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-Deficient TCR/CD3 Complex in Immortalized Mature CD4+ and CD8+ T Lymphocytes1
Inmunología, Facultad de Medicina, Universidad Complutense, Madrid, Spain
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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deficiency
using Herpesvirus saimiri. The accessibility of the
mutant TCR/CD3 complex to different Abs was consistently lower in
immortalized CD8+ cells when compared with CD4+
cells, relative to their corresponding CD3-sufficient controls.
Several TCR/CD3-induced downstream activation events, immediate
(calcium flux), early (cytotoxicity and induction of surface CD69 or
CD40L activation markers or intracellular TNF-
) and late
(proliferation and secretion of TNF-), were normal in -deficient
cells, despite the fact that their TCR/CD3 complexes were significantly
less accessible than those of controls. In contrast, the accumulation
of intracellular IL-2 or its secretion after CD3 triggering was
severely impaired in -deficient cells. The defect was upstream of
protein kinase C activation because addition of transmembrane stimuli
(PMA plus calcium ionophore) completely restored IL-2 secretion in
-deficient cells. These results suggest that the propagation of
signals initiated at the TCR itself can result in a modified downstream
signaling cascade with distinct functional consequences when is
absent. They also provide evidence for the specific participation of
the CD3 chain in the induction of certain cytokine genes in both
CD4+ and CD8+ human mature T cells. These
immortalized mutant cells may prove to be useful in isolating cytosolic
signaling pathways emanating from the TCR/CD3
complex. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, 
, 
, and 
, have been
reported. CD3 proteins are believed to maintain TCR/CD3 expression and
to participate in the delivery of signals that drive T cell maturation
or apoptosis in the thymus and T cell activation or anergy in the
periphery (1). During early T cell development, some CD3 chains may act
alone or assist immature TCR ensembles, such as those containing
pre-TCR (2). However, their relative contribution to the signals
that are propagated through the cytoplasm and that result in distal
activation events is a matter for discussion. CD3 proteins may have
partially overlapping functions, as all CD3 components display a shared
amino acid motif called ITAM4
(immunoreceptor tyrosine-based activation motif) in their cytoplasmic
domains, which can by itself transduce several T cell differentiation
and activation signals (for a review, see 3 . Alternatively, they
may have specialized functions, as ITAMs belonging to different CD3
chains show different affinities for downstream signaling molecules
(for a review, see 4 . Isolated CD3 or - ITAMs cannot induce
mature T cell proliferation (5) and ablation of CD3 blocks ß,
but not T cell development (6).
We have attempted to address this question for the CD3 chain by
studying the functional behavior of human mature T cells derived from a
natural selective CD3 deficiency (7). To circumvent the inherent
difficulties of growing primary T cells and our inability to obtain
CD8+ T cell lines (8), we have used Herpesvirus
saimiri (HVS), a common lymphotropic virus of squirrel monkeys,
known to immortalize both CD4+ and CD8+ human T
lymphocytes (9, 10). Immortalized cells remain IL-2-dependent, but
become Ag- and mitogen-independent for their continued growth (11).
However, they do display normal downstream functional responses
(proliferation, cytokine synthesis, induction of activation markers,
cytotoxicity, etc.) when their TCR/CD3 activation pathway is triggered
(12, 13).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HVS-transformed T cell lines were derived from PBL of a healthy
congenital CD3-deficient individual (DSF or III-2) (7, 14) and
normal donors, as previously described (10). Briefly, PBLs were
resuspended (2 x 106 cells/ml) in a mixture (1:1
proportions) of two culture media [RPMI 1640 medium from Biochrom
(Berlin, Germany) and cell growth (CG) medium from Vitromex (Vilshofen,
Germany)] supplemented with 10% FCS (Flow Laboratories,
Rockville, MD), 1% L-glutamine (BioWhitaker, Berkshire,
U.K.), 50 IU/ml human rIL-2 (Hoffmann-La Roche, Nutley, NJ), and 1
µg/ml phytohemagglutinin (PHA; Difco Laboratories, Detroit, MI). The
same day of isolation for CD8+ cells, or at day 3 for
CD4+ cells, they were resuspended at 2 x
106 cells/ml in CG/RPMI medium containing 50 IU/ml human
rIL-2 and exposed once to 1 ml of HVS supernatant in 24-well plates
(Costar, Cambridge, MA). Thereafter, medium was replaced every 3 to 4
days (no PHA, only rIL-2). An immortalized phenotype was indicated by
the death of control cultures (i.e., non-HVS-exposed) vs the sustained
growth, presence of HVS genomes, and T lymphoblast cell morphology of
test cultures, as described (9, 15). HVS-exposed T cells had been
maintained in long-term culture for more than 16 mo when the
experiments reported here were performed. Vß usage analyses were
performed as described elsewhere (16).
Phenotypical analyses
The following mAbs were used for cytofluorometric analyses: Leu4
(anti-CD3), Leu2a (anti-CD8), Leu3a (anti-CD4), Leu19
(anti-CD56), Leu23 (anti-CD69), Leu45R0 (anti-CD45R0),
Leu11c (anti-CD16), Leu16 (anti-CD20), and HLA-DR from Becton
Dickinson (Mountain View, CA); MsIgG (used as negative control) and
phycoerythrin (PE)-conjugated goat anti-mouse IgG (H+L) (used as
secondary Ab) from Caltag (South San Francisco, CA); IOT18
(anti-CD18), IOT3b (anti-CD3), and BMA031 (anti-TCRß)
from Immunotech (Marseille, France); 2H4 (anti-CD45RA) and T11
(anti-CD2) from Coulter Clone (Hialeah, FL); anti-CD5 from
Serotec (Sussex, U.K.); KOLT-2 (anti-CD28) from CLB
(Amsterdam, The Netherlands); OKT3 (anti-CD3), and OKT3a
(anti-TCRß) from Ortho Diagnostic (Raritan, NJ); TCR1
(anti-TCR) from T Cell Science (Cambridge, MA); anti-IL-2
and anti-TNF- from R&D Systems (Abingdon, U.K.); anti-CD154
(CD40L) from PharMingen (San Diego, CA). TG5 (an anti-CD3 rabbit
antiserum raised against the CD3 C-terminal peptide GLQGNQLRRN) and
X35 (anti-CD3) were kindly provided by D. Alexander (Babraham
Institute, Cambridge, U.K.) and D. Bourel (Centre Regional de
Transfusion Sanguine, Rennes, France), respectively. ßF1
(anti-TCRß) and OKT6 (anti-CD1) mAb ascites were kindly
provided by M. L. Toribio (Centro de Biología Molecular,
Madrid, Spain).
For single- and two-color immunofluorescence, 1 x 106 cells were incubated for 30 min at 4°C with appropriate FITC- or PE-conjugated mAb in PBS/EDTA buffer containing 1% FCS. After two washes with PBS, cells were analyzed in a Epics Elite Analyzer cytofluorometer (Coulter). Isotype-matched irrelevant Abs were used to define background fluorescence. For intracellular stainings using TG5, ßF1, or OKT6, cells were first permeabilized and fixed as explained below (cytokine synthesis assays).
The precise quantification of CD3 molecules per T cell was conducted by
indirect immunofluorescence in parallel with calibrated beads,
following the manufacturer’s protocol (Qifikit, Biocytex, Marseille,
France). Briefly, 1 x 106 PBL were first stained with
PE-conjugated CD4 (Leu3a) or CD8 (Leu2a) mAb for 30 min at 4°C. After
two washes with PBS, PBL, or HVS, cells were incubated with or without
(negative control) an anti-CD3 mAb (Leu4) for 30 min at 4°C.
Cells and different calibrated beads (with 0 (negative control), 5,000,
13,000, 26,000, 59,000, 110,000, 220,000, and 600,000 Fc-binding sites
per bead) were washed twice with PBS and incubated with FITC-conjugated
anti-mouse IgG for 45 min at 4°C. After two washes with PBS,
cells and calibrated beads were analyzed by flow cytometry. For each
positive bead, its mean fluorescence intensity (MFI) value was recorded
and corrected by subtracting the MFI of the negative control. A
standard curve was then calculated by plotting the number of sites per
bead against the corresponding corrected MFI value of each bead type,
and a linear regression analysis was performed (Inplot; GraphPad
Software, San Diego, CA). The correlation coefficient was always
>0.98. The number of bound mAb molecules per cell as a function of the
corrected MFI (Leu4 MFI - negative control MFI) was determined
by extrapolating from the standard curve. The result of such
calculation is the mean number of cell-bound mAb molecules per cell in
the considered population (or subpopulation).
Data were collected on 2 to 5 x 104 viable cells as determined by electronic gating on forward scatter and side scatter light parameters.
TCR/CD3 internalization assays
The assays were done as described by Dietrich et al. (17).
Briefly, cells were washed twice in PBS, resuspended (5 x
105 cells/ml) in CG/RPMI rIL-2-free medium and incubated at
37°C for 30 min in the presence or absence of 20 ng/ml PMA. After
stimulation, cells were washed in PBS containing 1% FCS and stained
with Leu4 mAb (anti-CD3) for 30 min at 4°C. Then, cells were
washed in PBS/1% FCS buffer and analyzed by flow cytometry as
described above. Results were expressed as the percentage of MFI of
control cells incubated without PMA.
Functional assays
Intracellular calcium release was induced in cells loaded with the fluorescent dye Fluo-3AM (Sigma, St. Louis, MO) according to a standard procedure (14). Briefly, 2 x 106 cells were washed twice and resuspended in Ca2+-free medium (Sigma) at a final concentration of 1 x 106 cells/ml. Then, cells were incubated in a stirring bath at 37°C for 30 min with 4 µM Fluo-3AM, washed once more with Ca2+-free medium, and resuspended at a final concentration of 0.5 to 1 x 106 cells/ml for flow cytometry analysis. Changes in relative fluorescence intensity were recorded as a function of time before and after the sequential addition of the following reagents: 1) anti-CD3 mAb (IOT3b from Immunotech, 100 µl at 12.5 µg/ml); and 2) cross-linking reagent, human-adsorbed goat anti-mouse IgG (H+L) from Caltag, 40 µl at 1.25 mg/ml.
Proliferation was measured by standard [3H]thymidine uptake assays (14). Immortalized cells were starved for 7 days in the absence of IL-2, adjusted to a final concentration of 5 x 105 cell/ml in fresh mixed medium, and incubated for 48 h in round-bottom 96-well plates (180 µl of cells/well) previously coated with different concentrations of anti-CD3 (IOT3b; Immunotech). Then cells were pulsed with [3H]thymidine (1 µCi/well, Amersham, Buckinghamshire, U.K.) for another 16 to 18 h and harvested onto glass fiber filters. Thymidine incorporation into cellular DNA was evaluated as cpm in a scintillation ß counter (Packard, Meriden, CT). Other stimuli (10 ng/ml PMA (Sigma); 7 million SRBC/well (BioMérieux, Charbonnier les Bains, France); 750 ng/ml ionomycin (Sigma)) were also assayed. All experiments were done in triplicate wells and expressed as median cpm values.
To measure CD3-mediated cytotoxicity, HVS-effector cells (E) were
starved (no rIL-2) overnight in CG/RPMI medium containing 5% FCS,
washed, and subsequently resuspended in CG/RPMI medium containing 10%
FCS without rIL-2 at a final concentration of 2.5 x
106 cells/ml. Target (T) cells (P815 mouse mastocytoma),
previously loaded with 51Cr, were resuspended in the same
medium at a concentration of 1 x 105 cells/ml. A
total of 5 x 103 P815 cells were used in all E:T
ratios (25:1, 10:1, 5:1, and 1:1). Effector and target cells were
incubated at 37°C/5% CO2 for 4 h in the presence of
0.2 µg/ml anti-CD3 (IOT3b) (positive test), in the absence of
anti-CD3 Ab (spontaneous lysis), or in the presence of an
isotype-matched irrelevant Ab (0.2 µg/ml w6/32, anti-HLA class
I). Lysis was measured as 51Cr release in a counter
(Packard). Values are given as [(E/T cpm -
basal cpm)/(max cpm - basal
cpm)] x 100, where E/T cpm is the median cpm value
for each E:T ratio assayed, max cpm indicates
51Cr-maximum release (median value, induced by addition of
100 µl/well, 2% SDS), and basal cpm denotes the amount of
51Cr released spontaneously from P815 cells in the absence
of E cells. Experiments were done in 96-well connical bottom-plates and
each combination was assayed in triplicate.
To measure CD69 and CD154 (CD40L) induction after stimulation, cells were starved in CG/RPMI medium without human rIL-2 for 7 days and resuspended at 5 x 105 cells/ml in 96-well plates in the absence or presence of 1 µg/ml plastic-bound anti-CD3 mAb (IOT3b) for 6 h at 37°C. Then, cells were washed twice in PBS, stained with anti-CD69 or anti-CD154 mAb for 30 min at 4°C, washed twice in PBS, and analyzed by flow cytometry as described above.
Cytokine synthesis assays
To analyze intracellular cytokine induction, rIL-2-starved cells
were resuspended at 5 x 105/ml in 96-well plates and
stimulated for 6 h with or without 1 µg/ml immobilized
anti-CD3 mAb (IOT3b). For the last 2 h, 10 µg/ml Brefeldin A
(Sigma) was added to the cultures to block secretion. Cells were
harvested, washed twice in PBS buffer, and fixed with 500 µl of 4%
formaldehyde in PBS for 20 min at room temperature. Then, the cells
were stained intracellularly for cytokine content using a modified
method based on that described by Assenmacher et al. (18). Briefly,
cells were washed twice in PBS containing 0.1% saponin (Sigma),
incubated with anti-IL-2 or anti-TNF- FITC-conjugated mAb in
100 µl of PBS containing 1% saponin for 30 min at room temperature,
and washed with PBS/0.1% saponin buffer. The cytometric analyses were
performed in an Epics Elite Analyzer as described above.
To determine cytokine secretion after stimulation, rIL-2-starved cells
were resuspended at 5 x 105 cells/ml in CG/RPMI
medium without IL-2 in a 96-well plate (105 cells/well)
with or without 1 µg/ml plastic-bound anti-CD3 mAb (IOT3b) or 10
ng/ml PMA plus 750 ng/ml ionomycin. To block autocrine use of IL-2, 10
ng/ml anti-CD25 mAb (Coulter) was added to the relevant cultures.
After 48 h, duplicate culture supernatants were collected and
assayed for their cytokine content (IL-2, TNF-, IFN-, IL-5, or
IL-6) by commercial ELISA assays (Bender MedSystems, Vienna, Austria).
The limits of detection were 15 pg/ml, 16 pg/ml, 5 pg/ml, 4 pg/ml, and
4 pg/ml, respectively.
Statistical analysis
Student’s t test was used for all comparisons. Only p values below 0.05 were considered significant. Data are presented as mean ± SD, except where indicated.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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After infection of PBLs from the CD3-deficient individual and
two unrelated normal donors with HVS supernatant, four different
HVS-immortalized T cell lines were obtained: two -deficient HVS T
cell lines termed DSF4 (CD4+) and DSF8 (CD8+),
and two -sufficient HVS T cell lines termed CTC4 and CTO8, with the
equivalent phenotypes. All cell lines rapidly reached a stable growth
rate (doubling times ranged 3.2 to 4.3 days). As expected, all cell
lines contained HVS genomes, as shown by PCR analysis using
HVS-specific primers (not shown).
All cell lines showed a characteristic mature activated T cell
profile (Table I
and 19 . However,
as shown in Figure 1A and
Table I, DSF4 and DSF8 T cells showed a selective impaired
accessibility of TCR/CD3-associated epitopes, ranging from virtually no
accessibility of TCRß framework epitopes (around 6- and 12-fold
less than controls using BMA031, respectively) to around 3-fold and
6-fold, respectively, less accessibility of CD3 epitopes (Leu4) as
compared with controls. Quantitative analysis of the cytofluorometric
data obtained with Leu4 essentially confirmed these findings, and
revealed an absolute number of 45,000 and 30,000 accessible sites per
cell in DSF4 and DSF8 cells, respectively, compared with 150,000 and
125,000 accessible sites per cell in CTC4 and CTC8, respectively (Table II). CD3-sufficient HVS T cells,
whether CD4+ or CD8+, increased their
accessibility to Leu4 around 3-fold as compared with fresh peripheral
blood T lymphocytes (Table II). In contrast, -deficient HVS T cells
increased their accessibility to Leu4 by a factor of 5 in the case of
CD4+ cells and, interestingly, by a factor of 10 in
CD8+ cells.
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protein in -deficient T cells,
permeabilized cells were stained intracellularly with an
anti-CD3 antiserum (TG5) and, as a positive control, with an
anti-TCRß mAb (ßF1). The results confirmed the lack of
TG5-reactive material in -deficient T cell lines (Fig. 1
B) Similar results were obtained by immunoprecipitation
(data not shown).
To test whether the HVS T cells obtained were representative of a few
TCR ß+ clones or, alternatively, they represented a
polyclonal population, Vß 1–20 regions were analyzed by RT-PCR after
more than 6 mo of culture. Results revealed that more than 85% of the
Vß genes tested (1–4, 5.1, 5.2/3, 6.1/3, 7–12, 13.1, 13.2, 14–20)
were expressed in CD4+ HVS T cells (not shown). These
patterns are compatible with the variable predominance of Vß gene
usage in HVS-immortalized peripheral T cells previously described
(20).
PMA-induced internalization of the TCR/CD3 complex was selectively
impaired in CD3-deficient T cells
Previous reports have shown that CD3 is crucial for
protein kinase C-dependent TCR/CD3 down-modulation (17, 21, 22). It was
thus relevant to test PMA-induced TCR/CD3 internalization in the
immortalized -deficient T cell lines, to further substantiate their
lack of CD3. As described, both CD4+ and
CD8+ CD3-sufficient HVS T cells lost around half of
their TCR/CD3 complexes from the membrane after stimulation (Fig. 2). CD3-deficient cells, in
contrast, were essentially unperturbed in their relative TCR/CD3
surface expression by the same stimulus, and the difference with
CD3-sufficient cells was statistically significant. These data
cannot be explained by a complete inability of CD3-deficient cells
to modulate their surface TCR/CD3 complexes or by the fact that they
already expressed fewer surface complexes, because Ab-mediated
internalization, which is believed to be protein kinase C-independent
(22), was completely normal (19).
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To assess qualitatively the signals propagated by the mutant
TCR/CD3 complex, we next assayed a number of functional
parameters on CD3-deficient and -sufficient HVS T cells using
TCR/CD3-dependent (anti-CD3 mAb), and -independent (PMA plus
ionomycin) stimuli.
First, calcium flux, an immediate activation event after
TCR/CD3 engagement was tested and found to be
undistinguishable in -sufficient vs -deficient HVS cell lines
(2% vs 3% as basal calcium content levels, 2% vs 4% in the presence
of soluble anti-CD3 mAb, and 75% vs 80% after CD3 cross-linking,
respectively (Fig. 3).
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A). It is
noteworthy that all lines showed a normal dose-dependent proliferation
in response to increasing amounts of plastic-bound anti-CD3 (Fig. 4B), although CD3-deficient T cells expressed 3- to
5-fold less accessible CD3 epitopes than CD3-sufficient ones.
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, both -deficient and
-sufficient CD8+ cells showed comparable cytotoxicity
responses at different E:T cell ratios.
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, unstimulated cells, both -deficient
and -sufficient, expressed comparable levels of CD154. After CD3
activation, all cells significantly up-regulated CD154 expression by a
factor of 6 (CD4+ cells) or 9 (CD8+ cells). In
contrast to CD154, CD69 expression was undetectable in these particular
HVS T cells (Fig. 6B), although this is not always the case
(Ref. 23, and our unpublished results). After CD3 engaging, however,
comparable levels of CD69 induction were recorded in all T cell lines.
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chain
of the TCR/CD3 complex was dispensable for several
TCR/CD3-induced activation events: immediate (calcium flux),
early (cytotoxicity, induction of CD69 or CD154), and late
(proliferation).
TCR/CD3-induced IL-2, but not TNF-, secretion was impaired in
the absence of CD3
The secretion of different lymphokines after stimulation was next
assayed by ELISA. As shown in Figure 7,
both -deficient and -sufficient unstimulated T cells produced basal
levels of IL-2 but, when stimulated with anti-CD3, only the latter
significantly increased IL-2 production by a factor of 6 to 8. By
contrast, IL-2 production after CD3 stimulation was impaired both in
CD4+ and in CD8+ -deficient HVS T cells
(p < 0.05 vs -sufficient cells). To rule
out the existence of an intrinsic (that is, TCR/CD3-independent) defect
of IL-2 production in -deficient HVS T cells, they were stimulated
with PMA plus ionomycin, which by-passes membrane signals. This strong
stimulant mixture discriminates between potentially inducible genes
(responsive) and irreversibly blocked genes (unresponsive), as shown in
Th1 vs Th2 cytokine profiles (24). In these conditions, all T cell
lines, both -deficient and -sufficient, produced similar amounts of
IL-2 (19- to 32-fold more than their corresponding basal values, Fig. 7). The observed defect in CD3-induced cytokine induction was selective
for IL-2, because TNF- synthesis after anti-CD3 stimulation was
comparable between -deficient and -sufficient T cell lines within
each T cell phenotype (3- to 4-fold in CD4+ cells, 10- to
23-fold in CD8+ cells), although high basal TNF-
production was observed in both CD4+ cell lines (Fig. 7).
High basal TNF- levels in HVS T cell has also been reported
previously (9, 13). IFN- production was also analyzed and shown to
be similar in all unstimulated cell lines, but it did not increase
after stimulation with anti-CD3 or PMA plus ionomycin, precluding
additional functional studies. To further rule out the possibility that
CD3-deficient T cells were derived from Th2 cells, we analyzed IL-5
and IL-6 synthesis. As shown in Figure 7, none of the tested HVS T
cells produced significant levels of IL-5 (or IL-6, data not shown)
under any culture conditions.
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(upper),
essentially no intracellular IL-2+ (iIL-2+)
cells were induced in -deficient HVS T cells after CD3 triggering
(<5%). In contrast, around 35% (32 to 37%) of -sufficient cells
became iIL-2+ under similar conditions. Again, the defect
was selective for IL-2 induction, because similar numbers of
TNF-+ cells (70 to 85%) were observed in both
-deficient and -sufficient cells in comparison to basal levels of
6 to 10%, irrespectively of their CD3 protein content (Fig. 8, lower).
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-deficient
cells.
Taken together, these data suggest that the CD3 chain of the TCR/CD3
complex was dispensable for the induction of certain cytokines (like
TNF-), but not for others (like IL-2), in human CD4+ and
CD8+ HVS-immortalized polyclonal T lymphocytes. Therefore,
the propagation of signals through a -deficient TCR/CD3 complex can
result in different functional consequences when compared with a
-sufficient TCR/CD3 complex.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is not absolutely required for the
surface expression of the TCR/CD3 complex in human T lymphocytes. This
finding is in contrast with several mutant T cells (17, 21, 25, 26, 27, 28),
but supports the data of others in normal T or non-T cells (29, 30), in
CD3-deficient mice (6) and in other CD3-deficient humans (Ref.
31; and M. J. D. van Tol, personal communication). This
discrepancy could be due to the existence of residual CD3 chains, or
fragments thereof, in the CD3-deficient cells, although our
available biochemical evidence does not support this possibility (Ref.
14, and our unpublished results). However, it must be born in mind that
all mutant T cells were clones, frequently derived from tumors
(Jurkat), and artificially selected in vitro on the basis of the
absence of surface TCR/CD3 expression and, for the reconstitution of
TCR expression by transfection with CD3 or - plasmids, again
selected specifically for expression at endogenous levels. Natural
mutant T cells, in contrast, have been probably positively selected in
vivo on the basis of their adequate and functional TCR/CD3 expression
(see Note added in proof).
Mature CD4+ or CD8+ T lymphocytes lacking
CD3 have been previously shown to suffer a dissimilar TCR/CD3
accessibility defect (8). However, it was difficult to draw firm
conclusions from those data due to the scant peripheral
CD8+ T cells of the donor. HVS immortalization has
confirmed that the lack of CD3 affects TCR/CD3 accessibility in
CD8+ T cells consistently more than in CD4+ T
cells, as revealed with several mAbs (Table I). This finding may be the
reflection of a hitherto unrecognized biochemical difference between
TCR/CD3 complexes in CD4+ vs CD8+ T cells,
revealed only when CD3 is absent. Alternatively, as suggested
previously (8), peripheral CD8+ T cells from this CD3
deficiency, and thus their immortalized progeny, may belong to a minor
population (in normals) that is relatively expanded in the absence of
CD3.
It is noteworthy that whereas -sufficient T cells up-regulated
their Leu4 binding sites two- to threefold upon immortalization,
-deficient cells up-regulated them 5-fold (CD4+ cells)
or 10-fold (CD8+ cells). This result may suggest that
TCR/CD3 surface levels or accessibility are deliberately maintained at
the levels that have been determined in vivo so that the available T
cells (particularly CD8+ cells) adapt to the lack of
CD3. Alternatively, it may be a HVS-associated effect, because HVS
specifically targets TCR-linked signal transduction pathways (Tip/Lck,
Stp, and C/Ras; Refs. 32 and 33). When cultured with
CD3-deficient cells, HVS may serve to immortalize only those cells
that maintain TCR levels above a certain threshold. The observed
increase in the level of expression of CD45RO or CD8 (Table I) in
-deficient cells may be a further compensatory mechanism to ensure
immortalization.
Asymmetric effects on TCR expression or function of CD4+ vs CD8+ T cells have been reported previously (34, 35, 36) and may be due to the preferential association of p56lck with CD4 (37, 38, 39). This association, in turn, may regulate the targeting of TCR/CD3 to the endosomal compartment thereby regulating TCR/CD3 surface levels.
We previously showed that, although primary CD4+ cells from
this patient proliferated in response to phytohemagglutinin and
allogeneic feeders, we were not able to induce the growth of primary
CD8+ cells (8). This was one of the reasons for trying HVS
immortalization. We believe that the proliferation of CD8+
cells following HVS-transformation may be due to one (or several) of
the following reasons: 1) Conventional T cell lines require frequent
TCR-mediated restimulation, and it has been shown that T cells respond
only when a threshold of 
8000 engaged TCRs have been reached (40).
As -deficient CD8+ PBL (but not CD4+) have
less than 3000 Leu4 sites (Table II), it is possible that they are at a
disadvantage in this particular system. 2) In contrast, autocrine
proliferation following HVS immortalization takes place through
CD2/CD58 homotypic interactions (11), which are intact in -deficient
CD8+ cells (Ref. 41, Table I, Fig. 4A, and our
unpublished results). 3) Exposure to HVS has been shown to be very
effective in rescuing even minute numbers of mature T lymphocytes in in
vitro differentiation assays (15).
Our data and published functional results (14, 19) indicate that CD3
in HVS-immortalized T cells is dispensable for the following
TCR/CD3-induced functions: calcium flux, TCR/CD3 down-regulation,
cytotoxicity, CD69 or CD40L up-regulation, TNF- synthesis, and
proliferation. This result may help us understand the survival of the
-deficient donor (presently healthy and in his teens) and of other
-deficient individuals (31), despite a susceptibility to bacterial
and particularly viral infections (like viral meningitis). In contrast,
TCR/CD3-induced synthesis of IL-2, as well as PMA-induced TCR/CD3
down-regulation, was severely impaired in -deficient cells.
Therefore, the cytosolic signals initiated by the TCR itself are
propagated differentially when CD3 is absent and can result in
distinct functional outcomes.
These data confirm previous reports indicating that the cytoplasmic
tail of CD3 is dispensable for CD3-induced TCR/CD3 down-modulation,
cytolysis and TNF- or IFN- synthesis (21), but not for
PMA-induced TCR/CD3 down-modulation. IL-2 synthesis was not tested in
those experiments, but a deficiency in IL-2 induction was reported in
an unimmortalized CD4+ T cell line from the same
-deficient donor (14) and in PBL (41). Calcium flux, however, was
partially impaired in that particular T cell line, although this may
perhaps have been due to clonal variation, as it has not been confirmed
in primary (our unpublished results) or immortalized T cells (present
results).
Taken together, the data suggest that CD3 may play a specialized
role in coupling the remaining TCR/CD3 chains with downstream
intracellular signaling circuitries. However, as we are looking at
mature T lymphocytes that presumably have been rescued in the thymus,
it is possible that they have been selected to be IL-2-uninducible
through their -deficient TCR/CD3 complex. Definitive proof will
require reconstitution of IL-2 secretion by CD3 gene transfer into
-deficient cells. The observed selective cytokine induction defect
may affect several coregulated Th1 genes and may explain the reported
IgG2 and polysaccharide Ab response deficiency in -deficient
individuals (42) or their reduced peripheral T cell pool (8). Indeed,
IgG2 switch has been proposed to be dependent on Th1 cytokines (43)
some of which, including IL-2, may be uninducible by the -deficient
TCR/CD3 complex.
HVS-immortalized mutant T cells from human CD3 deficiency were shown
to faithfully preserve the phenotypical and functional features of the
original T cells (Ref. 19, and present results). This finding has been
also shown for several other human immunodeficiencies, including
X-linked severe combined immunodeficiency (44), CD95 deficiency (23),
Wiskott-Aldrich syndrome (45), MHC class II deficiency (46), and
Ataxia-Telangiectasia (47). Thus, the immortalization method is clearly
a valid experimental approach to generate clean model systems of
natural mutant T cells. However, there are certain intrinsic
limitations: irregular phenotypes (CD25, CD56, CD69, CD154; Refs. 9,
10, 13, and 23), uninducible cytokines in certain conditions (like
IFN- in this report), and low relative responses to specific Ags due
to high background proliferation (13). Similar models and further
studies on this and other T cell deficiencies (in CD3, Zap70, CD40L,
ADA, PNP, lymphokine induction, Ca2+ influx, etc.; 48
would be useful to define specific pathways linking the biochemical
signals arising from surface receptors to their associated
transcriptional machinery.
Note added in proof. After submission of this paper,
CD3-deficient mice have been reported (Haks M., P. Krimpenfort, J.
Borst and A. Kruisbeek. 1998. The CD3 chain is essential for
development of both the TCRß and TCR lineages. EMBO J.
7:1871), which are essentially similar to available natural human
cases (Ref. 7, and van Tol, M. J. D., Ö. Sanal, R. Langlois van
den Bergh, Y. van den Wal, M. T. L. Roos, A. I. Berkel, J. M. Vossen,
and F. Koning. 1997. CD3 chain deficiency leads to a cellular
immunodeficiency with mild clinical presentation. Immunologist
S1:41.) and which confirm that TCR/CD3 accessibility is more
impaired in peripheral CD8+ T lymphocytes than in
CD4+ cells when is absent.
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Acknowledgments |
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Footnotes |
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2 A.P.-C. and D.A.-Z. are joint first authors.
3 Address correspondence and reprint requests to Dr. José R. Regueiro, Inmunología, Facultad de Medicina, Universidad Complutense, 28040 Madrid, Spain. E-mail address:
4 Abbreviations used in this paper: ITAM, immunoreceptor tyrosine-based activation motif; HVS, Herpesvirus saimiri C-488; CG, cell growth medium; PE, phycoerythrin; MFI, mean fluorescence intensity.
Received for publication December 4, 1997. Accepted for publication May 6, 1998.
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) and
-sufficient (
) HVS T cells in response to increasing amounts of
anti-CD3 (IOT3b) mAb. Data are given as mean cpm values ± SD
of five independent experiments.
) of four
independent experiments. For IL-2, the numbers above each bar indicate
the cytokine levels normalized to that of cells incubated in medium.
*Denotes p < 0.05 as compared with the
corresponding 