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><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&amp;amp;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...

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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

1.2K Views.  Memorial Sloan Kettering Cancer Center. Met deze methode kunnen gebruikers eenvoudig T-cellen in vivo volgen om de kinetiek van homing te evalueren en de persistentie van T-cellen te controleren. Het belangrijkste voordeel van deze techniek is dat het op meerdere tijdstippen op hetzelfde dier kan worden herhaald zonder dat er offers nodig zijn in flowcytometrie of immunohistochemie. Deze techniek kan ook worden aangepast voor het volgen van andere soorten immuuncellen.Om 293T-cellen te kweken, bereidt u een liter DMEM voor d...

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 ...

Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

Although in vitro screens are essential for the initial identification of candidate therapeutic agents, a rigorous assessment of the drug's ability to inhibit tumor growth must be performed in a suitable animal model. The type of animal model that is best for this purpose is a topic of intense discussion. Some evidence indicates that preclinical trials examining drug effects on tumors arising in transgenic mice are more predictive of clinical outcome1and so candidate therapeutic agents are often tested in these models. Unfortunately, transgenic models are not available for many tumor types. Further, transgenic models often have other limitations such as concerns as to how well the mouse tumor models its human counterpart, incomplete penetrance of the tumor phenotype and an inability to predict when tumors will develop.
Consequently, many investigators use xenograft models (human tumor cells grafted into immunodeficient mice) for preclinical trials if appropriate transgenic tumor models are not available. Even if transgenic models are available, they are often partnered with xenograft models as the latter facilitate rapid determination of therapeutic ranges. Further, this partnership allows a comparison of the effectiveness of the agent in transgenic tumors and genuine human tumor cells. Historically, xenografting has often been performed by injecting tumor cells subcutaneously (ectopic xenografts). This technique is rapid and reproducible, relatively inexpensive and allows continuous quantitation of tumor growth during the therapeutic period2. However, the subcutaneous space is not the normal microenvironment for most neoplasms and so results obtained with ectopic xenografting can be misleading due to factors such as an absence of organ-specific expression of host tissue and tumor genes. It has thus been strongly recommended that ectopic grafting studies be replaced or complemented by studies in which human tumor cells are grafted into their tissue of origin (orthotopic xenografting)2. Unfortunately, implementation of this recommendation is often thwarted by the fact that orthotopic xenografting methodologies have not yet been developed for many tumor types.
Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive sarcomas that occur sporadically or in association with neurofibromatosis type 13and most commonly arise in the sciatic nerve4. Here we describe a technically straightforward method in which firefly luciferase-tagged human MPNST cells are orthopically xenografted into the sciatic nerve of immunodeficient mice. Our approach to assessing the success of the grafting procedure in individual animals and subsequent non-biased randomization into study groups is also discussed.

Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

A method for reliably grafting luciferase-tagged human malignant peripheral nerve sheath tumor cells into the sciatic nerve of immunodeficient mice is described. The use of bioluminescence imaging to demonstrate proper establishment of tumor grafts and criteria for random segregation of animals into study groups are also discussed.

Although in vitro screens are essential for the initial identification of candidate therapeutic agents, a rigorous assessment of the drug's ability to ...

Medicine

A Syngeneic Mouse B-Cell Lymphoma Model for Pre-Clinical Evaluation of CD19 CAR T Cells

The astonishing clinical success of CD19 chimeric antigen receptor (CAR) T-cell therapy has led to the approval of two second generation chimeric antigen receptors (CARs) for acute lymphoblastic leukemia (ALL) andnon-Hodgkin lymphoma (NHL). The focus of the field is now on emulating these successes in other hematological malignancies where less impressive complete response rates are observed. Further engineering of CAR T cells or co-administration of other treatment modalities may successfully overcome obstacles to successful therapy in other cancer settings.
We therefore present a model in which others can conduct pre-clinical testing of CD19 CAR T cells. Results in this well tested B-cell lymphoma model are likely to be informative CAR T-cell therapy in general.
This protocol allows the reproducible production of mouse CAR T cells through calcium phosphate transfection of Plat-E producer cells with MP71 retroviral constructs and pCL-Eco packaging plasmid followed by collection of secreted retroviral particles and transduction using recombinant human fibronectin fragment and centrifugation. Validation of retroviral transduction, and confirmation of the ability of CAR T cells to kill target lymphoma cells ex vivo, through the use of flow cytometry, luminometry and enzyme-linked immunosorbent assay (ELISA), is also described.
Protocols for testing CAR T cells in vivo in lymphoreplete and lymphodepleted syngeneic mice, bearing established, systemic lymphoma are described. Anti-cancer activity is monitored by in vivo bioluminescence and disease progression. We show typical results of eradication of established B-cell lymphoma when utilizing 1st or 2nd generation CARs in combination with lymphodepleting pre-conditioning and a minority of mice achieving long term remissions when utilizing CAR T cells expressing IL-12 in lymphoreplete mice.
These protocols can be used to evaluate CD19 CAR T cells with different additional modification, combinations of CAR T cells and other therapeutic agents or adapted for the use of CAR T cells against different target antigens.

Here, we present a protocol for the production and pre-clinical testing of murine CD19 CAR T cells by retroviral transduction and utilization as a therapy against established syngeneic A20 B-cell lymphoma in BALB/c mice with or without lymphodepleting pre-conditioning.

The astonishing clinical success of CD19 chimeric antigen receptor (CAR) T-cell therapy has led to the approval of two second generation chimeric antigen ...

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

Immunology and Infection

A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex (pMHC) proteins expressed on the surface of antigen presenting cells (APCs). The TCR complex is composed of the ligand binding TCR&alpha;&beta; heterodimer that associates non-covalently with CD3 dimers (the &#949;&delta; and &#949;&gamma; heterodimers and the &zeta;&zeta; homodimer)1. Upon engagement of the receptor, the CD3 &zeta; chains are phosphorylated by the Src family kinase, Lck. This leads to the recruitment of the Syk family kinase, Zap70, which is then phosphorylated and activated by Lck. After that, Zap70 phosphorylates the adapter proteins LAT and SLP76, initiating the formation of the proximal signaling complex containing a large number of different signaling molecules2. 
The formation of this complex eventually results in calcium and Ras-dependent transcription factor activation and the consequent initiation of a complex series of gene expression programs that give rise to T cell differentiation2. TCR signals (and the resulting state of differentiation) are modulated by many other factors, including antigen potency and crosstalk with co-stimulatory/co-inhibitory, chemokine, and cytokine receptors 3-4. Studying the spatial and temporal organization of the proximal signaling complex under various stimulation conditions is, therefore, key to understanding the TCR signaling pathway as well as its regulation by other signaling pathways.
One very useful model system to study signaling initiated by the TCR at the plasma membrane in T cells is glass-supported lipid bilayers, as described previously5-6. They can be utilized to present antigenic pMHC complexes, adhesion, and co-stimulatory molecules to T cells-serving as artificial APCs. By imaging the T cells interacting with the lipid bilayer using total internal reflection fluorescence microscopy (TIRFM), we can restrict the excitation to within 100 nm of the space between the glass and the cell surface 7-8. This allows us to image primarily the signaling events occurring at the plasma membrane. As we are interested in imaging the recruitment of signaling proteins to the TCR complex, we describe a two-camera TIRF imaging system wherein the TCR, labeled with fluorescent Fab (fragment antigen binding) fragments of the H57 antibody (purified from hybridoma H57-597, ATCC, ATCC Number:HB-218) which is specific for TCR&beta;, and signaling proteins, tagged with GFP, may be imaged simultaneously and in real time. This strategy is necessary due to the highly dynamic nature of both the T cells and of the signaling events that are occurring at the TCR. This imaging modality has allowed researchers to image single ligands 9-11 as well as recruitment of signaling molecules to activated receptors and is an excellent system to study biochemistry in-situ12-16.

The compartmentalization of proteins either within the plasma membrane or into intracellular locations is one regulatory mechanism that can greatly influence signaling outcomes; hence, to understand signaling it is important to study the spatial and temporal behavior of the proteins involved. We describe here a TIRF microscopy based system to study signal transduction in T cells, but is broadly applicable.

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex ...

Real-time Live Imaging of T-cell Signaling Complex Formation

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating the activation and responses of multiple immune cells 3,4. T-cell activation is dependent on the recognition of specific antigens displayed by antigen presenting cells (APCs). The T-cell antigen receptor (TCR) is specific to each T-cell clone and determines antigen specificity 5. The binding of the TCR to the antigen induces the phosphorylation of components of the TCR complex. In order to promote T-cell activation, this signal must be transduced from the membrane to the cytoplasm and the nucleus, initiating various crucial responses such as recruitment of signaling proteins to the TCR;APC site (the immune synapse), their molecular activation, cytoskeletal rearrangement, elevation of intracellular calcium concentration, and changes in gene expression 6,7. The correct initiation and termination of activating signals is crucial for appropriate T-cell responses. The activity of signaling proteins is dependent on the formation and termination of protein-protein interactions, post translational modifications such as protein phosphorylation, formation of protein complexes, protein ubiquitylation and the recruitment of proteins to various cellular sites 8. Understanding the inner workings of the T-cell activation process is crucial for both immunological research and clinical applications.
Various assays have been developed in order to investigate protein-protein interactions; however, biochemical assays, such as the widely used co-immunoprecipitation method, do not allow protein location to be discerned, thus precluding the observation of valuable insights into the dynamics of cellular mechanisms. Additionally, these bulk assays usually combine proteins from many different cells that might be at different stages of the investigated cellular process. This can have a detrimental effect on temporal resolution. The use of real-time imaging of live cells allows both the spatial tracking of proteins and the ability to temporally distinguish between signaling events, thus shedding light on the dynamics of the process 9,10. We present a method of real-time imaging of signaling-complex formation during T-cell activation. Primary T-cells or T-cell lines, such as Jurkat, are transfected with plasmids encoding for proteins of interest fused to monomeric fluorescent proteins, preventing non-physiological oligomerization 11. Live T cells are dropped over a coverslip pre-coated with T-cell activating antibody 8,9, which binds to the CD3/TCR complex, inducing T-cell activation while overcoming the need for specific activating antigens. Activated cells are constantly imaged with the use of confocal microscopy. Imaging data are analyzed to yield quantitative results, such as the colocalization coefficient of the signaling proteins.

We describe a live-cell imaging method that provides insight into protein dynamics during the T-cell activation process. We demonstrate the combined usage of the T-cell spreading assay, confocal microscopy and imaging analysis to yield quantitative results to follow signaling complex formation throughout T-cell activation.

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

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 ...

Radial Mobility and Cytotoxic Function of Retroviral Replicating Vector Transduced, Non-adherent Alloresponsive T Lymphocytes

We report a novel adaptation of the Radial Monolayer Cell Migration assay, first reported to measure the radial migration of adherent tumor cells on extracellular matrix proteins, for measuring the motility of fluorescently-labeled, non-adherent human or murine effector immune cells. This technique employs a stainless steel manifold and 10-well Teflon slide to focally deposit non-adherent T cells into wells prepared with either confluent tumor cell monolayers or extracellular matrix proteins. Light and/or multi-channel fluorescence microscopy is used to track the movement and behavior of the effector cells over time. Fluorescent dyes and/or viral vectors that code for fluorescent transgenes are used to differentially label the cell types for imaging. This method is distinct from similar-type in vitro assays that track horizontal or vertical migration/invasion utilizing slide chambers, agar or transwell plates. The assay allows detailed imaging data to be collected with different cell types distinguished by specific fluorescent markers; even specific subpopulations of cells (i.e., transduced/nontransduced) can be monitored. Surface intensity fluorescence plots are generated using specific fluorescence channels that correspond to the migrating cell type. This allows for better visualization of the non-adherent immune cell mobility at specific times. It is possible to gather evidence of other effector cell functions, such as cytotoxicity or transfer of viral vectors from effector to target cells, as well. Thus, the method allows researchers to microscopically document cell-to-cell interactions of differentially-labeled, non-adherent with adherent cells of various types. Such information may be especially relevant in the assessment of biologically-manipulated or activated immune cell types, where visual proof of functionality is desired with tumor target cells before their use for cancer therapy.

We describe a protocol to monitor radial mobility of non-adherent immune cells in vitro using a cell sedimentation manifold/slide apparatus. Cell migration is tracked on monolayers of tumor cells or on extracellular matrix proteins. Examination by light and fluorescence microscopy allows for observation of cell mobility and cytotoxic functionality.

We report a novel adaptation of the Radial Monolayer Cell Migration assay, first reported to measure the radial migration of adherent tumor cells on ...

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte cell culture and 64Cu-antibody receptor targeting of the cells. In vitro evaluation of the radiolabel and non-invasive in vivo cell tracking in an animal model of an airway delayed-type hypersensitivity reaction (DTHR) by PET/CT are described.
In detail, the conjugation of a mAb with the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) is shown. Following the production of radioactive 64Cu, radiolabeling of the DOTA-conjugated mAb is described. Next, the expansion of chicken ovalbumin (cOVA)-specific CD4+ interferon (IFN)-&#947;-producing T helper cells (cOVA-TH1) and the subsequent radiolabeling of the cOVA-TH1 cells are depicted. Various in vitro techniques are presented to evaluate the effects of 64Cu-radiolabeling on the cells, such as the determination of cell viability by trypan blue exclusion, the staining for apoptosis with Annexin V for flow cytometry, and the assessment of functionality by IFN-&#947; enzyme-linked immunosorbent assay (ELISA). Furthermore, the determination of the radioactive uptake into the cells and the labeling stability are described in detail. This protocol further describes how to perform cell tracking studies in an animal model for an airway DTHR and, therefore, the induction of cOVA-induced acute airway DHTR in BALB/c mice is included. Finally, a robust PET/CT workflow including image acquisition, reconstruction, and analysis is presented.
The 64Cu-antibody receptor targeting approach with subsequent receptor internalization provides high specificity and stability, reduced cellular toxicity, and low efflux rates compared to common PET-tracers for cell labeling, e.g.64Cu-pyruvaldehyde bis(N4-methylthiosemicarbazone) (64Cu-PTSM). Finally, our approach enables non-invasive in vivo cell tracking by PET/CT with an optimal signal-to-background ratio for 48 h. This experimental approach can be transferred to different animal models and cell types with membrane-bound receptors that are internalized.

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo  Cell Trafficking by PET/CT

Following the preparation of a 64Cu-modified monoclonal antibody binding to a transgenic murine T cell receptor, T cells are radiolabeled in vivo, analyzed for viability, functionality, labeling stability and apoptosis, and adoptively transferred into mice with an airway delayed-type hypersensitivity reaction for non-invasive imaging by positron emission tomography/computed tomography (PET/CT).

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte ...

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and temporal specificity2. Chemotaxis is a critical function of lymphocytes and other immune cells that can be quantitatively assessed in vitro. This manuscript describes methods that permit the evaluation of chemotaxis, both in vitro and in vivo, for diverse cell types including cell lines and native cells. The in vitro, plate-based format permits the comparison of several conditions simultaneously in real-time, and can be completed within 1-4 h. In vitro assay conditions can be manipulated to introduce agonists and antagonists, as well as differentiate chemotaxis from chemokinesis, which is random movement. For in vivo trafficking assessments, immune cells can be labeled with multiple fluorescent dyes and used for adoptive transfer. The differential labeling of cells allows for mixed cell populations to be introduced into the same animal, thereby decreasing variance and reducing the number of animals required for an adequately powered experiment. Migration into lymphoid tissue occurs in as little as 1 h, and multiple tissue compartments can be sampled. Flow cytometry following tissue harvest allows for a rapid and quantitative analysis of the migratory patterns of multiple cell types.

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Protocols for quantitative assessment of lymphocyte chemotaxis and migration are important tools for immunology research. Here, an in vitro protocol is described that permits real-time, multiplexed evaluation of cell migration, as well as a complementary in vivo technique enabling tracking of native cells to spleen.

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and ...

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

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