Home › Immunology › A Syngeneic Mouse B-Cell Lymphoma Model for Pre-Clinical Evaluation of CD19 CAR T Cells
Immunology JoVE (Open Access) Citable · DOI

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

DOI: 10.3791/58492-v
What you'll learn
  • ✓ Generate CD19 CAR T cells via retroviral transduction in murine T cells
  • ✓ Evaluate CAR T cell efficacy against established A20 B-cell lymphoma in vivo
  • ✓ Compare treatment outcomes with and without lymphodepleting pre-conditioning
Protocol

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

Difficulty
advanced
Total time
~14 days (5 days in vitro transduction + 9+ days tumor challenge and monitoring)
Model organism
Mouse BALB/c; A20 B-cell lymphoma cell line
Biosafety
BSL-2

Steps

1
Prepare and transfect Plat-E retroviral packaging cells

Culture Plat-E cells and transfect with CD19 CAR construct using standard lipofection. Incubate transfected cells for 48 hours to allow viral protein expression.

▶ 00:55
2
Harvest virus supernatant and activate splenic T cells

Collect virus-containing supernatant from transfected Plat-E cells. Simultaneously isolate mouse splenic T cells and activate in vitro using anti-CD3/CD28 stimulation.

▶ 02:11
3
Perform first round CAR T cell transduction

Expose activated mouse T cells to retroviral supernatant under spinoculation conditions to introduce CD19 CAR construct into target cells.

▶ 04:27
4
Execute second round CAR T cell transduction

Repeat transduction protocol on Day 5 to increase CAR T cell frequency and enhance transgene integration in expanded T cell population.

▶ 06:43
5
Validate CAR T cell activity in vitro

Assess CAR T cell cytotoxicity, activation markers, and cytokine production against A20 target cells using flow cytometry and functional assays.

▶ 08:33
6
Evaluate CD19 CAR T cells against lymphoma in vivo

Challenge BALB/c mice with established A20 B-cell lymphoma, then treat cohorts with CAR T cells with or without lymphodepleting pre-conditioning. Monitor tumor burden and survival.

▶ 09:53
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