L tryptophan trp

Stock solutions of human serum albumin (HSA, Sigma-Aldrich, St. Louis, MO, USA), L-tyrosine (Tyr, Acros Organics, Morris Plains, NJ, USA), L-tryptophan (Trp, Sigma-Aldrich, USA) and glycyl-L-tryptophan (Gly-Trp, Sigma-Aldrich, USA) were prepared by dissolving weighed portions in a 100 mM phosphate-buffered solution with pH 7.4 (PBS, KH₂PO₄, Sigma-Aldrich, USA). A weighed portion of γ-globulins (Sigma-Aldrich, USA) was dissolved in 0.9% NaCl (solution for infusion, Asfarma Ltd., Moscow, Russia). A stock solution of uric acid (UA, Fluka, New York, NY, USA) was prepared in 32 mM KOH (Sigma-Aldrich, USA); the working solution was prepared by diluting the initial PBS with subsequent pH adjustment to 7.4.
Blood plasma was obtained from a healthy donor in a clinical laboratory on the day of the experiment. Blood (≈6 mL) was drawn into Li-heparin vacutainers by qualified personnel. The samples were centrifuged in test tubes at 1000× g for 10 min. During the day, the plasma was stored at 4 °C. The donor signed informed consent. The design was approved by the Ethics Committee of the Research Centre for Medical Genetics (Protocol #5, May 2019).

P. syringae strains were grown in NYG medium with Rif in overnight cultures. Cells were collected by centrifugation from each overnight culture, washed twice with 10 mM MgCl₂, re-suspended at a density of ~1 x 10⁷ cells mL^-1 in HS minimal media containing 10 mM citrate and incubated with shaking for 48 hrs at 28°C. The culture medium was supplemented with 0.25 mM L-Tryptophan (Trp, Sigma Aldrich, Cat No. T-0254), Indole-3-acetamide (IAM, Sigma Aldrich, Cat No. 286281), 3-Indoleacetonitrile (IAN, Sigma Aldrich, Cat No. 129453), Tryptamine hydrochloride (TAM, Sigma Aldrich, Cat No 246557) or Indole-3-acetaldehyde–sodium bisulfite addition compound (IAAld, Sigma Aldrich, Cat No. I1000), as indicated. One mL samples were taken at 24 and 48 hrs after incubation, centrifuged to pellet the cells and the resulting supernatants frozen in liquid nitrogen and stored at -80°C. Growth of cultures was monitored by reading the OD₆₀₀ at regular intervals with a spectrophotometer. The samples were prepared and analyzed for IAA production by LC-MS/MS as described in Supplemental Information (S1 Text).

Trp (l-tryptophan) and Kyn (l-kynurenine) were purchased from Sigma-Aldrich (St. Louis, MO, USA) at reagent grade > 98% (HPLC). A fresh stock solution, containing both Trp and Kyn at concentrations of 20 mM, was prepared in Milli-Q water the same day as a permeation experiment was initiated. The fresh stock solution was kept at − 20 °C for a maximum of 2 days and used to prepare calibration solutions for HPLC–UV analysis.
Methanol of HPLC gradient grade was obtained from VWR International (Fontenay-sous-Bois, France). Formic acid (> 99%) was purchased from Merck (Darmstadt, Germany). NaN₃, 30% HCl, and Na₂HPO₄·2H₂O were obtained from Sigma-Aldrich (St. Louis, MO, USA). KCl, NaCl, while KH₂PO₄ and NaH₂HPO₄·H₂O were purchased from Merck (Darmstadt, Germany). Phosphate buffered saline (PBS, pH = 7.4) was prepared from Milli-Q water with concentrations of 130.9 mM NaCl, 5.1 mM Na₂HPO₄·2H₂O, and 1.5 mM KH₂PO₄.

B cells were co-cultured in IMDM-10%FBS with a cocktail to mimic antigen and T cell help: 10 mg/ml F(ab)2 anti-IgM (Jackson, ImmunoResearch laboratories, Inc., West Grove, PA, USA), 10³ IU IL-2 (Proleukin, Prometheus laboratories Inc., San Diego, CA, USA), and 5 mg/ml anti-CD40 agonistic monoclonal antibody (Bioceros, Utrecht, The Netherlands). In some of the experiments, 200 µM tryptophan (TRP, l-tryptophan, Sigma-Aldrich) was added to the stimulation cocktail to counteract the activity of IDO.