Dissociation the Immunological Synapse Upon Cellular T Cells

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Peptide-Specific Intercellular Transfer of MHC Class II to CD4 + T Cells Directly from the Immunological Synapse upon Cellular Dissociation This information is current as of September 26, 2021. Scott A. Wetzel, Timothy W. McKeithan and David C. Parker J Immunol 2005; 174:80-89; ; doi: 10.4049/jimmunol.174.1.80 http://www.jimmunol.org/content/174/1/80 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Peptide-Speciﬁc Intercellular Transfer of MHC Class II to CD4؉ T Cells Directly from the Immunological Synapse upon Cellular Dissociation1

Scott A. Wetzel,2* Timothy W. McKeithan,† and David C. Parker3*

The transfer of membrane proteins from APC to T cells was initially described in the 1970s, and subsequent work has described two mechanisms of transfer: APC-derived exosomes and direct transfer of small packets, while cells remain conjugated. Using fibroblast APC expressing a GFP-tagged I-Ek molecule with covalently attached antigenic peptide, we observed a third mechanism in live cell imaging: T cells spontaneously dissociating from APC often capture MHC:peptide complexes directly from the immunological synapse. Using two I-Ek-restricted murine TCR transgenic T cells with different peptide specificity, we show in this study that the MHC transfer is peptide specific. Using blocking Abs, we found that MHC:peptide transfer in this system requires Downloaded from direct TCR-MHC:peptide interactions and is augmented by costimulation through CD28-CD80 interactions. Capture of the GFP-tagged MHC:peptide complexes correlates with an activated phenotype of the T cell, elevated CD69 with down-modulated TCR. The transferred MHC:peptide molecules transferred to the T cell are associated with molecules that imply continued TCR signaling; p56lck, phosphotyrosine, and polarization of the actin cytoskeleton. The Journal of Immunology, 2005, 174: 80–89.

lymphocyte recognition of cognate MHC:peptide com- some/vesicle mediated (17–19) or requiring direct cell-cell contact. http://www.jimmunol.org/ plexes results in the accumulation of cytoplasmic and Recently, several groups have shown that for TCR-mediated cap- T membrane-bound proteins to the contact region (1, 2). ture, the immunological synapse is the location of transfer, while These molecules are spatially and temporally segregated into dis- the cells remain conjugated (20–23). This transfer is Ag dependent tinct supramolecular activation complexes (SMACs)4 (3). The cen- (20, 22), but it is unknown whether transfer involves only specific tral supramolecular activation complex (c-SMAC) contains en- MHC:peptide molecules or whether nonspecific MHC:peptide li- gaged TCR/MHC:peptide, CD28/CD80, and signaling-associated gands are also transferred. The specificity of transfer may be crit- ␪ molecules such as protein kinase C (PKC)- and tyrosine-phos- ical in controlling immune responses in vivo, as T cells functioning phorylated proteins surrounded by a peripheral SMAC containing, as APCs in vivo can induce anergy (24, 25). The stripping of among others, adhesion molecules such as ICAM-1/LFA-1 (3, 4). specific MHC:peptide complexes from an APC may also provide by guest on September 26, 2021 The role of this immunological synapse is still unclear. It is widely a mechanism for T cell competition for MHC:peptide ligands, thus accepted that it functions in sustaining TCR-mediated intracellular increasing the affinity of a specific response (26). The potential signaling (4–7), but data from others have suggested that its main role of the T cell immunological synapse in MHC transfer is function is the polarized secretion of effector molecules (8–12). strengthened by the fact that both B cells (27, 28) and NK cells Alternatively, Shaw and colleagues (13) have hypothesized that (29–32) form immunological synapses and capture membrane pro- the synapse regulates TCR down-modulation and endocytosis. teins directly from them. Another potential role of the immunological synapse is the capture of membrane-bound molecules from APCs. The literature on The mechanism of direct cell-to-cell, contact-dependent transfer acquisition of MHC molecules by T cells from APC dates back in T-APC conjugates is unknown. It has been characterized by the over 20 years (14–16). Capture has been characterized as exo- transfer of multiple small packets of APC membrane containing surface proteins such as MHC (20), CD80 (33), and OX40L (34), while the cells remain conjugated. It is possible that this form of

*Department of Molecular Microbiology and Immunology, Oregon Health & Science transfer is the result of exosomal release from the APC and sub- University, Portland, OR 97239; and †Department of Pathology and Microbiology, sequent uptake in the region of the immunological synapse. This is University of Nebraska Medical Center, Omaha, NE 68198 supported by a recent report from Boes et al. (35) showing polar- Received for publication April 26, 2004. Accepted for publication October 22, 2004. ized MHC class II delivery to the T cell-dendritic cell interface. The costs of publication of this article were defrayed in part by the payment of page Additionally, electron microscopy data from Patel et al. (19) dem- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. onstrate the presence of APC-derived exosomes in the space be- 1 This work was supported by Grant AI29544 from the National Institutes of Health tween T cells and APCs at the T-APC interface. However, this is and a grant from the Oregon Health Sciences Foundation. not the only method of transsynaptic capture. Stinchcombe et al. 2 Current address: Division of Biological Sciences and Center for Environmental (11) showed by electron microscopy that during the process of Health Sciences, University of Montana, Missoula, MT 59812. spontaneous CTL release from target cells, small membrane 3 Address correspondence and reprint requests to Dr. David C. Parker, Department of bridges formed and that MHC class I molecules and yellow fluo- Molecular Microbiology and Immunology, L220, Oregon Health Sciences University, 3181 SW Sam Jackson Park Road, Portland, OR 97239. E-mail address: rescent protein-tagged APC membrane were subsequently trans- [email protected] ferred to the T cell upon dissociation. Such dissociation-associated 4 Abbreviations used in this paper: SMAC, supramolecular activation complex; c- transfer is consistent with the finding that during transendothelial SMAC, central SMAC; DIC, differential interference contrast microscopy; FKBP, FK506-binding protein; Hb, murine hemoglobin peptide; MCC, moth cytochrome c migration, T cells acquire endothelial membrane and membrane peptide; MFI, mean fluorescence intensity; PKC, protein kinase C. proteins such as CD31, CD49d, CD54, CD61, and CD62E (36).

Using fibroblasts expressing a GFP-tagged covalent MHC:pep- resuspended in RPMI 1640 (Invitrogen Life Technologies) containing sup- tide construct and costimulatory molecules (37) as APC, in this plements, as described for complete DMEM (complete RPMI 1640). Cells ␮ study we have characterized the transfer of MHC:peptide com- were primed in vitro with 2.5 M peptide (MCC 88–103 for AD10; Hb 64–76 for 3.L2) for 4–6 days with addition of 10 U/ml exogenous IL-2 on plexes from APC to T cells directly from the immunological syn- day 2. Lymphocytes were isolated from primed cultures by density centrifu- apse as a result of spontaneous cellular dissociation. In this system, gation using Lympholyte M (Cedarlane Laboratories). T cells were resus- MHC transfer is mediated by TCR/MHC interactions and is aug- pended at 5 ϫ 106/ml in complete phenol red-free RPMI 1640 for use. mented by CD28/CD80 costimulation. Using T cells from TCR Live cell microscopy transgenic mice specific for a different antigenic peptide presented 5 by I-Ek, we show in this work for the first time that the transsyn- For live cell microscopy, 2.5 ϫ 10 APCs were seeded into 0.17-mm Delta T culture dishes (Bioptechs) 1 day before the experiment in 1 ml of com- aptic transfer of MHC following Ag recognition is specific for plete DMEM. Dishes were fitted into a Bioptechs TC3 heated stage adapter MHC molecules loaded with cognate peptide. The extent of MHC: and maintained at 37°C for the duration of the imaging. After adding 2.5 ϫ peptide transfer to the T cells is associated with an activated phe- 105 AD10 T cells to the dish, alternating ϫ400 or ϫ600 green fluorescent notype (CD69high, TCR down-modulated). Finally, we report that (528 nm) and differential interference contrast (DIC) images were taken the transferred MHC:peptide molecules are found on the surface of every 8–12 s for 45 min with the Applied Precision Instruments Delta- Vision image restoration system. This includes the Applied Precision the T cell associated with the TCR and molecules involved in TCR Instruments chassis with precision motorized XYZ stage, a Nikon TE200 signaling. The data further suggest that the MHC:peptide mole- inverted fluorescent microscope with standard filter sets, halogen illumi- cules are in their native orientation on the surface of the T cell, nation with Applied Precision Instruments light homogenizer, a CH350L which correlates well with previous reports showing that after camera (500 kHz, 12-bit, 2 Megapixel, liquid cooled), and DeltaVision software. MHC transfer from APC, T cells can present Ag to other T cells Downloaded from (20, 22, 24, 25), and has implications for the initiation and control MHC transfer experiments of an immune response. To assess MHC transfer, unless otherwise stated, 1 ϫ 106 MCC:GFP cells were plated into individual wells of a six-well plate and incubated over- Materials and Methods night at 37°C before addition of 2.5 ϫ 106 in vitro primed T cells. In Ab Animals blockade experiments, blocking reagents were added to the APC or T cells 1 h before addition of T cells to the wells. After a 90-min incubation at

ϩ http://www.jimmunol.org/ Heterozygous AD10 TCR transgenic mice (V␤3 ), specific for pigeon 37°C, T cells were recovered from the cultures by rinsing with PBS. No cytochrome c fragment 88–104 (38) and reactive against moth cytochrome additional dissociating reagents were added (e.g., EDTA or trypsin) to aid c (MCC) fragment 88–103 on a B10.BR (H-2k) background, were kindly in T cell recovery. After washing with PBS, cells were aliquoted for fixed provided by S. Hedrick (University of California, San Diego, CA) by way cell microscopy or flow cytometry. of P. Marrack (National Jewish Medical Center, Denver, CO). Homozy- gous 3.L2 TCR transgenic mice (V␤8.3ϩ), specific for peptide 64–76 of Fixed cell microscopy murine hemoglobin d allele (Hb), were kindly provided by P. Allen (Wash- ington University, St. Louis, MO) (39). The mice were bred and main- T cells recovered from APC-containing wells, as above, were incubated for tained in specific pathogen-free conditions in the Oregon Health & Science 10 min on poly-L-lysine-coated LabTek II eight-chambered 0.15-mm cover University animal care facility. AD10 TCR transgenic mice were identified glasses in PBS at room temperature. Cells were fixed by addition of ice- cold fixative (4% paraformaldehyde, 0.5% glutaraldehyde in PBS) and in-

by PCR and flow cytometry. by guest on September 26, 2021 cubated for 30 min at room temperature in the dark. For intracellular stain- Abs and staining reagents ing, cells were permeabilized for 5 min with 0.1% Triton X-100 in PBS after fixation. Cultures were then stained with primary Abs at 10 ␮g/ml in The following conjugated or unconjugated Abs were purchased from BD PBS, or phalloidin (1/500 dilution in PBS), for2hatroom temperature in k Pharmingen: anti-I-E (17-3-3 and 14-4-4s), anti-CD80 (16-10A1), anti- a humidified chamber. Following three PBS washes of 10 min each, cells ICAM-1 (3E2), anti-CD69 (H1.2F3), anti-V␤3 (KJ25), anti-V␤8.3 were incubated with secondary Abs at 5 ␮g/ml for2hatroom temperature. (1B3.3), anti-TCR␤ (H57), anti-CD3 (145-2C11), anti-CD4 (Gk1.5), anti- After three more PBS washes, SlowFade Light antifade reagent (Molecular CD25 (3C7), and anti-CD71 (C2), in addition to streptavidin-allophyco- Probes) was added to the wells. Cells were stored at 4°C, protected from cyanin, and streptavidin-CyChrome. The following reagents were pur- light until imaged. chased from Molecular Probes (Eugene, OR): AlexaFluor 594-conjugated Cells to be imaged were chosen based upon morphology in DIC and phalloidin, anti-GFP AlexaFluor 594, chicken anti-rabbit IgG AlexaFluor presence of a GFP signal. A stack of 50–90 fluorescent images spaced 0.2 594, and anti-goat IgG AlexaFluor 350. Anti-PKC␪ (C18-G) was pur- ␮m apart in the z-axis was obtained at ϫ600 on the DeltaVision system and chased from Santa Cruz Biotechnology. Anti-phosphotyrosine (4G10) was deconvolved using an iterative, constrained algorithm. Deconvolution and purchased from Upstate Biotechnology. Rabbit polyclonal anti-Lck (2752) three-dimensional reconstructions were performed on an SGI Octane work- was purchased from Cell Signaling Technology. Anti-␥-tubulin (GTU-88) station (Applied Biosystems) using the Applied Precision Instruments Soft- was purchased from Sigma-Aldrich. Anti-mouse IgG PE was purchased Worx software package. Further image analysis and preparation of images from Southern Biotechnology Associates, and Texas Red-conjugated don- for publication were performed using SlideBook 4 software (Intelligent key anti-goat IgG and Cy5-conjugated anti-mouse IgG were purchased Imaging Innovations, Denver, CO) on a Macintosh G4 (Apple Computer). from Jackson ImmunoResearch Laboratories. Flow cytometry APCs T cells were stained with the indicated reagents for 30 min at 4°C in FACS MCC:GFP fibroblasts expressing enhanced GFP-tagged I-Ek ␤-chain with buffer (PBS plus 2% FBS plus 0.1% NaN3). After three washes, cells were covalent antigenic MCC were described previously (37). As a control in stained for 20 min with secondary reagents in FACS buffer. After three some experiments, a second transfected fibroblast line, MCC:FKBP additional washes, cells were analyzed on a four-color FACSCalibur (BD (FK506-binding protein), was used. This cell line expresses similar levels Biosciences) without fixation. Data were analyzed with CellQuest 3.3 soft- k of CD80 and ICAM-1 and surface MCC:I-E to MCC:GFP. It differs in that ware (BD Biosciences). the MCC:I-Ek ␤-chain is fused to three repeats of the FKBP (Ariad) rather than GFP. The MCC:FKBP cells express no GFP molecules. Cells were Results maintained in DMEM (Invitrogen Life Technologies) containing 10% FBS (HyClone) and supplemented with 1 mM L-glutamine, sodium pyruvate (100 Intercellular transfer of GFP-tagged MHC:peptide complexes mg/ml), 50 ␮M 2-ME, essential and nonessential amino acids (Invitrogen Life from APC to T cells via the immunological synapse upon Technologies), 100 U/ml penicillin G, 100 U/ml streptomycin, and 50 ␮g/ml spontaneous T-APC dissociation gentamicin (complete DMEM). Using fibroblasts expressing GFP-tagged MHC:peptide complexes In vitro T cell priming as surrogate APC in live cell imaging experiments, we observed in Single cell suspensions of splenocytes from 6- to 12-wk-old AD10 or 3.L2 ϳ10% of the T-APC interactions leading to mature immunological TCR transgenic mice were depleted of erythrocytes by hypotonic lysis and synapse formation that the T cells spontaneously dissociated from 82 PEPTIDE-SPECIFIC MHC TRANSFER FROM IMMUNOLOGICAL SYNAPSE

In each case, there is transfer of MHC:peptide from the APC to the T cell directly from the immunological synapse. After dissociation, the region of increased GFP intensity on the APC where the immunological synapse had formed diffuses back into the membrane and becomes indistinguishable from other regions of the APC membrane. This is best seen with the spot from the T cell marked with the pink arrow, where the spot on the APC disappears between 11 and 17 min, but the process can also be seen on the region of the APC interacting with the T cell marked with the gray arrow over the same time span. However, unlike the GFP regions on the APC, the spots on the T cell do not diffuse into the membrane, but remain as cohesive spots.

MHC:peptide complexes transferred to T cells associate with TCR, signaling molecules, and the cytoskeleton FIGURE 1. Upon dissociation from APC, T cells capture GFP-tagged The live cell data demonstrated that the MCC:I-Ek:GFP transferred MHC:peptide complexes directly from the immunological synapse. This live from the APC to the T cell via the immunological synapse upon cell imaging data show three individual T cells (indicated by the colored ar- spontaneous T-APC dissociation remained a focused, distinct spot rows) interacting with a single APC. GFP (in green) is overlaid on DIC images on the T cell surface. To examine the possibility that the trans- Downloaded from (blue). An immunological synapse forms at each T-APC interface, and a large patch of GFP is transferred to the T cell when it subsequently spontaneously ferred MHC:peptide complexes are associated with other mole- dissociates from the APC. The GFP signal on the T cell surface remains a cules on or within the T cell, T cells were recovered from a co- focused spot, while on the APC, the remnants of the immunological synapse culture with APC after 90 min, and after a 10-min incubation on diffuse back into the plasma membrane. The on-line supplement includes poly(L-lysine)-coated coverslips at 37°C, were ﬁxed and stained. quicktime movie of this live-cell imaging sequence. Images taken at ϫ600. An early time point was chosen to correlate with the duration of

Bar ϭ 10 ␮m. our live cell imaging experiments, where we observed the direct http://www.jimmunol.org/ APC to T cell MHC transfer (Fig. 1). Because we were interested only in the cells that had spontaneously detached from the APC, no dissociating agents, such as EDTA, were used in the T cell recov- the APC during the 1-h imaging period (37). This cellular disso- ery process. T cells were recovered by simply rinsing the cultures ciation was accompanied by the intercellular transfer of GFP- with PBS, leaving behind the adherent MCC:GFP cells and any tagged MHC:peptide molecules from the fibroblast APC to the T cells. In this study, we have characterized the phenomenon of intercellular MHC:peptide transfer from the fibroblast APC to the T cell during spontaneous cellular dissociation. by guest on September 26, 2021 The images in Fig. 1 are representative of Ͼ50 transfer events that we have observed in Ͼ20 live cell imaging experiments. The on-line supplement contains a movie of the entire image sequence (Movie 1).5 In this set of images, three separate T cells (indicated by the three different arrows) interact with a single MCC:GFP cell. Each T cell forms an immunological synapse, as shown by the significant increase in GFP intensity at the T-APC interface, and subsequently dissociates from the APC. At time 0 in Fig. 1, the T cell indicated by the yellow arrow has already formed a mature immunological synapse, and 2 min and 36 s later it has dissociated from the APC, carrying with it a GFP spot. This spot remains visible for the duration of imaging, although the intensity appears to decrease (which could be owing to movement of the T cell and associated spot out of the plane of focus). The second T cell, indicated by the gray arrow, makes contact with the APC at ϳ40 s (data not shown), and by 2 min and 36 s, there is a clear accumulation of MHC:peptide at the T-APC interface. This nascent synapse continues to grow, and by 7 min and7savery bright accumulation of GFP is present, signifying the formation of a mature immunological synapse. At 11 min and 13 s, the T cell has dissociated from the APC, carrying with it a GFP spot directly from the FIGURE 2. MHC:peptide complexes captured by T cells are associated immunological synapse. Again, the GFP is visible as a coherent with molecules involved in intracellular signaling and the actin cytoskel- spot on the T cell surface over 6 min later. The third T cell in Fig. eton. T cells were incubated with MCC:GFP cells for 90 min at 37°C 1 (pink arrow) makes contact and induces accumulation of GFP at before PBS wash to remove unconjugated T cells. T cells were fixed and the interface rapidly (ϳ2 min), but just as rapidly it dissociates stained directly with anti-GFP and anti-I-Ek (A). Alternatively, after fixa- from the APC without forming a mature synapse, carrying a large tion, T cells were permeabilized with 0.1% Triton X-100 before staining ␤ GFP spot with it. Dissociation and transfer of MHC precede ma- with: B, anti-GFP and anti-V 3; C, anti-GFP and anti-phosphotyrosine; D, anti-p56Lck and anti-phosphotyrosine; or E, phalloidin to stain F-actin. Im- ture synapse formation in ϳ20% of transfer events. ages are shown en face after three-dimensional image reconstructions. The on-line supplement includes movies of 3D rotations corresponding to A, B, 5 The on-line version of this article contains supplemental material. D, and E. Bar ϭ 5 ␮m. The Journal of Immunology 83 conjugated T cells. At least 20 cells were imaged for each staining Detection of MCC:I-Ek:GFP and CD80 transfer from APC to combination in three separate experiments, and representative im- the T cells by flow cytometry ages are found in Fig. 2. In the majority of T cells imaged (147 of The imaging data in Figs. 1 and 2 demonstrate that when T cells 165), the GFP on the T cell was found as a single spot or a few dissociate from APC, they capture MHC:peptide molecules di- tightly clustered regions on the T cell surface, in complete agree- rectly from the immunological synapse, and that the transferred ment with the live cell imaging data (Fig. 1). In the remaining T material colocalizes with signaling-associated molecules. We cells, the GFP was found as multiple smaller regions of increased turned to flow cytometry to evaluate the frequency of transfer, time GFP intensity randomly distributed on the T cell. The intensity of and dose dependency, molecular requirements, and peptide spec- these individual spots was generally lower compared with the large ificity of transfer. Ͻ Ͼ GFP spots, being 2-fold above background vs 3-fold for the In this and subsequent flow cytometry experiments, T cells were large spots. When stained with Abs, these smaller, significantly cultured with APC for 90 min before recovery by PBS wash, 30 less intense spots associated with several of the stains, but the min longer than the live cell imaging experiments to increase the numbers of individual cells for each staining regimen were too frequency of transfer events. With incubation times longer than 90 small to attach significance to the phenotype. min, the level of transfer detected increased, but significant levels To confirm that the green spot on the T cell surface was com- of APC death were observed after 6 h (data not shown). Cellular prised of GFP-tagged MHC:peptide molecules, we stained the re- debris can efficiently be captured by the T cells, as shown by use k covered T cells with anti-GFP and anti-I-E without detergent per- of freeze/thawed cell extracts (Fig. 3E). The 90-min incubation meabilization of the T cell. In these studies, we defined specific was chosen to optimize direct cell to cell transfer, but avoid the Downloaded from accumulation as being 3-fold or more above background compared killing of the APC and concomitant release and potential uptake of with other regions of the same T cell. We observed that the green apoptotic debris. k spot colocalized with anti-I-E , but not with anti-GFP in the non- In Fig. 3, representative of seven separate experiments, the T permeabilized T cells (Fig. 2A). However, after permeabilization cells became GFP positive (Fig. 3A) after a 90-min incubation with in 0.1% Triton X-100, there was very good colocalization of the APC, showing that the transfer of GFP and I-Ek from the APC to green spot with anti-GFP (Fig. 2, B and C). Taken together, the the T cells is detectable by flow cytometry. The mean fluorescence k http://www.jimmunol.org/ anti-GFP and anti-I-E staining confirms that the GFP spot trans- intensity increased on the T cells by 1.93-fold over the unstimu- k ferred to the T cell contains MCC:I-E :GFP molecules acquired lated controls (shaded histogram). This correlated very well with from the APC. Furthermore, the inaccessibility of the GFP to Ab the 1.92-fold increase observed in surface I-Ek staining (Fig. 3B). in intact cells indicates that the GFP is on the cytoplasmic side of the These data confirmed that T cells capture MHC:peptide complexes k plasma membrane. Combined with the surface I-E staining on intact directly from APC. Of note is the profile of both the GFP and the cells, this suggests that the transferred MHC:peptide molecules are surface I-Ek on the T cells. It might be expected that the transfer present on the T cell surface as transmembrane molecules. would result in the appearance of GFP-positive and GFP-negative When permeabilized cells were stained with anti-GFP and anti- populations. However, the profiles clearly show a heterogeneous ␤ V 3 (specific for the TCR transgene), we observed that the TCR monomodal population (Fig. 3, A and B), implying a continuum of by guest on September 26, 2021 k also colocalized with the MCC:I-E :GFP (Fig. 2B) in 29 of 34 cells the amounts of material transferred to T cells recovered after 90 imaged (85%). The fact that the MHC:peptide captured by the T min, when the majority of recovered T cells had interacted with cell is still in close association with the TCR raises the possibility APC, as shown by up-regulation of CD69 (see below, Fig. 4). that these molecules may still be interacting on the T cell surface, To determine whether other APC membrane proteins, particu- continuing to generate intracellular signals. To examine this possibil- larly members of the c-SMAC, were transferred to the T cells ity, cells were stained with an Ab specific for phosphotyrosine, and it along with the MHC:peptide complexes, T cells were also stained was observed that the transferred MCC:I-Ek:GFP molecules and for the presence of CD80. It has previously been established that phosphotyrosine colocalized in 29 of 33 cells (88%) (Fig. 2, C and D). murine T cells do not express CD80 endogenously, but instead In addition, when the cells were permeabilized and stained with anti- capture it from APC (33). We observed that like the MHC:peptide, p56lck, we observed that the GFP spot on the T cell colocalizes with CD80 is efficiently transferred to the T cells during dissociation, Lck as well as increased levels of phosphorylated tyrosine (Fig. 2D). with a mean fluorescence intensity (MFI) increase of 2.93-fold vs The Lck staining pattern in Fig. 2D is representative of 24 of the 31 unstimulated controls (Fig. 3C). Thus, transfer is not limited to just T cells imaged (77%) in which GFP and Lck colocalize over three MHC:peptide complexes, but includes other membrane proteins separate experiments. In three other T cells, there is a faint Lck ring found in the c-SMAC. surrounding the GFP (data not shown) reminiscent of the pattern seen To further characterize the MHC transfer event, we examined for phosphorylated Lck (40). We also observed a weak colocalization the dose dependency of transfer by the numbers of APC available of PKC-␪ with the GFP spot, but it was Ͻ2-fold above background to the T cells. When the number of APC is increased with a con- (data not shown), and thus did not reach the 3-fold threshold we set stant number of T cells (2.5 ϫ 106), there is a dose-dependent for specific accumulation. The accumulation of GFP-tagged MHC: increase in the amount of GFP transferred (Fig. 3D). With 105 peptide, TCR, Lck, phosphotyrosine, and weakly accumulated PKC-␪ APC (an APC:T ratio of 1:25), the GFP MFI increased by only strongly suggests that the GFP spot is an area of sustained T cell 28% above unstimulated controls. At 5 ϫ 105 MCC:GFP (1:5 intracellular signaling. APC:T ratio), the increase was 54%, while at 106 APC (the 1:2.5 Finally, we examined the T cell cytoskeleton to determine APC:T ratio used in all flow cytometry experiments), the MFI whether there was polarization toward the captured MHC:peptide increased by 1.76, similar to the values in Fig. 3A. Finally, at 5 ϫ complexes. When the T cells were stained with phalloidin, it was 106 APC (2:1 APC:T ratio), the GFP MFI was 2.8-fold above observed in Ͼ60% of T cells that F-actin preferentially accumu- background. lated under the GFP (Fig. 2E, 90° merge). The en face view (Fig. To exclude the possibility that the GFP capture measured by 2E, merge) showed that the F-actin surrounds the GFP region on flow cytometry is due to acquisition of fibroblast-derived exo- the T cell. Unlike the actin cytoskeleton, there was no microtubule- somes and/or cellular debris, the T cells were incubated with a organizing center polarization toward the GFP (data not shown). 24-h fibroblast-conditioned medium or with medium containing 84 PEPTIDE-SPECIFIC MHC TRANSFER FROM IMMUNOLOGICAL SYNAPSE Downloaded from http://www.jimmunol.org/

FIGURE 3. Transfer of MHC:peptide and CD80 from APC to T cells requires direct TCR-MHC interaction and is dose dependent. A total of 2.5 ϫ 106 T cells was cultured with 106 MCC:GFP cells for 90 min at 37°C before recovery of the T cells by PBS wash. GFP (A) and I-Ek (B) levels on the surface of live unstimulated (shaded) and APC-stimulated (line) T cells show similar levels of capture. C, T cells also capture CD80 from APC during culture. The shaded histogram is unstimulated and the filled by guest on September 26, 2021 line is MCC:GFP-stimulated cells. D, A total of 2.5 ϫ 106 T cells was added to wells containing increasing numbers of MCC:GFP cells. After 90-min coculture, T cells were examined for GFP levels, indicative of MHC:peptide capture. E, To quantitate passive capture of MHC:peptide from medium or cellular debris, T cells were incubated with 24-h fibroblast-conditioned medium (gray) or fibroblast freeze/thaw cellular debris (black). F–I, MCC:GFP cells or T cells were preincubated with 10 ␮g/ml indicated Abs for 1 h before initiation of coculture. Cells stimulated by MCC:GFP cells alone significantly increased GFP expression (black line in F-I) vs unstimulated cells (shaded in F–I). GFP profiles for treatment groups are shown in gray lines in F–I. F, Control anti-CD71 Ab does not affect MHC capture. G, Anti-CD80 clearly reduces, but does not block GFP-tagged MHC:peptide transfer to the T cells. H and I, Blockade of TCR/MHC interactions with anti-I-Ek (H) or anti-CD3 (I) prevents MHC: peptide transfer to the T cells.

FIGURE 4. MHC:peptide is preferentially captured by activated T cells. cellular debris after multiple rounds of freezing and thawing (Fig. T cells were incubated with MCC:GFP cells for 90 min to allow for trans- 3E). The data clearly show that the T cells become only faintly fer. T cells were recovered by PBS wash, as above, and stained for CD4, GFP positive when incubated with conditioned medium (17% in- V␤3 (TCR), and CD69 levels. A–C, Left column, Shows GFP vs TCR crease in MFI vs unstimulated), suggesting that this passive ac- levels; right column, CD69 vs TCR levels. A, Unstimulated cells express quisition of vesicles/exosomes is not responsible for the increase in uniformly high levels of TCR and are GFP negative. They are also CD69low. B, Left, T cells stimulated by MCC:GFP cells down-modulate GFP observed by flow cytometry. ϩ Thus, the data from Fig. 3 show that the transfer of MHC:pep- TCR, and cells with down-modulated TCR preferentially become GFP . C, Left, T cells stimulated with GFPϪ fibroblast APC (MCC:FKBP) have tide from APC to T cells occurs alongside the transfer of another ϩ similar levels of TCR down-modulation, but do not become GFP . B and c-SMAC component CD80 (Fig. 3C), and that transfer is depen- C, Right, CD69 vs TCR plots for the APC-stimulated cells show three dent upon Ag dose (Fig. 3D). The amount of transfer is also distinct populations (TCRhigh CD69low, TCRhigh CD69high, TCRlow dependent on the time of incubation (data not shown) and is not CD69high). D, Table shows GFP mean fluorescence values for each of these due to the passive acquisition of exosomes released from the populations in right column of A–C. E, GFP MFI (line) as a function of TCR fibroblasts. down-modulation. Shaded histogram shows cell number at each TCR level. The Journal of Immunology 85

Transfer is mediated by TCR-MHC interactions the GFP expression. The mean of the population increases, and there is a loss of the very dim cells. The GFP MFI difference The finding that the capture of MHC:peptide from the APC was high low directly from the immunological synapse (Fig. 1) and that transfer between the TCR and TCR populations in Fig. 4B is 2.1- was dose dependent (Fig. 3D) suggests that MHC:peptide transfer fold. The GFP signal in Fig. 4B is not due to an increase in is mediated by TCR-MHC interactions. To determine the molec- green autofluorescence due to T cell activation, because the T ular requirements for MHC:peptide transfer, APC or T cells were cells stimulated with the GFP-negative APC (MCC:FKBP) dis- preincubated with blocking Abs specific for MHC class II, CD3, play a similar level of TCR down-modulation, but no increase CD80, or CD71 (transferrin receptor) for 1 h before the initiation in GFP (Fig. 4C). of the coculture. The blocking Abs were present for the duration of To further examine the T cell phenotype, we compared CD69 the coculture. The fibroblast APC used in this study are Fc receptor and TCR levels (right column of Fig. 4, A–C). As in the left panel of Fig. 4A, the unstimulated T cells are TCRhigh and are CD69low, negative, and so do not display Abs in a multivalent fashion on intermediate their surface. At the end of the 90-min coculture, the T cells were with a very small population being CD69 . However, recovered, gated on CD4ϩ,V␤3ϩ cells, and examined by flow when the T cells were stimulated with either MCC:GFP or MCC: FKBP cells, two additional populations appeared, TCRhigh cytometry for GFP levels. The data in Fig. 3, F–I, representative of high low high three separate experiments, show the GFP levels on unstimulated CD69 and TCR CD69 (right panels in Fig. 4, B and C). cells (shaded) or on MCC:GFP cells without (black line) or with Each population was gated, and the GFP mean fluorescence inten- blocking Abs (gray line). Without any blocking reagents present, sity was determined. The GFP MFI value for each population is the GFP MFI of the T cells after incubation with the MCC:GFP shown in Fig. 4D. cells increased from 8.6 to 12.5, a 1.45-fold increase. To control The table in Fig. 4D shows that there is a correlation between T Downloaded from cell activation phenotype and the expression of captured GFP- for potential steric effects of an Ab bound to the surface of either k ϩ the T cell or the APC, we used anti-CD71 (Fig. 3F). When this Ab tagged MCC:I-E . For all CD4 T cells, there is a 64% increase in was present, there was no effect on GFP capture. GFP MFI for the MCC:GFP-stimulated cells vs unstimulated, 44% To assess the importance of costimulation to capture, we higher than the MCC:FKBP-stimulated T cells. When the results blocked with anti-CD80 (Fig. 3G). Blockade of CD80/CD28 in- are broken down into the three TCR vs CD69 phenotypes, the teractions reduced the GFP MFI by a modest 17%. The costimu- results are more striking. There are only small differences in GFP http://www.jimmunol.org/ levels for the nonactivated TCRhigh CD69low populations, but for lation results contrast sharply with the data involving blockade of high high Ag recognition. When TCR/MHC:peptide interactions are in- the clearly activated TCR CD69 population, the difference k between unstimulated and MCC:GFP is fairly large (58% increase hibited by addition of anti-I-E (Fig. 3H), there is a significant high reduction in the amount of MHC:peptide transferred to the T in MFI). The biggest difference is observed with the CD69 cells cells. Anti-CD3 completely inhibits MHC:peptide transfer to that have down-modulated TCR, greater than 2-fold. Thus, based the T cells (Fig. 3I). upon TCR and CD69 levels, there is a clear positive correlation Taken together, the results of the blocking experiments shown in between GFP-tagged MHC:peptide complex capture and the T cell Fig. 3 show that the transfer of MHC:peptide from APC to T cells activation state. by guest on September 26, 2021 in this system requires TCR engagement of the cognate MCC:I-Ek The correlation between activation state and MHC:peptide cap- ligand. These results also show that the interaction of CD80 with ture is dependent upon both CD69 expression and TCR down- CD28 (or CTLA-4), while augmenting TCR-mediated transfer, is modulation. This would predict that T cells with the most highly not essential for MHC:peptide capture. down-modulated TCR levels would be the brightest for GFP; however, the relationship between TCR level and GFP capture is more complex. Careful examination of Fig. 4B (left column) shows that Expression of GFP on T cells correlates with T cell activation the T cells that had down-modulated TCR ϳ5-fold were the phenotype brightest for GFP. However, as the TCR levels continued to de- We next turned to characterizing the activation phenotype of the T crease, GFP expression decreases until cells with 15- to 20-fold cells that had captured MHC:peptide. Knowing the activation state less TCR were essentially GFP negative. This is clearer in the of the T cells in the culture could provide important information histogram in Fig. 4E, in which the GFP level is plotted as a func- about the cells that captured MHC:peptide from the APC upon tion of TCR level. As the cells down-modulate receptor, the GFP dissociation. It is possible that the T cells that dissociated and level increases sharply, reaching a maximum at ϳ4-fold less TCR, captured MHC:peptide were not fully activated and the transfer then sharply declines. This implies that as TCR is internalized was a result of abortive activation. To assess their activation state, beyond a threshold of ϳ4- to 5-fold, MHC:peptide disappears and T cells were cocultured with APC for 90 min and recovered, as may also be internalized. previously described. As a control in these experiments, T cells Based upon these data, we conclude that the levels of GFP- were stimulated with a fibroblast APC transfected with a construct tagged MHC:peptide captured from the APC found on T cells are encoding the same extracellular complex, but with a nonfluores- a function of the TCR down-modulation and the activation state of cent cytoplasmic domain, MCC:FKBP. The extent of T cell pro- the T cell. Cells that have down-modulated TCR to a moderate liferation and induction of activation markers such as CD69 and amount (5- to 10-fold) and are CD69high express the highest CD25 induced by MCC:FKBP cells is comparable to MCC:GFP amount of GFP. Further down-modulation of the TCR is associ- cells. The T cells were stained with Abs to CD4, V␤3 (specific for ated with a reduction of GFP expression. These data also suggest the transgenic TCR), and CD69. The flow cytometry results for that capture is not the result of incomplete or abortive activation. CD4ϩ cells, representative of five separate experiments, are shown in Fig. 4. The left column of Fig. 4, A–C, shows TCR vs GFP levels. The MHC:peptide transfer to T cells is peptide specific unstimulated cells (Fig. 4A) are TCRhigh and GFP negative. When The process of direct intercellular transfer of MHC:peptide from stimulated by the MCC:GFP cells for 90 min, a substantial pro- APC to T cells in this study is TCR dependent, raising the possi- portion of the cells down-modulates their TCR (Fig. 4B), as ex- bility that transfer is peptide specific. To test this hypothesis, we pected. The TCR down-modulated population has a clear shift in used the fact that in addition to expressing the GFP-tagged I-Ek: 86 PEPTIDE-SPECIFIC MHC TRANSFER FROM IMMUNOLOGICAL SYNAPSE

cells with or without 20 ␮M Hb peptide prepulse. If MHC:peptide transfer is peptide specific, the Hb-specific 3.L2 T cells should not pick up the irrelevant MCC:I-Ek:GFP molecules from the APC. If, in constrast, transfer is not peptide specific, the 3.L2 T cells, re- sponding to Hb-loaded unlabeled MHC:peptide complexes, would be expected to capture GFP-tagged I-Ek:MCC complexes and should become GFPϩ. The results, representative of five separate experiments, are shown in Fig. 5A. There is no observable difference in green fluorescence on the 3.L2 T cells whether they were cultured on Hb-loaded MCC:GFP cells or Hb-negative MCC:GFP cells. Thus, the Hb-specific T cells do not pick up irrelevant GFP- tagged I-Ek:MCC molecules from the APC. In Fig. 5, B and C, MCC-specific T cells were mixed with Hb- specific T cells at a 1:1 ratio and then added to MCC:GFP cells that had been preloaded with 20 ␮M Hb by overnight incubation. After a 90-min coculture, both MCC-specific and Hb-specific T cells were recovered, gated for their respective TCRs, and examined by flow cytometry. Fig. 5B shows that the two T cells are easily sep-

arated based upon V␤ expression. In Fig. 5C, Ag is present for Downloaded from both types of T cells, but only the MCC-specific T cells capture MCC:I-Ek:GFP from the APC. The Hb-specific T cells, while they are stimulated to express CD69 (data not shown) and proliferate by the Hb-loaded MCC:GFP cells (37), do not capture any of the MCC-loaded, GFP-tagged I-Ek. Based upon these findings, we

conclude that MHC:peptide transfer in this system is peptide http://www.jimmunol.org/ speciﬁc.

Discussion In this study, we have described the transfer of specific MHC: peptide complexes from transfected fibroblast APC to T cells directly from the immunological synapse upon spontaneous cellular dissociation. Imaging (Fig. 2) and flow cytometry examination of T cells recovered from a 90-min coculture with APC (Fig. 3) suggest that the transferred MHC:peptide molecules are incorporated by guest on September 26, 2021 into a membrane on the surface of the T cell. Specifically, an anti-I-Ek Ab stained intact cells (Figs. 2A and 3B), but anti-GFP stained only after detergent permeabilization of the T cell’s plasma membrane. These findings are in agreement with the previously reported ability of T cells to act as APC after capturing MHC (19, 20, 22, 25, 33, 41, 42) and may have important implications in controlling an ongoing immune response, as discussed below. The I-Ek molecules may be incorporated into the plasma membrane of the T cell, or could be retained on membrane vesicles physically associating with the surface of the T cells via engaged TCR. The capture of plasma membrane and membrane-associated proteins from APC has previously been described for B cells (27, FIGURE 5. Transfer of MHC:peptide complexes to T cells is peptide 28), NK cells (29–31, 43), ␥␦ T cells (23), as well as CD4ϩ and specific. As above, T cells were incubated with MCC:GFP cells for 90 min CD8ϩ ␣␤ T cells. For T and NK cells, the mechanism usually falls before T cells were recovered from the culture, stained, and assessed by into two categories, vesicle-mediated transfer involving exosomes flow cytometry. A, CD4- and V␤8.3-gated Hb-specific 3.L2 T cells do not capture GFP-tagged MCC:I-Ek from APC, even when cells were preloaded from APC (17, 18, 44, 45) or, as described in this study, direct T with 20 ␮M Hb peptide. B and C, MCC-specific AD10 (V␤3ϩ) and Hb- cell-APC contact (46). The contact-dependent capture that we de- specific 3.L2 (V␤8.3ϩ) T cells were mixed at a 1:1 ratio and incubated for scribe differs from previous reports in that transfer occurs as the k 90 min with MCC:GFP prepulsed with 20 ␮M Hb peptide. B, After re- removal of a large patch of GFP-tagged I-E :MCC (along with covery, CD4ϩ T cells were stained with specific anti-V␤ Abs and were CD80) from the immunological synapse during dissociation, rather gated on their particular V␤ and examined for GFP expression. C, Only the than by transfer of multiple small packets of material when the T MCC-specific AD10 T cells captured the GFP-tagged MCC:I-Ek; the Hb- cells and APC are tightly conjugated (20). It is reminiscent of the specific 3.L2 cells did not. transfer of yellow fluorescent protein-tagged membrane during CD8ϩ CTL-APC dissociation described by Stinchcombe et al. (11). Using transmission electron microscopy, they described the MCC complexes, the MCC:GFP cells also express unlabeled wild- formation of small regions of fused T-APC membranes (“bridges”) type I-Ek molecules that can be exogenously loaded with murine only during the process of dissociation. The resolution of these hemoglobin (Hb64–76) peptide and used to stimulate T cell prolif- T-APC fusion events leads to transfer of APC surface molecules eration (37). To determine whether MHC transfer is peptide spe- onto T cells. Although we consider it highly unlikely, based upon cific, Hb-specific 3.L2 T cells were cocultured with MCC:GFP images in Fig. 1, it is nevertheless possible that the transfer event The Journal of Immunology 87 observed in this study is due to directed secretion of exosomes that The activation phenotype is also relevant when considering the are subsequently captured by the T cells. spontaneous dissociation of the T cells from the APC. In two pre- When examined by flow cytometry (Fig. 3), the level of GFP vious in vitro imaging studies, CD4ϩ T cells were seen to repeat- and anti-I-Ek staining is similar to that reported by Hudrisier et al. edly associate, then dissociate from macrophages (48) or dendritic (22), but appears relatively low compared with other previously cells in a three-dimensional collagen matrix (49). One interpreta- reported data for CD4ϩ T cells (15, 17, 19, 25, 45, 47). This is tion was that the cells were interacting with multiple APC partners, most likely due to differences in experimental details, as the MCC: summing the activation signals until full activation was achieved. I-Ek:GFP level on the surface of our MCC:GFP cells is relatively An alternate explanation for the spontaneous dissociation was that low. The GFP signal is ϳ7-fold above background (37). Also, T it was due to an abortive activation event leaving the cells partially cells were incubated with the APC for only 90 min before recovery activated. In this study, the T cells formed mature immunological from the culture without the use of any dissociating reagents (e.g., synapses, expressed high levels of CD69, and displayed significant EDTA) to insure that we were examining only cells that had spon- TCR down-modulation, suggesting that the T cells that dissociated taneously dissociated from the APC. Although transfer efficiency from the MCC:GFP cells were fully activated. Importantly, the increases with incubation duration (25), we found that there was rapid association-dissociation cycling seen previously in vitro (37, significant death of APC after6hofcoculture (data not shown), 48, 49) is very similar to the phenotype seen recently by Mempel and Fig. 3E shows that T cells can become GFP positive when et al. (50) and Miller et al. (51) using two-photon imaging of T-DC cultured with cellular debris, calling into question the mechanism interactions during the initial phases of T cell priming in lymph of transfer at later time points. nodes in vivo, suggesting that rather than being an artifact, the in vitro It is unknown whether the phenomenon described in this work phenomenon reflects a process that is physiologically relevant. Downloaded from with fibroblast APC reflects the situation with physiologic APC. Previous studies have shown that transfer is Ag dependent (20, However, 5 days after adoptive transfer into transgenic mice ex- 22), and that only the restricting MHC is transferred, but the pep- pressing a similar MHC:peptide construct on professional APC, tide specificity of transfer has not been resolved (15, 20). Several TCR transgenic T cells with significant amounts of captured MHC: studies have inferred peptide specificity using indirect measure- peptide can be detected by flow cytometry (S. Wetzel and C. Hud- ments such as cross-reactivity of CTL to the T cells after capturing dleston, unpublished observation). MHC (20) or absence of a decrease in detectable expression of http://www.jimmunol.org/ irrelevant MHC class I on the surface of a dendritic cell in vivo Ab-blocking experiments showed that the transfer described in (26), but the system used in this study allowed us to directly ad- this study was TCR mediated. Physical blockade of TCR or MHC dress this question. We used the fact that the transfected fibroblast prevented appreciable transfer onto the T cells (Fig. 3, G and H). k ϩ APCs express GFP-tagged I-E :MCC covalent molecules along These findings are in line with previously published data for CD8 with unlabeled wild-type I-Ek that can be exogenously loaded with T cells, which showed that Ag-dependent transfer required TCR/ Hb peptide to present Ag to Hb-specific T cells. The Hb-specific T MHC interaction and subsequent TCR signaling (20, 22). In con- k Ϫ Ϫ cells did not capture GFP-tagged irrelevant I-E :MCC from the trast to studies using CD28 / T cells showing that MHC class I ϩ APC during the culture period, but MCC-specific T cells did (Fig. transfer to CD8 CTL required CD28/CD80 interactions (21, 33), by guest on September 26, 2021 5, A and C). Thus, peptide-specific MHC transfer predominates our Ab-blocking experiments showed that transfer was reduced, during dissociation of the T cells from the APC. Although appar- but not prevented when CD28/CD80 interactions were blocked ently only MHC loaded with the cognate peptide ligand is trans- (MCC:GFP cells do not express CD86 (37)). This suggests that ferred, the transfer of another c-SMAC component CD80 suggests CD28/CD80 interactions augment the transfer, but are not neces- that the specific MHC:peptide molecules are not plucked from the sary for transfer to occur. These findings are consistent with our APC surface. It must be noted that the immunological synapse previous report showing that blockade of CD28/CD80 ligation sig- formed between the Hb-specific T cells and Hb-pulsed MCC:GFP nificantly reduced synapse size and organization, inhibiting the cells is enriched only for specific MHC:peptide (37), consistent formation of a mature immunological synapse, but not preventing with results from Wu¨lfing et al. (52), showing that at high peptide MHC:peptide accumulation at the T-APC interface (37). concentrations, accumulation of MHC molecules at the immuno- We have also shown that MHC transfer correlated with an ac- logical synapse is peptide specific. tivated phenotype of the T cells. T cells expressing the highest Perhaps the most intriguing data in this study is the imaging levels of CD69 were also the brightest with regard to transferred done 10 min after recovery of the T cells from the 90-min cocul- k MCC:I-E :GFP (Fig. 4B). Captured GFP levels are also related to ture with APC (Fig. 2), which showed that the GFP-tagged I-Ek: another marker of T cell activation, TCR down-modulation (Fig. 4, MCC molecules remain associated with the TCR upon capture as B and E). However, the relationship between TCR down-modula- a distinct, focused spot (Fig. 2). Furthermore, the GFP-tagged I-Ek: tion and GFP capture was more complex. As T cells down-mod- MCC colocalize with the src family kinase p56Lck and high levels ulated TCR, they had increasing levels of captured GFP, but be- of tyrosine-phosphorylated proteins. These findings suggest that yond a critical TCR threshold, ϳ5-fold lower than resting T cell the transferred MHC:peptide molecules may be interacting with blasts, the levels of GFP began to precipitously drop (Fig. 4E). All the colocalized TCR and sustaining intracellular signaling. If of the cells with TCR down-modulation were CD69high, indicating MCC:I-Ek:GFP molecules are in their native orientation in the T that they were acutely activated. Internalization of the TCR may cell plasma membrane, it implies that the transferred MHC:peptide lead to internalization and degradation of the associated GFP- molecules, along with the c-SMAC component CD80, are in- tagged MHC:peptide complexes. This is supported by the colocal- volved in autopresentation by the T cells. Although the data in Fig. ization of TCR and GFP that we observed (Fig. 2B) along with 2A, showing that the TCR and transferred MHC class II colocalize, time course data that showed that GFP levels dropped by ϳ50% are similar to imaging showing that CD8 and transferred MHC during the first 2 h after removal from APC before stabilizing and class I colocalize (11), this would appear to be contrary to recent slowly decaying over the next 46 h (data not shown). These results evidence that on a CTL membrane TCR and potential MHC:pep- are consistent with those of Huang et al. (20), who showed that tide ligands are spatially segregated to prevent autolysis (53). transferred MHC class I was internalized and delivered to lyso- However, the later study dealt with endogenously synthesized mol- somes for degradation. ecules, while in this study the MHC class II:peptide complexes are 88 PEPTIDE-SPECIFIC MHC TRANSFER FROM IMMUNOLOGICAL SYNAPSE drawn onto the T cells as a result of interactions with the TCR. 4. Grakoui, A., S. K. Bromley, C. Sumen, M. M. Davis, A. S. Shaw, P. M. Allen, When the CD8ϩ T cells were treated with agents that disrupted cell and M. L. Dustin. 1999. The immunological synapse: a molecular machine controlling T cell activation. Science 285:221. surface charge, TCR, CD8, and MHC all colocalized, leading to 5. Delon, J., and R. N. Germain. 2000. Information transfer at the immunological cell death via suicide, consistent with autopresentation (53). It is synapse. Curr. Biol. 10:R923. 6. Bromley, S. K., W. R. Burack, K. G. Johnson, K. Somersalo, T. N. Sims, plausible, therefore, that T cells might sustain intracellular signal- C. Sumen, M. M. Davis, A. S. Shaw, P. M. Allen, and M. L. Dustin. 2001. The ing via the TCR after MHC:peptide capture from an APC. immunological synapse. Annu. Rev. Immunol. 19:375. The results in this study, along with several previous reports, 7. Huppa, J. B., M. Gleimer, C. Sumen, and M. M. Davis. 2003. Continuous T cell receptor signaling required for synapse maintenance and full effector potential. suggest that MHC:peptide capture by T cells may be important in Nat. Immunol. 4:749. controlling an immune response. First, removal of specific MHC: 8. Kupfer, A., S. L. Swain, and S. J. Singer. 1987. 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