Fig. 5.

Potential signaling mechanisms that sculpt selectively attenuated CD69 activation in response to specific oscillatory signals. (A) optoCAR stimulation was perturbed in three ways: 1) inhibition of MEK in the Erk MAPK pathway using 50 nM PD0352901, 2) inhibition of calcineurin in the NFAT pathway using 10 μM cyclosporin A, and 3) gene KO of the regulatory phosphatase PTPN22. (B) Inhibition of NFAT reduced CD69 and Tim3 expression evenly at all oscillation periods, whereas inhibition of Erk reduced CD69 expression selectively on either side of the attenuation frequency. PTPN22 KO had a relatively minor impact on the induction of CD69 and Tim3 expression. n = 2 technical replicates, representative of three independent experiments and two donors. (C) A proposed qualitative model of temporal filtering. Oscillatory inputs (e.g., 7.5-min versus 30-min pulses) stimulate an upstream signaling module, here represented by ITAMs, Erk, and NFAT. Differential filtering downstream of Erk—for example, via differences in one or more of the individual rate constants (τ’_off, τ’’_off, τ’’’_off)—contributes to band-stop versus all-pass filtering profiles of CD69 and Tim3. The aggregate off-rate, τ_off,, is a function of the individual rate constants (τ_off = f(τ’_off ; τ’’_off ; τ’’’_off)). Filtering may also be impacted by Erk negative feedback. (D) A proposed model of in vivo temporal filtering. The timescale of T cell–SELF-presenting APCs is strikingly similar to the timescale of CD69 expression attenuation.