Diagnostic, prognostic, and immunological roles of CD177 in cervical cancer.

BACKGROUND: CD177, an indicator of prognosis in diverse cancers, is involved in the physiological processes of various tumor cells, and acts as an immune molecule with novel functions in cancer pathogenesis. However, the diagnostic, prognostic, and immunological role of CD177 in cervical cancer remains unclear. METHODS: Utilizing publicly available databases and integrating several bioinformatics analysis methods, we evaluated the expression level of CD177 in cervical cancer by GENT2, HPA, and GEO databases. And the experiments of western blot and immunohistochemical staining were used to test the hypothesis. The Kaplan-Meier Plotter database, Xena Shiny, and the constructed nomogram were clearly demonstrated its prognostic value for patients. Gene set enrichment analysis explored the relationship between CD177 and cervical cancer immune responses and immune cells infiltration level. In addition, we investigated the association between CD177 expression and stromal score, immune score, immune checkpoint, and drug sensitivity by TCGA RNA-seq data. RESULTS: CD177 was apparently expressed at low levels in cervical cancer and predicted a poor survival rate for patients. CD177 significantly activated immune-related signaling pathways and had a positive relationship with immune cell infiltration level. The high CD177 expression group possessed the high stromal score and immune score. CD177 had potential interactions with CTLA4, CD27, BLTA, CD200R1, CD80, NRP1, TNFRSF25, TIGIT, ICOS, and TNFSF9 checkpoint markers. And CD177 expression was positively relevant with drug sensitivity for Lapatinib, Belinostat, ATRA, Gefitinib, Navitoclax, and Tamoxifen. SIGNIFICANCE: These findings may shed light on the vital role of CD177 in cervical cancer diagnosis, prognosis, and immunological functions, and it may be a promising predictor and potential factor for cervical cancer patients.

Suppression of macrophage-mediated xenogeneic rejection by the ectopic expression of human CD177.

Cellular xenogeneic rejection by the innate immune system is a major immunological obstruction that needs to be overcome for the successful clinical use of xenografts. Our focus has been on macrophage-mediated xenogeneic rejection, since suppressing macrophage function has considerable potential for practical applications in the area of xenotransplantation. We report herein on an investigation of the suppressive effect of human CD177 (hCD177) against macrophage-mediated xenogeneic rejection. Wild type swine aortic endothelial cell (SEC) and an SEC transfectant with hCD177 (SEC/hCD177) were co-cultured with macrophages, and the degree of cytotoxicity was evaluated by WST-8 assays, and phagocytosis was examined using Calcein-AM labeling methods. The expression of anti/pro-inflammatory cytokines was evaluated by RT-qPCR and the phosphorylation of SHP-1 on macrophages in co-culture was evaluated by Western blotting. The result of cytotoxicity assays indicated that hCD177 suppressed M1 macrophage-mediated xenogeneic rejection (vs. SEC, p < 0.0001). Similarly, the result of phagocytosis assays indicated that hCD177 suppressed it (vs. SEC, p < 0.05). In addition, hCD177 significantly suppressed the expression of IL-1ß, a pro-inflammatory cytokine, in M1 macrophages (vs. SEC, p < 0.01). Luciferase assays using THP1-Lucia NF-kB also showed a significant difference in NF-kB activation (vs. SEC, p < 0.001). In addition, hCD177 was found to induce the phosphorylation of SHP-1 in M1 macrophages (vs. SEC, p < 0.05). These findings indicate that hCD177 suppresses M1 macrophage-mediated xenogeneic rejection, at least in part via in the phosphorylation of SHP-1.

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.

Competitively disrupting the neutrophil-specific receptor-autoantigen CD177:proteinase 3 membrane complex reduces anti-PR3 antibody-induced neutrophil activation.

CD177 is a neutrophil-specific receptor presenting the proteinase 3 (PR3) autoantigen on the neutrophil surface. CD177 expression is restricted to a neutrophil subset, resulting in CD177pos/mPR3high and CD177neg/mPR3low populations. The CD177pos/mPR3high subset has implications for antineutrophil cytoplasmic autoantibody (ANCA)-associated autoimmune vasculitis, wherein patients harbor PR3-specific ANCAs that activate neutrophils for degranulation. Here, we generated high-affinity anti-CD177 monoclonal antibodies, some of which interfered with PR3 binding to CD177 (PR3 "blockers") as determined by surface plasmon resonance spectroscopy and used them to test the effect of competing PR3 from the surface of CD177pos neutrophils. Because intact anti-CD177 antibodies also caused neutrophil activation, we prepared nonactivating Fab fragments of a PR3 blocker and nonblocker that bound specifically to CD177pos neutrophils. We observed that Fab blocker clone 40, but not nonblocker clone 80, dose-dependently reduced anti-PR3 antibody binding to CD177pos neutrophils. Importantly, preincubation with clone 40 significantly reduced respiratory burst in primed neutrophils challenged with either monoclonal antibodies to PR3 or PR3-ANCA immunoglobulin G from ANCA-associated autoimmune vasculitis patients. After separating the two CD177/mPR3 neutrophil subsets from individual donors by magnetic sorting, we found that PR3-ANCAs provoked significantly more superoxide production in CD177pos/mPR3high than in CD177neg/mPR3low neutrophils, and that anti-CD177 Fab clone 40 reduced the superoxide production of CD177pos cells to the level of the CD177neg cells. Our data demonstrate the importance of the CD177:PR3 membrane complex in maintaining a high ANCA epitope density and thereby underscore the contribution of CD177 to the severity of PR3-ANCA diseases.

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.

Decreased CD177pos neutrophils in myeloid neoplasms is associated with NPM1, RUNX1, TET2, and U2AF1 S34F mutations.

A decreased percentage of CD177pos neutrophils is frequently present in MDS and AML and is a useful flow cytometry (FCM) marker for the identification of MDS. The underlying mechanism leading to the low percentage of CD177pos neutrophils in MDS has not been explained. The aim of this study was to identify whether specific somatic mutations in myeloid neoplasms are associated with the low percentage of CD177pos neutrophils. 507 myeloid neoplasms with one or more pathogenic molecular abnormality identified by NGS and in which CD177 expression was assessed were evaluated. Correlation with CD177 expression was determined for 39 variables (including genes mutated, diagnostic groups and gender) using a 40 % cutoff level for low CD177 expression. In multivariate analysis mutations involving NPM1 (OD 0.26), RUNX1 (OD 0.39), TET2 (OD 0.58), and U2AF1 S34F (OD 0.25) were associated with low percentage of CD177pos neutrophils when all cases were evaluated. JAK2 (OD 2.5) alteration was associated with increased percentage of CD177pos neutrophils. Differences were noted between diagnostic subgroups with no single mutation associated with decreased CD177pos neutrophils in MDS and CCUS. The findings demonstrate an association between the percentage of CD177pos neutrophils and somatically acquired mutations involving several genes.

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.

CD177 modulates the function and homeostasis of tumor-infiltrating regulatory T cells.

Regulatory T (Treg) cells are one of the major immunosuppressive cell types in cancer and a potential target for immunotherapy, but targeting tumor-infiltrating (TI) Treg cells has been challenging. Here, using single-cell RNA sequencing of immune cells from renal clear cell carcinoma (ccRCC) patients, we identify two distinct transcriptional fates for TI Treg cells, Fate-1 and Fate-2. The Fate-1 signature is associated with a poorer prognosis in ccRCC and several other solid cancers. CD177, a cell surface protein normally expressed on neutrophil, is specifically expressed on Fate-1 TI Treg cells in several solid cancer types, but not on other TI or peripheral Treg cells. Mechanistically, blocking CD177 reduces the suppressive activity of Treg cells in vitro, while Treg-specific deletion of Cd177 leads to decreased tumor growth and reduced TI Treg frequency in mice. Our results thus uncover a functional CD177+ TI Treg population that may serve as a target for TI Treg-specific immunotherapy.

Inflammatory signature in acute-on-chronic liver failure includes increased expression of granulocyte genes ELANE, MPO and CD177.

Acute-on-Chronic Liver Failure (ACLF) is associated with innate immune dysfunction and high short-term mortality. Neutrophils have been identified to influence prognosis in ACLF. Neutrophil biology is under-evaluated in ACLF. Therefore, we investigated neutrophil-specific genes and their association with ACLF outcomes. This is an observational study. Enriched granulocytes, containing neutrophils, isolated from study participants in three groups- ACLF(n = 10), chronic liver disease (CLD, n = 4) and healthy controls (HC, n = 4), were analysed by microarray. Differentially expressed genes were identified and validated by qRT-PCR in an independent cohort of ACLF, CLD and HC (n = 30, 15 and 15 respectively). The association of confirmed overexpressed genes with ACLF 28-day non-survivors was investigated. The protein expression of selected neutrophil genes was confirmed using flow cytometry and IHC. Differential gene expression analysis showed 1140 downregulated and 928 upregulated genes for ACLF versus CLD and 2086 downregulated and 1091 upregulated genes for ACLF versus HC. Significant upregulation of neutrophilic inflammatory signatures were found in ACLF compared to CLD and HC. Neutrophil enriched genes ELANE, MPO and CD177 were highly upregulated in ACLF and their expression was higher in ACLF 28-day non-survivors. Elevated expression of CD177 protein on neutrophil surface in ACLF was confirmed by flow cytometry. IHC analysis in archival post mortem liver biopsies showed the presence of CD177+ neutrophils in the liver tissue of ACLF patients. Granulocyte genes ELANE, MPO and CD177 are highly overexpressed in ACLF neutrophils as compared to CLD or HC. Further, this three-gene signature is highly overexpressed in ACLF 28-day non-survivors.

Combined utility of CD177, P53, CD105 and c-kit immunohistochemical stains improves the detection of myelodysplastic syndrome.

The diagnosis of myelodysplastic syndrome (MDS) relies primarily on identifying peripheral blood cytopenia and morphologic dysplasia as well as detecting cytogenetic aberrations in a subset of patients. Accumulating data points to the importance of examining certain immunophenotypic changes characteristic of MDS, most of which are tested by flow cytometry. The role of immunohistochemistry in the diagnostic workup of MDS is less known. In this study, we used immunohistochemistry to survey the expression patterns of CD177, P53, CD105 and c- kit in a cohort of MDS bone marrow specimens (n = 57) and compared the results with a control group of patients who had cytopenia for other benign conditions (n = 49). MDS cases showed significant higher rates of: CD177-loss (13/57, 23% vs 1/49, 2%; P = .0016), P53 overexpression (8/57, 14% vs none; P = .005) and the presence of clusters of CD105-positive cells (6/57, 11% vs none; P = .021). Increased c-kit-positive cells was more common in MDS patients, but not statistically significant (17/57, 30% vs 8/49, 16%; P = .102). On multivariate analysis, only loss of CD177 expression was significantly higher in MDS group (P = .014). These findings suggest that a panel of immunohistochemical stains could serve as an adjunct tool in investigating unexplained cytopenias and warrant further comparative studies with flow cytometry.

Triggering Receptor Expressed on Myeloid Cells-1 Agonist Regulates Intestinal Inflammation via Cd177+ Neutrophils.

Triggering receptor expressed on myeloid cell-1 (TREM-1) signaling is expressed on neutrophils and monocytes that is necessary for the successful antimicrobial response and resolution of inflammation in the gut. In this study, we determined the effect of an anti-TREM-1 agonistic antibody (α-TREM-1) on colitis and identify its underlying mechanism of action. Administration of α-TREM-1 alleviated colitis in mice and resolved dysbiosis, which required TLR4/Myd88 signaling. α-TREM-1 increased the production of neutrophil extracellular traps and interleukin-22 by CD177+ neutrophils, which led to pathogen clearance and protection of the intestinal barrier. TREM-1 activation using an α-TREM-1 antibody protects against colitis by rebalancing the microbiota and protecting the epithelium against the immune response as well as modulates the function of neutrophils and macrophages. These results highlight the importance of the TREM-1 pathway in intestinal homeostasis and suggest that α-TREM-1 treatment may be an effective therapeutic strategy for inflammatory bowel disease.

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.

The neutrophil subset defined by CD177 expression is preferentially recruited to gingival crevicular fluid in periodontitis.

In recent years, the concept of distinct subpopulations of human neutrophils has attracted much attention. One bona fide subset marker, exclusively expressed by a proportion of circulating neutrophils in a given individual, and therefore dividing neutrophils in two distinct subpopulations, is the glycoprotein CD177. CD177 is expressed on the plasma and granule membranes of 0-100% of circulating neutrophils depending on the donor. Several in vitro studies have linked CD177 to neutrophil transmigration, yet very few have looked at the role of CD177 for tissue recruitment in vivo. We investigate whether the CD177+ and CD177- neutrophil subsets differ in their propensity to migrate to both aseptic- and microbe-triggered inflamed human tissues. Microbe-triggered neutrophil migration was evaluated in samples of gingival crevicular fluid (GCF) from patients with periodontitis, whereas neutrophil migration to aseptic inflammation was evaluated in synovial fluid from patients with inflammatory arthritis, as well as in exudate from experimental skin chambers applied on healthy donors. We found that the proportion of CD177+ neutrophils was significantly higher in GCF from patients with periodontitis, as compared to blood from the same individuals. Such accumulation of CD177+ neutrophils was not seen in the two models of aseptic inflammation. Moreover, the proportion of CD177+ neutrophils in circulation was significantly higher in the periodontitis patient group, as compared to healthy donors. Our data indicate that the CD177+ neutrophil subset is preferentially recruited to the gingival crevice of periodontitis patients, and may imply that this subtype is of particular importance for situations of microbe-driven inflammation.

Peptides Derived From Mismatched Paternal Human Leukocyte Antigen Predicted to Be Presented by HLA-DRB1, -DRB3/4/5, -DQ, and -DP Induce Child-Specific Antibodies in Pregnant Women.

Predicted Indirectly ReCognizable Human Leukocyte Antigen (HLA) Epitopes (PIRCHE) are known to be a significant risk factor for the development of donor HLA-specific antibodies after organ transplantation. Most previous studies on PIRCHE limited their analyses on the presentation of the HLA-DRB1 locus, although HLA-DRB3/4/5, -DQ, and -DP are also known for presenting allopeptides to CD4+ T cells. In this study, we analyzed the impact of predicted allopeptides presented by these additional loci on the incidence of HLA-specific antibodies after an immunization event. We considered pregnancy as a model system of an HLA immunization and observed child-specific HLA antibody (CSA) development of 231 mothers during pregnancy by samples being taken at delivery. Our data confirm that PIRCHE presented by HLA-DRB1 along with HLA-DRB3/4/5, -DQ, and -DP are significant predictors for the development of CSA. Although there was limited peptidome overlap observed within the mothers' presenting HLA proteins, combining multiple presenting loci in a single predictor improved the model only marginally. Prediction performance of PIRCHE further improved when normalizing scores by the respective presenters' binding promiscuity. Immunogenicity analysis of specific allopeptides could not identify significant drivers of an immune response in this small cohort, suggesting confirmatory studies.

Bioinformatic prediction of the structure and characteristics of human sperm acrosome membrane-associated protein 1 (hSAMP32) and evaluation of its antifertility function in vivo.

Human sperm acrosome membrane-associated protein 1 (hSAMP32) plays an important role in the acrosome reaction, sperm-egg primary binding, secondary binding and fusion processes. However, its spatial structural and invivo antifertility function remain unknown. In this study, we first analysed the physical and chemical characteristics and antigenic epitopes of immunised mice using bioinformatics. Then, we constructed the prokaryotic expression vector pcDNA3.1-hSAMP32 to immunise BALB/c mice invivo. IgG antibodies in the serum were detected, and the litter size of female mice and the number of the hamster eggs penetrated were counted. hSAMP32 was found to contain six hydrophilic regions and a signal peptide beginning at amino acid position 29. The transmembrane region of hSAMP32 was located within amino acids 217-239 with α-helices and random coil structures. We predicted five antigenic epitopes. The molecular weight of hSAMP32 was 59 kDa. Moreover, the results of invivo studies revealed that 56 days after the first immunisation, the litter size was significantly smaller for female pcDNA-3.1(+)-hSAMP32-immunised (mean±s.d. 4.33±1.21) than control mice (9.50±0.55), indicating that the immunocontraception vaccine had an antifertility effect. This experiment presents a theoretical and experimental basis for in-depth study of the hSAMP32 mechanism within the sperm-egg fusing process and for the screening of antigenic epitopes with immunocontraceptive properties.

Dynamics of Cardiac Neutrophil Diversity in Murine Myocardial Infarction.

RATIONALE: After myocardial infarction, neutrophils rapidly and massively infiltrate the heart, where they promote both tissue healing and damage. OBJECTIVE: To characterize the dynamics of circulating and cardiac neutrophil diversity after infarction. METHODS AND RESULTS: We employed single-cell transcriptomics combined with cell surface epitope detection by sequencing to investigate temporal neutrophil diversity in the blood and heart after murine myocardial infarction. At day 1, 3, and 5 after infarction, cardiac Ly6G+ (lymphocyte antigen 6G) neutrophils could be delineated into 6 distinct clusters with specific time-dependent patterning and proportions. At day 1, neutrophils were characterized by a gene expression profile proximal to bone marrow neutrophils (Cd177, Lcn2, Fpr1), and putative activity of transcriptional regulators involved in hypoxic response (Hif1a) and emergency granulopoiesis (Cebpb). At 3 and 5 days, 2 major subsets of Siglecfhi (enriched for eg, Icam1 and Tnf) and Siglecflow (Slpi, Ifitm1) neutrophils were found. Cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) analysis in blood and heart revealed that while circulating neutrophils undergo a process of aging characterized by loss of surface CD62L and upregulation of Cxcr4, heart infiltrating neutrophils acquired a unique SiglecFhi signature. SiglecFhi neutrophils were absent from the bone marrow and spleen, indicating local acquisition of the SiglecFhi signature. Reducing the influx of blood neutrophils by anti-Ly6G treatment increased proportions of cardiac SiglecFhi neutrophils, suggesting accumulation of locally aged neutrophils. Computational analysis of ligand/receptor interactions revealed putative pathways mediating neutrophil to macrophage communication in the myocardium. Finally, SiglecFhi neutrophils were also found in atherosclerotic vessels, revealing that they arise across distinct contexts of cardiovascular inflammation. CONCLUSIONS: Altogether, our data provide a time-resolved census of neutrophil diversity and gene expression dynamics in the mouse blood and ischemic heart at the single-cell level, and reveal a process of local tissue specification of neutrophils in the ischemic heart characterized by the acquisition of a SiglecFhi signature.

Diversity Outbred Mice Reveal the Quantitative Trait Locus and Regulatory Cells of HER2 Immunity.

The genetic basis and mechanisms of disparate antitumor immune response was investigated in Diversity Outbred (DO) F1 mice that express human HER2. DO mouse stock samples nearly the entire genetic repertoire of the species. We crossed DO mice with syngeneic HER2 transgenic mice to study the genetics of an anti-self HER2 response in a healthy outbred population. Anti-HER2 IgG was induced by Ad/E2TM or naked pE2TM, both encoding HER2 extracellular and transmembrane domains. The response of DO F1 HER2 transgenic mice was remarkably variable. Still, immune sera inhibited HER2+ SKBR3 cell survival in a dose-dependent fashion. Using DO quantitative trait locus (QTL) analysis, we mapped the QTL that influences both total IgG and IgG2(a/b/c) Ab response to either Ad/E2TM or pE2TM. QTL from these four datasets identified a region in chromosome 17 that was responsible for regulating the response. A/J and NOD segments of genes in this region drove elevated HER2 Ig levels. This region is rich in MHC-IB genes, several of which interact with inhibitory receptors of NK cells. (B6xA/J)F1 and (B6xNOD)F1 HER2 transgenic mice received Ad/E2TM after NK cell depletion, and they produced less HER2 IgG, demonstrating positive regulatory function of NK cells. Depletion of regulatory T cells enhanced response. Using DO QTL analysis, we show that MHC-IB reactive NK cells exert positive influence on the immunity, countering negative regulation by regulatory T cells. This new, to our knowledge, DO F1 platform is a powerful tool for revealing novel immune regulatory mechanisms and for testing new interventional strategies.

Characteristics of bull sperm acrosome associated 1 proteins.

An atypical distribution of sperm acrosomal tyrosine-phosphorylated proteins [which include sperm acrosome associated 1 (SPACA1) proteins] may be related to the relatively lesser pregnancy rates when semen of some bulls are used for artificial insemination (AI). There may also be these associations with bull SPACA1 proteins that are translocated from the equatorial segment to the anterior part in the acrosomes during sperm maturation in the normally functioning epididymis. The aim of the present study, therefore, was assessment of the characteristics of bull SPACA1 proteins. Results from immunocytochemical evaluations indicate there were large variations in sperm percentages with typically distributed SPACA1 proteins in acrosomes of cauda epididymal sperm samples (7%-95%). These values were positively correlated with percentages of epididymal spermatozoa with typically distributed acrosomal tyrosine-phosphorylated proteins (r=0.8564, P<0.001). Results indicate there are individual differences in translocation of SPACA1 proteins in the epididymis during sperm maturation, and that SPACA1 protein is one of the main determinants for the typical distribution of acrosomal tyrosine-phosphorylated proteins. In addition, conception rates as a result of AI using cryopreserved spermatozoa tended to be associated with percentages of epididymal spermatozoa with typically distributed SPACA1 proteins. Results from sucrose gradient centrifugation fractionation experiments indicate SPACA1 proteins are sperm membrane raft-associated proteins. Based on these results, it is hypothesized that there is an association between bull subfertility when semen is used for AI and epididymal dysfunctions in the arrangement of membrane lipid rafts during sperm maturation.

Uterine CD11c+ cells induce the development of paternal antigen-specific Tregs via seminal plasma priming.

Tolerogenic dendritic cells (tDCs) play a central role in the development of paternal antigen-specific regulatory T cells (Tregs) during pregnancy. We examined whether uterine CD11c+ antigen presenting cells (APC) induced paternal antigen-specific tolerance in allogeneic pregnant mice. Female BALB/c mice were mated with male DBA/2 mice, and their surface markers of APCs were studied using flow cytometry. After allogeneic mating, the uterine APCs exhibited significantly decreased expression of major histocompatibility complex (MHC) class II on day 3.5 post-coitus (pc) and day 5.5 pc. To analyze how seminal fluid affects surface markers of APCs, female BALB/c mice were mated with male mice that had undergone seminal vesicle excision (SVX). No reductions of MHC class II expression on APCs were seen in these mice. To analyze APC functions, a mixed lymphoid reaction (MLR) assay to paternal splenocytes was performed. Uterine APCs from allogeneic pregnant mice significantly suppressed the MLR reaction, but APCs from SVX mated mice did not suppress the MLR reaction Uterine APCs induced paternal antigen (Mls1a)-specific Treg development in vitro, but not in mice that mated with allogeneic SVX mice. These findings suggest that seminal fluid priming expands the paternal antigen-specific Treg population by inducing APCs development.

Fam20C regulates protein secretion by Cab45 phosphorylation.

The TGN is a key compartment for the sorting and secretion of newly synthesized proteins. At the TGN, soluble proteins are sorted based on the instructions carried in their oligosaccharide backbones or by a Ca2+-mediated process that involves the cargo-sorting protein Cab45. Here, we show that Cab45 is phosphorylated by the Golgi-specific protein kinase Fam20C. Mimicking of phosphorylation translocates Cab45 into TGN-derived vesicles, which goes along with an increased export of LyzC, a Cab45 client. Our findings demonstrate that Fam20C plays a key role in the export of Cab45 clients by fine-tuning Cab45 oligomerization and thus impacts Cab45 retention in the TGN.