Role of the Actin Cytoskeleton in T Cell
Absorption and Internalization of Ligands
from APC
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J Immunol 2001; 166:5099-5107; ;
doi: 10.4049/jimmunol.166.8.5099
http://www.jimmunol.org/content/166/8/5099
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References
Inkyu Hwang and Jonathan Sprent
Role of the Actin Cytoskeleton in T Cell Absorption and
Internalization of Ligands from APC1
Inkyu Hwang and Jonathan Sprent2
T
cell recognition of Ag on APC rapidly leads to tight conjugate formation and clustering of TCR and other molecules at the site of T-APC interaction (1– 4). At this “synapse,” TCR interaction with MHC-peptide complexes on APC
causes T cell activation and differentiation into effector cells; such
activation generally requires costimulation of T cells via interaction of CD28 with B7 (B7-1, B7-2) on APC (5, 6).
Especially with high-affinity peptides, TCR-MHC-peptide interaction is often followed by TCR down-regulation and internalization (7–9). Until recently, TCR down-regulation was presumed to
depend on TCR dissociation from MHC-peptide complexes. However, it is well established that TCR-MHC-peptide interaction can
cause T cells to rapidly absorb MHC and other molecules, including B7, from APC (10 –16). More recently, it has been shown that
T cell absorption of molecules from APC is followed by internalization of these molecules (15, 16). Hence, TCR down-regulation
may reflect internalization of intact TCR-MHC-peptide complexes. Surprisingly, T cell absorption and internalization of molecules from APC can also occur via CD28-B7 interaction, although not via LFA-1-ICAM-1 interaction (16).
The significance of T cell absorption and internalization of ligands derived from APC is uncertain. With high concentrations of
specific peptide, T cell absorption of MHC-peptide complexes
from APC renders T cells sensitive to fratricidal lysis by neighboring CTL (15). This mechanism could explain the exhaustion of
T cells responding to high concentrations of viruses (17, 18) and
may serve to guard against immunopathology resulting from overexuberant immune responses. Alternatively, T cell internalization
Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037
Received for publication December 7, 2000. Accepted for publication February
15, 2001.
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.
1
This work was supported by Grants CA38355, AI21487, AI46710, and AG01743
from the U.S. Public Health Service. This is Publication 13671-IMM from The
Scripps Research Institute.
2
Address correspondence and reprint requests to Dr. Jonathan Sprent, Department of
Immunology, IMM4, The Scripps Research Institute, 10550 North Torrey Pines
Road, La Jolla, CA 92037. E-mail address: [email protected]
Copyright © 2001 by The American Association of Immunologists
of APC-derived ligands could be important for allowing T/APC
dissociation, thus permitting effector T cells to leave lymphoid
tissues and enter the circulation (16).
The form in which APC-derived molecules are absorbed by T
cells is still unclear. In this respect it is notable that absorption
includes bystander molecules on APC (16). Thus, when CD28⫺/⫺
TCR-transgenic T cells are cultured with APC, addition of specific
peptide causes the T cells to absorb (and internalize) not only the
specific ligand recognized, i.e., MHC molecules, but also adjacent
molecules on the APC, e.g., B7. Likewise, CD28-mediated uptake
of B7 from APC (which is TCR independent) causes T cell uptake
of both B7 and MHC molecules. It is likely, therefore, that T cells
absorb APC-derived molecules as membrane fragments or vesicles. Whether these fragments/vesicles then fuse with the T cell
membrane has yet to be studied.
In this paper, we investigated whether T cell absorption of molecules from APC requires direct T-APC interaction or can occur via
uptake of cell fragments. Using latrunculin B (Lat B)3 (19, 20), we
also examined the role of the cytoskeleton in T cell absorption and
ligand internalization. The results show that the requirements for T
cell absorption to the cell surface are highly complex and depend on
the status of the T cells (resting vs activated), the T cell molecules
involved (TCR vs CD28), and, for TCR-mediated absorption, the
concentration of peptide on the APC. Likewise, T cell absorption
requires the cytoskeleton in some situations but not in others. By
contrast, the cytoskeleton plays a crucial role in ligand internalization.
Materials and Methods
Animals
C57BL/6J (B6) mice were purchased from The Jackson Laboratory (Bar
Harbor, ME). CD28⫺/⫺ mice (21) (originally purchased from The Jackson
Laboratory), 2C ␣␤ TCR-transgenic mice (22), and 2C.CD28⫺/⫺ mice
were bred at The Scripps Research Institute (La Jolla, CA). B7-2⫺/⫺ mice
were provided by Arlene Sharp (Harvard Medical School, Boston, MA)
(23) and maintained at The Scripps Research Institute. B7-2⫺/⫺CD28⫺/⫺
double knockout mice were bred at The Scripps Research Institute. All
mice were on a B6 background except for B7-2⫺/⫺ mice, which were on
a 129 background.
3
Abbreviations used in this paper: Lat B, latrunculin B; DC, dendritic cells; CD,
cytochalasin D.
0022-1767/01/$02.00
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A feature of T-APC interaction is that, via either TCR or CD28, T cells can absorb molecules from APC on to the cell surface and
then internalize these molecules. Here, using both normal and TCR-transgenic T cells, we investigated the mechanism of T cell
absorption of molecules from APC and the role of the cytoskeleton. The results show that although activated T cells could absorb
APC molecules in the form of cell fragments, uptake of molecules by resting T cells required direct T-APC interaction. Based on
studies with latrunculin B, surface absorption of molecules by resting T cells was crucially dependent upon the actin cytoskeleton
for both CD28- and TCR-mediated absorption. Significantly, however, TCR-mediated absorption became strongly resistant to
latrunculin B when the concentration of MHC-bound peptide on APC was raised to a high level, implying that the actin cytoskeleton is only important for absorption when the density of receptor/ligand interaction is low. By contrast, in all situations tested,
the actin cytoskeleton played a decisive role in controlling T cell internalization of ligands from the cell surface. The Journal of
Immunology, 2001, 166: 5099 –5107.
5100
Cell lines and media
Drosophila APC were generated and maintained as described previously (24).
RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 2 mM
glutamine, 5 ⫻ 10⫺5 M 2-ME, and antibiotics was used for T cell culture.
Peptides
QL9 (QLSPFPFDL) and P1A (LPYLGWLVF) peptides were synthesized
and purified as described previously (24). Ld-expressing APC were loaded
with the peptides for 2 h at room temperature before use. For measuring
CD28-mediated uptake of Ld, Ld expression on APC was stabilized by
addition of 10 ␮M P1A peptide; this peptide binds strongly to Ld, but is not
recognized by 2C TCR molecules.
Cell purification
Preparation of cell sonicates
meabilized with saponin (0.05% in PBS) for 10 min. The fixed cells were
treated with a blocking solution (1% BSA and 5% horse serum in PBS) for
1 h at room temperature and stained with mAbs for 30 min at room temperature. For staining for CD8, B7-1, B7-2, and IAb, directly conjugated
mAbs (Cy5-CD8, FITC-B7-1, FITC-B7-2, and Alexa 568-MHC class II
mAbs) were used. For Ld staining, Rhodamine Red X-conjugated donkey
anti-mouse polyclonal Ab was used as a secondary Ab. The stained cells
were observed using an Axiovert S100 TU inverted microscope (Zeiss,
New York, NY). LaserSharp (Bio-Rad, Hercules, CA) software was used
for creation and analysis of the images.
Results
To examine the requirements for T cells to absorb molecules from
APC, purified lymph node T cells were cultured with intact APC,
either in direct contact with APC or separated from APC in Transwells; in some experiments T cells were cultured with APC sonicates. After culture, T cells were stained for APC-derived molecules and then analyzed by or examined by confocal microscopy.
For most experiments, transfected Drosophila cells expressing
B7-1, ICAM-1, and MHC class I Ld molecules (B7-1.ICAM-1.Ld
APC) were used as a source of APC. T cells were prepared from
normal B6 mice or TCR-transgenic mice on a B6 background. Since
B6 (H2b) T cells are B7-1⫺ ICAM-1⫹ Ld⫺, we examined T cell
absorption of B7-1 and Ld molecules from APC; previous work with
Sonicates of APC were prepared by using a Misonix sonicator (Misonix,
Farmingdale, NY). Drosophila APC (5 ⫻ 106/ml) were subjected four
times to a 5-s pulse of sonication (power 2, 15% input). Complete disruption of the cells was monitored by observing the sonicated cell suspension
under the microscope.
Culture conditions
For the experiments using intact APC, 100 ␮l of purified resting T cells
(8 ⫻ 106/ml) or preactivated T cells (5 ⫻ 106/ml) was incubated with 100
␮l of APC (5 ⫻ 106/ml) in a 96-well plate for the indicated period of time.
To ensure immediate contact among T cells and APC, the plates were
briefly spun at low rpm (200 ⫻ g for 15 s). For the experiments using
sonicates, 100 ␮l of resting (8 ⫻ 106/ml) or activated (5 ⫻ 106) T cells was
incubated in a 96-well plastic tissue culture plate with 100 ␮l of sonicates
prepared from the same number of APC as when intact APC were used. For
the experiments using Transwells (Costar, Corning, NY), 200 ␮l of resting
(8 ⫻ 106/ml) or preactivated (5 ⫻ 106/ml) T cells was added to a 24-well
plate. Then, a Transwell (3-␮m pore size, 6.5-mm diameter; Costar) was
inserted, inside of which 200 ␮l of APC (1.5 ⫻ 107/ml) was added. For the
experiments using Lat B (Calbiochem, CA), T cells were pretreated with
the indicated dose of Lat B for 1 h at 37°C before use. Lat B was also kept
in the culture during incubation of T cells with APC.
Abs and flow cytometric analysis
Anti-Ld (30-5-7) (27), anti-CD8 (YTS-169), and anti-MHC class II (M5/
114) mAbs were prepared in ascites form in our laboratory. FITC-conjugated anti-CD4 (L3T4), anti-B7-1 (16-10A1), anti-B7-2 (GL1), anti-TCR
␤ (H57-597), PE-conjugated anti-B7-1 (16-10A1), anti-B7-2 (GL1), antiIAb (AF6-120.1), and anti-CD28 (37.51) mAbs were purchased from
PharMingen (San Diego, CA). PE-conjugated donkey anti-mouse IgG(H ⫹
L) (catalogue no. 715-116-151) and Rhodamine Red-X-conjugated donkey
anti-mouse IgG(H ⫹ L) (catalogue no. 715-295-151) were purchased from
Jackson ImmunoResearch (West Grove, PA). Cy-5-conjugated anti-CD8
(YTS.169) and Alexa 568-conjugated anti-MHC class II (M5/114) mAbs
were prepared in our laboratory by using Cy-5 (Amersham, Arlington
Heights, IL) and Alexa 568 protein labeling kit (Molecular Probes, Eugene,
OR), respectively. For staining of Lat B-treated cells, Lat B was included
during the staining procedure and was also present in the wash buffer. A
FACSCalibur (Becton Dickinson, Mountain View, CA) was used for flow
cytometry, and CellQuest (Becton Dickinson) was used for data analysis.
Laser scanning confocal microscopy
Specimens for confocal microscopy were prepared as described previously
(16). Briefly, 100 ␮l of Lat B-treated and nontreated resting or preactivated
T cells (8 ⫻ 106/ml) was incubated with 100 ␮l of Drosophila APC (8 ⫻
106/ml) or DC (8 ⫻ 106) on poly-L-lysine-coated coverslips in a 37°C
humidified CO2 incubator for the indicated period of time. After incubation, cells on the coverslips were sequentially fixed with freshly prepared
paraformaldehyde (2% in PBS) for 30 min at room temperature and per-
FIGURE 1. CD28-mediated T cell absorption of molecules from APC following direct culture of T cells and APC or separation of T cells and APC in
Transwells. Purified resting (A) or preactivated (B) normal B6 T cells were
cultured for 60 min at 37°C either alone or with Ld.B7-1.ICAM-1 Drosophila
APC; with APC, T cells were either in direct contact with APC or separated
in Transwells (pore size, 3 ␮m). In parallel, T cells were cultured with sonicates of the Drosophila APC. Before culture with T cells, Ld molecules on
Drosophila APC were stabilized by addition of P1A peptide (10 ␮M). After
culture of T cells and APC, cells were stained for CD4, CD8, B7-1, and Ld and
analyzed by FACS. The data show staining for B7-1 and Ld (thick lines) on
gated CD4⫹ and CD8⫹ cells; background staining with an isotype-matched
control mAb is also shown (thin lines). T/APC conjugates were gated out on
the basis of forward and side scatter.
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T cells were purified by sequential mAb plus C killing as described previously
(25). Briefly, total lymph node cells were treated with anti-HSA (J11D) and
anti-IAb (28-16-8s) mAbs plus a combination of guinea pig and rabbit complement for 40 min at 37°C followed by Ficoll gradient centrifugation. For the
purification of 2C CD8⫹ T cells, anti-CD4 (RL174) mAb was included in the
killing process along with J11D and 28-16-8s mAbs. Preactivated T cells were
prepared by culturing purified T cells overnight with PMA (10 nM; Sigma)
and ionomycin (370 ng/ml; Sigma) in a 37°C humidified CO2 incubator. Dendritic cells (DC) were prepared as described previously (16, 26).
T CELL ABSORPTION OF LIGANDS FROM APC
The Journal of Immunology
5101
T cells from ICAM-1⫺/⫺ mice showed that in addition to B7 and
MHC molecules, T cells can absorb ICAM-1 from APC (16).
CD28-mediated absorption
As documented previously, T cell absorption of molecules from
APC involves at least two receptor-ligand interactions, i.e.,
CD28-B7 and TCR-MHC-peptide interactions (15, 16). With culture of polyclonal normal B6 T cells, the absorption of molecules
from APC is largely a reflection of CD28-B7 interaction, with little
or no involvement of TCR molecules.
In confirmation of previous findings (16), short-term (60-min)
culture of normal B6 T cells with B7-1.ICAM-1.Ld Drosophila
APC led to prominent absorption of both B7-1 and Ld molecules
from the APC as detected by FACS analysis (Fig. 1). Such absorption applied to both CD4⫹ cells (Fig. 1) and CD8⫹ cells (data
not shown) and affected resting T cells (Fig. 1A) as well as preactivated T cells (T cells exposed to PMA and ionomycin; Fig.
1B). Confirming the crucial role of CD28 in absorption, T cell
uptake of both B7-1 and Ld from APC was almost undetectable
with CD28⫺/⫺ T cells (Fig. 1B). This finding implies that for absorption, CD28 binds B7-1 molecules that are physically linked to
Ld molecules. It should be noted that uptake of B7-1 and Ld molecules from APC was substantially higher with activated T cells
than with naive T cells (compare Fig. 1, A vs B) As discussed
previously (16, 28, 29), this finding correlates with the much
higher expression of CD28 on activated T cells.
A priori, T cell absorption of molecules from APC could reflect
either direct T-APC interaction or uptake of small membrane fragments released from APC. To examine this second possibility, T
cells were separated from APC in Transwell plates or cultured with
APC sonicates. With resting T cells, absorption of B7-1 and Ld
through the Transwell membrane was almost undetectable, even
with a relatively large pore size of 3 ␮m (Fig. 1A). Likewise,
uptake of these molecules was very low following T cell culture
with APC sonicates (Fig. 1A). In marked contrast, activated T cells
showed prominent absorption of B7-1 and Ld molecules in Transwells and also with APC sonicates (Fig. 1B); for Transwells, absorption required a high density of APC, i.e., 3-fold higher than for
direct T-APC interaction. In both situations, absorption was low or
undetectable with CD28⫺/⫺ T cells.
To examine the role of the cytoskeleton in T cell absorption of
APC-derived molecules, T cells were cultured with APC in the
presence of Lat B, an inhibitor of actin polymerization (19, 20).
For resting T cells, addition of Lat B at 20 ␮g/ml during direct
T-APC interaction almost totally inhibited T cell binding of Ld and
B7-1 (Figs. 2A and 3A); this finding applied to binding measured
at 10 – 60 min of culture (Fig. 3A) and was observed even with
quite low doses of Lat B, i.e., 0.25 ␮g/ml (Fig. 2B). Different
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FIGURE 2. Capacity of Lat B to inhibit CD28-mediated T cell absorption of molecules from Drosophila
APC. As in Fig. 1, normal resting (A and B) or preactivated (C) B6 T cells were cultured with Drosophila APC,
either directly or in Transwells; Lat B was added to the
cultures at 20 ␮g/ml (A and C) or in titrated concentrations (B). Cells were stained, as described in Fig. 1, after
culture for 60 min. The data show staining for B7-1 and
Ld on gated CD4⫹ and CD8⫹ cells. B, The data are
shown as the change in mean fluorescence intensity (⌬
MFI), i.e., as MFI values obtained after subtracting the
background MFI observed with an isotype-matched control mAb.
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T CELL ABSORPTION OF LIGANDS FROM APC
B7-2, and IAb after short-term, direct culture with B6 DC was
strong for B7-2⫺/⫺ CD28⫹ T cells, but weak, although significant,
for B7-2⫺/⫺ CD28⫺/⫺ T cells (Fig. 4A), indicating a marked, although not exclusive, role for CD28 in absorption from DC. Significantly, as with Drosophila APC, T cell uptake of molecules
from DC was almost totally inhibited by Lat B (Fig. 4A). This
finding refers to resting T cells. With activated T cells (Fig. 4B),
Lat B caused only partial inhibition of absorption, i.e., as for activated T cells and Drosophila APC. When activated T cells and
DC were separated in Transwells, by contrast, T cell absorption of
molecules from DC (which was lower than with Drosophila APC;
see Discussion) was totally inhibited by Lat B (Fig. 4C). Collectively, these data with DC as APC were thus in close accord with
the data with Drosophila APC.
TCR-mediated absorption
findings occurred with preactivated T cells (Figs. 2C and 3A). With
these T cells, even high doses of Lat B (20 ␮g/ml) caused only
partial inhibition of T cell binding following direct T-APC interaction. By contrast, Lat B strongly inhibited T cell binding in
Transwell cultures (Figs. 2C and 3B).
The above findings make two main points. First, whereas both resting and activated T cells can absorb B7-1 and Ld molecules through
direct contact with intact APC, only activated T cells absorb these
molecules when cultured with cell fragments. Second, based on the
effects of adding Lat B, both resting and activated T cells require actin
polymerization for CD28-mediated binding of APC-derived molecules; for activated T cells, however, the requirement for actin polymerization is much more stringent for absorption of cell fragments
than for absorption following direct T-APC interaction.
To examine whether the above findings with Drosophila APC
apply to normal APC, T cells were cultured with syngeneic B6 DC
as APC; the T cells were then stained for three molecules on the
APC, i.e., B7-1, B7-2, and IAb. Since T cells are B7-1⫺ B7-2⫹
IA⫺, the T cells were prepared from B7-2⫺/⫺ mice; these mice
were on a 129 (H2b) background. B7-2⫺/⫺ CD28⫺/⫺ T cells were
used as a control. For resting CD4⫹ cells, absorption of B7-1,
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FIGURE 3. Influence of culture time on the capacity of Lat B to inhibit
CD28-mediated T cell absorption of B7-1 and Ld molecules from Drosophila APC. Normal resting or activated B6 T cells were cultured with or
without Lat B (20 ␮g/ml) with Drosophila APC either directly (A) or in
Transwells (B), as described in the legend to Fig. 1, and then stained at 10,
30, or 60 min (A) or after 60 min (B). The data show the change in mean
fluorescence intensity (⌬ MFI) for staining of CD4⫹ cells for B7-1 and Ld.
Because of prominent absorption of APC-derived molecules via
CD28-B7 interaction, examining the role of TCR-MHC-peptide
interaction in absorption is best studied with TCR transgenic T
cells on a CD28⫺/⫺ background. Confirming previous findings
(16), direct culture of resting CD28⫺/⫺ 2C-transgenic CD8⫹ cells
(H2b) with Ld.B7-1.ICAM-1 Drosophila APC in the presence of
the highly immunogenic Ld-associated QL9 peptide (10 ␮M) led
to strong uptake of both Ld and B7-1 molecules from the APC.
Data for uptake of B7-1 are shown in Fig. 5A; no uptake occurred
with nonimmunogenic P1A peptide (data not shown). As for
CD28-mediated absorption by resting normal B6 T cells (Fig. 1),
uptake of Ld and B7-1 molecules by resting CD28⫺/⫺ 2C CD8⫹
cells required direct T-APC interaction, i.e., only very minimal
uptake occurred when T cells and APC were separated in Transwell plates (see below). In marked contrast to CD28-mediated absorption by normal resting B6 T cells (Figs. 2 and 3), however,
peptide-dependent absorption by resting CD28⫺/⫺ 2C CD8⫹ cells
was only partly inhibited by Lat B (Fig. 5A).
These data refer to addition of QL9 peptide at a high concentration, i.e., 10 ␮M. The effects of titrating the concentration of
peptide on uptake of B7-1 by resting CD28⫺/⫺ 2C cells following
direct T-APC interaction are shown in Fig. 5B. As expected, uptake of B7-1 was proportional to the concentration of QL9 peptide
added (reflecting that B7-1 uptake depends on TCR-Ld-QL9 interaction). With a high concentration of peptide (10 ␮M), inhibition of B7-1 absorption by Lat B was mild, i.e., ⬃50%. With a low
concentration of peptide (0.001 ␮M), by contrast, Lat B totally
blocked B7-1 absorption. Similar results were obtained with preactivated CD28⫺/⫺ 2C cells (Fig. 5, D and E). Note that, as for
CD28-mediated absorption (Fig. 1), peptide-dependent absorption
by CD28⫺/⫺ 2C cells was higher for activated than resting T cells.
The above findings indicated that TCR-mediated uptake of molecules from APC became relatively resistant to Lat B when the concentration of peptide was raised to a high level. These findings applied
to absorption via direct T-APC interaction. With T-APC separation in
Transwells, by contrast, absorption by T cells was totally inhibited by
Lat B even with a high concentration of peptide. This finding applied
to both resting and activated CD28⫺/⫺ 2C cells (Fig. 5, C and F); for
resting T cells, absorption via Transwells was very weak and apparent
only with a high concentration of peptide.
In the above experiments, the use of CD28⫺/⫺ T cells precluded a
possible contribution of CD28 to TCR-mediated absorption. The effects of using normal CD28⫹ 2C cells are shown in Fig. 6. In this
situation, 2C cell coabsorption of B7-1 and Ld from APC in the presence of QL9 peptide presumably involves both CD28-B7 and TCRMHC-peptide interaction; each of these interactions causes direct
binding of the specific ligand and, because of the physical association
between B7-1 and Ld, indirect binding of the other ligand.
The Journal of Immunology
5103
As expected, with CD28⫹ 2C cells direct T-APC interaction
without peptide led to appreciable absorption of B7-1 (Fig. 6A) and
Ld (data not shown), reflecting CD28-B7-mediated absorption;
with activated CD8⫹ 2C cells (Fig. 6D), absorption of B7-1 (and
Ld) was higher than with resting 2C cells (Fig. 6A), reflecting the
much higher density of CD28 on activated T cells. With addition
of QL9 peptide, the absorption of B7-1 (and Ld) increased considerably, paralleling the concentration of peptide added (Fig. 6, A
and D); this applied to both resting and activated 2C cells. Confirming the results with normal T cells (Figs. 2 and 3), addition of
Lat B abolished TCR-independent, CD28/B7-dependent uptake,
i.e., the uptake observed in the absence of QL9 peptide (Fig. 6, A
and D). With addition of QL9 peptide, uptake became Lat B resistant with high concentrations of peptide (Fig. 6, A and D). This
resistance to Lat B was most apparent when mean fluorescence
index values for cells cultured in the absence of peptide were subtracted, i.e., the data were expressed as the change in the mean
fluorescence index induced by addition of peptide (Fig. 6, B and
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FIGURE 4. CD28-mediated absorption by
T cells cultured with syngeneic DC. Purified
resting (A) or activated (B) T cells prepared
from B7-2 knockout (KO) or combined CD28
and B7-2 double-knockout (DKO) 129 mice
were cultured with purified DC prepared from
normal B6 mice. T cells and DC were cultured
either directly (A and B) or, for activated T
cells, separated in Transwells (C). T cells and
DC were cultured for 60 min with or without
Lat B (20 ␮g/ml) and stained for B7-1, B7-2,
and IAb. The data show staining of gated
CD4⫹ cells.
E). By contrast, as for CD28⫺/⫺ cells, absorption by CD28⫹ 2C
cells via Transwells was abolished by Lat B (data not shown).
Direct comparison of TCR-mediated uptake of B7-1 by CD28⫺/⫺
vs CD28⫹ 2C cells following direct T-APC interaction is shown in
Fig. 6, C and F. In the absence of Lat B, it can be seen that peptideinduced B7-1 absorption by CD28⫹ and CD28⫺/⫺ 2C cells was virtually identical for both resting (Fig. 6C) and activated (Fig. 6F) 2C
cells; similar findings applied to the absorption of Ld (data not
shown). However, with addition of Lat B to activated T cells, absorption induced by a high concentration of peptide was appreciably (and
reproducibly) higher with CD28⫹ than CD28⫺/⫺ cells; this difference
was not seen with resting T cells. Thus, CD28 had little, if any, effect
to enhance TCR-mediated absorption, but, if only for activated T
cells, was nevertheless able to promote resistance to Lat B.
Ligand internalization
In previous studies, uptake of APC-derived molecules onto the surface of T cells was shown to be followed by rapid internalization of
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T CELL ABSORPTION OF LIGANDS FROM APC
FIGURE 5. Lat B sensitivity of TCR-mediated absorption of B7-1 from APC by CD28⫺/⫺ 2C cells. A and
D, Purified CD28⫺/⫺ naive (A) or activated (D) 2C
CD8⫹ cells were cultured with or without QL9 peptide
(10 ␮M) plus Ld.B7-1.ICAM-1 Drosophila APC; cells
were cultured with (E) or without (F) Lat B at 20 ␮g/
ml. B and E, Same as for A and D, except that QL9
peptide was added in graded concentrations. C and F,
Same as for B and E, except that T cells and APC were
separated in Transwells. Cells were stained after culture
for 30 min (A, B, D, and E) or 60 min (C and F). The
data show B7-1 staining of gated CD8⫹ cells.
The above findings refer to CD28-mediated absorption. Comparable data applied to TCR-mediated absorption. This is illustrated in Fig. 7, F and G, where culturing either resting (Fig. 7F)
or activated (Fig. 7G) CD28⫺/⫺ 2C CD8⫹ cells with Ld.B71.ICAM-1 Drosophila APC plus a high concentration of QL9 peptide led to both surface and intracellular staining of T cells for Ld
(pink) in the absence of Lat B, but only surface staining in the
presence of Lat B. Similar findings applied to CD28⫹ 2C cells
(data not shown).
Discussion
As described here and previously, T cell absorption of molecules
from APC is receptor (TCR or CD28) specific, evident with both
transfected cell lines (Drosophila cells) and DC as APC, and occurs in vivo as well as in vitro (15, 16); for the molecules studied,
transfer from APC to T cells is unidirectional. Since the molecules
absorbed include not only the specific ligand recognized but also
adjacent bystander molecules on the APC, the molecules are presumably absorbed in the form of cell fragments/vesicles.
As shown here, the absorption of molecules from APC proved
to be most efficient when T cells and APC were in direct contact.
This requirement for direct cell-cell contact was most stringent for
resting T cells. Thus, for both CD28-mediated and TCR-mediated
absorption, uptake of molecules from APC by resting T cells was
clearly apparent after direct T-APC interaction, but was very low
or undetectable when T cells and APC were separated in Transwells or T cells were incubated with APC sonicates. Interestingly,
however, activated T cells behaved differently. Thus, unlike resting T cells, activated T cells were able to absorb molecules from
APC via Transwells and also from APC sonicates; as for direct cell
contact, T cell uptake of this shed material from APC was TCR or
CD28 dependent.
With regard to the mechanism of absorption, in other studies we
have found that absorption of shed material from APC by activated
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the ligands (15, 16). As for absorption to the cell surface, internalization reflected by either CD28-B7 or TCR-MHC-peptide interaction
and applied to both the specific ligand recognized and adjacent ligands on APC. Using confocal microscopy, we examined whether T
cell internalization of ligands from APC was Lat B sensitive.
In all situations examined, T cell internalization of ligands from
APC proved to be highly sensitive to Lat B. The key finding was
that Lat B blocked internalization in situations where Lat B only
partly inhibited absorption on to the cell surface; this applied to
both CD28-mediated and TCR-mediated uptake.
Data for CD28-mediated absorption of B7-1 from Ld.B71.ICAM-1 Drosophila APC by normal activated B6 CD8⫹ cells
are shown in Fig. 7A. In the absence of Lat B, fixation and permeabilization of nondissociated cultures of T cells and APC followed by staining for B7-1 (green) and CD8 (blue) revealed intracellular distribution of B7-1 in the T cells; cultures were
examined after 60 min. In the presence of Lat B, by contrast,
intracellular B7-1 staining of the T cells was undetectable. In these
undisturbed cultures, conjugate formation between T cells and
APC made it difficult to detect B7-1 staining on the T cell surface.
When the cultures were dissociated before staining, B7-1 staining
of T cells in the absence of Lat B was apparent both on the cell
surface and intracellularly (Fig. 7B). With addition of Lat B during
culture, however, B7-1 expression was limited to the cell surface;
note that with Lat B, surface staining of B7-1 on T cells was
blue-green, implying close association with the cell membrane.
For CD28-mediated absorption of B7-1 via Transwells, B7-1
(pink) staining of T cells was apparent both intracellularly and on
the cell surface when cells were cultured without Lat B (Fig. 7C).
With addition of Lat B, B7-1 staining of T cells was undetectable
on the cell surface (confirming the FACS data in Fig. 2) or intracellularly (Fig. 7C). Lat B also blocked internalization of B7-2
(Fig. 7D) and IAb (Fig. 7E) when activated CD28⫹ B7-2⫺/⫺
CD8⫹ cells were cultured with B6 DC.
The Journal of Immunology
5105
FIGURE 6. Lat B sensitivity of TCR-mediated absorption of B7-1 from APC by CD28⫹/⫹ 2C cells. A and
D, Same as for Fig. 5, B and E, except that CD28⫹/⫹ 2C
cells were used; as in Fig. 5, cells were cultured with (䡺)
or without (f) Lat B (20 ␮g/ml). B and E, Data from A
and D plotted as the change in mean fluorescence intensity (⌬ MFI) induced by addition of peptide. C and F, A
comparison of the data for CD28⫺/⫺ (F and E) vs
CD28⫹/⫹ (f and 䡺) 2C cells (Fig. 5, B and E, vs Fig. 6,
B and E).
question of whether this sequence of events involves the actin
cytoskeleton. Since the cytoskeleton controls receptor-mediated
endocytosis of soluble molecules, e.g., transferrin (36), and is also
important for T cell activation by peptide-pulsed APC (8, 37, 38),
we expected the actin cytoskeleton to play a key role in both T cell
absorption and internalization of molecules from APC. For internalization of ligands, this was indeed the case. Thus, in all situations tested Lat B totally inhibited internalization of ligands from
APC. For absorption of APC molecules onto the surface of T cells,
however, the cytoskeleton was required in some situations but not
in others. Here, for direct T-APC interaction, two factors were
important, 1) the activation status of the T cells, and 2) in the case
of TCR-mediated absorption, the concentration of peptide added to
APC. To address these points, CD28- and TCR-mediated absorptions need to be considered separately.
For T cell uptake via CD28-B7 interaction, absorption was highly
sensitive to Lat B for resting T cells, but was comparatively resistant
for activated T cells. This difference could reflect greater mobility of
CD28 in the membrane of activated than resting T cells. The possibility we favor, however, is that Lat B sensitivity is largely a reflection
of CD28 density, the expression of CD28 being much higher on activated than resting T cells (5, 29). For resting cells, the low density
of CD28 on these cells may severely limit contact with B7 on APC
unless CD28 molecules are drawn into the T/APC contact site, presumably by an actin-dependent mechanism. For activated T cells, by
contrast, mobilization of CD28 may be largely unnecessary because
the pre-existing local density of CD28 at the contact site is sufficiently
high to mediate B7 absorption; this model does not exclude the possibility that the CD28 molecules at the contact site form actin-independent microclusters (see below).
The effects of Lat B on absorption mediated by TCR-MHCpeptide interaction were unexpected. Here, the notable finding was
that sensitivity of absorption to Lat B was inversely related to
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T cells is under the critical control of LFA-1-ICAM-1 interaction
and occurs only with ICAM-1⫹ APC (our unpublished observations). Thus, in contrast to the Ld.B7-1.ICAM-1 Drosophila APC
used here, TCR- or CD28-mediated absorption from Drosophila
APC expressing Ld and B7-1 but lacking ICAM-1 required direct
T-APC interaction and did not occur via Transwells or from APC
sonicates. This finding indicates that LFA-1-ICAM-1 interaction is
not required for absorption following direct T-APC interaction, but
is crucial for absorption of shed material from APC. Presumably,
T cell binding of shed material is relatively weak, and for both
TCR-and CD28-mediated absorption, such binding has to be stabilized by LFA-1-ICAM-1 interaction. In other systems it has been
shown that the affinity of LFA-1 for ICAM-1 increases after T cell
activation (30 –32). Hence, for absorption of shed molecules from
APC, the much stronger absorption by activated than resting T
cells could be largely a reflection of enhanced LFA-1-ICAM-1
interaction by activated T cells. Note that T cell absorption of shed
molecules from APC has a precedent in the old observation that T
cells activated to MHC alloantigens were able to absorb these Ags
from APC sonicates (33, 34).
It should be emphasized that for activated T cells, absorption of
shed material from APC was much less than during direct T-APC
interaction. Nevertheless, absorption of shed material from APC
could be physiologically relevant because peptide-pulsed DC are
reported to shed exosomes, which are highly immunogenic in vivo
(35). Interestingly, exosome release in this study was much higher
with immature than mature DC. This finding might account for
why in our studies T cell absorption via Transwells was much
lower with DC as APC than with Drosophila cells. Thus, mature
DC were used in these studies, and we have yet to examine absorption from immature DC.
The observation that T cell uptake of APC-derived molecules is
followed by rapid internalization of these molecules raised the
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T CELL ABSORPTION OF LIGANDS FROM APC
peptide concentration. Thus, absorption was strongly Lat B sensitive
with low concentrations of peptide, but was relatively resistant with
high concentrations. The implication, therefore, is that the actin
cytoskeleton does control TCR-mediated absorption, but only when
the concentration of MHC-bound peptide on APC is relatively low.
On this point there are close similarities with the role of the actin
cytoskeleton in T cell activation. Thus, for T cell clones cultured with
peptide-pulsed APC, a rise in the intracellular Ca2⫹ concentration and
IFN-␥ production can be inhibited by cytochalasin D (CD), a drug
that, like Lat B, disrupts the actin cytoskeleton (37). Significantly,
however, the inhibitory effects of CD on T cell activation are much
less apparent with high concentrations of peptide.
In considering these findings, it is notable that although CD
inhibits stable “synapses” at the site of T/APC interaction, CD
does not block the formation of small clusters of molecules (CD3␨)
at the contact site (39). Hence, with a high concentration of peptide
on APC, actin-independent microclustering of TCR molecules interacting with a high density of MHC/peptide at the T/APC contact
site may be sufficient to induce T cell activation and also to allow
TCR-dependent absorption from the APC. Thus, under these particular conditions the actin cytoskeleton is not required.
This scenario could also apply to CD28-B7 interaction mediated
by activated T cells (see above). However, the situation may be
different for LFA-1-ICAM-1 interaction. As discussed earlier,
LFA-1-ICAM-1 interaction is largely unnecessary for absorption
following direct T-APC interaction, but is crucial for absorption of
shed material from APC. For absorption of shed material, there is
thus an interesting correlation between the requirement for LFA1-ICAM-1 interaction and the marked sensitivity to Lat B. To explain this correlation, one could postulate that LFA-1-ICAM-1 interaction is crucially dependent upon LFA-1 associating with the
actin cytoskeleton. Indirect support for this idea is provided by the
finding that treating T cells (but not APC) with CD prevents redistribution of ICAM-1 on APC during T-APC interaction (40).
As described above, there are striking parallels between the role of
the actin cytoskeleton in T cell activation and TCR-mediated absorption to the cell surface. An obvious question here is whether T cell
absorption (and internalization) of MHC and other molecules from
APC plays a significant role in T cell triggering. This possibility
seems unlikely in view of the evidence that T cells can be triggered in
situations where T cell absorption of MHC/peptide presumably cannot occur, e.g., when T cells are stimulated by MHC/peptide conjugated to beads or plastic (38, 41). It is also notable that CD28 plays a
crucial costimulatory role in T cell triggering, but does not seem to be
required for TCR-mediated absorption. Thus, peptide-induced absorption of B7-1 and Ld molecules from APC was not higher with CD28⫹
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FIGURE 7. Effects of Lat B on T cell internalization
of molecules absorbed from APC. A, Activated normal
B6 T cells were incubated with or without Lat B (20 ␮g/
ml) with Ld.B7-1.ICAM-1 Drosophila APC on poly-Llysine-coated coverslips for 1 h before staining for B7-1
(green) and CD8 (blue). B and C, Cells were cultured as
described in A, but either in direct contact with APC (B)
or separated from APC in Transwells (C). After culture,
dissociated T cells were allowed to settle on coverslips for
15 min, then fixed and permeabilized for staining for B7-1
(green for B, pink for C) and CD8 as in A. B, T cells
cultured without APC are shown as a control. D and E,
Activated 129.B7-2⫺/⫺ CD8⫹ cells were cultured directly
with B6 DC with or without Lat B and then, after fixation,
stained for B7-2 (D, green) and CD8 (D, blue) or IAb (E,
pink) and CD8 (E, blue). F, Resting CD28 ⫺/⫺ 2C CD8⫹
cells with or without Lat B were cultured on coverslips
with Ld.B7-1.ICAM-1 Drosophila plus QL9 peptide (10
␮M) for 30 min, then fixed and stained for Ld (red) and
CD8 (blue). G, Left, same as for F except that activated
CD28⫺/⫺ 2C CD8⫹ cells were used. G, Right, same as for
G, left, except that T cells were cultured in suspension in
a 96-well plate for 30 min before cell dissociation and
transfer to coverslips, followed by fixation and staining
for Ld.
The Journal of Immunology
Acknowledgments
We thank Dr. Arlene Sharp (Harvard Medical School) for kindly providing
the B7-2 knockout mice and Barbara Marchand for typing this manuscript.
References
1. Dusin, M. L., and J. A. Cooper. 2000. The immunological synapse and the actin
cytoskeleton: molecular hardware for T cell signaling. Nat. Immunol. 1:23.
2. Monks, C. R., B. A. Freiberg, H. Kupfer, N. Sciaky, and A. Kupfer. 1998. Threedimensional segregation of supramolecular activation clusters in T cells. Nature
395:82.
3. Wulfing, C., and M. M. Davis. 1998. A receptor/cytoskeletal movement triggered
by costimulation during T cell activation. Science 282:2266.
4. Viola, A., S. Schroeder, Y. Sakakibara, and A. Lanzavecchia. 1999. T lymphocyte costimulation mediated by reorganization of membrane microdomains. Science 283:680.
5. Lenschow, D. J., T. L. Walunas, and J. A. Bluestone. 1996. CD28/B7 system of
T cell co-stimulation. Annu. Rev. Immunol. 14:233.
6. Cai, Z., A. B. Brunmark, A. T. Luxembourg, K. C. Garcia, M. Degano, L. Teyton,
I. Wilson, P. A. Peterson, J. Sprent, and M. R. Jackson. 1998. Probing the activation requirements for naive CD8⫹ T cells with Drosophila cell transfectants as
antigen presenting cells. Immunol. Rev. 165:249.
7. Valitutti, S., S. Muller, M. Dessing, and A. Lanzavecchia. 1996. Different responses are elicited in cytotoxic T lymphocytes by different levels of T cell
receptor occupancy. J. Exp. Med. 183:1917.
8. Cai, Z., H. Kishimoto, A. Brunmark, M. R. Jackson, P. A. Peterson, and J. Sprent.
1997. Requirements for peptide-induced TCR downregulation on naive CD8⫹ T
cells. J. Exp. Med. 185:641.
9. San Jose, E., A. Borroto, F. Niedergang, A. Alcover, and B. Alarcon. 2000.
Triggering the TCR complex causes the downregulation of nonengaged receptors
by a signal transduction-dependent mechanism. Immunity 12:161.
10. Hudson, L., and J. Sprent. 1976. Specific adsorption of IgM antibody onto H-2activated mouse T lymphocytes. J. Exp. Med. 143:444.
11. Nagy, Z., B. E. Elliott, and M. Nabholz. 1976. Specific binding of K- and I-region
products of the H-2 complex to activated thymus-derived (T) cells belonging to
different Ly subclasses. J. Exp. Med. 144:1545.
12. Sharrow, S. O., B. J. Mathieson, and A. Singer. 1981. Cell surface appearance of
unexpected host MHC determinants on thymocytes from radiation bone marrow
chimeras. J. Immunol. 126:1327.
13. Lorber, M. I., M. R. Loken, A. M. Stall, and F. W. Fitch. 1982. I-A antigens on
cloned alloreactive murine T lymphocytes are acquired passively. J. Immunol.
128:2798.
14. Patel, D. M., P. Y. Arnold, G. A. White, J. P. Nardella, and M. D. Mannie. 1999.
Class II MHC/peptide complexes are released from APC and are acquired by T
cell responders during specific antigen recognition. J. Immunol. 163:5201.
15. Huang, J.-F., Y. Yang, H. Sepulveda, W. Shi, I. Hwang, P. A. Peterson,
M. R. Jackson, J. Sprent, and Z. Cai. 1999. TCR-mediated internalization of
peptide-MHC complexes acquired by T cells. Science 286:952.
16. Hwang, I., J.-F. Huang, H. Kishimoto, A. Brunmark, P. A. Peterson,
M. R. Jackson, C. D. Surh, Z. Cai, and J. Sprent. 2000. T cells can use either TCR
or CD28 receptors to absorb and internalize cell-surface molecules derived from
antigen-presenting cells. J. Exp. Med. 191:1137.
17. Pircher, H., K. Burki, R. Lang, H. Hengartner, and R. M. Zinkernagel. 1989.
Tolerance induction in double specific T cell receptor transgenic mice varies with
antigen. Nature 342:559.
18. Moskophidis, D., F. Lechner, H. Pircher, and R. Zinkernagel. 1993. Virus persistence in acutely infected immunocompetent mice by exhaustion of antiviral
cytotoxic effector T cells. Nature 362:758.
19. Spector, I., N. R. Shochet, Y. Kashman, and A. Groweiss. 1983. Latrunculins:
novel marine toxins that disrupt microfilament organization in cultured cells.
Science 219:493.
20. Spector, I., N. R. Shochet, D. Blasberger, and Y. Kashman. 1989. Latrunculins:
novel marine macrolides that disrupt microfilament organization and affect cell
growth. I. Comparison with cytochalasin D. Cell. Motil. Cytoskeleton 13:127.
21. Shahinian, A., K. Pfeffer, K. P. Lee, T. M. Kundig, K. Kishihara, A. Wakeham,
K. Kawai, P. S. Ohashi, C. B. Thompson, and T. W. Mak. 1993. Differential T
cell costimulatory requirements in CD28-deficient mice. Science 261:609.
22. Sha, W. C., C. A. Nelson, R. D. Newberry, D. M. Kranz, J. H. Russell, and
D. Y. Loh. 1988. Positive and negative selection of an antigen receptor on T cells
in transgenic mice. Nature 336:73.
23. Borriello, F., M. P. Sethna, S. D. Boyd, A. N. Schweitzer, E. A. Tivol, D. Jacoby,
T. B. Strom, E. M. Simpson, G. J. Freeman, and A. H. Sharpe. 1997. B7-1 and
B7-2 have overlapping, critical roles in immunoglobulin class switching and
germinal center formation. Immunity 6:303.
24. Cai, Z., A. Brunmark, M. R. Jackson, D. Loh, P. A. Peterson, and J. Sprent. 1996.
Transfected Drosophila cells as a probe for defining the minimal requirements for
stimulating unprimed CD8⫹ T cells. Proc. Natl. Acad. Sci. USA 93:14736.
25. Cai, Z., and J. Sprent. 1994. Resting and activated T cells display different requirements for CD8 molecules. J. Exp. Med. 179:2005.
26. Inaba, K., M. Pack, M. Inaba, H. Sakuta, F. Isdell, and R. M. Steinman. 1997.
High levels of a major histocompatibility complex II-self peptide complex on
dendritic cells from the T cell areas of lymph nodes. J. Exp. Med. 186:665.
27. Hansen, T. H., N. B. Myers, and D. R. Lee. 1988. Studies of two antigenic forms
of Ld with disparate ␤2-microglobulin (␤2m) associations suggest that ␤2m facilitates the folding of the ␣1 and ␣2 domains during de novo synthesis. J. Immunol. 140:3522.
28. Gause, W. C., V. Mitro, C. Via, P. Linsley, J. F. Urban, Jr., and R. J. Greenwald.
1997. Do effector and memory T helper cells also need B7 ligand costimulatory
signals? J. Immunol. 159:1055.
29. Turka, L. A., J. A. Ledbetter, K. Lee, C. H. June, and C. B. Thompson. 1990.
CD28 is an inducible T cell surface antigen that transduces a proliferative signal
in CD3⫹ mature thymocytes. J. Immunol. 144:1646.
30. van Kooyk, Y., P. van de. Wiel-van Kemenade, P. Weder, T. W. Kuijpers, and
C. G. Figdor. 1989. Enhancement of LFA-1-mediated cell adhesion by triggering
through CD2 or CD3 on T lymphocytes. Nature 342:811.
31. Dustin, M. L., and T. A. Springer. 1989. T-cell receptor cross-linking transiently
stimulates adhesiveness through LFA-1. Nature 341:619.
32. Lub, M., Y. van Kooyk, and C. G. Figdor. 1995. Ins and outs of LFA-1. Immunol.
Today 16:479.
33. Elliott, B. E., Z. Nagy, M. Nabholz, and B. Pernis. 1977. Antigen recognition by
T cells activated in the mixed lymphocyte reaction: specific binding of allogeneic
cell material after removal of surface-bound antigen by trypsin. Eur. J. Immunol.
7:287.
34. Elliott, B. E., B. Takacs, and Z. Nagy. 1979. Specific binding of radiolabeled
membrane vesicles by T cells activated in the mixed lymphocyte reaction. Eur.
J. Immunol. 9:646.
35. Zitvogel, L., A. Regnault, A. Lozier, J. Wolfers, C. Flament, D. Tenza,
P. Ricciardi-Castagnoli, G. Raposo, and S. Amigorena. 1998. Eradication of established murine tumors using a novel cell-free vaccine: dendritic cell-derived
exosomes. Nat. Med. 4:594.
36. Lamaze, C., L. M. Fujimoto, H. L. Yin, and S. L. Schmid. 1997. The actin
cytoskeleton is required for receptor-mediated endocytosis in mammalian cells.
J. Biol. Chem. 272:20332.
37. Valitutti, S., M. Dessing, K. Aktories, H. Gallati, and A. Lanzavecchia. 1995.
Sustained signaling leading to T cell activation results from prolonged T cell
receptor occupancy: role of T cell actin cytoskeleton. J. Exp. Med. 181:577.
38. Luxembourg, A. T., A. B. Brunmark, Y. Kong, M. R. Jackson, P. A. Peterson,
J. Sprent, and Z. Cai. 1998. Requirements for stimulating naive CD8⫹ T cells via
signal 1 alone. J. Immunol. 161:5226.
39. Krummel, M. F., M. D. Sjaastad, C. Wulfing, and M. M. Davis. 2000. Differential
clustering of CD4 and CD3␨ during T cell recognition. Science 289:1349.
40. Wulfing, C., M. D. Sjaastad, and M. M. Davis. 1998. Visualizing the dynamics
of T cell activation: intracellular adhesion molecule 1 migrates rapidly to the T
cell/B cell interface and acts to sustain calcium levels. Proc. Natl. Acad. Sci. USA
95:6302.
41. Goldstein, J. S., T. Chen, M. Brunswick, H. Mostowsky, and S. Kozlowski. 1998.
Purified MHC class I and peptide complexes activate naive CD8⫹ T cells independently of the CD28/B7 and LFA-1/ICAM-1 costimulatory interactions. J. Immunol. 160:3180.
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
2C cells than with CD28⫺/⫺ cells. In fact, for these cells the only
difference observed was that for absorption by activated cells, CD28⫹
cells showed greater Lat B resistance than CD28⫺/⫺ cells (which is
difficult to explain). Despite the lack of correlation between TCRmediated absorption and T cell triggering, there is an interesting correlation between TCR-mediated absorption and TCR down-regulation (8). Thus, following direct contact with APC, both processes
occur in parallel and are largely CD28 and LFA-1 independent. The
implication, therefore, is that, like TCR down-regulation, the extent of
TCR-mediated absorption may directly reflect the intensity of TCRMHC-peptide interaction. Thus, rather than correlating to T cell stimulation (which requires a combination of signals 1 and 2), TCR-mediated absorption may be a good guide to the strength of signal 1.
With regard to biological significance, we view T cell absorption of molecules during peptide-specific T-APC interactions as a
manifestation of strong receptor/ligand interactions at the T/APC
synapse. As mentioned earlier, internalization of the absorbed molecules may serve to disrupt the synapse after T cell activation, thus
allowing stimulated T cells to dissociate from APC and migrate
elsewhere to mediate their effector functions. A similar scenario
may apply to CD28-mediated absorption. Thus, movement of normal resting T cells through the lymphoid tissues could be impeded
by continuous contact of CD28 with B7 on APC. T cell absorption
and ingestion of B7 could resolve this problem. In favor of this
idea, T cell uptake of molecules from host cells in vivo is apparent
for cells harvested from spleen and LN, but not from blood, implying that the absorbed molecules are rapidly internalized (or
shed) before the cells enter the circulation (our unpublished
observations).
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