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Department of Pathology, University of Massachusetts Medical School, Worcester, MA 01655
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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1, leading to calcium mobilization and extracellular
signal-regulated kinase/mitogen-activated protein kinase activation.
These observations suggested that Itk might play an important role in
positive selection and CD4/CD8 lineage commitment during T cell
development in the thymus. To test this, we crossed Itk-deficient mice
to three lines of TCR transgenics and analyzed progeny on three
different MHC backgrounds. Analysis of these mice revealed that fewer
TCR transgenic T cells develop in the absence of Itk. In addition,
examination of multiple T cell development markers indicates that
multiple stages of positive selection are affected by the absence of
Itk, but the T cells that do develop appear normal. In contrast to the
defects in positive selection, CD4/CD8 lineage commitment seems to be
intact in all the TCR transgenic
itk-/- lines tested. Overall, these
data indicate that altering TCR signals by the removal of Itk does not
affect the appropriate differentiation of thymocytes based on their MHC
specificity, but does impact the efficiency with which thymocytes
complete their maturation process. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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- and 
-chain gene
rearrangements, and therefore express a mature TCR signaling complex on
their surface. At this stage in development, thymocytes must undergo
two concurrent, but distinct, developmental processes to become
functional and tolerant mature T cells. One of these processes is known
as repertoire selection, which ensures that mature T cells can
recognize foreign peptides presented by self-MHC molecules, but will
not become activated when they encounter self-peptide/self-MHC
complexes. The second process, lineage commitment, is the process by
which thymocytes that recognize peptide in the context of MHC class I
become CD8+ cytotoxic T cell precursors, and
thymocytes that recognize peptide in the context of MHC class II
molecules become CD4+ Th cell precursors. Both
these processes are known to be dependent on TCR signaling; however, it
has often been debated how the same initial signals can give rise to
these different and opposing developmental outcomes. Current theories propose that the strength of the signal received by a DP thymocyte through its TCR and/or coreceptor can determine the developmental fate of the thymocyte with respect to both selection and lineage commitment. In the case of selection, it is believed that the avidity of the TCR for self-peptide/self-MHC complexes in the thymus will influence the intensity of the TCR signal. Thus, thymocytes that weakly bind self-peptide/self-MHC complexes receive weak signals that induce positive selection and the generation of T cells that would be useful to the individual. In contrast, if the thymocytes bind strongly to self-peptide/self-MHC complexes, a strong signal would be transduced, leading to negative selection and the elimination of potentially autoreactive cells. Consistent with this idea, there is extensive evidence that both TCR avidity for self-peptide/self-MHC and TCR signal strength influence the repertoire selection process (1). Further, it has recently been demonstrated that there is a clear link between TCR-MHC/peptide avidity and the ensuing strength of the TCR signal (2, 3).
The events or signals that direct thymocytes into a specific lineage have been more difficult to dissect. Initial studies focused on the CD4 and CD8 coreceptors, including efforts to identify unique signaling molecules/events downstream of each of these coreceptors; however, little progress was made from these approaches. More recent data indicate that alterations in the activities of certain signaling molecules downstream of the coreceptor/TCR may influence lineage commitment. Although still somewhat controversial, it has been shown that both the activity of Lck (4, 5) and the extracellular signal-regulated kinase (Erk)/mitogen-activated protein kinase (MAPK) signaling pathways (6, 7, 8) affect lineage commitment, with high levels of both of these signals leading to CD4 commitment and low levels of these signals leading to CD8 commitment. These studies are consistent with early data indicating that the cytoplasmic tail of CD4 binds more efficiently to Lck that that of CD8 (9, 10, 11, 12). Putting these observations together, one popular model of lineage commitment suggests that when thymocytes recognize class II MHC/peptide complexes, engaging both the TCR and CD4, stronger Lck signals would be induced compared with engagement of TCR and CD8 during recognition of MHC class I/peptide complexes. In addition, it has been shown that agonist signals through the TCR induce CD4 development, whereas antagonist signals induce CD8 development (13, 14). Thus, similar to repertoire selection, lineage commitment may also be dependent on the strength of TCR plus coreceptor signaling.
In this study we were interested in determining the role of the Tec
family tyrosine kinase, Itk, in both the processes of TCR repertoire
selection and lineage commitment. Work from our laboratory and others
has suggested that Itk is important for efficient TCR signaling.
Studies in peripheral T cells have determined that cross-linking of the
TCR in Itk-deficient cells results in reduced PLC- phosphorylation,
reduced inositol trisphosphate generation, no sustained
Ca2+ flux (15), and reduced
translocation of NFAT into the nucleus (16). In addition,
Itk has been suggested to be important for full activation of the
Erk/MAPK signaling pathway (17). From these studies it
appears that although Itk is not required for the activation of these
signaling pathways, in its absence, signals downstream of the TCR are
reduced. Therefore, it seemed likely that both selection and lineage
commitment would be altered in the absence of Itk.
The analysis of itk-/- mice crossed to either a class I- or a class II-restricted TCR transgenic line revealed that virtually no TCR transgenic T cells developed in the absence of Itk (18). Based on this original observation and recent studies with these same TCR transgenics crossed to both itk-/- and rlk-/-itk-/- mice, which lack an additional Tec family member, it has been proposed that Tec family kinases play a role in setting thresholds for thymocyte development (19). To better understand the role of Tec family members in thymocyte development, we crossed itk-/- mice to additional TCR transgenic lines that express TCRs with different avidities for their selecting ligands in the thymus. These mice would allow us to determine whether the requirement for Tec family signals is influenced by the avidity of the TCR on the developing thymocyte. In addition, these mice provide the opportunity to determine whether signaling thresholds for both selection and lineage commitment are shifted in the absence of Itk.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Itk-deficient mice were generated in our laboratory previously
(15). These mice were crossed to 2B4 (20),
5C.C7 (21), and AND (22) TCR transgenic mice,
all of which were on the B10.BR (H-2k) genetic
background. The itk-/- mice crossed to
the TCR transgenic mice had a mixed 129/B10 genetic background
(H-2b). Since the 129 strain carries two
superantigens that delete V3-expressing T cells, a PCR screen was
designed to distinguish mice that carried at least one SAg from those
that were SAg negative; SAg-negative mice were used for all
experiments. The nontransgenic itk+/- and
itk-/- mice in all figures were
backcrossed to B10 mice at least eight times. Mice were analyzed
between 6 and 12 wk of age, and all TCR transgenic control mice are
itk+/- littermates. Neonates were
analyzed 22–40 h postbirth. When examined, there were no significant
phenotypic differences in the lymphocyte profiles of TCR transgenic
itk+/- and
itk+/+ mice (data not shown). Mice were
bred and maintained in a specific pathogen-free facility.
Antibodies
The following mAbs, purchased from BD Pharmingen (San
Diego, CA), were used for staining cells: anti-CD4-PE (H129.19),
anti-CD4-CyChrome (anti-CD4-Cy; H129.19), anti-CD8-FITC (53-6.7),
anti-CD8-Cy (53-6.7), anti-CD8-allophycocyanin (53-6.7),
anti-TCR-Cy (H57-597), anti-V3-PE (KJ25), anti-V11.1,
11.2b,d-FITC (RR8.1), anti-CD5-Cy (53-7.3),
anti-CD69-bio (H1.2F3), anti-CD24 (heat-stable Ag (HSA))-bio
(M1/69), purified hamster anti-mouse Bcl-2 (3F11), and purified hamster
anti-trinitrophenol (107.3). Hamster Abs were detected by goat
F(ab')2 anti-hamster IgG (H+L)-FITC (Caltag
Laboratories, Burlingame, CA). In most cases the 2B4 TCR transgenic was
detected by staining with the A2B4-2 Ab (23), which was
purified and conjugated to FITC in our laboratory. Biotinylated Abs
were detected using strepavidin-allophycocyanin (BD Pharmingen).
Cell preparation, staining, and flow cytometry
Lymphocyte cell suspensions were made from thymus, spleen, and lymph nodes (LNs; pooled inguinal, axillary, brachial, and cervical) by dissociation between two frosted slides in RPMI and 10% FCS. RBC were lysed by incubation in Tris-ammonium chloride for 5 min at room temperature. Cells were washed, resuspended in FACS buffer (1x HBSS, 2% FCS, and 0.01% NaN3), and plated in microwell staining plates at 5 x 105 to 2 x 106 cells/well. Biotinylated Abs were added, and cells were incubated on ice for 15–30 min. The cells were washed and incubated with a mixture of directly conjugated Abs and strepavidin-allophycocyanin on ice for 30 min. After washing, cells were fixed by the addition of 30 µl 4% paraformaldehyde. Fixed cells were analyzed 12–36 h later on a FACSCalibur (BD Biosciences, Mountain View, CA) flow cytometer. In general, 10,000–50,000 live (based on forward vs side scatter profiles) events/sample were collected for the analysis of CD4, CD8, and TCR staining in LN, spleen, and thymus preparations. For marker analysis on thymic subsets 100–200,000 events/sample were collected. Data were analyzed using CellQuest software (BD Biosciences).
Intracellular Bcl-2 staining
Cells were prepared and stained with Abs to cell surface molecules as described above. After extracellular staining, the cells were washed and fixed/permeabilized by incubation in 100 µl Cytofix/Cytoperm (BD Pharmingen) on ice for 20 min. The samples were then washed with Perm/Wash buffer (BD Pharmingen), and a hamster mAb to Bcl-2 was added. Duplicate cell samples were also stained with an irrelevant hamster Ab (anti-trinitrophenol). Cells were incubated on ice for 30 min. After washing, the hamster Abs were detected by incubation with a goat anti-hamster Ab conjugated to FITC on ice for 30 min. Cells were again washed and analyzed immediately. For samples stained with anti-Bcl-2, 200,000 events/sample were collected; 50,000 events/sample were collected for samples stained with the control hamster Ab.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The examination of itk-/- mice
generated in our laboratory (15) suggested that Itk plays
a role in T cell development. As previously reported (18, 19), we observed altered CD4+ and
CD8+ T cell populations in the thymus, spleen,
and LN of itk-/- mice (Fig. 1
). Although we did not observe
significant differences in lymphocyte cellularity in these organs, the
proportions of CD4+ and
CD8+ T cells differ from those in control mice
(itk+/+ or
itk+/-). In the thymus,
itk-/- mice have a slight reduction in
the percentage of CD4+CD8-
(SP) cells and at least a 2-fold increase in the percentage of
CD8+ SP cells (Fig. 1A). However, in
the periphery the percentage of CD4+ cells is, on
the average, half that in control mice, while the percentage of
CD8+ T cells is essentially normal (Fig. 1B).
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Development of T cells expressing 2B4, 5C.C7, or AND TCR in H-2k mice is affected by the absence of Itk
We crossed itk-/- mice to three
different lines of TCR transgenics, specifically, 2B4
(20), 5C.C7 (21), and AND (22)
transgenic mice. Each of these transgenics expresses a TCR that uses
V11 and V3 gene segments and is specific for a moth cytochrome
c peptide (88–103) bound to the MHC class II molecule
I-Ek. However, each TCR is thought to have a
different avidity for selecting ligand(s) in the thymus based on
differences in the efficiency of positive selection (2B4 <
5C.C7 < AND) (24). Thus, the use of these TCR
transgenic lines would allow us to determine whether the absence of Itk
would differentially affect the development of cells bearing TCRs
presumed to have varying avidities for selecting ligands. We chose to
use class II-restricted TCR transgenics because the development of
CD4+ T cells seemed to be more affected by the
absence of Itk. In addition, it has been proposed that signaling
strength may be a potential mechanism for T cell lineage commitment
(25) and that CD8+ T cells develop
as a result of weaker signals. Thus, we were interested in determining
whether the absence of Itk would lead to lineage switching in one or
more of these TCR transgenic lines. This outcome would be visible as
the development of MHC class II-restricted
itk-/- thymocytes into
CD8+ T cells instead of
CD4+ T cells, due to reduced signaling in the
absence of Itk.
Analysis of the 2B4, 5C.C7, and AND H-2k
itk-/- mice revealed that the ability of
the different TCR transgenic thymocytes to develop and populate the
periphery in the absence of Itk varies in a manner consistent with the
proposed avidity of each TCR for its selecting ligand(s) in the thymus.
In the 2B4 and 5C.C7 transgenic itk-/-
mice, the percentages and absolute numbers of
CD4+ SP cells in the thymus are dramatically
reduced compared with the corresponding
itk+/- TCR transgenic (Fig. 1A
and Table I). In the AND
itk-/- mice, the average percentage of
CD4+ SP cells in the thymus is reduced.
However, as a reflection of the slight increase in thymic cellularity
in AND itk-/- mice, the absolute numbers
of CD4+ SP cells are comparable to that seen in
AND itk+/- mice (Table I).
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B), and correlates with the presumed
avidity of each TCR for selecting ligands. In
itk-/- mice that express the 2B4 TCR,
which is thought to be a low avidity TCR, there are very few
CD4+ cells that express the transgenic TCR. In
contrast, there are many more TCR transgenic CD4+
T cells in the itk-/- mice
that express a high avidity TCR, such as 5C.C7 or AND. In fact, in the
AND itk-/- mice, the percentage and
absolute number of these cells are comparable to those in wild-type
AND mice.
It is important to note that the mice represented in Fig. 1 are all
8 wk of age. In mice that are younger (4 wk), the differences in
the percentage of peripheral CD4+ transgenic T
cells between itk-/- mice and wild-type
controls are more substantial in all three transgenic lines. This is
indicative of the lower percentages of CD4+ SP
being generated in the thymus of these mice. As these mice age, the
numbers of peripheral CD4+ transgenic T cells in
AND itk-/- mice increase to numbers
equivalent to those found in itk+/- mice;
however, this is not the case for the 2B4 and 5C.C7
itk-/- mice. These observations suggest
that age is not the only factor that allows the AND
itk-/- mice to accumulate as many
CD4+ transgenic T cells as wild-type AND mice
(see below).
Development of AND itk-/- T cells is affected by the strength and density of selecting ligands expressed by APC in the thymus
Based on the analysis of 2B4, 5C.C7, and AND itk-/- mice, it appears that the presumed avidity of the TCR for its selecting ligand(s) in the thymus plays an important role in the development of T cells in the absence of Itk. However, it is known that the development of T cells in each TCR transgenic line can be somewhat idiosyncratic, due in part to the fact that transgene expression can affect development before thymocytes reach the DP stage (26). Therefore, it is possible that the developmental efficiency of thymocytes in each of the transgenics is not solely based on their given avidities for the selecting ligands. To address this concern, we chose to analyze itk-/- mice that express one of the transgenic TCRs on different MHC backgrounds in which varying levels of two different selecting ligands are expressed.
The AND TCR can be positively selected on both
I-Ek and I-Ab molecules
(27). However, the generation of mature
CD4+ V11high T cells is
greatly reduced in mice that only express the
I-Ab molecule. These observations strongly
suggest that the AND TCR has a higher avidity for
I-Ek vs I-Ab MHC molecules
plus the self peptide(s) that mediates its selection. Therefore, we
decided to compare the selection of AND
itk-/- T cells on the
H-2k, H-2b, and
H-2k/b MHC backgrounds.
Analysis of lymphocytes from wild-type AND mice on all these
backgrounds confirms the selection hierarchy:
ANDb/b < ANDk/b <
ANDk/k (Fig. 2).
The percentage and perhaps the absolute number of
CD4+ cells in the thymus of wild-type
ANDk/k mice are slightly lower than those in
wild-type ANDk/b mice (Fig. 3A and Table I), but the
ANDk/k mice have more peripheral
CD4+ V11high cells (Fig. 3B and Table I). A probable explanation for this phenotype
is that, as previously suggested (27), the interaction
between AND thymocytes and their selecting ligand(s) in the thymus of
ANDk/k mice is of such high avidity that some of
the cells are actually deleted in these mice. This is supported by the
fact that ANDk/k mice generally have lower total
thymocyte numbers and fewer DPs compared with
ANDk/b or ANDb/b mice
(Table I).
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), very few of the CD4+ cells in the
periphery express high levels of the transgenic TCR, as demonstrated by
staining for V11 (Fig. 2B). In contrast, AND
itk-/- mice that express
I-Ek molecules have significant numbers
of CD4+ V11+ T cells in
both the thymus and periphery. As shown in the previous section, the
generation of transgenic T cells in the thymus of
ANDk/k itk-/- mice
appears to be slightly less efficient than in
ANDk/k itk+/- mice,
but the percentage and number of peripheral transgenic T cells are
comparable. Although time may lead to the accumulation of transgenic T
cells in the itk-/- mice, it is also
likely that the ANDk/k
itk-/- mice develop as many peripheral
cells as wild-type AND mice, because the deletion of some of the AND
thymocytes that takes place in wild-type ANDk/k
mice does not occur, or occurs to a lesser extent, in the absence
of Itk.
Wild-type ANDk/b mice have the highest number of
CD4+ SP T cells in the thymus of all three
backgrounds examined, presumably because they express the strongly
selecting I-Ek molecules on thymic stromal cells,
but due to the lower density of this selecting molecule, the partial
deletion that occurs in ANDk/k mice is absent.
The percentage of CD4+
V11high T cells in both the thymus and lymph
nodes of ANDk/b
itk-/- mice is one-half to one-third the
number of these cells found in ANDk/b
itk+/- mice (Fig. 2 and Table I). This phenotype is, as expected, intermediate between that of
ANDb/b itk-/-
mice, which have almost no mature transgenic T cells, and that of
ANDk/k itk-/-
mice, which have normal numbers of transgenic T cells. Thus, the
strength and density of selecting ligands or the avidity of the
interactions between the T cell and thymic stromal cells influence the
outcome of positive selection in the absence of Itk.
Analyses of various developmental markers suggest that the early stages of positive selection are impaired in itk-/- thymocytes, but that the SP thymocytes generated are normal
The analysis of each of the TCR transgenic itk-/- mice revealed that the accumulation of mature CD4+ TCR transgenic cells in both the thymus and periphery of these mice is impaired by the absence of Itk. We hypothesized that this lack of accumulation is due to a defect in the generation or positive selection of these cells. However, it remained possible that mature CD4+ cells were being generated normally, but their survival was impaired in the absence of Itk. Therefore, we were interested in determining whether the survival factor, Bcl-2 is properly up-regulated in itk-/- thymocytes.
It has been shown that Bcl-2 is up-regulated in a subset of
TCRhigh DP thymocytes and remains high in SP
cells (28). This expression pattern correlates with cells
that have received or are receiving positive selection signals. Fig. 3
depicts intracellular staining for Bcl-2 in
itk-/- thymocytes compared with their
wild-type littermates. In non-TCR transgenic
itk-/- cells, the levels of Bcl-2 are
up-regulated in both the DP TCRhigh and
CD4+ SP populations to the same extent as in
thymocytes from a wild-type littermate (Fig. 3A). The same
is true in the analogous populations of TCR transgenic thymocytes on
all the selecting backgrounds (Fig. 3B and data not shown).
These data indicate that itk-/- cells in
which the early stages of positive selection are properly initiated, as
determined by up-regulation of TCR, also properly up-regulate the
survival factor Bcl-2. However, within the entire DP population from
ANDk/b itk-/-
thymi, a greater proportion of DP cells have background (very low)
levels of Bcl-2. This is probably due to fewer cells in these mice
receiving the initial signals for selection.
Analysis of the Bcl-2 levels in cells undergoing selection and in cells
that have completed selection suggested that
itk-/- thymocytes properly up-regulated
this important survival factor and thus would have comparable viability
as their wild-type counterparts. Therefore, we were interested in
determining whether the regulation of additional markers is normal
in itk-/- thymocytes
undergoing selection; this analysis would allow us to
identify the stage(s) of selection in which
itk-/- thymocytes are defective. Fig. 4A depicts the stages of
positive selection based on the expression of the TCR, CD69, HSA, and
the coreceptors, CD4 and CD8.
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B). Thymocytes that have up-regulated CD69 are in either
stage C (TCRhigh DP) or stage D (SP)
(29). Comparing the percentage of cells in these
intermediate stages of selection from wild-type AND mice on each
selecting background revealed that there are fewer
CD69high cells in the presence of the strongly
selecting IEk ligand. One interpretation of these
data is that thymocytes receiving stronger signals up-regulate CD69 and
then progress through stages C and D more efficiently than cells
receiving weaker signals. If this interpretation is correct, we would
expect that a higher percentage of TCRhigh DP and
SP thymocytes in AND itk-/- mice would
have high levels of CD69 compared with their wild-type littermates. As
shown in Fig. 4B, this is, in fact, the case, suggesting
that in the absence of Itk, thymocytes do not progress through these
stages as efficiently as cells that express Itk. Finally, we examined
HSA levels on the CD4+ SP cells from these mice
(Fig. 4C). HSA is down-regulated in the final stage of
thymocyte selection and differentiation, such that the most mature
cells are HSA- (30). Although more
of the CD4+ SP thymocytes in AND
itk-/- mice are still
CD69high compared with those in control mice, it
appears that the same proportion of the CD4+ SPs
have begun to down-regulate HSA in both
itk+/- and
itk-/- mice. This suggests that the
down-regulation of HSA can take place while TCR signaling is still
occurring and that this happens normally even in the absence of
Itk. The expression of CD5, a marker for TCR signal strength, is lower on thymocytes from itk-/- mice
CD5 surface expression is regulated throughout T cell development
and correlates with the strength of the TCR signal received by the
developing thymocyte (31). The studies of peripheral
itk-/- T cells (15) and
thymocytes (32) indicated that T cells get a weaker signal
through their TCR in the absence of Itk; thus, we would expect that CD5
levels would be lower on thymocytes from
itk-/- mice. This prediction was first
verified by staining thymocytes from non-TCR transgenic
itk-/- mice, which revealed that DP
thymocytes have significantly lower levels of CD5 compared with DPs
from itk+/- mice (Fig. 5A).
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B). Similar to the non-TCR
transgenic mice, all the transgenic mice lacking Itk had lower surface
levels of CD5 on their DP thymocytes with the exception of
ANDk/k itk-/-
mice. Comparison of ANDk/k,
ANDk/b, and ANDb/b mice
shows that as the strength of the selecting background increases, the
differences in CD5 surface expression between DPs that express Itk and
those that do not is diminished. Interestingly, the CD5 surface levels
do not seem to vary significantly among the wild-type AND mice with
different selecting backgrounds. Unlike the non-TCR transgenic
itk-/- mice, all the TCR transgenic
itk-/- mice have lower levels of CD5 on
their CD4+ SP thymocytes, further indicating that
the strength of the signal through the same TCR is lower in cells that
lack Itk. Staining with the anti-V11 Ab confirms that the
differences in CD5 expression are not due to lower TCR levels on
itk-/- thymocytes (Fig. 5B). Lineage commitment is not affected by reduced TCR signaling in the absence of Itk
In light of increasing evidence that the strength of the TCR signal influences CD4/CD8 lineage commitment, and the suggestion that Tec family kinases are important for regulating TCR signaling thresholds (19), we were interested in determining whether lineage commitment would be affected in itk-/- mice. This model of lineage commitment proposes that strong signals downstream of the TCR induce DPs to differentiate into CD4+ T cells, while weak signals downstream of the TCR cause DPs to differentiate into CD8+ T cells (25). The TCR transgenic itk-/- mice that we generated to examine the role of Itk in positive selection also provided us with an ideal system to examine how lineage commitment would be affected in the absence of Itk, as these mice represent a range of TCR avidities for selecting ligands. Specifically, we anticipated that lineage switching might occur in some of the TCR transgenics, particularly those with low selection efficiencies. In such an instance, the loss of transgenic CD4+ SP cells would be compensated for by an increase in transgenic CD8+ SP cells. This expectation would be consistent with the increased percentage of CD8+ SP cells seen in non-TCR transgenic itk-/- mice.
As shown in Figs. 1 and 2 there were a small number of
CD8+ SP cells in the thymus and LN of all TCR
transgenic mice. The percentage and absolute number of these cells that
express high levels of the transgenic TCR are shown in Table I. In
virtually all cases there was no significant increase in the absolute
number of transgenic TCRhigh
CD8+ SPs in the absence of Itk as determined by
Student’s t test (p < 0.05). The
only exception was in the LN of ANDb/b mice, in
which the number of CD8+ cells was slightly
increased. Therefore, the data from the TCR transgenic mice suggest
that although the selection of CD4+ T cells is
reduced, lineage commitment occurs normally.
If this is indeed the case, the issue of why non-TCR transgenic
itk-/- mice have such a large number of
CD8+ SP cells in the thymus and an altered
CD4:CD8 ratio in the periphery remained unresolved. To address this
question we analyzed the expression of multiple thymocyte and T cell
markers on the CD8+ SP cells in
itk-/- mice (Fig. 6A). The phenotype that we
observe is that the majority of the CD8+ cells in
the thymus of itk-/- mice appeared to be
mature CD8+ T cells rather than newly developed
CD8+ SP cells. For instance, the vast majority of
CD8+ SP thymocytes in
itk-/- mice are
HSAlow/-, and thus resemble peripheral T cells
rather than maturing thymocytes (Fig. 6A). Likewise, the
analysis of Bcl-2 (Figs. 3A and 6) and CD5 (Figs. 5A and 6A) expression on the
CD8+ SPs of itk-/-
mice demonstrates that the majority of these cells have high levels of
both of these proteins, similar to the levels found on the most mature
thymocytes or peripheral T cells.
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A, the majority of
CD8+ SP thymocytes in
itk-/- mice are
CD44high, while in wild-type mice the majority of
these cells are CD44low. Based on these results
it appears that the CD8+ SP thymocytes in
itk-/- mice are not activated, but may
have been previously. These data leave open the possibility that
activated CD8+ cells recirculate from the
periphery to the thymus in itk-/- mice,
but in a steady-state analysis show up largely as previously activated
cells.
Alternatively, it is possible that the CD8+ SP
cells acquire this phenotype in the thymus due to irregular
development. To address this, we analyzed CD8+ SP
thymocytes from neonatal Itk-deficient mice to determine whether these
cells have a mature phenotype before CD8+ T cells
have populated the periphery. The CD4 vs CD8 profiles from neonatal
itk+/- and
itk-/- mice show that there are
significantly fewer CD4+ SPs in the Itk-deficient
mice (Fig. 6B). However, in contrast to adolescent mice,
there does not appear to be an increase in CD8+
SPs, a phenotype that is more obvious when examining the profiles of
mature TCRhigh cells. In addition analysis of
CD44 and HSA expression on the CD8+ SPs indicate
that the first few CD8+ SPs that are developing
in itk-/- mice have a normal phenotype,
as shown by comparison with itk+/-
littermates. Thus, if the CD8+ SPs in
Itk-deficient mice acquire the phenotype shown in Fig. 6A
while still in the thymus, it must occur postnatally during later waves
of CD8+ T cell maturation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 activation leading to sustained
Ca2+ mobilization and Erk/MAPK activation in
response to TCR stimulation (15, 19). Although these
pathways are reduced by the absence of Itk, signaling is not completely
abolished, possibly due to weak compensation by other Tec family
kinases, such as Rlk/Txk (19). Thus,
itk-/- mice provide an ideal opportunity
to determine how decreased activation of these signaling pathways
affects T cell development. For our studies we crossed itk-/- mice to three lines of MHC class II-specific TCR transgenics on three different MHC backgrounds. A previous study has addressed the role of Tec kinases in setting the signaling threshold between positive and negative selection using a single MHC class I TCR transgenic system. Here we address the effect of decreased TCR signaling on positive selection over a range of TCR avidities as well as on CD4 vs CD8 lineage commitment. Our results, based on analyses of thymocytes, show that the efficiency of positive selection is reduced in all cases in the absence of Itk. Interestingly, we did not observe some TCR/MHC combinations in which positive selection was completely abolished and others that were unaffected by the absence of Itk.
These studies extend previous reports of thymic selection in Itk-deficient mice by substantiating the conclusion that reduced CD4+ T cell numbers result from defective positive selection, as opposed to reduced CD4+ cell survival. First, we demonstrated that the survival factor, Bcl-2, is up-regulated to an equivalent degree in DP thymocytes, SP thymocytes, and peripheral T cells from itk-/- mice compared with control mice. In addition, the expression of other surface markers that define the final stage of thymocyte maturation, such as HSA, CD69, CD44, and CD62 ligand, is similar on cells generated in wild-type vs itk-/- TCR transgenic mice. In the course of this analysis we also observed that wild-type AND TCR transgenic mice expressing the highly selecting ligand, IEk, had fewer thymocytes with high levels of CD69. Thus, in an environment where thymocytes are receiving strong selection signals, there are fewer cells in the CD69high transition stage, and there is an increase in the more mature CD69- CD4+ SP subset. In the Itk-deficient AND mice there is a decrease in the overall number of TCRhigh thymocytes; however, among those cells, there is a dramatic increase in the proportion of CD69high transition cells. This suggests that thymocytes receiving weaker signals during positive selection may require longer to complete the selection process. Together, these findings support the conclusion that in the absence of Itk, fewer thymocytes undergo positive selection, and those that do take longer to become fully mature CD4+ SP cells.
Overall, these data reinforce the view of positive selection as an inherently stochastic process, where only a fraction of thymocytes with appropriate TCRs receive sufficient signals to undergo selection before they succumb to programmed cell death (death by neglect). Positive selection is thought to take 1.5–2 days of continuous low level signaling through the TCR (35), and it has been proposed that there are a limiting number of niches that are present in the thymus that will support selection (36). This concept has been used to explain why, in TCR transgenic mice in which all developing thymocytes express an appropriate TCR, the majority of cells do not actually get selected. Thus, one explanation for the less efficient development of itk-/- thymocytes is that these cells take longer to receive sufficient signals, and due to the limiting niches, fewer cells are successfully selected before undergoing cell death. This probabilistic model of positive selection is also consistent with the outcomes we observed in itk-/- mice with a range of avidities of TCR-MHC interactions (e.g., ANDb/b vs ANDk/b or ANDk/k), where the higher the TCR avidity, the higher the probability that even cells lacking Itk will achieve sufficient signals to promote maturation and survival.
Signals downstream of TCR complexes on thymocytes are also important
for developmental processes in addition to positive selection. At the
pro- to pre-T cell transition, signals downstream of the pre-TCR are
required for proliferation, survival, differentiation, and allelic
exclusion. Recent evidence suggests an important role for PLC- in
pre-TCR signaling (37). This observation suggests a role
for Itk in this signaling pathway, as Itk is important for full
activation of PLC-1 in peripheral T cells (15). In
fact, we did observe an increase in the percentage of
CD4- CD8- thymocytes in a
number of the TCR-transgenic itk-/- mice
(Figs. 1A and 2A). However, initial
characterization of the CD4-
CD8- CD3- (TN) thymocyte
populations in non-TCR transgenic Itk-deficient mice indicates that
there is no overt defect at this stage of development, as determined by
CD25 and CD44 analysis (J. Lucas and L. Berg, unpublished
observations). Therefore, we believe the increase in TN thymocytes is
more reflective of a loss of DP and/or CD4+ SPs
in these mice. Consistent with this is the fact that a significant
increase in the percentage of TN cells is only observed in
itk-/- TCR transgenic mice that have an
overall reduction in thymic cellularity.
This study also demonstrated that the majority of CD8+ thymocytes in itk-/- mice have a phenotype that is not consistent with newly generated CD8+ SPs, but instead resembles peripheral T cells with a memory phenotype (CD44high). Corroborating this observation, we found that the majority of CD8+ T cells in the periphery of itk-/- mice are also CD44high (data not shown). One interpretation of these data is that the CD44high CD8+ SP thymocytes in itk-/- mice are previously activated peripheral cells that have reentered the thymus. An alternative possibility is that these cells could be the products of homeostatic expansion, although this seems unlikely, as itk-/- mice are not lymphopenic. A third possibility is that the CD8+ SP thymocytes acquire this mature phenotype in the thymus and accumulate there before being exported to the periphery. We have begun to address this by looking at the phenotype of the CD8+ SPs in neonatal mice. The examination of the very earliest CD8+ SPs suggest that they do not acquire the mature phenotype early in development. A more detailed kinetic analysis of CD8+ SP maturation and thymocyte emigration is ongoing and should help shed light on the nature of these cells. However, at this point we believe that our initial hypothesis that CD4 development is more affected by the absence of Itk may need revision, as the defects in CD8+ T cell development may have been masked by expansion (in the thymus and/or the periphery) of the few cells that are selected.
The most unexpected result that we observed is the lack of effect on CD4/CD8 lineage commitment in Itk-deficient mice. The initial analyses of non-TCR transgenic itk-/- mice revealed that T cells of both lineages develop, but that the normal ratio of CD4+ to CD8+ cells is altered in favor of increased CD8+ cells. An attractive hypothesis to explain this observation was that stronger selection signals induce CD4+ differentiation, and that in the absence of Itk, class II-specific thymocytes would develop into CD8+ T cells due to weaker selection signals. This potential explanation for the phenotype of itk-/- mice was strengthened by recent studies identifying signaling molecules that appear to influence CD4/CD8 lineage decisions in the thymus. For instance, the level of Lck activity in developing thymocytes has been shown to directly affect the outcome of lineage commitment (4). As Itk is directly downstream of Lck, alterations in Lck activity should be reflected in concomitant changes in Itk activity. More importantly, the activity of the Erk/MAPK signaling pathway has also been shown to influence CD4/CD8 lineage commitment and as stated above, itk-/- T cells have reduced phosphorylation of Erk1/2 following CD3 cross-linking (19). Therefore, because molecules that are thought to be both upstream and downstream of Itk signaling are implicated in CD4/CD8 lineage commitment, it seemed likely that Itk would also be involved in this process.
Nonetheless, our data argue that Itk is not involved in CD4/CD8 lineage commitment. In non-TCR transgenic itk-/- mice the bulk of CD8+ SP thymocytes present do not represent newly differentiating cells. Furthermore, in all five different TCR transgenic/MHC backgrounds that we examined we never observed a substantial increase in the number of CD8+ cells developing in the absence of Itk. It is possible that we did not see a difference because the alteration in the strength of the TCR signal in these cases was either too large or too small without Itk, such that development of the transgenic T cells was nearly abolished (too large) or was reduced, but not enough to influence lineage commitment (too small). However, with the number of combinations examined, we do not think that this explanation is likely. Rather, we believe that the activity of Itk is not required for the signal that induces commitment to either specific lineage.
These observations support the interesting possibility that unique signals downstream of the coreceptor are responsible for lineage commitment and that Itk is not involved in this signaling pathway. Consistent with this idea, previous studies have shown that altering the coreceptor, Lck activity, or Erk/MAPK signaling can have an effect on lineage commitment, but that simply reducing TCR signaling does not (25). An alternative view of lineage commitment has proposed that CD4/CD8 lineage decisions are influenced by the kinetics/duration of TCR/coreceptor signaling (38, 39), rather than the strength, with CD4 lineage commitment requiring sustained TCR signals. Thus, it is possible that we did not observe lineage switching in the class II-restricted TCR transgenic itk-/- mice because these thymocytes may have received sustained signals for a sufficient period of time to ensure proper differentiation. Finally, it will be interesting to determine how development of the CD8 lineage is altered in thymocytes that lack Itk. Although we did not observe lineage switching, the CD8+ SPs in itk-/- mice have an unusual phenotype, which may suggest some alteration in the maturation of these cells. Therefore, we are currently crossing itk-/- mice to class I-restricted TCR transgenic mice to further examine this issue.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Leslie J. Berg, Department of Pathology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655. E-mail address: leslie.berg{at}umassmed.edu
3 Abbreviations used in this paper: DP, CD4+CD8+ thymocyte; Cy, CyChrome; Erk, extracellular signal-regulated kinase; HSA, heat-stable Ag; LN, lymph node; MAPK, mitogen-activated protein kinase; PLC, phospholipase C; SAg, superantigen; SP, CD4+CD8- or CD4-CD8+ thymocyte; TN, CD4-CD8-CD3-.
Received for publication November 21, 2001. Accepted for publication April 10, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T-cell receptor controls positive selection by modulating ERK activity. Nature 406:422.[Medline]
transgenes on early thymocyte development. Eur. J. Immunol. 23:1699.[Medline]
B and NFAT. Nat. Immunol. 2:403.[Medline]
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