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Tofacitinib is an oral Janus kinase inhibitor that is effective for the treatment of rheumatoid arthritis and shows encouraging therapeutic effects in several other autoimmune diseases. A prominent adverse effect of tofacitinib therapy is the increased risk of viral infections. Despite its advanced stage of clinical development, the modes of action that mediate the beneficial and adverse effects of tofacitinib in autoimmune diseases remain unclear. Interferon alfa (IFNα) produced by plasmacytoid dendritic cells (PDCs) is critically involved in the pathogenesis of many systemic autoimmune diseases and in immunity to viral infections. Using in vitro culture models with human cells, we studied the effects of tofacitinib on PDC survival and IFNα production, and on arthrogenic and antiviral effects of IFNα. Tofacitinib inhibited the expression of antiapoptotic BCL-A1 and BCL-XL in human PDC and induced PDC apoptosis. TLR7 stimulation upregulated the levels of antiapoptotic Bcl-2 family members and prevented the induction of PDC apoptosis by tofacitinib. However, tofacitinib robustly inhibited the production of IFNα by toll like receptor-stimulated PDC. In addition, tofacitinib profoundly suppressed IFNα-induced upregulation of TLR3 on synovial fibroblasts, thereby inhibiting their cytokine and protease production in response to TLR3 ligation. Finally, tofacitinib counteracted the suppressive effects of IFNα on viral replication. Tofacitinib inhibits PDC survival and IFNα production and suppresses arthrogenic and antiviral effects of IFNα signaling. Inhibition of the IFNα pathway at 2 levels may contribute to the beneficial effects of tofacitinib in autoimmune diseases and explain the increased viral infection rates observed during tofacitinib treatment., (Copyright © 2016 Elsevier Inc. All rights reserved.)

High-dose i.v. Ig (IVIg) is a prominent immunomodulatory therapy for various autoimmune and inflammatory diseases. Recent mice studies suggest that IVIg inhibits myeloid cell function by inducing a cascade of IL-33-Th2 cytokine production causing upregulation of the inhibitory FcγRIIb, as well as by modulating IFN-γ signaling. The purpose of our study was to explore whether and how these mechanisms are operational in IVIg-treated patients. We show that IVIg in patients results in increases in plasma levels of IL-33, IL-4, and IL-13 and that increments in IL-33 levels correlate with rises in plasma IL-4 and IL-13 levels. Strikingly, no upregulation of FcγRIIb expression was found, but instead a decreased expression of the activating FcγRIIa on circulating myeloid dendritic cells (mDCs) after high-dose, but not after low-dose, IVIg treatment. In addition, expression of the signaling IFN-γR2 subunit of the IFN-γR on mDCs was downregulated upon high-dose IVIg therapy. In vitro experiments suggest that the modulation of FcγRs and IFN-γR2 on mDCs is mediated by IL-4 and IL-13, which functionally suppress the responsiveness of mDCs to immune complexes or IFN-γ. Human lymph nodes and macrophages were identified as potential sources of IL-33 during IVIg treatment. Interestingly, stimulation of IL-33 production in human macrophages by IVIg was not mediated by dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN). In conclusion, high-dose IVIg treatment inhibits inflammatory responsiveness of mDCs in humans by Th2 cytokine-mediated downregulation of FcγRIIa and IFN-γR2 and not by upregulation of FcγRIIb. Our results suggest that this cascade is initiated by stimulation of IL-33 production that seems DC-SIGN independent., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Intravenous immunoglobulin (IVIg), a therapeutic preparation containing pooled human immunoglobulin (Ig) G, has been suggested to inhibit differentiation and maturation of dendritic cells (DCs); however, controversies exist on this issue. We aimed to reinvestigate the effects of IVIg on human DC maturation and cytokine production, and to determine whether an artifactual determinant is involved in the observed effects. Human monocyte-derived DCs or freshly isolated blood myeloid DCs were cultured in the presence of IVIg in vitro, and the expression of maturation markers CD80, CD86, CD83, and Human Leukocyte Antigen-DR were determined by flow cytometry, whereas production of interleukin (IL)-12 and IL-10 was measured by enzyme-linked immunosorbent assay, and T-cell stimulatory capacity was determined in cocultures with allogeneic CD4(+) T cells. Interestingly, we observed that IVIg did not inhibit, but instead stimulated, spontaneous maturation and T-cell stimulatory ability of human DCs, while leaving lipopolysaccharide-induced DC maturation and cytokine production unaffected. Strikingly, prevention of IVIg binding to culture plate surface, or blocking of the activating Fcγ receptor IIa on DC, abrogated the stimulatory effect of IVIg on costimulatory molecule expression and on T-cell stimulatory capacity of DCs, suggesting that IVIg activates DCs on IgG adsorption to the plastic surface. This study warrants for careful study design when performing cell culture studies with IVIg to prevent artifactual effects, and shows that IVIg does not modulate directly costimulatory molecule expression, cytokine production, or allogeneic T-cell stimulatory capacity of human DCs., (Copyright © 2014 Mosby, Inc. All rights reserved.)

Most studies on the effects of naturally occurring autoantibodies (NAbs) on immune cells have been performed in the context of research on the immunomodulatory effects of intravenous immunoglobulin (IVIG). Among others, IVIG inhibits the differentiation, maturation and functions of dendritic cells (DC), thereby suppressing T-cell activation. In addition, IVIG stimulates expansion and suppressive function of regulatory T cells (Treg) carrying the antigens CD4, CD25 and Foxp3. Current data on the immunomodulatory effects of IVIG on DC and Treg are summarized, and possible molecular interactions between NAbs and DC or Treg that mediate these effects are discussed.

Allo-reactive memory T cells are a major barrier for induction of immunological tolerance to allografts in humans. Here, we report that stimulation of unfractionated human T cells with TLR-stimulated allogeneic plasmacytoid dendritic cells (pDCs) induces CD8(+) regulatory T cells (Tregs) that inhibit T-cell allo-responses, including those of memory T cells. CD3(+) T cells were primed for 7 days with allogeneic pDCs that had been pre-stimulated with TLR-7 or TLR-9 ligands. While the T cells proliferated and produced cytokines during the priming culture, they were profoundly hypo-responsive to re-stimulation with the same allo-antigen in a second culture. Moreover, T cells primed by pDCs exerted donor-specific suppression on allo-responses of both unfractionated and memory CD3(+) T cells. The regulatory capacity of pDC-primed T cells was confined to CD8(+) LAG-3(+) Foxp3(+) CTLA-4(+) T cells, which suppressed allogeneic T-cell responses through a CTLA-4-dependent mechanism. Induction of CD8(+) Tregs by pDCs could be partially prevented by 1-methyl tryptophan, an inhibitor of indoleamine 2,3-dioxygenase. In conclusion, stimulation of human T cells by TLR-stimulated allogeneic pDCs induces CD8(+) Tregs that inhibit allogeneic T-cell responses, including memory T cells. Donor-derived pDCs may be considered as an immunotherapeutic tool to prevent activation of the recipient allo-reactive (memory) T-cell repertoire after allogeneic transplantation., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

It is thought, but there is no evidence, that myeloid dendritic cells (MDCs) of donor origin migrate into the recipient after clinical organ transplantation and sensitize the recipient's immune system by the direct presentation of donor allo-antigens. Here we show prominent MDC chimerism in the recipient's circulation early after clinical liver transplantation (LTx) but not after renal transplantation (RTx). MDCs that detach from human liver grafts produce large amounts of pro-inflammatory [tumor necrosis factor alpha and interleukin 6 (IL-6)] and anti-inflammatory (IL-10) cytokines upon activation with various stimuli, express higher levels of toll-like receptor 4 than blood or splenic MDCs, and are sensitive to stimulation with a physiological concentration of lipopolysaccharide (LPS). Upon stimulation with LPS, MDCs detaching from liver grafts prime allogeneic T cell proliferation and production of interferon gamma but not of IL-10. Soluble factors secreted by liver graft MDCs amplify allogeneic T helper 1 responses. In conclusion, after clinical LTx, but not after RTx, prominent numbers of donor-derived MDCs migrate into the recipient's circulation. MDCs detaching from liver grafts produce pro-inflammatory and anti-inflammatory cytokines and are capable of stimulating allogeneic T helper 1 responses, and this suggests that MDC chimerism after clinical LTx may contribute to liver graft rejection rather than acceptance.

Myeloid dendritic cells (MDC) play an important role in antigen-specific immunity and tolerance. In transplantation setting donor-derived MDC are a promising tool to realize donor-specific tolerance. Current protocols enable generation of tolerogenic donor MDC from human monocytes during 1-week cultures. However, for clinical application in transplantation medicine, a rapidly available source of tolerogenic MDC is desired. In this study we investigated whether primary human blood MDC could be transformed into tolerogenic MDC using dexamethasone (dex) and lipopolysaccharide (LPS). Human blood MDC were cultured with dex and subsequently matured with LPS in the presence or absence of dex. Activation of MDC with LPS after pretreatment with dex did not prevent maturation into immunostimulatory MDC. In contrast, simultaneous treatment with dex and LPS yielded tolerogenic MDC, that had a reduced expression of CD86 and CD83, that poorly stimulated allogeneic T-cell proliferation and production of T helper 1 (Th1) cytokines, and primed production of the immunoregulatory cytokine interleukin-10 (IL-10) in T cells. In vitro, however, these tolerogenic MDC did not induce permanent donor-specific hyporesponsiveness in T cells. Importantly, tolerogenic MDC obtained by LPS stimulation in the presence of dex did not convert into immunostimulatory MDC after subsequent activation with different maturation stimuli. In conclusion, these findings demonstrate that combined treatment with dex and LPS transforms primary human blood MDC into tolerogenic MDC that are impaired to stimulate Th1 cytokines, but strongly prime the production of the immunoregulatory cytokine IL-10 in T cells, and are resistant to maturation stimuli. This strategy enables rapid generation of tolerogenic donor-derived MDC for immunotherapy in clinical transplantation.

The modes of action of intravenous immunoglobulins (IVIgs) in exerting their immunomodulatory properties are broad and not fully understood. IVIgs can modulate the function of various immune cells, including suppressing the capacity of dendritic cells (DCs) to stimulate T cells. In the present study, we showed that DCs matured in the presence of IVIgs (IVIg-DCs) activated NK cells, and increased their interferon-gamma production and degranulation. The activated NK cells induced apoptosis of the majority of IVIg-DCs. In consequence, only in the presence of NK cells, IVIg-DCs were 4-fold impaired in their T-cell priming capacity. This was due to NK-cell-mediated antibody-dependent cellular cytotoxicity (ADCC) to IVIg-DCs, probably induced by IgG multimers, which could be abrogated by blockade of CD16 on NK cells. Furthermore, IVIg-DCs down-regulated the expression of NKp30 and KIR receptors, and induced the generation of CD56(bright)CD16(-)CCR7(+) lymph node-type NK cells. Our results identify a novel pathway, in which IVIgs induce ADCC of mature DCs by NK cells, which downsizes the antigen-presenting pool and inhibits T-cell priming. By influencing the interaction between DCs and NK cells, IVIgs modulate the ability of the innate immunity to trigger T-cell activation, a mechanism that can "cool down" the immune system at times of activation.

Background: Prophylactic administration of anti-HBs intravenous immunoglobulins (IVIg) in hepatitis B infected-liver transplant patients protects against acute rejection. To explore the suitability of intravenous immunoglobulins (IVIg) as prophylaxis of acute rejection and graft-versus-host disease (GVHD) after allograft transplantation, the effects of IVIg and calcineurin inhibitors (CNI) on human blood-derived T-cells and DC were compared., Methods: T-cells were stimulated with phytohemagglutinin (PHA) or allogeneic spleen antigen-presenting cells (APC) and T-cell proliferation and cytokine production were determined in presence or absence of IVIg or CNI. Immature blood dendritic cells (DC) were stimulated in presence or absence of IVIg or CNI, and allogeneic T-cell stimulatory capacity, cell death, and phenotypic maturation were established., Results: IVIg and CNI equally inhibited T-cell proliferation and IFN-gamma production after PHA stimulation or allogeneic stimulation. CD8+ T-cells were preferentially affected by both IVIg and CNI after allogeneic stimulation. Like CNI, addition of IVIg at later time points after T-cell activation suppressed mitotic progression of responding T-cells. IVIg-treated DC were suppressed in their capacity to stimulate allogeneic T-cell proliferation by 73+/-12%, whereas DC-function was not affected by CNI. The decreased allogeneic T-cell stimulatory capacity of IVIg-treated DC correlated to induction of cell death in DC and decreased up-regulation of CD40 and CD80., Conclusions: In vitro IVIg functionally inhibit the two principal immune cell-types involved in rejection and GVHD, i.e. T-cells and DC, whereas CNI only suppress T-cells. By targeting both T-cells and DC, IVIg may be a promising candidate for immunosuppressive treatment after allograft transplantation.

Patients with hepatocellular carcinoma (HCC) are characterized by a weak T-cell response to their tumor, and chronic carriers of hepatitis B virus or hepatitis C virus have a poor T-cell response against the virus. These inadequate T-cell responses may be due to insufficient activation of the T cells by dendritic cells (DCs). Because lymph nodes (LNs) are the primary site of antigen-specific T-cell activation, we hypothesized that hepatic LNs of patients with HCC and/or chronic viral hepatitis might have aberrant compositions of their DC populations. To address this hypothesis, we enumerated mature myeloid DCs (MDCs) and plasmacytoid DCs (PDCs) in hepatic LNs by quantitative immunohistochemistry. Patients with HCC and chronic viral hepatitis and patients with chronic viral hepatitis without HCC were compared with patients with liver inflammation of nonviral etiology and with organ donors with healthy livers. The numbers of PDCs and mature MDCs in hepatic LNs of patients with chronic viral hepatitis did not differ from those of patients with liver inflammation of nonviral etiology nor from individuals with healthy livers. However, hepatic LNs of patients with HBV or HCV infection complicated by HCC showed a 1.5-fold reduction in numbers of mature MDCs and a 4-fold increase in numbers of PDCs in their T-cell areas compared with those of patients with viral hepatitis only (P <.01). In conclusion, patients with HCC have an aberrant composition of the DC population in their hepatic LNs. This may be one of the causes of the inadequate T-cell response against HCC in these patients.

It is generally accepted that donor myeloid dendritic cells (MDC) are the main instigators of acute rejection after organ transplantation. The aim of the present study was to characterize MDC in human donor livers using liver grafts and perfusates as a source. Perfusates were collected during ex vivo vascular perfusion of liver grafts pretransplantation. MDC, visualized in wedge biopsies by immunohistochemistry with anti-BDCA-1 monoclonal antibody (mAb), were predominantly observed in the portal fields. Liver MDC, isolated from liver wedge biopsies, had an immature phenotype with a low expression of CD80 and CD83. Perfusates were collected from 20 grafts; perfusate mononuclear cells (MNC) contained 1.5% (range, 0.3-6.6%) MDC with a viability of 97 +/- 2%. Perfusates were a rich source of hepatic MDC since 0.9 x 10(6) (range, 0.11-4.5 x 10(6)) MDC detached from donor livers during vascular perfusion pretransplantation. Perfusate MDC were used to further characterize hepatic MDC. Perfusate MDC expressed less DC-LAMP (P = 0.000), CD80 (P = 0.000), CD86 (P = 0.003), and CCR7 (P = 0.014) than mature hepatic lymph node (LN) MDC, and similar CD86 (P = 0.140) and CCR7 (P = 0.262) as and more DC-LAMP (P = 0.007) and CD80 (P = 0.002) than immature blood MDC. Perfusate MDC differed from blood MDC in producing significantly higher amounts of interleukin (IL)-10 in response to lipopolysaccharide (LPS), and in being able to stimulate allogeneic T-cell proliferation. In conclusion, human donor livers contain exclusively immature MDC that detach in high numbers from the liver graft during pretransplantation perfusion. These viable MDC have the capacity to stimulate allogeneic T-cells, and thus may represent a major player in the induction of acute rejection., (Copyright 2006 AASLD)

It was investigated whether positive immunomagnetic selection with two novel DC-specific mAb allowed purification of functional myeloid dendritic cells (MDC) and plasmacytoid dendritic cells (PDC) from the human lymph nodes (LN). The results were compared with enrichment of DC by low-density Nycodenz gradient centrifugation followed by immunomagnetic depletion of residual B- and T-cells (Nycodenz method). MDC were selected from inguinal LN cell suspensions using CD1c mAb and PDC using anti-BDCA-4 mAb. Immunomagnetic selection with anti-CD1c mAb yielded highly pure MDC preparations (90 +/- 3% MDC; n = 7), provided that the B-cells were thoroughly depleted by using CD19 magnetic beads and Large Depletion (LD) columns prior to selection of MDC. The purified MDC comprised both mature and immature cells and were functional, secreting large amounts of cytokines upon stimulation and strongly stimulating allogeneic T-cell proliferation. Immunomagnetic selection with anti-BDCA-4 mAb enriched PDC 70-fold to a purity of 59 +/- 26% (n = 8). The contamination consisted mainly of BDCA-4(+) T- and NK-cells. The previously used Nycodenz method yielded mixtures of MDC and PDC, not allowing functional studies of MDC and PDC separately. In conclusion, positive immunomagnetic selection with CD1c mAb from human LN cell suspensions yields almost pure MDC preparations, which are, in contrast to those obtained by the Nycodenz method, not contaminated with PDC. Moreover, these MDC are functionally intact. Selection with anti-BDCA-4 mAb does enrich PDC from human LN, but the resulting preparations are contaminated with T- and NK-cells.

Dendritic cells (DC) play an important role in the induction of T-cell responses. We hypothesize that the hampered antiviral T-cell response in chronic hepatitis B patients is a result of impaired dendritic cell function. In this study, we compared the number, phenotype and functionality of two important blood precursor DC, myeloid DC (mDC) and plasmacytoid DC (pDC), of chronic hepatitis B patients with healthy volunteers. No differences in percentages of mDC and pDC in peripheral blood mononuclear cells were observed between chronic hepatitis B patients and healthy controls. The allostimulatory capacity of isolated and in vitro matured mDC, but not of pDC, was significantly decreased in patients compared to controls. Accordingly, a decreased percentage of mDC expressing CD80 and CD86 was observed after maturation, compared to controls. In addition, mDC of patients showed a reduced capacity to produce tumor necrosis factor alpha after a stimulus with synthetic double-stranded RNA and interferon gamma. Purified pDC from patients produced less interferon alpha, an important antiviral cytokine, in response to stimulation with Staphylococcus aureus Cowan strain I than pDC isolated from controls. In conclusion, mDC and pDC are functionally impaired in patients with chronic hepatitis B. This might be an important way by which hepatitis B virus evades an adequate immune response, leading to viral persistence and disease chronicity., (Copyright 2004 American Association for the Study of Liver Diseases)

CD4+type 1 T regulatory (Tr1) cells have a crucial role in inducing tolerance. Immune regulation by these cells is mainly mediated through the secretion of high amounts of IL-10. Several studies have suggested that this regulatory population may be involved in tumor-mediated immune-suppression. However, direct evidence of a role for Tr1 cells in human solid tumors is lacking. Usingex vivoisolated cells from individuals with hepatocellular carcinoma (HCC;n= 39) or liver metastases from colorectal cancer (LM-CRC;n= 60) we identify a CD4+FoxP3−IL-13−IL-10+T cell population in tumors of individuals with primary or secondary liver cancer that is characterized as Tr1 cells by the expression of CD49b and the lymphocyte activation gene 3 (LAG-3) and strong suppression activity of T cell responses in an IL-10 dependent manner. Importantly, the presence of tumor-infiltrating Tr1 cells is correlated with tumor infiltration of plasmacytoid dendritic cells (pDCs). pDCs exposed to tumor-derived factors enhance IL-10 production by Tr1 cells through up-regulation of the inducible co-stimulatory ligand (ICOS-L). These findings suggest a role for pDCs and ICOS-L in promoting intra-tumoral immunosuppression by Tr1 cells in human liver cancer, which may foster tumor progression and which might interfere with attempts of immunotherapeutic intervention. [ABSTRACT FROM AUTHOR]