The authors would like to thank Dr. Vladimir Ponomarev for sharing the luciferase constructs used in the experiments described in the representative results section of this article.

The following procedures have been evaluated and approved by Memorial Sloan Kettering&#39;s Institutional Animal Care and Use Committee.
1. Transfection of 293T cells with luciferase and harvest of viral supernatant
<ol>
	<li>Culture of 293T cells
	<ol>
		<li>Prepare media by adding the following to a liter of DMEM each: 110 mL of heat-inactivated fetal bovine serum (FBS), 11 mL of penicillin-streptomycin.</li>
		<li>Thaw 5 x 106 293T cells and transfer them into a T175 flask with the media prepared as described above.</li>
		<li>Split the 293T cells 1:10 every 3 days for 6 days. Do not allow the cells to become more than 90% confluent.</li>
	</ol>
	</li>
	<li>Transfection of 293T cells 
	NOTE: The following quantities of reagents are calculated for transfecting one T175 flask of 293T cells. Adjust accordingly if more flasks are to be transduced.
	<ol>
		<li>Transfer 1.5 mL of media to each of the two 50 mL tubes using a pipette. To one tube, add 10 &#181;g of VSV-G plasmid, 20 &#181;g of Gag/pol, and 20 &#181;g of click beetle red TD tomato (CBR-TDR) luciferase plasmid and mix. To the other tube, add 100 &#181;L of DNA in vitro transfection reagent and mix. Incubate both tubes at room temperature (RT) for 5 min. 
		NOTE: All plasmids described in this manuscript were provided by Dr. Vladimir Ponomarev.</li>
		<li>Transfer the media containing the DNA in vitro transfection reagent dropwise to the tube containing the DNA plasmids (over approximately 30 s) and mix gently with a pipette. Incubate at RT for 20 min.</li>
		<li>During this 20 min incubation, detach the 293T cells by adding 5-10 mL of 0.05% trypsin. Once the cells have detached, add 10 mL of media and centrifuge at 800 x g for 5 min. Resuspend the cells in 1.5 mL of media.</li>
		<li>Add the media containing the DNA in vitro transfection reagent and the DNA plasmids to the 293T cells and incubate at 37 &#176;C for 30 min.</li>
		<li>Transfer to a T175 flask and add 18 mL of media. Incubate at 37 &#176;C overnight.</li>
		<li>The next day, carefully aspirate the media with a glass pipette without detaching the cells. Replace with 18 mL of fresh media. Incubate at 37 &#176;C overnight in an incubator.</li>
	</ol>
	</li>
	<li>Harvesting viral supernatant
	<ol>
		<li>Remove the viral supernatant using a pipette put on ice. Centrifuge at 800 x g for 5 min to pellet the cells and debris. 
		NOTE: If the cell pellet is large, the cells were likely disrupted from the flask when the supernatant was removed. These cells can be plated again in a new flask with fresh media.</li>
		<li>Filter the viral supernatant using a 0.22 &#181;m filter.</li>
		<li>Use the viral supernatant immediately. Keep it on ice or at 4 &#176;C for up to 24 h, or freeze it at -80&#176; C for long-term storage.</li>
	</ol>
	</li>
</ol>
2. Expansion and transduction of activated human T cells with luciferase
<ol>
	<li>Activation of human T cells in vitro
	<ol>
		<li>Wash the beads by adding 10 mL of phosphate buffered saline (PBS) containing 0.1% bovine serum albumin (BSA) and 2 mM ethylenediaminetetraacetic acid (EDTA) to a sterile 15 mL tube, adding beads (one bead per two T cells), placing in a magnet rack for 2 min, and suctioning out the PBS.</li>
		<li>Prepare media as described in step 1.1.1, then add IL-2 to a final concentration of 30 IU/mL and add the washed beads. Make 2.5 mL of media for every two million T cells to be activated.</li>
		<li>Count the T cells (freshly purified or previously purified, frozen, thawed, and washed) using a hemocytometer and trypan blue stain. Add T cells to the media prepared in step 2.1.2 and gently invert the tube to mix. The T cells will expand at least 20x depending on the source and general health of the cells. Determine the number of T cells to activate by calculating the number needed to treat the mice and dividing by 20. Here, 20 x 106 T cells per mouse were used based on preliminary experiments.</li>
		<li>Plate 2.5 mL of media containing two million T cells, beads, and IL-2 in each well of a 24-well tissue culture plate. Culture at 37 &#176;C in a humidified 5% CO2 incubator.</li>
		<li>Examine the T cells under a 20x microscope every day for the next 3 days to determine when to transduce with luciferase. The cells are ready when they clump together and deplete the media, turning it from red/pink to light orange.</li>
	</ol>
	</li>
	<li>Preparation of retronectin plates 
	NOTE: Retronectin is used to coat the plates used in the transduction as it enhances gene transduction15.
	<ol>
		<li>Add 2 mL of 20 &#181;g/mL retronectin in PBS to a well of an untreated 6-well plate. Incubate for 2 h at RT or 1 h at 37 &#176;C. One retronectin-coated well is required for every two million T cells to be transduced.</li>
		<li>Aspirate the retronectin without scratching the bottom of the plate.</li>
		<li>Add 3 mL of 2% BSA in PBS (sterile filtered) per well in the 6-well plate. Incubate for 30 min at RT.</li>
	</ol>
	</li>
	<li>Spinoculation
	<ol>
		<li>Aspirate the BSA without scratching the bottom of the plate.</li>
		<li>Wash the wells once with 3 mL of PBS per well. Continue or replace with fresh PBS and leave the plate covered at 4 &#176;C overnight.</li>
		<li>Add 2 mL of CBR-TDR luciferase viral supernatant (collected in step 1.3) per well.</li>
		<li>Centrifuge at 1,240 x g for 90 min at 32 &#176;C.</li>
	</ol>
	</li>
	<li>Transduction
	<ol>
		<li>Count the T cells.</li>
		<li>Aspirate the viral supernatant without scratching the bottom of the plate.</li>
		<li>Plate two million T cells per well in 6 mL of Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) containing 30 IU/mL human IL-2.</li>
		<li>Examine the T cells daily. The T cells are ready to be expanded when they are nearly confluent, usually after 2 days.</li>
		<li>When the cells are nearly confluent, gently wash them off the bottom of the plate using a pipette and transfer each well to a separate T75 flask. Add 9 mL of media for a total of 15 mL of media per flask.</li>
		<li>The next day, add an additional 15 mL of media. The cells should be ready to inject into mice the day after this addition of media. Confirm successful transduction by performing flow cytometry (the luciferase construct contains a TD tomato fluorophore that is visible in the PE channel)16.</li>
	</ol>
	</li>
</ol>
3. Engraftment of luciferase-transduced T cells in immunocompromised mice
<ol>
	<li>Prepare and count the T cells for injection.
	<ol>
		<li>Remove the T cells from the flasks and count. The cells are ready to inject when they have expanded 20x-30x, usually by day 7 post-activation.</li>
		<li>Centrifuge the T cells at 800 x g for 5 min. Resuspend in 2 mL of media and remove the beads using a magnet rack.</li>
		<li>For experiments where T cells will be injected separately from bispecific antibodies, count the T cells and resuspend so that the final concentration is 20 million T cells per 100 &#181;L of media. Skip to step 3.2. For experiments using ex vivo armed T cells (EATs), skip to step 3.1.4.</li>
		<li>For experiments using ex vivo armed T cells, count the T cells and separate them into individual 1.5 mL tubes for each group, with 20 million cells per mouse. For example, if there are three groups of five mice each, prepare three 1.5 mL tubes with 100 million T cells each.</li>
		<li>Centrifuge the 1.5 mL tubes at 800 x g for 5 min. Remove the media carefully without disturbing the T cell pellet. Resuspend in no more than 50 &#181;L of media containing the bispecific antibodies. Incubate at RT for 30 min. 
		NOTE: For the purposes of experiments conducted in this study, proprietary IgG-[L]-scFv T cell-engaging bispecific antibodies generated in the lab were used. For T cell arming, use 5 &#181;g of antibody per 20 x 106 T cells. Commercially available bispecific antibodies could also be used.</li>
		<li>Wash away excess antibodies by adding 1,450 &#181;L of media to each tube. Centrifuge at 800 x g for 5 min, carefully aspirate the media, and resuspend at a concentration of 20 million armed T cells per 100 &#181;L media.</li>
	</ol>
	</li>
	<li>Injection and engraftment into mice
	<ol>
		<li>Anesthetize the mice in a chamber supplying 3.5% (v/v) inhaled isoflurane in 1 L/min oxygen. The mice are fully anesthetized when the hind limb pedal withdrawal reflex has disappeared. 
		NOTE:&#160;Provide thermal support throughout the procedure.</li>
		<li>Using a 26 G needle, inject 20 million T cells in 100 &#181;L of media retroorbitally per mouse.</li>
		<li>Administer 1,000 U recombinant IL-2 subcutaneously to support T cell survival in vivo.</li>
		<li>Administer the bispecific antibodies retroorbitally (into the eye not used for T cell injection) or intraperitoneally. Allow the mice to recover from anesthesia and return to their cages. 
		&#8203;NOTE: Again, here, proprietary antibodies that were generated in the lab were used, but commercially available antibodies could be used instead. In the experiments here, 0.3-10 &#181;g of antibody per mouse per dose was administered. The antibodies were administered twice per week for 3-4 weeks.</li>
	</ol>
	</li>
</ol>
4. In vivo imaging of mice engrafted with luciferase-transduced T cells
NOTE: This step is to be performed on the day of imaging, which is not necessarily the same day that the T cells and/or antibodies are administered to the mice. Typically, we perform imaging 24 h after administration of the luciferase-transduced T cells.
<ol>
	<li>Preparation of D-luciferin
	<ol>
		<li>Dissolve 1 g of D-luciferin in 33.3 mL of sterile PBS (final concentration: 30 mg/mL).</li>
		<li>Aliquot the dissolved D-luciferin into 33 x 1.5 mL microcentrifuge tubes and keep the tubes at -20 &#176;C.</li>
		<li>On the day of imaging, thaw enough aliquots of 30 mg/mL D-luciferin for the number of animals to be imaged. One tube is enough for 10 animals. 
		NOTE: Refreeze the remaining D-luciferin after use. D-luciferin is stable for at least five freeze/thaw cycles.</li>
	</ol>
	</li>
	<li>Administration of D-luciferin to mice 
	NOTE: Before administering D-luciferin to the mice, open the imaging software and initialize the system. The camera may take several minutes to cool, and this should be done before the mice are injected with D-luciferin to ensure that imaging can take place 5 min after administration of the substrate.
	<ol>
		<li>Anesthetize up to five mice in a chamber supplying 3.5% (v/v) inhaled isoflurane in 1 L/min oxygen. The mice are fully anesthetized when the hind limb pedal withdrawal reflex has disappeared.</li>
		<li>Once the mice are fully anesthetized, administer 100 &#181;L of 30 mg/mL D-luciferin per mouse (3 mg per mouse) by retroorbital injection with a 26 G needle. Image this group of mice before administering D-luciferin to the next group. Place the next group of mice in the isoflurane chamber to be anesthetized while the previous group is being imaged.</li>
	</ol>
	</li>
	<li>Imaging luciferase-transduced T cells
	<ol>
		<li>Move the anesthetized mice to the light-tight chamber of the imager and continue administering 3% isoflurane at 0.5 L/min. Place the mice on their sides such that the flank bearing the xenograft is facing up toward the camera. Take another set of images with the mice in a supine position to evaluate T cell presence in the lungs.</li>
		<li>Using the Acquisition Control Panel, select Luminescent, Photograph, and Overlay. Set the exposure time to auto, the binning to medium, and F/Stop to 1. Acquire images. After the first image, set the exposure time to match that which was automatically calculated for the first image so that subsequent images may be compared directly. It may be useful to capture images with multiple exposure times to determine the optimal exposure time for achieving the brightest signal without pixel saturation.</li>
		<li>Remove the mice from isoflurane, return to their cages, and observe until they are awake and ambulatory.</li>
		<li>Repeat the steps from step 4.2.1 until all groups have been imaged.</li>
	</ol>
	</li>
</ol>

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NKC reports receiving commercial research grants from Y-mAbs Therapeutics. NKC is the inventor and owner of issued patents licensed by MSK to Y-mAbs Therapeutics, Biotec Pharmacon/Lallemand, and Abpro-labs. MSK and NKC have financial interest in Y-mAbs. NKC reports receiving stock options from Eureka Therapeutics. HFG and MEC have no relevant disclosures.

While the T-BsAb blinatumomab has been approved for CD19-positive hematological malignancies, the successful implementation of T-BsAbs in solid tumors has proven much more difficult. Catumaxomab, a T-BsAb directed against epithelial cell adhesion molecule (EPCAM), was approved for the treatment of malignant ascites in ovarian cancer patients, but production of the drug was subsequently halted for commercial reasons19. No other T-BsAbs have been approved for solid tumors, underlining the challenges...

T cell-engaging bispecific antibodies (T-BsAbs) are engineered antibodies used to provide artificial specificity to polyclonal T cells by engaging T cells through one binding arm and a tumor antigen through another binding arm. This technology has been successfully applied to hematological cancers (CD19-targeting blinatumomab1), and numerous T-BsAbs are in preclinical and clinical development for a variety of solid tumors as well2. T-BsAbs engage polyclonal T cells in a major histocompatibility complex (MHC)-independent manner, and therefore even tumors that downregulate human leukocyte antigens (HLAs) are susceptible to this type of therapy3,4. T-BsAbs have been developed in dozens of different formats, with differences in the valency and spatial arrangement of the T cell and tumor binding arms, interdomain distances, and the inclusion of an Fc domain, which affects the half-life and can induce effector functions if present5. Previous work in our lab has shown that these factors significantly affect the anti-tumor efficacy of T-BsAbs, with up to 1,000-fold differences in potency6. Through this work, we identified the IgG-[L]-scFv format as the ideal platform for T-BsAbs (see Representative Results section for more detail regarding T-BsAb formats), and have applied this platform to targets including GD2 (neuroblastoma), HER2 (breast cancer and osteosarcoma), GPA33 (colorectal cancer), STEAP1 (Ewing Sarcoma), CD19 (B cell malignancies), and CD33 (B cell malignancies)7,8,9,10,11,12,13.
One of the major challenges to successfully implementing T-BsAb therapy in solid tumors is overcoming an immunosuppressive tumor microenvironment (TME) to drive T cell trafficking to tumors14. The factors affecting T-BsAb efficacy described above have a significant impact on the ability of T-BsAbs to effectively induce T cell homing to tumors, but this effect is difficult to evaluate in an in vivo system in real time. This manuscript provides a detailed description of the use of luciferase-transduced T cells in preclinical studies of T-BsAbs to evaluate T cell trafficking to various tissues in experimental immunocompromised mouse models during treatment. The overall goal of this method is to provide a means to evaluate T cell infiltration in tumors and other tissues, as well as real-time insight into T cell homing kinetics and persistence, without the need to sacrifice animals during treatment. For the increasing number of researchers focusing on cellular immunotherapies, the ability to track T cells in vivo in preclinical animal models is crucial. We aim to provide a thorough, detailed description of the method we have employed for tracking luciferase-transduced T cells to enable other researchers to easily replicate this technique.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>293T cells</td>
			<td>ATCC</td>
			<td>CRL-11268</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma Aldrich</td>
			<td>A7030-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>CD3/CD28 beads</td>
			<td>Gibco (ThermoFisher)</td>
			<td>11161D</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Luciferin, Potassium Salt</td>
			<td>Goldbio</td>
			<td>LUCK-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco (ThermoFisher)</td>
			<td>11965092</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA in vitro transfection reagent (polyjet)</td>
			<td>SignaGen Laboratories</td>
			<td>SL100688</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma Aldrich</td>
			<td>E9884-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco (ThermoFisher)</td>
			<td>10437028</td>
			<td></td>
		</tr>
		<tr>
			<td>Gag/pol plasmid</td>
			<td>Addgene</td>
			<td>14887</td>
			<td></td>
		</tr>
		<tr>
			<td>GFP plasmid</td>
			<td>Addgene</td>
			<td>11150-DNA.cg</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicilin-Streptomycin</td>
			<td>Gibco (ThermoFisher)</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human IL-2</td>
			<td>R&#38;D Systems</td>
			<td>202-IL-010/CF</td>
			<td></td>
		</tr>
		<tr>
			<td>Retronectin</td>
			<td>Takara</td>
			<td>T100B</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Gibco (ThermoFisher)</td>
			<td>25-300-120</td>
			<td></td>
		</tr>
		<tr>
			<td>VSV-G plasmid</td>
			<td>Addgene</td>
			<td>8454</td>
			<td></td>
		</tr>
	</tbody>
</table>

tracking bispecific antibody-induced t cell trafficking using luciferase-transduced human t cells

T cell-engaging bispecific antibodies (T-BsAbs) are in various stages of preclinical development and clinical testing for solid tumors. Factors such as valency, spatial arrangement, interdomain distance, and Fc mutations affect the anti-tumor efficacy of these therapies, commonly by influencing the homing of T cells to tumors, which remains a major challenge. Here, we describe a method to transduce activated human T cells with luciferase, allowing in vivo tracking of T cells during T-BsAb therapy studies. The ability of T-BsAbs to redirect T cells to tumors can be quantitatively evaluated at multiple time points during treatment, allowing researchers to correlate the anti-tumor efficacy of T-BsAbs and other interventions with the persistence of T cells in tumors. This method alleviates the need to sacrifice animals during treatment to histologically assess T cell infiltration and can be repeated at multiple time points to determine the kinetics of T cell trafficking during and after treatment.

As described in step 4.3, mice can be oriented in different positions during imaging to evaluate the presence of T cells in different tissues. Supine positioning allows for the assessment of T cells in the lungs, which is common at early time points after injection. Lateral positioning with the subcutaneous xenograft facing up is used to best assess T cell trafficking to the tumor. Female C.Cg-Rag2tm1Fwa Il2rgtm1Sug/JicTac mice were used for all experiments described in this manuscript. <st...

Watch this Scientific Journal Video about Tracking Bispecific Antibody-Induced T Cell Trafficking Using Luciferase-Transduced Human T Cells at JoVE.com

Tracking Bispecific Antibody-Induced T Cell Trafficking Using Luciferase-Transduced Human T Cells

Here, we describe a method for transducing human T cells with luciferase to facilitate in vivo tracking of bispecific antibody-induced T cell trafficking to tumors in studies to evaluate the anti-tumor efficacy and mechanism of T cell-engaging bispecific antibodies.

T cell-engaging bispecific antibodies (T-BsAbs) are in various stages of preclinical development and clinical testing for solid tumors. Factors such as ...

tracking-bispecific-antibody-induced-t-cell-trafficking-using

Research

JoVE Journal

Cancer Research

1.1K Views.  Memorial Sloan Kettering Cancer Center. Here, we describe a method for transducing human T cells with luciferase to facilitate in vivo tracking of bispecific antibody-induced T cell trafficking to tumors in studies to evaluate the anti-tumor efficacy and mechanism of T cell-engaging bispecific antibodies. 

Video: Tracking Bispecific Antibody-Induced T Cell Trafficking Using Luciferase-Transduced Human T Cells

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

CD8 T cell are key players in the fight against cancer. In order for CD8 T cells to kill tumor cells they need to enter into the tumor, migrate within the tumor microenvironment and respond adequately to tumor antigens. The recent development of improved imaging approaches, such as 2-photon microscopy, and the use of powerful mouse tumor models have shed light on some of the mechanisms that regulate anti-tumor T cell activities. Whereas such systems have provided valuable insights, they do not always predict human responses. In human, our knowledge in the field mainly comes from a description of fixed tumor samples from human patients, as well as in vitro studies. However, in vitro models lack the complex three-dimensional tumor milieu and, therefore, are incomplete approximations of in vivo T cell activities. Fresh slices made from explanted tissue represent a complex multi-cellular tumor environment that can act as an important link between co-cultured studies and animal models. Originally set up in murine lymph nodes1 and previously described in a JoVE article2, this approach has now been transposed to human tumors to examine the dynamics of both plated3&#160;as well as resident&#160;T cells4.
Here, a protocol for the preparation of human lung tumor slices, immunostaining of resident CD8 T and tumor cells, and tracking of CD8 T lymphocytes within the tumor microenvironment using confocal microscopy is described. This system is uniquely placed to screen for novel immunotherapy agents favoring T cell migration in tumors.

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

This protocol describes a method to image resident tumor-infiltrating CD8 T cells labeled with fluorescently coupled antibodies within human lung tumor slices. This technique permits real-time analyses of CD8 T cell migration using confocal microscopy.

CD8 T cell are key players in the fight against cancer. In order for CD8 T cells to kill tumor cells they need to enter into the tumor, migrate within the ...

A Spheroid Killing Assay by CAR T Cells

Immunotherapy has become a field of growing interest in the fight against cancer otherwise untreatable. Among all immunotherapeutic methods, chimeric antigen receptor (CAR) redirected T cells obtained the most spectacular results, in particular with pediatric B-acute lymphoblastic leukemia (B-ALL). Classical validation methods of CAR T cells rely on the use of specificity and functionality assays of the CAR T cells against target cells in suspension and in xenograft models. Unfortunately, observations made in vitro are often decoupled from results obtained in vivo and a lot of effort and animals could be spared by adding another step: the use of 3D culture. The production of spheroids out of potential target cells that mimic the 3D structure of the tumor cells when they are engrafted into the animal model represents an ideal alternative. Here, we report an affordable, reliable and easy method to produce spheroids from a transduced colorectal cell line as a validation tool for adoptive cell therapy (exemplified here by CD19 CAR T cells). This method is coupled with an advanced live imaging system that can follow spheroid growth, effector cells cytotoxicity and tumor cell apoptosis.

This protocol is designed to assess immunotherapeutic redirected T-cell (CAR T-cell) cytotoxicity against 3D structured cancerous cells (spheroids) in real time.

Immunotherapy has become a field of growing interest in the fight against cancer otherwise untreatable. Among all immunotherapeutic methods, chimeric ...

Using CRISPR/Cas9 to Knock Out GM-CSF in CAR-T Cells

Chimeric antigen receptor T (CAR-T) cell therapy is a cutting edge and potentially revolutionary new treatment option for cancer. However, there are significant limitations to its widespread use in the treatment of cancer. These limitations include the development of unique toxicities such as cytokine release syndrome (CRS) and neurotoxicity (NT) and limited expansion, effector functions, and anti-tumor activity in solid tumors. One strategy to enhance CAR-T efficacy and/or control toxicities of CAR-T cells is to edit the genome of the CAR-T cells themselves during CAR-T cell manufacturing. Here, we describe the use of CRISPR/Cas9 gene editing in CAR-T cells via transduction with a lentiviral construct containing a guide RNA to granulocyte macrophage colony-stimulating factor (GM-CSF) and Cas9. As an example, we describe CRISPR/Cas9 mediated knockout of GM-CSF. We have shown that these GM-CSFk/o CAR-T cells effectively produce less GM-CSF while maintaining critical T cell function and result in enhanced anti-tumor activity in vivo compared to wild type CAR-T cells.

Here, we present a protocol to genetically edit CAR-T cells via a CRISPR/Cas9 system.

Chimeric antigen receptor T (CAR-T) cell therapy is a cutting edge and potentially revolutionary new treatment option for cancer. However, there are ...

Manufacturing Chimeric Antigen Receptor (CAR) T Cells for Adoptive Immunotherapy

Adoptive immunotherapy holds promise for the treatment of cancer and infectious disease. We describe a simple approach to transduce primary human T cells with chimeric antigen receptor (CAR) and expand their progeny ex vivo. We include assays to measure CAR expression as well as differentiation, proliferative capacity and cytolytic activity. We describe assays to measure effector cytokine production and inflammatory cytokine secretion in CAR T cells. Our approach provides a reliable and comprehensive method to culture CAR T cells for preclinical models of adoptive immunotherapy.

Manufacturing Chimeric Antigen Receptor CAR T Cells for Adoptive Immunotherapy

We describe an approach to reliably generate chimeric antigen receptor (CAR) T cells and test their differentiation and function in vitro and in vivo.

Adoptive immunotherapy holds promise for the treatment of cancer and infectious disease. We describe a simple approach to transduce primary human T cells ...

Using Human Induced Pluripotent Stem Cells for the Generation of Tumor Antigen-specific T Cells

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of patients with advanced cancer. Adoptive T cell transfer (ACT) of highly enriched tumor antigen-specific T cells has been shown to cause durable regression of metastatic cancer in some patients. However, during expansion, these cells may become exhausted or senescent, limiting their effector function and persistence in vivo. Induced pluripotent stem cell (iPSC) technology may overcome these obstacles by leading to in vitro generation of large numbers of less differentiated tumor antigen-specific T cells. Human iPSC (hiPSC) have the capacity to differentiate into any type of somatic cell, including lymphocytes, which retain the original T cell receptor (TCR) genomic rearrangement when a T cell is used as a starting cell. Therefore, reprogramming of human tumor antigen-specific T cells to hiPSC followed by redifferentiation to T cell lineage has the potential to produce rejuvenated tumor antigen-specific T cells. Described here is a method for generating tumor antigen-specific CD8&#945;&#946;+ single positive (SP) T cells from hiPSC using OP9/DLL1 co-culture system. This method is a powerful tool for in vitro T cell lineage generation and will facilitate the development of in vitro derived T cells for use in regenerative medicine and cell-based therapies.

This article describes a method to generate functional tumor antigen-specific induced pluripotent stem cell-derived CD8&#945;&#946;+ single positive T cells using OP9/DLL1 co-culture system.

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of ...

Isolation and Expansion of Cytotoxic Cytokine-induced Killer T Cells for Cancer Treatment

Adoptive cellular immunotherapy focuses on restoring cancer recognition via the immune system and improves effective tumor cell killing. Cytokine-induced killer (CIK) T cell therapy has been reported to exert significant cytotoxic effects against cancer cells and to reduce the adverse effects of surgery, radiation, and chemotherapy in cancer treatments. CIK can be derived from peripheral blood mononuclear cells (PBMCs), bone marrow, and umbilical cord blood. CIK cells are a heterogeneous subpopulation of T cells with CD3+CD56+ and natural killer (NK) phenotypic characteristics that include major histocompatibility complex (MHC)-unrestricted antitumor activity. This study describes a qualified, clinically applicable, flow cytometry-based method for the quantification of the cytolytic capability of PBMC-derived CIK cells against hematological and solid cancer cells. In the cytolytic assay, CIK cells are co-incubated at different ratios with prestained target tumor cells. After the incubation period, the number of target cells are determined by a nucleic acid-binding stain to detect dead cells. This method is applicable to both research and diagnostic applications. CIK cells possess potent cytotoxicity that could be explored as an alternative strategy for cancer treatment upon its preclinical evaluation by a cytometer setup and tracking (CS &#38; T)-based flow cytometry system.

Here, we present a protocol to perform the isolation and expansion of peripheral blood mononuclear cells-derived cytokine-induced CD3+CD56+ killer cells and illustrate their cytotoxicity effect against hematological and solid cancer cells by using an in vitro diagnosis flow cytometry system.

Adoptive cellular immunotherapy focuses on restoring cancer recognition via the immune system and improves effective tumor cell killing. Cytokine-induced ...

Monitoring Cancer Cell Invasion and T-Cell Cytotoxicity in 3D Culture

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited number of assays allow for direct monitoring and mechanistic insights into the interactions between tumor and immune cells, amongst which, T-cells play a significant role in executing the cytotoxic response of the adaptive immune system to cancer cells. Most assays are based on two-dimensional (2D) co-culture of cells due to the relative ease of use but with limited representation of the invasive growth phenotype, one of the hallmarks of cancer cells. Current three-dimensional (3D) co-culture systems either require special equipment or separate monitoring for invasion of co-cultured cancer cells and interacting T-cells.
Here we describe an approach to simultaneously monitor the invasive behavior in 3D of cancer cell spheroids and T-cell cytotoxicity in co-culture. Spheroid formation is driven by enhanced cell-cell interactions in scaffold-free agarose microwell casts with U-shaped bottoms. Both T-cell co-culture and cancer cell invasion into type I collagen matrix are performed within the microwells of the agarose casts without the need to transfer the cells, thus maintaining an intact 3D co-culture system throughout the assay. The collagen matrix can be separated from the agarose cast, allowing for immunofluorescence (IF) staining and for confocal imaging of cells. Also, cells can be isolated for further growth or subjected to analyses such as for gene expression or fluorescence activated cell sorting (FACS). Finally, the 3D co-culture can be analyzed by immunohistochemistry (IHC) after embedding and sectioning. Possible modifications of the assay include altered compositions of the extracellular matrix (ECM) as well as the inclusion of different stromal or immune cells with the cancer cells.

The presented approach simultaneously evaluates cancer cell invasion in 3D spheroid assays and T-cell cytotoxicity. Spheroids are generated in a scaffold-free agarose multi-microwell cast. Co-culture and embedding in type I collagen matrix are performed within the same device which allows to monitor cancer cell invasion and T-cell mediated cytotoxicity.

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited ...

Isolating Human Peripheral Blood Mononuclear Cells and CD4+ T cells from S&#233;zary Syndrome Patients for Transcriptomic Profiling

Cutaneous T-cell lymphomas (CTCL) are derived from the transformation and uncontrolled proliferation of mature skin-homing T cells, and mycosis fungoides (MF) and S&#233;zary syndrome (SS) represent the most common subtypes. Despite a number of studies on characterizing gene expression, genetic alterations, and epigenetic abnormalities of CTCL, the molecular pathogenesis of MF/SS remains unclear. MF refers to the more common CTCL with a skin-predominance, and is usually limited to skin, whereas&#160;SS is an aggressive leukemic variant of CTCL with widespread skin involvement and is characterized by neoplastic distribution mainly involving blood, skin, and lymph node. Focusing on clinical practice, the identification of gene expression biomarkers has&#160;enormous potential to improve diagnosis and treatment of MF/SS. Indeed, recent transcriptomic studies have identified potential diagnostic biomarkers from differences in gene expression between normal and malignant T cells, which may improve our understanding of SS biology, and reveal potential therapeutic targets. This manuscript describes a detailed reproducible protocol for the isolation of peripheral blood mononuclear cells from fresh whole blood from patients diagnosed with SS, selection of CD4+ memory T cells (CD4+CD45RO+ T cells), chemical stimulation, and preparation of RNA suitable for transcriptomic profiling to discover novel prognostic molecular markers to gain additional insight in disease etiology. The stimulation using chemical agonist to activate nuclear regulation provides more specific assessment for pathways important in the dynamic transcription regulation and gene expression and eliminates confounding defects that may arise from upstream signaling defects arising from TCR antigen loss at the cell membrane. The data obtained from comparison of transcriptome of unstimulated to stimulated SS T cells unmasks functional regulatory gene expression defects not evident from analysis of quiescent unstimulated cells. Furthermore, the method outlined from this approach can be adapted for studying T cell gene expression defects in other T cell immune diseases.

We present a simple protocol for the isolation of peripheral blood mononuclear cells from whole blood obtained from patients diagnosed with S&#233;zary Syndrome, followed by selection of CD4+ T cells, their stimulation with phorbol12-myristate13-acetate and A23187 ionophore, and preparation of RNA for transcriptomic profiling.

Cutaneous T-cell lymphomas (CTCL) are derived from the transformation and uncontrolled proliferation of mature skin-homing T cells, and mycosis fungoides ...

Dynamic Imaging of Chimeric Antigen Receptor T Cells with [18F]Tetrafluoroborate Positron Emission Tomography/Computed Tomography

T cells genetically engineered to express chimeric antigen receptors (CAR) have shown unprecedented results in pivotal clinical trials for patients with B cell malignancies or multiple myeloma (MM). However, numerous obstacles limit the efficacy and prohibit the widespread use of CAR T cell therapies due to poor trafficking and infiltration into tumor sites as well as lack of persistence in vivo. Moreover, life-threatening toxicities, such as cytokine release syndrome or neurotoxicity, are major concerns. Efficient and sensitive imaging and tracking of CAR T cells enables the evaluation of T cell trafficking, expansion, and in vivo characterization and allows the development of strategies to overcome the current limitations of CAR T cell therapy. This paper describes the methodology for incorporating the sodium iodide symporter (NIS) in CAR T cells and for CAR T cell imaging using [18F]tetrafluoroborate-positron emission tomography ([18F]TFB-PET) in preclinical models. The methods described in this protocol can be applied to other CAR constructs and target genes in addition to the ones used for this study.

Dynamic Imaging of Chimeric Antigen Receptor T Cells with [18F]Tetrafluoroborate Positron Emission Tomography/Computed Tomography

This protocol describes the methodology for non-invasively tracking T cells genetically engineered to express chimeric antigen receptors in vivo with a clinically available platform.

T cells genetically engineered to express chimeric antigen receptors (CAR) have shown unprecedented results in pivotal clinical trials for patients with B ...

Expansion and Enrichment of Gamma-Delta (&#947;&#948;) T Cells from Apheresed Human Product

Although V&#947;9V&#948;2 T cells are a minor subset of T lymphocytes, this population is sought after for its ability to recognize antigens in a major histocompatibility complex (MHC)-independent manner and develop strong cytolytic effector function that makes it an ideal candidate for cancer immunotherapy. Due to the low frequency of Gamma-Delta (&#947;&#948;) T cells in the peripheral blood, we developed an effective protocol to greatly expand a highly pure &#947;&#948; T cells drug product for first-in-human use of allogeneic &#947;&#948; T cells in patients with acute myeloid leukemia (AML). Using healthy donor apheresis as an allogenic cell source, the lymphocytes are isolated using a validated device for a counterflow centrifugation method of separating cells by size and density. 
The lymphocyte-rich fraction is utilized, and the &#947;&#948; T cells are preferentially activated with zoledronic acid (FDA-approved) and interleukin (IL)-2 for 7 days. Following the preferential expansion of &#947;&#948; T cells, a clinical-grade magnetic cell-separation device and TCR&#945;&#946; beads are used to deplete contaminating T-cell receptor (TCR)&#945;&#946; T cells. The highly enriched &#947;&#948; T cells then undergo a second expansion using engineered artificial antigen-presenting cells (aAPCs) derived from K562 cells-genetically engineered to express single-chain variable fragment (scFv) for CD3 and CD28, 41BBL (CD137L) and IL15-RA-together with zoledronic acid and IL-2. Seeding all day-7 enriched &#947;&#948; T cells in co-culture with the aAPCs facilitates the manufacture of highly pure &#947;&#948; T cells with an average fold expansion of &gt;229,000-fold from healthy donor blood.

Expansion and Enrichment of Gamma-Delta &#947;&#948; T Cells from Apheresed Human Product

Presented is a protocol for the expansion of Gamma-Delta (&#947;&#948;) T cell drug product. Lymphocytes are isolated by elutriation and &#947;&#948;-enriched with zoledronic acid and interleukin-2. Alpha-beta T cells are depleted using a clinical-grade magnetic separation device. The &#947;&#948; cells are co-cultured with K562-derived, artificial antigen-presenting cells and expanded.

Although V&#947;9V&#948;2 T cells are a minor subset of T lymphocytes, this population is sought after for its ability to recognize antigens in a major ...