*2.5. Stimulation*

Isolated and collected splenocytes were seeded (4 × 105 cells per well) into 24 well culture plates (Nunc, Roskilde, Denmark) and stimulated in vitro with Concanavalin A (Con A), final concentration of 10 μg/mL (Sigma, Stockholm, Sweden) and PIPOx (final concentration of 5 mg/mL) for 24 h in a 37 ◦C incubator (5% CO2, humidified atmosphere). Following the exposition, the culture media were stored at −20 ◦C for determination of interleukins and growth factors.

Isolated splenocytes in complete RPMI-1640 (1 × 106 cells per ml) were seeded into 6-well culture plates (Nunc, Denmark). Afterwards, the cells were incubated for 1 h at 37 ◦C (humidified incubator, 5% CO2). Following this 1st incubation period, non-adherent cells were taken away and seeded into new 6-well culture plates. The adherent cells from the 1st incubation period were washed 3 times to take out all non-adherent cells and were adjusted with 500 μL of fresh complete RPMI-1640 medium. The first isolated non-adherent cells were allowed to adhere overnight in the complete RPMI-1640 medium at 37 ◦C, 5% CO2 in humidified incubator. Following this 2nd adherence period splenocytes depleted of adherent cells were collected and the adherent cells after 2nd adherence period were washed 3 times to eliminate any non-adherent cells.

The adherent cells (following the 1st and the 2nd incubation period) were harvested. Adherent cells obtained in this fashion after the 1st and the 2nd adherence period together with non-adherent cells were subjected to the immunocytometric analysis (FC500, Beckman Coulter, Fullerton, CA, USA) using Anti-Mouse CD3-FITC (clone KT3, Rat IgG2a), Anti-Mouse CD4-PE (clone YTS, Rat IgG2b), Anti-Mouse CD8α-PE (clone KT15, Rat IgG2a), Anti-Mouse CD11c-FITC (clone N418, Armenian Hamster IgG) (Antigenix America Inc., Melville, NY, USA), Anti-Mouse CD14-PE (clone rmC5-3 (RUO), Rat LOU/M IgG1, κ, BD Pharmingen). Phenotyping analysis of adherent cells after the 1st adherence period revealed enrichments in DCs and monocytes/macrophages with reduced proportion of lymphocytes (Table 1). Adherent cells obtained after the 2nd adherence period were more enriched in DCs with fewer monocytes/macrophages in comparison with enrichment after the 1st adherence period, as resulted from the immunocytometric assay of CD11c+ and CD14+ immunocytes.

The adherent and non-adherent cells from sequential adherent phases underwent in vitro stimulation with Con A and PIPOx in the same way as previous stimulation of splenocytes was conducted. Co-stimulation of adherent cells (1 × <sup>10</sup><sup>6</sup> cells/mL, adherent cells after the 1st and the 2nd adherence period, unstimulated or stimulated with Con A and PIPOx) with non-adherent splenocytes (1 × <sup>10</sup><sup>6</sup> cells/mL) was carried out by cocultivation in fresh complete RPMI-1640 medium for 4 days (37 ◦C humidified incubator, 5% CO2 atmosphere).

**Table 1.** Characterization of cell populations enriched by a sequential adherence method. Differentiation markers of cell populations: CD3+-T-cells, CD4+-helper T-cells (Th), CD8+-cytotoxic T-lymphocytes (CTLs), CD11c+-DCs, monocytes, granulocytes, CD14+-macrophages, monocytes.


*2.6. Cytokine Secretion Assay, Immunocytometry, and ELISA*

As previously described, washed and lysed splenocytes were adjusted by growth medium approximately to 4 × 107 cell/mL and 400 <sup>μ</sup>L aliquots were stimulated with PIPOx (concentration of 5 mg/mL) and plated in 24-well tissue culture plates (Nunc, Denmark). Polyclonal cell stimulation with mitogen Con A (10 μg/mL) was included into experiment as a positive control. 24-well tissue culture plates were allowed at 37 ◦C, 5% CO2 incubator for 24 h. Afterwards, the cell suspension was centrifuged at 800× *g* for 10 min at 4 ◦C and the cell pellet was resuspended in cold PBS pH 7.2 containing 0.5% bovine serum albumin and 2mM EDTA. The IL-10, IL-4, IFN-γ and IL-17 secretion assays were processed according to manufacturer's recommendation (MACS Cytokine Secretion Assay, Miltenyi Biotec, GmbH, Bergisch Gladbach, Germany). Counterstaining of CD4+ T-cells was performed by using rat anti-mouse CD4 FITC conjugate (Antigenix America). 10,000 viable cells were acquired by immunoflowcytometry using a Beckman Coulter FC 500 flow cytometer (Beckman Coulter Inc., Fullerton, CA, USA) equipped with a 488 nm argon laser and a 637 nm HeNe collinear laser, and controlled by CXP software [39]. A lymphocyte gate based on forward scatter vs. side scatter dot plot discrimination and settings was activated prior to further gating to exclude debris. The samples were assayed twice. The values are expressed as percentage of cytokine positive cells among CD4<sup>+</sup> cells ± SD. Quantitative detection of mouse IL-4, IL-10, IL-17 and IFN-γ cytokines in cell culture media supernatants following PIPOx and Con A specific stimulation of isolated splenocytes (see Section 2.5) was conducted by enzyme-linked immunosorbent assay Mouse Instant ELISA (Thermofisher Scientific, Waltham, MA, USA) according to manufacturer's recommendations. All samples were measured twice. The data are expressed as average ± SD.

#### *2.7. Phagocytosis*

Determination of cell phagocytosis, based on the ingestion of FITC-labeled *C. albicans* cells, was assayed under controlled conditions, following incubation with fluorescein isothiocyanate (FITC)-labeled *C. albicans* and RAW 264.7 macrophages for 30 min at 37 ◦C. Following treatment, the reaction was stopped by ice cooling the samples. The total amount of phagocyting cells, i.e., cells ingested at least one *C. albicans* cell was determined by immunocytometric assay, using a Beckman Coulter FC 500 flow cytometer (Beckman Coulter Inc., Fullerton, CA, USA).
