miR-17-92 expression in differentiated T cells - implications for cancer immunotherapy

Abstract

Background: Type-1 T cells are critical for effective anti-tumor immune responses. The recently discovered microRNAs (miRs) are a large family of small regulatory RNAs that control diverse aspects of cell function, including immune regulation. We identified miRs differentially regulated between type-1 and type-2 T cells, and determined how the expression of such miRs is regulated.

Methods: We performed miR microarray analyses on in vitro differentiated murine T helper type-1 (Th1) and T helper type-2 (Th2) cells to identify differentially expressed miRs. We used quantitative RT-PCR to confirm the differential expression levels. We also used WST-1, ELISA, and flow cytometry to evaluate the survival, function and phenotype of cells, respectively. We employed mice transgenic for the identified miRs to determine the biological impact of miR-17-92 expression in T cells.

Results: Our initial miR microarray analyses revealed that the miR-17-92 cluster is one of the most significantly over-expressed miR in murine Th1 cells when compared with Th2 cells. RT-PCR confirmed that the miR-17-92 cluster expression was consistently higher in Th1 cells than Th2 cells. Disruption of the IL-4 signaling through either IL-4 neutralizing antibody or knockout of signal transducer and activator of transcription (STAT)6 reversed the miR-17-92 cluster suppression in Th2 cells. Furthermore, T cells from tumor bearing mice and glioma patients had decreased levels of miR-17-92 when compared with cells from non-tumor bearing counterparts. CD4+ T cells derived from miR-17-92 transgenic mice demonstrated superior type-1 phenotype with increased IFN-gamma production and very late antigen (VLA)-4 expression when compared with counterparts derived from wild type mice. Human Jurkat T cells ectopically expressing increased levels of miR-17-92 cluster members demonstrated increased IL-2 production and resistance to activation-induced cell death (AICD).

Conclusion: The type-2-skewing tumor microenvironment induces the down-regulation of miR-17-92 expression in T cells, thereby diminishing the persistence of tumor-specific T cells and tumor control. Genetic engineering of T cells to express miR-17-92 may represent a promising approach for cancer immunotherapy.

Figure 1

Microarray analysis demonstrates up-regulation of miR-17-92 in Th1 cells. (A), Intracellular IFN-γ vs. IL-4 expression of Th1 and Th2 cells induced from mouse CD4⁺ splenic T cells in vitro. (B), Differentially expressed miRs were analyzed by hierarchical clustering of the log2 value of Th1/Th2 pair of miR microarray signal. Red indicates up-regulation in Th1; green, up-regulation in Th2. (C), miRs were ranked by relative fold expression in Th1/Th2 cells. Arrows indicate members of the miR-17-92 cluster or paralog clusters. miRs with a relative expression of >2.35 fold in Th1 are shown. (B and C), hsa- and mmu- indicate human and mouse miR probes, respectively. Hsa-probes can hybridize with most mouse miR due to the high homology and mmu-signals are shown only when murine miR has unique sequence compared to its human counterpart. (D), Ideogram of mouse chromosome 14 showing the location and order of the miR-17-92 cluster (adapted from NCBI Blast).

Figure 2

Enhanced expression of miRs from the miR-17-92 cluster in Th1 cells. Data represent relative expression of mature miRs in Th1 compared with Th2 cells. SNO202 was used as the internal control and ΔΔC_T method was used to examine expression relative to the Th2 cell value. Relative expression is shown for (A), miR-17-92 cluster members or (B), representative paralog cluster members, miR-106a and -106b. Error Bars indicate standard deviation of the triplicate samples. Each experiment was repeated at least 3 times. Up-regulation in Th1 vs. Th2 is significant in (A) with p < .01 for miR-92 and p < .0001 for all other miRs and in (B), with p < .001 for miR-106a and p < .05 for miR106b using the student t test.

Figure 3

Modulation of miR-17-92 expression by IL-4 signaling. (A) Immuno-magnetically isolated mouse splenic CD4⁺ T cells were cultured with 5 μg/ml plated anti-CD3, feeder cells and 100 U/ml hIL-2 ("Neutral" condition). Anti-IL-4 (2.5 μg/ml) or isotype control mAb was added to the appropriate wells and cultured for 5 days prior to extraction of total RNA. Statistical analysis was carried out using the student t test. The blockade of IL-4 up-regulated miR-17-5p and miR-92 significantly with p < .001 and p < .005, respectively. (B), CD4⁺ T cells were cultured with anti-CD3, feeder cells, and hIL-2 and varying amounts of IL-4 for 5 days. Total RNA was extracted and analyzed by RT-PCR for miR-17-5p expression. The dose dependent decrease of miR-17-92 expression was analyzed using post test for linear trend and was significant (p < .001). (C), Th1 and Th2 cells were induced from splenic CD4⁺ T cells isolated from either wild-type or STAT6^-/- mice. Total RNA was extracted and RT-PCR was performed using specific primers against miR-17-5p and miR-92. Columns represent the mean of triplicates from one of 2 two experiments with similar results, and error bars represent standard deviations. STAT6^-/- cells demonstrated significantly higher levels of miR-17-5p and miR-92 compared with wild type (WT) cells in both Th1 and Th2 conditions (p < .001) using the student t test.

Figure 4

Tumor bearing conditions down-regulate miR-17-5p expression in T cells. SPCs were harvested from C57BL/6 or STAT6^-/- mice bearing day 15 subcutaneous B16 melanoma (T+) or control non-tumor bearing mice (T-). (A), CD4⁺ and CD8⁺ T cells were isolated by immuno-magnetic bead separation, and evaluated for miR17-5p expression. (B), 1 × 10⁶ CD4⁺ cells from WT mice were briefly stimulated with anti-CD3 mAb for 6 hours. Concentration of IFN-γ secreted in culture media was evaluated by specific ELISA. (C), CD4⁺ T cells were isolated from healthy donor-derived peripheral blood mononuclear cells (PBMC) and stimulated with 5 μg/ml plated anti-CD3, feeder cells (irradiated PBMC) and 100 IU/ml hIL2 in the presence or absence of hIL-4 (10 ng/ml) for 5 days prior to extraction of total RNA. (D), Non-stimulated CD4⁺ and CD8⁺ T cells were isolated by immuno-magnetic beads from PBMC derived from healthy donors (n = 6) or patients with GBM (n = 8) and miR-17-5p expression was analyzed by RT-PCR. Data in (A), (B) and (C), are representative of 2 identical experiments with similar results. Columns represent the mean of triplicates from a single experiment and error bars represent standard deviation. * indicates p < 0.01 and ** indicates p < 0.05 between the two groups using the student t test.

Figure 5

T cells from miR-17-92 transgenic mice demonstrate enhanced Th1 phenotype. Splenic CD4⁺ T cells were immuno-magnetically isolated from miR-17-92 TG/TG or control animals. (A), miR-17-5p expression was analyzed in total RNA extracted from these freshly isolated cells. (B), Flow analysis was carried out on these freshly isolated cells for surface expression of CD49d, a subunit composing VLA-4. The grey-shaded region represents CD4⁺ T cells isolated from control wild type animals and the unshaded region with the solid line represents CD4⁺ T cells from miR-17-92 TG/TG mice. Dotted lines represent samples stained with isotype control Rat IgG2b. As the background staining with the isotype IgG2b was equally very low in the two cell types, the corresponding histograms are barely distinguishable each other. (C), Isolated cells were stimulated in Th1 skewing condition for 9 days and 5 × 10⁶cells were then plated in fresh media for 24 hours, at which point supernatant was collected and analyzed for IFN-γ by ELISA. Both in (A), and (C), values in the two groups were statistically different with p < .01 using the student t test.

Figure 6

Ectopic expression of miR-17-92 cluster members in the human Jurkat T cell line confers increased IL-2 production and resistance to AICD. Jurkat cells were transduced by either one of the following pseudo typed lentivirus vectors: 1) control vector encoding GFP; 2) the 17-92-1 expression vector encoding miR-17 18 and 19a, or 3) the 17-92-2 expression vector encoding miR 20, 19b-1, and 92a-1. (A), Transduced Jurkat cells (5 × 10⁴) in the triplicate wells were stimulated with PMA (10 ng/ml) and ionomycin (500 nM) for overnight and supernatant was harvested and tested for the presence of IL-2 by specific ELISA. The figure shows mean values and standard deviations of the amount of IL-2 released from each group. Statistical analysis was carried out using the student t test, and significant (p < .005) increase of IL-2 production was confirmed in both 17-92-1 and the 17-92-2 transduced groups compared with the control group. (B), Transduced Jurkat cells were treated with the AICD inducing condition (10 μg/ml anti-CD3 mAb) or in complete media (No Tx) for 24 hrs. Then, the relative numbers of viable cells were evaluated by 4 hour WST-1 assays. The figure shows mean values and standard deviations of 8 wells/group each containing 5 × 10⁵ cells. For each group, the relative OD readings at 450 nm of AICD-treated cells compared with control Jurkat cells without AICD-treatment is indicated. * indicates p < 0.05 between the two groups using student t test.