We thank L. Brockmann for critical review of the manuscript. This work was supported by NIH 1R01EB030352 and UL1 TR001873.

All animal procedures were performed with approval from the Institutional Animal Care and Use Committee (Columbia University, protocols AC-AABQ5551 and AC-AAAZ4470) using 6-8-week-old female BALB/c or CF57BL/6 mice weighing between 20-25 g. The animals were obtained from a commercial source (see Table of Materials). This protocol is structured around the &#39;days post-activation&#39; of murine T cells, and viral production begins on Day -2. Retrovirus can be stored at -80 &#176;C following initial production, and for future use of this protocol, one may commence with step 2, T cell isolation and activation on Day 0.
1. Retroviral vector production
NOTE: The viral products have been made replication-defective by separation of the packaging genes into two separate plasmids (see Table of Materials), greatly reducing the likelihood of recombination events and inadvertent production of replication-competent virus.
<ol>
	<li>Prepare Phoenix Eco cells one day prior to transfection (Day -2 procedure).
	<ol>
		<li>Plate approximately 1 x 107 cells in a 15 cm TC-treated plate or T150 culture flask, using 30 mL of culture medium (Phoenix Eco culture medium, as detailed in Table 1).</li>
		<li>Incubate overnight at 37 &#176;C. After 18-24 h, the cells should be approximately 70% confluent and uniformly distributed to ensure a high viral yield without overgrowth. 
		NOTE: For optimal results, use cells with a low passage and passage them the day before plating for viral production. Do not allow Phoenix Eco cells to overgrow during routine culture.</li>
	</ol>
	</li>
	<li>Prepare the transfection mix containing the lipofection and enhancer reagents, pCL-Eco (Gag/Pol), and pMSCV expression plasmid (pMSCV_PGK_mGFP28z) in reduced-serum media (see Table of Materials) (Day -1 procedure). 
	NOTE: Co-transfection should be performed at a 1:1 ratio of pMSCV and pCL-Eco.
	<ol>
		<li>Prepare Tube A: Dilute 105 &#181;L of the transfection reagent in 3.75 mL of reduced serum medium per 15 cm plate and mix thoroughly by vortexing or pipetting up and down.</li>
		<li>Prepare Tube B: Dilute pMSCV expression plasmid (21 &#181;g) and pCL-Eco (21 &#181;g) with 90 &#181;L of enhancer reagent into 3.75 mL of reduced serum medium per 15 cm plate. Mix well by pipetting up and down. 
		NOTE: If generating multiple viral products, set up a master mix containing pCL-Eco and the enhancer reagent.</li>
		<li>Add Tube A to Tube B and mix thoroughly by pipetting. Incubate for 10-20 min at room temperature, resulting in a total volume of approximately 7.5 mL.</li>
		<li>Carefully remove 10 mL of cell culture media from the Phoenix Eco plate(s) and add the entire volume of the transfection mix to the remaining media by tilting the plate and pipetting dropwise. Return cells to a 37 &#176;C incubator.</li>
	</ol>
	</li>
	<li>Change the media on transfected Phoenix Eco cells 16-20 h post-transfection (Day 0 procedure).
	<ol>
		<li>Pre-warm serum and &#946;-mercaptoethanol (2-ME, see Table of Materials)-free murine T cell medium (mTCM-viral harvest, as listed in Table 1) to 37 &#176;C using a water or bead bath.</li>
		<li>Carefully remove the Phoenix Eco culture medium by tilting the plate and placing the pipette tip at the bottom corner, using a vacuum if possible. Avoid over-drying the cells.</li>
		<li>Gently add the warmed mTCM-viral harvest medium to the side of the tilted plate by pipetting 30 mL on the slowest setting. Return cells to a 37 &#176;C incubator. 
		NOTE: This step can cause Phoenix Eco cells to detach from the plate and reduce viral titers. Pre-warmed media and gentle pipetting will minimize disruption.</li>
	</ol>
	</li>
	<li>Harvest the viral supernatant 48 h post-transfection (Day 1 procedure).
	<ol>
		<li>Harvest the viral supernatant and filter it through 0.45 &#181;m PVDF filters (see Table of Materials) to remove cells and debris. Aliquot and freeze the virus at -80 &#176;C or proceed directly to Day 1 of step 3 if using fresh virus.</li>
		<li>Determine the viral titer (transducing units/mL) by flow cytometry, as previously described9. This step is optional.
		<ol>
			<li>Determine the viral titer by small-scale transduction of 1 x 105 activated murine T cells in non-tissue culture (TC)-treated 96-well plates, pre-coated with retronectin (see Table of Materials) and loaded with threefold serial dilutions of retroviral supernatant in a final volume of 100 &#181;L of mTCM-viral harvest medium. 
			NOTE: See step 2 and step 3 below for detailed instructions on T cell isolation, activation, and transduction.</li>
			<li>Determine CAR surface expression 4-5 days post-transduction by flow cytometry.</li>
			<li>Calculate the viral titer according to the formula10: (N x F x D)/V, where N is the number of cells transduced, F is the frequency of CAR-positive cells, D is the dilution factor, and V is the transduction volume in mL to obtain transducing units (TU)/mL. 
			NOTE: Viral titer may decrease upon freeze/thaw; therefore, viral titer is ideally determined on frozen and subsequently thawed virus.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Murine T cell isolation
<ol>
	<li>Isolate and activate murine T cells (Day 0 procedure). 
	NOTE: These steps can be performed at room temperature (RT) or 4 &#176;C and should be carried out in a sterile environment.
	<ol>
		<li>Harvest the spleen(s) from the mouse strain of interest (e.g., Balb/c, C57BL/6) as previously described11 and obtain a single-cell suspension through mechanical dissociation.</li>
		<li>Using the back of a sterile syringe, crush the spleen(s) through a 70-100 &#181;m cell strainer into a 50 mL conical tube.</li>
		<li>After setting the syringe aside, wash the strainer with 5 mL of T cell isolation buffer (phosphate-buffered saline, PBS, supplemented with 2% fetal bovine serum, FBS).</li>
		<li>Repeat the mashing step and wash the strainer once more with 5 mL of T cell isolation buffer. Bring the final volume to 50 mL with T cell isolation buffer.</li>
		<li>Count live cells using a manual hemocytometer or an automatic cell counter by diluting at a 1:20 ratio and then mixing at 1:1 with trypan blue.</li>
		<li>Pellet the cells by centrifugation for 10 min at 450 x g, at 4 &#176;C (or RT), and re-suspend spleenocytes at 1 x 108/mL if using the recommended T cell isolation kit (see Table of Materials). 
		NOTE: Spleenocytes can be re-strained through a 40-70 &#181;m strainer at this stage to remove clumps.</li>
	</ol>
	</li>
	<li>Isolate CD3+ T cells by negative selection following the manufacturer&#39;s instructions (see Table of Materials). After magnetic separation, transfer the isolate to a 15 mL conical tube and perform a final live cell count.</li>
	<li>Pellet the isolated T cells by centrifugation for 10 min at 450 x g, at 4 &#176;C (or RT), and re-suspend them at 1 x 106/mL in mTCM-activation medium (Table 1).</li>
	<li>Activate T cells by adding murine anti-CD3/anti-CD28 monoclonal antibody-coated magnetic beads (see Table of Materials) at a ratio of 25 &#181;L/1 x 106 T cells and 100 U/mL IL-2.</li>
	<li>Place the cells in an incubator at 37 &#176;C and leave them overnight. 
	NOTE: Due to the small size of T cells, a more accurate live T cell count may be achieved by diluting at a 1:10-1:20 ratio and mixing at 1:1 with trypan blue for manual counting using a hemocytometer. Purity may be determined by flow cytometry by staining cells with a fluorescently conjugated &#945;CD3 antibody. Each spleen will yield between 7-10 x 106 T cells depending on the age and strain of the mouse.</li>
</ol>
3. Murine T cell transduction
<ol>
	<li>Prepare plates for transduction (Day 0 procedure).
	<ol>
		<li>Pre-coat non-treated sterile 24-well plates with 0.5 mL of human fibronectin transduction enhancer reagent (see Table of Materials) at a final concentration of 20-40 &#181;g/mL by diluting it in sterile PBS and store at 4 &#176;C overnight. 
		NOTE: This step may also be performed on Day 1 by coating non-treated 24-well plates with the transduction reagent and incubating them at room temperature for 2 h.</li>
	</ol>
	</li>
	<li>Perform T cell transduction (Day 1 procedure).
	<ol>
		<li>Prepare the pre-coated plate for transduction. Remove the transduction reagent from each well of the pre-coated 24-well plate and block with an equivalent volume of sterile-filtered PBS + 2% bovine serum albumin (BSA) (0.5 mL) for 30 min at RT.</li>
		<li>Wash once with 0.5-1 mL of PBS.</li>
	</ol>
	</li>
	<li>Add 0.5-1 mL of neat retrovirus from step 1 or diluted based on viral titer to each pre-coated well and centrifuge for 90 min at 2,000 x g and 32 &#176;C.</li>
	<li>Add 1 mL of activated T cells to each virally loaded well and centrifuge for 10 min at 450 x g and 32 &#176;C. Return cells to a 37 &#176;C incubator overnight.</li>
	<li>24 h post-transduction, remove 1-1.5 mL of cell culture media and replace it with 1-1.5 mL of mTCM-complete and 10 ng/mL of recombinant human IL-7 and IL-15 (see Table of Materials). Return cells to a 37 &#176;C incubator (Day 2 procedure). 
	NOTE: During ex vivo culture, 10 ng/mL of cytokines must be added to the culture every 48 h, and T cells should not be diluted beyond 1 x 106/mL.</li>
	<li>48 h post-transduction, transfer cells from the transduction plate into a fresh 24-well or 6-well plate and return cells to a 37 &#176;C incubator (Day 3 procedure). 
	NOTE: T cell viability may improve by transferring cells on 24 h to 2 days post-transduction, depending on starting T cell viability and viral titer.</li>
	<li>De-bead the T cells and confirm CAR expression (Day 5-6 procedure).
	<ol>
		<li>Thoroughly re-suspend cells to dissociate activated T cells from the &#945;CD3/CD28-coated beads (see Table of Materials) and place the cell suspension on a magnet for 30 s. Transfer the cell suspension to the desired ex vivo culture vessel and return it to a 37 &#176;C incubator. 
		NOTE: T cells may be cultured in culture flasks or deep well culture plates. It is recommended to plate a minimum of 5 x 106 T cells in 30 mL of mTCM-complete medium per well in a deep well of a 6-well format plate (see Table of Materials).</li>
		<li>Determine CAR expression by flow cytometry8. 
		NOTE: GFP-CAR expression was determined by incubation with 100 ng/mL of purified GFP. Incorporation of an N-terminal epitope tag will facilitate the detection of alternative CAR constructs.</li>
	</ol>
	</li>
	<li>Perform ex vivo culture of CAR-T cells (Day 7-10 procedure).
	<ol>
		<li>During ex vivo culture, maintain the cells by removing 50% of the culture medium and replacing it with fresh mTCM-complete + 2x 10 ng/mL of IL-7 and IL-15 every 48 h. 
		NOTE: Murine CAR-T cells are ready for use in downstream applications 7 days post-activation and should not be cryopreserved due to poor viability post-thaw.</li>
	</ol>
	</li>
</ol>

Streamlined Protocol for Mouse CAR-T Cell Generation in Syngeneic Models

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No conflicts of interests declared.

This protocol describes the steps and reagents necessary for the retroviral transduction of murine T cells to generate CAR-T cells for in vivo studies. Optimizing retroviral transduction conditions achieves robust CAR expression without the need for viral concentration through ultracentrifugation or additional reagents. However, there are multiple modifications that can be applied to this methodology.
While this protocol describes the example generation of a GFP-specific CAR, these methods can be adapted to generate mouse CAR-T cells targeting any extracellular antigen of interest. The pMSCV_PGK retroviral vector used in this study allows for the efficient release of the GFP-specific nanobody sequence by enzymatic digestion with NotI and BamHI and can be replaced with a user-defined antigen recognition domain such as an scFv, nanobody, or ligand-binding domain (Supplementary File 1). To facilitate the assessment of alternate CAR expression, it is recommended to design CARs with an N-terminal epitope tag, such as Myc (EQKLISEEDL) or Flag (DYKDDDDK) tags, which can be detected using fluorescently conjugated antibodies12. Moreover, the current study demonstrates the production of a single transgene; however, the incorporation of self-cleaving peptides (2A sequences) or internal ribosome entry sites (IRES) would enable the co-production of multiple transgenes, including additional technologies to enhance T cell function in vivo8,13,14.
Although this protocol was developed to eliminate the need for specialized equipment by omitting ultracentrifugation (24,000 x g, 2 h, 4 &#176;C), it is worth noting that concentration of the viral supernatant can achieve higher viral titers and reduce the volume of virus required to attain high CAR expression8. This protocol additionally streamlines retroviral vector production by achieving reproducibly high viral titers (~2 x 107 TU/mL) and steady CAR expression without the need for viral titer determination prior to use. However, in addition to facilitating standardization, determination of viral titer prior to T cell transduction may also increase T cell viability by reducing potential toxicity caused by unnecessarily high viral concentrations10,15.
Supplementation with IL-7 and IL-15 is recommended to promote T cell expansion and preserve T cell memory populations; however, recombinant human IL-2 may be used as an alternative to decrease the number of necessary reagents for ex vivo culture16,17,18,19. Nonetheless, the methods for CAR-T cell generation outlined here remain limited by the relatively poor expansion of murine T cells. However, the measures to improve T cell viability discussed above could be further enhanced by including additional supplements and 2-ME in the mTCM culture medium throughout the transduction process20. While these modifications may improve T cell expansion from 15-fold to up to 20-fold20, they may result in lower transduction efficiency and reduced CAR expression.
Ultimately, the production of murine CAR-T cells enables the development of syngeneic models to evaluate CAR-T cell function within an intact immune system - facilitating a context-specific assessment of their potency, durability, and safety. Whereas models in immunodeficient mice allow for important preclinical studies of a human T cell product targeting a human tumor antigen. The combination of these complementary model systems will be indispensable for understanding the complexity of CAR-T cell biology, optimizing new CAR designs, and ultimately translating preclinical findings into effective clinical therapies.

Adoptive T cell therapies expressing chimeric antigen receptors (CARs) have revolutionized the field of cancer immunotherapy by harnessing the power of the adaptive immune system to specifically target and eliminate antigen-positive cancer cells1. While the success of CAR-T cell therapies targeting B cell malignancies has been clinically validated, preclinical studies performed in animal models remain vital for the development of new CARs targeting solid tumors. However, limited clinical efficacy has been demonstrated in solid tumor indications thus far, and it is becoming increasingly apparent that individual preclinical models do not accurately predict the pharmacodynamics and clinical efficacy of a living medicine2,3. Therefore, investigators have begun to expand the preclinical study of CAR-T cell products to include parallel assessments in xenograft and syngeneic models of human and murine cancers, respectively.
Unlike xenograft models, where human tumors and T cells are engrafted into immunodeficient mice, syngeneic models enable the examination of CAR-T cell responses in the context of a functional immune system. Specifically, immune-competent mice bearing syngeneic tumors provide a system to study the interaction between adoptively transferred T cells and context-specific milieus - including tumor-associated macrophages (TAMs) and regulatory T cells (Tregs) known to suppress T cell function in the tumor microenvironment (TME)4,5,6. Moreover, syngeneic models offer an additional platform to assess on-target, off-tumor toxicity, and CAR-T cell interaction with host factors that may lead to additional toxicities, including cytokine release syndrome7.
Despite these advantages, the number of syngeneic CAR-T cell studies remains limited. Notably, syngeneic models require autologous engineering of CAR-T cells from the same mouse strain and thus present an additional challenge due to the lack of methodology for efficient murine T cell transduction and ex vivo expansion2,8. This protocol outlines the methods to achieve stable CAR expression through the production of retroviral vectors and optimized T cell transduction. A schematic of the entire process is shown in Figure 1. The use of this approach demonstrates efficient retroviral transduction of murine CAR-T cells and the achievement of high CAR expression without the need for viral concentration through ultracentrifugation. Strategies to change the antigen-specificity of the CAR construct are discussed in addition to the co-expression of additional transgenes.

<table><tbody><tr><td>0.45 &amp;mu;m filters</td><td>MilliporeSigma</td><td>SLHVR33RS</td><td></td></tr><tr><td>1 mL syringe&amp;nbsp;</td><td>Fisher Scientific&amp;nbsp;</td><td>14-955-450</td><td></td></tr><tr><td>1.5 mL microcentrifuge tubes&amp;nbsp;</td><td>Fisher Scientific&amp;nbsp;</td><td>05-408-135</td><td></td></tr><tr><td>10 mL syringe&amp;nbsp;</td><td>BD</td><td>14-823-16E</td><td></td></tr><tr><td>100 &amp;mu;m strainer</td><td>Corning</td><td>07-201-432</td><td></td></tr><tr><td>15 cm TC treated cell culture dishes</td><td>ThermoFisher Scientific&amp;nbsp;</td><td>130183</td><td></td></tr><tr><td>15 mL conical tubes&amp;nbsp;</td><td>Falcon</td><td>14-959-70C</td><td></td></tr><tr><td>40 &amp;mu;m strainer&amp;nbsp;</td><td>Corning</td><td>07-201-430</td><td></td></tr><tr><td>50 mL conical tubes&amp;nbsp;</td><td>Falcon</td><td>14-959-49A</td><td></td></tr><tr><td>70 &amp;mu;m strainer</td><td>Corning</td><td>07-201-431</td><td></td></tr><tr><td>Attune NxT Flow Cytometer&amp;nbsp;</td><td>ThermoFisher Scientific&amp;nbsp;</td><td></td><td></td></tr><tr><td>BALB/C, 6-8 week old&amp;nbsp;</td><td>Jackson Laboratory</td><td>651</td><td></td></tr><tr><td>B-Mercaptoethanol&amp;nbsp;</td><td>Gibco</td><td>21985023</td><td></td></tr><tr><td>Bovine Serum Albumin&amp;nbsp;</td><td>GOLDBIO</td><td>A-420-500</td><td></td></tr><tr><td>DMEM Medium</td><td>Gibco</td><td>11965092</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline (PBS), without Calcium and Magnesium&amp;nbsp;</td><td>Gibco</td><td>14-190-250</td><td></td></tr><tr><td>DynaMag-2 Magnet&amp;nbsp;</td><td>Invitrogen</td><td>12-321-D</td><td></td></tr><tr><td>EasySep Magnet&amp;nbsp;</td><td>Stemcell Technologies</td><td>18000</td><td></td></tr><tr><td>EasySep Mouse T cell Isolation Kit</td><td>Stemcell Technologies</td><td>19851</td><td></td></tr><tr><td>FACS buffer&amp;nbsp;</td><td>BD</td><td>BDB554657</td><td></td></tr><tr><td>Fetal bovine serum (FBS)&amp;nbsp;</td><td>Corning</td><td>MT35011CV</td><td></td></tr><tr><td>GlutaMAX</td><td>Gibco</td><td>35-050-061</td><td></td></tr><tr><td>G-Rex6</td><td>Wilson Wolf</td><td>80240M&amp;nbsp;</td><td></td></tr><tr><td>HEPES Buffer Solution&amp;nbsp;</td><td>Gibco</td><td>15-630-080</td><td></td></tr><tr><td>Human recombinant IL-15&amp;nbsp;</td><td>Miltenyi Biotec</td><td>130-095-765</td><td></td></tr><tr><td>Human recombinant IL-2</td><td>Miltenyi Biotec</td><td>130-097-748</td><td></td></tr><tr><td>Human recombinant IL-7</td><td>Miltenyi Biotec</td><td>130-095-363</td><td></td></tr><tr><td>Lipofectamine 3000</td><td>Invitrogen</td><td>L3000008</td><td></td></tr><tr><td>MEM Non-Essential Amino Acids Solution&amp;nbsp;</td><td>Gibco</td><td>11140-050</td><td></td></tr><tr><td>Mouse Anti-CD3 BV421</td><td>Biolegend</td><td>100228</td><td></td></tr><tr><td>Mouse Anti-CD3/CD28 Dynabeads</td><td>Gibco</td><td>11-453-D</td><td></td></tr><tr><td>Mouse Anti-CD4 BV605</td><td>BD</td><td>563151</td><td></td></tr><tr><td>Mouse Anti-CD44 APC&amp;nbsp;</td><td>Biolegend</td><td>103011</td><td></td></tr><tr><td>Mouse Anti-CD62L PE-Cy7</td><td>Tonbo</td><td>SKU 60-0621-U025</td><td></td></tr><tr><td>Mouse Anti-CD8 APC-Cy7</td><td>Tonbo</td><td>SKU 25-0081-U025</td><td></td></tr><tr><td>Nikon Ti2 with Prime 95B camera&amp;nbsp;</td><td>Nikon</td><td></td><td></td></tr><tr><td>Non-treated 24 well plates&amp;nbsp;</td><td>CytoOne</td><td>CC7672-7524</td><td></td></tr><tr><td>Opti-MEM</td><td>Gibco</td><td>31-985-062</td><td></td></tr><tr><td>pCL-Eco</td><td>Addgene</td><td>#12371</td><td></td></tr><tr><td>Penicillin/Streptomycin Solution</td><td>Gibco</td><td>15-070-063</td><td></td></tr><tr><td>Phoenix Eco cells</td><td>ATCC</td><td>CRL-3214</td><td></td></tr><tr><td>pMDG.2</td><td>Addgene</td><td>#12259</td><td></td></tr><tr><td>pMSCV_PGK_GFP28z</td><td>N/A</td><td>Produced by R.LV.</td><td></td></tr><tr><td>Purified sfGFP</td><td>N/A</td><td>Produced by R.LV.</td><td></td></tr><tr><td>RetroNectin (&#039;transduction reagent&#039;)</td><td>Takara Bio</td><td>T100B</td><td></td></tr><tr><td>RPMI 1640</td><td>Gibco</td><td>21875</td><td></td></tr><tr><td>Serological pipette 10 mL</td><td>Fisher Scientific&amp;nbsp;</td><td>13-678-11E</td><td></td></tr><tr><td>Serological pipette 25 mL</td><td>Fisher Scientific&amp;nbsp;</td><td>13-678-11</td><td></td></tr><tr><td>Serological pipette 5 mL</td><td>Fisher Scientific&amp;nbsp;</td><td>13-678-11D</td><td></td></tr><tr><td>Sodium Pyruvate</td><td>Gibco</td><td>11-360-070</td><td></td></tr><tr><td>TC-treated 24 well plates&amp;nbsp;</td><td>Corning</td><td>08-772-1</td><td></td></tr><tr><td>Trypan blue&amp;nbsp;</td><td>Gibco</td><td>15-250-061</td><td></td></tr></tbody></table>

efficient generation of murine chimeric antigen receptor (car)-t cells

Engineered cell therapies utilizing chimeric antigen receptor (CAR)-T cells have achieved remarkable effectiveness in individuals with hematological malignancies and are presently undergoing development for the treatment of diverse solid tumors. So far, the preliminary evaluation of novel CAR-T cell products has predominantly taken place in xenograft tumor models using immunodeficient mice. This approach is chosen to facilitate the successful engraftment of human CAR-T cells in the experimental setting. However, syngeneic mouse models, in which tumors and CAR-T cells are derived from the same mouse strain, allow evaluation of new CAR technologies in the context of a functional immune system and comprehensive tumor microenvironment (TME). The protocol described here aims to streamline the process of mouse CAR-T cell generation by presenting standardized methods for retroviral transduction and ex vivo T cell culture. The methods described in this protocol can be applied to other CAR constructs beyond the ones used in this study to enable routine evaluation of new CAR technologies in immune-competent systems.

The protocol described here aims to standardize the process of murine T cell transduction for the generation of mouse CAR-T cells. Figure 1 provides a detailed description of the steps involved. The process begins with the production of retroviral vectors via co-transfection of viral components into Phoenix Eco cells. Figure 2 provides an image of the optimal density of Phoenix Eco cells on the day of transfection. Isolated T cells are then activated 24 h post-transfection, or &#39;Day 0&#39; of this protocol, in preparation for transduction on Day 1. Following transduction, CAR-T cells are cultured in the presence of recombinant human IL-7 and IL-15 to achieve sufficient fold expansion while preserving stem cell memory populations.
The use of the recommended murine T cell isolation kit (see Table of Materials) with this protocol yields T cell purities of &#60;98% prior to transduction, as determined by flow cytometry (Figure 3A). Moreover, reproducible CAR expression rates of 65%-75% can be achieved using this protocol and the pMSCV retroviral vector modified to express the CAR construct from a PGK promoter (Figure 3B,C). Determination of retroviral titer reveals titers of up to ~2 x 107 TU/mL and CAR expression of 74% with 1/3rd of the viral supernatant harvested following co-transfection of pMSCV with pCL-Eco (Supplementary Figure 1).
Finally, ex vivo culture with 10 ng/mL of IL-7 and IL-15 leads to approximately 15-fold expansion, resulting in ~150 x 106 T cells by Day 10 post-activation from a single spleen (Figure 3D). Culture with IL-7 and IL-15 leads to comparable CD8+ and CD4+ T cell frequencies (Figure 3E) and preserved stem cell memory populations determined by CD62L+CD44- expression following 10 days of ex vivo culture (Figure 3F).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65887/65887fig01.jpg" /> 
Figure 1: Protocol overview. Top panel: A schematic overview of the protocol timeline. Bottompanel: Step-by-step instructions for retroviral vector production and mouse T cell transduction. <a href="https://www.jove.com/files/ftp_upload/65887/65887fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65887/65887fig02.jpg" /> 
Figure 2: Optimal cell density for transfection. Brightfield microscopy image of Phoenix Eco cells at the optimal density before transfection with pMSCV and pCL-Eco. Captured using a 10x objective lens. Scale bar = 200 &#181;m. <a href="https://www.jove.com/files/ftp_upload/65887/65887fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65887/65887fig03.jpg" /> 
Figure 3: Murine CAR-T cell production and ex vivo culture. (A) Flow cytometry plot showing the frequency of BALB/c CD3+ cells post-murine T cell isolation. (B) Schematic representation of the pMSCV retroviral vector used for GFP-CAR expression in murine T cells. (C) Flow cytometry histograms demonstrating CAR surface expression on mouse T cells after incubation with purified sfGFP. UT, untransduced. (D) Fold expansion of CAR-T cells by day 10 post-activation and culture with recombinant human IL-17 and IL-15. Approximately 8 x 106 murine T cells were activated before transduction and reached approximately 1.28 x 108 by day 10 post-activation. (E) Flow cytometry plot showing the frequency of CD8+ and CD4+ CAR-T cells on day 10 post-activation. (F) Flow cytometry plot demonstrating the frequency of CD8+ T cell memory populations determined by CD62L and CD44 surface expression. Stem cell memory (TSCM) is characterized by CD62L+CD44-, central memory (TCM) by CD62L+CD44+, and effector memory (TEM) by CD62L-CD44+. <a href="https://www.jove.com/files/ftp_upload/65887/65887fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Cell culture media formulation. The formulation details for cell culture media used in the protocol. <a href="https://www.jove.com/files/ftp_upload/65887/65887_Table 1.xlsx" target="_blank">Please click here to download this Table.</a>
Supplementary Figure 1: CAR surface expression and retroviral titers. (A) Flow cytometry histograms showing CAR surface expression on BALB/c T cells after incubation with purified sfGFP. Retrovirus was generated by transfecting Phoenix Eco cells with pMSCV_PGK_mGFP28z in combination with pCL-Eco and pMD2.G (I), pCL-Eco (II), or alone (III). T cells were transduced with diluted retrovirus as indicated. (B) Retroviral titers determined by CAR surface expression of transduced T cells. Transducing units (TU)/mL were calculated using the formula: (N&#8729;F&#8729;D)/V, where N is the number of cells transduced (1 x 105), F is the frequency of CAR+ cells, D is the dilution factor, and V is the transduction volume in mL (0.2 mL). <a href="https://www.jove.com/files/ftp_upload/65887/Supplementary Figure 1.zip" target="_blank">Please click here to download this File.</a>
Supplementary File 1: pMSCV expression vector sequence. The sequence of the GFP28z CAR and flanking sequences (italics) in the pMSCV expression vector, with NotI and BamHI enzyme sites highlighted in blue. <a href="https://www.jove.com/files/ftp_upload/65887/Supplementary File 1.zip" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Enhancing CAR-T Cell Function in Syngeneic Tumor Models at JoVE.com

Author Spotlight: Enhancing CAR-T Cell Function in Syngeneic Tumor Models 

This protocol streamlines retroviral vector production and murine T cell transduction, facilitating the efficient generation of mouse CAR-T cells.

Efficient Generation of Murine Chimeric Antigen Receptor (CAR)-T Cells

Engineered cell therapies utilizing chimeric antigen receptor (CAR)-T cells have achieved remarkable effectiveness in individuals with hematological ...

efficient-generation-of-murine-chimeric-antigen-receptor-car-t-cells

Nanyang Technological University

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3.1K Views.  Columbia University. This protocol streamlines retroviral vector production and murine T cell transduction, facilitating the efficient generation of mouse CAR-T cells.

Video: Author Spotlight: Enhancing CAR-T Cell Function in Syngeneic Tumor Models 

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

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

Manufacturing Chimeric Antigen Receptor CAR T Cells for Adoptive Immunotherapy

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

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

Cancer Research

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.

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 ...

Immunology and Infection

Functional Validation of Hypoxia-Sensitive CAR-T Cells for Selective Tumors

Generation and Functional Verification of Hypoxia-Sensitive Chimeric Antigen Receptor-T Cells

Extensive studies have proven the promise of chimeric antigen receptor T (CAR-T) cell therapy in treating hematological malignancies. However, treating solid tumors remains challenging, as exemplified by the safety concerns that arise when CAR-T cells attack normal cells expressing the target antigens. Researchers have explored various approaches to enhance the tumor selectivity of CAR-T cell therapy. One representative strategy along this line is the construction of hypoxia-sensitive CAR-T cells, which are designed by fusing an oxygen-dependent degradation domain to the CAR moiety and are strategized to attain high CAR expression only in a hypoxic environment-the tumor microenvironment (TME). This paper presents a protocol for the generation of such CAR-T cells and their functional characterization, including methods to analyze the changes in CAR expression and killing capacity in response to different oxygen levels established by a mobile incubator chamber. The constructed CAR-T cells are anticipated to demonstrate CAR expression and cytotoxicity in an oxygen-sensitive manner, thus supporting their capability to distinguish between hypoxic TME and normoxic normal tissues for selective activation.

Author Spotlight: Advancements in Hypoxia-Sensitive CAR-T Therapy for Enhanced Cancer Immunotherapy

Here we present a protocol for the generation and functional verification of hypoxia-sensitive chimeric antigen receptor (CAR)-T cells. This protocol presents the lentivirus-based generation of hypoxia-sensitive CAR-T cells and their characterization, including the validation of hypoxia-dependent CAR expression and selective cytotoxicity.

Extensive studies have proven the promise of chimeric antigen receptor T (CAR-T) cell therapy in treating hematological malignancies. However, treating ...

A Nonviral Approach to Generate Transient Chimeric Antigen Receptor T Cells Using mRNA for Cancer Immunotherapy

Chimeric antigen receptor (CAR) T cell therapy has emerged as a pioneering cancer treatment, achieving unprecedented success in treating certain hematological malignancies such as lymphomas and leukemias. However, as more cancer patients receive CAR-T cell therapies, treatment-associated secondary primary malignancies are increasingly being reported partly due to unexpected CAR transgene insertion, raising serious safety concerns. To address this issue, we describe here a nonviral, non-integrating approach to generate transient CAR-T cells using mRNA. We electroporated T cells with modified mRNA encoding a human epidermal growth factor receptor 2 (HER2)-specific CAR and generated transient HER2-targeted CAR-T cells. The CAR was efficiently expressed on the T cell surface 1 day after electroporation, increased by day 2, and dramatically declined by day 5. The transient CAR-T cells exhibited potent cytotoxicity against HER2-positive SKOV-3 ovarian cancer cells and secreted high levels of IFN-&#978;. This protocol provides a step-by-step guide for developing small-scale transient CAR-T cells without permanent CAR transgene integration, describing detailed procedures for preparation of CAR mRNA, activation and transfection of T cells, assessment of CAR expression, and in vitro analysis of CAR-T cell function. This method is suitable for transient CAR-T cell generation in both preclinical and clinical studies.

This protocol outlines a detailed procedure for generating transient chimeric antigen receptor (CAR) T cells using non-integrating mRNA for cancer immunotherapy and provides reliable methods for evaluating CAR-T cells and their cytotoxic function.

Chimeric antigen receptor (CAR) T cell therapy has emerged as a pioneering cancer treatment, achieving unprecedented success in treating certain ...

A Real-time Potency Assay for Chimeric Antigen Receptor T Cells Targeting Solid and Hematological Cancer Cells

Chimeric antigen receptor (CAR) T-cell therapy for cancer has achieved significant clinical benefit for resistant and refractory hematological malignancies such as childhood acute lymphocytic leukemia. Efforts are currently underway to extend this promising therapy to solid tumors in addition to other hematological cancers. Here, we describe the development and production of potent CAR T cells targeting antigens with unique or preferential expression on solid and liquid tumor cells. The in vitro potency of these CAR T cells is then evaluated in real-time using the highly sensitive impedance-based xCELLigence assay. Specifically, the impact of different costimulatory signaling domains, such as glucocorticoid-induced tumor necrosis factor receptor (TNFR)-related protein (GITR), on the in vitro potency of CAR T cells is examined. This report includes protocols for: generating CAR T cells for preclinical studies using lentiviral gene transduction, expanding CAR T cells, validating CAR expression, and running and analyzing xCELLigence potency assays.

We describe a quantitative real-time in vitro cytolysis assay system to evaluate the potency of chimeric antigen receptor T cells targeting liquid and solid tumor cells. This protocol can be extended to assess other immune effector cells, as well as combination treatments.

Chimeric antigen receptor (CAR) T-cell therapy for cancer has achieved significant clinical benefit for resistant and refractory hematological ...

High-Efficiency Generation of Antigen-Specific Primary Mouse Cytotoxic T Cells for Functional Testing in an Autoimmune Diabetes Model

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the spontaneous non-obese diabetes (NOD) mouse model, insulin is the primary target, and genetic manipulation of these animals to remove a single key insulin epitope prevents disease. Thus, selective elimination of professional antigen presenting cells (APCs) bearing this pathogenic epitope is an approach to inhibit the unwanted insulin-specific autoimmune responses, and likely has greater translational potential.
Chimeric antigen receptors (CARs) can redirect T cells to selectively target disease-causing antigens. This technique is fundamental to recent attempts to use cellular engineering for adoptive cell therapy to treat multiple cancers. In this protocol, we describe an optimized T-cell retrovirus (RV) transduction and in vitro expansion protocol that generates high numbers of functional antigen-specific CD8 CAR-T cells starting from a low number of naive cells. Previously multiple CAR-T cell protocols have been described, but typically with relatively low transduction efficiency and cell viability following transduction. In contrast, our protocol provides up to 90% transduction efficiency, and the cells generated can survive more than two weeks in vivo and significantly delay disease onset following a single infusion. We provide a detailed description of the cell maintenance and transduction protocol, so that the critical steps can be easily followed. The whole procedure from primary cell isolation to CAR expression can be performed within 14 days. The general method may be applied to any mouse disease model in which the target is known. Similarly, the specific application (targeting a pathogenic peptide/MHC class II complex) is applicable to any other autoimmune disease model for which a key complex has been identified.

This article describes a protocol for the generation of antigen-specific CD8 T cells, and their expansion in vitro, with the aim of yielding high numbers of functional T cells for use in vitro and in vivo.

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the ...

Engineering and Expansion of Human CAR Tregs for Immune Regulation Therapies

Generation of Human Chimeric Antigen Receptor Regulatory T Cells

Chimeric antigen receptor (CAR) T-cell therapy has reshaped the face of cancer treatment, leading to record remission rates in previously incurable hematological cancers. These successes have spurred interest in adapting the CAR platform to a small yet pivotal subset of CD4+ T cells primarily responsible for regulating and inhibiting the immune response, regulatory T cells (Tregs). The ability to redirect Tregs&#39; immunosuppressive activity to any extracellular target has enormous implications for creating cell therapies for autoimmune disease, organ transplant rejection, and graft-versus-host disease. Here, we describe in detail methodologies for bona fide Treg isolation from human peripheral blood, genetic modification of human Tregs utilizing either lentivirus or CRISPR/Cas9-aided knock-in using adeno-associated virus-mediated homologous directed repair (HDR) template delivery, and ex vivo expansion of stable human CAR Tregs. Lastly, we describe the assessment of human CAR Treg phenotypic stability and in vitro suppressive function, which provides insights into how the human CAR Tregs will behave in preclinical and clinical applications.

This protocol provides a streamlined workflow to generate and test human chimeric antigen receptor regulatory T cells (CAR Tregs).

Chimeric antigen receptor (CAR) T-cell therapy has reshaped the face of cancer treatment, leading to record remission rates in previously incurable ...

Droplet-based Cytotoxicity Assay to Assess Chimeric Antigen Receptor T cells at the Single-cell Level

The assessment of the cytotoxic potential of T cell-based therapies, such as chimeric antigen receptor (CAR) T cell treatments, is instrumental in assessing their efficacy and is a prerequisite for clinical application. However, traditional cytotoxicity assays are conducted as bulk assays and do not provide detailed information about the functional heterogeneity of the CAR T cell population. In this study, we describe a double-emulsion droplet-based method that allows for large-scale co-encapsulation of single effector CAR T cells with single target cells while enabling dual quantification of both cytotoxic effector molecules from T cells and cell death of the target cell. The protocol outlines a method for the generation and purification of CD19-specific CAR T cells, followed by their co-encapsulation in droplets with the CD19+ cell line JeKo-1, along with reagents to visualize cytotoxic effector molecule secretion (Granzyme B) and cell death (using propidium iodide, PI). We demonstrate how to generate droplets containing single CAR T and target cells using a commercially available microfluidics device for generating double emulsion droplets. Additionally, we provide examples of how to assay the functional diversity of CAR T cells in droplets using standard flow cytometry equipment. Finally, we briefly describe the temporal kinetics and heterogeneity of CD19-specific CAR T-cell killing. While this method focuses on cell death following a CAR T cell attack, it is also adaptable for examining other types of T cells, cytotoxic immune cells, and effector cell functions, such as cytokine secretion.

Here, we describe a method for creating double-emulsion droplets encapsulating one T cell and a cancer target cell to examine cell killing at the single-cell level. This method allows for dual quantification of both cytotoxic molecules and target-cell apoptosis within a large population of T cells.

The assessment of the cytotoxic potential of T cell-based therapies, such as chimeric antigen receptor (CAR) T cell treatments, is instrumental in ...

An Ex Vivo Technique to Manufacture Chimeric Antigen Receptor T Cells

Source: Ghassemi, S., et al. <a href="https://app.jove.com/v/59949">Manufacturing Chimeric Antigen Receptor (CAR) T Cells for Adoptive Immunotherapy.</a> J. Vis. Exp. (2019)
This video demonstrates a technique to manufacture chimeric antigen receptor (CAR) T cells ex vivo. Human T cells are incubated with magnetic beads coated with anti-CD3 and anti-CD28 antibodies, along with a medium supplemented with interleukin-2 (IL-2), leading to T cell activation and proliferation. Transgenic lentiviral vectors are introduced, allowing the transgenic RNA to integrate into the host genome and express the transgene. This results in the transduced T cells expressing chimeric antigen receptors (CARs) on their surface. The T cells are harvested at defined time points to assess CAR expression and cryopreservation.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. T Cell Activation, Transduction, ...

JoVE Encyclopedia of Experiments

Immunology

Immunotherapy

Cellular Immunotherapy

An Ex Vivo Technique to Generate Tumor-Specific Chimeric Antigen Receptor T Cells

Source: Riccione, K., et al. <a href="https://app.jove.com/v/52397">Generation of CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care.</a> J. Vis. Exp. (2015).
This video demonstrates a technique for the ex vivo generation of tumor antigen-specific chimeric antigen receptor (CAR) T cells. Retroviral vectors carrying transgenic RNA are mixed with spleen cells in a medium supplemented with interleukin-2 to promote T-cell survival and proliferation. The mixture is then transferred to a plate coated with recombinant human fibronectin fragments and centrifuged to facilitate the fusion of the virus with the cell, allowing the RNA to integrate into the host genome and produce surface-bound CARs targeting the tumor antigen.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...