1. Isolation of T cells with microbubbles using positive selection
NOTE: This protocol details a small-scale CD3+ positive selection approach using SAMBs.
<ol>
	<li>Incubate 3 x 108 commercially obtained PBMCs in 2.5 mL of separation buffer with biotinylated anti-CD3 (OKT3) antibody at a concentration of 25 ng of antibody per 1 million cells (25 ng/M). Gently mix by pipetting up and down, and incubate at room temperature for 10 min.</li>
	<li>Add streptavidin microbubbles (SAMBs) at a ratio of 0.5 (SAMB quantity):1 (cell quantity) according to the manufacturer&#39;s reported SAMB concentration.</li>
	<li>Mix using a commercial end-over-end (EOE) rotator at 20 rpm for 10-15 min at room temperature. Centrifuge for 5 min at 400 x g at room temperature.</li>
	<li>After centrifugation, the positively selected cells will be at the top of the suspension with the SAMBs. The remaining non-selected cells will be in the cell pellet at the bottom of the tube. Using a 9 in glass pipette, insert the tip below the bubble-cell layer to the bottom of the tube, aspirate the cell pellet and subnatant with an electronic pipette and transfer them to a new tube.</li>
</ol>
2. Co-stimulation (activation) of the positively selected T cells
<ol>
	<li>Resuspend the bubble-cell layer remaining in the original tube in 1 mL of complete T cell medium (or another desired medium).</li>
	<li>Count the cells in the subnatant with brightfield microscopy using an automated cell counter, and subtract this value from the starting cell number to determine the number of cells captured in the bubble-cell layer.</li>
	<li>Prior to this step, mix the biotinylated anti-CD28 antibody with commercial SAMBs for a minimum of 2 h to create the conjugated anti-CD28 SAMBs. Contact the SAMBs manufacturer to determine the amount of anti-CD28 antibody needed for conjugation. Add the anti-CD28 conjugated SAMBs to the bubble-cell suspension resulting from step 2.1 using a ratio of 1.5 (anti-CD28 SAMBs):1 (cells).</li>
	<li>Mix using EOE rotation for 15 min, and then adjust the total volume to 2 million cells per mL with complete T cell medium or another desired medium according to the cell number obtained in step 2.2.</li>
</ol>
3. Expansion of the co-stimulated cells in cell culture medium
<ol>
	<li>Distribute 1 mL of activated cells from step 2.4 at a concentration of 2 M/mL per well in a 24-well plate. Incubate in a humidified 5% CO2 incubator at 37 &#176;C.</li>
	<li>After 24 h, add 50 U/mL of IL-2 and 25 ng/M of soluble anti-CD3 (OKT3) to further encourage expansion, as calculated using the initial number of cells plated on day 0. Place the cell plate back into the humidified CO2 incubator, and let it incubate overnight at 37 &#176;C.</li>
</ol>
4. Optional: Transduction of activated T cells with lentivirus
NOTE: The approach used here is adapted from Prommersberger et al.10.
<ol>
	<li>Thaw the lentivirus at room temperature, and briefly mix by pipetting.</li>
	<li>Gently remove the mid-natant, 600 &#181;L from each well, without touching the daughter cells that are now at the bottom of the well or the bubble layer that has remained at the surface of the solution.</li>
	<li>Add 5 &#181;g/mL hexadimethrine bromide per well to enhance viral transduction. Add the lentiviral particles at a multiplicity of infection (MOI) of 3 (lentiviral particles per cell).</li>
	<li>Centrifuge the plate for 45 min at 800 x g and 32 &#176;C using slow acceleration and no break for deceleration. Incubate the cells for 4 h in a humidified CO2 incubator at 37 &#176;C.</li>
	<li>After 4 h, add 600 &#181;L of commercially available, fresh complete T cell medium and 50 U/mL of IL-2 to each well, and place the cell plate back into the humidified CO2 incubator at 37 &#176;C for T cell expansion.</li>
</ol>
5. Expansion of the T cells (with or without prior transduction)
<ol>
	<li>Every 2 days, remove half of the medium from the mid-natant, replace it with fresh, complete T cell medium, and add IL-2 at a concentration of 50 U/mL.</li>
	<li>Count the T cells twice per week to assess the cell density. When the cell density exceeds 2 x 106-2.5 x 106 cells/mL, transfer the cells into a bigger vessel, diluting them to 5 x 105 cells/mL.</li>
</ol>
6. Harvesting the T cells and flow cytometry
<ol>
	<li>Gently mix the contents of each well by pipetting up and down. Remove the entire contents of the well, including the microbubbles, and transfer them into a 1.5 mL tube.</li>
	<li>Wash each well with 400 &#181;L of calcium-free and magnesium-free DPBS (&#8722;/&#8722;), and transfer the solution into a 1.5 mL tube. Centrifuge at 400 x g for 5 min at room temperature.</li>
	<li>Aspirate the supernatant, and resuspend the cell pellet in 50 &#181;L of separation buffer.</li>
	<li>Stain with an activation and exhaustion antibody/staining cocktail, and incubate for 10 min at room temperature in the dark. Prepare the staining cocktails as follows- activation cocktail: AF700-CD3, PE/Dazzle-CD4, PE/Cy7-CD8, BV510-CD25, PE-CD69; exhaustion cocktail: AF700-CD3, PE/Dazzle-CD4, PE/Cy7-CD8, PE-PD-1.</li>
	<li>Add 1 mL of separation buffer, and gently mix. Centrifuge at 400 x g for 5 min at room temperature to wash out excess antibodies. Aspirate the supernatant completely.</li>
	<li>Resuspend the cell pellet in 1 mL of separation buffer, and transfer to an appropriate vessel (e.g., a FACS tube, a 96-well plate, etc.) for the flow cytometry analysis. The recommended flow cytometry analysis gating scheme is detailed in Figure 1.</li>
</ol>

<ol>
	<li>Albinger, N., Hartmann, J., Ullrich, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+status+and+perspective+of+CAR-T+and+CAR-NK+cell+therapy+trials+in+Germany.">Current status and perspective of CAR-T and CAR-NK cell therapy trials in Germany.</a> Gene Therapy. 28 (9), 513-527 (2021).</li><li>Tyagarajan, S., Spencer, T., Smith, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+CAR-T+cell+manufacturing+processes+during+pivotal+clinical+trials.">Optimizing CAR-T cell manufacturing processes during pivotal clinical trials.</a> Molecular Therapy. Methods &amp; Clinical Development. 16, 136-144 (2019).</li><li>Stock, S., Schmitt, M., Sellner, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+manufacturing+protocols+of+chimeric+antigen+receptor+T+cells+for+improved+anticancer+immunotherapy.">Optimizing manufacturing protocols of chimeric antigen receptor T cells for improved anticancer immunotherapy.</a> International Journal of Molecular Sciences. 20 (24), 6223 (2019).</li><li>Rohaan, M. W., Wilgenhof, S., Haanen, J. B. A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adoptive+cellular+therapies:+The+current+landscape.">Adoptive cellular therapies: The current landscape.</a> Virchows Archiv. 474 (4), 449-461 (2019).</li><li>Abou-El-Enein, M., 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=Scalable+manufacturing+of+CAR+T+cells+for+cancer+immunotherapy.">Scalable manufacturing of CAR T cells for cancer immunotherapy.</a> Blood Cancer Discovery. 2 (5), 408-422 (2021).</li><li>Poltorak, M. P., 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=Expamers:+A+new+technology+to+control+T+cell+activation.">Expamers: A new technology to control T cell activation.</a> Scientific Reports. 10, 17832 (2020).</li><li>Kagoya, 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=Transient+stimulation+expands+superior+antitumor+T+cells+for+adoptive+therapy.">Transient stimulation expands superior antitumor T cells for adoptive therapy.</a> JCI Insight. 2 (2), 89580 (2017).</li><li>Snow, T., Roussey, J., Wegner, C., McNaughton, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+No.+63/326,446.">Application No. 63/326,446.</a> US Patent. , (2022).</li><li>McNaughton, B., 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=Application+No.+16/004,874.">Application No. 16/004,874.</a> US Patent. , (2018).</li><li>Prommersberger, S., Hudecek, M., Nerreter, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody-based+CAR+T+cells+produced+by+lentiviral+transduction.">Antibody-based CAR T cells produced by lentiviral transduction.</a> Current Protocols in Immunology. 128 (1), 93 (2020).</li><li>Wijewarnasuriya, D., Bebernitz, C., Lopez, A. V., Rafiq, S., Brentjens, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excessive+costimulation+leads+to+dysfunction+of+adoptively+transferred+T+cells.">Excessive costimulation leads to dysfunction of adoptively transferred T cells.</a> Cancer Immunology Research. 8 (6), 732-742 (2020).</li><li>Li, Y., Kurlander, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+anti-CD3+and+anti-CD28-coated+beads+with+soluble+anti-CD3+for+expanding+human+T+cells:+Differing+impact+on+CD8+T+cell+phenotype+and+responsiveness+to+restimulation.">Comparison of anti-CD3 and anti-CD28-coated beads with soluble anti-CD3 for expanding human T cells: Differing impact on CD8 T cell phenotype and responsiveness to restimulation.</a> Journal of Translational Medicine. 8, 104 (2010).</li></ol>

All authors, at the time of submission, are employees of Akadeum Life Sciences, which manufactures and sells microbubble separation products.

The described protocol allows for the isolation of T cells from PBMC samples and the activation of suspended T cells in culture media with microbubbles. This method relies on functionalized microbubbles whose inherent buoyancy offers a unique opportunity to introduce co-stimulatory signals to cells and activate them while they are suspended in a culture medium, thereby reducing the exposure of the expanding cells to prolonged stimulation; such overstimulation can result in the increased expression of T cell exhaustion ma...

More than 500 chimeric antigen receptor (CAR)-T cell therapy clinical trials are currently being conducted across the world, and four CAR-T cell therapy products are available on the market1. However, numerous CAR-T cell research and manufacturing needs still exist that must be addressed to improve the efficacy, scalability, and long-term success of these potentially curative therapies2,3,4,5. Adoptive CAR-T cell clinical research and manufacturing begins with T cell isolation from a peripheral blood sample and the subsequent stimulation, transduction, and expansion of the isolated cells. Parameters such as T cell recovery, purity, and activation/exhaustion signals require careful consideration when choosing the cell isolation and stimulation techniques for CAR-T cell research and manufacturing3,4,6. Importantly, improvement in the therapeutic persistence of CAR-T cell therapies by minimizing the biological impediments that result from the current manufacturing processes, such as T cell exhaustion, is needed to enhance the therapeutic efficacy6,7.
As an alternative to traditional cell isolation methods such as fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS), here, buoyancy-activated cell sorting (BACS) with microbubbles for T cell isolation is demonstrated. Microbubble separation uses buoyant, hollow microspheres (microbubbles) to bind the targets and float them to the surface of fluid samples8,9. By functionalizing microbubbles with antibodies (i.e., anti-CD3), the desired T cell populations can be positively selected from peripheral blood samples. Subsequently, the use of a different population of antibody-functionalized microbubbles (i.e., anti-CD28) to co-stimulate and activate positively selected T cells in suspension is demonstrated in this work. Microbubbles offer a simple and highly tunable isolation and activation workflow that generates T cells ready for suspended cell culture and downstream applications such as genetic modification and expansion. Critically, buoyant cell activation with microbubbles promotes restrained cell stimulation to prevent excessive T cell exhaustion7.
For this study, flow cytometry was the primary tool used to analyze the isolation, activation, and transduction success of the functionalized microbubbles, as well as to provide detailed information about the specific subpopulations present during the growth and expansion phases post-transduction. In addition to flow cytometry, brightfield and fluorescence microscopy were used to confirm the cell health, morphology, and transduction success. Based on these results, the microbubble technology and protocol provide a more tunable and gentler alternative to the traditional isolation and activation methods currently in use today; in particular, microbubble-activated cells show notably lower expression of T cell exhaustion markers than that typically observed with industry-standard tools and kits.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >2-Mercaptoethanol</td>
			<td >Gibco</td>
			<td >21985-023</td>
			<td >CAS: 60-24-2</td>
		</tr>
		<tr >
			<td >Biologix Multi-Well Culture Plates 24-well plates</td>
			<td >VWR&#160;</td>
			<td>76081-560</td>
			<td></td>
		</tr>
		<tr >
			<td >Biotin anti-human CD28 (28.2) Antibody</td>
			<td >Biolegend</td>
			<td >302904</td>
			<td></td>
		</tr>
		<tr >
			<td >Biotin anti-human CD3 (OKT3) Antibody</td>
			<td >Biolegend</td>
			<td >317320</td>
			<td></td>
		</tr>
		<tr >
			<td >DPBS, no calcium, no magnesium</td>
			<td >Gibco</td>
			<td >14190-136</td>
			<td></td>
		</tr>
		<tr >
			<td >GlutaMAX Supplement</td>
			<td >Thermofisher</td>
			<td >35050061</td>
			<td></td>
		</tr>
		<tr >
			<td >Human Recombinant IL2&#160;</td>
			<td >BioVision (vwr)</td>
			<td >10006-122</td>
			<td></td>
		</tr>
		<tr >
			<td >Lentiviral Particle rLV.EF1.zsGreen1-9</td>
			<td >Takara Bio</td>
			<td >0038VCT</td>
			<td></td>
		</tr>
		<tr >
			<td >Leukopak</td>
			<td >BioIVT</td>
			<td>HUMANLMX100-0001129</td>
			<td></td>
		</tr>
		<tr >
			<td >Normal Human PBMCs</td>
			<td >BioIVT</td>
			<td>HUMANHLPB-0002562</td>
			<td></td>
		</tr>
		<tr >
			<td >Penicillin/Streptomycin 100X for tissue culture</td>
			<td >VWR</td>
			<td >97063-708</td>
			<td>CAS: 8025-06-7</td>
		</tr>
		<tr >
			<td >Polybrene Infection/Transfection Reagent</td>
			<td >Millipore Sigma</td>
			<td >TR-1003-G</td>
			<td>CAS:28728-55-4</td>
		</tr>
		<tr >
			<td >Pooled Human AB Serum Plasma Derived Heat Inactivated</td>
			<td >Innovative Research</td>
			<td >ISERABHI100mL</td>
			<td></td>
		</tr>
		<tr >
			<td >RPMI 1640 Medium, GlutaMAX Supplement, HEPES</td>
			<td >Gibco</td>
			<td >72400047</td>
			<td></td>
		</tr>
		<tr >
			<td >Streptavidin Microbubble Kit (includes Akadeum&#39;s separation buffer)</td>
			<td >Akadeum</td>
			<td>11110-000</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

T cells were isolated from purchased PBMCs and plated for activation as described in the protocol. The negative control samples (purchased PBMCs) were not activated. These control samples were included to demonstrate the effect that the microbubble activation process had on the experimental samples as compared to the untouched and unstimulated T cell controls, ensuring that the activation markers observed were the result of the added activation factors and were not inherent to the T cells themselves. As per the experimen...

Watch this Scientific Journal Video about Flotation-Based T Cell Isolation, Activation, and Expansion from Human Peripheral Blood Mononuclear Cell Samples Using Microbubbles at JoVE.com

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

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 ...

flotation-based-t-cell-isolation-activation-expansion-from-human

Research

JoVE Journal

Immunology and Infection

2.4K Views.  Akadeum Life Sciences. The objective of this study is to demonstrate the feasibility of flotation-based separation to isolate, activate, and expand primary human T cells.

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

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 ...

Artificial Antigen Presenting Cell (aAPC) Mediated Activation and Expansion of Natural Killer T Cells

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike classical T cells, NKT cells recognize lipid antigen in the context of CD1 molecules2. NKT cells express an invariant TCR&alpha; chain rearrangement: V&alpha;14J&alpha;18 in mice and V&alpha;24J&alpha;18 in humans, which is associated with V&beta; chains of limited diversity3-6, and are referred to as canonical or invariant NKT (iNKT) cells. Similar to conventional T cells, NKT cells develop from CD4-CD8- thymic precursor T cells following the appropriate signaling by CD1d 7. The potential to utilize NKT cells for therapeutic purposes has significantly increased with the ability to stimulate and expand human NKT cells with &alpha;-Galactosylceramide (&alpha;-GalCer) and a variety of cytokines8. Importantly, these cells retained their original phenotype, secreted cytokines, and displayed cytotoxic function against tumor cell lines. Thus, ex vivo expanded NKT cells remain functional and can be used for adoptive immunotherapy. However, NKT cell based-immunotherapy has been limited by the use of autologous antigen presenting cells and the quantity and quality of these stimulator cells can vary substantially. Monocyte-derived DC from cancer patients have been reported to express reduced levels of costimulatory molecules and produce less inflammatory cytokines9,10. In fact, murine DC rather than autologous APC have been used to test the function of NKT cells from CML patients11. However, this system can only be used for in vitro testing since NKT cells cannot be expanded by murine DC and then used for adoptive immunotherapy. Thus, a standardized system that relies on artificial Antigen Presenting Cells (aAPC) could produce the stimulating effects of DC without the pitfalls of allo- or xenogeneic cells12, 13. Herein, we describe a method for generating CD1d-based aAPC. Since the engagement of the T cell receptor (TCR) by CD1d-antigen complexes is a fundamental requirement of NKT cell activation, antigen: CD1d-Ig complexes provide a reliable method to isolate, activate, and expand effector NKT cell populations.

Artificial Antigen Presenting Cell aAPC Mediated Activation and Expansion of Natural Killer T Cells

Here we describe a method for activating and expanding human NKT cells from bulk T cell populations using artificial antigen presenting cells (aAPC). The use of CD1d-based aAPC provides a standardized method for generating high numbers of functional NKT cells.

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike ...

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-&gamma; receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of &gamma;-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

T cells expressing a CD19-specific chimeric antigen receptor (CAR) are infused as investigational treatment of B-cell malignancies in our first-in-human gene therapy trials. We describe genetic modification of T cells using the Sleeping Beauty (SB) system to introduce CD19-specific CAR and selective propagation on designer CD19+ artificial antigen presenting cells.

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer  a biologic product with the potential for ...

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

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 ...

Spatial and Temporal Control of T Cell Activation Using a Photoactivatable Agonist

T lymphocytes engage in rapid, polarized signaling, occurring within minutes following TCR activation. This induces formation of the immunological synapse, a stereotyped cell-cell junction that regulates T cell activation and directionally targets effector responses. To study these processes effectively, an imaging approach that is tailored to capturing fast, polarized responses is necessary. This protocol describes such a system, which is based on a photoactivatable peptide-major histocompatibility complex (pMHC) that is non-stimulatory until it is exposed to ultraviolet light. Targeted decaging of this reagent during videomicroscopy experiments enables precise spatiotemporal control of TCR activation and high-resolution monitoring of subsequent cellular responses by total internal reflection (TIRF) imaging. This approach is also compatible with genetic and pharmacological perturbation strategies. This allows for the assembly of well-defined molecular pathways that link TCR signaling to the formation of the polarized cytoskeletal structures that underlie the immunological synapse.

This protocol describes an imaging-based method to activate T lymphocytes using photoactivatable peptide-MHC, enabling precise spatiotemporal control of T cell activation.

T lymphocytes engage in rapid, polarized signaling, occurring within minutes following TCR activation. This induces formation of the immunological ...

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 ...

Separation of Immune Cell Subpopulations in Peripheral Blood Samples from Children with Infectious Mononucleosis

Infectious mononucleosis (IM) is an acute syndrome mostly associated with primary Epstein&#8211;Barr virus (EBV) infection. The main clinical symptoms include irregular fever, lymphadenopathy, and significantly increased lymphocytes in peripheral blood. The pathogenic mechanism of IM is still unclear; there is no effective treatment method for it, with mainly symptomatic therapies being available. The main question in EBV immunobiology is why only a small subset of infected individuals shows severe clinical symptoms and even develop EBV-associated malignancies, whilemost individuals are asymptomatic for life with the virus.
B cells are first involved in IM because EBV receptors are presented on their surface. Natural killer (NK) cells are cytotoxic innate lymphocytes that are important for killing EBV-infected cells. The proportion of CD4+ T cells decreases while that of CD8+ T cells expands dramatically during acute EBV infection, and the persistence of CD8+ T cells is important for lifelong control of IM. Those immune cells play important roles in IM, and their functions need to be identified separately. For this purpose, monocytes are separated first from peripheral blood mononuclear cells (PBMCs) of IM individuals using CD14 microbeads, a column, and a magnetic separator.
The remaining PBMCs are stained with peridinin-chlorophyll-protein (PerCP)/Cyanine 5.5 anti-CD3, allophycocyanin (APC)/Cyanine 7 anti-CD4, phycoerythrin (PE) anti-CD8, fluorescein isothiocyanate (FITC) anti-CD19, APC anti-CD56, and APC anti-CD16 antibodies to sort CD4+ T cells, CD8+ T cells, B cells, and NK cells using a flow cytometer. Furthermore, transcriptome sequencing of five subpopulations was performed to explore their functions and pathogenic mechanisms in IM.

We describe a method combining immunomagnetic beads and fluorescence-activated cell sorting to isolate and analyze defined immune cell subpopulations of peripheral blood mononuclear cells (monocytes, CD4+ T cells, CD8+ T cells, B cells, and natural killer cells). Using this method, magnetic and fluorescently labeled cells can be purified and analyzed.

Infectious mononucleosis (IM) is an acute syndrome mostly associated with primary Epstein&#8211;Barr virus (EBV) infection. The main clinical symptoms ...

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 ...