Biomedical subjects

Antonius G Rolink

Publications and source records attributed to Antonius G Rolink.

At least 19 recordsLinked to original sources

PubMed2006

Early lymphocyte development in bone marrow and thymus.

Haematopoietic stem cells (HSCs), a very rare cell type in the bone marrow, are responsible for the life-long production of all cells of the blood including T and B cells. Until recently, it was thought that the differentiation of HSCs into the various haematopoietic cells was rather hierarchical in that differentiation along a given lineage was associated with a progressive loss of potential to give rise to other blood cell lineages. The recent development of very sensitive and quantitative in vitro assays, together with the identification of new progenitor subpopulations, has challenged this idea. Thus, lymphocyte progenitors can be shown to keep their developmental potential to give rise to myeloid, dendritic and NK cells until just prior to their final commitment stage. Here we review these new findings and concepts.

Animals↗

PubMed2006

Peripheral T cell lymphopenia and concomitant enrichment in naturally arising regulatory T cells: the case of the pre-Talpha gene-deleted mouse.

In pre-Talpha (pTalpha) gene-deleted mice, the positively selectable CD4+ CD8+ double-positive thymocyte pool is only 1% that in wild-type mice. Consequently, their peripheral T cell compartment is severely lymphopenic with a concomitant increase in proportion of CD25+ FoxP3+ regulatory T cells. Using mixed bone marrow chimeras, where thymic output was 1% normal, the pTalpha(-/-) peripheral T cell phenotype could be reproduced with normal cells. In the pTalpha(-/-) thymus and peripheral lymphoid organs, FoxP3+ CD4+ cells were enriched. Parabiosis experiments showed that many pTalpha(-/-) CD4+ single-positive thymocytes represented recirculating peripheral T cells. Therefore, the enrichment of FoxP3+ CD4+ single-positive thymocytes was not solely due to increased thymic production. Thus, the pTalpha(-/-) mouse serves as a model system with which to study the consequences of chronic decreased thymic T cell production on the physiology of the peripheral T cell compartment.

Animals↗

PubMed2006

The potential involvement of Notch signaling in NK cell development.

NK cells constitute an essential element of the innate immune system; however, the cellular and molecular mechanisms that guide their early development are still poorly understood. Here, we demonstrate that in addition to its known crucial role in T cell development, Notch signaling can also be involved in NK cell development. Thus, upon co-culture on OP9 stroma expressing the Notch ligand Delta-like 1 (OP9-DL1), Pax5-deficient pro-B cells, which have multi-lineage potential, efficiently differentiate into T and NK cells. Upon DL-1 signaling, Pax5-deficient pro-B cells down-regulate both surface CD93 expression and transcripts for B cell-specific genes and concomitantly up-regulate T lineage gene transcripts. Subsequent transfer of DL-1-signaled Pax5-deficient pro-B cells onto OP9 stroma in the presence of IL-2 leads to their efficient differentiation into NK1.1(+), functional NK cells. Moreover, bone marrow early progenitor with lymphoid and myeloid differentiation potential (EPLM), which we have previously described as the normal in vivo-equivalent of Pax5-deficient pro-B cells, also gain the ability to differentiate into effector NK cells following transient DL1 Notch-mediated signaling. The potential involvement of Notch signaling in the generation of the NK cell repertoire in vivo is discussed.

Animals↗

PubMed2006

Increasing Flt3L availability alters composition of a novel bone marrow lymphoid progenitor compartment.

We have recently described a CD19(-) B220(+)CD117(low) bone marrow subpopulation with B, T, and myeloid developmental potential, which we have called "early progenitors with lymphoid and myeloid potential" or EPLM. These cells also expressed Fms-like tyrosine kinase 3, Flt3, or CD135. Treatment of mice with the corresponding ligand, Flt3L, showed a 50-fold increase in EPLM. In addition to the expected increase in dendritic cell numbers, Flt3L treatment had a reversible inhibitory effect on B lymphopoiesis. Limiting dilution analysis of sorted EPLM from Flt3L-treated mice showed that B-lymphocyte progenitor activity was reduced 20-fold, but that myeloid and T-cell progenitor activity was largely preserved. EPLM from treated mice transiently reconstituted the thymus and bone marrow of recipient mice, generating cohorts of functional T and B cells in peripheral lymphoid organs. Thus, Flt3L treatment results in a dramatic increase in a novel bone marrow cell with lymphoid and myeloid progenitor activity.

Animals↗

PubMed2006

Critical role for c-kit (CD117) in T cell lineage commitment and early thymocyte development in vitro.

The precise roles played by the transmembrane receptor tyrosine kinase c-kit and its ligand stem cell factor in early T cell development are difficult to study. Using cloned Pax5-deficient progenitor B cells, we show that following Notch signaling, which induces their commitment to the T cell developmental pathway, c-kit expression is rapidly up-regulated at both the transcriptional and cell surface level. Using either an anti-c-kit monoclonal antibody or Gleevec, a pharmacological inhibitor of c-kit signaling, we show that the Notch-induced T cell differentiation of either Pax5-deficient progenitor B cells, or the equivalent cell from the bone marrow of normal mice, is strictly dependent on c-kit signaling, whereas the differentiation of normal progenitors into the B cell lineage is not. Moreover, we show that the Notch and IL-7 signaling-induced proliferation and differentiation of CD44+CD25-c-kit(high) and CD44+CD25+c-kithigh thymocytes along the T cell, but not natural killer cell or macrophage, pathway also requires c-kit signaling, whereas the Notch-induced proliferation and differentiation of CD44-CD25+c-kitint cells along the T cell pathway is independent of c-kit. These results further highlight the complex inter-relationships existing between c-kit, Notch and IL-7 receptor signaling that control the proliferation and differentiation of early T cell progenitors.

Animals↗

PubMed2006

Regulatory T cells control autoimmunity following syngeneic bone marrow transplantation.

Sublethally irradiated, immunodeficient, C57BL/6 RAG-2 gene-deleted recipient mice reconstituted with T cell-depleted bone marrow (BM) grafts frequently developed diarrhea, lost weight and showed signs of autoimmunity, dying between 4 and 7 weeks after reconstitution. Mice died despite evidence of efficient donor-derived hemato-lymphoid reconstitution, and disease was associated with the presence of IgG anti-nuclear antibodies. Autoimmunity was initiated by T cells, but could be prevented by transfer of naturally arising regulatory T cells. In contrast, lethally irradiated, BM-reconstituted immunocompetent, C57BL/6 mice survived without signs of autoimmunity. Survival of immunocompetent mice was shown to be due to the presence of residual, extra-thymically located, radio-resistant, functional regulatory T cells. The importance of regulatory T cells was further shown by the reduced survival of immunocompetent BM recipients whose CD25+ T cells had been depleted prior to bone marrow transplantation. The implications of these results in the context of syngeneic graft-versus-host disease following BM transplantation are discussed.

Animals↗

PubMed2005

Homeostatic and functional analysis of mature B cells in lambda5-deficient mice.

The peripheral B-cell pool is in dynamic equilibrium and is controlled by a variety of factors. The rate of generation of B cells can influence both the composition and size of the peripheral B-cell compartment. Mice deficient for lambda5 gene expression have a block in early B-cell development leading to a markedly reduced number of peripheral B cells. To address the question of how this early developmental block influences the composition of the B-cell pool, we have analyzed mature B-cell subpopulations in lambda5-deficient mice. In analogy with other situations of B lymphopenia, the proportion was increased but not the absolute number of marginal-zone B cells, whereas those of follicular B cells were decreased. Immunohistology revealed that B-cell follicles were smaller in overall size and contained a prominent B-cell replete marginal zone. BrdU labelling kinetics showed slower turnover of follicular as well as of marginal-zone B cells. Functionally, lambda5(-/-) mice were able to mount not only primary but also secondary thymus-dependent as well as thymus-independent responses, albeit mostly at reduced levels.

Animals↗

PubMed2005

The role of Notch and IL-7 signaling in early thymocyte proliferation and differentiation.

We have analyzed the roles of Notch and IL-7 signaling in the proliferation and differentiation of mouse progenitor thymocyte subpopulations cultured on Notch delta-like-1 ligand-expressing OP9 stromal cells. Using bulk and limiting dilution cultures, we show that DN1 and DN2 cells require both Notch and IL-7 signaling for efficient proliferation and differentiation into cytoplasmic TCRbeta and surface TCRalpha/beta and TCRgamma/delta expressing T cells. Selection for cytoplasmic TCRbeta-positive cells is dependent on preTalpha expression. Both gamma/delta and alpha/beta TCR expressing T cells arising in culture can be efficiently stimulated by anti-CD3 cross-linking, suggesting that they might be functional. The differentiation of adult, but not fetal, DN1 and DN2 thymocytes into CD4 and/or CD8 expressing cells is inhibited by IL-7. Finally, efficient proliferation and differentiation of DN3 cells requires Notch signaling and preTCR expression, but is independent of IL-7.

Cell Differentiation↗

PubMed2005

Rapid in vivo analysis of mutant forms of the LAT adaptor using Pax5-Lat double-deficient pro-B cells.

Following injection into recombinase-activating gene-deficient (Rag1(-/-)) mice, pro-B cells lacking the Pax5 transcription factor (Pax5(-/-)) develop into most major hematopoietic lineages, with the notable exception of B cells. We assessed whether Pax5(-/-) pro-B cells that were also rendered deficient for the linker for activation of T cells (LAT), an adaptor essential for T cell receptor signaling, can be used for the rapid in vivo analysis of mutant forms of LAT. We showed that Pax5(-/-) Lat(-/-) pro-B cell lines can be infected with recombinant retroviruses expressing a LAT cDNA and sorted for the expression of LAT. When injected into Rag1(-/-) mice, they restore normal intrathymic T cell development and give rise to functional peripheral T cells. Considering that the handling of Pax5(-/-) pro-B cell lines is easier than that of bone marrow hematopoietic precursors, we used them for the rapid functional analysis of a novel Lat allelic series. When compared to knock-in and transgenic approaches, a major advantage of our Pax5(-/-) pro-B cell-based experimental approach consists in the production of mice bearing a given mutation within 2-3 months. Therefore, it constitutes a powerful first-line screen for mutations worth fastidious knock-in approaches.

Adaptor Proteins, Signal Transducing↗

PubMed2005

A B220+ CD117+ CD19- hematopoietic progenitor with potent lymphoid and myeloid developmental potential.

In this report, we identify in the bone marrow (BM) of normal mice a subpopulation of B220+ CD117+ CD19- NK1.1- cells with potent lymphoid and myeloid developmental potential. These cells represent 0.1-0.2% of nucleated BM cells. By limiting dilution analysis in the presence of the appropriate combination of stromal cells and cytokines, 1 in 5-10 sorted cells formed B cells, 1 in 10-15 formed T cells and 1 in 5-10 generated macrophages. When cultured on a mixture of OP9 stroma and OP9 stromal cells expressing the Notch ligand Delta-like-1, single cells generated both T and B cells. Following intravenous infusion, freshly sorted cells transiently reconstituted both the T and B cell progenitor compartments, generating cohorts of mature T and B lymphocytes. The relationship between B220+ CD117+ CD19- NK1.1- cells of wild-type mice and other multi-lineage BM progenitors is discussed.

Animals↗

PubMed2005

Transcriptional networks in developing and mature B cells.

The development of B cells from haematopoietic stem cells proceeds along a highly ordered, yet flexible, pathway. At multiple steps along this pathway, cells are instructed by transcription factors on how to further differentiate, and several check-points have been identified. These check-points are initial commitment to lymphocytic progenitors, specification of pre-B cells, entry to the peripheral B-cell pool, maturation of B cells and differentiation into plasma cells. At each of these regulatory nodes, there are transcriptional networks that control the outcome, and much progress has recently been made in dissecting these networks. This article reviews our current understanding of this exciting field.

Animals↗

PubMed2004

The earliest subpopulation of mouse thymocytes contains potent T, significant macrophage, and natural killer cell but no B-lymphocyte potential.

The B-lymphocyte potential of progenitor thymocytes and whether the thymus is colonized by common lymphocyte progenitor cells have been subjects of considerable debate. Herein, we have used limiting dilution analysis to determine the lineage potential of phenotypically defined subpopulations of CD4-CD8- double-negative thymocytes. Culture systems used showed single-hit kinetics and had a high plating efficiency for B-, myeloid, and natural killer cell development. The T-cell potential of sorted cells was confirmed by transferring cells to fetal thymus organ cultures. Our results indicate that the earliest population of CD117+ double-negative cells, although containing potent T-cell developmental potential and significant myeloid and natural killer potential, does not have any residual B-cell potential. Gene transcription analysis also indicated that these double-negative cells contained abundant T and myeloid, but not B cell-specific transcripts. The implications of these results within the context of current models of thymocyte development are discussed.

Animals↗

PubMed2004

The anti-apoptotic factor Bcl-2 can functionally substitute for the B cell survival but not for the marginal zone B cell differentiation activity of BAFF.

The TNF family ligand B cell-activating factor (BAFF, BLyS, TALL-1) is an essential factor for B cell development. BAFF binds to three receptors, BAFF-R, transmembrane activator and CAML interactor (TACI), and B cell maturation antigen (BCMA), but only BAFF-R is required for successful survival and maturation of splenic B cells. To test whether the effect of BAFF is due to the up-regulation of anti-apoptotic factors, TACI-Ig-transgenic mice, in which BAFF function is inhibited, were crossed with transgenic mice expressing FLICE-inhibitory protein (FLIP) or Bcl-2 in the B cell compartment. FLIP expression did not rescue B cells, while enforced Bcl-2 expression restored peripheral B cells and the ability to mount T-dependent antibody responses. However, many B cells retained immaturity markers and failed to express normal amounts of CD21. Marginal zone B cells were not restored and the T-independent IgG3, but not IgM, response was impaired in the TACI-IgxBcl-2 mice. These results suggest that BAFF is required not only to inhibit apoptosis of maturating B cells, but also to promote differentiation events, in particular those leading to the generation of marginal zone B cells.

Animals↗

PubMed2004

Molecular mechanisms guiding late stages of B-cell development.

In mice, large numbers of immature B cells are continuously produced in the bone marrow. To enter the pools of mature B cells, these immature B cells have to pass two checkpoints. First, B cells have to migrate from the bone marrow to the spleen. The second checkpoint involves the immature B cells differentiating to mature B cells within the spleen. As the net result of this selection and maturation, only a fraction of the newly produced B cells enters the mature B-cell pool. Recent advances in the understanding of the molecular mechanisms that operate at these two checkpoints are described and discussed.

Animals↗

PubMed2004

B-cell development and pre-B-1 cell plasticity in vitro.

B-lymphopoiesis in vivo is a very complex process that is influenced by cooperation between cells, cytokines, and other receptor-ligand interactions, which developmentally occur at different cellular stages. Various in vitro models have been very useful in unraveling this complex process of B-cell development. Here the protocols for how to grow and to study the differentiation of mouse pre-B-1, as developed in our laboratory, are described. Moreover, the protocols of how to grow and to test the plasticity of Pax-5-deficient pre-B-1 cells are also outlined.

Animals↗

PubMed2002

In vitro and in vivo plasticity of Pax5-deficient pre-B I cells.

In mice deficient for the transcription factor Pax-5, B cell development is blocked at the pre-B I cell stage. Like wild type, Pax-5-/- pre-B I cells can be grown long-term in vitro in the presence of stromal cells and IL-7. However, unlike their wild type in vitro grown counterparts, Pax-5-/- pre-B I cells posses an extraordinary developmental plasticity showing hematopoeitic stem cell features such as multipotency and self renewing capacity. Here we review and discuss this in vitro and in vivo plasticity of Pax-5-/- pre-B I cells.

Animals↗

PubMed2002

Extensive in vivo self-renewal, long-term reconstitution capacity, and hematopoietic multipotency of Pax5-deficient precursor B-cell clones.

Self-renewal, pluripotency, and long-term reconstitution are defining characteristics of single hematopoietic stem cells. Pax5(-/-) precursor B cells apparently possess similar characteristics. Here, using serial transplantations, with in vitro recloning and growth of the bone marrow-homed donor cells occurring after all transplantations, we analyzed the extent of self-renewal and hematopoietic multipotency of Pax5(-/-) precursor B-cell clones. Moreover, telomere length and telomerase activity in these clones was analyzed at various time points. Thus far, 5 successive transplantations have been performed. Clones transplanted for the fifth time, which have proliferated for more than 150 cell divisions in vitro, still repopulate the bone marrow with precursor B cells and reconstitute these recipients with lymphoid and myeloid cells. During this extensive proliferation, Pax5(-/-) precursor B cells shorten their telomeres at 70 to 90 base pairs per division. Their telomerase activity remains at 3% of that of HEK293 cancer cells during all serial in vivo transplantations/in vitro expansions. Together, these data show that Pax5(-/-) precursor B-cell clones possess extensive in vivo self-renewal capacity, long-term reconstitution capacity, and hematopoietic multipotency, with their telomeres shortening at the normal rate.

Animals↗

PubMed2002

Multiple hematopoietic cell lineages develop in vivo from transplanted Pax5-deficient pre-B I-cell clones.

Pax5-deficient pre-B I-cell clones, transplanted into natural killer (NK)-cell-deficient RAG2(-/-) IL-2Rgamma(-/-) hosts, populate the NK-cell compartment with functional NK cells. NK-cell generation from Pax5(-/-) pre-B I cells is also observed in NK-cell-proficient Balb/c RAG2(-/-) hosts. In the same Balb/c RAG2(-/-) hosts, Pax5(-/-) pre-B I-cell clones not only populate the pre-B I-cell compartment and fill the deficient T-cell-lineage compartment in the thymus and the periphery of all hosts, as shown before, they also generate CD8alpha(-) and CD8alpha(+) dendritic cells (DCs), macrophages, and granulocytes in vivo in approximately half the hosts. In some recipients, practically all the mature myeloid cells are of Pax5(-/-) origin, indicating the effectiveness by which Pax5(-/-) pre-B I cells can compete with endogenous myeloid precursors. In a smaller percentage of hosts, the generation of Pax5(-/-) pre-B I-cell-derived erythrocytes is observed 4 to 6 months after transplantation. The results indicate that Pax5(-/-) pre-B I cells can develop in vivo in hosts that have undergone transplantation to erythroid, myeloid, and lymphoid cell lineages. Hence, the Pax5(-/-) mutation introduces an unusual instability of differentiation in pre-B I cells so that they appear to dedifferentiate as far back as the pluripotent hematopoietic stem cell.

Animals↗