Edwin F. E. de Haas

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 Dδ2-Dδ3 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 (pTα) expression pattern show that human TCRβ-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.

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 DH-JH rearrangements, which are completed with VH-DJH 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.

Wnt Target Genes Identified by DNA Microarrays in Immature CD34+ Thymocytes Regulate Proliferation and Cell Adhesion

The thymus is seeded by very small numbers of progenitor cells that undergo massive proliferation before differentiation and rearrangement of TCR genes occurs. Various signals mediate proliferation and differentiation of these cells, including Wnt signals. Wnt signals induce the interaction of the cytoplasmic cofactor β-catenin with nuclear T cell factor (TCF) transcription factors. We identified target genes of the Wnt/β-catenin/TCF pathway in the most immature (CD4−CD8−CD34+) thymocytes using Affymetrix DNA microarrays in combination with three different functional assays for in vitro induction of Wnt signaling. A relatively small number (∼30) of genes changed expression, including several proliferation-inducing transcription factors such as c-fos and c-jun, protein phosphatases, and adhesion molecules, but no genes involved in differentiation to mature T cell stages. The adhesion molecules likely confine the proliferating immature thymocytes to the appropriate anatomical sites in the thymus. For several of these target genes, we validated that they are true Wnt/β-catenin/TCF target genes using real-time quantitative PCR and reporter gene assays. The same core set of genes was repressed in Tcf-1-null mice, explaining the block in early thymocyte development in these mice. In conclusion, Wnt signals mediate proliferation and cell adhesion, but not differentiation of the immature thymic progenitor 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‐β 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‐β/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‐γ production. GATA3 additionally constrained Th17 differentiation in vitro through IL‐4‐independent mechanisms, involving downregulating transcription of STAT3, STAT4, NFATc2 and the nuclear factor RORγt, 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.

Sustained cross-presentation capacity of murine splenic dendritic cell subsets in vivo: Nataschja I. Ho et al.

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.