Dead Cells Induce Innate Anergy via Mertk after Acute Viral Infection.

Infections can result in a temporarily restricted unresponsiveness of the innate immune response, thereby limiting pathogen control. Mechanisms of such unresponsiveness are well studied in lipopolysaccharide tolerance; however, whether mechanisms of tolerance limit innate immunity during virus infection remains unknown. Here, we find that infection with the highly cytopathic vesicular stomatitis virus (VSV) leads to innate anergy for several days. Innate anergy is associated with induction of apoptotic cells, which activates the Tyro3, Axl, and Mertk (TAM) receptor Mertk and induces high levels of interleukin-10 (IL-10) and transforming growth factor ß (TGF-ß). Lack of Mertk in Mertk-/- mice prevents induction of IL-10 and TGF-ß, resulting in abrogation of innate anergy. Innate anergy is associated with enhanced VSV replication and poor survival after infection. Mechanistically, Mertk signaling upregulates suppressor of cytokine signaling 1 (SOCS1) and SOCS3. Dexamethasone treatment upregulates Mertk and enhances innate anergy in a Mertk-dependent manner. In conclusion, we identify Mertk as one major regulator of innate tolerance during infection with VSV.

High Frequencies of Anti-Host Reactive CD8+ T Cells Ignore Non-Hematopoietic Antigen after Bone Marrow Transplantation in a Murine Model.

BACKGROUND: Graft versus host disease (GvHD) occurs in 20% of cases with patients having an MHC I matched bone marrow transplantation (BMT). Mechanisms causing this disease remain to be studied. METHODS: Here we used a CD8+ T cell transgenic mouse line (P14/CD45.1+) and transgenic DEE mice bearing ubiquitously the glycoprotein 33-41 (GP33) antigen derived from the major lymphocytic choriomeningitis virus (LCMV) epitope to study mechanisms of tolerance in anti-host reactive CD8+ T cells after BMT. RESULTS: We found that anti-host reactive CD8+ T cells (P14 T cells) were not negatively selected in the thymus and that they were present in wild type (WT) recipient mice as well as in DEE recipient mice. Anti-host reactive CD8+ T cells ignored the GP33 antigen expressed ubiquitously by host cells but they could be activated ex vivo via LCMV-infection. Lipopolysaccharides (LPS) induced transient cell damage in DEE mice bearing anti-host reactive CD8+ T cells after BMT, suggesting that induction of host inflammatory response could break antigen ignorance. Introducing the GP33 antigen into BM cells led to deletion of anti-host reactive CD8+ T cells. CONCLUSION: We found that after BMT anti-host reactive CD8+ T cells ignored host antigen in recipients and that they were only deleted when host antigen was present in hematopoietic cells. Moreover, LPS-induced immune activation contributed to induction of alloreactivity of anti-host reactive CD8+ T cells after BMT.

HLA-C, DRB1 and DQB1 alleles involved in genetic predisposition to psoriasis vulgaris in the Slovak population.

Psoriasis vulgaris is a complex chronic skin disease with immunological and genetic background. The most important predisposing genetic factors in psoriasis are genes of the human leukocyte antigen (HLA) region. Accumulative evidence has shown that several HLA alleles are closely associated with psoriasis; however, they tend to vary in different racial and ethnic backgrounds. One hundred forty-seven unrelated Slovak patients with psoriasis vulgaris (average age at onset 28 ± 14 years) were genotyped for the HLA-C, DQB1 and DRB1 alleles by the polymerase chain reaction using sequence-specific primers. Allele frequencies observed in the group of psoriatic patients were compared to those obtained in the ethnically matched control group comprising 194 subjects with no history of psoriasis. Susceptibility to psoriasis vulgaris in our study group is significantly associated with HLA-C*06 (odds ratio (OR) = 3.85), DRB1*07 (OR = 2.56) and DQB1*02 (OR = 1.09), respectively, whereas DRB*01 (OR = 0.05) is associated negatively. Hereby, we provide the first report on the association of HLA-C, DRB1 and DQB1 alleles with psoriasis in the Slovak population. Our findings confirm HLA-C*06 and DRB1*07 as the most important genetic risk factors for psoriasis. However, the role of HLA genes as causative in the pathogenesis of the disease remains unclear. Identification of genetic factors that increase the risk of psoriasis is a precondition that helps to elucidate the pathogenesis of this troubling disease and identify targets for a more specific and effective therapy.

Genetic, immunological and clinical risk factors for biliary strictures following liver transplantation.

BACKGROUND: Biliary strictures after liver transplantation (LT) are a major cause of morbidity and reduced graft survival. AIMS: The purpose of this study was to investigate genetic, immunological and clinical risk factors for the occurrence of post-LT ischaemic type biliary lesions (ITBLs) and biliary anastomotic strictures (AS). METHODS: Clinical and laboratory data, chemokine receptor (CCR) genotypes, chemotactic cytokines and anti-major-histocompatibility complex antibodies in serum were investigated in 162 LT patients. RESULTS: In the univariate analysis, older donor and recipient age, partial LT, high peak aspartate aminotransaminase (AST) levels and CC chemokine receptor 5 delta32 loss-of-function mutation (CCR5Δ32) were associated with ITBL, whereas LT for acute liver failure (ALF), ABO-compatible non-identical LT, presence of donor-specific anti-human leucocyte antigen (HLA) class II antibodies and fractalkine receptor (CX3CR1)-249II allele were associated with AS. In the multivariate analysis, CCR5Δ32 was an independent risk factor for ITBL, whereas LT for ALF, ABO-compatible non-identical LT, and CX3CR1-249II allele remained predictive for AS. Serum levels of interferon-gamma and interleukin (IL)-6 as well as IL-10 were significantly increased in patients with biliary strictures. CONCLUSION: Specific chemokine receptor polymorphisms of the recipient are associated with development of post-LT biliary strictures. Altered cytokine profile may contribute to enhanced fibrotic tissue remodelling and biliary stricture formation. Screening of anti-HLA antibodies might be useful for early identification of at-risk patients who could benefit from closer surveillance and tailored immunosuppressive regimen. Our findings may have relevance for prediction and management of post-LT biliary strictures.

Polymorphisms in the genes encoding TGF-beta1, TNF-alpha, and IL-6 show association with type 1 diabetes mellitus in the Slovak population.

Numerous cytokines have been shown to participate in the pathogenesis of type 1 diabetes (T1D). As gene polymorphisms can influence cytokine production or function, they may potentially contribute to genetic predisposition to the disease. The aim of this study was therefore to investigate the role of 22 single nucleotide polymorphisms (SNPs) in 13 cytokine and cytokine receptor genes in genetic susceptibility to T1D. Polymerase chain reaction with sequence-specific primers was used to genotype cytokine SNPs and HLA-DRB1 alleles in 151 diabetics and 140 healthy individuals of Slovak origin. Univariate analysis showed that transforming growth factor (TGF)-beta1 codon 10 TT homozygotes were significantly more susceptible to developing T1D than C allele carriers (P (c) = 0.0066, OR = 2.46). Furthermore, tumor necrosis factor (TNF)-alpha -308 A allele carriers were also significantly overrepresented among the diabetics (P (c) = 0.0031, OR = 2.62); however, the association of the -308 A allele with T1D might be due to its strong linkage disequilibrium with the susceptibility allele HLA-DRB1*0301. An association was also found with interleukin (IL)-6 -174 G/C and nt565 G/A SNPs; however, its significance was lost when statistical correction was applied. These data suggest that the TGF-beta1 codon 10 SNP is among numerous genetic variations with small individual effects on T1D development. Moreover, a possible role of TNF-alpha and IL-6 SNPs cannot be ruled out, although their association with T1D was due to strong LD with the HLA class II susceptibility allele or did not withstand statistical correction, respectively.

Association of common polymorphisms in known susceptibility genes with rheumatoid arthritis in a Slovak population using osteoarthritis patients as controls.

INTRODUCTION: Both genetic and environmental factors contribute to rheumatoid arthritis (RA), a common and complex autoimmune disease. As well as the major susceptibility gene HLA-DRB1, recent genome-wide and candidate-gene studies reported additional evidence for association of single nucleotide polymorphism (SNP) markers in the PTPN22, STAT4, OLIG3/TNFAIP3 and TRAF1/C5 loci with RA. This study was initiated to investigate the association between defined genetic markers and RA in a Slovak population. In contrast to recent studies, we included intensively-characterized osteoarthritis (OA) patients as controls. METHODS: We used material of 520 RA and 303 OA samples in a case-control setting. Six SNPs were genotyped using TaqMan assays. HLA-DRB1 alleles were determined by employing site-specific polymerase chain reaction (PCR) amplification. RESULTS: No statistically significant association of TRAF1/C5 SNPs rs3761847 and rs10818488 with RA was detected. However, we were able to replicate the association signals between RA and HLA-DRB1 alleles, STAT4 (rs7574865), PTPN22 (rs2476601) and OLIG3/TNFAIP3 (rs10499194 and rs6920220). The strongest signal was detected for HLA-DRB1*04 with an allelic P = 1.2*10-13 (OR = 2.92, 95% confidence interval (CI) = 2.18 - 3.91). Additionally, SNPs rs7574865STAT4 (P = 9.2*10-6; OR = 1.71, 95% CI = 1.35 - 2.18) and rs2476601PTPN22 (P = 9.5*10-4; OR = 1.67, 95% CI = 1.23 - 2.26) were associated with susceptibility to RA, whereas after permutation testing OLIG3/TNFAIP3 SNPs rs10499194 and rs6920220 missed our criteria for significance (Pcorr = 0.114 and Pcorr = 0.180, respectively). CONCLUSIONS: In our Slovak population, HLA-DRB1 alleles as well as SNPs in STAT4 and PTPN22 genes showed a strong association with RA.

Relevance of C1 and C2 epitopes for hemopoietic stem cell transplantation: role for sequential acquisition of HLA-C-specific inhibitory killer Ig-like receptor.

Killer Ig-like receptors (KIR) and HLA class I ligands were studied in unrelated hemopoietic stem cell transplantation for chronic myeloid leukemia (n = 108). Significantly improved overall survival was observed in patients, which were homozygous for HLA-C-encoded group 1 (C1) ligands compared with those with group 2 (C2) ligands. Favorable outcome in the former patient group was an early effect that was highly significant in patients transplanted with G-CSF-mobilized peripheral blood and patients with advanced disease stages. In contrast, presence of C1 ligands in the donor was associated with significantly reduced patient survival. The differential roles of the two HLA-C ligands are explained in the context of a biased NK cell reconstitution, which is generally dominated by the presence of C1- but absence of C2-specific NK cells. The clinical observations are corroborated by in vitro experiments showing that NK cells derived from hemopoietic progenitor cells generally acquire the C1-specific inhibitory KIR2DL2/3 at earlier time points and with higher frequency than the C2-specific KIR2DL1. These findings define a novel determinant for understanding the role of NK cells in clinical hemopoietic stem cell transplantation.

Increased frequencies of CD8+ T lymphocytes recognizing wild-type p53-derived epitopes in peripheral blood correlate with presence of epitope loss tumor variants in patients with hepatocellular carcinoma.

Wild-type (WT) sequence p53 peptides are attractive candidates for broadly applicable cancer vaccines. The aim of this study was to evaluate the potential of a WT p53-based immunotherapeutic approach for patients with hepatocellular carcinoma (HCC). Circulating CD8+ T cells specific for WT p53(149-157) and WT p53(264-272) HLA-A*0201 restricted epitopes were directly identified in the peripheral blood by the use of peptide/HLA-A2.1 tetramers in 24 HCC patients. Cytotoxic T lymphocyte (CTL) activity after WT p53 peptide-specific stimulation was assessed by analysis of granzyme B and interferon-gamma mRNA transcription, using a quantitative real-time polymerase chain reaction assay. Tumor immunophenotyping was performed to evaluate the p53 status, the expression of major histocompatibility complex (MHC) and costimulatory molecules in freshly isolated tumor cells. HCC patients exhibited significantly higher frequencies of WT p53-specific memory CD8+ T cells and stronger WT p53-specific CTL activity, when compared with healthy controls. Increased frequencies of p53-specific CD8+ T cells and their activity correlated with selective HLA-A2 allele loss and reduced costimulatory molecule expression of tumor cells. Moreover, augmented numbers of p53-specific T cells coincided with high MHC class II expression in tumor cells but were inversely related to the T status of the tumor node metastasis staging system. Our results indicate the existence of natural immunosurveillance and tumor immune evasion, involving a T cell response against WT p53 tumor antigen in patients with HCC. These findings may have important implications for the future development of cancer vaccines.

Genotypic inhibitory killer immunoglobulin-like receptor ligand incompatibility enhances the long-term antileukemic effect of unmodified allogeneic hematopoietic stem cell transplantation in patients with myeloid leukemias.

It remains controversial whether alloreactive donor-derived natural killer (NK) cells display graft-versus-leukemia reactions after unmodified allogeneic hematopoietic stem cell transplantation (HSCT). The present study evaluated the role of inhibitory killer immunoglobulin-like receptor (KIR) ligand incompatibility using a well-defined and uniform setting of unmodified allogeneic HSCT in 374 patients with myeloid leukemias. The most striking finding was a significant heterogeneity in the 5-year estimates of hematologic leukemic relapse after human leukocyte antigen (HLA)-identical (n = 237; 22%), HLA class I-disparate (n = 89; 18%), and KIR ligand-incompatible transplantations (n = 48; 5%) (P < .04). Multivariate analysis confirmed that the relative relapse risk (RR) was influenced by HLA class I disparity alone (RR 0.49), but was lowest after HLA class I-disparate, KIR ligand-incompatible transplantations (RR 0.24) (P < .008). The primary graft failure rates, however, increased from 0.4% after HLA class I-identical to 2.3% after HLA class I-disparate, and to 6.3% after KIR ligand-incompatible transplantations, respectively (P < .02). Unlike some other reports, no beneficial effect of KIR ligand incompatibility on other major endpoints of allogeneic HSCT (transplantation-related mortality, and overall and event-free survival) was detectable in the present study. In conclusion, unmodified allogeneic HSCT from KIR ligand-incompatible donors provides a superior long-term antileukemic efficacy in patients with myeloid malignancies.

Impact of disparity of minor histocompatibility antigens HA-1, CD31, and CD49b in hematopoietic stem cell transplantation of patients with chronic myeloid leukemia with sibling and unrelated donors.

Despite human leukocyte antigen (HLA) identity between donor and recipient, several patients develop acute graft-versus-host disease (aGVHD) after hematopoetic stem cell transplantation (HSCT) because of minor histocompatibility antigen (mHag) incompatibilities. The impact of multiple mHag disparities on the clinical outcome after HSCT still remains to be determined. We studied the genomic polymorphisms of HA-1, CD31, and CD49b and correlated mHag distribution with the occurrence of aGVHD after HSCT from HLA-matched sibling and unrelated donors. All 163 patients examined in our single-center study underwent HSCT for chronic myeloid leukemia in the first chronic phase. HA-1 and CD31 disparities are associated with increased aGVHD incidence in a subgroup of patients who test HLA-B44 supertype positive in univariate analysis. However, in a multivariate analysis, only increased patient age was confirmed as an independent aGVHD risk factor. Our findings indicate that the impact of mHag disparity on aGVHD development in HSCT from HLA-matched sibling and unrelated donors seems to be subordinated to classic aGVHD risk factors.

Hepatitis B virus-induced defect of monocyte-derived dendritic cells leads to impaired T helper type 1 response in vitro: mechanisms for viral immune escape.

Dendritic cells (DC) are the most potent antigen-presenting cells and play a central role in the induction of antiviral immune responses. Recently, we have shown that monocyte-derived DC (MoDC) from patients with chronic hepatitis B virus (HBV) infection are functionally impaired. In our present study MoDC from healthy subjects were propagated in vitro and inoculated with HBV particles to investigate the precise mechanisms that underly MoDC dysfunction. T-cell proliferation assays revealed an impaired allostimulatory capacity of HBV-inoculated MoDC (HBV-MoDC) as well as a lower potential of stimulating autologous T cells against a recall antigen in comparison to control-MoDC. Interleukin-2, tumour necrosis factor-alpha and interferon-gamma production by T cells in proliferation assays with HBV-MoDC was significantly lower than with control-MoDC and correlated with lower IL-12 production in HBV-MoDC cultures. The presence of the nucleoside analogue lamivudine (3TC), an inhibitor of HBV replication, restored impaired allostimulatory function of HBV-MoDC and up-regulated major histocompatibility complex class II expression. These results show that HBV infection compromises the antigen-presenting function of MoDC with concomitant impairment of T helper cell type 1 responses. This may play an important role for viral immune escape leading to chronic HBV infection. However, 3TC treatment can overcome HBV-MoDC-related T-cell hyporeactivity and this underscores its important role in enhanced immune responses to HBV.

Hematopoietic stem cell transplantation: contrasting the outcome of transplantations from HLA-identical siblings, partially HLA-mismatched related donors, and HLA-matched unrelated donors.

Allogeneic hematopoietic stem cell transplantation (HSCT) is a proven curative therapy for many hematologic malignancies. HSCT from HLA-identical sibling donors (ISDs) is still the golden standard. For the remaining 70% of the patients lacking an ISD, alternative (partially) HLA-matched family donors (MFDs) and HLA-matched unrelated donors (MUDs) are now widely accepted. However, it is presently unclear whether outcome after HSCT from an MFD or an MUD is superior. Thus, the classical clinical end points after HSCT from an ISD (n = 138), MFD (n = 86), and MUD (n = 101) were compared by means of univariate and multivariate statistical analyses. MFD transplantations with HLA class II (DRB1 +/- DQB1) mismatches in graft-versus-host (GVH) direction showed an increased risk of grades II to IV graft-versus-host disease, and MFD transplantations with more than a single HLA class I (A +/- B +/- C) mismatch in host-versus-graft (HVG) direction were associated with a higher risk of graft failure. However, no significant difference in overall survival was detectable among the 3 study groups after adjustment for the main predictors of transplantation outcome. Thus, for patients lacking an ISD, an already identified MFD with an HLA-DRB1 +/- DQB1 mismatch in GVH or a combined HLA-A +/- B +/- C mismatch in HVG direction should be accepted only in clinically urgent settings that leave no time to identify an MUD.

Role of G protein beta3 subunit C825T and HLA class II polymorphisms in the immune response after HBV vaccination.

The G protein beta3 (GNB3) subunit and HLA are candidate genes predictive of immune response capacity. We therefore studied the influence of both gene systems on cellular and humoral immunity against hepatitis B virus (HBV) in 79 HBV booster-vaccinated healthy volunteers and an independent group of 77 probands after HBV basic immunization. Following booster vaccination, lymphocyte in vitro proliferation after stimulation with HBV surface antigen was 2.5-fold increased in GNB3 825T (TC + TT) vs CC allele carriers (P = 0.01) and was not influenced by HLA-DRB1 or DQB1 alleles. In addition, anti-HBs antibody titers in both groups were 2-fold increased in TC vs CC and decreased in TT vs CC allele carriers. However, antibody titers after HBV booster immunization were elevated in HLA-DQB1*0301 carriers (P corrected = 0.027). In summary, the GNB3 825T allele appears as a marker particularly predictive of cellular and HLA-DQB1*0301 of humoral immune responses following HBV vaccination.