Immunoprofiling of early, untreated rheumatoid arthritis using mass cytometry reveals an activated basophil subset inversely linked to ACPA status.

BACKGROUND: Autoantibody production is a hallmark of rheumatoid arthritis (RA). Anti-citrullinated protein antibodies (ACPA) are highly disease-specific, and their presence is associated with more severe disease and poor prognosis compared to ACPA-negative patients. However, the immune cell composition associated with antibody-positive/negative disease is incompletely defined. Mass cytometry (MC) is a high-dimensional technique offering new possibilities in the determination of the immune cell composition in rheumatic diseases. Here, we set up a broad phenotyping panel to study the immune cell profile of early untreated RA to investigate if specific immune cell subsets are associated with ACPA+ versus ACPA- RA. METHODS: Freshly obtained PBMCs of early, untreated RA patients (8 ACPA+ and 7 ACPA-) were analysed using a 36-marker MC panel, including markers related to various immune lineages. Data were processed using Cytosplore for dimensional reduction (HSNE) and clustering. Groups were compared using Cytofast. A second validation cohort of cryopreserved PBMCs obtained from early RA patients (27 ACPA+ and 20 ACPA-) was used to confirm MC data by flow cytometry (FC). FC data were processed and analysed using both an unsupervised analysis pipeline and through manual gating. RESULTS: MC indicated no differences when comparing major immune lineages (i.e. monocytes, T and B cells), but highlighted two innate subsets: CD62L+ basophils (p = 0.33) and a subset of CD16- NK cells (p = 0.063). Although the NK cell subset did not replicate by FC, FC replication confirmed the difference in CD62L+ basophil frequency when comparing ACPA+ to ACPA- patients (mean 0.32% vs. 0.13%; p = 0.01). CONCLUSIONS: Although no differences in major lineages were found between early ACPA+ and ACPA- RA, this study identified the reduced presence of activated basophils in ACPA-negative disease as compared to ACPA-positive disease and thereby provides the first evidence for a connection between activated basophils and ACPA status.

Antibodies and B cells recognising citrullinated proteins display a broad cross-reactivity towards other post-translational modifications.

OBJECTIVE: Autoantibodies against antigens carrying distinct post-translational modifications (PTMs), such as citrulline, homocitrulline or acetyllysine, are hallmarks of rheumatoid arthritis (RA). The relation between these anti-modified protein antibody (AMPA)-classes is poorly understood as is the ability of different PTM-antigens to activate B-cell receptors (BCRs) directed against citrullinated proteins (CP). Insights into the nature of PTMs able to activate such B cells are pivotal to understand the 'evolution' of the autoimmune response conceivable underlying the disease. Here, we investigated the cross-reactivity of monoclonal AMPA and the ability of different types of PTM-antigens to activate CP-reactive BCRs. METHODS: BCR sequences from B cells isolated using citrullinated or acetylated antigens were used to produce monoclonal antibodies (mAb) followed by a detailed analysis of their cross-reactivity towards PTM-antigens. Ramos B-cell transfectants expressing CP-reactive IgG BCRs were generated and their activation on stimulation with PTM-antigens investigated. RESULTS: Most mAbs were highly cross-reactive towards multiple PTMs, while no reactivity was observed to the unmodified controls. B cells carrying CP-reactive BCRs showed activation on stimulation with various types of PTM-antigens. CONCLUSIONS: Our study illustrates that AMPA exhibit a high cross-reactivity towards at least two PTMs indicating that their recognition pattern is not confined to one type of modification. Furthermore, our data show that CP-reactive B cells are not only activated by citrullinated, but also by carbamylated and/or acetylated antigens. These data are vital for the understanding of the breach of B-cell tolerance against PTM-antigens and the possible contribution of these antigens to RA-pathogenesis.

Complement component C1q is produced by isolated articular chondrocytes.

OBJECTIVE: Inflammation and innate immune responses may contribute to development and progression of Osteoarthritis (OA). Chondrocytes are the sole cell type of the articular cartilage and produce extracellular-matrix molecules. How inflammatory mediators reach chondrocytes is incompletely understood. Previous studies have shown that chondrocytes express mRNA encoding complement proteins such as C1q, suggesting local protein production, which has not been demonstrated conclusively. The aim of this study is to explore C1q production at the protein level by chondrocytes. DESIGN: We analysed protein expression of C1q in freshly isolated and cultured human articular chondrocytes using Western blot, ELISA and flow cytometry. We examined changes in mRNA expression of collagen, MMP-1 and various complement genes upon stimulation with pro-inflammatory cytokines or C1q. mRNA expression of C1 genes was determined in articular mouse chondrocytes. RESULTS: Primary human articular chondrocytes express genes encoding C1q, C1QA, C1QB, C1QC, and secrete C1q to the extracellular medium. Stimulation of chondrocytes with pro-inflammatory cytokines upregulated C1QA, C1QB, C1QC mRNA expression, although this was not confirmed at the protein level. Extracellular C1q bound to the chondrocyte surface dose dependently. In a pilot study, binding of C1q to chondrocytes resulted in changes in the expression of collagens with a decrease in collagen type 2 and an increase in type 10. Mouse articular chondrocytes also expressed C1QA, C1QB, C1QC, C1R and C1S at the mRNA level. CONCLUSIONS: C1q protein can be expressed and secreted by human articular chondrocytes and is able to bind to chondrocytes influencing the relative collagen expression.

Targeted lipidomics reveals activation of resolution pathways in knee osteoarthritis in humans.

OBJECTIVE: To investigate the presence of inflammation and resolution pathways in osteoarthritis (OA). DESIGN: Tissues were obtained from knee OA patients and control rheumatoid arthritis (RA) patients. Cells in synovial fluid (SF) were visualized by flow cytometry. Cytokines and chemokines were measured by multiplex assay. Lipid mediators (LMs) were determined by targeted lipidomics using liquid-chromatography mass spectrometry. RESULTS: SF of OA patients contained less cells, especially neutrophils, less cytokines and comparable levels of chemokines compared to RA controls. Thirty-seven lipids were detected in the soluble fraction of SF, including polyunsaturated fatty acids (PUFAs) and their pro-inflammatory and pro-resolving lipoxygenase (LOX) and cyclooxygenase (COX) pathway markers in both OA and RA patients. Among these, pro-inflammatory LM such as prostaglandin E2 (PGE2) and thromboxane B2, as well as precursors and pathway markers of resolution such as 17-HDHA and 18-HEPE were detected. Interestingly, the pro-resolving lipid RvD2 could also be detected, but only in the insoluble fraction (cells and undigested matrix). Ratios of metabolites to their precursors indicated a lower activity of 5-LOX and 15-LOX in OA compared to RA, with no apparent differences in COX-derived products. Interestingly, synovial tissue and SF cells could produce 5-LOX and 15-LOX metabolites, indicating these cells as possible source of LM. CONCLUSIONS: By using a state-of-the-art technique, we show for the first time that resolution pathways are present in OA patients. A better understanding of these pathways could guide us to more effective therapeutic approaches to inhibit inflammation and further structural damage in OA and RA.

Prednisolone does not affect direct-acting antivirals against hepatitis C, but inhibits interferon-alpha production by plasmacytoid dendritic cells.

BACKGROUND: Chronic hepatitis C virus (HCV) infection compromises long-term outcomes of liver transplantation. Although glucocorticosteroid-based immunosuppression is commonly used, discussion is ongoing on the effect of prednisolone (Pred) on HCV recurrence and response to antiviral therapy post transplantation. Recently, new drugs (direct-acting antivirals) have been approved for the treatment of HCV, however, it remains unknown whether their antiviral activity is affected by Pred. The aim of this study was to investigate the effects of Pred on the antiviral activity of asunaprevir (Asu), daclatasvir (Dac), ribavirin (RBV), and interferon-alpha (IFN-α), and on plasmacytoid dendritic cells (PDCs), the main IFN-α-producing immune cells. METHODS: The effects of Pred and antiviral compounds were tested in both a subgenomic and infectious HCV replication model. Furthermore, effects were tested on human PDCs stimulated with a Toll-like receptor-7 ligand. RESULT: Pred did not directly affect HCV replication and did not inhibit the antiviral action of Asu, Dac, RBV, or IFN-α. Stimulated PDCs potently suppressed HCV replication. This suppression was reversed by treating PDCs with Pred. Pred significantly decreased IFN-α production by PDCs without affecting cell viability. When Asu and Dac were combined with PDCs, a significant cooperative antiviral effect was observed. CONCLUSION: This study shows that Pred acts on the antiviral function of PDCs. Pred does not affect the antiviral action of Asu, Dac, RBV, or IFN-α. This implies that there is no contraindication to combine antiviral therapies with Pred in the post-transplantation management of HCV recurrence.

Tumour antigen expression in hepatocellular carcinoma in a low-endemic western area.

BACKGROUND: Identification of tumour antigens is crucial for the development of vaccination strategies against hepatocellular carcinoma (HCC). Most studies come from eastern-Asia, where hepatitis-B is the main cause of HCC. However, tumour antigen expression is poorly studied in low-endemic, western areas where the aetiology of HCC differs. METHODS: We constructed tissue microarrays from resected HCC tissue of 133 patients. Expression of a comprehensive panel of cancer-testis (MAGE-A1, MAGE-A3/4, MAGE-A10, MAGE-C1, MAGE-C2, NY-ESO-1, SSX-2, sperm protein 17), onco-fetal (AFP, Glypican-3) and overexpressed tumour antigens (Annexin-A2, Wilms tumor-1, Survivin, Midkine, MUC-1) was determined by immunohistochemistry. RESULTS: A higher prevalence of MAGE antigens was observed in patients with hepatitis-B. Patients with expression of more tumour antigens in general had better HCC-specific survival (P=0.022). The four tumour antigens with high expression in HCC and no, or weak, expression in surrounding tumour-free-liver tissue, were Annexin-A2, GPC-3, MAGE-C1 and MAGE-C2, expressed in 90, 39, 17 and 20% of HCCs, respectively. Ninety-five percent of HCCs expressed at least one of these four tumour antigens. Interestingly, GPC-3 was associated with SALL-4 expression (P=0.001), an oncofetal transcription factor highly expressed in embryonal stem cells. SALL-4 and GPC-3 expression levels were correlated with vascular invasion, poor differentiation and higher AFP levels before surgery. Moreover, patients who co-expressed higher levels of both GPC-3 and SALL-4 had worse HCC-specific survival (P=0.018). CONCLUSIONS: We describe a panel of four tumour antigens with excellent coverage and good tumour specificity in a western area, low-endemic for hepatitis-B. The association between GPC-3 and SALL-4 is a novel finding and suggests that GPC-3 targeting may specifically attack the tumour stem-cell compartment.

CMV Primary Infection Is Associated With Donor-Specific T Cell Hyporesponsiveness and Fewer Late Acute Rejections After Liver Transplantation.

Viral infections, including cytomegalovirus (CMV), abrogate transplantation tolerance in animal models. Whether this also occurs in humans remains elusive. We investigated how CMV affects T cells and rejection episodes after liver transplantation (LT). Phenotype and alloreactivity of peripheral and allograft-infiltrating T cells from LT patients with different CMV status were analyzed by flow cytometry. The association of CMV status with early and late acute rejection was retrospectively analyzed in a cohort of 639 LT patients. CMV-positivity was associated with expansion of peripheral effector memory T cell subsets after LT. Patients with CMV primary infection showed donor-specific CD8(+) T cell hyporesponsiveness. While terminally differentiated effector memory cells comprised the majority of peripheral donor-specific CD8(+) T cells in CMV primary infection patients, they were rarely present in liver allografts. Retrospective analysis showed that R(-) D(+) serostatus was an independent protective factor for late acute rejection by multivariate Cox regression analysis (hazard ratio [HR] = 0.18, 95% CI = 0.04-0.86, p = 0.015). Additionally, CMV primary infection patients showed the highest Vδ1/Vδ2 γδ T cell ratio, which has been shown to be associated with operational tolerance after LT. In conclusion, our data suggest that CMV primary infection may promote tolerance to liver allografts, and CMV status should be considered when tapering or withdrawing immunosuppression.

SMAD4 exerts a tumor-promoting role in hepatocellular carcinoma.

Further understanding of the molecular biology and pathogenesis of hepatocellular carcinoma (HCC) is crucial for future therapeutic development. SMAD4, recognized as an important tumor suppressor, is a central mediator of transforming growth factor beta (TGFB) and bone morphogenetic protein (BMP) signaling. This study investigated the role of SMAD4 in HCC. Nuclear localization of SMAD4 was observed in a cohort of 140 HCC patients using tissue microarray. HCC cell lines were used for functional assay in vitro and in immune-deficient mice. Nuclear SMAD4 levels were significantly increased in patient HCC tumors as compared with adjacent tissues. Knockdown of SMAD4 significantly reduced the efficiency of colony formation and migratory capacity of HCC cells in vitro and was incompatible with HCC tumor initiation and growth in mice. Knockdown of SMAD4 partially conferred resistance to the anti-growth effects of BMP ligand in HCC cells. Importantly, simultaneous elevation of SMAD4 and phosphorylated SMAD2/3 is significantly associated with poor patient outcome after surgery. Although high levels of SMAD4 can also mediate an antitumor function by coupling with phosphorylated SMAD1/5/8, this signaling, however, is absent in majority of our HCC patients. In conclusion, this study revealed a highly non-canonical tumor-promoting function of SMAD4 in HCC. The drastic elevation of nuclear SMAD4 in sub-population of HCC tumors highlights its potential as an outcome predictor for patient stratification and a target for personalized therapeutic development.

Role of the immune system in pancreatic cancer progression and immune modulating treatment strategies.

Traditional chemotherapeutics have largely failed to date to produce significant improvements in pancreatic cancer survival. One of the reasons for the resilience of pancreatic cancer towards intensive treatment is that the cancer is capable of high jacking the immune system: during disease progression the immune system is converted from a system that attacks tumor cells into a support structure for the cancer, exerting trophic actions on the cancer cells. This turn-around of immune system action is achieved through mobilization and activation of regulatory T cells, myeloid derived suppressor cells, tumor-associated macrophages and fibroblasts, all of which suppress CD8 T cells and NK cells. This immune suppression occurs both through the expression of tolerance-inducing cell surface molecules, such as PD-L1, as well as through the production of "tolerogenic" cytokines, such as IL-10 and TGF-ß. Based on the accumulating insight into the importance of the immune system for the outcome of pancreatic cancer patients multiple new immunotherapeutic approaches against pancreatic cancer are being currently tested in clinical trials. In this review we give an overview of both the immune escaping mechanisms of pancreatic cancer as well as the new immune related therapeutic strategies currently being tested in pancreatic cancer clinical trials.

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.

Patients treated with high-dose intravenous immunoglobulin show selective activation of regulatory T cells.

Intravenous immunoglobulin (IVIg) is used to treat autoimmune and systemic inflammatory diseases caused by derailment of humoral and cellular immunity. In this study we investigated whether IVIg treatment can modulate regulatory T cells (Tregs ) in humans in vivo. Blood was collected from IVIg-treated patients with immunodeficiency or autoimmune disease who were treated with low-dose (n = 12) or high-dose (n = 15) IVIg before, immediately after and at 7 days after treatment. Percentages and activation status of circulating CD4(+) CD25(+) forkhead box protein 3 (FoxP3(+)) Tregs and of conventional CD4(+) FoxP3(-) T-helper cells (Tconv) were measured. The suppressive capacity of Tregs purified from blood collected at the time-points indicated was determined in an ex-vivo assay. High-dose, but not low-dose, IVIg treatment enhanced the activation status of circulating Tregs , as shown by increased FoxP3 and human leucocyte antigen D-related (HLA-DR) expression, while numbers of circulating Tregs remained unchanged. The enhanced activation was sustained for at least 7 days after infusion, and the suppressive capacity of purified Tregs was increased from 41 to 70% at day 7 after IVIg treatment. The activation status of Tconv was not affected by IVIg. We conclude that high-dose IVIg treatment activates Tregs selectively and enhances their suppressive function in humans in vivo. This effect may be one of the mechanisms by which IVIg restores imbalanced immune homeostasis in patients with autoimmune and systemic inflammatory disorders.

Genetic polymorphisms in innate immunity receptors do not predict the risk of bacterial and fungal infections and acute rejection after liver transplantation.

INTRODUCTION: We studied the influence of a broad range of genetic variants in recipient and donor innate immunity receptors on bacterial and fungal infections and acute rejection after liver transplantation (LT). METHODS: Seventy-six polymorphisms in TLR 1-10, NOD2, LBP, CD14, MD2, SIGIRR, Ficolins 1, -2, and -3, MASP 1, -2, and -3, and the complement receptor C1qR1 were determined in 188 LT recipients and 135 of their donors. Associations with clinically significant infections and acute rejection were analyzed for 50 polymorphisms. Significant associations were validated in an independent cohort of 181 recipients and 167 donors. RESULTS: Three recipient polymorphisms and 3 donor polymorphisms were associated with infections in the identification cohort, but none of these associations were confirmed in the validation cohort. Three donor polymorphisms were associated with acute rejection in the identification cohort, but not in the validation cohort. CONCLUSION: In contrast to their effect in the general population, 50 common genetic variations in innate immunity receptors do not influence susceptibility to bacterial/fungal infections after LT. In addition, no reproducible associations with acute rejection after LT were observed. Likely, transplant-related factors play a superior role as risk factors for bacterial/fungal infections and acute rejection after LT.

The infrapatellar fat pad of patients with osteoarthritis has an inflammatory phenotype.

OBJECTIVES: Obesity is a risk factor for the development of osteoarthritis (OA) in hands and knees. Adipose tissue can secrete different adipokines with powerful immunomodulatory effects. The infrapatellar fat pad (IFP) is an intra-articular organ in the vicinity of the synovium and cartilage. It is hypothesised that IFP-derived soluble factors could contribute to pathological processes in the knee joint. A study was therefore undertaken to compare the release of inflammatory mediators in the IFP and subcutaneous adipose tissue (ScAT) and to characterise the adipocytes and immune cell infiltrate in these tissues. METHODS: Paired IFP and ScAT samples were obtained from 27 patients with primary OA. The stromal vascular cell fraction (SVF) was isolated and characterised by fluorescence activated cell sorting. Cytokine and adipokine release in fat- and adipocyte-conditioned media was measured by luminex. RESULTS: IFP secreted higher levels of inflammatory mediators such as interleukin 6 (IL-6), adipsin, adiponectin and visfatin than ScAT. This could be due to differences in the phenotype of adipocytes and/or in the composition and phenotype of the SVF cells. IFP adipocyte-conditioned media showed a trend towards more IL-6 and adipsin than ScAT. Moreover, the SVF fraction of IFP contained more cells/g tissue, a lower percentage of T cells and a higher percentage of mast cells than ScAT. In addition, T cells had a predominantly pro-inflammatory phenotype while macrophages had a mixed pro- and anti-inflammatory phenotype in the IFP. CONCLUSION: There are profound differences in secreted inflammatory factors and immune cell composition between the IFP and ScAT. These data indicate that IFP-derived soluble mediators could contribute to pathophysiological processes in the OA knee joint.

Results of a two-center study comparing hepatic fibrosis progression in HCV-positive liver transplant patients receiving cyclosporine or tacrolimus.

A 2-center retrospective analysis was performed in 60 patients undergoing liver transplantation for hepatitis C virus (HCV)-related disease (cyclosporine in 20, tacrolimus in 40). Mean (±SEM) follow-up was 23.6 ± 22.5 and 22.3 ± 13.7 months in patients receiving cyclosporine or tacrolimus, respectively. Clinically indicated biopsies were performed in 15/20 cyclosporine patients (75%) and 22/40 tacrolimus patients (55%; P = .17). The Ishak fibrosis score was significantly lower in cyclosporine-treated patients versus tacrolimus-treated patients (mean 1.7 ± 0.4 vs 3.1 ± 0.4; P = .023), as was percentage of fibrosis grade Ishak ≥4 (7% vs 41%; P = .028). The mean time to moderate fibrosis (Ishak score ≥3) was 38.2 ± 15.1 months in cyclosporine patients (4/15) and 23.5 ± 12.6 months in tacrolimus patients (14/22); the difference was not statistically significant (P = .09). This retrospective study suggests that cyclosporine-based immunosuppression is associated with less severe hepatic fibrosis in HCV-positive liver transplant recipients compared with tacrolimus-based regimens, but a larger prospective comparative trial is necessary to confirm these findings.

Impairment of circulating myeloid dendritic cells in immunosuppressed liver transplant recipients.

The aim of the present study was to elucidate the impact of liver transplantation (LTX) on myeloid dendritic cell (MDC) homeostasis. We observed a threefold reduction of circulating CD1c(+) MDC immediately after LTX (n = 16; P < 0.01), and normalization between 3 and 12 months after LTX. This decline was not due to recruitment of MDC into the liver graft, as numbers of MDC in post-LTX liver graft biopsies were not increased compared to pre-LTX biopsies (n = 7). Moreover, no change in chemokine receptor expression on circulating MDC was observed, suggesting that their homing properties were not altered. Normalization of circulating MDC was associated with withdrawal of corticosteroid therapy, and not with changes in calcineurin inhibitor intake, indicating that corticosteroids are responsible for the observed changes in numbers of circulating MDC. During high-dose corticosteroid treatment early after LTX, circulating MDC showed a lowered maturation status with decreased expression of human leucocyte antigen D-related (HLA-DR) and CD86 compared to pre-LTX values (P < 0.01). However, when MDC from blood of LTX recipients were matured ex vivo, they up-regulated HLA-DR and co-stimulatory molecules to a comparable extent as MDC from healthy individuals. In addition, ex vivo matured MDC from both groups had equal allogeneic T cell stimulatory capacity. In conclusion, during the first months after LTX numbers and maturational status of circulating MDC are impaired significantly, probably due to a suppressive effect of corticosteroids on MDC. However, corticosteroid therapy does not imprint MDC with an intrinsic resistance to maturation stimuli.

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.

Polymorphisms in the T cell regulatory gene cytotoxic T lymphocyte antigen 4 influence the rate of acute rejection after liver transplantation.

BACKGROUND: The cytotoxic T lymphocyte antigen 4 (CTLA-4) gene encodes for a membrane bound (mCTLA-4) and a soluble (sCTLA-4) isoform, which are both involved in regulation of T cell function. The CTLA-4 +49A/G single nucleotide polymorphism (SNP) influences expression of mCTLA-4; +6230G/A SNP affects the production of sCTLA-4. AIM: To examine whether these functional SNPs influence the rate of rejection after liver transplantation. PATIENTS AND METHODS: Liver graft recipients (n = 483) were genotyped for both SNPs, and haplotypes were reconstructed. Association with rejection was tested by the log rank test using the Kaplan-Meier method with time to the first acute rejection episode as outcome. Multiple analysis of SNPs together with demographic factors was performed by Cox regression. RESULTS: Three haplotypes were observed in the cohort: +49A/+6230A, +49A/+6230G, and +49G/+6230G. The +49A/+6230G haplotype was significantly and dose dependently associated with acute rejection (p = 0.01). Of the demographic factors tested, only underlying liver disease was significantly associated with rejection. Adjusted for underlying liver disease, each additional +49A/+6230G haplotype allele resulted in a significantly higher risk of acute rejection (risk ratio 1.34 (95% confidence interval 1.04-1.72); p = 0.02). Patients who lacked this haplotype had the lowest, carriers an intermediate, and homozygotes the highest risk of acute rejection. CONCLUSION: The CTLA-4 +49A/+6230G haplotype, which encodes for normal mCTLA-4 expression but reduced sCTLA-4 production, is a co-dominant risk allele for acute rejection after clinical liver transplantation. This implies that even under immunosuppression, CTLA-4 is critically involved in the regulation of the human immune response to allogeneic grafts.

Hepatitis B immunoglobulins inhibit dendritic cells and T cells and protect against acute rejection after liver transplantation.

The efficacy of anti-viral intravenous immunogobulins (anti-HBs Ig and anti-CMV Ig) in preventing acute rejection after liver transplantation was assessed in a retrospective analysis, and correlated to their effects on immune cells in vitro. HBs Ag-positive liver graft recipients (n = 40) treated prophylactically with anti-HBs Ig had a significantly lower incidence of acute rejection compared with recipients without viral hepatitis (n = 147) (12% vs. 34%; p = 0.012), while the incidence of rejection in HCV-positive recipients (n = 29) was similar to that in the control group. Treatment with anti-CMV Ig (n = 18) did not protect against rejection. In vitro, anti-HBs Ig suppressed functional maturation of and cytokine production by human blood-derived dendritic cells (DC) at concentrations similar to the serum concentrations reached during anti-HBs Ig treatment of liver graft recipients. In addition, anti-HBs Ig inhibited allo-antigen- and lectin-stimulated proliferation of peripheral T cells. Anti-CMV Ig suppressed functional DC-maturation and alloantigen-stimulated T-cell proliferation, but not lectin-driven T-cell proliferation. In conclusion, anti-HBs Ig protects against acute rejection after liver transplantation, probably by functional inhibition of the two principal immune cells involved in allograft rejection, DC and T cells.