Modulating the differentiation status of ex vivo-cultured anti-tumor T cells using cytokine cocktails - PubMed (original) (raw)

Modulating the differentiation status of ex vivo-cultured anti-tumor T cells using cytokine cocktails

Shicheng Yang et al. Cancer Immunol Immunother. 2013 Apr.

Abstract

The genetic modification of CD8+ T cells using anti-tumor T-cell receptors (TCR) or chimeric antigen receptors is a promising approach for the adoptive cell therapy of patients with cancer. We previously developed a simplified method for the clinical-scale generation of central memory-like (Tcm) CD8+ T cells following transduction with lentivirus encoding anti-tumor TCR and culture in the presence of IL-2. In this study, we compared different cytokines or combinations of IL-2, IL-7, IL-12, IL-15, and IL-21 to expand genetically engineered CD8+ T cells. We demonstrated that specific cytokine combinations IL-12 plus IL-7 or IL-21 for 3 days followed by withdrawal of IL-12 yielded the phenotype of CD62L(high)CD28(high) CD127(high)CD27(high)CCR7(high), which is associated with less-differentiated T cells. Genes associated with stem cells (SOX2, NANOG, OCT4, and LIN28A), were also up-regulated by this cytokine cocktail. Moreover, the use of IL-12 plus IL-7 or IL-21 yielded CD8 T cells showing enhanced persistence in the NOD/SCID/γc-/- mouse model. This defined cytokine combination could also alter highly differentiated TIL from melanoma patients into cells with a less-differentiated phenotype. The methodology that we developed for generating a less-differentiated anti-tumor CD8+ T cells ex vivo may be ideal for the adoptive immunotherapy of cancer.

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Conflict of interest statement

The authors declare that they have no competing financial interests.

Figures

Fig. 1

Fig. 1

The screening for optimal combination of cytokines to generating less-differentiated anti-tumor CD8 T cells. a The schema for cytokine cocktails and time line during in vitro culture. All cultures were initially stimulated and maintained in IL-2 until day 4. LVV, lentiviral vector transduction; w/o, without. b The phenotype of in vitro-cultured CD8 T cells in the presence IL-12 and other cytokines. The phenotype of cells was evaluated using a panel of antibodies at day 12 of culture. c The phenotype of in vitro-cultured CD8 T cells following withdrawal of IL-12 at day 7. The phenotype of cells was evaluated at day 12 of culture. The mean ± SD from three donors was calculated, and t test was used for statistical analysis. An asterisk indicates p < 0.01 compared to the IL-2 group

Fig. 2

Fig. 2

Cytokine combinations lead to a less-differentiated phenotype in vitro. a Schema of cytokine in vitro culture. All cultures were initially stimulated and maintained in IL-2 until day 4. LVV, lentiviral vector transduction; w/o, without. b Phenotype of in vitro-cultured anti-tumor CD8 T cells grown in defined cytokine combinations at day 12. Following FACS, the percentage of each marker was calculated (left) and the MFI presented (right). c Gene expression of in vitro-cultured anti-tumor CD8 T cells. Total RNA from the cells was extracted and reverse-transcribed; the level of mRNA representing each candidate gene was measured using Q-RT-PCR. The level of mRNA at IL-2 group was set up as 1; the level of mRNA from other groups was normalized accordingly. The mean ± SD from three donors was calculated, and t test was used for statistical analysis. An asterisk indicates p < 0.05 compared to the IL-2 group

Fig. 3

Fig. 3

Function of cytokine-treated anti-tumor antigen CD8 T cells. T cells grown as described in the schema in Fig. 2a and then tested for anti-tumor reactivity at day 10 or 13 in the different cytokine combinations as shown. Co-cultures were with melanoma lines (mel 526, MART-1 +/HLA-A2+ and mel 938, MART-1 +/HLA-A2−) followed by determination of a IFN-γ and b IL-2. c The number of viable cell in each cytokine combination was determined and the fold expansion plotted

Fig. 4

Fig. 4

CD8 T cells cultured with IL-7 and IL-21 show enhanced engraftment in NOD/SCID/γc−/− mice. The CD8 T cells were transduced and maintained in the presence of IL-2. At day 4, the cells were split, cultured in IL-2 alone or IL-12 plus IL-7 or IL-21 until day 7, at which time the cells were split and the cultures continued without IL-12. At day 10, 1 × 106 CD8 T cells mixed with 3 × 105 unmanipulated CD4 T cells isolated from the same donor were injected intravenously per mouse. Forty days later, mice were killed, and lymphocytes were extracted from lymph nodes. FACS was performed using anti-human CD8 antibody on left; the total number of engrafted human CD8 were calculated and plotted on right. Control group indicates only CD4 T cells were injected. The mean ± SD from three mice was calculated

Fig. 5

Fig. 5

Effect of cytokine cocktails on TIL. a The schematic illustration of cytokine addition during 6-day in vitro culture. TILs were rapidly expanded until day 15 of growth, then harvested and washed, and the cytokines or their combinations were added as depicted. The concentration of IL-2 was 300 IU/ml, and the other cytokines were at 10 ng/ml. At day 3, the supernatant from the TIL culture was removed, and other cytokines were added without the use of IL-12. b The viability and fold expansion during 6-day in vitro culture. c The morphological variation of TILs cultured with different combinations of cytokines at day 6. Shown are representative photomicrograph fields, scale bar indicates 50 μm

Fig. 6

Fig. 6

The phenotype of TIL grown with different combinations of cytokines. a The phenotype of TILs at day 6 was analyzed by FACS using a panel of antibodies as shown. b Gene expression of cytokine-cultured TILs at day 6. The total RNA from the cells was extracted and reverse–transcribed; the level of mRNA representing each candidate gene was measured using Q-RT-PCR assay. The level of mRNA at IL-2 group was set up as 1; the level of mRNA from other groups was normalized accordingly. The mean ± SD from six donors was calculated. An asterisks indicate p < 0.01 compared to the IL-2 group

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