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. 2020 Sep 16;23(10):101570. doi: 10.1016/j.isci.2020.101570

Figure 2.

Figure 2

Nivolumab Binds PD-1 and Activates ERK to Suppress GABA Transmission in SDH Neurons

(A) Representative images showing localization of Pd1 mRNA by fluorescent in situ hybridization in the superficial SDH in WT (left) and Pd1−/− mice (right). Middle: higher magnification image of the dashed box. SDH neurons are labeled with Nissl staining. Note that Pd1 mRNA expression is lost in Pd1−/− mice (right).

(B) Left: representative currents induced by first GABA and second application of GABA (0.3 mM) in spinal lamina II neurons. Middle, same as left panel, but the second GABA application was combined with PD-L1 (30 ng/mL). Right: same as left panel, but the second GABA application was combined with PD-L1 and the SHP-1 inhibitor SSG (11 μM).

(C) Quantification of the amplitude of second GABA-induced currents (ratio of the first one) in vehicle/ACSF group (n = 8 neurons from 3 mice), PD-L1-treated group (n = 11 from 6 mice), and PD-L1- and SSG-treated group (n = 10 from 5 mice). (D–E) Nivolumab-induced pERK expression in spinal cord slices. (D) SDH immunostained for pERK following IgG or Nivo (300 ng/mL) incubation for 10 min (left) or 2 h (middle). (E) Quantification of the number of pERK-positive neurons in the SDH after IgG or Nivo incubation. n = 4 mice per group.

(F and G) Left: double staining of pERK and Nivolumab in SDH from WT mice (F) and Pd1−/− mice (G) after Nivo treatment (1000 ng/mL, 2 hr). Higher magnification images of box-1 and box-2 are indicated in the right panel in F, with each channel displayed separately.

(H and I) Effects of U0126 on GABA-induced currents in lamina II neurons of spinal cord slices. (H) Example traces of GABA (1 mM)-induced currents after 2 h incubation with IgG and vehicle, Nivo (300 ng/mL) and vehicle, IgG and U0126 (1 μM), and Nivo (300 ng/mL), and U0126 (1 μM). (I) Unit currents of GABA currents after incubation with IgG and vehicle (n = 6 from 4 mice), Nivo and vehicle (n = 11 from 5 mice), IgG and U0126 (n = 7 from 3 mice), or Nivo and U0126 (n = 8 from 3 mice). Gray arrowheads indicate the application of GABA (1 mM). VH = 0 mV.

One-way ANOVA followed by Bonferroni post-hoc test. Data displayed indicate the mean ± SEM.