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. 2005 Apr 1;174(7):4415-23.
doi: 10.4049/jimmunol.174.7.4415.

Primary human lymphocytes transduced with NY-ESO-1 antigen-specific TCR genes recognize and kill diverse human tumor cell lines

Yangbing Zhao  1 , Zhili Zheng, Paul F Robbins, Hung T Khong, Steven A Rosenberg, Richard A Morgan
Affiliations

Primary human lymphocytes transduced with NY-ESO-1 antigen-specific TCR genes recognize and kill diverse human tumor cell lines

Yangbing Zhao et al. J Immunol. .

Abstract

cDNAs encoding TCR alpha- and beta-chains specific for HLA-A2-restricted cancer-testis Ag NY-ESO-1 were cloned using a 5'RACE method from RNA isolated from a CTL generated by in vitro stimulation of PBMC with modified NY-ESO-1-specific peptide (p157-165, 9V). Functionality of the cloned TCR was confirmed by RNA electroporation of primary PBL. cDNA for these alpha- and beta-chains were used to construct a murine stem cell virus-based retroviral vector, and high titer packaging cell lines were generated. Gene transfer efficiency in primary T lymphocytes of up to 60% was obtained without selection using a method of precoating retroviral vectors onto culture plates. Both CD4(+) and CD8(+) T cells could be transduced at the same efficiency. High avidity Ag recognition was demonstrated by coculture of transduced lymphocytes with target cells pulsed with low levels of peptide (<20 pM). TCR-transduced CD4 T cells, when cocultured with NY-ESO-1 peptide pulsed T2 cells, could produce IFN-gamma, GM-CSF, IL-4, and IL-10, suggesting CD8-independent, HLA-A2-restricted TCR activation. The transduced lymphocytes could efficiently recognize and kill HLA-A2- and NY-ESO-1-positive melanoma cell lines in a 4-h (51)Cr release assay. Finally, transduced T cells could efficiently recognize NY-ESO-1-positive nonmelanoma tumor cell lines. These results strongly support the idea that redirection of normal T cell specificity by TCR gene transfer can have potential applications in tumor adoptive immunotherapy.

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Figures

FIGURE 1
FIGURE 1
RNA electroporation of stimulated PBL. PBLs stimulated with OKT3 Ab plus IL-2 for 3 days were electroporated with in vitro transcribed RNA at 2 μg/1 × 106 cells. NY-ESO-1 and Mart-1 TCR α- and β-chain RNAs were generated by in vitro transcription of PCR-amplified templates bearing T7 promoter and poly(A) tail at the 5′ and 3′ ends, respectively (15). Twenty hours after electroporation, gene expression was determined by FACS analysis. GFP expression of RNA-electroporated PBL was compared with PBL electroporated with same amount of GFP RNA and additional β-chain RNA (8FB6; 2 μg/1 × 106; A). Mart-1 TCR and NY-ESO-1 TCR α- and β-chain RNA were electroporated into PBLs, Vβ12 (Mart-1) and Vβ8 (NY-ESO-1) expression were detected after electroporation.
FIGURE 2
FIGURE 2
IFN-γ production of TCR RNA electroporated PBL. Three-day stimulated PBL were electroporated with NY-ESO-1 TCR RNA (NY-ESO-1), Mart1 TCR RNA (Mart-1), or GFP RNA (GFP) and cocultured with peptide-pulsed autologous dendritic cells (DC) or T2 cells. The peptides used were HLA-A2-restricted NY-ESO-1 (p157–165V; ESO-1) and Mart-1 (Mart) and HLA-DP4-restricted NY-ESO-1 (p161–180; ESO II).
FIGURE 3
FIGURE 3
NY-ESO-1 TCR retroviral vectors. Diagrams of two retroviral vectors used to transfer and express the TCR genes from CTL clone TE8-1-8F. In vector MSGE1AIB (AIB), the expression of both α- and β-chains is mediated by the LTR, with translation coupled by the use of an IRES element. In vector MSGE1APB (APB), the expression of the α-chain is mediated by the vector LTR gene promoter, whereas β-chain expression is driven by an internal PGK gene promoter. Restriction enzymes used for cloning of the vector are indicated.
FIGURE 4
FIGURE 4
Transduction of PBLs with TCR retroviral vectors. FACS profile of PBL from a melanoma transduced with supernatant from TCR vector clone AIB38 (AIB) and clone APB30 (APB). Cells were double-stained with anti-Vβ8 and anti-CD8, and the percentage of positive cells is indicated.
FIGURE 5
FIGURE 5
Vβ8 expression of APB-transduced PBLs. PBLs from three patients were transduced with TCR vector APB. Vβ8 and CD8 expressions of transduced PBLs from these three donors are shown; the percent positive cells is indicated. Background staining for Vβ12 in PBL3 was <1%.
FIGURE 6
FIGURE 6
Cytokine secretion staining of transduced CD8 T cells. A, Vβ8 staining of NY-ESO-1 TCR-transduced PBL (APB) and nontransduced PBL (NV) used in the cytokine secretion assay. B, The resultant FACS dot plots for control PBL (NV) or PBL transduced with NY-ESO-1 TCR vector (APB). Cells were cocultured with T2 cells pulsed with either HLA-A- specific gp100 peptide 209 –217 (210M) or NY-ESO-1 peptide 157–165v. Cells were gated for CD3-plus CD8-positive cells. The percentage of positive cells for IFN-γ secretion is shown.
FIGURE 7
FIGURE 7
Melanoma reactivity of TCR-transduced PBL. APB TCR vector-transduced PBL were cocultured with a panel of melanoma cell lines. After coculture, IFN-γ levels were determined in culture medium from TCR vector-transduced PBL (APB) or control vector-transduced PBL (MSGIN). NY-ESO-1 and HLA-A2 expressions of the melanoma cell lines are indicated.
FIGURE 8
FIGURE 8
Lysis of melanoma cell lines. TCR-transduced PBL was tested in a 51Cr release assay. A, TCR- (APB) or control vector- (GIN) transduced PBL were cocultured with 51Cr-labeled, NY-ESO-1 peptide-pulsed (ESO, p157–165) T2 cells or gp100 peptide-pulsed (GP100) as a negative control. B and C, The NY-ESO-1+,HLA-A2+ melanoma cell line 1390mel (B) or 624.28mel (C) was labeled with 51Cr, then cocultured with TCR- (APB) or control vector- (GIN) transduced PBL. Two melanoma cell lines, 526mel (HLA-A2+,NY-ESO-1−) and 586mel (HLA-A2−,NY-ESO-1+), were used as negative control. Cells were incubated at the indicated E:T cell ratio for 4 h, after which the percent lysis of target cells was calculated.
FIGURE 9
FIGURE 9
Nonmelanoma reactivity of TCR-transduced PBL. TCR-(APB) or control vector- (MSGIN, GIN) transduced PBL were cocultured with a panel of nonmelanoma tumor cell lines. IFN-γ release from the coculture supernatant was determined by ELISA. HLA-A2 (A2) and NY-ESO-1 (ESO) expressions of each cell line tested are indicated.
FIGURE 10
FIGURE 10
Lysis of nonmelanoma tumor lines by TCR-transduced PBL. NY-ESO-1 TCR APB-transduced PBL (B) or MSGIN-transduced PBL (A) were cocultured with 51Cr-labeled target cells as indicated. Cells were cocultured at the indicated E:T cell ratio for 4 h, after which the percent lysis of the target cells was calculated.
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