*4.3. Isolation, Stimulation and Culture of CD4<sup>+</sup> T Cells*

Peripheral blood mononuclear cells (PBMC) were isolated from the buffy coat from healthy volunteers from the local blood drive by Histopaque®-1077 (Sigma-Aldrich, St. Louis, MO, USA) density gradient centrifugation. Then, CD4<sup>+</sup> T cells were isolated using CD4<sup>+</sup> Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the detailed protocol provided by the manufacturer. The purity of isolated cells was >90% as evaluated by flow cytometry analysis (see below). A representative flow cytometry analysis of CD4<sup>+</sup> T cell purity is shown in Supplementary Figure S1.

Isolated CD4<sup>+</sup> T cells were resuspended in RPMI 1640 culture medium supplemented with 10% Fetal Bovine Serum, 1% HEPES buffer and 1% Pen-Strep (all from Invitrogen, ThermoFisher Scientific, Waltham, MA, USA) and subjected or not to stimulation with Dynabeads™ Human T-Activator CD3/CD28 [27,28] at bead-to-cell ratio 1:1 and 30 U/mL rIL-2 (all from Invitrogen, TermoFisher Scientific) according to the protocol provided by the manufacturer. Then, 1 <sup>×</sup> <sup>10</sup><sup>6</sup> of unstimulated or stimulated cells were cultured for 5 days in the medium alone or in the medium with control or endometriotic PF at 1:1 ratio without medium refreshment in the wells of 12-well plates (Corning Inc., Corning, NY, USA) at 37 ◦C and 5% CO<sup>2</sup> atmosphere.

The following culture cell-free supernatants were collected and stored frozen at −70 ◦C until used for cytokine and chemokine quantification. The cells were also harvested, and their phenotype was evaluated by flow cytometry as described below.

Peritoneal fluids containing particular cytokines or chemokines at concentrations far exceeding interquartile range values were not used in the experiments. Baseline concentration ranges of each tested cytokines and chemokines present in control and endometriotic PF used in the experiments are given in the legends to Figures 2 and 3. As seen, these baseline concentrations of cytokines and chemokines were relatively very low compared to those found in the cell-free supernatants following CD4<sup>+</sup> T cell cultures and therefore may be considered as negligible.
