Name: tracking bispecific antibody-induced t cell trafficking using luciferase-transduced human t cells
Uploaded: 2023-05-12T12:05:00
Description: Watch this Scientific Journal Video about Tracking Bispecific Antibody-Induced T Cell Trafficking Using Luciferase-Transduced Human T Cells at JoVE.com

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 ...

<ol>
	<li>G&#246;kbuget, N., 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=Blinatumomab+for+minimal+residual+disease+in+adults+with+B-cell+precursor+acute+lymphoblastic+leukemia.">Blinatumomab for minimal residual disease in adults with B-cell precursor acute lymphoblastic leukemia.</a> Blood. 131 (14), 1522-1531 (2018).</li><li>Runcie, K., Budman, D. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+regular+cytolytic+T+cell+synapses+by+bispecific+single-chain+antibody+constructs+on+MHC+class+I-negative+tumor+cells.">Induction of regular cytolytic T cell synapses by bispecific single-chain antibody constructs on MHC class I-negative tumor cells.</a> Molecular Immunology. 43 (6), 763-771 (2006).</li><li>Santich, B. H., Cheung, N. V., Klein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Editorial:+Bispecific+antibodies+for+T-cell+based+immunotherapy.">Editorial: Bispecific antibodies for T-cell based immunotherapy.</a> Frontiers in Oncology. 10, 628005 (2020).</li><li>Santich, B. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interdomain+spacing+and+spatial+configuration+drive+the+potency+of+IgG-[L]-scFv+T+cell+bispecific+antibodies.">Interdomain spacing and spatial configuration drive the potency of IgG-[L]-scFv T cell bispecific antibodies.</a> Science Translational Medicine. 12 (534), eaax1315 (2020).</li><li>Wang, L., Hoseini, S. S., Xu, H., Ponomarev, V., Cheung, N. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silencing+Fc+domains+in+T+cell-engaging+bispecific+antibodies+improves+T-cell+trafficking+and+antitumor+potency.">Silencing Fc domains in T cell-engaging bispecific antibodies improves T-cell trafficking and antitumor potency.</a> Cancer Immunology Research. 7 (12), 2013-2024 (2019).</li><li>Park, J. A., Cheung, N. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GD2+or+HER2+targeting+T+cell+engaging+bispecific+antibodies+to+treat+osteosarcoma.">GD2 or HER2 targeting T cell engaging bispecific antibodies to treat osteosarcoma.</a> Journal of Hematology &amp; Oncology. 13 (1), 172 (2020).</li><li>Wu, Z., Guo, H. F., Xu, H., Cheung, N. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+tetravalent+anti-GPA33/anti-CD3+bispecific+antibody+for+colorectal+cancers.">Development of a tetravalent anti-GPA33/anti-CD3 bispecific antibody for colorectal cancers.</a> Molecular Cancer Therapeutics. 17 (10), 2164-2175 (2018).</li><li>Lin, T. Y., Park, J. A., Long, A., Guo, H. F., Cheung, N. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+potent+anti-STEAP1+bispecific+antibody+to+redirect+T+cells+for+cancer+immunotherapy.">Novel potent anti-STEAP1 bispecific antibody to redirect T cells for cancer immunotherapy.</a> Journal for Immunotherapy of Cancer. 9 (9), e003114 (2021).</li><li>Hoseini, S. S., Espinosa-Cotton, M., Guo, H. F., Cheung, N. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overcoming+leukemia+heterogeneity+by+combining+T+cell+engaging+bispecific+antibodies.">Overcoming leukemia heterogeneity by combining T cell engaging bispecific antibodies.</a> Journal for Immunotherapy of Cancer. 8 (2), e001626 (2020).</li><li>Hoseini, S. S., Guo, H., Wu, Z., Hatano, M. N., Cheung, N. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+potent+tetravalent+T-cell-engaging+bispecific+antibody+against+CD33+in+acute+myeloid+leukemia.">A potent tetravalent T-cell-engaging bispecific antibody against CD33 in acute myeloid leukemia.</a> Blood Advances. 2 (11), 1250-1258 (2018).</li><li>Hoseini, S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+engaging+bispecific+antibodies+targeting+CD33+IgV+and+IgC+domains+for+the+treatment+of+acute+myeloid+leukemia.">T cell engaging bispecific antibodies targeting CD33 IgV and IgC domains for the treatment of acute myeloid leukemia.</a> Journal for Immunotherapy of Cancer. 9 (5), e002509 (2021).</li><li>Li, H., Er Saw, P., Song, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenges+and+strategies+for+next-generation+bispecific+antibody-based+antitumor+therapeutics.">Challenges and strategies for next-generation bispecific antibody-based antitumor therapeutics.</a> Cellular and Molecular Immunology. 17 (5), 451-461 (2020).</li><li>Rajabzadeh, A., Hamidieh, A. A., Rahbarizadeh, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinoculation+and+retronectin+highly+enhance+the+gene+transduction+efficiency+of+Mucin-1-specific+chimeric+antigen+receptor+(CAR)+in+human+primary+T+cells.">Spinoculation and retronectin highly enhance the gene transduction efficiency of Mucin-1-specific chimeric antigen receptor (CAR) in human primary T cells.</a> BMC Molecular and Cell Biology. 22 (1), 57 (2021).</li><li>Kleeman, 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=A+guide+to+choosing+fluorescent+protein+combinations+for+flow+cytometric+analysis+based+on+spectral+overlap.">A guide to choosing fluorescent protein combinations for flow cytometric analysis based on spectral overlap.</a> Cytometry Part A. 93 (5), 556-562 (2018).</li><li>Park, J. A., Santich, B. H., Xu, H., Lum, L. G., Cheung, N. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potent+ex+vivo+armed+T+cells+using+recombinant+bispecific+antibodies+for+adoptive+immunotherapy+with+reduced+cytokine+release.">Potent ex vivo armed T cells using recombinant bispecific antibodies for adoptive immunotherapy with reduced cytokine release.</a> Journal for Immunotherapy of Cancer. 9 (5), e002222 (2021).</li><li>Park, J. A., Wang, L., Cheung, N. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulating+tumor+infiltrating+myeloid+cells+to+enhance+bispecific+antibody-driven+T+cell+infiltration+and+anti-tumor+response.">Modulating tumor infiltrating myeloid cells to enhance bispecific antibody-driven T cell infiltration and anti-tumor response.</a> Journal of Hematology &amp; Oncology. 14 (1), 142 (2021).</li><li>Str&#246;hlein, M. A., Heiss, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+trifunctional+antibody+catumaxomab+in+treatment+of+malignant+ascites+and+peritoneal+carcinomatosis.">The trifunctional antibody catumaxomab in treatment of malignant ascites and peritoneal carcinomatosis.</a> Future Oncology. 6 (9), 1387-1394 (2010).</li><li>Rabinovich, B. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+fewer+than+10+mouse+T+cells+with+an+enhanced+firefly+luciferase+in+immunocompetent+mouse+models+of+cancer.">Visualizing fewer than 10 mouse T cells with an enhanced firefly luciferase in immunocompetent mouse models of cancer.</a> Proceedings of the National Academy of Sciences. 105 (38), 14342-14346 (2008).</li><li>Skovgard, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+CAR+T-cell+kinetics+in+solid+tumors:+Translational+implications.">Imaging CAR T-cell kinetics in solid tumors: Translational implications.</a> Molecular Therapy Oncolytics. 22, 355-367 (2021).</li></ol>

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 ...

This method allows users to easily track T cells in vivo to evaluate the kinetics of homing and to monitor T cell persistence. The main advantage of this technique is that it can be repeated at multiple time points on the same animal without requiring sacrifice in flow cytometry or immunohistochemistry. This technique could be modified for tracking other types of immune cells as well.To culture 293T cells, prepare one liter of DMEM by adding 110 milliliters of heat-inactivated fetal bovine serum and 11 milliliters of penicillin streptomycin. Thaw five times 10 to the sixth 293T cells and transfer them into a T175 flask containing DMEM. Split the cells one to 10 every three days for six days before the cells become more than 90%confluent.For transfection, transfer 1.5 milliliters of media to two 50 milliliter tubes using a pipette. To one tube, add 10 micrograms of VSVG plasmid, 20 micrograms of gag pol, and 20 micrograms of click beetle red TDtomato or CBRTDR luciferase plasmid and mix. Add 100 microliters of DNA in vitro transfection reagent to another tube and mix.Incubate both tubes at room temperature for five minutes. Transfer the media containing the DNA in vitro transfection reagent dropwise to the tube containing the DNA plasmids and mix gently with a pipette. Incubate at room temperature for 20 minutes.During incubation, detach the 293T cells by adding five to 10 milliliters of 0.05%trypsin. Once the cells have detached, add 10 milliliters of media and centrifuge at 800 G for five minutes. Resuspend the cells in 1.5 milliliters of media.Add the media containing the DNA in vitro transfection reagent and the DNA plasmids to the 293T cells and incubate at 37 degrees Celsius for 30 minutes. After incubation, transfer the suspension to a T175 flask. Add 18 milliliters of media and incubate at 37 degrees Celsius overnight.The next day, carefully aspirate the media with a glass pipette without detaching the cells and replace it with 18 milliliters of fresh media. Again, incubate overnight at 37 degrees Celsius. The following day, remove the viral supernatant using a pipette put on ice and centrifuge at 800 G for five minutes to pellet the cells and debris.Filter the viral supernatant using a 0.22 micron filter and immediately place it on ice. For in vitro activation of human T cells, add 10 milliliters of PBS containing 0.1%bovine serum albumin or BSA and two millimolar EDTA to a sterile 15 milliliter tube. Then to wash the beads, add beads into a tube and place the tube in a magnetic rack.After two minutes, remove the PBS from the tube. After preparing DMEM, add washed beads and interleukin-2 or IL-2 to a final concentration of 30 international units per milliliter. Count the T cells using a hemo cytometer and Trypan blue stain.After counting, add desired number of T cells to the media and gently invert the tube to mix. Plate 2.5 milliliters of media containing two million T cells, beads, and IL-2 in each well of a 24-well tissue culture plate. Culture at 37 degrees Celsius in humidified 5%carbon dioxide.Examine the T cells under a 20X objective every day for three days to determine when to transduce with luciferase. Next, to prepare a RetroNectin plate, add two milliliters of 20 micrograms per milliliter RetroNectin in PBS to a well of an untreated six-well plate and incubate for two hours at room temperature. Aspirate the RetroNectin without scratching the bottom of the plate and add three milliliters of 2%BSA in PBS per well in the six-well plate.Incubate for 30 minutes at room temperature. Next, for spinoculation, aspirate the BSA and wash the wells once with three milliliters of PBS. Continue or replace with fresh PBS and leave the plate covered at four degrees Celsius overnight.The next day, add two milliliters of CBRTDR luciferase viral supernatant per well and centrifuge at 1, 240 G for 90 minutes at 32 degrees Celsius. For transduction, after counting the T cells, aspirate the viral supernatant and plate two million T cells per well in six milliliters of DMEM containing 30 international units per milliliter of human IL-2. Examine the T cells for two days.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 nine milliliters of media for a total of 15 milliliter volume per flask. The next day, add 15 milliliters of media and confirm successful transduction by performing flow cytometry.After anesthetizing the mouse, using a 26 gauge needle, inject 20 million T cells in 100 microliters of media retroorbitally. Administer 1, 000 units of recombinant IL-2 subcutaneously to support T cell survival in vivo. Then administer the bispecific antibody retroorbitally or intraperitoneally.For in vivo imaging, administer 100 microliters of three milligrams of D-luciferin by retroorbital injection with a 26 gauge needle in an anesthetized mouse. To capture images, place the mouse in the light tight chamber of the imager such that the flank bearing the xenograft is facing up toward the camera. Using the acquisition control panel, select luminescent, photograph, and overlay.Set the exposure time to auto, the binning to medium, and f-stop to one and acquire images. After the first image, set the exposure time to match that automatically calculated for the first image so that subsequent images may be compared directly. Take another set of images with the mouse supine to evaluate T cell presence in the lungs.Armed T cells trafficked to the tumor faster than unarmed T cells, leaving the lungs two days sooner. Quantitation of T cell infiltration into tumors over time was measured by bioluminescence and expressed as total flocks or radiance per pixel integrated over the tumor contour. T cell trafficking could be monitored for 28 days post-injection of the luciferase-transduced T cells, allowing for tracking T cell homing and persistence throughout treatment.T cell trafficking into HER2 positive human breast cancer patient-derived xenografts in DKO mice is shown. Treatment with the HER2 bispecific antibody with an unsilenced FC had no effect on tumor growth compared to a controlled bispecific antibody. In contrast, treatment with HER2 bispecific antibody containing N297A mutation alone or in combination with a K322A mutation was able to halt tumor growth completely.For groups with the silencing mutations, the T cell luminescence signal reached a higher peak on day three post-injection and persisted at a higher level compared to the controlled bispecific antibody and unsilenced HER2 bispecific antibody. Mice bearing neuroblastoma patient-derived xenografts demonstrated depletion of Ly6C positive macrophages significantly increased T cell homing to tumors, resulting in decreased tumor growth and prolonged survival. The effect was more pronounced in osteosarcoma patient-derived xenografts.Among bispecific antibodies targeting the tumor antigen STEAP1, the IGGL SCFV format resulted in significantly more T cell infiltration on day six after the first dose of T cells which persisted for the duration of treatment. Such observations were not predicted by in vitro cytotoxicity assays. After tracking T cells in vivo, users can still perform immunohistochemistry to determine more specifically where the T cells are located within the tumor or other normal tissues.

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