Challenges of accounting nitrous oxide emissions from agricultural crop residues.

Crop residues are important inputs of carbon (C) and nitrogen (N) to soils and thus directly and indirectly affect nitrous oxide (N2 O) emissions. As the current inventory methodology considers N inputs by crop residues as the sole determining factor for N2 O emissions, it fails to consider other underlying factors and processes. There is compelling evidence that emissions vary greatly between residues with different biochemical and physical characteristics, with the concentrations of mineralizable N and decomposable C in the residue biomass both enhancing the soil N2 O production potential. High concentrations of these components are associated with immature residues (e.g., cover crops, grass, legumes, and vegetables) as opposed to mature residues (e.g., straw). A more accurate estimation of the short-term (months) effects of the crop residues on N2 O could involve distinguishing mature and immature crop residues with distinctly different emission factors. The medium-term (years) and long-term (decades) effects relate to the effects of residue management on soil N fertility and soil physical and chemical properties, considering that these are affected by local climatic and soil conditions as well as land use and management. More targeted mitigation efforts for N2 O emissions, after addition of crop residues to the soil, are urgently needed and require an improved methodology for emission accounting. This work needs to be underpinned by research to (1) develop and validate N2 O emission factors for mature and immature crop residues, (2) assess emissions from belowground residues of terminated crops, (3) improve activity data on management of different residue types, in particular immature residues, and (4) evaluate long-term effects of residue addition on N2 O emissions.

Sustained cross-presentation capacity of murine splenic dendritic cell subsets in vivo.

An exclusive feature of dendritic cells (DCs) is their ability to cross-present exogenous antigens in MHC class I molecules. We analyzed the fate of protein antigen in antigen presenting cell (APC) subsets after uptake of naturally formed antigen-antibody complexes in vivo. We observed that murine splenic DC subsets were able to present antigen in vivo for at least a week. After ex vivo isolation of four APC subsets, the presence of antigen in the storage compartments was visualized by confocal microscopy. Although all APC subsets stored antigen for many days, their ability and kinetics in antigen presentation was remarkably different. CD8α+ DCs showed sustained MHC class I-peptide specific CD8+ T-cell activation for more than 4 days. CD8α- DCs also presented antigenic peptides in MHC class I but presentation decreased after 48 h. In contrast, only the CD8α- DCs were able to present antigen in MHC class II to specific CD4+ T cells. Plasmacytoid DCs and macrophages were unable to activate any of the two T-cell types despite detectable antigen uptake. These results indicate that naturally occurring DC subsets have functional antigen storage capacity for prolonged T-cell activation and have distinct roles in antigen presentation to specific T cells in vivo.

C1q-Dependent Dendritic Cell Cross-Presentation of In Vivo-Formed Antigen-Antibody Complexes.

Dendritic cells (DCs) are specialized in Ag engulfment via a wide variety of uptake receptors on their cell surface. In the present study we investigated Ag uptake and presentation of in vivo-formed Ag-Ab complexes by i.v. injecting mice with Ag-specific Abs followed by the cognate Ag. We show by this natural Ab-mediated Ag targeting system that uptake by splenic APC subsets is severely hampered in mice lacking complement factor C1q (C1qa-/-). Moreover, no detectable Ag cross-presentation by CD8α+ DCs from C1qa-/- mice was found. On the contrary, Ag uptake was not hampered by APCs in FcγRI/II/III/IV-deficient (FcγR quadruple-/-) mice, and the cross-presentation ability of CD8α+ DCs was not affected. In conclusion, we show that C1q rather than FcγRs controls the Ab-mediated Ag uptake and its presentation by spleen APC subsets to T cells.

Development of a diverse human T-cell repertoire despite stringent restriction of hematopoietic clonality in the thymus.

The fate and numbers of hematopoietic stem cells (HSC) and their progeny that seed the thymus constitute a fundamental question with important clinical implications. HSC transplantation is often complicated by limited T-cell reconstitution, especially when HSC from umbilical cord blood are used. Attempts to improve immune reconstitution have until now been unsuccessful, underscoring the need for better insight into thymic reconstitution. Here we made use of the NOD-SCID-IL-2Rγ(-/-) xenograft model and lentiviral cellular barcoding of human HSCs to study T-cell development in the thymus at a clonal level. Barcoded HSCs showed robust (>80% human chimerism) and reproducible myeloid and lymphoid engraftment, with T cells arising 12 wk after transplantation. A very limited number of HSC clones (<10) repopulated the xenografted thymus, with further restriction of the number of clones during subsequent development. Nevertheless, T-cell receptor rearrangements were polyclonal and showed a diverse repertoire, demonstrating that a multitude of T-lymphocyte clones can develop from a single HSC clone. Our data imply that intrathymic clonal fitness is important during T-cell development. As a consequence, immune incompetence after HSC transplantation is not related to the transplantation of limited numbers of HSC but to intrathymic events.

Long term impact of residue management on soil organic carbon stocks and nitrous oxide emissions from European croplands.

Application of crop residues to agricultural fields is a significant source of the greenhouse gas nitrous oxide (N2O) and an essential factor affecting the soil organic carbon (SOC) balance. Here we present a biogeochemical modelling study assessing the impact of crop residue management on soil C stocks and N2O fluxes for EU-27 using available information on soils, management and climate and by testing various scenarios of residue management. Three biogeochemical models, i.e. CERES-EGC, LandscapeDNDC and LandscapeDNDC-MeTrx, were used in an ensemble approach on a grid of 0.25° × 0.25° spatial resolution for calculating EU-27 wide inventories of changes in SOC stocks and N2O emissions due to residue management for the years 2000-2100 using different climate change projections (RCP4.5 and RCP8.5). Our results show, that climate change poses a threat to cropping systems in Europe, resulting in potential yield declines, increased N2O emissions and loss of SOC. This highlights the need for adapting crop management to mitigate climate change impacts, e.g. by improved residue management. For a scenario with 100% residues retention and reduced tillage we calculated that in average SOC stocks may increase over 50-100 years by 19-23% under RCP8.5 and RCP4.5. However, complete retention of crop residues also resulted in an increase of soil N2O emissions by 17-30%, so that climate benefits due to increases in SOC stocks were eventually compensated by increased N2O emissions. The long-term EFN2O for residue N incorporation was 1.18% and, thus slightly higher as the 1% value used by IPCC. We conclude that residue management can be an important strategy for mitigating climate change impacts on SOC stocks, though it requires as well improvements in N management for N2O mitigation.

New insights on human T cell development by quantitative T cell receptor gene rearrangement studies and gene expression profiling.

To gain more insight into initiation and regulation of T cell receptor (TCR) gene rearrangement during human T cell development, we analyzed TCR gene rearrangements by quantitative PCR analysis in nine consecutive T cell developmental stages, including CD34+ lin- cord blood cells as a reference. The same stages were used for gene expression profiling using DNA microarrays. We show that TCR loci rearrange in a highly ordered way (TCRD-TCRG-TCRB-TCRA) and that the initiating Ddelta2-Ddelta3 rearrangement occurs at the most immature CD34+CD38-CD1a- stage. TCRB rearrangement starts at the CD34+CD38+CD1a- stage and complete in-frame TCRB rearrangements were first detected in the immature single positive stage. TCRB rearrangement data together with the PTCRA (pTalpha) expression pattern show that human TCRbeta-selection occurs at the CD34+CD38+CD1a+ stage. By combining the TCR rearrangement data with gene expression data, we identified candidate factors for the initiation/regulation of TCR recombination. Our data demonstrate that a number of key events occur earlier than assumed previously; therefore, human T cell development is much more similar to murine T cell development than reported before.

Wnt3a deficiency irreversibly impairs hematopoietic stem cell self-renewal and leads to defects in progenitor cell differentiation.

Canonical Wnt signaling has been implicated in various aspects of hematopoiesis. Its role is controversial due to different outcomes between various inducible Wnt-signaling loss-of-function models and also compared with gain-of-function systems. We therefore studied a mouse deficient for a Wnt gene that seemed to play a nonredundant role in hematopoiesis. Mice lacking Wnt3a die prenatally around embryonic day (E) 12.5, allowing fetal hematopoiesis to be studied using in vitro assays and transplantation into irradiated recipient mice. Here we show that Wnt3a deficiency leads to a reduction in the numbers of hematopoietic stem cells (HSCs) and progenitor cells in the fetal liver (FL) and to severely reduced reconstitution capacity as measured in secondary transplantation assays. This deficiency is irreversible and cannot be restored by transplantation into Wnt3a competent mice. The impaired long-term repopulation capacity of Wnt3a(-/-) HSCs could not be explained by altered cell cycle or survival of primitive progenitors. Moreover, Wnt3a deficiency affected myeloid but not B-lymphoid development at the progenitor level, and affected immature thymocyte differentiation. Our results show that Wnt3a signaling not only provides proliferative stimuli, such as for immature thymocytes, but also regulates cell fate decisions of HSC during hematopoiesis.

Beyond livestock carrying capacity in the Sahelian and Sudanian zones of West Africa.

We applied the process-based model, LandscapeDNDC, to estimate feed availability in the Sahelian and Sudanian agro-ecological zones of West Africa as a basis for calculating the regional Livestock Carrying Capacity (LCC). Comparison of the energy supply (S) from feed resources, including natural pasture, browse, and crop residues, with energy demand (D) of the livestock population for the period 1981-2020 allowed us to assess regional surpluses (S > D) or deficits (S < D) in feed availability. We show that in the last 40 years a large-scale shift from surplus to deficit has occurred. While during 1981-1990 only 27% of the area exceeded the LCC, it was 72% for the period 2011-2020. This was caused by a reduction in the total feed supply of ~ 8% and an increase in feed demand of ~ 37% per-decade, driven by climate change and increased livestock population, respectively. Overall, the S/D decreased from ~ 2.6 (surplus) in 1981 to ~ 0.5 (deficit) in 2019, with a north-south gradient of increasing S/D. As climate change continues and feed availability may likely further shrink, pastoralists either need to source external feed or significantly reduce livestock numbers to avoid overgrazing, land degradation, and any further conflicts for resources.

Flow Cytometry and Confocal Imaging Analysis of Low Wnt Expression in Axin2-mTurquoise2 Reporter Thymocytes.

Measuring Wnt expression levels is essential when trying to identify or test new Wnt therapeutic targets. Previous studies have shown that canonical Wnt signaling operates via a dosage-driven mechanism, motivating the need to study and measure Wnt signaling in various cell types. Although several reporter models have been proposed to represent physiological Wnt expression, either the genetic context or the reporter protein highly influenced the validity, accuracy, and flexibility of these tools. This paper describes methods for acquiring and analyzing data obtained with the Axin2-mTurquoise2 mouse Wnt reporter model, which contains a mutated Axin2em1Fstl allele. This model facilitates the study of endogenous canonical Wnt signaling in individual cells over a wide range of Wnt activity. This protocol describes how to fully appreciate Axin2-mTurquoise2 reporter activity using cell population analysis of the hematopoietic system, combined with cell surface markers or ß-catenin intracellular staining. These procedures serve as a base for implementation and reproduction in other tissues or cells of interest. By combining fluorescence-activated cell sorting and confocal imaging, distinct canonical Wnt expression levels can be visualized. The recommended measurement and analysis strategies provide quantitative data on the fluorescent expression levels for precise assessment of canonical Wnt signaling. These methods will be useful for researchers who want to use the Axin2-mTurquise2 model for canonical Wnt expression patterns.

Isolation of human and mouse hematopoietic stem cells.

Hematopoietic stem cells (HSC) are rare with estimated frequencies of 1 in 10,000 bone marrow cells and 1 in every 100,000 blood cells. The most important characteristic of HSC is their capacity to provide complete restoration of all blood cell lineages after bone marrow ablation. Therefore they are considered as the ideal targets for various clinical applications including stem cell transplantation and gene therapy. In adult mice and men, the main stem cell source is the bone marrow. For clinical applications HSC derived from umbilical cord blood (UCB) and G-CSF mobilized peripheral blood (PB) have been demonstrated to have several advantages compared to bone marrow; therefore, they are slowly replacing BM as alternative source of stem cells. The mouse is the model organism of choice for immunological and hematological research; therefore, studies of murine HSC are an important research topic. Here we described the most often used protocols and methods to isolate human and mouse HSC to high purity.

Estimating nitrogen flows of agricultural soils at a landscape level - A modelling study of the Upper Enns Valley, a long-term socio-ecological research region in Austria.

This paper explores the fate of reactive nitrogen (Nr) on the landscape scale of present agricultural production practice on arable and grassland soils. We use the soil modelling tool LandscapeDNDC (landscape scale DeNitrification-DeComposition model) to quantify resulting flows of Nr distributed to the atmosphere, hydrosphere and the crops. Test area is a watershed in the Austrian Alps characterized by arable production in the low-lying areas and grassland in the mountains. The approach considers an overall budget of nitrogen, and determines the nitrogen use efficiency for individual crops and crop rotations, with average levels found at 85% for the arable area and 68-98% for the grassland areas. Modelled Nr flows are compared to the values resulting from the national emission factor (EF) method used for the Austrian emission inventory. For the arable part of the study region, the annual sum of released Nr emissions derived from LandscapeDNDC modelling is lower than the result of the EF method by about 13% (or 7 kg N ha-1). Model results are lower also for other Nr species, yet nitrate leaching rates as well as ammonia emissions contribute a major share. For grassland areas, nitrate leaching values estimated by LandscapeDNDC greatly depend on local specifics and substantially exceed EF estimates. All other modelled Nr species are lower than the EF results. The model set-up allows to characterize spatially explicit effects of mitigation measures. As an example, we identify nitrous oxide (N2O) hot spots in the study region, and we quantify the N2O emission saving potential if focusing reduction efforts to such hot spots. Reducing fertilization of hot spots by half could remove 14% of N2O emission for 5% less crop yield and a loss of grassland yield by <1% when extrapolated to the whole study area.

Development of an in vivo model to study clonal lineage relationships in hematopoietic cells using Brainbow2.1/Confetti mice.

Hematopoietic stem cells maintain the homeostasis of all blood cell progeny during development and repopulation-demanding events. To study the lineage relationships during hematopoiesis, increasingly complex cell tracing models are being developed. In this study, we describe adaptations to the original R26R-Confetti mouse model, which subsequently offers a relatively easy approach to study low complexity clonality during hematopoiesis, with special focus on B and T lymphocyte development. This protocol employs spatiotemporal Cre expression controlled by gammaretroviral transduction for efficient fluorescent protein cell marking. Transplantation of fluorescently marked Lin- cKit+ hematopoietic progenitor cells into Rag1-/- mice, resulted in the visualization of differentially contributing stem cell clones to various lineages. Our methodology is useful to study questions in fundamental and preclinical hematopoietic research and in vivo B- and T-cell development.

A modeling study on mitigation of N2O emissions and NO3 leaching at different agricultural sites across Europe using LandscapeDNDC.

The identification of site-specific agricultural management practices in order to maximize yield while minimizing environmental nitrogen losses remains in the center of environmental pollution research. Here, we used the biogeochemical model LandscapeDNDC to explore different agricultural practices with regard to their potential to reduce soil N2O emissions and NO3 leaching while maintaining yields. In a first step, the model was tested against observations of N2O emissions, NO3 leaching, soil micrometeorology as well as crop growth for eight European cropland and grassland sites. Across sites, LandscapeDNDC predicts very well mean N2O emissions (r(2)=0.99) and simulates the magnitude and general temporal dynamics of soil inorganic nitrogen pools. For the assessment of site-specific mitigation potentials of environmental nitrogen losses a Monte Carlo optimization technique considering different agricultural management options (i.e., timing of planting, harvest and fertilization, amount of applied fertilizer as well as residue management) was used. The identified optimized field management practices reduce N2O emissions and NO3 leaching from croplands on average by 21% and 31%, respectively. Likewise, average reductions of 55% for N2O emissions and 16% for NO3 leaching are estimated for grasslands. For mitigating environmental loss - while maintaining yield levels - it was most important to reduce fertilizer application rates by in average 10%. Our analyses indicate that yield scaled N2O emissions and NO3 leaching indicate possible improvements of nitrogen use efficiencies in European farming systems. Moreover, the applied optimization approach can be used also in a prognostic way to predict optimal timings and fertilization options (rates and splitting) upon accurate weather forecasts combined with the knowledge of modeled soil nutrient availability and plant nitrogen demand.

Overexpression of LMO2 causes aberrant human T-Cell development in vivo by three potentially distinct cellular mechanisms.

Overexpression of LMO2 is known to be one of the causes of T-cell acute lymphoblastic leukemia (T-ALL) development; however, the mechanisms behind its oncogenic activity are incompletely understood. LMO2-overexpressing transgenic mouse models suggest an accumulation of immature T-cell progenitors in the thymus as the main preleukemic event. The effects of LMO2 overexpression on human T-cell development in vivo are unknown. Here, we report studies of a humanized mouse model transplanted with LMO2-transduced human hematopoietic stem/progenitor cells. The effects of LMO2 overexpression were confined to the T-cell lineage; however, initially, multipotent cells were transduced. Three effects of LMO2 on human T-cell development were observed: (1) a block at the double-negative/immature single-positive stage, (2) an accumulation of CD4(+)CD8(+) double-positive CD3(-) cells, and (3) an altered CD8/CD4 ratio with enhanced peripheral T lymphocytes. Microarray analysis of sorted double-positive cells overexpressing LMO2 led to the identification of an LMO2 gene set that clustered with human T-ALL patient samples of the described "proliferative" cluster. In this article, we demonstrate previously unrecognized mechanisms by which LMO2 alters human T-cell development in vivo; these mechanisms correlate with human T-ALL leukemogenesis.

Estimation and mitigation of N2O emission and nitrate leaching from intensive crop cultivation in the Haean catchment, South Korea.

Considering intensive agricultural management practices and environmental conditions, the LandscapeDNDC model was applied for simulation of yields, N2O emission and nitrate leaching from major upland crops and temperate deciduous forest of the Haean catchment, South Korea. Fertilization rates were high (up to 314 kg N ha(-1) year(-1)) and resulted in simulated direct N2O emissions from potato, radish, soybean and cabbage fields of 1.9 and 2.1 kg N ha(-1) year(-1) in 2009 and 2010, respectively. Nitrate leaching was identified as the dominant pathway of N losses in the Haean catchment with mean annual rates of 112.2 and 125.4 kg N ha(-1) year(-1), causing threats to water quality and leading to substantial indirect N2O emissions of 0.84 and 0.94 kg N ha(-1) year(-1) in 2009 and 2010 as estimates by applying the IPCC EF5. Simulated N2O emissions from temperate deciduous forest were low (approx. 0.50 kg N ha(-1) year(-1)) and predicted nitrate leaching rates were even negligible (≤0.01 kg N ha(-1) year(-1)). On catchment scale more than 50% of the total N2O emissions and up to 75% of nitrate leaching originated from fertilized upland fields, only covering 24% of the catchment area. Taking into account area coverage of simulated upland crops and other land uses these numbers agree well with nitrate loads calculated from discharge and concentration measurements at the catchment outlet. The change of current agricultural management practices showed a high potential of reducing N2O emission and nitrate leaching while maintaining current crop yields. Reducing (39%) and splitting N fertilizer application into 3 times was most effective and lead to about 54% and 77% reducing of N2O emission and nitrate leaching from the Haean catchment, the latter potentially contributing to improved water quality in the Soyang River Dam, which is the major source of drinking water for metropolitan residents.

Canonical wnt signaling regulates hematopoiesis in a dosage-dependent fashion.

Canonical Wnt signaling has been implicated in the regulation of hematopoiesis. By employing a Wnt-reporter mouse, we observed that Wnt signaling is differentially activated during hematopoiesis, suggesting an important regulatory role for specific Wnt signaling levels. To investigate whether canonical Wnt signaling regulates hematopoiesis in a dosage-dependent fashion, we analyzed the effect of different mutations in the Adenomatous polyposis coli gene (Apc), a negative modulator of the canonical Wnt pathway. By combining different targeted hypomorphic alleles and a conditional deletion allele of Apc, a gradient of five different Wnt signaling levels was obtained in vivo. We here show that different, lineage-specific Wnt dosages regulate hematopoietic stem cells (HSCs), myeloid precursors, and T lymphoid precursors during hematopoiesis. Differential, lineage-specific optimal Wnt dosages provide a unifying concept that explains the differences reported among inducible gain-of-function approaches, leading to either HSC expansion or depletion of the HSC pool.

Enforced expression of GATA3 allows differentiation of IL-17-producing cells, but constrains Th17-mediated pathology.

The zinc-finger transcription factor GATA3 serves as a master regulator of T-helper-2 (Th2) differentiation by inducing expression of the Th2 cytokines IL-4, IL-5 and IL-13 and by suppressing Th1 development. Here, we investigated how GATA3 affects Th17 differentiation, using transgenic mice with enforced GATA3 expression. We activated naïve primary T cells in vitro in the presence of transforming growth factor-beta and IL-6, and found that enforced GATA3 expression induced co-expression of Th2 cytokines in IL-17-producing T cells. Although the presence of IL-4 hampered Th17 differentiation, transforming growth factor-beta/IL-6 cultures from GATA3 transgenic mice contained substantial numbers of IL-17(+) cells, partially because GATA3 supported Th17 differentiation by limiting IL-2 and IFN-gamma production. GATA3 additionally constrained Th17 differentiation in vitro through IL-4-independent mechanisms, involving downregulating transcription of STAT3, STAT4, NFATc2 and the nuclear factor RORgammat, which is crucial for Th17 differentiation. Remarkably, upon myelin oligodendrocyte glycoprotein immunization in vivo, GATA3 transgenic mice contained similar numbers of IL-17-producing T cells in their lymph nodes as wild-type mice, but were not susceptible to autoimmune encephalomyelitis, possibly due to concomitant production of IL-4 and IL-10 induction. We therefore conclude that although GATA3 allows Th17 differentiation, it acts as an inhibitor of Th17-mediated pathology, through IL-4-dependent and IL-4-independent pathways.

Human thymus contains multipotent progenitors with T/B lymphoid, myeloid, and erythroid lineage potential.

It is a longstanding question which bone marrow-derived cell seeds the thymus and to what level this cell is committed to the T-cell lineage. We sought to elucidate this issue by examining gene expression, lineage potential, and self-renewal capacity of the 2 most immature subsets in the human thymus, namely CD34+ CD1a- and CD34+ CD1a+ thymocytes. DNA microarrays revealed the presence of several myeloid and erythroid transcripts in CD34+ CD1a- thymocytes but not in CD34+ CD1a+ thymocytes. Lineage potential of both subpopulations was assessed using in vitro colony assays, bone marrow stroma cultures, and in vivo transplantation into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. The CD34+ CD1a- subset contained progenitors with lymphoid (both T and B), myeloid, and erythroid lineage potential. Remarkably, development of CD34+ CD1a- thymocytes toward the T-cell lineage, as shown by T-cell receptor delta gene rearrangements, could be reversed into a myeloid-cell fate. In contrast, the CD34+ CD1a+ cells yielded only T-cell progenitors, demonstrating their irreversible commitment to the T-cell lineage. Both CD34+ CD1a- and CD34+ CD1a+ thymocytes failed to repopulate NOD/SCID mice. We conclude that the human thymus is seeded by multipotent progenitors with a much broader lineage potential than previously assumed. These cells resemble hematopoietic stem cells but, by analogy with murine thymocytes, apparently lack sufficient self-renewal capacity.

Wnt signaling in the thymus is regulated by differential expression of intracellular signaling molecules.

Wnt signaling is essential for T cell development in the thymus, but the stages in which it occurs and the molecular mechanisms underlying Wnt responsiveness have remained elusive. Here we examined Wnt signaling activity in both human and murine thymocyte populations by determining beta-catenin levels, Tcf-reporter activation and expression of Wnt-target genes. We demonstrate that Wnt signaling occurs in all thymocyte subsets, including the more mature populations, but most prominently in the double negative (DN) subsets. This differential sensitivity to Wnt signaling was not caused by differences in the presence of Wnts or Wnt receptors, as these appeared to be expressed at comparable levels in all thymocyte subsets. Rather, it can be explained by high expression of activating signaling molecules in DN cells, e.g., beta-catenin, plakoglobin, and long forms of Tcf-1, and by low levels of inhibitory molecules. By blocking Wnt signaling from the earliest stage onwards using overexpression of Dickkopf, we show that inhibition of the canonical Wnt pathway blocks development at the most immature DN1 stage. Thus, responsiveness to developmental signals can be regulated by differential expression of intracellular mediators rather than by abundance of receptors or ligands.

Ig gene rearrangement steps are initiated in early human precursor B cell subsets and correlate with specific transcription factor expression.

The role of specific transcription factors in the initiation and regulation of Ig gene rearrangements has been studied extensively in mouse models, but data on normal human precursor B cell differentiation are limited. We purified five human precursor B cell subsets, and assessed and quantified their IGH, IGK, and IGL gene rearrangement patterns and gene expression profiles. Pro-B cells already massively initiate D(H)-J(H) rearrangements, which are completed with V(H)-DJ(H) rearrangements in pre-B-I cells. Large cycling pre-B-II cells are selected for in-frame IGH gene rearrangements. The first IGK/IGL gene rearrangements were initiated in pre-B-I cells, but their frequency increased enormously in small pre-B-II cells, and in-frame selection was found in immature B cells. Transcripts of the RAG1 and RAG2 genes and earlier defined transcription factors, such as E2A, early B cell factor, E2-2, PAX5, and IRF4, were specifically up-regulated at stages undergoing Ig gene rearrangements. Based on the combined Ig gene rearrangement status and gene expression profiles of consecutive precursor B cell subsets, we identified 16 candidate genes involved in initiation and/or regulation of Ig gene rearrangements. These analyses provide new insights into early human precursor B cell differentiation steps and represent an excellent template for studies on oncogenic transformation in precursor B acute lymphoblastic leukemia and B cell differentiation blocks in primary Ab deficiencies.