A modular framework for the development of targeted Covid-19 blood transcript profiling panels.

BACKGROUND: Covid-19 morbidity and mortality are associated with a dysregulated immune response. Tools are needed to enhance existing immune profiling capabilities in affected patients. Here we aimed to develop an approach to support the design of targeted blood transcriptome panels for profiling the immune response to SARS-CoV-2 infection. METHODS: We designed a pool of candidates based on a pre-existing and well-characterized repertoire of blood transcriptional modules. Available Covid-19 blood transcriptome data was also used to guide this process. Further selection steps relied on expert curation. Additionally, we developed several custom web applications to support the evaluation of candidates. RESULTS: As a proof of principle, we designed three targeted blood transcript panels, each with a different translational connotation: immunological relevance, therapeutic development relevance and SARS biology relevance. CONCLUSION: Altogether the work presented here may contribute to the future expansion of immune profiling capabilities via targeted profiling of blood transcript abundance in Covid-19 patients.

High multi-cytokine levels are not a predictive marker of alloimmunization in transfused sickle cell disease patients.

Patients with sickle cell disease (SCD) receive multiple red blood cell (RBC) transfusions for both prevention of and therapy for disease-related complications. In some patients, transfusion results in development of both allo- and auto-antibodies to RBC antigens. What precipitates the antibody formation is currently unclear. It has been hypothesized that a pro-inflammatory state preceding the therapeutic transfusion may be a predisposing factor. Plasma levels of ten cytokines were evaluated upon recruitment to the study of 83 children with SCD undergoing therapeutic RBC transfusions. The levels of cytokines were correlated with development of anti-RBC antibodies prior, or during seven years post recruitment. Twelve subjects displayed significantly higher levels of all cytokines examined, with pro-, as well as anti-inflammatory properties. Surprisingly, the elevated levels of cytokines were preferentially found in patients without anti-RBC allo- and/or auto-antibodies. Further, presence of high cytokine levels was not predictive of anti-RBC antibody development during the subsequent seven year follow up. These data suggest that the increased concentration of multiple cytokines is not a biomarker of either the presence of or susceptibility to the development of RBC alloimmunization.

Genetics of transfusion recipient alloimmunization: can clues from susceptibility to autoimmunity pave the way?

The search for genetic determinants of alloimmunization in sickle cell disease transfusion recipients was based on two premises: i) that polymorphisms responsible for stronger immune and/or inflammatory responses and hemoglobin ß(S) mutation were co-selected by malaria; and ii) that stronger responder status contributes to development of lupus. We found a marker of alloimmunization in the gene encoding for Ro52 protein, also known as Sjögren syndrome antigen 1 (SSA1) and TRIM21. Surprisingly, the nature of the association was opposite of that with lupus; the same variant of a polymorphism (rs660) that was associated with lupus incidence was also associated with induction of tolerance to red blood cell antigens during early childhood. The dual function of Ro52 can explain this apparent contradiction. We propose that other lupus/autoimmunity susceptibility loci may reveal roles of additional molecules in various aspects of alloimmunization induced by transfusion as well as during pregnancy.

The association of CD81 polymorphisms with alloimmunization in sickle cell disease.

The goal of the present work was to identify the candidate genetic markers predictive of alloimmunization in sickle cell disease (SCD). Red blood cell (RBC) transfusion is indicated for acute treatment, prevention, and abrogation of some complications of SCD. A well-known consequence of multiple RBC transfusions is alloimmunization. Given that a subset of SCD patients develop multiple RBC allo-/autoantibodies, while others do not in a similar multiple transfusional setting, we investigated a possible genetic basis for alloimmunization. Biomarker(s) which predicts (predict) susceptibility to alloimmunization could identify patients at risk before the onset of a transfusion program and thus may have important implications for clinical management. In addition, such markers could shed light on the mechanism(s) underlying alloimmunization. We genotyped 27 single nucleotide polymorphisms (SNPs) in the CD81, CHRNA10, and ARHG genes in two groups of SCD patients. One group (35) of patients developed alloantibodies, and another (40) had no alloantibodies despite having received multiple transfusions. Two SNPs in the CD81 gene, that encodes molecule involved in the signal modulation of B lymphocytes, show a strong association with alloimmunization. If confirmed in prospective studies with larger cohorts, the two SNPs identified in this retrospective study could serve as predictive biomarkers for alloimmunization.

Age-related accumulation of T cells with markers of relatively stronger autoreactivity leads to functional erosion of T cells.

BACKGROUND: Thymic involution is a prominent characteristic of an aging immune system. When thymic function is reduced/absent, the peripheral T cell pool is subject to the laws of peripheral T cell homeostasis that favor survival/expansion of T cell receptors with relatively higher functional avidity for self-peptide/MHC complexes. Due to difficulties in assessing the TCR avidity in polyclonal population of T cells, it is currently not known whether high avidity T cells preferentially survive in aging individuals, and what impact this might have on the function of the immune system and development of autoimmune diseases. RESULTS: The phenotype of T cells from aged mice (18-24 months) indicating functional TCR avidity (CD3 and CD5 expression) correlates with the level of preserved thymic function. In mice with moderate thymic output (> 30% of peripheral CD62L(hi) T cells), T cells displayed CD3(low)CD5(hi) phenotype characteristic for high functional avidity. In old mice with drastically low numbers of CD62L(hi) T cells reduced CD5 levels were found. After adult thymectomy, T cells of young mice developed CD3(low)CD5(hi) phenotype, followed by a CD3(low)CD5(low) phenotype. Spleens of old mice with the CD3(low)/CD5(hi) T cell phenotype displayed increased levels of IL-10 mRNA, and their T cells could be induced to secrete IL-10 in vitro. In contrast, downmodulation of CD5 was accompanied with reduced IL-10 expression and impaired anti-CD3 induced proliferation. Irrespective of the CD3/CD5 phenotype, reduced severity of experimental allergic myelitis occurred in old mice. In MTB TCRß transgenic mice that display globally elevated TCR avidity for self peptide/MHC, identical change patterns occurred, only at an accelerated pace. CONCLUSIONS: These findings suggest that age-associated dysfunctions of the immune system could in part be due to functional erosion of T cells devised to protect the hosts from the prolonged exposure to T cells with high-avidity for self.

A population study of clinically actionable genetic variation affecting drug response from the Middle East.

Clinical implementation of pharmacogenomics will help in personalizing drug prescriptions and alleviate the personal and financial burden due to inefficacy and adverse reactions to drugs. However, such implementation is lagging in many parts of the world, including the Middle East, mainly due to the lack of data on the distribution of actionable pharmacogenomic variation in these ethnicities. We analyzed 6,045 whole genomes from the Qatari population for the distribution of allele frequencies of 2,629 variants in 1,026 genes known to affect 559 drugs or classes of drugs. We also performed a focused analysis of genotypes or diplotypes of 15 genes affecting 46 drugs, which have guidelines for clinical implementation and predicted their phenotypic impact. The allele frequencies of 1,320 variants in 703 genes affecting 299 drugs or class of drugs were significantly different between the Qatari population and other world populations. On average, Qataris carry 3.6 actionable genotypes/diplotypes, affecting 13 drugs with guidelines for clinical implementation, and 99.5% of the individuals had at least one clinically actionable genotype/diplotype. Increased risk of simvastatin-induced myopathy could be predicted in ~32% of Qataris from the diplotypes of SLCO1B1, which is higher compared to many other populations, while fewer Qataris may need tacrolimus dosage adjustments for achieving immunosuppression based on the CYP3A5 diplotypes compared to other world populations. Distinct distribution of actionable pharmacogenomic variation was also observed among the Qatari subpopulations. Our comprehensive study of the distribution of actionable genetic variation affecting drugs in a Middle Eastern population has potential implications for preemptive pharmacogenomic implementation in the region and beyond.

Organizing training workshops on gene literature retrieval, profiling, and visualization for early career researchers.

Early-career researchers must acquire the skills necessary to effectively search and extract information from biomedical literature. This ability is for instance crucial for evaluating the novelty of experimental results, and assessing potential publishing opportunities. Given the rapidly growing volume of publications in the field of biomedical research, new systematic approaches need to be devised and adopted for the retrieval and curation of literature relevant to a specific theme. In this context, we present a hands-on training curriculum aimed at retrieval, profiling, and visualization of literature associated with a given topic. The curriculum was implemented in a workshop in January 2021. Here we provide supporting material and step-by-step implementation guidelines with the ISG15 gene literature serving as an illustrative use case. Workshop participants can learn several skills, including: 1) building and troubleshoot PubMed queries in order to retrieve the literature associated with a gene of interest; 2) identifying key concepts relevant to given themes (such as cell types, diseases, and biological processes); 3) measuring the prevalence of these concepts in the gene literature; 4) extracting key information from relevant articles, and 5) developing a background section or summary on the basis of this information. Finally, trainees can learn to consolidate the structured information captured through this process for presentation via an interactive web application.

Clinical features, epidemiology, autoantibody status, HLA haplotypes and genetic mechanisms of type 1 diabetes mellitus among children in Qatar.

To describe the clinical features, epidemiology, autoantibody status, HLA haplotypes and genetic mechanisms of type 1 diabetes mellitus (T1DM). Patients (0-18 years) with diabetes were recruited. Clinical data was collected, autoantibodies and c-peptide were measured. Whole Genome Sequencing was performed. Genomic data analysis was compared with the known genes linked with T1DM and HLA alleles were studied. 1096 patients had one or more antibody positivity. The incidence of T1DM in 2020 was 38.05 per 100,000 children and prevalence was 249.73. GADA was the most common autoantibody followed by IAA. Variants in GSTCD, SKAP2, SLC9B1, BANK1 were most prevalent. An association of HLA haplotypes DQA1*03:01:01G (OR = 2.46, p value = 0.011) and DQB1*03:02:01G (OR = 2.43, p value = 0.022) was identified. The incidence of T1DM in Qatar is the fourth highest in the world, IA2 autoantibody was the most specific with some patients only having ZnT8 or IA2 autoantibodies thus underlining the necessity of profiling all 4 autoantibodies. The genes associated with T1DM in the Arab population were different from those that are common in the Caucasian population. HLA-DQ was enriched in the Qatari patients suggesting that it can be considered a major risk factor at an early age.

Adaptable TCR avidity thresholds for negative selection.

Central tolerance plays a significant role in preventing autoimmune diseases by eliminating T cells with high and intermediate avidity for self. To determine the manner of setting the threshold for deletion, we created a unique transgenic mouse strain with a diverse T cell population and globally increased TCR avidity for self-peptide/MHC complexes. Despite the adaptations aimed at reducing T cell reactivity (reduced TCR levels and increased levels of TCR signaling inhibitor CD5), transgenic mice displayed more severe experimental allergic encephalomyelitis and lupus. The numbers and activity of natural (CD4(+)CD25(+)) regulatory T cells were not altered. These findings demonstrate that the threshold for deletion is adaptable, allowing survival of T cells with higher avidity when TCR avidity is globally increased.

A Neutrophil-Driven Inflammatory Signature Characterizes the Blood Transcriptome Fingerprint of Psoriasis.

Transcriptome profiling approaches have been widely used to investigate the mechanisms underlying psoriasis pathogenesis. Most researchers have measured changes in transcript abundance in skin biopsies; relatively few have examined transcriptome changes in the blood. Although less relevant to the study of psoriasis pathogenesis, blood transcriptome profiles can be readily compared across various diseases. Here, we used a pre-established set of 382 transcriptional modules as a common framework to compare changes in blood transcript abundance in two independent public psoriasis datasets. We then compared the resulting "transcriptional fingerprints" to those obtained for a reference set of 16 pathological or physiological states. The perturbations in blood transcript abundance in psoriasis were relatively subtle compared to the changes we observed in other autoimmune and auto-inflammatory diseases. However, we did observe a consistent pattern of changes for a set of modules associated with neutrophil activation and inflammation; interestingly, this pattern resembled that observed in patients with Kawasaki disease. This similarity between the blood-transcriptome signatures in psoriasis and Kawasaki disease suggests that the immune mechanisms driving their pathogenesis might be partially shared.

Protective effect of HLA-DQB1 alleles against alloimmunization in patients with sickle cell disease.

BACKGROUND: Alloimmunization or the development of alloantibodies to Red Blood Cell (RBC) antigens is considered one of the major complications after RBC transfusions in patients with sickle cell disease (SCD) and can lead to both acute and delayed hemolytic reactions. It has been suggested that polymorphisms in HLA genes, may play a role in alloimmunization. We conducted a retrospective study analyzing the influence of HLA-DRB1 and DQB1 genetic diversity on RBC-alloimmunization. STUDY DESIGN: Two-hundred four multi-transfused SCD patients with and without RBC-alloimmunization were typed at low/medium resolution by PCR-SSO, using IMGT-HLA Database. HLA-DRB1 and DQB1 allele frequencies were analyzed using logistic regression models, and global p-value was calculated using multiple logistic regression. RESULTS: While only trends towards associations between HLA-DR diversity and alloimmunization were observed, analysis of HLA-DQ showed that HLA-DQ2 (p=0.02), -DQ3 (p=0.02) and -DQ5 (p=0.01) alleles were significantly higher in non-alloimmunized patients, likely behaving as protective alleles. In addition, multiple logistic regression analysis showed both HLA-DQ2/6 (p=0.01) and HLA-DQ5/5 (p=0.03) combinations constitute additional predictor of protective status. CONCLUSION: Our data suggest that particular HLA-DQ alleles influence the clinical course of RBC transfusion in patients with SCD, which could pave the way towards predictive strategies.

Are Immune Modulating Single Nucleotide Polymorphisms Associated with Necrotizing Enterocolitis?

Necrotizing enterocolitis (NEC) is a devastating gastrointestinal emergency. The purpose of this study is to determine if functional single nucleotide polymorphisms (SNPs) in immune-modulating genes pre-dispose infants to NEC. After Institutional Review Board approval and parental consent, buccal swabs were collected for DNA extraction. TaqMan allelic discrimination assays and BglII endonuclease digestion were used to genotype specific inflammatory cytokines and TRIM21. Statistical analysis was completed using logistic regression. 184 neonates were analyzed in the study. Caucasian neonates with IL-6 (rs1800795) were over 6 times more likely to have NEC (p = 0.013; OR = 6.61, 95% CI 1.48-29.39), and over 7 times more likely to have Stage III disease (p = 0.011; OR = 7.13, (95% CI 1.56-32.52). Neonates with TGFß-1 (rs2241712) had a decreased incidence of NEC-related perforation (p = 0.044; OR = 0.28, 95% CI: 0.08-0.97) and an increased incidence of mortality (p = 0.049; OR = 2.99, 95% CI: 1.01 - 8.86). TRIM21 (rs660) was associated with NEC-related intestinal perforation (p = 0.038; OR = 4.65, 95% CI 1.09-19.78). In premature Caucasian neonates, the functional SNP IL-6 (rs1800795) is associated with both the development and increased severity of NEC. TRIM21 (rs660) and TGFß-1 (rs2241712) were associated with NEC- related perforation in all neonates in the cohort. These findings suggest a possible genetic role in the development of NEC.

Genomics in premature infants: a non-invasive strategy to obtain high-quality DNA.

We used a cost-effective, non-invasive method to obtain high-quality DNA from buccal epithelial-cells (BEC) of premature infants for genomic analysis. DNAs from BEC were obtained from premature infants with gestational age ≤ 36 weeks. Short terminal repeats (STRs) were performed simultaneously on DNA obtained from the buccal swabs and blood from the same patient. The STR profiles demonstrated that the samples originated from the same individual and exclude any contamination by external DNAs. Whole exome sequencing was performed on DNAs obtained from BEC on premature infants with and without necrotizing enterocolitis, and successfully provided a total number of reads and variants corroborating with those obtained from healthy blood donors. We provide a proof of concept that BEC is a reliable and preferable source of DNA for high-throughput sequencing in premature infants.

rs660 polymorphism in Ro52 (SSA1; TRIM21) is a marker for age-dependent tolerance induction and efficiency of alloimmunization in sickle cell disease.

Patients with sickle cell disease (SCD) who receive red blood cell (RBC) transfusions have a higher rate of anti-RBC (allo and auto) antibody development than other transfused subjects. We hypothesized that an incidence and/or kinetics of RBC-specific antibody formation in SCD patients is influenced by a linked inheritance of the hemoglobin beta S (HbbetaS) allele and a polymorphism rs660C/T in the neighboring Ro52 gene. We found that 75% of C/T heterozygous and only 30.8% of T/T homozygous patients that developed antibodies were first transfused before the age of five. In addition, there was a significant inverse correlation between time of exposure to antigen or number of transfusions received and the age when T/T patients received first transfusion, indicating progressive development of competence of their immune system. In contrast, this correlation was not observed in patients with C/T genotype. Finally, increased expression of Ro52 was associated with the presence of the T/T genotype. These results suggest that rs660 polymorphism is a marker of efficiency of tolerance induction in early childhood and immune competence development to RBC antigens in SCD patients of pre-teen/teen age.

CD4-dependent signaling is required for a late checkpoint during Th2 development associated with resistance to activation-induced cell death.

Previous studies have found that class II-restricted T cells from CD4-deficient mice reconstituted with a tail-less CD4 transgene have a specific defect in the development of Th2 effector cells; however, the reason for this defect was not clear. Following stimulation with a high potency peptide and exogenous IL-4, CD4-dependent signaling is required for optimal generation of a Th2 effector population. However, initial IL-4 and GATA-3 transcription is appropriately induced, suggesting that the initial stages of Th2 development are intact and independent of CD4 after priming with a strong agonist peptide. In addition to the defect in Th2 development, CD4 mutant T cells are also relatively resistant to activation-induced cell death (AICD). Furthermore, inhibition of AICD in wild-type T cells causes a defect in Th2 development similar to that seen in the CD4 mutant T cells. These data support the hypothesis that CD4-dependent signaling pathways regulate a distinct checkpoint in the expansion and commitment phase of Th2 development, which is related to dysregulation of AICD.

Acquisition of CD80 by human T cells at early stages of activation: functional involvement of CD80 acquisition in T cell to T cell interaction.

The interaction between CD28 on T cells and CD80 on APCs intensifies the linkage between TCR and MHC at the site of contact between T cells and APCs. In this study, we demonstrate that during human T cell/human APC interaction, the autologous or allogeneic human CD4(+) T cells become positive for the detection of CD80 at an early stage of activation (24 h). This detection of CD80 is attributable to the acquisition of CD80 from APCs, as opposed to the up-regulation of endogenous CD80, as demonstrated by CD4(+) T cells treated with cyclohexamide. Furthermore, no CD80 mRNA could be detected at 24 h in T cells that had acquired CD80 from APCs. CD80 acquisition by T cells from APCs was enhanced upon TCR engagement. The amount of CD80 acquisition by CD4(+) T cells was shown to be related to the expression of CD80 on APCs. Using soluble fusion proteins (soluble CTLA-4, CD28, and CD80) to block either CD28 on the surface of T cells or CD80 on the surface of APCs, it was demonstrated that CD80 acquisition by T cells is mediated through its receptors, possibly CD28 interaction. Moreover, we demonstrate that T cells that have acquired CD80 have the ability to stimulate other T cells. These data thus suggest that CD80 acquisition by human T cells might play a role in the immunoregulation of T cell responses.