p53 Hinders CRISPR/Cas9-Mediated Targeted Gene Disruption in Memory CD8 T Cells In Vivo

Abstract

CRISPR/Cas9 technology has revolutionized rapid and reliable gene editing in cells. Although many cell types have been subjected to CRISPR/Cas9-mediated gene editing, there is no evidence of success in genetic alteration of Ag-experienced memory CD8 T cells. In this study, we show that CRISPR/Cas9-mediated gene editing in memory CD8 T cells precludes their proliferation after Ag re-encounter in vivo. This defect is mediated by the proapoptotic transcription factor p53, a sensor of DNA damage. Temporarily inhibiting p53 function offers a window of opportunity for the memory CD8 T cells to repair the DNA damage, facilitating robust recall responses on Ag re-encounter. We demonstrate this by functionally altering memory CD8 T cells using CRISPR/Cas9-mediated targeted gene disruption under the aegis of p53siRNA in the mouse model. Our approach thus adapts the CRISPR/Cas9 technology for memory CD8 T cells to undertake gene editing in vivo, for the first time, to our knowledge.

Figure 1.. CRISPR/Cas9-mediated p53 gene disruption enables expansion of transfected memory CD8 T cells in mice.

(A) Schematic depicting the experimental model used to generate genetically modified, congenically distinct, defined antigen-specific, primary memory CD8 T cells. Memory P14 or OT-IeGFP TCR transgenic (CD90.1⁺) CD8 T cells generated in donor (CD90.2⁺) mice (d90 post infection, p.i.) were selectively enriched, transfected by nucleofection with either plasmid encoding recombinant Cas9 (rCas9) and separate sgRNA or recombinant Cas9/sgRNA complexes and adoptively transferred immediately into recipient (CD90.2⁺) mice. The recipients were challenged with LCMV-Armstrong or Lm-Ova on d3 post-transfer (p.t.) of cells. Transferred (CD90.1⁺) CD8 T cells were assessed in blood by flow cytometry at d12p.t. (B) Representative flow-plots depicting frequencies of OT-IeGFP TCR transgenic (CD90.1⁺) CD8 T cells, transfected with rCas9 and the indicated sgRNAs before adoptive transfer to (CD90.2+) recipient mice and challenged with Lm-Ova, and examined at d9 p.i. Numbers inset represent the frequencies of the CD90.1 gated OT-I population presented as mean ± SEM from at least 2 independent experiments with ≥ 3 recipient mice per group. (C) Representative histograms of p53 expression levels in CD8 T cells co-transfected with rCas9 and ctrl sgRNA (grey) or p53sgRNA (red) and expanded in vivo as shown in 1A. Numbers inset represent the gMFI values of p53 expression. Data representative of 2 separate experiments.

Figure. 2.. CRISPR/Cas9 mediated ablation of eGFP expression in CD8 T cells.

(A) Schematic depicting the experimental model to ablate eGFP from primary OT-IeGFP TCR transgenic (CD90.1⁺) CD8 T cells. Memory OT-IeGFP TCR transgenic (CD90.1⁺) CD8 T cells generated in donor (CD90.2⁺) mice (d90 post infection, p.i.) were enriched, transfected with rCas9/sgRNA complexes by nucleofection and adoptively transferred immediately into recipient (CD90.2⁺) mice. The recipients were subsequently challenged with Lm-Ova on d3 post-transfer (p.t.) and the OT-I TCR transgenic (CD90.1⁺) CD8 T cells in circulation examined by flow cytometry on d7 and d530 p.i. The OT-I TCR transgenic (CD90.1⁺) CD8 T cells were subsequently adoptively transferred to secondary recipients (CD90.2+) on d530 p.i and challenged with Lm-Ova on d3 p.t. OT-I (CD90.1⁺) CD8 T cells in the secondary recipients were analyzed on d45 p.i. (B) Representative flow-plots depicting frequencies of OT-IeGFP TCR transgenic (CD90.1⁺) CD8 T cells and (C) frequencies of GFP expressing OT-I in groups transfected with the indicated reagents, in primary and secondary recipient B6 mice challenged with Lm-ova, analyzed at the indicated time-points. Data summarized as bar graphs in the right panel with each data point representing an individual sample, represents 1 of 3 independent experiments with ≥ 3 mice per group and analyzed with t-tests comparing the eGFPsgRNA and control transfected groups at each time point. *p ≤ 0.05, **p ≤ 0.01.

Figure. 3.. CRISPR/Cas9 mediated loss of IFNγ in murine primary memory CD8 T cells

(A) Representative flow plots displaying the frequencies of OT-I TCR transgenic (CD90.1⁺) CD8 T cells transfected with indicated reagents, in recipient B6 mice challenged with Lm-ova, observed at the indicated effector and memory time-points, as well as after adoptive-transfer to secondary recipients, similar to in Fig 2A. Data summarized as bar graphs in the right panel. (B) Representative flow plots showing IFNγ expression at the indicated time points in response to PMA/ionomycin stimulation, in the corresponding gated populations depicted in Fig 3A. Data summarized as bar graphs in the right panel with each data point representing an individual sample, represents one of three independent experiments (n > 3 mice per group) and analyzed with t-tests comparing the IFNgsgRNA or control transfected groups at each time point ** = p < 0.01.

Figure. 4.. CRISPR/Cas9 mediated loss of IFNγ in murine primary memory CD8 T cells limits the ability to control L. monocytogenes infection.

(A) Schematic depicting the experimental model for the challenge of IfngKO B6 recipient mice who received memory OT-I TCR transgenic (CD90.1⁺) CD8 T cells transfected with the indicated reagents. The IfngKO B6 recipient mice were challenged with virulent Lm-Ova the next day, sacrificed and organs harvested on 4 days later to determine control of bacterial infection. (B) Bacterial burden determined from the indicated groups by colony counts (top) and qPCR (bottom) for L. monocytogenes (hly gene) from the indicated organ homogenates. Dashed line indicates the limit of detection. Data representative of two independent experiments (n ≥ 3 mice per group) with each data point representing an individual sample, analyzed using one-way ANOVA comparing the indicated groups. ** = p < 0.01, n.s = p > 0.05