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. 2010 Jan;21(1):75-86.
doi: 10.1089/hum.2009.122.

4-1BB and CD28 signaling plays a synergistic role in redirecting umbilical cord blood T cells against B-cell malignancies

Syam Tammana  1 , Xin Huang, Marianna Wong, Michael C Milone, Linan Ma, Bruce L Levine, Carl H June, John E Wagner, Bruce R Blazar, Xianzheng Zhou
Affiliations

4-1BB and CD28 signaling plays a synergistic role in redirecting umbilical cord blood T cells against B-cell malignancies

Syam Tammana et al. Hum Gene Ther. 2010 Jan.

Abstract

Umbilical cord blood (UCB) T cells can be redirected to kill leukemia and lymphoma cells by engineering with a single-chain chimeric antigen receptor (CAR) and thus may have general applications in adoptive cell therapy. However, the role of costimulatory molecules in UCB T-cell activation and effector functions in context with CAR remains elusive. To investigate the effect of costimulatory molecules (4-1BB and CD28) on UCB T cells, we transduced UCB T cells with lentiviral vectors expressing Green Fluorescent Protein (GFP) and CAR for CD19 containing an intracellular domain of the CD3zeta chain and either a 4-1BB (UCB-19BBzeta) or a CD28 intracellular domain (UCB-1928zeta), both (UCB-1928BBzeta), or neither (UCB-19zeta). We found that UCB-19BBzeta and UCB-28BBzeta T cells exhibited more cytotoxicity to CD19(+) leukemia and lymphoma cell lines than UCB-19zeta and UCB-1928zeta, although differences in secretion of interleukin-2 and interferon-gamma by these T cells were not evident. In vivo adoptive transfer of these T cells into intraperitoneal tumor-bearing mice demonstrated that UCB-19BBzeta and UCB-1928BBzeta T cells mounted the most potent antitumor response. The mice adoptively transferred with UCB-1928BBzeta cells survived longer than the mice with UCB-19BBzeta. Moreover, UCB-1928BBzeta T cells mounted a more robust antitumor response than UCB-19BBzeta in a systemic tumor model. Our data suggest a synergistic role of 4-1BB and CD28 costimulation in engineering antileukemia UCB effector cells and implicate a design for redirected UCB T-cell therapy for refractory leukemia.

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Figures

FIG. 1.
FIG. 1.
Schematic representation of the chimeric antigen receptors (CARs) for CD19 and the lentiviral vector used in this study. (A) CD19 CARs: 19ζ, 19BBζ, 1928ζ, and 1928BBζ. (B) A lentiviral vector with a bidirectional promoter of human EF1α and mCMV expressing CD19 CARs (19ζ, 19BBζ, 1928ζ, and 1928BBζ) and GFP. CARs, chimeric antigen receptors; CMV, human cytomegalovirus; cPPT, central polypurine tract; CTE, constitutive element from the Mason-Pfizer monkey virus; EFIα, elongation factor 1α; eGFP, enhanced green fluorescent protein; GA, 5′ portion of the gag gene; mCMV, minimal core promoter element; RRE, Rev-response element; SA, splice acceptor; SD, splice donor; VH, variable domain of heavy chain; VL, variable domain of light chain; WPRE, woodchuck hepatitis virus posttranscriptional regulatory element.
FIG. 2.
FIG. 2.
Cell surface expression of CARs after transduction. (A) Expression of CAR and GFP in UCB1 T cells after transduction and cell sorting for GFP. Cy5-conjugated goat anti-mouse F(ab)2 antibody was used to visualize the surface expression of CD19 CARs. Similar data were obtained for UCB2, UCB3, and UCB4 T cells (not shown). It should be noted that frequent background CAR staining in UCB-GFP cells or untransduced UCB mock cells was observed, but this was not evident in peripheral blood lymphocytes (data not shown) (Huang et al., 2008). (B). Western blotting of CAR expression in transduced UCB T cells. One out of at least five representative data is shown. UCB, umbilical cord blood. Color images available online at www.liebertonline.com/hum.
FIG. 3.
FIG. 3.
Engineered T cells specifically kill CD19+ leukemia and lymphoma cells. A 4-hr 51Cr-release assay was utilized to assess cytotoxicity of UCB T cells against CD19+ target cells. One out of four representative data from UCB1 (A) and UCB2 (B) T cells is shown. E:T ratio, effector-to-target ratio.
FIG. 4.
FIG. 4.
IL-2 and IFN-γ production profile by engineered T cells. (A) IFN-γ secretion. (B) IL-2 production. As a positive control, UCB-derived T cells were stimulated in medium with phorbol myristate acetate (PMA) (50 ng/mL) and ionomycin (1 μM). Background cytokine production was determined from T cells and tumor cells alone. Both IFN-γ and IL-2 assays were conducted simultaneously by ELISA. IL-2 production was also examined by intracellular cytokine staining (BD Biosciences) (data not shown). One out of two representative data is shown. IFN-γ, interferon-γ; IL-2, interleukin-2.
FIG. 5.
FIG. 5.
In vivo antitumor responses by the engineered T cells in a local tumor model. (A) The experimental schedule. (B) BLI of tumor growth in NOD/SCID mice (six groups, n = 4 each) treated with the engineered UCB1 T cells expressing 19ζ, 19BBζ, 1928ζ, 1928BBζ, and GFP. One mouse each in UCB-19ζ (day 6) and UCB-1928ζ (day 10) groups died because of the anesthetic procedure for imaging. (C) Bioluminescent intensity of the mice treated with the engineered UCB. Establishment of luciferase-expressing CD19+ Daudi cells was described previously (Huang et al., 2008). (D) Animal survival after T-cell therapy (three groups, n = 5–7 each). BLI, bioluminescent imaging.
FIG. 6.
FIG. 6.
In vivo antitumor responses by the engineered T cells in a systemic mouse model. (A) The experimental schedule. (B) BLI of tumor growth in NOD/SCID mice (four groups, n = 7 each) treated with the engineered UCB2 T cells expressing 19BBζ, 1928BBζ, and GFP. Raji cells were transduced with a lentiviral vector expressing humanized firefly luciferase and truncated NGFR (hffluc/ΔNGFR) and subsequently sorted for NGFR expression. (C) Bioluminescent intensity of the mice treated with the engineered UCB2 T cells. NGFR, nerve growth factor receptor.
FIG. 6.
FIG. 6.
In vivo antitumor responses by the engineered T cells in a systemic mouse model. (A) The experimental schedule. (B) BLI of tumor growth in NOD/SCID mice (four groups, n = 7 each) treated with the engineered UCB2 T cells expressing 19BBζ, 1928BBζ, and GFP. Raji cells were transduced with a lentiviral vector expressing humanized firefly luciferase and truncated NGFR (hffluc/ΔNGFR) and subsequently sorted for NGFR expression. (C) Bioluminescent intensity of the mice treated with the engineered UCB2 T cells. NGFR, nerve growth factor receptor.
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