The authors would like to acknowledge Dr. Laura Johnson and Dr. Richard Morgan for providing the CAR retroviral construct. The authors also thank Giao Ngyuen for her assistance with dosimetry for whole brain irradiation. This work was supported by an NIH NCI grant 1R01CA177476-01.

This protocol is based on an experimental design where 10 mice are treated with 107 CAR T cells each. This means that 108 CAR T cells will be needed; the yield should be overestimated by 5 x 107-1 x 108 to account for loss in viability. The following protocol is scaled to generate approximately 200 x 106 cells. The cells are then administered intravenously to female C57BL/6 mice with 9 day established syngeneic EGFRvIII-positive intracranial tumors, developed from the existing KR158B astrocytoma or B16 melanoma cell lines. Concomitantly with CAR T cell generation, tumor-bearing mice are administered clinically relevant doses of lymphodepletive TMZ (60 mg/kg) and WBI (16.5 Gy).
Mice were maintained and bred under pathogen-free conditions at Duke University Medical Center (DUMC). All animal experiments were performed according to protocols approved by the Duke University Institutional Animal Care and Use Committee (IACUC).&#160;
1. Administration of Temozolomide to Tumor-bearing Mice
Days 0 through 4:
<ol>
	<li>Calculate the total number of mice to be injected and multiply this by 0.5 to determine the volume of TMZ solution needed (e.g., 30 mice x 0.5 ml = 15 ml TMZ) and add 1.5 ml to this volume to account for spillover (16.5 ml TMZ).</li>
	<li>Multiply this total volume by 3 mg/ml to determine the weight of lyophilized TMZ needed (e.g., 16.5 x 3 = 49.5 mg TMZ). Weigh this into a 50 ml conical. 
	CAUTION: TMZ is toxic by inhalation, ingestion, and contact with eyes, skin, and mucosal membranes.&#160;Consult with institution&#8217;s occupational safety and/or environmental health and wellness department for recommendations on reducing risk of exposure.&#160;</li>
	<li>Multiply the total volume by 0.15 to determine the volume of dimethyl sulfoxide (DMSO) needed (e.g., 16.5 x 0.15 = 2.475 ml DMSO).&#160;Filter sterilize this volume of DMSO (plus an additional 2 ml to account for spillover) through a 0.2 &#181;m filter into a sterile tube. Add 2.475 mL of sterile DMSO to the TMZ powder.</li>
	<li>Place the TMZ/DMSO solution in a beaker of water over a hot plate at 65 &#176;C for 10-15 min. Once TMZ is fully dissolved, the solution should become pale yellow in color; if the solution becomes pink, then the TMZ is degraded, and a new solution should be prepared with a fresh lot of TMZ.</li>
	<li>Calculate the appropriate volume of sterile saline needed by multiplying 0.85 by the total volume (0.85 x 16.5 ml = 14.025 ml saline). Add 15 ml of sterile&#160;saline to a 50 ml conical. While TMZ is dissolving in the DMSO, heat saline to 65 &#176;C.</li>
	<li>Vortex the TMZ/DMSO solution to ensure that the TMZ powder is fully dissolved and slowly add warmed saline to the TMZ/DMSO solution.</li>
	<li>Immediately after preparation, fully load 15 x 1 ml tuberculin syringes with TMZ solution for administration to 4 day established tumor bearing mice.</li>
	<li>Repeat this procedure on days 1 through 4 for a total of five TMZ administrations.</li>
</ol>
2. Whole Brain Irradiation of Tumor-bearing Mice
Days 2 through 4:
<ol>
	<li>Power on the X-ray irradiator and allow it to warm-up to full voltage. Ensure that the appropriate filter is in place and input the proper voltage and current settings (Figure 2A). 
	NOTE: The dosimetry of the irradiator should be established beforehand to determine the appropriate voltage settings and grid layout (Figure 2A,B).</li>
	<li>Calculate the length of time that is necessary to result in 5.5 Gy X-ray irradiation. For example, if X-rays are delivered at a rate of 2 Gy/min, then 2.75 min is necessary to deliver a total of 5.5 Gy. Input the appropriate time duration. 
	NOTE: Table 2 provides mouse WBI doses and their clinical equivalents in humans. In the context of high-grade gliomas, 60 Gy fractionated WBI (2 Gy fractions, 5 days/week for 6 weeks) is the clinical standard of care, and the murine equivalent used here is 16.5 Gy (5.5 Gy x 3).</li>
	<li>Prepare a fresh&#160;solution of ketamine/xylazine for systemic anesthesia by adding 2 ml ketamine and 1 ml xylazine to 17 ml saline such that the final solution is 10 mg/ml ketamine and 1 mg/ml xylazine.&#160;</li>
	<li>Weigh mice and administer 10 &#956;l of ketamine/xylazine intraperitoneally per gram of body weight such that animals receive a dose of ketamine at 100 mg/kg and 10 mg/kg xylazine. Mice should be fully sedated and visibly breathing within 2 min of ketamine/xylazine administration.</li>
	<li>To maintain ophthalmic moisture in sedated animals, gently rub a small amount of artificial tears ointment on each eye.</li>
	<li>Place sedated mice on the grid such that heads are positioned in the area that receives the highest X-ray intensity (Figure 2B,C). Shield bodies from the neck down with appropriately sized lead tubing to block systemic X-ray delivery (Figure 2D).</li>
	<li>Place positioned mice under the X-ray beam such that the laser denoting the focal point of the X-ray beam is at the coordinate (0,0) on the grid layout. Begin X-ray delivery.</li>
	<li>When X-ray delivery is complete, remove animals from the irradiator and place on a warm heating pad. Do not house sedated animals with conscious animals until animals have regained sufficient consciousness to maintain sternal recumbence. Once animals can maintain sternal recumbence, place conscious animals back in their appropriate cages.</li>
	<li>Repeat this procedure on days 3 and 4 for a total of three fractionated doses of radiation.</li>
</ol>
3. Transfection
Day -1:
<ol>
	<li>Prepare D10 media by adding 50 ml fetal bovine serum (FBS) to 500 ml of Dulbecco&#8217;s Modified Eagle Medium (DMEM).</li>
	<li>Prepare T cell media (TCM) by adding 5.5 ml L-Glutamine, 5.5 ml sodium pyruvate, 5.5 ml non-essential amino acids, and 5.5 ml pencillin/streptomycin (pen/strep) to 500 ml RPMI-1640 media. Next, add 550 &#956;l of 2-mercaptoethanol and 550 &#956;l gentamicin. Finally, add 50 ml FBS.</li>
	<li>Harvest in vitro cultured HEK293T cells and bring to a concentration of 7.5 x 106 cells/ml in D10 media.</li>
	<li>Plate 10 ml D10 media and add 1 ml of HEK293T cell suspension in each of 16 x 10 cm poly-D-lysine (PDL) coated plates.</li>
	<li>Incubate overnight at 37 &#176;C with 5% CO2.</li>
</ol>
Day 0:
<ol start="6">
	<li>Replace media with 10 ml fresh D10 at least 30 min before transfecting cells.</li>
	<li>Determine the amount of vector plasmid, pCL-Eco vector, and liposomal transfection reagent required for transfection by multiplying the total number of plates + 1 (16 + 1 = 17) by 14.1 &#956;g vector plasmid (14.1 x 17 = 239.7 &#956;g), 9.9 &#956;g pCL-Eco vector (9.9 x 17 = 168.3 &#956;g), and 60 &#956;l liposomal transfection reagent (60 x 17 = 1,020 &#956;l). All reagents should be at room temperature before preparing solutions.</li>
	<li>Label two tubes A and B. In tube A, add 239.7 &#956;g vector plasmid and 168.3 &#956;g pCL-Eco vector to (1.5 x 17) ml reduced-serum modified eagle&#8217;s medium (RS-MEM). In tube B, add 1020 &#956;l liposomal transfection reagent to (1.5 x 17) ml RS-MEM.</li>
	<li>Incubate A and B separately for 5 min at room temperature.</li>
	<li>Mix A and B together gently (vortex for 1-2 sec or invert several times) and incubate for 20 min at room temperature to form lipid/DNA complex.</li>
	<li>Add lipid/DNA complex drop wise to HEK293T cells in 10 cm dish.</li>
	<li>Incubate at 37 &#176;C for 6-8 hr or overnight (do not exceed 24 hr).</li>
	<li>After 6-8 hr incubation, replace medium with 12 ml of fresh TCM for viral production. This plated media will be used on Day 2 as viral supernatant for T cell transduction.</li>
</ol>
4. Splenectomy and T Cell Preparation
Day 0:
<ol>
	<li>Pour 10 ml of TCM into a 50 ml conical and place on ice for spleen collection.</li>
	<li>Sacrifice the appropriate number of animals by CO2 asphyxiation and secondary decapitation: Place animals in a cage receiving CO2 at a flow rate of&#160;10-30% cage volume/minute, per American Veterinary Medical Association guidelines&#160;&#160;(no more than 5 animals can be sacrificed simultaneously) until respiration terminates and for two minutes thereafter. Remove animals from the CO2 chamber and decapitate. 
	NOTE: One spleen of a 6-12 week old female C57BL/6 mouse will yield approximately 4.5-5 x 107 splenocytes. Here, 4 spleens will be harvested for approximately 200 x 106 cells.</li>
	<li>Lay the mouse such that its right side is facing up, and spray with 70% ethanol. With the forceps, grab a thin fold of skin below the left ribcage and cut a slight incision with the scissors. Peel back the skin, carefully grab a thin fold of the peritoneum with forceps, and cut a small cavity.</li>
	<li>The spleen is a small, elongated, dark red organ that resembles a flattened bean; delicately grab the spleen with the forceps and excise by cutting away the surrounding connective tissue. Place the excised spleens into the conical containing 10 ml of TCM on ice.</li>
	<li>Pour spleens over a 70 &#956;m mesh cell strainer and disaggregate by mashing with the blunt end of the inside of a 5 ml syringe to generate a single-cell suspension. A maximum of two spleens should be disaggregated per mesh strainer.</li>
	<li>Use a small volume of TCM to carefully wash the strainer following disaggregation to collect any remaining splenocytes. Pool all disaggregated spleens into a single-cell suspension. Bring final volume to 50 ml with TCM for one wash and spin at 300 x g for 10 min.</li>
	<li>Prepare a solution of 1x lysis buffer by adding 5 ml 10x lysis buffer to 45 ml sterile water. To eliminate red blood cells, resuspend pellet in 5 ml of 1x lysis buffer per spleen in a 50 ml conical, mixing well by gently pipetting up and down. If exceeding 5 spleens, use a 250 ml centrifuge tube. Place conical or centrifuge tube in a 37 &#176;C water bath for 5 min.</li>
	<li>Remove the lysis reaction from the water bath and add TCM at a 1:1 ratio with lysis buffer to neutralize the reaction. Wash by spinning at 300 x g for 10 min.</li>
	<li>Aspirate supernatant and fully resuspend pellet in TCM (2 ml/spleen, 8 ml total for 4 spleens) by pipetting up and down.</li>
	<li>Count cells by adding 10 &#956;l of cell suspension to 190 &#956;l trypan blue (1:20 dilution). Multiply the number obtained in one of the four gridded squares by 20 x 104 x total volume (8 ml) to obtain the total cell number (e.g., 125 cells x 20 x 104 x 8 ml = 200 x 106 splenocytes).</li>
	<li>Dilute cells to a concentration of 2 x 106 cells/ml in TCM, supplemented with 2 &#956;g/ml concanavalin A (ConA) and 50 IU/ml recombinant human interleukin-2 (rhIL-2). Thus, for 200 x 106 splenocytes, add 92 ml media to 8 ml cells, 200 &#956;g ConA, and 5,000 IU rhIL-2.</li>
	<li>Add 2 mL cells to each well of 24-well tissue-culture treated plates, such that 4 x 106 cells are in each well (e.g., 100 ml of cells will require approximately 4 plates).</li>
	<li>Incubate overnight at 37 &#176;C with 5% CO2.</li>
</ol>
5. Transduction
Day 1:
<ol>
	<li>Calculate the number of non-tissue culture treated 24-well plates needed for transduction by multiplying the number of spleens harvested by 2 (8 plates are needed for 4 spleens).</li>
	<li>Next, calculate the required volume of recombinant human fibronectin fragment (RHFF) solution needed to coat plates by multiplying (total number of wells + 3) x 0.5 ml (8 plates x 24 wells = 192 + 3 = 195) x 0.5 ml = 97.5 ml.</li>
	<li>Prepare 97.5 ml phosphate-buffered saline (PBS) containing RHFF at a concentration of 25 &#956;g/ml by multiplying the total volume by 25 &#956;g (97.5 x 25 = 2437.5 &#956;g). Add this amount of RHFF to 97.5 ml PBS.</li>
	<li>Coat non-tissue culture treated 24-well plates by adding 0.5 ml PBS/RHFF solution per well.</li>
	<li>Incubate overnight at 4 &#176;C.</li>
</ol>
Day 2:
<ol start="6">
	<li>Dump PBS/RHFF solution from non-tissue culture treated 24-well plates.</li>
	<li>Add 1 ml/well 2% bovine serum albumin (BSA) in PBS and incubate at room temperature for 30 min.</li>
	<li>Remove BSA by firmly upending plate. Wash by adding 2 ml PBS.</li>
	<li>Collect viral supernatant by transferring media from each HEK293T PDL plate into a 250 ml centrifuge tube and spin 10 min at 500 x g.</li>
	<li>Carefully transfer viral supernatant into a fresh 250 ml centrifuge tube, being sure not to disturb the cell pellet that may have formed.</li>
	<li>Add fresh TCM to the viral supernatant such that the final volume is 3 ml more than the amount needed for RHFF-coated wells. For example, for 192 RHFF-coated wells, bring the volume of viral supernatant to 192 + 3 ml = 195 ml.</li>
	<li>Remove cultured splenocytes from the incubator and resuspend by gently pipetting up and down 2 - 3 times in each well. Transfer to a 250 ml conical. If using a multichannel pipette, using a sterile reservoir prior to transfer will help expedite this step. Count as previously described and spin at 300 x g for 10 min.</li>
	<li>Add rhIL-2 to viral supernatant at a concentration of 50 IU/ml. For example, add 50 x 195 = 9,750 IU rhIL-2 to 195 ml of viral supernatant.</li>
	<li>Resuspend splenocytes in viral supernatant at 1 x 106 cells/ml</li>
	<li>Add 1 ml/well of splenocyte suspension to RHFF-coated 24-well plates.</li>
	<li>Spin for 90 min according to the following settings: 770 x g, acceleration = 4, brake/deceleration = 0, 32 &#176;C.</li>
	<li>Prepare a TCM solution with 50 IU/ml rhIL-2. For example, prepare a 200 TCM solution by adding 10,000 IU rhIL-2. Add 1 ml of rhIL-2/TCM to each well after centrifugation.</li>
	<li>Culture overnight at 37 &#176;C in 5% CO2.</li>
</ol>
6. CAR T cell Culture and Harvest
Days 3 and 4:
<ol>
	<li>If T cells achieve &#62;80% confluence, cells may be split (this usually occurs by day 3 or day 4). To split cells, gently pipet up and down in each well 2-3 times, and move 1 ml from each well into new wells of a fresh 24-well tissue-culture treated plate. Then, add 1 ml of fresh TCM with 50 IU/ml IL-2 to each well such that final volume is 2 ml in all wells.</li>
	<li>If cells do not reach &#62;80% confluence, perform a half media change by slowly pipetting off 1 ml of media from the top of each well. Avoid disturbing cells settled on the bottom while removing media. Add 1 ml of fresh TCM containing 50 IU/ml rhIL-2.</li>
</ol>
Day 5:
<ol start="3">
	<li>Resuspend CAR T cells by gently pipetting up and down 3 times in each well and transfer to a 250 ml centrifuge tube. Spin cells at 300 x g for 10 min.</li>
	<li>Completely aspirate supernatant without disturbing pellet. Wash once with PBS, count cells as previously described, and wash with PBS a second time. A typical CAR T cell yield is approximately 1 x 106 cells per well (8 plates x 24 wells = 192 x 106 CAR T cells).</li>
	<li>Resuspend cells in PBS at a concentration of 5 x 107/ml for an injection of 1 x 107 in a volume of 200 &#956;g (e.g., for 192 x 106 CAR T cells, resuspend washed pellet in 3.84 ml PBS).</li>
</ol>
7. CAR T cell Administration to Tumor-bearing Mice
Day 5:
<ol>
	<li>Transport cells on ice to animal facility for intravenous injection. Load 500-1,000 &#956;l into insulin syringe with 27 - 31 G needle, ensuring that all bubbles are expelled.</li>
	<li>Grab a mouse at the base of the tail and place in tube restrainer.</li>
	<li>Pull the tail taut with the vein facing upward, and glide the needle approximately 1-2 mm under the skin into the vein by inserting it parallel to the tail vein. Slowly expel 200 &#956;l into the vein. If the needle is correctly inserted into the vein, the volume will easily flow in; otherwise there will be resistance and the needle will need to be removed from the tail and re-inserted correctly.</li>
</ol>

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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=An+in+vivo+immunotherapy+screen+of+costimulatory+molecules+identifies+Fc-OX40L+as+a+potent+reagent+for+the+treatment+of+established+murine+gliomas.">An in vivo immunotherapy screen of costimulatory molecules identifies Fc-OX40L as a potent reagent for the treatment of established murine gliomas.</a> Clin Cancer Res. 18 (17), 4657-4668 (2012).</li><li>Newcomb, E. W., 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=Radiotherapy+enhances+antitumor+effect+of+anti-CD137+therapy+in+a+mouse+Glioma+model.">Radiotherapy enhances antitumor effect of anti-CD137 therapy in a mouse Glioma model.</a> Radiat Res. 173 (4), 426-432 (2010).</li><li>Su, Y. B., Krown, S. E., Livingston, P. O., Wolchok, J. D., Chapman, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+lymphotoxic+is+dose-intensified+temozolomide?+The+glioblastoma+experience.">How lymphotoxic is dose-intensified temozolomide? The glioblastoma experience.</a> J Clin Oncol. 23 (18), 4235-4236 (2005).</li><li>Neyns, B., Tosoni, A., Hwu, W. J., Reardon, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dose-dense+temozolomide+regimens:+antitumor+activity,+toxicity,+and+immunomodulatory+effects.">Dose-dense temozolomide regimens: antitumor activity, toxicity, and immunomodulatory effects.</a> Cancer. 116 (12), 2868-2877 (2010).</li><li>Muranski, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+intensity+lymphodepletion+and+adoptive+immunotherapy-how+far+can+we+go.">Increased intensity lymphodepletion and adoptive immunotherapy-how far can we go.</a> Nat Clin Pract Oncol. 3 (12), 668-681 (2007).</li><li>Wrzesinski, C., Restifo, N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Less+is+more:+lymphodepletion+followed+by+hematopoietic+stem+cell+transplant+augments+adoptive+T-cell-based+anti-tumor+immunotherapy.">Less is more: lymphodepletion followed by hematopoietic stem cell transplant augments adoptive T-cell-based anti-tumor immunotherapy.</a> Current Opinion in Immunology. 17 (2), 195-201 (2005).</li><li>Dudley, M. E., 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=Adoptive+cell+therapy+for+patients+with+metastatic+melanoma:+evaluation+of+intensive+myeloablative+chemoradiation+preparative+regimens.">Adoptive cell therapy for patients with metastatic melanoma: evaluation of intensive myeloablative chemoradiation preparative regimens.</a> J Clin Oncol. 26 (32), 5233-5239 (2008).</li><li>Sanchez-Perez, L. 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=Myeloablative+Temozolomide+Enhances+CD8(+)+T-Cell+Responses+to+Vaccine+and+Is+Required+for+Efficacy+against+Brain+Tumors+in+Mice.">Myeloablative Temozolomide Enhances CD8(+) T-Cell Responses to Vaccine and Is Required for Efficacy against Brain Tumors in Mice.</a> Plos One. 8 (3), (2013).</li><li>Su, Y. 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=Selective+CD4++lymphopenia+in+melanoma+patients+treated+with+temozolomide:+a+toxicity+with+therapeutic+implications.">Selective CD4+ lymphopenia in melanoma patients treated with temozolomide: a toxicity with therapeutic implications.</a> J Clin Oncol. 22 (4), 610-616 (1200).</li><li>Dummer, W., 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+homeostatic+proliferation+elicits+effective+antitumor+autoimmunity.">T cell homeostatic proliferation elicits effective antitumor autoimmunity.</a> J Clin Invest. 110 (2), 185-192 (2002).</li><li>Gattinoni, L., 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=Removal+of+homeostatic+cytokine+sinks+by+lymphodepletion+enhances+the+efficacy+of+adoptively+transferred+tumor-specific+CD8++T+cells.">Removal of homeostatic cytokine sinks by lymphodepletion enhances the efficacy of adoptively transferred tumor-specific CD8+ T cells.</a> J Exp Med. 202 (7), 907-912 (2005).</li><li>Wrzesinski, C., 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=Increased+intensity+lymphodepletion+enhances+tumor+treatment+efficacy+of+adoptively+transferred+tumor-specific+T+cells.">Increased intensity lymphodepletion enhances tumor treatment efficacy of adoptively transferred tumor-specific T cells.</a> J Immunother. 33 (1), 1-7 (2010).</li><li>Hughes, 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=Transfer+of+a+TCR+gene+derived+from+a+patient+with+a+marked+antitumor+response+conveys+highly+active+T-cell+effector+functions.">Transfer of a TCR gene derived from a patient with a marked antitumor response conveys highly active T-cell effector functions.</a> Hum Gene Ther. 16 (4), 457-472 (2005).</li><li>Morgan, R. 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=Recognition+of+glioma+stem+cells+by+genetically+modified+T+cells+targeting+EGFRvIII+and+development+of+adoptive+cell+therapy+for+glioma.">Recognition of glioma stem cells by genetically modified T cells targeting EGFRvIII and development of adoptive cell therapy for glioma.</a> Hum Gene Ther. 23 (10), 1043-1053 (2012).</li><li>Kerkar, S. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+engineering+of+murine+CD8++and+CD4++T+cells+for+preclinical+adoptive+immunotherapy+studies.">Genetic engineering of murine CD8+ and CD4+ T cells for preclinical adoptive immunotherapy studies.</a> J Immunother. 34 (4), 343-352 (2011).</li></ol>

The authors have no conflicts of interests to declare.

The treatment timeline described here was designed to model clinical standard of care and leverage its effects for CAR adoptive therapy. CAR T cell doses, TMZ regimens, and radiotherapy administration can be modified to enhance in vivo T cell activity, lymphodepletion, and tumor killing. TMZ regimens can be increased to yield host myeloablation and increased expansion of adoptively transferred cells30. Furthermore, the lymphodepletive effects of TMZ can be recapitulated by low-dose (4 - 6 Gy) single-f...

Glioblastoma (GBM) is the most common primary malignant brain tumor and is invariably fatal. Surgical resection coupled with non-specific standard of care chemotherapy and radiotherapy fails to completely eliminate malignant cells, resulting in a dismal prognosis of less than 15 months in patients with this disease1. In contrast, immunotherapy offers a precise approach for specifically targeting tumor cells, and thus has the potential to serve as a highly effective treatment platform with reduced risk of collateral toxicity2-4. T cells engineered ex vivo to express chimeric antigen receptors (CARs) offer a versatile strategy for tumor immunotherapy. CARs are generated by fusing the extracellular variable region of an antibody with one or more intracellular T cell signaling molecule(s), in lieu of a full-length major histocompatibility complex (MHC)-restricted T cell receptor5. This mode of antibody-like antigen recognition allows for reactive antigen-specific T cells to recognize and respond to tumor antigens in the absence of MHC and can be adapted for a virtually infinite antigen repertoire.
CAR T cells engineered against a variety of tumor antigens have shown preclinical efficacy and outstanding promise in the clinic6-9. Specifically, in the context of GBM, a CAR T cell platform targeting epidermal growth factor receptor variant III (EGFRvIII), a tumor-specific mutation expressed on the cell surface10, was shown to prolong survival in glioma-bearing mice11. Despite their versatility, however, the clinical benefit of CAR adoptive therapy has not been fully realized, due in part to tumor-associated immunosuppression and immune evasion12-16 as well as challenges in establishing and maintaining antigen-specific T cells in vivo. Leveraging standard of care (SOC) with immunotherapy can potentially overcome several of these limitations, resulting in enhanced efficacy in both the preclinical and clinical setting.
SOC for post-resection GBM consists of high-dose temozolomide (TMZ), a DNA alkylating agent17, and whole brain irradiation (WBI)1. These treatments are presumed to synergize with tumor vaccines via upregulation of tumor MHC expression18-20 and the shedding of antigens by dead tumor cells17,19,21,22. Indeed, the addition of TMZ20,23 or WBI18,24 leads to enhanced antitumor efficacy of immune-based treatments in the preclinical setting. Furthermore, like many non-specific cytotoxic chemotherapeutics, TMZ is known to cause systemic lymphopenia25,26, which can be leveraged as a means of host-conditioning for adoptive therapy platforms27-29. TMZ-mediated lymphodepletion has been shown to enhance the frequency and function of antigen-specific T cells, leading to increased efficacy of an adoptive therapy platform against intracranial tumors30. In the context of CAR therapy, lymphodepletion serves as a means of host-conditioning by both reducing the number of endogenous suppressor T cells31, and inducing homeostatic proliferation32 via reduced competition for cytokines33, thus enhancing antitumor activity11,34. Given the synergistic relationship between GBM SOC and immunotherapy platforms, evaluating novel adoptive therapies and vaccine platforms in the context of SOC is critical for drawing meaningful conclusions regarding efficacy.
In this protocol, we outline a method for the generation and intravenous administration of murine EGFRvIII-specific CAR T cells alongside TMZ and WBI in mice bearing EGFRvIII-positive intracranial tumors (see Figure 1 for treatment timeline). Briefly, CAR T cells are made ex vivo by retroviral transduction. Human embryonic kidney (HEK) 293T cells are transfected using a DNA/lipid complex (containing the CAR vector and pCL-Eco plasmids) to produce virus, which is then used to transduce activated murine splenocytes that are harvested and cultured in parallel. During the course of CAR generation, murine hosts bearing EGFRvIII-positive intracranial tumors are administered fractionated whole-brain X-ray irradiation and systemic TMZ treatment at doses comparable to clinical SOC. CAR T cells are then delivered intravenously to lymphodepleted hosts.
The following procedure is described in seven separate phases: (1) Administration of Temozolomide to Tumor-bearing Mice, (2) Whole Brain Irradiation of Tumor-bearing Mice, (3) Transfection, (4) Splenectomy and T cell Preparation, (5) Transduction, (6) CAR T cell Culture and Harvest, and (7) CAR T cell administration to Tumor-bearing Mice. These phases consist of several steps that span 6-7 days and are performed concurrently.

<table border="0" cellpadding="0" cellspacing="0" width="1130">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">Name of Material</td>
			<td style="width:172px;">Company</td>
			<td style="width:125px;">Catalog Number</td>
			<td style="width:449px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pCL-Eco Retrovirus Packaging Vector</td>
			<td>Imgenex</td>
			<td>10045P</td>
			<td>Helper vector for generating CAR retrovirus</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Concanavalin A</td>
			<td>Sigma Aldrich</td>
			<td>C2010</td>
			<td>Non-specific mitogen to induce T cell proliferation and viral transduction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Retronectin</td>
			<td>ClonTech/Takara</td>
			<td>T100B</td>
			<td>Facilitates retroviral transduction of T cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lipofectamine 2000</td>
			<td>Life Technologies</td>
			<td>11668-019</td>
			<td>Transfection reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM, high glucose, pyruvate</td>
			<td>Life technologies</td>
			<td>11995-065</td>
			<td>HEK293 culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640</td>
			<td>Life Technologies</td>
			<td>11875-093</td>
			<td>T cell culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Opti-MEM I Reduced Serum Medium</td>
			<td>Life technologies</td>
			<td>11058-021</td>
			<td>Transfection media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">200 mM L-Glutamine&#160;</td>
			<td>Life technologies</td>
			<td>25030-081</td>
			<td>T cell culture media supplement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100 mM Sodium Pyruvate</td>
			<td>Life technologies</td>
			<td>11360-070</td>
			<td>T cell culture media supplement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100X MEM Non-Essential Amino Acids Solution&#160;</td>
			<td>Life technologies</td>
			<td>11140-050</td>
			<td>T cell culture media supplement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">55 mM 2-Mercaptoethanol&#160;</td>
			<td>Life technologies</td>
			<td>21985-023</td>
			<td>Reducing agent to remove free radicals</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Life technologies</td>
			<td>15140-122</td>
			<td>T cell culture media supplement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gentamicin (50 mg/mL)&#160;</td>
			<td>Life technologies</td>
			<td>15750-060</td>
			<td>T cell culture media supplement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GemCell U.S. Origin Fetal Bovine Serum</td>
			<td>Gemini Bio Products</td>
			<td>100-500</td>
			<td>Provides growth factors and nutrients for in vitro cell growth&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine Serum Albumin (BSA), Fraction V&#8212;Standard&#160;Grade&#160;</td>
			<td>Gemini Bio Products</td>
			<td>700-100P</td>
			<td>Blocks non-specific binding of retrovirus to retronectin-coated plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pharm Lyse (10X concentrate)&#160;</td>
			<td>BD Biosciences</td>
			<td>555899</td>
			<td>Lyses red blood cells during splenocyte processing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70 &#181;m Sterile Cell Strainers</td>
			<td>Corning</td>
			<td>352350</td>
			<td>Filters away large tissue particles during splenocyte processing&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100 mm BioCoat Culture Dishes with Poly-D-Lysine&#160;</td>
			<td>Corning</td>
			<td>356469</td>
			<td>Promotes HEK293 cell adhesion to maximize proliferation after transfection&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Temozolomide</td>
			<td>Best Pharmatech</td>
			<td>N/A</td>
			<td>Lyophilized powder prepared on the day of administration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dimethyl Sulfoxide</td>
			<td>Sigma Life Sciences</td>
			<td>D2650</td>
			<td>Necessary for complete dissolution of temozolomide</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Saline</td>
			<td>Hospira</td>
			<td>IM 0132 (5/04)</td>
			<td>Solvent for temozolomide and ketamine/xylazine&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ketathesia HCl</td>
			<td>Henry Schein Animal Health</td>
			<td>11695-0701-1</td>
			<td>Ketamine solution&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AnaSed</td>
			<td>Lloyd Inc</td>
			<td>N/A</td>
			<td>Xylazine sterile solution 100 mg/mL&#160;</td>
		</tr>
	</tbody>
</table>

generation of car t cells for adoptive therapy in the context of glioblastoma standard of care

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo to express chimeric antigen receptors specific for glioma antigens (CAR T cells). The expansion and function of adoptively transferred CAR T cells can be potentiated by the lymphodepletive and tumoricidal effects of standard of care chemotherapy and radiotherapy. We describe a method for generating CAR T cells targeting EGFRvIII, a glioma-specific antigen, and evaluating their efficacy when combined with a murine model of glioblastoma standard of care. T cells are engineered by transduction with a retroviral vector containing the anti-EGFRvIII CAR gene. Tumor-bearing animals are subjected to host conditioning by a course of temozolomide and whole brain irradiation at dose regimens designed to model clinical standard of care. CAR T cells are then delivered intravenously to primed hosts. This method can be used to evaluate the antitumor efficacy of CAR T cells in the context of standard of care.

CAR T cells are generated by transduction with the EGFRvIII CAR retroviral vector11. This vector, MSGV1, was developed from the SFGtcLuc_ITE4 vector35, which contains the murine stem cell virus (MSCV) long terminal repeats, the extended gag region and envelope splice site (splice donor, sd, and splice acceptor, sa), and viral packaging signal (&#968;). The EGFRvIII CAR containing the human anti-EGFRvIII single-chain variable fragment (scFv) 139, in tandem with murine CD8TM, CD28, 4-1BB, and CD3&#950...

Watch this Scientific Journal Video about Generation of CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care at JoVE.com

Generation of CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care

The lymphodepletive and immunomodulatory effects of chemotherapy and radiation standard of care can be leveraged to enhance the antitumor efficacy of T cell immunotherapy. We outline a method for generating EGFRvIII-specific chimeric antigen receptor (CAR) T cells and administering them in the context of glioblastoma standard of care.

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo ...

generation-car-t-cells-for-adoptive-therapy-context-glioblastoma

Research

JoVE Journal

Immunology and Infection

21.3K Views.  Duke University. The lymphodepletive and immunomodulatory effects of chemotherapy and radiation standard of care can be leveraged to enhance the antitumor efficacy of T cell immunotherapy. We outline a method for generating EGFRvIII-specific chimeric antigen receptor (CAR) T cells and administering them in the context of glioblastoma standard of care. 

Video: Generation of CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully generated and infused T-cells specific for Epstein Barr virus (EBV), cytomegalovirus (CMV) and Adenovirus (Adv) using monocytes and EBV-transformed lymphoblastoid cell (EBV-LCL) gene-modified with an adenovirus vector as antigen presenting cells (APCs). As few as 2x105/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs after infusion and appeared to prevent and treat even severe viral disease resistant to other available therapies. The broader implementation of this encouraging approach is limited by high production costs, complexity of manufacture and the prolonged time (4-6 weeks for EBV-LCL generation, and 4-8 weeks for CTL manufacture &#x2013; total 10-14 weeks) for preparation. To overcome these limitations we have developed a new, GMP-compliant CTL production protocol. First, in place of adenovectors to stimulate T-cells we use dendritic cells (DCs) nucleofected with DNA plasmids encoding LMP2, EBNA1 and BZLF1 (EBV), Hexon and Penton (Adv), and pp65 and IE1 (CMV) as antigen-presenting cells. These APCs reactivate T cells specific for all the stimulating antigens. Second, culture of activated T-cells in the presence of IL-4 (1,000U/ml) and IL-7 (10ng/ml) increases and sustains the repertoire and frequency of specific T cells in our lines. Third, we have used a new, gas permeable culture device (G-Rex) that promotes the expansion and survival of large cell numbers after a single stimulation, thus removing the requirement for EBV-LCLs and reducing technician intervention. By implementing these changes we can now produce multispecific CTL targeting EBV, CMV, and Adv at a cost per 106 cells that is reduced by &gt;90%, and in just 10 days rather than 10 weeks using an approach that may be extended to additional protective viral antigens. Our FDA-approved approach should be of value for prophylactic and treatment applications for high risk allogeneic HSCT recipients.

A rapid, simple and cost-effective protocol for the generation of donor-derived multivirus-specific CTLs (rCTL) for infusion to allogeneic hematopoietic stem cell transplant (HSCT) recipients at risk of developing CMV, Adv or EBV infections. This manufacturing process is GMP-compliant and should ensure the broader implementation of T-cell immunotherapy beyond specialized centers.

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&amp;#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells

A basic necessity for researchers studying adaptive immunity with in vivo experimental models is an ability to identify T cells based on their T cell antigen receptor (TCR) specificity. Many indirect methods are available in which a bulk population of T cells is stimulated in vitro with a specific antigen and epitope-specific T cells are identified through the measurement of a functional response such as proliferation, cytokine production, or expression of activation markers1. However, these methods only identify epitope-specific T cells exhibiting one of many possible functions, and they are not sensitive enough to detect epitope-specific T cells at naive precursor frequencies. A popular alternative is the TCR transgenic adoptive transfer model, in which monoclonal T cells from a TCR transgenic mouse are seeded into histocompatible hosts to create a large precursor population of epitope-specific T cells that can be easily tracked with the use of a congenic marker antibody2,3. While powerful, this method suffers from experimental artifacts associated with the unphysiological frequency of T cells with specificity for a single epitope4,5. Moreover, this system cannot be used to investigate the functional heterogeneity of epitope-specific T cell clones within a polyclonal population.
	The ideal way to study adaptive immunity should involve the direct detection of epitope-specific T cells from the endogenous T cell repertoire using a method that distinguishes TCR specificity solely by its binding to cognate peptide:MHC (pMHC) complexes. The use of pMHC tetramers and flow cytometry accomplishes this6, but is limited to the detection of high frequency populations of epitope-specific T cells only found following antigen-induced clonal expansion. In this protocol, we describe a method that coordinates the use of pMHC tetramers and magnetic cell enrichment technology to enable detection of extremely low frequency epitope-specific T cells from mouse lymphoid tissues3,7. With this technique, one can comprehensively track entire epitope-specific populations of endogenous T cells in mice at all stages of the immune response.

This protocol describes the use of peptide:MHC tetramers and magnetic microbeads to isolate low frequency populations of epitope-specific T cells and analyze them by flow cytometry. This method enables the direct study of endogenous T cell populations of interest from in vivo experimental systems.

A  basic necessity for researchers studying adaptive immunity with in vivo experimental models is an  ability to identify T cells based on their T cell ...

Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of human Type 1 diabetes (T1D). Cell transfer studies in irradiated recipient mice have established that T cells are pivotal in T1D pathogenesis in this model. We describe herein a simple method to rapidly induce T1D by adoptive transfer of purified, primary CD4+ T cells from pre-diabetic NOD mice transgenic for the islet-specific T cell receptor (TCR) BDC2.5 into NOD.SCID recipient mice. The major advantages of this technique are that isolation and adoptive transfer of diabetogenic T cells can be completed within the same day, irradiation of the recipients is not required, and a high incidence of T1D is elicited within 2 weeks after T cell transfer. Thus, studies of pathogenesis and therapeutic interventions in T1D can proceed at a faster rate than with methods that rely on heterogenous T cell populations or clones derived from diabetic NOD mice.

We provide a reproducible method to induce type 1 diabetes (T1D) in mice within two weeks by the adoptive transfer of islet antigen-specific, primary CD4+ T cells.

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection occurring after allogeneic hematopoietic stem-cell transplantation. Viral-specific T cells from allogeneic donors, including third party donors, can be propagated ex vivo in compliance with current good manufacturing practice (cGMP), employing repeated rounds of antigen-driven stimulation to selectively propagate desired T cells. The identification and isolation of antigen-specific T cells can also be undertaken based upon the cytokine capture system of T cells that have been activated to secrete gamma-interferon (IFN-&#947;). However, widespread human application of the cytokine capture system (CCS) to help restore immunity has been limited as the production process is time-consuming and requires a skilled operator. The development of a second-generation cell enrichment device such as CliniMACS Prodigy now enables investigators to generate viral-specific T cells using an automated, less labor-intensive system. This device separates magnetically labeled cells from unlabeled cells using magnetic activated cell sorting technology to generate clinical-grade products, is engineered as a closed system and can be accessed and operated on the benchtop. We demonstrate the operation of this new automated cell enrichment device to manufacture CMV pp65-specific T cells obtained from a steady-state apheresis product obtained from a CMV seropositive donor. These isolated T cells can then be directly infused into a patient under institutional and federal regulatory supervision. All the bio-processing steps including removal of red blood cells, stimulation of T cells, separation of antigen-specific T cells, purification, and washing are fully automated. Devices such as this raise the possibility that T cells for human application can be manufactured outside of dedicated good manufacturing practice (GMP) facilities and instead be produced in blood banking facilities where staff can supervise automated protocols to produce multiple products.

The goal of this protocol is to manufacture pathogen-specific clinical-grade T cells using a bench-top, automated, second generation cell enrichment device that incorporates a closed cytokine capture system and does not require dedicated staff or use of a GMP facility. The cytomegalovirus pp65-specific-T cells generated can be directly administered to patients.

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection ...

Stereotactic Adoptive Transfer of Cytotoxic Immune Cells in Murine Models of Orthotopic Human Glioblastoma Multiforme Xenografts

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce efficient therapies have been proposed over the last decade. Among the promising candidates for designing novel therapeutic strategies, cellular immunotherapies have been targeted to eliminate highly invasive and chemo-radioresistant tumor cells, likely involved in a rapid and fatal relapse of this cancer. Thus, administration(s) of allogeneic GBM-reactive immune cell effectors, such as human V&#978;9V&#948;2 T lymphocytes, in the vicinity of the tumor would represents a unique opportunity to deliver efficient and highly concentrated therapeutic agents directly into the site of brain malignancies. Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a preclinical proof-of-concept for both the feasibility and the antitumor efficacy of these cellular immunotherapies that rely on stereotactic injections of allogeneic human lymphocytes within intrabrain tumor beds.

Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a proof-of-concept for both the feasibility and the antitumor efficacy of intrabrain-delivered cellular immunotherapies.

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce ...