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. 2020 Feb 24;9:e52668. doi: 10.7554/eLife.52668

Figure 4. PD-1 inhibition increases secretion of multiple cytokines and growth factors.

PD-1/PD-L1 signalling was inhibited by Spartalizumab, a humanized IgG4 anti-PD1 monoclonal antibody, in Mtb-infected microspheres at 20 and 200µ/ml in normoxia. Supernatants were collected at day 14 and accumulation of cytokines and growth factors was analysed by Luminex 35-multiplex assay. Concentrations were normalized to secretion by Mtb infected microspheres to demonstrate relative fold change, and individual concentrations are shown in Figure 4—figure supplement 1. The experiment was performed twice with three replicates. Red font: **p<0.001 for Spartalizumab versus isotype control.

Figure 4.

Figure 4—figure supplement 1. Cytokine accumulation around microspheres after inhibition of PD-1/PD-L1 signalling with Spartalizumab, a humanized IgG4 anti-PD1 monoclonal antibody at 20 and 200 µg/ml in normoxia (N) and 1% hypoxia (H).

Figure 4—figure supplement 1.

Supernatants were collected at day 14 and a Luminex 35-multiplex assay was performed. The experiment was performed twice with three replicates. Concentrations are in pg/ml. Labels in red correspond to significantly raised cytokine values. Red font: Significance **p<0.001.
Figure 4—figure supplement 2. PD-1 inhibition increases secretion of multiple cytokines and growth factors in 1% hypoxia.

Figure 4—figure supplement 2.

Supernatants were collected at day 14 and accumulation of cytokines and growth factors was analysed by Luminex 35-multiplex assay. Concentrations were normalized to secretion by Mtb infected microspheres to demonstrate relative fold change, and individual concentrations are shown in Figure 4—figure supplement 1. The experiment was performed twice with three replicates. Red font: **p<0.001 for Spartalizumab versus Isotype control.
Figure 4—figure supplement 3. Spartalizumab induces TNF-α secretion in uninfected and infected microspheres, which is neutralised by anti-TNF-α.

Figure 4—figure supplement 3.

PBMCs which were uninfected or infected with Mtb were encapsulated in alginate-collagen matrix after pre- incubation in Spartalizumab at 200 μg/ml. The supernatant was collected to measure TNF-α secretion at day 7. (A) Spartalizumab induces TNF-α secretion in uninfected cells above background, which is accentuated with Mtb infection. (B) Anti-PD-1 antibody increases TNF-α secretion from Mtb infected cells above isotype, and the detectable levels are suppressed by anti-TNF antibody (50 μg/ml).