Host genetic factors in susceptibility to herpes simplex type 1 virus infection: contribution of polymorphic genes at the interface of innate and adaptive immunity.

HSV-1 establishes life-long latency that can result in clinical relapses or in asymptomatic virus shedding. Although virtually all adults have been exposed to HSV-1, the clinical course varies remarkably. Genetic host variability could be related to this clinical diversity. In this study, we analyzed the contribution of gene families in chromosomes 1, 6, 12, and 19, which encode key regulators of the innate and adaptive immunity, in a cohort of 302 individuals. Class I and class II alleles of the HLA system, the copy-number variation of NK cell receptor genes (KIR and NKG2C), the combinations of killer cell Ig-like receptor and their HLA ligands, and CD16A and CD32A allotypes of variable affinity for IgG subclasses were all studied. Although no major susceptibility locus for HSV-1 was identified, our results show that the risk of suffering clinical HSV-1 infection is modified by MHC class I allotypes (B*18, C*15, and the group of alleles encoding A19), the high-affinity receptor/ligand pair KIR2DL2/HLA-C1, and the CD16A-158V/F dimorphism. Conversely, HLA class II and CD32A polymorphisms and NKG2C deletion did not seem to influence the clinical course of herpetic infection. Collectively, these findings support an important role in host defense against herpetic infection for several polymorphic genes implicated in adaptive immunity and in surveillance of its subversion. They confirm the crucial role of cytotoxic cells (CTL and NK) and the contribution of genetic diversity to the clinical course of HSV-1 infection.

Donor Lung Preservation at 10°C: Clinical and Logistical Impact.

INTRODUCTION: Cold static donor lung preservation at 10°C appears to be a promising method to safely extend the cold ischemic time (CIT) and improve lung transplant (LTx) logistics. METHODS: LTx from November 2021 to February 2023 were included in this single institution, prospective, non-randomized study comparing prolonged preservation at 10°C versus standard preservation on ice. The inclusion criteria for 10°C preservation were suitable grafts for LTx without any donor retrieval concerns. PRIMARY ENDPOINT: primary graft dysfunction (PGD) grade-3 at 72-h. Secondary endpoints: clinical outcomes, cytokine profile and logistical impact. RESULTS: Thirty-three out of fifty-seven cases were preserved at 10°C. Donor and recipient characteristics were similar across the groups. Total preservation times (h:min) were longer (p<0.001) in the 10°C group [1st lung: median 12:09 (IQR 9:23-13:29); 2nd: 14:24 (12:00-16:20)] vs. standard group [1st lung: median 5:47 (IQR 5:18-6:40); 2nd: 7:15 (6:33-7:40)]. PGD grade-3 at 72-h was 9.4% in 10°C group vs. 12.5% in standard group (p=0.440). Length of mechanical ventilation (MV), ICU and hospital stays were similar in both groups. Thirty and ninety-day mortality rates were 0% in 10°C group (vs. 4.2% in standard group). IL-8 concentration was significantly higher 6-h post-LTx in the standard group (p=0.025) and IL-10 concentration was increased 72-h post-LTx in the 10°C group (p=0.045). CONCLUSIONS: Preservation at 10°C may represent a safe and feasible strategy to intentionally prolong the CIT. In our center, extending the CIT at 10°C may allow for semi-elective LTx and improve logistics with similar outcomes compared to the current standard preservation on ice.

SARS-COV-2 seroprevalence among all healthcare workers in a tertiary hospital in Spain.

BACKGROUND: Healthcare workers (HCWs) have been a critical and vulnerable population during SARS-CoV-2 pandemic. The aim of this study was to determine the overall seroprevalence and to evaluate occupational risk factors among HCWs in one of the countries most affected by this pandemic. METHODS: We conducted a seroprevalence study for SARS-CoV-2 in a tertiary hospital in Madrid (Spain) between 24 April and 8 May 2020. A total of 4894 HCWs were invited for serologic testing. Serum samples were tested for SARS-CoV-2 IgM and IgG antibodies using Enzyme Immunoassay (ELISA) and Electro-Chemiluminescence Immunoassay (ECLIA) techniques. We calculated odds ratios to assess association between demographic and occupational characteristics with SARS-CoV-2 seroconversion. RESULTS: We processed 4324 serum samples. Overall, seroprevalence was of 16.6% (95% CI: 15.5-17.7). We found statistically significant differences in SARS-CoV-2 seroprevalence by type of employee, professional category, department and type of activity performed during the pandemic period, while no differences were identified between the personnel working in the COVID-19 wards compared to those working in non-COVID-19 wards. We confirmed 268 (26.7%) infections among 1005 hospital staff members tested by PCR. 60.5% of HCWs infected by SARS-CoV-2, assessed either by PCR or serology, could be considered asymptomatic or paucisymptomatic. CONCLUSIONS: HCWs have an increased risk of SARS-CoV-2 infection but COVID-19 patient exposure was not a determining factor. Universal mask wearing should be mandatory in healthcare settings given the important number of asymptomatic and paucisymptomatic cases.

Haplotype-Based Analysis of KIR-Gene Profiles in a South European Population-Distribution of Standard and Variant Haplotypes, and Identification of Novel Recombinant Structures.

Inhibitory Killer-cell Immunoglobulin-like Receptors (KIR) specific for HLA class I molecules enable human natural killer cells to monitor altered antigen presentation in pathogen-infected and tumor cells. KIR genes display extensive copy-number variation and allelic polymorphism. They organize in a series of variable arrangements, designated KIR haplotypes, which derive from duplications of ancestral genes and sequence diversification through point mutation and unequal crossing-over events. Genomic studies have established the organization of multiple KIR haplotypes-many of them are fixed in most human populations, whereas variants of those have less certain distributions. Whilst KIR-gene diversity of many populations and ethnicities has been explored superficially (frequencies of individual genes and presence/absence profiles), less abundant are in-depth analyses of how such diversity emerges from KIR-haplotype structures. We characterize here the genetic diversity of KIR in a sample of 414 Spanish individuals. Using a parsimonious approach, we manage to explain all 38 observed KIR-gene profiles by homo- or heterozygous combinations of six fixed centromeric and telomeric motifs; of six variant gene arrangements characterized previously by us and others; and of two novel haplotypes never detected before in Caucasoids. Associated to the latter haplotypes, we also identified the novel transcribed KIR2DL5B*0020202 allele, and a chimeric KIR2DS2/KIR2DL3 gene (designated KIR2DL3*033) that challenges current criteria for classification and nomenclature of KIR genes and haplotypes.

Association of DDX58 177 C > T polymorphism with decreased risk of Epstein-Barr virus-related nodular sclerosis classical Hodgkin lymphoma.

Classical Hodgkin lymphoma (cHL) is frequently related to Epstein-Barr virus (EBV) infection. Its malignant capacity is attributed to disruption of an EBV-host balance influenced by environmental and genetic drivers. EBV structures activate Type I interferon (IFN) pathway of the innate immunity, therefore, genetic polymorphisms could influence this response. We explored the impact of four single nucleotide polymorphisms (SNPs) on EBV-associated cHL susceptibility. Toll-like receptors 9 (TLR9_rs5743836), and 3 (TLR3_rs3775291), Interleukin-28B (IL28B_rs12979860), and DEAD-box polypeptide 58 (DDX58_rs10813831) were genotyped in 73 EBV-positive and 106 EBV-negative cHL patients and 396 controls. Only DDX58_rs10813831 T-allele was decreased among EBV-positive cHL compared to controls. A stratified analysis in EBV-positive cHL showed that the reduced rate was associated with younger age and nodular sclerosis. In conclusion, DDX58_rs10813831 T-allele may be associated with a reduced risk of nodular sclerosis EBV-related cHL, which suggests a role for RIG-I (retinoic acid-inducible gene I), encoded by DDX58, in these cases.

Biallelic ATM alterations detected at diagnosis identify a subset of treatment-naïve chronic lymphocytic leukemia patients with reduced overall survival similar to patients with p53 deletion.

The prognostic impact of biallelic ATM abnormalities (ATM mutation and concurrent 11q deletion) remains unknown. We studied ATM, BIRC3, SF3B1, and NOTCH1 genes in 118 treatment-naïve CLL patients at diagnosis. Patients with biallelic ATM alteration had a similar time to first treatment (TTFT) and shorter overall survival (OS) compared with patients with isolated 11q deletion and shorter TTFT and OS when compared to patients with wild-type ATM. Furthermore, biallelic ATM alteration (HR: 6.4; p ≤ 0.007) was significantly associated with an increased risk of death similar to p53 deletion (HR: 6.1; p ≤ 0.004), superior to 11q deletion alone (HR: 2.8; p ≤ 0.022) and independent of other significant parameters such as age, advanced clinical stage, and complex karyotype. Our results suggest the identification of ATM mutations in CLL patients with 11q deletion at diagnosis is clinically relevant and predicts disease progression, poor response to the treatment, and reduced OS independent of other molecular prognostic factors.

HLA Allele E*01:01 Is Associated with a Reduced Risk of EBV-Related Classical Hodgkin Lymphoma Independently of HLA-A*01/*02.

BACKGROUND: An inefficient immune response against Epstein-Barr virus (EBV) infection is related to the pathogenesis of a subgroup of classical Hodgkin lymphomas (cHL). Some EBV immune-evasion mechanisms target HLA presentation, including the non-classical HLA-E molecule. HLA-E can be recognized by T cells via the TCR, and it also regulates natural killer (NK) cell signaling through the inhibitory CD94/NKG2A receptor. Some evidences indicate that EBV-infected B-cells promote the proliferation of NK subsets bearing CD94/NKG2A, suggesting a relevant function of these cells in EBV control. Variations in CD94/NKG2A-HLA-E interactions could affect NK cell-mediated immunity and, consequently, play a role in EBV-driven transformation and lymphomagenesis. The two most common HLA-E alleles, E*01:01 and E*01:03, differ by a single amino acid change that modifies the molecule function. We hypothesized that the functional differences in these variants might participate in the pathogenicity of EBV. AIM: We studied two series of cHL patients, both with EBV-positive and-negative cases, and a cohort of unrelated controls, to assess the impact of HLA-E variants on EBV-related cHL susceptibility. RESULTS: We found that the genotypes with at least one copy of E*01:01 (i.e., E*01:01 homozygous and heterozygous) were underrepresented among cHL patients from both series compared to controls (72.6% and 71.6% vs 83%, p = 0.001). After stratification by EBV status, we found low rates of E*01:01-carriers mainly among EBV-positive cases (67.6%). These reduced frequencies are seen independently of other factors such as age, gender, HLA-A*01 and HLA-A*02, HLA alleles positively and negatively associated with the disease (adjusted OR = 0.4, p = 0.001). Furthermore, alleles from both HLA loci exert a cumulative effect on EBV-associated cHL susceptibility. CONCLUSIONS: These results indicate that E*01:01 is a novel protective genetic factor in EBV-associated cHL and support a role for HLA-E recognition on the control of EBV infection and lymphomagenesis.

Vascular endothelial growth factor A (VEGFA) gene polymorphisms have an impact on survival in a subgroup of indolent patients with chronic lymphocytic leukemia.

Vascular endothelial growth factor (VEGF)-mediated angiogenesis contributes to the pathogenesis of B-cell chronic lymphocytic leukaemia (CLL). We investigated the impact of VEGFA gene diversity on the clinical outcome of patients with this disease. A VEGFA haplotype conformed by positions rs699947 (-1540C>A), rs833061 (-460T>C) and rs2010963 (405C>G) and two additional single-nucleotide polymorphisms (SNPs), rs3025039 (936C>T) and rs25648 (1032C>T), were analysed in 239 patients at the time of their CLL diagnosis. Here, we showed that homozygosity for rs699947/rs833061/rs2010963 ACG haplotype (ACG+/+ genotype) correlated with a reduced survival in CLL patients (ACG+/+ vs other genotypes: HR = 2.3, p = 0.002; recessive model). In multivariate analysis, the ACG+/+ genotype was identified as a novel independent prognostic factor (HR = 2.1, p = 0.005). Moreover, ACG homozygosity subdivided patients with CLL with otherwise indolent parameters into prognostic subgroups with different outcomes. Specifically, patients carrying the ACG+/+ genotype with mutated IgVH, very low and low-risk cytogenetics, initial clinical stage, CD38 negative status or early age at diagnosis showed a shorter survival (ACG+/+ vs other genotypes: HR = 3.5, p = 0.035; HR = 3.4, p = 0.001; HR = 2.2, p = 0.035; HR = 3.4, p = 0.0001 and HR = 3.1, p = 0.009, respectively). In conclusion, VEGFA ACG+/+ genotype confers an adverse effect in overall survival in CLL patients with an indolent course of the disease. These observations support the biological and prognostic implications of VEGFA genetics in CLL.

KIR typing by non-sequencing methods: polymerase-chain reaction with sequence-specific primers.

The killer-cell immunoglobulin-like receptors (KIR), which enable NK cells to detect allogeneic target cells and abnormalities in the expression of self-HLA molecules, are encoded by genes that display extensive copy number variation. These variations in the KIR genotype are relevant for multiple aspects of human health, including therapy of cancer. PCR with sequence-specific primers (SSP) is simplest and most widely used among techniques for studying KIR genotypes. Here, we present a protocol that details the critical steps of a method for KIR genotyping by PCR-SSP.

KIR2DL5: An Orphan Inhibitory Receptor Displaying Complex Patterns of Polymorphism and Expression.

A recently developed anti-KIR2DL5 (CD158f) antibody has demonstrated KIR2DL5 expression on the surface of NK and T lymphocytes, making it the last functional KIR identified in the human genome. KIR2DL5 belongs to an ancestral lineage of KIR with Ig-like domains of the D0-D2 type, of which KIR2DL4, an HLA-G receptor, is the only other human member. Despite KIR2DL4 and KIR2DL5 being encoded by genes with similar domain usage, several KIR2DL5 functions resemble more closely those of KIR recognizing classical HLA class I molecules - surface-expressed KIR2DL5 inhibits NK cells through the SHP-2 phosphatase and displays a clonal distribution on NK and T lymphocytes. No activating homolog of KIR2DL5 has been described in any species. The genetics of KIR2DL5 is complicated by duplication of its gene in an ancestor of modern humans living ∼1.7 million years ago. Both KIR2DL5 paralogs have undergone allelic diversification; the centromeric gene is most often represented by alleles whose expression is silenced epigenetically through DNA methylation, thus providing a natural system to investigate the regulation of KIR transcription. The role of KIR2DL5 in immunity is not completely understood, in spite of different attempts to define its ligand. Here we revisit the most relevant characteristics of KIR2DL5, an NK-cell receptor possessing a unique combination of genetic, structural, and functional features.

New mutations in chronic lymphocytic leukemia identified by target enrichment and deep sequencing.

Chronic lymphocytic leukemia (CLL) is a heterogeneous disease without a well-defined genetic alteration responsible for the onset of the disease. Several lines of evidence coincide in identifying stimulatory and growth signals delivered by B-cell receptor (BCR), and co-receptors together with NFkB pathway, as being the driving force in B-cell survival in CLL. However, the molecular mechanism responsible for this activation has not been identified. Based on the hypothesis that BCR activation may depend on somatic mutations of the BCR and related pathways we have performed a complete mutational screening of 301 selected genes associated with BCR signaling and related pathways using massive parallel sequencing technology in 10 CLL cases. Four mutated genes in coding regions (KRAS, SMARCA2, NFKBIE and PRKD3) have been confirmed by capillary sequencing. In conclusion, this study identifies new genes mutated in CLL, all of them in cases with progressive disease, and demonstrates that next-generation sequencing technologies applied to selected genes or pathways of interest are powerful tools for identifying novel mutational changes.

Human KIR2DL5 is an inhibitory receptor expressed on the surface of NK and T lymphocyte subsets.

Human NK cells, by means of a repertoire of clonally distributed killer cell Ig-like receptors (KIR), survey the expression of individual self HLA class I molecules, which is often altered in infections and tumors. KIR2DL5 (CD158f) is the last identified KIR gene and, with KIR2DL4, constitutes a structurally divergent lineage conserved in different primate species. Research on KIR2DL5 has thus far been limited to its genetic aspects due to a lack of reagents to detect its product. We report here the identification and characterization of the receptor encoded by KIR2DL5 using a newly generated specific mAb that recognizes its most commonly expressed allele, KIR2DL5A*001. KIR2DL5 displays a variegated distribution on the surface of CD56(dim) NK cells. This contrasts with the expression pattern of its structural homolog KIR2DL4 (ubiquitous transcription, surface expression restricted to CD56(bright) NK cells) and resembles the profile of KIR recognizing classical HLA class I molecules. Like other MHC class I receptors, KIR2DL5 is also found in a variable proportion of T lymphocytes. KIR2DL5 is detected on the cell surface as a monomer of approximately 60 kDa that, upon tyrosine phosphorylation, recruits the Src homology region 2-containing protein tyrosine phosphatase-2 and, to a lesser extent, Src homology region 2-containing protein tyrosine phosphatase-1. Ab-mediated cross-linking of KIR2DL5 inhibits NK cell cytotoxicity against murine FcR+ P815 cells. KIR2DL5 is thus an inhibitory receptor gathering a combination of genetic, structural, and functional features unique among KIR, which suggests that KIR2DL5 plays a specialized role in innate immunity.

Epigenetic silencing of potentially functional KIR2DL5 alleles: Implications for the acquisition of KIR repertoires by NK cells.

NK cells detect altered patterns of HLA expression in infections and tumors using a variegated repertoire of killer cell Ig-like receptors (KIR). Each clone surveys different HLA molecules by expressing a limited subset of the KIR encoded in its genome, which is maintained throughout cell divisions by epigenetic mechanisms (methylation of the nonexpressed genes). How KIR repertoires are acquired remains, however, unexplained. Human KIR2DL5 is a useful model for studying KIR expression because it has alleles with similar coding regions, but drastically divergent expression - whilst some are transcribed in a typically clonal manner, others, with distinctive promoter polymorphisms, are nonexpressed. Here we investigate the relationship between the sequence diversity of KIR2DL5, including three novel alleles, and its variable transcription. The promoters of the transcribed alleles recruit the transcriptional regulator RUNX3, whilst a mutation shared by all silent alleles precludes this binding. However, all promoters are functional in vitro, and pharmacological DNA demethylation of NK cells rescues the transcription of silent alleles, indicating that only epigenetic mechanisms prevent their inclusion in a normal KIR repertoire. Our results are consistent with a model in which RUNX factors could function as switch elements in the acquisition of KIR repertoires by NK cell precursors.

The silent KIR3DP1 gene (CD158c) is transcribed and might encode a secreted receptor in a minority of humans, in whom the KIR3DP1, KIR2DL4 and KIR3DL1/KIR3DS1 genes are duplicated.

Killer-cell Ig-like receptors (KIR) are structurally and functionally diverse, and enable human NK cells to survey the expression of individual HLA class I molecules, often altered in infections and tumors. Multiple events of non-reciprocal recombination have contributed to the rapid diversification of KIR. We show that approximately 4.5% of the individuals of a Caucasoid population bear a recombinant allele of KIR3DP1, officially designed KIR3DP1*004, that associates tightly with gene duplications of KIR3DP1, KIR2DL4 and KIR3DL1/KIR3DS1. The KIR3DP1 gene is normally silent, but the recombinant allele carries a novel promoter sequence and, as a consequence, is transcribed in all tested individuals. Messenger RNA of KIR3DP1*004 is made up of six exons; of these, exons 1-5 are similar to, and spliced like, those encoding the leader peptide and Ig-domains of KIR3D. By contrast, exon 6 is homologous to no other human KIR sequence, but only to possible homologs in chimpanzees and rhesus macaques, and encodes a short hydrophilic tail. The putative KIR3DP1*004 product, like those of the related genes LAIR-2 and LILRA3/ILT6/LIR4, is predicted to be secreted to the extracellular medium rather than anchored to the cell membrane.

Three structurally and functionally divergent kinds of promoters regulate expression of clonally distributed killer cell Ig-like receptors (KIR), of KIR2DL4, and of KIR3DL3.

The generation of killer cell Ig-like receptor (KIR) expression patterns in NK cells involves variegated silencing of KIR genes by DNA methylation. To identify regulatory elements involved in KIR gene activation, upstream regions of KIR genes were functionally characterized in NK3.3 cells as well as in primary NK cells. Three kinds of KIR promoters were defined, controlling clonally expressed KIR genes, the constitutively active KIR2DL4, and the weakly expressed KIR3DL3. Upstream of a short core promoter common to all KIR genes, a region containing functionally divergent elements was characterized. Although this region had no impact on the activity of the KIR2DL3 promoter, an inhibitory element was identified in the KIR2DL4 promoter and an activating element was found in the KIR3DL3 promoter. Upon treatment with a methyltransferase inhibitor, KIR3DL3 expression could be readily induced showing that the low levels of KIR3DL3 expression in peripheral blood are due to sustained DNA methylation of an otherwise fully functional promoter. Analysis of transcription factor binding sites identified a functional acute myeloid leukemia (AML) site common to all three KIR promoters. Mutation of this site led to a substantial increase in activity of all KIR promoters. Among the different members of the AML family, AML-2 was identified as the predominant KIR binding factor. The present study suggests that AML-2 acts as a repressor of KIR expression in mature NK cells and opens the possibility that AML factors and associated cofactors are involved in regulation of KIR expression during NK cell development.

Recognition of HLA-G by the NK cell receptor KIR2DL4 is not essential for human reproduction.

A central issue of reproductive immunology in mammals is why a semi-allogeneic embryo is not rejected by the pregnant mother. This is particularly intriguing since, in different species, the early pregnant uterus is infiltrated by numerous maternal lymphocytes, predominantly NK cells. The human NK cell receptor KIR2DL4 has been implicated in the maternal tolerance to the embryo due to its recognition of HLA-G, a non-classical MHC molecule expressed preferentially in the placenta. Killer cell Ig-like receptors (KIR) are believed to participate in the natural immunity to infection and tumors, but KIR2DL4 has unique structural, functional and genetic features that could confer it a different role. However, we demonstrate here that the KIR2DL4:HLA-G interaction is not essential for human reproduction by showing that a multiparous woman lacks a KIR2DL4 gene.

Imprint of human cytomegalovirus infection on the NK cell receptor repertoire.

Expression of the activating CD94/NKG2C killer lectin-like receptor (KLR) specific for HLA-E was analyzed in peripheral blood lymphocytes (PBLs) from healthy adult blood donors; the expression of other natural killer (NK) cell receptors (ie, CD94/NKG2A, KIR, CD85j, CD161, NKp46, NKp30, and NKG2D) was also studied. Human cytomegalovirus (HCMV) infection as well as the HLA-E and killer immunoglobulin-like receptor (KIR) genotypes were considered as potentially relevant variables associated with CD94/NKG2C expression. The proportion of NKG2C(+) lymphocytes varied within a wide range (<0.1% to 22.1%), and a significant correlation (r = 0.83; P < .001) between NKG2C(+) NK and T cells was noticed. The HLA-E genotype and the number of activating KIR genes of the donors were not significantly related to the percentage of NKG2C(+) lymphocytes. By contrast, a positive serology for HCMV, but not for other herpesviruses (ie, Epstein-Barr and herpes simplex), turned out to be strongly associated (P < .001) with increased proportions of NKG2C(+) NK and T cells. Remarkably, the CD94/NKG2C(+) population expressed lower levels of natural cytotoxicity receptors (NCRs) (ie, NKp30, NKp46) and included higher proportions of KIR(+) and CD85j(+) cells than CD94/NKG2A(+) cells. Altogether, these data support that HCMV infection selectively shapes the natural killer cell receptor (NKR) repertoire of NK and T cells from healthy carrier individuals.

Some human KIR haplotypes contain two KIR2DL5 genes: KIR2DL5A and KIR2DL5B.

Killer-cell immunoglobulin-like receptors (KIR) comprise a family of structurally diverse proteins encoded by a compact cluster of genes located in human Chromosome 19q13.4. The most recently described member of the KIR family, KIR2DL5, is represented in human populations by at least four gene variants, whose exons differ by two to eight nucleotides. We show here that these structurally similar variants are encoded by alleles of two different loci, KIR2DL5A and KIR2DL5B, which map to different regions of the KIR-gene cluster. Regarding KIR2DL5, four groups of KIR haplotypes can be distinguished: those having both KIR2DL5A and KIR2DL5B, those having either KIR2DL5A or KIR2DL5B, and those lacking KIR2DL5. Positive association between KIR2DL5A and KIR2DL5B was detected but did not reach statistical significance. These results are consistent with a model in which KIR2DL5A and KIR2DL5B are products of a gene duplication, which through the action of subsequent recombination have became separated on some haplotypes.