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. 2005 Dec 1;175(11):7226-34.
doi: 10.4049/jimmunol.175.11.7226.

Primary human T lymphocytes engineered with a codon-optimized IL-15 gene resist cytokine withdrawal-induced apoptosis and persist long-term in the absence of exogenous cytokine

Cary Hsu  1 , Marybeth S Hughes, Zhili Zheng, Regina B Bray, Steven A Rosenberg, Richard A Morgan
Affiliations

Primary human T lymphocytes engineered with a codon-optimized IL-15 gene resist cytokine withdrawal-induced apoptosis and persist long-term in the absence of exogenous cytokine (V体育官网)

Cary Hsu (VSports最新版本) et al. J Immunol. .

Abstract (VSports手机版)

IL-15 is a common gamma-chain cytokine that has been shown to be more active than IL-2 in several murine cancer immunotherapy models. Although T lymphocytes do not produce IL-15, murine lymphocytes carrying an IL-15 transgene demonstrated superior antitumor activity in the immunotherapy of B16 melanoma. Thus, we sought to investigate the biological impact of constitutive IL-15 expression by human lymphocytes. In this report we describe the generation of a retroviral vector encoding a codon-optimized IL-15 gene. Alternate codon usage significantly enhanced the translational efficiency of this tightly regulated gene in retroviral vector-transduced cells VSports手机版. Activated human CD4+ and CD8+ human lymphocytes expressed IL-15Ralpha and produced high levels of cytokine upon retroviral transduction with the IL-15 vector. IL-15-transduced lymphocytes remained viable for up to 180 days in the absence of exogenous cytokine. IL-15 vector-transduced T cells showed continued proliferation after cytokine withdrawal and resistance to apoptosis while retaining specific Ag recognition. In the setting of adoptive cell transfer, IL-15-transduced lymphocytes may prolong lymphocyte survival in vivo and could potentially enhance antitumor activity. .

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Conflict of interest statement

Disclosures

The authors have no financial conflict of interest.

"VSports注册入口" Figures

FIGURE 1
FIGURE 1
Amino acid sequence of the mature IL-15 protein, illustrating codon optimization. The mature protein contains 114 aa. Sixty-three codons were replaced with alternate sequences coding for the same amino acid. Nineteen of the substitutions resulted in a shift from a rarely used codon (<20%) to a more frequently used codon.
FIGURE 2
FIGURE 2
IL-15 expression by transfected cell lines. NIH-3T3, TE671, and 293T cells were transfected by lipofection with wild-type or CO IL-15 retroviral vector plasmids. The tissue culture plates were subsequently washed and refreshed with complete medium. After 24 h, the cell culture medium was tested for IL-15. Data are representative of two experiments.
FIGURE 3
FIGURE 3
Cytokine production and vector integration by IL-15-transduced cells. NIH-3T3 cells were transduced in triplicate with three separate preparations of serially diluted retroviral supernatants derived from either wild-type or CO IL-15 retroviral vectors. A, After 48 h in culture with fresh medium, the cell culture supernatants were harvested and assayed for IL-15. B, Genomic DNA was isolated from cell lysates. PCR amplification was conducted using primers flanking the insert as well as primers for β-actin. The PCR products were run on an agarose gel; the individual bands were then quantitated, and the ratio of retroviral insert to β-actin was calculated. Data are representative of two independent experiments.
FIGURE 4
FIGURE 4
Cytokine production and growth of IL-15-transduced PBLs after withdrawal from exogenous cytokine support. OKT3-activated PBLs from five patients were transduced with IL-15. Control cells were not transduced. A, On culture day 7, cytokine production was measured in the presence or the absence of plate-bound OKT3. B, The PBLs were washed extensively, and 5 × 106 cells from each culture were plated in fresh medium in the absence of exogenous cytokine. Viable cells were enumerated every 4–7 days by trypan blue exclusion; concurrently, the cell culture medium was refreshed by replacing half the conditioned medium with fresh medium. C, A similar experiment was performed in which the culture was conducted for >180 days. AIB, lymphocytes transduced with a control vector.
FIGURE 5
FIGURE 5
Cell surface molecule profile of OKT3-activated lymphocytes. A, IL-15Rα expression in OKT3-activated lymphocytes. Day 10 untransduced lymphocytes were grown in the presence of IL-2 (300 IU/ml) or IL-15 (100 ng/ml). IL-15-transduced lymphocytes from the same patient were grown in the absence of exogenous cytokine. After 3 days in culture, flow cytometric analysis was performed using FITC-coupled CD4 or CD8 and biotinylated anti-IL-15Rα secondarily stained with streptavidin-PE. The unshaded histograms represent isotype control staining, and the shaded histograms represent IL-15Rα staining for the indicated condition. B, CD27, CD28, and CD62L expression in lymphocytes after prolonged culture. After activation with OKT3, untransduced lymphocytes were grown in either IL-2 (300 IU/ml) or IL-15 (10 ng/ml), whereas IL-15-transduced lymphocytes were withdrawn from IL-2 after transduction. After 3 wk in culture, FACS analysis was performed on CD3-gated cells. The numbers in each histogram represent the percentages of cells exhibiting positive staining, followed by the mean fluorescence intensity in parentheses.
FIGURE 6
FIGURE 6
Thymidine incorporation assay. PBLs were untransduced (UT), control vector-transduced (AIB), or transduced with IL-15. On culture day 7, the cells were washed extensively and plated in triplicate in fresh medium with or without exogenous IL-2 (300 IU/ml). The cells were cultured for 4 days and were pulsed with [3H]thymidine during the final 16 h of culture. Data represent the mean ± SEM.
FIGURE 7
FIGURE 7
IL-2 withdrawal-induced apoptosis. OKT3-stimulated PBLs were untransduced, transduced with control vector (AIB), or transduced with IL-15. For 3 consecutive days, starting on culture day 7, a fraction of cells from each transduction condition was washed extensively and replated in the absence of exogenous IL-2. On day 10, the cells were stained with 7-AAD/annexin V and analyzed by flow cytometry. For each plot, the upper right sextant contains necrotic cells, the middle right contains late apoptotic cells, and the lower right contains early apoptotic cells. The numbers to the right of each plot represent the percentage of cells in the corresponding sextant.
FIGURE 8
FIGURE 8
Bcl-2 and Bcl-xL expression in activated lymphocytes. OKT3-stimulated PBLs were untransduced, transduced with a control vector (AIB), or transduced with IL-15. On culture day 7, a fraction of the cells was washed extensively and plated in fresh medium without IL-2. Forty-eight hours later, the cells were stained with anti-CD3 and anti-CD8 Abs. Subsequently, intracellular staining was performed with either anti-Bcl-2 (A) or anti-Bcl-xL (B). Lymphocytes were forward and side scatter gated to exclude dead cells. Subsequently, staining in CD3 and CD8 populations was used to delineate CD4+ and CD8+ cells. The unshaded histogram depicts cells maintained in medium containing IL-2, and the shaded histogram represents cells after IL-2 withdrawal. Tables below each set of histograms show the percentages of cells exhibiting positive staining for Bcl-2 or Bcl-xL, respectively.
FIGURE 9
FIGURE 9
Peptide-specific reactivity is maintained in IL-15-transduced lymphocytes after withdrawal from IL-2. The gp100-reactive PBL cultures were generated and subsequently untransduced (UT), transduced with the MART TCR vector (AIB), or transduced with IL-15. These lymphocytes were tested for peptide reactivity by coculturing them with peptide-pulsed T2 cells at a 1:1 ratio. Cell culture supernatants were harvested and assayed for IFN-γ content 24 h after cocultures were initiated. A, On culture day 7, a peptide titration curve was established by coculturing lymphocytes with T2 cells that were pulsed with serially diluted gp100 peptide. B, To evaluate specific reactivity, culture day 7 lymphocytes were cocultured with T2 cells pulsed with 1 μg of peptide derived from influenza virus (Flu), MART, or gp100. C, In a similar fashion, to evaluate peptide reactivity after cytokine withdrawal, day 7 cultured cells were withdrawn from IL-2 and subsequently tested for reactivity to peptide-pulsed T2 cells on culture day 12.
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References

    1. Burton JD, Bamford RN, Peters C, Grant AJ, Kurys G, Goldman CK, Brennan J, Roessler E, Waldmann TA. A lymphokine, provisionally designated interleukin T and produced by a human adult T-cell leukemia line, stimulates T-cell proliferation and the induction of lymphokine-activated killer cells. Proc Natl Acad Sci USA. 1994;91:4935–4939. - PMC - PubMed
    1. Grabstein KH, Eisenman J, Shanebeck K, Rauch C, Srinivasan S, Fung V, Beers C, Richardson J, Schoenborn MA, Ahdieh M, et al. Cloning of a T cell growth factor that interacts with the β chain of the interleukin-2 receptor. Science. 1994;264:965–968. - PubMed
    1. Dudley ME, Rosenberg SA. Adoptive-cell-transfer therapy for the treatment of patients with cancer. Nat Rev Cancer. 2003;3:666–675. - PMC - PubMed
    1. Waldmann TA. IL-15 in the life and death of lymphocytes: immuno-therapeutic implications. Trends Mol Med. 2003;9:517–521. - VSports app下载 - PubMed
    1. Malek TR, Bayer AL. Tolerance, not immunity, crucially depends on IL-2. Nat Rev Immunol. 2004;4:665–674. - PubMed

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