Prevalence and risk factors of undiagnosed diabetes mellitus among gastroenterological patients: a HbA1c-based single center experience - Prevalence of undiagnosed diabetes in gastroenterological patients.

BACKGROUND: Diabetes mellitus is a major risk factor for microvascular disease, leading to chronic kidney injury or cardiovascular disease, but there is a tremendous proportion of patients worldwide who suffer from undiagnosed diabetes. Until now, little is known about the prevalence of undiagnosed diabetes in gastroenterology inpatients. OBJECTIVE: To improve detection of undiagnosed diabetes, a routine screening procedure for gastroenterology inpatients, based on hemoglobin A1c (HbA1c) and fasting plasma glucose (FPG) measurement, was established. METHODS: We conducted a retrospective analysis of the implemented diabetes screening. Diabetes mellitus was diagnosed according to the guideline of the German Diabetes Association in patients with an HbA1c of ≥6.5% anld/or fasting plasma glucose (FPG) ≥126 mg/dL. Univariate and multivariate analyses were performed to identify independent risk factors for undiagnosed diabetes. RESULTS: Within a 3-month period, 606 patients were eligible for a diabetes screening. Pre-existing diabetes was documented in 120 patients (19.8 %), undiagnosed diabetes was found in 24 (3.9%), and 162 patients (26.7%) met the definition for prediabetes. Steroid medication use, age, and liver cirrhosis due to primary sclerosing cholangitis (PSC) were identified as risk factors for undiagnosed diabetes. CONCLUSION: The prevalence of undiagnosed diabetes in gastroenterology inpatients is markedly elevated in comparison to the general population, and a substantial number of inpatients are in a prediabetic status, underlining the need for diabetes screening. In addition to previously described risk factors of patient age and steroid medication use, we identified PSC-related liver cirrhosis (but not liver cirrhosis due to another etiology) as an independent risk factor for undiagnosed diabetes.

A Genome-Wide CRISPR/Cas9-Based Screen Identifies Heparan Sulfate Proteoglycans as Ligands of Killer-Cell Immunoglobulin-Like Receptors.

While human leukocyte antigen (HLA) and HLA-like proteins comprise an overwhelming majority of known ligands for NK-cell receptors, the interactions of NK-cell receptors with non-conventional ligands, particularly carbohydrate antigens, is less well described. We previously found through a bead-based HLA screen that KIR3DS1, a formerly orphan member of the killer-cell immunoglobulin-like receptor (KIR) family, binds to HLA-F. In this study, we assessed the ligand binding profile of KIR3DS1 to cell lines using Fc fusion constructs, and discovered that KIR3DS1-Fc exhibited binding to several human cell lines including ones devoid of HLA. To identify these non-HLA ligands, we developed a magnetic enrichment-based genome-wide CRISPR/Cas9 knock-out screen approach, and identified enzymes involved in the biosynthesis of heparan sulfate as crucial for the binding of KIR3DS1-Fc to K562 cells. This interaction between KIR3DS1 and heparan sulfate was confirmed via surface plasmon resonance, and removal of heparan sulfate proteoglycans from cell surfaces abolished KIR3DS1-Fc binding. Testing of additional KIR-Fc constructs demonstrated that KIR family members containing a D0 domain (KIR3DS1, KIR3DL1, KIR3DL2, KIR2DL4, and KIR2DL5) bound to heparan sulfate, while those without a D0 domain (KIR2DL1, KIR2DL2, KIR2DL3, and KIR2DS4) did not. Overall, this study demonstrates the use of a genome-wide CRISPR/Cas9 knock-out strategy to unbiasedly identify unconventional ligands of NK-cell receptors. Furthermore, we uncover a previously underrecognized binding of various activating and inhibitory KIRs to heparan sulfate proteoglycans that may play a role in NK-cell receptor signaling and target-cell recognition.

KIR3DS1 directs NK cell-mediated protection against human adenovirus infections.

Human adenoviruses (HAdVs) are a major cause for disease in children, in particular after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Currently, effective therapies for HAdV infections in immunocompromised hosts are lacking. To decipher immune recognition of HAdV infection and determine new targets for immune-mediated control, we used an HAdV infection 3D organoid system, based on primary human intestinal epithelial cells. HLA-F, the functional ligand for the activating NK cell receptor KIR3DS1, was strongly up-regulated and enabled enhanced killing of HAdV5-infected cells in organoids by KIR3DS1+ NK cells. In contrast, HLA-A and HLA-B were significantly down-regulated in HAdV5-infected organoids in response to adenoviral E3/glycoprotein19K, consistent with evasion from CD8+ T cells. Immunogenetic analyses in a pediatric allo-HSCT cohort showed a reduced risk to develop severe HAdV disease and faster clearance of HAdV viremia in children receiving KIR3DS1+/HLA-Bw4+ donor cells compared with children receiving non­KIR3DS1+/HLA-Bw4+ cells. These findings identify the KIR3DS1/HLA-F axis as a new target for immunotherapeutic strategies against severe HAdV disease.

Clinical Presentation and Disease Course of 37 Consecutive Cases of Progressive Multifocal Leukoencephalopathy (PML) at a German Tertiary-Care Hospital: A Retrospective Observational Study.

Background: Progressive multifocal leukoencephalopathy (PML) caused by JCV is a rare but frequently fatal disease of the central nervous system, usually affecting immunocompromised individuals. Our study aims to expand the data on patient characteristics, diagnosis, clinical course, possible PML-directed treatment, and outcome of patients with PML at a German tertiary-care hospital. Methods:In this single-center observational cohort study, 37 consecutive patients with a confirmed diagnosis of PML seen at the University Medical Center Hamburg-Eppendorf from 2013 until 2019 were retrospectively analyzed by chart review with a special focus on demographics, risk factors, and clinical aspects as well as PML-directed treatment and survival. Results:We identified 37 patients with definite, probable, and possible PML diagnosis. 36 patients (97%) had underlying immunosuppressive disorders such as HIV/AIDS (n = 17; 46%), previous treatment with monoclonal antibodies (n = 6; 16%), hematological or oncological malignancies (n = 6; 16%), sarcoidosis (n = 5; 14%), solid organ transplantation (n = 1; 3%), and diagnosis of mixed connective tissue disease (n = 1; 3%). In only one patient no evident immunocompromised condition was detected (n = 1; 3%). Treatment attempts to improve the outcome of PML were reported in 13 patients (n = 13; 35%). Twenty seven percent of patients were lost to follow-up (n = 10). Twenty four-month survival rate after diagnosis of PML was 56% (n = 15). Conclusion: This interdisciplinary retrospective study describes epidemiology, risk factors, clinical course, and treatment trials in patients with PML at a German tertiary-care hospital. Acquired immunosuppression due to HIV-1 constituted the leading cause of PML in this monocenter cohort.

Upregulation of HLA-F expression by BK polyomavirus infection induces immune recognition by KIR3DS1-positive natural killer cells.

BK polyomavirus-associated nephropathy is a common complication after kidney transplantation leading to reduced graft function or loss. The molecular pathogenesis of BK polyomavirus-induced nephropathy is not well understood. A recent study had described a protective effect of the activating natural killer cell receptor KIR3DS1 in BK polyomavirus-associated nephropathy, suggesting a role of NK cells in modulating disease progression. Using an in vitro cell culture model of human BK polyomavirus infection and kidney biopsy samples from patients with BK polyomavirus-associated nephropathy, we observed significantly increased surface expression of the ligand for KIR3DS1, HLA-F, on BK polyomavirus-infected kidney tubular cells. Upregulation of HLA-F expression resulted in significantly increased binding of KIR3DS1 to BK polyomavirus-infected cells and activation of primary KIR3DS-positive natural killer cells. Thus, our data provide a mechanism by which KIR3DS-positive natural killer cells can control BK polyomavirus infection of the kidney, and rationale for exploring HLA-F/KIR3DS1 interactions for immunotherapeutic approaches in BK polyomavirus-associated nephropathy.

Primary HIV-1 Strains Use Nef To Downmodulate HLA-E Surface Expression.

Human immunodeficiency virus type 1 (HIV-1) has evolved elaborate ways to evade immune cell recognition, including downregulation of classical HLA class I (HLA-I) from the surfaces of infected cells. Recent evidence identified HLA-E, a nonclassical HLA-I, as an important part of the antiviral immune response to HIV-1. Changes in HLA-E surface levels and peptide presentation can prompt both CD8+ T-cell and natural killer (NK) cell responses to viral infections. Previous studies reported unchanged or increased HLA-E levels on HIV-1-infected cells. Here, we examined HLA-E surface levels following infection of CD4+ T cells with primary HIV-1 strains and observed that a subset downregulated HLA-E. Two primary strains of HIV-1 that induced the strongest reduction in surface HLA-E expression were chosen for further testing. Expression of single Nef or Vpu proteins in a T-cell line, as well as tail swap experiments exchanging the cytoplasmic tail of HLA-A2 with that of HLA-E, demonstrated that Nef modulated HLA-E surface levels and targeted the cytoplasmic tail of HLA-E. Furthermore, infection of primary CD4+ T cells with HIV-1 mutants showed that a lack of functional Nef (and Vpu to some extent) impaired HLA-E downmodulation. Taken together, the results of this study demonstrate for the first time that HIV-1 can downregulate HLA-E surface levels on infected primary CD4+ T cells, potentially rendering them less vulnerable to CD8+ T-cell recognition but at increased risk of NKG2A+ NK cell killing.IMPORTANCE For almost two decades, it was thought that HIV-1 selectively downregulated the highly expressed HLA-I molecules HLA-A and HLA-B from the cell surface in order to evade cytotoxic-T-cell recognition, while leaving HLA-C and HLA-E molecules unaltered. It was stipulated that HIV-1 infection thereby maintained inhibition of NK cells via inhibitory receptors that bind HLA-C and HLA-E. This concept was recently revised when a study showed that primary HIV-1 strains reduce HLA-C surface levels, whereas the cell line-adapted HIV-1 strain NL4-3 lacks this ability. Here, we demonstrate that infection with distinct primary HIV-1 strains results in significant downregulation of surface HLA-E levels. Given the increasing evidence for HLA-E as an important modulator of CD8+ T-cell and NKG2A+ NK cell functions, this finding has substantial implications for future immunomodulatory approaches aimed at harnessing cytotoxic cellular immunity against HIV.

A subset of HLA-DP molecules serve as ligands for the natural cytotoxicity receptor NKp44.

Natural killer (NK) cells can recognize virus-infected and stressed cells1 using activating and inhibitory receptors, many of which interact with HLA class I. Although early studies also suggested a functional impact of HLA class II on NK cell activity2,3, the NK cell receptors that specifically recognize HLA class II molecules have never been identified. We investigated whether two major families of NK cell receptors, killer-cell immunoglobulin-like receptors (KIRs) and natural cytotoxicity receptors (NCRs), contained receptors that bound to HLA class II, and identified a direct interaction between the NK cell receptor NKp44 and a subset of HLA-DP molecules, including HLA-DP401, one of the most frequent class II allotypes in white populations4. Using NKp44ζ+ reporter cells and primary human NKp44+ NK cells, we demonstrated that interactions between NKp44 and HLA-DP401 trigger functional NK cell responses. This interaction between a subset of HLA-DP molecules and NKp44 implicates HLA class II as a component of the innate immune response, much like HLA class I. It also provides a potential mechanism for the described associations between HLA-DP subtypes and several disease outcomes, including hepatitis B virus infection5-7, graft-versus-host disease8 and inflammatory bowel disease9,10.

Interactions Between KIR3DS1 and HLA-F Activate Natural Killer Cells to Control HCV Replication in Cell Culture.

Killer-cell immunoglobulin-like receptors (KIRs) are transmembrane glycoproteins expressed by natural killer (NK) cells. Binding of KIR3DS1 to its recently discovered ligand, HLA-F, activates NK cells and has been associated with resolution of hepatitis C virus (HCV) infection. We investigated the mechanisms by which KIR3DS1 contributes to the antiviral immune response. Using cell culture systems, mice with humanized livers, and primary liver tissue from HCV-infected individuals, we found that the KIR3DS1 ligand HLA-F is up-regulated on HCV-infected cells, and that interactions between KIR3DS1 and HLA-F contribute to NK cell-mediated control of HCV. Strategies to promote interaction between KIR3DS1 and HLA-F might be developed for treatment of infectious diseases and cancer.

Natural Killer Cell Interactions with Classical and Non-Classical Human Leukocyte Antigen Class I in HIV-1 Infection.

Natural killer (NK) cells are effector lymphocytes of the innate immune system that are able to mount a multifaceted antiviral response within hours following infection. This is achieved through an array of cell surface receptors surveilling host cells for alterations in human leukocyte antigen class I (HLA-I) expression and other ligands as signs of viral infection, malignant transformation, and cellular stress. This interaction between HLA-I ligands and NK-cell receptor is not only important for recognition of diseased cells but also mediates tuning of NK-cell-effector functions. HIV-1 alters the expression of HLA-I ligands on infected cells, rendering them susceptible to NK cell-mediated killing. However, over the past years, various HIV-1 evasion strategies have been discovered to target NK-cell-receptor ligands and allow the virus to escape from NK cell-mediated immunity. While studies have been mainly focusing on the role of polymorphic HLA-A, -B, and -C molecules, less is known about how HIV-1 affects the more conserved, non-classical HLA-I molecules HLA-E, -G, and -F. In this review, we will focus on the recent progress in understanding the role of non-classical HLA-I ligands in NK cell-mediated recognition of HIV-1-infected cells.

HIV-1-Mediated Downmodulation of HLA-C Impacts Target Cell Recognition and Antiviral Activity of NK Cells.

It was widely accepted that HIV-1 downregulates HLA-A/B to avoid CTL recognition while leaving HLA-C unaltered in order to prevent NK cell activation by engaging inhibitory NK cell receptors, but it was recently observed that most primary isolates of HIV-1 can mediate HLA-C downmodulation. Now we report that HIV-1-mediated downmodulation of HLA-C was associated with reduced binding to its respective inhibitory receptors. Despite this, HLA-C-licensed NK cells displayed reduced antiviral activity compared to their unlicensed counterparts, potentially due to residual binding to the respective inhibitory receptors. Nevertheless, NK cells were able to sense alterations of HLA-C expression demonstrated by increased antiviral activity when exposed to viral strains with differential abilities to downmodulate HLA-C. These results suggest that the capability of HLA-C-licensed NK cells to control HIV-1 replication is determined by the strength of KIR/HLA-C interactions and is thus dependent on both host genetics and the extent of virus-mediated HLA-C downregulation.

Human Leukocyte Antigen F Presents Peptides and Regulates Immunity through Interactions with NK Cell Receptors.

Evidence is mounting that the major histocompatibility complex (MHC) molecule HLA-F (human leukocyte antigen F) regulates the immune system in pregnancy, infection, and autoimmunity by signaling through NK cell receptors (NKRs). We present structural, biochemical, and evolutionary analyses demonstrating that HLA-F presents peptides of unconventional length dictated by a newly arisen mutation (R62W) that has produced an open-ended groove accommodating particularly long peptides. Compared to empty HLA-F open conformers (OCs), HLA-F tetramers bound with human-derived peptides differentially stained leukocytes, suggesting peptide-dependent engagement. Our in vitro studies confirm that NKRs differentiate between peptide-bound and peptide-free HLA-F. The complex structure of peptide-loaded ß2m-HLA-F bound to the inhibitory LIR1 revealed similarities to high-affinity recognition of the viral MHC-I mimic UL18 and a docking strategy that relies on contacts with HLA-F as well as ß2m, thus precluding binding to HLA-F OCs. These findings provide a biochemical framework to understand how HLA-F could regulate immunity via interactions with NKRs.

Peptide-specific engagement of the activating NK cell receptor KIR2DS1.

The activating NK cell receptor KIR2DS1 has been shown to be involved in many disorders including autoimmune diseases, malignancies and pregnancy outcomes. However, the precise ligands and functions of this receptor remain unclear. We aimed to gain a better understanding of the factors involved in the binding of KIR2DS1 and its inhibitory counterpart KIR2DL1 to HLA class I molecules, and the consequences for KIR2DS1+ NK-cell function. A systematic screen that assessed binding to 97 HLA-I proteins confirmed that KIR2DS1-binding was narrowly restricted to HLA-C group 2 complexes, while KIR2DL1 showed a broader binding specificity. Using KIR2DS1ζ+ Jurkat reporter-cells and peptide-pulsed 721.221.TAP1KO-HLA-C*06:02 cells, we identified the synthetic peptide SRGPVHHLL presented by HLA-C*06:02 that strongly engaged KIR2DS1- and KIR2DL1-binding. Functional analysis showed that this HLA-C*06:02-presented peptide can furthermore activate primary KIR2DS1(+) NK cell clones. Thus, we demonstrated peptide-dependent binding of the activating NK cell receptor KIR2DS1, providing new insights into the underlying mechanisms involved in KIR2DS1-related disorders.

Open conformers of HLA-F are high-affinity ligands of the activating NK-cell receptor KIR3DS1.

The activating natural killer (NK)-cell receptor KIR3DS1 has been linked to the outcome of various human diseases, including delayed progression of disease caused by human immunodeficiency virus type 1 (HIV-1), yet a ligand that would account for its biological effects has remained unknown. We screened 100 HLA class I proteins and found that KIR3DS1 bound to HLA-F, a result we confirmed biochemically and functionally. Primary human KIR3DS1(+) NK cells degranulated and produced antiviral cytokines after encountering HLA-F and inhibited HIV-1 replication in vitro. Activation of CD4(+) T cells triggered the transcription and surface expression of HLA-F mRNA and HLA-F protein, respectively, and induced binding of KIR3DS1. HIV-1 infection further increased the transcription of HLA-F mRNA but decreased the binding of KIR3DS1, indicative of a mechanism for evading recognition by KIR3DS1(+) NK cells. Thus, we have established HLA-F as a ligand of KIR3DS1 and have demonstrated cell-context-dependent expression of HLA-F that might explain the widespread influence of KIR3DS1 in human disease.

Sequence variations in HCV core-derived epitopes alter binding of KIR2DL3 to HLA-C∗03:04 and modulate NK cell function.

BACKGROUND & AIMS: Both natural killer (NK) cells and human leukocyte antigen (HLA)/killer cell immunoglobulin like receptor (KIR) interactions have been shown to play an important role in the control, clearance and progression of hepatitis C virus (HCV) disease. Here we aimed at elucidating the effects of viral peptides derived from HCV on HLA stabilization, changes in KIR binding and primary NK cell function. METHODS: Transporter for antigen presentation-deficient 722.221 cells stably transfected with HLA-C∗03:04 were used to screen 200 overlapping peptides, covering the non-structural protein 3 (NS3) and core protein of HCV genotype 1, for their ability to bind and stabilize HLA-C∗03:04. Binding of KIR2DL3 to the HLA-peptide complex was assessed using a KIR2DL3-IgG fusion construct. Primary NK cells were isolated from healthy donors to investigate the effects of identified peptides on KIR2DL3(+) NK cell function. RESULTS: Thirty-one peptides able to stabilize HLA-C∗03:04 were identified. One 9mer peptide, YIPLVGAPL, resulted in significantly higher KIR2DL3 binding to HLA-C∗03:04(+) 722.221 cells and suppression of primary KIR2DL3(+) NK cell function. Interestingly this sequence exhibited a high frequency of mutations in different HCV genotypes. These genotype-specific peptides showed lower HLA-C∗03:04 stabilization, decreased binding of the inhibitory KIR2DL3 and lower inhibition of NK cell function. CONCLUSIONS: Taken together we show that a viral peptide derived from the core protein of HCV genotype 1 binding to HLA-C∗03:04 results in a sequence-dependent engagement of the inhibitory NK cell receptor KIR2DL3, while the large majority of the remaining 30 HLA-C∗03:04 binding HCV core peptides did not. These data show that sequence variations within HCV can modulate NK cell function, providing potential pathways for viral escape. LAY SUMMARY: We identified a HCV peptide that dampens NK cell responses, and thereby possibly prevents killing of infected cells through this part of the innate immune system. This is facilitated via presentation of the viral peptide on HLA∗03:04 to the inhibitory KIR receptor KIR2DL3 on NK cells. Naturally occurring sequence mutations in the peptide alter these interactions making the inhibition less efficient.

Selection of an HLA-C*03:04-Restricted HIV-1 p24 Gag Sequence Variant Is Associated with Viral Escape from KIR2DL3+ Natural Killer Cells: Data from an Observational Cohort in South Africa.

BACKGROUND: Viruses can evade immune surveillance, but the underlying mechanisms are insufficiently understood. Here, we sought to understand the mechanisms by which natural killer (NK) cells recognize HIV-1-infected cells and how this virus can evade NK-cell-mediated immune pressure. METHODS AND FINDINGS: Two sequence mutations in p24 Gag associated with the presence of specific KIR/HLA combined genotypes were identified in HIV-1 clade C viruses from a large cohort of infected, untreated individuals in South Africa (n = 392), suggesting viral escape from KIR+ NK cells through sequence variations within HLA class I-presented epitopes. One sequence polymorphism at position 303 of p24 Gag (TGag303V), selected for in infected individuals with both KIR2DL3 and HLA-C*03:04, enabled significantly better binding of the inhibitory KIR2DL3 receptor to HLA-C*03:04-expressing cells presenting this variant epitope compared to the wild-type epitope (wild-type mean 18.01 ± 10.45 standard deviation [SD] and variant mean 44.67 ± 14.42 SD, p = 0.002). Furthermore, activation of primary KIR2DL3+ NK cells from healthy donors in response to HLA-C*03:04+ target cells presenting the variant epitope was significantly reduced in comparison to cells presenting the wild-type sequence (wild-type mean 0.78 ± 0.07 standard error of the mean [SEM] and variant mean 0.63 ± 0.07 SEM, p = 0.012). Structural modeling and surface plasmon resonance of KIR/peptide/HLA interactions in the context of the different viral sequence variants studied supported these results. Future studies will be needed to assess processing and antigen presentation of the investigated HIV-1 epitope in natural infection, and the consequences for viral control. CONCLUSIONS: These data provide novel insights into how viruses can evade NK cell immunity through the selection of mutations in HLA-presented epitopes that enhance binding to inhibitory NK cell receptors. Better understanding of the mechanisms by which HIV-1 evades NK-cell-mediated immune pressure and the functional validation of a structural modeling approach will facilitate the development of novel targeted immune interventions to harness the antiviral activities of NK cells.

Sequence variations in HIV-1 p24 Gag-derived epitopes can alter binding of KIR2DL2 to HLA-C*03:04 and modulate primary natural killer cell function.

OBJECTIVE: The aim of this study was to assess the consequence of sequence variations in HLA-C03:04-presented HIV-1 p24 Gag epitopes on binding of the inhibitory natural killer (NK) cell receptor KIR2DL2 to HLA-C03:04. DESIGN: HIV-1 may possibly evade recognition by KIR+ NK cells through selection of sequence variants that interfere with the interactions of inhibitory killer cell immunoglobulin-like receptors (KIRs) and their target ligands on HIV-1 infected cells. KIR2DL2 is an inhibitory NK cell receptor that binds to a family of HLA-C ligands. Here, we investigated whether HIV-1 encodes for HLA-C03:04-restricted epitopes that alter KIR2DL2 binding. METHODS: Tapasin-deficient 721.220 cells expressing HLA-C03:04 were pulsed with overlapping peptides (10mers overlapped by nine amino acids, spanning the entire HIV-1 p24 Gag sequence) to identify peptides that stabilized HLA-C expression. The impact that sequence variation in HLA-C03:04-binding HIV-1 epitopes has on KIR2DL2 binding and KIR2DL2+ NK cell function was determined using KIR2DL2-Fc constructs and NK cell degranulation assays. RESULTS: Several novel HLA-C03:04 binding epitopes were identified within the HIV-1 p24 Gag consensus sequence. Three of these consensus sequence peptides (Gag144-152, Gag163-171 and Gag295-304) enabled binding of KIR2DL2 to HLA-C03:04 and resulted in inhibition of KIR2DL2+ primary NK cells. Furthermore, naturally occurring minor variants of epitope Gag295-304 enhanced KIR2DL2 binding to HLA-C03:04. CONCLUSION: Our data show that naturally occurring sequence variations within HLA-C03:04-restricted HIV-1 p24 Gag epitopes can have a significant impact on the binding of inhibitory KIR receptors and primary NK cell function.