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.

<table><tbody><tr><td>Phosphate-buffered saline</td><td>Various</td><td>Varies</td><td></td></tr><tr><td>Ethylenediamenetetraacetic acid, 0.5 M solution</td><td>Thermo Fisher Scientific</td><td>AM9260G</td><td></td></tr><tr><td>Triton X-100 nonionic detergent</td><td>Sigma-Aldrich</td><td>X100</td><td></td></tr><tr><td>Beta-mercaptoethanol</td><td>Sigma-Aldrich</td><td>M3148</td><td></td></tr><tr><td>Dimethyl sulfoxide</td><td>Sigma-Aldrich</td><td>D8418</td><td></td></tr><tr><td>Formaldehyde</td><td>Sigma-Aldrich</td><td>F1635</td><td>Solution is 37% formaldehyde and so must be diluted 25 times for 1.5% solution</td></tr><tr><td>Cell strainers, 70 &amp;mu;m pore size</td><td>Fisher Scientific</td><td>08-771-2</td><td></td></tr><tr><td>Phalloidin-fluorescein isothiocyanate</td><td>Sigma-Aldrich</td><td>P5282</td><td></td></tr><tr><td>7-aminoactinomycin D</td><td>Thermo Fisher Scientific</td><td>A1310</td><td>Reconstitute in DMSO</td></tr><tr><td>Allophycocyanin-conjugated anti-mouse CD90.2</td><td>eBioscience</td><td>17-0902</td><td></td></tr><tr><td>Pacific blue-conjugated anti-mouse CD11b</td><td>eBioscience</td><td>48-0112</td><td>Pacific blue has been replaced by eFluor 450</td></tr><tr><td>Biotinylated anti-mouse CD3</td><td>eBioscience</td><td>13-0032</td><td></td></tr><tr><td>Biotinylated anti-mouse MHC class II</td><td>eBioscience</td><td>13-5321</td><td></td></tr><tr><td>Biotinylated anti-mouse B220</td><td>eBioscience</td><td>13-0452</td><td></td></tr><tr><td>Biotinylated anti-mouse CD8</td><td>eBioscience</td><td>13-0081</td><td></td></tr><tr><td>Biotinylated anti-mouse CD19</td><td>eBioscience</td><td>13-0193</td><td></td></tr><tr><td>Anti-biotin microbeads</td><td>Miltenyi Biotec</td><td>130-090-485</td><td></td></tr><tr><td>LS columns</td><td>Miltenyi Biotec</td><td>130-042-401</td><td></td></tr><tr><td>MidiMACS magnetic cell separator</td><td>MIltenyi Biotec</td><td>130-042-302</td><td></td></tr><tr><td>recombinant mouse GM-CSF</td><td>Peprotech</td><td>315-03</td><td></td></tr><tr><td>recombinant human TGF&amp;beta;1</td><td>Peprotech</td><td>100-21</td><td>Human TGF&amp;beta;1 has activity on mouse cells</td></tr><tr><td>Amnis ImageStream X Mark II</td><td>Amnis/EMD Millipore</td><td>N/A</td><td>Imaging flow cytometer; details available at http://www.emdmillipore.com/</td></tr><tr><td>IDEAS Software</td><td>Amnis/EMD Millipore</td><td>N/A</td><td>Free download (registration required): https://www.amnis.com/index.php/page/Display/login%20%20</td></tr><tr><td>Cell culture medium&amp;nbsp;</td><td>Various</td><td>Varies</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Various</td><td>Varies</td><td></td></tr><tr><td>Cell culture plates</td><td>Various</td><td>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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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 ...

measurement-of-t-cell-alloreactivity-using-imaging-flow-cytometry

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Immunology and Infection

13.4K Views.  University of Toronto and University Health Network. This paper describes a method for measuring alloreactivity in a mixed population of T cells using imaging flow cytometry.

Video: Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry

Induction of Alloantigen-specific Anergy in Human Peripheral Blood Mononuclear Cells by Alloantigen Stimulation with Co-stimulatory Signal Blockade

Allogeneic hematopoietic stem cell transplantation (AHSCT) offers the best chance of cure for many patients with congenital and acquired hematologic diseases. Unfortunately, transplantation of alloreactive donor T cells which recognize and damage healthy patient tissues can result in Graft-versus-Host Disease (GvHD)1. One challenge to successful AHSCT is the prevention of GvHD without associated impairment of the beneficial effects of donor T cells, particularly immune reconstitution and prevention of relapse. GvHD can be prevented by non-specific depletion of donor T cells from stem cell grafts or by administration of pharmacological immunosuppression. Unfortunately these approaches increase infection and disease relapse2-4. An alternative strategy is to selectively deplete alloreactive donor T cells after allostimulation by recipient antigen presenting cells (APC) before transplant. Early clinical trials of these allodepletion strategies improved immune reconstitution after HLA-mismatched HSCT without excess GvHD5, 6. However, some allodepletion techniques require specialized recipient APC production6, 7and some approaches may have off-target effects including depletion of donor pathogen-specific T cells8and CD4 T regulatory cells9.One alternative approach is the inactivation of alloreactive donor T cells via induction of alloantigen-specific hyporesponsiveness. This is achieved by stimulating donor cells with recipient APC while providing blockade of CD28-mediated co-stimulation signals10.This "alloanergization" approach reduces alloreactivity by 1-2 logs while preserving pathogen- and tumor-associated antigen T cell responses in vitro11. The strategy has been successfully employed in 2 completed and 1 ongoing clinical pilot studies in which alloanergized donor T cells were infused during or after HLA-mismatched HSCT resulting in rapid immune reconstitution, few infections and less severe acute and chronic GvHD than historical control recipients of unmanipulated HLA-mismatched transplantation12. Here we describe our current protocol for the generation of peripheral blood mononuclear cells (PBMC) which have been alloanergized to HLA-mismatched unrelated stimulator PBMC. Alloanergization is achieved by allostimulation in the presence of monoclonal antibodies to the ligands B7.1 and B7.1 to block CD28-mediated costimulation. This technique does not require the production of specialized stimulator APC and is simple to perform, requiring only a single and relatively brief ex vivo incubation step. As such, the approach can be easily standardized for clinical use to generate donor T cells with reduced alloreactivity but retaining pathogen-specific immunity for adoptive transfer in the setting of AHSCT to improve immune reconstitution without excessive GvHD.

This paper describes a simple technique to induce alloantigen-specific anergy in human peripheral blood mononuclear cells. The technique can be applied clinically to generate non-alloreactive donor cells. Infusion of these cells could improve immune reconstitution and reduce toxicity after allogeneic hematopoietic stem cell transplantation.

Allogeneic hematopoietic stem cell transplantation (AHSCT) offers the best chance of cure for many patients with congenital and acquired hematologic ...

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells to kill CD4+ T cells loaded with their cognate peptide. Target CD4+ T cells are divided into two populations, labeled with two different concentrations of CFSE. One population is pulsed with the peptide of interest (CFSE-low) while the other remains un-pulsed (CFSE-high). Pulsed and un-pulsed CD4+ T cells are mixed at an equal ratio and incubated with an increasing number of purified CD8+ T cells. The specific killing of autologous target CD4+ T cells is analyzed by flow cytometry after coculture with CD8+ T cells containing the antigen-specific effector CD8+ T cells detected by peptide/MHCI tetramer staining. The specific lysis of target CD4+ T cells measured at different effector versus target ratios, allows for the calculation of lytic units, LU30/106 cells. This simple and straightforward assay allows for the accurate measurement of the intrinsic capacity of CD8+ T cells to kill target CD4+ T cells.

This protocol describes a sensitive, cell-based cytotoxicity assay. By enumerating the decrease in frequency of live target CD4+ T cells in the presence of an increasing number of effector CD8+ T cells, this assay allows for the direct assessment of cytolytic activity of antigen-specific CD8+ T cells.

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

The Use of Flow Cytometry to Assess the State of Chromatin in T Cells

During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to clonal proliferation of only those T cells that bear a receptor that recognizes the antigen. Chromatin decondensation is a hallmark of T cell activation and is required for T cells to acquire the ability to proliferate after antigen engagement. This change in chromatin condensation can be detected using antibodies raised against histone proteins. These antibodies cannot bind to their epitopes in na&#239;ve T cells as well as they can in activated T cells. We describe how to simultaneously stain T cell-specific surface markers, track viability with a fixable dead cell stain, and measure chromatin status via intracellular staining of Histone H3 proteins. Stained cells are analyzed by flow cytometry and chromatin condensation status is measured as the mean fluorescence intensity (MFI) of the Histone H3 stain. Chromatin decondensation during T cell activation is demonstrated as an increase in the MFI

Flow cytometry can be utilized to assess the state of chromatin within T cells. This protocol allows scientists to interpret evidence of chromatin decondensation during T cell activation demonstrated by an increase in the mean florescence intensity (MFI) of fluorescent Histone H3 antibodies

During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to ...

Analysis of Human T Cell Activity in an Allogeneic Co-Culture Setting of Pre-Treated Tumor Cells

Cytotoxic T cells play a key role in the elimination of tumor cells and are, therefore, intensively studied in cancer immunology. The frequency and activity of cytotoxic T cells within tumors and their tumor microenvironment (TME) are now well-established prognostic and predictive biomarkers for numerous tumor types. However, it is well-known that various tumor treatment modalities, including radiotherapy, chemotherapy, immunotherapy, and targeted therapy, modulate not only the immunogenicity of the tumor but also the immune system itself. Consequently, the interaction between tumor cells and T cells requires more intensive study in different therapeutic contexts to fully understand the complex role of T cells during tumor therapy. To address this need, a protocol was developed to analyze the activity and proliferative capacity of human cytotoxic (CD8+) T cells in co-culture with pre-treated tumor cells. Specifically, CD8+ T cells from healthy donors are stained with the non-toxic proliferation marker carboxyfluorescein diacetate succinimidyl ester (CFSE) and stimulated using CD3/CD28-coated plates. Subsequently, T cells are co-cultured with pre-treated tumor cells. As a readout, T cell proliferation is quantified by measuring CFSE signal distribution and assessing the expression of surface activation markers via flow cytometry. This can be further complemented by quantifying cytokine release using enzyme-linked immunosorbent assay (ELISA). This method facilitates the evaluation of treatment-induced changes in the interaction between tumor cells and T cells, providing a foundation for more detailed analyses of tumor treatment modalities and their immunogenicity in a human ex vivo setting. Additionally, it contributes to the reduction of pre-clinical in vivo analyses.

The present protocol describes an experimental workflow that allows for the ex vivo analysis of human T cell stimulation in an allogenic co-culture system with pre-treated tumor cells.

Cytotoxic T cells play a key role in the elimination of tumor cells and are, therefore, intensively studied in cancer immunology. The frequency and ...

Cancer Research

Qualitative and Quantitative Analysis of the Immune Synapse in the Human System Using Imaging Flow Cytometry

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins towards the immune synapse to assure a stable binding and signal exchange. Classical confocal, TIRF, or super-resolution microscopy have been used to study the immune synapse. Since these methods require manual image acquisition and time-consuming quantification, the imaging of rare events is challenging. Here, we describe a workflow that enables the morphological analysis of tens of thousands of cells. Immune synapses are induced between primary human T cells in pan-leukocyte preparations and Staphylococcus aureus enterotoxin B (SEB)-loaded Raji cells as APCs. Image acquisition is performed with imaging flow cytometry, also called In-Flow microscopy, which combines features of a flow cytometer and a fluorescence microscope. A complete gating strategy for identifying T cell/APC couples and analyzing the immune synapses is provided. As this workflow allows the analysis of immune synapses in unpurified pan-leukocyte preparations and hence requires only a small volume of blood (i.e., 1 mL), it can be applied to samples from patients. Importantly, several samples can be prepared, measured, and analyzed in parallel.

Here, we describe a complete workflow for the qualitative and quantitative analysis of immune synapses between primary human T cells and antigen-presenting cells. The method is based on imaging flow cytometry, which allows the acquisition and evaluation of several thousand cell images within a relatively short period of time.

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins ...

Discrimination of Seven Immune Cell Subsets by Two-fluorochrome Flow Cytometry

Immune cell characterization heavily relies on multicolor flow cytometry to identify subpopulations based on differential expression of surface markers. Setup of a classic multicolor panel requires high-end instruments, custom labeled antibodies, and careful study design to minimize spectral overlap. We developed a multiparametric analysis to identify major human immune populations (CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, NK cells and monocytes) in peripheral blood by combining seven lineage markers using only two fluorochromes. Our strategy is based on the observation that lineage markers are constantly expressed in a unique combination by each cell population. Combining this information with a careful titration of the antibodies allows investigators to record five additional markers, expanding the optical limit of most flow cytometers. Head-to-head comparison demonstrated that the vast majority of immune cell populations in peripheral blood can be characterized with comparable accuracy between our method and the classic &#34;one fluorochrome-one marker approach&#34;, although the latter is still more precise for identifying populations such as NKT cells and &#947;&#948; T cells. Combining seven markers using two fluorochromes allows for the analysis of complex immune cell populations and clinical samples on affordable 6-10 fluorochrome flow cytometers, and even on 2-3 fluorochrome field instruments in areas with limited resources. High-end instruments can also benefit from this approach by using extra fluorochromes to accomplish deeper flow cytometry analysis. This approach is also very well suited for screening several cell populations in the case of clinical samples with limited number of cells.

Here, we present a flow cytometric protocol to identify CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, NK cells and monocytes in human peripheral blood by using only two fluorochromes instead of seven. With this approach, five additional markers can be recorded on most flow cytometers.

Immune cell characterization heavily relies on multicolor flow cytometry to identify subpopulations based on differential expression of surface markers. ...

Analysis of T-cell Receptor-Induced Calcium Influx in Primary Murine T-cells by Full Spectrum Flow Cytometry

Calcium influx in response to T-cell receptor stimulation is a common measure of T-cell signaling. Several calcium indicator dyes have been developed to assess calcium signaling by band-pass flow cytometry. This protocol is designed to measure calcium responses in primary murine T-cells using full spectrum flow cytometry. Total splenocytes are labeled with the ratiometric calcium indicator dye Indo-1, along with a panel of fluorochrome-conjugated antibodies to cell surface molecules. Leveraging the capabilities of full spectrum flow cytometry provides a platform for utilizing a wide array of cell surface stains in combination with Indo-1. Cells are then analyzed in real-time at 37 &#176;C before and after the addition of an anti-CD3 antibody to stimulate the T-cell receptor. After unmixing the spectral signals, the ratio of calcium-bound to calcium-free Indo-1 is calculated and can be visualized over time for each gated population of splenocytes. This technique can allow for the simultaneous analysis of calcium responses in multiple cell populations.

Calcium influx, a measure of T-cell signaling, is an effective way to analyze responses to T-cell receptor stimulation. This protocol for multiplexing Indo-1 with panels of antibodies directed at cell surface molecules takes advantage of the highly flexible capabilities of full spectrum flow cytometry.

Calcium influx in response to T-cell receptor stimulation is a common measure of T-cell signaling. Several calcium indicator dyes have been developed to ...

Measuring Alloreactivity in a Mixed Population of T Cells

Source: Juvet, S. C. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/55283">Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry.</a>&#160;J. Vis. Exp.&#160;(2017)
This video demonstrates a method for assessing alloreactivity in mixed T cell populations by observing interactions with alloantigen-carrying dendritic cells, leading to the formation of immunological synapses. Differential staining and quantification of synapse-containing doublets via flow cytometry reveal T cell alloreactivity.

1. Prepare Reagents and Materials Required

	Prepare phosphate-buffered saline (PBS) containing 2% fetal bovine serum (FBS) (&#34;wash buffer&#34;). ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Cellular Immunodiagnostics

A Flow Cytometry Assay to Identify Immune Cell Subsets in Peripheral Blood Mononuclear Cells

Source: Torchia, M. L. G., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/58955">Discrimination of Seven Immune Cell Subsets by Two-fluorochrome Flow Cytometry.</a> J. Vis. Exp. (2019)
This video demonstrates a flow cytometry-based technique using two fluorochromes to identify CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, natural killer (NK) cells, and monocytes in human peripheral blood mononuclear cells (PBMCs). Upon incubating the cells with fluorochrome-conjugated antibodies against the surface markers, the cell subsets are identified using an optimal flow cytometric gating strategy.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
NOTE: This protocol has been tested ...