Inducible RNAi in vivo reveals that the transcription factor BATF is required to initiate but not maintain CD8+ T-cell effector differentiation

Abstract

The differentiation of effector CD8(+) T cells is critical for the development of protective responses to pathogens and for effective vaccines. In the first few hours after activation, naive CD8(+) T cells initiate a transcriptional program that leads to the formation of effector and memory T cells, but the regulation of this process is poorly understood. Investigating the role of specific transcription factors (TFs) in determining CD8(+) effector T-cell fate by gene knockdown with RNAi is challenging because naive T cells are refractory to transduction with viral vectors without extensive ex vivo stimulation, which obscures the earliest events in effector differentiation. To overcome this obstacle, we developed a novel strategy to test the function of genes in naive CD8(+) T cells in vivo by creating bone marrow chimera from hematopoietic progenitors transduced with an inducible shRNA construct. Following hematopoietic reconstitution, this approach allowed inducible in vivo gene knockdown in any cell type that developed from this transduced progenitor pool. We demonstrated that lentivirus-transduced progenitor cells could reconstitute normal hematopoiesis and develop into naive CD8(+) T cells that were indistinguishable from wild-type naive T cells. This experimental system enabled induction of efficient gene knockdown in vivo without subsequent manipulation. We applied this strategy to show that the TF BATF is essential for initial commitment of naive CD8(+) T cells to effector development but becomes dispensable by 72h. This approach makes possible the study of gene function in vivo in unperturbed cells of hematopoietic origin that are refractory to viral transduction.

Keywords: BATF; CD8 T cell; RNAi; transcription factor.

Fig. 1.

Transduced bone marrow progenitor populations efficiently reconstitute myeloid and lymphoid compartments and develop normally into functional CD8⁺ T cells. (A) Schematic diagram of transduction strategy. (B) Fraction of LSK cells (Left) transduced with GFP-expressing lentivirus at the time of transplant, and in lymphoid (Middle) and myeloid (Right) cell populations following engraftment. (C) Quantitation of fractions of transduced (GFP⁺) and untransduced (GFP^–), donor-derived T cells following engraftment as in B. (D) Fraction of transduced (GFP⁺) and untransduced (GFP^–), donor-derived naive P14 CD8⁺ T cells (Left) before adoptive transfer, and of effector (Right) P14 CD8⁺ T cells in tissues indicated 10 d following transfer and subsequent host infection with PR8-GP33 influenza virus. Representative data are shown from two to three experiments with four to five mice per group.

Fig. 2.

CD8⁺ T cells derived from transduced LSK cells are indistinguishable from untransduced naive CD8⁺ T cells. Expression of (A) naive surface markers, (B) cytokine receptors, and (C) Ki-67 by wild-type P14 CD8⁺ T cells (black); P14 CD8⁺ T cells derived from transduced LSKs (red); P14 CD8⁺ T cells stimulated by LCMV infection (green); by the cytokines IL-7 and IL-15 (blue); or by anti-CD3 and anti-CD28 antibodies (orange). (D) Relative transcript abundance of transcriptional regulators and effector molecules changes measured by quantitative RT-PCR in examined groups.

Fig. 3.

Novel shRNA vector enables efficient, inducible, and transient gene knockdown in vitro and in vivo. (A) Fraction of GFP-expressing Jurkat cells transduced with lentivirus expressing an shRNA targeting GFP under constitutive (white symbols) or inducible (black) promoters, cultured with 100 μM IPTG (gray box) for times indicated. BATF transcript levels (B) in anti-CD3/CD28-stimulated shBATF-naive CD8⁺ T cells cultured in vitro with (dark gray or black bars) or without (light gray) IPTG starting at the day indicated. Cells were continuously exposed to IPTG by in vivo exposure in bone marrow chimeric mice 3 d before T-cell sort (d −3) or 1 d following activation (d +1) and for the remainder of the experiment. (C) BATF protein abundance in anti-CD3/CD28-stimulated wild-type and shBATF–naive CD8⁺ T cells exposed to IPTG d −3 or incubated in medium alone. Numbers represent BATF densiometry values normalized to β-actin in shBATF relative to the wild-type cells. (D) Cells treated as in B were transferred into recipient mice that were also infected with LCMV and IPTG exposure was maintained by treating mice with 20 mM IPTG in drinking water starting 3 d prior to transfer (in bone marrow chimeras) or 1 d following transfer until 3 d following transfer. Batf mRNA level was normalized to Hprt and 2^-ΔCt values reported. Significance was assessed with one-way ANOVA; *P < 0.05, ***P < 0.001, ****P < 0.0001. Representative data are shown from two experiments.

Fig. 4.

BATF knockdown in vivo in primary CD8⁺ T cells impairs effector differentiation. (A) Relative fraction of P14 shBATF– (solid gates/lines) or shRFP– (dotted gates/lines) CD8⁺ T cells at the time of transfer or at d 7 p.i. with LCMV Armstrong in IPTG-treated animals (from d −3 on). Representative plots (Left and Middle) from a single animal and summary data from five mice (Right). (B) Ratio of P14 shBATF– or control shLacZ–effector CD8⁺ T cells to shRFP–effector CD8⁺ T cells at d 8 p.i. with IPTG exposure. Ratio at d 8 p.i. was normalized to ratio at d 0 and shown for three different shRNAs targeting BATF. (C) Apoptosis of shBATF– or shLacZ–effector CD8⁺ T cells measured by active caspase staining (FLICA) at d 5 p.i. Representative plots (Left) and summary data (Right). Significance was assessed with Student’s t test; **P < 0.01, ****P < 0.0001. Representative data are shown from three (A and B) or two (C) experiments with three to five mice per group.

Fig. 5.

BATF is required to initiate but not maintain effector differentiation. (A) Schematic diagram of timing of IPTG administration. (B) BATF transcript abundance in shBATF– and shRFP–effector CD8⁺ T cells on d 8 p.i. as described in A. (C) Ratio of shBATF– or shLacZ–effector CD8⁺ T cells to shRFP–effector CD8⁺ T cells at d 8 p.i. with continuous IPTG exposure initiated at the times indicated. Day 8 p.i. ratios were normalized to the d 0 ratio and Log2 transformed. Significance was assessed with one-way ANOVA; **P < 0.01, ***P < 0.001, ****P < 0.0001. Representative data are shown from three experiments with three to five mice per group.