| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
,
,
*
Program in Immunology,
Department of Microbiology and Immunology, and
Howard Hughes Medical Institute, Stanford University, Stanford, CA 94305; and
§
Department of Microbiology and Immunology, Emory University, Atlanta, GA 30322
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Positive selection is responsible for ensuring that all T cells recognize self MHC. To become positively selected, an immature thymocyte must express a TCR that can interact with self MHC peptide complexes, generate a signal, and initiate a program of differentiation that includes down-regulation of recombinase-activating gene-1 (RAG1)4 RAG2, HSA, and either CD4 or CD8 as well as transient up-regulation of CD69 (1, 2, 3). The process of selecting cells that react with self MHC peptide complexes, however, leads to a repertoire of mature T cells that includes autoreactive cells (4, 5). Thus, before thymocytes emigrate to the periphery, cells that might react with self MHC peptide complexes to a significant degree must be eliminated. This negative selection also requires the TCR expressed by a thymocyte to interact with MHC peptide complexes. As in positive selection, this binding event generates a signal and induces CD69 expression. Rather than inducing differentiation, this signal causes these autoreactive thymocytes to apoptose and eventually be eliminated from the thymus. Although positive and negative selections each result in different cell fates, these divergent outcomes are both triggered by TCR interactions with self MHC peptide complexes.
How does a thymocyte distinguish between the signals that induce positive selection/differentiation vs negative selection/death? One possibility is that a given thymocyte may respond differently to TCR stimulation by MHC peptide complexes depending on its developmental stage. In this scenario, TCR-mediated signals induce positive selection early in development, while similar signals induce negative selection later, after the threshold for signaling has been raised or after signaling has been coupled to different transcriptional events. In support of this model, CD4+8+ thymocytes contain a subset of TCR+ cells that cannot be deleted by TCR stimulation (6). Further evidence derives from studies of superantigen-mediated negative selection of cells bearing a particular TCR ß-chain. In these studies thymocytes expressing the appropriate Vß were present in the immature CD4+8+ populations, but were absent (or reduced in number) in the more mature CD4+8-/int populations (7). Because the CD8intCD4+ cells were already enriched for the TCR Vß-chains that were positively selected in their particular MHC background, in this system negative selection followed positive selection. Although this study is the only in vivo approach to examine nontransgenic thymocyte selection, the necessity of using superantigens may have biased the results. For example, superantigen-mediated deletion may occur later than peptide-mediated deletion either because the endogenous superantigens interact weakly with most TCRs or because these molecules are not highly expressed in the thymic cortex (8, 9).
Studies using TCR transgenic mice in which most thymocytes express the same TCR suggest an alternative hypothesis. Instead of developmental stage, the specificity and the strength of the TCR-MHC peptide interaction may be the primary determinants of developmental fate. For instance, immature CD4+8+ cells from some TCR transgenic mice are negatively selected early in thymic development (10, 11, 12) or on day 17 of gestation during fetal development, preceding the appearance of positively selected CD8+ T cells by several days (13). Furthermore, when immature thymocytes are removed from nonselecting thymuses and stimulated through their TCR/CD3 complex they can be induced to apoptose in the absence of prior positive selection (14, 15). These studies show that TCR transgenic thymocytes can be deleted early in development and/or ontogeny, but they also show that even under the control of endogenous promoters, transgene-encoded TCR genes are expressed at higher than normal levels in the immature CD4-8- and CD4+8+ cells in the thymus (13).
This aberrant TCR expression may lead to artifactual early deletion in
several ways. First, as Berg et al. showed (10), TCR
ß transgene expression can cause aberrant early CD3 expression
that may increase the sensitivity of immature cells to TCR stimulation
and lead to abnormally early deletion of cells. In fact, in these
studies the expression of both TCR chains, but not TCR ß alone,
results in massive deletion of
CD4+8+ thymocytes in mice
that expressed endogenous superantigens reactive to the
transgene-encoded Vß segment. Second, in some in vitro studies,
thymocytes removed from intact thymuses have been shown to increase TCR
expression (16). Again, aberrantly high TCR levels might
lead to nonphysiologically relevant negative selection. Finally, the
studies that document early negative selection employ transgenic T
cells bearing receptors derived from hybridomas or T cell lines that
were selected because they responded vigorously to Ag. Thus these cells
are likely to have a reasonably high avidity for the selecting Ag that
could lead to unusually efficient or early deletion. Taken together,
these arguments indicate that TCR transgenic models of early events in
thymic selection are potentially unreliable.
Because of the problems with these model systems, the relative timing
of positive and negative selections has remained controversial. In this
study we attempt to resolve this controversy by tracking the
development of a polyclonal, MHC peptide-specific population of
thymocytes in the presence or the absence of the deleting peptide. To
identify MHC peptide-specific cells, we have used an MHC
tetramer-staining reagent (17, 18) that permits the
detection and isolation of Ag-specific T cells even when these cells
comprise only a small fraction (0.1%) of the total thymocytes. By
crossing mice that are transgenic for a normally regulated TCR
ß-chain from an I-Ek+ moth cytochrome
c (MCC)-reactive T cell clone (5C.C7) to mice that express a
soluble hen egg lysozyme protein modified to include the antigenic
MCC88–103 peptide (HELCYT), we are able to
monitor positive and negative selection of Ag-specific T cells in vivo,
in the absence of early expression of TCR or CD3
(19, 20, 21). We show that cells with the highest capacity to
bind the negatively selecting ligand are deleted most efficiently, that
this deletion occurs throughout thymic development, and that positive
selection is not required for negative selection in vivo.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Soluble - and ß-chains of the I-Ek
MHC molecule were produced in Escherichia coli
(22). The -chain has been engineered to contain a short
biotinylation motif on the membrane-proximal section of the molecule.
After production and folding of the molecule with MCC peptide the
complexes were purified by affinity chromatography and specifically
biotinylated with the enzyme Bir A in the presence of protease
inhibitors (17, 18). The biotinylated molecules were
size-purified by fast protein liquid chromatography, then mixed in a
6-fold molar excess with fluorescently labeled streptavidin to form
peptide/MHC tetramers. Excess or unconjugated
MCC/I-Ek was washed away by centrifugation
through a Centricon 100K (Amicon, Beverly, MA).
Flow cytometry
Thymocytes were isolated by removing whole thymus into cold PBS
and disrupting the tissue by pushing through a metal sieve or plastic
mesh. Cells were incubated in red cell lysis buffer (0.74 M ammonium
chloride) for 5 min at 37°C, then washed three times in FACS wash
buffer (2% FCS/PBS, pH 7.4). Cells were maintained at 4°C for the
rest of the procedure, and all washes were performed with FACS wash
buffer. Cells were counted with a hemocytometer, and
106 cells were used for each combination of
staining reagents. The staining parameters of tetramer preparations
varied. Each preparation was titrated on 5C.C7 TCRß transgenic T
cells to determine the optimum staining concentration on the day before
analysis. The appropriate concentration of tetramer/Av-PE (
50
µg/ml of tetramer/Av-PE) or the equivalent amount of Av-PE alone was
added to cells from 2 to 4.5 h before analysis. All other Abs were
added 30–50 min before analysis. Dead cells were labeled by incubating
cells for 5 min with 10 µg/ml propidium iodide before washing several
times. Cells were analyzed by flow cytometry on a customized dual laser
Vantage (Becton Dickinson, Mountain View, CA) and analyzed using either
FACS Desk or FlowJo software (Beckman Center Shared FACS Facility,
Stanford, CA). All analyses excluded small cells, propidium
iodide-positive cells, and any cells that bound to CyChrome-conjugated
irrelevant Abs (B220-CyChrome). The percentage of tetramer positive
cells was computed by dividing the number of tetramer-positive cells by
the number of cells found in the next largest subset.
Antibodies
The following Abs were used: anti-B220-CyChrome (RA3-6B2), CD3-FITC (145-2C11), CD69-FITC (H1.2F3), and heat-stable Ag (HSA)/CD24- FITC (M1/69) all from PharMingen (San Diego, CA); Ultralite streptavidin-PE (Molecular Probes, Eugene Oregon); and anti-CD4-allophycocyanine (GK1.5), anti-CD8-BSA-Texas Red, and anti-Vß3-APC (KJ25; produced by members of our laboratory or received as a gift from the Weissman laboratory, and conjugated to fluorophores from Molecular Probes by standard procedures).
Mouse strains
The generation of 5C.C7 TCR ß transgenic and HELCYT transgenic mice has been previously described (19, 20). All mice were bred and maintained in a pathogen-free animal facility (DLAM, Stanford University). Both the HELCYT and 5C.C7 ß x B10.BR strains have been crossed >10 times to the B10.BR background.
Peptides
The antigenic peptides used in folding reactions and for injection of transgenic mice were synthesized in the PAN Facility (Beckman Center, Stanford University) and purified by HPLC. The MCC88–103 peptide used had the sequence ANERADLIAYLKQATK.
Single-cell RT-PCR
Single CD4+V11+
cells that were tetramer high, medium, or negative were sorted into
RT-PCR buffer as described previously (23). The rearranged
TCR V11 genes were amplified by RT-PCR using V11-specific nested
primers. These cDNAs were subcloned, and both strands were sequenced by
standard techniques.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To study the generation of Ag-specific T cells at the early stages
of thymocyte development, we examined thymocytes from 5C.C7 TCR
ß-chain transgenic mice (ß transgenics). The expression of the TCR
ß transgene modifies the normal TCR repertoire such that these mice
produce a detectable population of
MCC/I-Ek-specific cells without significantly
disrupting other developmental processes (10, 19, 20).
Three lines of evidence suggest that this is true. First, studies of
5C.C7 ß transgenic fetal thymocyte development show that TCR
ß-positive cells appear on the appropriate day of gestation (F. de
St. Groth, unpublished observations). Second, CD3 expression in
CD4-8- and
CD4+8+ cells from TCR ß
transgenics resembles expression in wild-type animals (Fig. 1
) (20). These expression
patterns are strikingly different from the aberrantly high level of CD3
expression exhibited by TCR ß
CD4-8- thymocytes; if
anything, CD3 expression is slightly decreased in TCR ß transgenics.
Third, TCR ß transgenic mice select a normal thymic repertoire of TCR
-chains at the appropriate time in development (19, 24). Although the presence of the ß transgene causes the
mature T cell repertoire to contain higher than normal numbers of
CD4+ cells, this skewing most likely reflects
normal thymic selection processes operating on cells that each express
a ß-chain that is more reactive with class II MHC than with class I
MHC. Thus, unlike the TCR ß transgenic model systems used for
earlier studies of the timing of positive and negative selections
(9, 10, 11, 12, 13), TCR ß transgenic mice provide an accessible,
yet physiologically relevant, model to study the thymic development of
small, polyclonal populations of Ag-specific T cells.
|
To identify and follow these Ag-specific thymocytes, we generated
a tetrameric MCC/I-Ek staining reagent (17, 18). This reagent consists of four biotinylated
MCC/I-Ek complexes that are multimerized by
binding them to a fluorescently labeled streptavidin molecule. These
tetramers label T cells that express receptors specific for the
MCC/I-Ek complex. In a typical experiment a
tetramer preparation labels 95% of total thymocytes from 5C.C.7 TCR
ß transgenic mice, 4.0% of thymocytes from 5C.C7 ß transgenic
mice, and <0.3% of thymocytes from B10.BR nontransgenic mice (Fig. 2A). Tetramer staining is TCR
specific because incubation with an Ab that binds to the TCR ß-chain
reduces the number of tetramer high cells to background levels (Fig. 2A). In addition, tetramer staining in ß and TCR-ß
transgenic mice is significantly higher than background staining with
streptavidin-PE (Av-PE) alone and is greatest in the
CD4+ cell subset (Fig. 2B).
|
B, thymocytes and lymph node T cells from a 5C.C7 ß
transgenic mouse were stained with tetramer as well as Abs against the
CD4, CD8, and V11 molecules. Nearly all tetramer binding cells also
expressed V11 (data not shown). Single
CD4+V11+ cells that were
tetramer high, low, or negative were sorted, and the rearranged TCR
V11 genes were amplified by RT-PCR, subcloned, and sequenced. As
shown in Fig. 3, tetramer-reactive cells
from both lymph node and thymus exhibited the characteristic CDR3 loop
length restriction seen in other
MCC/I-Ek-reactive cells, while tetramer-negative
V11-positive cells had a more diverse repertoire of CDR3 loop
lengths.
|
-chain in two cells from both
the tetramer high and tetramer low sorted cells but not in
tetramer-negative cells (Table I). In the
tetramer high cells the CDR3 loop initiates with the germline-encoded
glutamic acid at CDR3 position 1 followed by a small residue and then a
serine at position 3. This canonical motif has been found in many
peripheral MCC-reactive cells and is highly selected for in the
MCC/I-Ek-reactive memory T cell population
(23). The middle three residues contain either an N-T-N
series or two small residues, such as S, A, or G. Position 99 favors
the polar residues lysine or glutamine in 21/22 sequences, and the
final position is always occupied by hydrophobic leucine or valine.
Several sequences appeared more than once, including one
complementarity-determining region-3 amino acid sequence that occurred
11 times (although the nucleotide sequences coding for the amino acids
did vary). Thus, the pool of amino acid sequences that can bind well to
the tetramer is limited.
|
Endogenous expression of MCC induces the deletion of MCC/I-Ek-specific thymocytes.
To examine negative selection in this model system, we crossed
5C.C7ß transgenic mice to HELCYT transgenic mice. HELCYT animals
express a soluble fusion protein of hen egg lysozyme (HEL) protein and
residues 80–103 of the moth cytochrome c protein
(19). In the HELCYT x 5C.C7 ß double-transgenic
mice (HELCYT/ß), MCC-specific T cells fail to develop
(20). Splenocytes and thymocytes were isolated from three
age- and sex-matched mice from each treatment group. After gating out
dead cells and cells that stained with Abs to B220 and MHC class II
Ags, 500,000 events were collected, and tetramer staining was compared
with CD4 staining. Although splenocytes have a higher background
staining level than thymocytes, the presence of endogenous MCC reduced
the number of CD4+ tetramer high cells by 95%
and that of tetramer intermediate cells by 87% (Fig. 4). The presence of MCC also depleted
thymic CD4+tetramer+ cells by 97 and 83%,
respectively (Fig. 4). Importantly, neither the total number of cells
per organ nor the percentage of tetramer-negative cells was affected by
the presence of the HELCYT protein. Thus, MCC-specific cells have
probably been eliminated in the thymus or depleted in the periphery by
the products of the HELCYT transgene, most likely the MCC peptide
contained in the sequence 80–103.
|
To determine the mechanism and timing of the removal of these
potentially autoreactive cells, we examined the percentage of
tetramer-positive cells present at the stages of thymic development as
defined by the expression of the CD4 and CD8 coreceptors (7, 26). Although recent reports indicate that these subsets may
have a more complex precursor or lineage relationship than was
originally proposed, coreceptor expression provides an initial
approximation of thymocyte developmental stage (27, 28, 29, 30).
In Fig. 5A the gates used to
define the various thymic subsets and the tetramer-positive populations
are drawn on representative samples from a 5C.C7 TCRß transgenic
mouse.
|
B). The first
evidence of deletion occurs in the next detectable group of
tetramer-positive cells, the
CD4int8int subset. In this
subset HELCYT/ß mice retain only half as many tetramer-positive cells
as ß transgenics. Similarly, depletion of tetramer-positive cells is
about 50% in the CD4+8+
subset. In the CD4+8int and
CD4+8- subsets, the
percentage of tetramer-positive cells per subset increases 5- to
10-fold in the ß transgenic mice (Fig. 5B, right
panels). This increase suggests that cells that recognize
MCC/I-Ek are preferentially selected from the
CD4+8+ stage, or
alternatively, that these cells have an overall increase in TCR
expression. The percentage of tetramer-positive cells in the mature
(CD4+8-/int) subsets in
HELCYT/ß mice is greatly decreased compared with that in ß
transgenics; very few residual tetramer-positive cells remain.
Importantly, we observed no effect on tetramer-negative cells;
therefore, the HELCYT transgene deletes only cells that have
demonstrable avidity for MCC/I-Ek tetramers. In
fact, we observed efficient deletion in every thymocyte CD4/CD8 subset
that had detectable MCC/I-Ek-reactive TCR
expression. Thus, independent of the lineage relationship between these
subsets, negative selection must occur in a large percentage of the
least mature thymocytes. The extent and timing of deletion correlate with tetramer binding
To examine the influence of tetramer binding avidity and receptor
affinity on the extent and timing of deletion of
MCC/I-Ek-specific cells, we analyzed thymocyte
development in HELCYT/ß and ß transgenic mice as a function of the
tetramer staining level. Tetramer staining of mature T cells bearing
TCRs with known affinities for MHC peptide complexes has been shown to
reflect the avidity of the TCR tetramer interaction (25).
As in Fig. 5, thymocytes were first divided into three subsets based on
CD4 and CD8 staining, then subdivided by successive levels of tetramer
staining (Fig. 6, A and
B). To ensure that these populations were comparable with
respect to CD3 levels, the analysis of immature
(CD4+8+/4int8int)
cells was restricted to those with CD3 staining levels between 1 and 10
(on the log fluorescence scale Fig. 6D). Mature
CD4+8- cells were left
ungated as their CD3 levels were similar (average CD3 levels range
between 4 and 10 on the log fluorescence scale; Fig. 6E).
|
C). At the
earliest stage of development, the
CD4int8int cells were
65% deleted in tetramer high cells, but were only 30% deleted in
lower cells. This suggests that cells with higher avidity for tetramer
(and presumably higher TCR-MCC/I-Ek affinity) are
deleted more efficiently and at an earlier developmental stage than
other cells. Similarly, the extent of deletion in the
CD4+ 8+ subset was nearly
complete in the tetramer high (+++) population, was less complete in
the tetramer medium (++) subset and was only 66% in the tetramer low
(+) cells. The cells with highest avidity for tetramer (+++) were
completely eliminated before they reached the mature
CD4+8- stage; the percent
deletion was the same in these populations. In contrast, cells with
lower avidity for the tetramer (+ and ++) did not become fully deleted
until the CD4+8- stage
(Fig. 6C). The data for the tetramer very low (+/) cells
were difficult to assess, as these cells overlap significantly with
cells found in avidin-PE alone controls. Thus, deletion of Ag-specific
cells occurs more efficiently and earlier when the cells express TCR
complexes with high avidity for the I-Ek-MCC
complexes. Negative selection can occur before or during positive selection
These data suggest that negative selection is influenced by TCR
specificity and occurs at early developmental stages, but do not permit
the relative ordering of positive and negative selections. To determine
whether positive selection is required for negative selection in vivo,
we examined MCC-mediated deletion in the context of other hallmarks of
thymic development, including cell size, CD3 expression, and CD69
expression. Because the expression of CD4 and CD8 may not perfectly
reflect the maturation level of an individual thymocyte
(30, 31, 32), the
CD4+8+ and
CD4int8int populations
(populations 2 and 3 from Fig. 5) were pooled and divided into
subpopulations. Although the gates used to define the subpopulations
are based on positive and negative staining controls they are
necessarily somewhat arbitrary. To control for this we performed
analyses using several different gating strategies. Here we describe
representative results from robust analyses, defined as those that are
not influenced by subtle shifts in the definitions of these
populations. By comparing the percentage of tetramer-positive cells
(Fig. 7a) present in each
subset in HELCYT/ß mice to the cells per subset in ß transgenic
mice it is possible to determine whether deletion can precede positive
selection in vivo.
|
, a and d). In addition,
although small CD4+8+
tetramer-positive cells are present at or near background levels, the
few detectable tetramer-positive cells in ß transgenics are reduced
to background levels in HELCYT/ß mice. Thus, negative
selection can efficiently remove potentially autoreactive cells from
both large and small
CD4+8+/CD4
int8int thymocyte
populations. Although the small cells may include cells that have not
yet been positively selected, they also seem to include cells that have
already failed positive selection and are on the pathway to death by
neglect (32). Because such a small percentage of these
cells stains with tetramer, the small cells that express low levels of
other coreceptors and are indistinguishable from dying cells have been
excluded from further analyses.
A well-studied marker of thymic subpopulations is the CD3 expression
level. For instance, in one study cells that had undergone positive
selection and were
CD3highCD4+8int
were the earliest detectable deleted subset, while
CD3lowCD4+8int
cells were unaffected by the presence of a deleting Ag
(7). In the present study although the
CD4+8+ and
CD3lowCD4+8int
precursors and the
CD3highCD4+8int
cells are efficiently deleted (Fig. 5 and data not shown), the
CD3lowCD4+8+
subset might still be partly resistant to deletion. By examining the
percentage of tetramer-positive cells in three mice from each treatment
group we found that while ß transgenic mice have clearly detectable
populations of tetramer-positive cells in both the
CD3high and large CD3low
subsets, these populations are significantly reduced in HELCYT/ß mice
(Fig. 7, b, e, and f). The number of
tetramer-positive cells in the small CD3low
subset did not differ significantly from background staining of
B10.Br mice. These data suggest that MCC peptide-mediated deletion
occurs at the earliest detectable stage of TCR/CD3 expression and
during or before positive selection.
Perhaps the most rigorous way to identify a pre-positive selection
population is to identify cells that have not yet received TCR-mediated
signals. To distinguish between cells that have received such signals
and those that have not, we examined the appearance of the T cell
activation marker CD69. In the thymus CD69 is up-regulated rapidly and
transiently on CD4+8+ and
CD4+8- thymocytes when
they have undergone either positive or negative selection (1, 34). The percentage of tetramer-positive cells present in the
CD69lowCD4+8+/CD4int8int
population (composed of cells that have not yet received TCR-mediated
signals for positive selection) in HELCYT/ß mice was reduced to
background levels (Fig. 7, c, g, and
h). Deletion appeared equally efficient in the
CD69lowCD4+8+
and CD4int8int populations
(data not shown). Therefore, these preselected cells can be efficiently
deleted. Again, the number of tetramer-positive cells in the small CD69
low populations was similar in B10.Br,
HELCYT/ß, and ß transgenic mice (data not shown). Given our present
ability to define pre-positive selection subsets, these data show that
an appreciable amount of negative selection can precede or coincide
with positive selection.
Negative selection also occurs later in thymocyte development
Although the increasing efficiency of deletion in later stages of
development suggests that deletion does occur at more than one
developmental time point, other studies of TCR transgenic mice have
suggested that in vivo, deletion occurs within a restricted
developmental window (14, 35, 36). To determine whether
deletion also occurs at later stages of development in HELCYT/ß
transgenic mice, we reasoned that if all deletion occurs at or before
the immature CD4+8+ stage,
the ratio of mature tetramer-positive cells to immature
tetramer-positive CD4+8+
cells should remain constant in the presence or the absence of deleting
Ag. Instead, as is shown in Table II, the
HELCYT/ß mice have a decreased ratio of mature/immature tetramer
positive thymocytes. In addition, deletion induced by a single
administration of peptide Ag either 14 or 26 h before removal of
the thymocytes also results in a decrease in this ratio compared with
that in ß transgenics. The increased ratio of the injected mice
compared with the HELCYT/ß mice suggests either that the injection of
peptide produces less efficacious complexes, or that there is a more
mature population that is resistant to deletion by injected
peptide.
|
a) and
subdivided into subsets based on three markers of differentiation: CD8,
HSA, and CD69. As CD4+8-
thymocytes mature they down-regulate all three markers (although they
first up-regulate CD69 if given a TCR-mediated signal) (1, 2, 39). To determine whether any of these markers identified a
particularly resistant thymic population, we calculated the percent
deletion in each treatment group for each of nine different subsets
(Fig. 8, a, c, and e). In eight of these, the
presence of the HELCYT Ag induced more complete deletion than did
peptide injection. The percent deletion was relatively constant in all
CD8 subsets, indicating that these subsets are functionally similar
with respect to negative selection (Fig. 8b). In contrast,
the HSA+ subsets, although equally deleted in
HELCYT/ß mice, showed a differential susceptibility to injected
peptide Ag. In contrast to other studies, the
HSAlow mature cells are most efficiently deleted
(Fig. 8, c and d), suggesting that these cells
are not resistant to deletion and that HSA is not required for the
induction of negative selection. Similarly,
CD69low cells are more efficiently deleted than
the presumably less mature CD69int cells (Fig. 8, e and f). As the time for progression from
CD4+8+ to
CD4+8- mature thymocytes
is thought to be 3 days (40), it is unlikely that all of
this deletion could be accounted for by precursor depletion. Instead,
these data suggest that even putatively mature populations of
thymocytes can undergo Ag-induced cell death, perhaps by mechanisms
similar to those that regulate activation-induced cell death of mature
peripheral T cells.
|
e). In HELCYT/ß mice
the percentage of these CD69high cells that are
tetramer positive is indistinguishable from that in PBS-injected ß
transgenics. In contrast, MCC-injected mice exhibit a 200-fold increase
in the relative number of CD69high cells in the
mature CD4+8- compartment
14 h after injection and a 30-fold increase over that in
PBS-injected mice 26 h after injection (Fig. 8g). These
cells are likely to be the cells that have recently received a strong
TCR-mediated signal but have not yet fully executed their apoptotic
program or that have not yet been eliminated by phagocytosis. One
explanation for this result is that the injection of peptide may
produce extensive simultaneous deletion that temporarily overwhelms the
phagocytic cells found in the thymus. We conclude that negative
selection by endogenous MHC peptide complexes can occur throughout
thymic development. |
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
) and CD3 expression (Fig. 6). In
addition, the tetramer-positive large
CD4+8+ cells, which are
enriched for cells still subject to positive selection, and the
tetramer-positive small
CD4+8+ cells, which have
been recently shown to contain pre-positively selected cells, are each
deleted in HELCYT/ß transgenic mice (Fig. 6). Finally, immature cells
that have not yet up-regulated CD69 in response to the TCR-mediated
signaling involved in positive selection are also deleted in HELCTY/ß
mice (Fig. 6). By injecting mice with peptide just 14 h before
sacrifice, we also find that deletion can occur in later stages of
thymic development. Recent studies have suggested that positive selection may also occur at an earlier stage than was previously thought (30), and that immature thymic subsets are likely to have more complex lineage relationships than those outlined in this paper (29, 31). Thus, the early subsets that we have analyzed may contain some positively selected cells. To address this possibility, we performed additional analyses on smaller subsets defined by CD4, CD8, size, and either CD69 or CD3 and were unable to identify a population of tetramer-positive cells that was not deleted in the presence of the HELCYT transgene. The overwhelming extent of the HELCYT-induced negative selection observed in every thymocyte subset that contains detectable tetramer-positive cells suggests that if there is a detectable population of pre-positively selected cells, they are most likely eliminated in the HELCYT/ß transgenic mice.
Although it is formally possible that in this study, each negatively
selected cell receives a positively selecting signal just before
deletion or as a component of its negatively selecting signal, several
observations argue against these models. When immature
CD4+8+/4int8int
thymocytes from either TCR ß transgenic or nontransgenic mice are
removed from positively selecting thymuses and cultured, very few of
these immature thymocytes become
CD4+8- cells. Instead,
unless they are cultured in the presence of thymic stromal cells
bearing the correct MHC peptide complexes, they remain at the
CD4+8+ stage and eventually
die of neglect (32, 41, 42). Thus, even in the presence of
a rearranged TCR and expression of the appropriate MHC molecules in
vivo, the majority (>90%) of large
CD4+8+/4int8int
cells have not yet been positively selected. Because we observe >50%
deletion in each early thymocyte subset, the deleted cells must include
some cells that have not undergone positive selection.
These observations argue strongly against the hypothesis that all T cells in the thymus are first positively selected in the cortex, then negatively selected in the thymic medulla (7). Interestingly, despite evidence showing that cortical thymic epithelial cells are poor or inefficient mediators of negative selection (4, 43), we observed efficient negative selection in cell populations that reside primarily in the thymic cortex. One possible explanation for this is that the endogenous expression of the soluble HELCYT protein permits unusually high expression of deleting complexes on cortical epithelial cells. This possibility is weakened by the fact that cultured thymic epithelial cells from HELCYT mice fail to induce negative selection or enhanced calcium signaling in 5C.C7 TCR transgenic cells in vitro unless exogenous MCC peptide is added to the culture medium (K. K. Baldwin, manuscript in preparation) (20). Alternatively, cortical thymocytes may encounter interdigitating bone marrow-derived cells or subcapsular APCs in the cortex that capture, process, and present the circulating HELCYT protein and subsequently induce deletion of MCC/I-Ek-specific cells. In either case, these data show that early deletion of Ag-specific cells can occur even when TCR expression is developmentally normal, and the population of interest is a minor component of the entire T cell repertoire.
The fact that some Ag-specific cells are eliminated at later stages of
thymic development (Fig. 8 and Table II) indicates that early deletion
is incomplete and that, as others have shown, medullary deletion is
necessary to ensure the complete elimination of autoreactive cells
(4). An interesting question with relevance to
autoimmunity is: how do these tetramer-specific cells escape early
deletion? One possibility is that they do not encounter the appropriate
APCs until the thymocytes enter the deleting cell-rich medulla.
Alternatively, the escaping cells may have lower expression levels of
coreceptors that enhance deletion (although none of the coreceptors we
examined correlated with deletion). Finally, the TCRs expressed by the
resistant thymocytes may have a lower avidity for the deleting MHC
peptide complexes. Our data suggest that TCR affinity and/or thymocyte
avidity for tetramer can play a role in the timing of deletion.
Thymocytes that have the highest tetramer binding ability are deleted
somewhat more efficiently overall than tetramer low cells (90 vs
77%). Similarly, higher binding cells are already 66% deleted by the
CD4int8int stage compared
with 40% in the less avid tetramer-specific population (Fig. 6C). Thus, the cells most likely to initiate an autoimmune
response are eliminated most efficiently and earliest in the
thymus.
Even so, TCR avidity as measured by tetramer binding ability is
probably not the sole determinant of the timing of negative selection.
A large number of the tetramer low (but specific) cells are eliminated
even at the earliest developmental stage, and a few cells that stain
brightly with the tetramer persist throughout thymic development (Figs. 5 and 6). These cells are of particular interest as they may represent
potentially autoreactive cells. Some tetramer high cells are detectable
in the periphery of HELCYT/ß double-transgenic mice as well (Fig. 4),
yet the mice are healthy and exhibit no obvious autoimmunity. Thus,
these self-reactive cells may somehow be rendered unresponsive by
peripheral tolerance mechanisms such as anergy. Alternatively, as has
been shown in other systems, these autoreactive cells may be induced to
undergo Ag-induced cell death in the periphery (reviewed in Ref.
44). Using the peptide/MHC tetramer to track these cells
and follow them during the course of immune challenge with MCC or
related Ags may provide further insight into the mechanisms of thymic
and peripheral tolerance induction.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Current address: Howard Hughes Medical Institute/Center for Neurobiology and Behavior, Columbia College of Physicians and Surgeons, New York, NY 10032
3 Address correspondence and reprint requests to Dr. Mark M. Davis, Howard Hughes Medical Institute, Stanford University, 279 Campus Drive, Stanford, CA 94305-5323. E-mail address:
4 Abbreviations used in this paper: RAG, recombinase-activating gene; MCC, moth cytochrome c; HSA, heat-stable Ag; HEL, hen egg lysozyme.
Received for publication February 1, 1999. Accepted for publication April 30, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
ß+ T cells: one population is deleted by ligation of ß TCR. Cell 58:1047.[Medline]
ß versus ß T cell receptor transgenic mice undergoing negative selection. Nature 340:559.[Medline]
This article has been cited by other articles:
|
|
 |
|
  M. Stojakovic, L. I. Salazar-Fontana, Z. Tatari-Calderone, V. P. Badovinac, F. R. Santori, D. Kovalovsky, D. Sant'Angelo, J. T. Harty, and S. Vukmanovic Adaptable TCR Avidity Thresholds for Negative Selection J. Immunol., November 15, 2008; 181(10): 6770 - 6778. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. C. M. Davalos-Misslitz, T. Worbs, S. Willenzon, G. Bernhardt, and R. Forster Impaired responsiveness to T-cell receptor stimulation and defective negative selection of thymocytes in CCR7-deficient mice Blood, December 15, 2007; 110(13): 4351 - 4359. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. R. Santori, Z. Popmihajlov, V. P. Badovinac, C. Smith, S. Radoja, J. T. Harty, and S. Vukmanovic TCRbeta Chain That Forms Peptide-Independent Alloreactive TCR Transfers Reduced Reactivity with Irrelevant Peptide/MHC Complex J. Immunol., May 15, 2007; 178(10): 6109 - 6114. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Yu, J. Mao, Y. Wu, H. Luo, and J. Wu Ephrin-B1 Is Critical in T-cell Development J. Biol. Chem., April 14, 2006; 281(15): 10222 - 10229. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Cheunsuk, Z.-X. Lian, G.-X. Yang, M. E. Gershwin, J. R. Gruen, and C. L. Bowlus Prss16 Is Not Required for T-Cell Development Mol. Cell. Biol., January 15, 2005; 25(2): 789 - 796. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Zhan, J. F. Purton, D. I. Godfrey, T. J. Cole, W. R. Heath, and A. M. Lew Without peripheral interference, thymic deletion is mediated in a cohort of double-positive cells without classical activation PNAS, February 4, 2003; 100(3): 1197 - 1202. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. J. Cho, S. G. Edmondson, A. D. Miller, M. Sellars, S. T. Alexander, S. Somersan, and J. A. Punt Cutting Edge: Identification of the Targets of Clonal Deletion in an Unmanipulated Thymus J. Immunol., January 1, 2003; 170(1): 10 - 13. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Terra, N. Labrecque, and C. Perreault Thymic and Extrathymic T Cell Development Pathways Follow Different Rules J. Immunol., July 15, 2002; 169(2): 684 - 692. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. B. Sant'Angelo and C. A. Janeway Jr. Negative selection of thymocytes expressing the D10 TCR PNAS, May 14, 2002; 99(10): 6931 - 6936. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Le Campion, B. Lucas, N. Dautigny, S. Leaument, F. Vasseur, and C. Penit Quantitative and Qualitative Adjustment of Thymic T Cell Production by Clonal Expansion of Premigrant Thymocytes J. Immunol., February 15, 2002; 168(4): 1664 - 1671. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Quarsten, S. N. McAdam, T. Jensen, H. Arentz-Hansen, K. E. A. Lundin, and L. M. Sollid Staining of Celiac Disease-Relevant T Cells by Peptide-DQ2 Multimers J. Immunol., November 1, 2001; 167(9): 4861 - 4868. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Viret, D. B. Sant'Angelo, X. He, H. Ramaswamy, and C. A. Janeway Jr. A Role for Accessibility to Self-Peptide-Self-MHC Complexes in Intrathymic Negative Selection J. Immunol., April 1, 2001; 166(7): 4429 - 4437. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. P. Riley, D. M. Cerasoli, M. S. Jordan, A. L. Petrone, F. F. Shih, and A. J. Caton Graded Deletion and Virus-Induced Activation of Autoreactive CD4+ T Cells J. Immunol., November 1, 2000; 165(9): 4870 - 4876. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Viret, O. Lantz, X. He, A. Bendelac, and C. A. Janeway Jr. A NK1.1+ Thymocyte-Derived TCR {beta}-Chain Transgene Promotes Positive Selection of Thymic NK1.1+ {alpha}{beta} T Cells J. Immunol., September 15, 2000; 165(6): 3004 - 3014. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
