SARS-CoV-2 infection in the first trimester and the risk of early miscarriage: a UK population-based prospective cohort study of 3041 pregnancies conceived during the pandemic.

STUDY QUESTION: Does maternal infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the first trimester affect the risk of miscarriage before 13 week's gestation? SUMMARY ANSWER: Pregnant women with self-reported diagnosis of SARS-CoV-2 in the first trimester had a higher risk of early miscarriage. WHAT IS KNOWN ALREADY: Viral infections during pregnancy have a broad spectrum of placental and neonatal pathology. Data on the effects of the SARS-CoV-2 infection in pregnancy are still emerging. Two systematic reviews and meta-analyses reported an increased risk of preterm birth, caesarean delivery, maternal morbidity and stillbirth. Data on the impact of first trimester infection on early pregnancy outcomes are scarce. This is the first study, to our knowledge, to investigate the rates of early pregnancy loss during the SARS-CoV-2 outbreak among women with self-reported infection. STUDY DESIGN, SIZE, DURATION: This was a nationwide prospective cohort study of pregnant women in the community recruited using social media between 21 May and 31 December 2020. We recruited 3545 women who conceived during the SARS-CoV-2 pandemic who were <13 week's gestation at the time of recruitment. PARTICIPANTS/MATERIALS, SETTING, METHODS: The COVID-19 Contraception and Pregnancy Study (CAP-COVID) was an on-line survey study collecting longitudinal data from pregnant women in the UK aged 18 years or older. Women who were pregnant during the pandemic were asked to complete on-line surveys at the end of each trimester. We collected data on current and past pregnancy complications, their medical history and whether they or anyone in their household had symptoms or been diagnosed with SARS-CoV-2 infection during each trimester of their pregnancy. RT-PCR-based SARS-CoV-2 RNA detection from respiratory samples (e.g. nasopharynx) is the standard practice for diagnosis of SARS-CoV-2 in the UK. We compared rate of self-reported miscarriage in three groups: 'presumed infected', i.e. those who reported a diagnosis with SARS-CoV-2 infection in the first trimester; 'uncertain', i.e. those who did not report a diagnosis but had symptoms/household contacts with symptoms/diagnosis; and 'presumed uninfected', i.e. those who did not report any symptoms/diagnosis and had no household contacts with symptoms/diagnosis of SARS-CoV-2. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 3545 women registered for the CAP-COVID study at <13 weeks gestation and were eligible for this analysis. Data for the primary outcome were available from 3041 women (86%). In the overall sample, the rate of self-reported miscarriage was 7.8% (238/3041 [95% CI, 7-9]). The median gestational age (GA) at miscarriage was 9 weeks (interquartile range 8-11). Seventy-seven women were in the 'presumed infected' group (77/3041, 2.5% [95% CI 2-3]), 295/3041 were in the uncertain group (9.7% [95% CI 9-11]) and the rest in the 'presumed uninfected' (87.8%, 2669/3041 [95% CI 87-89]). The rate of early miscarriage was 14% in the 'presumed infected' group, 5% in the 'uncertain' and 8% in the 'presumed uninfected' (11/77 [95% CI 6-22] versus 15/295 [95% CI 3-8] versus 212/2669 [95% CI 7-9], P = 0.02). After adjusting for age, BMI, ethnicity, smoking status, GA at registration and the number of previous miscarriages, the risk of early miscarriage appears to be higher in the 'presumed infected' group (relative rate 1.7, 95% CI 1.0-3.0, P = 0.06). LIMITATIONS, REASONS FOR CAUTION: We relied on self-reported data on early pregnancy loss and SARS-CoV-2 infection without any means of checking validity. Some women in the 'presumed uninfected' and 'uncertain' groups may have had asymptomatic infections. The number of 'presumed infected' in our study was low and therefore the study was relatively underpowered. WIDER IMPLICATIONS OF THE FINDINGS: This was a national study from the UK, where infection rates were one of the highest in the world. Based on the evidence presented here, women who are infected with SARS-CoV-2 in their first trimester may be at an increased risk of a miscarriage. However, the overall rate of miscarriage in our study population was 8%. This is reassuring and suggests that if there is an effect of SARS-CoV-2 on the risk of miscarriage, this may be limited to those with symptoms substantial enough to lead to a diagnostic test. Further studies are warranted to evaluate a causal association between SARS-CoV-2 infection in early pregnancy and miscarriage risk. Although we did not see an overall increase in the risk of miscarriage, the observed comparative increase in the presumed infected group reinforces the message that pregnant women should continue to exercise social distancing measures and good hygiene throughout their pregnancy to limit their risk of infection. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by a grant from the Elizabeth Garrett Anderson Hospital Charity (G13-559194). The funders of the study had no role in study design, data collection, data analysis, data interpretation or writing of the report. J.A.H. is supported by an NIHR Advanced Fellowship. A.L.D. is supported by the National Institute for Health Research University College London Hospitals Biomedical Research Centre. All authors have completed the ICMJE uniform disclosure form at www.icmje.org/coi_disclosure.pdf and declare: support to J.A.H. and A.L.D. as above; no financial relationships with any organizations that might have an interest in the submitted work in the previous 3 years; no other relationships or activities that could appear to have influenced the submitted work. TRIAL REGISTRATION NUMBER: N/A.

Targeting the Spleen as an Alternative Site for Hematopoiesis.

Bone marrow is the main site for hematopoiesis in adults. It acts as a niche for hematopoietic stem cells (HSCs) and contains non-hematopoietic cells that contribute to stem cell dormancy, quiescence, self-renewal, and differentiation. HSC also exist in resting spleen of several species, although their contribution to hematopoiesis under steady-state conditions is unknown. The spleen can however undergo extramedullary hematopoiesis (EMH) triggered by physiological stress or disease. With the loss of bone marrow niches in aging and disease, the spleen as an alternative tissue site for hematopoiesis is an important consideration for future therapy, particularly during HSC transplantation. In terms of harnessing the spleen as a site for hematopoiesis, here the remarkable regenerative capacity of the spleen is considered with a view to forming additional or ectopic spleen tissue through cell engraftment. Studies in mice indicate the potential for such grafts to support the influx of hematopoietic cells leading to the development of normal spleen architecture. An important goal will be the formation of functional ectopic spleen tissue as an aid to hematopoietic recovery following clinical treatments that impact bone marrow. For example, expansion or replacement of niches could be considered where myeloablation ahead of HSC transplantation compromises treatment outcomes.

Clinical Germline Genome Editing: When Will Good be Good Enough?

Ensuring experimental outcomes are of the highest clinical caliber is crucial prior to the introduction of germline genome editing. However, if we are to police scientific progress using probability or the potential to go wrong, then we must account for the specious standards of human reproduction. With 15% of clinically recognized pregnancies estimated to end in spontaneous miscarriage within the first trimester, and 25% of all pregnancies ending in miscarriage, human reproduction has a high failure rate. These figures, coupled with the percentage of all births with congenital defects and the number of these who will die in the first year of life, paint two scenarios: one, that evolutionary checkpoints are cruel but critical, and two, that for the seemingly inevitable 3%, or 8 million babies born annually with congenital disorders, perhaps more must be done for prevention, when methods exist for prediction. Unifying progress in three coevolving technologies-assisted reproduction, genome editing, and genome sequencing-could produce revolutionary clinical changes in the harsh global statistics of hereditary disease. A historical perspective on the rocky foundations upon which IVF was built suggests that lessons should be learned from the misalignment of research and clinical practice due to funding and research restrictions. At present, it seems likely that history will repeat itself, and that progress in research will be hampered by hypocritical hesitation.

Normal Levels of Sox9 Expression in the Developing Mouse Testis Depend on the TES/TESCO Enhancer, but This Does Not Act Alone.

During mouse sex determination, transient expression of the Y-linked gene Sry up-regulates its direct target gene Sox9, via a 3.2 kb testis specific enhancer of Sox9 (TES), which includes a core 1.4 kb element, TESCO. SOX9 activity leads to differentiation of Sertoli cells, rather than granulosa cells from the bipotential supporting cell precursor lineage. Here, we present functional analysis of TES/TESCO, using CRISPR/Cas9 genome editing in mice. Deletion of TESCO or TES reduced Sox9 expression levels in XY fetal gonads to 60 or 45% respectively relative to wild type gonads, and reduced expression of the SOX9 target Amh. Although human patients heterozygous for null mutations in SOX9, which are assumed to have 50% of normal expression, often show XY female sex reversal, mice deleted for one copy of Sox9 do not. Consistent with this, we did not observe sex reversal in either TESCO-/- or TES-/- XY embryos or adult mice. However, embryos carrying both a conditional Sox9 null allele and the TES deletion developed ovotestes. Quantitative analysis of these revealed levels of 23% expression of Sox9 compared to wild type, and a significant increase in the expression of the granulosa cell marker Foxl2. This indicates that the threshold in mice where sex reversal begins to be seen is about half that of the ~50% levels predicted in humans. Our results demonstrate that TES/TESCO is a crucial enhancer regulating Sox9 expression in the gonad, but point to the existence of additional enhancers that act redundantly.

Correction: Normal Levels of Sox9 Expression in the Developing Mouse Testis Depend on the TES/TESCO Enhancer, but This Does Not Act Alone.

[This corrects the article DOI: 10.1371/journal.pgen.1006520.].

A novel myeloid cell in murine spleen defined through gene profiling.

A novel myeloid antigen presenting cell can be generated through in vitro haematopoiesis in long-term splenic stromal cocultures. The in vivo equivalent subset was recently identified as phenotypically and functionally distinct from the spleen subsets of macrophages, conventional (c) dendritic cells (DC), resident monocytes, inflammatory monocytes and eosinophils. This novel subset which is myeloid on the basis of cell surface phenotype, but dendritic-like on the basis of cell surface marker expression and antigen presenting function, has been tentatively labelled "L-DC." Transcriptome analysis has now been employed to determine the lineage relationship of this cell type with known splenic cDC and monocyte subsets. Principal components analysis showed separation of "L-DC" and monocytes from cDC subsets in the second principal component. Hierarchical clustering then indicated a close lineage relationship between this novel subset, resident monocytes and inflammatory monocytes. Resident monocytes were the most closely aligned, with no genes specifically expressed by the novel subset. This subset, however, showed upregulation of genes reflecting both dendritic and myeloid lineages, with strong upregulation of several genes, particularly CD300e. While resident monocytes were found to be dependent on Toll-like receptor signalling for development and were reduced in number in Myd88-/- and Trif-/- mutant mice, both the novel subset and inflammatory monocytes were unaffected. Here, we describe a novel myeloid cell type closely aligned with resident monocytes in terms of lineage but distinct in terms of development and functional capacity.

Improved cryopreservation of spermatozoa using vitrification: comparison of cryoprotectants and a novel device for long-term storage.

STUDY QUESTION: Does cryoprotection of spermatozoa using a vitrification protocol with improved cryoprotective agents and a novel device for large storage lead to better outcomes than conventional slow freezing? Vitrification of human sperm using sucrose and dextran-based cryoprotectant (CPA4) with a new vitrification device resulted in significantly better sperm motility and progressive motility and improved DNA integrity with lower DNA fragmentation compared with conventional slow freezing. WHAT IS KNOWN ALREADY: A major limitation to clinical implementation of vitrification is the right balance between the volume of spermatozoa suspension cryopreserved and a standardised use of CPAs for survival of spermatozoa. STUDY DESIGN, SIZE, DURATION: This was a control versus current clinical practice study using 30 fresh human semen samples to carry out the different cryoprotectant analyses followed by a further 23 semen samples to test the novel vitrification protocol. PARTICIPANTS/MATERIALS, SETTING, METHODS: All human specimens fulfilled the following criteria: > 5 million spermatozoa/mL, > 20% total motility, ≥ 1.8 mL in volume, with all participants falling within the age range of 25-45 inclusively. The concentration, progressive motility, non-progressive motility, immotility, and various morphokinetic variables including DAP, DCL, DSL, LIN, and STR were then determined using the IVOS II™ Clinical CASA system (Hamilton Thorne, Beverly, MA, USA) on the basis of the 5th Edition of WHO Laboratory Manual for the Examination and Processing of Human Semen. MAIN RESULTS AND THE ROLE OF CHANCE: Among the 6 cryopreservation methods in this study, vitrification with the funnel-shaped device using CPA4 best preserves the 13 sperm parameters evaluated by CASA system. Conventional slow freezing and vitrification with the device using seminal plasma also protects sperm quality, but the overall motilities are statistically lower in comparison with the novel vitrification approach with cryoprotective media using the device. DNA fragmentation significantly increased after cryopreservation through the method of conventional slow freezing (p = 0.07). There was no significant difference in DNA fragmentation between fresh control and vitrification (p = 1.000). LIMITATIONS, REASONS FOR CAUTION: Extensive training is required to minimise the human error in using the vitrification device to perform cryopreservation. Each operator can only handle one sample at a time with device vitrification, whereas several samples can be processed without the need for special training with conventional slow freezing. WIDER IMPLICATIONS OF THE FINDINGS: The presented study shows that a new vitrification method could improve survival sperm rate. Human sperm vitrification using our novel protocol gives higher motility and progression and lower percentage of DNA fragmentation than conventional slow freezing. Our findings indicate that this method could supersede the current clinical practice in particular for patients with oligospermia as it reduces osmotic damage, time, and cost.

MCSF drives regulatory DC development in stromal co-cultures supporting hematopoiesis.

BACKGROUND: Splenic stroma overlaid with hematopoietic progenitors supports in vitro hematopoiesis with production of dendritic-like cells. Co-cultures of murine lineage-depleted bone marrow over the 5G3 stromal line produce two populations of cells, characterised as CD11b+CD11c+MHC-II- dendritic-like 'L-DC', and CD11b+CD11c+MHC-II+ cells, resembling conventional dendritic cells (cDC). To date, the functional capacity of these two subsets has not been clearly distinguished. RESULTS: Here we show both the L-DC and cDC-like subsets can be activated and induce proliferation of OT-I CD8+ T cells, being strong inducers of IL-2 and IFN-γ production. Both subsets lack ability to induce proliferation of OT-II CD4+ T cells. The cDC-like population is shown here to resemble regulatory DC in that they induce FoxP3 expression and IL-10 production in OT-II CD4+ T cells, in line with their function as regulatory DC. L-DC did not activate or induce the proliferation of CD4+ T cells and did not induce FoxP3 expression in CD4+ T cells. L-DC can be distinguished from cDC-like cells through their superior endocytic capacity and expression of 4-1BBL, F4/80 and Sirp-α. A comparison of gene expression by the two subsets was consistent with L-DC having an activated or immunostimulatory DC phenotype, while cDC-like cells reflect myeloid dendritic cells with inflammatory and suppressive properties, also consistent with functional characteristics as regulatory DC. When a Transwell membrane was used to prevent hematopoietic cell contact with stroma, only cDC-like cells and not L-DC were produced, and cell production was dependent on M-CSF production by stroma. CONCLUSION: Co-cultures of hematopoietic progenitors over splenic stroma produce two distinct subsets of dendritic-like cells. These are here distinguished phenotypically and through gene expression differences. While both resemble DC, there are functionally distinct. L-DC activate CD8+ but not CD4+ T cells, while the cDC-like population induce regulatory T cells, so reflecting regulatory DC. The latter can be enriched through Transwell co-cultures with cell production dependent on M-CSF.

Antigen presenting capacity of murine splenic myeloid cells.

BACKGROUND: The spleen is an important site for hematopoiesis. It supports development of myeloid cells from bone marrow-derived precursors entering from blood. Myeloid subsets in spleen are not well characterised although dendritic cell (DC) subsets are clearly defined in terms of phenotype, development and functional role. Recently a novel dendritic-like cell type in spleen named 'L-DC' was distinguished from other known dendritic and myeloid cells by its distinct phenotype and developmental origin. That study also redefined splenic eosinophils as well as resident and inflammatory monocytes in spleen. RESULTS: L-DC are shown to be distinct from known splenic macrophages and monocyte subsets. Using a new flow cytometric procedure, it has been possible to identify and isolate L-DC in order to assess their functional competence and ability to activate T cells both in vivo and in vitro. L-DC are readily accessible to antigen given intravenously through receptor-mediated endocytosis. They are also capable of CD8+ T cell activation through antigen cross presentation, with subsequent induction of cytotoxic effector T cells. L-DC are MHCII- cells and unable to activate CD4+ T cells, a property which clearly distinguishes them from conventional DC. The myeloid subsets of resident monocytes, inflammatory monocytes, neutrophils and eosinophils, were found to have varying capacities to take up antigen, but were uniformly unable to activate either CD4+ T cells or CD8+ T cells. CONCLUSION: The results presented here demonstrate that L-DC in spleen are distinct from other myeloid cells in that they can process antigen for CD8+ T cell activation and induction of cytotoxic effector function, while both L-DC and myeloid subsets remain unable to activate CD4+ T cells. The L-DC subset in spleen is therefore distinct as an antigen presenting cell.

The B subunit of Escherichia coli heat-labile toxin alters the development and antigen-presenting capacity of dendritic cells.

Escherichia coli's heat-labile enterotoxin (Etx) and its non-toxic B subunit (EtxB) have been characterized as adjuvants capable of enhancing T cell responses to co-administered antigen. Here, we investigate the direct effect of intravenously administered EtxB on the size of the dendritic and myeloid cell populations in spleen. EtxB treatment appears to enhance the development and turnover of dendritic and myeloid cells from precursors within the spleen. EtxB treatment also gives a dendritic cell (DC) population with higher viability and lower activation status based on the reduced expression of MHC-II, CD80 and CD86. In this respect, the in vivo effect of EtxB differs from that of the highly inflammatory mediator lipopolysaccharide. In in vitro bone marrow cultures, EtxB treatment was also found to enhance the development of DC from precursors dependent on Flt3L. In terms of the in vivo effect of EtxB on CD4 and CD8 T cell responses in mice, the interaction of EtxB directly with DC was demonstrated following conditional depletion of CD11c(+) DC. In summary, all results are consistent with EtxB displaying adjuvant ability by enhancing the turnover of DC in spleen, leading to newly mature myeloid and DC in spleen, thereby increasing DC capacity to perform as antigen-presenting cells on encounter with T cells.

Expression of TCR-Vß peptides by murine bone marrow cells does not identify T-cell progenitors.

Germline transcription has been described for both immunoglobulin and T-cell receptor (TCR) genes, raising questions of their functional significance during haematopoiesis. Previously, an immature murine T-cell line was shown to bind antibody to TCR-Vß8.2 in absence of anti-Cß antibody binding, and an equivalent cell subset was also identified in the mesenteric lymph node. Here, we investigate whether germline transcription and cell surface Vß8.2 expression could therefore represent a potential marker of T-cell progenitors. Cells with the TCR phenotype of Vß8.2(+) Cß(-) are found in several lymphoid sites, and among the lineage-negative (Lin(-)) fraction of hematopoietic progenitors in bone marrow (BM). Cell surface marker analysis of these cells identified subsets reflecting common lymphoid progenitors, common myeloid progenitors and multipotential progenitors. To assess whether the Lin(-) Vß8.2(+) Cß(-) BM subset contains hematopoietic progenitors, cells were sorted and adoptively transferred into sub-lethally irradiated recipients. No T-cell or myeloid progeny were detected following introduction of cells via the intrathymic or intravenous routes. However, B-cell development was detected in spleen. This pattern of restricted in vivo reconstitution disputes Lin(-) Vß8.2(+) Cß(-) BM cells as committed T-cell progenitors, but raises the possibility of progenitors with potential for B-cell development.

Characterization of the effect of LPS on dendritic cell subset discrimination in spleen.

The Gram-negative bacterial endotoxin lipopolysaccharide (LPS) is a potent inflammatory mediator and a leading cause of bacterial sepsis. While LPS is known to activate antigen-presenting cells, here we find that LPS down-regulates expression of CD11c and CD11b on splenic dendritic cell subsets, thus confounding the ability to identify these subsets following treatment. This has implications with regard to tracking the response to LPS in terms of the cell subsets involved, and should be considered whenever such studies are undertaken.

Skeletal stem/progenitor cells provide the niche for extramedullary hematopoiesis in spleen.

In bone marrow, the niche which supports hematopoiesis and nurtures hematopoietic stem cells (HSCs) contains perivascular reticular cells representing a subset of skeletal stem/progenitor cells (SSPCs). These stromal cells which provide the niche are lost or become inadequate during stress, disease or ageing, such that HSCs leave bone marrow and enter spleen and other peripheral sites to initiate extramedullary hematopoiesis and particularly myelopoiesis. Spleen also maintains niches for HSCs under steady-state conditions, evident since neonatal and adult spleen contain HSCs in low number and provide low-level hematopoiesis. In spleen, HSCs are found in the sinusoidal-rich red pulp region also in the vicinity of perivascular reticular cells. These cells resemble to some extent the known stromal elements reflecting HSC niches in bone marrow, and are investigated here for their characteristics as a subset of SSPCs. The isolation of spleen stromal subsets and the generation of cell lines which support HSCs and myelopoiesis in vitro has led to the identification of perivascular reticular cells which are unique to spleen. Analysis of gene and marker expression, as well as differentiative potential, identifies an osteoprogenitor cell type, reflective of one of several subsets of SSPCs described previously in bone, bone marrow and adipose tissue. The combined information supports a model for HSC niches in spleen involving perivascular reticular cells as SSPCs having osteogenic, stroma-forming capacity. These associate with sinusoids in red pulp to form niches for HSCs and to support the differentiation of hematopoietic progenitors during extramedullary hematopoiesis.

Investigating the significance of segmental aneuploidy findings in preimplantation embryos.

Segmental aneuploidies (SAs) are structural imbalances, namely, gains or losses, involving a chromosomal segment. Most preimplantation genetic testing platforms can detect segmental imbalances greater than 5-10 Mb, either full or mosaic; however, questions remain about clinical significance. An in-depth review was performed to determine the accuracy, frequency, and types of SAs detected in preimplantation embryos. A comprehensive search of the literature revealed an incidence of approximately 8.15% in preimplantation embryos, compared with a prevalence of 3.55% in prenatal diagnosis samples. Several studies have used rebiopsy analysis to validate the accuracy and reproducibility of such findings in blastocyst-stage embryos. A comparison of these studies yielded a mean confirmation rate of SAs slightly higher than 30%. This result could be attributed to their mitotic origin as well as to the technical limitations of preimplantation genetic testing. In addition, the few available studies in which embryos with a segmental finding were transferred in utero are analyzed to discuss the reproductive competence of such embryos. Except for 1 study, all outcomes were described for segmental embryos in a mosaic state. As a result, there is still insufficient evidence to provide accurate information about the effect of segmental imbalances on embryonic reproductive competence and to determine gestational and newborn risks.

Myelopoiesis in spleen-producing distinct dendritic-like cells.

Dendritic cells (DC) represent a heterogeneous class of antigen presenting cells (APC). Previously we reported a distinct myeloid dendritic-like cell present in spleen, as an in vivo counterpart to cells produced in murine spleen long-term cultures (LTC-DC). These cells, named 'L-DC', were found to be functionally and phenotypically distinct from conventional (c)DC, plasmacytoid (p)DC and monocytes. These results suggested that spleen may represent a niche for development of L-DC from endogenous progenitors. Adult murine spleen has now been investigated for the presence of L-DC progenitors. Lineage-negative (Lin)(-) ckit(lo) and Lin(-) ckit(hi) progenitor subsets were identified as candidate populations, and tested for ability to produce L-DC; in vitro upon co-culture with the spleen stromal line STX3, and in vivo after adoptive therapy into mice. Both subsets colonized STX3 stroma in vitro for L-DC production, indicating that they contained either a common or two distinct progenitors for L-DC. However, only the Lin(-) ckit(hi) subset gave progeny cells after adoptive transfer into lethally irradiated mice. In vivo development was however multilineage and not restricted to L-DC development. Multilineage reconstitution reflects long-term reconstituting haematopoietic stem cells (LT-HSC), suggesting a close relationship between L-DC progenitors and LT-HSC. L-DC were however produced in vivo in much higher number than monocytes/macrophages and cDC, indicating the presence of a specific L-DC progenitor within the Lin(-) ckit(hi) subset. A model is advanced for development of L-DC directly from haematopoietic progenitors in spleen and dependent on the spleen microenvironment.

Murine spleen contains a diversity of myeloid and dendritic cells distinct in antigen presenting function.

The spleen contains multiple subsets of myeloid and dendritic cells (DC). DC are important antigen presenting cells (APC) which induce and control the adaptive immune response. They are cells specialized for antigen capture, processing and presentation to naïve T cells. However, DC are a heterogeneous population and each subset differs subtly in phenotype, function and location. Similarly, myeloid cell subsets can be distinguished which can also play an important role in the regulation of immunity. This review aims to characterize splenic subsets of DC and myeloid cells to better understand their individual roles in the immune response.

Hematopoiesis leading to a diversity of dendritic antigen-presenting cell types.

Hematopoietic stem cells (HSCs) undergo expansion and differentiation, giving rise to all terminally differentiated blood cells throughout life. HSCs are found in distinct anatomical sites during development, and in adults, hematopoiesis occurs predominantly on the luminal side of the bone cavity in bone marrow. Millions of newly formed blood cells are generated per second to accommodate the short half-life of hematopoietic cells. For this to happen, HSCs must sustain their self-renewal capacity as well as their capability to commit and differentiate toward multiple cell lineages. Development of the hematopoietic system is finely regulated as the animal ages, so that it does not become exhausted or misdirected. This review covers aspects of hematopoietic development from the embryonic period through adult life in relation to development of dendritic cells. It also considers a role for HSCs in extramedullary sites and their possible role in myelopoiesis, with formation of tissue-specific antigen-presenting cells.

Delineation of precursors in murine spleen that develop in contact with splenic endothelium to give novel dendritic-like cells.

Hematopoietic cell lineages are best described in terms of distinct progenitors with limited differentiative capacity. To distinguish cell lineages, it is necessary to define progenitors and induce their differentiation in vitro. We previously reported in vitro development of immature dendritic-like cells (DCs) in long-term cultures (LTCs) of murine spleen, and in cocultures of spleen or bone marrow (BM) over splenic endothelial cell lines derived from LTCs. Cells produced are phenotypically distinct CD11b(hi)CD11c(lo)CD8(-)MHC-II(-) cells, tentatively named L-DCs. Here we delineate L-DC progenitors as different from known DC progenitors in BM and DC precursors in spleen. The progenitor is contained within the lineage-negative (Lin)(-)c-kit(+) subset in neonatal and adult spleen. This subset has multipotential reconstituting ability in mice. In neonatal spleen, the progenitor is further enriched within the c-kit(lo) and CD34(+) subsets of Lin(-)c-kit(+) cells. These cells seed cocultures of splenic endothelial cells, differentiating to give L-DCs that can activate T cells. L-DC progenitors are distinguishable from described splenic CD11c(lo) DC precursors and from Fms-like tyrosine kinase 3(+) DC progenitors in BM. Overall, this study confirms that LTCs are a physiologically relevant culture system for in vitro development of a novel DC type from spleen progenitors.