Supported by the National Institutes of Health (U19 CA100625 and R21 CA137645). JKD was supported by the Leukemia &amp; Lymphoma Society and the American Society of Blood and Marrow Transplantation/OtsukaNew Investigator Award.

1. Preparation of PBMC
<ol>
	<li>Procedures for good aseptic technique and universal precautions must be adhered to during the conduct of this protocol.</li>
	<li>Isolate PBMC from healthy volunteer donors by density gradient centrifugation using Ficoll-Hypaque. Alternatively cryopreserved PBMC can be resuscitated.</li>
	<li>Count viable PBMC after staining with Trypan Blue using a hemocytometer. Resuspend PBMC in complete culture media (CM) at a concentration of 1 million viable cells/mL in a 15mL Falcon tube.</li>
</ol>
2. Setting Up the Bulk Alloanergizing Co-culture
<ol>
	<li>PBMCs from two separate donors are needed to set up the alloanergization cultures. Cells from one donor will serve as the responder and cells from another donor will serve as the stimulator (termed the First Party stimulator).</li>
	<li>Place 10 million stimulator PBMC at 1 million/ml in a 15 mL Falcon tube and add 100mg of anti-B7.1 and 100mg of anti B7.2 antibodies to block costimulation. Gently agitate the tube and incubate for 30 minutes. Incubation conditions for this and all subsequent steps are 37&deg;C/5% CO2/80% humidity</li>
	<li>Irradiate stimulator PBMC (33 Gy) and add to a T-25 50cm3 cell culture flask with a gas-permeable cap. Label the flask "Bulk alloanergizing co-culture".</li>
	<li>Add 10mL of responder PBMC (at 1 million/ml in CM) to the flask.</li>
	<li>Add a further 100mg each of anti-B7.1 and anti B7.2 antibodies and mix gently prior to placing the flask upright in an incubator for 72 hours</li>
</ol>
3. Setting Up the Bulk Control Co-culture
<ol>
	<li>Place 10 million stimulator PBMC (at 1 million/ml) in a 15mL Falcon tube.</li>
	<li>Irradiate 10 million stimulator PBMC (33 Gy).and add to a 50cm3 cell culture flask. Label "Bulk control co-culture".</li>
	<li>Add 10mL of responder PBMC at 1 million/ml in CM to the flask and mix gently prior to placing the flask upright in an incubator for 72 hours.</li>
</ol>
4. Setting Up the Primary Mixed Lymphocyte Reaction (MLR) to Measure the Efficacy of Co-stimulatory Blockade
<ol>
	<li>The efficacy of costimulatory blockade is measured in a primary MLR which is set up using PBMC from the bulk alloanergizing and control co-cultures</li>
	<li>The primary MLR is set up on the same day that the bulk cultures have been set up</li>
	<li>First label one U bottomed 96 well plate "Primary MLR" for each of the three time points to be measured (Day 4, 5 and 6 after plates are set up).</li>
	<li>Decant 5mL from each of the bulk control and alloanergizing co-cultures into 15mL Falcon tubes.</li>
	<li>Pipette 200mL of cell suspension from each Falcon tube into triplicate wells on each plate. Label these wells "no costimulatory blockade (CSB)" for the cells from the bulk control co-cultures and "CSB" for the cells from the bulk alloanergizing co-cultures</li>
	<li>Add 200mL of CM to 6 wells on each plate as negative controls. 200mL PBS is added to all empty wells to reduce evaporation. Place plates in the incubator.</li>
</ol>
5. Setting Up the Secondary MLR to Measure the Efficacy of Alloanergization
<ol>
	<li>72 hours after setting up the bulk co-cultures, residual alloresponses in alloanergized PBMCs are measured in a secondary MLR. In the secondary MLR, PBMC from both the bulk alloanergizing co-culture and the bulk control co-culture are washed and restimulated with irradiated allostimulator PBMC</li>
	<li>To set up the secondary MLR label one U bottomed 96 well plate "Secondary MLR" for each of the three time points to be measured (Day 3, 5 and 7 after set up).</li>
	<li>Remove 5mL from each of the bulk control and alloanergizing co-cultures, transfer to 15mL Falcon tubes and centrifuge for 5-10 minutes at 2000 rpm.Aspirate the supernatant carefully, disrupt the pellet, add 10mL of CM and centrifuge the cells again. Repeat this washing step.</li>
	<li>Resuspend the cells in 1mL of CM. Count the viable cells using Trypan Blue staining, and adjust the cell concentration to 1 million viable responder cells/mL.</li>
	<li>Pipette 100mL of cell suspension from each Falcon tube into triplicate wells of each plate. Label these wells "First Party (FP) allorestimulation"</li>
	<li>Pipette 100mL of cell suspension from each Falcon tube into a further 3 wells on each plate and label these wells "CD3/28 stimulation"</li>
	<li>To prepare stimulator cells for the secondary MLR, isolate PBMC from fresh blood (or resuscitate cryopreserved PBMC) from the same healthy donor used to supply first party stimulator PBMCs in the alloanergizing and control cultures.</li>
	<li>Suspend PBMC from the FP donor at a concentration of 1 million/mL and irradiate (33Gy)</li>
	<li>Add 100mL of irradiated stimulator cells to wells labeled "FP allorestimulation"</li>
	<li>For positive controls, add 1 mL each of CD3 and CD28 monoclonal antibodies and 98mL of CM to wells labeled "CD3/28 stimulation". Add 200 mL of CM to 6 wells on each plate as a negative control and 200mL of PBS to empty wells. Place the plates in the incubator.</li>
</ol>
6. Measuring the Specificity of Alloanergization
<ol>
	<li>The specificity of alloanergization is determined by comparing proliferation of responder cells taken from the completed alloanergizing and control co-cultures after stimulation with irradiated PBMC obtained from a "third party" healthy donor (i.e. a different donor to the First Party) in a secondary MLR.</li>
	<li>Label three 96 well plates "Secondary MLR Specificity" Day 3, 5 and 7.</li>
	<li>Remove 5mL from each of the bulk control and alloanergizing co-cultures transfer to 15mL Falcon tubes and centrifuge for 5-10 minutes at 2000 rpm. Aspirate the supernatant carefully, disrupt the pellet, add 10mL of CM and centrifuge the cells again. Repeat this washing step.</li>
	<li>Resuspend the cells in 1mL of CM. count using Trypan Blue staining, and adjust the cell concentration to 1 million viable responder cells/mL.</li>
	<li>To prepare third party stimulator cells for the secondary MLR, isolate PBMC from fresh blood (or resuscitate cryopreserved PBMC) from a new healthy donor. Pipette 100mL of cell suspension from each Falcon tube into triplicate wells of each plate. Label these wells "TP allostimulation"</li>
	<li>Suspend PBMC from a TP donor at 1 million/mL and irradiate (33 Gy)</li>
	<li>Add 200mL of irradiated TP stimulator cells to wells labeled "TP allostimulation"</li>
	<li>The specificity of alloanergization may also be determined by comparing proliferation of responder cells taken from the completed alloanergizing and control co-cultures after stimulation with an infectious pathogen such as CMV. For this step it is essential to use responder PBMC from donors with previously demonstrated proliferative responses to CMV lysate.</li>
	<li>Label three 96 well plates "Secondary MLR CMV" Day 3, 5 and 7.</li>
	<li>To prepare the responder cells for the secondary MLR to measure CMV responses, transfer 5 ml from each of the bulk control and alloanergizing co-cultures to 15 ml tubes and wash, count and resuspend at 1 million cells/ml in CM as previously described.Pipette 100mL of cell suspension from each Falcon tube into a 3 wells on each plate.</li>
	<li>Add 0.1 mL of CMV lysate in 100ul CM to wells</li>
	<li>Add 200 mL of CM to 6 wells on all plate as a negative control and add 200mL of PBS to empty wells. Place the plates in the incubator.</li>
</ol>
7. Measuring Proliferation in the Primary and Secondary MLRs
<ol>
	<li>Responder cell proliferation is measured by thymidine incorporation assay in the primary and secondary MLRs. Proliferation is measured at Day 4, 5 and 6 after the Primary MLR plates are set up and at Day 3, 5 and 7 after the Secondary MLR plates are set up.</li>
	<li>1mCi of tritiated thymidine is added to wells 16 hours prior to harvesting</li>
	<li>After the addition of tritiated thymidine, the plate is incubated for a further16 hours then harvested onto a filter mat using a Tomtec harvester (or similar). Air-dry the filtermat for one hour then place in a sample bag, add 5 mL scintillation fluid and seal. Read the plate in a Wallac Micro beta scintillation counter or similar.</li>
</ol>
8. Calculating the Efficacy of Co-stimulatory Blockade in the Primary MLR
<ol>
	<li>The percentage inhibition (PI) of primary alloresponses is calculated as 100 x [mean cpm in allostimulation wells (bulk alloanergy co-culture PBMC) - mean cpm in allostimulation wells (bulk control co-culture PBMC)} 
<img src="/files/ftp_upload/2673/2673eq1.jpg" alt="Equation 1" /></li>
</ol>
9. Calculating the Efficacy of Alloanergization in the Secondary MLR
The PI of secondary alloresponses is calculated as 
<img src="/files/ftp_upload/2673/2673eq2.jpg" alt="Equation 2" />
10. Calculating the Specificity of Alloanergization in the Secondary MLR
<ol>
	<li>After restimulation of cells with CD3 and CD28, TP allostimulators or CMV antigen, the PI of responses to these stimuli may also be calculated using this formula. This gives a measure of the specificity of the alloanergization process</li>
</ol>
11. Generating Alloanergized Donor PBMC for Clinical Use After Allogeneic Hematopoietic Stem Cell Transplantation (HSCT)
<ol>
	<li>Alloanergized donor PBMC may be generated for clinical use after HLA-mismatched allogeneic HSCT using the protocol outlined above</li>
	<li>When generating cells for clinical use, responder PBMC are obtained from the HSCT donor and stimulator PBMC from the HSCT recipient prior to transplant.</li>
	<li>When generating PBMC for clinical use, all steps are performed under Good Manufacturing Process conditions in accredited stem cell processing facilities.</li>
	<li>Additional quality control assays are performed including endotoxin assays and gram stain and culture to meet safety criteria prior to the release of cells for clinical use</li>
</ol>
12. Representative Results
Using HLA-mismatched stimulator and responder PBMC, the presence of costimulatory blockade in the primary MLR reduces mean alloproliferation of responder PBMC at Day 5 to around 30% (+/- 10%, mean +/- standard deviation) of that seen in control primary MLR in the absence of costimulatory blockade. This is equivalent to a mean inhibition of primary alloproliferation of around 70% (+/- 10%) at D5, Figure 1 A and B.
In the secondary MLR at Day 5, FP alloproliferation of alloanergized PBMC is typically 10-15% (+/- 10%) of that seen with control PBMC equivalent to a mean inhibition of proliferation of 85-90% (+/- 10%), Figure 2A and B. This demonstrates that alloanergized PBMC are hyporesponsive to FP allostimulators.
In contrast alloanergized PBMC typically retain 70-100% of their proliferation to mitogens (CD3/CD28 antibodies), TP allostimulators and CMV lysate (in CMV-reactive donors). This demonstrates that hyporesponsiveness of alloanergized PBMC is specific to FP alloantigens.
<img src="/files/ftp_upload/2673/2673fig1.jpg" alt="Figure 1" /> 
Figure 1. A.Proliferation (determined by thymidine incorporation) in a Primary Mixed Lymphocyte Reaction using HLA-mismatched stimulator and responder peripheral blood mononuclear cells (PBMC) in the absence or the presence of costimulatory blockade using humanized monoclonal anti-B7.1 and -B7.2 antibodies. Results are shown as mean (+/-sd) for 8 representative experiments using unique stimulator-responder pairs. B. Percentage inhibition of alloproliferation in primary MLRs performed in the presence of costimulatory blockade using humanized monoclonal anti-B7.1 and -B7.2 antibodies. Results are shown as mean (+/-sd) for the same 8 unique stimulator-responder pairs depicted in Figure 1A.
<img src="/files/ftp_upload/2673/2673fig2.jpg" alt="Figure 2" /> 
Figure 2. A. Proliferation (determined by thymidine incorporation) in secondary MLRs where responder PBMC from primary MLRs performed in the absence (control PBMC) or the presence of costimulatory blockade (alloanergized PBMC) are restimulated with irradiated first party stimulators. Results are shown as mean (+/-sd) for the 8 unique stimulator-responder pairs depicted in Figure 1. B. Percentage inhibition of First Party alloproliferation in secondary MLRs where responder PBMC from primary MLRs performed in the absence (control PBMC) or the presence of costimulatory blockade (alloanergized PBMC) are restimulated with irradiated first party stimulators. Results are shown as mean (+/-sd) for the 8 unique stimulator-responder pairs depicted in Figure 1 and Figure 2A.

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	<li>Ferrara, J. L., Levy, R., Chao, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiologic+mechanisms+of+acute+graft-vs.-host+disease.">Pathophysiologic mechanisms of acute graft-vs.-host disease.</a> Biol Blood Marrow Transplant. 5, 347-356 (1999).</li><li>Keever, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+reconstitution+following+bone+marrow+transplantation:++Comparison+of+recipients+of+T-cell+depleted+marrow+with+recipients+of+conventional+marrow+grafts.">Immune reconstitution following bone marrow transplantation: Comparison of recipients of T-cell depleted marrow with recipients of conventional marrow grafts.</a> Blood. 73, 1340-1350 (1989).</li><li>Bacigalupo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+risk+of+leukemia+relapse+with+high-dose+cyclosporine+A+after+allogeneic+marrow+transplantation+for+acute+leukemia.">Increased risk of leukemia relapse with high-dose cyclosporine A after allogeneic marrow transplantation for acute leukemia.</a> Blood. 77, 1423-1428 (1991).</li><li>Horowitz, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Graft-versus-leukemia+reactions+after+bone+marrow+transplantation.">Graft-versus-leukemia reactions after bone marrow transplantation.</a> Blood. 75, 555-562 (1990).</li><li>Andre-Schmutz, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+reconstitution+without+graft-versus-host+disease+after+haemopoietic+stem-cell+transplantation:+a+phase+1/2+study.">Immune reconstitution without graft-versus-host disease after haemopoietic stem-cell transplantation: a phase 1/2 study.</a> Lancet. 360, 130-137 (2002).</li><li>Amrolia, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adoptive+immunotherapy+with+allodepleted+donor+T-cells+improves+immune+reconstitution+after+haploidentical+stem+cell+transplantation.">Adoptive immunotherapy with allodepleted donor T-cells improves immune reconstitution after haploidentical stem cell transplantation.</a> Blood. 108, 1797-1808 (2006).</li><li>Amrolia, P. 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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+blockade+of+B7+family-mediated+costimulation+is+necessary+to+induce+human+alloantigen-specific+anergy:+a+method+to+ameliorate+graft-versus-host+disease+and+extend+the+donor+pool.">Complete blockade of B7 family-mediated costimulation is necessary to induce human alloantigen-specific anergy: a method to ameliorate graft-versus-host disease and extend the donor pool.</a> Blood. 87, 4887-4893 (1996).</li><li>Davies, J. K., Yuk, D., Nadler, L. M., Guinan, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+alloanergy+in+human+donor+T+cells+without+loss+of+pathogen+or+tumor+immunity.">Induction of alloanergy in human donor T cells without loss of pathogen or tumor immunity.</a> Transplantation. 86, 854-864 (2008).</li><li>Davies, J. 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No conflicts of interest declared.

The induction of alloantigen-specific hyporesponsiveness, or anergy, in donor PBMC via allostimulation in the presence of co-stimulatory blockade is a simple technique for the generation of donor PBMC with reduced alloreactivity. We have developed the process in the laboratory and are currently applying the strategy in the clinic to generate donor PBMC with reduced alloreactivity for infusion after HLA-mismatched allogeneic HSCT. The aim of using such therapy is to improve immune reconstitution without excess toxicity. Although our current clinical application of the strategy is limited to the setting of allogeneic HSCT, the approach could be applied to other settings where tissue damage is caused by unwanted T cell responses, such as rejection of solid organ transplantation or autoimmune conditions.
Several reagents can be used for blockade of CD28-mediated co-stimulatory signals during the alloanergization co-culture process. Here we describe our current protocol using clinical-grade humanized murine monoclonal antibodies directed against the ligands of CD28 (B7.1 and B7.2). Non-clinical grade murine anti-human B7.1 and B7.2 antibodies are commercially available from several manufacturers. Alternatively, the fusion protein Cytotoxic T Lymphocyte Antigen (CTLA) 4-Immunoglobulin (Ig) can be used to block CD28-costimulation during the alloanergization process. CTLA4-Ig, which consists of the extracellular portion of the CTLA4 molecule linked to the IgG Fc gamma receptor, binds with high affinity to B7.1 and B7.2. The use of CTLA4-Ig results in similar efficacy and specificity of alloanergization of human PBMCs.
The technique of alloanergization is simple to perform. Fresh or previously cryopreserved stimulator PBMCs can be used with no significant variation in the outcome of the process. The requirement for only a single relatively brief ex vivo incubation step with no cell sorting procedures minimizes potential for cell death and bacterial contamination of cells prior to infusion which makes the strategy relatively simple to apply at a clinical scale.
One limitation of the approach we describe (and other approaches to selectively reduce alloreactivity of human cells) is the absence of a real-time assay to determine residual alloreactivity. Proliferation assays take 3-5 days to perform to confirm the reduction of alloreactivity and cells generated for clinical use must be infused prior to the results of these assays being available. Furthermore, measurement of proliferation of PBMCs by thymidine incorporation, although easy to perform, measures proliferation of B cells and other cell subsets in addition to T cell proliferation. CFSE dye dilution of responder PBMCs can be used as an alternate assay and permits determination of T cell subset-specific alloproliferation. One way to improve the clinical application of our strategy approach would be to develop and validate an assay of alloreactivity that takes only a few hours to perform which could be performed prior to release of alloanergized cells for clinical use.
In addition to demonstrating the efficiacy of the strategy it is also important to confirm the specificity alloanergization process. This can easily be done by determination of the relative preservation of alloresponses specific to third party allostimulators and when CMV-reactive donor cells are being alloanergized, by the preservation of proliferative responses to CMV

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Equipment</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>Ficoll-Hypaque PLUS</td>
			<td>GE Life Sciences</td>
			<td> &nbsp; 17-1440-02</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Invitrogen</td>
			<td>11875-093</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hepes 1M</td>
			<td>Invitrogen</td>
			<td>15630-080</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;L-Glutamine 200mM</td>
			<td>Invitrogen</td>
			<td>25030-081</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Invitrogen</td>
			<td>15750-060</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Human AB serum (heat inactivated)</td>
			<td>Gemini Bio-Products</td>
			<td>100-512</td>
			<td>Use validated batches</td>
		</tr>
		<tr>
			<td>Complete Culture Media (500ml)</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>440ml RPMI 1640, 50ml AB serum, 5ml Hepes, 5 ml Glutamine, 167uL Gentamicin</td>
		</tr>
		<tr>
			<td>Irradiator</td>
			<td>GammaCell 1000</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>T-25 Cell Culture Flasks with gas permeable caps</td>
			<td>Corning, Inc.</td>
			<td>3056</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Humanized Monoclonal anti-B7.1 and anti B7.2 antibodies</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>See protocol text</td>
		</tr>
		<tr>
			<td>U bottomed 96 well culture plates</td>
			<td>BD Labware</td>
			<td>353077</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Monoclonal CD3 antibody</td>
			<td>Beckman Coulter</td>
			<td>IM0178</td>
			<td>Reconstitute with 200ml distilled water</td>
		</tr>
		<tr>
			<td>Monoclonal CD28 antibody</td>
			<td>Beckman Coulter</td>
			<td>IM1376</td>
			<td>Reconstitute with 200ml distilled water</td>
		</tr>
		<tr>
			<td>CMV grade 2 antigen</td>
			<td>Microbix</td>
			<td>EL-01-02</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tritiated Thymidine</td>
			<td>NEN</td>
			<td>NET027</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Scintillation Fluid</td>
			<td>PerkinElmer, Inc.</td>
			<td>1205-440</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Harvester</td>
			<td>Tomtec</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Printed Filtermat A</td>
			<td>PerkinElmer, Inc.</td>
			<td>1450-421</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Filter Bag</td>
			<td>PerkinElmer, Inc.</td>
			<td>1450-432</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1450 MicroBeta TriLux Scintillation Counter</td>
			<td>Wallac</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

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.

Watch this Scientific Journal Video about Induction of Alloantigen-specific Anergy in Human Peripheral Blood Mononuclear Cells by Alloantigen Stimulation with Co-stimulatory Signal Blockade at JoVE.co

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

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 ...

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Research

JoVE Journal

Immunology and Infection

14.4K Views.  Dana Farber Cancer Institute. 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.

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

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

Expansion of Human Peripheral Blood  &gamma;&delta; T Cells using Zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and anti-infective activity.1 The majority of &gamma;&delta; T cells in peripheral blood have the V&gamma;9V&delta;2 T cell receptor. These cells recognize antigen in a major histocompatibility complex-independent manner and develop strong cytolytic and Th1-like effector functions.1Therefore, &gamma;&delta; T cells are attractive candidate effector cells for cancer immunotherapy. V&gamma;9V&#x03B4;2 T cells respond to phosphoantigens such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which is synthesized in bacteria via isoprenoid biosynthesis;2 and isopentenyl pyrophosphate (IPP), which is produced in eukaryotic cells through the mevalonate pathway.3 In physiological condition, the generation of IPP in nontransformed cell is not sufficient for the activation of &gamma;&delta; T cells. Dysregulation of mevalonate pathway in tumor cells leads to accumulation of IPP and &gamma;&delta; T cells activation.3 Because aminobisphosphonates (such as pamidronate or zoledronate) inhibit farnesyl pyrophosphate synthase (FPPS), the enzyme acting downstream of IPP in the mevalonate pathway, intracellular levels of IPP and sensitibity to &gamma;&delta; T cells recognition can be therapeutically increased by aminobisphosphonates. IPP accumulation is less efficient in nontransfomred cells than tumor cells with a pharmacologically relevant concentration of aminobisphosphonates, that allow us immunotherapy for cancer by activating &gamma;&delta; T cells with aminobisphosphonates. 4 Interestingly, IPP accumulates in monocytes when PBMC are treated with aminobisphosphonates, because of efficient drug uptake by these cells. 5 Monocytes that accumulate IPP become antigen-presenting cells and stimulate V&gamma;9V&delta;2 T cells in the peripheral blood.6 Based on these mechanisms, we developed a technique for large-scale expansion of &gamma;&delta; T cell cultures using zoledronate and interleukin-2 (IL-2).7 Other methods for expansion of &gamma;&delta; T cells utilize the synthetic phosphoantigens bromohydrin pyrophosphate (BrHPP)8 or 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP).9 All of these methods allow ex vivo expansion, resulting in large numbers of &gamma;&delta; T cells for use in adoptive immunotherapy. However, only zoledronate is an FDA-approved commercially available reagent. Zoledronate-expanded &gamma;&delta; T cells display CD27-CD45RA- effector memory phenotype and thier function can be evaluated by IFN-&gamma; production assay. 7

Expansion of Human Peripheral Blood  &amp;#947;&amp;#948; T Cells using Zoledronate

A method to expand &gamma;&delta; T cells from peripheral blood mononuclear cells (PBMC) is described. PBMC-derived &gamma;&delta; T cells are stimulated and expanded using zoledronate and interleukin-2 (IL-2). Large scale expansion of &gamma;&delta; T cells can be applied to autologous cellular immunotherapy of cancer.

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

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 ...

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Flotation-Based T Cell Isolation, Activation, and Expansion from Human Peripheral Blood Mononuclear Cell Samples Using Microbubbles

The process of isolating T cells from peripheral blood mononuclear&#160;cells (PBMCs) to establish ex vivo cultures is crucial for research, clinical testing, and cell-based therapies. In this study, a simple, novel protocol to isolate, activate, and expand T cells from PBMCs ex vivo is presented. This study utilizes functionalized buoyancy-activated cell sorting (BACS) technology to isolate and activate T cells. Briefly, the protocol involves the positive selection of CD3+ cells from leukopak-derived PBMCs, followed by a 48 h co-stimulation with pre-conjugated anti-CD28-bound streptavidin microbubbles (SAMBs) prior to transduction in 24-well plates. Functionalized microbubbles offer a unique opportunity to buoyantly activate cells, leading to proliferative phenotypes that allow for expansion with minimal exhaustion. This technique offers reduced exhaustion because the co-stimulatory microbubbles remain buoyant and return to the top of the culture medium, thus reducing the amount of time that the expanding cells are in contact with the co-stimulatory factors. The results indicate that the isolated and cultured T cells receive enough stimulation to activate and proliferate but not to an extent that leads to overactivation, which then leads to exhaustion, as demonstrated by the presence of excessive PD-1.

The objective of this study is to demonstrate the feasibility of flotation-based separation to isolate, activate, and expand primary human T cells.

The process of isolating T cells from peripheral blood mononuclear&#160;cells (PBMCs) to establish ex vivo cultures is crucial for research, clinical ...

PBMC Collection and Cryostorage for Mitochondrial Bioenergetics Analysis

Cryopreservation and Bioenergetic Evaluation of Human Peripheral Blood Mononuclear Cells

The physiological functions of eukaryotic cells rely on energy mainly provided by mitochondria. Mitochondrial dysfunction is linked to metabolic diseases and aging. Oxidative phosphorylation plays a decisive role, as it is crucial for the maintenance of energetic homeostasis. PBMCs have been identified as a minimally invasive sample to measure mitochondrial function and have been shown to reflect disease conditions. However, measurement of mitochondrial bioenergetic function can be limited by several factors in human samples. Limitations are the amount of samples taken, sampling time, which is often spread over several days, and locations. Cryopreservation of the collected samples can ensure consistent collection and measurement of samples. Care should be taken to ensure that the parameters measured are comparable between cryopreserved and freshly prepared cells. Here, we describe methods for isolating and cryopreserving PBMCs from human blood samples to analyze the bioenergetic function of the mitochondria in these cells. PBMC cryopreserved according to the protocol described here show only minor differences in cell number and viability, adenosine triphosphate levels, and measured respiratory chain activity compared with freshly harvested cells. Only 8-24 mL of human blood is needed for the described preparations, making it possible to collect samples during clinical studies multicentrally and determine their bioenergetics on site.

Author Spotlight: Preservation of Bioenergetic Parameters in Peripheral Blood Mononuclear Cells After Cryopreservation 

Isolated peripheral blood mononuclear cells can be used for the analysis of immune functions and disorders, metabolic diseases, or mitochondrial functions. In this work, we describe a standardized method for the preparation of PBMCs from whole blood and the subsequent cryopreservation. Cryopreservation makes this time and place independent.

The physiological functions of eukaryotic cells rely on energy mainly provided by mitochondria. Mitochondrial dysfunction is linked to metabolic diseases ...