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The Journal of Immunology, 2005, 175: 7829-7836.
Copyright © 2005 by The American Association of Immunologists

CD80 Cytoplasmic Domain Controls Localization of CD28, CTLA-4, and Protein Kinase C{theta} in the Immunological Synapse1

Su-Yi Tseng*, Mengling Liu{dagger} and Michael L. Dustin2,*

* New York University School of Medicine, Skirball Institute, and {dagger} Division of Biostatistics, New York University Cancer Institute, New York, NY 10016


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The binding of costimulatory ligand CD80 to CD28 or CTLA-4 on T cells plays an important role in the regulation of the T cell response. We have examined the role of the cytoplasmic domain of CD80 in murine T cell costimulation and its organization in the immunological synapse (IS). Removal of CD80 cytoplasmic tail decreased its effectiveness in costimulating T cell proliferative response and early IL-2 production in response to agonist MHC-peptide complexes. Immunofluorescent study showed a decreased tailless CD80 accumulation in the IS of naive T cells. The two forms of CD80 accumulated differently at the IS; the tailless CD80 was colocalized with the TCR whereas the full-length CD80 was segregated from the TCR. In addition, we showed that CD80, CD28, and protein kinase C{theta} colocalized in the presence or absence of the CD80 cytoplasmic tail. Thus, the cytoplasmic tail of CD80 regulates its spatial localization at the IS and that of its receptors and T cell signaling molecules such as protein kinase C{theta}, and thereby facilitates full T cell activation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Costimulatory ligand CD80 and CD86, expressed primarily on APCs, have the ability to augment T cell responses through interactions with CD28 (1) or inhibit T cell responses through interaction with CTLA-4 (2, 3). CD80 and CD86 are type I transmembrane proteins with two Ig-like domains each and short cytoplasmic domains. The cytoplasmic domain of CD80 has been implicated in effective signaling to T cells suggesting that active processes in the APC may contribute to the delivery of costimulatory signals (4, 5, 6, 7). This finding is consistent with other evidence for an important role of the dendritic cell (DC)3 cytoskeleton in T cell stimulation (8, 9, 10).

CD28/CTLA-4 interaction with CD80/CD86 take place in the contact area between T cells and APC in parallel with TCR interaction with MHC-peptide complexes and LFA-1 interaction with ICAM-1 (11, 12, 13). In many situations a specialized pattern called the immunological synapse (IS) forms consisting of a central supermolecular activation cluster (cSMAC) containing TCR interacting with MHC-peptide complexes surrounded by a peripheral SMAC (pSMAC) containing LFA-1 and ICAM-1 interacting pairs (14, 15). Protein kinase C (PKC){theta} localizes in the IS and in large part defines the cSMAC (12, 15). PKC{theta} is required for normal activation of NF-{kappa}B and AP-1 transcription factors during T cell activation (16) and full activation of PKC{theta} depends upon CD28 engagement (17, 18). It is not known, however, whether the cytoplasmic domain of CD80 controls the pattern of CD28, CTLA-4, or PKC{theta} in the IS.

CD28 expressed on T cells delivers a positive costimulatory signal upon ligation with CD80 or CD86. Ligation augments T cell activation by reducing the number of TCRs that must be triggered for T cell activation (19, 20), and enhances the production of IL-2 (21). CD28 ligation also stimulates cytoskeletal transport of molecular complexes toward the IS (22), enhances accumulation of lipid rafts in the IS (23), promotes formation of the IS (24), and increases the duration of Lck signaling (13). CD28 can accumulate in the IS without ligand, but only appears to signal when engaged by CD80 or CD86 (25).

CTLA-4 is an important inhibitory molecule, as exhibited by the lethal lymphoproliferative disease in CTLA4–/– mice (26, 27). CTLA-4 is not detected on the cell surface until 24 h after T cell activation, peaking at 48–72 h (28, 29, 30). CTLA-4 ligation to CD80 or CD86 antagonizes CD28 signals by blocking IL-2 gene transcription and prevents the entry of T cells into the cell cycle beyond G1/G0 leading to inhibition of T cell proliferation (29, 31). In addition, CTLA-4 inhibits proximal signaling through the TCR (32). CTLA-4 is localized in an intracellular pool in activated T cells and its recruitment to the IS is dependent on the strength of the TCR signal (12). CTLA-4 accumulation in the IS is completely dependent upon ligation by CD80 or CD86 (25).

To assess the importance of CD80 cytoplasmic tail in orchestrating costimulation, we generated full-length CD80 (CD80FL) and cytoplasmic tail deleted CD80 (CD80TL) each tagged with yellow fluorescent protein (YFP) and expressed these in Chinese hamster ovary (CHO) cells that also expressed I-Ek and ICAM-1. We examined the functional responses of TCR transgenic naive T cells to antigenic peptide-pulsed CHO cells expressing CD80FL with C-terminal YFP (CD80FL-YFP) or CD80TL with C-terminal YFP (CD80TL-YFP) and found that the T cell proliferated more and made IL-2 earlier when the CD80 cytoplasmic tail was intact. To test the hypothesis whether this functional difference was due to changes in the organization of CD80 and its receptors in the IS, we performed confocal imaging of T cell-APC interactions. We found that in naive T cells CD80TL-YFP is recruited in a different pattern than CD80FL-YFP. CD80FL-YFP and its receptors accumulated in a pattern in which they were segregated from the TCR in the cSMAC, whereas CD80TL-YFP and its receptors accumulated in a pattern colocalized with the TCR in the cSMAC. In addition, we found that PKC{theta} colocalized with CD80FL-YFP and CD28, not with the TCR. Thus, a previously unappreciated feature of CD80 is that it may function to segregate CD28 and CTLA-4 away from the TCR to create a distinct costimulation zone in the IS near the boundary between the cSMAC and the pSMAC.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cells and constructs

CHO cells transfected with I-Ek were kindly provided by A. van der Merwe (Oxford University, Oxford, U.K.) and M. Davis (Stanford University, Palo Alto, CA) (33). This CHO line expressed endogenous hamster ICAM-1, and I-Ek was selected with 400 µg/ml G418. CD80 was fused into enhanced YFP N-1 vector (Clontech Laboratories) at SacII restriction enzyme site to generate in frame C-terminal YFP fusion constructs. The stop codon of CD80 molecule was removed by PCR and fused to the enhanced YFP cDNA sequence and subcloned into the pBudCE4.1 vector (Invitrogen Life Technologies). The primers used for this PCR were 5'-CCCGCTATGGCTTGCAATTGTCAG-3' and 5'-CCCGCGGAAGGAAGACGGTCTGTTCAG-3'. For the CD80TL, the tail was removed where the transmembrane region ends at amino acid 275 via PCR with primers 5'-GCTAGAATTCGCTATGGCTTGCAATTGT-3' and 5'-GCTACCTCGGGTGCTTACAGAAGCATTTG-3', and fused in frame with a 10 amino acid spacer to the enhanced YFP. Following confirmation of DNA sequence, constructs were transfected into CHO cells using Lipofectamine 2000 (Invitrogen Life Technologies) CHO cells were cultured in RPMI 1640 (Invitrogen Life Technologies) medium supplemented with 10% FBS (HyClone Laboratories), Nonessential amino acids, L-glutamate, sodium pyruvate (Cellgro). CD80FL is selected with 4 µg/ml zeocin and CD80TL is selected with 4 µg/ml blasticidin. After stable CHO cell lines were established, they were sorted for various expression levels by the New York University (NYU) Cancer Institute Flow Cytometry facility (NYU Medical Center, New York, NY).

Mice and T cell activation

B10.A 5C.C7 TCR (V{alpha}11/V{beta}3) transgenic mice were purchased from Taconic Farms and bred and maintained in the NYU mouse facility. Mice were housed under specific pathogen-free conditions at the Skirball Institute (NYU Medical Center, New York, NY) animal care facility in accordance with Institutional Animal Care and Use Committee guidelines. 5C.C7 T cells are activated by a peptide of moth cytochrome c (MCC) 88-103 in the context of I-Ek. MCC 88-103 (ANERADLIAYLKQTK) was synthesized from Dana-Farber peptide synthesis facility (Boston, MA). T cells were cultured in the medium described for CHO cells plus 2 µM 2-ME (Sigma-Aldrich).

Proliferation and cytokine assays

CHO cells were treated with mitomycin C (Sigma-Aldrich) at 0.1 mg/1 x 106 CHO cells for 20 min at 37°C and plated at 2 x 104 cells/well (96-well flat-bottom plates). MCC 88-103 peptide was then added. Lymph nodes were obtained from 6- to 10-wk-old 5C.C7 TCR transgenic mice. RBCs were lysed using ACK buffer and CD4 T cells were negatively purified with anti-CD8-{alpha} (Ly2), anti-NK1.1 (DC5), and anti-MHC class II MACS beads (Miltenyi Biotec). Typical purities were >90% CD4-positive T cells. T cells were plated at 1.5 x 105 cells/well. After 48 h of incubation, 10 µl of [3H]thymidine 1 µCi/well (General Electric-Amersham Biosciences) was added to each well and incubated for an additional 16 h before harvest with a 96-well Cell Harvester (Tomtec), and filter paper was analyzed by liquid scintillation for determination of the amount of incorporated radioactive counts.

For CFSE proliferation, purified T cells were washed twice in HEPES-buffered saline and labeled with CFSE at 0.1 µM/107 T cells/ml (Invitrogen Life Technologies and Molecular Probes) for 10 min at room temperature. Labeling was quenched immediately by adding culture medium with 10% FBS. The cells were counted and used for proliferation assay as described. The percentage of cells in each division was determined using a proliferation-fitting model from FlowJo (Tree Star). For restimulation assay, 5C.C7 purified naive T cells were stimulated with CHO transfected with CD80FL with 100 nM MCC peptide in 96-well plate as described. After 48 h, the viable T cells were enriched using Lympholyte-M centrifugation (Cederlane Laboratories) and washed three times with media and plated in 24-well plates. After 4–5 days of resting, these T cells, termed primed T cells, were washed twice and used.

IL-2 and IFN-{gamma} cytokine capture kits (Miltenyi Biotec) were used to determine cytokine production by T cells, following manufacturer’s instruction.

FACS analysis, Abs, and DC purification

Abs used include: 14-4-4s, anti-CD28, and anti-CTLA-4 (BD Pharmingen), 1610A (eBioscience), anti-PKC{theta} polyclonal Ab (Santa Cruz Biotechnology), and anti-hamster ICAM-1 Ab (clone J5-3F9) provided by V. Kuchroo and colleagues (34). Alexa Fluor 546 conjugated secondary goat anti-Syrian hamster and goat anti-rabbit (Molecular Probes), and Cy5-conjugated goat anti-Armenian hamster, and anti-mouse (Jackson ImmunoResearch Laboratories). D4 mAb was generously provided by M. Davis and colleagues (35). CD11c+ splenic DCs were purified using CD11c beads (Miltenyi Biotec) and cultured overnight in the presence of 10 ng/ml LPS (Sigma-Aldrich) and 1 µg/ml anti-CD40 (BD Pharmingen).

Fluorescence photobleaching recovery

CHO cells expressing CD80FL or CD80FL-YFP were plated on a Delta-T dish and were labeled with Cy5-conjugated anti-CD80 Ab at 4°C to limit internalization. Photobleaching was performed at 24°C, and the fluorescence recovery monitored over a 200 s time period to determine the percentage of CD80FL vs CD80FL-YFP that was laterally mobile. CHO cells expressing CD80FL-YFP or CD80TL-YFP were plated on Delta-T dishes (Bioptechs) and photobleaching of YFP was done at 37°C. For photobleaching, regions of interest drawn on the cells were exposed to 100% laser power at 633 nm for 50 s or 514 nm for 25 s to bleach the fluorophores Cy5 and YFP, respectively. Within 1 s of bleaching, an image was acquired that could be used to determine the initial postbleach intensity. A series of images was then acquired until the spot reached a steady state. The percentage of laterally mobile receptors was calculated as [(prebleach intensity – final postbleach intensity)/(prebleach intensity – initial postbleach intensity)] x 100. The diffusion coefficient (D) was calculated using D = r2/(4x{tau}), where {tau} is the graphically determined half-time for recovery, and the average radius (r) is 2.63 µm for CD80FL-YFP and 2.33 µm for CD80TL-YFP.

Fluorescence microscopy and image analysis

For live cell images, purified naive 5C.C7 T cells specific for I-Ek, H57-Fab-Alexa Fluor 568 and H155-Fab-Cy5 (H57 and H155 are nonblocking Abs to the TCR and LFA-1, respectively) were added to a monolayer of CHO plus I-Ek transfectants pulsed overnight with agonist MCC peptide 88-103. These interactions were imaged at 37°C with in a 1:1 mixture of Leibovitz’s L-15 medium (Invitrogen Life Technologies) and Ex Vivo 15 medium (Cambrex Bio Science Walkersville), and supplemented with nonessential amino acids, L-glutamine, and sodium pyruvate. Imaging experiments were performed with an objective heater (Bioptechs).

For fixed cell images, cells were prepared as follows: after T cells-CHO cells interacted for 30 and 60 min at 37°C and 5% CO2 in a Lab-Tek II Chamber No. 1.5 coverglass system eight-well glass-bottom slide (Nalge Nunc International), they were briefly washed with PBS to wash away excessive T cells that did not form conjugate with the CHO cells. These cells were fixed with 4% methanol-free formaldehyde/PBS for 10 min (for permeabilized membrane) or 2% methanol-free formaldehyde/PBS for 2 min (for nonpermeabilized membrane) at 24°C. The cells were washed three times with 1x HBSS and incubated with primary Abs for 1 h at 24°C, washed and followed by a second Abs incubation for 1 h at 24°C.

Images were taken with LSM 510 Laser Scanning Confocal Microscope (Zeiss) and x100 PLAN 1.4 NA Apochromat objective. Image stacks consisted of 8–15 planes space 0.48 µm. The images were analyzed using the LSM and Volocity (Improvision) software. For three-dimensional visualization of intercellular contacts only those complexes were taken into consideration whose contact areas were flat enough to be contained in a rectangular volume for an en face projection. Accumulated CD80 was considered a cluster when the ratio of fluorescence intensity at the area of interest was 1.5 times greater than the surrounding area. The threshold for the images was set where the average baseline CD80 intensity outside contact areas was considered as background and was set to 0 pixel value. In a given experiment the brightest CD80 clusters were set to a pixel value of 255 for ease of visualization.

Statistical method for analysis of contingency table

The observations from the experiments consisted of counts in the cells of a contingency table formed by the cross-classification of stimulus factors (experiment number, cell type, and time point) and response factors (no accumulation vs accumulation, colocalized vs segregated). Stimulus factors had their marginal totals fixed in advance and the main interest was in the conditional probabilities of the response factor given the stimulus factors. The Poisson log-linear model allowed the modeling of multinomial data with multiplicative probability specification to be fitted and tested. The log-linear model consisted of main effects from each factor, first-order interactions from two factors and second-order interaction from three factors with sum-to-zero constraints to account for the many parameters. The models were focused on the evaluation of the effects of the stimulus factors on the response factors. All the stimulus factors and their interaction must be included for the analysis to respect the fixed totals over response factors. The main effect of response factors and interactions between the response and stimulus factors were selected into the model to see whether the effects of factors and their interactions were enough to explain the observed data. The final model was reached when the {chi}2 test showed the consistency between the observed data and the expected number from the model. SE was calculated as: sqrt(p*(1–p)/n).

Hypothesis testing was based on likelihood ratio tests following the central {chi}2 distribution with degrees of freedom determined by the model. All results reported were obtained using S-PLUS v.6.2 (generalized linear model command) (36, 37).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Deletion of CD80 cytoplasmic tail decreased T cell proliferative response

The contribution of the cytoplasmic domain of CD80 in costimulation and formation of IS was studied using CHO cells as APCs. CHO cells were chosen because they expressed endogenous hamster ICAM-1 to provide adhesion and no endogenous CD80. In naive T cell proliferation assay, the addition of anti-hamster ICAM-1 Ab eliminated T cell proliferation stimulated by CHO cells transfected with I-Ek (data not shown). This result is consistent with a previous study in which hamster ICAM-1 had costimulatory activity (34). We have generated CHO cells stably expressing I-Ek together with CD80FL, CD80FL-YFP, or CD80TL-YFP. These CHO cell lines were sorted by flow cytometry for equivalent expression levels of ICAM-1, I-Ek, and CD80 (Fig. 1A). The CD80 expression levels were similar to that of LPS-activated CD11c+ spleen DCs (Fig. 1B). At 100 nM Ag dose, the loading efficiency of MCC 88-103 peptide into CHO cell I-Ek was 1% based on staining with the MHC-peptide complex specific mAb D4 (data not shown).



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  		FIGURE 1. Fusion of YFP to CD80 does not alter its function or membrane mobility. A, FACS analysis of surface expression of CHO APCs. CHO cells lines I-Ek-CHO, CD80FL-CHO, CD80FL-YFP-CHO, and CD80TL-YFP-CHO were sorted to equivalent levels of expression for I-Ek, hamster ICAM-1, and variants of CD80. Anti-CD80, anti-hamster ICAM-1, and anti-I-Ek (gray line) are shown with control (black line). B, CD80 expression levels on CHO transfected cell lines and LPS activated CD11c+ DCs are equivalent. FACS plot indicates the values (inset) for mean fluorescence intensity of anti-CD80 staining. Splenic DCs were purified using CD11c beads and stimulated overnight with 10 µg/ml LPS and 1 µg/ml anti-CD40 Ab. The DC populations displayed were gated on CD11chigh cells. C, The fusion of YFP to CD80 did not alter the ability of CD80 to costimulate T cell proliferative response. CHO transfectants of CD80FL-CHO (•) and CD80FL-YFP-CHO ({square}). At 48 h, [3H]thymidine was added for 16 h before harvesting. Error bars represent SD from triplicate data sets. Data are representative of two independent experiments. D, Fluorescence recovery after photobleaching at 24°C of CD80 vs CD80FL-YFP membrane mobility. CD80FL (•) and CD80FL-YFP ({square}). The regions of interest for bleaching had a radius (r) of 2.5 µm. E, Fluorescence recovery after photobleaching at 37°C of CD80FL-YFP ({square}), where r = 2.63 µm vs CD80TL-YFP ({blacktriangleup}), where r = 2.33 µm membrane mobility with the number of cells (n) shown. Data are representative of two independent experiments.

 
A comparison of CD80FL and CD80FL-YFP showed that CD80FL-YFP costimulated 5C.C7 T cell proliferation in a manner indistinguishable from wild-type CD80FL (Fig. 1C). To directly test the effect of chimeric fusion of YFP to CD80 on lateral mobility of CD80 on CHO cells, photobleaching recovery experiments were performed. The recovery curves were similar for CD80FL and CD80FL-YFP (Fig. 1D). Therefore, the attachment of YFP did not alter the function or mobility of full-length CD80.

We also characterized whether the removal of CD80 cytoplasmic tail, CD80TL-YFP, affected CD80 lateral mobility by fluorescence recovery after photobleaching. The removal of CD80 cytoplasmic tail did not alter the apparent half-time or fractional mobility (Fig. 1E). CD80FL-YFP and CD80TL-YFP had similar calculated diffusion coefficients, D = 0.027 ± 0.0019 µm2/s and D = 0.021 ± 0.0013 µm2/s, respectively. Therefore, the cytoplasmic tail of CD80 does not appear to regulate its lateral mobility.

We compared the function of CD80FL-YFP with CD80TL-YFP using proliferation and cytokine production assays. CD80FL-YFP increased the amplitude of naive 5C.C7 T cell proliferation at every peptide dose tested when compared with CHO cells lacking any CD80 (Fig. 2A). CD80TL-YFP was intermediate between CD80FL-YFP and no CD80 across the entire dose range (Fig. 2A). This hierarchy was observed in five independent experiments. CFSE dye dilution also showed a decreased number of naive 5C.C7 T cells with three divisions in response to CD80TL-YFP CHO compared with CD80FL-YFP CHO cells when each were pulsed with 100 nM MCC 88-103 (Fig. 2B). More naive 5C.C7 T cells produced IL-2 when stimulated by I-Ek CHO cells expressing CD80FL-YFP than identically Ag-pulsed cells expressing CD80TL-YFP at 8 h (Fig. 2C). CD80FL-YFP or CD80TL-YFP expressing I-Ek CHO stimulated similar levels of IL-2 by naive T cells at 48 h (data not shown). Thus, the cytoplasmic domain of CD80 regulates proliferation and early IL-2 production by naive T cells.



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  		FIGURE 2. CD80 cytoplasmic domain deletion decreased T cell response to Ag. A, CD80 cytoplasmic tail deletion affected naive T cell proliferation. A total of 1.5 x 105 purified 5C.C7 TCR transgenic T cells in Rag–/– background were added to 2 x 104 CHO transfectants. At 48 h, [3H]thymidine was added for 16 h before harvesting. I-Ek-CHO ({circ}), CD80FL-YFP-CHO ({square}), and CD80TL-YFP-CHO ({blacktriangleup}). Data are representative of five independent experiments. Error bars represent SD from triplicate data sets. B, CFSE-labeled 5C.C7 T cells were added to CHO transfectants pulsed with 100 nM MCC peptide (gray line) or no peptide (black line) and analyzed by flow cytometry at 72 h. Data are representative of two independent experiments. C, IL-2 and IFN-{gamma} production by T cells stimulated with CHO transfectants and antigenic peptide at indicated dose (right). T cells were stained for cytokine production 8 h after CHO-T cell interactions. Data are representative of two independent experiments.

 
Recruitment of CD80 to the IS of naive T cells

The differences in activity of CD80FL-YFP and CD80TL-YFP in proliferation and IL-2 production by naive T cells led us to examine their distribution in the IS. We used live-cell confocal microscopy at 37°C to examine the location of CD80, TCR, and LFA-1 within stable interfaces formed between naive 5C.C7 T cells and Ag-pulsed I-Ek-CHO cells, CD80FL-YFP CHO cells, or CD80TL-YFP CHO cells. We imaged stable IS defined as T cell-CHO cell interfaces present at 30–60 min after introduction of T cells where the T cell remained in place during the time required to obtain a complete confocal data set (~2 min) and had LFA-1 accumulation. TCR-{beta} subunit was visualized with monovalent Alexa Fluor 568-conjugated H57 Fab and LFA-1 with monovalent Cy5-conjugated H155 Fab, whereas CD80 was visualized using YFP fluorescence. We have previously shown that H57 and H155 Fab do not impair CD4+ T cell functional responses (38). Optical sections acquired at 0.48-µm intervals were reconstructed into three-dimensional objects and rotated to obtain an en face view of the interface between T cell and CHO-APC. IS were further subdivided based on TCR accumulation in the cSMAC and LFA-1 accumulation in the pSMAC. In the absence of CD80, two patterns of TCR accumulation were observed: "multifocal," with small TCR clusters throughout the interface (Fig. 3A), and "central," with central TCR accumulation in the cSMAC (Fig. 3B). At 30 min, 74% of IS observed displayed multifocal TCR accumulation whereas only 26% displayed central TCR accumulation (Fig. 3C). In contrast, TCR was localized in a well-defined cSMAC in 100% of stable interactions in the presence of CD80FL-YFP or CD80TL-YFP (Fig. 3C). This result is consistent with earlier studies demonstrating a role for CD80 in establishing a central TCR cluster (22, 24, 39).



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  		FIGURE 3. CD80-CD28 engagement facilitated TCR accumulation in the cSMAC. IS of I-Ek-CHO and naive 5C.C7 T cells. The stable synapses exhibited both a central (A) and multifocal (B) TCR accumulation pattern in the IS. The color overlay images showed LFA-1 in blue and TCR in red. Images are a three-dimensional reconstruction rotated in an en face view with T cell on top of the CHO cell. Scale bar indicates a distance of 2 µm. C, Quantitation of TCR localization in the absence of CD80. Counts were taken during a 30 min time frame of I-Ek-CHO and T cell interactions. The percentages of the cells observed with each pattern were the calculated as the mean of three independent experiments of a total of n = 63 cells. The error bars indicate the mean SE.

 
To address the spatial localization of CD80FL-YFP and CD80TL-YFP, three accumulation patterns were scored on >50 live-cell contacts in three independent experiments. En face views of LFA-1 and TCR fluorescence were used to identify the pSMAC and cSMAC of the stable IS. CD80 clusters segregated from the TCR cluster were scored as "segregated" and a single CD80 cluster colocalized with the TCR cluster was scored as "colocalized." No CD80 clusters in the interface were scored as none, which may reflect no CD80 redistribution or redistribution at a level that cannot be distinguished from the basal CD80 density at the plasma membrane.

We observed that CD80FL-YFP localization was segregated from the TCR in 87% (SE = 4%) of stable IS (61–120 min), where individual clusters could be close to the TCR cluster, but did not overlap (Fig. 4A and C). In contrast, only 4% (SE = 2%) of IS displayed a central cluster of CD80FL-YFP that colocalized with the TCR. The CD80 accumulation was most visible after TCR clustered in the center of the IS. The percentage of cells that have CD80FL-YFP clusters segregated from the TCR increased with time. CD80 accumulation in the interface was Ag driven because no CD80 accumulation was observed in the absence of MCC 88-103 peptide in rare stable interfaces formed over a 4-h observation period (data not shown).



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  		FIGURE 4. CD80 cytoplasmic domain deletion resulted in decreased and differently localized CD80 recruitment to the IS with naive T cells. In the presence of 100 nM MCC peptide, naive 5C.C7 T cells and CHO cell interactions resulted in CD80FL-YFP and CD80TL-YFP molecules differentially accumulated in the IS. A, CD80FL-YFP accumulation was segregated from the TCR at the interface of the IS. B, CD80TL-YFP accumulation colocalized with the TCR in a central cluster in the IS. The color overlay images show TCR in red and CD80 in green. Areas of TCR and CD80 overlap are in yellow. Images are shown as a cross-section of a three-dimensional plane rotated in an en face view with T cell on top of the CHO APC. Scale bar = 2 µm. Quantitation of the accumulation patterns of CD80FL-YFP (C) vs CD80TL-YFP (D) in the IS over a 2-h time course. No CD80 accumulation in the cSMAC ({square}), CD80 segregated from the TCR, segregation ({blacksquare}), and CD80 colocalized with the TCR in a central cluster ({cjs2108}) are shown. Only cells with an LFA-1 ring (pSMAC) were scored. The percentage of cells in each accumulation pattern represents the mean percentage of three independent experiments. The hypothesis that there was no association between cell type and pattern of accumulation was rejected with a value *, p < 0.0001 from the three experiments combined or each experiment individually. They each suggested significant association between cell types and patterns. The conditional independence between the segregated and colocalized and two cell types was also tested and rejected with a value for p < 0.0001 (whether or not the value was corrected for multiple testing).

 
In contrast, CD80TL-YFP tends to be coclustered with the TCR in the cSMAC (Fig. 4, B and D). At 30 min, no IS had yet accumulated CD80FL-YFP in a central cluster, but 30% (SE = 5%) of IS already had CD80TL-YFP accumulated in a central cluster. This trend continued for 2 h. IS with CD80TL-YFP clusters segregated from the TCR were 3- to 4-fold less frequent than CD80FL-YFP clusters segregated from the TCR at all times. Thus, CD80TL-YFP appeared to cluster in a different pattern than CD80FL-YFP in the IS.

A striking difference between CD80FL-YFP and CD80TL-YFP was in the degree of detectable clusters. By 120 min, 90% of IS has CD80FL-YFP clustered, whereas only 50% of IS had CD80TL-YFP clustered (Fig. 4, C and D). This apparent decrease in CD80TL-YFP accumulation in the IS and differential accumulation patterns compared with CD80FL-YFP are correlated with the lower T cell proliferation and IL-2 production in response to CD80TL-YFP compared with CD80FL-YFP.

Statistical testing on the differences in the CD80 patterns of naive T cell IS was performed to determine whether these differences were significant. We considered the hypothesis that there was no association between CHO cell type and pattern of accumulation; this hypothesis was rejected with a value of p < 0.0001 from the three experiments combined (Fig. 4, C vs D; p < 0.0001). We also performed this test for each of the three experiments individually, and they each yielded significant association between cell types and patterns. The distinction between segregated and colocalized patterns between CD80FL-YFP and CD80TL-YFP, independent of the "none" category, was also of interest to us. Thus the null hypothesis of conditional independence between the two patterns and two cell types was also tested. The null hypothesis was rejected with an uncorrected value of p < 0.0001 (corrected value was also p < 0.0001, in which the correction was done for the multiple testing), so the patterns of segregated and colocalized differed significantly between CD80FL-YFP and CD80TL-YFP at all times.

Segregation of CD80 and TCR in the IS of previously activated primary T cell

We next examined patterns of TCR and CD80 accumulation during restimulation of 5C.C7 T cells after primary stimulation with CHO cells expressing I-Ek and CD80FL-YFP. In this model, we also observed different patterns of accumulation between CD80FL-YFP (Fig. 5,A and C) and CD80TL-YFP (Fig. 5, B and C). The more prominent CD80 clusters with primed T cells were likely due to the higher levels of CD28 and CTLA-4 expression on activated T cells (29, 40, 41). In addition, the accumulation pattern of CD80FL-YFP appeared to form a partial ring around the central TCR accumulation.



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  		FIGURE 5. CD80 cytoplasmic domain regulated CD80 segregation from the TCR in previously activated primary CD4 T cell. IS of CHO pulsed with 100 nM MCC peptide and previously activated primary 5C.C7 T cells was determined. A, CD80FL-YFP. B, CD80TL-YFP. Images were acquired at 37°C. The color overlay images showed TCR in red and CD80 in green. Areas of TCR and CD80 overlap are in yellow. Images are a cross-section of a three-dimensional plane rotated in an en face view with the T cell on top of the CHO APC. Scale bar = 2 µm. C, Quantification of the accumulation patterns of CD80FL-YFP (FL) vs CD80TL-YFP (TL). CD80 segregated from the TCR ({blacksquare}) and CD80 colocalized with the TCR in a central cluster ({cjs2108}) are shown. The percentage of cells in each accumulation pattern represents the mean percentage of two independent experiments. *, Statistical analysis shows p < 0.0001.

 
In contrast to naive T cells, previously activated primary T cells have similar pattern of distribution for the different time points studied, suggesting that the accumulation of CD80 takes place faster with previously activated T cells. We summarized the 0–30 min time point. CD80FL-YFP clusters segregated from the TCR in 76% (SE = 10%) of IS and colocalized with the TCR in 24% (SE = 10%) of IS. In contrast, CD80TL-YFP clusters segregated from the central TCR in 28% (SE = 7%) and colocalized with the TCR in 72% (SE = 7%) of IS (Fig. 5C). For this data set, the test for independence between accumulation patterns and the cell types also suggested that there was a significant increased association of segregated and colocalized patterns with CD80FL-YFP and CD80TL-YFP CHO cells, respectively, with a value of p < 0.0001 (Fig. 5C). These data suggested that the cytoplasmic tail of CD80 played a role in orchestrating the spatial localization of CD80 on the APC side of the IS.

CD28 and CTLA-4 spatial accumulation in the IS are regulated by CD80

To delineate the relationship of CD80 accumulation pattern to potential receptor interactions, we examined the accumulation of CD28 and CTLA-4 in the naive T cell IS by fixed-cell immunofluorescence staining and three-dimensional confocal microscopy. At 30 min, CD28 clusters colocalized with CD80FL-YFP (Fig. 6A) and CD80TL-YFP (Fig. 6B) based on examination of red/green pseudocolored images that showed extensive yellow areas on merging. At the 30 min time point, CTLA-4 staining was not detected with naive 5C.C7 T cells (data not shown).



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  		FIGURE 6. CD80 cytoplasmic domain regulated both CD28 and CTLA-4 segregation from the TCR. Both naive and previously activated primary 5C.C7 T cells were stimulated CD80FL-YFP or CD80TL-YFP CHO pulsed with 100 nM MCC 88-103 peptide. Cells were fixed after T cell-CHO cell had been interacting for 30 min at 37°C. At this time point, CTLA-4 was not detected on naive T cells. The color overlay images show CTLA-4 in red, CD28 in red, and CD80 in green. The white dashes represent the outer boundary of the T cell. Images are a cross-section of a three-dimensional plane rotated to en face view. Scale bar = 2 µm. The localization of CD80FL (A) and CD80TL (B) modulated the localization of CD28 with respect to CD80 in the IS of naive T cells. The localization of CD80FL-YFP (C) and CD80TL-YFP (D) modulated the localization of CD28 and CTLA-4 with respect to CD80 in the IS of previously activated primary T cells. 5C.C7 T cells (d6-d8) were used.

 
Localization of CD28, CTLA-4, CD80FL-YFP, and CD80TL-YFP was examined in previously activated T cell. In fixed-cell imaging, CD80FL-YFP, CD28, and CTLA-4 accumulated in a ring-like pattern in previously activated T cell IS. However, CD28 and CTLA-4 accumulation patterns were only partly colocalized in the IS. CTLA-4 was consistently localized toward the center of the CD80FL-YFP ring, whereas CD28 was localized toward the periphery of this ring. CD80TL-YFP localized in a single central cluster in which components of CD28 and CTLA-4 in the IS were highly colocalized. Overall, the accumulation of CD28 and CTLA-4 followed the accumulation pattern of CD80 whether CD80 is in a ring-like structure or in a central cluster (Fig. 6, C and D). When the cells were fixed and permeabilized, previously activated T cells contained CTLA-4 in intracellular membranes that was polarized to the interface in response to IS formation in addition to CTLA-4 that was in the IS itself. These two locations of CTLA-4 were distinguished by generating three-dimensional volumes and rotating between en face and side views.4

PKC{theta} colocalized with CD28 and CD80 in the cSMAC

We next determined the location of PKC{theta}. PKC{theta} was detected by immunofluorescence staining of activated 5C.C7 T cells forming in the IS with MCC-pulsed I-Ek CHO cells expressing CD80FL-YFP or CD80TL-YFP. PKC{theta} was colocalized with CD28 and CD80FL-YFP (Fig. 7A) or CD80TL-YFP (Fig. 7B) at 30 min. As shown in Fig. 7C, >80% of PKC{theta} colocalized with CD80 and CD28, and <10% of the cells analyzed did not have detectable PKC{theta} clusters. Thus, by definition, the ring of CD80FL-YFP, CD28, and CTLA-4 is part of the cSMAC even though it is segregated from TCR.



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  		FIGURE 7. PKC{theta} colocalized with CD28 in the IS. Recruitment of PKC{theta} to the cSMAC appears to be spatially regulated by CD80. Cells were fixed after T cell-CHO cell had been interacting for 30 min at 37°C. The color overlay images show CD80 in green, CD28 in red, and PKC{theta} in red. A, PKC{theta} localization in the presence of CD80FL-YFP. B, PKC{theta} localization in the presence of CD80TL-YFP. C, quantitation of PKC{theta} localization in the cSMAC with respect to CD80FL-YFP vs CD80TL-YFP. Segregated and colocalized categories represent the spatial localization of CD80. Colocalization of CD80, CD28, and PKC{theta} ({square}) and absence of PKC{theta} ({blacksquare}) are represented. Cells that did not have CD28 and PKC{theta} colocalized ({cjs2108}) are also shown. Data represent two independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
An earlier study on cytoplasmic domain-deficient human CD80 demonstrated that CHO cells expressing tailless CD80 were ineffective in providing costimulation for anti-CD3-induced proliferation of peripheral blood T cells, whereas CHO cells expressing full-length CD80 provided effective costimulation (4). Using CHO cells to present agonist MHC-peptide complexes to Ag-specific T cells, we found quantitative differences between CHO cells expressing CD80 with and without the cytoplasmic domain. Although we did not see a null function for tailless CD80, the differences between the results of Doty and Clark (4) and our study likely arise from differences between the anti-CD3 and agonist MHC-peptide driven responses or CD80 expression level. Notably, Doty and Clark (4) observed that high-level expression of tailless CD80 gained costimulatory activity in their system. Because we carefully chose a CD80 expression level similar to in vitro activated DC, we feel our results are physiologically relevant.

Costimulatory and TCR-based signaling is required for full T cell activation, but it has not been proven that TCR and CD28 must be colocalized to function. Although CD28 and CD80 colocalize with TCR in the cSMAC when planar bilayers or B cell lines are used as APC, it has also been shown that T cells can integrate Ag receptor and costimulatory signals when the agonist MHC-peptide complex and CD80 are presented on different cells (42, 43). Consistent with both of these findings, we observed that the intact cytoplasmic domain of CD80 promoted segregation of itself, CD28, and CTLA-4 from the TCR in the cSMAC, demonstrating that precise colocalization of costimulatory and Ag signals are unnecessary for full function. Further, because this segregation depends on the cytoplasmic tail of CD80, tailless CD80 permitted colocalization of CD28 and TCR, which correlated with diminished activation, suggesting that the normal segregation of costimulatory and TCR signals is actually critical for their proper function. Notably, we demonstrate that CD28, not TCR, colocalizes with the intracellular signaling molecule PKC{theta}.

Previous studies have attempted to determine whether APCs play an active role in regulating the formation of the IS; however, results have been mixed and the answer may depend on cell type. B cell lines can be treated with cytochalasin D and other cytoskeletal inhibitors without altering efficiency of Ag presentation (44). In contrast, treatment of DC with cytochalasin D reduces the efficiency of Ag presentation (8), and Rac1/2–/– DCs with intrinsic cytoskeletal defect were unable to activate T cells (10). Although CHO cells have been used widely as model APC, there are no studies that have addressed the role of the CHO cell cytoskeleton in Ag presentation. Using CHO cells as APC, we concur with Egen and Allison (12) that CD28 and CTLA-4 are recruited to the IS, but we only observed CD28 and CTLA-4 predominantly in the central cluster colocalizing with the TCR when the CHO cells expressed tailless CD80, whereas full-length CD80 recruited CD28 and CTLA-4 to a domain of the cSMAC separate from that occupied by the TCR. This segregation may be accomplished by strong CHO cytoskeleton interactions with the CD80 cytoplasmic domain, but not the MHC class II cytoplasmic domains. This situation would allow TCR-MHC complexes to translocate freely to the center under control of the T cells, but not the translocation of CD80 and its receptors, which would be under the control of the CHO cell. These mechanisms may be shared by cells that spread or extend cytoskeletal projections, like adherent CHO and DC, but lacking in B cells.

How does segregating CD80 and its receptors from TCR increase T cell activation? The cSMAC has been associated with down-regulation of TCR signaling (45, 46), and removal of CD28 signaling from the vicinity of the TCR in the cSMAC may prolong CD28 signaling. Alternatively, if some TCR signaling persists in the cSMAC, physically segregating CTLA-4 and associated phosphatases from TCR may augment this residual TCR signaling (32, 47).

Another possibility for the reduced costimulation by tailless CD80 is the loss of reverse signaling by its cytoplasmic tail upon engagement to CD28/CTLA-4 through cytokine production. Grohmann et al. (7) and Orabona et al. (6) showed that CTLA-4 and CD28 are able to reverse signal through B7 to DC. This reverse signaling resulted in cytokine production by the DC that can regulate the activation status of a T cell. CTLA-4 engagement with B7 resulted in IDO and IFN-{gamma} production, which is inhibitory for T cell activation (5, 7), and CD28 engagement with B7 resulted in the production of IL-2 and the activation of suppressor of cytokine signaling 3 by DC (48). There is no evidence that CHO cells can make relevant cytokine, but this possibility cannot be excluded.

Although CHO cells are engineered rather than physiological APCs, it is likely that the CD80 cytoplasmic domain-dependent mechanism revealed in our experiments is relevant to APC in which the cytoskeleton or reverse signaling has been shown to play a role in Ag presentation, including DC. The ability to regulate CD80 localization could be controlled developmentally by altered expression of the function of cytoskeletal adapters that link CD80 to filament systems or by alteration in the CD80 cytoplamic domain itself. It is possible that professional APCs may be able to selectively alter this CD80 tail-dependent segregation or signaling process depending on parameters such as the maturation state and the local inflammatory milieu, thereby fine-tuning T cell activation in a subtle but potentially important way. Interference with the contribution of the CD80 cytoplasmic tail to activation of T cells may be useful in therapeutic suppression of costimulation.


   Acknowledgments
 
We thank Rajat Varma and Guy Shakhar for advice on image analysis. We thank Tom Cameron for critical reading of the manuscript. We acknowledge the NYU Cancer Institute Biostatistics Shared Core facility. We thank M. Davis and V. Kuchroo for valuable reagents.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grants AI43542 and GM64900 (to M.L.D.). Irene Diamond Foundation supports M.L.D. This work is also supported by Grant 5456-04 from the Leukemia and Lymphoma Society Fellowship (to S.-Y.T.), and by the National Cancer Institute Cancer Center Project No. 5 P30 CA016087-24 (to M.L.). Back

2 Address correspondence and reprint requests to Dr. Michael L. Dustin, New York University School of Medicine, Skirball Institute, 540 First Avenue, SK2-4, New York, NY 10016. E-mail address: dustin{at}saturn.med.nyu.edu Back

3 Abbreviations used in this paper: DC, dendritic cell; IS, immunological synapse; YFP, yellow fluorescent protein; CHO, Chinese hamster ovary; PKC, protein kinase C; cSMAC, central supermolecular activation cluster; pSMAC, peripheral supermolecular activation cluster; MCC, moth cytochrome c. Back

4 The online version of this article contains supplemental material. Back

Received for publication July 1, 2005. Accepted for publication September 27, 2005.


   References
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 Introduction
 Materials and Methods
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 Discussion
 Disclosures
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