This work was supported by grants to S.K. from the Japan Society for the Promotion of Science KAKENHI (JP17K16045) and the Japan Agency for Medical Research and Development (JP18cm0106335 and 19cm0106310).

Sampling was performed during routine clinical procedures. All human samples were obtained after informed consent was provided by the subjects, in accordance with the Declaration of Helsinki and with the approval of the ethical review board of the Graduate School of Medicine, Osaka University, Japan (15383 and 752).
1. Whole Blood Sample Preparation and Staining
<ol>
	<li>Collect whole blood samples into blood collection tubes containing ethylene diamine tetra-acetic acid (EDTA). 
	NOTE: Blood collection can be performed using either a regular needle or a blood lancet.</li>
	<li>Transfer 20 &#181;L of whole blood samples to 5 mL round-bottom polystyrene flow cytometry tubes. 
	NOTE: To reduce non-specific binding of cells to tubes, 1 mL of 2% fetal bovine serum (FBS) in phosphate buffered saline (PBS) is added into tubes and vortexed for 10 s before application to samples.</li>
	<li>Add 20 &#181;L of 2% FBS in PBS.</li>
	<li>Add 10 &#181;L of human-specific FcR blocking reagent. Mix well and incubate for 15 min at room temperature.</li>
	<li>Add 500 &#181;L of red blood cell lysis buffer. Mix well and incubate for 10 min at room temperature.</li>
	<li>Add 4 mL of 2% FBS in PBS and spin down cells at 400 x g (1,500 rpm) for 5 min at 4 &#176;C. Remove supernatant by aspiration.</li>
	<li>Repeat the wash and aspiration process described in step 1.6.</li>
	<li>Re-suspend cells in 100 &#181;L of 2% FBS in PBS and divide into two tubes of 50 &#181;L each.</li>
	<li>Add surface marker antibodies (Table 1). Mix well and incubate for 20 min at room temperature in the dark. 
	NOTE: When profiling the T cell immune status, the number of markers can be increased based on the flow cytometry machine quality.</li>
	<li>Wash samples 2x as described in step 1.6.</li>
	<li>Resuspend cells in 200 &#181;L of 2% FBS in PBS.</li>
</ol>
2. Flow Cytometric Analysis
<ol>
	<li>Insert tubes into the flow cytometer and acquire cells, basically following the recommended protocol9.</li>
	<li>Record 10,000 events as the lymphocyte gate (Figure 1A) and export flow data as .fcs files for analysis.</li>
	<li>Open files in the analysis software. Visualize the cells on a forward scatter (FSC) (A) vs. side scatter (SSC) (A) plot and gate lymphocytes (Figure 1A).</li>
	<li>After selecting single cells using FSC (H) vs. FSC (W) and SSC (H) vs. SSC (W) (Figure 1B) and displaying them on a CD3 vs. CD8 or CD3 vs. CD4 plot, gate the CD8 T cells and CD4 T cells, respectively (Figure 1C).</li>
	<li>After selecting the gated cells and displaying them on a PD-1 vs. human IgG4 plot, identify PD-1&#8211;blocking antibody&#8211;bound CD8 and CD4 T cells based on isotype control (Figure 1D).</li>
</ol>

<ol>
	<li>Gong, J., Chehrazi-Raffle, A., Reddi, S., Salgia, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+PD-1+and+PD-L1+inhibitors+as+a+form+of+cancer+immunotherapy:+a+comprehensive+review+of+registration+trials+and+future+considerations.">Development of PD-1 and PD-L1 inhibitors as a form of cancer immunotherapy: a comprehensive review of registration trials and future considerations.</a> Journal for ImmunoTherapy of Cancer. 6 (1), 8 (2018).</li><li>Borghaei, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nivolumab+versus+Docetaxel+in+Advanced+Nonsquamous+Non-Small-Cell+Lung+Cancer.">Nivolumab versus Docetaxel in Advanced Nonsquamous Non-Small-Cell Lung Cancer.</a> The New England Journal of Medicine. 373 (17), 1627-1639 (2015).</li><li>Brahmer, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nivolumab+versus+Docetaxel+in+Advanced+Squamous-Cell+Non-Small-Cell+Lung+Cancer.">Nivolumab versus Docetaxel in Advanced Squamous-Cell Non-Small-Cell Lung Cancer.</a> The New England Journal of Medicine. 373 (2), 123-135 (2015).</li><li>Herbst, R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pembrolizumab+versus+docetaxel+for+previously+treated,+PD-L1-positive,+advanced+non-small-cell+lung+cancer+(KEYNOTE-010):+a+randomised+controlled+trial.">Pembrolizumab versus docetaxel for previously treated, PD-L1-positive, advanced non-small-cell lung cancer (KEYNOTE-010): a randomised controlled trial.</a> The Lancet. 387 (10027), 1540-1550 (2016).</li><li>Postow, M. A., Sidlow, R., Hellmann, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune-Related+Adverse+Events+Associated+with+Immune+Checkpoint+Blockade.">Immune-Related Adverse Events Associated with Immune Checkpoint Blockade.</a> The New England Journal of Medicine. 378 (2), 158-168 (2018).</li><li>Weber, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+Profile+of+Nivolumab+Monotherapy:+A+Pooled+Analysis+of+Patients+With+Advanced+Melanoma.">Safety Profile of Nivolumab Monotherapy: A Pooled Analysis of Patients With Advanced Melanoma.</a> Journal of Clinical Oncology. 35 (7), 785-792 (2017).</li><li>Champiat, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Management+of+immune+checkpoint+blockade+dysimmune+toxicities:+a+collaborative+position+paper.">Management of immune checkpoint blockade dysimmune toxicities: a collaborative position paper.</a> Annals of Oncology. 27 (4), 559-574 (2016).</li><li>Brahmer, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+I+study+of+single-agent+anti-programmed+death-1+(MDX-1106)+in+refractory+solid+tumors:+safety,+clinical+activity,+pharmacodynamics,+and+immunologic+correlates.">Phase I study of single-agent anti-programmed death-1 (MDX-1106) in refractory solid tumors: safety, clinical activity, pharmacodynamics, and immunologic correlates.</a> Journal of Clinical Oncology. 28 (19), 3167-3175 (2010).</li><li>Bommareddy, P. K., Lowe, D. B., Kaufman, H. L., Rabkin, S. D., Saha, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-parametric+flow+cytometry+staining+procedure+for+analyzing+tumor-infiltrating+immune+cells+following+oncolytic+herpes+simplex+virus+immunotherapy+in+intracranial+glioblastoma.">Multi-parametric flow cytometry staining procedure for analyzing tumor-infiltrating immune cells following oncolytic herpes simplex virus immunotherapy in intracranial glioblastoma.</a> Journal of Biological Methods. 6 (2), 112 (2019).</li><li>Osa, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+implications+of+monitoring+nivolumab+immunokinetics+in+non-small+cell+lung+cancer+patients.">Clinical implications of monitoring nivolumab immunokinetics in non-small cell lung cancer patients.</a> JCI Insight. 3 (19), 59125 (2018).</li><li>Zelba, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+quantification+of+T-cells+expressing+PD-1+in+patients+on+anti-PD-1+immunotherapy.">Accurate quantification of T-cells expressing PD-1 in patients on anti-PD-1 immunotherapy.</a> Cancer Immunology, Immunotherapy. 67 (12), 1845-1851 (2018).</li><li>Simoni, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bystander+CD8(+)+T+cells+are+abundant+and+phenotypically+distinct+in+human+tumour+infiltrates.">Bystander CD8(+) T cells are abundant and phenotypically distinct in human tumour infiltrates.</a> Nature. 557 (7706), 575-579 (2018).</li><li>Chiu, H. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+general+method+for+quantifying+IgG-based+therapeutic+monoclonal+antibodies+in+human+plasma+using+protein+G+purification+coupled+with+a+two+internal+standard+calibration+strategy+using+LC-MS/MS.">Development of a general method for quantifying IgG-based therapeutic monoclonal antibodies in human plasma using protein G purification coupled with a two internal standard calibration strategy using LC-MS/MS.</a> Analytica Chimica Acta. 1019, 93-102 (2018).</li></ol>

The authors have nothing to disclose.

In this article, we report a method using a flow cytometer to detect PD-1&#8211;blocking antibodies bound to T cells derived from a drop of peripheral blood, which we originally developed for nivolumab detection10. Although this technique is very simple and easy to perform, two important points should be noted in order to obtain accurate results. One is that to detect PD-1 molecules, an appropriate antibody that competes with nivolumab and pembrolizumab should be used. This issue was evaluated in a previous study11. The other is that RBC lysis should be performed thoroughly before surface staining. As long as an isotype control is established for each assay to determine the gate of the IgG4-positive cluster, the frequency is unaffected. However, with this protocol, when RBC lysis is insufficient there may be reductions of both the T cell count in the lymphocyte gate and of the intensity of each surface marker. In addition, the RBC lysis step must be performed before surface staining, otherwise the anti-IgG4 antibody (HP6025) does not function properly.
The limitation of this method is that the population of T cells bound to PD-1&#8211;blocking antibodies include specific T cell clones responsible for therapeutic effects and irAEs; however, the frequencies of these specific clones among the antibody-bound population are quite low. Therefore, we still need to enrich the specific target using certain markers, for instance CD3912. Another limitation is that the fluorescence intensity of IgG4 is not high in some cases, which makes it difficult to determine binding status (i.e., PB and LB).
Other studies have monitored therapeutic PD-1 antibodies in the blood to evaluate pharmacokinetics. Measuring the plasma concentration of nivolumab or pembrolizumab is essential to determine how the residual amount of these antibodies in blood correlates with time. However, we previously reported that the concentration of nivolumab does not completely correlate with residual binding to T cells10. Compared to the measurement of plasma antibody concentration13, our method may be more appropriate for evaluating the residual binding of anti&#8211;PD-1 antibodies to T cells. The original method of monitoring nivolumab binding to T cells in blood required incubation with fluorescence-labeled nivolumab8. We simplified this approach and succeeded in substantially reducing the assay time and the amount of blood needed for analysis.
Frozen cells can also be analyzed using this assay, suggesting that this method is a good fit for multicenter studies. The usefulness of this approach will be further enhanced by combining monitoring binding status with other immunological markers, such as Ki-67, of T cells bound to PD-1&#8211;blocking antibodies10. Future studies should use our method in conjunction with single-cell RNA sequencing and T cell receptor sequencing to try to identify and characterize the specific subsets of T cells that are bound to PD-1&#8211;blocking antibodies and are responsible for irAEs.

PD-1&#8211;blocking antibodies have become the standard choice for treatment of various types of cancer, including non-small cell lung cancer (NSCLC)1,2,3,4. They show a remarkable therapeutic effect in a subset of cancer patients who have not responded to conventional cytotoxic chemotherapies. However, immune checkpoint inhibitors (ICIs), which include PD-1&#8211;blocking antibodies, can cause a unique and distinct spectrum of adverse events, termed immune-related adverse events (irAEs)5. Although irAEs can affect almost all tissues, they are most commonly observed in the gastrointestinal tract, endocrine glands, skin, and liver, and they can cause pruritus, rash, nausea, diarrhea, and thyroid disorders6,7. In general, most irAEs appear within 1 to 2 months after the initiation of ICIs. However, in some cases, they can occur later than 1 year after the beginning of treatment or even after treatment cessation6,7. They also cause various symptoms that may be difficult to discriminate from other pathologies. Thus, it can be challenging to promptly diagnose irAEs and treat them appropriately. irAEs can affect all tissues, and their onset is strongly influenced by circulating immune cells, especially T cells bound to PD-1&#8211;blocking antibodies. Therefore, a straightforward and minimally invasive method to monitor antibody-targeted T cells is important in clinical settings.
Here, we developed a simple method to assess the binding of PD-1&#8211;blocking antibodies to T cells using a drop of peripheral whole blood from cancer patients who received nivolumab or pembrolizumab. Using this approach, we were able to monitor each of the following: 1) the duration of antibody binding to T cells, 2) the occupancy of T cell PD-1 molecules by therapeutic antibodies, and 3) the activation status and immunological features of T cells. This method is a modification of a previously reported technique8. The amount of blood required is almost the same as that needed for a glucose test, and the approach does not require mononuclear cell enrichment or co-culturing with PD-1&#8211;blocking antibodies. We confirmed that this method can also be performed using frozen samples, including peripheral blood mononuclear cells (PBMCs) and cells from pleural effusion, pericardial effusion, bronchoalveolar lavage fluid, and cerebrospinal fluid, suggesting that this strategy may be useful in the context of a multicenter study. This method may facilitate the early diagnosis of irAEs, and also help to determine the appropriate immunosuppressive treatments to control their symptoms and to identify the optimal times to initiate subsequent therapies after PD-1 inhibitors.

<table><tbody><tr><td>10X RBC Lysis Buffer (Multi-species)</td><td>Thermo Fisher Scientific</td><td>00-4300-54</td><td>50 mL</td></tr><tr><td>APC/Cyanine7 anti-human CD4 Antibody</td><td>BioLegend</td><td>300518</td><td>Clone RPA-T4</td></tr><tr><td>BD FACS Canto II Flow Cytometer</td><td>BD</td><td></td><td></td></tr><tr><td>Brilliant Violet 510 anti-human CD8a Antibody</td><td>BioLegend</td><td>301048</td><td>Clone RPA-T8</td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline</td><td>nacalai tesque</td><td>14249-95</td><td>500 mL</td></tr><tr><td>Falcon Round-Bottom Polystyrene Tubes</td><td>STEMCELL Technologies</td><td>352058</td><td>5 mL</td></tr><tr><td>FcR Blocking Reagent, human</td><td>Miltenyi Biotec</td><td>130-059-901</td><td>2 mL</td></tr><tr><td>FLOWJO</td><td>BD</td><td></td><td></td></tr><tr><td>Gibco Fetal Bovine Serum</td><td>Thermo Fisher Scientific</td><td>12676029</td><td>500 mL</td></tr><tr><td>Mouse IgG1 monoclonal - Isotype control</td><td>abcam</td><td>ab81200</td><td></td></tr><tr><td>Mouse monoclonal Anti-Human IgG4 Fc</td><td>abcam</td><td>ab99825</td><td>Clone HP6025</td></tr><tr><td>Pacific Blue Mouse Anti-Human CD3</td><td>BD</td><td>558117</td><td>Clone UCHT1</td></tr><tr><td>PE-Cy7 Mouse anti-Human CD279 (PD-1)</td><td>BD</td><td>561272</td><td>Clone EH12.1</td></tr><tr><td>PE-Cy7 Mouse IgG1 &amp;kappa; Isotype Control</td><td>BD</td><td>557646</td><td></td></tr></tbody></table>

monitoring pd-1-blocking antibodies bound to t cells derived from a drop of peripheral blood

Immune checkpoint inhibitors, including PD-1&#8211;blocking antibodies, have significantly improved treatment outcomes in various types of cancer. The pharmacological efficacy of these immunotherapies is long lasting, extending even beyond the discontinuation of their injections, due to persistent blood concentrations. Here we developed a simple flow cytometry assay to evaluate the T cell binding status of the PD-1&#8211;blocking antibodies nivolumab and pembrolizumab. Like a glucose test, this assay requires just a single drop of peripheral blood. Visualizing antibody binding on T cells is more reliable than measuring antibody blood concentrations. In addition, if necessary, we can potentially analyze many distinctive immune-related markers on T cells bound to PD-1&#8211;blocking antibodies. Thus, this is a simple and minimally invasive strategy to analyze the pharmacological effect of PD-1&#8211;blocking antibodies in cancer patients.

The gating strategy and flow cytometry analysis (Figure 1) can detect PD-1&#8211;blocking antibody binding to T cells obtained from a drop of NSCLC patient peripheral blood. Before PD-1&#8211;blocking antibody is administered, no human IgG4-positive CD8 or CD4 T cells are present, and PD-1 expression can be confirmed by a PD-1&#8211;detecting antibody (EH12.1) (Figure 2A). After nivolumab or pembrolizumab administration, IgG4 (nivolumab, pembrolizumab) can be detected on T cells by anti-IgG4 antibody (HP6025) whereas the PD-1&#8211;detecting antibody EH12.1 does not recognize any PD-1 on T cells because therapeutic PD-1 antibodies disturb EH12.1 binding. This means that we are indirectly measuring the therapeutic binding of PD-1&#8211;blocking antibody based on the lack of binding of PD-1&#8211;detecting antibody. Representative data show the different binding statuses of PD-1&#8211;blocking antibody (Figure 2A). Nivolumab and pembrolizumab binding and occupancy of PD-1 on T cells decrease over time10, and there is partial binding (PB), which is shown in the double-positive area, and finally complete loss of binding (LB) (Figure 2B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60608/60608fig01.jpg" /> 
Figure 1: Representative gating strategy to evaluate PD-1&#8211;blocking antibody binding to T cells from a drop of peripheral blood. (A) FSC (A) vs. SSC (A) plot and gating of lymphocytes. (B) Doublets are excluded by drawing gates around the main cell population on plots of FSC (H) vs. FSC (W) and SSC (H) vs. SSC (W). (C) CD3 vs. CD8 plot (upper) and CD3 vs. CD4 plot (lower) and gating of CD8 T cells and CD4 T cells, respectively. (D) PD-1 vs. human IgG4 plot and detection of the binding of nivolumab (a PD-1&#8211;blocking antibody) to CD8 and CD4 T cells. Orange dots and black dots indicate anti-IgG4 antibody and isotype control staining, respectively. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60608/60608fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60608/60608fig2a.jpg" /> 
Figure 2: Representative flow cytometry analysis indicating the change in binding status of PD-1&#8211;blocking antibodies. (A) Staining of PD-1 and human IgG4 in CD8 T cells from ICI-pretreated patient blood was evaluated by flow cytometry (left). Ten microliters of pretreated blood was treated with serial dilution of nivolumab for 15 min, and steps 1.4 to 1.10 of the protocol were completed. Complete binding (CB) (red), partial binding (PB) (blue), and loss of binding (LB) (green) are defined by the indicated gates. (B) The status of PD-1&#8211;blocking antibody binding to CD8 T cells was analyzed at the follow-up time points, as indicated, in NSCLC patients who discontinued nivolumab and pembrolizumab. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60608/60608fig2alarge.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></td>
			<td>PE</td>
			<td>BV421</td>
			<td>PE-Cy7</td>
			<td>APC-Cy7</td>
			<td>BV510</td>
		</tr>
		<tr>
			<td>Binding evaluation</td>
			<td>IgG4</td>
			<td>CD3</td>
			<td>PD-1</td>
			<td>CD4</td>
			<td>CD8</td>
		</tr>
		<tr>
			<td>Isotype control</td>
			<td>Isotype control</td>
			<td>CD3</td>
			<td>Isotype control</td>
			<td>CD4</td>
			<td>CD8</td>
		</tr>
	</tbody>
</table>
Table 1: Antibodies used in flow cytometric analysis.

Watch this Scientific Journal Video about Monitoring PD-1-Blocking Antibodies Bound to T Cells Derived from a Drop of Peripheral Blood at JoVE.com

Monitoring PD-1-Blocking Antibodies Bound to T Cells Derived from a Drop of Peripheral Blood

We developed a simple flow cytometry assay for evaluating the binding of PD-1&#8211;blocking antibodies to T cells, requiring only a drop of peripheral blood from cancer patients.

Immune checkpoint inhibitors, including PD-1&#8211;blocking antibodies, have significantly improved treatment outcomes in various types of cancer. The ...

monitoring-pd-1-blocking-antibodies-bound-to-t-cells-derived-from

Nanyang Technological University

Research

JoVE Journal

Medicine

5.6K Views.  Osaka University Graduate School of Medicine. We developed a simple flow cytometry assay for evaluating the binding of PD-1–blocking antibodies to T cells, requiring only a drop of peripheral blood from cancer patients.

Video: Monitoring PD-1-Blocking Antibodies Bound to T Cells Derived from a Drop of Peripheral Blood

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 ...

Immunology and Infection

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are upregulated upon this ongoing antigen exposure and contribute to loss of proliferation, reduced cytolytic function, and impaired cytokine production by CD4 and CD8 T cells. In the murine model of LCMV infection, administration of blocking antibodies against these two pathways augmented T cell responses. However, there is currently no in vitro assay to measure the impact of such blockade on cytokine secretion in cells from human samples. Our protocol and experimental approach enable us to accurately and efficiently quantify the restoration of cytokine production by HIV-specific CD4 T cells from HIV infected subjects.
Here, we depict an in vitro experimental design that enables measurements of cytokine secretion by HIV-specific CD4 T cells and their impact on other cell subsets. CD8 T cells were depleted from whole blood and remaining PBMCs were isolated via Ficoll separation method. CD8-depleted PBMCs were then incubated with blocking antibodies against PD-L1 and/or IL-10R&#945; and, after stimulation with an HIV-1 Gag peptide pool, cells were incubated at 37 &#176;C, 5% CO2. After 48 hr, supernatant was collected for cytokine analysis by beads arrays and cell pellets were collected for either phenotypic analysis using flow cytometry or transcriptional analysis using qRT-PCR. For more detailed analysis, different cell populations were obtained by selective subset depletion from PBMCs or by sorting using flow cytometry before being assessed in the same assays. These methods provide a highly sensitive and specific approach to determine the modulation of cytokine production by antigen-specific T-helper cells and to determine functional interactions between different populations of immune cells.

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

We developed an in vitro assay to investigate the role of immunoregulatory pathways in the regulation of cytokine secretion by HIV-specific CD4 T cells.

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are ...

Flow Analysis of PDL1-Vaxx Antibody Blockade Using Magnetic Fluorescent Beads

Magnetic Fluorescent Bead-Based Dual-Reporter Flow Analysis of PDL1-Vaxx Peptide Vaccine-Induced Antibody Blockade of the PD-1/PD-L1 Interaction

The inhibition of checkpoint receptors (PD-1, PD-L1, and CTLA-4) with monoclonal antibodies has shown great benefit in clinical trials for treating cancer patients and has become a mainstay approach in modern cancer immunotherapy. However, only a subset of patients respond to checkpoint monoclonal antibody immunotherapy. Therefore, it is urgent to develop new therapeutic strategies against cancer. A novel B-cell peptide epitope PDL1 (programmed death ligand 1) cancer vaccine has been developed, with amino acids 130-147 linked to the MVF peptide (&#34;promiscuous&#34; T-cell measles virus fusion protein) via a GPSL linker. Preclinical testing has indicated that this PDL1 vaccine (PDL1-Vaxx) effectively stimulates highly immunogenic antibodies in animals. Animals immunized with PDL1-Vaxx show reduced tumor burden and extended survival rates in various animal cancer models. The mechanisms of action indicate that vaccine-elicited antibodies inhibit tumor cell proliferation, induce apoptosis, and block the PD-1/PD-L1 interaction. This manuscript introduces a magnetic bead-based assay that uses a dual-reporter flow analysis system to evaluate the PD-1/PD-L1 interaction and its blockade by the anti-PDL1 antibodies raised against the PDL1-Vaxx.

Author Spotlight: Magnetic Fluorescent Bead-Based Dual-Reporter Flow Analysis of PDL1-Vaxx Peptide Vaccine-Induced Antibody Blockade of the PD-1/PD-L1 Interaction 

Checkpoint inhibitors are important targets in developing therapies for the battle against cancer. This report introduces a novel PDL1 peptide-based cancer vaccine, PDL1-Vaxx, which induces neutralizing polyclonal antibody production that blocks PD-1/PDL1 complex formation. This work also details the development and testing of a fluorescent bead-based assay for analyzing this activity.

The inhibition of checkpoint receptors (PD-1, PD-L1, and CTLA-4) with monoclonal antibodies has shown great benefit in clinical trials for treating cancer ...

Cancer Research

Detection of Antibodies That Neutralize the Cellular Uptake of Enzyme Replacement Therapies with a Cell-based Assay

The administration of enzyme replacement therapies (ERTs) and other biologic therapies to patients may elicit an anti-drug immune response. The characterization of these anti-drug antibodies (ADA), especially those that may neutralize the biological activity of the drug, termed neutralizing antibodies (NAbs), is crucial in understanding the effects of these antibodies on the drug&#39;s pharmacological profile. This protocol describes a cell-based flow cytometry method to detect factors that neutralize the cellular uptake of a representative lysosomal ERT in human matrix. The protocol consists of three procedures: screening, a confirmatory step, and titer assays to detect, identify, and establish the relative level of neutralizing antibody titer in subject samples.
In this method, samples are first mixed with the fluorophore-conjugated ERT product, then incubated with cells [e.g., human T lymphocytes (Jurkat cells)] that express a cell-surface cation-independent mannose 6-phosphate receptor (CI-M6PR), and finally, analyzed with a flow cytometer. A sample without NAbs will result in the uptake of the fluorophore-conjugated ERT product via CI-M6PR, whereas, the presence of NAbs will bind to the drug and interfere with the CI-M6PR binding and uptake. The amount of the fluorophore-conjugated ERT internalized by the Jurkat cells is measured by flow cytometry and evaluated as the percentage (%) signal inhibition compared to the response obtained in the presence of a representative drug-na&#239;ve matrix. In the confirmatory step, the samples are pre-incubated with ERT-conjugated magnetic beads to deplete drug-specific factors that bind to the drug (such as NAbs) prior to an incubation with cells. Samples that screen and confirm positive for drug-specific NAbs in the assay are then serially diluted to generate an antibody titer. Semi-quantitative antibody titers may be correlated with measurements of drug safety and efficacy.

Here we present a cell-based flow cytometry method to detect neutralizing antibodies or other factors that interfere with the cellular uptake of enzyme replacement therapies in a human matrix, such as cerebral spinal fluid (CSF) or human serum.

The administration of enzyme replacement therapies (ERTs) and other biologic therapies to patients may elicit an anti-drug immune response. The ...

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of glass-supported planar bilayers and in vitro-derived T-cell clones or lines1,2,3,4. How these findings apply to the primary human T cells isolated from blood or lymphoid tissues is not known, partly due to significant difficulties in obtaining a sufficient number of cells for analysis5. Here we address this through the development of a technique exploiting multichannel flow slides to build planar lipid bilayers containing activating and adhesion molecules. The low height of the flow slides promotes rapid cell sedimentation in order to synchronize cell:bilayer attachment, thereby allowing researchers to study the dynamic of the synaptic interface formation and the kinetics of the granules release. We apply this approach to analyze the synaptic interface of as few as 104 to 105 primary cryopreserved T cells isolated from lymph nodes (LN) and peripheral blood (PB). The results reveal that the novel planar lipid bilayer technique enables the study of the biophysical properties of primary human T cells derived from blood and tissues in the context of health and disease.

The protocol describes a technique to study the ability of primary polyclonal human T cells to form synaptic interfaces using planar lipid bilayers. We use this technique to show the differential synapse formation capability of human primary T cells derived from lymph nodes and peripheral blood.

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of ...

Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes

Receptor-associated enzymes are the major mediators of cellular activation. These enzymes are regulated, at least in part, by physical interactions with cytoplasmic tails of the receptors. The interactions often occur through specific protein domains and result in activation of the enzymes. There are several methods to study interactions between proteins. While co-immunoprecipitation is commonly used to study domains that are required for protein-protein interactions, there are no assays that document the contribution of specific domains to activity of the recruited enzymes at the same time. Accordingly, the method described here combines co-immunoprecipitation and an on-bead enzymatic activity assay for simultaneous evaluation of interactions between proteins and associated enzymatic activation. The goal of this protocol is to identify the domains that are critical for physical interactions between a protein and enzyme and the domains that are obligatory for complete activation of the enzyme. The importance of this assay is demonstrated, as certain receptor protein domains contribute to the binding of the enzyme to the cytoplasmic tail of the receptor, while other domains are necessary to regulate the function of the same enzyme.

Here, we present a protocol for co-immunoprecipitation and an on-bead enzymatic activity assay to simultaneously study the contribution of specific protein domains of plasma membrane receptors to both enzyme recruitment and enzyme activity.

Receptor-associated enzymes are the major mediators of cellular activation. These enzymes are regulated, at least in part, by physical interactions with ...

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte (CTL) responses elicited against tumor- and pathogen-derived peptides. They offer several advantages over traditional killing assays. First, they permit the monitoring of CTL-mediated cytotoxicity within architecturally intact secondary lymphoid organs, typically in the spleen. Second, they allow for mechanistic studies during the priming, effector and recall phases of CTL responses. Third, they provide useful platforms for vaccine/drug efficacy testing in a truly in vivo setting. Here, we provide an optimized protocol for the examination of concomitant CTL responses against more than one peptide epitope of a model tumor antigen (Ag), namely, simian virus 40 (SV40)-encoded large T Ag (T Ag). Like most other clinically relevant tumor proteins, T Ag harbors many potentially immunogenic peptides. However, only four such peptides induce detectable CTL responses in C57BL/6 mice. These responses are consistently arranged in a hierarchical order based on their magnitude, which forms the basis for TCD8 &#8220;immunodominance&#8221; in this powerful system. Accordingly, the bulk of the T Ag-specific TCD8 response is focused against a single immunodominant epitope while the other three epitopes are recognized and responded to only weakly. Immunodominance compromises the breadth of antitumor TCD8 responses and is, as such, considered by many as an impediment to successful vaccination against cancer. Therefore, it is important to understand the cellular and molecular factors and mechanisms that dictate or shape TCD8 immunodominance. The protocol we describe here is tailored to the investigation of this phenomenon in the T Ag immunization model, but can be readily modified and extended to similar studies in other tumor models. We provide examples of how the impact of experimental immunotherapeutic interventions can be measured using in vivo cytotoxicity assays.

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

We describe here a flow cytometry-based in vivo killing assay that enables examination of immunodominance in cytotoxic T lymphocyte (CTL) responses to a model tumor antigen. We provide examples of how this elegant assay may be employed for mechanistic studies and for drug efficacy testing.

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte ...

Profiling of the Human Natural Killer Cell Receptor-Ligand Repertoire

Natural killer (NK) cells are among the first responders to viral infections. The ability of NK cells to rapidly recognize and kill virally infected cells is regulated by their expression of germline-encoded inhibitory and activating receptors. The engagement of these receptors by their cognate ligands on target cells determines whether the intercellular interaction will result in NK cell killing. This protocol details the design and optimization of two complementary mass cytometry (CyTOF) panels. One panel was designed to phenotype NK cells based on receptor expression. The other panel was designed to interrogate expression of known ligands for NK cell receptors on several immune cell subsets. Together, these two panels allow for the profiling of the human NK cell receptor-ligand repertoire. Furthermore, this protocol also details the process by which we stain samples for CyTOF. This process has been optimized for improved reproducibility and standardization. An advantage of CyTOF is its ability to measure over 40 markers in each panel, with minimal signal overlap, allowing researchers to capture the breadth of the NK cell receptor-ligand repertoire. Palladium barcoding also reduces inter-sample variation, as well as consumption of reagents, making it easier to stain samples with each panel in parallel. Limitations of this protocol include the relatively low throughput of CyTOF and the inability to recover cells after analysis. These panels were designed for the analysis of clinical samples from patients suffering from acute and chronic viral infections, including dengue virus, human immunodeficiency virus (HIV), and influenza. However, they can be utilized in any setting to investigate the human NK cell receptor-ligand repertoire. Importantly, these methods can be applied broadly to the design and execution of future CyTOF panels.

Here we design two complementary mass cytometry (CyTOF) panels and optimize a CyTOF staining protocol with the aim of profiling the natural killer cell receptor and ligand repertoire in the setting of viral infections.

Natural killer (NK) cells are among the first responders to viral infections. The ability of NK cells to rapidly recognize and kill virally infected cells ...

Preparation of Peripheral Blood Mononuclear Cell Pellets and Plasma from a Single Blood Draw at Clinical Trial Sites for Biomarker Analysis

Analysis of biomarkers in peripheral blood is becoming increasingly important in clinical trials to establish proof of mechanism to evaluate effects of treatment, and help guide dose and schedule setting of therapeutics. From a single blood draw, peripheral blood mononuclear cells can be isolated and processed to analyze and quantify protein markers, and plasma samples can be used for the analysis of circulating tumor DNA, cytokines, and plasma metabolomics. Longitudinal samples from a treatment provide information on the evolution of a given protein marker, the mutational status and immunological landscape of the patient. This can only be achieved if the processing of the peripheral blood is carried out effectively in clinical sites and samples are properly preserved from the bedside to bench. Here, we present an optimized general-purpose protocol that can be implemented at clinical sites for obtaining PBMC pellets and plasma samples in multi-center clinical trials, that will enable clinical professionals in hospital laboratories to successfully provide high quality samples, regardless of their level of technical expertise. Alternative protocol variations are also presented that are optimized for more specific downstream analytical methods. We apply this protocol for studying protein biomarkers against DNA damage response (DDR) on X-ray irradiated blood to demonstrate the suitability of the approach in oncology settings where DDR drugs and/or radiotherapy have been practiced as well as in preclinical stages where mechanistic hypothesis testing is required.

This protocol details clinically implementable preparation of high quality PBMC and plasma biosamples at the clinical trial site that can be used for translational biomarker analysis.

Analysis of biomarkers in peripheral blood is becoming increasingly important in clinical trials to establish proof of mechanism to evaluate effects of ...

Immunoprecipitation Assay to Study Enzyme Receptor Interactions in Transfected Cells

Source: Strazza, M., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/58433">Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes.</a> J. Vis. Exp. (2018).
In this video, we demonstrate a technique to perform the co-immunoprecipitation of GFP-tagged wild-type and mutated PD-1 proteins using beads coated with anti-GFP antibodies. The bead-bound PD-1-GFP proteins are incubated with SHP2 proteins that interact with PD-1, enabling the identification of specific structural motifs in PD-1 that play a critical role in SHP2 interaction.

Source: Strazza, M., et al. Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes. J. Vis. Exp. (2018).
In this ...

Monitoring PD-1-Blocking Antibodies Bound to T Cells Derived from a Drop of Peripheral Blood

Summary

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Chapters in this video

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

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

Magnetic Fluorescent Bead-Based Dual-Reporter Flow Analysis of PDL1-Vaxx Peptide Vaccine-Induced Antibody Blockade of the PD-1/PD-L1 Interaction

Detection of Antibodies That Neutralize the Cellular Uptake of Enzyme Replacement Therapies with a Cell-based Assay

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue

Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8⁺ T Cell Responses

Profiling of the Human Natural Killer Cell Receptor-Ligand Repertoire

Preparation of Peripheral Blood Mononuclear Cell Pellets and Plasma from a Single Blood Draw at Clinical Trial Sites for Biomarker Analysis

Immunoprecipitation Assay to Study Enzyme Receptor Interactions in Transfected Cells