SMA20/PMIS2 Is a Rapidly Evolving Sperm Membrane Alloantigen with Possible Species-Divergent Function in Fertilization.

Immunodominant alloantigens in pig sperm membranes include 15 known gene products and a previously undiscovered Mr 20,000 sperm membrane-specific protein (SMA20). Here we characterize SMA20 and identify it as the unannotated pig ortholog of PMIS2. A composite SMA20 cDNA encoded a 126 amino acid polypeptide comprising two predicted transmembrane segments and an N-terminal alanine- and proline (AP)-rich region with no apparent signal peptide. The Northern blots showed that the composite SMA20 cDNA was derived from a 1.1 kb testis-specific transcript. A BLASTp search retrieved no SMA20 match from the pig genome, but it did retrieve a 99% match to the Pmis2 gene product in warthog. Sequence identity to predicted PMIS2 orthologs from other placental mammals ranged from no more than 80% overall in Cetartiodactyla to less than 60% in Primates, with the AP-rich region showing the highest divergence, including, in the extreme, its absence in most rodents, including the mouse. SMA20 immunoreactivity localized to the acrosome/apical head of methanol-fixed boar spermatozoa but not live, motile cells. Ultrastructurally, the SMA20 AP-rich domain immunolocalized to the inner leaflet of the plasma membrane, the outer acrosomal membrane, and the acrosomal contents of ejaculated spermatozoa. Gene name search failed to retrieve annotated Pmis2 from most mammalian genomes. Nevertheless, individual pairwise interrogation of loci spanning Atp4a-Haus5 identified Pmis2 in all placental mammals, but not in marsupials or monotremes. We conclude that the gene encoding sperm-specific SMA20/PMIS2 arose de novo in Eutheria after divergence from Metatheria, whereupon rapid molecular evolution likely drove the acquisition of a species-divergent function unique to fertilization in placental mammals.

The CD177 c.1291A Allele Leads to a Loss of Membrane Expression and Mimics a CD177-Null Phenotype.

CD177 is a glycosyl phosphatidyl inositol (GPI)-linked, neutrophil-specific glycoprotein that in 3-5% of normal individuals is absent from all neutrophils. The molecular mechanism behind the absence of CD177 has not been unravelled completely. Here, we analyse the impact of the recently described CD177 c.1291G>A variant on CD177 expression. Recombinant CD177 c.1291G>A was expressed in HEK293F cells and its expression on the cell surface, inside the cell, and in the culture supernatant was investigated. The CD177 c.1291G>A protein was characterised serologically and its interaction with proteinase 3 (PR3) was demonstrated by confocal laser scanning microscopy. Our experiments show that CD177 c.1291G>A does not interfere with CD177 protein biosynthesis but affects the membrane expression of CD177, leading to very low copy numbers of the protein on the cellular surface. The mutation does not interfere with the ability of the protein to bind PR3 or human polyclonal antibodies against wild-type CD177. Carriers of the c.1291G>A allele are supposed to be phenotyped as CD177-negative, but the protein is present in soluble form. The presence of CD177 c.1291A leads to the production of an unstable CD177 protein and an apparent "CD177-null" phenotype.

Comparing ovarian expression of sperm acrosome associated 3 protein in young and adult queens.

The purpose of this research was to quantify sperm acrosome associated 3 protein expression in the ovaries of young (3.0 ± 0.9 months, n = 11) and adult (10.4 ± 2.8 months, n = 11) queens. Immunohistochemistry was performed on formalin-fixed, paraffin-embedded feline ovarian sections. Ovaries were obtained following routine ovariohysterectomy of queens. Cellular expression of sperm acrosome associated 3 protein was measured in primordial, primary, secondary, and tertiary follicles using an image-analysis software's red, green, and blue stack and manual thresholding functions. The oocyte nucleus, ooplasm, granulosa cells, and theca cells were outlined using the freehand selection tool and mean grey value was recorded. Results from each cellular location were compared between age groups using a Student's t-test and between follicle stages using an analysis of variance. Compared to adult queens, younger queens had significantly greater sperm acrosome associated 3 protein expression in granulosa cells of primary, secondary, and tertiary follicles. Also, theca cells of secondary and tertiary follicles had significantly greater sperm acrosome associated 3 protein expression in younger queens compared to adult queens. The oocyte nucleus of primordial, primary, and secondary follicles had significantly greater sperm acrosome associated 3 protein expression in younger queens compared to adult queens. However, sperm acrosome associated 3 protein expression within the ooplasm did not differ significantly between age groups of any follicle type. More research is needed to determine what role sperm acrosome associated 3 protein may play in female fertility in animals as well as what mechanisms regulate ovarian sperm acrosome associated 3 protein expression over time.

An accurate genetic assay to identify human neutrophil antigen 2 deficiency.

OBJECTIVE: We aimed to develop accurate and user-friendly genetic assays to identify the inherited neutrophil antigen-2 (HNA-2) deficiency in humans. BACKGROUND: HNA-2 is one of the most important neutrophil antigens implicated in a number of human disorders. HNA-2 deficiency or HNA-2 null is a common phenotype observed in 3%-5% Americans. HNA-2 null individuals are at risk to produce isoantibodies (or alloantibodies) that play important roles in transfusion-related acute lung injury, immune neutropenia, and bone marrow graft failure. We previously demonstrated that the CD177 coding SNP 787A > T (c.787A > T) is the most important genetic determinant for HNA-2 deficiency. However, reliable genetic assays are not available for routine clinical laboratory application up to now. STUDY DESIGN AND METHODS: A novel polymerase chain reaction (PCR) strategy was used to determine genotypes of the CD177 SNP c.787A > T. In the simplified PCR assay, all allele specific primers and internal control primers were included in the same reaction, which ensures reliability of the assay. In addition, a novel high-throughput nested TaqMan assay was developed to determine genotypes of c.787A > T for large population genetic analysis of HNA-2 deficiency. RESULTS: CD177 SNP c787A > T genotypes of 396 subjects were 100% concordant among the single PCR reaction method, the nested TaqMan assay, and Sanger Sequencing analysis. Out of 396 subjects, all 18 donors with the CD177 STP homozygous genotype were HNA-2 null. CONCLUSION: The novel PCR-based genotyping assay is accurate to identify HNA-2 deficient individuals and is suitable for clinical laboratories. In addition, the innovative high-throughput nested TaqMan assay will be useful for large-scale population screens and genetic studies of HNA-2 deficiency.

Human neutrophil antigen-1, -3, -4, and -5 allele and genotype frequencies in the Croatian blood donor population and their clinical significance.

OBJECTIVES: Human neutrophil antigens (HNAs) and antibodies play an important role in allo- and autoimmunity associated with immune neutropenia and transfusion reactions. The aim of this study was to determine the HNA-1, -3, -4 and -5 allele and genotype frequencies in the Croatian blood donor population to assess the role of HNA-1, -3, -4, and -5 alleles in the development of neonatal alloimmune neutropenia and antibody-mediated transfusion-related acute lung injury. MATERIAL AND METHODS: A total of 371 blood samples from unselected healthy blood donors were analyzed. Samples from all 371 donors were genotyped for HNA-1, samples from 160 donors were genotyped for HNA-3, and samples from 142 donors were genotyped for HNA-4 and HNA-5 using the polymerase chain reaction with sequence-specific primers (PCR-SSP) method. RESULTS: The frequencies of the FCGR3B*01, FCGR3B*02 and FCGR3B*03 HNA-1 alleles were 0.393, 0.607 and 0.022, and of the SLC44A2*01 and SLC44A2*02 HNA-3 alleles 0.781 and 0.219, respectively. The frequencies of the ITGAM*01 and ITGAM*02 HNA-4 alleles were 0.796 and 0.204, and of the ITGAL*01 and ITGAL*02 HNA-5 alleles 0.718 and 0.282, respectively. CONCLUSION: These are the first results on the HNA allele and genotype frequencies in the Croatian blood donor population. We observed no deviations from previous reports on Caucasian populations. Determination of the HNA antigen frequencies in the population is important to estimate the risk of alloimmunization to HNA, especially the risk of fetal-maternal incompatibility and alloantibody production by transfusion of the HNA incompatible blood components.

Neonatal alloimmune neutropenia: diagnosis and management of 31 Italian patients.

BACKGROUND: In neonates, antibody-mediated destruction of neutrophils may occur as a consequence of auto- or isoimmune disorders. There are few studies on this topic, and particularly on neonatal alloimmune neutropenia (NAN). MATERIALS AND METHODS: We retrospectively evaluated the clinical and molecular/serological findings of 83 neutropenic infants referred to our Reference Laboratory for diagnostic evaluation of NAN, from 2008 to 2021. We also genotyped 260 Italian healthy subjects for the four principal human neutrophil antigens (HNA). RESULTS: The diagnosis of NAN was confirmed in 31 cases. The other 52 cases were autoimmune neutropenia (n=21), neutropenia caused by maternal neutrophil autoantibodies (n=8), neutropenia of non-immune origin (n=17), and cases in which a diagnosis could not be reached (n=6). The median age at neutropenia onset and absolute neutrophil count (ANC) were significantly lower in NAN than in non-NAN cases (1 vs 30 days, p<0.005; 330 vs 580/µL, p=0.003, respectively). About 74% of NAN cases developed neutropenia within the first week of life and laboratory investigations were required within 2 weeks. In five patients the neutropenia was discovered at the end of the first month of life and they were referred to our laboratory 1-2 months later when neutropenia had already resolved. Infections were seen in 19% of NAN cases. The median time to resolution of NAN was 31 days. About 50% of NAN cases were due to alloantibodies against HNA-1b, the most frequent allele of HNA-1 in the Italian population (allele frequency 0.63). In five cases of NAN the mothers had an HNA-1 null phenotype, a frequency higher than that observed in our Italian cohort. DISCUSSION: NAN should be considered by clinicians in infants with neutropenia onset within 5-7 days of life, even though there can be other reasons for a low ANC. If neutropenia is detected later, benign neutropenia seems more likely, although persistence of maternal alloantibodies cannot be ruled out.

Genetic landscape of human neutrophil antigen variants in India from population-scale genomes.

Antibodies against human neutrophil antigens (HNAs) play a significant role in various clinical conditions such as neonatal alloimmune neutropenia, transfusion-related acute lung injury, and other nonhemolytic transfusion reactions. This study aims to identify the genotype and allele frequencies of HNAs in the healthy Indian population. Ten genetic variants in four human genes encoding alleles of HNAs class I-V approved by the International Society of Blood Transfusion-Granulocyte Immunobiology Working Party were used in the analysis. Genetic variants from whole genome sequences of 1029 healthy Indian individuals corresponding to HNA alleles were analyzed. The frequencies of the variants were compared with global population datasets using an in-house computational pipeline. In HNA class I, allele frequencies of FCGR3B*01, FCGR3B*02, and FCGR3B*03 encoding HNA-1a, HNA-1b, and HNA-1c were 0.07, 0.8, and 0.13, respectively. HNA class 3 alleles namely SLC44A2*01 (encoding HNA-3a) and SLC44A2*02 (encoding HNA-3b) were found at allele frequencies of 0.78 and 0.22, respectively. The frequencies of ITGAM*01 encoding HNA-4a and ITGAM*02 encoding HNA-4a were 0.95 and 0.05, respectively. Furthermore, allele frequencies of HNA class 5 alleles were 0.32 for ITGAL*01 (encoding HNA-5a) and 0.68 for ITGAL*02 (encoding HNA-5b). Interestingly, it was also found that rs2230433 variant deciding the HNA class 5 alleles, was highly prevalent (78.2%) in the Indian population compared with other global populations. This study presents the first comprehensive report of HNA allele and genotype frequencies in the Indian population using population genome datasets of 1029 individuals. Significant difference was observed in the prevalence of HNA5a and HNA5b in India in comparison to other global populations.

Human neutrophil antigen 3 genotype impacts neutrophil-mediated endothelial cell cytotoxicity in a two-event model of TRALI.

BACKGROUND: Antibodies against human neutrophil antigen (HNA)-3a are associated with severe cases of transfusion-related acute lung injury (TRALI). The HNA-3 system is located on choline transporter-like 2 (CTL-2) protein. CTL-2 is encoded by the gene SLC44A2 and a single-nucleotide polymorphism c.461G>A results in two antigens: HNA-3a and HNA-3b. Three HNA-3 genotypes/ phenotypes exist: HNA-3aa, HNA-3bb, and HNA-3ab. Two different pathways of anti-HNA-3a TRALI have been described: a two-hit neutrophil-dependent pathway and a one-hit neutrophil-independent pathway. However, it is not clear whether HNA-3ab heterozygous patients have a lower risk of anti-HNA-3a-mediated TRALI compared to HNA-3aa homozygous patients. MATERIALS AND METHODS: Healthy volunteers were genotyped for HNA-3 by real-time polymerase chain reaction, and phenotyped for HNA-3a by granulocyte immunofluorescence test (GIFT) and granulocyte agglutination test (GAT) against two donor sera containing anti-HNA-3a antibodies. The two sera were also used in in vitro models of human pulmonary microvascular endothelial cell (HLMVEC) cytotoxicity to investigate pathways of TRALI development. RESULTS: For both anti-HNA-3a sera, GIFT results matched the genotype, with a lower GIFT ratio for HNA-3ab neutrophils compared to HNA-3aa neutrophils, whereas GAT results showed no difference in agglutination. HLMVEC cytotoxicity was not observed in a one-hit neutrophil-independent model but was observed in a two-hit neutrophil-dependent model. Differences in cytotoxicity were observed between the two anti-HNA-3a sera used. Consistent with reduced HNA-3a antigen density as measured by GIFT, HNA-3ab neutrophils mediated less HLMVEC cytotoxicity than HNA-3aa neutrophils. CONCLUSION: HNA-3 genotype and HNA-3a antigen expression impacted the severity of anti-HNA-3a-mediated HLMVEC cytotoxicity in a two-hit neutrophil-dependent model of TRALI. Different HNA-3a antibodies might also impact the magnitude of HLMVEC cytotoxicity.

In Vitro Model of Suppression of the Alloantigen Response by Tolerogenic Dendritic Cells Transfected with Personalized DNA Constructs Encoding HLA Epitopes.

BACKGROUND: A search for efficient graft rejection modulation techniques for the promotion of durable engraftment remains to be a matter of close study all over the world. Despite the variety of immunosuppressive drugs, the schemes currently used show a lack of selectivity and have a number of side effects. Here we investigated an approach for the induction of antigen-specific tolerance in a human "stimulator-responder" model in vitro, using dendritic cells (DCs) transfected with designed DNA constructs encoding the stimulator's major histocompatibility complex (MHC) epitopes. METHODS: The object of the study is peripheral blood mononuclear cells (PBMCs) from 10 healthy donors. To induce antigen-specific tolerance, personalized DNA constructs were created for five responder-stimulator pairs, based on the sequences of donors' and recipients' MHCs. DNA sequencing was performed to select epitopes for incorporation into genetic constructs. A mixed lymphocyte culture assay was used (i) to assess the proliferative response in both directions for all possible stimulator-responder pairs (90 reactions) and (ii) to assess the tolerogenic properties of the generated transfected DCs (5 reactions). RESULTS: A significant increase in the amounts of FoxP3+ CD4+CD25+ cells and in IL-10 production was shown in culture of donor mononuclear cells after co-cultivation with the responder's dendritic cells transfected with donor-specific plasmids. The tolerogenic cultures generated using tolerogenic DCs transfected with MHC epitopes had a significantly greater ability to inhibit the proliferation of autologous MNCs in response to an allogeneic MHC stimulus. CONCLUSIONS: The produced DCs transfected with DNA constructs against HLA stimulating epitopes exhibited tolerogenic properties and may be used to develop antigen-specific tolerance. Thus, we proposed a perspective approach to the induction of antigen-specific tolerance, which should subsequently be studied for use in clinical practice.

Human neutrophil antigen 2 sequence-based typing: Joining the hunt for the CD177 answer.

BACKGROUND AND OBJECTIVES: Isoantibodies to human neutrophil antigen 2 (CD177) have been associated with several clinical conditions but to date the molecular basis for altered or non-expression has not been determined. Reliance on phenotyping and crossmatch to investigate these neutropenic clinical cases are inconvenient for the patients and demanding of resources within the laboratory. Therefore, a molecular approach has been introduced to address both issues. MATERIALS AND METHODS: A DNA panel of 100 randomly selected blood donors were collected and supplemented with 18 DNA samples from blood donors previously shown to be CD177 null. All DNA samples were sequence-based typed for all exons and observed polymorphisms recorded. The DNA from two families previously investigated for neonatal alloimmune neutropenia due to CD177 isoantibodies were also analysed. RESULTS: The incidence of CD177 null could be associated with a known exon 7 single-nucleotide polymorphism in 16/21 known CD177 null samples, which is consistent with previously published findings. Two additional mutations that may lead to null expression were also identified, of which one may be novel. In both family investigations, this same mutation could also be observed in the maternal DNA sample. CONCLUSION: Based on these observations, introduction of CD177 genotyping into routine use would identify null expression in over 75% (16/21) of associated cases. In turn, this could significantly reduce the need for supplementary testing and associated inconvenience to patients while permitting increased efficiency of laboratory testing. An added benefit would potentially elucidate other clinically relevant mutations and associated antigenic targets.

The neutrophil protein CD177 is a novel PDPN receptor that regulates human cancer-associated fibroblast physiology.

The cancer-associated fibroblast (CAF) marker podoplanin (PDPN) is generally correlated with poor clinical outcomes in cancer patients and thus represents a promising therapeutic target. Despite its biomedical relevance, basic aspects of PDPN biology such as its cellular functions and cell surface ligands remain poorly uncharacterized, thus challenging drug development. Here, we utilize a high throughput platform to elucidate the PDPN cell surface interactome, and uncover the neutrophil protein CD177 as a new binding partner. Quantitative proteomics analysis of the CAF phosphoproteome reveals a role for PDPN in cell signaling, growth and actomyosin contractility, among other processes. Moreover, cellular assays demonstrate that CD177 is a functional antagonist, recapitulating the phenotype observed in PDPN-deficient CAFs. In sum, starting from the unbiased elucidation of the PDPN co-receptome, our work provides insights into PDPN functions and reveals the PDPN/CD177 axis as a possible modulator of fibroblast physiology in the tumor microenvironment.

Marginal zone B cells mediate a CD4 T-cell-dependent extrafollicular antibody response following RBC transfusion in mice.

Red blood cell (RBC) transfusions can result in alloimmunization toward RBC alloantigens that can increase the probability of complications following subsequent transfusion. An improved understanding of the immune mechanisms that underlie RBC alloimmunization is critical if future strategies capable of preventing or even reducing this process are to be realized. Using the HOD (hen egg lysozyme [HEL] and ovalbumin [OVA] fused with the human RBC antigen Duffy) model system, we aimed to identify initiating immune factors that may govern early anti-HOD alloantibody formation. Our findings demonstrate that HOD RBCs continuously localize to the marginal sinus following transfusion, where they colocalize with marginal zone (MZ) B cells. Depletion of MZ B cells inhibited immunoglobulin M (IgM) and IgG anti-HOD antibody formation, whereas CD4 T-cell depletion only prevented IgG anti-HOD antibody development. HOD-specific CD4 T cells displayed similar proliferation and activation following transfusion of HOD RBCs into wild-type or MZ B-cell-deficient recipients, suggesting that IgG formation is not dependent on MZ B-cell-mediated CD4 T-cell activation. Moreover, depletion of follicular B cells failed to substantially impact the anti-HOD antibody response, and no increase in antigen-specific germinal center B cells was detected following HOD RBC transfusion, suggesting that antibody formation is not dependent on the splenic follicle. Despite this, anti-HOD antibodies persisted for several months following HOD RBC transfusion. Overall, these data suggest that MZ B cells can initiate and then contribute to RBC alloantibody formation, highlighting a unique immune pathway that can be engaged following RBC transfusion.

Evaluation of multi-epitope recombinant protein as a candidate for a contraceptive vaccine.

Contraceptive vaccine (CV) is a valuable, non-invasive, and alternative method for purposeful contraception. Sperm antigens are useful targets for producing CVs due to their specialized expression in sperm. In this study, a recombinant protein containing three main sperm epitopes (IZUMO1, SACA3, and PH-20) was designed and evaluated as CV to control fertility in male mice. The chimeric recombinant protein was expressed and purified in E. coli. Male mice were immunized by 100 µg purified protein and sera were collected to assess IgG antibodies. Evaluating the reproductive performance, immunized male mice mated with normal-fertile female mice and mating rate and the number of newborns was studied. Immunized mice were sacrificed and necropsy and histopathology studies were conducted. The results revealed that the designed chimeric protein stimulated the immune system of the mice effectively. The level of IgG antibody was significantly higher in vaccinated mouse rather than control mouse. Eighty percent of the vaccinated mice became infertile and in the remaining ones, the number of children decreased to 4-6 offspring instead of 10-12 in normal mice. Histopathological studies showed that no organs including heart, brain, lung, liver, kidney and intestine were damaged. However, Normal spermatogenesis has been disrupted and necrotic spermatogonia cells were reported in Seminiferous tubules. We concluded that the designed chimeric protein containing IZUMO1, SACA3, and PH-20 epitopes can stimulate the immune system and cause male contraception without any side effects.

A host-based two-gene model for the identification of bacterial infection in general clinical settings.

OBJECTIVES: In this study, we aimed to develop a simple gene model to identify bacterial infection, which can be implemented in general clinical settings. METHODS: We used a clinically availablereal-time quantitative polymerase chain reaction platform to conduct focused gene expression assays on clinical blood samples. Samples were collected from 2 tertiary hospitals. RESULTS: We found that the 8 candidate genes for bacterial infection were significantly dysregulated in bacterial infection and displayed good performance in group classification, whereas the 2 genes for viral infection displayed poor performance. A two-gene model (S100A12 and CD177) displayed 93.0% sensitivity and 93.7% specificity in the modeling stage. In the independent validation stage, 87.8% sensitivity and 96.6% specificity were achieved in one set of case-control groups, and 93.6% sensitivity and 97.1% specificity in another set. CONCLUSIONS: We have validated the signature genes for bacterial infection and developed a two-gene model to identify bacterial infection in general clinical settings.

Antibodies to Low-Copy Number RBC Alloantigen Convert a Tolerogenic Stimulus to an Immunogenic Stimulus in Mice.

Red blood cells expressing alloantigens are well known to be capable of inducing robust humoral alloantibody responses both in transfusion and pregnancy. However, the majority of transfusion recipients and pregnant women never make alloantibodies, even after repeat exposure to foreign RBCs. More recently, RBCs have been used as a cellular therapeutic-very much like transfusion, engineered RBCs are highly immunogenic in some cases but not others. In animal models of both transfusion and RBC based therapeutics, RBCs that do not induce an immune response also cause tolerance. Despite a robust phenomenology, the mechanisms of what regulates immunity vs. tolerance to RBCs remains unclear. However, it has been reported that copy number of alloantigens on the RBCs is a critical factor, with a very low copy number causing non-responsiveness (in both humans and mice) and also leading to tolerance in mice. Recently, we reported that an IgG2c specific for an RBC antigen can substantially enhance the humoral immune response upon transfusion of RBCs expressing that antigen. Herein, we report that an IgG2c converts RBCs with low antigen copy number from a tolerogenic to an immunogenic stimulus. These findings report the first known stimulus that induces humoral alloimmunization to a low copy number RBC alloantigen and identify a previously undescribed molecular switch that has the ability to affect responder vs. non-responder phenotypes of transfusion recipients.

Impaired adhesion of neutrophils expressing Slc44a2/HNA-3b to VWF protects against NETosis under venous shear rates.

Genome-wide association studies linked expression of the human neutrophil antigen 3b (HNA-3b) epitope on the Slc44a2 protein with a 30% decreased risk of venous thrombosis (VT) in humans. Slc44a2 is a ubiquitous transmembrane protein identified as a receptor for von Willebrand factor (VWF). To explain the link between Slc44a2 and VT, we wanted to determine how Slc44a2 expressing either HNA-3a or HNA-3b on neutrophils could modulate their adhesion and activation on VWF under flow. Transfected HEK293T cells or neutrophils homozygous for the HNA-3a- or HNA-3b-coding allele were purified from healthy donors and perfused in flow chambers coated with VWF at venous shear rates (100 s-1). HNA-3a expression was required for Slc44a2-mediated neutrophil adhesion to VWF at 100 s-1. This adhesion could occur independently of ß2 integrin and was enhanced when neutrophils were preactivated with lipopolysaccharide. Moreover, specific shear conditions with high neutrophil concentration could act as a "second hit," inducing the formation of neutrophil extracellular traps. Neutrophil mobilization was also measured by intravital microscopy in venules from SLC44A2-knockout and wild-type mice after histamine-induced endothelial degranulation. Mice lacking Slc44a2 showed a massive reduction in neutrophil recruitment in inflamed mesenteric venules. Our results show that Slc44a2/HNA-3a is important for the adhesion and activation of neutrophils in veins under inflammation and when submitted to specific shears. The fact that neutrophils expressing Slc44a2/HNA-3b have a different response on VWF in the conditions tested could thus explain the association between HNA-3b and a reduced risk for VT in humans.

CRISPR/Cas9-mediated knockout of clinically relevant alloantigenes in human primary T cells.

BACKGROUND: The ability of CRISPR/Cas9 to mutate any desired genomic locus is being increasingly explored in the emerging area of cancer immunotherapy. In this respect, current efforts are mostly focused on the use of autologous (i.e. patient-derived) T cells. The autologous approach, however, has drawbacks in terms of manufacturing time, cost, feasibility and scalability that can affect therapeutic outcome or wider clinical application. The use of allogeneic T cells from healthy donors may overcome these limitations. For this strategy to work, the endogenous T cell receptor (TCR) needs to be knocked out in order to reduce off-tumor, graft-versus-host-disease (GvHD). Furthermore, CD52 may be knocked out in the donor T cells, since this leaves them resistant to the commonly used anti-CD52 monoclonal antibody lymphodepletion regimen aiming to suppress rejection of the infused T cells by the recipient. Despite the great prospect, genetic manipulation of human T cells remains challenging, in particular how to deliver the engineering reagents: virus-mediated delivery entails the inherent risk of altering cancer gene expression by the genomically integrated CRISPR/Cas9. This is avoided by delivery of CRISPR/Cas9 as ribonucleoproteins, which, however, are fragile and technically demanding to produce. Electroporation of CRISPR/Cas9 expression plasmids would bypass the above issues, as this approach is simple, the reagents are robust and easily produced and delivery is transient. RESULTS: Here, we tested knockout of either TCR or CD52 in human primary T cells, using electroporation of CRISPR/Cas9 plasmids. After validating the CRISPR/Cas9 constructs in human 293 T cells by Tracking of Indels by Decomposition (TIDE) and Indel Detection by Amplicon Analysis (IDAA) on-target genomic analysis, we evaluated their efficacy in primary T cells. Four days after electroporation with the constructs, genomic analysis revealed a knockout rate of 12-14% for the two genes, which translated into 7-8% of cells showing complete loss of surface expression of TCR and CD52 proteins, as determined by flow cytometry analysis. CONCLUSION: Our results demonstrate that genomic knockout by electroporation of plasmids encoding CRISPR/Cas9 is technically feasible in human primary T cells, albeit at low efficiency.

Distinct Gene Expression Signatures Characterize Strong Clinical Responders Versus Nonresponders to Canakinumab in Children With Systemic Juvenile Idiopathic Arthritis.

OBJECTIVE: Canakinumab is a human anti-interleukin-1ß (anti-IL-1ß) blocking agent that effectively neutralizes IL-1ß-mediated signaling for treatment of systemic juvenile idiopathic arthritis (JIA). While many patients have dramatic clinical response to IL-1 blockade, approximately one-third fail to respond, but there are currently no validated clinical or immunologic predictors of response. We undertook this study to characterize distinct gene signatures for treatment response and nonresponse to canakinumab in systemic JIA patients. METHODS: We performed a secondary analysis of whole-blood gene expression microarrays using blood samples obtained from healthy controls and systemic JIA patients at baseline and on day 3 after canakinumab treatment (GEO accession no. GSE80060). Patients were considered strong clinical responders if they met the ACR90 response (exhibited ≥90% improvement in the American College of Rheumatology [ACR] JIA response criteria; nonresponders were those who met ACR30 [exhibiting ≤30% improvement in the ACR JIA response criteria]). A random-effects model with patient identity as the random variable was used for differential expression analysis. RESULTS: We identified a distinct gene expression signature in patients with a strong clinical response to canakinumab treatment as compared to nonresponders, mediated by up-regulation of neutrophil- and IL-1-associated genes and characterized by increasing divergence from control transcriptomes with increasing clinical response. We also identified a signature including up-regulated CD163 expression that was associated with canakinumab nonresponse. Intriguingly, canakinumab treatment induced either up- or down-regulation of type I interferon (IFN) genes, independent of clinical response. CONCLUSION: Here, we identify a gene signature in systemic JIA patients prior to receiving treatment that distinguishes strong responders to canakinumab from nonresponders. Further prospective studies are needed to assess the utility of these insights for treatment decisions in systemic JIA and to track the association of up-regulated type I IFN signatures with systemic JIA complications.

Human neutrophil antigens: Nature, clinical significance and detection.

Granulocytes are an essential part of both the innate and adaptive immune systems. Human neutrophil antigens (HNAs) are a family of epitopes that are located on glycoproteins that are mostly expressed on human granulocytes. Antibodies that recognize these epitopes have been associated with neutropenia, transfusion complications, haematopoietic stem cell transplant nonengraftment and renal transplant rejection. Currently, there are fourteen recognized HNA alleles across five antigen systems (HNA-1 through HNA-5), the molecular basis of which are located on the genes FCGR3B, CD177, SLC44A2, ITGAM and ITGAL, respectively. Elucidation of the associated genes has permitted the development of testing strategies for HNA typing and aided understanding of the associated epitopes. This review will outline the associated clinical conditions that require HNA investigation and how these are performed in specialized laboratories. Investigations provided are both reactive for patients with a variety of existing or suspected neutropenias and proactive in the testing of blood component donors in order to reduce the potential risk to patients who require transfusion.

Antigen-specific B cell depletion for precision therapy of mucosal pemphigus vulgaris.

Desmoglein 3 chimeric autoantibody receptor T cells (DSG3-CAART) expressing the pemphigus vulgaris (PV) autoantigen DSG3 fused to CD137-CD3ζ signaling domains, represent a precision cellular immunotherapy approach for antigen-specific B cell depletion. Here, we present definitive preclinical studies enabling a first-in-human trial of DSG3-CAART for mucosal PV. DSG3-CAART specifically lysed human anti-DSG3 B cells from PV patients and demonstrated activity consistent with a threshold dose in vivo, resulting in decreased target cell burden, decreased serum and tissue-bound autoantibodies, and increased DSG3-CAART engraftment. In a PV active immune model with physiologic anti-DSG3 IgG levels, DSG3-CAART inhibited antibody responses against pathogenic DSG3 epitopes and autoantibody binding to epithelial tissues, leading to clinical and histologic resolution of blisters. DSG3 autoantibodies stimulated DSG3-CAART IFN-γ secretion and homotypic clustering, consistent with an activated phenotype. Toxicology screens using primary human cells and high-throughput membrane proteome arrays did not identify off-target cytotoxic interactions. These preclinical data guided the trial design for DSG3-CAART and may help inform CAART preclinical development for other antibody-mediated diseases.