Figure 4.

No cytokine or cell surface marker induction was observed after either SpCas9 or SaCas9 stimulation

Peripheral blood mononuclear cells (PBMCs) were collected from both transplanted and non-transplanted animals and stimulated with SpCas9 or SaCas9 (10 μg/mL) and analyzed for (A) interferon-gamma (IFN-γ), interleukin-2 (IL-2), or tumor necrosis factor alpha (TNF-α) or (B) CD154 or CD137 positive CD3+ T cell frequencies. PMA/I (phorbol-12-myristate-13-acetate/ionomycin) was used as a positive control. No Stim, no stimulation. (C) PBMCs from two human and eight rhesus donors were left unstimulated (NC) or treated with phytohemagglutinin (PHA, 5 μg/mL) as a positive control or Cas9 antigens (20 μg/mL) and tested by IFN-γ ELISpot. ns, not significant, and ∗∗∗p < 0.001.