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Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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ß T cells. Expression of the CD81 molecule on T cells
increases following activation, raising the possibility of a role for
this molecule in progression of the activation process. Using an in
vitro costimulation assay, we show that CD81 can function as a
costimulatory molecule on both CD4+ and CD8+ T
cells. This costimulation functions independently of CD28, and unlike
costimulation through CD28, is susceptible to inhibition by cyclosporin
A. Strikingly, the pattern of cytokine production elicited by
costimulation via CD81 is unique. IL-2 production was not up-regulated,
whereas both IFN-
and TNF- expression significantly increased.
Together our results demonstrate an alternate pathway for costimulation
of T cell activation mediated by CD81. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ligation of the TCR initially results in full phosphorylation of
tyrosine residues in the 
-chain of the CD3 complex, leading to
recruitment and activation of nonreceptor tyrosine kinases such as
ZAP-70 (7). Here, intracellular signaling pathways
diverge, but they ultimately converge once again in the nucleus.
Evidence indicates that the signal transduction pathway regulated by
CD28 is distinct from that generated through the TCR (5, 8). Although not yet fully understood, signals through CD28
appear to be mediated by phosphatidylinositol
(PI)3 3-kinase
(5, 9, 10, 11, 12, 13) and sphingomyelinase (14, 15), and
also involve many other signaling cascades including activation of
phospholipase C (16, 17, 18), p21ras(19), and c-Raf-1 (20). CD28
pathways are believed to not control gene transcription directly, but
to converge with TCR signals to fully activate transcription factors
and thereby induce cytokine synthesis. One model suggests that both
pathways are integrated on the level of the protein kinases JNK1 (c-Jun
N-terminal kinase) and JNK2, because these kinases need both TCR and
CD28 signals for full activation (21). A recent report
suggests that CD28 in fact amplifies TCR-induced ZAP-70 activity,
thereby regulating the intersection of the TCR and CD28 signaling
pathways (22). Another school of thought favors CD28
engagement leading to the redistribution and clustering of membrane and
intracellular kinase-rich raft microdomains at the site of TCR
engagements, resulting in higher and more stable tyrosine
phosphorylation of substrates (23).
Despite the tremendous focus on CD28, analysis of mice deficient for this molecule indicated that CD28 was in fact not the only molecule capable of providing a costimulatory signal to T cells. Although CD28-deficient mice do show a reduced response to lectins and T helper activity, they can mount efficiently an immune response to virus and are able to reject allografts (24, 25). Clearly, other molecules can provide costimulatory activity for T cells. Several molecules have in fact been identified, including CD43 (26), HSA (27), CD2 (28, 29), CD5 (30, 31), CD44 (32), CD29 (33, 34), CD11a (28, 35), members of the TNF receptor family such as 4-1BB (36), GPI-anchored molecules such as Thy-1 (37), and the tetraspanins CD82 (38) and CD9 (39, 40). More recently, two novel molecules have been described, the signaling lymphocytic activation molecule (SLAM) and inducible costimulator (ICOS), both of which appear to play a role in T cell activation distinct from that of CD28 (41, 42, 43). The precise mechanism of costimulation by any of these molecules is unknown and at present, for the majority of these molecules, it is unknown whether or not they function independently of CD28.
CD81, also known as TAPA-1 (target of antiproliferative Ab-1), is a member of the tetraspanin or transmembrane 4 superfamily (TM4SF) (44, 45). It is expressed on a wide variety of tissues and cell types, including both B and T cells as well as epithelial cells, and has the capacity to associate with other cell surface proteins in a cell type-specific manner (46, 47). On B cells, CD81 forms part of the CD19/CD21 B cell Ag receptor-coreceptor complex (48, 49, 50). This complex functions in a similar manner to that of CD4 or CD8 in T cells, lowering the threshold for activation (51). In fact, mice deficient for the CD81 molecule show decreased expression of this CD19/CD21 complex and subsequently reduced B cell proliferation and Ab production (52, 53, 54).
In human T cell lines, CD81 can associate with the CD4 and CD8 coreceptors (55, 56). Interestingly, CD81 appears to bind to the cytoplasmic region of CD4, and its binding is inhibited by bound p56lck (56). However, the function of this association with CD4 and CD8 is unknown. What is known is that CD81 expression increases after mitogenic T cell activation (W. L. Havran and R. Boismenu, unpublished observations). Furthermore, CD81 expressed on human thymocytes can function as a costimulatory molecule for these cells, with simultaneous cross-linking of CD81 and CD3 promoting a vigorous proliferative response (57).
In fact, while structurally distinct from CD28, several features of CD81 do suggest an important intracellular signaling function for this molecule. It has hydrophilic amino- and carboxyl-terminal cytoplasmic domains and four membrane-spanning regions (46, 58). Most sequence diversity found between species is contained within the large extracellular loop located between the third and fourth transmembrane regions (47). The transmembrane domains and cytoplasmic regions are highly conserved (47). CD81 lacks immunoreceptor tyrosine-phosphorylated activation motifs, thus the possible mechanism of signaling by the CD81 molecule remains to be defined.
CD81 is also associated with a number of integrins, including
3ß1 (59)
and 6ß1
(60), and recent evidence suggests that TM4SF proteins may
in fact act as linkers between the extracellular -chain domains and
intracellular signaling molecules such as PI 4-kinase and protein
kinase C (61, 62, 63). It is thus possible that CD81 and other
tetraspanins do not in fact signal, but exert, their effects by
inducing signaling through their associated molecules.
In this report, we demonstrate that murine CD81 can indeed function as
a T cell costimulatory molecule on both the CD4+
and CD8+ subsets of ß T cells. This
costimulation functions independently of CD28, and in fact appears to
follow a different pathway to T cell activation resulting in the
generation of a cytokine profile distinct from that elicited by
CD28-mediated costimulation. This suggests that CD81 may play a unique
role in ß T cell immune responses.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 mice were raised in our breeding colony at The Scripps Research Institute (TSRI; La Jolla, CA). Mutant mice with a disrupted CD28 gene (CD280/0; Ref. 24) were obtained from The Jackson Laboratory (Bar Harbor, ME) and had been backcrossed several generations onto the C57BL/6 background. RAG0/0DO11.10 TCR transgenic mice (64) obtained from Dr. S. Webb (TSRI) and RAG0/0OT-1 TCR transgenic mice (65) obtained from Dr. C. Surh (TSRI) were used as a source of exclusively naive CD4+ and CD8+ T cells, respectively. In all experiments, mice were used at 6–8 wk of age.
Antibodies
Anti-CD3
mAb (2C11), anti-CD4, and anti-CD8 were
obtained from PharMingen (San Diego, CA) and used as purified Ab. The
anti-CD28 (37.51) was kindly provided by Dr. James Allison and has
been described previously (66). 2F7 is a hamster mAb
produced in our laboratory, directed against murine CD81
(67). 1F3 is a hamster mAb produced from the same fusion
as the 2F7 mAb. It recognizes an unknown Ag on epithelial cells, not
expressed on lymphocytes. Either 1F3 or hamster Ig were used as the
control Ab in all experiments. Purified anti-mouse IgG + IgM Abs
used for B cell depletions were obtained from Caltag (Burlingame, CA).
Anti-CD4, -CD8, -CD25, -CD69, and -Thy1.2 Abs used for FACS analysis
were obtained from PharMingen directly conjugated to FITC, PE,
Cychrome, or allophycocyanin.
T cell purification
Single cell suspensions of spleen cells were isolated on density gradients of Lympholyte-M (Cedarlane Laboratories, Ontario, Canada). T cells were purified from these suspensions by depletion of B cells on anti-mouse IgG- + IgM-coated flasks as described previously (68). Briefly, spleen cell suspensions were incubated at room temperature for 1 h in flasks that had been coated overnight with anti-mouse IgG + IgM Abs (Caltag). The purity of the cell population harvested from the flasks was analyzed by flow cytometry. Typically >95% of recovered cells were Thy1+ and >90% CD44low.
For the purification of CD4+ and CD8+ subsets of T cells following panning, T cells were further purified using negative selection on magnetic beads. Briefly, T cells were incubated with purified anti-mouse CD4 mAb (RM4-5; PharMingen) or anti-mouse CD8 mAb (53.6.7; PharMingen) for 30 min on ice. Cells were washed once with DMEM supplemented with 2% FCS and then incubated with anti-rat Ig-coated magnetic beads (BioMag; PerSeptive Biosystems, Framingham, MA) at 1 ml beads per 107 cells for 30 min on ice with occasional mixing. Magnetic bead-coated cells were then removed using a magnet (Advanced Magnetics; Cambridge, MA), and the unbound cells were washed once in DMEM supplemented with 2% FCS. Purity of the population was assessed by flow cytometry. Typically, purified CD4+ cells contained <1% CD8+ cells and purified CD8+ cells contained <3% CD4+ cells.
Costimulation assays
Abs were diluted in ELISA coating buffer (50 mM Tris, 150 mM NaCl; pH 8.0 at room temperature) and immobilized to individual wells of 96-well flat-bottom microtiter ELISA plates in a final volume of 100 µl. The plates were incubated at 4°C overnight. Before the addition of cells, the plates were washed twice with ELISA coating buffer and blocked for 15 min with 100 µl DMEM supplemented with 10% heat-inactivated FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 10 mM HEPES, and 50 µM 2-ME.
Purified T cells or T cell subsets were cultured at 1 x 105 cells per well. In some cases cyclosporin A (CsA) (Sigma, St. Louis, MO) or herbimycin A (Sigma) were also added at various concentrations. Unless otherwise stated, cells were pulsed with 0.5 µCi [3H]thymidine at 58 h after initiation of culture, and harvested 14 h later. For time course experiments, [3H]thymidine was added for the final 14 h of each time point. Cells were harvested onto glass fiber filters (Cambridge Technology, Watertown, MA) and 2 ml scintillation fluid was added to each sample. Counts were read on a Beckman LS3801 scintillation counter (Beckman Coulter, Fullerton, CA). Cell viabilities were determined at various time points by trypan blue dye exclusion. All data points were performed in triplicate and are presented as mean ± SD.
Immunofluorescence staining and flow cytometry
Cells were harvested from in vitro cultures 48 h following the initiation of culture. Cells were washed once in PBS containing 2% FCS and 0.02% sodium azide (wash buffer), and resuspended in wash buffer. The following mAbs were used for immunofluorescence staining as FITC, PE, or Cychrome conjugates: CD4, CD8, CD25, and CD69. mAbs to CD28 and CD81 were used conjugated to biotin and revealed with streptavidin-coupled Red670. Cells were incubated with the appropriate mAbs for 15 min on ice, and washed with wash buffer between staining reagents. Labeled cells were analyzed on a FACsort flow cytometer using CellQuest software (Becton Dickinson, Mountain View, CA).
Analysis of cytokine production
Purified T cells and T cell subsets were cultured as described above. At 48 h following initiation of culture, cells were harvested from the wells and analyzed for cytokine production by RNase protection assay (RPA). RNA was isolated from cells using TRIzol reagent (Life Technologies; Gaithersburg, MD). Briefly, cells were pelleted and resuspended in 1 ml TRIzol reagent. After 5 min at room temperature, 200 µl CHCl3 was added, samples were mixed well, and then centrifuged at 12,000x g for 15 min at 4°C. The aqueous phase was transferred to a fresh tube and the RNA was precipitated with 500 µl isopropanol. After a 10-min incubation at room temperature, RNA was pelleted and washed with 70% ethanol. RNA pellets were air-dried and resuspended in sterile RNase-free water. RPA was performed on 3 µg of RNA per reaction using a multiprobe RPA (PharMingen) as per manufacturer’s instructions.
For analysis of TNF- production, 50 µl of supernatant was
harvested from cultures at 24 h and analyzed by ELISA using a
Quantikine M kit (R&D Systems, Minneapolis, MN) as per manufacturer’s
instructions.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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During the initial characterization of the anti-CD81 mAb, 2F7, it was found that CD81 is expressed on the surface of mature lymphoid T cells and that this expression is increased upon mitogenic T cell stimulation (W. L. Havran and R. Boismenu, unpublished observations), indicating a possible role for this molecule in T cell activation. This, together with a reported function of CD81 as a costimulatory molecule on human thymocytes (57), raised the possibility that CD81 might also function as a costimulatory molecule on murine mature T cells.
To address this possibility, purified splenic T cells, of which >90%
were CD44low, were cultured in microtiter plates
that had been coated with anti-CD3 and anti-CD81 mAbs.
Coimmobilization of these two Abs induced potent proliferation of the
cultured T cells (Fig. 1
A).
This stimulation was comparable to that obtained with coimmobilized
anti-CD3 and anti-CD28 mAbs or mitogenic stimulation with PMA
and ionomycin (Fig. 1A). In contrast, a control mAb (1F3)
was unable to induce T cell proliferation when coimmobilized with
anti-CD3 mAb, and none of the mAbs alone elicited any stimulation
of the T cells (Fig. 1A). Comparable costimulation was
obtained with splenic T cells from C57BL/6 and BALB/c mice (not shown).
Furthermore, costimulation through either CD81 or CD28 induced similar
morphology changes in the cultured cells. The cells became enlarged and
somewhat irregular shaped, whereas cells ligated with anti-CD3,
anti-CD28, or anti-CD81 alone did not show any such changes in
morphology. They remained small and round in appearance, as is
characteristic of resting T cells (data not shown).
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, B and C).
Thus, CD81 appears to be able to provide the costimulatory signal
necessary to both initiate and sustain a T cell response.
We then established that costimulation through CD81 was functional in
both the CD4+ and CD8+
subsets of T cells. Isolated CD4+ or
CD8+ T cells were cultured in microtiter plates
that had been coated previously with anti-CD3 and anti-CD81 or
anti-CD28. As illustrated in Fig. 2A, both
CD4+ and CD8+ T cells
responded equally well to costimulation whether it was provided by the
CD28 or CD81 molecule. Again, a control mAb (1F3) elicited no
costimulation in either subset nor did any of the mAbs alone (Fig. 2A). This result was confirmed using
RAG0/0DO11.10 TCR transgenic
CD4+ T cells and RAG0/0OT-1
TCR transgenic CD8+ T cells (Fig. 2B).
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The similarity of the costimulation by anti-CD81 to that seen
with anti-CD28 led to the possibility that CD81 may in fact be
acting through CD28. This was a distinct possibility in light of the
fact that of the many other molecules reported as being costimulatory
for T cells, only a few have been shown to act independently of CD28
(26, 39, 41). To address this, we examined the
costimulatory ability of anti-CD81 in CD28-deficient
(CD280/0) T cells. As shown in Fig. 3A, there was no defect in
CD81-mediated costimulation in CD280/0 cells when
compared with wild-type cells. This was also true of CD81-deficient T
cells; there was no defect in CD28-mediated costimulation in the
absence of the CD81 molecule (data not shown). Clearly then, CD81 does
not require the presence of CD28 to function, thus costimulation
through CD81 functions independently of CD28.
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B, using immobilized
anti-CD3 with suboptimal amounts (2.5 µg/ml) of anti-CD81 and
anti-CD28 together induced proliferation in excess of what was seen
with immobilized anti-CD3 in conjunction with either anti-CD28
or anti-CD81 alone. In fact, this simultaneous costimulation
induced an additive effect on proliferation, again demonstrating
independent stimulation through the CD81 and CD28 molecules.
Although CD28 and CD81 clearly act independently, the possibility
remained that these two molecules served the same function and that the
intracellular signaling events following ligation of CD28 and CD81 were
the same. To address this possibility we made use of two inhibitors,
CsA and herbimycin A, which have been reported to differentially effect
CD28-mediated costimulation. CsA has been shown to be ineffective as an
inhibitor of CD28-mediated costimulation (70), whereas
this stimulation is sensitive to the protein tyrosine kinase (PTK)
inhibitor, herbimycin A (71, 72, 73). Here we found (Fig. 4) that at the concentrations tested,
addition of CsA to cultures of freshly isolated splenic T cells had no
effect on CD28-mediated costimulation, consistent with previous reports
(70). In contrast, increasing concentrations of CsA had a
marked effect on costimulation through CD81, inhibiting proliferation
by almost half at only 10 ng/ml CsA. Higher concentrations of CsA than
shown here were found to be toxic to T cells, and so were not used in
these experiments. This differential sensitivity to CsA indicates that
not only do CD81 and CD28 function independently, but that their
intracellular signaling pathways leading to T cell activation also
differ.
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). This inhibitory effect also functions for CD81-mediated
costimulation as well as mitogenic stimulation with PMA and ionomycin.
This indicates a requirement for PTKs in T cell activation via both
CD81 and CD28. However, we cannot exclude the possibility that this
effect of herbimycin A is in fact on the signals generated through the
TCR-CD3 complex, signals that have been well documented to involve PTKs
(74). Phenotype of T cells stimulated via CD3 and CD81
Despite the independence of the two signals generated through CD81
and CD28, costimulation of T cells with either anti-CD81 or
anti-CD28 or with PMA and ionomycin led to a significant increase
in surface expression of both CD81 and CD28, regardless of the stimulus
(Fig. 5A). A control mAb,
which did not induce proliferation (Fig. 1A), also did not
cause any increase in CD28 or CD81 expression (Fig. 5A).
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B, very similar activation profiles were induced
regardless of the stimulus. Costimulation via CD81 or CD28 as well as
mitogenic stimulation with PMA and ionomycin all led to an increase in
expression of the activation markers CD69 and CD25 (Fig. 5B)
as well as CD44 and a decrease in L-selectin
expression (not shown). Cytokine production following costimulation through CD81
Although the phenotype of cells activated via CD81 or via CD28 was
similar, the question remained whether or not these two distinct
stimuli translate into equivalent activation of the cells. The
differential inhibitory effect of CsA (Fig. 4) suggests that some
difference does exist. Furthermore, it has been found in murine
(W. L. Havran and R. Boismenu, unpublished observations) and human
(75) lymphocytes that ligation of CD81 alone can lead to
TNF- production, whereas this has not been shown for CD28,
indicating another difference in the signals generated through these
two molecules. To address further this question of differential
activation pathways, we examined cytokine production by the activated T
cells. In agreement with previous reports, TNF- was clearly
detectable in culture supernatants from cells stimulated with CD81
alone (Fig. 6A), and there was
some augmentation of this production with CD3 and CD81 costimulation.
In contrast, while costimulation through CD3 and CD28 also led to
TNF- production, no TNF- was produced following stimulation with
either CD3 or CD28 alone (Fig. 6A).
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B).
Although activation of T cells with anti-CD3 together with
anti-CD28 led to production of TNF-, IL-2, IFN-, and some
IL-9, costimulation through CD81 induced only TNF- and IFN-
transcription (Fig. 6B). No IL-2 and subsequently no IL-9
was detectable following costimulation through CD81 (Fig. 6B). This result was confirmed by an IL-2-dependent CTLL
assay, intracellular cytokine staining, and flow cytometry as well as
by RT-PCR (not shown). In addition, IL-2-deficient T cells responded
normally to CD81-mediated costimulation and exhibited a diminished
response to CD28-mediated costimulation (data not shown). It is thus
unclear what cytokine is sustaining the proliferative response to
CD81-mediated costimulation. By examining costimulation in
IL-10-deficient T cells, we were able to eliminate this cytokine as a
possible candidate (data not shown). Nevertheless, taken together, the
differential cytokine production following costimulation through CD81
and CD28 suggests distinct functional outcomes of activation via these
two molecules. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß T cells. Splenic T cells
were cultured with coimmobilized anti-CD3 and anti-CD81 mAbs.
This led to strong proliferation of both CD4+ and
CD8+ T cells, which was comparable in strength
and duration to the proliferation elicited by coimmobilized
anti-CD3 and anti-CD28 mAbs or mitogenic stimulation with PMA
and ionomycin. Consequently we were able to compare and contrast T cell
costimulation mediated by CD81 and CD28, and to characterize the
mechanism of costimulation through CD81. The first issue we addressed was the independence of costimulation through CD81 and CD28. Since the identification of CD28 as a costimulatory molecule for T cells, a number of other molecules have been reported to be capable of providing this function (26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 38, 39, 40). Analysis of mice deficient for CD28 strengthened this idea, as these mice were still capable of mounting efficiently an immune response to several pathogens (24, 25). Nevertheless, of all the alternative molecules described to date, very few have been shown to actually function independently of CD28 (26, 39, 41), although such molecules were presumed to exist given the relatively subtle phenotype of the CD28-deficient mice. We found that costimulation through CD81 did indeed function independently of CD28, and in fact simultaneous costimulation through these two molecules augmented proliferation equivalent to an additive effect of costimulation through CD28 and CD81 independently. Perhaps costimulation via CD81 can account for the ability of CD28-deficient mice to still respond to virus infections and reject allografts. It would be interesting to follow such responses in vivo in animals lacking the CD81 molecule.
Identification of costimulatory molecules functioning independently of CD28 had, to date, been limited to the observation that such molecules can provide a costimulatory signal in cells deficient for the CD28 molecule (26, 39, 41). In this paper we extended our analysis to downstream signals generated through CD81 and CD28. In agreement with what has been reported previously, we found that costimulation through CD28 was insensitive to inhibition by CsA. On the other hand, we observed a strong inhibitory effect of CsA on costimulation through CD81. The transcriptional activation of several cytokine genes (76) and cell surface molecule genes such as CD40L (77) is initiated by NF-AT activation. This activation is blocked by CsA (76, 77). Thus, the inhibitory effect of CsA on costimulation through CD81 indicates a requirement for the Ca2+/calcineurin pathway and NF-AT family members in activation of T cells via CD3 and CD81. As such, it appears that there are different intracellular targets for the signals generated through CD28 and CD81. Some similarities do exist, however. Both costimulatory pathways are sensitive to the PTK inhibitor, herbimycin A. As mentioned earlier, this similarity could be in the signals generated through the TCR rather than through the costimulatory molecule, as PTKs are well documented as being involved in TCR-mediated signaling events (74).
We also observed similarities in the phenotype of the T cells activated via CD3 and CD28 or CD81. CD69, CD25, and CD44 were up-regulated and CD62L was down-regulated regardless of the stimulus used. Interestingly, costimulation through CD81 led to an increase in expression of both CD81 and CD28. Conversely, costimulation through CD28 also resulted in increased expression of both molecules. Together these data demonstrate that costimulation through CD81 results in an activated phenotype, apparently indistinguishable from the phenotype of cells activated by costimulation through CD28.
The phenotypic similarities, yet divergence of intracellular signaling
pathways following engagement of CD81 and CD28, led us to question
whether the functional outcomes of stimulation through CD81 and CD28
would also diverge. Indeed, cytokine production following ligation of
CD81 was quite distinct from that of CD28 engagement. We observed
significant TNF- production following ligation of CD81 alone. This
is in agreement with previous observations (W. L. Havran and R.
Boismenu, unpublished observations; Ref. 75). No TNF-
was detected following ligation of CD28 alone. This suggests that
engagement of the CD81 molecule activates intracellular signals that
are unresponsive to CD28 engagement.
IL-2 production is one of the hallmarks of CD28-mediated costimulation (78). Although we also observed strong IL-2 production following costimulation through CD28, we were unable to detect any IL-2 message or protein following costimulation via CD81, again indicating divergence of the signals generated through CD81 and CD28. The lack of IL-2 production following costimulation is not unique to CD81 (41, 43, 69), but is generally associated with an ability to initiate, but not to sustain a proliferative response (69, 79). In the present study, however, costimulation via CD81 led to a vigorous response that was equivalent in extent and duration to a CD28-mediated or mitogenic response. It is unclear what cytokine elicits the proliferative response initiated by costimulation through CD81. From the RPA data, clearly IL-4 and IL-15 are not responsible. We also eliminated IL-10 as a candidate, and thus the molecule or molecules that are responsible for the sustained proliferation and cell survival elicited by CD81-mediated costimulation have yet to be defined. Experiments are currently in progress to characterize further this unique response and its implications in in vivo responses to viruses and other pathogens.
In conclusion, we have demonstrated that CD81 can function as a
costimulatory molecule on murine ß T cells. This costimulation
functions independently of CD28 and, in fact, initiates a different
pathway to activation, resulting in a unique functional outcome.
Identification of the intracellular pathways activated by CD81 and of a
ligand for this molecule on APC would provide insight into its role in
in vivo immune responses. It is possible, however, that no ligand
exists for CD81, and that the costimulatory effects of CD81 are in fact
due to cross-linking of a larger complex. Indeed, CD81 is known to
associate with a number of different molecules in human T cells;
including CD4, CD8 (55, 56), other TM4SF members, and
integrins (44). Thus, the possibility exists that ligation
of CD81 leads to signaling through one of its associated molecules
rather than directly through CD81 itself. In fact, recent evidence does
suggest that TM4SF proteins may act as linkers between extracellular
integrin -chain domains and intracellular signaling molecules such
as PI 4-kinase and protein kinase C (61, 62, 63).
Whatever the precise mechanisms of CD81-mediated costimulation, clearly CD81 and CD28 act independently and have distinct functional outcomes. However, it does remain unclear whether or not their activation is initiated by the same stimulus or whether they work together in an in vivo immune response. Interestingly, both CD28- and CD81-deficient mice do have defects in T helper activity (24, 25, 80) suggesting some overlap in their function. Further analysis of immune responses in these deficient animals and in animals lacking both molecules may help clarify their individual roles.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Wendy L. Havran, Department of Immunology, IMM8, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037.
3 Abbreviations used in this paper: PI, phosphatidylinositol; TM4SF, transmembrane 4 superfamily; CsA, cyclosporin A; RPA, RNase protection assay; PTK, protein tyrosine kinase; TSRI, The Scripps Research Institute.
Received for publication September 13, 1999. Accepted for publication June 5, 2000.
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A. B. van Spriel, K. L. Puls, M. Sofi, D. Pouniotis, H. Hochrein, Z. Orinska, K.-P. Knobeloch, M. Plebanski, and M. D. Wright A Regulatory Role for CD37 in T Cell Proliferation J. Immunol., March 1, 2004; 172(5): 2953 - 2961. [Abstract] [Full Text] [PDF]
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R. M. Aspalter, M. M. Eibl, and H. M. Wolf Regulation of TCR-mediated T cell activation by TNF-RII J. Leukoc. Biol., October 1, 2003; 74(4): 572 - 582. [Abstract] [Full Text] [PDF]
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M. Mittelbrunn, M. Yanez-Mo, D. Sancho, A. Ursa, and F. Sanchez-Madrid Cutting Edge: Dynamic Redistribution of Tetraspanin CD81 at the Central Zone of the Immune Synapse in Both T Lymphocytes and APC J. Immunol., December 15, 2002; 169(12): 6691 - 6695. [Abstract] [Full Text] [PDF]
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 J. M. Tarrant, J. Groom, D. Metcalf, R. Li, B. Borobokas, M. D. Wright, D. Tarlinton, and L. Robb The Absence of Tssc6, a Member of the Tetraspanin Superfamily, Does Not Affect Lymphoid Development but Enhances In Vitro T-Cell Proliferative Responses Mol. Cell. Biol., July 15, 2002; 22(14): 5006 - 5018. [Abstract] [Full Text] [PDF]
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 J. Deng, R. H. Dekruyff, G. J. Freeman, D. T. Umetsu, and S. Levy Critical role of CD81 in cognate T-B cell interactions leading to Th2 responses Int. Immunol., May 1, 2002; 14(5): 513 - 523. [Abstract] [Full Text] [PDF]
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K. L. Clark, Z. Zeng, A. L. Langford, S. M. Bowen, and S. C. Todd PGRL Is a Major CD81-Associated Protein on Lymphocytes and Distinguishes a New Family of Cell Surface Proteins J. Immunol., November 1, 2001; 167(9): 5115 - 5121. [Abstract] [Full Text] [PDF]
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 C. S. Stipp, D. Orlicky, and M. E. Hemler FPRP, a Major, Highly Stoichiometric, Highly Specific CD81- and CD9-associated Protein J. Biol. Chem., February 9, 2001; 276(7): 4853 - 4862. [Abstract] [Full Text] [PDF]
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