Dicer controls CD8+ T-cell activation, migration, and survival

Abstract

The RNaseIII enzyme Dicer is required for mature microRNA production. Although extensive investigation has been carried out to determine the role of Dicer/miRNAs in the immune system, their function in mature CD8(+) T cells has not been examined. We deleted Dicer in mature polyclonal and TCR transgenic CD8(+) T cells using either tat-cre or the distal lck promoter, which drives cre expression after the stage of positive selection. Following antigenic challenge by a pathogen infection in vivo, Dicer-deleted CD8(+) T cells failed to accumulate at the usual peak of the response. Surprisingly however, we found that deletion of Dicer in mature CD8(+) T cells allowed them to respond more rapidly than control cells to TCR stimuli in vitro. In response to anti-CD3 plus anti-CD28 stimulation, Dicer-deleted T cells up-regulated CD69 faster and entered the first mitosis earlier than control T cells. In addition, activated Dicer(-/-) cells failed to rapidly down-regulate CD69 when removed from the TCR stimulus. As a probable consequence of this sustained CD69 expression, Dicer(-/-) T cells showed defective migration out of the central lymphoid organs in vivo. We identify miR-130/301, which are dramatically up-regulated following T-cell activation, as able to down-regulate CD69 expression via binding to a conserved site in the 3'UTR of CD69 mRNA. Thus, cellular functions dependent on Dicer expression are not required for the early steps in CD8(+) T-cell activation, but are essential for their survival and accumulation.

Fig. 1.

Dicer is essential for the CD8⁺ T-cell response to pathogen in vivo. (A) Two days after in vitro culture, YFP expression in tat-cre treated OT-1 cells was examined. FACS profiles were pregated on 7AAD⁻ live cells. (B and C) Seven days after LM-ova infection, host mice were killed and donor OT-1 cells in the spleen, lymph node (LN), and liver were analyzed. The number of YFP⁺ cells was calculated based on total organ cellularity and the percentage of YFP⁺ cells. Each symbol in C represents an individual mouse.

Fig. 2.

Characterization of Dicer deletion driven by expression of dlck-cre. (A) Thymus and (B) lymph nodes from 7-wk-old Dicer^f/+ Rosa-YFP^f dLck-cre (Dicer^+/−) and Dicer^f/f Rosa-YFP^f dLck-cre (Dicer^−/−) were analyzed by flow cytometry. (C) RNA was isolated from CD8⁺CD62L⁺CD44⁻ naive cells from control and Dicer^−/− mice, and from YFP⁺ and YFP⁻ OT-1 CD8⁺ splenocytes from Dicer^−/− mice and subjected to real-time PCR analysis.

Fig. 3.

Defective polyclonal CD8⁺ T-cell responses in Dicer^−/− mice. Dicer^+/− and Dicer^−/− mice were infected with 2,000 cfu LM-ova i.v. and 7 d postimmunization, the CD8⁺ effector T-cell response was determined. (A) Tetramer staining of splenocytes for K^b/ova-specific cells. (B) Splenocytes were restimulated with ova peptide and the YFP⁺ CD8⁺ population was analyzed by intracellular cytokine staining.

Fig. 4.

Faster activation, proliferation and prolonged expression of CD69 in Dicer^−/− CD8⁺ T cells following in vitro stimulation. Dicer^+/− and Dicer^−/− CD8⁺ T cells (CD45.2) were mixed in a 1:1 ratio with CD45.1 B6 CD8⁺ T cells, labeled with CFSE, and stimulated with αCD3/CD28. (A) Two days after stimulation, CD45.1 and CD45.2 viable cells were analyzed for CFSE dilution and CD69 expression. (B) After 2 d, cells were washed and transferred to medium containing the indicated cytokines. One day later, viable cells were analyzed as above.

Fig. 5.

Prolonged CD69 surface expression and defective migration of Dicer^−/− CD8⁺ T cells responding to infection in vivo. (A) YFP expression profiles of OT-1 T cells before transfer. (B) At day 3.5 postinfection, donor OT-1 cells in spleen and liver, gated as CD8⁺CD45.2⁺, were analyzed by flow cytometry. (C) Percentage of CD69⁺ cells among the OT-1 T-cell population is shown for various organs. (D) Percentage of YFP⁺ OT-1 cells among total lymphocyte population from each organ. Each point represents an individual mouse.

Fig. 6.

MicroR-130/301 regulates CD69 expression in activated CD8⁺ T cells. (A) Alignment of CD69 mRNA 3′UTR to show the conserved miR-130/301 binding site. (B) Expression of miR-130/301 in WT CD8⁺ T cells during in vitro activation. Purified WT CD8⁺ T cells were activated with αCD3/CD28 for 2 d, washed, and transferred to IL-2 for up to another 3 d. At the indicated time points, cells were harvested. RNA was extracted and subjected to real-time PCR analysis. (C) FACS profile of sorted FITC⁺ cells post culture, gated on 7-AAD⁻ live cells.