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TCR Comodulation of Nonengaged TCR Takes Place by a Protein Kinase C and CD3 Di-Leucine-Based Motif-Dependent Mechanism ¹

Charlotte Menné Bonefeld^*, Anette B. Rasmussen^*, Jens Peter H. Lauritsen^*, Marina von Essen^*, Niels Ødum^*, Peter S. Andersen and Carsten Geisler^2,*

^* Institute of Medical Microbiology and Immunology, Panum Institute, University of Copenhagen, Copenhagen; and Symphogen, Lyngby, Denmark

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
One of the earliest events following TCR triggering is TCR down-regulation. However, the mechanisms behind TCR down-regulation are still not fully known. Some studies have suggested that only directly triggered TCR are internalized, whereas others studies have indicated that, in addition to triggered receptors, nonengaged TCR are also internalized (comodulated). In this study, we used transfected T cells expressing two different TCR to analyze whether comodulation took place. We show that TCR triggering by anti-TCR mAb and peptide-MHC complexes clearly induced internalization of nonengaged TCR. By using a panel of mAb against the Ti chain, we demonstrate that the comodulation kinetics depended on the affinity of the ligand. Thus, high-affinity mAb (K_D = 2.3 nM) induced a rapid but reversible comodulation, whereas low-affinity mAb (K_D = 6200 nM) induced a slower but more permanent type of comodulation. Like internalization of engaged TCR, comodulation was dependent on protein tyrosine kinase activity. Finally, we found that in contrast to internalization of engaged TCR, comodulation was highly dependent on protein kinase C activity and the CD3 di-leucine-based motif. Based on these observations, a physiological role of comodulation is proposed and the plausibility of the TCR serial triggering model is discussed.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The TCR comprises the clonotypic Ti and chains and the invariant CD3, , , and chains (1, 2, 3, 4, 5, 6). The - and -chains are responsible for the specific recognition of peptide-MHC complexes and the signaling events following Ag recognition are mediated by the CD3 and -chains (7, 8, 9).

Regulation of TCR expression levels is most probably a very important mechanism that allows T cells to calibrate their responses to different levels of stimuli. TCR internalization takes place both in resting T cells as part of constitutive TCR cycling and following TCR triggering and protein kinase C (PKC) ³ activation. During constitutive internalization, TCR cycling between the cell surface and the endosomes results in a steady-state distribution of the TCR with 70–85% of the cycling TCR pool at the cell surface and 15–30% of the pool inside the cell (10, 11, 12, 13). Recently, it has been shown that constitutive TCR cycling is dependent on the di-leucine-based (LL-based) motif in CD3 (14). This motif consists of the DxxxLL sequence and the mechanism whereby it mediates TCR internalization has been described in detail previously (15, 16). In completely assembled TCR complexes, the CD3 LL-based motif is not fully exposed. However, subsequent to phosphorylation of CD3 serine126 the LL-based motif becomes exposed and the clathrin-associated AP-2 binds to the motif. Binding of AP-2 leads to internalization of the TCR by the clathrin-dependent internalization machinery (16). In addition to constitutive TCR cycling, both PKC- and ligand-induced TCR down-regulation are dependent on the CD3 LL-based motif (14, 17, 18). During constitutive TCR cycling and following PKC-mediated TCR internalization, the TCR are recycled back to the cell surface in a functional state (19). In contrast, when the TCR are internalized by the ligand-induced mechanism, which is dependent on protein tyrosine kinase (PTK) activity, a large fraction of the TCR is targeted for lysosomal degradation (17, 20, 21, 22, 23).

After contact between a T cell and an APC loaded with the relevant peptide, TCR are actively recruited to the contact zone between the T cell and the APC (24). A well-organized structure, the immunological synapse (IS), is formed within minutes of contact, with adhesion molecules in the periphery and TCR located at the center of the IS (25, 26). Only a small fraction of the TCR in the IS appears to be bound to specific peptide-MHC complexes (24). This is interesting taking into consideration that up to 90% of the TCR on the surface of the T cell can be rapidly internalized after ligand stimulation. Based on such observations, Lanzavacchia and coworkers (27) proposed the TCR serial triggering model in which one peptide-MHC complex is able to stimulate and down-regulate a large number of TCR (up to 200) within a relatively short period. However, some studies have challenged the TCR serial triggering model by showing that TCR down-regulation might include both directly engaged as well as nonengaged TCR (28, 29, 30, 31, 32). A few studies suggested that a physical association of multiple TCR might be the mechanism behind comodulation (28, 29), whereas other studies have indicated that trans-acting phosphorylation of nonengaged TCR plays a crucial role (30, 31, 32). Still other studies have not been able to detect TCR comodulation (3, 27, 33). Thus, whether comodulation of nonengaged TCR actually takes place and if so the mechanisms behind comodulation are still not fully known.

The aim of this study was to investigate whether comodulation of nonengaged TCR takes place and if so to determine the role of ligand affinity and the mechanisms involved in TCR comodulation.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cells and reagents

The V8⁺ chicken OVA/I-A^d-specific mouse T cell hybridoma DO11.10 (34), the I-A^d+ mouse B cell lymphoma M12.B5 (35), the V8⁺ human Jurkat T cells with SV40 virus large T Ag (JTag), and the human T cell Jurkat variant JBN, which lacks the Ti chain, were cultured in complete medium (RPMI 1640 medium supplemented with 0.5 IU/L penicillin, 500 mg/L streptomycin, and 10% FBS) at 37°C in 5% CO₂. The anti-mouse V8.2 mAb F23.1 and the F23.1 variants 20 and 32 (36) were kindly provided by Dr. K. Karjalainen (Institute for Research in Biomedicine, Bellinzona, Switzerland). The anti-human V8 mAb MX6 was kindly provided by Dr. A. Boylston (University of Leeds, Leeds, U.K.). The PE-, FITC-, and nonconjugated anti-mouse V8 mAb (F23.1), anti-mouse V2 mAb (B20.6), PE-conjugated anti-human V8 mAb (BV8), and anti-human V3 mAb (BV3S1) were obtained from BD PharMingen (San Diego, CA). Protein A was purchased from Amersham Pharmacia Biotech (Uppsala, Sweden). The chicken OVA peptide OVA_323–339 was obtained from Schafer-N (Copenhagen, Denmark). 2',7'-bis-(2-carboxyethyl)-5-(and 6)-carboxyfluorescein acetoxymethyl ester (BCECF/AM) was purchased from Molecular Probes (Eugene, OR). The broad PKC inhibitor Ro 31-8220 (37) was kindly provided by Dr. D. Bradshaw (Roche Research Center, Welwyn Garden City, U.K.). The scr family-specific inhibitor PP1 and the broad PKC inhibitor bisindolylmaleimide I were obtained from Calbiochem (San Diego, CA).

Transfection

DO11.10 cells were transfected with the expression vector pCA134 (38) coding for the mouse V2 chain (kindly provided by Dr. B. Malissen, Marseille, France) and JBN cells were transfected with the expression vector pcDNA6-HA1.7-TCR-5 (39) coding for the human V3 chain (kindly provided by Dr. L. Wedderburn, London, U.K.) as previously described (40). Cells were plated at 1 x 10⁴ and 5 x 10⁴ cells/ml in 96-well tissue culture plates in complete medium containing either 1 mg/ml G418 sulfate (DO11.10) or 5 µg/ml blasticidin (JBN). After 3–4 wk of selection G418- and blasticidin-resistant clones were expanded and maintained in complete medium.

For lipotransfection of JTag cells, 0.5 ml of RPMI 1640 containing DMRIE-C reagent (16 µg/ml; Invitrogen, Carlsbad, CA) and 0.5 ml of RPMI 1640 containing the expression vectors pcDNA6-HA1.7-TCR-5 (4 µg/ml) and either pEGFP-HCD3-WT-1 or pEGFP-HCD3-LLAA-3 (4 µg/ml) were mixed in each well of a six-well plate. The pEGFP-HCD3-WT-1 coded for chimeric wild-type (WT) CD3-green fluorescent protein (GFP; CD3WT-GFP) and pEGFP-HCD3-LLAA-3 coded for CD3LLAA-GFP mutated in the LL-based motif as described previously (14). To allow formation of lipid-DNA complexes, the plates were incubated at room temperature for 30 min. Subsequently, 2 x 10⁶ JTag cells were added to each well. Following 4 h of incubation at 37°C in 5% CO₂, 2 ml of RPMI 1640 containing 15% FBS was added to each well. After 24 h, 1 ml of complete medium was added to each well and the cells were used for experiments 48 h after lipotransfection.

TCR comodulation

For experiments with the V2⁺V8⁺ DO11.10 transfectants, Maxisorb plates (Nunc, Roskilde, Denmark) were coated with protein A or rabbit anti-rat Ig (10 µg/ml) overnight at 4°C. The plates were washed in PBS and subsequently blocked for 1 h with 2% BSA in PBS at room temperature. Following blocking, the plates were washed in PBS and incubated with various concentrations of anti-mouse V8 or anti-mouse V2 mAb diluted in PBS with 0.2% BSA for 2 h at room temperature. The cells were adjusted to 4 x 10⁵ cells/ml and transferred to the coated plates, centrifuged for 1 min at 500 rpm, and incubated at 37°C. At the indicated time, cells were transferred to ice. The cells were stained directly with PE-conjugated anti-mouse V2 or anti-mouse V8 mAb and analyzed by flow cytometry. For stimulation with peptide/MHC, the APC cells M12.B5 were pulsed for 2 h with the indicated concentrations of OVA_323–339 peptide, stained with BCECF/AM for 10 min, washed four times, and cocultured with DO11.10 cells at a ratio of 1:1 for the time indicated. The cells were subsequently transferred to 4°C and analyzed for TCR expression by incubation with PE-conjugated anti-mouse V2 or anti-mouse V8 mAb and gating out cells with green fluorescence (BCECF/AM). For the experiments using kinase inhibitors, cells were preincubated with PP1 (16 µM, 30 min), Ro 31-8220 (40 µM, 15 min) or bisindolylmaleimide (10 nM,; 15 min) at 37°C. The TCR down-regulation experiments were performed as described above. However, cells treated with the PKC inhibitors were stained with FITC-conjugated Abs.

For experiments with JTag transfectants, plates were coated with anti-human V8 mAb (250 ng/ml) overnight at 4°C and subsequently washed in PBS. The cells were adjusted to 5 x 10⁵ cells/ml and transferred to the coated plates, centrifuged for 1 min at 500 rpm, and placed at 37°C. At the indicated time, cells were transferred to ice. The cells were stained directly with PE-conjugated anti-human V3 or anti-human V8 mAb and analyzed by flow cytometry. TCR down-regulation was determined as ((mean fluorescence intensity (MFI) of stimulated cells)/(MFI of nonstimulated cells)) x 100%.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Coexpression of V2 and V8 TCR at the surface of transfected T cells

To obtain cells expressing two distinct TCR, we transfected the V8⁺ DO11.10 T cell hybridomas with plasmids coding for the mouse V2 chain. Double-positive transfectants that stained equally bright for V2 and V8 were selected for subsequent experiments (Fig. 1, A and B). When using Ti-specific mAb in comodulation analysis, it is mandatory to know whether the mAb used for stimulation cross-reacts with the other type of Ti chain expressed by the double-positive transfectants. Thus, to analyze for possible cross-reaction of the stimulating mAb, the transfectants were preincubated with either nonconjugated anti-mouse V2 or anti-mouse V8 mAb for 2 h at 12°C. The cells were next incubated with either PE-conjugated anti-mouse V2 or anti-mouse V8 mAb for 30 min on ice and subsequently analyzed by flow cytometry. The nonconjugated anti-mouse V8 mAb was able to blocked subsequent binding of PE-conjugated anti-V8 mAb but did not affect the binding of PE-conjugated anti-mouse V2 mAbs (Fig. 1, C and D). Likewise, the nonconjugated anti-mouse V2 mAb blocked subsequent binding of PE-conjugated anti-mouse V2 mAb but did not affect the binding of PE-conjugated anti-mouse V8 mAb (Fig. 1, C and D).

View larger version (30K): [in this window] [in a new window]   FIGURE 1. Coexpression of V2 and V8 at the surface of transfected DO11.10 T cell. A, V8+ DO11.10 T cell hybridomas were transfected with the expression vector pCA134 coding for the mouse V2 chain. Coexpression of V2 and V8 was analyzed by two-color flow cytometry. B, In addition, the expression levels of V2 (light gray, empty), V8 (black, empty), and CD3 (dark gray, empty) were analyzed by one-color flow cytometry. C and D, Blocking studies. Cells preincubated with nonconjugated anti-mouse V8 mAb (dashed line marked V8), anti-mouse V2 mAb (dashed line marked V2), or PBS containing 0.1% NaN3, 5% mouse serum, and 5% rat serum (full line) were subsequently stained with either PE-conjugated anti-mouse V8 mAb (C) or anti-V2 mouse mAb (D) and analyzed by flow cytometry. The blocking mAb are given within the histograms and the staining mAb are given below the histograms. The abscissa gives the fluorescence intensity in a logarithmic scale in arbitrary units and the ordinate gives the relative cell number.

Both stimulation with V-specific mAb and with specific peptide-MHC complexes induce comodulation

Some studies have shown that TCR triggering can induce comodulation of nonengaged TCR (30, 31, 32). In contrast, other studies did not observe comodulation of nonengaged TCR following stimulation of T cells expressing two different TCR (3, 27, 33). To analyze whether engaged TCR induced comodulation of nonengaged TCR, the V2⁺V8⁺ DO11.10 transfectants were stimulated with plate-bound V8- or V2-specific mAb for various periods of time. Stimulation of the cells with anti-mouse V8 mAb induced down-regulation of both the specifically engaged TCR (V8) and the nonengaged TCR (V2) (Fig. 2A). Likewise, stimulation of the cells with anti-mouse V2 mAb induced down-regulation of both the specifically engaged TCR (V2) and the nonengaged TCR (V8) (Fig. 2B). The kinetics whereby engaged TCR and nonengaged TCR were down-regulated differed. The engaged TCR were rapidly down-regulated and reached a plateau of 30% after 1 h. The nonengaged TCR were down-regulated to a lesser degree and with a slightly slower kinetics than engaged receptors. To analyze whether comodulation also took place during stimulation with natural peptide/MHC ligands, the V2⁺V8⁺ DO11.10 transfectants were incubated for various times with I-A^d+ M12.B5 cells pulsed with OVA_323–339. Although down-regulation of the directly triggered TCR (V8) was not as efficient as seen after stimulation with the anti-V mAb, comodulation of V2 clearly took place (Fig. 2C). Taken together, these experiments clearly demonstrated that comodulation of nonengaged TCR took place both following triggering with high-affinity ligands in the form of mAb and following triggering with low-affinity natural ligands in the form of peptide-MHC complexes.

View larger version (14K): [in this window] [in a new window]   FIGURE 2. TCR triggering with mAb and peptide-MHC complexes induces comodulation of nonengaged receptors. V2+V8+ DO11.10 transfectants were stimulated with plate-bound anti-mouse V8 mAb (1 µg/ml, A), plate-bound anti-mouse V2 mAb (1 µg/ml, B), or M12.B5 cells (C) pulsed with 10 µM OVA323–339 at 37°C. At the times indicated, the cells were transferred to ice. Subsequently, the cells were stained with anti-mouse V2 mAb (•) or anti-mouse V8 mAb () and analyzed by flow cytometry. TCR down-regulation was determined as ((MFI of stimulated cells)/(MFI of nonstimulated cells)) x 100%. The results of one representative experiment of three independently performed experiments are shown.

It has been shown that the affinity between the ligand and the TCR affects the activation levels of the T cell (36, 41, 42), and we speculated whether the observed differences in TCR down-regulation observed after stimulation with mAb vs peptide-MHC complexes could be due to the 300-fold higher affinity of the mAb compared with the peptide-MHC complexes. To investigate the role of affinity on TCR comodulation, we used a panel of variants of the anti-mouse V8 mAb F23.1 with known affinities (36). V2⁺V8⁺ DO11.10 transfectants were stimulated with the plate-bound Abs for various times and were subsequently tested for the expression of V2 and V8. All mAb applied induced TCR down-regulation of both V2 and V8 (Fig. 3). However, the affinity of the stimulating mAb had a great impact on the degree of TCR down-regulation of both engaged and nonengaged TCR. For the mAb with the highest affinity (K_D = 2.3 nM), an optimum was reached after 30 min for nonengaged TCR and after 60 min for engaged TCR. Interestingly, after reaching a comodulation optimum following 30 min of stimulation, the level of comodulation started to decline. After 2 h of V8 stimulation, the expression level of nonengaged TCR were similar to the steady-state level of unstimulated cells, whereas the engaged TCR were permanently down-regulated to a level of 30% of unstimulated cells (Fig. 3A). This experiments demonstrated that comodulation of nonengaged TCR takes places in a time-dependent and reversible manner following stimulation with high-affinity ligands. This might explain why some groups did observe comodulation whereas other groups did not observe this phenomenon.

View larger version (15K): [in this window] [in a new window]   FIGURE 3. The role of TCR ligand affinity in TCR comodulation. V2+V8+ DO11.10 transfectants were stimulated with variants of the anti-mouse V8 mAb (1 µg/ml) with different affinities (A, KD = 2.3 nM; B, KD = 28 nM; C, KD = 6200 nM). At the times indicated, the cells were transferred to ice. Subsequently, the cells were stained with anti-mouse V8 mAb () or anti-mouse V2 mAb (•) and analyzed by flow cytometry. TCR down-regulation was determined as ((MFI of stimulated cells)/(MFI of nonstimulated cells)) x 100%. The results are given as mean ± SD of three or four independent experiments. A, The V2 expression level at the 30-min time point differed significantly (p < 0.02) from the expression level at the 120-min time point.

Both PTK and PKC activation is required for TCR comodulation

Previous studies have demonstrated that ligand-mediated TCR internalization is dependent on PTK activity (17, 20, 21, 22). In addition, a recent study demonstrated that efficient ligand-mediated TCR internalization also depended on PKC activity (18). We therefore wished to investigate the role of PTK and PKC in comodulation of the TCR. Accordingly, TCR comodulation experiments were performed in the presence of either the scr family-specific inhibitor PP1 or the broad PKC inhibitors Ro 31-8220 and bisindolylmaleimide. V2⁺V8⁺ DO11.10 transfectants were pretreated with either PP1, Ro 31-8220, or bisindolylmaleimide or were left untreated. The cells were next stimulated with plate-bound anti-mouse V8 mAb as described above in the presence or absence of inhibitors and tested for the expression of V2 and V8. As plate-bound anti-mouse V8 mAb, either the low-affinity (K_D = 6200 nM) or the high-affinity (K_D = 2.3 nM) anti-mouse V8 mAb was used. Both PP1 and Ro 31-8220 completely inhibited comodulation of V2 following stimulation with the low-affinity anti-mouse V8 mAb (Fig. 4, A–C). Likewise, down-regulation of engaged V8 was completely inhibited by PP1, whereas treatment with Ro 31-8220 only partially inhibited V8 down-regulation (Fig. 4, A–C). When using the high-affinity mAb, V2 comodulation was almost completely inhibited in cells treated with PKC inhibitor, whereas V8 down-regulation was only partially inhibited (Fig. 4, D and E). PP1 did not significantly inhibit V8 down-regulation or V2 comodulation following stimulation with the high-affinity mAb (data not shown) as previously observed by others following stimulation with high-affinity ligands (32). Taken together, these data indicated that TCR comodulation is strongly dependent on PKC activity whereas down-regulation of engaged TCR only partially depends on PKC activity. Furthermore, both TCR comodulation and down-regulation of engaged TCR are dependent on PTK activity following stimulation with low-affinity ligands.

View larger version (27K): [in this window] [in a new window]   FIGURE 4. TCR comodulation is dependent on both PKC and PTK activity. V2+V8+ DO11.10 transfectants were preincubated with PP1 (B, 16 µM for 30 min), Ro 31-8220 (C, 40 µM for 15 min), or bisindolylmaleimide (E, 10 nM for 15 min) or were left untreated (A and D) at 37°C. The cells were subsequently stimulated with plate-bound anti-mouse V8 mAb (A–C, KD = 6200 nM, 1 µg/ml; D and E, KD = 2.3 nM, 0.25 µg/ml) at 37°C. The cells were transferred to ice at the time indicated and examined for expression of V2 (•) and V8 () by flow cytometry. TCR down-regulation was determined as ((MFI of stimulated cells)/(MFI of nonstimulated cells)) x 100%. The results are given as mean ± SD of three independent experiments.

TCR comodulation is dependent on the CD3 LL-based motif

A previous study on TCR double-positive cells has demonstrated that the CD3 chain of both engaged and nonengaged TCR becomes phosphorylated following specific stimulation of one of the TCR types expressed (30). Several studies have demonstrated the important role of PKC-mediated activation of the LL-based motif in CD3 during PKC-induced TCR down-regulation (16, 17, 43). Taken together with the present results on the dependence of TCR comodulation on PKC activity, this implied that the CD3 LL-based motif might play a central role in TCR comodulation. To test this, the V8⁺ Jurkat T cell line JTag was cotransfected with V3 and chimeric constructs of WT CD3WT-GFP or mutated CD3LLAA-GFP. To exclude cross-reactivity between the anti-human V8 mAb and V3, the Ti chain-negative Jurkat variant JBN was transfected with V3. V3⁺ JBN cells and V8⁺ JTag cells were then stimulated with plate-bound anti-human V8 mAb and subsequently analyzed for the expression of V3 and V8, respectively. Stimulation with anti-human V8 mAb induced TCR down-regulation in V8⁺ Jurkat cells, whereas no effect was seen on V3⁺ JBN cells, demonstrating that the anti-human V8 mAb did not cross-react with V3 (Fig. 5A). Subsequently, the V3⁺V8⁺ JTag transfectants coexpressing either CD3WT-GFP or CD3LLAA-GFP were stimulated with plate-bound anti-human V8 mAb for various times and analyzed for the expression of V8 and V3. In V3⁺V8⁺ transfectants coexpressing CD3WT-GFP, both engaged and nonengaged TCR were down-regulated (Fig. 5B). However, the engaged TCR were down-regulated with faster kinetics and to a greater extent than the nonengaged TCR. In V3⁺V8⁺ transfectants coexpressing CD3LLAA-GFP, down-regulation of engaged TCR was partially inhibited whereas comodulation of nonengaged TCR was almost completely inhibited (Fig. 5C). These results indicated that comodulation is strongly dependent on the LL-based motif of CD3 and further confirm previous studies indicating that efficient ligand-mediated TCR down-regulation is dependent on this motif (18).

View larger version (13K): [in this window] [in a new window]   FIGURE 5. TCR comodulation is dependent on the LL-based motif of CD3. A, V3+ JBN cells and V8+ JTag cells were stimulated with plate-bound anti-human V8 mAb (250 ng/ml) at 37°C for the times indicated. Subsequently, JBN cells were examined for V3 expression (•) and JTag cells were examined for expression of V8 (). B and C, Cotransfection studies. V8+ JTag cells were transiently transfected with the V3 chain and either CD3WT-GFP (B) or CD3LLAA-GFP (C). Subsequently, cells were stimulated with plate-bound anti-human V8 mAb (250 ng/ml) at 37°C. At the times indicated, the cells were transferred to ice, stained with anti-human V3 mAb (•) or anti-human V8 mAb () and analyzed by flow cytometry. Only cells expressing high levels of GFP were analyzed and the level of TCR down-regulation was determined as ((MFI of stimulated cells)/(MFI of nonstimulated cells)) x 100%. The results are given as mean ± SD of three independent experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

There has been some disagreement in the literature concerning TCR comodulation. In some studies comodulation were observed (28, 30, 31, 32), whereas other studies failed to detect comodulation (3, 27, 33). Hou et al. (3) examined for comodulation following 20 h of stimulation with a high-affinity mAb and found no comodulation. However, we found re-expression of comodulated receptors following 2 h of stimulation with a high-affinity mAb, and it is therefore likely that Hou et al. (3) did not observe any comodulation because they analyzed for comodulation too late. Furthermore, some degree of comodulation is actually seen in the studies by Stotz et al. (33) and Valitutti et al. (27), although the contrary was reported by these authors.

We found that comodulation was highly dependent on the affinity of the stimulating ligand. Following stimulation with a high-affinity ligand, a rapid down-regulation of engaged as well as nonengaged receptors was observed. Whereas the expression level of engaged TCR remained low during the experiments, comodulation was reversible in that nonengaged receptors became re-expressed at the cell surface after 30 min of stimulation. In contrast, when T cells were stimulated with low-affinity ligands, the engaged and nonengaged receptors followed approximately the same kinetics for down-regulation. In agreement with these results, a recent study observed recycling of nonengaged receptors following stimulation with a high-affinity ligand and, furthermore, found approximately the same kinetics for engaged and nonengaged receptors following stimulation with a low-affinity ligand (32). The observation that comodulation is reversible argues against the suggestion that physical association between engaged and nonengaged TCR may be the mechanism behind comodulation as proposed by others (28, 29) based on studies suggesting that each TCR complex comprises two Ti dimers. According to these studies, reversible comodulation of V2 might be explained by down-regulation of V2/V8-containing TCR complexes and the isolated reappearance of V2/V2-containing TCR complexes. However, we do not think that the reversible comodulation of V2 observed following stimulation with the high-affinity anti-V8 mAb can be explained by accumulation of V2-V2 complexes at the cell surface during the rather short time interval of the experiments. It is known that TCR down-regulated by direct triggering do not recycle but are degraded. If the observed V2 down-regulation was caused by down-regulation of V2-V8 complexes, the reappearance of V2 should be caused solely by new synthesis of V2. The rate constant for TCR new synthesis is low (0.001 min^-1, C.G., unpublished data) and it has been shown that re-establishment of normal TCR levels following TCR triggering with anti-TCR mAb takes >24 h (44). However, in our experiments normal levels of V2 are observed 90 min after maximal comodulation. This could easily be explained by recycling of internalized V2, as the recycling rate constant is 50 times higher (0.05 min^-1) than the rate constant for new synthesis (0.001 min^-1) (13). The question is then how could the comodulation of V2 be reversible. Stimulation with the high-affinity anti-V8 mAb causes a rapid and profound down-regulation of V8. It may be suggested that TCR signaling through V8 is significantly reduced after 1 h due to the induced low levels of V8. Whereas the directly triggered V8 are degraded, the comodulated V2 quickly recycles back to the surface when PKC activity returns to basic prestimulation levels (19).

In this study, the V2 chain used to transfect DO11.10 cells was obtained from the H-2K^b-specific KB5-C20 T cell line (38). With regard to the comodulation experiments using specific peptide/MHC stimulation, we cannot formally exclude the possibility that the TCR generated by pairing of the transfected V2 chain and the endogenous DO11.10 TCR chain recognized OVA_323–339/I-A^d although we find this unlikely.

We found that PTK is required for TCR down-regulation of both engaged and nonengaged receptors following stimulation with a low-affinity ligand. A previous study found that down-regulation of nonengaged receptors was dependent on PTK, whereas down-regulation of engaged receptors was independent of PTK (32). The divergence from our observations could be explained both by the use of a high-affinity ligand and by the fact that a TT chimera was used as model for one of the TCR in this study (32). Indeed, we have data showing that stimulation with high-affinity ligands can induce TCR down-regulation independently of PTK (data not shown).

We have recently shown that efficient down-regulation of engaged TCR is dependent on PKC and the LL-based motif in CD3 (18). In the present study, we elaborated on this observation and found that in addition to the requirement for efficient down-regulation of engaged TCR, PKC activity and the LL-based motif in CD3 are required for comodulation of nonengaged TCR. In agreement with this, it has been shown that stimulation of the TCR leads to phosphorylation of CD3 both in engaged and nonengaged receptors (30). Comodulation of V3 in JTag cells cotransfected with CD3LLAA-GFP was not completely abolished. This was probably due to the fact that although CD3LLAA-GFP was overexpressed in the JTag cells they still expressed WT CD3. Thus, not all of the V3⁺ TCR expressed at the cell surface contained CD3LLAA-GFP but a small fraction contained the WT CD3 and therefore maintained the ability to be comodulated. Inhibition of PKC activity led to down-regulation of engaged receptors with a slower kinetics compared with untreated cells. This indicated a role of PKC early in the TCR internalization. The requirements of PKC and the LL-based motif for down-regulation of engaged receptors could either be a direct effect or an indirect effect because it must be expected that a fraction of the Ab-specific receptors were comodulated rather than down-regulated by direct engagement.

The role of TCR down-regulation of both engaged and nonengaged TCR is still unknown. It could play a role in receptor revision and tolerance induction during T cell development as suggested by Fink and McMahan (45). Furthermore, it may serve as a protective mechanism to avoid overstimulation of the T cell or it may be a part of the signaling mechanism following TCR triggering. We have recently shown that PKC activation increases the endocytic rate of the TCR without affecting the exocytic rate (13). It is therefore likely that TCR triggering would adjust a new kinetic equilibrium for nonengaged TCR with reduced levels of TCR expressed on the surface and an increased pool of intracellular TCR. The recycling ability of this pool of nonengaged TCR allows directed exocytosis of nontriggered TCR, which may suggest that down-regulation of nonengaged receptors serves as a recruitment mechanism for TCR to the IS (Fig. 6). This could explain how a large proportion of the TCR are rapidly concentrated in the IS after contact between the T cell and the APC (24).

View larger version (28K): [in this window] [in a new window]   FIGURE 6. Model for comodulation. A, At steady state, the TCR cycles constitutively between the cell surface and the endosomes with a distribution of 80% of the TCR at the surface and 20% in the endosomes. B, Following stimulation, an increase in PKC activity changes the steady-state distribution of nonengaged TCR by increasing the endocytic rate. This leads to a new surface:endosome distribution ratio of nonengaged TCR. The internalized, nonengaged TCR might subsequently be actively transported to the IS contributing to the recruitment of fresh TCR to the IS.

	Acknowledgments

 
The technical help of Bodil Nielsen is gratefully acknowledged.

	Footnotes

 
¹ This work was supported by the Danish Medical Research Council, the Carlsberg Foundation, the Foundation of Vilhelm Pedersen and Wife by recommendation of the Novo Nordisk Foundation, the A. P. Møller Foundation for the Advancement of Medical Sciences, and the Astrid Thaysen Foundation for Basic Medical Sciences. C.M.B., J.P.H.L., and M.v.E. were recipients of Ph.D. scholarships from the University of Copenhagen.

² Address correspondence and reprint requests to Dr. Carsten Geisler, Institute of Medical Microbiology and Immunology, Panum Institute, University of Copenhagen, Building 22.5, Blegdamsvej 3C, DK-2200 Copenhagen, Denmark. E-mail address: c.geisler{at}immi.ku.dk

³ Abbreviations used in this paper: PKC, protein kinase C; LL, di-leucine based; PTK, protein tyrosine kinase; IS, immunological synapse; WT, wild type; GFP, green fluorescent protein; MFI, mean fluorescence intensity.

Received for publication January 27, 2003. Accepted for publication July 17, 2003.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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