Quantification of CD4+ T Cell Alloreactivity and Its Control by Regulatory T Cells Using Time‐Lapse Microscopy and Immune Synapse Detection

Assays designed to select transplant recipients for immunosuppression withdrawal have met with limited success, perhaps because they measure events downstream of T cell–alloantigen interactions. Using in vitro time‐lapse microscopy in a mouse transplant model, we investigated whether transplant outcome would result in changes in the proportion of CD4+ T cells forming prolonged interactions with donor dendritic cells. By blocking CD4–MHC class II and CD28–B7 interactions, we defined immunologically relevant interactions as those ≥500 s. Using this threshold, T cell–dendritic cell (T‐DC) interactions were examined in rejection, tolerance and T cell control mediated by regulatory T cells. The frequency of T‐DC contacts ≥500 s increased with T cells from mice during acute rejection and decreased with T cells from mice rendered unresponsive to alloantigen. Regulatory T cells reduced prolonged T‐DC contacts. Importantly, this effect was replicated with human polyclonally expanded naturally occurring regulatory T cells, which we have previously shown can control rejection of human tissues in humanized mouse models. Finally, in a proof‐of‐concept translational context, we were able to visualize differential allogeneic immune synapse formation in polyclonal CD4+ T cells using high‐throughput imaging flow cytometry.

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Assays designed to select transplant recipients for immunosuppression withdrawal have met with limited success, perhaps because they measure events downstream of T cell-alloantigen interactions. Using in vitro time-lapse microscopy in a mouse transplant model, we investigated whether transplant outcome would result in changes in the proportion of CD4 + T cells forming prolonged interactions with donor dendritic cells. By blocking CD4-MHC class II and CD28-B7 interactions, we defined immunologically relevant interactions as those ≥500 s. Using this threshold, T cell-dendritic cell (T-DC) interactions were examined in rejection, tolerance and T cell control mediated by regulatory T cells. The frequency of T-DC contacts ≥500 s increased with T cells from mice during acute rejection and decreased with T cells from mice rendered unresponsive to alloantigen. Regulatory T cells reduced prolonged T-DC contacts. Importantly, this effect was replicated with human polyclonally expanded naturally occurring regulatory T cells, which we have previously shown can control rejection of human tissues in humanized mouse models. Finally, in a proof-of-concept translational context, we were able to visualize differential allogeneic immune synapse formation in polyclonal CD4 + T cells using high-throughput imaging flow cytometry.

Introduction
Marked patient-to-patient differences exist in the immunosuppression required to prevent allograft rejection (1,2). Many assays have been developed in an attempt to predict rejection or to identify operationally tolerant patients (3). The mixed leukocyte reaction, which measures recipient T cell proliferation in response to donor antigens, is poorly predictive (4,5), although deep sequencing of recipient TCRs in pretransplant mixed leukocyte reactions was recently found to be predictive of tolerance in a small group of patients (6). Limiting dilution assays, cytokine enzyme-linked immunospot assays and the transvivo assay are either impractical or measure a narrow range of phenomena that may inadequately reflect donor reactivity (7)(8)(9)(10)(11)(12). Transcriptomics methods have shown promise in several cohorts (13)(14)(15)(16)(17)(18), but important differences across studies (19) raise questions about the practicality of this approach. Better tools to assess donor reactivity in individual patients are urgently needed to allow informed decisions about immunosuppression minimization.
In many transplant models, sustained allograft survival depends on regulatory T cells (Tregs) (20)(21)(22)(23), and immunosuppression weaning in some patients might involve such populations. Tregs control autoimmunity by inhibiting stable immune synapse formation between T cells and dendritic cells (DCs) (24), partly because autoreactive Tregs make prolonged contacts with DCs (25), depriving effector T cells (Teffs) of the sustained contacts required for activation (24,26). Whether these phe-nomena characterize alloreactive T cell and Treg behavior has not been studied systematically but is important because Treg cellular therapy is currently the subject of a phase I/IIa clinical trial in renal transplant recipients (27).
Using in vitro time-lapse microscopy to examine mouse T cell-DC (T-DC) interactions, we tested the hypothesis that the frequency of prolonged contacts between recipient CD4 + T cells and donor DCs in vitro reflects allograft rejection and tolerance. Using antibodies or costimulatory blockade, we demonstrated that a threshold of 500 s distinguishes brief sampling interactions from those that drive a productive T cell response. Allograft rejection and tolerance were associated with predictable changes in the proportion of interaction events ≥500 s. Furthermore, human CD4 + T cells exhibited similar behavior in response to allogeneic DCs. Finally, we showed that imaging flow cytometry can be used to examine CD4 + T cell alloreactivity, suggesting that the state of T cell responsiveness to donor antigens could be evaluated in a high-throughput manner. Our observations imply that measuring the frequency of prolonged T-DC contact or immune synapse formation in vitro might be a useful measure of donor reactivity in transplant recipients.

Tolerance induction and transplantation
CBA Foxp3-GFP mice were untreated or received YTS177 200 lg intravenously on days À28 and À27 and/or 200 lL B6 heparinized whole blood as a donor-specific transfusion (DST) on day À27 and sometimes also on day À1. Some mice received PC61 1 g intraperitoneally on day À14. At day 0, mice received a heterotopic B6 cardiac allograft, as described (28), or were euthanized to obtain CD4 + T cells. Cessation of cardiac pulsation signified rejection (approximately day 8 in untreated animals). For skin grafting, B6 tail skin grafts were applied to the left flank of CBA or CBA RAG À/À mice. Dressings were removed at day 6, and grafts were monitored two or three times weekly for rejection.

Human PBMCs and expanded Tregs
Buffy coats (UK National Blood Service, Watford, UK) were separated on Ficoll-Paque (GE Healthcare, Little Chalfont, UK). PBMCs were cryopreserved prior to recovery and sorting. Separately, autologous CD4 + CD25 hi CD127 lo Tregs were sorted and expanded prior to cryopreservation, as described (29). Recovered Tregs were labeled with 1 lM CMPTX (Life Technologies, Paisley UK). All blood donors provided informed consent (UK National Research Ethics Service, Oxford, UK).

Time-lapse microscopy and image analysis
Time-lapse microscopy was performed as described by Sarris et al (32) with minor modifications. T cells (total 2 9 10 5 , in some cases 1 9 10 5 conventional T cells and 1 9 10 5 Tregs) were added to 1 9 10 5 (5 9 10 4 for human MoDCs) allogeneic DCs in 200 lL indicator-free RPMI containing 20 mM HEPES (Sigma-Aldrich, St. Louis, MO) in eight-well chambers (Lab-Tek II; Nalge Nunc International, Roskilde, Denmark). Imaging was performed on a Deltavision Elite Imaging System (Applied Precision; Imaging Solutions, Preston, UK). Polarized light, green fluorescent protein (GFP) and cyan fluorescent protein (CFP) images (920) were acquired sequentially every 20 s for 40 min (120 frames). The number of contiguous frames in which individual T cells and DCs were in contact was enumerated and expressed as contact duration (in seconds). For each recording, all T cells contacting 16 individual DCs were analyzed, typically providing ≥100 T-DC interaction events. T cells that appeared to be caught passively in DC clusters were excluded from the analysis.

Statistical analysis
Statistical analysis was performed using GraphPad Prism 5 (GraphPad Software, San Diego, CA). Proportions of T-DC interaction times ≥500 s were compared by Fisher exact test or v 2 test. Graft survival data were analyzed using the log-rank test. Means of two groups were compared using paired or unpaired t-tests according to the experimental design.

Results
A threshold of 500 s discriminates immunologically relevant T-DC interactions in a polyclonal T cell population To measure allogeneic T-DC contact duration, we sorted CD4 + T cells from CBA Foxp3-GFP mice, combined them with B6 CFP BMDCs and recorded serial 920 images every 20 s over 40 min. This allowed us to distinguish Foxp3 À T cells (colorless), Foxp3 + Tregs (green) and DCs (blue) (Figure 1A and Video S1). Although most effector T-DC interactions were short-lived, contacts lasting the entire length of the recording were also seen ( Figures 1B-D).
T cell activation requires prolonged TCR signaling (33,34), triggering a "stop" signal that arrests T cells on antigenpresenting cells (APCs) (35). We reasoned that blockade of TCR-MHC or costimulatory interactions could be used to define a minimum contact duration required for T cell activation. Consequently, we assessed the effect of a CD4 antibody (YTS177) that blocks CD4 + T cell activation by alloantigen (36) and CTLA4-Ig (abatacept), which blocks CD28 costimulation (37), on T-DC contact durations. Both reagents markedly reduced long T-DC interactions ( Figure 1D) but had no discernable effect on interactions <500 s (dotted line in Figure 1D). Based on this observation and similar findings with MHC class II À/ À DCs (25), we defined a threshold of 500 s to discriminate immunologically irrelevant, short-lived contacts from prolonged interactions likely to result in T cell activation.
We reasoned that T-DC contact times would differ substantially if T cells were given the opportunity to interact with either allogeneic or syngeneic DCs. To assess this possibility, we combined CBA T cells with a 1:1 mixture of B6 (CFP + ) and CBA (CFP À ) DCs. As expected, there were proportionately more prolonged allogeneic T-DC contacts than syngeneic ones ( Figure 1E), indicating that this method is capable of distinguishing alloreactivity from self-reactivity.
Graft rejection depends on alloreactive T cell expansion. We predicted that CD4 + T cells from animals that had rejected an allograft would exhibit an increased proportion of prolonged T-DC interactions. Untreated CBA Fox-p3-GFP mice were untransplanted or given B6 cardiac allografts. Rejection was confirmed by palpation on day 8. To allow acute inflammation to subside, CD4 + T-DC interaction times were measured on day 14 (timeline in Figure 1F). Rejecting animals exhibited a twofold increase in the proportion of long interactions, from 5.6% to 11.1% ( Figure 1G and Video S2). Consequently, an increased proportion of prolonged T-DC contacts detected in vitro correlates with allograft rejection in vivo.

An increase in long T-DC interactions precedes the onset of visible allograft rejection
Allograft biopsies are invasive, and because rejection is a diffuse process, multiple biopsies are often required (38). Allograft rejection is often assessed using clinical indices of organ function (38), but these usually reflect rather than predict organ damage. Detection of increased alloreactivity before organ dysfunction develops could provide an enhanced opportunity for therapeutic intervention.
To test the hypothesis that an increase in long T-DC interactions might predict rejection, na€ ıve CBA mice were given B6 skin grafts. Median graft survival time was 11 days (Figures 2A and B). On day 7, before visible signs of rejection ( Figure 2B, left panel), T-DC interaction times were measured using CD4 + T cells from the draining and nondraining axillary lymph nodes of three mice. In each animal, there was a two-to threefold (mean 2.6fold) increase in prolonged interactions made by draining versus nondraining lymph node CD4 + T cells ( Figure 2C). Moreover, the proportion of prolonged contacts made by nondraining lymph node CD4 + T cells was similar to that of CD4 + T cells from a nontransplanted mouse ( Figure 2C, first column). In this model, an increase in prolonged T-DC interactions in vitro preceded allograft rejection.
Tolerance induction results in a decrease in the proportion of stable T-DC contacts Identification of operationally tolerant patients remains challenging; therefore, we asked whether changes in the proportion of prolonged T-DC interactions might reflect donor unresponsiveness. CBA mice were rendered unresponsive to B6 cardiac allografts with a nondepleting anti-CD4 antibody (YTS177) and DST ( Figure 3A). Such animals exhibit long-term allograft survival without further therapy (39). Controls received a B6 cardiac allograft with no pretreatment. Seven days later, recipient splenic CD4 + T-DC interaction times were measured. Figures 3(B) and (C) show that whereas CD4 + T cells from nonpretreated mice exhibited many prolonged T-DC interactions (mean 26.8%), cells from YTS177/DST-treated mice exhibited a proportion of prolonged T-DC interactions comparable to those of na€ ıve mice (mean 8.3%; v 2 test, p < 0.0001, and t-test, p = 0.002). Consequently, demonstrable tolerance to alloantigens is associated with a reduction in prolonged T-DC contacts.
Decreased T-DC interactions in animals with induced tolerance are dependent on Tregs Treatment of mice with either YTS177 alone or DST alone fails to result in long-term cardiac allograft survival  (39). We predicted that CD4 + T cells from mice given only one of these treatments would not exhibit the reduction in prolonged interactions seen with cells from mice given both. To provide an alloantigen challenge, mice received a second DST on day À1 ( Figure 4A). T-DC interactions were measured on day 0. YTS177 alone or DST alone had a modest effect on the proportion of prolonged T-DC interactions compared with untreated mice. In contrast, tolerance induction reduced the frequency of prolonged interactions by >50% ( Figure 4B).
The YTS177/DST protocol generates alloreactive Tregs that arise from the pretreatment alone and are responsi-ble for long-term allograft survival in both adoptive transfer and primary transplant recipients (20,22,40). The effect of YTS177/DST on prolonged T-DC contacts (Figure 4B) is consistent with regulation by alloreactive Tregs but also could be explained by direct effects on Teffs. To distinguish these possibilities, Foxp3-GFP mice were given YTS177/DST or left untreated. On day 0, CD4 + GFP À T cells were transferred at two doses to CBA RAG À/À mice, which then received B6 skin grafts the following day. Figure 4C shows that untreated and 177/ DST GFP À T cells rejected grafts with similar kinetics, arguing against an independent effect of tolerance induction on Teffs. That the reduction in stable contacts shown in Figure 4B is due to Tregs is confirmed by the observation that Treg depletion prior to microscopy completely eliminated the effect of pretreatment on T-DC interactions ( Figure 4D). Consequently, alloantigeninduced Tregs that regulate allograft rejection in vivo also control allogeneic T-DC interactions in vitro.
Treg depletion at day À14 during the YTS177/DST protocol abrogates tolerance (40); therefore, we asked whether this would alter the frequency of prolonged T-DC interactions on day 0 in vitro. CBA Foxp3-GFP mice received YTS177/DST and were either left untreated or were given anti-CD25 mAb PC61 on day À14. Importantly, by day À14, alloantigen-activated Teffs have downregulated CD25 and so are not depleted by PC6l (40). Treg depletion was %50% at day 0 ( Figure 4E). On day 0, spleen and lymph node total CD4 + T cells were sorted, and the interaction times of non-Treg GFP À T cells with B6 DCs were measured. In vivo depletion of Tregs resulted in a 2.2-fold increase in the proportion of prolonged T-DC contacts ( Figure 4F). Taken together, these data show that in vitro timelapse microscopy can model tolerance induction in vivo.
We then examined the ability of Tregs from na€ ıve and YTS177/DST-treated animals to control allogeneic T-DC contacts in vitro. T-DC interaction times of CD4 + GFP À T cells from na€ ıve mice were determined in the presence or absence of Tregs from either na€ ıve or YTS177/ DST-treated mice (1:1 ratio of Tregs:Teffs). The rate of prolonged contact formation for CD4 + GFP À T cells (10.0%) ( Figure 5A, first column) was higher than that seen in Figure 1D, consistent with the fact that Tregs were present in the latter experiment but not in the former. Both Treg populations reduced prolonged T-DC interactions ( Figure 5A, second and third columns). Interactions between Tregs themselves and DCs were not significantly different between the two populations ( Figure 5B).
Total T cell numbers were kept constant in these experiments (1 9 10 5 CD4 + GFP À and 1 9 10 5 Tregs vs. 2 9 10 5 Teffs) to avoid crowding. Conceivably, this could have reduced the opportunity for Teffs to make prolonged contacts with DCs owing to their decreased frequency in the well; however, we observed only a modest (0-30%) reduction in the number of T-DC contacts made by GFP À T cells in the presence of Tregs ( Figure 5C), despite a threefold reduction in the proportion of prolonged contacts ( Figure 5A). Despite their decreased density when Tregs were added, GFP À T cells had a similar opportunity to form prolonged DC contacts, confirming a Treg-dependent inhibition of prolonged contact formation.
In autoimmune models, Tregs can displace conventional T cells from DCs (41). Within cultures containing Tregs, we compared T-DC interaction times between DCs visited and not visited by Tregs. We observed a nonsignificant decrease in the lengths of prolonged (≥500 s) interactions with DCs visited and not visited by Tregs (median 1330 vs. 884 s, respectively), but there was no difference in the proportion of interactions ≥500 s (both %3%) (data not shown).
Taken together, these data are consistent with previous observations (24,26,41) and the results in Figure 4. They also concur with our previous work showing that naturally occurring Tregs can be as effective as YTS177/DSTdriven Tregs at preventing skin graft rejection, with potency differences emerging only at high Teff:Treg ratios (40).

Human T cells exhibit allogeneic T-DC interactions that can be controlled by Tregs
We next sought to determine whether alloreactivity in polyclonal human CD4 + T cells could be quantified in a similar manner. Interactions of human CD4 + T cells from three healthy donors with allogeneic MoDCs were recorded using time-lapse microscopy. Similar to our findings with mouse cells, the addition of a CD4 antibody, TRX1 (42), that prevents human T cell activation ( Figure 6A, second and fourth columns) or abatacept ( Figure 6A, sixth column) indicated that a threshold of 500 s allowed discrimination between productive and nonproductive contacts.
Our laboratory has reported a method to expand highly potent human CD4 + CD25 + CD127 lo Tregs that can be cryopreserved and recovered without losing function (29,43).
Freshly thawed expanded human CD4 + CD25 + CD127 lo Tregs were labeled with the dye CMTPX. Sorted CD4 + CD25 À T cells were incubated with allogeneic MoDCs in the presence or absence of Teffautologous CMTPX-labeled Tregs ( Figure 6B), and interaction times were determined. As shown in Figure 6C, CD4 + CD25 À T cells from three individual patients had >500 s T-DC interaction frequencies of 12.2%, 13.3%, and 7.1%. Autologous expanded Tregs dramatically reduced these contacts, and in one case, they were eliminated completely (Figures 6C and D). Figure 5A, the total T cell number in the wells was kept constant to avoid crowding. Conventional T cells might thus have shown a reduction in prolonged interactions due to a diminished opportunity to interact with MoDCs; however, we observed either no such reduction or only a modest reduction in the number of T cell contacts with each DC ( Figure 6E). Consequently, the profound inhibition of prolonged contact formation in the presence of Tregs was not due to a reduced T-DC contact opportunity, confirming that alloreactive human T-DC interactions can be modeled with this method. More important, the impact of expanded Tregs on T-DC interactions in vitro reflects their ability to control human allograft rejection in functional in vivo models (29,44,45).

As in the experiments shown in
Alloreactive immune synapse formation can be detected with imaging flow cytometry Time-lapse microscopy requires lengthy manual data analysis, an approach unlikely to be clinically applicable. Imaging flow cytometry has been used to examine immune synapses formed by monoclonal T cell populations (46)(47)(48)(49) or T cells driven by superantigens (50). We hypothesized that this technique could be used to enumerate alloreactivity in a polyclonal CD4 + T cell population. CBA CD4 + T cells were incubated with B6 DCs for 4 h; fixed and stained for T cell and DC markers, intracellular actin, and nuclear DNA; and then examined on an Image-Stream IS100 imaging flow cytometer (Amnis Corp).
Cell doublets containing one DC and one T cell were identified (the full gating strategy is shown in Figure S1). Doublets making an immune synapse could be  distinguished from those making simple membrane contact based on phalloidin-FITC staining that identified cytoskeleton rearrangements at the T-DC interface (compare with Figure 7A center and lower panels). As in Figure 1, the addition of anti-CD4 antibody (YTS 177) was used to determine whether imaging flow cytometry could distinguish between productive and nonproductive T-DC contacts. YTS177 caused a %1.7-fold reduction in the frequency of synapses in the appropriate contact gate (Figures 7B and C), providing a proof-of-concept demonstration that CD4 + T cell alloreactivity can be quantified in a semiautomated manner.
Finally, to ascertain whether induction of allograft tolerance could be detected or predicted using imaging flow cytometry, CBA mice were left untreated or had tolerance induced using the 177/DST protocol before transplantation with B6 cardiac allografts. Seven days later, CD4 + splenocytes were isolated from individual mice; incubated with B6 DCs for 4 h; and then processed, stained and acquired on the IS100 imaging flow cytometer. The data were then analyzed in a blinded fashion, as described in Figures 7 and S1. Strikingly, the frequency of synapse formation was as high as 30% in nonpretreated animals, whereas YST177/DST-treated animals exhibited a frequency of ≤10% (Figures 8A and B). These data are remarkably similar to those shown in Figure 3(B) and validate the imaging flow cytometry approach as a measure of CD4 + T cell alloreactivity.

Discussion
T cells that subsequently become activated must arrest and remain in contact with APCs and form a mature immunological synapse (24,26,33,35,51,52) characterized by actin polymerization and polarization (53). In this study, we assessed whether stable interactions between CD4 + T cells and allogeneic donor DCs in vitro could be used as a surrogate parameter for allograft rejection and unresponsiveness.
In vitro time-lapse microscopy readily identified interactions between naive T cells and allogeneic DCs in both mouse and human. Anti-CD4 antibody and CTLA4-Ig, both known to block T cell activation (25,37,54), provided an unbiased threshold of 500 s for productive alloreactive T-DC interactions. This is very similar to the figure of 400 s obtained by Sarris et al (25), who used MHC class II À/À DCs to distinguish between immunologically relevant and irrelevant self-restricted T cell responses. More recently, Dilek et al examined the effect of CD28 blockade on interaction times between a human CD4 + T cell clone and allogeneic B cells (55). CD28 blockade reduced T cell and B cell contact times to <300 s compared with >600 s in its absence (55). Our data are highly comparable with independent studies and provide a justification for measuring alloreactive T-DC contact times in vitro.
Allograft rejection requires clonal expansion of alloreactive T cells driven by allogeneic APCs in graft-draining secondary lymphoid organs (56). The data shown in Figure 2(C) demonstrate that this phenomenon can be detected in vitro by video microscopy and, importantly, that the technique can distinguish between responses in the draining and nondraining lymph nodes. Furthermore, the technique can also distinguish between mice rejecting an allograft and those in which tolerance was induced with a protocol that leads to long-term graft survival ( Figure 3) (20,39).
Using two-photon imaging of pancreatic lymph nodes, Tang and colleagues showed that Tregs reactive to an islet autoantigen reduced contact times between APCs and Teffs reactive to the same autoantigen (24). Similar findings were reported by Tadokoro et al in experimental autoimmune encephalomyelitis (26). In both cases, Tregs prevented Teff "swarming" and arrest on APCs. We have shown in this study that both mouse and human Tregs can reduce the frequency of stable T cell contacts with allogeneic DCs (Figures 5 and 6). Critically, when tested in the context of in vivo responses, this effect correlates with Treg function in a well-characterized model of tolerance induction (Figures 4C-F). Consequently, a phenomenon previously shown to be relevant to Treg control of autoimmunity in vivo also occurs in an in vitro setting and reflects Treg-mediated regulation of alloreactivity. Clinical trials of Treg cellular therapy are being conducted in hematopoietic stem cell transplantation (54,57) and kidney transplantation (27), but methods for assessing their immunological impact have not been firmly established (58). Our data suggest a possible means of evaluating Treg efficacy.
CD4 + T cells from na€ ıve mice ( Figures 1D and F, 4B and D, and 5) and humans ( Figures 6A and C) exhibited a frequency of prolonged T-DC contacts of 5-15%. This is strikingly similar to the precursor frequency estimates, also 5-15%, of alloreactive T cells (59)(60)(61)(62). Significantly, Figure 8: Discrimination of tolerized and rejecting mice using imaging flow cytometry. CBA mice were given YTS177/ DST or no pretreatment followed by a B6 cardiac allograft, as in Figure 3. After 7 days, recipient splenic CD4 + T cells were cocultured with B6 DCs for 4 h and prepared for imaging flow cytometry, as described in Figure 7. (A) Representative plots of CD90 intensity in the DC object mask versus CD11b intensity in the T cell object mask are shown for a nonpretreated animal (left panel) and a YTS177/DST-treated animal (right panel). Synapses (large dots) within the membrane contact gate were identified manually by an analyst blinded to treatment assignment. Synapse frequencies in the membrane contact gate are shown above each plot. (B) Synapse frequencies in the membrane contact gate for 6 animals per group in two independent experiments are shown. Unpaired t-test, p = 0.0002. DC, dendritic cell; DST, donor-specific transfusion. Figure 3(B) shows that tolerance induction that leads to long-term cardiac allograft survival (39) results in a reduction but not an abrogation of prolonged contacts. From a practical perspective, this emphasizes that if such an approach were to be considered for clinical evaluation, it would be essential to obtain baseline data before transplant to provide the reference for longitudinal follow-up of rejection and quiescence. It should be noted that the data presented in this study were obtained using cryopreserved DCs (all results) and cryopreserved human T cells (Figure 6), so it is possible to envision conducting such assays in either deceased or living donor transplantation. Donor cells could be cryopreserved and then recovered at important junctures, such as during weaning of immunosuppression to assess antidonor T-DC interactions.
We focused on CD4 + T cell responses principally because in the B6 to CBA mouse strain combination, long-term graft survival can be achieved by manipulating only the CD4 + T cell compartment (39) and because graft survival in this model is dependent on CD4 + Tregs (20,22,63). These techniques, however, would lend themselves to an examination of alloreactive CD8 + T cell responses. Whether T cells interacting with autologous DCs via the indirect pathway could be detected using this method is unknown and was not tested in this study. The indirect pathway of alloantigen presentation is particularly important in the setting of chronic allograft dysfunction and is more relevant at later points after transplant (64,65). Far fewer T cells are indirectly rather than directly alloreactive, and it seems unlikely that they could be quantified using time-lapse microscopy. A highthroughput strategy such as imaging flow cytometry might allow enumeration of these relatively rare events.
Enumeration of T cell-APC contacts by time-lapse microscopy provides an important proof of concept but is very time consuming and could not be used clinically in its present form. Clinical translation will require semiautomated methods for detecting T-DC contacts. The data shown in Figures 7 and 8 demonstrate that imaging flow cytometry could provide a high-throughput alternative. This will require further evaluation and development, but our data support the concept that identifying a change in the frequency of prolonged T-DC contacts or immune synapses could be useful in the management of transplant patients.

Supporting Information
Additional Supporting Information may be found in the online version of this article. Figure S1: Gating strategy used to identify doublets with membrane contact by imaging flow cytometry. (A) Gates used to identify T cells in contact with DCs are shown. In-focus events (top left) were plotted according to brightfield aspect ratio versus area to identify doublets. The latter were then plotted according to CD11b and CD90 staining. Double-positive events were then further analyzed to include those containing only one DC and only one T cell (middle two plots). Finally, CD11b + CD90 + doublets were further refined by including only those events with two 7AAD spots (i.e. nuclei) (bottom left histogram). Within this gate, T cells and DCs with physical membrane contact were identified by plotting CD90 staining intensity (T cell marker) in the DC object mask against CD11b staining intensity (DC marker) in the T cell object mask (bottom right plot). Synapses were identified within this gate by manual tagging of events with prominent polarization of actin staining within the T cell. (B) Images of a doublet are shown. The DC object mask is shown overlying the CD11b eF450 image, and the T cell object mask is shown overlying the CD90 APC image. Nuclear staining with 7AAD (yellow) is also shown. Merged image without masks is shown at right. The actin image (phalloidin-fluorescein isothiocyanate) is omitted for clarity. APC, allophycocyanin; DC, dendritic cell.