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.

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Early human T cell development: analysis of the human thymus at the time of initial entry of hematopoietic stem cells into the fetal thymic microenvironment.

Journal of Experimental Medicine ◽

10.1084/jem.181.4.1445 ◽

1995 ◽

Vol 181 (4) ◽

pp. 1445-1458 ◽

Cited By ~ 96

Author(s):

B F Haynes ◽

C S Heinly

Keyword(s):

Stem Cells ◽

T Cell ◽

Hematopoietic Stem Cells ◽

T Cell Development ◽

Fold Increase ◽

Cell Receptor ◽

Cell Development ◽

Hematopoietic Stem ◽

Human Thymus ◽

Human T Cell

To determine events that transpire during the earliest stages of human T cell development, we have studied fetal tissues before (7 wk), during (8.2 wk), and after (9.5 wk to birth) colonization of the fetal thymic rudiment with hematopoietic stem cells. Calculation of the approximate volumes of the 7- and 8.2-wk thymuses revealed a 35-fold increase in thymic volumes during this time, with 7-wk thymus height of 160 microM and volume of 0.008 mm3, and 8.2-wk thymus height of 1044 microM and volume of 0.296 mm3. Human thymocytes in the 8.2-wk thymus were CD4+ CD8 alpha+ and cytoplasmic CD3 epsilon+ cCD3 delta+ CD8 beta- and CD3 zetta-. Only 5% of 8-wk thymocytes were T cell receptor (TCR)-beta+, < 0.1% were TCR-gamma+, and none reacted with monoclonal antibodies against TCR-delta. During the first 16 wk of gestation, we observed developmentally regulated expression of CD2 and CD8 beta (appearing at 9.5 wk), CD1a,b, and c molecules (CD1b, then CD1c, then CD1a), TCR molecules (TCR-beta, then TCR-delta), CD45RA and CD45RO isoforms, CD28 (10 wk), CD3 zeta (12-13 wk), and CD6 (12,75 wk). Whereas CD2 was not expressed at the time of initiation of thymic lymphopoiesis, a second CD58 ligand, CD48, was expressed at 8.2 wk, suggesting a role for CD48 early in thymic development. Taken together, these data define sequential phenotypic and morphologic changes that occur in human thymus coincident with thymus colonization by hematopoietic stem cells and provide insight into the molecules that are involved in the earliest stages of human T cell development.

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Disregulated expression of the transcription factor ThPOK during T-cell development leads to high incidence of T-cell lymphomas

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1424104112 ◽

2015 ◽

Vol 112 (25) ◽

pp. 7773-7778 ◽

Cited By ~ 9

Author(s):

Hyung-Ok Lee ◽

Xiao He ◽

Jayati Mookerjee-Basu ◽

Dai Zhongping ◽

Xiang Hua ◽

...

Keyword(s):

Transcription Factor ◽

T Cell ◽

Lymphoblastic Leukemia ◽

T Cell Development ◽

Cell Receptor ◽

Cell Development ◽

Thymocyte Development ◽

Tcr Signaling ◽

Double Positive ◽

Human T Cell

The transcription factor T-helper-inducing POZ/Krueppel-like factor (ThPOK, encoded by the Zbtb7b gene) plays widespread and critical roles in T-cell development, particularly as the master regulator of CD4 commitment. Here we show that mice expressing a constitutive T-cell–specific ThPOK transgene (ThPOKconst mice) develop thymic lymphomas. These tumors resemble human T-cell acute lymphoblastic leukemia (T-ALL), in that they predominantly exhibit activating Notch1 mutations. Lymphomagenesis is prevented if thymocyte development is arrested at the DN3 stage by recombination-activating gene (RAG) deficiency, but restored by introduction of a T-cell receptor (TCR) transgene or by a single injection of anti-αβTCR antibody into ThPOKconst RAG-deficient mice, which promotes development to the CD4+8+ (DP) stage. Hence, TCR signals and/or traversal of the DN (double negative) > DP (double positive) checkpoint are required for ThPOK-mediated lymphomagenesis. These results demonstrate a novel link between ThPOK, TCR signaling, and lymphomagenesis. Finally, we present evidence that ectopic ThPOK expression gives rise to a preleukemic and self-perpetuating DN4 lymphoma precursor population. Our results collectively define a novel role for ThPOK as an oncogene and precisely map the stage in thymopoiesis susceptible to ThPOK-dependent tumor initiation.

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Modeling of human T cell development in vitro as a read-out for hematopoietic stem cell multipotency

Biochemical Society Transactions ◽

10.1042/bst20210144 ◽

2021 ◽

Author(s):

Steven Strubbe ◽

Tom Taghon

Keyword(s):

Stem Cell ◽

T Cell ◽

Hematopoietic Stem Cell ◽

T Cell Development ◽

Adult Life ◽

Cell Development ◽

In Vitro Models ◽

Hematopoietic Stem ◽

Human T Cell

Hematopoietic stem cells (HSCs) reside in distinct sites throughout fetal and adult life and give rise to all cells of the hematopoietic system. Because of their multipotency, HSCs are capable of curing a wide variety of blood disorders through hematopoietic stem cell transplantation (HSCT). However, due to HSC heterogeneity, site-specific ontogeny and current limitations in generating and expanding HSCs in vitro, their broad use in clinical practice remains challenging. To assess HSC multipotency, evaluation of their capacity to generate T lymphocytes has been regarded as a valid read-out. Several in vitro models of T cell development have been established which are able to induce T-lineage differentiation from different hematopoietic precursors, although with variable efficiency. Here, we review the potential of human HSCs from various sources to generate T-lineage cells using these different models in order to address the use of both HSCs and T cell precursors in the clinic.

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Separation of Notch1 Promoted Lineage Commitment and Expansion/Transformation in Developing T Cells

Journal of Experimental Medicine ◽

10.1084/jem.194.1.99 ◽

2001 ◽

Vol 194 (1) ◽

pp. 99-106 ◽

Cited By ~ 121

Author(s):

David Allman ◽

Fredrick G. Karnell ◽

Jennifer A. Punt ◽

Sonia Bakkour ◽

Lanwei Xu ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Development ◽

Cell Receptor ◽

Cell Development ◽

Surface Proteins ◽

Lineage Commitment ◽

Double Positive ◽

Hematopoietic Stem ◽

Multipotent Progenitor Cells

Notch1 signaling is required for T cell development. We have previously demonstrated that expression of a dominant active Notch1 (ICN1) transgene in hematopoietic stem cells (HSCs) leads to thymic-independent development of CD4+CD8+ double-positive (DP) T cells in the bone marrow (BM). To understand the function of Notch1 in early stages of T cell development, we assessed the ability of ICN1 to induce extrathymic T lineage commitment in BM progenitors from mice that varied in their capacity to form a functional pre-T cell receptor (TCR). Whereas mice repopulated with ICN1 transduced HSCs from either recombinase deficient (Rag-2−/−) or Src homology 2 domain–containing leukocyte protein of 76 kD (SLP-76)−/− mice failed to develop DP BM cells, recipients of ICN1-transduced Rag-2−/− progenitors contained two novel BM cell populations indicative of pre-DP T cell development. These novel BM populations are characterized by their expression of CD3ε and pre-Tα mRNA and the surface proteins CD44 and CD25. In contrast, complementation of Rag-2−/− mice with a TCRβ transgene restored ICN1-induced DP development in the BM within 3 wk after BM transfer (BMT). At later time points, this population selectively and consistently gave rise to T cell leukemia. These findings demonstrate that Notch signaling directs T lineage commitment from multipotent progenitor cells; however, both expansion and leukemic transformation of this population are dependent on T cell–specific signals associated with development of DP thymocytes.

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Expression of T Cell Receptor Associated Proteins During Human T Cell Development

Progress in Immunology ◽

10.1007/978-3-642-83755-5_170 ◽

1989 ◽

pp. 1276-1279 ◽

Cited By ~ 1

Author(s):

D. Campana ◽

E. Coustan-Smith ◽

L. Wong ◽

G. Janossy

Keyword(s):

T Cell ◽

T Cell Receptor ◽

T Cell Development ◽

Cell Receptor ◽

Cell Development ◽

Human T Cell ◽

Associated Proteins

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

Journal of Experimental Medicine ◽

10.1084/jem.20042524 ◽

2005 ◽

Vol 201 (11) ◽

pp. 1715-1723 ◽

Cited By ~ 221

Author(s):

Willem A. Dik ◽

Karin Pike-Overzet ◽

Floor Weerkamp ◽

Dick de Ridder ◽

Edwin F.E. de Haas ◽

...

Keyword(s):

Gene Expression ◽

T Cell ◽

Gene Expression Profiling ◽

T Cell Receptor ◽

Expression Profiling ◽

Gene Rearrangement ◽

T Cell Development ◽

Cell Receptor ◽

Cell Development ◽

Human T Cell

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.

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Characterization in vitro and engraftment potential in vivo of human progenitor T cells generated from hematopoietic stem cells

Blood ◽

10.1182/blood-2008-10-187013 ◽

2009 ◽

Vol 114 (5) ◽

pp. 972-982 ◽

Cited By ~ 89

Author(s):

Génève Awong ◽

Elaine Herer ◽

Charles D. Surh ◽

John E. Dick ◽

Ross N. La Motte-Mohs ◽

...

Keyword(s):

Stem Cells ◽

T Cell ◽

Hematopoietic Stem Cells ◽

T Cell Development ◽

Cell Development ◽

Human Umbilical Cord Blood ◽

Human Umbilical Cord ◽

Hematopoietic Stem ◽

Human T Cell

T-cell development follows a defined set of stage-specific differentiation steps. However, molecular and cellular events occurring at early stages of human T-cell development remain to be fully elucidated. To address this, human umbilical cord blood (UCB) hematopoietic stem cells (HSCs) were induced to differentiate to the T lineage in OP9-DL1 cocultures. A developmental program involving a sequential and temporally discrete expression of key differentiation markers was revealed. Quantitative clonal analyses demonstrated that CD34+CD38− and CD34+CD38lo subsets of UCB contain a similarly high T-lineage progenitor frequency, whereas the frequency in CD34+CD38+/hi cells was 5-fold lower. Delta-like/Notch-induced signals increased the T-cell progenitor frequency of CD34+CD38−/lo cells differentiated on OP9-DL1, and 2 distinct progenitor subsets, CD34+CD45RA+CD7++CD5−CD1a− (proT1) and CD34+CD45RA+CD7++CD5+CD1a− (proT2), were identified and their thymus engrafting capacity was examined, with proT2 cells showing a 3-fold enhanced reconstituting capacity compared with the proT1 subset. Furthermore, in vitro–generated CD34+CD7++ progenitors effectively engrafted the thymus of immunodeficient mice, which was enhanced by the addition of an IL-7/IL-7 antibody complex. Taken together, the identification of T-progenitor subsets readily generated in vitro may offer important avenues to improve cellular-based immune-reconstitution approaches.

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Synergistic Effects of Interleukin-7 and Pre-T Cell Receptor Signaling in Human T Cell Development

Journal of Biological Chemistry ◽

10.1074/jbc.m112.380113 ◽

2012 ◽

Vol 287 (40) ◽

pp. 33826-33835 ◽

Cited By ~ 6

Author(s):

Ekta S. Patel ◽

Lung-Ji Chang

Keyword(s):

T Cell ◽

T Cell Receptor ◽

Synergistic Effects ◽

T Cell Development ◽

Cell Receptor ◽

Cell Development ◽

Interleukin 7 ◽

Human T Cell ◽

T Cell Receptor Signaling ◽

Cell Receptor Signaling

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Rac GTPase isoforms Rac1 and Rac2 play a redundant and crucial role in T-cell development

Blood ◽

10.1182/blood-2008-01-132068 ◽

2008 ◽

Vol 112 (5) ◽

pp. 1767-1775 ◽

Cited By ~ 62

Author(s):

Fukun Guo ◽

Jose A. Cancelas ◽

David Hildeman ◽

David A. Williams ◽

Yi Zheng

Keyword(s):

T Cell ◽

T Cell Development ◽

Cell Lineage ◽

Cell Receptor ◽

Cell Development ◽

Rac Gtpase ◽

Developmental Defects ◽

Hematopoietic Stem ◽

And Migration ◽

Specific Deletion

Abstract Rac GTPases have been implicated in the regulation of diverse functions in various blood cell lineages, but their role in T-cell development is not well understood. We have carried out conditional gene targeting to achieve hematopoietic stem cell (HSC)– or T-cell lineage–specific deletion of Rac1 or Rac1/Rac2 by crossbreeding the Mx-Cre or Lck-Cre transgenic mice with Rac1loxp/loxp or Rac1loxp/loxp;Rac2−/− mice. We found that (1) HSC deletion of both Rac1 and Rac2 inhibited production of common lymphoid progenitors (CLPs) in bone marrow and suppressed T-cell development in thymus and peripheral organs, whereas deletion of Rac1 moderately affected CLP production and T-cell development. (2) T cell–specific deletion of Rac1 did not affect T-cell development, whereas deletion of both Rac1 and Rac2 reduced immature CD4+CD8+ and mature CD4+ populations in thymus as well as CD4+ and CD8+ populations in spleen. (3) The developmental defects of Rac1/Rac2 knockout T cells were associated with proliferation, survival, adhesion, and migration defects. (4) Rac1/Rac2 deletion suppressed T-cell receptor–mediated proliferation, IL-2 production, and Akt activation in thymocytes. Thus, Rac1 and Rac2 have unique roles in CLP production and share a redundant but essential role in later stages of T-cell development by regulating survival and proliferation signals.

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