Molecular dissection of the miR-17-92 cluster's critical dual roles in promoting Th1 responses and preventing inducible Treg differentiation - PubMed (original) (raw)
Molecular dissection of the miR-17-92 cluster's critical dual roles in promoting Th1 responses and preventing inducible Treg differentiation
Shan Jiang et al. Blood. 2011.
Abstract
Mir-17-92 encodes 6 miRNAs inside a single polycistronic transcript, the proper expression of which is critical for early B-cell development and lymphocyte homeostasis. However, during the T-cell antigen response, the physiologic function of endogenous miR-17-92 and the roles of the individual miRNAs remain elusive. In the present study, we functionally dissected the miR-17-92 cluster and revealed that miR-17 and miR-19b are the key players controlling Th1 responses through multiple coordinated biologic processes. These include: promoting proliferation, protecting cells from activation-induced cell death, supporting IFN-γ production, and suppressing inducible regulatory T-cell differentiation. Mechanistically, we identified Pten (phosphatase and tensin homolog) as the functionally important target of miR-19b, whereas the function of miR-17 is mediated by TGFβRII and the novel target CREB1. Because of its vigorous control over the Th1 cell-inducible regulatory T cell balance, the loss of miR-17-92 in CD4 T cells results in tumor evasion. Our results suggest that miR-19b and miR-17 could be harnessed to enhance the efficacy of T cell-based tumor therapy.
Figures
Figure 1
The functionality of miR-19b and miR-17 in antigen-stimulated CD4+ T-cell proliferation and activation-induced cell death. (A,C) Assessment of miR-17-92 relative expression in CD4+ T cells by qPCR. Data were normalized to a reference small RNA U6. Bar graph shows means ± SEM from 3 independent experiments. (A) LN T cells from 5C.C7 TCR-transgenic mice were primed by syngeneic APCs loaded with agonist peptide MCC (10μM) and transduced with retrovirus encoding GFP alone (mock) or the mir-17-92 gene with GFP. Three days later, CD4+GFP+ T cells were FACS sorted, and the expression of miRNA was analyzed by qPCR. Data were normalized to the mock group. (B) mir-17-92 genes were introduced into 5C.C7 T cells as described in panel A. Cells were selected by puromycin for 48 hours, and restimulated with MCC-loaded CH27 APCs (10μM) for 24 hours. 5-ethynyl-2′-deoxyuridine (EdU) was supplied into the culture medium 3 hours before cell fixation. The percentage of CD4+GFP+ T cells in the S phase was measured with the Click-iT EdU flow cytometry assay (Invitrogen). Top: representative FACS plot; bottom: statistical analysis of 5 independent experiments. (C) Expression of miRNAs in CD4+ T cells from LNs and spleens of miR-17-92f/fCD4-Cre− (WT), miR-17-92f/+CD4-Cre+ (f/+), and miR-17-92f/fCD4-Cre+ (KO) littermates. Data were normalized to WT. (D) CD4+CD25− T cells from LNs and spleens of WT, f/+, and KO littermates were labeled for 10 minutes at 37°C with CFSE at a ratio of 3 × 106 cells/4μM chemical, followed by washes with complete culture medium, and were then activated by 1 μg/mL of plate-bound anti-CD3 and anti-D28 Abs (eBioscience) for 72 hours. Left: representative FACS plot showing CFSE dilution. Tinted peaks represent CFSE-stained T cells without stimulation; right: statistical analysis of 3 independent experiments. (E) CD4+CD25− T cells from WT or KO mice were primed and transduced with indicated retroviruses. After 2 days of puromycin selection, cells were restimulated with anti–CD3/CD28 for 24 hours, and the number of T cells in the S phase was determined by staining of pulsed EdU. Bar graphs summarize means ± SEM from 4 independent experiments and data were normalized to WT-mock. The statistical significance was assessed in comparison with the KO-mock group. (F) LN T cells from 5C.C7 TCR-transgenic mice were primed and transduced with retrovirus encoding GFP or miR-17-92 as described in panel A. Three days later, viable cells were enriched by density gradient centrifugation and then restimulated with CH27 loaded with MCC (10μM) for 24 hours, and the status of AICD was assessed by annexin V and 7AAD staining (BioLegend). The bar graph summarizes the means ± SEM from 4 independent experiments. (G) CD4+CD25− T cells from LNs and spleens of WT, f/+, and KO littermates were stimulated with anti–CD3/CD28 Abs for 72 hours, and the percentages of CD4+ T cells undergoing apoptosis were assessed by annexin V and 7AAD staining. The bar graph summarizes means ± SEM from 3 independent experiments. (H) CD4+CD25− T cells from LNs and spleens of KO mice were primed and transduced with retrovirus encoding individual miRNAs of miR-17-92 as described in panel E. Three days after transduction, viable CD4+ T cells were enriched and restimulated with anti-CD3/CD28 for 24 hours. The profiles of restimulation-induced apoptosis were measured by annexin V staining. Bar graphs summarize the means ± SEM from 3-5 independent experiments. The statistical significance was assessed in comparison with the KO-mock group. *P < .05; **P < .01; ***P < .001; and ns, no significance.
Figure 2
miR-19b is indispensible for IFNγ production from differentiated Th1 cells. (A-B) CD4+CD25− T cells were sorted from LNs and spleens of WT, f/+, and KO littermates, activated by anti–CD3/CD28 Abs under Th1-skewing conditions for 4 days with 50 ng/mL of recombinant mouse IL-12 (Peprotech), 10 μg/mL of purified anti–IL-4 (11B11), and 50 U/mL of recombinant mouse IL-2 (Peprotech). (A) The percentage of viable cells producing IFN-γ and the mean florescence intensity (MFI) of IFN-γ were determined by intracellular staining after 4 hours of stimulation with 0.9nM PMA and 0.5 μg/mL of ionomycin (Sigma-Aldrich) in the presence of 5 μg/mL of brefeldin A (Sigma-Aldrich) and 2μM monensin (eBioscience). The Abs used were anti–IFNγ-APC (BioLegend) and anti–IL-4-PE (BD). The bar graph summarizes the means ± SEM from 3 independent experiments. (B) The mRNA levels of T-bet and IFN-γ from CD4+ T cells differentiated under the Th1-skewing conditions were quantified by qPCR. Data were normalized to a reference gene, SDHA, and are shown as relative to WT. The bar graph shows means ± SEM from 3 independent experiments. (C) As described in Figure 1E, CD4+CD25− conventional T cells of KO mice were primed and transduced with retrovirus encoding individual miRNAs within the miR-17-92 cluster, and then cultured under the Th1-skewing condition for 4 days. The percentages of IFN-γ- or IL-4–producing cells and the MFI of IFN-γ signal were measured by intracellular cytokine staining. Left: representative FACS plot; right: means ± SEM from 3 independent experiments. Statistical analysis was done by comparison with mock. ***P < .001.
Figure 3
miR-19b and miR-17 enhance DTH responses in vivo. (A) WT and KO mice were immunized subcutaneously with 100μg of KLH in CFA (Sigma-Aldrich) and 8 days later, were injected with 50μg of KLH in 1 footpad and PBS in the contralateral footpad. Increase in footpad thickness was measured for both groups at 48 hours after secondary challenge (n = 4). (B-E) WT B.10A mice were transferred through the tail vein with 0.5 × 106 CD4+ T cells from 5C.C7 Rag2−/− mice infected with GFP-, miR-17-, or miR-19b–expressing retrovirus and immunized subcutaneously with 20 μg of MCC peptide in CFA. Five days after immunization, mice were injected with 20 μg of MCC and PBS in each lateral footpad. Seventy-two hours later, the swelling of footpads (in panel B) and the number of total lymphocytes from the popliteal LN (in panel C) were measured (n ≥ 5). The percentage of IFN-γ–producing cells within the GFP+CD4+ population in the DLN was assayed (in panel D) by intracellular staining (n ≥ 4). Representative images of footpad tissues with H&E staining were shown in panel E. *P < .05; **P < .01; ***P < .001; and ns, no significance. Each experiment was repeated 3 times.
Figure 4
miR-19b and miR-17 suppress iTreg differentiation. (A) 5C.C7 T cells were primed, transduced with retrovirus encoding GFP alone or miR-17-92/GFP, and cultured under iTreg-differentiation conditions for 6 days with 0.2-1.0 ng/mL of recombinant human TGFβ (Peprotech), 50 U/mL of recombinant mouse IL-2 (Peprotech), 10 μg/mL of anti–IL-4 (11B11), 10 μg/mL of anti–IFN-γ (XMG1.2), and 10 μg/mL of anti–IL-6 (BD Biosciences). The percentage of Treg cells within the CD4+GFP+ population was measured by Foxp3 staining (eBioscience). The bar graph summarizes means ± SEM from 4 independent experiments. (B) CD4+CD25− T cells sorted from LNs and spleens of WT, f/+, and KO littermates were cultured under iTreg-differentiation conditions for 6 days with the indicated TGF-β doses, and the percentage of CD25+Foxp3+ Treg cells was assessed. Left: representative FACS plot; right: statistical analysis of 4 independent experiments at the indicated TGF-β dose. (C) 5C.C7 T cells were transduced with individual miRNAs from the miR-17 or miR-19 families, and cultured under iTreg-differentiation conditions for 6 days. The percentage of CD25+Foxp3+ cells was measured by flow cytometry. Bar graphs summarize the means ± SEM of 3 independent experiments. *P < .05; **P < .01; ***P < .00; and ns, no significance.
Figure 5
Pten is the primary target of miR-19b in regulating CD4+ T cell effector functions. (A) Schematic illustration of the predicted targeting sites for miR-19b and miR-17 within the 3′UTR of pten mRNA. (B) The full-length 3′UTR of pten (Pten-WT) or 3′UTR with mutations at the 2 miR-19b target sites (Pten-MU) were cloned downstream of a luciferase reporter and transfected into NIH3T3 cell lines stably expressing the indicated miRNAs. The luciferase activity was measured 72 hours after transfection. Bar graphs show the means ± SD of 3 independent experiments. (C) 5C.C7 T cells transduced with mock, mir-17-92, or mir-19b were sorted by FACS, and total RNA and protein were extracted for qPCR and Western blot. (D) Relative expression of Pten mRNA in CD4+CD25− T cells from LNs and spleens of WT, f/+, and KO mice was measured by qPCR. T cells activated with plate-bound anti–CD3/CD28 Abs for 72 hours were lysed for protein quantification. qPCR data were normalized to SDHA and are shown as relative to mock or WT. Bar graph shows the means ± SEM for 3 independent experiments. (E) miR-19b–mediated regulation on PI3K signaling on antigen engagement was visualized by fluorescence video microscopy. As described previously, the PH domain of Akt kinase was fused with GFP as an imaging probe to monitor the production of PIP3 through PI3K activation. miR-19b or mock vectors were expressed simultaneously with this PH-GFP–imaging probe in 5C.C7 T cells, which were challenged by CH27 APCs preloaded with MCC agonist peptide. Live-cell imaging was performed to monitor the initial signaling strength and the duration of PI3K activation. T cells with PH-GFP expression and stimulated by contact with a single APC were monitored for > 1 hour in a 3D manner, and the best focus plane was used for data analysis. The activity of PI3K was represented by measuring the ratio of the average probe fluorescent intensity in the synaptic region versus the average intensity in the rest cell area. Top panels: representative montages from the DIC channel; bottom panels: representative montages from the GFP channel. (F) The highest level of probe synaptic accumulation within the first 10 minutes of T:APC contact was used as the mark for the maximal activity of PI3K activation in the initiation stage (before the formation of mature immunologic synapse) of TCR signaling. (G) A similar measurement was performed at 1 hour after the initiation of TCR signaling. *P < .05; **P < .01; and ***P < .001.
Figure 6
miR-17 modulates CD4+ T cell effector responses by targeting TGFβRII and CREB1. (A) Schematic representation of the putative miR-17–binding sites in the 3′UTR of tgfbr2 and creb1. (B-C) A portion of the 3′UTR of tgfbr2 (NM009371 position 2171-2552) or creb1 (NM133828 position 3442-3792 containing site 3644 or position 6758-7144 containing site 6984) was cloned downstream of a luciferase reporter and transfected into an NIH3T3 cell line stably expressing miR-17, miR-20a, or mock control. The luciferase activity was measured 72 hours after transfection. Bar graphs show the means ± SD of 3 (B) or 6 (C) independent experiments. (D-E) CD4+CD25− T cells from WT or KO mice were transduced with indicated retrovirus, and CD4+GFP+ T cells were FACS sorted and total RNA and protein were extracted for qPCR (D) and Western blot (E). The bar graph shows means ± SEM from 3 independent experiments. (F) T cells were transduced with indicated virus and cultured under iTreg-differentiation conditions for 5 days. CD4+GFP+ T cells were then sorted, lysed, and analyzed for Smad3 Ser423/425 phosphorylation by Western blot. (G-I) 5C.C7 T cells were primed and transduced with retrovirus containing both the CREB1-IRES-GFP expression cassette and the indicated miR-17 expression cassette, miR-17 alone with GFP marker, or GFP only. (G) Assessment of CREB1 expression in 5C.C7 T cells by qPCR. The graph shows means ± SEM from 3 independent experiments. (H) Death profile of CD4+GFP+ T cells after restimulation with anti–CD3/CD28 Abs. Left: representative FACS plot; right: bar graphs showing means ± SEM from 3 independent experiments. (I) 5C.C7 T cells were transduced as indicated and cultured under iTreg-differentiation conditions for 5 days. The percentage of CD25+Foxp3+ cells inside of the CD4+ T-cell populations and the MFI of the intracellular Foxp3 staining were measured. The numbers on the left corner show the means ± SEM of the percentage of iTreg cells from 3 independent experiments. *P < .05; **P < .01; and ***P < .001.
Figure 7
In vivo, the miR-17-92 cluster is essential for the T cell–mediated antitumor response. (A) WT (n = 5), f/+ (n = 8), and KO (n = 7) littermates were injected subcutaneously with 2 × 105 B16/F10 melanoma cells. The tumor volume was measured each day from 7 days after injection up to 16 days and plotted against time. Left panel: the representative tumor growth curve; right panel: compiled data from 3 independent experiments. Individual dot represents the relative tumor volume normalized to that of the WT at 12 days after melanoma cell injection. (B) Lymphocytes from the dLNs of tumor-carrying mice were isolated 16 days after B16/F10 melanoma inoculation and stimulated with 1 μg/mL of anti–CD3/CD28 Abs for 24 hours. Supernatants from the cultures were assayed for the concentration of indicated Th1, Th2, and Th17 cytokines using a cytokine bead array (BD Biosciences and Bender). Each dot represents data obtained from an individual mouse (n = 3). (C) WT, f/+, and KO littermates were injected with 3 × 105 OVA-secreting B16/F0 cells. Sixteen days after injection, T cells were enriched from the dLNs, labeled with CFSE, and stimulated with LB27.4 APCs loaded with 10μM OVA peptide (323-339) for 48 hours. Antigen-specific responses (proliferation, AICD, and IFN-γ production) were measured as described above. Each data point represents the sample from an individual mouse (n = 3). Each experiment was repeated at least 3 times. *P < .05; **P < .01; and ns, no significance.
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