The immunomodulatory properties of the selected Lactobacillus bacteria were assessed by measuring the induction of innate and adaptive cytokine production, proliferation and cell death of unstimulated, polyclonal stimulated and allergen-specific stimulated hPBMC. The Lactobacillus strains studied showed an overall stimulating effect on IL-10, decreased prototypical Th2 cytokines and differentially stimulated signature Th1 cytokine induction. Blood was collected from five birch pollen-allergic patients, two grass pollen-allergic patients

and one adult healthy control. All birch- and grass-allergic patients reported having rhinoconjunctivitis during the birch or grass pollen season, respectively, and had serum-specific IgE to birch

or grass pollen Caspase inhibitor of at least class 4 (except for one person who had class 3), measured by ImmunoCAP NVP-LDE225 (Phadia AB, Uppsala, Sweden). The healthy donor displayed no birch or grass pollen-specific IgE in his sera (<0.35 kU L−1/class 0). Blood was obtained outside the pollen season in September, and none of the patients showed allergic symptoms at the time of investigation. Furthermore, none of the patients had received allergen-specific immunotherapy or used antihistamines or corticosteroids in the month before the blood drawing. All participants gave their informed consent and the performed experiments were approved by the local ethical committee (Commissie Mensgebonden Onderzoek, regio Wageningen). Six Lactobacillus strains (Table 1) of the species Lactobacillus acidophilus, Lactobacillus plantarum and Lactobacillus fermentum were selected from our culture collection on the basis of high survival rates under conditions of low pH and/or the presence of bile, isolation from gastrointestinal tract, or were strains from species that are among the predominant Lactobacillus populations in the human gut. Further selection, including 70 strains, was based on IL-10-inducing capacities in 24-h hPBMC cultures of a healthy donor according to standardized procedures in our laboratories. Furthermore,

a mixture of strains B2261 and B633 was included, further referred to as a mixture of B2261 and B633. The choice for this mixture was based on combining the highest IL-10-inducing strain (B633) and the highest GPX6 IL-12-inducing strain (B2261), of the 70 strains included in this initial screening. Strains were cultured for 24 h at 37 °C in Man Rogosa Sharpe (MRS) broth (Merck, Darmstadt, Germany), after which fresh broth was inoculated with 1% (v/v) overnight culture. After an additional 24 h of incubation at 37 °C, bacterial cells were harvested by centrifugation at 1000 g, washed twice with phosphate-buffered saline (PBS), and resuspended in PBS. The bacterial cell numbers were determined by plate counting on MRS agar, and OD was measured at a wavelength of 600 nm.