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.

Lymphatic endothelial cell-mediated accumulation of CD177+Treg cells suppresses antitumor immunity in human esophageal squamous cell carcinoma.

Regulatory T (Treg) cells are critical in shaping an immunosuppressive microenvironment to favor tumor progression and resistance to therapies. However, the heterogeneity and function of Treg cells in esophageal squamous cell carcinoma (ESCC) remain underexplored. We identified CD177 as a tumor-infiltrating Treg cell marker in ESCC. Interestingly, expression levels of CD177 and PD-1 were mutually exclusive in tumor Treg cells. CD177+ Treg cells expressed high levels of IL35, in association with CD8+ T cell exhaustion, whereas PD-1+ Treg cells expressed high levels of IL10. Pan-cancer analysis revealed that CD177+ Treg cells display increased clonal expansion compared to PD-1+ and double-negative (DN) Treg cells, and CD177+ and PD-1+ Treg cells develop from the same DN Treg cell origin. Importantly, we found CD177+ Treg cell infiltration to be associated with poor overall survival and poor response to anti-PD-1 immunotherapy plus chemotherapy in ESCC patients. Finally, we found that lymphatic endothelial cells are associated with CD177+ Treg cell accumulation in ESCC tumors, which are also decreased after anti-PD-1 immunotherapy plus chemotherapy. Our work identifies CD177+ Treg cell as a tumor-specific Treg cell subset and highlights their potential value as a prognostic marker of survival and response to immunotherapy and a therapeutic target in ESCC.

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.

Immunoprotection of cellular transplants for autoimmune type 1 diabetes through local drug delivery.

Type 1 diabetes mellitus (T1DM) is an autoimmune condition that results in the destruction of insulin-secreting ß cells of the islets of Langerhans. Allogeneic islet transplantation could be a successful treatment for T1DM; however, it is limited by the need for effective, permanent immunosuppression to prevent graft rejection. Upon transplantation, islets are rejected through non-specific, alloantigen specific, and recurring autoimmune pathways. Immunosuppressive agents used for islet transplantation are generally successful in inhibiting alloantigen rejection, but they are suboptimal in hindering non-specific and autoimmune pathways. In this review, we summarize the challenges with cellular immunological rejection and therapeutics used for islet transplantation. We highlight agents that target these three immune rejection pathways and how to package them for controlled, local delivery via biomaterials. Exploring macro-, micro-, and nano-scale immunomodulatory biomaterial platforms, we summarize their advantages, challenges, and future directions. We hypothesize that understanding their key features will help identify effective platforms to prevent islet graft rejection. Outcomes can further be translated to other cellular therapies beyond T1DM.

The Entangled World of Memory T Cells and Implications in Transplantation.

Memory T cells that are specific for alloantigen can arise from a variety of stimuli, ranging from direct allogeneic sensitization from prior transplantation, blood transfusion, or pregnancy to the elicitation of pathogen-specific T cells that are cross-reactive with alloantigen. Regardless of the mechanism by which they arise, alloreactive memory T cells possess key metabolic, phenotypic, and functional properties that render them distinct from naive T cells. These properties affect the immune response to transplantation in 2 important ways: first, they can alter the speed, location, and effector mechanisms with which alloreactive T cells mediate allograft rejection, and second, they can alter T-cell susceptibility to immunosuppression. In this review, we discuss recent developments in understanding these properties of memory T cells and their implications for transplantation.

Prior viral infection primes cross-reactive CD8+ T cells that respond to mouse heart allografts.

Introduction: Significant evidence suggests a connection between transplant rejection and the presence of high levels of pre-existing memory T cells. Viral infection can elicit viral-specific memory T cells that cross-react with allo-MHC capable of driving allograft rejection in mice. Despite these advances, and despite their critical role in transplant rejection, a systematic study of allo-reactive memory T cells, their specificities, and the role of cross-reactivity with viral antigens has not been performed. Methods: Here, we established a model to identify, isolate, and characterize cross-reactive T cells using Nur77 reporter mice (C57BL/6 background), which transiently express GFP exclusively upon TCR engagement. We infected Nur77 mice with lymphocytic choriomeningitis virus (LCMV-Armstrong) to generate a robust memory compartment, where quiescent LCMV-specific memory CD8+ T cells could be readily tracked with MHC tetramer staining. Then, we transplanted LCMV immune mice with allogeneic hearts and monitored expression of GFP within MHC-tetramer defined viral-specific T cells as an indicator of their ability to cross-react with alloantigens. Results: Strikingly, prior LCMV infection significantly increased the kinetics and magnitude of rejection as well as CD8+ T cell recruitment into allogeneic, but not syngeneic, transplanted hearts, relative to non-infected controls. Interestingly, as early as day 1 after allogeneic heart transplant an average of ~8% of MHC-tetramer+ CD8+ T cells expressed GFP, in contrast to syngeneic heart transplants, where the frequency of viral-specific CD8+ T cells that were GFP+ was <1%. These data show that a significant percentage of viral-specific memory CD8+ T cells expressed T cell receptors that also recognized alloantigens in vivo. Notably, the frequency of cross-reactive CD8+ T cells differed depending upon the viral epitope. Further, TCR sequences derived from cross-reactive T cells harbored distinctive motifs that may provide insight into cross-reactivity and allo-specificity. Discussion: In sum, we have established a mouse model to track viral-specific, allo-specific, and cross-reactive T cells; revealing that prior infection elicits substantial numbers of viral-specific T cells that cross-react to alloantigen, respond very early after transplant, and may promote rapid rejection.

BSHI and BTS UK guideline on the detection of alloantibodies in solid organ (and islet) transplantation.

Solid organ transplantation represents the best (and in many cases only) treatment option for patients with end-stage organ failure. The effectiveness and functioning life of these transplants has improved each decade due to surgical and clinical advances, and accurate histocompatibility assessment. Patient exposure to alloantigen from another individual is a common occurrence and takes place through pregnancies, blood transfusions or previous transplantation. Such exposure to alloantigen's can lead to the formation of circulating alloreactive antibodies which can be deleterious to solid organ transplant outcome. The purpose of these guidelines is to update to the previous BSHI/BTS guidelines 2016 on the relevance, assessment, and management of alloantibodies within solid organ transplantation.

Enrichment of effector memory T cells in the CD4 and CD8 T cell compartment during chronic graft versus host disease in children.

BACKGROUND: During allogeneic Hematopoietic stem cell transplantation (HSCT), frequent pathological scenarios include graft versus host disease (GVHD) and viral infections. We hypothesized if exogenous stimulus as alloantigen and viral antigens might impact on central and effector memory T cells in pediatric recipients. PATIENTS AND METHODS: Subjects included 21 pediatric recipients and 20 healthy children (control group). Peripheral blood samples of patients were collected along the first 712 days post-HSCT. T cell phenotyping of naïve, central, and effector memory T cells (TCMs and TEMs, respectively) was conducted using flow cytometry. Viral nucleic acids were detected using real-time PCR. RESULTS: T cell reconstitution was not reached after 1 year post-HSCT. Chronic GVHD was associated with increased numbers of naïve CD4 T cells (p < 0.05) as well as an increase in TEM and TCM cells of the CD4 (p < 0.0001 and p < 0.05, respectively) and CD8 T cell TEM (p < 0.0001). and TCM (p < 0.001) populations too. Moreover, BK and Epstein-Barr viruses were the main viral pathogens detected (<104 copies), which were associated with a decrease in all T cell compartments. CONCLUSION: During chronic GVHD, alloantigen persistence generates TEM cell enrichment among CD4 and CD8 T cells, and viral infections are associated with deficient recovery of T cells after HSCT.

Production of recombinant humanized monoclonal anti-human neutrophil antigen (HNA) antibodies with potential applicability as standard antibodies.

BACKGROUND: Antibodies against human neutrophil antigen (HNA) are involved in the pathogenesis of neonatal alloimmune neutropenia, autoimmune neutropenia, and transfusion-related acute lung injury. The present methods for anti-HNA antibody identification strongly depend on the presence of standard antisera with known allo/isospecificities. Here, we aimed to produce recombinant humanized antibodies to HNA from available mouse monoclonal antibodies (MoAbs). STUDY DESIGN AND METHODS: RNAs were extracted from available hybridoma cells producing mouse anti-HNA antibodies recognizing HNA-1a (TAG-1), -1b (TAG-2), -2 (TAG-4), and FcγRIIIb, and the cDNA was synthesized. Recombinant fragments consisting of the variable regions of the H and L chains of the mouse MoAb ligated to the constant region of human IgG were incorporated into an expression vector and transfected into CHO cells. Antibody specificity of the selected humanized monoclonal antibodies was confirmed, and tested by the participants of the ISBT Granulocyte Immunobiology Working Party (GIWP) workshop 2020. RESULTS: GIFT results confirmed the specific reactivity of TAGH-1 to -4, except for a cross-reactivity of TAGH-2 with HNA-1a/a neutrophils, only in flow-cytometry. MAIGA results showed clear specificity of all humanized antibodies, but the selection of the appropriate capture monoclonal antibody was essential for the test. The results of the ISBT GIWP showed high concordance among the labs. CONCLUSIONS: These are the first humanized monoclonal antibodies to HNA-1 and HNA-2 antigens produced and they will be important standard reagents for laboratories testing for neutrophil antibodies. We plan to have these humanized MoAbs available through WHO.

Alloantigen Infusion Activates the Transcriptome of Type 2 Conventional Dendritic Cells.

Recent studies have revealed novel molecular mechanisms by which innate monocytic cells acutely recognize and respond to alloantigen with significance to allograft rejection and tolerance. What remains unclear is the single-cell heterogeneity of the innate alloresponse, particularly the contribution of dendritic cell (DC) subsets. To investigate the response of these cells to exposure of alloantigen, C57BL/6J mice were administered live allogenic BALB/cJ splenic murine cells versus isogenic cells. In parallel, we infused apoptotic allogenic and isogenic cells, which have been reported to modulate immunity. Forty-eight hours after injection, recipient spleens were harvested, enriched for DCs, and subjected to single-cell mRNA sequencing. Injection of live cells induced a greater transcriptional change across DC subsets compared with apoptotic cells. In the setting of live cell infusion, type 2 conventional DCs (cDC2s) were most transcriptionally responsive with a Ccr2+ cDC2 subcluster uniquely responding to the presence of alloantigen compared with the isogenic control. In vitro experimentation confirmed unique activation of CCR2+ cDC2s following alloantigen exposure. Candidate receptors of allorecognition in other innate populations were interrogated and A type paired Ig-like receptors were found to be increased in the cDC2 population following alloexposure. These results illuminate previously unclear distinctions between therapeutic infusions of live versus apoptotic allogenic cells and suggest a role for cDC2s in innate allorecognition. More critically, these studies allow for future interrogation of the transcriptional response of immune cells in the setting of alloantigen exposure in vivo, encouraging assessment of novel pathways and previously unexamined receptors in this setting.

Hunting for the elusive target antigen in gestational alloimmune liver disease (GALD).

The prevailing concept is that gestational alloimmune liver disease (GALD) is caused by maternal antibodies targeting a currently unknown antigen on the liver of the fetus. This leads to deposition of complement on the fetal hepatocytes and death of the fetal hepatocytes and extensive liver injury. In many cases, the newborn dies. In subsequent pregnancies early treatment of the woman with intravenous immunoglobulin can be instituted, and the prognosis for the fetus will be excellent. Without treatment the prognosis can be severe. Crucial improvements of diagnosis require identification of the target antigen. For this identification, this work was based on two hypotheses: 1. The GALD antigen is exclusively expressed in the fetal liver during normal fetal life in all pregnancies; 2. The GALD antigen is an alloantigen expressed in the fetal liver with the woman being homozygous for the minor allele and the father being, most frequently, homozygous for the major allele. We used three different experimental approaches to identify the liver target antigen of maternal antibodies from women who had given birth to a baby with the clinical GALD diagnosis: 1. Immunoprecipitation of antigens from either a human liver cell line or human fetal livers by immunoprecipitation with maternal antibodies followed by mass spectrometry analysis of captured antigens; 2. Construction of a cDNA expression library from human fetal liver mRNA and screening about 1.3 million recombinants in Escherichia coli using antibodies from mothers of babies diagnosed with GALD; 3. Exome/genome sequencing of DNA from 26 presumably unrelated women who had previously given birth to a child with GALD with husband controls and supplementary HLA typing. In conclusion, using the three experimental approaches we did not identify the GALD target antigen and the exome/genome sequencing results did not support the hypothesis that the GALD antigen is an alloantigen, but the results do not yield basis for excluding that the antigen is exclusively expressed during fetal life., which is the hypothesis we favor.

Heterogeneity in allospecific T cell function in transplant-tolerant hosts determines susceptibility to rejection following infection.

Even when successfully induced, immunological tolerance to solid organs remains vulnerable to inflammatory insults, which can trigger rejection. In a mouse model of cardiac allograft tolerance in which infection with Listeria monocytogenes (Lm) precipitates rejection of previously accepted grafts, we showed that recipient CD4+ TCR75 cells reactive to a donor MHC class I-derived peptide become hypofunctional if the allograft is accepted for more than 3 weeks. Paradoxically, infection-induced transplant rejection was not associated with transcriptional or functional reinvigoration of TCR75 cells. We hypothesized that there is heterogeneity in the level of dysfunction of different allospecific T cells, depending on duration of their cognate antigen expression. Unlike CD4+ TCR75 cells, CD4+ TEa cells specific for a peptide derived from donor MHC class II, an alloantigen whose expression declines after transplantation but remains inducible in settings of inflammation, retained function in tolerant mice and expanded during Lm-induced rejection. Repeated injections of alloantigens drove hypofunction in TEa cells and rendered grafts resistant to Lm-dependent rejection. Our results uncover a functional heterogeneity in allospecific T cells of distinct specificities after tolerance induction and reveal a strategy to defunctionalize a greater repertoire of allospecific T cells, thereby mitigating a critical vulnerability of tolerance.

Clinical significance of human neutrophil antigen-1 antibodies in children with neutropenia.

Primary autoimmune neutropenia in young children is characterized by chronic neutropenia and positivity for antibodies against human neutrophil antigens (HNAs). This study analyzed the clinical characteristics of 402 children with neutropenia to identify differences between those with and without HNA-1 antibodies (HNA1abs). HNAabs in sera were detected by granulocyte immunofluorescence testing using flow cytometry. Relative fluorescence intensity (RFI) values were used to divide patients into positive (PG, n = 302), borderline (BG, n = 34), and negative (NG, n = 66) groups. The antibodies reacted to HNA-1a alone (59%), HNA-1b alone (1%), and HNA-1a/1b (40%). The PG had a significantly lower absolute neutrophil count before definitive diagnosis and a 1.6- to 2-times greater risk of hospitalization during neutropenia than the other groups. The median duration of neutropenia was longest in the PG at 25 months, followed by 20 months in the BG and 14 months in the NG. This large-scale cohort characterizes clinically distinct groups using the RFI value for HNA1abs in young children with neutropenia. Detection of HNA1abs may aid in understanding the clinical characteristics of children with neutropenia.

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.

Antibody-mediated antigen loss switches augmented immunity to antibody-mediated immunosuppression.

Antibodies against fetal red blood cell (RBC) antigens can cause hemolytic disease of the fetus and newborn (HDFN). Reductions in HDFN due to anti-RhD antibodies have been achieved through use of Rh immune globulin (RhIg), a polyclonal antibody preparation that causes antibody-mediated immunosuppression (AMIS), thereby preventing maternal immune responses against fetal RBCs. Despite the success of RhIg, it is only effective against 1 alloantigen. The lack of similar interventions that mitigate immune responses toward other RBC alloantigens reflects an incomplete understanding of AMIS mechanisms. AMIS has been previously attributed to rapid antibody-mediated RBC removal, resulting in B-cell ignorance of the RBC alloantigen. However, our data demonstrate that antibody-mediated RBC removal can enhance de novo alloimmunization. In contrast, inclusion of antibodies that possess the ability to rapidly remove the target antigen in the absence of detectable RBC clearance can convert an augmented antibody response to AMIS. These results suggest that the ability of antibodies to remove target antigens from the RBC surface can trigger AMIS in situations in which enhanced immunity may otherwise occur. In doing so, these results hold promise in identifying key antibody characteristics that can drive AMIS, thereby facilitating the design of AMIS approaches toward other RBC antigens to eliminate all forms of HDFN.

Analysis of T-cell alloantigen response via a direct pathway in kidney transplant recipients with donor-specific antibodies.

Donor-specific antibodies (DSAs) are the main cause of graft loss over time. The direct pathway of alloantigen recognition is important in the pathogenesis of acute rejection. Recent studies have suggested that the direct pathway also contributes to the pathogenesis of chronic injury. Nevertheless, there are no reports on T-cell alloantigen response via the direct pathway in kidney recipients with DSAs. We analyzed the T-cell alloantigen response via the direct pathway in kidney recipients with DSAs (DSA+) or without DSAs (DSA-). A mixed lymphocyte reaction assay was implemented to assess the direct pathway response. DSA+ patients showed significantly higher CD8+ and CD4+ T cell responses to donor cells than DSA- patients. Furthermore, proliferating CD4+ T cells showed a marked increase in Th1 and Th17 responses in DSA+ patients than in DSA- patients. In a comparison between anti-donor and third-party responses, the anti-donor CD8+ and CD4+ T cell response was significantly lower than the anti-third-party response. In contrast, the donor-specific hyporesponsiveness was absent in DSA+ patients. Our study demonstrated that DSA+ recipients have a greater potential for developing immune responses against the donor tissues via the direct alloantigen recognition pathway. These data contribute to an understanding of DSAs pathogenicity during kidney transplantation.

T cell specific deletion of IRF4 with Ox40-Cre impairs effector and memory T cell responses in heart transplantation.

BACKGROUND: IRF4 is the pioneer factor for effector T cell maturation. Here we investigated the function of IRF4 in maintaining OX40-related T cell responses following alloantigen activation in a mouse heart transplantation model. METHODS: Irf4flox/flox mice were bred with Ox40cre/+ mice to generate Irf4flox/floxOx40cre/+ mice. Wild type C57BL/6, Irf4flox/floxOx40cre/+ mice were transplanted with BALB/c heart allografts, with or without BALB/c skin-sensitization. CD4+ TEa T cells co-transfer experiments and flow cytometric analysis were conducted to investigate the amount of CD4+ T cells and the percentage of the T effector subset. RESULTS: Irf4flox/floxOx40cre/+ and Irf4flox/floxOx40cre/+ TEa mice were constructed successfully. IRF4 ablation in activated OX40-mediated alloantigen specific CD4+ TEa T cells reduced effector T cell differentiation (CD44hiCD62Llo, Ki67, IFN-γ), which caused long-term allograft survival (> 100 d) in the chronic rejection model. In the donor skin-sensitized heart transplantation model, the formation and function of alloantigen-specific memory CD4+ TEa cells were also impaired in Irf4flox/floxOx40cre/+ mice. Additionally, deletion of IRF4 after T cell activation in Irf4flox/floxOx40cre/+ mice reduced T cell reactivation in vitro. CONCLUSIONS: IRF4 ablation after OX40-related T cell activation could reduce effector and memory T cell formation and inhibit their function in response to alloantigen stimulation. These findings could have significant implications in targeting activated T cells to induce transplant tolerance.

70 analyte PFAS test method highlights need for expanded testing of PFAS in drinking water.

In this community-led pilot study we sought to investigate the utility of expanded per- and polyfluoroalkyl substances (PFAS) testing for drinking water, using a targeted analysis for 70 PFAS and the Total Oxidizable Precursor (TOP) Assay which can indicate the presence of precursor PFAS. PFAS were detected in 30 out of 44 drinking water samples across 16 states; 15 samples would exceed US EPA's proposed maximum contaminant levels for six PFAS. Twenty-six unique PFAS were identified, including 12 not covered by either US EPA Methods 537.1 or 533. An ultrashort chain PFAS, PFPrA, had the highest frequency of detection, occurring in 24 of 30 samples. It was also the PFAS reported at the highest concentration in 15 of these samples. We created a data filter to model how these samples would be reported under the upcoming fifth Unregulated Contaminant Monitoring Rule (UCMR5) requirements. All of the 30 samples with PFAS quantified by the 70 PFAS test had one or more PFAS present that would not be captured if the UCMR5 reporting requirements were followed. Our analysis suggests the upcoming UCMR5 will likely underreport PFAS in drinking water, due to limited coverage and higher minimum reporting limits. Results were inconclusive on the utility of the TOP Assay for monitoring drinking water. The results from this study provide important information to community participants regarding their current PFAS drinking water exposure. In addition, these results suggest gaps that need to be addressed by regulatory and scientific communities, in particular, the need for expanded targeted analysis of PFAS, the development of a sensitive, broad spectrum PFAS test, and further investigation into ultrashort chain PFAS.

Alloantigen Copy Number as a Critical Factor in RBC Alloimmunization.

RBC alloimmunization remains a significant barrier to ongoing transfusion therapy leading to morbidity, and in extreme cases mortality, due to delayed or insufficient units of compatible RBCs. In addition, the monitoring and characterization of alloantibodies, often with multiple specificities in a single patient, consumes substantial health care resources. Extended phenotypic matching has mitigated, but not eliminated, RBC alloimmunization and is only logistically available for specialized populations. Thus, RBC alloimmunization remains a substantial problem. In recent decades it has become clear that mechanisms of RBC alloimmunization are distinct from other antigens and lack of mechanistic understanding likely contributes to the fact that there are no approved interventions to prevent RBC alloimmunization from transfusion. The combination of human studies and murine modeling have identified several key factors in RBC alloimmunization. In both humans and mice, immunogenicity is a function of alloantigen copy number on RBCs. Murine studies have further shown that copy number not only changes rates of immunization but the mechanisms of antibody formation. This review summarizes the current understanding of quantitative and qualitative effects of alloantigen copy number on RBC alloimmunization.