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What’s in a name?: Sub-fractionation of common lymphoid progenitors
Linköping University, Department of Clinical and Experimental Medicine, Experimental Hematology . Linköping University, Faculty of Health Sciences.
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The hematopoietic system is a highly dynamic organ developed in many multi-cellular organisms to provide oxygen, prevent bleeding and to protect against microorganisms. The blood consist of many different specialized cells that all derive from rare hematopoietic stem cells (HSCs) located in the bone marrow in mice and humans. Blood cell production from HSCs occurs in a stepwise manner through development of intermediate progenitors that gradually loose lineage potentials. This is a tightly regulated process with complex regulatory mechanisms and many checkpoints that ensure a high and balanced production of blood cells. One of the fundamental questions in hematopoiesis relates to how the maturation of the cells is controlled and driven towards defined cell fates. The understanding of these processes is largely facilitated by isolation of intermediate populations of cells at defined stages of development.

This thesis is focused on the regulatory mechanisms that regulate the maturation of B-lymphocytes constituting an important part of adaptive immunity by being responsible for the production of antibodies. It has been suggested that all the lymphoid cells have a common lineage restricted ancestor defined as a Lin-KitloSca1loFlt3+IL7R+ common lymphoid progenitor (CLP). These cells are believed retain the combined potentials for B, T and NK cells and it has been presumed that commitment of CLPs to B lineage is associated with expression of CD19 and B220 on progenitor B-cells.

The aim of this thesis has been to identify the point of no return in B-cell development in order to allow for a better understanding of lineage restriction events in early lymphopoesis.

To this end, we have used reporter transgenic mice where marker gene expression has been controlled by the transcription regulatory elements from one early lymphoid marker (Rag1) and one B-lymphoid restricted gene (λ5, Igll1). This allowed us to identify three functionally distinct sub-populations within the conventional CLP compartment. The cells were identified as CLPRaglowλ5- cells retaining B, T, Nk and a limited myeloid potential while up-regulation of Rag1 to generate CLPRaghighλ5- cells, was associated with loss of Nk potential as well as of the residual myeloid potential. Ultimately expression of λ5 in the CLPRag1highλ5+ compartment identifies the first committed B cells. Hence, our data suggest that the point of no return in B-cell development can be found within the CD19- CLP compartment. Using this new model for B-cell development, we investigated the instructive vs. permissive role of IL7 signaling in B cell commitment. Our results show that in absence of IL7, CLP maturation is impaired and generation of the earliest committed B-lineage cells is severely impaired. CLP maturation could not be rescued by ectopic expression of the anti-apoptotic Bcl2 protein even though the cells were able to generate normal B lineage cells after restoration of the IL7 signal. These findings suggest that Il7 is crucial for the maturation of lineage restricted CLPs and provide support for an instructive role of IL7 in early Bcell development.

This thesis highlights the importance of precise identification of the point of commitment in B cell development and provides insight to the hematopoietic hierarchical model with the potential to serve as a map to better understand the mechanisms of hematopoietic disorders.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press , 2010. , 57 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1214
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-61590ISBN: 978-91-7393-284-4 (print)OAI: oai:DiVA.org:liu-61590DiVA: diva2:370521
Public defence
2010-11-29, Linden, Hälsouniversitetet, Campus US, Linköpings Universitet, Linköping, 13:00 (English)
Opponent
Supervisors
Available from: 2010-11-17 Created: 2010-11-17 Last updated: 2010-12-14Bibliographically approved
List of papers
1. EBF1 is essential for B-lineage priming and establishment of a transcription factor network in common lymphoid progenitors
Open this publication in new window or tab >>EBF1 is essential for B-lineage priming and establishment of a transcription factor network in common lymphoid progenitors
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2008 (English)In: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 181, no 5, 3364-3372 p.Article in journal (Refereed) Published
Abstract [en]

Development of B-lymphoid cells in the bone marrow is a process under strict control of a hierarchy of transcription factors. To understand the development of a B-lymphoid-restricted functional network of transcription factors, we have investigated the cell autonomous role of the transcription factor EBF1 in early B cell development. This revealed that even though transplanted EBF1-deficient fetal liver cells were able to generate common lymphoid progenitors (CLPs) as well as B220(+)CD43(+)AA4.1(+) candidate precursor B cells, none of these populations showed signs of B lineage priming. The isolated CLPs were able to generate T lymphocytes in vitro supporting the idea that the phenotype of EBF1-deficient mice is restricted to the development of the B lineage. Furthermore, EBF deficient CLPs displayed a reduction in Ig H chain recombination as compared with their wild-type counterpart and essentially lacked transcription of B-lineage-associated genes. Among the genes displaying reduced expression in the EBF1 deficient CLPs were the transcription factors Pax5, Pou2af1 (OcaB), and FoxO1 that all appear to be direct genetic targets for EBF1 because their promoters contained functional binding sites for this factor. This leads us to suggest that EBF1 regulates a transcription factor network crucial for B lineage commitment.

Keyword
Animals B-Lymphocytes/*cytology Cell Lineage Down-Regulation *Gene Regulatory Networks Lymphoid Progenitor Cells/*cytology Mice Precursor Cells, B-Lymphoid/cytology T-Lymphocytes/cytology Trans-Activators/*physiology Transcription Factors/genetics
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-43426 (URN)73828 (Local ID)73828 (Archive number)73828 (OAI)
Available from: 2009-10-10 Created: 2009-10-10 Last updated: 2017-12-13
2. B-lineage commitment prior to surface expression of B220 and CD19 on hematopoietic progenitor cells
Open this publication in new window or tab >>B-lineage commitment prior to surface expression of B220 and CD19 on hematopoietic progenitor cells
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2008 (English)In: Blood, ISSN 0006-4971, E-ISSN 1528-0020, Vol. 112, no 4, 1048-1055 p.Article in journal (Refereed) Published
Abstract [en]

Commitment of hematopoietic progenitor cells to B-lymphoid cell fate has been suggested to coincide with the development of PAX5-expressing B220 +CD19+ pro-B cells. We have used a transgenic reporter mouse, expressing human CD25 under the control of the B-lineage- restricted IgII1 (λ5) promoter to investigate the lineage potential of early progenitor cells in the bone marrow. This strategy allowed us to identify a reporter expressing LIN-B220- CD19-CD127 +FLT3+ SCA1lowKITlow population that displays a lack of myeloid and a 90% reduction in in vitro T-cell potential compared with its reporter-negative counterpart. Gene expression analysis demonstrated that these lineage-restricted cells express B- lineage-associated genes to levels comparable with that observed in pro-B cells. These data suggest that B-lineage commitment can occur before the expression of B220 and CD19. © 2008 by The American Society of Hematology.

Keyword
Animals Antigens, CD19/*analysis Antigens, CD45/*analysis B-Lymphocytes/chemistry/*cytology Bone Marrow Cells *Cell Lineage Hematopoietic Stem Cells/chemistry/*cytology Humans Interleukin-2 Receptor alpha Subunit Mice Mice, Inbred C57BL Mice, Transgenic
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-43397 (URN)10.1182/blood-2007-11-125385 (DOI)73745 (Local ID)73745 (Archive number)73745 (OAI)
Available from: 2009-10-10 Created: 2009-10-10 Last updated: 2017-12-13
3. Single-cell analysis of the common lymphoid progenitor compartment reveals functional and molecular heterogeneity
Open this publication in new window or tab >>Single-cell analysis of the common lymphoid progenitor compartment reveals functional and molecular heterogeneity
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2010 (English)In: Blood, ISSN 0006-4971, E-ISSN 1528-0020, Vol. 115, no 13, 2601-2609 p.Article in journal (Refereed) Published
Abstract [en]

To investigate molecular events involved in the regulation of lymphoid lineage commitment, we crossed lambda 5 reporter transgenic mice to Rag1-GFP knockin mice. This allowed us to subfractionate common lymphoid progenitors and pre-pro-B (fraction A) cells into lambda 5(-)Rag1(low), lambda 5(-)Rag1(high), and lambda 5(+)Rag1(high) cells. Clonal in vitro differentiation analysis demonstrated that Rag1(low) cells gave rise to B/T and NK cells. Rag1(high) cells displayed reduced NK-cell potential with preserved capacity to generate B- and T-lineage cells, whereas the lambda 5(+) cells were B-lineage restricted. Ebf1 and Pax5 expression was largely confined to the Rag1high populations. These cells also expressed a higher level of the surface protein LY6D, providing an additional tool for the analysis of early lymphoid development. These data suggest that the classic common lymphoid progenitor compartment composes a mixture of cells with relatively restricted lineage potentials, thus opening new possibilities to investigate early hematopoiesis.

Place, publisher, year, edition, pages
American Society of Hematology, 2010
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-54870 (URN)10.1182/blood-2009-08-236398 (DOI)000276201000009 ()
Available from: 2010-04-16 Created: 2010-04-16 Last updated: 2017-12-12
4. Functional and molecular analysis of B-lineage commitment suggests an instructive role for Il-7 in the earliest lymphoid restricted cells
Open this publication in new window or tab >>Functional and molecular analysis of B-lineage commitment suggests an instructive role for Il-7 in the earliest lymphoid restricted cells
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Deficiencies in the Il-7 signaling pathway result in severe disruptions of lymphoid development in adult mice. In order to understand how Il-7 deficiency impacts early lymphoid development we have investigated lineage restriction events within the CLP compartment of Il-7 knock out mice. This revealed that while Il-7 deficiency had a minor impact on the development and functional properties of LY6D- multipotent CLPs, the formation of the lineage restricted LY6D+ CLP population was dramatically reduced. This was reflected in a low level transcription of B-lineage genes as well as in a loss of functional B-cell commitment in developing progenitors. The defect could not be rescued by ectopic expression of Bcl-2 suggesting that the cytokine act in an instructive manner in early lymphoid development. This clarifies the role of Il-7 in early lymphoid development and puts emphasis on the relevance of the recently defined lineage restricted progenitor cells in lymphoid differentiation.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-61589 (URN)
Available from: 2010-11-17 Created: 2010-11-17 Last updated: 2010-11-17

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