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, But Not by Surface Pre-TCR Complexes, Are Able to Induce Maturation of an Early Thymic Lymphoma In Vitro1
Fox Chase Cancer Center, Immunobiology Working Group, Division of Basic Sciences, Philadelphia, PA 19111
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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molecules (a signaling
chimera that mimics pre-TCR signaling in vivo) by undergoing changes in
gene expression observed following pre-TCR activation in normal
thymocytes. These changes include down-regulation of CD25,
recombinase-activating gene (RAG)-1, RAG-2, and pT
; and the
up-regulation of TCR germline transcripts. We term this complete set
of changes in gene expression, in vitro maturation. Interestingly,
Scid.adh undergoes only a subset of these changes in gene expression
following Ab engagement of the pre-TCR. Our findings make two important
points. First, because TAC:CD3 stimulation of Scid.adh induces
physiologically relevant changes in gene expression, Scid.adh is an
excellent cellular system for investigating the molecular requirements
for pre-TCR signaling. Second, Ab engagement of CD3 signaling
domains in isolation (TAC:CD3) promotes in vitro maturation of
Scid.adh, whereas engagement of CD3 molecules contained within the
complete pre-TCR fails to do so. Our current working hypothesis is that
CD3 fails to promote in vitro maturation when in the context of an
Ab-engaged pre-TCR because another pre-TCR subunit(s), possibly TCR
,
qualitatively alters the CD3 signal. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) of the TCR. These rearrangement events occur as
CD4-CD8- double-negative
(DN)3 thymocytes
develop through the CD8low immature
single-positive (ISP) stage to the
CD4+CD8+ double-positive
(DP) stage. The transition from the DN to DP stage is governed by a
checkpoint termed ß selection, which stipulates that only those
precursors that have maintained the translational reading frame of the
rearranged TCRß gene are allowed to progress to the DP stage
(3). ß selection is exerted on a DN thymocyte
subpopulation that is
HSAhighCD44lowCD25+.
Upon expression of the TCRß protein product, these cells undergo
proliferative expansion, down-regulate CD25, progress to the ISP stage
by up-regulating CD8, and finally develop into DP thymocytes. Those
precursors failing to productively rearrange the TCRß locus die by
apoptosis (3, 4).
The precise mechanism whereby maturation of thymocytes to the DP stage
is linked to productive rearrangement of the TCRß locus is unclear,
but appears to involve signaling through a surrogate form of the TCR
termed the pre-TCR complex (5, 6). The pre-TCR complex
comprises CD3
, 
, , and signaling subunits in association
with a heterodimer of TCRß disulfide linked to an invariant 33-kDa
subunit termed pre-T (pT) (7, 8). The ability of the
pre-TCR to promote development of DN thymocytes to the DP stage is
attenuated in mice lacking any of the receptor subunits (except CD3)
and in mice with mutations preventing rearrangement of the TCRß gene
locus (e.g., recombinase-activating gene (RAG) deficiency or the
scid mutation in DNA-dependent protein kinase) (6, 8, 9, 10, 11, 12, 13). The developmental arrest of RAG-deficient thymocytes can
be overcome (i.e, development to the DP stage is restored) by Ab
engagment of: 1) chimeric transgenes consisting of the (human IL-2R
(TAC)) exodomain fused to either the CD3 or TCR cytosolic
signaling domains (TAC:CD3 and TAC:TCR) (14); or 2)
partial CD3 complexes termed clonotype-independent CD3 (CIC) complexes
that are expressed on the surface of DN thymocytes
(15, 16, 17). Thus, Ab stimulation of CIC as well as the
TAC:CD3 and TAC:TCR chimeras can mimic pre-TCR function.
Most cell surface receptor complexes are activated by ligand
engagement, for which Ab stimulation frequently serves as an effective
surrogate. However, this does not appear to be true for the pre-TCR. Ab
engagement of surface pre-TCR complexes in vivo fails to promote
maturation of thymocytes to the DP stage, instead arresting their
development at the CD8 ISP stage (18, 19, 20). Moreover,
removal of the potential ligand-binding exodomains of TCRß and pT
does not abrogate the ability of the pre-TCR to support development of
thymocytes to the DP stage (21, 22). Taken together, these
data suggest that ligand engagement of surface pre-TCR complexes may
not be responsible for pre-TCR activation in vivo. Ligand-independent
models of pre-TCR triggering have also been proposed, and these can be
subdivided into two categories. The first proposes that assembly of
TCRß with the remaining pre-TCR subunits and subsequent deposition of
the complete complex on the cell surface are sufficient to trigger
pre-TCR signaling, even in the absence of ligand engagement
(22). The second proposes that surface expression of
pre-TCR complexes may not be necessary and that pre-TCR signaling is
instead triggered internally, while the pre-TCR is still en route to
the cell surface (23, 24). These models are currently
unresolved.
A more precise understanding of how pre-TCR signals are triggered and
initiate thymocyte differentiation has been hindered by the lack of an
in vitro system amenable to both biochemical and genetic analysis. Such
in vitro systems have been indispensable tools in the study of T cell
activation (25), T cell development from the DP to
single-positive stage (26, 27, 28), and B cell development
(29, 30, 31, 32). In this study, we establish the Scid.adh thymic
lymphoma as an in vitro model system with which to investigate proximal
pre-TCR signal transduction and associated changes in gene expression.
Scid.adh is phenotypically similar to an immature thymocyte just before
ß selection
(HSAhighCD44lowCD25+)
and exhibits the ability to differentiate in vitro. In response to
stimulation with the TAC:CD3 chimera, which promotes maturation of
normal DN thymocytes to the DP stage in vivo (14),
Scid.adh down-regulates mRNAs encoding pT, RAG-1, and RAG-2
(33, 34, 35), and down-regulates surface expression of CD25,
one of the earliest phenotypic indicators of pre-TCR signaling in vivo
(36, 37). In addition, TAC:CD3 stimulation of Scid.adh
induces expression of germline transcripts from the TCR locus
associated with the maturation of DN thymocytes to the DP stage
(34, 38). We term this collective set of changes in gene
expression, "in vitro maturation". Importantly, Ab engagement of
pre-TCR complexes expressed on the surface of Scid.adh induces only a
subset of the changes in gene expression defined as in vitro
maturation, despite the fact that pre-TCR complexes contain CD3
subunits. The inability of Ab engagement of CD3 to fully induce in
vitro maturation despite the presence of CD3 within the pre-TCR
complex suggests that another pre-TCR subunit(s) may alter the CD3
signal and prevent it from coupling to the requisite downstream
signaling cascades. Moreover, the failure of Ab engagement of surface
pre-TCR complexes to fully induce in vitro maturation of Scid.adh is
not consistent with ligand-dependent models of pre-TCR activation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Scid.adh, SL-12, and SL343 are all spontaneous thymic lymphomas
isolated from mice bearing the scid mutation (scid mice) and
adapted to growth in culture. SL-12 (TCR-)
(39) and SL-343 (TCR-)
(40) were obtained from Dr. M. Bosma (Fox Chase Cancer
Center, Philadelphia, PA). SL-12ß.12 was generated by electroporation
of pXS-2B4ß into SL-12, as previously described (7). The
DP /ßTCR+ thymic lymphoma VL3-3 M2 was
obtained from Dr. C. Guidos (Hospital for Sick Children, Toronto,
Canada) (41). The retroviral producer line 
2 (NIH 3T3)
was obtained from Dr. P. Tsichlis (Thomas Jefferson University,
Philadelphia, PA) (42). The anti-TAC mAb-producing
hybridoma hd245/332 was obtained from the American Type Culture
Collection (Manassas, VA) with the permission of Dr. T. Waldman
(National Institute of Child Health and Human Development (NICHD),
National Cancer Institute, Bethesda, MD) (43). All lines
were maintained in RPMI supplemented, as previously described
(44). The following mAbs were used to stimulate cells in
culture: anti-TCRß (H57-597) (45); anti-TAC
(hd245/332) (43). The following fluorochrome-conjugated
mAbs (PharMingen, San Diego, CA) were used in flow cytometry:
anti-CD5-PE (53-7.3), anti-CD25-FITC (7D4),
anti-TAC-FITC (M-A251), anti-TCRß-FITC (H57-597), and
anti-human CD3-FITC (Leu4) (UCHT1). The following Abs were used in
immunoprecipitations: anti-TCRß (H57-597), anti-CD3/
(7D6), anti-CD3 (R9), and anti-TCR (551). Anti-pT Ab
used in the recapture assay has been previously described
(7).
Cell stimulation and flow cytometry
All stimulations were conducted on plate-bound Ab at 37°C. Cells were harvested at early- to mid-log phase, counted, and plated in 2 ml of RPMI (2–3 x 105/well) in 24-well tissue culture plates that had been precoated overnight at 37°C with either anti-TCRß or anti-TAC at 5 µg/ml in PBS. After the indicated time of stimulation, cells were removed from the wells by repeated pipetting. For each sample, 106 cells were washed in staining buffer (0.1% BSA/0.1% sodium azide/HBSS), stained for 30 min at 4°C with the indicated fluorochrome-conjugated mAb, washed twice in staining buffer, and analyzed by flow cytometry using a FACScan cytometer (Becton Dickinson, San Jose, CA) and CELLQUEST software (Becton Dickinson). Dead cells were excluded using the vital dye propidium iodide. For RT-PCR analysis, stimulated and unstimulated cells were prepared for flow cytometry as above and isolated using a dual laser/dye laser flow cytometer (FACStarPlus; Becton Dickinson).
Retroviral gene transfer
A 2 retroviral packaging cell line expressing the MFG-2B4 ß
retroviral vector was obtained from Dr. L. Spain (Wistar Institute,
Philadelphia, PA) (46). The murine stem cell virus
(MSCVneo) expression vector was obtained from Dr.
R. Hawley (University of Toronto, Toronto, Canada). pXS-TAC and
pXS-TAC:CD3 were obtained from Dr. J. Bonifacino (NICHD,
National Institute of Health, Bethesda, MD). The TAC:CD3 chimera
contains the TAC exo- and transmembrane domains linked to the
cytoplasmic tail of CD3, thus eliminating the motifs in CD3 that
promote assembly with other TCR components (47). The TAC
and TAC:CD3 inserts were shuttled from pXS through the PCR cloning
vector pCR2.1 (Invitrogen, SanDiego, CA) into
MSCVneo by PCR using standard methodology
(48). Briefly, the coding regions of TAC and TAC:CD3
were amplified using oligonucleotide primers that flank the
translational start and stop sites, and that were appended with linkers
encoding the HpaI (5') and XhoI (3') restriction
sites. The amplified inserts were verified by sequencing and
directionally subcloned into MSCVneo using
standard methodology. MSCVneo,
MSCVneo-TAC, and
MSCVneo-TAC:CD3 were separately transfected
into the 2 retroviral packaging line using lipofectamine, according
to manufacturer’s specifications (Life Technologies, Grand Island,
NY). Stable viral-producing cells were selected in 500 µg/ml G418.
The specified thymic lymphoma lines were retrovirally infected by
coculture. Briefly, in a single well of a six-well culture dish, 3
x 105 lymphoma cells and
106 2 retroviral producer cells were seeded in
3 ml RPMI containing 4 µg/ml polybrene, and incubated for 48
h at 37°C. Infected thymic lymphomas were removed from the
producer monolayer and selected in 500 µg/ml G418. Infected thymic
lymphoma cells expressing homogenously high levels of TCRß, TAC, or
TAC:CD3 were isolated by flow cytometry as above.
Semiquantitative RT-PCR and Southern blot analysis
For each treatment condition, a total of 5 x
106 thymic lymphoma cells was sorted into
microfuge tubes by flow cytometry. Total RNA was isolated into a final
volume of 50 µl of RNase-free H2O using the
RNeasy RNA purification system (Qiagen, Valencia, CA). All RNA samples
were treated with DNase I (Life Technologies) before performing first
strand cDNA synthesis (1 µg RNA/reaction) using random primers and
the Superscript preamplification system (Life Technologies). Titrated
amounts of cDNA were amplified by PCR (19 cycles) using primers
specific for: ß-actin, RAG-1, RAG-2, pT, and TCR-C (see Table I
). Forty percent of each PCR reaction
was resolved on a 1.5% agarose electrophoresis gel and blotted onto
Nytran membranes (Scheicher & Schuell, Keene, NH), which were rinsed in
phosphate buffer (111 mM
Na2HPO4, 87 mM
NaH2PO4-H20,
pH 6.8) and allowed to air dry overnight. Prehybridization of membranes
was conducted for 4 h at 67°C in pre-hyb solution (5x SSC, 5x
Denhardt solution, 0.5% SDS, 1 mM EDTA, 0.1 mg/ml salmon sperm DNA).
cDNA probes were labeled with [32P]dCTP using
the Prime-It II random primer labeling kit (50 ng probe/reaction;
Stratagene, La Jolla, CA) and purified on NucTrap probe purification
columns (Stratagene). Labeled probe was added directly to membranes in
pre-hyb solution (106cpm/ml), and probe
hybridization was allowed to proceed overnight at 67°C. Blots were
washed sequentially in 2x and 1x SSC/0.5% SDS before quantification
using a Fuji phospor imager and Fuji MacBas V2.2 software (Fuji Photo
Film, Tokyo, Japan).
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Scid.adh cells were biotin labeled, as described, after which
cell viability was consistently >98% (49). Cell lysis,
immunoprecipitation, recapture assays, electrophoresis, and
immunoblotting were as described previously (7). Briefly,
digitonin extracts of biotin-labeled Scid.adh cells were exhaustively
cleared of biotin-labeled proteins by sequential passage over
streptavidin-agarose (Pierce, Rockford, IL). The SDS-eluted proteins
were resolved by SDS-PAGE, blotted onto Immobilon polyvinylidene
difluoride membranes (NEN Lifescience Products, Boston, MA), and probed
with anti-CD3 Ab (HMT3-1). Bound Ab was visualized with
HRP-conjugated protein A (HRP-protein A; Kirkegard and Perry,
Gaithersburg, MD), followed by chemiluminescence (Renaissance, NEN
Lifescience Products).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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signals induce maturation of the Scid.adh thymic lymphoma in
vitro
Ab stimulation of a transgenic TAC:CD3 chimera elicits signals
capable of substituting for the pre-TCR complex, as evidenced by the
ability of such signals to promote development of pre-TCR-deficient DN
thymocytes to the DP stage (14). In contrast, Ab
triggering of the pre-TCR complex itself arrests development at the CD8
ISP stage (18, 19, 20). As a first step toward understanding
why Ab engagement of the pre-TCR fails to promote development to the DP
stage, we sought to establish an in vitro system in which to study
pre-TCR function. A series of scid thymic lymphomas was evaluated to
identify cell lines that exhibit changes in gene expression in response
to TAC:CD3 stimulation similar to those observed following pre-TCR
signaling (triggered by productive rearrangement of TCRß) in normal
thymocytes in vivo. We reasoned that studying proximal signaling events
in a cell line in which physiologically relevant gene expression
changes are induced is critical, because those changes in gene
expression attest to the relevance of the proximal signals. Scid thymic
lymphomas were chosen because scid thymocytes are unable to mediate
V(D)J recombination, and so their development is arrested just before
ß selection at the
HSA+CD44-CD25+
stage (50). Consequently, thymic lymphomas from
scid mice, being phenotypically similar to their
nontransformed counterparts, might retain the ability to respond to
pre-TCR signals in a physiologically relevant manner. We identified
such a cell line, Scid.adh. The Scid.adh lymphoma, although
CD4-CD8+, resembles most
closely the phenotype of a DN thymocyte in that it is
HSAhighCD44lowCD25+
(Fig. 1A), expresses message
for pT, and does not express a functional TCRß gene, as confirmed
by both biochemical and genetic analysis (data not shown). After
24 h of stimulation with plate-bound anti-TAC mAb, surface
expression of the activation Ag CD5 was markedly increased on
TAC:CD3-expressing Scid.adh cells (Fig. 1B), as was
expression of other differentiation Ags including CD27 and the
costimulatory molecule CD28; however, surface expression of the CD4 and
CD8 coreceptor molecules was not altered (Fig. 1B). More
importantly, TAC:CD3 stimulation sharply down-regulated the surface
expression of CD25 (Fig. 1B), which is a hallmark of pre-TCR
activation in vivo (8, 51). It should be noted that CD25
down-regulation was only observed after stimulation of Scid.adh
lymphoma cells, whereas TAC:CD3 stimulation failed to do so in three
other lymphomas tested: 1) SL12-ß12, DN,
pre-TCR+; 2) SL-343, DP,
pre-TCR-; and 3) VL3-3 M2, DP,
ßTCR+ (data not shown).
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stimulation of RAG-deficient DN
thymocytes) greatly reduces the abundance of pT and RAG mRNA
(33, 34, 35), and up-regulates sterile TCR transcripts,
which precedes the initiation of gene rearrangement at the TCR locus
(34, 35, 38). To determine whether TAC:CD3 stimulation
alters gene expression in Scid.adh in a similar way,
TAC:CD3-expressing Scid.adh cells were cultured on plate-bound
anti-TAC mAb for 24 h, following which those that had fully
(CD25low, Fig. 2A) or partially
(CD25int; Fig. 2A) down-modulated CD25
were isolated using flow cytometry. The effect of TAC:CD3
stimulation on expression of pT, RAG-1, RAG-2, and TCR-C mRNA in
these populations was assessed using RT-PCR (Fig. 2B and
Table II). In TAC:CD3-stimulated
CD25low Scid.adh cells, mRNA encoding full-length
pTa was decreased 30-fold, while that of RAG-1
and RAG-2 were each decreased 
20-fold. Moreover, the
CD25low cells exhibited a 30-fold increase in
germline TCR-C transcripts as well as a 40-fold increase in the ISP
stage-specific transcript J49 (data not shown) (38).
Curiously, while the up-regulation of TCR-C mRNA in
CD25int Scid.adh cells was comparable with that
in CD25low cells, the down-regulation of
pTa and RAG-1/2 mRNA in the
CD25int cells was modest (Table II). Thus, the
extent to which RAG and pT mRNA levels were down-regulated
correlates tightly with the extent to which CD25 surface expression was
reduced, but not with the extent of TCR-C induction. Taken together,
the changes in surface phenotype and in molecular markers induced
following TAC:CD3 stimulation of Scid.adh are consistent with the
way a normal thymocyte responds to ß selection.
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While Ab engagement of surface TAC:CD3 molecules is able to
mimic pre-TCR function in vivo and promote development of DN thymocytes
to the DP stage (14), engagement of surface pre-TCR
complexes using anti-TCRß mAb fails to do so, instead arresting
thymocytes at the CD8 ISP stage (18, 19, 20). Consequently, we
asked whether anti-TCRß stimulation of surface pre-TCR complexes
on Scid.adh would induce in vitro maturation. Because of the
scid defect in V(D)J recombination, Scid.adh is unable to
rearrange its endogenous TCRß locus (39). To facilitate
expression of surface pre-TCR complexes, Scid.adh cells were
retrovirally transduced with a cDNA encoding the 2B4 TCRß subunit
(Scid.adh-TCRß; Figs. 3 and
4). The TCRß cDNA induced Scid.adh to
express TCRß on the cell surface, as measured by flow cytometry (Fig. 4). Moreover, biochemical analysis of surface biotin-labeled
Scid.adh-TCRß cells revealed that pT-TCRß heterodimers were
coprecipitated with Ab to CD3, CD3, and TCR, indicating
that the pre-TCR complexes expressed by these cells comprised all of
the known pre-TCR subunits (TCRß, pT, CD3 and ) (Fig. 3). Stimulation of the pre-TCR-expressing Scid.adh cells with
plate-bound anti-TCRß mAb induced a substantial increase in
expression of the activation Ag CD5 (Fig. 4), verifying that the
anti-TCRß mAb did indeed trigger the pre-TCR. However,
anti-TCRß-induced signals caused only a marginal down-modulation
of CD25 relative to that caused by TAC:CD3 stimulation (Fig. 4).
Furthermore, the inability of Ab engagement of the pre-TCR to
down-modulate CD25 was not unique to anti-TCRß mAb, as
stimulation of the pre-TCR complex with anti-CD3 mAb also failed
to down-modulate CD25 (data not shown). Because anti-TCRß
stimulation of Scid.adh-TCRß cells did not significantly alter CD25
expression, we wanted to determine how anti-TCRß stimulation
affected other genes whose expression is altered during in vitro
maturation: pT, RAG-1, RAG-2, and TCR-C (Fig. 2
B).
Semiquantitative RT-PCR analysis revealed that anti-TCRß
stimulation of Scid.adh-TCRß cells markedly increased the levels of
TCR-C transcript (70-fold) (Fig. 5
and Table II) as well as the ISP stage-specific transcript J49 by
50-fold (data not shown) (38); however, the induction of
TCR-C levels is not well correlated with CD25 down-modulation even
in TAC:CD3-stimulated cells (compare CD25low
to CD25int; Table II). In agreement with its
failure to down-modulate CD25, anti-TCRß stimulation was unable
to significantly down-modulate pT or RAG mRNA levels (Fig. 5 and
Table II). Taken together, these data demonstrate that Ab engagement of
the complete pre-TCR complex is unable to induce in Scid.adh the
complete set of changes in gene expression defined as in vitro
maturation. More specifically, these data demonstrate that Scid.adh
maturation can be induced by CD3 signals when CD3 is engaged in
isolation as a TAC:CD3 chimera, but not when CD3 is engaged in
the context of the complete pre-TCR complex. Furthermore, the inability
of pre-TCR ligation to fully promote in vitro maturation does not
appear to be specific to the Vß3 TCRß transgene used above, as Ab
engagement of Vß8-containing pre-TCR complexes expressed in Scid.adh
also fails to induce in vitro maturation (data not shown).
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complexes on the cell
surface does not disrupt TAC:CD3-driven in vitro maturation of
Scid.adh
There are three potential explanations as to why in vitro
maturation of Scid.adh can be fully induced by signals from isolated
CD3 molecules, but only partially by signals from CD3 molecules
within the pre-TCR complex. First, the number of CD3 subunits
expressed on the surface of Scid.adh as part of the pre-TCR complex may
be significantly lower than the number expressed on the surface in the
form of the TAC:CD3 chimera, resulting in a signal that falls below
the threshold required to induce in vitro maturation. Second,
expression of TCRß in Scid.adh may render the cell refractory to
induction of in vitro maturation. Third, the association of CD3 with
the other pre-TCR subunits may alter its ability to signal maturation.
We tested these possibilities by introducing a TCRß cDNA into cells
already expressing TAC:CD3. First, to assess the ratio of surface
CD3 expressed as TAC:CD3 to that expressed in a
pre-TCR-associated form, surface biotin-labeled proteins were isolated
using avidin-agarose and then blotted with anti-CD3 Ab. Because
the amount of immunoreactivity found in the TAC:CD3 form was
approximately equivalent to that of the CD3 monomer, we conclude
that the expression levels of TAC:CD3 and pre-TCR-associated CD3
are equivalent (Fig. 6). When these
dual-expressing Scid.adh cells were stimulated with anti-TCRß
mAb, they were unable to down-modulate CD25 (Fig. 7). Consequently, differences in
expression level do not explain the inability of the pre-TCR to fully
induce in vitro maturation. Second, to assess whether expression of
TCRß renders Scid.adh unable to undergo in vitro maturation, we
stimulated the dual-expressing Scid.adh line with anti-TAC mAb.
Similar to cells expressing only the TAC:CD3 chimera, TAC:CD3
stimulation induced in vitro maturation of Scid.adh as defined by
down-modulation of CD25, indicating that expression of TCRß does not
render Scid.adh unable to undergo in vitro maturation (Fig. 7). The
remaining possibility that association of CD3 with the other
subunits of the pre-TCR alters its signaling ability could be
manifested in the following two ways: 1) CD3 transduces a dominant
signal that arrests in vitro maturation; or 2) CD3 transduces a
limited signal that neither promotes development nor interferes with
induction of development by another stimulus. To distinguish between
these possibilities, we simultaneously stimulated Scid.adh via
TAC:CD3 and the pre-TCR. In response to costimulation with both
anti-TAC and anti-TCRß Ab, Scid.adh down-modulated CD25
expression, indicating that coengagement of the pre-TCR was not able to
disrupt in vitro maturation of Scid.adh in response to TAC:CD3
stimulation (Fig. 7). This suggests that the failure of CD3
molecules to induce in vitro maturation within the context of the
pre-TCR is not due to transduction of a dominant-negative signal.
Rather, it appears that Ab engagement of the pre-TCR transduces a
limited signal that is incompetent to induce full in vitro maturation.
Taken together, these data serve to underscore three important points.
First, of the thymic lymphomas examined, Scid.adh was the only one
whose responses to TAC:CD3 stimulation paralleled those of normal
thymocytes following ß selection in vivo. Indeed, TAC:CD3
stimulation, which mimics pre-TCR function in vivo by driving
development of DN thymocytes to the DP stage (14), induces
down-modulation of CD25, pT, RAG-1, and RAG-2 in Scid.adh in vitro.
Second, while engagement of isolated CD3 molecules (i.e., the
TAC:CD3 chimera) is capable of inducing the complete set of changes
in gene expression associated with in vitro maturation of Scid.adh,
engagement of surface pre-TCR complexes is not, despite the fact that
the pre-TCR also contains CD3. This suggests that another pre-TCR
component(s) may alter the ability of CD3 to promote maturation when
it is engaged while in the context of the complete pre-TCR complex.
Third, coligation of the intact pre-TCR with TAC:CD3 does not
disrupt the ability of TAC:CD3 to induce in vitro maturation,
suggesting that Ab engagement of the pre-TCR does not induce a
dominant-negative signal.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, which is able to mimic pre-TCR function in vivo
(14), is able to drive in vitro maturation of Scid.adh, as
defined by the following effects: 1) down-regulation of CD25 surface
expression; 2) down-regulation of mRNA encoding pT, RAG-1, and
RAG-2; and 3) up-regulation of TCR germline transcripts. All of the
effects listed above have been shown to occur following pre-TCR
activation (or a stimulus mimicking pre-TCR activation) in normal
thymocytes in vivo (1, 33, 34, 35, 38, 51). Interestingly,
however, Ab engagement of pre-TCR complexes expressed on the surface of
Scid.adh fails to induce the complete set of gene expression changes
associated with in vitro maturation. It is particularly curious that Ab
engagement of surface pre-TCR complexes fails to fully drive in vitro
maturation of Scid.adh because pre-TCR complexes contain the CD3
subunit that, when engaged in the form of an isolated TAC:CD3
signaling chimera, is quite capable of inducing Scid.adh
maturation.
Models attempting to explain how pre-TCR signaling is initiated can be
divided into two categories: ligand dependent and ligand independent.
Currently, there are several pieces of evidence that are inconsistent
with a ligand-dependent model of pre-TCR triggering. First, analysis of
the efficiency with which TCRß transgenic DN precursors differentiate
to the DP stage suggests that this transition is not constrained by a
limiting number of intrathymic "niches" or extracellular ligands,
as is true of the Ag-driven selection events that promote maturation of
DP thymocytes to the CD4+ or
CD8+ single-positive stage (52).
Second, the absence of peptide-bearing major histocompatibility Ags
(the ligand of the TCRß complex) does not disrupt pre-TCR function
(53). Third, the ability of the pre-TCR to promote
maturation of DN thymocytes to the DP stage does not require the
potential ligand-binding exodomains of either pT or TCRß
(21, 22, 54). Finally, as mentioned above, Ab engagement
of surface pre-TCR complexes not only blocks development at the CD8 ISP
stage (18, 19, 20), but also fails to induce in vitro
maturation of Scid.adh. Taken together, these observations are most
consistent with ligand-independent triggering of pre-TCR signals.
Ligand-independent pre-TCR activation might be triggered by pre-TCR
complexes at the cell surface or alternatively, during assembly or
intracellular transport. At present, there is no clear evidence to
distinguish these possibilities.
Although Ab engagement of surface pre-TCR complexes fails to fully
induce in vitro maturation of Scid.adh or complete maturation of normal
thymocytes to the DP stage, there is an important difference in these
responses. In vitro, Ab engagement of complete pre-TCR complexes on
Scid.adh fails to down-regulate CD25 or RAG expression, whereas these
molecules are down-regulated following anti-TCRß stimulation of
normal thymocytes in vivo (19, 20). One possible
explanation for this discrepancy is that Ab-induced pre-TCR signals in
vivo may be augmented by costimulatory interactions between the
thymocyte and the thymic stroma, which might mask the inadequacy of the
pre-TCR signals. Indeed, we have recently found that Ab engagement of
the pre-TCR complex on Scid.adh more effectively down-modulates CD25
expression when coengaged with CD2 (data not shown). Importantly, CD2
is expressed by thymocytes at the stage of ß selection (55, 56) and has been shown to act synergistically with ßTCR
signals to promote later stages of thymocyte development
(57). Thus, to gain insight into the precise role that
pre-TCR signaling plays in promoting development, it becomes necessary
to separate the contribution of pre-TCR signals from that of stromal
costimulation. It is curious that Ab stimulation of surface pre-TCR
complexes (comprising known signaling subunits CD3 and TCR)
is not able to mimic pre-TCR function, because Ab ligation of isolated
CD3 subunits (i.e., TAC:CD3 or CIC) is able to do so
(14, 15, 16, 17). The mechanistic basis for this difference is
currently unclear, but is likely to be related to the content of
signaling subunits in each of the complexes. TAC:CD3 contains only
the cytoplasmic tail of CD3 and its single immunoreceptor
tyrosine-based activation motif (ITAM), whereas each pre-TCR complex
contains multiple ITAMs from four different signaling subunits, and
potentially other motifs in the cytoplasmic tail of pT
(58). Indeed, association of CD3 with the signaling
subunits in the pre-TCR may alter the resultant signal either
quantitatively or qualitatively. Regarding quantitative effects, we
have excluded the trivial explanation in Scid.adh that the level of
expression of the isolated TAC:CD3 differs from that of CD3 in
the context of the pre-TCR complex (Fig. 6). In addition, we have
determined that Ab-induced ligation of either TAC:CD3 or pre-TCR in
Scid.adh results in an equivalent level of phosphorylation of the
tyrosine kinase ZAP70 (manuscript in preparation). Moreover, the
induction of CD5 expression following ligation of the pre-TCR complex
exceeds that induced following TAC:CD3 stimulation (compare Figs. 1A and 4). Taken together, these data indicate that the
signal intensity produced by Ab engagement of the pre-TCR is at least
as great as that induced by TAC:CD3 stimulation. Yet, pre-TCR
ligation fails to fully drive in vitro maturation, irrespective of the
level at which the pre-TCR complex is expressed on the cell surface
(data not shown). Thus, a more likely explanation for the inability of
Ab-stimulated pre-TCRs to drive in vitro maturation of Scid.adh is that
the resultant signal is qualitatively distinct from that produced by
TAC:CD3.
If association of CD3 with other pre-TCR subunits qualitatively
alters the resultant signal, which subunit(s) is responsible for this
effect? We propose that TCR is the most attractive candidate for
four reasons. First, the binding of phosphorylated CD3 with ZAP70
following Ab stimulation is enhanced in TCR complexes lacking or
its cytoplasmic signaling domain (59). Second, Ab
engagement of CIC (heterodimers of CD3 and CD3 that lack
TCR) promotes development of DN thymocytes all the way to the DP
stage (15, 16, 17). Third, while Ab engagement of the pre-TCR
complex arrests thymocyte development at the CD8 ISP stage
(18, 19, 20), Ab stimulation of the pre-TCR complex expressed
on thymocytes in TCR-deficient mice appears to promote development
to the DP stage (60). Fourth, it was demonstrated recently
that productive stimulation of the ßTCR complex results in
complete phosphorylation of TCR, while stimuli producing
unresponsiveness are unable to induce complete phosphorylation of
TCR (61). These differences in phosphorylation may be a
determining factor in the nature of the resultant signal
(61). For all of these reasons, we think that the TCR
subunit is the most likely candidate to be responsible for limiting the
ability of CD3 molecules in Ab-engaged pre-TCR complexes to promote
maturation. Interestingly, it would appear that this proposed
inhibitory effect of TCR can occur only from within the context of
the pre-TCR complex, as a TAC:TCR chimera can drive thymocyte
development as efficiently as TAC:CD3 (14).
It is paradoxical that the presence of TCR in Ab-engaged pre-TCR
complexes might be inhibitory because TCR is required for the
pre-TCR to promote development of thymocytes to the DP stage (10, 62, 63). However, this apparent paradox might be explained in
the following manner. Currently, the preponderance of evidence suggests
that pre-TCR signaling is not induced by ligand engagement. Thus, it is
possible that the requirement of TCR for pre-TCR function may be
either to stabilize pre-TCR structure or alternatively to promote
transport of the pre-TCR to the cell surface, where its signaling is
triggered in response to nonspecific, low affinity interactions with
thymic stroma. Consistent with this viewpoint, truncated TCR
transgenes that lack ITAMs are able to compensate for TCR deficiency
and restore development to the DP stage (64). In contrast,
engagement of surface pre-TCR complexes by high affinity ligands (e.g.,
anti-TCRß Ab) could induce complete TCR phosphorylation and
signal amplification, possibly perceived by the developing thymocyte as
autoreactivity and resulting in developmental arrest. In the absence of
TCR, even such high affinity ligands would be unable to induce the
same extent of signal amplification. If the Ab-engaged pre-TCR
complexes are inducing inhibitory signals, at least in Scid.adh, those
inhibitory signals cannot disrupt the productive signals induced by
TAC:CD3. Experiments are in progress to assess the role of TCR
phosphorylation in pre-TCR function.
Progress in understanding how pre-TCR signaling is triggered in vivo
has been hampered by the lack of an in vitro model system in which
pre-TCR function can be studied both genetically and biochemically. Our
analysis of the Scid.adh thymic lymphoma represents the first
description of an in vitro tumor model system that responds to stimuli
that mimic pre-TCR function in vivo (i.e., TAC:CD3) by altering gene
expression in a fashion that closely parallels that of normal
thymocytes undergoing ß selection in vivo. In fact, the chief utility
of Scid.adh is that the induced changes in gene expression are
physiologically relevant, providing confidence that the proximal
signaling events are also representative of those which occur in normal
thymocytes. It should be noted that while the in vitro maturation of
Scid.adh does closely parallel the behavior of normal thymocytes,
Scid.adh differs from normal thymocytes in that Scid.adh does not
up-regulate CD4 to become DP following TAC:CD3 stimulation. Another
tumor model system that has been used to study proximal pre-TCR
signaling events is the scid thymic lymphoma SCB.29
(65), but in that study the authors did not attempt to
determine whether the proximal pre-TCR signals produced physiologically
relevant changes in gene expression. An earlier study, however, did
demonstrate that Ab engagement of pre-TCR complexes expressed on SCB.29
resulted in CD25 up-regulation, rather than the down-regulation that
results from pre-TCR signaling in normal thymocytes (66).
Consequently, the appropriateness of SCB.29 as a model system to study
pre-TCR signaling and its downstream effects on gene expression remains
to be established.
Using the Scid.adh model system, we intend to dissect the signaling
pathways that lie downstream of the pre-TCR and to investigate the way
in which these pathways are linked to the changes in gene expression
responsible for promoting thymocyte development. By comparing the
signaling events triggered by a stimulus that fully induces in vitro
maturation of Scid.adh (Ab engagement of TAC:CD3) with one that does
so only partially (Ab engagement of the complete pre-TCR), we should
gain insight into the molecular requirements for productive pre-TCR
signaling.
|
Acknowledgments |
|---|
2
-MFG-2B4ß-expressing retroviral producer cell line, Dr. R. Hawley for
the retroviral vector MSCVneo, Dr. T. Waldman for
the anti-TAC hybridoma (hd245/332), and Dr. J. Bonifacino for
TAC and TAC:CD3 cDNA. We thank Drs. K. Campbell,
D. Kappes, G. Koretzky, J. Clements,
J. Punt, L. Spain, and A. Singer for critical
review of this manuscript. |
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Michael Carleton, Fox Chase Cancer Center, Immunobiology Working Group, Division of Basic Sciences, 7701 Burholme Avenue, Philadelphia, PA 19111. E-mail address:
3 Abbreviations used in this paper: DN, double negative; CIC, clonotype-independent CD3; DP, double positive; HSA, heat stable Ag; ISP, immature single positive; ITAM, immunoreceptor tyrosine-based activation motif; MSCV, murine stem cell virus; RAG, recombinase-activating gene; (TAC), IL-2R.
Received for publication February 17, 1999. Accepted for publication June 25, 1999.
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stimulation as well as Ab engagement of the
pre-TCR complex1
