Name: measurement of t cell alloreactivity using imaging flow cytometry
Uploaded: 2017-04-19T13:00:00
Duration: 9 min 4 s
Description: Watch this Scientific Journal Video about Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry at JoVE.com

S.C.J. was supported by an International Society for Heart and Lung Transplantation Research Fellowship and a Royal College of Physicians and Surgeons of Canada Detweiler Traveling Fellowship.S.M. was supported in part by an International Society for Heart and Lung Transplantation Career Development Award (to S.C.J.). S.S. was supported by the National Institutes of Health Research Oxford Biomedical Research Centre.J.H. is the recipient of a Kidney Research U.K. Senior Non-Clinical Fellowship. This work was funded by the following grants to A.B. and K.W.: a Wellcome Trust Programme Grant (082519Z07Z), a British Heart Foundation Programme Grant (PG/10/62.28504), and the EU Framework Program 7 (ONE Study; BioDRIM). The authors wish to thank Michael Parsons and the Flow Cytometry Core Facility at the Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto for providing access to and support with the ImageStream Mark X instrument.

The authors have nothing to disclose.

Imaging flow cytometry has been used to demonstrate immune synapse formation between monoclonal T cells and APCs or in the presence of superantigens24,25,26,27,28. This method takes advantage of the fact that after a productive T cell-APC contact, the T cell rearranges its actin cytoskeleton, polarizing it toward the site of contact21. This rearrangement does not occur without TCR signaling, and it is therefore an early correlate of T-cell activation19,20,21. The method presented here adapts this approach to the measurement of alloreactive T-cell frequency in polyclonal T-cell populations. As such, it may in the future serve as the basis for the development of assays for donor reactivity in clinical transplantation.
Although direct comparisons have not yet been made, the detection of alloreactive immune synapses appears to have superior predictive power than the conventional MLR. For example, previous work has shown that, in the tolerizing protocol described above, the results of an MLR fail to reliably correlate with graft outcome2.
A number of assays have been developed for the operationally tolerant state in humans9,10,11, although these do not measure effector cell function in response to alloantigen. In contrast, IFN&#947; ELISPOT assays8 measure effector T-cell function but cannot capture the full spectrum of cytokine secretion that may be relevant to acute and chronic allograft rejection, such as IL-1738,39. The limiting dilution assay4, which is labor intensive, and the trans-vivo assay6, which requires mice, have significant practical limitations that would hinder their application in a clinical setting. Recent improvements on the analysis of proliferating cells using TCR sequence analysis of T cells responding in the MLR may be of value, but like the assay presented here, will require further validation in clinical studies18,40.
Further development of the immune synapse detection assay will require that a number of important questions be answered. First, the assay as developed only measures direct alloreactivity. The direct pathway involves the presentation of allogeneic MHC/peptide complexes on donor-derived APCs. The latter are generally eliminated quickly after transplantation, and further alloantigen presentation is carried out by recipient APCs presenting intact donor MHC (semi-direct pathway) or processed donor antigens on self MHC (indirect pathway). The indirect pathway is an important driver of chronic allograft rejection33,41.
In principle, it should be possible to detect indirect immune synapses using this assay, but indirectly alloreactive T cells have a much lower frequency than direct ones42,43, meaning that the analysis of a larger number of events will be required. A second consideration is that we have only tested this assay using CD4+ T cells, whereas CD8+ T cells are also an important component of the anti-donor response. Again, it should be possible to detect CD8+ T cell-APC synapses using this method. Another limitation is that the method requires the manual review and analysis of cell images in the final membrane contact gate, and we are currently working on the automation of this step.
Finally, the method requires testing and development in human subjects, and preliminary studies with human samples are currently being performed. Further phenotypic T-cell subset analysis (i.e., effector, memory, regulatory, etc.) in combination with the detection of immune synapses in transplant recipients would represent a powerful approach for characterizing the alloreactive T-cell repertoire and will be an important focus for future work.

Solid organ transplantation has transformed the care of patients with end-stage diseases of the kidney, liver, heart, and lungs. Owing to disparities in major and minor histocompatibility antigens, however, allografts are promptly rejected by recipient T cells if immunosuppressive drugs are not used. These agents have numerous adverse effects, including risks for cancer and organ dysfunction. A major clinical goal is therefore to lower the dose of immunosuppression to the minimum level required to prevent allograft rejection. This level is likely to vary depending on the degree of activation of the innate immune system; the degree of donor-recipient alloantigen mismatch; and inter-patient differences in immune function, pharmacokinetics, and pharmacodynamics.
Unfortunately, transplant clinicians do not have any tools for accurately assessing donor reactivity in individual patients1. The mixed leukocyte reaction (MLR) can detect donor reactivity, but it fails to reliably predict graft outcome2,3. Limiting dilution assays, cytokine ELISPOTs, and the trans-vivo assay either measure a limited range of responses or are not practical4,5,6,7,8.Gene expression profiles have revealed signatures related to operational tolerance9,10,11,12 and rejection13,14,15, but these are not always generalizable across populations16 and may ultimately have limited usefulness in individual patients. Sequence-based analyses of the T-cell receptor (TCR) of T cells in the peripheral blood17 or proliferating in the MLR18 have also been developed but require further validation.
Conceptually, it would be desirable to have an assay that detects the earliest requisite steps in recipient T-cell activation by a donor antigen. Since culturing cells over days (as in the MLR) can introduce artefacts, such a test would ideally not require the measurement of downstream events, such as proliferation or effector function. Equally, however, it would also be desirable for the test to depend on some element of T-cell function, since purely descriptive assessments (e.g., TCR sequencing) may be unable to distinguish between anergic and functional T cells.
Numerous studies have indicated that prolonged T-APC contact is required for the formation of an immune synapse, which is an essential first step in the T-cell response19,20,21,22. We recently reported that, during dynamic in vitro time lapse imaging, about 5 - 10% of mouse CD4+ T cells form long-lasting contacts with allogeneic bone marrow-derived dendritic cells (BMDCs)23. The frequency of prolonged contact was increased in animals that rejected a graft, whereas in mice previously rendered tolerant to the same antigens, it remained at levels seen in untransplanted mice23. Prolonged interactions were reduced in the presence of recipient Tregs and increased in their absence, and we observed similar phenomena using human T cells and allogeneic monocyte-derived DCs (MoDCs)23.
However, the enumeration of prolonged contacts made within a polyclonal T-cell population is time-consuming and labor-intensive. We therefore made use of imaging flow cytometry to examine allogeneic immune synapse formation. Imaging flow cytometry incorporates the multiparametric data acquisition and analysis capabilities of conventional flow cytometry with the single-cell imaging abilities of fluorescence microscopy. This technique has been used by other investigators to study immune synapse formation by monoclonal T cells24,26,27or in the presence of superantigens28. In such settings, however, the frequency of responding T cells ranges from 30-100%, whereas alloreactive T cells are generally estimated to represent 5-15% of the total T-cell repertoire29,30,31,32. Importantly, we showed that imaging flow cytometry can produce a very comparable measure of alloreactive T-cell frequency23 and that changes in synapse frequency within a polyclonal T-cell population are predictive of graft outcome23. Currently, this approach has been optimized to measure the direct alloreactivity of CD4+ T cells, but, in principle, it could also be developed to examine CD8+ T cells and the indirect pathway. Indirect alloreactivity is believed to become increasingly relevant at longer times post-transplant33. We are currently developing this method to use human cells, which will allow for testing in patients. Thus, in the future, the overall approach may be useful for the functional evaluation of T-cell responses in transplant recipients before transplant; immediately after transplant; and in the long-term, when drug minimization becomes an important goal.

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			<td align="left" height="21" style="height:21px;width:287px;">Phosphate-buffered saline</td>
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			<td></td>
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			<td align="left" height="21" style="height:21px;width:287px;">Dimethyl sulfoxide</td>
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			<td align="left" style="width:119px;">D8418</td>
			<td></td>
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			<td align="left" height="21" style="height:21px;width:287px;">Formaldehyde</td>
			<td align="left" style="width:133px;">Sigma-Aldrich</td>
			<td align="left" style="width:119px;">F1635</td>
			<td align="left">Solution is 37% formaldehyde and so must be diluted 25 times for 1.5% solution</td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;width:287px;">Cell strainers, 70 &#956;m pore size</td>
			<td align="left" style="width:133px;">Fisher Scientific</td>
			<td align="left" style="width:119px;">08-771-2</td>
			<td></td>
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			<td align="left" height="21" style="height:21px;width:287px;">Phalloidin-fluorescein isothiocyanate</td>
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			<td align="left" style="width:119px;">P5282</td>
			<td></td>
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			<td align="left" height="42" style="height:42px;width:287px;">7-aminoactinomycin D</td>
			<td align="left" style="width:133px;">Thermo Fisher Scientific</td>
			<td align="left" style="width:119px;">A1310</td>
			<td align="left">Reconstitute in DMSO</td>
		</tr>
		<tr height="42">
			<td align="left" height="42" style="height:42px;width:287px;">Allophycocyanin-conjugated anti-mouse CD90.2</td>
			<td align="left" style="width:133px;">eBioscience</td>
			<td align="left" style="width:119px;">17-0902</td>
			<td></td>
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			<td align="left" height="42" style="height:42px;width:287px;">Pacific blue-conjugated anti-mouse CD11b</td>
			<td align="left" style="width:133px;">eBioscience</td>
			<td align="left" style="width:119px;">48-0112</td>
			<td align="left">Pacific blue has been replaced by eFluor 450</td>
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			<td align="left" height="21" style="height:21px;width:287px;">Biotinylated anti-mouse CD3</td>
			<td align="left" style="width:133px;">eBioscience</td>
			<td align="left" style="width:119px;">13-0032</td>
			<td></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;width:287px;">Biotinylated anti-mouse MHC class II</td>
			<td align="left" style="width:133px;">eBioscience</td>
			<td align="left" style="width:119px;">13-5321</td>
			<td></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;width:287px;">Biotinylated anti-mouse B220</td>
			<td align="left" style="width:133px;">eBioscience</td>
			<td align="left" style="width:119px;">13-0452</td>
			<td></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;width:287px;">Biotinylated anti-mouse CD8</td>
			<td align="left" style="width:133px;">eBioscience</td>
			<td align="left" style="width:119px;">13-0081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;width:287px;">Biotinylated anti-mouse CD19</td>
			<td align="left" style="width:133px;">eBioscience</td>
			<td align="left" style="width:119px;">13-0193</td>
			<td></td>
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			<td align="left" height="21" style="height:21px;width:287px;">Anti-biotin microbeads</td>
			<td align="left" style="width:133px;">Miltenyi Biotec</td>
			<td align="left" style="width:119px;">130-090-485</td>
			<td></td>
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			<td align="left" height="21" style="height:21px;width:287px;">LS columns</td>
			<td align="left" style="width:133px;">Miltenyi Biotec</td>
			<td align="left" style="width:119px;">130-042-401</td>
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			<td align="left" height="21" style="height:21px;width:287px;">MidiMACS magnetic cell separator</td>
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			<td align="left" style="width:119px;">130-042-302</td>
			<td></td>
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		<tr height="21">
			<td align="left" height="21" style="height:21px;width:287px;">recombinant mouse GM-CSF</td>
			<td align="left" style="width:133px;">Peprotech</td>
			<td align="left" style="width:119px;">315-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;width:287px;">recombinant human TGF&#946;1</td>
			<td align="left" style="width:133px;">Peprotech</td>
			<td align="left" style="width:119px;">100-21</td>
			<td align="left">Human TGF&#946;1 has activity on mouse cells</td>
		</tr>
		<tr height="42">
			<td align="left" height="42" style="height:42px;width:287px;">Amnis ImageStream X Mark II</td>
			<td align="left" style="width:133px;">Amnis/EMD Millipore</td>
			<td align="left" style="width:119px;">N/A</td>
			<td align="left">Imaging flow cytometer; details available at http://www.emdmillipore.com/</td>
		</tr>
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			<td align="left" height="42" style="height:42px;width:287px;">IDEAS Software</td>
			<td align="left" style="width:133px;">Amnis/EMD Millipore</td>
			<td align="left" style="width:119px;">N/A</td>
			<td align="left">Free download (registration required): https://www.amnis.com/index.php/page/Display/login%20%20</td>
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			<td align="left" height="21" style="height:21px;width:287px;">Cell culture medium&#160;</td>
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			<td align="left" height="21" style="height:21px;width:287px;">Cell culture plates</td>
			<td align="left" style="width:133px;">Various</td>
			<td align="left" style="width:119px;">Varies</td>
			<td></td>
		</tr>
	</tbody>
</table>

measurement of t cell alloreactivity using imaging flow cytometry

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal of immunosuppression. The mixed leukocyte reaction (MLR), limiting dilution assays, and trans-vivo delayed-type hypersensitivity (DTH) assay have all been applied to this question, but these methods have limited predictive ability and/or significant practical limitations that reduce their usefulness. Imaging flow cytometry is a technique that combines the multiparametric quantitative powers of flow cytometry with the imaging capabilities of fluorescent microscopy. We recently made use of an imaging flow cytometry approach to define the proportion of recipient T cells capable of forming mature immune synapses with donor antigen-presenting cells (APCs). Using a well-characterized mouse heart transplant model, we have shown that the frequency of in vitro immune synapses among T-APC membrane contact events strongly predicted allograft outcome in rejection, tolerance, and a situation where transplant survival depends on induced regulatory T cells. The frequency of T-APC contacts increased with T cells from mice during acute rejection and decreased with T cells from mice rendered unresponsive to alloantigen. The addition of regulatory T cells to the in vitro system reduced prolonged T-APC contacts. Critically, this effect was also seen with human polyclonally expanded, naturally occurring regulatory T cells, which are known to control the rejection of human tissues in humanized mouse models. Further development of this approach may allow for a deeper characterization of the alloreactive T-cell compartment in transplant recipients. In the future, further development and evaluation of this method using human cells may form the basis for assays used to select patients for immunosuppression minimization, and it can be used to measure the impact of tolerogenic therapies in the clinic.

This method was used to investigate CD4+ T-cell alloreactivity in mice rendered tolerant to donor alloantigens before heterotopic cardiac allograft transplantation. CBA mice (H-2k) were given a tolerizing protocol consisting of a donor-specific (B6, H-2b) blood transfusion combined with a non-depleting CD4 antibody one month prior to receiving a B6 cardiac transplant. This protocol results in long-term allograft survival that is dependent on Foxp3+ regulatory T cells36,37. Seven days post-transplant, splenic CD4+ T cells were obtained from tolerized and non-tolerized recipients of B6 cardiac allografts and were co-incubated with B6 bone marrow-derived DCs according to this protocol. Figure 2 shows representative data from this experiment. The membrane contact gate is shown in Figure 2A, with green crosshairs placed on a synaptic event (left panel, 1) and on a non-synaptic event (right panel). Figure 2B shows the brightfield and fluorescence channels for this event. To reduce bias, data were analyzed by an observer blinded to treatment assignment23. As shown in several examples in Figure 3, both from non-tolerized (Figure 3A-B) and tolerized (Figure 3C-D) CBA recipients of B6 hearts, synapses are easily distinguished from non-synaptic contacts by the presence of a dense FITC-positive ridge at the T-APC interface. These results show that visual detection of immune synapses made by recipient T cells tracks with the degree of alloreactivity in the recipient.
<img alt="Figure 2" src="/files/ftp_upload/55283/55283fig2.jpg" /> 
Figure 2. Identification of T-APC Doublets with Membrane Contact and Immune Synapse Formation. Events in the final doublet gate (Figure 1F) are analyzed. A. T-cell marker fluorescence in the APC object mask is plotted against APC marker fluorescence in the DC object mask. Some doublet events have an APC and a T cell without cell-cell contact and appear in the lower left corner of the plot (images not shown). A membrane contact gate can thus be drawn that includes only doublets in which T cells and APCs are in contact. Images of each event in this gate are reviewed for evidence of actin cytoskeletal rearrangement in the phalloidin-FITC channel and can be tagged using the analysis software. The left panel indicates an immune synapse event (labeled 1 and indicated by green crosshairs), whereas the right panel indicates a membrane contact event without immune synapse formation (labeled 2 and indicated by green crosshairs). The determination of synapse formation requires manual review of these images, shown in B. B. The top row shows brightfield and fluorescence channel images for a doublet with an immune synapse (corresponds to event 1 in A); the bottom row shows a doublet with membrane contact but lacking synapse formation (corresponds to event 2 in A). The data were analyzed in a blinded fashion with respect to treatment assignment and are from a previously published experiment23. <a href="https://www.jove.com/files/ftp_upload/55283/55283fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55283/55283fig3.jpg" /> 
Figure 3. Examples of T-APC Synapse Formation. CBA mice received cardiac allografts from B6 donors after either no pre-treatment (A-B) or after tolerance induction with B6 whole blood under the cover of a non-depleting anti-CD4 antibody (C-D). After 7 days, splenic CD4+ T cells were tested for synapse formation with B6 DCs. A. Three examples of non-synaptic doublets with membrane contact from a non-tolerized animal. B. Three examples of immune synapses from a non-tolerized animal. C. Three examples of non-synaptic doublets with membrane contact from a tolerized animal. D. Three examples of immune synapses from a tolerized animal. Synapse formation is indicated by the presence of a bright, FITC-positive ridge at the T-APC interface (Ch03). The data were analyzed in a blinded fashion with respect to treatment assignment and are from a previously published experiment23. <a href="https://www.jove.com/files/ftp_upload/55283/55283fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antibody/Dye</td>
			<td>Fluorochrome</td>
			<td>Channel</td>
			<td>Concentration</td>
		</tr>
		<tr>
			<td>CD11c</td>
			<td>eF450</td>
			<td>Ch02</td>
			<td>5 &#181;g/mL, titrate empirically</td>
		</tr>
		<tr>
			<td>CD90.2</td>
			<td>APC</td>
			<td>Ch06</td>
			<td>5 &#181;g/mL, titrate empirically</td>
		</tr>
		<tr>
			<td>Phalloidin</td>
			<td>FITC</td>
			<td>Ch03</td>
			<td>0.05 - 0.5 &#181;g/mL</td>
		</tr>
		<tr>
			<td>7-AAD</td>
			<td>-</td>
			<td>Ch05</td>
			<td>25 &#181;g/mL</td>
		</tr>
	</tbody>
</table>
Table 1. Antibodies and Dyes Used in this Study. Fluorochrome-conjugated antibodies, dyes, suppliers, and recommended concentrations are presented in the table. The imaging flow cytometer channel that was used to detect each fluorochrome is also shown in the table.

Watch this Scientific Journal Video about Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry at JoVE.com

Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry

This paper describes a method for measuring alloreactivity in a mixed population of T cells using imaging flow cytometry.

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal ...

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