By
From the * Division of Hematologic Products, Center for Biologics Evaluation and Research, Food and
Drug Administration, Bethesda, Maryland 20892; and the Laboratory of Mammalian Genes and
Development, National Institute of Child Health and Human Development, National Institutes of
Health, Bethesda, Maryland 20892
Immature thymocytes undergo a selection process within the thymus based on their T cell antigen receptor (TCR) specificity that results either in their maturation into functionally competent, self-MHC-restricted T cells (positive selection) or their deletion (negative selection). The
outcome of thymocyte selection is thought to be controlled by signals transduced by the TCR that
vary in relation to the avidity of the TCR-ligand interaction. The TCR is composed of four
distinct signal transducing subunits (CD3-, -
, -
, and
) that contain either one (CD3-
, -
,
-
) or three (-
) signaling motifs (ITAMs) within their intracytoplasmic domains. A possible
function for multiple TCR ITAMs could be to amplify signals generated by the TCR during selection. To determine the importance of the multiple TCR-
chain ITAMs in thymocyte selection, transgenes encoding
/
TCRs with known specificity were bred into mice in which
chains lacking one or more ITAMs had been genetically substituted for endogenous
. A direct
relationship was observed between the number of
chain ITAMs within the TCR complex and the efficiency of both positive and negative selection. These results reveal a role for multiple
TCR ITAMs in thymocyte selection and identify a function for TCR signal amplification in
formation of the T cell repertoire.
During their development, thymocytes are subjected to
a selection process that results in the survival of functionally competent, self-MHC-restricted cells (positive selection), and the deletion of potentially autoreactive cells
(negative selection; reference 1). The fate of immature thymocytes is ultimately dictated by the specificity of their
TCRs (2) and is presumably controlled by TCR-mediated
signals that vary with the avidity of TCR-self ligand interactions within the thymus. The TCR complex contains multiple signal transducing subunits (CD3- A specific role for the multiple ITAM TCR structure
could be in selection of the T cell repertoire during thymocyte development. Recent data support the idea that
relatively low affinity TCR-ligand interactions can promote
the selection of immature thymocytes, whereas higher affinity interactions are required for mature T cell activation
(2, 16). Thus, the multiple Transgenic Mice.
Transgenes encoding full-length Proliferation Assays.
Single cell suspensions were prepared
from LNs from normal ( Flow Cytometry.
For multicolor flow cytometry (FCM), thymocytes or LN cells were first incubated with antibody to the Fc
receptor (mAb 2.4G2) to prevent FcR binding. For two- and threecolor FCM, cells were incubated with FITC-conjugated, PEconjugated, and biotinylated antibodies, followed by the addition of
red 670 streptavidin (GIBCO BRL, Gaithersburg, MD). The FCM
was performed on a Becton Dickinson Immunocytometry Systems
FACScan® using standard Cell Quest software. Data were collected
on 10-20 × 104 viable cells as determined by forward and side
light scatter. The majority of monoclonal antibodies used for FCM
analysis were purchased from PharMingen (San Diego, CA) and included biotinylated anti-CD4 (RM4.5) and PE-anti-CD8 (6.7),
FITC and PE-anti-CD3 (145-2C11), FITC-anti-TCRV Superantigen-induced Thymocyte Deletion.
4-8-wk-old mice were
injected intraperitoneally with PBS alone, 10 µg staphylococcus
enterotoxin B (SEB; Sigma Chemical Co., St. Louis, MO) in
PBS, or 20 µg SEB in PBS, every other day for 1 wk (three doses).
2 d after the final injection, mice were killed and thymocytes
were harvested. Thymocytes were stained with anti-V Reconstitution of
To examine the importance of the multiple TCR- Examination of
In female H-2Db, H-Y+/ Table 1.
Numbers of Thymocytes in H-Y+/
Despite the near absence of T3.70+, CD8+ LN T cells,
large numbers of T3.70 To assess the role of the We next examined LNs to determine the fate of the DP
thymocytes observed in male H-Y+/ To study the role of
In these studies, we investigated the role of the TCR- Our results with two Interestingly, incremenatal reduction in the number of
TCR- Our results demonstrate that TCR signal amplification
also plays an important role in negative selection; however, in
H-Y Tg mice, the impact of removing the That reduction in the number of TCR ITAMs quantitatively affects both positive and negative selection is consistent with the hypothesis that the outcome (positive or negative) of thymocyte selection is dictated by quantitative
rather than qualitative differences in the TCR signaling response (2). Although it might have been predicted, we did
not observe positive selection of T3.70+ thymocytes in
H-Y+ Finally, it has remained unclear how relatively small differences in the affinity or avidity of TCR self-ligand interactions can differentially lead either to positive or negative
selection (32). Our data suggest that the ability of the TCR
to elicit such developmentally distinct cell fate decisions stems
from its unique structure. Multiple ITAMs may serve to
greatly magnify TCR signals and thereby translate relatively
small differences in avidity into large signaling differences.
Thus, the unique configuration of the TCR, with its capacity for signal amplification, plays a pivitol role in determining the ultimate fate of developing thymocytes and in
shaping the mature T cell repertoire.
, -
, -
, and
) that share a common functional sequence, the immunoreceptor
tyrosine-based activation motif (ITAM)1 within their intracytoplasmic domains (3, 4). After TCR engagement, phosphorylation of ITAMs leads to the recruitment of SH2 domain-containing proteins (e.g., tyrosine kinases) to the TCR
complex and initiation of the T cell activation cascade (4).
The CD3 subunits each contain a single ITAM, whereas
contains three ITAMs within its longer cytoplasmic tail.
ITAM sequences are conserved but nonidentical, and it remains unclear whether individual motifs perform unique or
similar functions (8). It has been suggested that due to its
singular configuration,
chain may function as the predominant TCR signaling structure and that its triplicated
ITAMs may serve primarily to facilitate TCR signal amplification (9, 10).
chain is required for normal T cell development as
-deficient (
/
) mice have markedly reduced numbers of
both CD4+CD8+ (double positive, DP) and CD4+CD8
and CD4
CD8+ (single positive, SP) thymocytes (11).
However, the major developmental defects in
/
mice were
subsequently shown to be the result of impaired TCR surface expression rather than lack of
-mediated signals per se, as transgene-encoded
chain variants that lacked ITAM
sequences but could still facilitate TCR surface expression
(
-0 ITAM), restored T cell development in
/
mice
(15). These results demonstrated that
chain signals were
not specifically required for T cell development as signal transduction by TCR complexes that contain the CD3
subunit ITAMs, but not
chain ITAMs, was sufficient for
the generation of mature T cells.
chain ITAMs could be required to amplify TCR signals originating from the relatively weak interactions that mediate thymocyte selection. Since this question could not be easily addressed in mice
with a heterogeneous population of T cells with diverse
TCR specificities, we fixed the specificity of the TCRs expressed on developing thymocytes by introducing transgenes that encode productively rearranged TCR-
and -
chains (
/
-TCR Tg) into
/
mice (21, 22). We then
bred into the
/
-TCR Tg/
/
background transgenes
encoding either full-length
chain (3 ITAM),
chains
containing a single ITAM (1 ITAM), or a
chain lacking
all three ITAMs (
- 0 ITAM). The results of these studies reveal that
chain signals influence both positive and negative thymocyte selection and identify a role for the multiple
ITAM TCR structure in selection of the T cell repertoire.
chain (
-3
ITAM Tg) and
chain variants with intact extracellular and
transmembrane domains, but containing only a single ITAM (either the membrane proximal ITAM or the membrane distal
ITAM; [
-1 ITAM Tg]), or containing no ITAMs (
-0 ITAM
Tg) were generated using the identical (human CD2) promoter/
enhancer cassette. The generation of
/
mice,
transgenes, and
genetic reconstitution of
/
mice with
transgenes has been
described previously (11, 15). The
/
-TCR transgenes used in
these experiments encoded either an MHC class I-restricted TCR
specific for male antigen (H-Y; 21) or an MHC class II-restricted
TCR specific for pigeon cytochrome C (AND; 22).
/
TCR
transgenes were introduced into the
/
background by breeding and identified by Southern blotting or PCR analysis of tail
DNA. All mice used for these studies were bred and maintained
in a barrier (specific pathogen-free) facility.
+/+) or transgene-reconstituted
/
mice.
T cells were purified by incubating cells on rabbit anti-mouse Igcoated plates. Accessory cells and APCs (either for FcR-mediated antibody cross-linking or allogeneic stimulator cells) were prepared from spleen cell suspensions of C57BL/6 or BALB/c mice.
APCs were depleted of T cells with anti-Thy1.2 + C
and irradiated with 3,000 rads. 105 responder T cells were combined with
5 × 105 accessory cells in flat-bottom 96-well plates in the presence or absence of the indicated stimulants. After the specified
time, cells were pulsed for 8 h with 1 µCi [3H]thymidine and
harvested. For mitogen stimulation, Con A at 2 µg/ml was added
to the culture. For anti-CD3
stimulation, 25% tissue culture
supernatant from the 145-2C11 hybridoma was added to the culture. Cells were cultured in presence of syngeneic (C57BL/6) APCs and harvested after 48 h. For the mixed lymphocyte reaction, responder T cells were combined with either syngeneic
(C57BL/6) or allogeneic (BALB/c) APCs. Cells were harvested
after a total of 96 h.
14
(14-2), and FITC-anti-V
11 (RR8-1). Anti-TCRV
8 (F23.1) and
anti-H-Y clonotypic receptor (T3.70) were purified from cell culture supernatants and labeled with FITC.
8-FITC or
anti-V
14-FITC, followed by anti-CD3
-PE. Deletion was assessed by software gating on CD3high (SP cells) and determining
the percentage of cells expressing individual TCRV
chains. Percent deletion was determined by dividing the percentage of
CD3high, TCRV
8+ thymocytes remaining after SEB injection
by the percentage of CD3high, TCRV
8+ cells present in mice
injected with PBS alone.
/
mice with a transgene encoding
a
variant chain lacking all three
chain ITAMs (
-0
ITAM) restores TCR surface expression and promotes the
generation of large numbers of CD4+CD8
and CD4
CD8+
SP thymocytes and peripheral T cells (15). Significantly,
the T cells generated in
-0 ITAM Tg mice are functionally competent as assessed by their ability to respond to
TCR-dependent stimuli (Fig. 1; 23). These results demonstrate that TCR-
chain signals are not specifically required
for either the generation or activation of mature T cells.
Fig. 1.
Role of TCR- chain ITAMs in mature T cell function. (A)
Proliferative response of LN T cells to anti-CD3
and Con A stimulation. (B) Proliferative response of LN T cells to allogenic stimulator cells. LN
T cells were obtained from 4-8-wk-old nontransgenic (
+/+/) mice, or
/
mice that express transgenes encoding either full-length
chain (
-3
ITAM Tg) or
chain lacking ITAM sequences (
-0 ITAM Tg) and stimulated as described in Materials and Methods.
[View Larger Versions of these Images (56 + 55K GIF file)]
ITAMs in the selection of the T cell repertoire, we fixed
the specificity of the TCRs expressed on developing thymocytes by introducing a transgene that encodes productively rearranged TCR-
and -
chains specific for the
male H-Y antigen in the context of H-2Db (H-Y TCR;
21). This approach enabled us to examine both positive selection (H-2Db female mice) and negative selection (H-2Db
male mice) of thymocytes bearing a single
/
-TCR.
/
mice that express the H-Y transgene revealed that the developmental impairment observed
previously in
/
mice was also observed in both male and
female H-Y+/
/
mice (Fig. 2). These results were expected because of the extremely low level of TCR surface
expression in the absence of
chain (11) and are consistent with a requirement for the TCR for normal thymocyte development and selection. To address the importance of the
chain ITAMs in thymocyte selection, we next
examined H-Y+/
/
mice reconstituted with transgenes
encoding either full-length
chains (H-Y+/
-3 ITAM Tg
mice),
chains containing a single ITAM (H-Y+/
-1 ITAM
Tg mice), or
chains lacking all three ITAMs (H-Y+/
- 0 ITAM Tg mice) (15). Each of the
transgenes was capable of restoring TCR surface expression in mice lacking endogenous
chain (Fig. 3; 15). For these experiments transgenic founder lines were chosen that gave comparable levels of TCR surface expression that were equal to or greater
than that found on thymocytes from
+/+ mice (Fig. 3; 15).
Fig. 2.
Phenotype of H-Y/
/
and H-Y+/
/
mice. Data show
immunofluorescence and multicolor FCM analysis of thymocytes and LN
T cells from adult (6-12-wk-old) H-2Db mice. Three-color FCM analysis
was performed on cells stained with anti-H-Y clonotypic antibody (T3.70)
conjugated to FITC, anti-CD8-PE and anti-CD4-biotin, followed by
streptavidin-red 670 (16). CD4 versus CD8 two-color profiles are displayed on total thymocytes or total lymph node T cells. Single-color profiles (solid lines) depict T3.70 staining on total thymocytes. Dotted lines
reflect staining with negative control antibody.
[View Larger Version of this Image (43K GIF file)]
Fig. 3.
Role of TCR-mediated signals in thymocyte
positive and negative selection.
(A) Phenotype of thymocytes
from H-Y+/
/
;
Tg female
mice. (B) Phenotype of thymocytes from H-Y+/
/
;
Tg
male mice. H-Y+/
/
mice
were reconstituted with transgenes encoding either full-length
chains (
-3 ITAM Tg),
chains that contain a single
ITAM (either the first or third,
-1 ITAM Tg) or
chains that
lack ITAMs (
-0 ITAM Tg).
Data show immunofluorescence
and multicolor FCM analysis of
thymocytes from adult H-2Db
mice. Three-color FCM was
performed on cells stained with
anti-H-Y clonotypic antibody
(T3.70) conjugated to FITC,
anti-CD8-PE, and anti-CD4-
biotin, followed by streptavidin-
red 670 (16). Two-color plots
show total thymocytes or software-gated T3.70+ thymocytes.
Numbers in the quadrants reflect
the percentage of total thymocytes in that quadrant. Singlecolor profiles (solid lines) depict
T3.70 staining on either
CD4+CD8+ thymocytes (A) or
total thymocytes (B). Dotted
lines reflect staining with negative control antibody.
[View Larger Versions of these Images (62 + 64K GIF file)]
+/+ mice, interaction of the
H-Y TCR on immature thymocytes with an unidentified
ligand results in positive selection and generation of CD8
SP thymocytes that express high surface levels of the H-Y
TCR (detected by reactivity with the clonotype-specific
antibody, T3.70; reference 24; Fig. 3 A, Table 1 A). Importantly, a phenotype similar to that of H-Y+/
+/+ mice
was observed in female H-Y+/
/
mice reconstituted with
the full-length
transgene (H-Y+/
-3 ITAM Tg; Fig. 3 A,
Table 1 A). These mice also had large thymi that contained
a high percentage of T3.70+, CD8 SP cells indicating that
positive selection was effectively restored in
/
mice by
expression of a transgene-encoded full-length (3 ITAM)
chain (Fig. 3). However, in female H-Y+/
/
mice reconstituted with a transgene encoding the signaling-deficient
chain (H-Y+/
-0 ITAM Tg), positive selection of
T3.70+ thymocytes was markedly impaired as evidenced
by the extremely low percentage of T3.70+, CD8 SP thymocytes (Fig. 3 A, Table 1 A). Interestingly, H-Y+/
/
females reconstituted with transgenic
chains that contain
a single ITAM (either the first [membrane proximal] or
third [membrane distal]) exhibited an intermediate phenotype (Fig. 3 A). Although there were subtle differences in the
two H-Y+/
-1 ITAM Tg lines, the percentage of T3.70+,
CD8 SP thymocytes in both lines consistently fell between
those observed in mice reconstituted with either the
-3
ITAM or
-0 ITAM transgenes. The direct relationship
between the number of
chain ITAMs and the generation
of clonotypic (T3.70+) CD8 SP thymocytes was even more
evident when absolute numbers of T3.70+, CD8+ thymocytes in the various transgenic lines were compared (Table 1 A). Examination of LNs from H-Y+/
-0 ITAM Tg females also revealed a lower percentage of T3.70+, CD8+
T cells relative to H-Y+/
-3 ITAM Tg females (Fig. 4). A
similar relationship between the number of TCR-
ITAMs
and the efficiency of positive selection was also observed with
a class II-restricted TCR-
Tg (AND; reference 22; Fig. 5).
Together, these results demonstrate that TCR ligand interactions that can generate signals that promote positive selection in the presence of full-length
chains are unable to
generate these signals in the absence of
chain ITAMs.
+/+ Mice and H-Y+/
/
Mice Reconstituted with Transgenes Encoding the Full-length
Chain (
-3 ITAM Tg), a
Chain Containing the Third ITAM (
-1ITAM Tg) or a
Chain lacking ITAMs (
-0 ITAM Tg)
Females
+/+
n = 3
-3 ITAM Tg
n = 3
-1 ITAM Tg
n = 6
-0 ITAM Tg
n = 6
Total
Thymocytes
97 ± 42
166 ± 74
122 ± 45
56 ± 22
Total T3.70+
53 ± 25
117 ± 33
91 ± 51
47 ± 18
T3.70+ DN*
8.2 ± 3
6.2 ± 3
3.3 ± 2
1.9 ± 2
T3.70+ DP*
28 ± 10
89 ± 29
74 ± 26
42 ± 16
T3.70+ CD8 SP*
15.5 ± 12
17 ± 6
9.8 ± 5
0.6 ± 0.2
Males
n = 4
n = 5
n = 6
n = 7
Total
Thymocytes
6.0 ± 1.0
5.6 ± 2.7
7.6 ± 5.2
17 ± 10
Total T3.70+
5.1 ± 0.7
4.9 ± 2.4
6.6 ± 4.3
14.3 ± 9.3
T3.70+ DN*
3.9 ± 0.4
2.6 ± 1.8
0.9 ± 0.6
0.8 ± 0.9
T3.70+ DP*
0.5 ± 0.4
1.0 ± 0.7
4.0 ± 2.5
11.0 ± 7.6
Data are given in 106 cells as means ± SEM rounded to the nearest whole number. T3.70+ thymocyte subpopulations were calculated by multiplying the total T3.70+ thymocyte number by the percent of cells in a given quadrant as depicted in Fig. 2.
*
Double negative = CD4 CD8
; DP = CD4+CD8+; CD8 SP = CD4
CD8+.
Fig. 4.
Analysis of LN T cells from H-Y+/ Tg mice. Immunofluorescence and multicolor FCM analysis of LN cells from adult female and male H-Y+/
-3 ITAM Tg or H-Y+/
-0 ITAM Tg mice. Also shown
are LN cells from a nontransgenic C57BL/6 (H-Y
/
+/+) mouse. Threecolor FCM was performed on total LN cells after staining with FITC-
anti-H-Y clonotypic antibody (T3.70), anti-CD8-PE and anti-CD4-
biotin, followed by streptavidin-red 670. CD4 versus CD8 two-color
profiles are displayed on total (left) or gated T3.70+ (right) cells. Numbers
in the regions reflect the percentage of total cells found in that region.
[View Larger Version of this Image (50K GIF file)]
Fig. 5.
Positive selection in MHC class II-restricted /
TCR transgenic mice. AND+/
/
mice were generated by mating and then reconstituted with transgenes encoding either a full-length
chain (
-3 ITAM
Tg), a
chain containing a single (third) ITAM (
-1 ITAM Tg), or a
chain that lacks ITAMs (
- 0 ITAM Tg). Data show immunofluorescence
and multicolor FCM analysis of thymocytes from adult H-2Db mice.
FCM was performed on cells stained with FITC-conjugated antibody
specific for the transgenic V
chain (V
11), anti-CD8-PE and antiCD4-biotin, followed by streptavidin-red 670. Two-color plots show
software-gated V
11+ thymocytes. Numbers in the quadrants reflect the
percentage of total thymocytes in that quadrant. Single-color profiles
(solid lines) depict V
11 staining on total thymocytes. Dotted lines reflect
staining with negative control antibody.
[View Larger Version of this Image (49K GIF file)]
, CD8 SP thymocytes and T cells
were detected in H-Y+/
-0 ITAM Tg females (Figs. 3 A
and 4). Since these T3.70
CD8 SP cells expressed
/
-TCRs
composed of transgenic TCR-
chains paired with non-
transgenic, endogenously derived TCR-
chains, their TCR
specificity was not restricted to the H-Y antigen. Therefore,
the reduction in T3.70+, CD8 SP thymocytes and T cells
in H-Y+/
-0 ITAM Tg mice specifically reflected the failure of the H-Y TCR to promote positive selection of
T3.70+ DP thymocytes.
ITAMs in negative selection,
we next examined H-Y+/
Tg male mice. Both H-Y+/
+/+
males and H-Y+/
-3 ITAM males had small thymi with
few (<107) thymocytes which were predominantly CD4
CD8
(Fig. 3 B, Table 1 B). The marked reduction in DP
thymocytes in these mice is consistent with previous data
indicating that negative selection occurs before the DP
stage in H-Y Tg males (25). In contrast to mice expressing
full-length
chain, H-Y+/
-0 ITAM Tg males exhibited a
very different phenotype. Thymi from these mice were
three- to sixfold larger than H-Y+/
-3 ITAM Tg males
(Table 1 B) and contained predominantly T3.70+, DP thymocytes (Fig. 3 B). Again, the phenotype exhibited by both lines of H-Y+/
-1 ITAM Tg mice was intermediate
between H-Y+/
-3 ITAM Tg males and H-Y+/
-0 ITAM
Tg males. For example, thymi from H-Y+/
-1 ITAM Tg
males contained lower percentages of DP thymocytes than
did H-Y+/
-0 ITAM Tg males, but contained higher percentages of CD4
CD8low cells which are thought to be the
immediate precursors of DP thymocytes (26-28; Fig. 3 B,
Table 1 B). Thus, the early negative selection of H-Y clonotypic (T3.70+) thymocytes was attenuated in H-Y+/
-0
ITAM Tg and H-Y+/
-1 ITAM Tg males.
-0 ITAM Tg and
H-Y+/
-1 ITAM Tg mice. Whereas LN from both H-Y+/
+/+ males and H-Y+/
-3 ITAM Tg males contained almost no "conventional" CD4high or CD8high T cells, large
numbers of both CD4high and CD8high T cells were present
in H-Y+/
-0 ITAM Tg (Fig. 4) and H-Y+/
-1 ITAM Tg
males (data not shown). These CD4high and CD8high T cells expressed high levels of TCR which were uniformly T3.70low/
indicating that they were composed primarily of nontransgenic TCR-
chains. Therefore, although negative selection
of T3.70+ DP thymocytes was not abrogated in H-Y+/
-0
ITAM Tg and H-Y+/
-1 ITAM Tg males, some DP thymocytes were able to escape negative selection. Furthermore,
these thymocytes were also capable of being positively selected on the basis of non-H-Y restricted TCRs generated
from rearrangement and expression of endogenous TCR-
genes.
-mediated signals in deletion of
more mature (SP) thymocytes, we examined superantigenmediated thymocyte deletion in the various transgenic lines.
The superantigen SEB deletes SP thymocytes bearing TCRs
that include V
8 (29). Adult mice were injected with SEB
or PBS every other day for 1 wk and the percentage of
TCRV
8+ SP thymocytes remaining was determined by
FCM analysis. At similar doses of antigen, the efficiency of
SEB-induced thymocyte deletion in
-0 ITAM mice was
significantly reduced relative to
-3 ITAM Tg mice (Fig. 6).
Nevertheless, thymocyte deletion by SEB was detectable in
-0 ITAM Tg mice and increased with increasing SEB dosage (Fig. 6). The deletion of V
8+ thymocytes was specific
as the percent TCRV
14+ SP thymocytes (which are not
deleted by SEB) was similar in
-0 ITAM Tg and
-3
ITAM Tg mice (data not shown). Therefore, in the absence of TCR-
signals, deletion of SP thymocytes by SEB
was significantly impaired.
Fig. 6.
Superantigen-mediated deletion of TCRV8+ thymocytes. Mice were injected
with PBS alone, 10 µg SEB in
PBS, or 20 µg SEB. Thymocytes were stained with anti-V
8-FITC
or anti-V
14-FITC, followed by anti-CD3
-PE. Deletion was assessed by software gating on
CD3high (SP cells) and determining the percentage of cells expressing individual TCRV
chains. Deletion was specific for
cells expressing V
8 as cells expressing V
14 were not deleted (29). Results are given as mean ± SEM
and are from at least six different SEB injected mice per group compiled
from two to three separate experiments.
[View Larger Version of this Image (18K GIF file)]
chain ITAMs in thymocyte selection by fixing the specificity of the TCR and then varying the TCR signaling potential by altering the number of TCR-
chain ITAMs within
the TCR complex. The results reveal a direct relationship
between the number of
chain ITAMs in the TCR complex and the efficiency of both positive and negative thymocyte selection. In addition, they suggest that the multiple
chain ITAMs function primarily to amplify signals
generated by the TCR during thymocyte selection. In this
respect, our findings are consistent with in vitro results obtained from mature T cell clones which failed to identify a
specific function for individual
ITAMs, but found that
triplication of a single motif resulted in a quantitative enhancement of signaling (9, 10). However, our current results do not preclude a specific role for individual TCR-
ITAMs in more specialized T cell functions that have yet to
be examined.
/
-TCR Tg systems (H-Y and
AND) suggest that positive selection, which is thought to be
mediated by low avidity TCR-ligand interactions (2, 16)
is particularly dependent upon signal amplification by the
TCR-
chain. In H-Y+/
-0 ITAM Tg female mice, most
thymocytes that expressed only the H-Y-specific TCR failed
to be positively selected, whereas a large number of thymocytes that expressed non-H-Y-specific TCRs were capable of being positively selected. We predict that the mature T cells generated in both non-
/
-TCR Tg/
-0 ITAM
Tg (15) and H-Y+/
-0 ITAM Tg mice are selected on the
basis of TCRs with relatively high affinity for self ligands,
and that these TCRs might otherwise promote negative selection in
+/+ or
-3 ITAM Tg mice where the TCR
contains the full complement of ITAMs. Signal amplification by the
chain ITAMs may therefore not be absolutely
required for positive selection if the avidity of the positively
selecting TCR-ligand interaction is sufficiently high. In these
instances, signal transduction by the remaining ITAMs supplied by the CD3 chains (which are targeted to the cell surface in our model system) is apparently adequate for selection in the absence of
-mediated signals.
ITAMs resulted in graded effects on positive selection. Graded or quantitative effects on positive selection
have also been observed with increasing concentrations of
positively selecting ligand (16). These and other (20) data
suggest that a broad range of signaling responses below a
certain threshold may be capable of mediating positive selection, albeit with different efficiency.
ITAMs from the
TCR complex varied depending on the developmental stage.
Negative selection of early "transtitional" (CD8lowCD4
)
thymocytes was abrogated in the absence of
-mediated
signals, presumably because low CD8 surface density lowers the avidity of the ligand interaction (21, 25). These cells
could give rise to T3.70+ (CD4highCD8high) DP thymocytes,
but DP thymocytes were more susceptible to negative selection as demonstrated by their low numbers in H-Y+/
-0
ITAM Tg males. Nevertheless, negative selection was also impaired at the DP stage in H-Y+/
-0 ITAM Tg mice to
the extent that some DP thymocytes survived, rearranged,
and expressed endogenous TCR-
, and were positively selected on the basis of non-H-Y clonotypic TCRs. Superantigen-induced deletion of SP thymocytes was also affected in
-0 ITAM Tg mice, demonstrating that negative selection
which occurs at later stages of thymocyte developement is
also impaired in the absence of
-mediated signals.
0-ITAM Tg males. However, the signaling capacity of the H-Y TCR in H-Y+
0-ITAM Tg males may not
have been sufficiently reduced to observe such an outcome,
as negative selection, though impaired, was not abrogated
in these mice. Our findings suggest that signals generated from negative selecting interactions are more difficult to attenuate than those generated during positive selection, not
only because higher avidity TCR-ligand interactions are
thought to promote negative selection, but also because of
the effect of TCR signal amplification. Therefore, these results provide an alternative interpretation to studies suggesting that positive and negative selection are biochemically
distinguishable based primarily on the finding that approaches that disrupt positive selection fail to perturb negative selection (30, 31).
Address correspondence to Elizabeth W. Shores, Division of Hematologic Products, Center for Biologics, HFM 538, Bldg. 29A, Rm 2B23, 29 Lincoln Drive, MSC 4555, Bethesda, MD 20892-4555.
Received for publication 3 January 1997.
1Abbreviations used in this paper: DP, double positive; FCM, flow cytometry; ITAM, immunoreceptor tyrosine-based activation motif; SEB, Staphylococcus enterotoxin B; SP, single positive.We thank B.J Fowlkes, A.S. Rosenberg, A. Singer, and M. Vacchio for reading the manuscript and for helpful discussions.
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