Blood Cell Phenotypes Research Articles

Genomic and immune landscape of metastatic melanoma (MM) treated with pembrolizumab (PEM).

184 Background: Predictive biomarkers for PEM in MM is an active area of research. Methods: INSPIRE (NCT02644369) is a biomarker-driven Princess Margaret Cancer Centre initiative to evaluate genomic and immunologic changes in tumor and blood of patient (pts) treated with PEM at 200 mg IV Q3W. Baseline (BL) and on treatment (post 2-3 doses of PEM) fresh tumor biopsies were collected for DNA/RNA sequencing, immune-profiling, and tumor PD-L1 expression by immunohistochemistry (QualTek), with longitudinal blood collection for immunophenotyping. Results: As of 9/2017, 11 MM pts (9 cutaneous, 1 choroidal, 1 mucosal) were enrolled. Median treatment duration was 6 months (0.75 – 14.25); 7 pts remain on study. 7/11 (64%) pts had partial response (PR), 3/11 (27%) progressive disease (PD) and 1 was not evaluable (NE) per RECIST 1.1. Based on data for 8 pts (6 PR, 2 PD), higher percentage (%) PD-1 (mean 70 vs 12%), TIGIT (82 vs 43%) and 4-1BB (23 vs 4%) positivity on CD8 T cells in tumor vs blood was seen (all p < 0.05). In BL blood, lower % TIGIT positive CD8 T cells was detected in pts with PR vs PD (38 vs 58%, p < 0.05). T cell profile of 4 pts with paired BL and post PEM samples is shown in Table 1. Mean tumor PD-L1 score was 24% (0 - 90%), with no correlation to PEM response. Genomic analysis on 8 pts (5 PR, 2 PD, 1 NE) found mean tumor mutation burden (TMB) of 538. B2M deletion was seen in 2/2 PD and 0/6 PR pts. Among pts with PRs, 4/6 (67%) had TAP1 and TAP2 amplification post PEM therapy. HLA-A and HLA-B amplifications were each found in 3/6 (50%) PR pts, with only 1 present at BL. TMB was not associated with response to PEM. Conclusions: In this exploratory analysis of MM pts treated with PEM, there was significantly discordant T cell phenotype in tumor vs blood at BL. Our findings suggest B2M deletion to be associated with PEM resistance. The observed development of HLA-A/B and TAP1/2 amplification in 50-67% pts with PR post PEM suggests potential predictive significance, although further validation is warranted. Clinical trial information: NCT02644369. [Table: see text]

A Prospective Study of Humoral and Cellular Immune Responses to Hepatitis B Vaccination in Habitual Marijuana Smokers.

Exposure to Δ9-tetrahydrocannabinol (THC) in vitro and in animal models can significantly impair the differentiation, activation and function of dendritic cells, T cells and B cells. However, studies directly assessing the impact of marijuana smoking on human immunity are lacking. A prospective study of immune responses to a standard hepatitis B vaccination was therefore carried out in a matched cohort of 9 marijuana smokers (MS) and 9 nonsmokers (NS). In addition to their regular marijuana use, MS smoked four marijuana cigarettes in a monitored setting on the day of each vaccination. Blood samples were collected over time to assess the development of hepatitis B-specific immunity. The majority of subjects from both the NS (8) and MS (6) groups developed positive hepatitis B surface antibody titers (>10IU/L) and of these 6 NS and 5 MS were classified as high antibody (good) responders (>100IU/L). The development of a good response correlated with the presence of hepatitis B-specific T cell proliferation and cytokine production, resulting in a clear distinction regarding the immune status of good responders versus non-responders. However, even though there were slighter more non-responders in the MS cohort, there were no significant differences between MS and NS with respect to peripheral blood cell phenotypes or vaccination-related changes in hepatitis B responses. While a larger cohort may be required to rule out a small suppressive effect, our findings do not suggest that habitual marijuana smoking exerts a major impact on the development of systemic immunity to hepatitis B vaccination.

A Pilot Investigation of Natural Killer Cell Function and Phenotypes in Stable and Active Multiple Sclerosis Patients

Background: Previous studies have reported impaired cytotoxic activity in Natural Killer (NK) CD56Dim and CD56Bright cell phenotypes in peripheral blood associated with Multiple Sclerosis (MS). Recently it has been suggested that NK cell phenotype could be associated with new lesions on Magnetic resonance imaging (MRI) and may be used as an immunological indicator of disease activity. This project, for the first time, investigates NK cell cytotoxicity in MS patients with active and stable lesions.Methods: NK cell cytotoxic activity and NK CD56Dim and CD56Bright cell phenotypes were examined in MS patients using flow cytometry. Isolated NK cells were labelled with antibodies to determine CD56Dim and CD56Bright NK cells and cytotoxic function using target cells (K562). Seven patients (aged 38.0 ± 3.21) with stable Relapsing Remitting MS (RRMS) who had previously received alemtuzumab (Lemtrada®), five patients with active MS (aged 32.66 ± 5.17) on nil medication and five healthy controls (aged 34.4 ± 6.12) participated.Results: There were no significant differences for NK cell cytotoxic activity and NK CD56Dim and CD56Bright phenotypes between stable RRMS, active RRMS and healthy controls.Conclusion: Clear associations between NK cell cytotoxicity and clinical MS subtypes in this study were not identified.

Prediction of Extended Red Blood Cell Phenotype from Exome Next Generation Sequencing Data

Introduction:Accurate typing of patient and donor red blood cell (RBC) antigens is critical for safe transfusion practice. Although blood typing is traditionally accomplished by serology, genotyping methods to predict RBC antigens have proven valuable in a growing number of situations such as recently-transfused patients, scarcity of typing reagents, and indeterminate serologic results. However current RBC genotyping assays address a limited number of blood group genes and associated variants, and may not detect novel genetic changes and certain rare but clinically-significant variants. Next generation sequencing (NGS) technology provides an appealing alternative technology, allowing the user to examine a patient's entire genome or exome in a high-throughput manner. Whereas efforts are underway in multiple fields to apply exome sequencing (ES) for diagnostic, prognostic, and treatment purposes, Transfusion Medicine, with its extensive clinical genomic database, should find ready application from this approach. We describe here the creation of an algorithm to interpret NGS into a predicted extended RBC phenotype, and its application to analyze ES data from 245 participants of the ClinSeq® sequencing cohort.Methods:RyLAN (Red Cell and Lymphocyte Antigen prediction from NGS) was created as an open-source Python application that takes an NGS sorted binary alignment matrix (.bam) file and index as input. The software interacts with a non-relational database that encodes genomic blood group coordinates and phenotype interpretation rules, and yields a predicted extended RBC phenotype and quality parameters. Hard filters for mapping quality, depth, vcf QUAL, and fraction of alternate allele can be modified per individual genomic coordinate. The output is provided as a MongoDB document to facilitate advanced bulk queries and statistical analysis. We employed RyLAN to analyze 245 ES NGS files from the ClinSeq® cohort, using a database of 176 known antigenic, null, and weak blood group single nucleotide variants in 27 blood group genes as input.Results:The cohort consisted of 115 females and 130 males; 89% of participants self-described as white race, non- Hispanic ethnicity. Three percent of participants self-described as Hispanic or Latino, 4% as Asian, 2% with African ancestry, and the remaining as mixed or unknown race. From the total 176 genomic positions analyzed, 160 were not addressed by current commercially-available RBC genotyping platforms. The average read depth for the positions of interest was 78.2, and the average vcf QUAL value was 968. The highest variant nucleotide frequency was observed at the Fya/Fyb and Jka/Jkb loci (275 and 223 total haplotype variant calls, respectively).Among other phenotypes, RyLAN predicted 4 instances of heterozygosity for the KEL*02N.17 allele, 5 heterozygous individuals for the weak FY* X allele, 32 total heterozygous samples for various weak Kidd alleles, 2 homozygous individuals for weak Kidd expression, 1 heterozygosity for Lu6/Lu9, 1 SC:1,2 case, 1 Co(a-b+) predicted phenotype, and a total of 19 RHAG*01.04 and 47 KLF1*BGM12 alleles. Limited areas of the BCAM, KLF1, KEL, FUT7, ERMAP and CR1 genes failed quality filters repeatedly, and careful review indicated that these regions were not captured in the ES libraries. The ACKR1 promoter GATA-binding site variant was present in every sample and predicted all cases of self-reported African ancestry.Conclusions:We describe a new, open-source informatics tool to translate NGS data into a predicted extended RBC phenotype, and demonstrate its application through the analysis of 245 ClinSeq® ES files. Most predicted antigen frequencies were as expected for the ethnic composition of our cohort. We detected a higher frequency of the RHAG p.V270I and KLF1 p.S102P variants than expected, findings that are in agreement with the 1000 Genomes Project and warrant further study. Our analysis also corroborates the relative frequency of the JK*01W.01 allele, and the presence of the JK*01W.03 and JK*01W.04 alleles in the Caucasian population, which can lead to serologic discrepancies in other genotyping platforms. Serologic confirmation of these findings is being conducted. Further study of genomic data across multiple ethnic groups can help refine knowledge of blood group gene polymorphisms and their clinical association. DisclosuresNo relevant conflicts of interest to declare.

IL-6 production by monocytes is associated with graft function decline in patients with borderline changes suspicious for acute T-cell-mediated rejection: a pilot study

Although borderline changes (BL) suspicious for acute T-cell-mediated rejection represent a diagnostic category, its clinical relevance is questioned leading to heterogeneous therapeutic management. We hypothesized that measuring IL-6 secretion by peripheral blood mononuclear cells identifies patients with ongoing graft damage. We examined the association between secreted IL-6 and the change in estimated glomerular filtration rate at 6 months after the biopsy (ΔeGFR). We then conducted phenotypic and functional studies on patient and mouse innate immune cells in the blood and the kidney. In a training set, ΔeGFR was strongly associated with IL-6 levels, showing a clinically meaningful decline of 4.6 ± 1.5 ml/min per increase in log10 IL-6 (P = 0.001). These results were consistent after adjustment and were reproduced in a validation cohort. Phenotyping of peripheral blood cells revealed that the main source of IL-6 was CD14+ CD16- CCR2+ HLA-DR+ CD86+ CD11c+ inflammatory monocytes. There was a significant correlation between IL-6 secretion and interstitial dendritic cell density in the biopsy. Finally, characterization of mouse kidney dendritic cells revealed that they share features with macrophages and function as effector cells secreting IL-6. In conclusion, measuring IL-6 secreted by peripheral blood cells can be useful in the management of patients with BL in the absence of a concurrent inflammatory condition.

Development of a Multifunctional Nanobiointerface Based on Self-Assembled Fusion-Protein rSbpA/ZZ for Blood Cell Enrichment and Phenotyping.

We present a multifunctional nanobiointerface for blood cell capture and phenotyping applications that features both excellent antifouling properties and high antibody activity. Multifunctionality is accomplished by modifying polymeric materials using self-assembled S-layer fusion-protein rSbpA/ZZ to immobilize high density antibodies at the two protein A binding sites of the rSbpA/ZZ nanolattice structure. Controlled orientation and alignment of the antibodies reduced antibody consumption 100-fold and increased cell capture efficiency 4-fold over standard methodologies. Cell analysis in complex samples was made possible by the remarkable antifouling properties of the rSbpA domain, while at the same time reducing unspecific binding and forgoing tedious blocking procedures. An automated microfluidic in situ cell analysis platform for isolation and phenotyping of primary peripheral blood mononuclear cells was developed as practical application. Results obtained using our automated microfluidic cell analysis platform showed that the multifunctional nanobiointerface can discriminate among T helper and cytotoxic T cells, and thymocytes. Additionally, on-chip cell capture under flow conditions using a high affinity CD 3 selective nanobiointerface preferentially isolated cells with strong surface marker expression. This means that our dynamic microfluidic cell purification method allows the enrichment of 773 CD 8 positive cytotoxic T cells out of a total blood cell population of 7728 PBMCs, which is an increase in cell enrichment of 8-fold with a purity of 85%.

High output flow cytometry array profiling facilitates discriminant phenotyping of peripheral blood cells from patients with different forms of autoimmune uveitis

Abstract The information content of multi-parametric flow cytometry-based immune-phenotyping experiments is often underexploited given the paucity of adequate tools and strategies for large-scale unbiased data analysis. Here, we developed and applied a data analysis approach taking into account all mathematically possible combinations of markers in a given flow cytometry panel. We analyzed flow cytometry data generated from peripheral blood samples of healthy humans and patients with autoimmune uveitis in the setting of 2 different autoimmune diseases: Behcet’s disease and sarcoidosis. Original FACS data files were mined from Dryad Digital Repository http://dx.doi.org/10.5061/dryad.v6ste with reference to http://dx.doi.org/10.1371/journal.pcbi.1003215, and gated in bivariate plots. We employed combinatory mathematics to generate a matrix quantifying the representation of all possible cell populations using a given set of markers within the respective starting population and coded the algorithm to create a Java-based platform enabling the computation of large numbers of markers. Resulting metadata were visualized in a heat map approach. Our method enabled clustering of healthy vs diseased subjects using only 4 common markers (CD3, C8, CD197, and CD45), and of sarcoid vs Behcet’s patients with minimal error. Our approach demonstrates that multi-dimensional analysis of flow cytometry data allows meaningful large-scale screening of biologically relevant markers enabling classification and characterization of states of health and autoimmune disease. The approach is unbiased and has the potential to facilitate the discovery of cell populations with relevance as potential biomarkers or biological research targets.

Prevalência de aloimunização eritrocitária em pacientes portadores de anemia falciforme

Embora as transfusões de concentrado de hemácias sejam importantes para o tratamento de pacientes com anemia falciforme, elas acarretam riscos imunológicos tais como a aloimunização a antígenos eritrocitários. Aproximadamente 50% dos pacientes de anemia falciforme recebem transfusões no decorrer da vida, e entre 5 e 10% desses pacientes são submetidos a um programa de transfusão crônica. A aloimunização eritrocitária é uma complicação relativamente comum, podendo até mesmo levar a reações transfusionais hemolíticas tardias, contribuindo para aumentar as comorbidades da doença. Importantes medidas para prevenção dessas complicações nesses pacientes são o uso de hemácias previamente fenotipadas, além da fenotipagem do próprio receptor de concentrado de hemácias, determinando seu correto perfil fenotípico e possibilitando a escolha de concentrado de hemácias com antígenos correspondentes ao do paciente a ser transfundido. Extensa genotipagem eritrocitária profilática para selecionar doadores para pacientes que receberão repetidas transfusões durante um longo período é uma aplicação atraente de tipagem de sangue baseado em DNA. Isso é particularmente relevante para pacientes com doença falciforme, nos quais a taxa de aloimunização é elevada.

Differential effects of biological DMARDs on peripheral immune cell phenotypes in patients with rheumatoid arthritis.

The aim of this study was to assess the therapeutic effects of biological DMARDs (bDMARDs) on the diversity of immune cell phenotypes in peripheral blood of patients with RA. Peripheral immune cell phenotypes were determined in 108 RA patients who were non-responsive to conventional DMARDs and 33 healthy control subjects by eight-colour flow cytometry. We also examined the correlation between the phenotypes and clinical findings and assessed the effects of 24-week treatment with bDMARDs. The proportions of T follicular helper (Tfh) cells, IgD- CD27- double negative B cells and plasmacytoid dendritic cells (pDCs) were higher in patients with active RA than in healthy control. The percentages of memory T cells, Th17 and Tfh cells correlated with autoantibody titres, whereas that of plasmablasts correlated with disease activity scores. Treatment with TNF inhibitors reduced the proportion of pDCs, while tocilizumab reduced the proportion of double-negative B cells but increased naïve and activated Treg cells. Abatacept treatment resulted in marked decrease in the proportion of activated Tfh but slightly reduced Th17 and Treg cells. The proportion of Tfh cells was an independent and significant predictor of the response to abatacept therapy. Molecular targeted therapies induced different changes in different immune cell phenotypes. Among the phenotypes, Tfh cells seem a potential target for abatacept. Immunophenotypic analysis might be useful for prediction of the response to bDMARDs.

Assessment of common red blood cell pretreatments to yield an accurate serologic antigen phenotype compared with genotype-predicted phenotype.

For patients requiring multiple transfusions and patients with positive direct antiglobulin tests (DATs), an extended red blood cell (RBC) phenotype can provide valuable information and help to determine the risk of forming alloantibodies. In some instances, the phenotype may be used for prophylactic matching. Phenotyping in this patient population is often hindered by the presence of circulating donor cells and/or by a positive DAT. Several methods, such as EDTA glycine acid (EGA) treatment to remove IgG, hypotonic saline wash to separate autologous RBCs, or reticulocyte separation, are often used in these situations to isolate patient RBCs for serologic phenotyping. This study aimed to determine the accuracy of each RBC pretreatment method by comparing serologically determined antigen types with those predicted by RBC genotyping. Forty-eight peripheral blood samples from recently transfused patients were phenotyped for selected antigens in the Rh, Kell, MNS, Duffy, and Kidd systems. Treatment methods for the sample sets were reticulocyte separation (N = 12), EGA (N = 16), and hypotonic saline wash (N = 20). DNA was extracted using standard methods, and genotyping was performed using the HEA BeadChip panel. In addition, 21 samples positive for RBC-bound IgG were EGAtreated up to two times. These samples were analyzed pre- and post-EGA treatment for RBC-bound IgG by tube DAT and by flow cytometry with fluorescein isothiocyanate-labeled antihuman IgG. After reticulocyte separation, 3 of the 12 samples had discordant types with one antigen each: Fyb, N, and K; serologic results were negative compared with genotype-predicted positive phenotype results. The EGA-treated sample set showed one discordant type: Fyb; serologic results were negative compared with genotype-predicted positive phenotype results. Four of the 20 samples had discordant types involving the following antigens: Fyb, N, e, and M; serologic results were negative compared with genotype-predicted positive phenotype results. After EGA treatment of 21 samples, 14 (67%) were negative for RBC-bound IgG by tube DAT, and 7 remained positive. Using flow cytometry, EGA treatment rendered only 4 samples negative, and 17 remained positive. In the antigen testing sample set of 48 samples, 10 of 511 total antigen types tested were discordant. Discordant types were most frequent in the hypotonic saline wash sample set (N = 6). In the flow cytometry sample set, 48 percent of the samples negative by tube DAT after EGA elution had detectable RBCbound IgG by flow cytometry. These findings suggest that caution should be taken when using phenotype results from all pretreated RBCs and support the use of RBC genotyping to predict RBC antigen expression in samples from recently transfused patients.

Red blood cell phenotype prevalence in blood donors who self-identify as Hispanic

Molecular genotyping platforms provide a quick, high-throughput method for identifying red blood cell units for patients on extended phenotype-matching protocols, such as those with sickle cell disease or thalassemia. Most of the antigen prevalence data reported are for non-Hispanic populations. Therefore, this study sought to determine the phenotype prevalence in a single blood center's Hispanic population and to compare those results with previously reported rates in non-Hispanic donor populations. We performed a retrospective review of all serologic and molecular typing from donors who self-reported as Hispanic. The phenotype prevalence was reported and compared with rates from other racial/ethnic groups. A total of 1127 donors who selfidentified as Hispanic were screened by serologic methods for Rh and Kell antigens, and 326 were subsequently selected for molecular typing. The most prevalent probable Rh phenotypes were R1r (26.6%), R1R2 (21.5%), and R1R1 (20.7%); rr was found in 7.8 percent of donors tested. The percentage of K+ donors in this population was 2.8 percent. The most prevalent Duffy phenotypes were Fy(a+b+) (35.9%), Fy(a+b-) (35.6%), and Fy(a-b+) (27%). Of the donors studied, 15.3 percent had an FY GATA mutation. Only 1.5 percent of the donors were Fy(a-b-). The Jk(a+b+) phenotype was found in nearly half of the population. M+N+S+s+ was the most prevalent MNS phenotype from that group, constituting 22.4 percent. A total of 95.7 percent of the donors were Lu(a-b+), and Di(a-b+) was observed in 94.4 percent. The most prevalent Dombrock phenotype was Do(a+b+), constituting 46.9 percent, followed closely by Do(a-b+) at 40.5 percent. Hispanic donor antigen prevalence is distinctly different from other racial/ethnic groups and should be considered when attempting to find extended matched units for these patients.

Peripheral killer cells do not differentiate between asthma patients with or without fixed airway obstruction

ABSTRACTObjective: The three main types of killer cells – CD8+ T cells, NK cells and NKT cells – have been linked to asthma and chronic obstructive pulmonary disease (COPD). However, their role in a small subset of asthma patients displaying fixed airway obstruction (FAO), similar to that seen in COPD, has not been explored. The objective of the present study was to investigate killer cell numbers, phenotype and function in peripheral blood from asthma patients with FAO, asthma patients without FAO, and healthy individuals. Methods: Peripheral CD8+ T cells (CD8+CD3+CD56−), NK cells (CD56+CD3−) and NKT-like cells (CD56+CD3+) of 14 asthma patients with FAO (post-bronchodilator FEV/FVC <0.7, despite clinician-optimised treatment), 7 asthma patients without FAO (post-bronchodilator FEV/FVC ≥ 0.7), and 9 healthy individuals were studied. Results: No significant differences were seen between the number, receptor expression, MAPK signalling molecule expression, cytotoxic mediator expression, and functional cytotoxicity of peripheral killer cells from asthma patients with FAO, asthma patients without FAO and healthy individuals. Conclusions: Peripheral killer cell numbers or functions do not differentiate between asthma patients with or without fixed airway obstruction.

Large Scale Discoveries of Genes and Polymorphisms Associated with Variation in Hematologic Traits

Hematopoiesis generates mature blood cells from hematopoietic stem cells (HSC) in distinct lineages to release of trillions of mature cells each day into the peripheral blood stream to perform essential functions such as oxygen transport, hemostasis and host defense. The formation and turnover of blood cells are tightly controlled and so the properties of blood cells, including their volume and count, have large heritabilities and are easily influenced by genetic variation. Here we describe the most statistically powerful genome wide association study (GWAS) of blood cell indices to date. We tested associations of 29.5 million polymorphic DNA sequence variants derived using the the Affymetrix axiom array with interpolation of 20 million variants using the UK 10000 genome data with 36 different hematological indices of red cells, white cells and platelets, some of which, such as the reticulocyte count, have been explored for the first time. We discovered significant associations at thousands of associated genetic variants, including hundreds of associations for low frequency genetic variants, thus identifying associations with larger effects on indices than those reported for common variants by previous discovery studies. We have described detailed follow-up studies of the novel associations. Using cell type-specific epigenome and gene expression data generated by the BLUEPRINT project and results from chromatin conformation capture in major blood cell types, we can identify the likely causal variants and their functional impact at a large number of the novel loci. Finally, we have evaluated the contribution of genetic variants to common and complex diseases. In conclusion, we have interrogated phenotypes across the whole hematopoietic tree and increased the number of traits associated with blood cell phenotypes by an order of magnitude. Overall, our results demonstrate widespread and powerful genetic influences on the formation and regulation of the major human blood cell types, identifying many novel genes involved and show the value of genome-wide functional annotation from relevant primary cell populations for interpreting genetic association results. DisclosuresNo relevant conflicts of interest to declare.

Red Cell Alloimmunization Rates in Allogeneic Hematopoietic Stem Cell Transplant Recipients

Introduction: Donor selection for allogeneic hematopoietic stem cell transplant (allo-HSCT) is dependent on matching with the intended recipient HLA allele profile, but not blood group compatibility. Red blood cell (RBC) phenotype matching is not considered, even if recipient alloantibodies are present pre-HSCT. Historically, up to 3.7% of allo-HSCT recipients have been found to develop new RBC alloantibodies following allo-HSCT. We completed an audit of all adult and pediatric allo-HSCT recipients of the Alberta Bone Marrow and Blood Cell Transplant Program to define the rate of RBC alloimmunization, and evaluate the impact of this RBC alloantibody presence on donor marrow engraftment in our allo-HSCT recipient population.Methods:A retrospective review was completed including all allogeneic pediatric and adult HSCT recipients between January 1, 2007 and January 1, 2015. Data was obtained from review of cellular therapy laboratory electronic records with red cell alloantibody information extracted manually from the transfusion medicine laboratory information system.Results: A total of 674 patients, including 104 pediatric recipients (<18 years old), underwent 697 allo-HSCT procedures (591 peripheral blood, 45 marrow, 61 cord blood). The mean HSCT recipient age was 40 (range 0-66) and most common HSCT indication was acute myeloid leukemia. Myeloablative conditioning was given to all adults and 86% of pediatric recipients. Fully HLA matched grafts were provided to 77% of recipients. ABO compatibility status of allo-HSCT procedures included the following: 362 (52%) ABO identical grafts, 154 (22%) grafts with a minor incompatibility, 143 (21%) grafts with a major incompatibility, and 38 (5.0%) grafts with bidirectional incompatibility. Rh mismatches were present in 165 (24%) of donor-recipient pairs.A total of 47 allo-HSCT recipients, including 3 pediatric and 44 adult patients, were found to have RBC alloantibodies before or after allo-HSCT. A total of 45 (6.4%) of allo-HSCT recipients had detectable RBC alloantibodies pre-HSCT, with 69 individual alloantibodies identified. The most common RBC alloantibody was anti-E (30%). Antibody screen results available on the day of or following HSCT in 43 allo-HSCT recipients found: 12 (28%) with antibody disappearance pre-HSCT and a negative screen on the date of allo-HSCT, 15 (35%) with antibody waning to disappearance after allo-HSCT, and 11 (26%) with persistence of pre-HSCT antibodies following allo-HSCT. New post-HSCT RBC alloantibodies were detected in 3 adult recipients of peripheral blood collected stem cell grafts (anti-D; anti-Kpa; anti-K plus anti-E), with an overall rate of 0.4%. These patients all received myeloablative conditioning and grafts which were ABO identical or had a minor ABO incompatibility. The anti-D antibody developed post-transplant in an Rh positive recipient of an Rh negative graft. Thus, the calculated overall rate of anti-D development in Rh mismatched HSCT recipients was 0.6%. There was no observed impact on neutrophil and platelet engraftment comparing adult allo-HSCT recipients who did and did not have pre-HSCT RBC alloantibodies.Conclusion: The risk of post-HSCT RBC alloantibody development is very low, even in Rh mismatched donor-recipient pairs. ABO incompatibility does not affect the risk of post-HSCT alloantibody development. Allo-HSCT recipients infrequently have pre-HSCT RBC alloantibodies, which may disappear after myeloablative conditioning. The presence of RBC alloantibodies pre-HSCT does not appear to impact donor marrow engraftment. The results of our retrospective study are limited by the availability, timing and frequency of post-HSCT antibody screen investigations. The decision to perform an antibody screen post-HSCT is a clinical one, typically dependent on recipient transfusion needs. Further prospective research is required to more accurately determine the rate of new post-HSCT alloantibody development and duration of alloantibody persistence or disappearance in allo-HSCT recipients. Results of these studies may also help guide RBC transfusion decisions in HSCT recipients known to have pre-HSCT RBC alloantibodies with proven engraftment and a negative post-HSCT antibody screen. DisclosuresNo relevant conflicts of interest to declare.

The hide and seek of Plasmodium vivax in West Africa: report from a large-scale study in Beninese asymptomatic subjects.

Background Plasmodium vivax is considered to be absent from western Africa, where the prevalence of Duffy-negative red blood cell phenotype proves to be high. Several studies have, however, detected P. vivax infection cases in this part of Africa, raising the question of what is the actual prevalence of P. vivax in local populations. MethodsThe presence of P. vivax was investigated in a large population of healthy blood donors in Benin using microscopy, serology and molecular detection. The seroprevalence was measured with species-specific ELISA using two recombinant P. vivax proteins, namely rPvMSP1 and rPvCSP1. Specific molecular diagnosis of P. vivax infection was carried out using nested-PCR. The performances and cut-off values of both rPvCSP1 and rPvMSP1 ELISA were first assessed using sera from P. vivax-infected patients and from non-exposed subjects.ResultsAmong 1234 Beninese blood donors, no parasites were detected when using microscopy, whereas 28.7% (354/1234) of patients exhibited had antibodies against rPvMSP1, 21.6% (266/1234) against rPvCSP1, and 15.2% (187/1234) against both. Eighty-four samples were selected for nested-PCR analyses, of which 13 were positive for P. vivax nested-PCR and all Duffy negative. ConclusionThe results of the present study highlight an unexpectedly high exposure of Beninese subjects to P. vivax, resulting in sub-microscopic infections. This suggests a probably underestimated and insidious parasite presence in western Africa. While the vaccination campaigns and therapeutic efforts are all focused on Plasmodium falciparum, it is also essential to consider the epidemiological impact of P. vivax.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-016-1620-z) contains supplementary material, which is available to authorized users.

The Allelic Landscape of Human Blood Cell Trait Variation and Links to Common Complex Disease

SummaryMany common variants have been associated with hematological traits, but identification of causal genes and pathways has proven challenging. We performed a genome-wide association analysis in the UK Biobank and INTERVAL studies, testing 29.5 million genetic variants for association with 36 red cell, white cell, and platelet properties in 173,480 European-ancestry participants. This effort yielded hundreds of low frequency (<5%) and rare (<1%) variants with a strong impact on blood cell phenotypes. Our data highlight general properties of the allelic architecture of complex traits, including the proportion of the heritable component of each blood trait explained by the polygenic signal across different genome regulatory domains. Finally, through Mendelian randomization, we provide evidence of shared genetic pathways linking blood cell indices with complex pathologies, including autoimmune diseases, schizophrenia, and coronary heart disease and evidence suggesting previously reported population associations between blood cell indices and cardiovascular disease may be non-causal.

Antigen exposure shapes the ratio between antigen-specific Tregs and conventional T cells in human peripheral blood

The T-cell receptor (TCR) is required for maturation and function of regulatory T cells (Tregs), but the ligand specificities of Tregs outside the context of transgenic TCRs are largely unknown. Using peptide-MHC tetramers, we isolated rare specific Foxp3+ cells directly ex vivo from adult peripheral blood and defined their frequency and phenotype. We find that a proportion of circulating Tregs recognize foreign antigens and the frequency of these cells are similar to that of self-reactive Tregs in the absence of cognate infection. In contrast, the frequencies of Tregs that recognize some common microbial antigens are significantly reduced in the blood of most adults. Exposure to peripheral antigens likely has a major influence on the balance between Tregs and conventional T-cell subsets because a larger proportion of flu-specific T cells has a regulatory cell phenotype in the cord blood. Consistent with this finding, we show that lymphocytic choriomeningitis virus infection can directly modulate the ratio of virus-specific effectors and Tregs in mice. The resulting change in the balance within an antigen-specific T-cell population further correlates with the magnitude of effector response and the chronicity of infection. Taken together, our data highlight the importance of antigen specificity in the functional dynamics of the T-cell repertoire. Each specific population of CD4+ T cells in human peripheral blood contains a subset of Tregs at birth, but the balance between regulatory and effector subsets changes in response to peripheral antigen exposure and this could impact the robustness of antipathogen immunity.

Progressive changes in composition of lymphocytes in lung tissues from patients with non-small-cell lung cancer

Immune cell infiltration is a common feature of many human solid tumors. Innate and adaptative immune systems contribute to tumor immunosurveillance. We investigated whether tumors evade immune surveillance by inducing states of tolerance and/or through the inability of some immune subpopulations to effectively penetrate tumor nests. Immunohistochemistry and flow cytometry analysis were used to study the composition and distribution of immune subpopulations in samples of peripheral blood, tumor tissue (TT), adjacent tumor tissue (ATT), distant non-tumor tissue (DNTT), cancer nests, cancer stroma, and invasive margin in 61 non-small-cell lung cancer (NSCLC) patients. A significantly higher percentage of T and B cells and significantly lower percentage of NK cells were detected in TT than in DNTT. Memory T cells (CD4+CD45RO+, CD8+CD45RO+) and activated T cells (CD8+DR+) were more prevalent in TT. Alongside this immune activation, the percentage of T cells with immunosuppressive activity was higher in TT than in DNTT. B- cells were practically non-existent in tumor nests and were preferentially located in the invasive margin. The dominant NK cell phenotype in peripheral blood and DNTT was the cytotoxic phenotype (CD56+ CD16+), while the presence of these cells was significantly decreased in ATT and further decreased in TT. Finally, the immunologic response differed between adenocarcinoma and squamous cell carcinoma and according to the tumor differentiation grade. These findings on the infiltration of innate and adaptative immune cells into tumors contribute to a more complete picture of the immune reaction in NSCLC.

Genetic Screen in Drosophila Larvae Links ird1 Function to Toll Signaling in the Fat Body and Hemocyte Motility.

To understand how Toll signaling controls the activation of a cellular immune response in Drosophila blood cells (hemocytes), we carried out a genetic modifier screen, looking for deletions that suppress or enhance the mobilization of sessile hemocytes by the gain-of-function mutation Toll10b (Tl10b). Here we describe the results from chromosome arm 3R, where five regions strongly suppressed this phenotype. We identified the specific genes immune response deficient 1 (ird1), headcase (hdc) and possibly Rab23 as suppressors, and we studied the role of ird1 in more detail. An ird1 null mutant and a mutant that truncates the N-terminal kinase domain of the encoded Ird1 protein affected the Tl10b phenotype, unlike mutations that affect the C-terminal part of the protein. The ird1 null mutant suppressed mobilization of sessile hemocytes, but enhanced other Tl10b hemocyte phenotypes, like the formation of melanotic nodules and the increased number of circulating hemocytes. ird1 mutants also had blood cell phenotypes on their own. They lacked crystal cells and showed aberrant formation of lamellocytes. ird1 mutant plasmatocytes had a reduced ability to spread on an artificial substrate by forming protrusions, which may explain why they did not go into circulation in response to Toll signaling. The effect of the ird1 mutation depended mainly on ird1 expression in hemocytes, but ird1-dependent effects in other tissues may contribute. Specifically, the Toll receptor was translocated from the cell membrane to intracellular vesicles in the fat body of the ird1 mutant, and Toll signaling was activated in that tissue, partially explaining the Tl10b-like phenotype. As ird1 is otherwise known to control vesicular transport, we conclude that the vesicular transport system may be of particular importance during an immune response.

Frequencies of red blood cell major blood group antigens and phenotypes in the Chinese Han population from Mainland China.

Alloantibodies directed to red blood cell (RBC) antigens play an important role in alloimmune-mediated haemolytic transfusion reactions and haemolytic disease of the foetus and newborn. The frequencies and phenotypes of RBC antigens are different in populations from different geographic areas and races. However, the data on major blood group antigens in the Chinese Han population from Mainland China are still very limited; thus, we aimed to investigate them in this study. A total of 1412 unrelated voluntary Chinese Han blood donors were randomly recruited. All donors were typed for blood group antigens: D, C, c, E, e, C(w) , Jk(a) , Jk(b) ,M, N, S, s, Le(a) , Le(b) , K, k. Kp(a) , Kp(b) , Fy(a) , Fy(b) , Lu(a) , Lu(b) , P1 and Di(a) using serological technology. Calculations of antigen and phenotype frequencies were expressed as percentages and for allele frequencies under the standard assumption of Hardy-Weinberg equilibrium. Amongst the Rh antigens, D was the most common (98.94%) followed by e (92.28%), C (88.81%), c (58.43%), E (50.78%) and C(w) (0.07%) with DCe/DCe (R1 R1 , 40.72%) being the most common phenotype. In the Kell blood group system, k was present in 100% of the donors and a rare phenotype, Kp (a+b+), was found in 0.28% of the donors. For the Kidd and Duffy blood group systems, Jk (a+b+) and Fy (a+b-) were the most common phenotypes (44.05% and 84.35%, respectively). In the MNS blood group system, M+N+S-s+ (45.54%) was the most common, whereas M+N-S-s- and M-N+S-s- were not found. The rare Lu (a-b-) and Lu (a+b+) phenotypes were identified in 0.43% and 1.13% of the donors, respectively. Le(a) and Le(b) were seen in 17.92% and 63.03% of donors, respectively. The frequency of Di(a) was 4.75%, which was higher than in the Chinese population in Taiwan region or the Caucasian and Black populations (P<0.0001). This study systematically describes the frequencies of 24 blood group antigens in the Chinese Han population from Mainland China. The data can be helpful in creating a donor database for preparation of indigenous cell panels and providing antigen-negative blood to patients with multiple alloantibodies.