Shanta Mancham

Characterization of human liver dendritic cells in liver grafts and perfusates.

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 × 106 (range, 0.11‐4.5 × 106) 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. Liver Transpl 12: 384–393, 2006.

Prednisolone Suppresses the Function and Promotes Apoptosis of Plasmacytoid Dendritic Cells

Organ transplant recipients are highly susceptible to viral infections early after transplantation. Plasmacytoid dendritic cells (PDC) play a major role in antiviral immunity. Therefore, we determined the numbers of circulating PDC after liver transplantation (LTX) and established the effects of immunosuppressive drugs on PDC survival and function. PDC were determined longitudinally in 13 LTX recipients treated with prednisone and cyclosporin or tacrolimus. Purified PDC were cultured with or without clinically relevant concentrations of cyclosporin, tacrolimus or prednisolone. Apoptosis induction was monitored by determination of active caspase‐3, nuclear condensation and annexin‐V/7AAD staining. After LTX, a 4‐fold reduction in the number of circulating PDC was observed (p < 0.01), which recovered partially after discontinuation of prednisone treatment. In vitro, prednisolone induced apoptosis in PDC, while cyclosporin and tacrolimus did not. Higher doses of prednisolone were needed to induce apoptosis in Toll‐like receptor (TLR)‐stimulated PDC. However, non‐apoptosis inducing concentrations of prednisolone suppressed interferon‐alpha production, upregulation of co‐stimulatory molecules and allo‐stimulatory capacity of TLR‐stimulated PDC. In conclusion, prednisolone induces apoptosis in PDC, which explains the decline in circulating PDC numbers after transplantation. Moreover, prednisolone suppresses the functions of TLR‐stimulated PDC. Therefore, corticosteroid‐free immunosuppressive therapy may reduce the number and severity of viral infections after transplantation.

Liver grafts contain a unique subset of natural killer cells that are transferred into the recipient after liver transplantation

In contrast to other solid organ transplantations, liver grafts have tolerogenic properties. Animal models indicate that donor leukocytes transferred into the recipient after liver transplantation (LTX) play a relevant role in this tolerogenic phenomenon. However, the specific donor cell types involved in modulation of the recipient alloresponse are not yet defined. We hypothesized that this unique property of liver grafts may be related to their high content of organ‐specific natural killer (NK) and CD56+ T cells. Here, we show that a high proportion of hepatic NK cells that detach from human liver grafts during pretransplant perfusion belong to the CD56bright subset, and are in an activated state (CD69+). Liver NK cells contained perforin and granzymes, exerted stronger cytotoxicity against K562 target cells when compared with blood NK cells, and secreted interferon‐γ, but no interleukin‐10 or T helper 2 cytokines, upon stimulation with monokines. Interestingly, whereas the CD56bright subset is classically considered as noncytolytic, liver CD56bright NK cells showed a high content of cytolytic molecules and degranulated in response to K562 cells. After LTX, but not after renal transplantation, significant numbers of donor CD56dim NK and CD56+ T cells were detected in the recipient circulation for approximately 2 weeks. In conclusion, during clinical LTX, activated and highly cytotoxic NK cells of donor origin are transferred into the recipient, and a subset of them mixes with the recirculating recipient NK cell pool. The unique properties of the transferred hepatic NK cells may enable them to play a role in regulating the immunological response of the recipient against the graft and therefore contribute to liver tolerogenicity. Liver Transpl 16:895–908, 2010.

Superior immunomodulatory effects of intravenous immunoglobulins on human T-cells and dendritic cells: comparison to calcineurin inhibitors.

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-&ggr; 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.

Intravenous Immunoglobulin Treatment in Humans Suppresses Dendritic Cell Function via Stimulation of IL-4 and IL-13 Production

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.

Human plasmacytoid dendritic cells induce CD8+LAG-3+Foxp3+CTLA-4+ regulatory T cells that suppress allo-reactive memory T cells

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.

Dexamethasone transforms lipopolysaccharide-stimulated human blood myeloid dendritic cells into myeloid dendritic cells that prime interleukin-10 production in T cells

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.

Migration of allosensitizing donor myeloid dendritic cells into recipients after liver transplantation

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 recipients immune system by the direct presentation of donor allo‐antigens. Here we show prominent MDC chimerism in the recipients 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 recipients 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. Liver Transpl 16:12–22, 2010.

Rapamycin has suppressive and stimulatory effects on human plasmacytoid dendritic cell functions

Plasmacytoid dendritic cells (PDC) are involved in innate immunity by interferon (IFN)‐α production, and in adaptive immunity by stimulating T cells and inducing generation of regulatory T cells (Treg). In this study we studied the effects of mammalian target of rapamycin (mTOR) inhibition by rapamycin, a commonly used immunosuppressive and anti‐cancer drug, on innate and adaptive immune functions of human PDC. A clinically relevant concentration of rapamycin inhibited Toll‐like receptor (TLR)‐7‐induced IFN‐α secretion potently (−64%) but TLR‐9‐induced IFN‐α secretion only slightly (−20%), while the same concentration suppressed proinflammatory cytokine production by TLR‐7‐activated and TLR‐9‐activated PDC with similar efficacy. Rapamycin inhibited the ability of both TLR‐7‐activated and TLR‐9‐activated PDC to stimulate production of IFN‐γ and interleukin (IL)‐10 by allogeneic T cells. Surprisingly, mTOR‐inhibition enhanced the capacity of TLR‐7‐activated PDC to stimulate naive and memory T helper cell proliferation, which was caused by rapamycin‐induced up‐regulation of CD80 expression on PDC. Finally, rapamycin treatment of TLR‐7‐activated PDC enhanced their capacity to induce CD4+forkhead box protein 3 (FoxP3)+ regulatory T cells, but did not affect the generation of suppressive CD8+CD38+lymphocyte activation gene (LAG)‐3+ Treg. In general, rapamycin inhibits innate and adaptive immune functions of TLR‐stimulated human PDC, but enhances the ability of TLR‐7‐stimulated PDC to stimulate CD4+ T cell proliferation and induce CD4+FoxP3+ regulatory T cell generation.

PD-L1, Galectin-9 and CD8+ tumor-infiltrating lymphocytes are associated with survival in hepatocellular carcinoma

ABSTRACT Novel systemic treatments for hepatocellular carcinoma (HCC) are strongly needed. Immunotherapy is a promising strategy that can induce specific antitumor immune responses. Understanding the mechanisms of immune resistance by HCC is crucial for development of suitable immunotherapeutics. We used immunohistochemistry on tissue-microarrays to examine the co-expression of the immune inhibiting molecules PD-L1, Galectin-9, HVEM and IDO, as well as tumor CD8+ lymphocyte infiltration in HCC, in two independent cohorts of patients. We found that at least some expression in tumor cells was seen in 97% of cases for HVEM, 83% for PD-L1, 79% for Gal-9 and 66% for IDO. In the discovery cohort (n = 94), we found that lack of, or low, tumor expression of PD-L1 (p < 0.001), Galectin-9 (p < 0.001) and HVEM (p < 0.001), and low CD8+TIL count (p = 0.016), were associated with poor HCC-specific survival. PD-L1, Galectin-9 and CD8+TIL count were predictive of HCC-specific survival independent of baseline clinicopathologic characteristics and the combination of these markers was a powerful predictor of HCC-specific survival (HR 0.29; p <0.001). These results were confirmed in the validation cohort (n = 60). We show that low expression levels of PD-L1 and Gal-9 in combination with low CD8+TIL count predict extremely poor HCC-specific survival and it requires a change in two of these parameters to significantly improve prognosis. In conclusion, intra-tumoral expression of these immune inhibiting molecules was observed in the majority of HCC patients. Low expression of PD-L1 and Galectin-9 and low CD8+TIL count are associated with poor HCC-specific survival. Combining immune biomarkers leads to superior predictors of HCC mortality.