitro antigen restimulation assay. Results: MICCop cells were able to inhibit the harmful autoreactive T-cell response and prevented mice from further relapses without affecting general immune responses. Administered MICCop migrated to various organs leading to an increased infiltration of the spleen and the central nervous system with CD4+CD25+FoxP3+ cells displaying a suppressive cytokine profile and inhibiting T-cell responses. Conclusion: We describe a clinically applicable cell therapeutic approach for controlling relapses in autoimmune encephalomyelitis by specifically silencing the deleterious autoimmune response

Patients with idiopathic recurrent miscarriage have abnormally high TGFß+ blood NK, NKT and T cells in the presence of abnormally low TGFß plasma levels

Background: Previously, we demonstrated up-regulated activated CD4+ and CD8+ T lymphocytes as well as up-regulated cytotoxic NK cells in the blood of patients with idiopathic recurrent miscarriage. In the present study, we tried to identify deficiencies in counter-regulating immune mechanisms of these patients. Method: Cytokines were determined in NK cells and in plasma samples of 35 healthy controls, 33 patients with idiopathic recurrent miscarriage, 34 patients with end stage renal disease, 10 transplant patients early and 37 transplant patients late post-transplant using flow-cytometry and luminex. In addition, cytokines were studied in supernatants of cell cultures with peripheral blood mononuclear cells stimulated in-vitro with tumor cell line K562. Results: Patients with idiopathic recurrent miscarriage exhibited the highest absolute cell counts of circulating TGFß1+ NK, NKT and T lymphocytes and the lowest TGFß1 plasma levels of all study groups (for all p &lt; 0.050). In-vitro, peripheral blood mononuclear cells of patients with idiopathic recurrent miscarriage showed high spontaneous TGFß1 production that could not be further increased by stimulation with K562, indicating increased consumption of TGFß1 by activated cells in the cell culture. Moreover, patients with idiopathic recurrent miscarriage had abnormally high IL4+ as well as abnormally high IFNy+ NK cells (p &lt; 0.010) but similar IL10+ NK cell numbers as female healthy controls and showed the lowest plasma levels of IL10, TGFß3, IL1RA, IL1ß, IL5, IL6, IL8, IL17, TNFα, GM-CSF, TPO and VEGF and the highest plasma levels of G-CSF, FGF-basic, CCL3 and CXCL5 as compared to female HC and female transplant recipients (for all p &lt; 0.050). Conclusions: Patients with idiopathic recurrent miscarriage show an activated immune system that can hardly be stimulated further and cannot be efficiently down-regulated by up-regulated TGFß1+ and IL4+ NK, NKT and T lymphocytes which are present concomitantly in these patients. The strongly decreased TGFß and IL10 plasma levels indicate deficient down-regulation and reflect a dysbalance of the immune system in patients with idiopathic recurrent miscarriage. These findings may be relevant for explaining the pathogenesis of idiopathic recurrent miscarriage

Long- and short-term outcomes in renal allografts with deceased donors: A large recipient and donor genome-wide association study.

Improvements in immunosuppression have modified short-term survival of deceased-donor allografts, but not their rate of long-term failure. Mismatches between donor and recipient HLA play an important role in the acute and chronic allogeneic immune response against the graft. Perfect matching at clinically relevant HLA loci does not obviate the need for immunosuppression, suggesting that additional genetic variation plays a critical role in both short- and long-term graft outcomes. By combining patient data and samples from supranational cohorts across the United Kingdom and European Union, we performed the first large-scale genome-wide association study analyzing both donor and recipient DNA in 2094 complete renal transplant-pairs with replication in 5866 complete pairs. We studied deceased-donor grafts allocated on the basis of preferential HLA matching, which provided some control for HLA genetic effects. No strong donor or recipient genetic effects contributing to long- or short-term allograft survival were found outside the HLA region. We discuss the implications for future research and clinical application