Miranda R. M. Baert

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.

Gene-expression profiling of CD34+ cells from various hematopoietic stem-cell sources reveals functional differences in stem-cell activity

The replacement of bone marrow (BM) as a conventional source of stem cell (SC) by umbilical cord blood (UCB) and granulocyte‐colony stimulating factor‐mobilized peripheral blood SC (PBSC) has brought about clinical advantages. However, several studies have demonstrated that UCB CD34+ cells and PBSC significantly differ from BM CD34+ cells qualitatively and quantitatively. Here, we quantified the number of SC in purified BM, UCB CD34+ cells, and CD34+ PBSC using in vitro and in vivo assays for human hematopoietic SC (HSC) activity. A cobblestone area‐forming cell (CAFC) assay showed that UCB CD34+ cells contained the highest frequency of CAFCwk6 (3.6‐ to tenfold higher than BM CD34+ cells and PBSC, respectively), and the engraftment capacity in vivo by nonobese diabetic/severe combined immunodeficiency repopulation assay was also significantly greater than BM CD34+, with a higher proportion of CD45+ cells detected in the recipients at a lower cell dose. To understand the molecular characteristics underlying these functional differences, we performed several DNA microarray experiments using Affymetrix gene chips, containing 12,600 genes. Comparative analysis of gene‐expression profiles showed differential expression of 51 genes between BM and UCB CD34+ SC and 64 genes between BM CD34+ cells and PBSC. These genes are involved in proliferation, differentiation, apoptosis, and engraftment capacity of SC. Thus, the molecular expression profiles reported here confirmed functional differences observed among the SC sources. Moreover, this report provides new insights to describe the molecular phenotype of CD34+ HSC and leads to a better understanding of the discrepancy among the SC sources.

Ectopic retroviral expression of LMO2, but not IL2Rγ, blocks human T-cell development from CD34+ cells: Implications for leukemogenesis in gene therapy

The occurrence of leukemia in a gene therapy trial for SCID-X1 has highlighted insertional mutagenesis as an adverse effect. Although retroviral integration near the T-cell acute lymphoblastic leukemia (T-ALL) oncogene LIM-only protein 2 (LMO2) appears to be a common event, it is unclear why LMO2 was preferentially targeted. We show that of classical T-ALL oncogenes, LMO2 is most highly transcribed in CD34+ progenitor cells. Upon stimulation with growth factors typically used in gene therapy protocols transcription of LMO2, LYL1, TAL1 and TAN1 is most prominent. Therefore, these oncogenes may be susceptible to viral integration. The interleukin-2 receptor gamma chain (IL2Rγ), which is mutated in SCID-X1, has been proposed as a cooperating oncogene to LMO2. However, we found that overexpressing IL2Rγ had no effect on T-cell development. In contrast, retroviral overexpression of LMO2 in CD34+ cells caused severe abnormalities in T-cell development, but B-cell and myeloid development remained unaffected. Our data help explain why LMO2 was preferentially targeted over many of the other known T-ALL oncogenes. Furthermore, during T-cell development retrovirus-mediated expression of IL2Rγ may not be directly oncogenic. Instead, restoration of normal IL7-receptor signaling may allow progression of T-cell development to stages where ectopic LMO2 expression causes aberrant thymocyte growth.

Gene therapy: is IL2RG oncogenic in T-cell development?

Arising from: N.-B. Woods, V. Bottero, M. Schmidt, C. von Kalle & I. M. Verma 440, 1123 (2006); see also communication from Thrasher et al.; Woods et al. replyThe gene IL2RG encodes the γ-chain of the interleukin-2 receptor and is mutated in patients with X-linked severe combined immune deficiency (X-SCID). Woods et al. report the development of thymus tumours in a mouse model of X-SCID after correction by lentiviral overexpression of IL2RG and claim that these were caused by IL2RG itself. Here we find that retroviral overexpression of IL2RG in human CD34+ cells has no effect on T-cell development, whereas overexpression of the T-cell acute lymphoblastic leukaemia (T-ALL) oncogene LMO2 leads to severe abnormalities. Retroviral expression of IL2RG may therefore not be directly oncogenic — rather, the restoration of normal signalling by the interleukin-7 receptor to X-SCID precursor cells allows progression of T-cell development to stages that are permissive for the pro-leukaemic effects of ectopic LMO2.

Correction of murine Rag1 deficiency by self-inactivating lentiviral vector-mediated gene transfer

Severe combined immunodeficiency (SCID) patients with an inactivating mutation in recombination activation gene 1 (RAG1) lack B and T cells due to the inability to rearrange immunoglobulin (Ig) and T-cell receptor (TCR) genes. Gene therapy is a valid treatment option for RAG-SCID patients, especially for patients lacking a suitable bone marrow donor, but developing such therapy has proven challenging. As a preclinical model for RAG-SCID, we used Rag1−/− mice and lentiviral self-inactivating (SIN) vectors harboring different internal elements to deliver native or codon-optimized human RAG1 sequences. Treatment resulted in the appearance of B and T cells in peripheral blood and developing B and T cells were detected in central lymphoid organs. Serum Ig levels and Ig and TCR Vβ gene segment usage was comparable to wild-type (WT) controls, indicating that RAG-mediated rearrangement took place. Remarkably, relatively low frequencies of B cells produced WT levels of serum immunoglobulins. Upon stimulation of the TCR, corrected spleen cells proliferated and produced cytokines. In vivo challenge resulted in production of antigen-specific antibodies. No leukemia development as consequence of insertional mutagenesis was observed. The functional reconstitution of the B- as well as the T-cell compartment provides proof-of-principle for therapeutic RAG1 gene transfer in Rag1−/− mice using lentiviral SIN vectors.

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.

350. Ectopic Retroviral Expression of LMO2, but Not IL2Rγ, Blocks Human T-Cell Development from CD34+ Progenitor Cells: Implications for Gene Therapy

The occurrence of leukemia in 3 out of 11 patients in a gene therapy trial for SCID-X1 emphasized insertional mutagenesis as an adverse effect. In two out of three patients retroviral integration occurred near the T-cell acute lymphoblastic leukemia (T-ALL) oncogene LIM-only protein 2 (LMO2). It has remained unclear why LMO2 was preferentially targeted. We show here that of classical T-ALL oncogenes, mainly LMO2 is transcribed in CD34+ progenitor cells derived from umbilical cord blood (UCB). Upon stimulation with growth factors typically used in gene therapy protocols, transcription of LMO2, LYL1 and TAL1 increased 2|[ndash]|3 fold in CD34+ UCB cells, whereas the other T-ALL oncogenes remained unaffected. Similarly, when mobilized peripheral blood CD34+ cells were stimulated according to transduction protocols used for clinical SCID-X1 gene therapy trials, the same pattern of T-ALL oncogene transcription was observed, suggesting that cytokine stimulation leads to upregulation of proto-oncogenes irrespective of the source of CD34+ progenitor cells used. Transcription of LMO2, LYL1 and TAL1 is particularly high and their loci could therefore be preferentially accessible for viral integration. Retroviral overexpression of LMO2 in CD34+ cells led to severe abnormalities in human T-cell development, but B-cell, NK cell and myeloid development remained unaffected. The interleukin-2 receptor gamma chain (IL2R|[gamma]|), causing SCID-X1 when mutated, has been proposed to act as an oncogene cooperative to LMO2. However, we found that overexpression of IL2R|[gamma]| had no effect on T-cell development. Moreover, IL2R|[gamma]| normally is expressed at high levels in CD34+ progenitor cells and at even higher levels in the thymus. Thus, transduction of IL2R|[gamma]| in CD34+ progenitor cells does not cause ectopic expression of this gene. In conclusion, our data provide an explanation why LMO2 was preferentially targeted over other known T-ALL oncogenes. Furthermore, IL2R|[gamma]| does not appear to act as an oncogene. Rather, the normal IL2R|[gamma]| chain restores IL7 receptor signalling in the progeny of transduced SCID-X1 cells developing in the thymus, allowing T-cell development to progress to stages where ectopic LMO2 expression hampers T-cell development in the thymus, creating a probable pre-leukemic condition by accumulation of immature cells under intense proliferative pressure.