Reprogramming of T cells to natural killer-like cells upon Bcl11b deletion

Abstract

T cells develop in the thymus and are critical for adaptive immunity. Natural killer (NK) lymphocytes constitute an essential component of the innate immune system in tumor surveillance, reproduction, and defense against microbes and viruses. Here, we show that the transcription factor Bcl11b was expressed in all T cell compartments and was indispensable for T lineage development. When Bcl11b was deleted, T cells from all developmental stages acquired NK cell properties and concomitantly lost or decreased T cell-associated gene expression. These induced T-to-natural killer (ITNK) cells, which were morphologically and genetically similar to conventional NK cells, killed tumor cells in vitro, and effectively prevented tumor metastasis in vivo. Therefore, ITNKs may represent a new cell source for cell-based therapies.

Figures

Fig. 1

Bcl11b is essential for T cell development and for maintaining T cell identity. Thymocytes from flox/flox or flox/+ control mice were treated, or not, with OHT then sorted into DN1 or DN2 subsets, and cultured on OP9-DL1 stromal cells. (A) Flow cytometry profiles of cultured DN1 and DN2 thymocytes (+OHT) in the absence of IL-2. Numbers refer to percentage of cells in the gate. Data are representative of three experiments. (B) Flow cytometry profiles of cultured flox/flox DN3 thymocytes (±OHT) supplemented with IL-2. Data are representative of three experiments. (C) Killing of OP9-DLI stromal cells by OHT-treated flox/flox DN3 thymocytes. Scale bar, 40 μm. (D) DNA from purified NKp46+ cells was prepared and subjected to PCR to detect DJ (top) and V(D)J (bottom) recombination at the TCRβ locus. T, T cells growing from untreated DN3 thymocytes; N1 and N2, sorted NKp46+ cells growing from OHT-treated flox/flox DN3 thymocytes; Thy, wild-type whole thymocytes; B, B cells; and GL, germline band. H2O: no DNA template in PCR. Numbers indicate DJ recombination products. (E and F) Microarray analysis of gene expression in NKp46+CD3− ITNK cells from DN3 thymocytes (columns I1 to I4), IL-2–expanded NK cells (LAK; L1 to L4) and sorted DN3 flox/flox thymocytes (DN3; D1 to D4) were subjected to expression. (E) Two-way hierarchical cluster map of the array data. Column numbers (I1 to I4 for instance) refer to four independent RNA samples for each cell type, and rows represent individual transcripts. Scale indicates the log2 value of normalized signal level. (F) QRT-PCR validation of gene expression of selected genes among ITNKs, LAKs, and DN3 cells. Bars are means ± SD of three samples.

Fig. 2

Efficient reprogramming of T cells to ITNKs. (A) Representative flow cytometry profiles of ITNKs reprogrammed from single flox/flox DN3 cells. Numbers refer to percentage in total cells. T: T cells that did not have complete Bcl11b deletion. Data are representative of three experiments. (B) PCR genotyping of Bcl11b deletion in two representative T cell (T1 and T2) and ITNK (I1 and I2) wells. flox, floxed allele; del, deletion allele. −OHT: no OHT treatment; H2O: no template control. (C) DJ recombination at the TCRβ locus of five ITNK wells (I1 to I5) showing unique DJ recombination. L, DNA ladder; Thy, wild-type thymocytes. (D) Giemsa stain of parental DN3 thymocytes (T) and ITNK cells. Scale bar, 20 μm. (E) Transmission electron micrograph of an ITNK cell. 1, Nucleus; 2, Golgi body; 3, granule; 4, endoplasmic reticulum. Scale bar, 2 μm. (F) Cytotoxicity of ITNKs (labeled as “+OHT”) and LAKs measured in standard 51Cr-release assays with B16F10, RMA, and RMA-S tumor cell targets at the indicated effector-to-target (E:T) ratios. −OHT: flox/flox T cells. Data are means of triplicate wells.

Fig. 3

ITNKs reprogrammed in vivo are potent tumor cell killers. (A) Flow cytometric analysis of thymocytes and splenocytes from OHT-treated flox/flox and flox/+ mice. Numbers refer to the percentage in the lymphocyte gate. Data are representative of four mice. (B) Analysis of ITNKs from thymic γδ T cells in OHT-treated flox/flox mice. Data are representative of two mice. (C) ITNKs production in Rag2−/−Il2rg−/− recipients injected with flox/flox DP thymocytes. Two weeks after injection, donor (CD45.2+) and host (CD45.1+) splenocytes were analyzed. Numbers refer to the percentage of lymphocyte gate. Plots are representative of 15 mice from three independent experiments. (D) Ex vivo expansion of ITNKs in IL-2 from splenocytes of the recipient mice. Viable cells were counted (top) at the indicated time points and analyzed (bottom). Numbers refer to percentages. Most cells in the culture were ITNKs because they expressed NKp46, TCRβ, NK1.1, and NKG2D. Bars are means ± SD of four samples. Data are representative of three experiments. (E) The ex vivo expanded ITNKs (labeled as “+OHT”) were used in 51Cr-release killing assays with B16F10, RMA, and RMA-S tumor cell targets at the indicated effector-to-target (E:T) ratios. −OHT: flox/flox T cells. Data are means of triplicate wells. Results are representative of three experiments. (F) ITNKs prevented tumor metastasis. Rag2−/−Il2rg−/− recipients were first transplanted with treated (+OHT) or untreated (−OHT) flox/flox DP thymocytes or phosphate-buffered saline. Recipients were subsequently injected intravenously with 50,000 B16F10 melanoma cells. Lung tumor colonies were enumerated 2 weeks after tumor challenge. Data are from individual mice, and bars represent the means.

Fig. 4

Bcl11b is a direct downstream target gene of Notch signaling. (A) Bcl11b protein in T cells after OHT treatment detected by Western blot. (B) Schematic of the Bcl11b locus showing putative CSL binding sites (BS) and that of an irrelevant control binding site (CTL). (C) Genomic DNA was prepared from immunoprecipitation of thymocytes, by using CSL or control immunoglobulin G (IgG) antibodies, and was amplified by using primers flanking the putative CSL or the control binding sites at the Bcl11b locus. Three Bcl11b-binding regions: Region 1, about 1.8 kb from start of the transcription; region 2, 5.4 kb downstream of exon 1; region 3, about 600 base pairs downstream of exon 2. CSL, CSL antibody; IgG, control IgG. Fold-enrichment was calculated relative to the IgG control (set to 1). Bars are means ± SD of triplicate samples.