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. 2011 May;34(4):343-52.
doi: 10.1097/CJI.0b013e3182187600.

Genetic engineering of murine CD8+ and CD4+ T cells for preclinical adoptive immunotherapy studies

Sid P Kerkar  1 , Luis Sanchez-Perez, Shicheng Yang, Zachary A Borman, Pawel Muranski, Yun Ji, Dhanalakshmi Chinnasamy, Andrew D M Kaiser, Christian S Hinrichs, Christopher A Klebanoff, Christopher D Scott, Luca Gattinoni, Richard A Morgan, Steven A Rosenberg, Nicholas P Restifo
Affiliations

Genetic engineering of murine CD8+ and CD4+ T cells for preclinical adoptive immunotherapy studies

Sid P Kerkar et al. J Immunother. 2011 May.

Abstract

T-cell receptor (TCR) gene therapy enables for the rapid creation of antigen-specific T cells from mice of any strain and represents a valuable tool for preclinical immunotherapy studies. Here, we describe the superiority of γ-retroviral vectors compared with lentiviral vectors for transduction of murine T cells and surprisingly illustrate robust gene-transfer into phenotypically naive/memory-stem cell like (TN/TSCM; CD62L(hi)/CD44(low)) and central memory (TCM; CD62L(hi)/CD44(hi)) CD8+ T cells using murine stem cell-based γ-retroviral vectors (MSGV1). We created MSGV1 vectors for a major histocompatibility complex-class I-restricted TCR specific for the melanocyte-differentiation antigen, glycoprotein 100 (MSGV1-pmel-1), and a major histocompatibility complex-class II-restricted TCR specific for tyrosinase-related protein-1 (MSGV1-TRP-1), and found that robust gene expression required codon optimization of TCR sequences for the pmel-1 TCR. To test for functionality, we adoptively transferred TCR-engineered T cells into mice bearing B16 melanomas and observed delayed growth of established tumors with pmel-1 TCR engineered CD8+ T cells and significant tumor regression with TRP-1 TCR transduced CD4 T cells. We simultaneously created lentiviral vectors encoding the pmel-1 TCR, but found that these vectors mediated low TCR expression in murine T cells, but robust gene expression in other murine and human cell lines. These results indicate that preclinical murine models of adoptive immunotherapies are more practical using γ-retroviral rather than lentiviral vectors.

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Conflict of interest statement

The authors declare that there are no potential conflicts of interests

Figures

FIGURE 1
FIGURE 1
Flow diagram for gamma-retroviral and lentiviral production.
FIGURE 2
FIGURE 2
Flow diagram for gene transduction protocols
FIGURE 3
FIGURE 3
Optimization of gene transfer into T cells with gamma-retroviral vectors. A, Bulk C57BL/6 splenocytes were stimulated with either anti-CD3 and anti-CD28 or con A and IL-7 for 24 or 48 hours and transduced with the MSGV1-GFP vector. B, Congenically marked (CD45.1) CD8+ T cells isolated from C57/BL-6 spleens were stimulated with anti-CD3 and anti-CD28 and cultured in IL-2, IL-15, or IL-21 and transduced with the MSGV1-GFP vector. Transduction efficiencies for A and B were measured by flow cytometry 3 days following gene transfer (grey = non-transduced control). C, CD8+ T cells were stimulated with anti-CD3/anti-CD28 for 24 hrs, cultured in IL-2, transduced with a MSGV1-thy1.1 vector and examined by flow cytometry 3 and 5 days after transduction for CD62L and CD44 to define TN/SCM (CD62LHi CD44Low) and TCM (CD62LHi CD44Hi) populations. All results in A, B, and C are representative of at least 3 independent experiments.
FIGURE 4
FIGURE 4
Redirection of open-repertoire CD8+ and CD4+ T cells with MSGV1 vectors encoding the codon optimized pmel-1 and native TRP-1 TCRs. A, The α and β subunits of the pmel-1 TCR (Vα1/Vβ13) and the TRP-1 TCR (Vα3.2/Vβ14) were linked either by a sequence encoding the foot and mouth disease picornavirus 2A ribosomal skip peptide (upper panels) or an internal ribosomal entry site, IRES (lower panels) and cloned into the MSGV1 vector backbone (SD, splice done; SA, splice acceptor; LTR, long terminal repeat; Ψ, packaging sequence). B, C57BL?6 splenocytes were stimulated with anti-CD3/anti-CD28 for 24 hrs, cultured in IL-2, and transduced with the MSGV-1 vectors encoding for either the native or codon optimized pmel-1 TCR sequences containing IRES or 2A. Transduction efficiencies in CD8+ T cells were measured by flow cytometry for Vβ13 (upper panel) or gp10025-33 tetramer expression (lower panel). C, C57/BL-6 splenocytes were stimulated with anti-CD3/anti-CD28 for 24 hrs, cultured in IL-2, and transduced with the MSGV1-TRP-1-IRES and MSGV1-TRP-1-2A vectors. TCR transfer efficiencies in CD4+ T cells were measured by flow cytometry for Vβ14. Mock represents cells taken through the transduction protocol with complete media instead of viral supernatant. All results A, B, and C, are representative at least 3 independent experiments.
FIGURE 5
FIGURE 5
Anti-tumor immunity of CD8+ T cells engineered to express the pmel-1 TCR and CD4+ T cells transduced with the TRP-1 TCR. A, Tumor treatment following the adoptive transfer of 4×106 open repertoire CD8+ T cells transduced with the MSGV1-GFP, MSGV1-pmel-1-IRES, or MSGV1-pmel-1-2A vectors into sublethaly-irradiated (5 Gy) mice bearing B16 tumors (n=5) established for 10 days [*, ** = p<0.05 compared to no treatment (NT)] B, Anti-tumor immunity following the adoptive transfer of 4×106 open repertoire CD4+ T cells transduced with the MSGV1-GFP, MSGV1-TRP-1-IRES, or MSGV1-TRP-1-2A vectors into sublethaly-irradiated (5Gy) mice bearing B16 tumors (n=5) established for 10 days [*, ** = p<0.05 compared to no treatment (NT)]. All results for (A) and (B) are representative of at least two separate experiments.
FIGURE 6
FIGURE 6
Gene transfer with HIV based self-inactivated lentiviral vectors encoding the pmel-1 TCR. A, The HIV based lentiviral vector encoding the pmel-1 TCR linked by either a 2A linker (upper panel) or an internal ribosomal entry site, IRES (lower panel) (LTR, long terminal repeat; Ψ, packaging sequence; cPPT, central polypurine tract; RRE, rev response element; MSCV-U3, murine-stem-cell-virus-based promoter). B, Open-repertoire murine CD8+ T cells from spleens were stimulated with anti-CD3 and anti-CD28 and transduced with the LV-Pmel-2A and LV-Pmel-IRES vectors. Human jurkat T cells were transduced with the same viral supernatant. Transduction efficiencies were analyzed by flow cytometry for Vβ-13 expression. C, Transduced murine CD8+ T cells and human jurkat T cells from B, were analyzed by flow cytometry for gp10025-33 tetramer expression. Mock represent cells taken through the transduction protocol with complete media instead of viral supernatant. All results for A, B, and C, are representative of at least 2 independent experiments.
FIGURE 7
FIGURE 7
Comparative transductions between murine and human cells with lentiviral vectors. A, Murine fibroblasts, CD8+ and CD4+ T cells, along with human jurkat, CD8+ and CD4+ T cells were transduced with a HIV based lentivirus encoding the green flourescent protein (GFP). Murine and human T cells were stimulated similarily with anti-CD3 and anti-CD28 and cultured in IL-2. Transduction efficiencies in cells were analyzed by flow cytometry for GFP expression. Results are representative of at least 2 independent experiments. B, The mean flourescence intensity (MFI) was calcluated and quantified for GFP transduced murine and human CD8+ and CD4+ T cells (*, ** p <0.05; n=3). All results for A and B are representative of at least 2 independent experiments.
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References

    1. Rosenberg SA. Immunotherapy and gene therapy of cancer. Cancer Res. 1991;51:5074s–9s. - PubMed
    1. Hwu P, Rosenberg SA. The genetic modification of T cells for cancer therapy: an overview of laboratory and clinical trials. Cancer Detect Prev. 1994;18:43–50. - PubMed
    1. Clay TM, Custer MC, Sachs J, Hwu P, Rosenberg SA, Nishimura MI. Efficient transfer of a tumor antigen-reactive TCR to human peripheral blood lymphocytes confers anti-tumor reactivity. J Immunol. 1999;163:507–13. - PubMed
    1. Brenner MK, Heslop HE. Adoptive T cell therapy of cancer. Curr Opin Immunol. 22:251–7. - PMC - PubMed
    1. Morgan RA, Dudley ME, Wunderlich JR, et al. Cancer regression in patients after transfer of genetically engineered lymphocytes. Science. 2006;314:126–9. - PMC - PubMed

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