In vitro vascular differentiation system efficiently produces natural killer cells for cancer immunotherapies.

Background: Immunotherapeutic innovation is crucial for limited operability tumors. CAR T-cell therapy displayed reduced efficiency against glioblastoma (GBM), likely due to mutations underlying disease progression. Natural Killer cells (NKs) detect cancer cells despite said mutations - demonstrating increased tumor elimination potential. We developed an NK differentiation system using human pluripotent stem cells (hPSCs). Via this system, genetic modifications targeting cancer treatment challenges can be introduced during pluripotency - enabling unlimited production of modified "off-the-shelf" hPSC-NKs. Methods: hPSCs were differentiated into hematopoietic progenitor cells (HPCs) and NKs using our novel organoid system. These cells were characterized using flow cytometric and bioinformatic analyses. HPC engraftment potential was assessed using NSG mice. NK cytotoxicity was validated using in vitro and in vitro K562 assays and further corroborated on lymphoma, diffuse intrinsic pontine glioma (DIPG), and GBM cell lines in vitro. Results: HPCs demonstrated engraftment in peripheral blood samples, and hPSC-NKs showcased morphology and functionality akin to same donor peripheral blood NKs (PB-NKs). The hPSC-NKs also displayed potential advantages regarding checkpoint inhibitor and metabolic gene expression, and demonstrated in vitro and in vivo cytotoxicity against various cancers. Conclusions: Our organoid system, designed to replicate in vivo cellular organization (including signaling gradients and shear stress conditions), offers a suitable environment for HPC and NK generation. The engraftable nature of HPCs and potent NK cytotoxicity against leukemia, lymphoma, DIPG, and GBM highlight the potential of this innovative system to serve as a valuable tool that will benefit cancer treatment and research - improving patient survival and quality of life.

Conventional natural killer cells control vascular remodeling in the uterus during pregnancy by acidifying the extracellular matrix with a2V.

Vascular remodeling within the uterus immediately before and during early pregnancy increases blood flow in the fetus and prevents the development of gestational hypertension. Tissue-resident natural killer (trNK) cells secrete pro-angiogenic growth factors but are insufficient for uterine artery (UtA) remodeling in the absence of conventional natural killer (cNK) cells. Matrix metalloproteinase-9 (MMP9) is activated in acidic environments to promote UtA remodeling. We have previously shown that ATPase a2V plays a role in regulating the function of cNK cells during pregnancy. We studied the effect of a2V deletion on uterine cNK cell populations and pregnancy outcomes in VavCrea2Vfl/fl mice, where a2V is conditionally deleted in hematopoietic stem cells. Conventional NKcells were reduced but trNK cells were retained in implantation sites at gestational day 9.5, and UtA remodeling was inhibited despite no differences in concentrations of pro-angiogenic growth factors. The ratio of pro-MMP9 to total was significantly elevated in VavCrea2Vfl/fl mice, and MMP9 activity was significantly reduced. The pH of implantation sites was significantly elevated in VavCrea2Vfl/fl mice. We concluded that the role of cNK cells in the uterus is to acidify the extracellular matrix (ECM) using a2V, which activates MMP9 to degrade the ECM, release bound pro-angiogenic growth factors, and contribute to UtA remodeling. Our results are significant for the understanding of the development of gestational hypertension.

Ephrin-B2-expressing natural killer cells induce angiogenesis.

Background: Therapeutic angiogenesis aims to induce new blood vessel growth in ischemic tissues; however, previous clinical trials have had limited success. Studies of uterine angiogenesis revealed a specialized subset of natural killer (NK) cells, called uterine NK (uNK) cells, which have unique proangiogenic abilities. Methods: We show that uNK cells in mice express ephrin-B2, a regulator of angiogenesis, to induce tubule formation in an ex vivo coculture tubule formation assay. We next induced the expression of ephrin-B2 by splenic NK (sNK) cells harvested from male mice. Results: We showed that induced NK (iNK) cells can also instruct endothelial cells to form tubules using ephrin-B2. Conclusions: We concluded that Ephrin-B2 is a marker of proangiogenic uNK cells and that a proangiogenic phenotype characterized by ephrin-B2 can be induced in sNK cells to induce therapeutic angiogenesis.

IL-22 regulates endometrial regeneration by enhancing tight junctions and orchestrating extracellular matrix.

The uterine endometrium uniquely regenerates after menses, postpartum, or after breaks in the uterine layer integrity throughout women's lives. Direct cell-cell contacts ensured by tight and adherens junctions play an important role in endometrial integrity. Any changes in these junctions can alter the endometrial permeability of the uterus and have an impact on the regeneration of uterine layers. Interleukin 22 (IL-22) is a cytokine that is recognized for its role in epithelial regeneration. Moreover, it is crucial in controlling the inflammatory response in mucosal tissues. Here, we studied the role of IL-22 in endometrial recovery after inflammation-triggered abortion. Fecundity of mice was studied in consecutive matings of the same animals after lipopolysaccharide (LPS) (10 µg per mouse)-triggered abortion. The fecundity rate after the second mating was substantially different between IL-22 knockout (IL-22-/-) (9.1%) and wild-type (WT) (71.4%) mice (p < 0.05), while there was no difference between the groups in the initial mating, suggesting that IL-22 deficiency might be associated with secondary infertility. A considerable difference was observed between IL-22-/- and WT mice in the uterine clearance following LPS-triggered abortion. Gross examination of the uteri of IL-22-/- mice revealed non-viable fetuses retained inside the horns (delayed clearance). In contrast, all WT mice had completed abortion with total clearance after LPS exposure. We also discovered that IL-22 deficiency is associated with a decreased expression of tight junctions (claudin-2 and claudin-10) and cell surface pathogen protectors (mucin-1). Moreover, IL-22 has a role in the remodeling of the uterine tissue in the inflammatory environment by regulating epithelial-mesenchymal transition markers called E- and N-cadherin. Therefore, IL-22 contributes to the proper regeneration of endometrial layers after inflammation-triggered abortion. Thus, it might have a practical significance to be utilized as a treatment option postpartum (enhanced regeneration function) and in secondary infertility caused by inflammation (enhanced barrier/protector function).

The role of immunologic tests for subfertility in the clinical environment.

Unexplained subfertility and implantation failures not only are emotionally and physically distressing but also become a significant obstacle to reproductive-age couples who wish to build their family. Often, the currently recommended evaluation for these couples is significantly limited, and many of causes remain unexplained. To obtain an accurate diagnosis and treatment, proper evidence-based laboratory evaluation should be performed. Immune tests for women with subfertility and implantation failures are essential to recognize the immune etiology and appropriate therapeutic strategies. This review focuses on currently used immune tests for subfertile women.

Post-hoc evaluation of peripheral blood natural killer cell cytotoxicity in predicting the risk of recurrent pregnancy losses and repeated implantation failures.

Peripheral blood NK cytotoxicity assay (NKC) is one of the commonly utilized diagnostic tools for recurrent pregnancy losses (RPL) and repeated implantation failures (RIF). In this retrospective cohort study, we aimed to assess the cutoff values of NKC for RPL and RIF. A total of 883 women were included in this study; 24 nonpregnant fertile women, 604 nonpregnant women with three or more RPL, 163 nonpregnant women with two or more of RIF, 48 normal pregnant women, and 44 pregnant women with a history of RPL. Peripheral blood NKC assay was performed by flow cytometry. The differences between groups were analyzed using Student's t-test, a logistic regression analysis, and the area under the receiver operating characteristic curve analysis. Both nonpregnant fertile and normal pregnant women had significantly lower NKC at an effector to target cell ratio (E:T) of 50:1 (13.5 ± 1.1% and 12.9 ± 1.0%, respectively) when compared to women with RPL and RIF, and pregnant women with a history of RPL (23.6 ± 0.3%, 23.9 ± 0.5%, and 23.7 ± 1.0%, P < 0.0001 respectively). In addition, the area under the receiver operating characteristics curve for RPL and RIF using pre-conception NKC was 0.863 (P < 0.0001) and 0.879 (P < 0.0001), respectively, and for RPL using post-conception NKC was 0.736 (P = 0.001). These findings suggest that NKC significantly distinguishes nonpregnant women with RPL and RIF from fertile controls and pregnant RPLwomen from normal pregnant controls.

Decidualization score identifies an endometrial dysregulation in samples from women with recurrent pregnancy losses and unexplained infertility.

OBJECTIVE: To study decidualization-associated endometrial factors. DESIGN: Retrospective cohort study to compare endometrial gene expression patterns in women experiencing reproductive failure including recurrent pregnancy loss or unexplained infertility versus fertile controls. SETTING: University Reproductive Medicine Center. PATIENTS: Women experiencing recurrent reproductive failure including recurrent pregnancy loss or unexplained infertility (n = 42) and fertile controls (n = 18). INTERVENTIONS: Endometrial biopsy samples were analyzed with targeted ribonucleic acid sequencing via next-generation sequencing. MAIN OUTCOME MEASURES: The primary end point measurements were the expression of genes important for endometrial transformation during decidualization measured singly and in a combined/cumulative score approach. The secondary end point measurements were receiver operating curve analysis and comparisons between the specific biomarkers. RESULTS: The comparison revealed differential expression of factors associated with decidualization, tissue homeostasis, and immune regulation: FOXO1, GZMB, IL15, SCNN1A, SGK1, and SLC2A1. A combined evaluation of these 6 signature factors was designated as a decidualization score in which the maximal score was "6" and the minimal was "0". Among controls, 89% of the samples had a score ≥5 and 11% had a score of "4". A total of 76% of samples in the patient group had scores ≤4 and 19% had the lowest score of "0". A decidualization score <4 provided evidence of abnormality in the decidualization process with a sensitivity of 76% (95% CI 61%-88%) and specificity of 89% (95% CI 65%-99%). CONCLUSIONS: Decidualization scoring can determine whether the endometrial molecular profile is implantation-friendly. Further validation of this testing approach is necessary to determine a particular patient population in whom it could be used for selecting patients that require therapeutic actions to improve endometrial conditions prior to the in vitro fertilization procedure.

Clinical molecular genetics evaluation in women with reproductive failures.

Molecular diagnostics is a rapidly growing branch of the clinical laboratory and has accelerated the advance of personalized medicine in the fields of pharmacogenomics, pharmacogenetics, and nutrigenomics. The versatility of molecular biology allows it to be effective in several medical fields that include reproduction, immunogenetics, and virology. Implementation of molecular and sequencing technology in reproductive medicine can add another layer of understanding to better define the causes behind infertility and recurrent reproductive loss. In the following, we examine current molecular methods for probing factors behind reproductive pregnancy loss including reverse transcription polymerase chain reaction and next generation sequencing (NGS). We review several current and potential genetic (DNA) and transcriptional (RNA)-based parameters in women with infertility that can be significant in diagnosis and treatment. These molecular factors can be inferred either from genomic DNA or RNA locally within the endometrium. Furthermore, we consider infection-based abnormalities such as human herpesvirus-6 and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Finally, we present future directions as well as data demonstrating the potential role of human endogenous retroviruses in pregnancy loss. We hope these discussions will assist the clinician in delineating some of the intricate molecular factors that can contribute to infertility and recurrent reproductive failures.

Decreased HLA-C1 alleles in couples of KIR2DL2 positive women with recurrent pregnancy loss.

Specific killer cell immunoglobulin-like receptor (KIR) in women with recurrent pregnancy loss (RPL) and HLA ligands in couples invoke a susceptibility to RPL. However, the relationship between KIR2DL2 and its cognate ligand HLA-C1 has not been explored. In this prospective cohort study, 160 Caucasian women with RPL and 99 partners were included. KIR/HLA-C typing, NK assay, Th1/Th2 intracellular cytokine ratios, 25-(OH)-vitamin D level, and the presence of autoantibodies were analyzed. KIR2DL2 positive women (P = 0.023) and their partners (P = 0.017) had lower allele frequencies of HLA-C1 than those of KIR2DL2 negative women. KIR2DL2 positive women had significantly lower genotype frequency of HLA-C1C1 as compared to the North American Caucasian population controls (P < 0.05). In the partners of KIR2DL2 positive women, there was a substantially higher frequency of HLA-C2C2 than controls (P = 0.016). Besides, KIR2DL2 negative women had a higher prevalence of anti-ssDNA antibody as compared with that of KIR2DL2 positive women (P = 0.043). There were no differences in the distribution of HLA-C genotypes based on KIR2DL2, regardless of pregnancy outcome in women with RPL and their partners while on immunomodulation treatment. In conclusion, decreased ligands for inhibitory KIRs (inhKIR) could lead to insufficient inhibition of maternal uterine NK cells toward the trophoblast, thereby contributing to the pathogenesis of RPL. Specific KIR and HLA-C genotyping may predict the reproductive outcome of women with RPL.

Iodide Transporters in the Endometrium: A Potential Diagnostic Marker for Women with Recurrent Pregnancy Failures.

OBJECTIVE: The element iodine is an essential nutrient utilized by the thyroid glands, and deficiency of this element has been linked to reproductive failures. Iodide transporters are also present in reproductive tissues and cells of embryonic origin such as the endometrium and trophoblasts, respectively. The aim of this study is to understand if levels of iodide transporters are linked to pregnancy outcomes. SUBJECTS AND METHODS: RNA derived from endometrial biopsies from controls or women with recurrent reproductive failures was analyzed utilizing RT-PCR and targeted RNASeq. RESULTS: When compared to controls, women with 2 or more reproductive failures had a significant increase (>5 fold) in mRNA levels of the iodine transporters NIS and PENDRIN, but not thyroglobulin when probed vis RT-PCR. Targeted RNASeq analysis confirmed these findings when another group of patients were analyzed. CONCLUSION: These findings suggest possible abnormal iodine metabolism and a deficiency of iodine in endometrial tissues from some of the women with reproductive failures. We hypothesize from these findings that inorganic iodide and/or iodine is required for optimal cellular function in reproductive tissues, and that iodide transporters may potentially be used as a marker for infertility or for probing potential localized iodine deficiency that may not present in a typical thyroid panel analysis.

PD-1 and PD-L1 expression on T-cell subsets in women with unexplained recurrent pregnancy losses.

PROBLEM: Does programmed death-1 (PD-1)/programmed death ligand-1 (PD-L1) expression on the T-cell subsets such as T helper (Th) 1, Th17, and Treg cells differentiate women with recurrent pregnancy losses (RPL) from normal fertile women? METHOD OF STUDY: The study was designed as a prospective cohort study. Forty-five women with two or more RPL of unknown etiology and twenty fertile women who had at least one or more live-born infants were enrolled prospectively from Jan 2017 to Jul 2019. PD-1 and PD-L1 expression on T-cell subsets were measured by flow cytometric analysis. RESULTS: The proportions of PD-1+ Th1 (CD4+ /IFN-γ+ /CD279+ and CD4+ /TNF-α+ /CD279+ ) and PD-1+ Th17 cells (CD4+ /IL17+ /CD279+ ) were significantly lower in RPL group than those of controls (P < .05, respectively). The proportion of PD-1+ Tregs (CD4+ /CD25+ /CD127dim/- /CD279+ ) in RPL group was not different from that of controls. The proportion of PD-L1+ Th17 cells (CD4+ IL17+ CD274+ ) was significantly lower as compared with that of /controls (P < .05). However, the proportions of PD-L1+ Th1 (CD4+ /IFN-γ+ /CD274+ and CD4+ /TNF-α+ /CD274+ ) and PD-L1+ Treg (CD4+ /CD25+ /CD127dim/- /CD274+ ) cells were not different between the RPL group and controls (P > .05, respectively). In Th1, Th17 and Treg cells, the proportions of PD-L1+ (CD274+ ) cells were significantly higher than those of PD-1+ (CD279+ ) cells in both RPL group and controls (P < .05, respectively). CONCLUSION: PD-1 and PD-L1 expressions on Th17 cells as well as PD-1 expression on Th1 cells were significantly downregulated in women with RPL, which may lead to increased Th1 and Th17 immunity, and imbalance between Th17, Th1, and Treg cells in women with RPL.

Poor ovarian response is associated with serum vitamin D levels and pro-inflammatory immune responses in women undergoing in-vitro fertilization.

Poor ovarian response (POR1) limits the success of infertility treatment modality. In this study, we aim to investigate if POR is associated with serum 25(OH) vitamin D (VD2) levels and pro-inflammatory immune responses in infertile women with a history of in-vitro fertilization and embryo transfer failures. A retrospective cross-sectional study included 157 women with IVF failures. Study patients were divided into four groups based on serum 25(OH)VD level and ovarian responses during the most recent IVF cycle; low VD (LVD3) with POR, LVD with normal ovarian response (NOR4), normal VD (NVD5) with POR, and NVD with NOR. Serum 25(OH)VD level, cellular- and auto-immunity, and metabolic parameters, including homocysteine and plasminogen activator inhibitor-1 were investigated. Peripheral blood CD56+ NK cell levels (%) and NK cytotoxicity were significantly higher in POR-LVD when compared to the other groups (P < 0.05, respectively). CD19 + B and CD19+/5+ B-1 cell levels were significantly higher in women with POR-LVD as compared with those of NOR-LVD and POR-NVD (P < 0.05, respectively). TNF-α/IL-10 producing Th1/Th2 cell ratio of POR-LVD was significantly higher than those of POR-NVD and NOR-NVD (P < 0.05 respectively). Peripheral blood homocysteine level of POR-LVD was significantly higher than those of NOR-LVD and POR-NVD (P < 0.05 respectively). We conclude that assessment of cellular and autoimmune abnormalities and metabolic factors, such as homocysteine should be considered in women with POR and LVD. VD and folic acid supplementation may be explored further as a possible therapeutic option for POR with immune and metabolic etiologies.

Mast cell-induced immunopathology in recurrent pregnancy losses.

PROBLEM: Mast cells (MC) have been known to play an important role in inflammation and angiogenesis by secreting numerous mediators, such as proteases, gelatinases, and proteoglycans. Three different MC subtypes were found in the endometrial layers of the uterus. In this study, we aim to investigate the role of endometrial MCs in recurrent pregnancy losses (RPL). METHOD OF STUDY: Endometrial biopsy was performed 5-7 days post-ovulation (implantation window) in women with a history of two or more RPL (n = 46) and normal fertile women (n = 10). Quantitative RT-PCR was performed to detect the expression of various mast cell mediators. Endometrial samples were evaluated using immunohistochemistry for c-kit receptor (CD117) and tryptase (MC activation marker). RESULTS: Mast cells were present throughout the entire layers of the endometrium; their count was elevated in RPL patients as compared to controls. The gene expression of c-Kit receptor was not different between the study groups. There are significant increases in the mRNA expression of various mediators, that is, stem cell factor (P = 0.029), tryptase (P = 0.024), heparan sulfate (P = 0.0005), and MMP-2 (P < 0.0001) in women with RPL as compared to normal controls. Chymase gene expression was not detected in most of the endometrial samples. CONCLUSION: This study has shown that MCs are overactive in RPL patients by creating a pro-inflammatory milieu, suggesting a novel role in the immunopathology of RPL. Future studies are needed to better understand the role of MC in implantation and placental angiogenesis.

Dysregulated uterine natural killer cells and vascular remodeling in women with recurrent pregnancy losses.

PROBLEM: Angiogenesis and vascular remodeling in secretory endometrium represent one of the crucial steps in pregnancy establishment, for which uterine NK (uNK) cells have an important role. Impairment of these steps may proceed to implantation and instigate initial pathology of recurrent pregnancy losses (RPL). In this study, we aim to investigate vascular development and density of uNK cells in secretory endometrium of women with RPL. METHODS OF STUDY: Mid-secretory phase endometrial tissues from women with RPL (n = 15) and fertile controls (n = 7) were investigated. CD56+ and CD16+ uNK cells, CD31+ vascular endothelial cells and smooth muscle myosin (SMM)+ . Vascular smooth muscle cells (VSMC) expressing SMM were investigated using immunohistochemistry and western blot. High-throughput quantitative real-time polymerase chain reaction (qRT-PCR) was used as well. RESULTS: CD56+ uNK number was significantly higher in women with RPL compared to controls (P < 0.0001). uNK cell density by immunohistochemistry was positively correlated with CD56 mRNA expression by qRT-PCR (r2  = 0.43, P = 0.0137). The number of blood vessels represented by the expression of either CD31 or SMM was higher in women with RPL as compared to controls (P < 0.05 and P < 0.0001, respectively), and correlated with the number of uNK cell (r2  = 0.18, P < 0.04, and r2  = 0.65, P < 0.0001, respectively). The wall thickness of spiral arteries was significantly higher in women with RPL as compared with that of controls (P = 0.0027). CONCLUSION: Increased uNK cells in mid-secretory endometrium are associated with increased vascularization and defective vascular transformation of spiral arteries in women with RPL.

Inflammation induced preterm labor and birth.

Preterm birth which occurs before 37 weeks gestation is one of the most common obstetrical complication in humans. After many studies, it appears that "not one answer fits all" regarding the risk factors, causes and the treatments for this syndrome. However, it is becoming more evident that one of the major risk factors is inflammation and/or infection in the fetoplacental unit. In animal models (usually consisting of mice injected with lipopolysaccharide at 14 days of gestation), IL-22 and IL-6 have been identified as factors related to preterm birth. There are some clinical tests available to determine the risk for preterm labor and delivery, which can be identified before, during early, or at mid-gestation. However, treatment of preterm birth with antibiotics so far has not been "curable" and studies using anti-inflammatory treatments are not readily available. More studies regarding causes and treatments for preterm labor and delivery in humans are necessary to prevent neonatal deaths and/or developmental abnormalities associated with this common syndrome.

Application of small molecule CHIR99021 leads to the loss of hemangioblast progenitor and increased hematopoiesis of human pluripotent stem cells.

Improving our understanding of the intricacies of hematopoietic specification of induced or embryonic human pluripotent stem cells is beneficial for many areas of research and translational medicine. Currently, it is not clear whether, during human pluripotent stem cells hematopoietic differentiation in vitro, the maturation of definitive progenitors proceeds through a primitive progenitor (hemangioblast) intermediate or if it develops independently. The objective of this study was to investigate the early stages of hematopoietic specification of pluripotent stem cells in vitro. By implementing an adherent culture, serum-free differentiation system that utilizes a small molecule, CHIR99021, to induce human pluripotent stem cells toward various hematopoietic lineages, we established that, compared with the OP9 coculture hematopoietic induction system, the application of CHIR99021 alters the early steps of hematopoiesis such as hemangioblasts, angiogenic hematopoietic progenitors, and hemogenic endothelium. Importantly, it is associated with the loss of hemangioblast progenitors, loss of CD43+ (primitive hematopoietic marker) expression, and predominant development of blast-forming unit erythroid colonies in semisolid medium. These data support the hypothesis that the divergence of primitive and definitive programs during human pluripotent stem cells differentiation precedes the hemangioblast stage. Furthermore, we have shown that the inhibition of primitive hematopoiesis is associated with an increase in hematopoietic potential, which is a fruitful finding due to the growing need for lymphoid and myeloid cells in translational applications.

Prevalence of HHV-6 in endometrium from women with recurrent implantation failure.

PROBLEM: To study the prevalence of HHV-6 in endometrial biopsies among women experiencing recurrent implantation failure (RIF) after IVF/ET compared with controls. METHOD OF STUDY: Thirty women experiencing RIF after IVF/ET and 10 fertile women participated in the study. All women had endometrial biopsies taken in the luteal phase of their menstrual cycle for an endometrial immune profile (EIP) and HHV-6 mRNA as well as lymphocyte and granulocyte populations. The prevalence of HHV-6 in endometrial biopsies was determined, and biopsies for positive and negative expression of HHV-6 were compared with the results of their EIP and lymphocyte and granulocyte populations. RESULTS: Thirty-seven percentage of women with a history of RIF and 0% of controls demonstrated the presence of HHV-6 in their endometrial biopsies. No associations were found when the results of the endometrial immune profile were compared with the presence or absence of HHV-6. Significant increase in neutrophil-specific CD16b mRNA was found in HHV-6-positive samples, and the levels of B cells-related CD19 mRNA were lower in biopsies from women with RIF in comparison with normal controls. CONCLUSION: HHV-6 infection is an important factor in RIF.

Interleukin 22 prevents lipopolysaccharide- induced preterm labor in mice.

Preterm birth is widespread and causes 35% of all neonatal deaths. Infants who survive face potential long-term complications. A major contributing factor of preterm birth is infection. We investigated the role of interleukin 22 (IL22) as a potential clinically relevant cytokine during gestational infection. IL22 is an effector molecule secreted by immune cells. While the expression of IL22 was reported in normal nonpregnant endometrium and early pregnancy decidua, little is known about uterine IL22 expression during mid or late gestational stages of pregnancy. Since IL22 has been shown to be an essential mediator in epithelial regeneration and wound repair, we investigated the potential role of IL22 during defense against an inflammatory response at the maternal-fetal interface. We used a well-established model to study infection and infection-associated inflammation during preterm birth in the mouse. We have shown that IL22 is upregulated to respond to an intrauterine lipopolysaccharide administration and plays an important role in controlling the risk of inflammation-induced preterm birth. This paper proposes IL22 as a treatment method to combat infection and prevent preterm birth in susceptible patients.

A Role for Iodide and Thyroglobulin in Modulating the Function of Human Immune Cells.

Iodine is an essential element required for the function of all organ systems. Although the importance of iodine in thyroid hormone synthesis and reproduction is well known, its direct effects on the immune system are elusive. Human leukocytes expressed mRNA of iodide transporters (NIS and PENDRIN) and thyroid-related proteins [thyroglobulin (TG) and thyroid peroxidase (TPO)]. The mRNA levels of PENDRIN and TPO were increased whereas TG transcripts were decreased post leukocyte activation. Flow cytometric analysis revealed that both PENDRIN and NIS were expressed on the surface of leukocyte subsets with the highest expression occurring on monocytes and granulocytes. Treatment of leukocytes with sodium iodide (NaI) resulted in significant changes in immunity-related transcriptome with an emphasis on increased chemokine expression as probed with targeted RNASeq. Similarly, treatment of leukocytes with NaI or Lugol's iodine induced increased protein production of both pro- and anti-inflammatory cytokines. These alterations were not attributed to iodide-induced de novo thyroid hormone synthesis. However, upon incubation with thyroid-derived TG, primary human leukocytes but not Jurkat T cells released thyroxine and triiodothyronine indicating that immune cells could potentially influence thyroid hormone balance. Overall, our studies reveal the novel network between human immune cells and thyroid-related molecules and highlight the importance of iodine in regulating the function of human immune cells.

Predicting NK cell subsets using gene expression levels in peripheral blood and endometrial biopsy specimens.

PROBLEM: In molecular analysis of tissue biopsy specimens, one crucial aspect is characterization of immune cell populations. This is especially important for evaluation of uterine receptivity by assessing levels of lymphocyte populations including CD56bright CD16- uterine NK cells and CD56dim CD16+ conventional NK cells. Our objective was to investigate whether measuring total RNA transcripts from a tissue specimen would accurately reflect immune cell levels and be a new technique to assess immune cell subsets. METHOD OF STUDY: Peripheral blood mononuclear cells (PBMCs) and endometrial tissues were used. Flow cytometry was utilized for the analysis of lymphocyte subsets in PBMCs, and RT-qPCR was applied to quantify RNA transcripts indicative of lymphocyte and granulocyte populations. RESULTS: In PBMC specimens, there were significant correlations between gene expression levels and cell subsets. NK cells correlated with CD16A, NKp46, and CD56 transcripts, B cells correlated with EBF1, and CD8+ T cells correlated with CD8ß. Finally, endometrial tissues displayed high CD56 expression and very low CD3ε, CD16A, and NKp30, reflecting the characteristic endometrial NK cell subsets. CONCLUSION: Strong correlations between RT-qPCR data and levels of lymphocyte subsets indicate that gene expression analysis will be a useful technique for characterizing levels of CD56+ cells in endometrial tissues.