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Blood, Vol. 93 No. 3 (February 1), 1999:
pp. 974-990
Upregulated Expression of Fibronectin Receptors Underlines the
Adhesive Capability of Thymocytes to Thymic Epithelial Cells During the
Early Stages of Differentiation: Lessons From Sublethally Irradiated
Mice
By
Sergio R. Dalmau,
Claudia S. Freitas, and
Wilson Savino
From the Program of Experimental Medicine, Basic Research Center,
National Cancer Institute, Rio de Janeiro, Brazil; the Department of
Biochemistry, Biology Institute, University of the State of Rio de
Janeiro, Rio de Janeiro, Brazil; and Laboratory on Thymus Research, the
Department of Immunology, Oswaldo Cruz Institute-Oswaldo Cruz
Foundation, Rio de Janeiro, Brazil.
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ABSTRACT |
A 250-cGy whole-body  -radiation dose was used to induce thymus
regression in mice, and to study the expression and function of
extracellular matrix (ECM) receptors in distinct thymocyte subsets
emerging during repopulation of the organ. The onset of regeneration
was detected from day 2 to 3 postirradiation (P-Ir), when a remarkable
increase in the absolute counts of
CD3 CD25hiCD44+ and
CD3CD25in/hiCD44
cells occurred. Enhanced expression of L-selectin,  4, and 5 integrin chains (L-selhi 4hi
5hi) was also exhibited by these cells. This pattern of
expression was maintained until the
CD4+CD8+ (DP) young stage was achieved.
Afterward, there was a general downregulation of these ECM receptors in
DP as well as in CD4+ or CD8+ single
positive (SP) thymocytes (L-selin 4in
5in). In some recently generated SP cells, 4
expression was downregulated before the 5 chain, and L-selectin was
upregulated in half of more mature cells. The expression of the 6
integrin chain was downregulated only in maturing CD4+
cells. Importantly, the increased expression of L-selectin and 4 and
5 chains in thymocytes was strongly correlated with their adhesiveness to thymic epithelial cells (TEC) in vitro. Blocking experiments with monoclonal antibody or peptides showed the following: (1) that the LDV rather than the REDV cell attachment motif in the IIIC
segment of fibronectin is targeted by the 4 integrin during
thymocyte/TEC adhesion; (2) that the RGD motif of the
120-kD fragment of fibronectin, a target for 5
integrin, has a secondary role in this adhesion; and (3) that the YIGSR
cell attachment motif of the  1 chain of laminin/merosin recognized
by a nonintegrin receptor is not used for thymocyte adherence. In
conclusion, our results show that an upregulated set of receptors
endows CD25+ precursors and cells up to the young DP
stage with a high capability of interacting with thymic ECM components.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
SUSTAINED CONTACT of thymocytes with the
thymic microenvironment has been shown to be necessary for the
generation of CD4+CD8+ double-positive (DP)
thymocytes,1,2 as well as for a successful progression to
positive selection.3 An obvious conclusion from these
observations is that thymic environmental components deliver signals to
thymocytes, such as those mediated by soluble and/or membrane-bound cytokines,4,5 as well as by ligands present on the membrane of stroma cells or in the extracellular matrix (ECM)
compartment.6,7
One ECM component that seems to be implicated in thymocyte
differentiation is fibronectin (FN). In vitro, coculture of murine CD4CD8 double-negative (DN)
thymocytes with thymic microenvironmental cell lines promotes their
passage to the DP stage. In this system, addition of anti-FN antibodies
blocks the DN  DP transition.1,2 Moreover, murine
DN cells and human DN and DP cells represent the major subsets that
adhere to FN-coated plates.6,8 Additionally, FN supports
migration of murine DP thymocytes through the cortically located thymic
nurse cells9 and the migration of human CD4+ or
CD8+ single-positive (SP) thymocytes.10
Other thymic ECM components such as laminin (LN) and one particular
laminin variant, namely merosin (laminin-2), influence thymocyte
migration and differentiation. Laminin is involved in the migration of
immature thymocytes within thymic nurse cell complexes.11
Furthermore, in the presence of Mn2+, up to 50% of human
thymocytes can bind constitutively to LN- or LN-2-coated surfaces,
using  61 and, to a lesser extent, 31 integrins.12 A putative role for FN and LN in thymic
positive selection was suggested by results showing that these proteins can costimulate the CD3-driven thymocyte proliferation.8,12
Although consistent, the above results mostly derive from in vitro
studies. In this context, putative in vivo fluctuations of ECM
receptors along with thymocyte differentiation may provide clues for a
better understanding of how thymocytes sequentially interact with ECM.
Sublethal irradiation promotes a profound depletion of the thymic
lymphoid compartment. Depletion is followed by a wave of repopulation
solely dependent on intrathymic resident precursors, and being
completed by day 10 postirradiation (P-Ir).13,14 Therefore,
this "ontogenetic recapitulation" model is useful for studying
the expression of molecules in thymocytes at particular stages of their
development, under the influence of the stromal architecture of an
adult thymus. In this report we analyzed the following: (1) the
expression of ECM receptors throughout thymocyte differentiation after
exposure of adult mice to 250 cGy -radiation; (2) the correlation
between the expression of these receptors and the ECM adhesive status
of thymocytes; and (3) the participation of some ECM receptors in
mediating this adhesive process.
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MATERIALS AND METHODS |
Animals.
Male and female C57Bl/6 mice ages 6 to 8 weeks were obtained from the
animal care facilities of the Brazilian National Cancer Institute, Rio
de Janeiro, and kept on a 12-hour (6:00 AM to 6:00 PM) light regimen with food and water ad libitum.
After irradiation, animals were housed at a maximum of six to a cage.
In some experiments 15- to 17-day fetuses were used.
Antibodies and chemicals.
The following monoclonal (MoAb) and conventional antibodies were used:
phycoerythrin (PE)-conjugated anti-CD4 (clone GK1.5) and fluorescein
isothiocyanate (FITC)- or biotin-conjugated anti-CD8 (clone 53.6.7)
MoAbs were purchased from Becton Dickinson (Mountain View, CA); the
FITC-conjugated anti-CD3 (clone 145-2C11) was from Boehringer
Mannheim (Mannheim, Germany), and the biotin-conjugated anti-CD3
MoAb (clone 99B) was from GIBCO-BRL Life Technologies (Gaithersburg,
MD). Unlabeled (no azide/low endotoxin) or PE-conjugated anti-4
integrin chain (clone R1-2) and anti-5 integrin chain (clone
5H10-27) MoAb, as well as PE-conjugated anti-CD117 (clone 2B8),
anti-L-selectin (clone MEL-14) MoAbs were obtained from Pharmingen
(San Diego, CA); the anti-6 integrin chain MoAb (clone GoH3) was
from Immunotech (Marseille, France), and the FITC-bound anti-CD25
(clone AMT.13), the biotinylated anti-phosphotyrosine (clone PT-66),
the quantum red-coupled anti-CD4 (clone H129.19), anti-CD8
(clone 53-6.7), and the anti-CD44 MoAbs (clone IM7.8.1) were from Sigma
Chemical Co (St Louis, MO). The FITC-conjugated rabbit anti-rat Ig
serum was from W & L Immunochemicals (Rio de Janeiro, Brazil), and the
FITC-, PE-, or biotin-conjugated isotype-matched rat IgG1
or IgG2 antibody controls were purchased from Becton Dickinson, Pharmingen, or Sigma. Control goat, hamster, or mouse Ig
fractions were prepared in our laboratories. When using biotinylated antibodies, specific labeling was visualized with streptavidin-PE (Becton Dickinson) or streptavidin-Tricolor (Caltag Laboratories, San
Francisco, CA). The following peptides were purchased from Sigma: the
1-25 and 90-109 amino acid sequences of the alternatively spliced type
III connecting segment (CSIII) of FN (F-5007 and F-6398, respectively);
the Gly-Arg-Gly-Asp-Ser (GRGDS) sequence enclosed by the 120-kD
fragment of FN (G-4391), and the 925-933 sequence of the 1 chain of
LN that binds the nonintegrin 67-kD cellular receptor (G-0668). Unless
otherwise stated, all chemicals cited below were purchased from Sigma.
Irradiation schedule.
Mice were habituated to the presence of a 10 × 8 × 2-cm fenestrated acrylic box left in the cage for 24 hours.
Five or six mice were then gently forced to enter the box and were
whole-body irradiated between 5:00 and 7:00 AM. The radiation
dose was 250 cGy, delivered by a 60Co -ray source
(THERATRON 780C apparatus; Theratronics Int Ltd, Kanata, Ontario,
Canada) at a dose rate of 25 cGy/min.
Thymuses and thymocyte suspensions.
Thymuses were excised after bleeding and killing the mice by cervical
dislocation under deep anesthesia. After washing in Eagle's minimum
essential medium with Earle's base, pH 7.2 (MEM), thymuses were minced
in MEM diluted 1:2 with 0.9 g% NaCl (MEM/3) plus 5% fetal calf serum
(FCS; Fazenda Pigue, Nova Friburgo, RJ) and pressed through a 200 mesh
stainless steel grid with the aid of a rubber policeman. Thymocytes
were washed once in MEM/3-FCS (400g, 5 minutes, 10°C) and
suspended to 2 × 107 cells/mL, being kept on melting
ice before labeling. In some experiments, thymocytes were treated with
an NH4Cl solution for erythrocyte lysis.
Detection of apoptotic DNA.
Internucleosomal apoptotic DNA breaks were assessed by agarose (1.8%)
gel electrophoresis as reported by Swat et al.15 Genomic DNA extracted from 1 × 106 viable thymocytes
(viability determined by the criteria of birefringence under phase
contrast microscopy) was applied to each lane.
Nuclear staining for cell-cycle phase analysis.
For nuclear staining 0.5 to 1.0 × 106 thymocytes in
25 to 50 µL were transferred to appropriate flow cytometer tubes.
One-half milliliter of a 50 µg/mL propidium iodide
(PI)/4 mmol/L trisodium citrate/0.3% Triton X-100 solution was added
and the tubes were left to stand at room temperature in the dark for 15 minutes. The same volume of 100 µg/mL bovine pancreatic RNAse (5 to
10 Kunitz U/mL, R-5503) in 40 mmol/L trisodium citrate, pH 8.2, was then added and the tubes were left to stand under the same conditions for an additional 15 minutes.
Labeling of surface antigens.
Because of the poor cell yield obtained from day 2 and 3 P-Ir mice, we
opted to perform labelings with thymocyte pools from at least three
organs. One million, 1.5 × 106, or 2.0 × 106 thymocytes were labeled with relevant or irrelevant
antibodies diluted in MEM/3-FCS in 200-µL final volumes. Incubations
were done in 1-mL Eppendorf tubes on melting ice in the dark for 45 minutes. When one of the antibodies was biotinylated, the cells underwent a second step of labeling with streptavidin-Tricolor or
streptavidin-PE. Alternatively, when using an uncoupled antibody (ie,
anti-6 chain MoAb), dual or triple labelings were done in three
steps: the first using the relevant (or irrelevant) antibody, the
second with the fluorochrome-coupled anti-Ig antibody, and the third
with the other antigen-directed fluorochrome-conjugated antibody(ies).
At the end of each incubation step, 1 mL of MEM1/3-FCS was added and
the cells were spun down at 1,000g for 1.5 minutes. Cell
pellets were washed twice and then suspended in either 100 to 200 µL
(for the next labeling step) or 1.0 mL of this medium (for
cytofluorometric analysis). The DNA-specific fluorochrome, PI, was finally added at a final concentration of 2 µg/mL, to exclude dead cells (which are permeable to propidium
iodide) during analysis.
Labeling of intracellular phosphotyrosine (PTyr).
After surface immunostaining, cells were washed twice with
phosphate-buffered saline (PBS) and the cell pellet was suspended in
1.0 mL of a freshly prepared solution of 1.0% paraformaldehyde plus
0.01% Tween 20 in PBS, pH 7.2. The suspension was kept protected from
light in a refrigerator for 24 to 48 hours to allow full fixation and
permeabilization of the specimens. Cells were washed three times with
PBS-FCS and incubated with standardized amounts of conjugated anti-PTyr
MoAb in 200 µL PBS at room temperature in the dark for 1 hour. Cells
were finally washed two times with PBS-FCS and suspended in the same
solution for the cytofluorometric readings.
Flow cytometry.
Flow cytometry was performed in a FACSCAN II apparatus (Becton
Dickinson) equipped with an air-cooled 488 nm argon ion laser of 15 mW.
The green fluorescence emission of FITC was detected after passage
through a 530 nm band pass filter, the orange emission of PE or PI
(cell cycle) after passage through a 585 nm band pass filter, and red
emission of tricolor or quantum red after passage through a long band
pass filter above 650 nm. Dynamic adjustment of electronic compensation
among these reading channels was performed to remove overlap of
spectral emissions. Data were obtained with the LYSYS II software
program (Becton Dickinson Immunocytometry Systems) and stored in a
Hewlett-Packard computer (model HP9000/300). Twenty
thousand to 50,000 thymocytes (or nuclei) were acquired in each reading
by the initial criteria of conventional size (forward light scatter)
and granularity (side light scatter).
Thymocyte adhesion assay.
A thymic epithelial cell line 2BH4 derived from C57Bl/6
mice16 was used for the thymocyte adhesion assays. These
cells secrete, among other ECM components, FN and LN, as assessed by
immunostaining in our laboratories. Cells in exponential growth were
detached by trypsinization and seeded in 25-cm2 Falcon
tissue culture flasks (5 × 105 viable cells per
flask) in 5 mL of RPMI 1640 medium plus 10% FCS. Twenty-four hours
later, the culture medium was removed and the nonconfluent 2BH4
monolayers were washed four times with warm FCS-free medium. A total of
1.5 to 2.5 × 107 thymocytes, suspended in 1 mL medium
without serum, were then added to the monolayers and the bottles left
to stand for 1 hour at 37°C in an incubator. After incubation,
loose thymocytes were aspired and the bottles gently washed with three
changes of 5 mL warm medium to remove the remaining nonadherent
thymocytes. Adherent thymocytes were recovered in 1.5 mL cold medium
with 10% FCS after beating the bottles horizontally followed by
pipette flushing under microscope observation. When testing for
antibody or peptide blocking activity, thymocytes were preincubated in their presence for 30 minutes in an ice bath before the addition to
2BH4 monolayers (see Table 2 for concentrations of each antibody or peptide).
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RESULTS |
Thymus cellularity after a 250-cGy exposure.
As depicted in Fig 1A, the percentage of
cells recovered from thymuses after a 250-cGy whole-body radiation dose
rapidly decreased, so that on days 2 and 3 P-Ir, only 1% to 2% of the
control values were recovered. This accounted for a reduction of more
than 80% (from 64.6 ± 15.3 mg to 11.1 ± 2.0 mg) in thymus
weight on these days. This thymocyte death occurred by apoptosis
because the typical DNA ladder in agarose gel electrophoresis was
strongly enhanced between 2 and 12 hours P-Ir (data not shown). Thymus
cellularity remained essentially unchanged by days 2-3 P-Ir, after
which repopulation started. The onset of repopulation was characterized
by a tremendous increase in the number of cycling cells (Fig 1B).
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| Fig 1.
Percentage and cycling status of thymocytes obtained from
control or irradiated mice. (A) The percentages ± SD of cells
recovered from the thymus (2 lobes) of control mice (day 0) or of mice
killed on different days after irradiation. The number of mice used per
point is shown in parenthesis. The range of absolute thymocyte count
recovery for control mice is indicated in the graph. (B) The
distribution of thymocytes according to their cycling status. In this
experiment, except for six individually analyzed control mice
(G0/G1 = 90.75 ± 0.86, S = 8.17 ± 0.73, and G2/M = 1.08 ± 0.16), percentage values
were obtained with the labeling of pooled thymocytes from at least
three animals per point.
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Evolution of thymocyte subpopulations after irradiation.
Absolute thymocyte counts showed that the first subpopulations starting
recovery 2 days P-Ir comprised the DN, the CD8+, and, to a
lesser extent, CD4+ phenotypes, all in the
CD3/lo stage (Fig 2).
Then, by day 3 P-Ir, CD3/loDP,
CD3in/hiDN, and CD3in/hiDP cells started to
increase. More radioresistant, the CD3in/hiCD4+
or CD3in/hiCD8+ subsets, which progressively
decreased in number until day 5 P-Ir, probably because of their exit to
the periphery, started to show an increase after this day (for the
CD4+) or after day 7 P-Ir (for the CD8+). Taken
together, these results confirm previous reports showing that in the
C57Bl/6 mouse strain the DN DP transition occurs preferentially by the acquisition of the CD8 antigen,17
with more time being expended for the generation of CD8+
than for CD4+ mature cells.18
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| Fig 2.
Absolute values of thymocyte subpopulations, as defined
by CD3/CD4/CD8 triple-labeling, found in control or irradiated mice.
For simplification, CD4/CD8-defined thymocyte subsets were divided into
 /lo or in/hi according to their level of CD3 expression. For
examples for this arbitrary CD3 division, see Figs 12 and 13. Values
were obtained with the labeling of pooled thymocytes from at least
three animals per point.
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CD25, CD44, and CD117 (c-kit) expression on thymocytes.
The surface expression of CD25 and/or CD44 has been shown to
define distinct stages within early differentiating CD3
thymocytes.19 As expected from the literature, in control
C57Bl/6 mice, less than 2% of CD25+ (ie,
CD25hi) cells and less than 3% of CD44+ (ie,
CD44in/hi) cells were found among CD3/lo
thymocytes (Fig 3). Maximal relative
increases in CD44+ and CD25+ cells were
attained on days 2 and 3 P-Ir, respectively. On day 3 P-Ir, when
CD3/lo thymocytes were seen mostly in DN and
CD8+ subsets, CD25hi plus CD25in
cells represented 70% of all CD3/lo thymocytes.
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| Fig 3.
CD25 and CD44 expression in CD3 and
CD3lo thymocytes in control or irradiated mice. Major
quadrants: upper left, CD25CD44+ cells;
upper right, CD25+(hi)CD44+ cells; lower
left, CD25+CD44 plus
CD25inCD44 (middle gate) cells; lower right;
CD25+CD44 cells. Numbers represent the
percentage of thymocytes in each quadrant or the gate. Values were
obtained with the labeling of pooled thymocytes from at least three
animals per point.
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An increase of CD25+ cells at the onset of P-Ir thymocyte
recovery has been previously reported.20 Our data
(Fig 4) show that
CD25+CD44+ and
CD25+CD44 cells, both restricted to the
CD3 stage, are the first to start recovery from day
2 to day 3 P-Ir. CD25+CD44+ cells exhibited a
higher expansion rate (ie, slope) between days 2 and 3 P-Ir, an early
decrease after this expansion (day 4 v day 7 P-Ir), and a
10-fold lower number throughout the recovery period, compared with
CD25+CD44 cells. This indicates that
CD25+CD44+ cells are precursors of
CD25+CD44 cells. In turn,
CD25CD44 cells started their
increase later, on day 4 P-Ir, when the first DP cells emerged (as seen
in Fig 2). Because they were present among both CD3
and CD3lo cells, a more advanced maturation status can be
ascribed to them when compared with the subsets mentioned earlier.
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| Fig 4.
CD25/CD44-defined thymocyte subsets obtained at distinct
stages of maturation (ie, level of CD3 expression) in control or
irradiated mice. Absolute values were obtained with the labeling of
pooled thymocytes from at least three animals per point.
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Considering that CD44 expression is shared by other leukocyte
types,21 the
CD3CD25CD44+ cells
seen in Figs 3 and 4 could putatively represent macrophages, dendritic
cells, B lymphocytes, and granulocytes, also discretely present in the
thymus.22 CD117, a stem cell factor receptor (c-kit), has
been shown to be expressed only in hematopoietic progenitors and its
levels decrease during thymocyte differentiation as early as in the
CD3CD4CD8
triple negative (TN) stage.19 Control and day 2 P-Ir
thymocytes showed over 20% and 45% CD117+ cells among
CD44+CD3 thymocytes, respectively
(Fig 5). Importantly, the labeling
intensity for CD117 was higher in CD44+ than in
CD44 cells, supporting the notion that the former
were in a more primitive stage of differentiation than the latter.
Taken together, these results support the phenotypic maturation
sequence CD25CD44+CD117++
CD25+CD44+CD117++
CD25+CD44CD117+
CD25CD44CD117
among CD3 cells, as observed during ontogeny or as
suggested by defective or genetically manipulated mice.19
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| Fig 5.
CD44 and CD117(c-kit) expression in CD3
thymocytes obtained from control or irradiated mice. Numbers represent
the percentage of thymocytes found in histogram selected region or dot
plot defined quadrant regions. In this typical experiment thymocyte
pools from 3 control or 6 day 2 P-Ir mice were labeled. Irrel. Ab,
irrelevant antibody.
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Expression of L-selectin and integrin 4 or 5 chains at
different CD3-defined thymocyte differentiation stages.
L-selectin (CD62L), a C-type lectin, recognizes sialil Lewis-x or
sialil Lewis-a sugar moieties properly anchored on
sialomucins.23 In control mice, most thymocytes exhibited a
positive level of staining, arbitrarily defined by us as intermediary
(L-selin), since a significant percentage of thymocytes
with a high level of L-selectin expression (L-selhi) were
also present (data not shown). The latter were found in all CD3 stages,
but their frequency was higher among CD3hi cells
(Fig 6). These L-selhi
thymocytes further increased among CD3hi cells until day 3 P-Ir.
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| Fig 6.
L-selectin expression as a function of CD3 surface level
in thymocytes obtained from control or irradiated mice. Numbers
represent the percentages found for L-sel (left region),
L-selin (middle region), and L-selhi (right
region) thymocytes. The area tagged with "irrl" shows the
labeling of control thymocytes with an irrelevant antibody. Values were
obtained with the labeling of pooled thymocytes from at least three
animals per point.
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Among CD3/lo thymocytes from control mice, about 20%
were L-selhi. In contrast, on day 3 P-Ir more than 50% of
CD3/lo thymocytes showed an L-selhi
profile. This was still seen on day 4 P-Ir, but on day 5 P-Ir and
thereafter thymocytes (except most CD3hi cells) regained a
predominant L-selin pattern, similar to control mice.
Analysis of integrins that bind to FN showed that 20% to 30% of bulk
control mouse thymocytes exhibited a high labeling density for 4 or
5 integrin chains, respectively (not shown). In contrast to
L-selhi cells, the frequency of 4hi and
5hi cells did not increase among CD3hi
thymocytes during the first days after irradiation (Figs
7 and 8). However, the
relative numbers of 4hi and 5hi cells in
CD3/lo thymocytes exceeded 65% from day 2 to 3 P-Ir. Cells bearing the 4hi and 5hi
phenotypes only reached a majority among CD3in thymocytes
on day 4 P-Ir. After day 5 P-Ir, the intermediary intensity of 4 or
5 labeling (4in or 5in), predominant
in CD3/lo/in thymocytes from control mice, was seen
again. Also noteworthy was the high proportion of
5hiCD3in/hi cells in control or day 10 P-Ir
mice in contrast to 4hiCD3in/hi cells,
indicating that 5hi expression is preserved for a longer
time than 4hi expression during thymocyte maturation.
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| Fig 7.
Integrin 4 chain expression as a function of CD3
surface level in thymocytes obtained from control or irradiated mice.
Numbers represent the percentages found for 4 (left
region), 4in (middle region), and 4hi
(right region) thymocytes. The area tagged with "irrl" shows the
labeling of control thymocytes with an irrelevant antibody. Values were
obtained with the labeling of pooled thymocytes from at least three
animals per point.
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| Fig 8.
Integrin 5 chain expression as a function of CD3
surface level in thymocytes obtained from control or irradiated mice.
Numbers represent the percentages found for 5 (left
region), 5in (middle region), and 5hi
(right region) thymocytes. The area tagged with "irrl" shows the
labeling of control thymocytes with an irrelevant antibody. Values were
obtained with the labeling of pooled thymocytes from at least three
animals per point.
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High expression of 4 and 5 chains is acquired at the
CD3CD25hi stage in adult mice and during
normal ontogeny.
The above results established temporal and numeric relationships
between the emergence of CD25in/hi cells and the increase
of L-selhi , 4hi, or 5hi
cells among CD3/lo thymocytes at the beginning of
reconstitution. Triple-labeling experiments with day 3 P-Ir thymocytes
confirmed that the vast majority of CD3/lo and
CD25in/hi thymocytes have 4hi or
5hi phenotypes (Fig 9, R2).
Interestingly, most CD25hi cells exhibited a
L-selin phenotype whereas most CD25/in
cells exhibited an L-selhi phenotype.
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| Fig 9.
CD3 and CD25 labeling profiles of 4hi,
5hi, and L-selhi cells from day 3 P-Ir
thymocytes. Thymocytes pooled from 20 irradiated mice were triple
stained with anti-CD3, anti-CD25, and anti-ECM receptor (anti-4
chain, anti-5 chain, or anti-L-selectin) MoAb cocktails, or with
irrelevant antibodies (Irrel. Ab). Thymocytes were arbitrarily split
into negative/intermediary (R1) or high (R2) staining for ECM receptor
expression (histograms) and then analyzed for CD3 and CD25 expression
(dot plots).
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Figure 10 shows that thymic
CD25in/hi cells from 17-day fetuses are present in a good
number, and most thymocytes have reached or are acquiring the DP
phenotype. Note the high expression of 4 or 5 chains as a
hallmark of CD25in/hi thymocytes. Integrin chain expression
started to decrease after CD25 downregulation and when the highest
level of CD8 expression (ie, DP phenotype) was reached. Similarly to
that observed on day 3 P-Ir, the L-selhi phenotype is
predominantly expressed by CD25in rather than
CD25hi cells.
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| Fig 10.
Expression of 4 and 5 integrin chains, and
L-selectin on day 17 fetal thymocytes. Thymocytes pooled from 15 17-day
fetuses were triple stained with anti-CD8, anti-CD25, and anti-ECM
receptor (anti-4 chain, anti-5 chain, or anti-L-selectin) MoAb
cocktails, or with irrelevant antibodies (Irrel. Ab).
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High expression of L-selectin and integrin 4 and 5 chains in
different CD4/CD8-defined thymocyte subsets.
In control mice, L-selhi, 4hi, and
5hi cells represented 45%, 70%, and 50% of DN
thymocytes, respectively. After a general decrease on day 1 P-Ir they
further increased, reaching 60%, 90%, and 70% on day 3 P-Ir,
respectively. The levels of 4hi or 5hi DN
cells were then roughly maintained at least until day 7 P-Ir, whereas
the levels of DN L-selhi cells decreased from day 4 to day
5 P-Ir and to levels close to those found in control mice thereafter
(Fig 11).
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| Fig 11.
Relative number of L-selhi,
4hi, and 5hi cells among CD4/CD8
thymocyte subsets. Values were obtained with the labeling of pooled
thymocytes from at least three animals per point.
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Similar profiles were found for 4hi or
5hi CD8+ cells. These cells decreased from
30% to 40% (control mice) to less than 20% up to day 2 P-Ir, then
exhibiting a recovery to 50% to 70% up to day 4 P-Ir. After another
transient decrease on day 5 P-Ir, they remained roughly in this range
until day 10 P-Ir. Nonetheless, L-selhi CD8+
cells (40% to 50% in control mice) did not exhibit a decrease after
irradiation, increasing to close to 80% on day 4 P-Ir, and behaving
like 4hi and 5hi CD8+ cells thereafter.
In control mice, 4hi cells represented only 5% of DP
thymocytes, while L-selhi or 5hi cells
represented roughly 10%. On day 1 P-Ir, these percentages increased,
exceeding 50% on days 3 and 4 P-Ir (when DP begins repopulation, as
seen in Fig 2), and suddenly decreased on day 5 P-Ir and to values
close to those found in control mice thereafter.
Finally, 4hi CD4+ cells were very poorly
represented in control mice (around 5%), with their percentages not
varying significantly after irradiation; only a mild increase was noted
between days 4 and 7 P-Ir. In turn, 5hi CD4+
cells were slightly better represented in control mice (about 15%).
After irradiation, they decreased to 5% at day 2 P-Ir and increased to
values close to 25% from day 5 to day 7 P-Ir, a percentage still
present on day 10 P-Ir. L-selhi CD4+ thymocytes
exhibited a continuous increase in relative values from 25% in control
mice to more than 50% on day 4 P-Ir. In contrast to
L-selhiCD8+ thymocytes,
L-selhiCD4+ cells later exhibited a gradual
decrease that peaked on day 7 P-Ir. Both the increase in
5hi CD4+ cells and the major decrease in
L-selhiCD4+ cells seen on day 7 P-Ir coincided
with the new generation of CD4+CD3in/hi cells
(as seen in Fig 2). Importantly, more than 95% of CD4+ or
CD8+ cells contributing to the increase of
L-selhi until day 3 or 2 p-Ir, respectively, were
CD3in/hi. Thereafter the relative increase of
L-selhi among these subsets was due to
CD3/lo thymocytes, mainly CD8+ ones
(Table 1).
In summary, the above results indicate that the high expression of
L-selectin and 4 and 5 integrin chains underlines the DN
DP transition.
Expression of integrin 6 chain within CD3- or CD4/CD8-defined
thymocyte subsets.
Immunolabeling of the 6 integrin chain allows the assessment of a
laminin receptor expression on thymocytes. As shown in Fig 12, thymocytes from control mice
exhibited a progressive decrease in 6 labeling, from the immature
CD3lo cells to the more mature CD3hi cells
(arbitrarily defined as 6hi to 6in). This
difference in 6 expression in the CD3lo CD3hi transition was sharper on day 3 P-Ir or even on day 4 P-Ir (data not shown). In keeping with this is the analysis of CD4/CD8
subsets (Fig 13). When DN cells were
represented mainly by CD3/lo cells, as in control or
day 4 and 6 P-Ir mice (Table 1), they exhibited a predominant
6hi profile. By contrast, when CD3in/hi
cells increased in number within them, as on day 2 P-Ir (40%), they
shifted to an 6in labeling profile. In control or day 2, 4, or 6 P-Ir mice, the vast majority of DP thymocytes maintained an
6hi profile (Fig 13). Regardless of the maturation
status, a predominant 6hi profile was also kept in
CD8+ cells; on day 2 P-Ir, when more than 98% of
CD8+ cells were found to be CD3in/hi, or
conversely on days 4 or 6 P-Ir, when 80% of them were found to be
CD3/lo, most exhibited 6hi labeling.
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| Fig 12.
Integrin 6 chain expression as a function of CD3
surface level in thymocytes obtained from control or day 3 P-Ir mice.
Numbers represent the percentages found for 6 (left
region), 6in (middle region), and 6hi
(right region) thymocytes. The area tagged with "irrl" shows the
labeling of control thymocytes with an irrelevant antibody. Values were
obtained with the labeling of pooled thymocytes from at least three
animals per point.
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| Fig 13.
Integrin 6 chain expression in CD4/CD8-defined
thymocyte subsets in control or day 2, 4, and 6 P-Ir. Numbers represent
the percentages found for 6 (left region),
6in (middle region), and 6hi (right
region) thymocytes. Values were obtained with the labeling of pooled
thymocytes from at least three animals per point. Note: in this typical
experiment, fluorescence intensities exhibited by subpopulations should
be compared only for each day because thymocyte labelings were done on
different days. Labeling with irrelevant antibody were done each day
(not shown) and defined the negative region (left region) in the
histograms.
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Differing from other subsets, CD4+ cells exhibited less
rigid 6 staining profiles. In control mice they were predominantly 6/in, a tendency that was emphasized until day 4 P-Ir. However, on day 6 P-Ir (ie, at the beginning of
CD3in/hi CD4+ cell expansion), they were seen
predominantly as 6hi cells. Moreover, when
CD4+ cells were arbitrarily split into two gates, those
cells enclosed in a gate closer to the CD4 axis in CD4 × CD8
plots (more mature) exhibited a 65% to 100% lower intensity of 6
labeling than those enclosed in a gate more distant from the CD4 axis
(less mature), both in control and different day P-Ir mice (not shown).
A similar split for CD8+ cells showed a less pronounced but
still detectable (35%) decay in 6 labeling for cells closer to the
CD8 axis as compared to those more distant from this axis (data not shown).
Upregulation of fibronectin receptors is associated with an
intracellular increase of phosphotyrosine levels.
Treatment of human thymocytes with antibodies against 1 or 5
integrin chains has been shown to induce protein tyrosine
phosphorylation.24 Therefore, we analyzed the levels of
intracellular PTyr in thymocytes between days 2 and 3 P-Ir, the period
when most CD3/lo cells with a high 4, 5, or
L-sel surface expression started to accumulate. In control mice, most
of the CD3/lo cells exhibited a PTyrin
labeling profile, while CD3in/hi cells shifted to a
PTyrhi profile. Thus, at first glance, elevation of
intracellular PTyr content seems to be correlated with thymocyte
maturation. However, in these animals 15% of CD3/lo
cells exhibited a PTyrhi label represented mainly by DN and
CD8+ cells (data not shown). The presence of such
CD3/lo PTyrhi cells was maximal at days
3-4 P-Ir when they corresponded to 75% of CD3-/lo cells
(data not shown).
Figure 14 shows that most
CD3/lo cells having acquired an 4hi
status also acquired PTyrhi labeling. Similar results were
obtained for 5hi acquisition related to
PTyrhi levels (not shown). Surprisingly, at day
21/2 P-Ir, the majority of
CD3/lo PTyrhi cells still exhibited
L-sel/in labeling (Fig
15), thus suggesting that the increase in intracellular PTyr was not
time-associated with L-sel upregulation in these cells.
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| Fig 14.
Intracellular phosphotyrosine content and integrin 4
chain surface expression in thymocytes from control or day
21/2 P-Ir mice. Plots show PTyr and integrin
4 chain levels according to the level of CD3 expression as defined
in the histograms. Background labeling with irrelevant antibodies
(Irrel. Ab) is also shown. Values were obtained with the labeling of
pooled thymocytes from at least three animals per point.
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| Fig 15.
Intracellular phosphotyrosine contents and L-selectin
surface expression in thymocytes from control or day
21/2 P-Ir mice. Plots show PTyr and L-sel
levels according to the level of CD3 expression as defined in the
histograms. Background labelings with irrelevant antibodies (Irrel. Ab)
are also shown. Values were obtained with the labeling of pooled
thymocytes from at least three animals per point.
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CD25+ cells are the most adherent to ECM; adhesion is
lost as CD3 expression is upregulated; enhanced ECM adhesion hallmarks
cells in the DN stage or transiting to the DP stage.
Taking into account the modulation of ECM receptors on thymocytes at
different stages of differentiation, as shown during the thymic
regression/reconstitution process after irradiation, and the
simultaneous increase in the intracellular phosphotyrosine levels and
FN receptors, we searched for the phenotypes of ECM interacting
thymocytes. For this purpose, the major histocompatibility complex
(MHC)-compatible 2BH4 thymic epithelial cell (TEC) line was used as a
source of ECM. Figure 16 shows the
phenotypes of adherent thymocytes recovered when control, day 3, or day
4 P-Ir thymocytes were allowed to bind to a subconfluent monolayer of 2BH4 cells. Adherent cells were enriched for
CD3/loCD25in/hi cells and depleted for
CD3hi cells. CD4/CD8 profiles showed a simultaneous
enrichment of DN cells among these thymocytes. It was noteworthy that,
despite no marked changes in the percentages of CD8+ cells
between total and adherent cells, the amounts of CD8+ cells
closer to the axis (enclosed in circles in Fig 16) were reduced. This
indicates that ECM adherent thymocytes were also enriched for cells
transiting to the DP stage.
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| Fig 16.
The phenotype of TEC adherent thymocytes. Thymocytes
(2.5 × 107 in 1 mL FCS-free medium) from control, day 3, or day 4 P-Ir were added to 2BH4 subconfluent monolayers in
25-cm2 flasks and incubated for 1 hour at 37°C.
Nonadherent thymocytes were removed by aspiration followed by three
gentle washes with warm medium. Adherent thymocytes were recovered in
cold medium with FCS and stained for the markers shown on the axis. The
circled region in the lower set of panels shows the percentage of
CD8+ cells highly negative for CD4 labeling (ie, mature
CD8 cells). Values were obtained with the labeling of pooled thymocytes
from at least three mice per point.
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4 Integrin rather than 5 integrin intervenes in the adhesion of
day 3 P-Ir thymocytes to thymic epithelial cells.
The above experiments showed that an increase in the expression of ECM
receptors on thymocytes was related to their adhesiveness to ECM
components. However, they did not prove that the ECM receptors studied
were responsible for this adhesiveness. To address this issue,
anti-4 or anti-5 chain MoAbs reported to block integrin binding
to FN or peptides that mimic FN and interfere with integrin binding to
FN were assayed for their capability to inhibit adhesion of day 3 P-Ir
thymocytes to 2BH4 monolayers (Table 2).
Adhesion was strongly blocked by anti-4 chain MoAb (60% inhibition)
and the FN 1-25 IIICS peptide which contains the critical LDV motif recognized by 41 integrin (70% inhibition). A FN 90-109 IIICS segment, which contains a critical REDV motif also recognized by
41 integrin,25 exhibited a poor inhibitory effect
(21%). Anti-5 chain MoAb or FN GRGDS peptide which contains the
critical RGD motif recognized by 51 integrin exhibited no or poor
(18%) anti-adhesive action, respectively.
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Abstract
Introduction