Primary Human Renal-Derived Tubular Epithelial Cells Fail to Recognize and Suppress BK Virus Infection.

BACKGROUND: BK polyomavirus (BKV)-associated nephropathy is a threat to kidney allograft survival affecting up to 15% of renal transplant patients. Previous studies revealed that tubular epithelial cells (TEC) show a limited response towards BKV infection. Here we investigated the interplay between BKV and TEC in more detail. In particular, we questioned whether BKV suppresses and/or evades antiviral responses. METHODS: Human primary TEC and peripheral blood mononuclear cells were infected with BKV Dunlop strain or other viruses. Moreover, TEC were stimulated with genomic double-stranded (ds)DNA or IFN. Viral replication and cellular responses were measured using quantitative real time PCR and multiplex assay. RESULTS: BKV infection of primary human TEC did not induce an antiviral response, whereas infection with influenza A virus, herpes simplex virus 1, or cytomegalovirus induced a strong antiviral response measured by upregulation of interferon-stimulated genes, such as CXCL10 and DAI. In addition, intracellular delivery of dsDNA or stimulation with IFN did elicit a rapid and pronounced response. However, BKV infection did not affect dsDNA-induced gene expression, indicating BKV did not modulate the antiviral response. Prestimulation of primary TEC with IFNα or dsDNA did not hamper replication of BKV, whereas influenza and herpes simplex virus 1 replication were clearly reduced. In contrast, BKV infection of leukocytes did elicit an antiviral response. CONCLUSIONS: BKV specifically evades innate immunity in TEC and is not susceptible to an intrinsic interferon response, which may facilitate latent presence of the virus in this cell type.

Clinically Relevant Reactivation of Polyomavirus BK (BKPyV) in HLA-A02-Positive Renal Transplant Recipients Is Associated with Impaired Effector-Memory Differentiation of BKPyV-Specific CD8+ T Cells.

Polyomavirus BK (BKPyV) frequently reactivates in immunosuppressed renal transplant recipients (RTRs) and may lead to graft loss due to BKPyV-induced interstitial nephritis (BKVN). Little is known on the differentiation of CD8+ T cells targeting BKPyV in RTRs. Here we investigated whether BKPyV-specific CD8+ T cell differentiation differs in RTRs with varying degrees of BKPyV reactivation and/or BKVN. Using combinatorial encoding with tetramers carrying BKPyV major capsid protein (VP1) and large T antigen protein (LTAG) epitopes, we investigated CD8+ T cell responses to BKPyV in longitudinally obtained PBMC samples from 46 HLA-A02-positive RTRs and 20 healthy adults. We were also able to isolate BKPyV-specific CD8+ T cells from five renal allografts, two of which were affected by BKVN. Before transplantation, BKPyV-specific CD8+ T cells targeting VP1 and LTAG epitopes appeared predominantly as central-memory and CD27+/CD28+ effector-memory (TEM), and naïve-like PD-1-expressing cells, respectively. After viral reactivation, BKPyV-specific CD8+ T cells assumed CD28- TEM and TEMRA states in patients who were able to control BKPyV, whereas differentiation lagged behind in patients with severe viral reactivation or BKVN. Furthermore, VP1-specific CD69+/CD103+ tissue-resident memory (TRM) cells accumulated in BKVN-affected allografts but lacked signs of effector differentiation. In contrast, granzyme B-expressing effector cells were detected in allografts not affected by BKVN. In conclusion, effector-memory differentiation of BKPyV-specific CD8+ T cells in patients with high viral load or BKVN is impaired. Further characterization of the specific mechanisms behind this altered cellular differentiation is necessary to develop therapies that can prevent the emergence of BKVN.

Phenotypic and functional characterization of circulating polyomavirus BK VP1-specific CD8+ T cells in healthy adults.

The human polyomavirus BK virus (BKV) establishes a latent and asymptomatic infection in the majority of the population. In immunocompromised individuals, the virus frequently (re)activates and may cause severe disease such as interstitial nephritis and hemorrhagic cystitis. Currently, the therapeutic options are limited to reconstitution of the antiviral immune response. T cells are particularly important for controlling this virus, and T cell therapies may provide a highly specific and effective mode of treatment. However, little is known about the phenotype and function of BKV-specific T cells in healthy individuals. Using tetrameric BKV peptide-HLA-A02 complexes, we determined the presence, phenotype, and functional characteristics of circulating BKV VP1-specific CD8(+) T cells in 5 healthy individuals. We show that these cells are present in low frequencies in the circulation and that they have a resting CD45RA(-) CD27(+) memory and predominantly CCR7(-) CD127(+) KLRG1(+) CD49d(hi) CXCR3(hi) T-bet(int) Eomesodermin(lo) phenotype. Furthermore, their direct cytotoxic capacity seems to be limited, since they do not readily express granzyme B and express only little granzyme K. We compared these cells to circulating CD8(+) T cells specific for cytomegalovirus (CMV), Epstein-Barr virus (EBV), and influenza virus (Flu) in the same donors and show that BKV-specific T cells have a phenotype that is distinct from that of CMV- and EBV-specific T cells. Lastly, we show that BKV-specific T cells are polyfunctional since they are able to rapidly express interleukin-2 (IL-2), gamma interferon (IFN-γ), tumor necrosis factor α, and also, to a much lower extent, MIP-1ß and CD107a.

Viral double-stranded RNA sensors induce antiviral, pro-inflammatory, and pro-apoptotic responses in human renal tubular epithelial cells.

Viral infection in the kidney is characterized by tubular injury induced directly by the virus and/or by cytotoxic lymphocytes. Previously, we found that human tubular epithelial cells express Toll-like receptor 3 (TLR3), melanoma differentiation-associated gene 5 (MDA5), and retinoic acid-inducible gene-I (RIG-I), all sensors of double-stranded RNA (dsRNA) and potent inducers of antiviral activity. Here, we demonstrate increased expression of these three dsRNA sensors in kidney transplant biopsies during cytomegalovirus or BK virus infection. In primary tubular epithelial cells, dsRNA sensor activation induced the production of pro-inflammatory TNF-α and antiviral IFN-ß. Notably, dsRNA also enhanced the expression of pro-apoptotic proteins; however, dsRNA alone did not cause cell death due to the expression of anti-apoptotic proteins. The dsRNA sensitized tubular epithelial cells to apoptosis induced by an agonistic antibody against the Fas receptor (CD95), an apoptotic pathway that eliminates infected cells. These findings indicate that tubular epithelial cells require at least two signals to undergo apoptosis, which can help preserve tubular integrity even under inflammatory conditions. Thus, sensors of viral dsRNA promote antiviral, pro-inflammatory, and pro-apoptotic responses in tubular epithelial cells, which may orchestrate the control of viral infection in the kidney.

Triggering of the dsRNA sensors TLR3, MDA5, and RIG-I induces CD55 expression in synovial fibroblasts.

BACKGROUND: CD55 (decay-accelerating factor) is a complement-regulatory protein highly expressed on fibroblast-like synoviocytes (FLS). CD55 is also a ligand for CD97, an adhesion-type G protein-coupled receptor abundantly present on leukocytes. Little is known regarding the regulation of CD55 expression in FLS. METHODS: FLS isolated from arthritis patients were stimulated with pro-inflammatory cytokines and Toll-like receptor (TLR) ligands. Transfection with polyinosinic-polycytidylic acid (poly(I:C)) and 5'-triphosphate RNA were used to activate the cytoplasmic double-stranded (ds)RNA sensors melanoma differentiation-associated gene 5 (MDA5) and retinoic acid-inducible gene-I (RIG-I). CD55 expression, cell viability, and binding of CD97-loaded beads were quantified by flow cytometry. RESULTS: CD55 was expressed at equal levels on FLS isolated from patients with rheumatoid arthritis (RA), osteoarthritis, psoriatic arthritis and spondyloarthritis. CD55 expression in RA FLS was significantly induced by IL-1ß and especially by the TLR3 ligand poly(I:C). Activation of MDA5 and RIG-I also enhanced CD55 expression. Notably, activation of MDA5 dose-dependently induced cell death, while triggering of TLR3 or RIG-I had a minor effect on viability. Upregulation of CD55 enhanced the binding capacity of FLS to CD97-loaded beads, which could be blocked by antibodies against CD55. CONCLUSIONS: Activation of dsRNA sensors enhances the expression of CD55 in cultured FLS, which increases the binding to CD97. Our findings suggest that dsRNA promotes the interaction between FLS and CD97-expressing leukocytes.

SerpinB9 expression in human renal tubular epithelial cells is induced by triggering of the viral dsRNA sensors TLR3, MDA5 and RIG-I.

BACKGROUND: Serine protease inhibitor B9 (serpinB9) protects against granzyme B-mediated apoptosis and could help to reduce tubular damage under inflammatory conditions like interstitial nephritis. Previously, we found that tubular serpinB9 expression was increased during subclinical rejection. Here, we studied the regulation of serpinB9 expression in tubular epithelial cells (TECs) under inflammatory conditions. METHODS: SerpinB9 expression was analysed on messenger RNA (mRNA), and protein levels in primary human TECs were stimulated with various cytokines and pattern recognition receptor ligands and in kidney transplant biopsies obtained during different types of viral infection. RESULTS: Of the inflammatory stimuli tested, only the double-stranded RNA (dsRNA) analogue poly(I:C) promoted serpinB9 mRNA and protein expression. We found that TECs express the viral dsRNA receptors Toll-like receptor 3 (TLR3), melanoma differentiation-associated gene 5 (MDA5) and retinoic acid-inducible gene-I (RIG-I). dsRNA receptor ligands enhanced serpinB9 expression, which involved nuclear factor-kappaB (NF-κB) activation, did not require Type I interferon production and was a direct result of dsRNA receptor-induced gene transcription. In kidney transplants, serpinB9 transcription was increased during infection with cytomegalovirus, Epstein-Barr virus or BK virus compared to stable grafts. Immunohistochemistry showed that tubuli and lymphocytes expressed the inhibitor. CONCLUSION: SerpinB9 expression in human TECs is induced by triggering of the viral dsRNA sensors TLR3, MDA5 and RIG-I. Viral dsRNA may increase the threshold for granzyme B-mediated apoptosis in TECs via serpinB9 upregulation and thus help to protect the kidney against cytotoxic insults during viral infection.

Urinary granzyme A mRNA is a biomarker to diagnose subclinical and acute cellular rejection in kidney transplant recipients.

The distinction between T-cell-mediated rejection (TCMR) and other causes of kidney transplant dysfunction such as tubular necrosis requires biopsy. Subclinical rejection (SCR), an established risk factor for chronic allograft dysfunction, can only be diagnosed by protocol biopsy. A specific non-invasive biomarker to monitor immunological graft status would facilitate diagnosis and treatment of common transplantation-related complications. To identify possible markers, we measured urinary mRNA levels of several cytolytic proteins by quantitative PCR. Our cohort of 70 renal transplant recipients had biopsy proven type I and type II TCMR, acute tubular necrosis, SCR, calcineurin inhibitor-toxicity, cytomegalovirus infection, and stable graft function with normal histology. Granzyme A (GzmA) mRNA was significantly higher in subclinical and acute cellular rejection compared to patients with stable grafts or those with tubular necrosis with 80% sensitivity and up to 100% specificity. Granzyme B and perforin mRNA levels could significantly discriminate acute rejection from stable or tubular necrosis, but were not significantly elevated during SCR. Importantly, only GzmA mRNA remained below detection limits from grafts that were stable and most with tubular necrosis. Hence, the presented data indicate that urinary GzmA mRNA levels may entail a diagnostic non-invasive biomarker to distinguish patients with subclinical and acute cellular rejection from those with tubular necrosis or stable grafts.

Alteration of viral lipid composition by expression of the phospholipid floppase ABCB4 reduces HIV vector infectivity.

BACKGROUND: The presence of cholesterol in the Human Immunodeficiency Virus (HIV) lipid envelop is important for viral function as cholesterol depleted viral particles show reduced infectivity. However, it is less well established whether other viral membrane lipids are also important for HIV infection. The ABCB4 protein is a phosphatidyl choline (PC) floppase that mediates transport of PC from the inner to the outer membrane leaflet. This property enabled us to modulate the lipid composition of HIV vectors and study the effects on membrane composition and infection efficiency. RESULTS: Virus generated in the presence of ABCB4 was enriched in PC and cholesterol but contained less sphingomyelin (SM). Viral titers were reduced 5.9 fold. These effects were not observed with an inactive ABCB4 mutant. The presence of the ABC transport inhibitor verapamil abolished the effect of ABCB4 expression on viral titers. The ABCB4 mediated reduction in infectivity was caused by changes in the viral particles and not by components co purified with the virus because virus made in the presence of ABCB4 did not inhibit virus made without ABCB4 in a competition assay. Incorporation of the envelope protein was not affected by the expression of ABCB4. The inhibitory effect of ABCB4 was independent of the viral envelope as the effect was observed with two different envelope proteins. CONCLUSION: Our data indicate that increasing the PC content of HIV particles reduces infectivity.