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. Author manuscript; available in PMC: 2015 Jun 17.
Published in final edited form as: J Immunol. 2009 Mar 15;182(6):3650–3659. doi: 10.4049/jimmunol.0801532

FIGURE 6.

FIGURE 6

In vivo transfer of Ag-presenting capacity to MHC I-deficient CD8α+ DCs by migratory MHC I+ monocyte-derived DCs. A, CD45.1+ monocyte-derived cells collected from the peritoneal lavage of thioglycolate-treated wild-type C57BL/6 mice were loaded with OVA Ag and injected i.c. into the scapular skin of β2m-deficient CD45.2+ mice. The appearance of the CD45.1+ monocyte-derived cells into the recipient brachial lymph nodes was monitored by flow cytometry, where a population of CD45.1+ cells was detected (right panel, gated box) in contrast to the same lymph nodes from β2m-deficient mice that did not receive CD45.1+ monocyte-derived cells (left panel). B, CD8α+ and CD8α− CD45.2+ β2m-deficient lymph node DCs were sorted from lymph nodes that either did not receive monocyte-derived cells (not shown) or received WT CD45.1+ monocyte-derived cells that were loaded or not loaded (not shown) ex vivo with OVA peptide. Dot plots show the gates that were used to sort after initially gating on CD45.2+ cells. DCs from these sorts were cocultured with OT-I CFSE-labeled T cells in vitro and then proliferation was monitored 68 h later. a and b are used to match gates for DCs that were sorted (left) and later cocultured with T cells for assessment of proliferation (right). C, A similar experimental design as in A was conducted except OVA-B16 was loaded with ssRNA and injected into the peritoneum as a source of Ag, instead of peptide loading as in A. CD8α+ β2m-deficient DCs (gate c) and DCs from the injected monocyte-derived cells recovered in the draining lymph nodes (gate d) were purified by flow cytometric cell sorting and then cocultured with OT-I CFSE-labeled T cells in vitro for 4 days (right panel). These samples (black profiles, gates c or d) were compared with wild-type spleen DCs loaded with soluble OVA, as a positive control (open profiles, +OVA), or to CD8α+ DCs from β2m-deficient mice sorted from nondraining mesenteric lymph nodes and then cultured with OT-1 T cells in the absence (gray line) or presence of OVA257–264 peptide (thin lines, +Pep).