Characterization of Antigen-Presenting Cell Subsets in Human Liver-Draining Lymph Nodes.

T-cell immunity in the liver is tightly regulated to prevent chronic liver inflammation in response to antigens and toxins derived from food and intestinal bacterial flora. Since the main sites of T cell activation in response to foreign components entering solid tissues are the draining lymph nodes (LN), we aimed to study whether Antigen-Presenting Cell (APC) subsets in human liver lymph-draining LN show features that may contribute to the immunologically tolerant liver environment. Healthy liver LN, iliac LN, spleen and liver perfusates were obtained from multi-organ donors, while diseased liver LN were collected from explanted patient livers. Inguinal LN were obtained from kidney transplant recipients. Mononuclear cells were isolated from fresh tissues, and immunophenotypic and functional characteristics of APC subsets were studied using flowcytometry and in ex vivo cultures. Healthy liver-draining LN contained significantly lower relative numbers of CD1c+ conventional dendritic cells (cDC2), plasmacytoid DC (PDC), and CD14+CD163+DC-SIGN+ macrophages (MF) compared to inguinal LN. Compared to spleen, both types of LN contained low relative numbers of CD141hi cDC1. Both cDC subsets in liver LN showed a more activated/mature immunophenotype than those in inguinal LN, iliacal LN, spleen and liver tissue. Despite their more mature status, cDC2 isolated from hepatic LN displayed similar cytokine production capacity (IL-10, IL-12, and IL-6) and allogeneic T cell stimulatory capacity as their counterparts from spleen. Liver LN from patients with inflammatory liver diseases showed a further reduction of cDC1, but had increased relative numbers of PDC and MF. In steady state conditions human liver LN contain relatively low numbers of cDC2, PDC, and macrophages, and relative numbers of cDC1 in liver LN decline during liver inflammation. The paucity of cDC in liver LN may contribute to immune tolerance in the liver environment.

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 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.

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.

Allosuppressive donor CD4+CD25+ regulatory T cells detach from the graft and circulate in recipients after liver transplantation.

Organ transplantation (Tx) results in a transfer of donor leukocytes from the graft to the recipient, which can lead to chimerism and may promote tolerance. It remains unclear whether this tolerance involves donor-derived regulatory T cells (Tregs). In this study, we examined the presence and allosuppressive activity of CD4+CD25+Foxp3+ Tregs in perfusates of human liver grafts and monitored the cells presence in the circulation of recipients after liver Tx. Vascular perfusions of 22 liver grafts were performed with University of Wisconsin preservation and albumin solutions. Flow cytometric analysis revealed that perfusate T cells had high LFA-1 integrin expression and had a reversed CD4 to CD8 ratio compared with control blood of healthy individuals. These findings indicate that perfusate cells are of liver origin and not derived from residual donor blood. Further characterization of perfusate mononuclear cells showed an increased proportion of CD4+CD25+CTLA4+ T cells compared with healthy control blood. Increased percentages of Foxp3+ cells, which were negative for CD127, confirmed the enrichment of Tregs in perfusates. In MLR, CD4+CD25+ T cells from perfusates suppressed proliferation and IFN-gamma production of donor and recipient T cells. In vivo within the first weeks after Tx, up to 5% of CD4+CD25+CTLA4+ T cells in recipient blood were derived from the donor liver. In conclusion, a substantial number of donor Tregs detach from the liver graft during perfusion and continue to migrate into the recipient after Tx. These donor Tregs suppress the direct pathway alloresponses and may in vivo contribute to chimerism-associated tolerance early after liver Tx.

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 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.