Kirstin M. Heutinck

Serine proteases of the human immune system in health and disease.

Serine proteases form a large family of protein-cleaving enzymes that play an essential role in processes like blood coagulation, apoptosis and inflammation. Immune cells express a wide variety of serine proteases such as granzymes in cytotoxic lymphocytes, neutrophil elastase, cathepsin G and proteinase 3 in neutrophils and chymase and tryptase in mast cells. Regulation of proteolysis induced by these serine proteases is essential to prevent self-induced damage. Hence, there are specialized serine protease inhibitors, serpins, which are broadly distributed. Here, we discuss the function of human serine proteases in inflammation, apoptosis and tissue remodeling. Furthermore, we address their impact on development and progression of immune mediated-diseases. Understanding the mode of action of serine proteases will help to unravel molecular processes involved in immunological disorders and will facilitate the identification of new therapeutic targets.

Human Cytomegalovirus Induces Systemic Immune Activation Characterized by a Type 1 Cytokine Signature

Mechanisms underlying the onset and perpetuation of chronic immune activation in individuals without overt infectious or autoimmune diseases are unclear. Cytomegalovirus (CMV) is a persistent virus that induces a permanent increase of highly differentiated, interferon-gamma-secreting effector T cells. We hypothesized that, because of this increase, CMV also induces a systemic inflammatory response. We measured acute phase proteins, cytokines, and chemokines in serum samples from renal transplant recipients who developed a primary CMV infection and healthy CMV serum-positive or -negative individuals. Primary CMV infection induced a clear proinflammatory response that was maintained during latency. This response was characterized by increased levels of acute phase proteins, such as serum amyloid-A and C-reactive protein, and type 1 cytokines, such as interleukin-18, interferon-inducible protein-10, and interferon-gamma. This continuous activation of the immune system may play a role in the pathogenesis of chronic allograft rejection and potentially contribute to the acceleration of chronic diseases.

Treatment with everolimus is associated with a procoagulant state.

INTRODUCTION Renal transplant recipients are at increased risk of venous thromboembolic events, which is in part caused by their treatment with maintenance immunosuppressive drugs. Because we observed an increased incidence of venous thromboembolic events in renal transplant recipients treated with the mTOR inhibitor (mTORi) everolimus, we aimed to identify prothrombotic mechanisms of this immunosuppressive drug. MATERIALS AND METHODS In a single center study, nested in a multi-center randomized controlled trial, we measured parameters of coagulation, anti-coagulation and fibrinolysis in renal transplant recipients, receiving the mTORi everolimus (n=16, mTOR group) and compared them to a similar patient group, receiving a calcineurin inhibitor and/or mycophenolate sodium (n=20, non-mTOR group). All patients were at least 6 months following transplantation with a stable transplant function. RESULTS The use of an mTORi was associated with significantly higher levels of von Willebrand factor, prothrombin fragment 1+2, thrombin-activatable fibrinolysis inhibitor and plasminogen activator inhibitor-1 as compared to a non-mTORi based immunosuppressive regimen. CONCLUSIONS Treatment with an mTORi leads to increased endothelial activation, thrombin formation and impaired fibrinolysis in renal transplant recipients. This suggests an increased risk of thrombotic events in renal transplant recipients treated with mTOR inhibitors. A prospective study to establish the precise risk of thrombotic events in these patients is urgently needed.

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.

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-1β 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.

Phenotypic and Functional Characterization of Circulating Polyomavirus BK VP1-Specific CD8+ T Cells in Healthy Adults

ABSTRACT 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+ CD49dhi CXCR3hi T-betint Eomesoderminlo 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-1β and CD107a.

Virus-Specific CD8(+) T Cells Cross-Reactive to Donor-Alloantigen Are Transiently Present in the Circulation of Kidney Transplant Recipients Infected With CMV and/or EBV.

T cells play a dual role in transplantation: They mediate transplant rejection and are crucial for virus control. Memory T cells generated in response to pathogens can cross‐react to alloantigen, a phenomenon called heterologous immunity. Virus‐specific CD8+ T cells cross‐reacting to donor‐alloantigen might affect alloimmune responses and hamper tolerance induction following transplantation. Here, we longitudinally studied these cross‐reactive cells in peripheral blood of 25 kidney transplant recipients with a cytomegalovirus and/or Epstein‐Barr virus infection. Cross‐reactive T cells were identified by flow cytometry as virus‐specific T cells that proliferate in response to donor cells in a mixed‐lymphocyte reaction. In 13 of 25 patients, we found cross‐reactivity to donor cells for at least 1 viral epitope before (n = 7) and/or after transplantation (n = 8). Cross‐reactive T cells were transiently present in the circulation, and their precursor frequency did not increase following transplantation or viral infection. Cross‐reactive T cells expressed interferon‐γ and CD107a in response to both alloantigen and viral peptide and resembled virus‐specific T cells in phenotype and function. Their presence was not associated with impaired renal function, proteinuria, or rejection. In conclusion, virus‐specific T cells that cross‐react to donor‐alloantigen are transiently detectable in the circulation of kidney transplant recipients.

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-β. 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.

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.

Allo‐HLA Cross‐Reactivities of Cytomegalovirus‐, Influenza‐, and Varicella Zoster Virus–Specific Memory T Cells Are Shared by Different Healthy Individuals

Virus‐specific T cells can recognize allogeneic HLA (allo‐HLA) through TCR cross‐reactivity. The allospecificity often differs by individual (private cross‐reactivity) but also can be shared by multiple individuals (public cross‐reactivity); however, only a few examples of the latter have been described. Because these could facilitate alloreactivity prediction in transplantation, we aimed to identify novel public cross‐reactivities of human virus‐specific CD8+ T cells directed against allo‐HLA by assessing their reactivity in mixed‐lymphocyte reactions. Further characterization was done by studying TCR usage with primer‐based DNA sequencing, cytokine production with ELISAs, and cytotoxicity with 51chromium‐release assays. We identified three novel public allo‐HLA cross‐reactivities of human virus‐specific CD8+ T cells. CMV B35/IPS CD8+ T cells cross‐reacted with HLA‐B51 and/or HLA‐B58/B57 (23% of tetramer‐positive individuals), FLU A2/GIL (influenza IMP[58‐66] HLA‐A*02:01/GILGFVFTL) CD8+ T cells with HLA‐B38 (90% of tetramer‐positive individuals), and VZV A2/ALW (varicella zoster virus IE62[593‐601] HLA‐A*02:01/ALWALPHAA) CD8+ T cells with HLA‐B55 (two unrelated individuals). Cross‐reactivity was tested against different cell types including endothelial and epithelial cells. All cross‐reactive T cells expressed a memory phenotype, emphasizing the importance for transplantation. We conclude that public allo‐HLA cross‐reactivity of virus‐specific memory T cells is not uncommon and may create novel opportunities for alloreactivity prediction and risk estimation in transplantation.