Staging and resetting T cell activation in SMACs


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Article

Nature Immunology 3, 911 - 917 (2002)
Published online: 3 September 2002; | doi:10.1038/ni836

Staging and resetting T cell activation in SMACs

Benjamin A. Freiberg^1,^2,⁶, Hannah Kupfer^1,⁶, William Maslanik¹, Joe Delli^1,², John Kappler³, Dennis M. Zaller⁴ & Abraham Kupfer^1,^2,⁵

¹ Division of Cell Biology, Department of Pediatrics, National Jewish Medical and Research Center, 1400 Jackson St., Denver, CO 80206, USA.

² Department of Immunology, University of Colorado Health Sciences Center, Denver, CO 80262, USA.

³ Howard Hughes Medical Institute, National Jewish Medical and Research Center, 1400 Jackson St., Denver, CO 80206, USA.

⁴ Merck Research Labs, Rahway, NJ 07065, USA.

⁵ Department of Cellular and Structural Biology, University of Colorado Health Sciences Center, Denver, CO 80262, USA.

⁶ These authors contributed equally to this work.

Correspondence should be addressed to Abraham Kupfer kupfera@njc.org

During the productive interaction of T cells with antigen-presenting cells (APCs), engaged receptors, including the T cell antigen receptors and their associated tyrosine kinases, assemble into spatially segregated supramolecular activation clusters (SMACs) at the area of cell contact. Here, we studied intracellular signaling in SMACs by three-dimensional immunofluorescence microscopic localization of CD3, CD45, talin, phosphotyrosine, Lck and phosphorylated ZAP-70 in T cell−APC conjugates. Two distinct phases of spatial-temporal activation, one before and one after SMAC formation, which were separated by a brief state of inactivation caused by CD45, were observed at the T cell−APC contact area. We propose that pre-SMAC signals are sufficient to activate cell adhesion, but not productive T cell responses, which require orchestrated signaling in SMACs.

The ligation of T cell antigen receptors (TCRs) activates the receptor-associated tyrosine kinase Lck, which then phosphorylates and modulates the functions of its protein substrates, including the tyrosine kinase ZAP-70¹. This increased tyrosine kinase activity is often used as an early biochemical indicator of T cell activation. Cross-linking of the TCRs by TCR antibodies also results in the rapid activation of these kinases, but it is not sufficient to cause productive cellular responses². Productive responses require additional signals that can be provided by the coclustering of additional receptors, such as CD28 and lymphocyte function−associated antigen 1 (LFA-1)². Such artificial activation requires extensive receptor clustering by high-affinity antibodies; this contrasts with physiological activation, which requires the ligation of just a few TCR molecules that have a much lower affinity for their ligand^3,^4,⁵.

The mechanistic basis for this differential sensitivity is not clear, but it appears that massive artificial clustering of the receptors may bypass the physiological mechanisms that have evolved to enable T cells to detect efficiently and respond selectively to low amounts of specific antigens. One possibility is that the regulated balance between tyrosine kinases and tyrosine phosphatases may be more efficiently modulated during T cell−antigen-presenting cell (APC) interactions. The activity of Lck depends on the TCR and on the receptor tyrosine phosphatase CD45 and is regulated by tyrosine phosphorylation of two primary sites¹. Lck with a COOH-terminal phosphorylated tyrosine (pY) (pY505 in mouse Lck) is largely inactive⁶. Dephosphorylation of pY505 is required for the unfolding of inactive kinase and for exposing its catalytic domain⁷. The unfolded Lck can become fully active by interactions with other proteins and by phosphorylation of a second pY residue (pY394 in murine Lck)^8,⁹. The receptor phosphatase CD45 can dephosphorylate both pY505 and pY394 on Lck^10,^11,^12,^13,¹⁴. The same phosphatase required for priming Lck activation can also inhibit the successful functioning of this kinase.

Several models of T cell activation propose that CD45 is physically separated from the engaged TCR and Lck and is copartitioned with other large receptors, including the beta

₂ integrin LFA-1^15,^16,^17,¹⁸. Such molecular segregation addresses the mechanism that may prevent dephosphorylation of pY394 and inactivation of a preactivated Lck. However, the proposed segregation of CD45 from Lck and the TCR does not explain how CD45 would be able to dephosphorylate the inhibitory pY505, an essential role for CD45 in initial activation of the kinase. The high concentration of CD45 at the cell membrane may explain the need for massive clustering of TCRs by antibodies, and it suggests that the interplay between kinases and phosphatases may be highly regulated during T cell−APC interactions.

To uncover such regulatory mechanisms, it is necessary to study the more physiological interactions between T cells and their APCs. In contrast to the random antibody-induced clustering of receptors, when T cells are activated by APCs, the engaged TCRs and the tyrosine kinases Lck and Fyn cocluster into a central supramolecular activation cluster (cSMAC), whereas LFA-1 and the cytoskeletal protein talin cluster into a peripheral SMAC (pSMAC)¹⁹. Engagement of the TCR with antagonist peptides that fail to activate T cells productively can deliver some initial signals and lead to recruitment of talin to the contact area, but fail to cause the assembly of SMACs¹⁹. This suggests that SMAC assembly may be required for productive activation, but the functional role played by SMACs and their potential involvement in kinase-phosphatase interaction are still unclear. It is also unclear whether any of the TCR-induced biochemical pathways—including critical activation of the Src family kinase Lck and its substrate, the tyrosine kinase ZAP-70¹—are linked to clustering of the TCR in the cSMAC.

To address whether these events occur in the cSMAC, we used three-dimensional (3D) immunofluorescence microscopy to follow the TCR, tyrosine kinases, the phosphatase CD45 and receptor proximal signaling during the interaction of T cells with APCs. We found that initial signaling at the contact area, as shown by clustering of talin, was seen before the formation of distinct SMACs. Although Lck was found in the cSMAC, at the site of engaged TCRs, pY labeling was absent from the early cSMAC, but pY reappeared there later. Spatial-temporal 3D analysis, and the use of peptide covalently linked to major histocompatibility complex (MHC) tetramers, showed that CD45 and Lck were initially recruited to the cSMAC via the TCR. CD45 was then cleared from the cSMAC and clustered into a distal SMAC (dSMAC). Tyrosine-phosphorylated ZAP-70, a substrate of active Lck, appeared transiently in early contact areas and reappeared in the cSMAC upon the local clearing of CD45; this indicated two distinct phases of spatial-temporal activation occurred in the contact area.

Results
Spatial-temporal clustering of talin and pY
TCR activation can be followed at the single cell level via TCR-dependent recruitment of talin to the point of T cell−APC contact²⁰. Labeling of talin acts as a sensitive reporter of activation and provides additional spatial information. The engagement of a small number of TCRs, which is not enough to cause T cell proliferation, is sufficient to cause recruitment of talin to the T cell−APC contact area and activation of LFA-1²⁰. In addition, 3D localization of talin within the T cell−APC contact area can indicate the presence or absence of organized SMACs and delineates the pSMACs and cSMACs¹⁹. T cell activation can also be followed at the single cell level with pY labeling. Because of the TCR-induced activation of Lck and ZAP-70, it is expected that T cell activation would cause the intracellular localized appearance of tyrosine-phosphorylated proteins at the T cell−APC contact area.

We mixed T cells from AD10 TCR−transgenic mice with CH12 B cells that had been incubated with the antigen pigeon cytochrome C (PCC). Beginning 45 s after cells were mixed, samples of these cells were removed and then fixed. The fixed cells were immunofluorescently labeled with affinity-purified guinea pig and rabbit polyclonal antibodies specific for talin and pY, respectively, and analyzed by digital-deconvolution 3D microscopy. Within 45 s of cell conjugation, talin was already clustered at the T cell−APC contact area in almost all of the conjugates (Fig. 1a−d). However, talin appeared to be randomly dispersed along the entire contact site, which indicated that organized SMACs had not yet formed. In the same cells, pY was observed all over the membrane of the T cells, with numerous small patches of pY in the contact area. By 3 min, talin was clustered in distinct pSMACs in most conjugates (Fig. 1e−h). In the same cells, the cSMAC—where the engaged TCRs and their associated activated tyrosine kinases were expected to be clustered¹⁹—showed no pY labeling; however, pY labeling was seen in other parts of the contact area, including the pSMAC (Fig. 1e−h). By 7 min, pY labeling was clustered in the cSMAC and pSMAC (Fig. 1i−l). At later time points (23 min), pY was again mostly absent from the cSMAC (Fig. 1m−p). Labeling of the cells with another pY monoclonal antibody (mAb) showed similar temporal localizations of pY and confirmed the absence of pY from the presumed early cSMAC (data not shown).

Figure 1. Spatial and temporal clustering of talin and pY at the 3D-reconstructed T cell−APC contact site.

	Antigen-specific AD10-CH12 cell conjugates were fixed 45 s (a−d), 3 min (e−h), 7 min (i−l) and 23 min (m−p) after cell mixing. The cells were labeled with affinity-purified guinea pig anti-talin (b,f,j,n) and rabbit anti-pY (c,g,k,o). (a,e,i,m) Nomarski (differential interference contrast) images of corresponding cells, and (b−d, f−h, j−l, n−p) 3D views of the cell contact sites are shown. (d,h,l,p) Composite labeling for talin (green) and pY (red) is also shown. These labeling patterns were seen in 78 10% of the conjugates at each time point. The reconstructed contact views were magnified $times$ 1.5. Scale bar, 5 m. Full Figure and legend (39K)

Most of the pY membrane labeling was likely not generated upon TCR activation by the APC. Labeling of unbound T cells, even in the absence of APCs, already showed pY labeling over the entire membrane of the T cells, whereas talin was mostly diffuse over the entire cell (data not shown). Immunoblotting of whole cell lysates from freshly isolated AD10 splenic T cells also showed the presence of pY labeling of multiple proteins, including Lck (data not shown). Thus, at the single cell level, the talin labeling implied that signaling occurred even before the assembly of SMACs. pY labeling was less informative for TCR-dependent signaling, as it detected both pre-existing and TCR-induced phosphorylation. However, pY labeling did reveal the unexpected absence of pY, both pre-existing and TCR-induced phosphorylation, from the early cSMAC.

Clustering of Lck and engaged TCR in early cSMACs
To determine whether the absence of pY from the presumed early cSMACs was simply due to the local absence of clustered TCR and Lck, AD10 T cell−CH12 conjugates were immunofluorescently labeled with anti-CD3 (mAb F500) and either anti-pY (mAb 4G10) or anti-Lck, then analyzed by digital-deconvolution 3D microscopy. We found that CD3 was indeed clustered in the cSMAC, but pY was not localized with the clustered CD3 (data not shown). In addition, and in agreement with published data¹⁹, Lck was similarly coclustered in the cSMAC with CD3, at both 3 and 7 min (data not shown). Colabeling of CD3, CD4 or talin with an affinity-purified antibody to phosphorylated Src (anti−phospho-Src) that recognizes Lck and other Src-related kinases that are phosphorylated on their activation loop, showed a similar lack of labeling in the cSMAC at 3 min (data not shown).

To show that clustered CD3 in the early cSMAC represented the peptide-MHC−engaged TCRs, the T cells were conjugated to CH12 cells that expressed recombinant moth cytochrome C peptide (MCC)−I-E^k linked to green fluorescent protein (GFP). Analysis of these conjugates demonstrated that GFP-MCC−I-E^k was clustered in the APC in a region that was juxtaposed to clustered CD3 in the cSMAC of the T cell (data not shown). These results showed that the early cSMACs already contained engaged TCRs, CD4 and Lck. Thus, the finding that the early cSMAC lacked pY and phosphorylated Src kinases was not expected.

CD45 recruitment to the early cSMAC
To determine the possible cause of the local absence of pY from the early cSMACs, we analyzed the spatial-temporal 3D localization of CD45, TCR, Lck and talin in T cell−APC conjugates. AD10 T cell−CH12 conjugates were prepared as before. The fixed cells were immunofluorescently labeled with anti-CD45 (mAb TIB122) and anti-Lck and analyzed by digital-deconvolution 3D microscopy (Fig. 2). By 45 s, CD45 and Lck were randomly distributed at the cell contact area. By 3 min, CD45 was clustered in the cSMAC along with Lck in 78% of the cell conjugates (Fig. 2aE−H, b). In contrast, by 7 min most conjugates still showed Lck in the cSMAC, but CD45 was excluded (Fig. 2aI−L, b). Labeling with a polyclonal antibody specific for the intracellular COOH-terminal domain of CD45 showed similar patterns of clustering of CD45 in the cSMAC (data not shown).

Figure 2. Spatial-temporal colocalization and segregation of Lck and CD45 at the 3D-reconstructed T cell−APC contact site.

	(a) Antigen-specific AND-CH12 conjugates were fixed 45 s (A−D), 3 min (E−H) and 7 min (I−L) after cell mixing. The cells were labeled with rabbit anti-Lck (C,G,K) and rat anti-CD45 (B,F,J). (A,E,I) Nomarski images, and (D,H,L) composite of labeling for CD45 (green) and Lck (red) at the cell contact sites are shown. Note that at 3 min, CD45 and Lck colocalize in the cSMAC; by 7 min, Lck and CD45 are segregated. (b) Quantification of the temporal clustering of CD45 and Lck in the cSMAC. One-hundred antigen-specific conjugates were analyzed per time point; the results shown are from a typical experiment. Full Figure and legend (44K)

Quantitative 3D analysis revealed that CD45 that was coclustered with the TCR represented a small fraction (1.9 plusminus

0.3%) of total CD45. When CD45 cleared the cSMAC, it was still segregated from the pSMAC. Even at 23 min, CD45 was clustered in an outer, previously unrecognized, dSMAC (Fig. 3). In control experiments, the cell conjugates were labeled with anti−MHC class I specific for H-2^k, which is not involved in this cellular interaction. H-2^k did not cluster with, nor was it excluded from, the cSMAC, pSMAC or dSMAC at any of the time points studied. These results demonstrated the existence of at least three distinct, activation-induced, contact domains—cSMACs, pSMACs and dSMACs (Fig. 3)—and established that CD45 can sequentially cluster in different SMACs. The persistent exclusion of clustered CD45 from the pSMAC was unexpected. However, it may explain the presence of pY there and may be necessary to maintain LFA-1 in its high-avidity state at the pSMAC, in accordance with CD45 being a key negative regulator of integrin adhesion²¹.

Figure 3. Spatial-temporal redistribution of CD45 into the dSMAC but not the pSMAC.

	Antigen-specific AD10-CH12 cell conjugates were fixed 23 min after cell mixing. The cells were triply labeled with hamster anti-CD3 (F500A2) (a,e), rat anti-CD45 (b,f) and guinea-pig anti-talin (c,g). (d) Nomarski image of cell conjugate, (e−g) reconstructed 3D contact views of each label; and (h) the corresponding overlays are shown. Note the formation of three distinct contact zones and the absence of clustered CD45 and TCR in the pSMAC. This labeling pattern was seen in >90% of the conjugates. Full Figure and legend (21K)

Recruitment of CD45 by engaged TCR
Recruitment of receptors such as TCR and LFA-1 to the cell contact site requires binding to their ligands on the membrane of the APC²². Because there are no defined ligands for CD45, it was important that we determined whether recruitment of CD45 to the engaged TCR was dependent on an unidentified CD45 ligand or was directly induced by the TCR. Thus, we conjugated T cells from AD10 TCR−transgenic mice to cell-sized beads that were coated with multivalent recombinant MCC−I-E^k complexes²³. The recombinant I-E^k was covalently linked to either an agonist MCC peptide (which consisted of amino acids 88−103 of MCC) or a control antagonist MCC99A peptide (in which lysine at position 99 changed to alanine)²³. Binding of the beads to the T cells was entirely dependent on the presence of antigenic peptide. The T cells stably bound beads that contained MCC−I-E^k, but not beads that contained MCC99A−I-E^k. In addition, CD3 and CD45 were rapidly recruited to the contact area between T cells and the bound beads (Fig. 4). By 1 min, the clustering of CD3 at the T cell−bead interface preceded CD45 clustering (Fig. 4a−c); this indicated that the recruited TCR was not stably associated with CD45 before binding of the TCR to the peptide-MHC complexes. During the next 6 min, both CD3 and CD45 were enriched at the T cell−bead interface (Fig. 4d−i). By 23 min, most of the TCRs were recruited to the T cell−bead contact site and CD45 was cleared from this region (Fig. 4j−l). In control experiments, the T cell−bead conjugates were doubly labeled with CD3 and H-2^k antibodies. H-2^k labeling delineated the T cell membrane but was neither enriched nor depleted at any of the time points analyzed (data not shown). Thus, recruitment and clearing of CD45 during T cell−APC interactions and in the T cell−bead conjugates were regulated by the engaged TCR and not by interactions of CD45 with unknown ligands on the APC.

Figure 4. Transient recruitment of CD45 to engaged TCRs induced by immobilized peptide−I-E^k tetramers.

	AD10 cells were mixed with streptavidin beads that were preincubated with purified recombinant biotinylated MCC−I-E^k. The cells were fixed after 1 min 20 s (a−c), 3 min (d−f), 7 min (g−i) and 23 min (j−l) and labeled with anti-CD3 (b,e,h,k) and anti-CD45 (c,f,i,l). (a,d,g,j) Nomarski images of T cell−bead conjugates are shown. Note that at 1 min, CD3 was recruited to the T cell−bead contact site before CD45. During the next 6 min, both CD3 and CD45 were clustered at the T cell−bead interface; however, by 23 min, CD45 was cleared from the bead-contact site, whereas most of the TCR remained there. These labeling patterns were observed in $approx$ 90% of the T cell−bead conjugates. Full Figure and legend (34K)

CD45-CD3 association in early cSMACs
To investigate the possibility that colocalized CD45 and CD3 were molecularly associated in the cSMAC, AD10-CH12 conjugates were formed and, after 3 min, were labeled with anti-CD3 and anti-CD45 followed by Indocarbocyanine (Cy3)- and Indodicarbocyanine (Cy5)-tagged secondary antibodies. Fluorescent resonance energy transfer (FRET) between the labels was assessed by the acceptor photobleaching method²⁴. Although CD45 and CD3 staining was detected on the entire T cell membrane, FRET between labeled CD45 and CD3—which indicated a distance of <100 Å between the donor and acceptor labels—was detected only in the cSMAC (Fig. 5). These findings confirmed that clustered CD45 was intimately associated with CD3 and was a molecular resident of the early cSMAC. Previous microscopic studies of CD45 have yielded conflicting data. Fluorescence microscopy of T cell−APC conjugates showed that starting at 5 min after cell conjugation, CD45 was stably excluded from the TCR²⁵; this is in agreement with our observations. No analysis of earlier time points was reported²⁵. In contrast, studies of T cell interactions with artificial lipid monolayers—which contained glycosyl-phosphatidyl inositol (GPI)-linked I-E^k and intercellular adhesion molecule 1 (ICAM-1)—showed that after initial exclusion that lasted several minutes, CD45 was recruited to and remained at the center of the contact area adjacent to the TCR²⁶. These findings may have resulted from the artificial method of activation, the lack of additional costimulation signals and the use of live T cells that were labeled with fluorescent CD45 antibodies to follow CD45. Use of the latter may have caused antibody-induced internalization of CD45 and a sustained intracellular pool of labeled CD45; this was not seen by us here, as we applied the antibodies after cell fixation.

Figure 5. Detection of FRET between CD3 and CD45 in the cSMAC.

	(a) Negative and positive controls for the FRET protocol. (A−F) AD10 T cells were fixed and doubly labeled with anti-CD3 (Cy3, red) (A,B) and anti-CD45 (Cy5, blue) (D,E) followed by Cy3- or Cy5-labeled antibodies to hamster and rat. (G,H) Fixed AD10 T cells were labeled with anti-CD45, followed with a mixture of Cy3− and Cy5−rat antibodies. (A,D,G,J) Initial labeling is shown. (B,E,H,K) Labeling after Cy5 was photobleached is shown. Note the increased labeling of Cy3 in H but not in B. FRET analysis data are shown in F and L and are pseudocolored from blue to red: blue indicates complete lack of FRET and red indicates maximal FRET values. The analysis was done as described²⁴. (b) FRET analysis of AD10-CH12 conjugates that were fixed after 3 min of interaction. The cells were labeled and analyzed as in the control (A−F). F shows the presence of FRET (red) only in the cSMAC. Nine random conjugates were analyzed and similar results were detected in seven of these conjugates. Full Figure and legend (42K)

Biochemical studies have indicated that CD45 was excluded from membrane fractions that contained the activated TCR^16,^17,¹⁸ but CD45 was detected in such membrane fractions by others²⁷. However, a transient biochemical association between a small fraction of CD45 and the TCR has also been reported²⁸. Because 98% of the CD45 was not localized with the engaged TCR, it is likely that interactions between a small number of such molecules in specific cellular locations would be missed by biochemical assays that analyze protein interactions in whole cell extracts.

Spatial-temporal localization of pY493−ZAP-70
Transient TCR-induced recruitment and association of CD45 with cSMAC can explain the absence of pY from the early cSMACs. It also suggests that TCR-associated tyrosine kinases may be temporarily inactivated rather than activated in the early cSMACs. ZAP-70 plays a key role in the initiation of T cell activation¹. Labeling of T cell−APCs with anti-talin and anti−ZAP-70 demonstrated that ZAP-70 was mostly absent from the cell contact area at 3 min, but was present in the cSMAC at 7 min (data not shown). The absence of ZAP-70 from the cSMAC at 3 min suggested that the absence of pY labeling was not simply due to a temporal and localized masking of the pY by intermolecular or intramolecular interactions.

ZAP-70 is phosphorylated and activated by active Lck²⁹. Although ZAP-70 can undergo phosphorylation on multiple tyrosine residues, Lck directly phosphorylates Y493 and Y492 of ZAP-70, which then becomes a highly active kinase^30,^31,^32,³³. We raised a mAb called AZ9.1 against a synthetic peptide—ALGADDSpYpYTARS—that corresponded to amino acids 485−497 of ZAP-70 (in which Y492 andY493 were phosphorylated), referred to hereafter as ZAP-70(485−497)pY492-pY493. AZ9.1 bound with high affinity to ZAP-70(485−497)pY492-pY493 and with a lower affinity to ZAP-70(485−497)pY493, but it did not bind to ZAP-70(485−497)pY492 or to unphosphorylated ZAP-70 (Fig. 6a,b). To examine the activation state of ZAP-70 at cell contact areas, cell conjugates were labeled with anti-talin and AZ9.1. Digital microscopic analysis showed small patches of AZ9.1 in the cell contact area 45 s after cell mixing (Fig. 6c, A−D). At 3 min there was no detectable labeling in the cell contact area (Fig. 6c, E−H); strong labeling was noted in the cSMAC at 7 min (Fig. 6c, I−L). By 23 min, AZ9.1 labeling was reduced again, but was clearly detectable and resembled the labeling seen at 45 s (data not shown).

Figure 6. The spatial-temporal appearance of phosphorylated ZAP-70 in the T cell−APC contact.

	(a) Immunoassay with mAb AZ9.1, which used microtiter plates coated with unphosphorylated, singly or doubly phosphorylated peptides. The AZ9.1 mAb binds with high affinity to ZAP-70(485−497)pY492-pY493 and ZAP-70(485−497)Y492-pY493, but does not bind to ZAP-70(485−497)pY492-Y493 or the unphosphorylated peptide. (b) Immunoblot analysis of lysates from control and pervandate-treated Jurkat T cells. The anti-pY mAb PY20 detects many bands from the pervandate lysates, whereas AZ9.1 (anti-phospho ZAP-70) detects only a 70-kD band. In addition, immunoprecipitation of these lysates with AZ9.1 followed by immunoblot analysis with an anti−ZAP-70 (mAb TL 29) demonstrates that this 70-kD band is indeed ZAP-70. (c) AND-CH12 conjugates were fixed at 45 s (A−D), 3 min (E−H) and 7 min (I−L). The cells were labeled with anti-talin (B,F,J) and AZ9.1 (C,G,K). 3D-rendered contacts are shown for talin (green) and AZ9.1 (red). (D,H,L) 3D overlays are shown. These labeling patterns were seen in 75 10% of conjugates. IP, immunoprecipitation. Full Figure and legend (73K)

These findings indicated that Lck phosphorylated ZAP-70 at the T cell−APC contact site before SMAC formation and in later cSMACs, but not in the early SMACs, where CD45 was present. Thus, Lck and ZAP-70 were active before SMAC formation, inactivated in the early cSMACs and reactivated in the cSMAC upon clearing of CD45.

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Discussion
The combined use of antibodies specific for TCR, CD45, Lck, pY, talin, ZAP-70 and phosphorylated ZAP-70 revealed the formation of several distinct 4-dimensional high-order molecular associations. These sequential combinatorial colocalizations likely indicated different TCR-dependent activation states. Based on these findings, we propose a staged model of T cell activation by APCs. Upon contacting an APC, the engagement of a small number of TCR molecules can initiate intracellular activation events. A primary consequence of these signals is the recruitment of talin and conversion of the beta

₂ integrin into its high-avidity state. This phase of signaling involves small-scale clusters, or just TCR dimers, but does not require large-scale clustering of the TCR and precedes the formation of detectable SMACs. While the cells are held together by activated integrins, more TCRs can find and bind additional specific peptide-MHC complexes that are present on the APC. Signals generated at this stage cause the formation of segregated SMACs. The TCR molecules that are at the cSMAC, transiently recruit CD45 to the cSMAC, causing the local tyrosine dephosphorylation of TCR-associated proteins. Once CD45 clears the cSMAC, new tyrosine phosphorylation will take place in the cSMAC. This includes the activation of primed Lck and the phosphorylation and activation of ZAP-70.

It should be noted that the kinase activity of Lck can also be regulated by the interaction of its SH3 domain with the proline-rich region of CD28, which increases the activity of Lck³⁴. During the early stages of T cell activation, CD28 does not colocalize with Lck and the clustering of CD28, compared to Lck or CD4, to the cSMAC is delayed (data not shown). An analysis of the interaction of naïve TCR transgenic T cells with splenic non-B−non T cells that acted as APCs also found that signaling precedes the formation of SMACs³⁵. The SMACs in these conjugates were formed very slowly and the TCR was rapidly cleared from the contact site; it was proposed that there was no signaling from the TCR and its associated tyrosine kinases in the cSMAC³⁵. These findings differ greatly from our findings of rapid SMAC formation and sustained expression of the TCR in the cSMAC. Differences may reflect the use of alternative cells, particularly the splenic APCs (non-B−non-T cells) used³⁵, which may have low amounts of costimulatory ligands, versus the B cells, which express high amounts of B7-2 and ICAM-1, that we used here.

This model addresses several key unresolved issues of T cell activation: the remarkably high sensitivity to low concentrations of antigen and the exquisite antigen specificity of the T cells. In our model, the first phase of activation would enable the T cells, upon initial recognition of just a few antigens, to rapidly and effectively bind to the APC. Although the initial activation events, before the formation of SMACs, may be sufficient to potentiate integrins adhesion—and may be required for subsequent SMAC formation—they may not be sufficient to cause T cell proliferation. We propose that new TCR-induced signals generated after the formation of SMACs would be required to trigger the productive activation of T cells by APCs. This may explain how the specificity of the T cell response is controlled: only cells that express sufficient amounts of agonist peptides would trigger the formation of SMACs and subsequent signaling.

The recruitment of CD45 to the initial cSMAC may be an important reset and control step that serves as a regulatory master switch of activation. Although the precise role played by this unexpected cSMAC dephosphorylation is still unclear, it may potentially serve several functions. Signaling and adaptor proteins that are initially recruited to the cSMAC may arrive at this contact site in different states of phosphorylation. For example, Lck or ZAP-70 may be phosphorylated on multiple regulatory sites, including activating and inhibitory sites. The dephosphorylation of all these sites would reset their state of phosphorylation so that upon clearing CD45, they would all be phosphorylated only on the appropriate sites. Such synchronized phosphorylation may induce more effective signals by a limited number of clustered kinases and may include additional molecular interactions that were not present during the earlier pre-SMAC signaling. The dephosphorylation of TCR-associated proteins may also provide a safety barrier between the requirement for high sensitivity and for antigen-specificity, by providing an inhibitory stage that would prevent each accidental initial activation event from progressing further.

Although this model focuses on TCR-associated signals, it is important to note that additional signals are most likely generated in other parts of the T cell−APC contact site. In fact we observed persistent labeling of pY in the pSMAC, which was consistent with the exclusion of CD45 from that area. Although the nature of the pSMAC signals is still unclear, such signals may at least be required for stable cell adhesion. Other functional studies support certain aspects of this proposed model. Antagonist peptides can trigger the clustering of talin and cell adhesion, but they fail to form stable SMACs, cannot recruit PKC- theta

and do not cause productive T cell activation^19,³⁶. Suboptimal concentrations of antigen cause activation of talin and integrin adhesion, but not T cell proliferation²⁰. We found that PP2, a specific inhibitor of most tyrosine kinases (excluding ZAP-70), can eliminate clustering of talin at the T cell−APC cell contact site and greatly diminish cell conjugation (data not shown). In the same experiments, we found that SU6656, a specific inhibitor of most Src kinases (except Lck), did not inhibit talin clustering at the contact site. Thus, recruitment of talin appears to depend on the activation of Lck. Additional extensive studies are required to test all the aspects of this proposed model.

We have shown here that TCR-mediated activation events occur at several spatially and temporally distinct stages. Signals are generated before the formation of SMACs and within the SMACs. These data reveal the dynamic and temporal complexity of molecular associations between receptors and signaling proteins within the different SMACs. The distinct, spatially and temporally regulated higher order molecular complexes enable key effector proteins to perform sequentially different functions and provide a mechanism for complex signal integration and propagation.

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Methods
Cells and cell conjugation.
AD10 TCR−transgenic mice were used as a source of T cells. Splenic cell suspensions were treated with PCC (50

g/ml) in the presence of recombinant interleukin 2 (IL-2). The cell cultures were used 2−4 weeks later, when all the remaining viable cells appeared to be small round resting transgenic T cells. Cell conjugates were formed by a 5-s cospin of equal numbers of T and CH12 cells. The cells were immediately incubated for 0−21 min at 37 °C, plated for 30 s on poly(D)lysine-coated coverslips and fixed. The CH12 B cell line was as described¹⁹.

Antibodies, peptide-MHC tetramers and labeling.
Antibodies, fixation and immunofluorescence labeling were as described¹⁹. Anti-CD45 (mAb TIB122) recognizes all isoforms of CD45, which differ in their extracellular domains but share an identical intracellular phosphatase domain¹⁰. The production and purification of soluble recombinant biotinylated MCC−I-E^k was as described²³. Multivalent MCC−I-E^k complexes were formed by incubating Dynabeads M-280 Streptavidin (Dynal Inc., Oslo, Norway) with saturating amounts of biotinylated MCC−I-E^k. The T cells were conjugated to the beads during a 5-s cospin and processed like T cell−APC conjugates.

Three-dimensional imaging and analysis.
The 3D immunofluorescence and corresponding Nomarski images of cells were recorded by a digital multidimensional fluorescence microscopy system (Intelligent Imaging Innovations, Denver, CO) as described¹⁹. SlideBook software (Intelligent Imaging Innovations) was used for 3D image capture, deconvolution, analysis and rendering¹⁹. The 3D rendering of cell contacts was generated as orthographic projections of the voxels that make up the T cell−APC contact. Clustering at the contact was defined by contact-voxels, whose intensities were higher than the noncontact voxels intensities at the rest of cell. Contact depletion was defined inversely. To determine the phenotypic labeling for each time point, 3D images were recorded for $approx$ 30 conjugates that were randomly selected, based only on Nomarski imaging, A labeling phenotype that was seen in at least 20 of these conjugates was defined as representative of that time point. These experiments were repeated at least four times to confirm the sequential redistribution of proteins. For quantitative analysis of CD45 and Lck, 100 cell conjugates were analyzed, at each time point, to determine the major labeling phenotype.

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Received 22 March 2002; Accepted 5 August 2002; Published online: 3 September 2002.

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Acknowledgments
We thank P. Marrack, A. Weiss and G. Koretzky for helpful comments and T. Potter for critical reading of the manuscript. Supported in part by grants from the NIH (to A. K.).

Competing interests statement: The authors declare competing financial interests.

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