CX3CR1⁺ LPCs fail to efficiently induce T cell proliferation in vitro and in vivo. (A) OT-I or OT-II cells were co-cultured with peptide-loaded CD103⁺ DC, CX3CR1^int LPC, or CX3CR1^high LPC at a ratio of 10:1 for 3 d. Proliferation was determined by thymidine incorporation assay and is depicted as a percentage of the mean T cell proliferation in CD103⁺ DC co-cultures (thymidine index). Results are the mean (SEM) of three or more independent experiments using pooled cells from four to five mice per experiment. Data were analyzed using one sample Student's t test, compared with CD103⁺ DC. Co-cultures of LPC/T cells at a 1:2 ratio showed a similarly inefficient induction of T cell proliferation compared with the 1:10 ratio (not depicted). *, P < 0.05; **, P < 0.01; ***, P < 0.001. (B) 500 µg of unlabeled or Cy5-labeled OVA was injected into the gut lumen of anaesthetized CX3CR1^+/GFP mice. 1 h later, mice were sacrificed and Cy5 fluorescence in CD103⁺ DC and CX3CR1⁺ LPC was determined by flow cytometry. Dot plots are gated on CD103⁺ DC or MHCII⁺CX3CR1⁺ LPC as indicated. Gates were adjusted to contain 0.1% of positive cells in controls (injected with unlabeled OVA) and numbers indicate the proportion of Cy5-positive cells. Data are representative of five mice analyzed in three independent experiments. CD103⁺ DC and CX3CR1^high LPC from OVA-Cy5–injected mice were sorted 1 h after injection and co-cultured with OT-II T cells for 3 d at a ratio of 1:10. Cells were pulsed with 1 µCi [³H]thymidine 18 h before collection, and thymidine incorporation was quantified in a β counter. Bars represent pooled data obtained in two independent experiments. Error bars indicate SEM. Results were obtained with a one-sample Student's t test. ***, P < 0.001. (C) 10⁷ CFSE-labeled DO11.10 cells were adoptively transferred to BALB/c recipients. 1 d later, 5,000 peptide-loaded CD103⁺ DCs or CX3CR1⁺ LPCs were injected into the MLN-afferent lymphatics. Proliferating antigen-specific T cells were detected in the MLN 3 d after injection. Plots are gated on live CD4⁺ cells. Control mice received 20 mg OVA by gavage. Results are representative of two independent experiments.