Limits...
Identification of a novel developmental stage marking lineage commitment of progenitor thymocytes.

Carlyle JR, Michie AM, Furlonger C, Nakano T, Lenardo MJ, Paige CJ, Zúñiga-Pflücker JC - J. Exp. Med. (1997)

Bottom Line: The identification and functional properties of such a progenitor population remain undefined.We report the identification of a novel developmental stage during fetal thymic ontogeny that delineates a population of T/NK-committed progenitors (NK1. 1(+)/CD117(+)/CD44(+)/CD25(-)).Thus, the restriction point to T and NK lymphocyte destinies from a multipotent progenitor stage is marked by a thymus-induced differentiation step.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, University of Toronto, Toronto, Ontario M5S 1A8, Canada.

ABSTRACT
Bipotent progenitors for T and natural killer (NK) lymphocytes are thought to exist among early precursor thymocytes. The identification and functional properties of such a progenitor population remain undefined. We report the identification of a novel developmental stage during fetal thymic ontogeny that delineates a population of T/NK-committed progenitors (NK1. 1(+)/CD117(+)/CD44(+)/CD25(-)). Thymocytes at this stage in development are phenotypically and functionally distinguishable from the pool of multipotent lymphoid-restricted (B, T, and NK) precursor thymocytes. Exposure of multipotent precursor thymocytes or fetal liver- derived hematopoietic progenitors to thymic stroma induces differentiation to the bipotent developmental stage. Continued exposure to a thymic microenvironment results in predominant commitment to the T cell lineage, whereas coculture with a bone marrow-derived stromal cell line results in the generation of mature NK cells. Thus, the restriction point to T and NK lymphocyte destinies from a multipotent progenitor stage is marked by a thymus-induced differentiation step.

Show MeSH

Related in: MedlinePlus

Temporal generation of FTNK cells from FTLP and FL precursors after in vitro transfer into fetal thymi. Sorted (NK1.1−/CD117+/ CD90lo/CD24lo/CD25−) day-14 fetal thymic lymphoid progenitor  (FTLP) and fetal liver (FL)–derived precursors were transferred into  dGuo-depleted fetal thymic lobes, cultured for 24 h in a hanging drop  configuration, and then cultured in standard FTOC for an additional 1, 3,  or 5 d. Flow cytometric analysis of cell surface expression of NK1.1 versus  CD117 shows that NK1.1 expression is induced on CD117+ precursors  (FTNK stage) as early as 2 d after exposure to thymic stroma, peaks by  day 4, and declines by day 6 with the appearance of NK1.1−/CD117−  cells. 3 × 104 sorted cells per dGuo-treated fetal thymus lobe (three  lobes/timepoint) were used; the above results are representative of at least  four independent trials. Cell yields ranged from 1–5 × 103, 0.5–1 × 104,  and 1–3 × 104 cells/lobe at days 2, 4, and 6, respectively. Plots display  1 × 104 live-gated events.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2198984&req=5

Figure 4: Temporal generation of FTNK cells from FTLP and FL precursors after in vitro transfer into fetal thymi. Sorted (NK1.1−/CD117+/ CD90lo/CD24lo/CD25−) day-14 fetal thymic lymphoid progenitor (FTLP) and fetal liver (FL)–derived precursors were transferred into dGuo-depleted fetal thymic lobes, cultured for 24 h in a hanging drop configuration, and then cultured in standard FTOC for an additional 1, 3, or 5 d. Flow cytometric analysis of cell surface expression of NK1.1 versus CD117 shows that NK1.1 expression is induced on CD117+ precursors (FTNK stage) as early as 2 d after exposure to thymic stroma, peaks by day 4, and declines by day 6 with the appearance of NK1.1−/CD117− cells. 3 × 104 sorted cells per dGuo-treated fetal thymus lobe (three lobes/timepoint) were used; the above results are representative of at least four independent trials. Cell yields ranged from 1–5 × 103, 0.5–1 × 104, and 1–3 × 104 cells/lobe at days 2, 4, and 6, respectively. Plots display 1 × 104 live-gated events.

Mentions: During early development, thymic progenitors transiently express various markers associated with activated mature T cells (38, 39) and NK cells (6, 13). These include lymphokine receptors such as CD25, several adhesion molecules such as intercellular adhesion molecules, very late activation antigens, and CD44, and other function-associated molecules such as CD16/32 (1–3, 6, 38, 39). It would appear that the NK1.1+ phenotype also corresponds to a temporary stage that T lymphocytes pass through on their way to maturity. To test this, we purified day-14 FTLP and FL cells by FACS® and followed their developmental progression to determine whether these isolated precursors could give rise to FTNK cells in reconstituted dGuo–FTOCs. Indeed, both FL and FTLP precursors directly give rise to substantial populations of FTNK cells within 48 h after transfer into FTOCs (Fig. 4). FTLPs appear to differentiate and reach the FTNK stage with faster kinetics than FL-derived progenitors (Fig. 4). This finding is in agreement with previous reports showing that fetal thymus–derived precursors show a faster reconstitution kinetics than FL-derived progenitors (2, 40). The appearance of FTNK cells peaks by day 4 after thymic reconstitution with FTLPs (Fig. 4; 83%), and declines by day 6. It is at these later timepoints that FTNK cells gain CD25 expression (Fig. 5 b; 37% of CD117+ gated cells coexpress NK1.1+/CD25+), and later lose NK1.1 expression as irrevocable commitment to the T cell lineage occurs and the CD25+/CD117+/NK1.1− stage is reached (Fig. 5 b) (4, 5). Again, the temporal kinetics of CD25+ expression by FTNK cells and loss of CD117 expression by developing thymocytes is more accelerated in dGuo–FTOCs reconstituted with FTLPs than with FL-derived progenitors (Fig. 5, a and b). Fig. 5 b also shows that by day 6 of reconstitution both precursor cell types give rise to CD117−/ CD25−/NK1.1+ cells, presumably pre-NK cells, while few CD117−/CD25+/NK1.1− (pre-T cells) are detected. These cells develop at later timepoints (data not shown). Finally, Fig. 5 b shows that nonreconstituted dGuo–FTOCs (control FTOC) remain devoid of lymphocytes. Thus, when purified FTLPs, which make up 4% of total day-14 fetal thymus, or FL cells are reintroduced to the thymic microenvironment a synchronized progression through the FTNK stage is clearly observed (Figs. 4 and 5), which occurs before the CD117+/ CD25+ stage of T cell development.


Identification of a novel developmental stage marking lineage commitment of progenitor thymocytes.

Carlyle JR, Michie AM, Furlonger C, Nakano T, Lenardo MJ, Paige CJ, Zúñiga-Pflücker JC - J. Exp. Med. (1997)

Temporal generation of FTNK cells from FTLP and FL precursors after in vitro transfer into fetal thymi. Sorted (NK1.1−/CD117+/ CD90lo/CD24lo/CD25−) day-14 fetal thymic lymphoid progenitor  (FTLP) and fetal liver (FL)–derived precursors were transferred into  dGuo-depleted fetal thymic lobes, cultured for 24 h in a hanging drop  configuration, and then cultured in standard FTOC for an additional 1, 3,  or 5 d. Flow cytometric analysis of cell surface expression of NK1.1 versus  CD117 shows that NK1.1 expression is induced on CD117+ precursors  (FTNK stage) as early as 2 d after exposure to thymic stroma, peaks by  day 4, and declines by day 6 with the appearance of NK1.1−/CD117−  cells. 3 × 104 sorted cells per dGuo-treated fetal thymus lobe (three  lobes/timepoint) were used; the above results are representative of at least  four independent trials. Cell yields ranged from 1–5 × 103, 0.5–1 × 104,  and 1–3 × 104 cells/lobe at days 2, 4, and 6, respectively. Plots display  1 × 104 live-gated events.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2198984&req=5

Figure 4: Temporal generation of FTNK cells from FTLP and FL precursors after in vitro transfer into fetal thymi. Sorted (NK1.1−/CD117+/ CD90lo/CD24lo/CD25−) day-14 fetal thymic lymphoid progenitor (FTLP) and fetal liver (FL)–derived precursors were transferred into dGuo-depleted fetal thymic lobes, cultured for 24 h in a hanging drop configuration, and then cultured in standard FTOC for an additional 1, 3, or 5 d. Flow cytometric analysis of cell surface expression of NK1.1 versus CD117 shows that NK1.1 expression is induced on CD117+ precursors (FTNK stage) as early as 2 d after exposure to thymic stroma, peaks by day 4, and declines by day 6 with the appearance of NK1.1−/CD117− cells. 3 × 104 sorted cells per dGuo-treated fetal thymus lobe (three lobes/timepoint) were used; the above results are representative of at least four independent trials. Cell yields ranged from 1–5 × 103, 0.5–1 × 104, and 1–3 × 104 cells/lobe at days 2, 4, and 6, respectively. Plots display 1 × 104 live-gated events.
Mentions: During early development, thymic progenitors transiently express various markers associated with activated mature T cells (38, 39) and NK cells (6, 13). These include lymphokine receptors such as CD25, several adhesion molecules such as intercellular adhesion molecules, very late activation antigens, and CD44, and other function-associated molecules such as CD16/32 (1–3, 6, 38, 39). It would appear that the NK1.1+ phenotype also corresponds to a temporary stage that T lymphocytes pass through on their way to maturity. To test this, we purified day-14 FTLP and FL cells by FACS® and followed their developmental progression to determine whether these isolated precursors could give rise to FTNK cells in reconstituted dGuo–FTOCs. Indeed, both FL and FTLP precursors directly give rise to substantial populations of FTNK cells within 48 h after transfer into FTOCs (Fig. 4). FTLPs appear to differentiate and reach the FTNK stage with faster kinetics than FL-derived progenitors (Fig. 4). This finding is in agreement with previous reports showing that fetal thymus–derived precursors show a faster reconstitution kinetics than FL-derived progenitors (2, 40). The appearance of FTNK cells peaks by day 4 after thymic reconstitution with FTLPs (Fig. 4; 83%), and declines by day 6. It is at these later timepoints that FTNK cells gain CD25 expression (Fig. 5 b; 37% of CD117+ gated cells coexpress NK1.1+/CD25+), and later lose NK1.1 expression as irrevocable commitment to the T cell lineage occurs and the CD25+/CD117+/NK1.1− stage is reached (Fig. 5 b) (4, 5). Again, the temporal kinetics of CD25+ expression by FTNK cells and loss of CD117 expression by developing thymocytes is more accelerated in dGuo–FTOCs reconstituted with FTLPs than with FL-derived progenitors (Fig. 5, a and b). Fig. 5 b also shows that by day 6 of reconstitution both precursor cell types give rise to CD117−/ CD25−/NK1.1+ cells, presumably pre-NK cells, while few CD117−/CD25+/NK1.1− (pre-T cells) are detected. These cells develop at later timepoints (data not shown). Finally, Fig. 5 b shows that nonreconstituted dGuo–FTOCs (control FTOC) remain devoid of lymphocytes. Thus, when purified FTLPs, which make up 4% of total day-14 fetal thymus, or FL cells are reintroduced to the thymic microenvironment a synchronized progression through the FTNK stage is clearly observed (Figs. 4 and 5), which occurs before the CD117+/ CD25+ stage of T cell development.

Bottom Line: The identification and functional properties of such a progenitor population remain undefined.We report the identification of a novel developmental stage during fetal thymic ontogeny that delineates a population of T/NK-committed progenitors (NK1. 1(+)/CD117(+)/CD44(+)/CD25(-)).Thus, the restriction point to T and NK lymphocyte destinies from a multipotent progenitor stage is marked by a thymus-induced differentiation step.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, University of Toronto, Toronto, Ontario M5S 1A8, Canada.

ABSTRACT
Bipotent progenitors for T and natural killer (NK) lymphocytes are thought to exist among early precursor thymocytes. The identification and functional properties of such a progenitor population remain undefined. We report the identification of a novel developmental stage during fetal thymic ontogeny that delineates a population of T/NK-committed progenitors (NK1. 1(+)/CD117(+)/CD44(+)/CD25(-)). Thymocytes at this stage in development are phenotypically and functionally distinguishable from the pool of multipotent lymphoid-restricted (B, T, and NK) precursor thymocytes. Exposure of multipotent precursor thymocytes or fetal liver- derived hematopoietic progenitors to thymic stroma induces differentiation to the bipotent developmental stage. Continued exposure to a thymic microenvironment results in predominant commitment to the T cell lineage, whereas coculture with a bone marrow-derived stromal cell line results in the generation of mature NK cells. Thus, the restriction point to T and NK lymphocyte destinies from a multipotent progenitor stage is marked by a thymus-induced differentiation step.

Show MeSH
Related in: MedlinePlus