By
From the * Basel Institute for Immunology, 4005 Basel, Switzerland; The Netherlands Cancer
Institute, 1066 CX Amsterdam, The Netherlands
Here, we report data concerning the discovery in adult human peripheral blood of a precursor
cell population able to differentiate into CD4+CD3++ mature T cells. These cells, which
represent 0.1-0.5% of total peripheral blood mononuclear cells (PBMC), express substantial levels of CD4, but lack CD3 surface expression. At a molecular level, they express the pre-T
cell receptor
(pT
) gene, CD3-
, CD-
and CD-
, and RAG-1 recombination enzyme and
have initiated rearrangements in the T cell receptor (TCR)-
locus (D-J). Moreover, low levels of CD3
protein, but not of TCR-
chain, can be detected in their cytoplasm. Our results
suggest that CD4+CD3
cells identified in peripheral blood are different from CD3
CD4+CD8
thymocytes and may contain precursors of an extrathymic T cell differentiation pathway.
In murine T cell development, early thymocytes that productively rearrange the TCR- Analysis of pT Using murine pT We have previously shown that murine pT Antibodies.
Cell staining was performed using the following
mouse anti-human mAbs: phycoerythrin-conjugated (R-PE)-CD4,
fluorescein-conjugated FITC-CD3, FITC-CD14, R-PE-CD5,
biotin-CD10, R-PE-CD33, biotinylated HLA-DR, and RPE-
CD34 (Becton Dickinson, San Jose, CA); tricolor CD4, biotinylated CD2 and RPE-CD7 (Caltag Laboratories, San Francisco, CA),
CD45RA (IgG2b) and B7-2 (IgG2b) (PharMingen, San Diego,
CA), FITC-pan-TCR- Surface Staining and Sorting of Lymphocytes.
PBMC were separated from 50-60 ml blood from 25-33-yr-old male volunteers
by standard Ficoll gradient centrifugation, resuspended in PBS
containing 2% FCS at 107 cells per ml, and stained following standard procedures. Stained samples were analyzed and sorted on either FACScan®, FACStar Plus®, or FACS Vantage®. Sorted populations were reanalyzed by the same machines to test purity.
Cell Depletion.
A single-cell suspension was first stained, as
described previously, with FITC-conjugated TR66 mAb and, subsequently, PBMC were depleted of CD3+ cells using Dynabeads
(Milan, Switzerland).
Cytoplasmic Staining.
Sorted cells (0.5-1 × 105) were washed
twice in PBS and fixed in 500 µl of 2% paraformaldehyde for 20 min at room temperature. Samples were washed again twice with
PBS and, subsequently, lysed in 1 ml Saponin buffer (0.5% Saponin, 10 mM Hepes, 5% FCS) for 10 min at room temperature.
Samples were then stained for 30 min at room temperature with
HB-9283 supernatant specific for TCR- Cell Cycle Analysis.
Sorted cells (0.5-1 × 105) were fixed in 2 ml 70% ethanol at 4°C overnight. Samples were than centrifuged
at 2,000 rpm for 5 min and resuspended in 250 µl RNAse A
(Sigma, Buchs, Switzerland) (0.5 mg/ml in 0.1 M Tris, pH 7.5, 0.1 M NaCl) and incubated for 30 min at 37°C. Without centrifuging, 250 µl pepsin (Sigma, Buchs, Switzerland) (1 mg/ml in
0.4% HCl) were added and samples were incubated for 15 min at
37°C. Without centrifuging, 500 µl ethidium bromide (0.02 mg/ml,
0.2 M Tris, pH 8-8.5, 0.5% BSA) were added and samples were
incubated for another 15 min at room temperature protected
from light with aluminum foil. At the end of incubation, samples
were kept on ice until analysis. Analysis was performed on a
FACScan®, equipped with the doublets discriminating module
(DDM).
Reverse Transcription-PCR.
In the analysis of pT DNA Extraction and PCR.
DNA was prepared from cells by
directly sorting them in PBS. Subsequently, 5 µl of proteinase K
(20 mg/ml; Merck, Darmstadt, Germany) was added and sample
were left at 55°C for 2 h. Phenol/chloroform extraction and
DNA precipitation followed, according to standard procedures.
PCR conditions were as described elsewhere (10) using TBF1
(upstream, the D Southern Blot Analysis.
Some of the gels containing PCR
products were blotted for 2 h with 0.4N NaOH and subsequently
hybridized, according to standard procedures, with the following
different oligonucleotides: for RAG-1 gene product with 5 Hybrid Human/Mouse Fetal Thymic Organ Cultures.
The in vitro
development of human T cells was studied using the hybrid human/mouse fetal thymic organ culture (FTOC) (11). Fetal thymi
from embryos of RAG-1-deficient mice were dissected on day
15-16 of gestation and precultured for 5 d in the presence of 1.35 mM 2-deoxyguanosine (Sigma, St. Louis, MO) to remove endogenous thymocytes. Next, the thymic lobes were cocultured
for 2 d in hanging drops in wells of a Terasaki plate with human
progenitor cells and transferred to nucleopore filters, which were
layered over gelfoam rafts in 6-well plates (Costar). The lobes
populated with human cells were cultured for the indicated number of days in Yssel's medium (12) supplemented with 2% normal
human serum and 5% FCS. To analyze differentiation of human
cells, the mouse thymi were dispersed into single cell suspensions
and stained with mAbs specific for human cell surface antigens.
Liquid Cultures in the Presence of Cytokines.
To test T cell differentiation potential, 1 × 104 sorted cells were cocultured with
irradiated (3,000 rads) allogeneic PBMC as feeder cells in U-bottomed 96-well plates in complete RPMI medium with 5% human
serum (HS), 0.1 µg/ml PHA, 1,000 U/ml IL-2, and 500 U/ml IL-7.
After 10 d, growing cells were stained and analyzed by FACS®.
Limiting Dilution Analysis.
Flow cytometry-sorted progenitor cells were carefully spun down and diluted in the same medium described above for T cell differentiation, containing 1 × 106/ml irradiated allogeneic PBMC as feeder cells. 1 cell/well
was distributed in Terasaki plates. After 10-15 d, growing wells
were transferred in U-bottomed 96-well plates and cells stained
and analyzed by FACS®.
By
PCR analysis of retrotranscribed mRNA of PBMC of adult
donors we were able to detect low levels of pT
Rearrangement of B cell
(13) and T cell (14) antigen receptor gene loci to form
functional VDJ gene segments is a key molecular event in
lymphocyte ontogeny. In the thymus, DJ Several DJ
To ascertain whether the presence of DJ Previous studies have shown that
analysis of the expression of RAG-1 and RAG-2 genes and
of TCR transcription are useful to trace early events during
T cell development both in murine and in human T cell
development (7, 16). As independent confirmation of DJ
Because CD3 CD4+CD3
Expression of CD3 Subunits but Not TCR-
locus are selected to
continue maturation. This happens because of the coupling
of the rearranged
chain with the invariant pre-TCR-
(pT
)1 protein (1, 2). This pre-TCR regulates early T cell
development; later stages are under the control of the
mature TCR, composed of the
and
chains (3). The
TCR-
-pT
heterodimer is associated with CD3 molecules (4), and signals triggered by the pre-TCR induce expansion and differentiation of immature precursor cells (5).
gene-deficient mice provided formal proof
that expression of the pre-TCR is required in the transition
of CD25+ double-negative (DN) T cell precursors into
small CD4+CD8+ thymocytes (5). This transition normally
occurs through a stage in which maturing thymocytes proliferate vigorously and lose the expression of CD25. Nevertheless, low levels of mature
T cells can be detected in
the periphery of pT
knockout mice, suggesting that the
expression of pT
is necessary for quantitative expansion of
maturing thymocytes, but not crucial for differentiation.
cDNA as a probe, we were able to
show that a comparable gene was expressed in human thymocytes (6). Amino acid sequence comparison between
human and mouse pT
cDNAs revealed high sequence
homology in the extracellular as well as the transmembrane region, but complete divergence in the cytoplasmic region.
Recently, another group cloned human pT
cDNA and
performed a comparison of the developmental regulation of
pT
, TCR-
, TCR-
, and RAG-1 gene expression, providing a picture of the maturational progression of early
human intrathymic stages (7).
expression
is exquisitely T lineage specific and occurs in pro-T cells
outside the thymus, in the earliest T cell precursors identified in the thymus, and in sites that support extrathymic T cell
development (gut and liver), (8). Consistent with these observations, pT
RNA could not be detected in human
non-T cells including B, NK, myeloid, and dendritic cells
(7). Here, we have used the expression of the pT
gene as
a tool to identify a human T cell precursor in the peripheral
blood of adult donors.
(T Cell Diagnostic, Inc., Boston, MA).
To deplete samples from CD3+ cells, we used TR66 mAb (9)
that was FITC conjugated according to standard procedures. For
TCR-
cytoplasmic staining we used HB-9283 supernatant (American Type Culture Collection). Second-step antibodies used were
goat anti-mouse IgG1 R-PE or FITC conjugated (Southern Biotechnology Associates, Inc.)
cytoplasmic chain or
CD3-FITC. After washing twice with Saponin buffer, goat anti-
mouse IgG1 R-PE antibody was added in the case of TCR-
cytoplasmic staining. As background controls, for the TCR-
cytoplasmic staining we used the second-step antibody alone (goat
anti-mouse IgG1 R-PE) and for CD3 cytoplasmic staining an irrelevant FITC-conjugated antibody (CD20; Becton Dickinson).
and CD3
expression in the total population, cells (0.5 × 105) were directly
sorted into 500 µl of RNAzol (Cinna Scientific, Houston, TX)
in Eppendorf tubes. Total RNA was extracted according to the
protocol of the manufacturer. cDNA was prepared with random hexamer primers and reverse transcribed with a Superscript kit (GIBCO BRL, Gaithersburg, MD). In the analysis of pT
expression in a limited amount of cells, we used the automatic cell
deposit unit technique (ACDU): cells (50, 20, 10, 5, and 1) were
directly sorted in 5 µl PBS in 96-well PC plates type H (Costar,
Cambridge, MA). After sorting, plates were immediately put on
dry ice and left for 30 min. cDNA was made directly in the plates
after heating for 2 min at 95°C using MMLV reverse transcriptase
and random hexamer primers at 37°C. Primers used were oligonucleotides specific for pT
(5
-GGCACACCCTTTCCTTCTCTG-3
and 5
-GCAGGTCCTGGCTGTAGAAGC-3
), for CD3
(5
-GTCTCCATCTCTGGAACCACAG-3
and 5
-GGCCTTTCTATTCTTGCTCCAG-3
), for CD3
(5
-GGAGGAATTCACTGACATGGAACAGGGGAAGG-3
and 5
-ACTCGAATTCCTGAGTTCAATTCCTCCTCAAC-3
), for CD3
(5
-GTGAATTGCAATACCAGCATC-3
and 5
-GCTGTACTGAGCATCATCTC-3
) and for RAG-1 (5
-CCAAATTGCAGACATCTCAAC-3
and 5
-CAACATCTGCCTTCACATCGATCC-3
) gene transcripts. PCRs were done in a 30 µl
reaction mixture containing 50 mM KCl, 10 mM Tris-HCl (pH
8.3), 1.5 mM MgCl2, 10 mM mixed dNTP, 10 pM of each oligonucleotide primer, and 1 U AmpliTaq DNA polymerase
(Roche Diagnostic System). DNA was amplified for 35 cycles at
an annealing temperature of 55°C for pT
and RAG-1 and 63°C
for CD3 with a thermal cycling machine (Perkin-Elmer Cetus). A 9-µl portion of each amplified product was examined by 1.3% agarose gel electrophoresis and stained with ethidium bromide. In
the case of ACDU procedure, a first PCR was done directly in the plates on total cDNA samples in 85 µl reaction at 55°C annealing temperature for 30 cycles using an Hybaid Omnigen
PCR machine. Subsequently, 2 µl of each sample was used for a
second PCR following the procedure described above. Primers
used were the same for both PCRs. Primers to detect DJ
rearrangements at a transcriptional level were the following: 5
TGGGAGGGGCTGTTTTTGT-3
and 5
-GATCTCATAGAGGATGGTGGC-3
.
1 segment) and TBR1 (downstream, the J
1-6
element) primers (10) for genomic DJ
amplification.
-ACCATCCACAGGACCATGGACTGG-3
, for DJ
rearrangements
of the TCR-
locus with TBR3 (10), for immature D-J-C transcripts from the
locus with a C
-specific oligo 5
-GAGCCATCAGAAGCAGAGATC-3
.
Identification of a pT+ Population in Peripheral Blood.
message
in all of the samples tested (data not shown; Fig. 1 B, lane 1).
Depletion of CD3+ T cells from PBMC did not abrogate
the detection of pT
expression by PCR. This result was
expected, because mature T cells have completely lost expression of pT
(6). In view of our previously demonstration (6) that the thymic subset expressing the highest levels of pT
is CD3
CD4+CD8
(immature single positive, ISP), we investigated whether expression of CD4 was
shared by pT
+ cells in PBMC. Fig. 1 demonstrates that
this is indeed the case, because sorted CD4+, CD3
cells
(Fig. 1 A, shown in the box, R2) expressed pT
(Fig. 1 B,
lane 2). No pT
message could be detected in total PBMC
depleted of CD4+CD3
CD14
cells (Fig. 1 B, lane 3). Monocytes, which also express low levels of CD4 but are
CD3
, were excluded from the selection by staining with a
specific mAb (CD14) and on the basis of their size (Fig. 1 A).
Interestingly, the physical parameters (FSC versus SSC) of
the pT
+ subset differ substantially from those of mature
lymphocytes and monocytes, in that pT
+ cells have a size
and a cell complexity intermediate between lymphocytes and
monocytes (Fig. 1 A, bottom). This cell population represents, in different healthy adult individuals analyzed (25-35 yr old), 3-9% of gated cells (R1) and 0.1-0.5% of the total
PBMC, obtained by Ficoll preparation.
Fig. 1.
(A) FACScan® analysis of presorted and sorted cells from PBMC. Mononuclear cells were separated from total PB by standard Ficoll gradient
centrifugation, CD3 depleted using FITC-TR66 mAb and stained with CD4-PE- and CD14-FITC-specific mAbs. Top left, CD3/CD14 versus CD4
staining pattern of cells gated through R1 (FSC versus SSC, bottom left). R1 determined by backgating R2 into FSC versus SSC (see also size pattern, bottom right). Top and bottom right, cells after sorting (purity close to 100%). (B) CD4+CD3CD14
sorted cells express pT
. RT-PCR analysis for the expression of pT
(top) and
-actin (bottom) was performed on total PBMC, CD4+CD3
CD14
sorted cells according to gates shown in A and total sorted
PBMC gating out CD4+ CD3
CD14
(PBMC, CD4+CD3
CD14
). To ascertain that PCR products shown were derived from pT
cDNA, gels were
blotted and probed with a pT
-specific oligonucleotide (data not shown).
[View Larger Versions of these Images (28 + 28K GIF file)]
Rearrangement and
Gene Transcription in
pT
+ Cells in Peripheral Blood.
rearrangements precede V(D)J
rearrangements (15). Therefore, the pattern of TCR
gene rearrangements represents a helpful
marker to assess the maturational stage of a given population and its possible commitment to the T cell lineage. To
analyze the rearrangement status of the TCR-
locus (DJ
versus germline configuration) in the pT
+ population observed in human peripheral blood, genomic DNA was isolated from CD4+CD3
CD14
pT
+ cells, and a recently
described (10) PCR-based method was used, to amplify the
DJ
1 gene region specifically. Relevant controls in which
DNA was obtained either from CD4+CD3+ mature T lymphocytes, CD14+ monocytes (obtained from FACS® sorting), or from unseparated PBMC were run in parallel. The primer combination used in this method allows to amplify,
in a single PCR reaction, a germline-derived DNA fragment of 3 kb and/or several shorter DNA products derived
from DJ
rearranged loci. PCR products are then blotted
and hybridized with an internal oligonucleotide probe.
rearrangements (DJ
1.3, DJ
1.5, and DJ
1.6)
could be demonstrated in genomic DNA from CD4+CD3
CD14
progenitors, in addition to a dominant germline
band (GL) (Fig. 2 A). As expected, monocytes (CD14+) were
characterized by the presence of an amplification product only in germline configuration, while in mature T cells
(CD4+CD3+) and total PBMC all the different DJ
rearrangements were present. As expected, a band corresponding to germline gene configuration also could be detected
in PBMC because of the presence of non-T populations.
This result suggests the presence of T cell lineage committed cells within the pT
+ CD4+CD3
population.
Fig. 2.
Detection of partial (D-J) rearrangements of the TCR- locus. (A) At DNA level. Genomic DNA was isolated from sorted
CD4+CD3
CD14
, CD4+CD3+, CD14+, and total PBMC and amplified by PCR using TBF1 and TBR1 primers to detect D
1-J
1 rearrangements. Normal PBMC and monocytes (CD14+) were used as positive and negative controls, respectively. PCR products were blotted and
hybridized with TBR3 probe. PCR products, ranging from 200 to 3,000 bp, and the J
segment used are indicated. GL, germline. (B) At RNA
level. Total RNA was first isolated from sorted CD4+CD3
CD14
,
CD14+, and from total PBMC and, subsequently, RT-PCR using TBF1
and C
primer set was performed. PCR products were blotted and
probed with a C
-specific primer.
[View Larger Version of this Image (46K GIF file)]
rearrangements was paralleled by transcription of the rearranged genes,
we tested for the presence of transcripts corresponding to
the partial DJ
rearrangements by RT-PCR. Total RNA
was isolated from sorted peripheral populations (CD4+CD3
CD14
and CD14+) or total PBMC and reverse transcribed into cDNA before amplification with a primer specific for the upstream D
1 sequence together with a C
specific primer. PCR products were analyzed by Southern blotting with an internal oligo specific for the C
region.
All cDNA were controlled for integrity with
-actin primers before D-J-C amplification (data not shown). Results
are shown in Fig. 2 B, in which the presence of a transcript
of 600 bp, corresponding to partial DJ
rearrangement, is
detected both in unfractionated PBMC and sorted CD4+
CD3
CD14
cells, but not in CD14+ monocytes. Taken
together, these results show that both rearrangement of,
and transcription from TCR-
loci are present in a fraction of the pT
+ cell population present in peripheral blood.
CD14
Cell Population Expresses RAG-1
Gene and Is Not Cycling.
rearrangement of the TCR-
locus of CD4+CD3
CD14
cells, we tested for RAG-1 gene expression in two different
preparations of CD4+CD3
CD14
cells from human peripheral blood. RAG-1 message could indeed be detected
in both preparations (Fig. 3 A, lanes 1 and 2), as well as in
DN cells from thymus, but not in mature CD4+CD3+ T
cells.
Fig. 3.
CD4+CD3CD14
cell population expresses RAG-1 and is
not cycling. (A) RT-PCR analysis on two different samples of sorted
CD4+CD3
CD14
PBMC (1, 2), mature T cells (CD4+CD3+) and DN
thymocytes (DN Thymus). PCR products were blotted and probed with a
RAG-1-specific oligonucleotide. (B) Cell cycle analysis on sorted
CD4+CD3
CD14
(top) and on Jurkat T cell line (bottom).
[View Larger Version of this Image (23K GIF file)]
CD4+CD8
immature thymocytes are
actively cycling, we then asked whether peripheral pT
+,
CD4+CD3
CD14
cells were also characterized by active
proliferation. Analysis of DNA content in the potential T cell
precursor population revealed that all of the cells were in
G0-G1 phase of the cell cycle, while TCR-
+ Jurkat T
cell line showed also G2-S phases (Fig. 3 B). Thus, CD4+
CD3
CD14
cells are in a resting state, express RAG-1
and pT
, and carry DJ rearrangements at the TCR-
locus.
CD14
cells
were isolated by flow cytometry as described above (see
Fig. 1) and subsequently stained with a set of monoclonal antibodies specific for cell surface markers. Fig. 4 shows
that sorted cells homogeneously expressed high levels of
CD45RA, intermediate levels of MHC class II HLA-DR,
low levels of the costimulatory molecule B7-2, but did not
express the hematopoietic progenitor cell surface antigen
CD34 or the neutral endopeptidase CD10, both present on
a T, B, NK, and DC cell precursor population recently described in human bone marrow (17). Only a very small
fraction, around 2-5% of sorted cells, expressed CD5 and
CD7, found on mature and immature T cells. A higher
fraction, 15-40% depending on the different individuals
analyzed, expressed the T- and NK-specific molecule
CD2. CD33, expressed on myeloid progenitors and monocytes, also presented a bimodal distribution with most of
the cells expressing low levels of the marker (CD33dim).
Both CD33+ and CD33
cells expressed pT
(data not
shown). CD4+CD3
CD14
cells did not exhibit detectable levels of the lineage-specific cell markers CD1a, CD8,
CD16, and CD56, CD19 and CD20,
TCR and CD83
(data not shown).
Fig. 4.
Cell surface analysis of pT+ cells. Mononuclear cells were
separated with standard Ficoll gradient and depleted of CD3+ cells with
FITC-TR66 mAb. Three-color analysis was then performed using CD4-
and CD14-specific mAbs in combination with a third antibody of interest
(histograms of which are shown in the figure): in the case of biotinylated
antibodies (CD2, CD10, and HLA-DR), CD4-R-PE and CD14-FITC
and, subsequently, APC were used; in the case of directly PE-labeled antibodies (CD5, CD7, CD33, and CD34), CD4-tricolor and CD14-FITC
were used. When staining with CD33 or B7-2 mAbs we had to sort
CD4+CD3
CD14
cells, as shown in Fig. 1, and restain them with one
of the two antibodies that were then detected with mouse anti-human
IgG2.
[View Larger Version of this Image (29K GIF file)]
Chain in
CD4+CD3
CD14
PBMC.
Although the pT
+ peripheral blood cell population does not express CD3 on the cell
surface, as assessed by FACS® analysis, we investigated
whether transcripts for the different components of this
TCR-associated signalling molecule could be detected. For
this purpose, cDNA from CD4+CD3
CD14
sorted cells
was amplified with three different sets of primers specific
for CD3-
, CD3-
, and CD3-
transcripts, respectively. Analysis of the PCR products showed that CD4+CD3
CD14
cells expressed all three CD3 components, as can
be observed in the T cell line MOLT 4 (Fig. 5 A); CD3-
was found to be expressed in low amount, because it appeared barely detectable on agarose gels after staining with
ethidium bromide; nonetheless, the presence of the message was readily detectable after hybridization with a specific internal probe of the blotted PCR products (data not shown).
Fig. 5.
(A) CD3-, CD3-
,
and CD3-
transcripts are expressed in pT
+ cells. RT-PCR
analysis with primers specific for
three components (
,
, and
)
of the CD3 signaling molecule
was performed on CD4+
CD3
CD14
cell population.
The T cell line MOLT4 and water were used as positive and
negative controls, respectively.
(B) CD3-
molecule, but not
TCR
chain, is present in the
cytoplasm of pT
+ cells. Cytoplasmic staining was performed
with a TCR-
-specific (top right
and left) or a CD3-
-specific (bottom right and left) mAbs on a T
cell clone (right top and bottom)
and on CD4+CD3
CD14
sorted PBMC (left top and bottom). In the case of TCR-
cytoplasmic staining, mouse anti-
human IgG1 was subsequently
used. Dotted histogram lines
represent negative controls that
were the following: for
-cytoplasmic, mouse anti-human
IgG1 alone and for CD3-cytoplasmic, CD20-FITC.
[View Larger Versions of these Images (24 + 50K GIF file)]
and
CD3 components in the CD4+CD3
CD14
cell population, together with the described rearrangement of TCR-
genes, raised the possibility that, at this stage, such cells
might assemble in their cytoplasm a putative human preTCR complex consisting of a TCR-
chain associated
with the pT
chain. To test this possibility we performed
cytoplasmic staining to detect the presence of TCR-
and
CD3-
proteins in CD4+CD3
CD14
cells. Flow cytometric analysis shown in Fig. 5 B revealed that while pT
+
cells do not express TCR-
chain in the cytoplasm, all cells within this population express CD3-
protein, but at lower
levels than in a mature human T cell clone. Specificity of
the CD3-
cytoplasmic staining was confirmed by relevant
controls, and in particular by the lack of any FACS® profile
shift in similarly stained B lymphocytes and dendritic cells
(data not shown). The absence of TCR-
chain in the cytoplasm of CD4+CD3
CD14
cells is consistent with lack
of VDJ
rearrangements at DNA level (data not shown).
These data indicate that while some components of such
putative human pre-TCR, and in particular CD3-
, are
not only transcribed, but also translated into proteins, others, like the TCR-
chain are only present in the form of
D-J transcript, while no translation can be detected.
To define whether all CD4+CD3CD14
cells or, on the
other hand, only a fraction of them expressed pT
, we performed PCR-based analysis of pT
expression on discrete
numbers of sorted CD4+CD3
CD14
PBMC obtained
with the ACDU technique. In brief, 50, 20, 10, or 5 CD4+CD3
CD14
cells were directly sorted in 96-well
plates and cDNA was synthesized directly in the plates.
Two rounds of PCR followed, a first one on the total
cDNA obtained and a second one on a fraction of the first
PCR product (2 µl). For both PCRs, the same pT
-specific primers were used.
When we initially restricted our analysis to duplicates of
each sorted cell aliquots (50, 20, 10, 5, cells; Fig. 6, top left), a positive reaction could be detected in all the wells, down
to the ones that contained 5 cells/well. To try and get a
more accurate and reliable estimate of the frequency, ten
separate PCR reactions were run, each on a well containing 5 cells (Fig. 6, top right). The analysis of the results
showed a positive signal in 50% of the samples. Assuming
that our PCR was 100% efficient on these 5-cell samples, a
frequency of pT+ cells of 1/7 can be calculated using Poisson's equation.
Next, we asked whether differences existed in the frequency of pT+ cells in the two subsets that could be identified by differential expression of CD2. Therefore, we separated our starting population into CD2+ and CD2
(see
Fig. 4) subsets and performed the same PCR-based analysis. This showed an enriched pT
+ cell frequency in the
CD2
subset where all samples containing 5 cells/well
scored positive, whereas only 50% of the samples containing 10 cells/well showed a positive PCR reaction in the
CD2+ subset (Fig. 6, bottom).
T Cell Differentiation Potential.
The fact that the CD4+CD3
CD14
PB cell population was found to express CD3-
,
CD3-
, and CD3-
, pT
, and RAG-1 transcripts, and has
partially rearranged the TCR-
locus strongly suggested that committed T cell precursors were present. Moreover,
the phenotype of these cells resembles that of the CD3
CD4+CD8
ISP thymocytes. These latter cells differentiate
into DP and CD4+TCR-
+ cells in mouse FTOC. To
analyze the T cell developmental potential, CD4+CD3
CD14
peripheral blood cells were introduced into FTOC.
After 3 wk of incubation only few human cells were recovered from the FTOC with CD4+CD3
CD14
peripheral
blood cells. To determine accurately the phenotype of these
cells, we stained with anti-CD45, anti-CD4, and anti-CD3 or anti-CD8. Fig. 7 shows that almost all human cells expressed high levels of CD3 and CD4. The CD4+ cells
completely lack CD8. This pattern of differentiation is distinct from what we observe with CD3
CD4+CD8
ISP
thymocytes, which give rise to a majority of DP and SP
cells (Fig. 7).
As the CD2
To characterize further the progeny obtained from CD4+CD3
In the present work, we describe a novel population of
T cell progenitors present in human peripheral blood. This
cell population lacks CD3 on the cell membrane, expresses
CD4, but not the early hematopoietic progenitor cell marker
CD34. The latter molecule has been shown to be expressed
on all of the early T cell progenitor populations described
until now, both inside (19) and outside the thymus (17,
20). Despite the absence of CD34, these cells have
characteristics of committed T cell progenitors. Specifically, these cells show partial TCR- locus rearrangements
(DJ but not VDJ) and have transcripts of genes of pT
and
RAG-1. Moreover, these cells express CD3-
, CD3-
, and
CD3-
genes. The fact that these CD4+CD3
cells do not
express CD34 and that they were found to be the only
fraction in total PBMC positive for pT
, is in contrast with some recently published data (7) in which CD34+ cells isolated from both adult and cord blood (CB) were found to
be pT
+. We failed to detect pT
message in CD34+ cells
present in either PB (data not shown) and CB (Blom, B., and H. Spits, manuscript in preparation). This discrepancy
may be due to differences in the purification of CD34+
cells; moreover, CD34+ cells were obtained with G-CSF
treatment (7); it is possible that in this way pT
+ cells are
mobilized from bone marrow, where we can detect pT
RNA in CD34+ cells (data not shown).
Cells within this population can develop into CD4+
TCR-+ cells in both FTOC as well as with IL-2, IL-7,
and PHA. This latter development is not due to selective
outgrowth of mature T cells, because the cloning efficiency
(5-10%) is much higher than the theoretical contamination
of cell surface CD3+ cells (0.1-0.5%). In addition, the possibility that CD4+CD3
CD14
cells are derived from activated mature CD4+ T cells that have downregulated the
TCR seems to be highly unlikely because, in contrast with
mature T cells, these cells do not express the TCR-
protein in the cytoplasm. Moreover, also the relatively low
levels of CD3-
protein in the cytoplasm, size and cell
complexity (FSC versus SSC) distinguish the CD4+CD3
CD14
cells from mature T cells.
The development of CD4+CD3CD14
peripheral blood
cells into CD4+TCR-
+ cells in both a human/mouse
hybrid FTOC as well as with IL-2, IL-7, and PHA clearly
demonstrates the presence of T cell precursors in adult
PBMC and could suggest that they are prothymocytes on their way to the thymus. Cells, similar to the CD4+ peripheral blood cells, with partial DJ
rearrangements and
pT
gene expression have been identified in day 15.5 murine fetal blood and were shown to be able to home in the
thymus and to give rise to DP and SP thymocytes (23). A
fraction (~65%) of these fetal blood murine cells also expressed CD4 (Rodewald, H.R., personal communication).
It is possible that the CD4+CD3
CD14
PBMC are the
direct precursors of the CD3
CD4+CD8
ISP cells in the
thymus. These latter cells are actively dividing, express
CD1, CD5, and CD7, and develop into DP and SP cells in an FTOC and in human thymic fragments transplanted
SCID mice. However, although CD4+CD3
CD14
cells
and CD3
CD4+CD8
ISP thymocytes share expression of
CD4 and of pT
, the two populations are distinct both
with respect to phenotypic and differentiation characteristics (Table 1). The most conspicuous phenotypic difference concerns the expression of CD1a, which is present on all
ISP thymocytes but not on the CD4+CD3
CD14
PBMC
and CD45RA, which has an opposite expression pattern.
More importantly, CD4+CD3
CD14
PBMC are not
able to develop in the hybrid human/mouse FTOC in double positive (DP) cells, but only into single-positive
(SP) CD4+CD3+ cells, whereas DP and few CD3+ SP are
obtained from ISP thymocytes in the same experimental conditions. Moreover, it is impossible to expand ISP thymocytes with IL-2, IL-7, PHA, and feeder cells as observed
for CD4+CD3
CD14
PBMC both in bulk culture and in
limiting dilution (results not shown). If these CD3
CD4+
PBMC are prothymocytes, one should assume that cells of
different developmental stages can enter the thymus, because the thymus also contains CD34+CD1
progenitor cells
with the TCR in germline configuration and the capacity
to develop into T cells, DC, and NK cells (18). One should
also expect to find a population that is intermediate between CD3
CD4+ PBMC and ISP thymocytes. Such a
population has not yet been described in the human thymus. Based on the fact that the thymus in adults is severely
involuted, it may be reasoned that there is not an influx of
new progenitors into the thymus in adults. This notion, together with the observations pointing to substantial differences with ISP thymic precursors, in particular with respect
to the capacity to develop into DP cells in a FTOC, could
raise the alternative possibility that CD4+CD3
CD14
cells represent an extrathymic pathway of T cell development. Studies with nude mice have shown that mature T cells
can develop in aged mice in the absence of a thymus (24),
indicating that this organ is not required for development
of some T cells. CD3
Thy-1low cells have been found in the
bone marrow of nude mice that have partial DJ rearrangements of the TCR-
locus (25). A transcript containing
TCR-
constant region sequences but not variable region sequences was amplified, suggesting that an unrearranged
TCR-
gene locus is transcriptionally active in this bone
marrow population. These characteristics of Thy-1lowCD3
cells in nude bone marrow cells are very similar to those of the CD3
CD4+ PBMC population described here. T cells
that develop in nude mice have a different TCR-V
repertoire than euthymic controls (26) and some TCR-V
can be
expressed on extrathymically developed cells that are deleted in euthymic controls (27). The TCR-V
repertoire
of the CD3+ cells that develop from the CD4+CD3
CD14
has not yet been investigated in detail. However,
we can exclude that these circulating PBMC are the precursors of those T cells that express a monomorphic TCR
(V
24, V
11) (28, 29), because none of the CD4+CD3+
cells developing from CD4+CD3
CD14
PBMC cells
were found to be V
24+V
11+ (data not shown).
Received for publication 24 October 1996 and in revised form 9 December 1996.
The Basel Institute for Immunology was founded and is supported by F. Hoffmann-La Roche, Limited, Basel, Switzerland.We thank H. von Boehmer for advice and support; K. Hafen for expert technical assistance; H.P. Strahlberger for art work; L. Inverardi, A. Kruisbeek, and H.R. Rodewald for critical reading of the manuscript; E. Ten Boeckel for suggestions concerning the PCR, T. Goebel, T. Winkler, G. Wiedle, A. Young, J. Bachl, J. Kirberg, T. Borgreffe, C. Schwaerzeler, C. Schaniel, R. Ceredig, M. Kopf, R. Mussman, M. Colonna, and volunteers working at the Basel Institute for Immunology for providing their blood.
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