By
From the Basel Institute for Immunology, CH-4005 Basel, Switzerland
Thymic T cell development is controlled by T cell receptor (TCR)-major histocompatibility complex (MHC) interactions, whereas a further dependence of peripheral mature T cells on TCR-MHC contact has not been described so far. To study this question, CD4 T cell survival was surveyed in mice lacking MHC class II expression and in mice expressing MHC class II exclusively on dendritic cells. Since neither of these mice positively select CD4 T cells in the thymus, they were grafted with MHC class II-positive embryonic thymic tissue, which had been depleted of bone marrow derived cells. Although the thymus grafts in both hosts were repopulated with host origin thymocytes of identical phenotype and numbers, an accumulation of CD4+ T cells in peripheral lymphoid organs could only be observed in mice expressing MHC class II on dendritic cells, but not in mice that were completely MHC class II deficient. As assessed by histology, the accumulating peripheral CD4 T cells were found to be in close contact with MHC class II+ dendritic cells, suggesting that CD4 T cells need peripheral MHC class II expression for survival and that class II+ dendritic cells might play an important role for the longevity of CD4 T cells.
Thymic positive selection is a process that generates mature CD4+ and CD8+ single-positive T lymphocytes
from CD4+CD8+ double-positive thymocytes. The mechanistic control of positive selection is the interaction between TCR on thymocytes and MHC-encoded molecules
on thymic epithelial cells. Mature CD4+ and CD8+ single-positive thymocytes, selected on MHC class II and I, respectively, subsequently leave the thymus and seed the peripheral
lymphoid organs (1). Consequently, CD4+ single-positive thymocytes and CD4+ peripheral T cells are nearly
absent in class II-deficient mice (4, 5).
The further survival of peripheral T cells seems not to be
dependent on antigen-specific TCR-MHC interactions.
Transfer experiments performed with T cells from TCR-transgenic mice in the presence or absence of antigen (6, 7)
showed that specific Ag is not necessary for T cell survival.
In another experimental model, Sprent et al. (8) demonstrated that when unseparated lymph node cell suspensions
were injected into H-2 identical SCID hosts, they formed a
self-sufficient pool of lymphocytes. T cells survived in this
system without reduction in numbers in the absence of antigen. However, conflicting results have been reported on
the survival of T cells in the absence of MHC molecules
expressed on hematopoietic cells. When irradiated normal
mice received bone marrow from class II-deficient mice,
normal CD4 T cell repopulation was observed in one study
(9). Others doing the same experiment could not detect reconstitution of the CD4 compartment in the MHC class
II-negative environment of such mice (10). Huss et al.
speculated that this discrepancy could have been caused by the different time spans of bone marrow inoculum in the
host mice used by the two groups or different bone marrow treatments (e.g., T cell depletion) before injection
(10). Therefore, these experiments could not definitely
clarify the question of whether peripheral CD4 T cell survival is dependent of peripheral MHC class II expression.
In a recent report, Takeda et al. (11) transplanted untreated
fetal thymi from MHC class II+ mice under the kidney
capsules of class II+, as well as class II-deficient, hosts. The
authors observed an identical initial donor type CD4+ T
cell accumulation in the periphery of both hosts. In comparison to the MHC class II+ mice, the class II-deficient
hosts showed faster declining numbers of peripheral CD4+
T cells. These results suggested that interactions between
CD4+ T cells and MHC class II+ peripheral cells are not
necessary for short-term survival, but might be important
for longevity of T cells. However, a potential contamination of the MHC class II-deficient peripheral organs of the
hosts with MHC class II+ donor type cells originating from
the transplanted thymi (thymic dendritic cells, B cells, macrophages) cannot be excluded when the thymus grafts
(TGs),1 are not depleted of hemopoietic cells before transplantation. Furthermore, the initial export of large numbers
of donor-type thymocytes from untreated grafts (12) might
not reflect the actual kinetics of thymocyte export from a
developing thymus.
To avoid the presence of donor-type thymocytes and to
exclude the possibility of contamination of the hosts with
thymus-derived MHC class II-positive cells, in this report
MHC class II+ fetal TGs were depleted of hematopoietic
cells before transplantation. Then survival of host-type
CD4 T cells in a host lacking MHC class II expression
completely (4) was compared to CD4 T cell survival in an
environment where only dendritic cells (DCs) express
MHC class II (13). Although TGs in both types of hosts
were repopulated with similar numbers of phenotypically
identical host-type thymocytes, only the mice expressing
MHC class II on DCs, but not the conventional MHC
class II-deficient hosts, showed accumulation of CD4 T
cells in blood and peripheral lymphoid organs. The conclusion from these experiments is that CD4 T cells, after having left the thymus, do need interaction with MHC class
II-positive peripheral cells for survival. MHC class II-positive DCs are sufficient to ensure this survival.
Mice.
Transgenic mice expressing the MHC class II I-E mAbs and Reagents.
The mAbs specific for CD4 (No. 09005),
CD8 (No. 01044), CD3 (No. 01085), heat stable antigen (HSA;
No. 01575), CD69 (No. 01505), V Isolation of DCs from Spleen.
Spleens of different mice were
digested with collagenase (CLSPA; Worthington Biochemical
Corp., Freehold, NJ) twice for 30 min at 37°C as described previously (13). Cells were then recovered by centrifugation at 300 g
for 5 min, resuspended in a 17% OptiprepTM (Nycomed Pharma,
Oslo, Norway) solution diluted in Hank's balanced salt solution
without Ca2+ and Mg2+, overlaid with 12% OptiprepTM diluted
in 0.88% (wt/vol) NaCl, 1 mM EDTA, 10 mM Hepes, 0.5%
BSA, pH 7.4, and 2 ml Hank's without Ca2+ and Mg2+, respectively. Cells were then centrifuged at 600 g for 15 min at 20°C.
The low density cells at the Hank's 12% OptiprepTM interface were harvested, washed twice, and used as a splenic DC-enriched fraction.
Transplantation of Fetal Thymus.
Fetal thymi from C57BL/6 × DBA/2 crosses were taken at day 14 of gestation (vaginal plug
was counted as day 0 of pregnancy). The thymic lobes were
placed on culture plate inserts (Millipore No. PIHA01250; Bedford, MA) floating on culture medium containing 1.35 mM 2 Immunohistology.
Fresh organs were embedded in O.C.T.
medium (No. 4583; Miles Inc., Elkhart, IN), snap frozen, and 6-mm
sections were cut with a cryostat. Sections were air dried (60 s),
acetone fixed (2 s), and finally air dried for a minimum of 12 h.
Sections were rehydrated in PBS containing 1% BSA and 10%
normal mouse serum. mAbs diluted in PBS/BSA/normal mouse
serum were added directly onto the sections and incubated for 60 s.
After washing, sections were either incubated with the second
step reagents or directly mounted in Fluoromount (Southern
Biotechnology Assoc. Inc., Birmingham, AL). For immunohistological analysis, we used biotinylated mAbs and alkaline phosphatase-conjugated streptavidin (No. RPN 1234; Amersham,
Buckinghamshire, UK). Color reaction was done with the Vector-Red Alkaline Phosphatase Substrate Kit (No. SK-5100; Vector Labs., Inc., Burlingame, CA) according to the manufacturer's
instructions.
Isolation and Analysis of Peripheral Blood Lymphocytes.
Mice were
bled at their tail veins and the blood was mixed immediately with
Heparin-containing PBS. Lymphocytes were then isolated over a
ficoll gradient, washed twice, and 105 viable cells were stained
with 20 mg/ml mAb that was directly labeled. After washing,
cells were analyzed using a FACScan®.
To investigate survival of CD4+ T cells in different environments, we studied CD4+ T cell repopulation of two
different mouse strains lacking endogeneous CD4+ T cells:
conventional MHC class II-deficient mice (B6I-A As described recently (13,
16), C57BL/6 mice expressing MHC class II I-E
To further characterize the cells expressing the I-E transgene, we isolated the light density cell fraction from collagenase-treated spleens as described earlier (18) and performed a three-color flow cytometric analysis (Fig. 1 b).
From all cells obtained in the low buoyant density fraction,
~80% were cells with relatively high forward scatter and
intermediate side scatter signals (data not shown), whereas
lymphocytes from the high density fraction had both low
forward and side scatter signals (data not shown). Analysis
of the cells from the low density fraction, which fulfilled the indicated forward/side scatter criteria, showed that
~70% were CD11c positive (Fig. 1 b). CD11c-positive
cells derived from B6I-A Taken together, these data indicate that the majority of
macrophages (>90%) do not express the transgene and that
the CD11c promoter used in this study drives expression of
the I-E transgene specifically to most of the DC in spleen
and lymph nodes. Due to absence of mature CD4+ T cells,
the B6CD11c-E To reconstitute B6CD11c-E
We then analyzed the expression of HSA. As described
previously, immature thymocytes express high levels of
HSA and loose this marker on their way to mature single
positive cells (27). Accordingly, thymocytes from TG of
B6I-A When CD69 expression in the thymocyes of different
origins was compared, we found the expected transient
CD69 upregulation (Fig. 2, right panel histograms, CD69
gate 2; reference 28). With further maturation, the percentage of CD69high thymocytes was decreasing again (Fig.
2, right panel histograms, CD69 gate 3).
These data demonstrate that the TG in B6I-A Because all mice (n = 2 × 20) in this experiment received the same identically treated embryonic thymi and
repopulated them with thymocytes with a similar efficiency, this experimental system seems to be ideal to study
the further development and survival of CD4 lineage T
cells in MHC class II disparate host environments.
In the B6CD11c-E
In the other experimental group, 20 MHC class II-negative B6I-A To check the possibility that the surviving CD4+ T cells
were an accumulation of (self) antigen-specific cells that had
been stimulated by I-E on DCs in B6CD11c-E We also examined the frequency of T cells expressing
various TCR V Immunohistochemical analysis of lymph nodes from transplanted
B6CD11c-E
In this report, two different mouse strains were used to
study CD4+ T cell survival in presence or absence of MHC
class II expression. Besides conventional MHC class II-deficient mice (B6I-A Benoist and Mathis (31) raised the question of which peripheral cells need to express MHC molecules to enhance
T cell survival. As shown in this and previous reports (13,
16), the CD11c promoter drives expression of the MHC
class II I-E transgene specifically to DCs of thymus (13),
spleen, and lymph nodes. Approximately 70-80% of all
subpopulations of DCs do express the transgenic I-E,
whereas B cells and the majority of macrophages (~90%)
are transgene negative. Relatively high levels of MHC class
II expression, together with costimulatory or adhesion
molecules on DCs (32), may predestine this cell type for
supporting peripheral T cell survival. Immunohistochemical analysis of frozen lymph node sections from the TG
transplanted B6CD11c-E The presented data suggest that mature CD4+ T cells,
once they have left the MHC class II-rich thymic environment, have to perpetually recognize MHC molecules in
the absence of specific antigen to survive. This peripheral
TCR-MHC interaction might create a baseline activation,
in the absence of which CD4+ T cells are extremely short
lived.
d
transgene under control of the mouse CD11c promoter were
bred to MHC class II
/
(B6 I-A
/
) mice (4, 13). The resulting
mice have been named B6CD11c-E
dI-A
/
in this study. Line
107.1 expressing I-E
d under the control of the MHC class II
promoter has been described earlier (14) and was crossed onto the
MHC class II
/
background (B6-E
dI-A
/
). All mice were
bred in the animal colony of the Basel Institute for Immunology
(Basel, Switzerland).
2 TCR (No. 01634), V
4
TCR (No. 01934), V
5.1/5.2 TCR (No. 01354), V
6 TCR
(No. 01364), V
7 TCR (No. 01424), V
8.1/8.2 TCR (No.
01344), V
11 TCR (No. 01374), V
13 TCR (No. 01394), I-E
(No. 09625), CD11c (No. 09705), CD62L (No. 01264), and
CD11b (No. 01712) were purchased from PharMingen (San Diego, CA). Anti-CD4 R613 and streptavidin-R613 (GIBCO BRL,
Gaithersburg, MD) were used for three-color fluorescence analysis. The rat anti-mouse mAbs 33D1 (TIB227; American Type Culture Collection, Rockville, MD) and NLDC-145 (reference
15; a kind gift of A. Schneeberger, Vienna, Austria) were used as culture supernatants. With these mAbs, flow cytometry was performed on a FACStar® (Becton Dickinson, Mountain View, CA)
instrument. Single cell preparation, staining and FACS® analysis
was done according to standard procedures.
-deoxyguanosine (dGuo; No. D-0901; Sigma Chemical Co., St.
Louis, MO). Thymi were incubated at 37°C, 6% CO2 for 5 d, and received an additional irradiation of 3,000 Rad when they were washed in a large volume of PBS to remove the drug. Each mouse received two thymic lobes under the kidney capsule.
/
; reference 4) or the same mice transgenically reconstituted for
MHC class II I-E expression on DCs (B6CD11c-E
dI-A
/
;
reference 13).
dI-A
/
Mice
Is Restricted to CD11c+ DCs.
d as a
transgene under the control of CD11c regulatory elements, display class II I-E expression only on DCs. When bred to
the class II-deficient (I-A
/
) background (4), the presence
of MHC class II I-E on DCs does not lead to positive selection of mature CD4+ thymocytes (13). By immunohistochemical (Fig. 1 a) and FACS® analysis of lymph nodes
and spleen (Fig. 1 b), I-E transgene expression correlates
with expression of the DC marker CD11c in B6CD11c-E
dI-A
/
mice. In lymph nodes of B6CD11c-E
dI-A
/
mice, transgenic I-E expression is restricted to DCs of the
paracortical T cell area, whereas the B cells in follicles, as
defined with an anti-IgM reagent, fail to express the transgene (Fig. 1 a). A similar I-E pattern is observed in the
spleen, where again B cell areas are negative for transgene
expression, whereas DCs in T areas of white pulp as well as
marginal zone DCs express I-E (Fig. 1 a). These findings are
in agreement with our recent reports, describing the absence of I-E transgene expression on CD19+ thymic B cells
(13) and B220+ splenic B cells (16). Macrophages as detected with anti-CD11b (Mac-1) reagents can be located
outside of the white pulp areas of the spleen and give a weak
staining scattered all over these regions (Fig. 1 a, Mac-1).
Furthermore, these cells can be localized in the outer regions of the marginal zone, whereas a weaker Mac-1 staining of the T areas of the white pulp might be due to Mac-1 expression on DCs, as reported previously (for review see
reference 17). In lymph nodes, Mac-1-positive macrophages could be found predominantly in the lateral regions
of the paracortex (Fig. 1 a). Since the I-E staining in lymph
node and spleen is localized in those regions that also stain
positive for CD11c, but not in those that are stained with
IgM and Mac-1 reagents, it seems that the CD11c regulatory elements drive expression of the transgenic I-E in a restricted manner to CD11c-positive cells. Previously, we reported that 2-7% of peritoneal IFN-
-treated cells of
B6CD11c-E
d mice would express the I-E transgene (13,
16). Those cells were Mac-1+ and Fc
III/II receptor+, and
could also be found in the B6CD11c-E
dI-A
/
mice used
in this study (references 13, 16, and data not shown).
Fig. 1.
(a) MHC class II I-E
transgene expression correlates to
CD11c expression in B6CD11c-EdI-A
/
transgenic mice. Immunohistochemical analysis of
normal adult lymph nodes (left)
and spleen (right) from
B6CD11c-E
dI-A
/
mice
(13). Biotinylated antibodies specific for CD11b (Mac-1), CD11c, MHC class II I-E, or
IgM were used on serial cryostat
sections of frozen organs and developed with streptavidin-
alkaline phosphatase. Transgenic
I-E expression correlates to
CD11c staining in lymph node
and spleen, but spares IgM-positive B cells and most of the Mac-1-positive macrophages. All sections were originally photographed at ×200. (b) Transgenic
I-E expression correlates with
CD11c expression on DCs and
can be found on less as well as
more mature DC subpopulations. Three-color flow cytometric analysis of low density spleen cells from a low buoyant density gradient was performed. Only cells meeting high forward scatter and intermediate
side scatter criteria are shown in this analysis. Cells falling within the gates shown in the top panels (CD11c, I-E) were further analyzed with the mAb
NLDC-145 and 33D1 (lower histograms, solid lines). The controls for the second step reagent are shown as dotted lines (no first step mAb).
[View Larger Versions of these Images (25 + 126K GIF file)]
/
mice were obviously negative
for the transgenic I-E (Fig. 1 b, left). In contrast, the corresponding cells derived from the transgenic B6CD11c-E
dI-A
/
mice showed expression of I-E on ~70-80% of the
CD11c-positive fraction as previously reported (16). To
identify more precisely the distribution of transgene expression in distinct splenic DC subpopulations, we used additional DC markers in a three-color FACS® analysis. The
DC-restricted mAb 33D1 (19) has been described to react
with at least 80% of all splenic DCs (20), whereas the remaining 33D1-negative DC fraction of the spleen corresponds to a minor subpopulation reacting with the mAb
NLDC-145 (20); NLDC-145 is a marker for interdigitating cells (IDC; reference 21). It has been proposed that the
bulk of DCs in spleen are the less mature CD11c+NLDC-145
(33D1+) cells and are located at the periphery of the
white pulp nodule. Those cells would mature into the
CD11c+NLDC-145+ (33D1
) IDC phenotype, and be
then located in the central area of the periarteriolar sheath
(17, 20, 22, 23). In concordance, the CD11c+I-E
DCs
from the transgene negative B6I-A
/
mice (Fig. 1 b, left)
were mostly NLDC-145
or 33D1+, whereas a minor
fraction was NLDC-145+ or 33D1
, respectively (Fig. 1 b,
left, lower histograms). To further analyze DCs from the
transgenic B6CD11c-E
dI-A
/
mice, the two gates shown
in Fig. 1 b (right upper plot) were set on either transgene expressing (CD11c+I-E+) or nonexpressing DC (CD11c+I-E
). This analysis revealed that the transgenic I-E is expressed in all types of DCs, since the CD11c+I-E+ subpopulation
could be further subdivided into NLDC-145+ and NLDC-145
cells. Similarly, the CD11c+I-E+ DCs are splitting up
into 33D1+ and 33D1
cells, respectively (Fig. 1 b, right,
CD11c+I-E+). The smaller CD11c+I-E
subpopulation,
which does not express the transgenic I-E, behaves identically to the CD11c+I-E+ fraction, namely representing DCs
from all phenotypes, NLDC-145+, NLDC-145
, 33D1+,
and 33D1
(Fig. 1 b, right, CD11c+I-E
). Taken together,
the immunohistochemical analysis (Fig. 1 a), the flow cytometric data (Fig. 1 b) and our previous report (16) demonstrate that expression of the transgenic I-E is found on ~70-80% of all DCs in lymph nodes and spleen. Transgene expression is not restricted to a certain DC subpopulation, since it can be found on all types of CD11c+ DCs of
the spleen including NLDC-145+ 33D1
IDCs in the T
areas as well as NLDC-145
33D1+ cells representing DCs
from peripheral white pulp areas (20). The absence of
transgene expression on 20-30% of CD11c+ DCs might be
due to missing regulatory elements that could be located
further upstream of the promoter-containing DNA segment used in the transgenic construct (13). Another possibility is the absence of enhancer elements that could be located in introns of the CD11c gene and that are therefore
not included in the transgenic construct.
dI-A
/
mice and the nontransgenic class
II-deficient B6I-A
/
mice offer a unique system to study
the role of MHC class II+ DCs in survival of mature CD4+
T cells.
dI-A
/
and B6I-A
/
mice with mature CD4+ T cells, both types of mice were
engrafted with fetal thymi from MHC class II+ mice. To
ensure that the fetal thymus would not release any MHC class II+ cells, the grafts were treated with 2-dGuo plus an
additional 3,000 Rad irradiation before transplantation,
procedures which have separately been described to efficiently and completely remove bone marrow-derived cells
from the thymus (24). Survival and function of the TG
was monitored every 3 wk by killing one animal per experimental group, reisolating the grafted thymic tissue from
the kidney capsule, and submitting the thymic cell suspension to FACS® analysis. The three-color FACS® analysis
shown in Fig. 2 was performed with reisolated thymic grafts at 17 wk after transplantation. First, the thymocytes
were analyzed according to their CD4 and CD8 expression
and gated in distinct developmental stages between CD4+
CD8+ double-positive and CD4+ single-positive thymocytes (Fig. 2, left, dot histograms, gates 1-3). The cells in
gates 1-3 were then further analyzed according to their expression of the classical thymocyte maturation markers CD3, HSA, and CD69 (27). Although CD3dull thymocytes could only be detected in gate 1 within the more
immature CD4+ CD8+ double-positive population (see
Fig. 2, right panel histograms, CD3 gate 1), CD3high thymocytes did accumulate with ongoing maturation (Fig. 2,
right panel histograms, CD3 gate 2 and CD3 gate 3).
Fig. 2.
Identical thymocyte repopulation of thymic grafts in B6CD11c-EdI-A
/
and B6I-A
/
mice. Three-color FACS® analysis of fetal thymic grafts at wk 17 after transplantation. CD4 lineage thymocytes were divided into the indicated gates 1, 2, and 3 and further analyzed for expression of
CD3, HSA, and CD69. In TG from both hosts, total thymocyte numbers were ~3 × 106 from wk 3-8 and ~106 in wk 17-21 after transplantation.
[View Larger Version of this Image (48K GIF file)]
/
as well as B6CD11c-E
dI-A
/
hosts were HSAhigh
only in their immature stage (Fig. 2, right panel histograms, HSA gate 1) and lost this marker gradually with maturation
(Fig. 2, right panel histograms, HSA gates 2 and 3).
/
as well
as in B6CD11c-E
dI-A
/
hosts were accepted, functional,
and had been efficiently repopulated by T cell precursors,
which gave rise to mature thymocytes as defined by a
CD4high, CD3high, HSAlow, CD69low phenotype (Fig. 2).
Thymocyte origin from host bone marrow was confirmed
by absence of staining for MHC class I Kd molecules (data
not shown). From weeks 3 to 21 after transplantation, a
normal development of thymocytes in the grafted embryonic thymi of the different host animals was observed as
described in Fig. 2 for week 17 post-transplantation thymi.
At the beginning of the experimental period, the TG contained ~3 × 106 thymocytes (3-10 wk after transplantation) with a tendency to decrease in cell numbers down to
1 × 106 in wk 17 (data not shown).
dI-A
/
Mice.
dI-A
/
group, 16 mice received embryonic thymus transplants under their
kidney capsules. Seven of these mice were killed during the
course of the experiment and their thymic transplants reisolated and analyzed by FACS® as shown in Fig. 2. Nine
mice from this group were kept during the whole experimental period of 21 wk and their peripheral blood lymphocytes analyzed as shown in Fig. 3 (Fig. 3, B6CD11c-E
dI-A
/
). Taken together, all 16 thymus-transplanted
B6CD11c-E
dI-A
/
mice showed a substantial peripheral
reconstitution with CD4+ peripheral T cells in blood (Fig.
3), lymph nodes (see Fig. 4) and spleen (data not shown).
Fig. 3.
Accumulation of
peripheral CD4+ T cells can be
detected in thymus-grafted
B6CD11c-EdI-A
/
mice, but
not in nontransgenic thymus-grafted B6I-A
/
mice. Animals
were bled, leukocytes purified by
ficoll gradient centrifugation,
stained with mAbs specific for
CD4 and CD8, and analyzed by
flow cytometry. The percentages
of CD4+ cells of total blood leukocytes from 9 individual mice/
group are shown.
[View Larger Version of this Image (19K GIF file)]
Fig. 4.
Analysis of lymph node cells from TG and TG+ B6I-A
/
, TG+ B6CD11c-E
dI-A
/
, and B6-E
dI-A
/
mice (control mice expressing transgenic I-E under control of the MHC class II promoter; references 13, 14). Cell suspensions from lymph nodes of the indicated mice were analyzed by flow cytometry. Three-color analysis with mAbs specific for CD4, CD8, and
/
-TCR is shown in the two left sets of panels. The histograms
for
/
-TCR staining and the Mel-14/CD69 staining was performed on gated CD4+ T cells. The percentages and total cell numbers falling within the
indicated regions from the Mel-14/CD69 stainings are shown on the right side and are an average of 3 animals/group (percentage, total cell numbers).
[View Larger Version of this Image (30K GIF file)]
/
mice had received thymic transplants under
their kidney capsule. 11 mice from this group, the data of
which are not included in Fig. 3, were killed at different
time points after transplantation to ensure repopulation of
the thymic graft with thymocytes. The data shown in Fig.
3 were derived from nine transplanted B6I-A
/
mice that
were killed after 21 wk and analyzed for presence of TGs.
As a result, all 20 transplanted B6I-A
/
mice analyzed had
accepted and repopulated the TGs with thymocytes as
shown in Fig. 2. Neverthless, the percentage of CD4+ T
cells among the peripheral blood lymphocytes of all 20 thymus-transplanted class II-deficient B6I-A
/
mice did at
no time differ from that observed in untransplanted B6I-A
/
mice (2-5%; Fig. 3, B6I-A
/
references 4, 5); neither could CD4+ T cells be found in spleen (data not
shown) or lymph nodes (see Fig. 4). Surprisingly, the identical development of mature CD4+ thymocytes in the TG
of both host types did not lead to a comparable accumulation of peripheral CD4+ T cells.
dI-A
/
mice, we performed FACS® analysis with markers discriminating between activated and resting T cells (Fig. 4).
CD4/CD8 staining of lymph node cells (Fig. 4) and splenocytes (data not shown) confirmed the presence of CD4+
T cells in transplanted B6CD11c-E
dI-A
/
, but not in
transplanted B6I-A
/
mice. CD4+ T cells from TG+ and
TG
B6I-A
/
mice (and untransplanted B6CD11c-E
dI-A
/
mice, data not shown) showed relatively low
/
-TCR
surface expression levels, as has been reported for the few
atypical CD4+ T cells of class II-deficient mice (5). In contrast, the CD4+ T cells from transplanted B6CD11c-E
dI-A
/
mice expressed normal TCR levels, resembling
CD4+ T cells from B6E
dI-A
/
control mice, which express MHC class II I-E under the control of a class II promoter and do positively select CD4+ T cells in their own
thymus (references 13, 14; Fig. 4, bottom row). Furthermore,
analysis of total CD4+ T cell numbers within the various
subsets as defined by staining with mAb-specific for Mel-14
and CD69 demonstrates that the accumulating CD4+ T
cells in transplanted B6CD11c-E
dI-A
/
mice distribute
equally into naive (Mel-14+/CD69
) and activated (Mel-14
/CD69+) subsets (Fig. 4). Similar results were obtained,
when a mAb-specific for pgp-1 was used (data not shown).
chains (V
2, 4, 5, 6, 7, 8, 11, and 13;
data not shown). This analysis confirmed that the CD4+ T
cells from transplanted B6CD11c-E
dI-A
/
mice were a
polyclonal population, and therefore an oligoclonal expansion of CD4+ T cells (e.g., in response to environmental
antigen presented in context of I-E on DCs) as the reason
for peripheral CD4+ T cell reconstitution in B6CD11c-E
dI-A
/
mice, could be excluded.
dI-A
/
Mice Are Located in T Cell Areas of Lymph Nodes.
dI-A
/
mice shows that the CD4+ T cells
are located in the paracortical area close to the I-E+ DCs
(Fig. 5 A). This is in contrast to the remnant CD4+ T cells
of class II-deficient mice, which had been reported to be
localized in the B cell follicles (5). Higher magnification reveals close T cell-DC contact (Fig. 5 B).
Fig. 5.
Peripheral CD4+ T cells accumulate in T cell areas of lymph nodes in close proximity to I-E+ DCs. Double immunofluorescense analysis of
a lymph node from a TG+ B6CD11c-EdI-A
/
mouse at 8 wk after thymus transplantation. (A) CD4+ T cells (anti-CD4-FITC, green) are found in
the paracortical area of the lymph node, whereas I-E-expressing DCs (anti-I-E-PE, red) are localized, but not in the primary B follicle (F) (original magnification: 200). (B) Higher magnification (1,000) of the same area.
[View Larger Version of this Image (69K GIF file)]
/
, (4) ), a transgenic strain was used
that is expressing an MHC class II I-E transgene under the
control of the CD11c promoter in the class II-deficient background (B6CD11c-E
dI-A
/
; references 13, 16). Both
mouse types used as thymus graft recipients were equally
devoid of mature peripheral CD4+ T cells (4, 13). When
groups of both mice were transplanted with MHC class II-
positive fetal thymic grafts, only in the B6CD11c-E
dI-A
/
group was an accumulation of mature CD4+ T cells detected, whereas in none of the transplanted B6I-A
/
mice
was a significant increase of peripheral CD4+ T cells found.
Further analysis demonstrated that the failure of reconstituting B6I-A
/
mice with mature CD4+ T cells was not
due to inefficient repopulation of the transplanted thymi,
since in all 20 transplanted B6I-A
/
animals, the thymic
grafts could be reisolated at different time points and had
been repopulated with host-type thymocytes. The total
numbers of thymocytes and their phenotype as analyzed by several thymic markers (CD3, CD4, CD8, HSA, and
CD69) was normal and identical in both experimental
groups. One possibility for the absence of reconstitution
could be a very rapid decay of CD4+ T cells when interactions with class II MHC in the periphery are not possible.
These results are in contrast with the reported survival of
CD4+ T cells for more than 6 mo in mice lacking MHC
class II (11). However, this discrepancy might be caused by
different experimental strategies; while Takeda et al. (11)
transplanted embryonic thymi without pretreatment, the
thymi in the present study were irradiated and dGuo
treated. Placing untreated embryonic thymi under the kidney capsule of normal mice has been reported to result in
considerable enlargement of the transplanted thymi for
prolonged periods (12). These enlarged thymic grafts contain up to 2 × 107 donor-derived thymocytes during the
initial 3 wk, and are releasing important numbers of mature
donor-type T cells into the periphery (11, 12). The pretreated thymi in this study at no point of the experiment
contained >3 × 106 thymocytes and showed, in accordance with previous studies, optimal thymocyte production
after ~3 wk of repopulation by host-type cells (12). These
relatively low cell numbers allow to survey more precisely
the subtle repopulation kinetics than an initially high, one
"wave" thymocyte output. As demonstrated in the case of
the B6CD11c-E
dI-A
/
recipients, the final repopulation
efficacy of this sytem is comparable to that reached with
the high output of untreated thymic grafts (11). Furthermore, when untreated thymi are transplanted, thymic cells
of hemopoietic origin (thymic DCs, B cells, macrophages, or precursors of those) might theoretically as well emigrate
from the TG and therefore render the initially class II-deficient environment at least partially MHC class II positive.
In the presented experiments, such a possibility could be
excluded by irradiation and dGuo treatment of the TG.
This was leading to a complete absence of MHC class II-
expressing cells in the transplanted B6I-A
/
recipients
which might be responsible for the fast decay of recent CD4+ thymic emigrants. When this paper was submitted,
another report on similar findings was published (30, 31).
Tanchot et al. (30) demonstrated that naive CD8+ T cells
are disappearing within a few days after transfer into a class
I-deficient environment, but survive for an extended period when the correct MHC molecules are expressed. A
very fast dead of CD4+ T cells due to complete absence of
MHC class II-expressing cells together with the relatively
low output of T cells from the treated TG in B6I-A
/
mice might explain why not even a low level reconstitution with CD4 T cells was observed.
dI-A
/
recipients revealed a
close proximity of the repopulating CD4+ T cells and DCs
in the paracortex. Whether other MHC class II+ cell types
have similar capacities remains to be explored. The localization of DCs in T cell areas of spleen and lymph nodes might specialize them for such T cell survival-inducing
functions, whereas, for example, MHC class II-expressing
B cells are located in separate follicles and might therefore
have fewer opportunities to interact with T cells in the absence of antigen.
Address correspondence to Thomas Brocker, Basel Institute for Immunology, Grenzacherstr. 487, CH-4005 Basel, Postfach, Switzerland. Phone: 41-61-6051331; FAX: 41-61-6051364; E-mail: BROCKER{at}bii.ch
Received for publication 4 June 1997 and in revised form 12 August 1997.
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