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IMMUNOBIOLOGY
Dissociation of thymic positive and negative selection in transgenic mice
expressing major histocompatibility complex class I molecules
exclusively on thymic cortical epithelial cells
Myriam Capone, Paola Romagnoli, Friedrich Beermann, H. Robson MacDonald, and Joost P. M. van Meerwijk
Thymic positive and negative selection of
developing T lymphocytes confronts us with
a paradox: How can a T-cell antigen receptor
(TCR)-major histocompatibility complex
(MHC)/peptide interaction in the former process lead to transduction of signals allowing for cell survival and in the latter induce
programmed cell death or a hyporesponsive state known as anergy? One of the
hypotheses put forward states that the outcome of a TCR-MHC/peptide interaction depends on the cell type presenting the select-
ing ligand to the developing thymocyte. Here
we describe the development and lack of
self-tolerance of CD8ⴙ T lymphocytes in
transgenic mice expressing MHC class I
molecules in the thymus exclusively on cortical epithelial cells. Despite the absence of
MHC class I expression on professional
antigen-presenting cells, normal numbers
of CD8ⴙ cells were observed in the periphery. Upon specific activation, transgenic
CD8ⴙ T cells efficiently lysed syngeneic
MHC class Iⴙ targets in vitro and in vivo,
indicating that thymic cortical epithelium (in contrast to medullary epithelium and antigen-presenting cells of hematopoietic origin) is incapable of
tolerance induction. Thus, compartmentalization of the antigen-presenting cells
involved in thymic positive selection
and tolerance induction can (at least in
part) explain the positive/negative selection paradox. (Blood. 2001;97:1336-1342)
© 2001 by The American Society of Hematology
Introduction
Because of the random nature of T-cell receptor alpha (TCR␣) and
TCR␤ gene rearrangements, the developing T-cell repertoire needs
to undergo positive and negative selection processes. Thymic
positive selection is thought to enrich for T cells expressing a
clonotypic TCR␣␤ capable of interaction with self-major histocompatibility complex (MHC) heterodimers, although alternative views
exist. Thymic negative selection eliminates or inactivates selfreactive T lymphocytes through induction of apoptosis and anergy.
Although negative selection probably rids the developing T-cell
repertoire of the majority of self-reactivity, peripheral tolerance
mechanisms survey remaining self-specific T-cell clones. The
resulting peripherally circulating T-cell repertoire is capable of
recognition of foreign, but not self-peptides presented by self-MHC
molecules.1-5
Because thymic positive and negative selection both involve
TCR-MHC/peptide interactions, an important issue is what determines the outcome of such an interaction. Initially, studies have
focused on the thymic stromal cell types involved in thymic
selection. It has been known that MHC/peptide complexes expressed on cortical epithelium are capable of positive selection of
conventional TCR␣␤⫹ thymocytes, whereas medullary epithelium
as well as antigen-presenting cells (APCs) of hematopoietic origin
cannot,6-8 although quantitatively minor exceptions to this rule
have been reported.9,10 On the other hand, negative selection is
known to be mediated primarily by APCs of hematopoietic origin
(reviewed in Anderson et al1 and Laufer et al11).
Given the fact that thymic epithelium can induce positive
selection while APCs cannot, it was conceivable that differences in
the repertoire of presented peptides play a role in the outcome of a
TCR-MHC/peptide interaction. Although peptide elution studies
from epithelial cells and APCs have thus far failed to confirm this
hypothesis,12 several lines of evidence indicate that thymic epithelial cells have specialized antigen presentation properties and may
present different peptides from those presented by professional
APC.13-18 Therefore, an interaction with MHC/peptide ligands
expressed by thymic epithelial cells may allow for positive
selection, and these cells would subsequently not be negatively
selected because the same ligand is not expressed on cells capable
of negative selection. However, in mice expressing MHC class II
molecules loaded with a single peptide CD4⫹ T lymphocytes
develop,19-23 demonstrating that possible differences in the
repertoire of peptides presented by positively and negatively
selecting cell types alone cannot fully explain the positive/negative
selection paradox.
An alternative hypothesis on the mechanism of thymic positive/
negative selection states that the intensity of TCR triggering
determines the selection outcome. A high avidity interaction would
lead to negative selection, a very weak signaling to death by
neglect, whereas an intermediate TCR-MHC/peptide avidity would
allow for positive selection. In fetal thymus organ cultures, it has
been shown that the low level expression of agonist MHC/peptide
ligand allows the survival of developing thymocytes, supporting
From the Ludwig Institute for Cancer Research, Lausanne Branch, University
of Lausanne, Epalinges, Switzerland; the Institut National de la Santé et de la
Recherche Médicale (INSERM), Toulouse, France; the Swiss Institute for
Experimental Cancer Research, Epalinges, Switzerland; and the Faculty of Life
Sciences (UFR-SVT), University Toulouse III, Toulouse, France.
Reprints: Joost P. M. van Meerwijk, INSERM U395 CHU Purpan, BP 3028,
31024 Toulouse Cedex 3, France; e-mail: joost.van-meerwijk@purpan.
inserm.fr.
Submitted September 18, 2000; accepted October 9, 2000.
Supported in part by grants from ARC (#7287), Région Midi Pyrénées
(RECH/97001940), FRM (10000121-10), and by institutional funds from
INSERM and University Toulouse III.
1336
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2001 by The American Society of Hematology
BLOOD, 1 MARCH 2001 䡠 VOLUME 97, NUMBER 5
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BLOOD, 1 MARCH 2001 䡠 VOLUME 97, NUMBER 5
the avidity model.24,25 However, when tested, the surviving mature
T cells could not be activated by using the same ligand, suggesting
that even low avidity interactions do not allow for functional
positive selection.26,27 Recent data on the capacity of modified
peptide ligands to differentially induce certain, but not other
effector functions, suggested that these ligands may also play a role
in positive selection (reviewed by Jameson et al28 and Germain and
Stefanova29). Studies on TCR-transgenic fetal thymus organ cultures supplemented with modified peptides have suggested a role
for these peptides in positive selection.30 However, the fact that
altered peptide ligands can also inhibit positive selection,31 and an
unexpected MHC/modified peptide ligand-induced mismatch between MHC class specificity and CD4/CD8 lineage outcome32 has
complicated the interpretation of altered peptide studies. Finally, a
differentiation-state dependent “interpretation” of TCR-mediated
signals has been proposed to play a role in positive/negative
selection.33,34
In an alternative approach to the paradox of positive/negative
selection, we and others have initiated a dissection of the 2
selection processes.8,35-38 We have previously generated irradiation
bone marrow chimeras in which radioresistant cells (which are
required for positive selection) express MHC molecules, but APCs
of hematopoietic origin do not. Although a 2- to 3-fold increase in
the rate of development of mature thymocytes was observed,
syngeneic reactivity of chimera-derived T cells was rather limited,
implying that a significant level of negative selection was still
operating in the absence of MHC expression on APCs. Here we
report data on transgenic mice expressing MHC class I ligands
exclusively on thymic cortical epithelial cells. Although positive
selection seems to occur normally in these transgenic mice, no
evidence for any negative selection could be observed using in
vitro and in vivo assays. Therefore, positive and negative selection
seem to be mediated by different cell types expressing self-MHC/
peptide ligands.
Materials and methods
Mice
C57BL/6 and DBA/2 mice were obtained from Harlan Netherlands
(Zeist, The Netherlands), whereas H-2Kb mutant B6.C-H2bm1- and
␤2-microglobulin (␤2m)-deficient C57BL/6-␤2m° mice39 were purchased from Jackson Laboratories (Bar Harbor, ME). The construct used
to generate K14-␤2m transgenic mice was made as follows: A 0.6kilobase (kb) SalI/NsiI ␤2m complementary DNA (cDNA) fragment
(generously provided by Dr D. Margulies, National Institutes of Health,
Bethesda, MD) was inserted into the BamHI site of pG3Z.K14
(generously provided by Dr E. Fuchs, Howard Hughes Medical Institute,
University of Chicago, IL), a vector containing the human keratin 14
(K14) promoter, ␤globin intron, and the K14 poly A addition signal.40
The 3.6-kb transgene was excised with KpnI and HindIII and microinjected into (B6 ␤2m° x C57BL/6)F1 zygotes. Transgenic founders were
backcrossed to C57BL/6-␤2m° mice to obtain K14-␤2m transgenic,
endogenous ␤2m-deficient mice.
DISSOCIATION OF THYMIC POSITIVE AND NEGATIVE SELECTION
1337
concentration for 30 minutes. After washing with PBS/0.01% Tween20
for 30 minutes, slides were incubated with FITC-labeled antirat IgG
F(ab⬘)2 antibody fragment (Immunotech, Marseille, France) for 30
minutes and subsequently washed for 30 minutes. After blocking with
mouse Ig (Pierce, Rockford, IL), slides were incubated with biotinylated
H-2Kb and H-2Db specific antibody 28-8-6 (Pharmingen), washed, and
finally incubated with rhodamine-conjugated streptavidine (Immunotech). After extensive washing, slides were mounted in Faramount
(Dako) and analyzed by immunofluorescence microscopy.
Flow cytometric analysis
Single cell suspensions were prepared from thymus, spleen, and pooled
mesenteric, brachial, inguinal, and axillary lymph nodes isolated from
K14-␤2m mice and ␤2m⫹/° littermates, as well as from bone marrow
from hematopoietic chimeras. Cells were preincubated with 2.4G2
culture supernatant to block Fc receptors,41 washed, and subsequently
incubated with antibody for 30 minutes in PBS/2% fetal calf serum
(FCS). The following antibodies were used: PE-conjugated anti-CD62L
and anti-B220 (Caltag, Burlingame, CA); FITC-conjugated anti-TCR␤
and anti-H-2Kb, PE-conjugated anti-CD4, anti-CD44, anti-CD69, antiMac1, and anti-CD11c, and Cy-chrome–labeled anti-CD8 (all from
Pharmingen). After 2 washes in PBS/2% FCS, cells were analyzed with
a FACScan flow cytometer (Becton Dickinson, San Jose, CA).
Cytotoxic T lymphocyte assays
Unseparated splenocytes derived from K14-␤2m transgenic or control
(C57BL/6, B6.C-H2bm1 or DBA/2) mice were cultured for 6 days in the
presence of T-cell–depleted (anti-Thy1 antibody AT8342 plus complement) irradiated (1000 Rad ␥) splenocytes in the presence of 30 U/mL
IL-2 (EL4 supernatant43). For lysis of RMA and EL-4 targets (C57BL/6
origin43,44), C57BL/6 APC-stimulated effector cells were used, whereas
P815 (DBA/2 origin45) lysis assays were performed by using T cells
stimulated with DBA/2 APCs. Targets (2000 cells per well) were labeled
with 51Cr, extensively washed, and mixed with effector cells in duplicate
at effector (viable cell)-to-target (E/T) ratios indicated. 51Cr release in
the supernatant was measured 4 hours later. Specific lysis is 51Cr release
above background as a percentage of maximum (as determined by acid
lysis of targets). For antibody-blocking experiments, effectors were
preincubated (30 minutes) with the indicated concentrations of anti-CD4
(GK1.546) or anti-CD8 (H3547) antibodies, then mixed with 51Cr-labeled
targets (2000 cells per well) at an E/T ratio required for half maximum lysis.
Limiting dilution assays
Splenocytes from K14-␤2m and control (C57BL/6 and B6.C-H2bm1)
mice were preincubated with 2.4G2 supernatant,41 and subsequently
doubly stained with PE-conjugated anti-CD8 and FITC-conjugated
anti-TCR␤ antibodies (Pharmingen). CD8⫹TCR⫹ cells were then sorted
(with a FACStar Plus sorter, Becton Dickinson, San Jose, CA) directly
into 96-well plates containing 2.5 ⫻ 106 irradiated (1000 Rad ␥)
C57BL/6 splenocytes per well. Cultures were maintained for 2 weeks in
200 ␮L Dulbecco modified Eagle medium supplemented with 30 U/mL
exogenous IL2 (EL4 supernatant). Half (100 ␮L) of the cultures were
used for standard cytotoxic assays, performed with 51Cr-labeled RMA
targets. Lysis was considered positive if 51Cr release exceeded mean ⫹ 3
SD of spontaneous release (measured in 48 control wells containing
targets and APCs). The frequency of RMA lysing precursors was
calculated as described.48
Immunohistology
Thymi were snap frozen in liquid nitrogen and subsequently placed in
OCT (Miles, Elkhard, IN). Cryostat sections (5 ␮m) were air-dried on
frosted microscope slides for at least 2 hours, placed in acetone at room
temperature (RT) for 5 minutes, and rehydrated in phosphate-buffered
saline (PBS). Blocking was performed for 15 minutes with antibody
diluent (Dako, Glostrup, Denmark). Sections were then incubated with
monoclonal antibody 6C3 (Pharmingen, San Diego, CA) at a saturating
Hematopoietic chimeras
Irradiation bone marrow chimeras were prepared essentially as described previously.49 Briefly, anti-NK1.1 antibody-treated hosts (100 ␮g
PK136 intraperitoneally50) were lethally irradiated (1000 Rad ␥) using a
137Cs source and intravenously reconstituted with 107 C57BL/6 plus
C57BL/6-␤2m° bone marrow cells (ratio 1:1) that had previously been
depleted of T cells using anti-Thy1 antibody AT8342 plus complement.
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1338
CAPONE et al
BLOOD, 1 MARCH 2001 䡠 VOLUME 97, NUMBER 5
Unseparated lymph node cells (107) from transgenic or control mice
were coinjected. In some experiments, transgenic lymph node cells were
depleted of CD4⫹ and/or CD8⫹ T cells before transfer by using
anti-CD4 antibody RL172.451 or anti-CD8 antibody 3.16852 plus
complement. Chimeras were kept on antibiotic-containing drinking
water (0.2% Bactrim, Roche, Basel, Switzerland) for the duration of the
experiment (2 weeks).
Results
Major histocompatibility complex class I expression
in K14-␤2m transgenic mice
We have generated transgenic mice that express murine ␤2m under
the control of the human keratin K14 promoter known to be
exclusively active in the basal layer of stratified squamous epithelia.40,53 Transgenic mice were crossed to C57BL/6 ␤2m-deficient
animals39 and K14-␤2m transgenic, endogenous ␤2m-deficient
mice identified (K14-␤2m). Flow cytometric analysis of K14-␤2m
T cells, B cells, macrophages, and dendritic cells from bone
marrow, spleen, liver, and thymus demonstrated that these cells do
not express detectable levels of MHC class I molecules (Figure 1A;
data not shown). In contrast, cortical (but not medullary) epithelial
cells in K14-␤2m transgenic thymi express MHC class I molecules
at levels approaching those observed in wild-type controls (Figure 1B).
Thymic cortical epithelial expression of major
histocompatibility complex class I allows
development of CD8ⴙ T lymphocytes
We next analyzed the development of mature CD8⫹ T lymphocytes in K14-␤2m transgenic mice. Compared with wildtype controls, thymi from transgenic mice contained a 2- to
3-fold increased percentage and an absolute number of mature
CD4⫺CD8⫹TCRhigh thymocytes (Figure 2A). In K14-␤2m
lymph nodes and spleens, normal numbers of CD8⫹ T lymphocytes were detected (Figure 2A). Moreover, on the basis
of the expression of the activation/memory markers CD44,
CD62L, and CD69, most CD8⫹ splenocytes from transgenic as
well as wild-type littermates had a naive phenotype (Figure
2B). This result is surprising in view of recent reports indicating that peripheral naive CD8⫹ T lymphocytes require
TCR interactions with MHC class I for their survival.54,55
Activated CD8ⴙ T lymphocytes from K14-␤2m mice efficiently
lyse syngeneic major histocompatibility complex class I
expressing targets in vitro
To analyze whether T lymphocytes derived from K14-␤2m transgenic mice had undergone negative selection, splenocytes were
cultured with irradiated syngeneic MHC class I and II expressing
(C57BL/6) APCs for 6 days in the presence of exogenous IL-2,
followed by an in vitro lysis assay that used C57BL/6-derived
RMA or EL-4 lymphomas as targets. K14-␤2m–derived effector T
cells lysed syngeneic targets as efficiently as (allogeneic) B6.CH2bm1- or DBA/2-derived T cells (Figure 3A). Similar results were
obtained with T lymphocytes derived from a second independent
K14-␤2m transgenic mouse line (data not shown). Lysis by both
K14-␤2m– and B6.C-H2bm1–derived effector T cells was completely CD8-dependent, as shown by anti-CD8 antibody blocking
(Figure 3B).
We next analyzed the frequency of autospecific CD8⫹
Figure 1. MHC class I expression in K14-␤2m mice. (A) Lack of H-2Kb
expression on K14-␤2m splenic B cells, T cells, macrophages, and dendritic cells.
Splenocytes from K14-␤2m mice (black lines) and ␤2m⫹/° littermates (gray lines)
were stained with anti–H-2Kb combined with anti-B220, anti-TCR␤, anti–Mac-1,
or anti-CD11c antibodies. Histograms of H-2Kb expression on electronically gated
B220⫹ (B cells), TCR␤⫹ (T cells), Mac-1⫹ (macrophages), or CD11c⫹ (dendritic
cells) subsets are representative of 3 independent experiments. (B) Thymic
cortical epithelial expression of MHC class I in K14-␤2m mice. Sections of
wild-type C57BL/6, C57BL/6-␤2m°, and K14-␤2m thymi were doubly stained with
FITC-conjugated antibody 6C3, which detects cortical epithelial cells, and
rhodamine-labeled H-2Kb– and H-2Db–specific antibody 28-8-6. Green and red
fluorescence of the same fields is shown.
K14-␤2m transgenic T lymphocytes by limiting dilution analysis. The minimal estimate of the H-2b reactive cytotoxic T
lymphocyte (CTL) precursor frequency among K14-␤2m–derived
CD8 T lymphocytes was 1 of 48, ie, 7-fold higher than that
observed among allogeneic B6.C-H2bm1 CTL (1 of 320), and
almost 100-fold higher than that of syngeneic wild-type C57BL/6
cells (1 of 3372) (Figure 3C). These data confirm the high level of
autoreactivity of K14-␤2m–derived CTL.
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BLOOD, 1 MARCH 2001 䡠 VOLUME 97, NUMBER 5
DISSOCIATION OF THYMIC POSITIVE AND NEGATIVE SELECTION
1339
syngeneic targets in vivo, we cotransferred C57BL/6 and C57BL/6␤2m° bone marrow as well as K14-␤2m or control C57BL/6 lymph
node T cells into lethally irradiated C57BL/6 hosts. Survival of
coinjected C57BL/6 and ␤2m° bone marrow cells was analyzed by
flow cytometry 2 weeks after transfer. In K14-␤2m T-cell–injected
hosts, no MHC class I positive bone marrow precursor cells had
survived, and only ␤2m° bone marrow had reconstituted the hosts
(Figure 4A). Moreover, antibody-depletion experiments demonstrated that CD8⫹ T cells from the K14-␤2m lymph node cell
inoculum were capable of in vivo C57BL/6 bone marrow lysis
(Figure 4B). Therefore, CD8⫹ T lymphocytes developing in
K14-␤2m transgenic mice are capable of lysing targets that express
syngeneic MHC class I ligands in vivo as well as in vitro.
Discussion
Analysis of the mechanisms responsible for thymic positive and
negative selection would be greatly facilitated by the dissection of
these processes. Here we have reported data on mice expressing
MHC class I molecules under control of the human K14 promoter.
In the thymus, MHC class I expression was limited to cortical
epithelial cells, and no expression by medullary epithelial cells, T
or B lymphocytes, dendritic cells, and macrophages was observed.
CD8⫹ T lymphocytes developed apparently normally and populated peripheral lymphoid organs. These cells could be stimulated
Figure 2. Development and peripheral maintenance of CD8ⴙ T lymphocytes in
K14-␤2m mice. (A) Thymocytes from K14-␤2m mice and ␤2m⫹/° littermates were
triply stained with anti-CD4, anti-CD8, and anti-TCR␤ antibodies. Percentages
(mean ⫾ SD, n ⫽ 6) of CD4⫺CD8⫺, CD4⫹CD8⫹, CD4⫹CD8⫺TCRhigh and
CD4⫺CD8⫹TCRhigh cells are indicated. Thymi from K14-␤2m mice and ␤2m⫹/°
littermates contained 127 ⫾ 45 ⫻ 106 and 116 ⫾ 25 ⫻ 106 cells, respectively. Spleen
and lymph node cell suspensions were stained with anti-CD8 and anti-TCR␤
antibodies (n ⫽ 3). K14-␤2m mice and ␤2m⫹/° littermates had 98 ⫾ 26 ⫻ 106 and
107 ⫾ 25 ⫻ 106 splenocytes and 20 ⫾ 6 ⫻ 106 and 28 ⫾ 2 ⫻ 106 lymph node cells,
respectively. (B) Expression of memory/activation markers by CD8⫹ splenocytes
from K14-␤2m mice and ␤2m⫹/° littermates. Cells were stained with anti-TCR␤,
anti-CD8, and anti-CD44, anti-CD62L or anti-CD69 antibodies. Representative
histograms of electronically gated TCR␤⫹CD8⫹ cells are shown.
CD8ⴙ T cells from K14-␤2m mice kill syngeneic major
histocompatibility complex class I–expressing
hematopoietic targets in vivo
T lymphocytes that can be activated by syngeneic MHC ligands in
vitro are not necessarily reactive to syngeneic cells in vivo.38,56-58
To assess whether K14-␤2m–derived T cells were capable of lysing
Figure 3. Activated CD8ⴙ T lymphocytes from K14-␤2m mice lyse MHC class Iⴙ
syngeneic targets in vitro. (A) Splenocytes from DBA/2, C57BL/6, B6.C-H2bm1 and
K14-␤2m mice were stimulated in vitro with irradiated C57BL/6 spleen cells in the
presence of exogenous IL-2. After 6 days of culture, effector cells were assayed for
cytolytic activity using C57BL/6-derived RMA lymphoma target cells at E/T ratios
indicated. Data are representative of 3 independent experiments. Similar results
were obtained by using C57BL/6-derived EL-4 thymoma cells as targets (data not
shown). (B) In vitro–stimulated effector cells from K14-␤2m and B6.C-H2bm1 mice
were incubated for 30 minutes with titrated concentrations of anti-CD4 or anti-CD8
antibodies before addition of labeled RMA target cells. Data are depicted as
percentage of lysis in the absence of antibody. (C) Precursor frequency analysis of
syngeneic target lysing CD8⫹ T lymphocytes. Indicated numbers of CD8⫹TCR␤⫹
splenocytes were electronically sorted into 96-well plates seeded with C57BL/6
APCs. Lysis of RMA targets was assessed 2 weeks later.
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CAPONE et al
Figure 4. CD8ⴙ T lymphocytes from K14-␤2m mice kill syngeneic MHC class Iⴙ
hematopoietic cells in vivo. Lethally irradiated C57BL/6 hosts were reconstituted
with a mixture of C57BL/6 and C57BL/6-␤2m° bone marrow cells that were
cotransferred with C57BL/6 or K14-␤2m lymph node (LN) cells. Where indicated, the
K14-␤2m lymph node cells were depleted of either CD4⫹ (LN ⌬CD4) or CD4⫹ plus
CD8⫹ (LN ⌬CD4⌬CD8) cells (by antibody plus complement treatment) before
transfer. Reconstitution by C57BL/6 bone marrow cells was assessed 2 weeks later
by 2-color flow cytometry of bone marrow with the use of anti-CD43 (detecting B cell
precursors) and anti–H-2Kb antibodies.
to lyse syngeneic targets in vitro as well as in vivo. Therefore,
dissection of the thymic positive and negative selection mechanisms can be achieved by using transgenic mice that express MHC
class I molecules exclusively on cortical epithelial cells.
Expression of transgenic ␤2m under control of the human K14
promoter in the thymus was limited to cortical epithelial cells,
consistent with the results obtained by Laufer and colleagues8 who
used transgenic mice that expressed the I-A␤ chain under control of
the same promoter construct. In transgenic mice expressing the
costimulatory molecule B7-1 under control of a human K14
promoter construct, the expression pattern seemed very different:
Medullary (but not cortical) epithelial cells expressed the transgene.59 However, the promoter construct was not identical to the
one used by us and by Laufer and colleagues.8,60 Finally, analysis of
murine K14 expression in the thymus revealed its exclusive
localization in medullary regions.61 Taken together, these data
suggest that cortical targeting achieved by us and by Laufer and
colleagues critically depends on the promoter construct and is not
an inherent characteristic of K14 expression.
Thymic cortical epithelial expression of MHC class I molecules
in our transgenic mice allowed for efficient positive selection of
mature CD8⫹ thymocytes. Similarly, MHC class II expression by
thymic cortical epithelial cells leads to positive selection of mature
CD4⫹ thymocytes.6,8 In contrast, in mice in which MHC class II
expression was limited to thymic medullary epithelium, no positive
BLOOD, 1 MARCH 2001 䡠 VOLUME 97, NUMBER 5
selection of CD4⫹ T lymphocytes occurred.6 Moreover, MHC class
I expression by APCs leads to positive selection of only a minute
population of cytotoxic T cells, whereas MHC class II expression
by APCs does not allow detectable positive selection of CD4⫹ T
cells.7,9,10,37 It therefore appears that only thymic cortical epithelium supports positive selection of significant numbers of both
CD4⫹6,8 and CD8⫹ T lymphocytes (our results).
In our K14-␤2m mice, thymic cortical epithelial expression of
MHC class I led to the development of 2- to 3-fold increased
numbers of mature CD8⫹ thymocytes. In the absence of MHC class
I expression by professional APCs (and therefore of an important
fraction of thymic clonal deletion), this increase in mature CD8⫹
thymocytes was to be expected. Indeed, we have previously shown
that in bone marrow chimeras expressing MHC by radioresistant
thymic epithelial cells, but lacking MHC expression by APCs, a 2to 3-fold increase in the rate of generation of mature CD8⫹
thymocytes occurs.37
Because peripheral naive CD8⫹ T lymphocytes are believed to
require TCR interactions with MHC class I for their survival,54,55
the persistence of normal numbers of peripheral K14-␤2m CD8⫹ T
cells in the absence of MHC class I expression by professional
APCs was rather unexpected. In contrast to naive T cells, memory
CD8⫹ T cells are thought to survive in the absence of continuous
TCR-MHC class I interactions,55 though alternative views exist.54
Nevertheless, flow cytometric analysis of K14-␤2m peripheral
CD8⫹ T cells revealed that the vast majority expressed a naive
resting phenotype. Therefore, it appears that even in the absence of
MHC class I expression by professional APCs,62 a quantitatively
normal CD8⫹ T-cell repertoire is maintained. The peripheral CD8⫹
T lymphocyte pool in K14-␤2m transgenic mice may be maintained by increased thymic egress of mature cells, or alternatively
may persist because of TCR interactions with MHC class I
expressed extrathymically on epithelial cells.40,53 Additional experiments will be required to distinguish between these 2 possibilities.
A significant proportion of peripheral CD8⫹ T lymphocytes
from K14-␤2m transgenic mice were autospecific and could be
induced to lyse syngeneic MHC-expressing target cells in vitro. In
bone marrow chimeras lacking MHC class I (J.V.M.V.M. and
H.R.M.D., unpublished data, 1997) or MHC class I and II
expression38 by hematopoietic cells, but expressing MHC on
radioresistant elements (eg, thymic cortical and medullary epithelium), T lymphocytes lysing host-type MHC-expressing target cells
in vitro could readily be generated as well. Syngeneic H-2b targets
were approximately 10-fold less efficiently lysed by chimeraderived CD8⫹ T lymphocytes than by allogeneic DBA/2 T cells
(our unpublished results, 1997, and van Meerwijk and MacDonald38). In contrast, K14-␤2m and allogeneic DBA/2 or B6.CH2bm1 CTL lysed H-2b targets equally efficiently. Moreover,
although K14-␤2m transgenic CTL lysed H-2b targets in vitro as
well as in vivo, in the case of P3F1 and MHC°3wild-type
chimeras, as well as in transgenic mice expressing tolerizing
ligands exclusively on medullary epithelium, in vitro T-cell reactivity to host or transgene-type MHC was accompanied by in vivo
tolerance, a phenomenon termed “split tolerance.”38,56-58 Taken
together, these data confirm a role for medullary epithelium in
negative selection.16,17,58,63,64 Moreover, because mature CD4⫹ T
lymphocytes that had artificially developed in the absence of any
MHC-TCR interaction (and therefore MHC-dependent positive or
negative selection) were equally reactive to H-2b and H-2d APCs,65
the fact that H-2b targets are lysed equally efficiently by K14-␤2m
as by allogeneic B6.C-H2bm1– and DBA/2-derived T cells strongly
suggest that cortical epithelium does not support negative selection.
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BLOOD, 1 MARCH 2001 䡠 VOLUME 97, NUMBER 5
DISSOCIATION OF THYMIC POSITIVE AND NEGATIVE SELECTION
Our data indicating that cortical epithelial cells are incapable of
CD8⫹ T lymphocyte tolerance induction are apparently contradictory to earlier reports showing that in mice expressing transgenic
MHC class I restricted TCRs autospecific (cortical) CD4⫹CD8⫹
thymocytes were absent (reviewed by Stockinger66). However,
deletion observed in these mice is not necessarily due to MHC
expressed by cortical epithelial cells and may be mediated by
cortical macrophages, a hypothesis consistent with the relatively
poor TCR-transgenic CD4⫹CD8⫹ thymocyte deletion by ligandexpressing thymic epithelial cells.67 Data suggesting that other
mechanisms may be involved in the depletion of autospecific TCR
transgenic CD4⫹CD8⫹ thymocytes have also been reported.68,69
The frequency of autoreactive CTL precursors in K14-␤2m
mice (as determined by limiting dilution analysis) was 1 of 48, a
value similar to that obtained by Laufer and colleagues for
autoreactive CD4⫹ T-cell precursors.8 These data suggest that only
2% of positively selectable thymocytes normally undergo tolerance
induction, a value well below the 50% to 70% obtained earlier by
us and by others analyzing T-hybrid specificity and kinetics of
mature CD4⫹ or CD8⫹ thymocyte development.22,37,65 However,
values obtained by limiting dilution analysis are necessarily
minimal estimates, and (in the absence of a valid correction
for plating efficiency) cannot be compared with kinetic and
T-hybrid data.
In conclusion, our data indicate that MHC class I expressed by
1341
cortical epithelial cells cannot induce negative selection of CD8⫹ T
lymphocytes, a conclusion consistent with earlier data on reactivity
of CD4⫹ T cells that had developed in mice expressing MHC class
II exclusively on cortical epithelium.8,35 Therefore, cortical epithelial cells appear to be specialized in thymic positive selection,
whereas medullary epithelial cells and intrathymic APCs of
hematopoietic origin are specialized in negative selection. Although certain in vitro cultured thymic epithelial cell lines are
capable of both positive and negative selection in vitro and when
injected intrathymically,70,71 the physiologic relevance of these
findings remains unclear. Whatever the explanation, the data
presented here and elsewhere8,35 on K14-MHC transgenic mice
clearly point to a general lack of tolerance induction by cortical
epithelial cells. The ability to dissect thymic positive and negative
selection by using these mice should facilitate analysis of the
responsible mechanisms.
Acknowledgments
We thank Dr E. Fuchs for the K14 promoter construct, Dr D.
Margulies for ␤2m cDNA, Dr C. L. Blanchard for constructing the
K14-␤2m vector, Dr M. Guttinger for helpful suggestions, and R.
Lees and G. Enault for expert technical help.
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2001 97: 1336-1342
doi:10.1182/blood.V97.5.1336
Dissociation of thymic positive and negative selection in transgenic
mice expressing major histocompatibility complex class I molecules
exclusively on thymic cortical epithelial cells
Myriam Capone, Paola Romagnoli, Friedrich Beermann, H. Robson MacDonald and Joost P. M. van
Meerwijk
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