Interleukin 4 (il 4)

RAW264.7 murine macrophage cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS and cultured in a 37 °C incubator with 5% CO₂. The cells were plated in 6-well plates (1 × 10⁶ cells/ml) and incubated with exosome-depleted DMEM conditioned media containing either 200 ng/μl LPS (Sigma-Aldrich, St. Louis, MO, USA), 40 ng/ml IL-4 (Sigma-Aldrich), 50 μg/ml T. pisiformis cysticercus-derived exosome-like vesicles, sterile PBS, or a combination with LPS + exosome-like vesicles, or IL-4 + exosome-like vesicles. All treatments were conducted in triplicate.

BV2, B6M7 and primary microglia were treated with LPS (100 ng/ml, E. coli, serotype 055: B5) + IFNγ (100 ng/ml) or IL-4 (20 ng/ml) (all from Sigma-Aldrich, Dorset, UK) for 24 h. The cells were termed as M(LPS + IFNγ) and M(IL-4) respectively. The supernatants were collected for cytokine/chemokine measurements (see below). Cells were collected for gene and protein expression studies.

Splenic B cells were cultured (10⁶ cells/ml) in RPMI (Euroclone) + 10% FCS (Euroclone) and 2-mercaptoethanol (50 mM; Life Technologies) for 72h with 20 μg/ml LPS (Sigma) ± 10 ng/ml IL-4 (Sigma) or for 48h with 10 μg/ml anti-CD40 (BD Pharmingen) ± 10 ng/ml IL-4 (Sigma). To perform fast stimulation 5x10⁶ B cells were resuspended in 400ul PBS without FCS and treated for 5 and 10 minutes with 10μg/ml anti-CD40 (BD Pharmingen) ± 10 ng/ml IL-4 or for 1, 5, 10 minutes with 10 μg/ml anti-Mouse IgM (Jackson Immuno Research); the incubation was performed in water bath at 37°C followed by the addiction of ice-cold PBS to stop the stimulation; cells were immediately pelleted and lysed. DLBCL cell lines OCI-Ly1, OCI-Ly18 and Pfeiffer were purchased from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Germany). OCI-Ly1 were maintained in IMDM (Invitrogen) + 20% FBS + 2-mercaptoethanol (50 mM); OCI-Ly18 in RPMI + 10% FBS + 2-mercaptoethanol (50 mM); Pfeiffer in RPMI + 10% FBS + Sodium Pyruvate 1 mM. The cells were maintained in an incubator at 37°C in a modified atmosphere with 5% CO2. Testing for Mycoplasma infection was carried out on a monthly basis. All procedures of handling were carried out under a sterile hood.

Normal donor monocytes were plated on day 1 in 6-well plates at 5 × 10⁶/ well in RPMI-10 FBS supplemented with 10 ng/ml IL-4 (Peprotech) and 800 IU/ml GM-CSF (Peprotech) and incubated at 37 °C overnight. On day 2, fresh media supplemented with 10 ng/ml IL-4 and 1600 IU/ml GM-CSF was added to the monocytes and incubated at 37 °C for another 48 h. On day 4, non-adherent cells were removed and immature dendritic cells washed and pulsed with 5 μM peptide in AIM-V-10 % FBS supplemented with 10 ng/ml IL-4, 800 IU/ml GM-CSF, 10 ng/ml LPS (Sigma-Aldrich), and 100 IU/ml IFN-γ (Peprotech) at 37°C overnight. Day 1 was repeated on days 4 and 8 to generate dendritic cells for the second and third stimulations on days 8 and 12, respectively. On day 5, normal donor-matched CD8+ T cells were co-cultured with the pulsed dendritic cells in AIM-V-10 % FBS. Day 5 protocol was repeated on day 8 and day 12 using dendritic cells generated on days 4 and 8 for the second and third stimulation, respectively.

Normal donor monocytes were plated on day 1 in 6-well plates at 5 × 10⁶ cells per well in RPMI-10 FBS supplemented with 10 ng ml⁻¹ IL-4 (Peprotech) and 800 IU ml⁻¹ GM-CSF (Peprotech) and incubated at 37 °C overnight. On day 2, fresh medium supplemented with 10 ng ml⁻¹ IL-4 and 1,600 IU ml⁻¹ GM-CSF was added to the monocytes and incubated at 37 °C for another 48 h. On day 4, non-adherent cells were removed, and immature dendritic cells washed and pulsed with 5 µM peptide in AIM-V-10% FBS supplemented with 10 ng ml⁻¹ IL-4, 800 IU ml⁻¹ GM-CSF, 10 ng ml⁻¹ lipopolysaccharide (Sigma-Aldrich), and 100 IU ml⁻¹ IFN-γ (Peprotech) at 37 °C overnight. Day 1 was repeated on days 4 and 8 to generate dendritic cells for the second and third stimulations on days 8 and 12, respectively.
On day 5, normal donor-matched CD8+ T cells were enriched using protein kinase inhibitor dasatinib (Sigma-Aldrich), dextramers, and anti-PE or anti-APC beads (Miltenyi Biotec) as previously described^{50 (link)}. Enriched T cells were co-incubated with the appropriate pulsed dendritic cells in AIM-V-10% FBS. The day 5 protocol was repeated on days 8 and 12 using dendritic cells generated on days 4 and 8 for the second and third stimulation, respectively. Expanded T cells were validated for antigen-specificity by staining with the appropriate dextramers and for activation marker 41BB/CD137 (BioLegend).

Normal donor monocytes were plated on day 1 in 6-well plates at 5 × 10⁶ cells per well in RPMI-10 FBS supplemented with 10 ng ml⁻¹ IL-4 (Peprotech) and 800 IU ml⁻¹ GM-CSF (Peprotech) and incubated at 37 °C overnight. On day 2, fresh medium supplemented with 10 ng ml⁻¹ IL-4 and 1,600 IU ml⁻¹ GM-CSF was added to the monocytes and incubated at 37 °C for another 48 h. On day 4, non-adherent cells were removed, and immature dendritic cells washed and pulsed with 5 µM peptide in AIM-V-10% FBS supplemented with 10 ng ml⁻¹ IL-4, 800 IU ml⁻¹ GM-CSF, 10 ng ml⁻¹ lipopolysaccharide (Sigma-Aldrich), and 100 IU ml⁻¹ IFN-γ (Peprotech) at 37 °C overnight. Day 1 was repeated on days 4 and 8 to generate dendritic cells for the second and third stimulations on days 8 and 12, respectively.
On day 5, normal donor-matched CD8+ T cells were enriched using protein kinase inhibitor dasatinib (Sigma-Aldrich), dextramers, and anti-PE or anti-APC beads (Miltenyi Biotec) as previously described^{50 (link)}. Enriched T cells were co-incubated with the appropriate pulsed dendritic cells in AIM-V-10% FBS. The day 5 protocol was repeated on days 8 and 12 using dendritic cells generated on days 4 and 8 for the second and third stimulation, respectively. Expanded T cells were validated for antigen-specificity by staining with the appropriate dextramers and for activation marker 41BB/CD137 (BioLegend).