The Journal of Immunology, 2002, 169: 4945-4950.
Copyright © 2002 by The American Association of Immunologists
The Role of Fibroblasts in Thymocyte-Positive Selection1
Mirjana Lili
*,
Fabio R. Santori*,
Eric G. Neilson
,
Alan B. Frey
and
Stanislav Vukmanovi2,*
Department of
* Pathology, Michael Heidelberger Division of Immunology and Kaplan Comprehensive Cancer Center, New York University School of Medicine, New York, NY 10016;
Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232; and
Department of Cell Biology and Kaplan Comprehensive Cancer Center, New York University School of Medicine, New York, NY 10016
 |
Abstract
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Mice with fibroblast-specific expression of TAP-1 were generated by
expressing the TAP-1 transgene under the control of the
fibroblast-specific protein (FSP) 1 promoter/enhancer on
TAP-1-deficient background. MHC class I expression in primary
fibroblast cultures isolated from the resulting strain mimicked that of
wild-type counterparts. MHC class I was detected in both types of
fibroblasts following treatment with IFN-
. Positive selection of
CD4-CD8+ thymocytes was observed in neither
adult nor fetal/neonatal thymus of transgenic mice. IFN--induced
expression of MHC class I rescued positive selection of
CD4-CD8+ T cells in fetal thymic organ
cultures, but not in adult mice. Contrary to previous suggestions, our
results indicate a limited role of fibroblasts in promoting positive
selection. In addition, the results suggest that positive selection may
occur by a different mechanism in fetal vs adult
thymus.
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Introduction
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During
differentiation in the thymus, immature T cells undergo positive
selection (1, 2). This process is based on the interaction
of their TCR with self-peptide-MHC complexes presented by thymic
stromal cells. Identification of thymic stromal cells that promote
positive selection has been recognized as a key to understanding this
process and has therefore been extensively studied. A variety of
approaches has indicated that thymic epithelial cells are the major
cell type influencing positive selection (3, 4, 5, 6, 7, 8). The
crucial consideration was: are cortical epithelial cells specialized
for promoting positive selection, or can this function be ascribed to
their unique anatomical positioning? Major support for the latter
notion was provided by studies suggesting that fibroblastoid cell lines
can induce positive selection following intrathymic injection as
efficiently as thymic epithelial cells (9, 10). However,
in the fetal thymic reaggregation cultures, fibroblasts were not able
to promote positive selection of thymocytes in the absence of thymic
epithelial cells (8). These disparate results could be
explained by possible injection of fibroblasts into an anatomically
incorrect microenvironment. Furthermore, the numbers of injected
fibroblasts likely exceeded normal representation of this cell type,
and expression of MHC molecules by the injected cells was ectopic. On
the other hand, the thymus reaggregation experiments might have exposed
the unique (dis)abilities of neonatal fibroblasts and/or thymic
epithelial cells. In addition, the possibility that
deaggregation/reaggregation procedures might have selectively disabled
certain properties of particular cell types cannot be excluded with
certainty.
In this study, we present a strategy that bypasses the difficulties
encountered in previous approaches. Transgenic mice expressing TAP-1
under the fibroblast-specific protein
(FSP)3 1
promoter/enhancer (11) were generated and bred to the
TAP-1-deficient mice (12) to produce mice with
fibroblast-selective expression of TAP-1. Studies of thymic selection
in these mice are not limited to any stage of development (neonatal or
adult) and microanatomical distribution of fibroblasts, as well as all
other thymic cell types, should not differ from wild type. Finally, the
levels of MHC class I in these mice should not be supraphysiological,
as the levels of other molecules involved in MHC class I assembly
(class I H chain, TAP-2, and 2-microglobulin)
should be limiting for the potentially excessive expression of
transgenic TAP-1.
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Materials and Methods
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Mice
The FSP-1 promoter/enhancer (11, 13) was flanked at
the 5'' end with cDNA encoding mouse TAP-1 (supplied by Dr. J. J.
Monaco, University of Cincinnati, Cincinnati, OH) and the exon-intron
sequence of the human -globin (Fig. 1
A). DNA injection into
C57BL/6 fertilized eggs was performed at the New York University School
of Medicine transgenic facility. Six founders were identified by
Southern blotting of tail DNA using the
BamHI-EcoRI DNA fragment (0.9 kb) from the human
-globin gene as a probe (Fig. 1B). Southern blot and
detection of fluorescein-labeled probes were performed using the Gene
Images CDP-Star detection module (Amersham Pharmacia Biotech,
Piscataway NJ). All founders were bred for two generations to
TAP-1-/- background to obtain FSP-1
promotor-specific TAP-1 expression (FSTE) mice. Mice were screened by a
combination of peripheral blood staining for
H-2Kb (for TAP-1 deficiency) and Southern
blotting and/or PCR of genomic tail DNA with primers specific for the
transgenic construct, sense = 5'-GCGGCTCCTGCAGACTGA-3'; antisense
= 5'-CTGATGCAATCATTCGTCTG-3'. TAP-1-/- mice
were purchased from The Jackson Laboratory (Bar Harbor, ME). C57BL/6
and H-Y TCR transgenic mice were purchased from Taconic Farms
(Germantown, NY).
Cells
To obtain primary fibroblast cultures, small pieces of skin from
tails or spleens were plated in RPMI 1640 (10% FCS) without
growth factors. Debris was removed after 1 wk and the adherent cells
were trypsinized and expanded. By day 15, only fibroblasts were
growing. We were able to subculture these fibroblasts for >30
doublings. Some fibroblast cultures were treated for 2 days with 500
U/ml IFN- (gift from Dr. D. Levy, New York University School of
Medicine).
Monoclonal Abs and immunofluorescence analysis
All mAbs were purchased from BD PharMingen (San Diego, CA).
Fibroblasts were stained with an
anti-H-2Kb-specific mAb (Y3) on ice for 30
min, washed in PBS (containing 1% BSA) followed by FITC-conjugated
anti-mouse Ig for an additional 30 min. Thymocytes were stained
using anti-mouse CD4-PE, CD8-CyChrome, and CD24-FITC. Cells were
fixed in 1% paraformaldehyde and analyzed using FACScan (BD
Biosciences, Mountain View, CA).
RT-PCR analysis
Total RNA was isolated from 5 x 106
cells using TRIzol reagent (Life Technologies, Gaithersburg, MD)
following the manufacturer’s protocol. cDNA was synthesized using the
SuperScript One-Step RT-PCR with Platinum Taq (Life Technologies). PCR
was conducted using PCR SuperMix (Life Technologies). Amplification was
conducted with primers specific for H-2Db and
-actin (14) and for TAP-1, TAP-2, and low molecular
weight protein 2 (LMP-2) (15).
Fetal thymus organ culture (FTOC)
FTOC was performed as described previously (16).
H-Y TCR transgenic mice in TAP-1-/- background
were bred with FSTE mice. The thymic lobes were removed from animals at
gestational day 16 and placed on sponge-supported filters (Millipore,
Bedford, MA) in medium with or without 500 U/ml IFN-. Cultures
were arranged so that one lobe was treated with IFN-, while the
other lobe from the same fetus was treated as control. After 10 days,
thymocytes were harvested and analyzed by flow cytometry. Fetuses were
screened by PCR of genomic DNA with primers specific for the TAP-1
transgene (described above) and the H-Y transgene, sense =
5'-AGGAGGAAACAGTGCCCAGTCCGT-3'; antisense = 5'-
CGCAGACCCTCCTTGATCCTGGCC-3'.
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Results
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Expression of the TAP-1 transgene
To determine expression of the transgene, adult skin fibroblasts
were isolated from F1 offsprings of transgenic
founders and TAP-1-/- mates. RT-PCR was
conducted using primers from the mouse TAP-1 (forward) and human
-globin (reverse) sequences 477 bp apart (Fig. 2A). Mice that expressed the
transgenic mRNA were further bred onto the
TAP-1-/- background. To analyze MHC class I
expression, primary embryonic or adult fibroblast cultures from
wild-type (C57BL/6), FSTE, and TAP-1-/- mice
were established and analyzed by flow cytometry. MHC class I molecules
were not detectable irrespective of the source of fibroblasts (Fig. 3 and data not shown). To determine
whether the transgene is functionally expressed in FSTE fibroblasts, we
treated primary culture fibroblasts with IFN-. After this
treatment, FSTE as well as wild-type fibroblasts expressed readily
detectable levels of MHC class I (Fig. 3).
To determine whether IFN- induced expression of endogenous and/or
transgenic genes involved in assembly of MHC class I, RT-PCR were
performed with primers specific for MHC class I H chain, TAP-1, TAP-2,
and LMP-2 molecules. The expression of TAP-2, and especially of MHC
class I H chain, depended on IFN- (Fig. 2
B). In
contrast, expression of the TAP-1 transgene driven by the FSP-1
promoter/enhancer was IFN- independent. Thus, up-regulation of
endogenous genes, but not of the transgene, appears to be the mechanism
of IFN--mediated induction of MHC class I expression in FSTE
fibroblasts. Thus, in the cell types that constitutively express genes
involved in the assembly, MHC class I expression should not depend on
IFN--treatment. Except for the cultured fibroblasts, we did not
observe MHC class I expression in several lymphoid tissues of FSTE mice
(spleen, peripheral blood, lymph nodes, or thymus; data not shown),
consistent with selective IFN--inducible expression by
fibroblasts.
Fibroblasts induce neither positive nor negative selection under
physiological conditions
FSTE mice were tested for the presence of
CD4-CD8+ thymocytes and
peripheral CD8+ T cells. Immunofluorescence
staining revealed enrichment in
CD4-CD8+ thymocytes
relative to TAP-1-/- counterparts was not
observed in adult mice (Fig. 4A) or 16–20 gestational day
embryos. The same finding was obtained in the periphery wherein no
enrichment of CD8+ spleen cells was found (Fig. 4B). These findings could be ascribed to either insufficient
MHC class I levels expressed by fibroblasts in vivo or to genuine
inability of fibroblasts to promote positive selection. To address the
former possibility, we bred FSTE mice to H-Y TCR transgenic background
and asked whether fibroblasts can promote deletion of
CD4+CD8+ thymocytes in male
mice. The CD4/CD8 profile of FSTE/H-Y mice was identical to that of
TAP-1-/-/H-Y mice (Fig. 5), indicating that levels of male Ag-MHC
complexes in FSTE mice are insufficient to induce negative
selection.
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FIGURE 4. Fibroblasts from FSTE mice fail to induce positive selection in vivo.
Cells isolated from thymi (A) or spleens
(B) of FSTE or TAP-1-/- mice were stained
with anti-CD8 and anti-CD4 mAbs and analyzed by flow
cytometry.
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FIGURE 5. Fibroblasts from FSTE mice fail to induce negative selection in vivo.
Cells isolated from thymi of H-Y TCR transgenic male mice on the
wild-type (WT), TAP-1-/-, or FSTE background were stained
with mAbs specific for CD8 and CD4 and analyzed by flow
cytometry.
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Up-regulation of MHC class I in FTOCs enables fibroblasts to induce
positive selection
Two approaches were taken to test whether fibroblasts have the
potential to induce positive selection of T cells, both based on the
ability of IFN- to induce MHC class I expression by fibroblasts
(see Fig. 3). First, FSTE mice were treated with poly(I:C), an agent
that induces massive endogenous IFN- secretion (17).
FSTE mice were injected with poly(I:C) or PBS alone twice with a 5-day
interval between the injections, and thymi from injected animals were
analyzed 5 days after the second injection. However, poly(I:C)
treatment could not induce positive selection of
CD4-CD8+ thymocytes in
FSTE mice, nor could it enhance the selection of the same cells in
wild-type mice, despite increasing the levels of MHC class I (data not
shown).
Second, FTOCs from TAP-1-/- or FSTE mice were
treated with IFN-. A modest, but consistent increase in the
numbers of
CD24lowCD4-CD8+
thymocytes was observed in IFN--treated FSTE, but not
TAP-1-/- FTOCs (Fig. 6). Immature
CD24highCD4-CD8+
thymocytes are more abundant in FTOCs than in the adult thymus, thus
presenting a relatively high background for detection of mature
CD4-CD8+ thymocytes. MHC
class I-restricted TCR transgenic mice have an inverted CD4:CD8 cell
ratio so that differences between positive and negative controls are
more pronounced. Therefore, FTOCs were established from H-Y TCR
transgenic mice on TAP-1-/- or FSTE
backgrounds. Interestingly, treatment of
TAP-1-/-/H-Y FTOCs with IFN- induced an
increase in the fraction of immature
CD24highCD4-CD8+
thymocytes (Fig. 7). We do not know the
reason for this phenomenon, but it is apparently related to the
presence of the H-Y TCR, since non-TCR transgenic cultures showed no
such increase (Figs. 6 and 7). When IFN--treated FSTE/H-Y lobes
were compared with TAP-1-/-/H-Y lobes, a
significant and selective increase in the numbers of mature
CD24lowCD4-CD8+
thymocytes was observed (Fig. 7). Since
CD4-CD8+ and not
CD4+CD8- thymocytes
express high levels of transgenic TCR (18), an appearance
of T3.70high cells in IFN-treated FSTE, but
not TAP1-/- FTOCs (Fig. 8) further suggested that positive
selection of mature
CD4-CD8+ thymocytes
occurred in FSTE IFN-treated FTOCs. Collectively, these results
suggest that fibroblasts have a very limited role in positive selection
physiologically, but that they have the potential for supporting
positive selection, at least during early ontogenesis.
|
Discussion
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To achieve selective expression of MHC class I molecules in
fibroblasts, we reconstituted an essential component in the MHC class I
assembly, TAP-1, in TAP-1-deficient mice. Reconstitution of TAP-1 is
advantageous in comparison to expression of
2-microglobulin or MHC class I H chain (in
appropriate deficient mice). The
2-microglobulin molecule does not have a
transmembrane region, thereby potentially allowing its dissociation
from cell surface MHC class I (19). This could create a
possibility wherein peptide-class I H chain complexes on bone
marrow-derived cells (or on any other cell type) could acquire
cell-free 2-microglobulin and stabilize a
functionally significant portion of cell surface MHC class I. This
scenario is impossible with an intracellular transmembrane molecule
such as TAP-1. The disadvantages to use of class I H chain are the
restriction to use only one class I allele and its potential
overexpression. In contrast, obtaining supraphysiological levels of MHC
class I by reconstituting TAP-1 is not a serious concern. The
functional TAP molecule is a heterodimer of TAP-1 and TAP-2 subunits
(20) and the limiting levels of endogenous levels of TAP-2
will prevent excessive formation of the TAP heterodimer, even under
conditions of potential overexpression of TAP-1. The limiting levels of
2-microglobulin and the class I H chain
further ensures that normal levels of MHC class I are not exceeded.
Indeed, levels of expression of MHC class I in IFN -treated
primary fibroblasts isolated from FSTE mice are certainly not higher
than in wild-type fibroblasts.
An important question is whether the undetectable levels of MHC class I
in both wild-type and FSTE primary fibroblast cultures are an artifact
of in vitro culture or faithfully reproduce in vivo conditions.
Although it is difficult to answer this question with certainty, three
pieces of information support the latter possibility. First, patterns
of expression of other genes can be stably maintained during fibroblast
cultures and can be quite distinct in fibroblasts isolated from
different tissues (21). Second, embryonal/neonatal
fibroblasts could affect positive selection after IFN- treatment
(Fig. 7), suggesting that the absence of positive selection in FTOCs
before IFN- treatment was attributable to insufficient levels of
MHC class I expression. Finally, we could detect no thymocyte-negative
selection in male H-Y/FSTE mice (Fig. 5).
Positive selection appears to require at least two signals: MHC
dependent that can be delivered by bone marrow-derived cells, and
MHC-independent signal that requires radioresistant thymic stroma
(22). Using the two-signal model of positive selection, at
least three explanations could account for differences in
embryonal/neonatal vs adult positive selection by fibroblasts. First,
embryonal/neonatal, but not adult fibroblasts, may share with cortical
epithelial cells a unique costimulation/adhesion molecular structure
required for positive selection. This unique structure could possibly
reflect the presence of putative positive selection-inducing
molecule(s) and/or the absence of putative positive
selection-inhibiting molecule(s). An example for the latter possibility
would be the B7-1 molecule, which can enhance signals, leading to
negative selection (23), and has a thymic distribution
consistent with a putative negative effect on positive selection
(24). Fibroblasts were reported to express homologues of
costimulatory molecules (25), but the ability of these
homologues to enhance negative selection is not yet established.
Second, selection of embryonal/neonatal and adult thymocytes may have
distinct requirements. This possibility thus far has little
experimental support and we consider it unlikely. Nevertheless, various
differences between embryonic/neonatal thymic differentiation have
previously been noted, including involvement of TdT in TCR gene
rearrangements (26) and maturation of regulatory T cells
(27). Finally, the third possibility is that
embryonal/neonatal but not adult thymic fibroblasts may be localized in
the vicinity of cortical thymic epithelial cells. Thus, in the
embryonal/neonatal thymus, fibroblasts may supply the MHC-dependent
signal, whereas cortical thymic epithelial cells could provide the
"second signal" in trans. Although the first two
possibilities remain to be formally excluded, the last scenario is most
consistent with existing literature that includes reports of positive
selection induced by intrathymic injection of fibroblast cell lines
(9, 10) or inefficient selection by bone marrow-derived
cells (28, 29).
In conclusion, to achieve selective expression of MHC class I molecules
in fibroblasts, we reconstituted an essential component in the MHC
class I assembly, TAP-1, in TAP-1-deficient mice. Results presented in
this report suggest that fibroblasts appear to be unable to
significantly impact on MHC-dependent aspects of T cell differentiation
under physiological conditions. This, however, does not challenge the
well-established role of fibroblasts in maintaining the structural
integrity of the thymus and promoting earlier MHC-independent stages of
thymocyte differentiation (30, 31).
|
Acknowledgments
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We thank John Hirst for FACS analysis, John Monaco for providing
the mouse TAP-1 cDNA, and David Levy for providing IFN- and for
helpful discussions.
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Footnotes
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1 This work was supported by the National Institutes of Health Grants AI41573 (to S.V.) and CA83687 (to A.B.F.) and National Cancer Institute Core Support Grant 5P30 CA16087. 
2 Address correspondence and reprint requests to Dr. Stanislav Vukmanovi, Department of Pathology, Michael Heidelberger Division of Immunology, New York University School of Medicine, 550 First Avenue, New York, NY 10016. E-mail address: vukmas01{at}med.nyu.edu
3 Abbreviations used in this paper: FSP, fibroblast-specific protein; FSTE, FSP-1 promotor-specific TAP-1 expression; FTOC, fetal thymic organ culture.
Received for publication July 9, 2002.
Accepted for publication August 27, 2002.
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Top
Abstract
Introduction
) providing two introns (
) and a
polyadenylation signal ([
); restriction sites shown in parentheses
have been destroyed by blunt-end cloning. B, BglII; BH,
BamHI; E, EcoRI; S, SalI;
Sp, SpeI. B, Southern blot analysis of
EcoRI-digested tail DNA from transgenic offspring. The
transgene is indicated by the 2.1-kb band. Southern blot analysis was
performed using a probe containing a portion of the human 
