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Blood, 1 December 2000, Vol. 96, No. 12, pp. 3988-3990
BRIEF REPORT
MHC class II and c-kit expression allows rapid
enrichment of T-cell progenitors from total bone marrow cells
Christiane Ody,
Catherine Corbel,
Dominique Dunon,
Olli Vainio, and
Beat A. Imhof
From the Department of Pathology, Centre Médical
Universitaire (CMU), Geneva, Switzerland; Institut d'Embryologie
Cellulaire et Moléculaire du Centre National de la Recherche
Scientifique (CNRS) et du Collège de France, Nogent/Marne,
France; UMR-CNRS 7622, Université Pierre et Marie Curie, Paris,
France; and the Department of Medical Microbiology, Turku University,
Turku, Finland.
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Abstract |
T-cell progenitors in the embryonic bone marrow express the
tyrosine kinase receptor c-kit. RR5, an anti-MHC class II
 chain monoclonal antibody, subdivides this c-kit positive
population. Intrathymic transfer experiments showed that most of the
T-cell progenitors belong to the MHC class
II+/c-kit+ bone marrow population
in the embryo and young adult. On transplantation, these bone marrow
progenitors lose this expression and differentiate into CD4 CD8 T
lymphocytes. In contrast, erythroid progenitors are restricted to the
MHC class II /c-kit+ population.
The MHC class II+/c-kit+ pro-T
cells are metabolically active, because they stain brightly with
rhodamin 123. Their cyclin A and B expression level suggests that they
are in the mitotic phase of the cell cycle. Thus, we define an easy
sorting protocol, which allows enrichment of T-cell progenitors from
total bone marrow hemopoietic cells.
(Blood. 2000;96:3988-3990)
© 2000 by The American Society of Hematology.
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Introduction |
T-lineage commitment precedes thymus
colonization.1,2 In birds, it has recently been shown that
T-cell progenitors from embryonic bone marrow (BM), which will migrate
to the thymus via the blood stream, express
c-kit.3-5 It is not yet clear at which stage of
commitment MHC class II is expressed on hemopoietic progenitors and
whether its simultaneous expression with other hemopoietic markers can
define irreversibly engaged progenitors. Some in vitro studies with
human BM cells show that primitive self-renewing multipotent
hemopoietic cells are CD34+ and MHC class
II,6 whereas other studies with human fetal
BM cells describe the CD34+, CD38, and MHC
class II+ positive cell population as
multipotent.7 Nevertheless, no study refers to the
presence of MHC class II molecules on T-cell progenitors. The results
presented here define T-cell progenitors as metabolically active cells
with a high expression level of the mitotic cyclins A and B with
preferential location in the MHC class II/c-kit
double-positive population of embryonic and adult BM. On in vitro
culture, these cells were also able to give rise to myeloid but not to
erythroid colonies. These results correlate well with the recent
demonstration of the presence of a common precursor able to
differentiate into myeloid and T cells.8
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Study design |
Outbred JA57 and White Leghorn chick embryos were obtained from
local facilities. The 2 congenic strains H.B19+ and
H.B19, which can be distinguished by the presence of the
ov alloantigen on T-lineage cells in the H.B19+
animals, were produced at our animal facilities. The following monoclonal antibodies (mAbs) were used: RR5, a mouse IgG2b, raised in
our laboratory (submitted for publication); a mouse antichicken c-kit conjugated to phycoerythrin (PE)3;
11A9, which recognizes the ov epitope expressed on T-lineage cells from
the H.B19+ chicken strain9; antichicken
CD4(2-6) and CD8(11-39) mAbs directly conjugated to fluorescein
isothiocyanate (FITC) or PE, respectively.10,11
Antichicken cyclin A (R28) and cyclin B (R18), rabbit polyclonal
antibodies, were kind gifts from Prof E. Nigg, University of Geneva,
Switzerland.12,13 In some cases, RR5 was used directly
coupled to Cy5 using the Fluorolink Cy5 reactive kit from Amersham
(Life Science, Arlington Heights, IL). Bone marrow cell suspensions,
immunostaining, flow cytometric analysis, and sorting were performed as
previously described.3 Three-color fluorescence-activated
cell sorter (FACS) analysis was performed on a FACSCalibur (Becton
Dickinson, San Jose, CA) using FL1, FL2, and FL4 with appropriate
compensations. Antibodies directly labeled with Cy5 or biotinylated
antibodies revealed by APC-streptavidin (Molecular Probe, Eugene, OR)
were detected on FL4. When necessary, the contribution of dead cells
was assessed by adding propidium iodide (PI; Calbiochem, Juro Supply,
Lucerne, Switzerland). For cyclin A and B staining, cells were fixed in 4% paraformaldehyde, permeabilized with 0.3% saponin and incubated with rabbit anticyclin polyclonal antibodies or with a control rabbit
serum diluted 1:500 in phosphate-buffered saline (PBS) containing 3%
bovine serum albumin (BSA) and 0.03% saponin. Goat antirabbit coupled
to FITC (Southern Biotechnology Associates) was used as a
second step antibody. Cells were incubated with rhodamine 123 (Rh)
(Molecular Probes) according to published protocols14,15 either directly or after staining with RR5cy5 and c-kitPE.
In vitro assay for hemopoietic progenitors in semisolid cultures was
performed as previously described.4 In vivo T-cell
progenitor assay by intrathymic injection was conducted as already
described.4
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Results and discussion |
The c-kit positive cell population from embryonic and
young adult bone marrow contains MHC class II positive cells
Recent evidence from experiments on mouse and chicken have shown
that the T-lineage commitment precede thymus colonization and that the
reconstitutive T-cell progenitors were among the c-kit
positive cells.2-5 The anti-MHC class II -chain mAb RR5 subdivided the c-kit positive population on FACS analysis
(Figure 1A). In
embryonic BM at day 13.5 (E13.5), c-kit positive cells accounted for more than 15% in the forward side scatter window defined
for progenitors and the RR5/c-kit double-positive population represented 3% to 7% of the gated cells ( 1.7% of total BM cells). In the young adult, the c-kit positive cells were less
abundant (7%) and the RR5/c-kit double-positive population
accounted for less than 2% of the gated cells.
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| Figure 1.
Analysis of bone marrow cells.
(A) Comparative flow cytometric analysis of embryonic and
adult bone marrow cells. Embryonic E13.5 and adult BM cells
were stained with the RR5 and c-kit mAbs and analyzed by flow cytometry
in the FSC/SSC progenitor window. The RR5/c-kit
double-positive cells shown in the window were sorted for intrathymic
injection and in vitro differentiation. (B) Analysis of myeloid and
erythroid potential of sorted cells from E13.5 BM. The 1000 cells from
each sorted population were cultured in duplicate in erythroid
differentiation medium. Results represent the mean number of colonies
scored in duplicate cultures from one representative experiment (of 5 experiments). M: macrophage; G: granulocyte; M/G:
macrophage/granulocyte; Eb/Ec: erythroblast/erythrocyte colonies. No
colonies were obtained from the double negative population.  indicates M,G, and M/G;  indicates Eb/Ec. (C) Rhodamine staining of
RR5/c-kit double-positive BM cells. Comparative Rh staining
of total bone marrow cells (hatched line) and
RR5+/c-kit+ cells (continuous line).
(D) Cyclin A and B staining of RR5/c-kit double-positive BM
cells. Cyclin staining of embryonic total bone marrow cells (thin line)
and RR5+/c-kit+ cells (bold line).
Control: normal rabbit serum; cyclin A: polyclonal rabbit antichicken
cyclin A serum; cyclin B: polyclonal rabbit antichicken cyclin B serum.
Rabbit antibodies were detected by a goat antirabbit antibody
conjugated to FITC.
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RR5+/c-kit+ bone marrow cells
harbor myelomonocytic, T-cell, but no erythroid progenitors
Semisolid cultures of sorted embryonic BM cells showed that, under
appropriate conditions, erythroid colonies developed almost exclusively
from RR5/c-kit+ cells (Figure 1B).
By contrast, myeloid colonies were recorded in similar numbers from the
RR5+/c-kit+ double-positive as well
as from the RR5/c-kit+ and
RR5+/c-kit cells (Figure 1B). The
T-cell differentiation potential as judged by intrathymic injection
into ov congenic animals, suggested that T-cell
progenitors were enriched in the
RR5+/c-kit+ BM population of the
embryo when compared with
RR5/c-kit+ cells (Table
1). In the young adult, the injection of
1000 double-positive sorted cells resulted in a thymic chimerism of
7.6% ± 2% (mean ± SEM). The in vitro studies showed that the
isolated progenitors were not multipotent because they have lost their
erythroid potential (Figure 1B). Most of the thymocytes recovered 14 days after grafting were CD4/CD8 double-positive with very few mature
single positive cells (data not shown). This proportion was similar to
that found in age-matched uninjected ov+ control chickens.
Analysis of MHC class II expression revealed that most of the
transferred T-cell progenitors lose their expression during T-cell
differentiation (data not shown).
RR5/c-kit double-positive T-cell progenitors are
metabolically active and express cyclin A and B
Rh 123 staining showed that in embryonic BM, 70% of the
double-positive cells were Rhhi, whereas in the adult BM,
these cells were exclusively in the Rhhi compartment
(Figure 1C, continuous lines). Injection of 1000 Rhlo
embryonic cells resulted in chimerism of 1.8% showing that the contribution of this population to the T-cell potential of the double-positive cells is minimal. Thus, the majority of the T-cell progenitors in the RR5+/c-kit+
population were metabolically active and belonged to the fraction of
nonquiescent hemopoietic cells, which, after total body irradiation, have been shown to mediate transient short-term
reconstitution.14,15 In the adult mouse, the prothymocytes
able to rapidly repopulate the thymus after intravenous injection were
in the c-kit+, Lin,
Ly6A/E+, and Rhhi fraction of the BM. The
corresponding Rhlo cell population had a delayed thymic
repopulation ability, and it was suggested that this delay was due to
the inability of these cells to seed the thymus. Seeding could only
take place through further maturation of these Rhlo cells
into more mature Rhhi cells, presumably in the
BM.16 These results confirmed previous data obtained by
single Rh staining and sorting experiments.14 To
investigate in the embryo the correlation of the Rh staining with the
cell cycle status of these cells, we studied the expression of cyclin A
and B by flow cytometry.17 Interestingly, all the RR5+/c-kit+ double-positive cells,
which included both the Rhlo and hi cells from embryonic
BM, were among the cyclin A and B high-expressing cells (Figure 1D,
bold lines). This indicated that the
RR5+/c-kit+ cells were engaged in
the mitotic phase of the cell cycle. To directly evaluate the T-cell
potential of the Rhhi population of embryonic BM, we sorted
c-kit- or RR5-labeled cells from the Rhhi
window to perform intrathymic injections. The results showed that
progenitors were preferentially located in the Rhhi window
of either c-kit or RR5 positive populations (Table 1). Thus,
MHC class II, a molecule that has always been considered as a hallmark
of definitive differentiation toward dendritic cells, B-cells, or
macrophages, is also present on T-cell progenitors and subsequently
disappears when the progenitors further differentiate in the thymic
environment. The molecule is coexpressed with c-kit on
short-term committed progenitors unable to give rise to erythroid colonies in vitro (Figure 1B). The progenitors are in a cycling status
characterized by high Rh retention (Figure 1C) and a high level of
expression of the so-called mitotic cyclins (Figure 1D).
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Acknowledgments |
We are indebted to Dominique Wohlwend, Viktor Hasler, Raija
Raulimo, Pierre Vaigot, and Suzanne Bissat for excellent technical assistance, Jean-Claude Rumbeli and Etienne Denkinger for photographic work, and Fabrice Bonaccorsi for computer assistance. Special thanks
goes to Caroline Johnson-Leger for critical reading and improvement of
the manuscript.
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Footnotes |
Submitted December 24, 1999; accepted August 1, 2000.
Supported by the Swiss National Science Foundation grant no.
21-49 241.96, Association pour la Recherche contre le Cancer (ARC-9122) and (ARC- 9738), Human Frontier HFSP-RG 366/96 and the
Academy of Finland (grant no. 4293).
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
Reprints: B. A. Imhof, Department of Pathology, Centre
Médical Universitaire (CMU), CH-1211Geneva 4, Switzerland.
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Abstract
Introduction