HDAC inhibition prevents transgene expression downregulation and loss-of-function in T-cell-receptor-transduced T cells

Abstract

T cells that are gene-modified with tumor-specific T cell receptors are a promising treatment for metastatic melanoma patients. In a clinical trial, we treated seven metastatic melanoma patients with autologous T cells transduced to express a tyrosinase-reactive T cell receptor (TCR) (TIL 1383I) and a truncated CD34 molecule as a selection marker. We followed transgene expression in the TCR-transduced T cells after infusion and observed that both lentiviral- and retroviral-transduced T cells lost transgene expression over time, so that by 4 weeks post-transfer, few T cells expressed either lentiviral or retroviral transgenes. Transgene expression was reactivated by stimulation with anti-CD3/anti-CD28 beads and cytokines. TCR-transduced T cell lentiviral and retroviral transgene expression was also downregulated in vitro when T cells were cultured without cytokines. Transduced T cells cultured with interleukin (IL)-15 maintained transgene expression. Culturing gene-modified T cells in the presence of histone deacetylase (HDAC) inhibitors maintained transgene expression and functional TCR-transduced T cell responses to tumor. These results implicate epigenetic processes in the loss of transgene expression in lentiviral- and retroviral-transduced T cells.

Keywords: HDAC inhibitors; TCR-transduced T cells; cancer immunotherapy; cell therapy; functional responses; gene silencing; gene-modified T cells; sodium butyrate; transgene silencing; vorinostat.

Graphical abstract

Figure 1

Transgene expression is reversibly downregulated in patients (A and B) Cryopreserved patient PBMCs were thawed and analyzed by flow cytometry for expression of CD34 on live CD3⁺CD4⁺CD34⁺ or live CD3⁺CD8⁺CD34⁺ T cells. (A) Expression of CD34 versus CD4 on live CD3⁺ T cells for a patient given lentivirally transduced T cells (top, LV) and retrovirally transduced T cells (bottom, RV). (B) MFI of CD34 on live CD34⁺ CD4⁺ (top) and CD34⁺ CD8⁺ (bottom) T cells. (C and D) Patient blood samples were thawed then rested or cultured in complete media with IL-2 (300 IU/mL) and IL-15 (100 ng/mL) for 2 days, then restimulated with Human T-Activator CD3/CD28 beads at a 1:1 cell:bead ratio. (C) Expression of CD34 and gating on CD34⁺CD4⁺ and CD34⁺CD8⁺ are shown. (D, top) The percent CD34⁺CD4⁺ (left) and CD34⁺CD8⁺ (right) are shown for every patient; (bottom) the MFI of CD34 on CD34⁺CD4⁺ (left) and CD34⁺CD8⁺ (right) T cells is shown for every patient. Patients receiving lentiviral-transduced T cells are shown in blue, while those receiving retroviral-transduced T cells are shown in black. p values were calculated by a ratio paired t test and summarized with ∗∗p < 0.01 and ∗p < 0.05.

Figure 2

Reversible transgene expression downregulation occurs in the absence of cytokines (A and B) T cells from the apheresis of patients 1–3 were activated, separated into two groups, and transduced with lentivirus or retrovirus and expanded. Transduced T cells were cultured with and without cytokines for 23 days. CD34 and Vβ12 expression was assessed by flow cytometry. (A) Example of CD34 versus Vβ12 expression on CD4⁺ and CD8⁺ T cells on day 23. (B) Time course of CD34 (left) and Vβ12 (right) expression on CD4⁺ (top) and CD8⁺ (bottom) T cells. Data in (B) were analyzed by 2-way ANOVA and Tukey’s multiple comparisons test. (C) Transduced T cells were cultured for 4 weeks without cytokines, then restimulated. CD34 and Vβ12 expression was assessed on CD4⁺ and CD8⁺ T cells prior to and after restimulation for 2 days with IL-2 and IL-15 or with CD3/CD28 beads as well as IL-2 and IL-15. Data in (C) were analyzed by paired t test. p values are summarized as ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, ∗∗p < 0.01, and ∗p < 0.05.

Figure 3

Culture in IL-15 reduces downregulation of transgene expression Healthy donor T cells were activated, transduced with lentivirus (pLVX-1383I, left) or retrovirus (SAMEN-1383I, RV, right), then CD34-sorted and rapidly expanded. (A) Lentiviral- and retroviral-transduced T cells were then cultured in the presence or absence of IL-2 and IL-15. Expression of CD34 and Vβ12 was assessed by flow cytometry on CD4⁺ or CD8⁺ CD3⁺ T cells at indicated time points. (B) Lentiviral- and retroviral-transduced T cells were cultured for 14 days in the absence or presence of T2 cells, T2 cells pulsed with tyrosinase, 624 MEL cells, or IL-2 (300 IU/mL) and IL-15 (100 ng/mL). Tumor cells were added at a ratio of 1:4 tumor:T cell. On day 12, tumor cells were no longer detectable, and a second dose of tumor cells was added. Error bars represent standard deviation. Significance is shown by Student’s t test with p values summarized as ∗∗p < 0.01, and ∗p < 0.05.

Figure 4

Culture with HDAC inhibitors reduces transgene expression downregulation Transduced T cells (from healthy donors) were cultured for 12 days with no cytokines or with IL-2 and IL-15 in the absence or presence of HDAC inhibitors, sodium butyrate (SB) or suberoylanilide hydroxamic acid (SAHA), or a DNA methyltransferase inhibitor, 5-aza-2′-deoxycytidine (5a2d). (A) Representative fluorescence-activated cell sorting (FACS) plots of CD34 versus vβ12 on CD4⁺ (top) and CD8⁺ (bottom) T cells. (B) Comparison of percentages CD34⁺Vβ12⁺ of CD4⁺ (top) and CD8⁺ (bottom) T cells. Results are representative of at least four donors and three independent experiments. Significance was determined by paired t test (pairing on donor and virus type) with p values summarized as ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, ∗∗p < 0.01, and ∗p < 0.05

Figure 5

Transduced CD4⁺ and CD8⁺ T cell responses to antigen are protected in the absence of cytokines by sodium butyrate Transduced T cells (from healthy donors) were cultured for 10 days with and without sodium butyrate and cytokines. After 10 days, cells were washed and cocultured with T2/tyrosinase or 624 MEL in the presence of Golgi blockers and a labeled anti-CD107a antibody. After the coculture, the cells were stained extracellularly for CD3, CD4, CD8, and CD34; stained with a viability dye; then fixed, permeabilized and stained intracellularly for TNF-α, IFN- γ, and IL-2. Statistics shown are Student’s t test of differences in total cells expressing 1 or more effector molecules (IL-2, IFN-γ, TNF-α, and CD107a). p values are summarized as ∗∗p < 0.01, and ∗p < 0.05.