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Opposite CD4/CD8 Lineage Decisions of CD4+8+
Mouse and Rat Thymocytes to Equivalent
Triggering Signals: Correlation with Thymic
Expression of a Truncated CD8 α Chain in Mice
But Not Rats
Rita Mitnacht, Astrid Bischof, Nora Torres-Nagel and Thomas
Hünig
J Immunol 1998; 160:700-707; ;
http://www.jimmunol.org/content/160/2/700
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The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 1998 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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References
Opposite CD4/CD8 Lineage Decisions of CD4181 Mouse and
Rat Thymocytes to Equivalent Triggering Signals: Correlation
with Thymic Expression of a Truncated CD8a Chain in Mice
But Not Rats1
Rita Mitnacht, Astrid Bischof, Nora Torres-Nagel, and Thomas Hünig2
T
cell repertoire selection is controlled by the interaction of
clonally distributed TCR molecules expressed on immature CD4181 thymocytes with MHC/peptide complexes
at the surface of thymic cortical epithelial cells. The rescue of cells
with self-MHC-restricted TCR is accompanied by lineage decision: thymocytes selected by recognition of MHC class I molecules down-regulate CD4 and become CTL precursors maintaining
the coreceptor CD8 (1, 2), whereas those selected by MHC class II
down-regulate CD8 and become Th cell precursors maintaining
the coreceptor CD4 (3, 4).
Based on experiments in mice expressing class I or class IIrestricted transgenic TCR and defective or transgenic MHC or coreceptor molecules, several models have been proposed to explain
the coordination of TCR class specificity with CD4/8 lineage commitment during positive selection (for a recent summary, see Ref.
5). These models contain elements that have been named “instructive” if MHC class recognition informs the CD4181 cell of the
appropriate lineage decision (6); “stochastic” if lineage commitment is independent of MHC class recognition and coreceptor en-
Institute for Virology and Immunobiology, University of Würzburg, Würzburg,
Germany
Received for publication July 14, 1997. Accepted for publication October
2, 1997.
The costs of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
gagement, requiring a “selective” element in which coligation of
the TCR with the appropriate coreceptor rescues only those cells
that made the right choice (7); and “default” if failure to engage the
TCR by one MHC class will result in commitment to the opposite
lineage (8).
The concept of an instructive element in CD4/8 lineage decision
has recently found support from coreceptor-domain shuffling experiments conducted in TCR-transgenic mice. In animals containing a chimeric coreceptor with an extracellular CD8a and an intracellular CD4 domain, CD4181 thymocytes expressing an MHC
class I-restricted transgenic TCR were diverted into the CD4 subset during positive selection (9). Like CD4 itself, this chimeric
coreceptor engages more of the nonreceptor kinase p56lck (Lck)3
than CD8 (10 –12). Since Lck plays a central role in the initiation
of TCR signaling (13, 14), it was proposed that the elevated
amounts of Lck delivered to the TCR complex by the cytoplasmic
CD4 domain of the chimeric coreceptor compared with the wildtype CD8 molecule during MHC I recognition resulted in an increased signaling strength that instructed the immature thymocyte
to initiate CD4 lineage commitment (9). A mechanism in which
quantitative differences in Lck delivery are used for MHC class
identification during positive selection may also provide an explanation for the puzzling finding that in addition to the full-length
CD8a molecule, mouse CD4181 thymocytes co-express a tailless isoform of CD8a generated by alternative splicing that is unable to bind Lck (15, 16). These CD8a9/b heterodimers are lost
1
This work was supported by the DFG through SFB 165, and by Fonds der
Chemischen Industrie e.V.
2
Address correspondence and reprint requests to Dr. T. Hünig, Institute for Virology and Immunobiology, Versbacher Straße 7, D-97078 Würzburg, Germany. E-mail address: [email protected]
Copyright © 1998 by The American Association of Immunologists
Abbreviations used in this paper: Lck, p56lck; PNA, peanut agglutinin; CASUP,
supernatant of Con A-stimulated peripheral T cells; FTOC, fetal thymic organ
culture.
3
0022-1767/98/$02.00
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Unselected CD4181 rat thymocytes, generated in vitro from their direct precursors, are readily converted to functional TCRhigh
T cells by stimulation with immobilized TCR-specific mAb plus IL-2. Lineage decision invariably occurs toward CD4281,
regardless of the timing of TCR stimulation after entry into the CD4181 compartment or the concentration of TCR-specific mAb
used for stimulation. CD4-specific mAb synergizes with suboptimal TCR-specific mAb in inducing T cell maturation, but lineage
decision remains exclusively CD4281. These results contrast with those obtained in mice, in which Abs to the TCR complex
were shown to promote CD4182 T cell maturation from CD4181 thymocytes. Surprisingly, when rat and mouse CD4181
thymocytes were stimulated with PMA/ionomycin under identical conditions, the opposite lineage commitment was observed,
i.e., mouse thymocytes responded with the generation of CD4182 and rat thymocytes with the generation of CD4281 cells. It
thus seems that CD4181 thymocytes of the two species respond with opposite lineage decisions to strong activating signals such
as given by TCR-specific mAb or PMA/ionomycin. A possible key to this difference lies in the availability of p56lck for coreceptorsupported signaling. We show that in contrast to mouse CD4181 thymocytes, which express both a complete and a truncated
CD8a-chain (CD8a*) unable to bind p56lck, rat thymocytes only express full-length CD8a molecules. Mice, but not rats,
therefore may use CD8a* as a “dominant negative” coreceptor chain to attenuate the CD8 signal, thereby facilitating MHC class
II recognition through the higher amount of p56lck delivered, and rats may use a different mechanism for MHC class distinction
during positive selection. The Journal of Immunology, 1998, 160: 700 –707.
The Journal of Immunology
View, CA). Routinely, 10,000 events were analyzed. Results are shown as
log10 fluorescence intensities on a four-decade scale displayed as dot plots
or histograms.
Preparation of cells
Immature rat CD4281 thymocytes were isolated by treating thymocyte
suspensions with saturating amounts of R73 and W3/25 mAbs and removing the labeled cells by rosetting with rabbit anti-mouse Ig (Dakopatts,
Hamburg, Germany)-coated sheep erythrocytes. The remaining cells were
treated with OX35 and OX44 mAb, followed by sheep anti-mouse Ig ferritin particles (Miltenyi Biotec GmbH, Bergisch-Gladbach, Germany), before being passed through a magnetic-activated cell sorter (Miltenyi
GmbH). The resulting population consisted of more than 99% immature
CD4281 cells with a viability of .90% as determined by trypan blue
exclusion. Peanut agglutinin (PNA) positive rat and mouse thymocytes
were purified by panning on PNA (Sigma)-coated plates (20 mg/ml, carbonate buffer pH 9.5 for 24 h) in PBS, 5% FCS at 1 3 107 cells in 10 ml
per 100 mm plate (Greiner, Sulzfeld, Germany) for 90 min at 4°C. After 3
washes, bound cells were eluted with 10 ml 0.2 M b-galactose in PBS/FCS
for 10 min at room temperature, and washed twice in PBS/FCS.
Cell culture
A total of 5 3 105 thymocytes/ml of supplemented RPMI 1640 (18) were
cultured in 24-well plates (Costar, Cambridge, MA). For TCR stimulation,
culture wells were precoated overnight with rabbit anti-mouse Ig (40 mg/ml
in carbonate buffer, pH 9.5) followed by a 2-h incubation with mAb R73
and/or OX35 in balanced salt solution at the concentrations given, and
extensive washing. In all, 500 U/ml of human rIL-2 (a kind gift of Hoechst
AG, Frankfurt, Germany) was added where given. Identical results were
obtained using recombinant rat IL-2 (the kind gift of Dr. Mason, Oxford,
U.K.). Where indicated, PMA and ionomycin (both from Sigma) were
added at 0.4 ng and 0.2 mg/ml, respectively. In experiments not shown, the
following cytokine preparations were included: 0.1% of rat IL-4 containing
supernatant prepared from transfected Chinese hamster ovary cells (26)
with a titer of 1025 in an MHC class II induction assay using rat B cells;
50% culture supernatant from mouse IL-7-transfected 3T3 cells (the kind
gift of Drs. Rolink and Melchers) that promoted recovery of immature rat
thymocytes when used at 30% final concentration; 20% supernatant of Con
A-stimulated peripheral T cells (CASUP) prepared from rat spleen stimulated for 24 h with 5 mg of Con A/ml at 107 cells/ml; and supernatant of
rat thymic stromal cells prepared by dispase digestion of lymphocyte-depleted thymus fragments and cultured for 24 h at 106/ml.
Coreceptor re-expression assay
Conditions for pronase-stripping and coreceptor re-expression followed the
protocol developed by Suzuki and colleagues (8).
Immunoprecipitation and Western blotting
Materials and Methods
Animals
Young adult Lewis rats and BALB/c mice of both sexes, bred at the Institute’s facilities, were used.
Antibodies
Rat-specific mAbs W3/25 and OX-35 (both anti-CD4), OX-8 (anti-CD8a),
341 (anti-CD8b), OX-44 (anti-CD53), and R73 (anti-TCRa/b), were obtained from PharMingen, San Diego, CA, and from Serotec, Oxford, U.K.,
as purified Ab or Ab conjugates. Mouse CD4-specific mAb L3T4 was from
Boehringer, Mannheim, Germany, and 53-6.7 (anti-CD8) and H57-597 (antiTCRa/b) were from PharMingen. Phycoerythrin-conjugated F(ab9)2 donkey anti-mouse Ig was obtained from Dianova GmbH (Hamburg, Germany), and streptavidin-Red670 was obtained from Life Technologies,
Eggenstein, Germany.
Immunofluorescence and flow cytometry
For 2- or 3-color FACS analysis, 5 3 104 to 2 3 105 cells in 100 ml
PBS/0.2% BSA/0.02% sodium azide were incubated for 15 min on ice with
an unlabeled mAb, followed by a 15-min treatment with F(ab9)2 donkey
anti-mouse phycoerythrin, 10 mg/ml normal mouse IgG (Sigma Chemicals
Co., St. Louis, MO), and FITC- or biotin-conjugated mAb to the second
marker. Finally, biotinylated mAb were developed with streptavidinRED670 (Life Technologies). Flow cytometry was performed with a
FACScan flow cytometer, using LYSYS II software for acquisition and
Cellquest software for analysis (all from Becton Dickinson, Mountain
Thymocytes (108/ml) or nylon wool-passed lymph node cells (6 3 107/ml)
were lysed with 1% Nonidet P-40 in the presence of 1 mM Na3VO4, 20
mM NaF, 1 mg/ml leupeptin, and 1 mg/ml aprotinin, precipitated with
OX-8 mAb or normal Ig using protein G-coupled Sepharose beads (Pharmacia, Uppsala, Sweden), and washed extensively with lysis buffer. Endo
F-treatment was performed as described in reference 15, using 1 U Endo F
(Boehringer, Mannheim, Germany) per sample. Reducing SDS-PAGE and
transfer to nitrocellulose membranes followed standard procedures. Blots
were probed with biotinylated OX-8 mAb and developed with horseradish
peroxidase-conjugated streptavidin and the enhanced chemiluminescence
system (Amersham, Braunschweig, Germany).
Results
Rat CD8 T cells induced from virgin CD4181 thymocytes
are de novo differentiated cells
Stimulation of rat virgin CD4181 thymocytes, generated in vitro
from their direct precursors, with immobilized TCR-specific mAb
and IL-2 converts about 50% of input cells within 2 days to phenotypically and functionally mature TCRhigh cells, which are exclusively CD4281 (18). Although the purity of input cells and
absence of measurable proliferation during this phenotypic conversion made an expansion of contaminating mature CD8 cells
highly unlikely, this is now formally excluded by the use of
LEW.1F rats as thymocyte donors: in that strain, 3% of immature
CD4181 cells express TCR utilizing the Va8.2 segment, whereas
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from the surface after positive selection and could act as “dominant negative” molecules to attenuate the Lck signal during class
I-mediated positive selection.
In our earlier studies on the development of rat thymocytes, we
employed an in vitro system in which “virgin” CD4181 thymocytes are generated by overnight incubation of their direct precursors (17). These cells can be followed as a single cohort of synchronously differentiating cells, which express cell surface TCR
and other markers of CD4181 thymocytes at the levels characteristic of their ex vivo isolated counterparts (18, 19). We have previously shown that stimulation of such “virgin” CD4181 thymocytes with immobilized TCR-specific mAb induces downregulation of CD4 and CD8 (18) and expression of IL-2Rb (20,
21). Inclusion of IL-2 rescues approximately 50% of input cells
and, without proliferation, converts them within 2 days to phenotypically and functionally mature TCRhigh cells that are exclusively of the CD4281 phenotype (18). In these studies, the CD8
isoform detected was predominantly CD8aa rather than CD8ab,
which characterizes thymus-derived T cells (20). However, as will
be shown below, CD8ab cells are efficiently and exclusively produced in this system if conditions for stimulation are appropriately
modified.
While in vitro generation of rat CD8 T cells from CD4181
precursors has proven very robust and efficient, we have been unsuccessful at reproducing these findings in mice using either
freshly isolated or in vitro-generated CD4181 thymocytes (our
unpublished observations). On the other hand, the differentiation of
CD4182, but not CD4281, lineage cells from mouse CD4181
precursors by stimulation with TCR-specific (hybrid) mAb has
been reported by a number of groups (22-25). To reconcile these
divergent results obtained with rats and mice, we have tried to
identify conditions for the in vitro induction of CD4 lineage commitment in rat CD4181 thymocytes and have, by replacing species-specific mAbs and cytokines with the nonspecific stimulants
PMA and ionomycin, directly compared the response of rat and
mouse CD4181 cells to a strong activating signal with regard to
lineage commitment. Finally, we have investigated whether rat
CD4181 thymocytes express CD8a9, a possible tool for MHC
class discrimination during repertoire selection in mice.
701
702
OPPOSITE LINEAGE DECISIONS OF RAT AND MOUSE THYMOCYTES
FIGURE 1. Rat CD8 T cells obtained in vitro from virgin CD4181 precursors by TCR plus IL-2 stimulation are de novo differentiated cells. Ex
vivo analysis of Va8.2 expression among LEW.1F lymph node CD8 T cells ( left panel ), of immature CD4181 thymocytes enriched by PNA binding
( center panel ), and of CD8 T cells generated in vitro from virgin CD4181 cells by 48-h stimulation with immobilized TCR-specific mAb plus IL-2,
and 24 h rest to allow TCR up-regulation ( right panel ). Note the same TCRhigh level but different frequencies of Va8.21 cells in left and right panels.
Some models of positive selection and lineage decision of
CD4181 thymocytes contain a “default” element in which a cell
that fails to be engaged in one differentiation pathway spontaneously opts for the second one (Ref. 8, reviewed in Ref. 5). We
therefore asked whether TCR engagement at different timepoints
after entry into the CD4181 compartment might reveal a loss of
responsiveness to CD8 lineage instruction, and possibly a gain of
CD4 lineage commitment. Immature CD4281 cells or their 1-, 2-,
or 3-day-old CD4181 progeny were stimulated with immobilized
TCR-specific mAb plus IL-2 and analyzed daily for coreceptor
expression. As can be seen in Figure 2, this stimulation protocol
invariably resulted in the induction of the CD4281 phenotype,
even if the cells had spent their maximum lifetime of 3 days as
CD4181 cells. Mature levels of TCR expression after release from
TCR engagement and stability of CD4/8 phenotype after surface
stripping and re-expression confirmed that lineage decision and
maturation had indeed occurred (not shown, but see below). Thus,
the capacity to enter the mature CD4281 subset is maintained in
CD4181 rat thymocytes for the entire observation period in vitro,
which coincides with the lifespan determined for the corresponding mouse subset in vivo (28, 29). Note also that CD4181 cells
maintained in medium remained homogeneously CD4high8high, indicating that at least outside the thymic microenvironment, spontaneous down-regulation of one or the other coreceptor does not
occur with time.
Thymic stromal cells, their supernatants, IL-4, IL-7, or
CASUP do not induce lineage decision toward CD4
As one factor determining the unidirectional lineage decision observed in the present system, IL-2 was considered. Accordingly,
we tested to determine whether other cytokines might allow the
generation of CD4 cells. As potential candidates, rat IL-4 and IL-7
were tested in recombinant form, and in a broader approach,
CASUP or supernatant obtained from freshly isolated thymic stromal cells were added to the cultures. With the exception of IL-7,
which slightly improved cell recovery, and CASUP, which contained IL-2 and had the same effect, none of these additions af-
FIGURE 2. Rat thymocytes maintain their potential for CD8 commitment for several days after entry into the CD4181 compartment.
Immature CD4281 thymocytes were cultured in medium to allow
conversion to CD4181 cells, or were transferred to anti-TCR-coated
plates in IL-2-containing medium at the time points indicated. In all,
2000 cells collected in a live gate are shown for each point.
fected the kinetics or outcome of in vitro differentiation, i.e., they
did not lead to the appearance of CD4/8 single positive or TCRhigh
cells when added by themselves or together with soluble or immobilized TCR-specific mAb, nor did they affect the differentiation of CD8 cells when added together with IL-2 (data not shown).
Thus, using a limited panel of available rat cytokines and crude
sources of unseparated supernatants, we have to date not been able
to identify a cofactor that will direct the response to TCR stimulation by immobilized mAb toward the generation of CD4182
cells.
Maintenance of CD8 lineage decision after reconstitution of
suboptimal TCR stimulation by CD4 engagement
Due to their MHC class specificity, the CD4 and CD8 coreceptors
may participate in lineage instruction of thymocytes undergoing
positive selection. To investigate whether mAb-mediated co-ligation of TCR and CD4 would induce lineage decision toward CD4
rather than CD8, virgin CD4181 thymocytes were stimulated with
immobilized TCR-specific mAb and IL-2 over a wide range of
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due to RT1f-driven thymic overselection, 14% of mature CD8 T
cells and thymocytes are Va8.21 (27). As shown in Figure 1, CD8
T cells derived by in vitro stimulation of LEW.1F virgin CD4181
thymocytes contain the same frequency of Va8.21 cells as their
immature precursors, indicating that they were not derived from
mature contaminants but generated by de novo differentiation.
Anti-TCR plus IL-2 induces CD8 lineage decision regardless
of timing after entry into the CD4181 compartment
The Journal of Immunology
Opposite lineage decisions induced in mouse and rat
CD4181 thymocytes by PMA/ionomycin
The ease with which mature CD4281 T cells are induced from
immature rat CD4181 precursors by stimulation with TCR-specific mAb plus IL-2 prompted us to repeat these experiments in
mice. Despite extensive variations in cytokine supplements, all
attempts to generate mature T cells from TCR-stimulated mouse
CD4181 thymocytes in single-cell culture remained unsuccessful
(data not shown). However, other groups have shown that mouse
TCR-specific mAb or hybrid Abs are able to induce maturation of
CD4 T cells in vivo (22, 23) or fetal thymic organ culture (FTOC)
(24), and that TCR-specific mAb coimmobilized with mAb to various thymocyte cell surface molecules induces CD4 lineage commitment in single-cell cultures of mouse CD4181 thymocytes
(25). Furthermore, transient treatment of CD4181 mouse thymocytes with the protein kinase C activator PMA plus the Ca-ionophor ionomycin was shown to result in the generation of CD4182
thymocytes from CD4181 precursors in vitro (30). Together, these
results suggested that a “strong” signal such as those provided by
the high-affinity interactions of Abs with TCR or by forced protein
kinase C activation and elevation of Ca21 levels is perceived by
mouse CD4181 thymocytes as a signal to enter the CD4 lineage,
and that rat CD4181 thymocytes might respond with the opposite
decision. To directly test this possibility, CD4181 mouse and rat
thymocytes were enriched by panning on PNA-coated plates and
subjected to a 20-h PMA/ionomycin pulse followed by 20 h of rest
in medium. After PMA/ionomycin treatment, an aliquot of the
cells was subjected to coreceptor stripping by pronase treatment
(8), and the re-expressed CD4 and CD8 molecules were analyzed
after the resting period. As shown in Figure 4, A and B, mouse and
rat CD4181 thymocytes responded to this artificial stimulus with
the expression of opposite CD4/8 phenotypes: in agreement with
published results, CD4182 cells were obtained from mouse
CD4181 thymocytes. The parallel rat cultures did not generate
this subset but yielded CD4281 cells. Again, these CD8 cells ex-
pressed both CD8a- and b-chains (data not shown). Interestingly,
CD4281 and CD4182 thymocytes generated in this fashion from
mouse and rat CD4181 cells, respectively, contained both
TCRhigh and TCRlow/negative cells (Fig. 4C). This suggests that
circumvention of TCR signaling by PMA/ionomycin induces lineage commitment even in those CD4181 cells that have failed to
express appropriately rearranged and pairing TCRa-chains, and
argues against selective survival or expansion of mature cells contained in the PNA1 thymocyte preparations, which always contain
some single positive cells. In the case of PMA/ionomycin-driven
differentiation of rat cells, the contribution of contaminating mature CD4281 cells to the observed enrichment of the CD4281
phenotype was further excluded by the use of the LEW.1F strain
as the thymocyte donor and verification of preselection Va8.2 usage as described above (not shown). These data indicate that immature CD4181 thymocytes from rat and mice are programmed
for opposite lineage decisions in response to the strong stimulus
provided by the pharmacologic agents PMA and ionomycin, and
possibly also to physiologic, TCR/coreceptor-mediated signals.
Rat thymocytes do not express a truncated form of CD8a
Based on the above results, we considered the possibility that
mouse CD4181 thymocytes are programmed to respond to a
strong signal with lineage decision toward CD4 because under
physiologic conditions, more Lck will be delivered by CD4 than
by CD8, and consequently, this may not hold true for their rat
counterparts. Since in mice, a tail-less CD8a9 isoform is exclusively expressed in immature mouse thymocytes (16), where it
may attenuate the CD8-mediated Lck signal at that stage, the existence of a truncated CD8a molecule was investigated in rats by
Western blotting using a mAb (OX-8) that recognizes the CD8a
membrane-proximal hinge region. Immunoprecipitates from lysates of rat thymocytes and of peripheral T cells were either directly examined or treated with endoglycosidase F after immunoprecipitation with OX-8 to improve resolution. As can be seen in
Figure 5, a single band migrating at the apparent m.w. expected for
the intact CD8a-chain was observed in both thymocytes and peripheral T cells. Thus, in contrast to results obtained with mouse
CD4181 thymocytes, where the CD8a9 molecule is readily detected biochemically (16), this molecular species is absent from
the rat.
Discussion
The experiments presented in this communication indicate that
CD4181 thymocytes from mice and rats differ in their interpretation of external stimuli that induce CD4/8 lineage commitment.
This was directly demonstrated by employing identical conditions
for isolation and stimulation of CD4181 cells from the two species, i.e., enrichment by PNA binding and activation with PMA
and ionomycin. This approach eliminates potential differences in
signal delivery that may be encountered with species-specific
mAbs or cytokines. Confirming the results of others, we found that
a 20-h pulse treatment with PMA/ionomycin induced down-regulation of CD8 in CD4181 mouse thymocytes (30); in contrast,
CD4181 thymocytes from rats down-regulated CD4 under the
same conditions.
These results are in agreement with those obtained with mAbmediated TCR stimulation. In the rat, maturation of both CD8aa
(18, 20) and CD8ab T cells (this paper) is easily induced by stimulating CD4181 thymocytes with TCR-specific mAb and IL-2
(18), but this protocol is unsuccessful in mice (our unpublished
observations). In that species, however, a number of studies have
demonstrated lineage decision toward CD4 if TCR-specific mAb
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concentrations in the presence or absence of coimmobilized CD4specific mAb. After 2 days of stimulation, cells were rested for 1
day to allow up-regulation of the TCR and analyzed for the expression of CD4, CD8a, CD8b, and TCR-ab. As shown in Figure
3, the fraction of CD4181 immature thymocytes induced to differentiate increased with the concentration of TCR-specific mAb
employed, resulting in the generation of CD4281 TCRhigh cells.
Moreover, while CD4281 T cells generated by low concentrations
of TCR-specific mAb were of the CD42 CD8a1b1 phenotype,
those obtained with high concentrations of TCR-specific mAb contained many CD428a1b2 cells. This is in line with our previously
published experiments in which even higher concentrations of
TCR-specific mAb were routinely used and led to the predominant
induction of the CD428a1b2 phenotype (18, 20). When suboptimal amounts of TCR-specific mAb were employed, the reduced
number of cells addressed was strongly increased by the coimmobilization of CD4-specific mAb. At the same time, however, CD4
costimulation did not induce a CD4182 phenotype but rather resulted in an increased yield of CD428a1b1 TCRhigh cells. In
additional experiments (not shown), coligation of TCR and CD4
by mAb was again combined with the cytokine supplements listed
above. With the exception of IL-2 and of CASUP, which allowed
the differentiation of TCRhigh CD4281 cells, none of these additions allowed T cell maturation of anti-TCR- plus antiCD4-stimulated cells in this in vitro system. Also, as expected, the
same synergism described in Figure 3 for TCR- and CD4-specific
Abs was also observed when instead of anti-CD4, anti-CD8 mAb
were employed (not shown).
703
704
OPPOSITE LINEAGE DECISIONS OF RAT AND MOUSE THYMOCYTES
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FIGURE 3. Anti-CD4 synergizes with suboptimal anti-TCR in the in vitro generation of rat CD8 T cells. Virgin CD4181 cells, obtained by
overnight culture of immature CD4281 thymocytes, were cultured in the presence of IL-2 for 2 days in wells coated with RaMIg followed by
TCR-specific mAb at the concentrations indicated. In the bottom row of each panel, remaining RaMIg binding sites were saturated with an excess
of CD4-specific mAb (OX-35). In all, 2000 cells collected in a live gate are shown for each point.
were employed to drive positive selection. In the experiments by
Müller and Kyewski, hybrid mAb cross-linking the TCR to the
surface of thymic epithelial cells efficiently induced CD4, but not
CD8 T cell maturation in vivo (31), even if MHC II or CD4 were
absent (23). In a similar approach employing FTOC from class
II-deficient mice, Takahama and coworkers found that TCR- and
CD3-specific mAb rescued CD4 T cell development (22). Finally,
Bommhardt et al. recently reported that cross-linking of CD3 to
The Journal of Immunology
705
FIGURE 4. Opposite CD4/8 lineage decisions of rat and mouse
CD4181 cells stimulated with PMA and ionomycin. Young adult
Lewis rat and BALB/c mouse thymocytes were enriched for CD4181
cells by panning on PNA-coated plates, and stimulated for 20 h with
0.4 ng/ml PMA and 0.2 mg/ml ionomycin or left unstimulated. An
aliquot of the cells was subjected to pronase stripping (8) and cultured
for another 20 h at 4°C or 37°C. A, B, Two-color CD4/CD8 analysis of
rat ( A ) and mouse ( B ) thymocytes. C, TCR expression of unstimulated
CD4181 cells or CD4281 rat and CD4182 mouse cells obtained by
PMA/ionomycin stimulation. Histograms were obtained from gated
CD4/CD8/TCR three-color analyses.
either CD4 or CD8 using recombinant F(ab9)2 Abs drives CD4 T
cell development in FTOC in an MHC-independent fashion (24).
In addition to these experiments in which TCR-specific mAb induce CD4 T cell differentiation in an intact thymic microenvironment, an in vitro differentiation system for mouse CD4181 thymocytes was recently described in which coimmobilization of
mAb with one of several thymocyte cell surface molecules supported anti-TCR-driven T cell maturation in single-cell culture.
Again, the phenotype of differentiated cells was exclusively
CD4182 (25).
At first sight, these and our present results, in which CD4- and
TCR-specific Ab synergized in the generation of CD8 T cells from
rat CD4181 thymocytes, contradict instructive models of lineage
commitment because they show that neither the “correct” MHC
class nor the relevant coreceptor need to be engaged for efficient
conversion of mouse or rat CD4181 precursors to CD4 or CD8 T
cells, respectively.
We do, however, interpret these results in support of the “signal
strength” hypothesis of lineage instruction, and postulate that
while mouse CD4181 thymocytes interpret a strong signal as
MHC II recognition and thus commit toward CD4, rat CD4181
thymocytes are differently programmed and respond to a strong
signal with CD8 commitment. This hypothesis is based on the
notion that due to the manyfold higher stability of TCR-mAb complexes compared with those formed between TCR and MHC/peptide in positive selection (32), stimulation with TCR-specific mAb
will lead to the formation of stable TCR aggregates on which the
intracellular signaling machinery, including Lck, will efficiently
assemble even in the absence of coreceptor engagement, thereby
providing a strong signal. Indeed, peripheral T cells, which require
coreceptor participation in Ag-driven responses, are readily activated with TCR-specific mAb without the need for recruitment of
coreceptors to the TCR complex (33), and synergism between
TCR and coreceptor-specific mAb only becomes apparent under
conditions of suboptimal stimulation by TCR-specific mAb (34).
Although not addressed experimentally, we consider it likely
that also in positive selection of thymocytes driven by TCR-specific mAb, the strong signal is delivered via Lck, even if the coreceptors are not engaged. Coreceptor-independent participation of
Lck in mAb-induced TCR signaling has been demonstrated in mature T cell lines lacking these molecules (35), and Lck is not exclusively associated with CD4 and CD8. In fact, CD4 T cell maturation through a strong, Lck-mediated signal may actually be
favored in experiments using TCR-specific mAb and MHC II-deficient mice (22, 23), because the amount of available free Lck is
increased in the absence of constitutive CD4/MHC II
interactions (36).
Even in thymic selection driven by the physiologic engagement
of the TCR by MHC molecules, coreceptor-mediated Lck delivery
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FIGURE 5. Rat thymocytes and lymph node cells express only one
species of CD8a. Lysates were immunoprecipitated with mAb OX-8,
blotted, and probed with the same Ab. Where indicated, precipitates
were treated with endoglycosidase F to improve resolution.
706
OPPOSITE LINEAGE DECISIONS OF RAT AND MOUSE THYMOCYTES
the induction of IL-2Rb expression on all TCR1CD4181 thymocytes by stimulation with TCR-specific mAb and their IL-2 dependent conversion to CD8 T cells indicate that together, these two
signals instruct CD8 lineage decision in rats and argue against
stochastic lineage precommitment in rat CD4181 thymocytes.
The mechanism by which IL-2R signaling promotes differentiation of rat CD8 T cells remains to be analyzed. In addition to the
induction of genes via pathways involving janus-kinases and signal transducers and activators of transcription (39), the activation
of Lck in signal transduction through the IL-2Rb-chain (40, 41)
might support or prolong the hypothetical strong Lck signal guiding CD8 development in rats. In the context of the divergent lineage decisions of rat and mouse CD4181 thymocytes presently
described, it is of interest that IL-2R lacking the a-chain (IL2Rbg) are functional in rats (20) and humans (42), but not in mice
(43). Therefore, rats and humans, but not mice, have the potential
to use signaling through the IL-2Rbg in CD8 commitment. From
this point of view, it is not surprising that an essential contribution
of the IL-2/IL-2R system to mouse CD8 T cell generation has been
excluded by gene targeting (44 – 47), but it may be premature to
generalize this result to other species. Moreover, it is possible that
IL-15, known to employ the IL-2Rbg for signal transduction (48)
and to be produced by thymic epithelial cells (49), is of relevance
in vivo, rather than IL-2.
In the system of in vitro differentiation of rat virgin CD4181
thymocytes (18) presently employed, a homogeneous population
of synchronously differentiating cells that has not received TCRmediated selection signals in vivo is followed over time. Although
lacking the natural microenvironment, this system closely mimics
the in vivo situation with regard to kinetics of constitutive and
inducible expression of cell surface receptors (19) and regulation
of recombination-associated gene-1 and TCR mRNA (H.-J. Park,
and T. Hünig, unpublished observations). Of note, none of the
phenotypic intermediates with regard to coreceptor expression that
have been observed in the mouse thymus and have given rise to
complex models of positive selection and lineage decision (7, 8,
50, 51) are spontaneously formed even during prolonged culture of
virgin CD4181 cells in vitro, suggesting that they are the result of
in vivo interactions with stromal cells. With regard to the kinetics
of CD4/8 commitment, we found that rat virgin CD4181 cells
retain the potential to be converted to a CD4281 cell for several
days, arguing against a default element in lineage decision in
which a cell would spontaneously switch to the opposite lineage
commitment in the absence of an instructing signal (8).
An interesting side aspect of the present work is that in vitro
stimulation of rat virgin CD4181 cells with increasing doses of
immobilized TCR-specific mAb and IL-2 results in a shift from the
CD8ab phenotype characteristic of thymus-derived T cells to the
CD8aa isoform expressed by many gut-associated T cells and by
activated rat CD4 T cells (52). Thus, a supraoptimal TCR-mediated signal may divert CD4181 in cells from their mainstream
differentiation pathway into the CD8aa subset.
In summary, mouse and rat CD4181 cells respond to stimulation with TCR and coreceptor-specific mAb and to PMA/ionomycin treatment with opposite lineage commitment, suggesting that
also under physiologic conditions of repertoire selection, the same
stimulus is interpreted differently. It will be of interest to see
whether, as is presently hypothesized, human and rat CD4181
cells generate a stronger signal in MHC I vs II recognition, and
how similar initial signals may be translated into opposite lineage
choices in the different species analyzed.
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is not an absolute prerequisite, as shown by the low-level thymic
maturation of CD4 T cells, which proceeds in mice expressing
CD4 without the Lck binding cytoplasmic domain (37), and by the
ability of an Lck mutant lacking CD4/8 interactions to promote
thymocyte maturation (38). Although the latter findings appear to
contradict a role of coreceptor-delivered Lck in lineage instruction,
it seems possible that in its absence, higher-affinity receptors are
selected that can initiate the signaling cascade by coreceptor-independent recruitment of Lck.
In mice, a difference in the amount of coreceptor-mediated Lck
delivery to the TCR may be achieved in two ways: an intrinsically
lower capacity of the intracytoplasmic domain of CD8a compared
with that of CD4 to bind this kinase, and the expression of a truncated CD8a isoform (CD8a9) on CD4181 mouse thymocytes. It is
intriguing that these molecules, which make up about 40% of
CD8a, are expressed at the cell surface exclusively in the selectable CD4181 compartment, but are intracellularly degraded in
mature CD8 T cells (15). We speculate that in mouse CD4181
thymocytes, they may act as dominant negative molecules to attenuate CD8-mediated Lck delivery and thereby increase the sensitivity of MHC class discrimination through signal strength. Indeed, in the experiments by Itano and colleagues, diversion of
thymocytes with class I-restricted transgenic TCR into the CD4
subset was not only observed in the presence of a chimeric CD8/4
molecule, but was, to a lesser degree, also facilitated by a CD8a
transgene unable to provide the truncated CD8a splice variant (9).
Our present finding that rat CD4181 thymocytes, which apparently respond to strong signaling with lineage decision toward
CD8, lack the truncated CD8a isoform, is compatible with the
hypothesis that sensing of MHC class through signal strength is
reversed between the two species. In addition to the biochemical
evidence presented, inspection of the CD8a sequence in rats and
humans reveals that in neither of the two species does the potential
exist for the generation of a tail-less CD8a isoform by alternative
splicing. Rather, omission of exon IV, which in mice results in the
formation of CD8a9 lacking both exons IV and V due to a stop
codon generated by frame-shift at the beginning of exon V, would,
in these species, yield very large cytoplasmic tails without recognizable homology to known protein domains (not shown). Therefore, neither rats nor humans attenuate the CD8-dependent Lck
signal in immature thymocytes by a truncated CD8a isoform, and
thus cannot use this hypothetical mechanism of MHC class discrimination in positive selection.
Although the finding that mAbs to the TCR and to CD4 synergize in the generation of rat CD8 T cells in vitro fits well with a
species-specific modification of the signal strength model as outlined above, the additional requirement for IL-2R signaling in that
system does not permit the assignment of the CD8 lineage decision
solely to the intensity of the TCR/coreceptor signal perceived.
Thus, we have previously shown that the in vitro generation of rat
CD8 T cells by stimulation with TCR-specific mAb and IL-2 can
be divided into two steps: first, TCR stimulation leads to downregulation of CD4 and CD8, and de novo expression of the IL2Rb-chain (20) in all TCR1CD4181 thymocytes (21). IL-2 then
rescues these cells and induces up-regulation of CD8, but not CD4,
as well as phenotypic and functional maturation (18, 20). Accordingly, one may argue that TCR stimulation primed TCR1CD4181
thymocytes for positive selection but was neutral with regard to
lineage decision, which was guided toward CD8 by IL-2, but
might have occurred toward CD4 if some different unknown second signal had been provided. Our failure to identify such an instructional second signal for rat CD4 commitment in cytokine
preparations from peripheral lymphoid cells and thymic stromal
cells does, of course, not exclude its existence. On the other hand,
The Journal of Immunology
Acknowledgments
We thank Anneliese Schimpl and Thomas Herrmann for critically reading
the manuscript and Uschi Bommhardt for reminding us of the existence of
CD8a9 in mouse thymocytes.
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