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. 2010 Jul-Aug;33(6):648-58.
doi: 10.1097/CJI.0b013e3181e311cb.

A simplified method for the clinical-scale generation of central memory-like CD8+ T cells after transduction with lentiviral vectors encoding antitumor antigen T-cell receptors

Shicheng Yang  1 , Mark E Dudley, Steven A Rosenberg, Richard A Morgan
Affiliations

A simplified method for the clinical-scale generation of central memory-like CD8+ T cells after transduction with lentiviral vectors encoding antitumor antigen T-cell receptors

Shicheng Yang et al. J Immunother. 2010 Jul-Aug.

Abstract

Adoptive transfer of antigen-specific CD8+ T cells can effectively treat patients with metastatic melanoma. Recent efforts have emphasized the in vitro generation of antitumor T cells by transduction of genes encoding antitumor T-cell receptors. At present, lentiviral vector-mediated transduction of CD8+ T cells relies on anti-CD3/CD28 bead stimulation; however, this method fails to efficiently expand CD8+ T cells. Herein we sought to establish a methodology for lentiviral vector transduction using optimal activating agents for efficient gene delivery and robust expansion of CD8+ T cells. To overcome the inability of anti-CD3/CD28 beads to efficiently expand CD8+ T cells, we evaluated alternative activating agents including feeder cells from allogeneic peripheral blood mononuclear cells and plate-bound anti-CD3 antibody. Analyses of gene transfer, cell phenotype, fold expansion, and biologic activities were used to determine the optimal methodology. Plate-bound anti-CD3 provided an ideal activation platform that afforded optimal lentiviral vector-mediated gene transfer efficiency (up to 90%), and coupled with peripheral blood mononuclear cells feeder cells yielded up to 600-fold expansion of CD8+ T cells within 12 days. The T-cell antigen receptor (TCR) engineered CD8+ T cells conferred specific antitumor activity and many displayed a central memory-like phenotype. The methodology described here could be readily applied for engineering CD8+ T cells with antitumor specificity for human adoptive immunotherapy.

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Conflict of interest statement

The authors have declared there are no financial conflicts of interest in regards to this work.

The authors declare that they have no competing financial interests.

Figures

FIGURE 1.
FIGURE 1.
Lentiviral vector-mediated transduction of CD8+ T cells activated by anti-CD3/CD28 beads. A, Schematic illustration of experimental design. Negative-selected CD8+ T cells were activated using anti-CD3/CD28 beads at a bead to cell ratio of 3:1 using 1 × 106 cell per well of a 24-well plate. One day later, cells were washed twice with PBS and transduced with lentiviral vector supernatant as described in methods. Transduced cells were fluorescence-activated cell sorter (FACS) analyzed at day 7 and day 12 posttransduction. At day 12, cells were subject to a second expansion (S2) using either anti-CD3/CD28 beads or PBMC feeder cells supplemented with 30 ng/mL OKT3. At day 24 poststimulation (S2d12), cells were analyzed by FACS and their function tested. B, T-cell antigen receptor (TCR) expression after lentiviral vector-mediated transduction. CD8+ T cells were from 3 donors PBMC (d1, d2, and d3), were analyzed for transgene TCR expression by staining with MART-1 tetramer and purity of CD8+ T cells was determined by anti-CD8 antibody. C, Functional analysis of transduced CD8+ T cells. The cells were counted at day 12 (left) and day 24 (right) posttransduction and the actual fold expansion determined. INF-γ induction was carried out by coculture with melanoma tumor lines Mel526, Mel 624 (HLA-A2+, MART-1+) and Mel 888, Mel 938 (HLA-A2−, MART-1+). Plotted were the means of triplicate values determined after overnight coculture.
FIGURE 2.
FIGURE 2.
Phenotypic analysis of CD8+ T cells from negative or positive selection. A and B, Phenotype of negatively or positively selected CD8+ T cells at day 1. CD8+ T cells from 2 donors (d7 and d8) were obtained by negative or positive selection and cultured in AIM-V medium containing 300 IU/mL IL-2 overnight. The phenotype of cells were evaluated by FACS using a panel of antibodies indicated on top of each image. The arrows in second column on ‘‘positive selection’’ groups indicate populations of CD3-CD8+ NK cells.
FIGURE 3.
FIGURE 3.
Feeder cells from PBMC support both gene delivery and robust expansion of CD8+ T cells derived from negative or positive selection. A, Schematic illustration of anti-CD3/CD28 beads or feeder cells-mediated activation and transduction. CD8+ T cells from 2 donors (d7 and d8) were obtained by negative or positive selection and activated by anti-CD3/CD28 beads or feeder cells (in the presence of 30 ng/mL OKT3) overnight. The next day the cells were washed twice and transduced with lentiviral vector by spinoculation in the presence of protamine sulfate, and cultured for 12 days. B, At day 12, the transgene expression and phenotype of cells were evaluated using a panel of antibodies as indicated. The fold expansion was listed on right of each group. Top panel represents the anti-CD3/CD28 beads activated CD8+ T cells, and lower panel represents feeder cells activated CD8+ T cells. C, At day 12, the function of transduced cells activated by anti-CD3/CD28 beads or feeder cells was tested for IFNγ (left) and IL2 (right) by coculture with melanomalines, where d7- and d8-indicate donor-specific CD8+ T cells from negative selection, whereas d7+, d8+ indicate CD8+ T cells frompositive selection.
FIGURE 4.
FIGURE 4.
Lentiviral vector-mediated transduction of CD8+ T cells using plate-bound OKT3 activation. A, Schematic illustration of plate-bound OKT3 activation and transduction. B, Phenotypic analysis of CD8+ T cells activated by plate-bound OKT3. Positive-selected CD8+ T cells were activated by plate-bound OKT3 overnight, and the next day cells were washed twice with PBS and transduced by spinoculation as described in methods. Six hours after transduction, feeder cells (feeders to CD8, 10:1) were added to 24-well plates and briefly resuspended by gentle mixing. At day 2, cell media was replenished with fresh media, and at day 4, cells were transferred to 6-well plates. At day 12, the transgene expression and phenotype of cells were evaluated using a panel of antibodies as indicated. The number representing fold expansion was listed on right. C, Cytokine production by transduced CD8+ T cells. At day 12, the function of transduced cells activated by aAPC or feeder cells was tested for cytokine induction by coculture with melanoma lines. d4, d5 indicate CD8+ T cells from donor 4 and donor 5.
FIGURE 5.
FIGURE 5.
Head-to-head comparison of plate-bound OKT3 to anti-CD3/CD28 beads in the activation and expansion of CD8+ T cells. A, Schematic illustration of plate-bound OKT3 activation or anti-CD3/CD28 beads. B, Phenotypic analysis of CD8+ T cells activated by plate-bound OKT3 and anti-CD3/CD28 beads. Positive-selected CD8+ T cells obtained from donor 6 (d6) were activated by plate-bound OKT3 or anti-CD3/CD28 beads overnight, and then transduced with the anti-MART-1 TCR vector. Six hours after transduction, feeder cells were added to the plate-bound OKT3 activated CD8+ T cells, for expansion. At day 12, the transgene expression and phenotype of cells were evaluated using a panel of antibodies. The number representing fold expansion was listed on right. C, Cytokine production by transduced CD8+ T cells. At day 12, the function of transduced cells was tested for IFN-γ cytokine induction following coculture with melanoma lines.
FIGURE 6.
FIGURE 6.
Efficient transduction and robust expansion of clinical-grade CD8+ T cells. A, Lentiviral vector-mediated transgene expression and expansion. CD8+ T cells from donor 6 (d6) were purified using the CliniMACS separation system from Miltenyi Biotec. CD8+ T cells were activated using plate-bound OKT3 for 1 day, and transduced the next day by spinoculation as described in methods using vectors containing MART-1 or gp100 TCRs or green fluorescent protein (GFP) as control. Six hours after transduction, PBMC feeder cells were added (feeder cells to CD8+, 10:1) and mixed. The cells were cultured in vitro for 12 days and the expression of 2 TCRs and GFP was measured by FACS (left side). The growth curve of DMF5 TCR transduced cells was plotted in the middle panel and the fold expansion of these transduced cells was calculated on right. B, Phenotypical analysis of transduced CD8+ T cells at day 12. A panel of differentiation markers were used as denoted on top of each FACS image. C, Cytokine induction. Transduced CD8+ T cells were cocultured with melanoma lines, and cytokine production of IFN-γ and IL-2 was measured by ELISA. D, Specific lysis of melanoma lines. The specific lytic activities of transduced CD8+ T cells were determined by coculture with 51Cr-labeled tumor cells at the indicated E: T ratios. The percent cell lysis was calculated using the formula [(specific release-spontaneous release)/(total release-spontaneous release)] × 100. Results representing mean of triplicate cultures were plotted.
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References

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