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Departments of
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Medicine and
Pathology, Washington University School of Medicine, St. Louis, MO 63110
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although many molecules have been implicated as costimulatory receptors, CD28 appears to be the most important. Engagement of CD28 either by its ligand on the APC, B7-1 (CD80) or B7-2 (CD86), or by Ab can strongly enhance TCR signaling responses (4). Importantly, blocking CD28 engagement inhibits T cell activation and may result in T cell anergy (5, 6, 7, 8). Surprisingly, studies of mice that lack expression of CD28 demonstrate that significant residual T cell function exists in the absence of CD28 (9, 10). Although T cells from CD28-deficient mice have decreased overall responses, they are still capable of mounting significant responses to mitogenic stimuli, and are capable of strong responses against transplanted tissues and some viral infections (10, 11). Thus, CD28 is not absolutely required for T cell function.
One potential explanation for these results is that CD28 may share
functions with other T cell membrane proteins. One candidate is the CD2
molecule. Like CD28, CD2 is a member of the Ig supergene family
(12). It is expressed primarily on T cells and binds
ligands expressed mainly on APC. When bound to their ligands, CD2 and
CD28 can help to span a distance of 
15 nm, the same distance spanned
by the TCR bound to its ligand, MHC/peptide (13). Because
co-engagement of CD2 with the TCR can strongly potentiate T cell
activation, CD2 has also been considered a costimulatory molecule.
Although T cells from mice lacking expression of CD2 were initially
reported to have normal activation parameters, a recent study suggests
that CD2 may have a small role in enhancing T cell activation
(14, 15).
The fact that T cells from mice lacking CD2 or CD28 are only mildly affected led us to explore whether CD2 and CD28 might share some overlapping functions. We therefore generated mice deficient in both CD2 and CD28 expression. Our data demonstrate that CD2/CD28 double-deficient T cells have a profound defect in activation and proliferation. These data suggest that CD2 and CD28 have redundant functions in T cell activation and together regulate some of the initial steps in T cell activation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CD28-deficient mice backbred five generations to C57BL/6 have previously been described (10). CD2-deficient mice were generously provided from N. Killeen (University of California, San Francisco) via J. Bromberg (University of Michigan, Ann Arbor). The mice were crossed to generate mice homozygous for the null alleles at CD2 and CD28. Littermates were bred to either CD2- or CD28-deficient mice to generate control mice. Genotypes were determined by PCR and confirmed by southern blotting. CD2/CD28-deficient mice described above were then crossed to CD28-deficient D011.10 mice (provided by S. Reiner and C. Thompson, University of Chicago). Pups deficient at both CD2 and CD28 were then screened for the presence of the TCR transgene by flow cytometry using the clonotypic mAb KJ1-26. MHC haplotype was also determined by flow cytometry of peripheral blood. Mice were 6–12 wk of age for all experiments, and were age and sex matched within each experiment. All mice are housed in specific pathogen free environments maintained by the Department of Comparative Medicine at the Washington University School of Medicine.
Antibodies
Anti-CD3 (145-2c11, hamster IgG) was provided by J. Bluestone, (University of Chicago). The clonotypic mAb KJ1-26 was generously provided by K. Murphy (Washington University, St. Louis, MO). All other Abs were purchased from PharMingen (San Diego, CA). Flow cytometric analysis was performed on a FACSCalibur flow cytometer using CellQuest software (Becton Dickinson Corporation, Mountain View, CA).
Proliferation assays
Lymph node cells or splenocytes were isolated, and single cell
suspensions were prepared following standard protocols. Cultures were
stimulated with the indicated doses of anti-CD3 mAb and pulsed for
the final 8 h of a 48-h culture with 1.0 µCi/well tritiated
thymidine ([3H]TdR; ICN, Costa Mesa, CA). Each
condition was plated in quadruplicate and the mean ± SD shown.
Exogenous IL-2 (100 U/ml) was added at the start of the culture as
indicated in the experiments. Purified T cells were prepared from lymph
node cells by negative selection with magnetic bead conjugated
anti-B220 and anti-MHC class II mAb according to the
manufacturers instructions (Miltenyi Biotec, Auburn, CA). Cells
prepared in this manner were 
99% Thy1.2 positive by FACS analysis
and unresponsive to Con A stimulation (data not shown). Purified lymph
node T cells were stimulated with plate immobilized anti-CD3
following standard methods. For stimulation of cells in the D011.10
background, 1.5 x 105 splenocytes were
stimulated with the OVA323–339 peptide in
round-bottom microtiter plates. Proliferation was determined by
[3H]TdR incorporation. Thymidine incorporation
was measured at 48, 72, and 96 h. All experiments were repeated a
minimum of three times, and representative data are presented.
Conjugate assay
The murine B cell line A20 that expresses I-Ad was pulsed with the indicated dose of Ag for 18 h and then labeled with Cell Tracker Green (0.5 µM; Molecular Probes, Eugene, OR) per the manufacturer’s protocol. Lymph node cells were isolated and labeled with Cell Tracker Orange (0.5 µM; Molecular Probes). The ability to form T cell/APC cell conjugates was assessed as previously described (16). Briefly, T cells and APC were resuspended in cold DMEM following labeling at 5 x 106 cells per ml. Twenty-five microliters of each were then placed into a 0.2 ml tube and the cells briefly pelleted at 4°C and then incubated at 37°C for 6 min. The cells were then resuspended in 2 ml of PBS containing 1% BSA and gently mixed. The presence of T cell:APC conjugates was assessed using two-color flow cytometric analysis on a FACSCalibur flow cytometer. Data are expressed as the percentage of conjugates relative to wild type, where the wild type is set at 100%. The mean of two independent experiments is presented.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To assess the role of CD2 and CD28 in T cell development, we bred
mice singly deficient in either CD2 or CD28 to obtain mice deficient in
both genes. The genotype of each mouse was determined by PCR analysis
using primers specific for the wild-type and knockout alleles, and
confirmed by Southern blotting. In addition, surface expression of CD2
and CD28 was assessed by flow cytometry for each experiment. Analysis
of peripheral T cell subsets demonstrated no significant alterations in
cell number or distribution (Fig. 1
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Thymocyte cellularity and subsets were also similar to wild-type mice.
The percentage of T cells and B cells in the lymph nodes as assessed by
Thy1.2 and B220 staining were similar in all genotypes examined (data
not shown). Thus, T cell development was not grossly altered in mice
lacking both CD2 and CD28.
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To determine whether T cells lacking both CD2 and CD28
were able to respond to TCR engagement, we stimulated bulk lymph node
cells with soluble anti-CD3 and measured proliferation by thymidine
incorporation (Fig. 2A).
Wild-type mice or mice deficient in only CD2 or CD28 mounted a robust
proliferative response to anti-CD3 stimulation. In contrast, T
cells from mice deficient in both CD2 and CD28 were virtually
unresponsive to CD3 engagement by soluble Ab. However, T cells from all
genotypes proliferated strongly to stimulation with PMA and ionomycin
(Fig. 2B). Similar results were obtained in experiments
performed with splenocytes or lymph node cells and when examined at 24,
48, and 72 h. No reproducible differences were observed in the
response of mice homozygous or heterozygous for the wild-type alleles
(data not shown).
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C). This
demonstrates that signal transduction pathways initiated through the
TCR are functional, and suggests that the defect may involve
interactions between the T cell and the APC. In support of this idea,
the CD2/CD28-deficient mice failed to respond to Con A treatment, which
is also dependent on interactions with accessory cells (Fig. 2B).
We also examined the expression of the activation markers CD69 and CD25
following stimulation of bulk lymph node cells with soluble
anti-CD3. Expression of both CD69 and CD25 were decreased in the
double knockout mice (Table I).
Consistent with the failure to induce CD25 expression, addition of
exogenous IL-2 did not restore the proliferative response of the
CD2/CD28-double deficient T cells (Fig. 2D).
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). Viability was >90%
for all genotypes at the initiation of the culture (data not shown). At
48 h following activation, there was a decrease in cell survival
for both the CD28 and CD2/CD28-deficent cells. However, the magnitude
of the difference is insufficient to account for the differences in
proliferation suggesting that induction of cell death alone cannot
explain the lack of proliferative response. Ag-specific proliferation of CD2/CD28-deficient T cells is impaired in a dose- and time-dependent manner
To examine the proliferative response following stimulation in a
more physiologically relevant context, we bred double-deficient mice to
the TCR transgenic mouse expressing the DO11.10 TCR. The D011.10 mouse
expresses a transgenic TCR specific for the
OVA323–339 peptide in the context of
I-Ad (18). Expression of the TCR
transgene as assessed by flow cytometry with a clonotype-specific
Ab revealed similar levels of TCR expression in all genotypes
(data not shown). Stimulation of CD2/CD28 double-deficient splenocytes
with OVA323–339 peptide demonstrated a dose- and
time-dependent impairment in the proliferative response (Fig. 3). At low Ag doses, no significant
proliferative response was generated in the double-deficient T cells.
Proliferation was detected only at the highest peptide dose used (3
µM). In addition, the impaired response of T cells from the double
knockout mice was most significant at later time points. Mice lacking
CD28 alone also demonstrated a time- and dose-dependent response to
peptide/MHC, but unlike the double knockout T cells, this impairment
was overcome at higher doses of Ag and was less significant at later
time points. CD2-deficient mice proliferate at levels similar to
wild-type mice (Ref. 15 and data not shown). No
significant differences were observed in stimulation of lymph node
cells or splenocytes (data not shown). Thus, T cells lacking both CD2
and CD28 exhibit a profound impairment to Ag activation.
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As both CD2 and CD28 bind to ligands expressed on APC, both
proteins may be involved in facilitating interactions between T cells
and APC. We tested this hypothesis by measuring the ability of
double-deficient cells to form T cell:APC conjugates. DO11.10 T cells
were co-incubated with Ag-pulsed APC and assessed for conjugate
formation by flow cytometry. As shown in Fig. 4, T cells lacking both CD2 and CD28 had
a marked reduction in the number of stable conjugates formed as
compared with either wild-type T cells or T cells from mice deficient
in only CD2 or CD28. At the highest dose of Ag examined, no difference
was observed between genotypes, consistent with the observation that
some proliferative response can be elicited at these Ag doses. This
suggests that both molecules work together to facilitate T cell
interactions with APC.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Surprisingly, we found that purified T cells from the double-deficient mice could be activated with immobilized anti-CD3. Differences in the mechanism by which soluble vs immobilized anti-CD3 activates T cells may therefore provide insights into how CD2 and CD28 function. Stimulation of T cells using soluble anti-CD3 relies on the presence of Fc-bearing cells to cross-link the TCR. In contrast, immobilized anti-CD3 results in activation in the absence of any accessory cells as the solid phase substrate on which the Ab is immobilized provides for high degrees of cross-linking. That immobilized anti-CD3 activates double-deficient T cells demonstrates that the intrinsic signaling capabilities of the TCR are intact. The discrepancy in the ability of soluble vs immobilized anti-CD3 to activate double-deficient T cells suggests that CD2 and CD28 may function primarily by stabilizing the interaction of the T cell with the APC. In the absence of CD2 and CD28, interactions with Fc-bearing B cells and macrophages might be impaired, resulting in ineffective TCR cross-linking.
Breeding the double knockout mouse to a TCR transgenic mouse enabled us to examine T cell activation by peptide Ag. Similar to soluble anti-CD3 stimulation, we found that Ag-induced T cell proliferation was significantly impaired. Given that the TCR is competent to signal in these cells, the most compelling explanation for the defect is the inability of T cells to form stable contacts with the APC. In confirmation of this, direct assay of the ability of T cells to form conjugates with APC demonstrated a marked reduction in T cell:APC pairs, particularly at submaximal Ag stimulation.
While both CD2 and CD28 contribute to the formation of the T cell:APC contact, this does not exclude other mechanisms, such as activation of intracellular signaling cascades, as potential mechanisms for either CD2 or CD28 function. Our data suggest that ligation of either CD2 or CD28 is required for the initial activation of T cells. However, their mechanism of action may be either similar or distinct, but in the absence of both, the T cell is severely impaired in its ability to respond to Ag.
Most current models of T cell activation subscribe to the two-signal hypothesis which proposes that two signals are required for T cell activation (3). The first is transduced by the TCR, and the second by a costimulatory molecule. Although many molecules have been implicated as costimulatory molecules, the best studied is CD28. CD28 is thought to transduce a required and specific biochemical signal that integrates with the signal initiated by the TCR to effect T cell activation. As T cells lacking CD28 can still be activated, either the signal transduced by CD28 can be delivered by another molecule on the surface of the T cell- or TCR-mediated signals alone may be sufficient.
Evidence is accumulating to support the latter possibility. These data suggest that CD28 may function mainly to enhance and amplify signals transduced by the TCR. Because CD28 is an adhesion molecule, it may function in concert with other adhesion molecules such as CD2 to stabilize the formation of a close membrane contact between the T cell and the APC. CD28 engagement also promotes a cytoskeletal mechanism that recruits lipid rafts to the T cell/APC contact (19, 20). These adhesive and cytoskeletal functions of CD28 result ultimately in promoting and stabilizing T cell interactions with APCs. This interaction is critical as TCR recognition of Ag is dependent upon formation of a stable and organized contact structure known as the immunological synapse (13, 21).
In support of this model, CD28 has been shown to be particularly important for Ags with a short half-life. CD28-deficient mice cannot respond to Ags that are rapidly cleared from the mouse, but can mount normal responses to Ags that are present in the mouse for several days (22, 23). These data suggest that one mechanism by which CD28 may regulate T cell proliferation is by enhancing TCR sensitivity and length of engagement rather than transducing a unique signal.
Like CD28, CD2 is thought to play an important role in facilitating interactions between the T cell and the APC. CD2 binds specific ligands expressed on a wide range of APC. In humans, the principle ligand for CD2 is CD58, also known as LFA-3 (24). In rodents, the related molecule CD48 is the most active natural ligand for CD2 (12). Although the affinity of CD2 for its ligands in vitro is relatively low (10-5), recent evidence suggest that this affinity is physiologically relevant (25).
As CD2 ligation can enhance signaling mediated by the TCR, it has been considered by some to be a costimulatory molecule (26, 27). CD2 also shares some structural features in common with CD28. Both are members of the Ig superfamily, and each contains Ig repeats in the extracellular domain. Bound to their ligands, they span the same distance, 15 nm, similar to the distance spanned by the TCR bound to peptide/MHC. Lastly, both contain relatively long cytoplasmic domains that contain potential sites of binding of Src homology 3 (SH3) domain containing proteins. Both molecules are likely to be involved in facilitating cell-cell contact between the T cell and the APC.
As mice lacking CD2 are relatively normal, it seemed likely that the role of CD2 in T cell biology was not essential and could be substituted by other molecules on the surface of the T cell (15). In this paper we present data supporting a model where CD2 and CD28 share some functions. We have shown previously that CD2 clustering plays an essential role in helping to form a specialized cell contact called the immunological synapse (21). Preliminary experiments suggest that CD28 can also induce protein clustering and segregation and thus may function in a manner similar to CD2 (S. Bromley and M. Dustin, personal communication). Furthermore, it is also possible that the cytoplasmic tails of CD2 and CD28 interact with the same or similar intracellular proteins. Although the precise mechanism by which CD2 and CD28 coordinately regulate the process of T cell activation remains to be defined, the data presented in this manuscript demonstrate that each may function to facilitate the interaction with APCs. The binding of CD2 and CD28 with their ligands would allow for efficient TCR engagement, especially in the setting of limiting Ag. Thus, the engagement of the accessory molecules CD2 and CD28 may effectively set the threshold for T cell activation by Ag.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jonathan M. Green, Washington University School of Medicine, Box 8052, 660 South Euclid Avenue, St. Louis, MO 63110. E-mail address:
Received for publication November 8, 1999. Accepted for publication January 27, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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