ThPOK is a critical multifaceted regulator of myeloid lineage development.

The transcription factor ThPOK (encoded by Zbtb7b) is well known for its role as a master regulator of CD4 lineage commitment in the thymus. Here, we report an unexpected and critical role of ThPOK as a multifaceted regulator of myeloid lineage commitment, differentiation and maturation. Using reporter and knockout mouse models combined with single-cell RNA-sequencing, progenitor transfer and colony assays, we show that ThPOK controls monocyte-dendritic cell versus granulocyte lineage production during homeostatic differentiation, and serves as a brake for neutrophil maturation in granulocyte lineage-specified cells through transcriptional regulation of lineage-specific transcription factors and RNA via altered messenger RNA splicing to reprogram intron retention.

Essential role of a ThPOK autoregulatory loop in the maintenance of mature CD4+ T cell identity and function.

The transcription factor ThPOK (encoded by the Zbtb7b gene) controls homeostasis and differentiation of mature helper T cells, while opposing their differentiation to CD4+ intraepithelial lymphocytes (IELs) in the intestinal mucosa. Thus CD4 IEL differentiation requires ThPOK transcriptional repression via reactivation of the ThPOK transcriptional silencer element (SilThPOK). In the present study, we describe a new autoregulatory loop whereby ThPOK binds to the SilThPOK to maintain its own long-term expression in CD4 T cells. Disruption of this loop in vivo prevents persistent ThPOK expression, leads to genome-wide changes in chromatin accessibility and derepresses the colonic regulatory T (Treg) cell gene expression signature. This promotes selective differentiation of naive CD4 T cells into GITRloPD-1loCD25lo (Triplelo) Treg cells and conversion to CD4+ IELs in the gut, thereby providing dominant protection from colitis. Hence, the ThPOK autoregulatory loop represents a key mechanism to physiologically control ThPOK expression and T cell differentiation in the gut, with potential therapeutic relevance.

Mouse models of neutropenia reveal progenitor-stage-specific defects.

Advances in genetics and sequencing have identified a plethora of disease-associated and disease-causing genetic alterations. To determine causality between genetics and disease, accurate models for molecular dissection are required; however, the rapid expansion of transcriptional populations identified through single-cell analyses presents a major challenge for accurate comparisons between mutant and wild-type cells. Here we generate mouse models of human severe congenital neutropenia (SCN) using patient-derived mutations in the GFI1 transcription factor. To determine the effects of SCN mutations, we generated single-cell references for granulopoietic genomic states with linked epitopes1, aligned mutant cells to their wild-type equivalents and identified differentially expressed genes and epigenetic loci. We find that GFI1-target genes are altered sequentially, as cells go through successive states of differentiation. These insights facilitated the genetic rescue of granulocytic specification but not post-commitment defects in innate immune effector function, and underscore the importance of evaluating the effects of mutations and therapy within each relevant cell state.

Human CD4+ iNKT cell adoptive immunotherapy induces anti-tumour responses against CD1d-negative EBV-driven B lymphoma.

Invariant natural killer T (iNKT) cells are a conserved population of innate T lymphocytes that are uniquely suitable as off-the-shelf cellular immunotherapies due to their lack of alloreactivity. Two major subpopulations of human iNKT cells have been delineated, a CD4- subset that has a TH1/cytolytic profile, and a CD4+ subset that appears polyfunctional and can produce both regulatory and immunostimulatory cytokines. Whether these two subsets differ in anti-tumour effects is not known. Using live cell imaging, we found that CD4- iNKT cells limited growth of CD1d+ Epstein-Barr virus (EBV)-infected B-lymphoblastoid spheroids in vitro, whereas CD4+ iNKT cells showed little or no direct anti-tumour activity. However, the effects of the two subsets were reversed when we tested them as adoptive immunotherapies in vivo using a xenograft model of EBV-driven human B cell lymphoma. We found that EBV-infected B cells down-regulated CD1d in vivo, and administering CD4- iNKT cells had no discernable impact on tumour mass. In contrast, xenotransplanted mice bearing lymphomas showed rapid reduction in tumour mass after administering CD4+ iNKT cells. Immunotherapeutic CD4+ iNKT cells trafficked to both spleen and tumour and were associated with subsequently enhanced responses of xenotransplanted human T cells against EBV. CD4+ iNKT cells also had adjuvant-like effects on monocyte-derived DCs and promoted antigen-dependent responses of human T cells in vitro. These results show that allogeneic CD4+ iNKT cellular immunotherapy leads to marked anti-tumour activity through indirect pathways that do not require tumour cell CD1d expression and that are associated with enhanced activity of antigen-specific T cells.

Suppression of Ca2+ signals by EGR4 controls Th1 differentiation and anti-cancer immunity in vivo.

While the zinc finger transcription factors EGR1, EGR2, and EGR3 are recognized as critical for T-cell function, the role of EGR4 remains unstudied. Here, we show that EGR4 is rapidly upregulated upon TCR engagement, serving as a critical "brake" on T-cell activation. Hence, TCR engagement of EGR4-/- T cells leads to enhanced Ca2+ responses, driving sustained NFAT activation and hyperproliferation. This causes profound increases in IFNγ production under resting and diverse polarizing conditions that could be reversed by pharmacological attenuation of Ca2+ entry. Finally, an in vivo melanoma lung colonization assay reveals enhanced anti-tumor immunity in EGR4-/- mice, attributable to Th1 bias, Treg loss, and increased CTL generation in the tumor microenvironment. Overall, these observations reveal for the first time that EGR4 is a key regulator of T-cell differentiation and function.

Functional Conservation of a Developmental Switch in Mammals since the Jurassic Age.

ThPOK is a "master regulator" of T lymphocyte lineage choice, whose presence or absence is sufficient to dictate development to the CD4 or CD8 lineages, respectively. Induction of ThPOK is transcriptionally regulated, via a lineage-specific silencer element, SilThPOK. Here, we take advantage of the available genome sequence data as well as site-specific gene targeting technology, to evaluate the functional conservation of ThPOK regulation across mammalian evolution, and assess the importance of motif grammar (order and orientation of TF binding sites) on SilThPOK function in vivo. We make three important points: First, the SilThPOK is present in marsupial and placental mammals, but is not found in available genome assemblies of nonmammalian vertebrates, indicating that it arose after divergence of mammals from other vertebrates. Secondly, by replacing the murine SilThPOK in situ with its marsupial equivalent using a knockin approach, we demonstrate that the marsupial SilThPOK supports correct CD4 T lymphocyte lineage-specification in mice. To our knowledge, this is the first in vivo demonstration of functional equivalency for a silencer element between marsupial and placental mammals using a definitive knockin approach. Finally, we show that alteration of the position/orientation of a highly conserved region within the murine SilThPOK is sufficient to destroy silencer activity in vivo, demonstrating that motif grammar of this "solid" synteny block is critical for silencer function. Dependence of SilThPOK function on motif grammar conserved since the mid-Jurassic age, 165 Ma, suggests that the SilThPOK operates as a silenceosome, by analogy with the previously proposed enhanceosome model.

Disregulated expression of the transcription factor ThPOK during T-cell development leads to high incidence of T-cell lymphomas.

The transcription factor T-helper-inducing POZ/Krueppel-like factor (ThPOK, encoded by the Zbtb7b gene) plays widespread and critical roles in T-cell development, particularly as the master regulator of CD4 commitment. Here we show that mice expressing a constitutive T-cell-specific ThPOK transgene (ThPOK(const) mice) develop thymic lymphomas. These tumors resemble human T-cell acute lymphoblastic leukemia (T-ALL), in that they predominantly exhibit activating Notch1 mutations. Lymphomagenesis is prevented if thymocyte development is arrested at the DN3 stage by recombination-activating gene (RAG) deficiency, but restored by introduction of a T-cell receptor (TCR) transgene or by a single injection of anti-αßTCR antibody into ThPOK(const) RAG-deficient mice, which promotes development to the CD4(+)8(+) (DP) stage. Hence, TCR signals and/or traversal of the DN (double negative) > DP (double positive) checkpoint are required for ThPOK-mediated lymphomagenesis. These results demonstrate a novel link between ThPOK, TCR signaling, and lymphomagenesis. Finally, we present evidence that ectopic ThPOK expression gives rise to a preleukemic and self-perpetuating DN4 lymphoma precursor population. Our results collectively define a novel role for ThPOK as an oncogene and precisely map the stage in thymopoiesis susceptible to ThPOK-dependent tumor initiation.

Adolescent maternal mortality at a district health services over a five year period in South Africa: A retrospective study.

OBJECTIVE: To determine the trend in adolescent maternal deaths and deliveries over a period of 5 years and 9 months (July 2014-March 2020) at the Ekurhuleni Health District in South Africa. METHODS: The present study was a retrospective review and secondary data analysis using data from the District Health Information System and clinical oversight data from the District Clinical Specialist Team. The study population was adolescent pregnant women aged 10-19 years who died at health facilities. Descriptive and inferential statistics were used for analysis. RESULTS: There was a total of 12 559 adolescent deliveries. Adolescent birth rate was lower than that of sub-Saharan Africa. Adolescent deaths (n = 37) contributed to around 8% of the total maternal deaths. Deliveries (97%) and deaths (98%) were most common among women aged 15-19 years. Six (16%) women had a repeat pregnancy. A total of 21 (57%) had booked for antenatal care. There were few antenatal visits (mean 4 ± SD 2.1). The main three causes of death were hypertension (35%) followed by hemorrhage (24%) and suicide (14%). Postpartum deaths (62%) were significantly (chi-square test, P = 0.02) higher than antepartum deaths (38%). The majority (73%) of newborns were born alive which was significantly (chi-square test, P = 0.002) higher than those which were stillborn (27%). CONCLUSION: The main challenges were the high number of adolescent deliveries, repeat pregnancies, and preventable causes of death. Multidisciplinary collaboration involving obstetricians, midwives, pediatricians, school health services, social workers and psychologists is indispensable for comprehensive management, prioritizing pregnancy prevention among this vulnerable group.

Interferon-gamma signaling promotes melanoma progression and metastasis.

Interferon-gamma (IFNG) has long been regarded as the flag-bearer for the anti-cancer immunosurveillance mechanisms. However, relatively recent studies have suggested a dual role of IFNG, albeit there is no direct experimental evidence for its potential pro-tumor functions. Here we provide in vivo evidence that treatment of mouse melanoma cell lines with Ifng enhances their tumorigenicity and metastasis in lung colonization allograft assays performed in immunocompetent syngeneic host mice, but not in immunocompromised host mice. We also show that this enhancement is dependent on downstream signaling via Stat1 but not Stat3, suggesting an oncogenic function of Stat1 in melanoma. The experimental results suggest that melanoma cell-specific Ifng signaling modulates the tumor microenvironment and its pro-tumorigenic effects are partially dependent on the γδ T cells, as Ifng-enhanced tumorigenesis was inhibited in the TCR-δ knockout mice. Overall, these results show that Ifng signaling may have tumor-promoting effects in melanoma by modulating the immune cell composition of the tumor microenvironment.

F1-adenosine triphosphatase displays properties characteristic of an antigen presentation molecule for Vgamma9Vdelta2 T cells.

Human Vgamma9Vdelta2 T lymphocytes are activated by phosphoantigens provided exogenously or produced by tumors and infected cells. Activation requires a contact between Vgamma9Vdelta2 cells and neighboring cells. We previously reported a role for cell surface F1-adenosine triphosphatase (ATPase) in T cell activation by tumors and specific interactions between Vgamma9Vdelta2 TCRs and purified F1-ATPase. 721.221 cells do not express surface F1-ATPase and do not support phosphoantigen responses unless they are rendered apoptotic by high doses of zoledronate, a treatment that promotes F1-expression as well as endogenous phosphoantigen production. By monitoring calcium flux in single cells, we show in this study that contact of T cells with F1-ATPase on polystyrene beads can partially replace the cell-cell contact stimulus during phosphoantigen responses. Triphosphoric acid 1-adenosin-5'-yl ester 3-(3-methylbut-3-enyl) ester, an adenylated derivative of isopentenyl pyrophosphate, can stably bind to F1-ATPase-coated beads and promotes TCR aggregation, lymphokine secretion, and activation of the cytolytic process provided that nucleotide pyrophosphatase activity is present. It also acts as an allosteric activator of F1-ATPase. In the absence of Vgamma9Vdelta2 cells, triphosphoric acid 1-adenosin-5'-yl ester 3-(3-methylbut-3-enyl) ester immobilized on F1-ATPase is protected from nucleotide pyrophosphatase activity, as is the antigenic activity of stimulatory target cells. Our experiments support the notion that Vgamma9Vdelta2 T cells are dedicated to the recognition of phosphoantigens on cell membranes in the form of nucleotide derivatives that can bind to F1-ATPase acting as a presentation molecule.

Maternal mortality from COVID 19 among South African pregnant women.

OBJECTIVE: To determine the COVID 19 related maternal deaths among South African pregnant women at Ekurhuleni health district in South Africa and to compare with similar studies. STUDY DESIGN: Retrospective review of all maternal deaths from all health care facilities from April to September 2020 were conducted using COVID registers and maternity case records. Human Research Ethics Committee of the University of Witwatersrand approved the study. Data included total live births, maternal mortality ratio (MMR), age, ethnicity, place of admission, parity, pregnancy status, antenatal complications, gestational age (GA) at delivery, GA at COVID 19 diagnosis, GA at death, symptoms, comorbidity, investigations (HIV, platelets, lymphocytes and LDH), and fetal outcome. Descriptive statistics (mean ± standard deviation, number and percentages) were calculated. RESULTS: Six women died from COVID. All were African. Mean age was 33.5 (SD ± 4.3) years majority (83%) were multiparous. The mean GA at the time of diagnosis was 35 (± 5.8) weeks. All had dyspnea at presentation. All had hypertension. HIV rate (50%) was higher than the national rate. High lactic dehydrogenase was the commonest laboratory abnormality. Rate of macerated stillborn (66%) was very high. CONCLUSION: To date, there are no African studies reporting on maternal mortality from COVID 19. This study provided valuable insight into maternal deaths due to COVID among South African women. COVID 19 is a novel cause of maternal death that has increased the death rate among South African pregnant women. Hypertensive women are at increased risk of death. They should be routinely tested for COVID. Women are at risk of death during the third trimester of pregnancy. High rate of stillborn is a concern. Decision to deliver earlier should be an option. High HIV rate and LDH count should alert health care workers to perform these tests among all COVID positive mothers.

An autonomous TCR signal-sensing switch influences CD4/CD8 lineage choice in mice.

How multipotential cells initiate distinct gene expression programs in response to external cues to instruct cell fate choice remains a fundamental question in biology. Establishment of CD4 and CD8 T cell fates during thymocyte development is critically regulated by T cell receptor (TCR) signals, which in turn control expression of the CD4-determining transcription factor ThPOK. However, the mechanism whereby differential TCR signals are molecularly interpreted to promote or antagonize ThPOK expression, and thereby CD4 versus CD8 lineage fates remains unknown. Here we show, using reverse genetic and molecular approaches that an autonomous, position-independent TCR-sensing switch is embedded within the ThPOK locus. Further, using an in vivo mutagenesis approach, we demonstrate that differential TCR signals are interpreted during lineage commitment by relative binding of EGR, NFAT and Ebox factors to this bistable switch. Collectively our study reveals the central molecular mechanism whereby TCR signaling influences differential lineage choice. Ultimately, these findings may provide an important new tool for skewing T cell fate to treat cancer and autoimmune diseases.

Specific requirements for Vgamma9Vdelta2 T cell stimulation by a natural adenylated phosphoantigen.

Human Vgamma9Vdelta2 T lymphocytes recognize phosphorylated alkyl Ags. Isopentenyl pyrophosphate (IPP) was previously proposed as the main Ag responsible for Vgamma9Vdelta2 T cell activation by cancer cells. However, triphosphoric acid 1-adenosin-5'-yl ester 3-(3-methylbut-3-enyl) ester (ApppI), a metabolite in which the isopentenyl moiety is linked to ATP, was reported in cells activated with aminobisphosphonates. The contribution of this compound to tumor-stimulatory activity was thus examined. ApppI induces selective expansion of Vgamma9Vdelta2 T cells from PBMCs. In the absence of APCs, however, ApppI has little stimulatory activity on Vgamma9Vdelta2 T cells, and optimal activation with ApppI requires addition of a nucleotide pyrophosphatase releasing IPP plus AMP. Thus, ApppI has no intrinsic stimulatory activity. Nevertheless, stimulation by ApppI is strengthened by the presence of APCs. Moreover, in contrast to IPP, ApppI can be efficiently pulsed on dendritic cells as well as on nonprofessional APCs. Pulsed APCs display stable and phosphatase-resistant stimulatory activity, indicative of Ag modification. HPLC analysis of tumor cell extracts indicates that latent phosphoantigenic activity is stored intracellularly in the Vgamma9Vdelta2 cell-sensitive tumor Daudi and can be activated by a nucleotide pyrophosphatase activity. The presence of ApppI in Daudi cell extracts was demonstrated by mass spectrometry. Nucleotidic Ags such as ApppI are thus a storage form of phosphoantigen which may represent a major source of phosphoantigenic activity in tumor cells. The unique properties of ApppI may be important for the design of Ags used in anticancer immunotherapeutic protocols using Vgamma9Vdelta2 cells.

Clinical Features and Outcomes of COVID-19 Infection among Pregnant Women in South Africa.

BACKGROUND AND OBJECTIVE: Studies among COVID positive pregnant women are limited. Previous studies reported high rate of symptoms, comorbidities and poor fetal/neonatal outcome among women of different ethnicity. To date, no study was reported among pregnant African women. This study was conducted to determine the clinical features and outcomes of COVID-19 positive pregnant women in Ekurhuleni District in South Africa. METHODS: Retrospective record review of 103 COVID-19 infected pregnant women during the period of April to September 2020 was done. Clinical features, symptoms, comorbidities, laboratory results, maternal and neonatal outcomes were analyzed. Descriptive statistics (mean + standard deviation, number and percentages) and inferential statistics (Chi-square test) were calculated. Ethical clearance was obtained from the Human Research Ethics Committee of the University of Witwatersrand, South Africa. RESULTS: Majority (90%) were of African ethnicity and symptomatic (53%). The commonest symptom, comorbidity and laboratory abnormality was cough (62%), hypertension (23%) and high LDH (20%), respectively. Thrombocytopenia and lymphocytopenia occurred among 9% and 15% of the women, respectively. Although association was not significant, symptoms were more common among comorbid, HIV positive and women with abnormal results. Commonest complications were preterm labor (8%) and macerated stillbirths (6%). Six (6%) mothers died. CONCLUSION AND GLOBAL HEALTH IMPLICATIONS: COVID positive pregnant South African women were commonly symptomatic but incidence of adverse fetal outcomes was low. High rate of preterm labor, macerated stillbirths and maternal deaths was a concern. This study has several strengths. It included all COVID positive women over a 6 months' period at all level of healthcare facilities such as clinics, community health centers/midwifery units, and district, regional and tertiary hospitals, conducted in an African country. Previous studies included mainly hospitals raising a high probability of including mainly severely ill women. The study might benefit the health care workers in other African countries.

RNF168-mediated localization of BARD1 recruits the BRCA1-PALB2 complex to DNA damage.

DNA damage prompts a diverse range of alterations to the chromatin landscape. The RNF168 E3 ubiquitin ligase catalyzes the mono-ubiquitination of histone H2A at lysine (K)13/15 (mUb-H2A), forming a binding module for DNA repair proteins. BRCA1 promotes homologous recombination (HR), in part, through its interaction with PALB2, and the formation of a larger BRCA1-PALB2-BRCA2-RAD51 (BRCA1-P) complex. The mechanism by which BRCA1-P is recruited to chromatin surrounding DNA breaks is unclear. In this study, we reveal that an RNF168-governed signaling pathway is responsible for localizing the BRCA1-P complex to DNA damage. Using mice harboring a Brca1CC (coiled coil) mutation that blocks the Brca1-Palb2 interaction, we uncovered an epistatic relationship between Rnf168- and Brca1CC alleles, which disrupted development, and reduced the efficiency of Palb2-Rad51 localization. Mechanistically, we show that RNF168-generated mUb-H2A recruits BARD1 through a BRCT domain ubiquitin-dependent recruitment motif (BUDR). Subsequently, BARD1-BRCA1 accumulate PALB2-RAD51 at DNA breaks via the CC domain-mediated BRCA1-PALB2 interaction. Together, these findings establish a series of molecular interactions that connect the DNA damage signaling and HR repair machinery.

Role of a second stage partogram in predicting the outcome of normal labour.

BACKGROUND: Management of the second stage of labour is dictated by arbitrary time limits rather than true measures of progress. No partogram is available for second stage of labour. OBJECTIVES: To evaluate a partogram designed for use for the second stage of labour. METHODS: This prospective cross-sectional analytical study included low-risk pregnant women with singleton fetuses with vertex presentations at term. From onset of the second stage, vaginal examinations were performed every 30 min until delivery. A scoring system developed by Sizer et al. was used based on station and position of fetal head. Scores were plotted on a second stage partogram and used to predict labour outcomes, such as duration of second stage and mode of delivery. RESULTS: Of 79 women examined, 73 had spontaneous vaginal delivery. Of the remaining six, four required oxytocin infusion and other two required vacuum extraction. The median durations of the second stage of labour for primigravidas (n = 34) and multigravidas (n = 45) were 35 and 25 min, respectively. The median Sizer's partogram score at the onset of second stage was 4. Multiple regression analysis showed that the partogram score (r2 = 0.27) and gravidity (r2 = 0.10) were independent predictors of duration of the second stage. There was a significant association between second stage progress plotted to the right of the partogram line and non-spontaneous delivery (P = 0.01). CONCLUSION: The second stage partogram score at onset can predict the duration of second stage. Poor progress plotted on the partogram is associated with non-spontaneous delivery.

Novel mutation in Teneurin 3 found to co-segregate in all affecteds in a multi-generation family with developmental dysplasia of the hip.

DDH is a debilitating condition characterized by incomplete formation of the acetabulum leading to dislocation of the hip, suboptimal joint function and accelerated wear of the articular cartilage resulting in early onset crippling arthritis of the hip in 20-40 year olds. Current diagnostic tests in newborns using physical manipulation of the femur or ultrasound either under or over-diagnose this condition. Developing an accurate, cost effective diagnostic test is a goal of this study. To better understand the biologic pathways involved in acetabular development, DNA from severely affected individuals in a four generation family that showed inter-generational transmission of the disorder was isolated and whole exome sequenced. A novel A to C transversion at position 183721398 on human chromosome four was found to co-segregate with the affected phenotype in this family. This mutation encodes a glutamine to proline change at position 2665 in the Teneurin 3 (TENM3) gene and was judged damaging by four prediction programs. Eight week old knock-in mutant mice show delayed development of the left acetabulum and the left glenoid fossa as shown by the presence of more Alcian blue staining on the socket rims of both the hip and the shoulder. We hypothesize that mutated TENM3 will slow chondrogenesis. MMP13 has been shown to impair extracellular matrix remodeling and suppress differentiation. Bone marrow cells from the knock-in mouse were found to overexpress MMP13 with or without BMP2 stimulation. This variant may elucidate pathways responsible for normal hip development and become part of an accurate test for DDH. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.