Enhanced surface TCR replenishment mediated by CD28 leads to ...

International Immunology, Vol. 12, No. 6, pp. 833–842

© 2000 The Japanese Society for Immunology

Enhanced surface TCR replenishment mediated by CD28 leads to greater TCR engagement during primary stimulation Adam G. Schrum, Andrew D. Wells and Laurence A. Turka University of Pennsylvania, 701 Clinical Research Bldg, 415 Curie Boulevard, Philadelphia, PA 19104, USA Keywords: cellular activation, co-stimulatory molecules, T lymphocytes

Abstract When T cells are stimulated with high concentrations of strong TCR agonist, engaged TCR are internalized and degraded, resulting in greatly reduced surface TCR levels for up to several days post-stimulation. It has been noted that surface TCR levels rise subsequently, even in the presence of continuing stimulation, but the role of CD28 co-stimulation in surface TCR replenishment has not been investigated. Here, we have examined the return of surface TCR following activation, the availability of these TCR for antigenic engagement and the role of CD28 in that process. We report that within 24 h of stimulation, the level of surface TCR expression becomes dependent on the degree of CD28 signaling provided during T cell activation. In addition, when cells are removed from stimulus after 24 h, surface TCR expression recovers to a stable level which exceeds that of unstimulated cells and is proportional to the degree of CD28 co-stimulation. TCR that replenish the plasma membrane during T cell activation can be down-regulated by receptor occupancy with the same efficiency as TCR on freshly stimulated cells. Thus, as a result of enhanced surface TCR replenishment, CD28-co-stimulated cells can engage and down-regulate more TCR than costimulation-deprived cells in the face of ongoing stimulation. Furthermore, engagement of newly expressed TCR on activated T cells re-induces CD69, suggesting participation of these replenishing TCR in continued T cell signaling. These data identify the augmentation of surface TCR replenishment during activation as a novel mechanism that likely contributes to the enhanced antigenic sensitivity of CD28-co-stimulated T cells. Introduction The quantity of surface TCR expressed has been shown to critically influence the activation potential of mature T cells. Cells with low surface TCR expression fail to demonstrate full activation under conditions of antigenic stimulation (1,2). Expression of low surface TCR levels has been implicated as a mechanism of peripheral tolerance induction to self antigens (3,4). As little as a 1.5-fold difference in surface TCR expression between T cells has been demonstrated to greatly affect activation and proliferative potential under conditions of antigenic challenge (1). Thus, the level of surface TCR expression is directly proportional to the probability that T cells will be able to achieve threshold signaling when presented with antigenic stimulus. During T cell stimulation via strong TCR agonists, surface TCR are triggered, internalized and degraded (5,6). High doses of strong TCR agonists have been reported to lower

surface TCR within 5 h by as much as 90% to a minimal level that is thought to be maintained for several days (1,7), after which time surface TCR levels rise, even in the presence of continuing stimulation (6). Internalization under these conditions results in a parallel decline and virtual disappearance of total cellular TCR via degradation of engaged receptors in lysosomal compartments (8,9). Since decreasing the dose of a strong agonist proportionally decreases TCR internalization, down-regulation has been proposed to serve as a clear indicator of the quantity of TCR that has been engaged (10,11). Previous in vitro studies involving agonist-induced TCR down-regulation have involved T cell activation regimens wherein the TCR agonist has not been overtly removed from culture throughout a time course (1,6,12,13). Therefore, it has not been demonstrated whether surface TCR replenishment fails to occur for several days or whether it occurs at levels

Correspondence to: L. A. Turka Transmitting editor: C. B. Thompson

Received 1 October 1999, accepted 16 February 2000

834 CD28 enhances surface TCR replenishment masked by rapid consumption via continued agonist-induced internalization. Nonetheless, surface TCR levels do eventually recover when high-dose agonist is still present in culture, raising the question of whether surface-replenishing TCR are immediately available for stimulation or if they experience a refractory period first. If TCR are recruited to the plasma membrane during the hours that succeed initial down-regulation, what role they might play in continued recognition of antigen during primary T cell activation is not well defined. Here, we have examined the dynamics of activationinduced surface TCR replenishment and the ability of replenishing TCR to be engaged and down-regulated by strong agonist encounter. Since CD28 co-stimulation is known to increase T cell antigenic sensitivity (1,7,12), we investigated the contribution of CD28 signaling to surface TCR expression for T cells activated by direct TCR interaction with strong agonist. We demonstrate that CD28 co-stimulation increases the quantity of TCR that replenish the membrane subsequent to initial down-regulation. These surface-replenishing TCR can be engaged and down-regulated by agonist with an efficiency similar to that observed during the initial stages of antigenic encounter. The enhanced surface TCR replenishment mediated by CD28 co-stimulation results in a greater number of receptors that are engaged during an ongoing stimulus. This is likely to represent a mechanism by which CD28 co-stimulation augments T cell antigenic sensitivity.

In vitro stimulation of BALB/c T cells with anti-CD3 mAb

Methods

Whole splenocyte single-cell suspensions were prepared from BALB/c mice with the aid of Nitex fabric (Tetko, Elmsford, NY) followed by erythrocyte lysis via hypotonic shock. Cells were resuspended in RPMI 1640 medium containing 10% FBS (Hyclone, Logan, UT), 2 mM L-glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin and 5 µM β-mercaptoethanol, and plated for culture at 1⫻106 cells/ml in 96-well round-bottom microtiter plates. T cells were activated with varying concentrations of soluble anti-CD3 mAb in the presence of agents which induced maximal CD28 co-stimulation (2 µg/ml antiCD28 and 10 µg/ml human Ig), CD28 co-stimulatory blockade (2 µg/ml hamster Ig and 10 µg/ml CTLA-4–Ig) or ‘physiologic’ CD28 co-stimulation provided by residual APC in the culture (control Ig: 2 µg/ml hamster Ig and 10 µg/ml human Ig). This T cell activation system allows for Fc receptor-mediated crosslinking of anti-CD3:TCR–CD3 ligand–receptor complexes (14–16). For some experiments, stimulus was removed after 24 h of culture by washing cell samples 5 times with 200 µl volumes of RPMI supplemented medium. Fresh medium was used to return volumes to 200 µl/well and tissue culture was resumed at 37°C. For a subset of these experiments, 50 µl of medium was decanted per well so as not to disturb the settled cells at 69 h post-stimulus. Then anti-CD3 mAb was added back to the cells in a 50 µl volume of medium per well and settled cells were resuspended to facilitate antibody binding. After a further 3 or 27 h (72 or 96 h post-stimulus respectively), cells were harvested and prepared for flow cytometric analysis.

Mice

In vitro stimulation of DO11.10/RAG2–/– T cells with pOVA

BALB/c mice were purchased from the Jackson Laboratory (Bar Harbor, ME). DO11.10/RAG2–/– mice on the BALB/c background were a gift from Dr W. Lee (State University of New York, Albany, NY). Congenic Thy1.1⫹ Thy1.2– mice on the BALB/c background were a gift from Dr R. W. Dutton (Trudeau Institute, Saranac Lake, NY). All mice were received, maintained and bred in our animal facility until their use at 8– 12 weeks of age.

Single-cell whole splenocyte suspensions were prepared from Thy1.2⫹ DO11.10/RAG2–/– (responder) and congenic Thy1.1⫹ Thy1.2– (stimulator) BALB/c mice. Stimulator splenocytes (1⫻106 cells/ml) were pulsed with increasing concentrations of pOVA and incubated overnight to facilitate peptide loading of MHC molecules. Responder splenocytes were harvested and placed into culture with stimulators at a 1:2 responder: stimulator ratio. Activation was further influenced by addition of anti-CD28 mAb, CTLA-4–Ig, anti-IL-2 mAb, rmIL-2 (R & D Systems, Minneapolis, MN), control Ig and/or PBS as indicated in figure legends. Differences at the Thy1 locus circumvented the need for both APC irradiation and cell selection mediated by antibodies that might unaccountably affect the in vitro stimulations.

Igs and peptides Purified antibodies used for in vitro stimulations included antiCD3ε mAb (hybridoma 145-2C11; provided by J. Bluestone, University of Chicago, Chicago, IL), anti-CD28 mAb (hybridoma 37.51; provided by J. Allison, University of California, Berkeley, CA), anti-IL-2 mAb (hybridoma S4B6-1; ATCC, Manassas, VA), and irrelevant hamster, human and rat γ-globulin (Jackson ImmunoResearch, West Grove, PA). The fusion protein CTLA-4–Ig was provided by R. Peach (BristolMyers Squibb, Princeton, NJ). The ovalbumin peptide 323– 339 (pOVA) was synthesized by the Protein Chemistry Lab (University of Pennsylvania, Philadelphia, PA). Additional mAb purchased from PharMingen (San Diego, CA) for use in staining for flow cytometry included anti-CD16/CD32 (2.4G2), anti-Thy1.2 (53-2.1), anti-Thy1.1 (OX-7), anti-TCRβ (H57-597), anti-hamster Ig, κ light chain (RG7/7.6), anti-CD69 (H1.2F3), anti-CD4 (L3T4) and anti-CD8 (Ly-2). The DO11.10 clonotypespecific mAb KJ1-26 was provided by Dr M. Jenkins (University of Minnesota, Minneapolis, MN).

Flow cytometric (FCM) analysis Cells were harvested from culture and washed in cold buffer containing PBS, 2% FBS and 0.01% NaN3. Cells stimulated with anti-CD3 mAb were incubated at 4°C with an additional 10 µg/ml anti-CD3 mAb to insure saturated binding of all surface CD3ε molecules. After washing away unbound mAb, bound anti-CD3 was detected with biotin- or FITC-conjugated anti-hamster Ig κ light chain (which does not detect the λ light chain of the anti-CD28 mAb, data not shown). Cells that had been stimulated with pOVA on APC were stained with phycoerythrin (PE)-conjugated anti-TCRβ. Other antibodies and reagents used for staining included: purified anti-CD16/ 32, PE-conjugated anti-CD69, PE- or allophycocyanin-conjug-

CD28 enhances surface TCR replenishment 835 ated anti-Thy1.2, PE- or allophycocyanin-conjugated streptavidin, and propidium iodide (PI) as a vital dye. Negative control stains for surface TCR were fluorochrome-conjugated irrelevant hamster and mouse Ig. Since subunits of TCR and CD3 are expressed together as part of a multimeric complex at the T cell surface (for review, see 17), the term ‘surface TCR’ in this report is used to denote measurements of either surface CD3ε or TCRβ. After staining, three- or four-color FCM was performed on Becton Dickinson Immunocytometry Systems (San Jose, CA) FACScan single-laser or FACSCalibur dual-laser cytometers calibrated with CaliBRITE beads. CellQuest software was used for data acquisition and analysis. At least 10,000 events were collected from each sample, and analysis was restricted to live T cells by gating on Thy1.2⫹ PI– cells. Semi-quantitative assessment of surface TCR expression via FCM and statistical analyses Geometric mean fluorescence intensities (MFI) were obtained corresponding to the level of CD3ε or TCRβ fluorescence on live T cells as calculated by CellQuest software. Within each experiment, all samples were performed in duplicate and error bars in the figure graphs represent SEM. The level of surface TCR of stimulated cells was then expressed as a percentage of that of unstimulated T cells: (mean geometric MFI, stimulated cells/mean geometric MFI, unstimulated cells)⫻100. Statistics Statistical significance was determined using P values generated by one- or two-tailed paired Student’s t-tests calculated with the use of Microsoft Excel software, and by generating confidence intervals using standard equations for parametric data. Results CD28 co-stimulation does not affect TCR engagement at early time points of stimulation To investigate the effects of CD28 co-stimulation on surface TCR expression, we cultured mouse splenocytes with increasing concentrations of anti-CD3 mAb, a strong TCR agonist, under conditions of either maximal (anti-CD28 mAb treated) or blocked (CTLA-4–Ig treated) CD28 co-stimulation. As previously reported (18), engagement of TCR by anti-CD3 mAb induced surface receptor down-regulation in a dose-dependent, CD28-independent fashion which was maximal at 5 h post-stimulus (Fig. 1). CD28 co-stimulation results in enhanced surface TCR levels by 24 h post-stimulus with anti-CD3-mAb To study the duration of TCR down-regulation in our activation system, we examined surface TCR expression at a later time point. At 24 h post-stimulus, surface TCR levels remained dependent on the dose of anti-CD3 mAb; however, T cells receiving maximal CD28 co-stimulation expressed ~1.5- to 2-fold higher surface TCR levels than did cells whose CD28 co-stimulation had been blocked during activation (Fig. 2). T cells receiving endogenous co-stimulation, via B7 molecules

expressed on APC in culture, exhibited intermediate surface TCR expression (Fig. 2B, control Ig). Thus the level of surface TCR expression 24 h post-stimulation depended not only on the presence or absence, but on the degree of CD28 costimulation. These differences do not reflect differences in viability or proliferation at this time point (data not shown). In the latter regard, T cells do not begin to proliferate until at least 40 h in this system (12). Both CD4⫹ and CD8⫹ T cell subsets are affected equally, and the effect appears indiscriminate of mouse gender or strain (data not shown). As expected, there was no difference in the percentage of T cells that were CD69⫹ between experimental groups, verifying that CD28 co-stimulation did not affect early events in T cell activation (data not shown). We conclude from these data that surface TCR levels become CD28 dependent as cellular activation progresses. One effect of CD28 co-stimulation on T cells is a marked enhancement of cell enlargement (19). It was possible that the higher surface TCR levels observed on co-stimulated cells were merely a result of the effect of co-stimulation on cell size. To explore this question, we examined surface TCR expression as a function of relative cell size as measured by forward scatter via FCM. We observed that large cells which had been treated with CTLA-4–Ig displayed lower surface TCR levels when compared to equivalently sized, anti-CD28treated cells (Fig. 3). The most pronounced CD28-mediated enhancement of surface TCR expression was seen for cells that had undergone considerable enlargement (Fig. 3). These trends are reproducible in nine replicate experiments (data not shown). Additionally, we note that regardless of costimulatory status, surface TCR levels and cell size have a positive correlation (Fig. 3), a trend which is absent in unstimulated cells (data not shown). We conclude that activation-induced cell enlargement correlates with increases in surface TCR expression and that CD28 co-stimulation further enhances this expression by an additional, unidentified mechanism. T cells stimulated with peptide-loaded MHC on APC display CD28-dependent surface TCR enhancement which can be attributed to the action of IL-2 To determine whether CD28 co-stimulation enhanced surface TCR levels during activation initiated by peptide-loaded MHC, we made use of mice expressing the DO11.10 transgenic TCR (20) which recognizes pOVA in the context of I-Ad. These mice were crossed onto a RAG2–/– background. Thus, the T cells from these mice are CD4⫹ and solely express the clonotypic TCR specificity (21). Thy1.1⫹ congenic BALB/c mice were used as donors for stimulator APC (see Methods) to allow the distinction of responder T cells via their expression of Thy1.2 instead of by their clonotypic TCR, which, upon engagement and down-regulation, could disappear from the cell surface. Whether assayed at early time points (6,9 and data not shown) or at 96 h (Fig. 4) post-stimulation, surface TCR downregulation had occurred in an antigenic dose-dependent manner, as had been observed when anti-CD3 mAb was used as TCR agonist (Figs 1 and 2). However, by 96 h, maximally co-stimulated T cells displayed greater surface TCR levels than did cells whose CD28 co-stimulation had

836 CD28 enhances surface TCR replenishment

Fig. 1. CD28 co-stimulation does not affect TCR engagement and down-regulation at early time points after the commencement of antigenic stimulation with anti-CD3 mAb. Cells were stimulated with increasing concentrations of anti-CD3 mAb in the presence of either anti-CD28 mAb, to maximize co-stimulation, or CTLA-4–Ig, to block co-stimulation, as indicated. After 1–9 h, cells were stained with a saturating concentration of anti-CD3 (10 µg/ml), followed by a FITC-conjugated secondary mAb. Surface TCR level is thus expressed as a percentage of the CD3-specific geometric MFI of unstimulated cells obtained from FCM analysis. Data is shown from one of three representative experiments. Results from both one- and two-tailed paired Student’s t-tests reveal no statistical difference between experimental groups based on co-stimulatory manipulation.

Fig. 2. Surface TCR levels are co-stimulation dependent by 24 h post-stimulus. (A) Freshly isolated BALB/c whole splenocytes were either kept unstimulated at 4°C (thick, solid line), or stimulated in vitro with 2 µg/ml anti-CD3 mAb ⫹ anti-CD28 mAb or 2 µg/ml anti-CD3 mAb ⫹ CTLA-4–Ig. After 24 h, some cells were stained with anti-CD3 and secondary mAb (as per Fig. 1) to determine surface TCR expression, while other cells were stained in parallel with negative control mAb for FCM analysis as described in Methods. (B) Cells were stimulated for 24 h with increasing doses of anti-CD3 mAb in the presence of either anti-CD28 mAb, irrelevant control mAb (to permit endogenous, B7-mediated co-stimulation from APC in the culture) or CTLA-4–Ig. The level of surface TCR is expressed as a percentage of the CD3-specific geometric MFI of unstimulated cells. The data represent one of nine experiments. Statistical significance between surface TCR levels of cells with maximal versus blocked co-stimulation is demonstrated by P ⬍ 0.001 using a two-tailed paired Student’s t-test and data from all nine experiments.

CD28 enhances surface TCR replenishment 837 stimulation with anti-CD3 mAb (data not shown). We conclude that CD28-dependent surface TCR enhancement can be demonstrated for cells stimulated with peptide-loaded MHC on APC and that the co-stimulatory mechanism by which this occurs can be attributed to the action of IL-2. CD28-mediated augmentation of surface TCR expression results from both quantitative and kinetic differences in TCR replenishment of the plasma membrane

Fig. 3. Activated T cells of similar size express different levels of surface TCR that depend on CD28 co-stimulation. Freshly isolated BALB/c whole splenocytes were activated in vitro with 2 µg/ml antiCD3 mAb in the presence of either anti-CD28 mAb or CTLA-4–Ig as indicated. After 24 h, cells were stained with anti-CD3 and secondary mAb to determine surface TCR expression. Forward scatter measurements were used to gate on cells of similar sizes in increments of 10 relative units. Geometric MFI, representing either surface TCR or negative control stain, is displayed for cells within each cell size gate. The data represent one of nine experiments.

been blocked during activation with most peptide doses (Fig. 4). This difference was not manifest at extremely high doses of pOVA stimulus (Fig. 4A) nor at time points even as late as 48 h post-stimulation (data not shown). Thus T cells stimulated by APC took longer to manifest a CD28-dependent enhancement of surface TCR expression when compared to cells stimulated with anti-CD3 mAb (Fig. 2). This probably reflects general differences in stimulatory systems, such as the fact that cell division also lags by at least 24 h for whole splenocyte APC-mediated T cell activation (12,21). Since a significant downstream effect of CD28 co-stimulation involves up-regulation of IL-2 (19,22), we examined the role of IL-2 in the activation-induced enhancement of surface TCR expression we had attributed to CD28 co-stimulation. We found that provision of exogenous rmIL-2 to CTLA-4–Igtreated cells resulted in a moderate to full enhancement of surface TCR expression compared to that of anti-CD28treated cells that had been stimulated with peptide-loaded MHC complexes on live APC (Fig. 4A). Likewise, blockade of IL-2 with an anti-IL-2 blocking mAb inhibited CD28-mediated enhancement of surface TCR levels 96 h post-stimulus (Fig. 4B). These same effects were seen at 24 or 96 h post-

To determine whether the lower level of surface TCR expression on cells stimulated with anti-CD3 mAb in the presence of CTLA-4–Ig represented a temporary lag in TCR up-regulation or a lasting phenotype, we examined later time points. BALB/c splenocytes were stimulated in vitro with increasing concentrations of anti-CD3 mAb in the presence of either antiCD28 mAb or CTLA-4–Ig and surface TCR levels examined throughout a time course out to 120 h post-stimulus. To assess the role that the persistent presence of TCR agonist could have on surface TCR levels throughout the time course, some cultures were thoroughly washed after 24 h in order to remove stimulating anti-CD3 mAb and then their incubation was continued in fresh media. Under conditions of continual TCR ligation, anti-CD28treated cells replenished surface TCR to a higher level than did CTLA-4–Ig-treated cells throughout the time course of the culture (Fig. 5A–C). This replenished level was maximal at 48 h, tending to decline thereafter. When T cells were relieved of TCR stimulation after 24 h, TCR levels rebounded significantly beyond the baseline of unstimulated T cells (i.e. ⬎100% in Fig. 5D–F). This effect was independent of co-stimulation, although cells which had received CD28 signals tended to replenish TCR more rapidly and to plateau at higher levels. Thus, at later time points of stimulation, surface TCR expression surpasses unstimulated levels and is further heightened by CD28 co-stimulation. TCR that replenish the plasma membrane are competent to be engaged by ligand and down-regulated during primary stimulation The results above also demonstrated that continuous antigenic stimulation negatively affected the level of surface TCR expression detected during 5 days of culture (Fig. 5). This suggests that the TCR which replenish the plasma membrane following initial agonist-induced down-regulation may be immediately available for ligand engagement, signal transduction and internalization. Alternatively, relief of TCR ligation may stimulate TCR synthesis and surface replenishment. To determine if surface-replenishing TCR could be downregulated by agonist during primary stimulation, splenocytes were stimulated for 24 h, washed to remove stimulus and returned to culture until 69 h post-stimulus to allow surface TCR replenishment to complete and stabilize (determined from Fig. 5). At 69 h, the cells were re-stimulated with increasing concentrations of anti-CD3 mAb, after which their relative surface TCR levels were determined. We found that TCR that replenished the plasma membrane were competent to engage anti-CD3 mAb and be internalized in a dosedependent fashion; this event was not influenced by CD28 co-stimulation during the initial stimulus (Fig. 6A).


Fig. 4. Cells stimulated with MHC–peptide on APC display CD28-dependent surface TCR enhancement that can be attributed to IL-2. Whole splenocytes from Thy1.1-congenic BALB/c mice were pulsed with 0.125–50 µg/ml pOVA overnight at 37°C, as indicated. Then whole splenocytes from DO11.10/RAG2–/– (Thy1.2) mice were added to cultures, responder:stimulator ⫽ 1:2, in the presence of combinations of (A) anti-CD28 mAb, CTLA-4–Ig, rmIL-2 (40 U/ml) and vehicle (PBS) or (B) anti-CD28 mAb, CTLA-4–Ig, anti-IL-2 mAb (50 µg/ml) and control Ig (rat). At 96 h post-stimulation, cells were stained with anti-Thy1.2 and anti-TCRβ mAb for FCM analysis. The surface TCR levels are reported as a percentage of the TCRβ-specific geometric MFI of unstimulated cells. Data is shown from one of two representative experiments. Twotailed paired Student’s t-tests reveal a statistically significant effect by CD28 on surface TCR levels (P ⬍ 0.001) using data from all pOVA dilutions from both experiments.

We next sought to determine if the efficiency of downregulation of replenished TCR might be influenced by prior co-stimulation history. Figure 6(B) compares the proportion of surface TCR internalized by agonistic ligation during the first 3 h of stimulation with the proportion internalized on cells that had been activated in the presence of either anti-CD28 mAb or CTLA-4–Ig. For each concentration of anti-CD3 mAb stimulus, the percentage of TCR down-regulated was equivalent regardless of whether or not the cells were co-stimulated. Thus, the efficiency of down-regulation of newly expressed TCR on activated T cells is similar to that observed on freshly stimulated, previously resting T cells and is not altered by concurrent or previous CD28 co-stimulation. We conclude, however, that as surface TCR replenishment is enhanced by CD28 co-stimulation, a greater number of TCR are engaged in the presence of an ongoing stimulus when compared to co-stimulation-deprived cells. Surface-replenishing TCR transduce signals upon engagement during primary stimulation In order to determine if signal transduction accompanied down-regulation of engaged surface-replenishing TCR, splenocytes were stimulated with anti-CD3 in the presence of either anti-CD28 or CTLA-4–Ig for 24 h, washed and returned to culture. By 69 h post-stimulus, CD69 expression had returned to basal levels. The cells were then re-stimulated with a graded concentration of anti-CD3 mAb for 3 or 24 h,

after which CD69 expression was assessed as an indicator of TCR signaling (23). At 3 h after re-stimulation with anti-CD3, we observed a dose-dependent increase in the percentage of CD69⫹ T cells, indicating that TCR re-engagement was accompanied by downstream T cell activation (Fig. 7A). The fact that CTLA-4–Ig-treated cells did not up-regulate CD69 to the same extent within 3 h was not due to an intrinsic defect in their ability to express CD69, since CD69 expression increased after 1 day of stimulus (Fig. 7B). The difference in the timing of CD69 up-regulation noted here may not be entirely attributable to differences in surface TCR levels between co-stimulatory conditions, since other functional differences are likely to exist between these cell populations. However, we conclude that surface-replenishing TCR transmit signals when engaged by agonist and that CD28 co-stimulation provides T cells with a greater number of TCR that likely contribute to enhanced antigenic sensitivity during ongoing primary stimulation. Discussion This work describes the impact of CD28 co-stimulation on the recruitment of TCR to the plasma membrane subsequent to activation of freshly harvested T cells via strong agonist encounter. We have observed that CD28 co-stimulation does not affect antigenic engagement early during stimulation, but increases the level to which subsequent surface TCR

CD28 enhances surface TCR replenishment 839

Fig. 5. Steady-state surface TCR expression increases as a result of T cell activation and is further enhanced by CD28 co-stimulation. Whole splenocytes were stimulated with anti-CD3 mAb in the presence of either anti-CD28 mAb or CTLA-4–Ig as indicated. (A–C) Cells were subjected to continuous stimulation for up to 5 days. Surface TCR levels were obtained via FCM periodically throughout the time course and are reported as a percentage of the CD3-specific geometric MFI of unstimulated cells. Data is shown from a single mouse spleen and represents one of four experiments. (D–F) After 24 h of stimulation (asterisk), cells were washed thoroughly to terminate stimulus and then returned to culture in fresh medium. Surface TCR levels were monitored throughout the time course. Data is shown from the same mouse spleen as in (A–C) and represents one of three experiments. Results from one-tailed paired Student’s ttests indicate significantly higher surface TCR at time points 48–120 h for maximally co-stimulated cells (P 艋0.005).

Fig. 6. The efficiency of surface TCR engagement by agonist does not change as a function of CD28 co-stimulatory history. (A) Cells were stimulated with 2 µg/ml anti-CD3 mAb in the presence of either anti-CD28 mAb or CTLA-4–Ig. After 24 h, stimulus was removed by washing cells extensively and returning them to culture in fresh media, as in Fig. 5(D–F). At 69 h post-stimulus, cells were introduced to an additional anti-CD3 mAb stimulus for 3 h in culture at the indicated concentrations. Surface TCR levels are reported as CD3specific geometric MFI obtained from FCM analysis. (B) The percent loss of surface TCR expression resulting from 3 h of anti-CD3 mAb treatment was calculated using the starting TCR geometric MFI (right panel; determined by staining with anti-CD3 and secondary mAb) for activated cells from part (A) (squares) and, for comparison, previously unstimulated T cells (circles). Confidence intervals (99%) for the estimation of percent TCR down-regulation are displayed instead of SE bars. Data represents one of three experiments.

replenishment occurs. Furthermore, TCR that replenish the plasma membrane are available for ligand engagement and signal transduction in response to a persistent stimulus. Thus we present a novel mechanism by which CD28 co-stimulation causes an increase in the number of TCR that can be engaged during primary stimulation. Our operational definition of ‘primary stimulation’ is one in which cells have been subjected to stimulatory agent(s), upon which cellular activation commences involving a response that peaks and wanes. We consider that a true resting period can only begin subsequent to the waning phase of the response. In our experiments using anti-CD3 mAb, all stimulated cells, including those from which stimulus was removed

after 24 h, progressed through activation such that proliferation commenced by ~48 h, peaked at 72 h and waned by 96 h (12 and data not shown). Thus our assays of the ability of newly arrived surface TCR to engage and be down-regulated by agonist (Figs 6 and 7) were accomplished on T cells during the peak of their proliferative response. We emphasize that none of the T cells in our experiments experienced a rest from cellular activation, and therefore confine our interpretations to the behavior of surface TCR replenishment and engagement on T cells undergoing primary stimulation. What is the likely source of TCR that replenish the surface following initial receptor down-regulation? Although surface TCR on resting T cells are known to circulate in a receptor


Fig. 7. Surface-replenishing TCR transduce signals upon engagement during primary stimulation. Splenocytes were initially stimulated with 2 µg/ml anti-CD3 mAb and either anti-CD28 mAb or CTLA-4–Ig. After 24 h, stimulus was removed as described in Figs 5(D–F) and 6. At 69 h post-stimulus, cells were introduced to an additional anti-CD3 mAb stimulus for either (A) 3 or (B) 27 h and the resultant CD69 upregulation was determined via FCM. Data is shown from one of two representative experiments.

recycling pathway at a low constitutive rate (24–26), antigenic stimulation directs triggered TCR to a degradative compartment (6) by a mechanism dominant to recycling (27), making multiple triggerings of a single TCR improbable. Since stimulation of quiescent T cells is known to result in a substantial induction of TCR subunit loci within 24 h (13,28,29), it is likely that most if not all surface-replenishing TCR in our experiments are synthesized de novo. Whatever the source of surface-replenishing TCR, their ability to be down-regulated by agonistic ligation suggests that they may provide T cells with the means to continue to monitor the quantity of antigen present throughout the progression of primary stimulation itself. We have shown that within 1 day of removal of 2 µg/ml anti-CD3 stimulus, surface TCR increases from 48 to 171% of original levels (Fig. 5D), while under continuous stimulation, levels increase from 48 to only 62% (Fig. 5A) for CD28-co-stimulated cells. This indicates that under conditions where continual TCR ligation is permitted, a co-stimulated T cell can express, engage and internalize an entire second ‘complement’ (~109%) of TCR during the second day of stimulus alone. The internalization (Figs 5 and 6) and consequent CD69 up-regulation (Fig. 7) induced by engagement of surface-replenishing TCR indicate that these receptors are capable of initiating receptor-mediated signal transduction (23,27,30–32). Ongoing studies involve the biochemical characterization of such signaling and its down-stream repercussion(s) on effector functions such as cytokine production and proliferation. We believe that the lag in full surface TCR recovery after initial engagement that we and others have observed actually results from a masking of surface replenishment mediated by rapid engagement and internalization of newly expressed receptors. This could explain why surface TCR replenishment is not detected when T cells are maintained in the persistent presence of certain high-dose agonists, as is the case for

DO11.10 T cells stimulated with MHC class II molecules loaded with up to 50 µg/ml pOVA (Fig. 4A). Thus the number of TCR engaged for a given concentration of TCR agonist cannot be properly evaluated without accounting for the expression and consumption of surface-replenishing TCR during the full time of stimulation. We are not aware of any prior studies of TCR engagement that have distinguished between receptors that were resident at the plasma membrane during quiescence and those that arrived as a consequence of cellular activation. By demonstrating that surface-replenishing TCR can participate in antigenic signaling during primary stimulation, we have come to favor a model in which all surface TCR are equivalently poised for receptor occupancy regardless of the time of their arrival to the plasma membrane. The mechanism by which CD28 co-stimulation enhances surface TCR replenishment depends on the action of IL-2 (Fig. 4). Signaling through the IL-2 receptor promotes the synthesis of a number of proteins relevant to T cell growth, including c-myc, c-fos and c-jun (33,34); it may also affect synthesis of TCR–CD3 subunits by some unidentified mechanism. Proliferation itself might facilitate recovery of surface TCR expression, though it is not required since anti-CD3-stimulated T cells begin to replenish surface TCR prior to cell division (Fig. 2). Alternatively, CD28 and IL-2 receptor signaling might alter the balance of phosphatase and protein kinase C activities, which has recently been demonstrated to play a role in the ‘fine-tuning’ of surface TCR levels in certain systems (27) and thus modulate surface TCR expression by a posttranslational mechanism. A combination of these and/or other mechanisms may account for our observations. CD28-mediated enhancement of surface TCR expression during activation, which can be considerably masked by the persistent presence of agonist, could contribute to the reported ability of co-stimulation to increase T cell sensitivity to antigen (1,7,12). A qualitative effect of CD28 on TCR engagement has been suggested by the co-stimulationdependent recruitment of membrane microdomains into concentrated sites of T cell–APC interaction (18). However, a CD28-mediated augmentation of TCR expression, with the resulting opportunity for engagement, has not been recognized previously. In a growing number of experimental systems, modest alterations in TCR expression have been shown to amount to substantial changes in the outcome of T cell stimulation (1–4,35). Thus, the mechanisms by which CD28 co-stimulation favors productive T cell activation may involve both qualitative and quantitative effects on TCR engagement. In conclusion, others have shown that the quantity of TCR triggered during antigenic encounter represents a critical parameter in determining the outcome of a T cell response to stimulus (1,36–38). The present data demonstrate that CD28 co-stimulation enhances the recruitment of TCR to the plasma membrane subsequent to an initial antigenic stimulus, making a greater number of TCR available for engagement and signaling. As a result, CD28-co-stimulated cells engage more TCR per dose of agonist than do co-stimulation-deprived cells in the face of an ongoing stimulus. This represents a novel means by which CD28 co-stimulation could enhance T cell responses to antigen. Understanding the precise functional and signaling roles of continued antigenic signaling as

CD28 enhances surface TCR replenishment 841 T cell activation progresses will provide further insights into how the outcome of T cell activation is decided. Acknowledgements We thank Drs Jonni S. Moore, Terri M. Laufer, Thomas A. Judge, Steven C. Eck and Hrefna Gudmundsdottir for critical review and help in preparation of this manuscript. This work was supported by NIH grants AI-41521 and AI-43620. L. A. T. is an Established Investigator of the American Heart Association. A. G. S. and A. D. W. are supported by NIH training grant CA 09140.

Abbreviations APC FCM MFI pOVA PE PI rmIL-2

antigen-presenting cell flow cytometry mean fluorescence intensity peptide derived from chicken ovalbumin amino acids 323–339 phycoerythrin propidium iodide recombinant murine interleukin 2

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References 1 Viola, A. and Lanzavecchia, A. 1996. T cell activation determined by T cell receptor number and tunable thresholds. Science 273:104. 2 Blichfeldt, E., Munthe, L. A., Rotnes, J. S. and Bogen, B. 1996. Dual T cell receptor T cells have a decreased sensitivity to physiological ligands due to reduced density of each T cell receptor. Eur. J. Immunol. 26:2876. 3 Schonrich, G., Kalinke, U., Momburg, F., Malissen, M., SchmittVerhulst, A. M., Malissen, B., Hammerling, G. J. and Arnold, B. 1991. Down-regulation of T cell receptors on self-reactive T cells as a novel mechanism for extrathymic tolerance induction. Cell 65:293. 4 Girgis, L., Davis, M. M. and de St Groth, B. F. 1999. The avidity spectrum of T cell receptor interactions accounts for T cell anergy in a double transgenic model. J. Exp. Med. 189:265. 5 Lanzavecchia, A. 1997. Understanding the mechanisms of sustained signaling and T cell activation. J. Exp. Med. 185:1717. 6 Cai, Z., Kishimoto, H., Brunmark, A., Jackson, M. R., Peterson, P. A. and Sprent, J. 1997. Requirements for peptide-induced T cell receptor downregulation on naive CD8⫹ T cells. J. Exp. Med. 185:641. 7 Iezzi, G., Karjalainen, K. and Lanzavecchia, A. 1998. The duration of antigenic stimulation determines the fate of naive and effector T cells. Immunity 8:89. 8 Cenciarelli, C., Hou, D., Hsu, K. C., Rellahan, B. L., Wiest, D. L., Smith, H. T., Fried, V. A. and Weissman, A. M. 1992. Activationinduced ubiquitination of the T cell antigen receptor. Science 257:795. 9 Valitutti, S., Muller, S., Salio, M. and Lanzavecchia, A. 1997. Degradation of T cell receptor (TCR)–CD3-zeta complexes after antigenic stimulation. J. Exp. Med. 185:1859. 10 Valitutti, S., Muller, S., Cella, M., Padovan, E. and Lanzavecchia, A. 1995. Serial triggering of many T-cell receptors by a few peptide–MHC complexes. Nature 375:148. 11 Bachmann, M. F., Oxenius, A., Speiser, D. E., Mariathasan, S., Hengartner, H., Zinkernagel, R. M. and Ohashi, P. S. 1997. Peptide-induced T cell receptor down-regulation on naive T cells predicts agonist/partial agonist properties and strictly correlates with T cell activation. Eur. J. Immunol. 27:2195. 12 Wells, A. D., Gudmundsdottir, H. and Turka, L. A. 1997. Following the fate of individual T cells throughout activation and clonal expansion. Signals from T cell receptor and CD28 differentially regulate the induction and duration of a proliferative response. J. Clin. Invest. 100:3173. 13 Bronstein-Sitton, N., Wang, L., Cohen, L. and Baniyash, M. 1999.

21

22

23

24

25

26

27

28

29

30

31

32

Expression of the T cell antigen receptor zeta chain following activation is controlled at distinct checkpoints. Implications for cell surface receptor down-modulation and re-expression. J. Biol. Chem. 274:23659. Leo, O., Sachs, D. H., Samelson, L. E., Foo, M., Quinones, R., Gress, R. and Bluestone, J. A. 1986. Identification of monoclonal antibodies specific for the T cell receptor complex by Fc receptormediated CTL lysis. J. Immunol. 137:3874. Leo, O., Foo, M., Sachs, D. H., Samelson, L. E. and Bluestone, J. A. 1987. Identification of a monoclonal antibody specific for a murine T3 polypeptide. Proc. Natl Acad. Sci. USA 84:1374. Samelson, L. E., O’Shea, J. J., Luong, H., Ross, P., Urdahl, K. B., Klausner, R. D. and Bluestone, J. 1987. T cell antigen receptor phosphorylation induced by an anti-receptor antibody. J. Immunol. 139:2708. Terhorst, C., Exley, M., Franco, R., Hall, C., Kang, J., Mueller, B., Sancho, J., She, J. and Wileman, T. 1993. Coupling of T-cell activation with T-cell receptor assembly. Year Immunol. 7:1. Viola, A., Schroeder, S., Sakakibara, Y. and Lanzavecchia, A. 1999. T lymphocyte co-stimulation mediated by reorganization of membrane microdomains. Science 283:680. June, C. H., Ledbetter, J. A., Gillespie, M. M., Lindsten, T. and Thompson, C. B. 1987. T cell proliferation involving the CD28 pathway is associated with cyclosporine-resistant interleukin 2 gene expression. Mol. Cell Biol. 7:4472. Murphy, K. M., Heimberger, A. B. and Loh, D. Y. 1990. Induction by antigen of intrathymic apoptosis of CD4⫹CD8⫹TCRlo thymocytes in vivo. Science 250:1720. Gudmundsdottir, H., Wells, A. D. and Turka, L. A. 1999. Dynamics and requirements of T cell clonal expansion in vivo at the singlecell level: effector function is linked to proliferative capacity. J. Immunol. 162:5212. Lindsten, T., June, C. H., Ledbetter, J. A., Stella, G. and Thompson, C. B. 1989. Regulation of lymphokine messenger RNA stability by surface-mediated T cell activation pathway. Science 244:339. Lucas, B., Stefanova, I., Yasutomo, K., Dautigny, N. and Germain, R. N. 1999. Divergent changes in the sensitivity of maturing T cells to structurally related ligands underlies formation of a useful T cell repertoire. Immunity 10:367. Krangel, M. S. 1987. Endocytosis and recycling of the T3– T cell receptor complex. The role of T3 phosphorylation. J. Exp. Med. 165:1141. Minami, Y., Samelson, L. E. and Klausner, R. D. 1987. Internalization and cycling of the T cell antigen receptor. Role of protein kinase C. J. Biol. Chem. 262:13342. Dietrich, J., Backstrom, T., Lauritsen, J. P., Kastrup, J., Christensen, M. D., von Bulow, F., Palmer, E. and Geisler, C. 1998. The phosphorylation state of CD3gamma influences T cell responsiveness and controls T cell receptor cycling. J. Biol. Chem. 273:24232. Lauritsen, J. P., Christensen, M. D., Dietrich, J., Kastrup, J., Odum, N. and Geisler, C. 1998. Two distinct pathways exist for downregulation of the TCR. J. Immunol. 161:260. Lindsten, T., June, C. H. and Thompson, C. B. 1988. Transcription of T cell antigen receptor genes is induced by protein kinase C activation. J. Immunol. 141:1769. Paillard, F., Sterkers, G. and Vaquero, C. 1990. Transcriptional and post-transcriptional regulation of TcR, CD4 and CD8 gene expression during activation of normal human T lymphocytes. EMBO J. 9:1867. Luton, F., Buferne, M., Davoust, J., Schmitt-Verhulst, A. M. and Boyer, C. 1994. Evidence for protein tyrosine kinase involvement in ligand-induced TCR/CD3 internalization and surface redistribution. J. Immunol. 153:63. Cenciarelli, C., Wilhelm, K. G., Jr, Guo, A. and Weissman, A. M. 1996. T cell antigen receptor ubiquitination is a consequence of receptor-mediated tyrosine kinase activation. J. Biol. Chem. 271:8709. D’Oro, U., Vacchio, M. S., Weissman, A. M. and Ashwell, J. D. 1997. Activation of the Lck tyrosine kinase targets cell surface T cell antigen receptors for lysosomal degradation. Immunity 7:619.

842 CD28 enhances surface TCR replenishment 33 Taniguchi, T. and Minami, Y. 1993. The IL-2/IL-2 receptor system: a current overview. Cell 73:5. 34 Ihle, J. N., Witthuhn, B. A., Quelle, F. W., Yamamoto, K. and Silvennoinen, O. 1995. Signaling through the hematopoietic cytokine receptors. Annu. Rev. Immunol. 13:369. 35 Korganow, A. S., Ji, H., Mangialaio, S., Duchatelle, V., Pelanda, R., Martin, T., Degott, C., Kikutani, H., Rajewsky, K., Pasquali, J. L., Benoist, C. and Mathis, D. 1999. From systemic T cell selfreactivity to organ-specific autoimmune disease via immunoglobulins. Immunity 10:451. 36 Valitutti, S., Muller, S., Dessing, M. and Lanzavecchia, A. 1996.

Different responses are elicited in cytotoxic T lymphocytes by different levels of T cell receptor occupancy. J. Exp. Med. 183:1917. 37 Itoh, Y. and Germain, R. N. 1997. Single cell analysis reveals regulated hierarchical T cell antigen receptor signaling thresholds and intraclonal heterogeneity for individual cytokine responses of CD4⫹ T cells. J. Exp. Med. 186:757. 38 Itoh, Y., Hemmer, B., Martin, R. and Germain, R. N. 1999. Serial TCR engagement and down-modulation by peptide:MHC molecule ligands: relationship to the quality of individual TCR signaling events. J. Immunol. 162:2073.