Induction of T cell development and establishment of T cell competence from embryonic stem cells differentiated in vitro

Schmitt, Thomas M.; Pooter, Renée F. de; Gronski, Matthew A; Cho, Sarah K.; Ohashi, Pamela S.; Zúñiga‐Pflücker, Juan Carlos

doi:10.1038/ni1055

Cited by 330 publications

(347 citation statements)

References 35 publications

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“…Git2 2/2 mice (21) on a C57BL/6 background were crossed to OT-II + mice, and heterozygous offspring were analyzed. OP9-DL1 cells have been described previously (22). Reconstituted mice were generated by transferring 2 3 10 6 bone marrow cells from TCR transgenic DO11.10 + wild type or aPix 2 BALB/C mice to lethally irradiated (1000 rad) hosts.…”

Section: Methodsmentioning

confidence: 99%

αPIX RhoGEF Supports Positive Selection by Restraining Migration and Promoting Arrest of Thymocytes

Korthals

Schilling

Reichardt

et al. 2014

The Journal of Immunology

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Thymocytes mature in a series of stages by migrating through specific areas of the thymus and interacting with other cells to receive the necessary developmental signals; however, little is known about the molecular mechanisms governing this migration. We report that murine thymocytes with a knockout mutation in α-PAK (p21-activated kinase)-interacting exchange factor (PIX; Arhgef6), an activator of Rho GTPases, showed greatly increased motility and altered morphology in two-dimensional migration on ICAM-1. αPIX was also required for efficient positive selection, but not negative selection, of thymocytes. TCR signaling was normal in αPix− thymocytes, indicating that the effects of αPIX on positive selection are largely independent of TCR signaling. αPix− thymocytes also paused less during migration in the thymic cortex, interacted less with ICAM-1 coated beads, and could overcome TCR stop signals, consistent with defective scanning behavior. These results identify αPIX as a regulator of thymocyte migration and subsequent arrest that is linked to positive selection.

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Section: Methodsmentioning

confidence: 99%

αPIX RhoGEF Supports Positive Selection by Restraining Migration and Promoting Arrest of Thymocytes

Korthals

Schilling

Reichardt

et al. 2014

The Journal of Immunology

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“…Several years ago Zuniga-Pflucker and colleagues generated an OP9 stromal cell line expressing DL1 [36,37]. This stromal cell line is able to promote differentiation into the T-cell lineage of early hematopoietic precursors [36,37].…”

Section: Introductionmentioning

confidence: 99%

“…This stromal cell line is able to promote differentiation into the T-cell lineage of early hematopoietic precursors [36,37]. This culture system has been of great utility in our understanding of the early stages of thymocyte development.…”

Section: Introductionmentioning

confidence: 99%

“…Recently, it was suggested that the transition from a Notch-dependent to Notch-independent stage of T-cell development, mediated by either Notch ligand Delta-like 1 (DL1) or DL4, occurred at the CD8 1 ISP stage [36]. However, the stages during T-cell development at which stage these signaling pathways are required, and how they synergize, are still not clear.Several years ago Zuniga-Pflucker and colleagues generated an OP9 stromal cell line expressing DL1 [36,37]. This stromal cell line is able to promote differentiation into the T-cell lineage of early hematopoietic precursors [36,37].…”

mentioning

confidence: 99%

“…In vitro culture systems trying to recapitulate this complex differentiation process were of great utility but have shown some limitations. In particular, the OP9 stromal cells, incapable of supporting myelopoiesis and made to express the Notch ligands DL1 or DL4, were clearly instrumental in revolutionizing the ability to study T-cell differentiation in vitro [36,37]. However, such culture systems are not ideal since the density of Notch ligand expression cannot be controlled and other signals may be delivered to the co-cultured cells.…”

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confidence: 99%

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The preTCR‐dependent DN3 to DP transition requires Notch signaling, is improved by CXCL12 signaling and is inhibited by IL‐7 signaling

et al. 2011

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The requirement for Notch signaling during T-cell development has been extensively studied. Nevertheless, the developmental stage at which it is required and whether additional signaling pathways are needed are still poorly understood. By using a stromalcell-free culture system, we show that sorted double-negative 3 (DN3) thymocytes only require a Delta-like-4-induced Notch signal to differentiate into double-positive (DP) cells. This differentiation process is preTCR-a dependent. DN3 cells undergo 4-5 proliferation cycles, and the addition of the chemokine CXCL12 improves proliferation. IL-7 blocks the differentiation of DN3 cells to DP cells but not the Notch-induced proliferation of cultured DN3 cells. The impaired differentiation correlates with an inhibition of Rag-2 upregulation. Overall, the in vitro stromal-cell-free culture system presented here also provides a powerful and unique tool for studying the mechanisms involved in the positive and negative selection of T cells.Key words: Delta-like 4 and preTCR . DN3 cells . Double-positive cells . Notch IntroductionTo maintain T lymphopoiesis, progenitor cells from the BM constantly seed the thymus. In transplantation settings, it has been shown that different progenitors can home to the thymus and generate T cells [1][2][3][4][5][6][7][8][9]. However, under physiological conditions the role of each of these progenitors is still unclear. It is generally believed that the so-called thymic seeding precursor (TSP) still has the capability of generating B cells, NK cells, DCs and other myeloid cells [2,5,6,10,11]. Upon receiving the Notch signal, the differentiation potential towards the B-cell lineage of the TSP is rapidly lost [2,10,11], whereas other lineage options are still maintained [2,5,6,10,11]. However, a recent study using an IL-7Ra reporter mouse indicated that the non-T-cell lineage differentiation of these progenitors is not, or only very rarely, occurring under physiologic conditions [12,13].Within the thymus, the earliest thymocytes are characterized by the absence of CD4 and CD8 expression and are therefore called double-negative (DN) cells. Based on the expression of CD25, CD44 and CD117, DN cells can be subdivided into four subpopulations. DN1 cells express high cell surface levels of CD44 and CD117, and are negative for CD25, whereas their DN2 progeny express all three markers [14][15][16]. In vitro, DN1 and DN2 cells still possess the capacity to differentiate into NK cells, DCs and other myeloid cells [5,6,11,17] suggesting that they are not yet restricted to the T-cell lineage. T-cell commitment is achieved at the DN3 stage. However, the mechanisms operating . It is at this stage of development that the rearrangement of the TCR-b chain locus is completed, and the cells are selected for a productive TCR-b chain rearrangement, also known as the b-selection checkpoint [18][19][20]. DN3 cells that have rearranged their TCR-b chain locus unproductively can either differentiate into g/d T cells or will otherwise undergo apoptosis. DN3 cells car...

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In Vitro Systems for the Study of T Cell Development: Fetal Thymus Organ Culture and OP9‐DL1 Cell Coculture

2006

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Most T cell development occurs within the thymus and includes a series of selection processes that are, in large part, still poorly understood. Studies of T cell development have been greatly advanced by the description of multiple phenotypic subsets of T cells and their maturational relationships. This unit describes a system for observing and modulating T cell development in vitro via the culture of entire mouse fetal thymic lobes. Methods are included for the isolation of fetal thymi and culture to allow for normal T cell development on either transwell plates or Gelfoam sponges. A method for depleting hematopoietic cells from thymic lobes using 2'-deoxyguanosine and subsequent reconstitution with precursor cells is also described. This protocol is valuable for the study of tolerance and T cell selection. A support protocol describing methods of altering and monitoring T cell development are outlined. In addition, methods for culturing fetal thymic lobes under high oxygen submersion conditions and for the preparation of reaggregate thymus organ cultures are provided. Finally, a simple and practical method that allows for the thymus-independent generation of T cells from defined sources of stem/progenitor cells by OP9-DL1 coculture is described.

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Induction of T cell development and establishment of T cell competence from embryonic stem cells differentiated in vitro

Cited by 330 publications

References 35 publications

αPIX RhoGEF Supports Positive Selection by Restraining Migration and Promoting Arrest of Thymocytes

αPIX RhoGEF Supports Positive Selection by Restraining Migration and Promoting Arrest of Thymocytes

The preTCR‐dependent DN3 to DP transition requires Notch signaling, is improved by CXCL12 signaling and is inhibited by IL‐7 signaling

In Vitro Systems for the Study of T Cell Development: Fetal Thymus Organ Culture and OP9‐DL1 Cell Coculture

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