Evaluation of Cross-presentation in Bone Marrow-derived Dendritic Cells in vitro and Splenic Dendritic Cells ex vivo Using Antigen-coated Beads

Abstract

Antigen presentation by MHC class I molecules, also referred to as cross-presentation, elicits cytotoxic immune responses. In particular, dendritic cells (DC) are the most proficient cross-presenting cells, since they have developed unique means to control phagocytic and degradative pathways. This protocol allows the evaluation of antigen cross-presentation both in vitro (by using bone marrow-derived DC) and ex vivo (by purifying CD8⁺ DC from spleen after incorporation of particulate antigen) using ovalbumin (OVA)-coupled particles. Cross-presentation efficiency is measured by three different readouts: the B3Z hybridoma T cell line (Karttunen et al., 1992) and stimulation of antigen-specific CD8⁺ T cells (OT-I) (Kurts et al., 1996), either analyzing OT-I activation by CD69 expression or OT-I proliferation after labeling them with carboxyfluorescein succinimidyl ester (CFSE). By using this approach, we could show recently that DCs are able to increase cross-presentation efficiency transiently upon engagement of TLR4 (Alloatti et al., 2015).

Figure 1. Scheme of an average 96-well plate loaded with BMDCs.

Plate 100,000 BMDCs/well in 50 μl of BMDC culture medium for B3Z readout, or 10,000 cells/well in 50 μl of BMDC culture medium for OT-I readouts. Add the antigen to the right concentration in 50 μl of BMDC culture medium for a final volume of 100 μl. In the scheme, sOVA and SIINFEKL peptide are also analyzed (see Notes 2 and 3). In the scheme, a minimum setup is shown: two different BMDC samples are analyzed, which are plated in duplicates. Additional replicates are suggested to increase robustness of the obtained data.

Figure 2. Gating strategy for cross-presentation analysis.

a. Activation of OT-I T cells by measuring CD69 expression at the cell surface. BMDC were incubated 5 h with bbOVA (upper panel) and sOVA (see Note 2) (lower panel), fixed and co-cultured with OT-I T cells. After 16 h, CD69 expression was measured. b. Proliferation of OT-I T cells. BMDC were incubated for 1 h in the presence of bbOVA (upper panel) and sOVA (lower panel) and co-cultured for 72 h with CFSE-labeled OT-I T cells. Proliferation was addressed by measuring loss of CFSE staining by flow cytometry and by calculating the proliferation index (right panel).

Figure 3. Workflow of the antigen cross-presentation approach in vivo

Figure 4. Gating strategy to sort CD8⁺ DC from spleen.

Total splenic cells were gated in order to exclude B, T and dead cells. Subsequently, gates were performed on CD11c⁺ and CD8⁺ cells. CD11c⁺ CD8⁺ cells that have phagocytosed at least one bead were sorted and co-cultured with OT-I T cells.