Fig 1.
Distinct distribution of injected fluid via intradermal or subcutaneous route.
(A) The ultrasound echographic images of injection site of skin were obtained immediately after 2 μl of India ink injected with 36G ID injection system for ID or 26G injection needle for SC. (B) Photomicrographs of skin histological sections. Immediately after injection via the ID or SC route, injected skin region was cut out and performed H&E staining. Lower panels are magnified view of boxed region in upper panels. Scale bar, 100 μm.
Fig 2.
Intradermal antigen injection preferentially diminishes IgE antibody formation.
BALB/c mice continuously received 6 times by intradermal (ID) or subcutaneous (SC) injection with 2 μg of OVA in 2 μl saline, or by patch and topical application (epicutaneous) with 200 μg of OVA in 200 μl saline. Blood samples were collected at 3 days before the first sensitization (day-3) and 18 days after the first sensitization (day 18). (A) Timeline for sensitization and blood sampling. (B–D) Concentrations of OVA-specific and total serum IgE (B), IgG1 (C) and IgG2a (D) were determined by ELISA. Each circle represents the concentration of individual 10 mice, and bar shows the mean ± SD. *P < 0.05, ***P < 0.001.
Fig 3.
Intradermal injection efficiently induces antigen-specific IgG production.
BALB/c mice continuously received 6 times by intradermal (ID) or subcutaneous (SC) injection with three different doses, either 0.2, 2 or 20 μg of OVA in 2 μl saline. Blood samples were collected at 3 days before the first sensitization (day-3) and 18 days after the first sensitization (day 18). (A) Timeline for injection and blood sampling. (B–D) Concentrations of OVA-specific serum IgE (B), IgG1 (C) and IgG2a (D) were determined by ELISA. Each circle represents the concentration of individual 7 mice, and bar shows the mean ± SD. *P < 0.05, ***P < 0.001.
Fig 4.
The dominant DC subset that migrates into the draining LN depends on the route of immunization.
BALB/c mice were injected with Alexa488 labeled OVA via the intradermal (ID) or subcutaneous (SC) route and draining LNs were harvested 24 hr after the injection. Analysis of Alexa488+ cells in draining LN was performed by flow cytometry. (A) Representative FACS plots of LN cells from ID or SC injected mice. The percentages of Alexa488+/MHC-IIhigh cells in each plot are indicated. (B and C) The percentage (B) and absolute number (C) of Alexa488+/MHC-IIhigh cells in draining LN cells from ID or SC injected mice. Each bar shows the mean ± SD of 10 mice per group. **P < 0.01 and ***P < 0.001. (D) Representative FACS plots showing cells gated on Alexa488+/MHC-IIhigh LN cells from ID or SC injected mice stained for the indicated markers. The percentages of CD301b+CD11c+ (CD301b+ dDC), EpCAM+CD11c+ (LC) and CD103+CD11c+ (CD103+ dDC) cells in each plot are indicated. (E and F) Each circle represents the percentage of each DC subset among Alexa488+/MHC-IIhigh cells (E) and total cell number of each DC subset in draining LN (F) of individual mice, and horizontal bar indicates the mean. The experiments were independently performed 18 times. *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 5.
Langerin+ cells that migrate into the draining LN are efficiently decreased by DT injection in Langerin-DTR mice.
(A) B6/J and Langerin-DTR (B6/J background) mice were administered DT via intraperitoneal injection one day before each OVA injection. Mice were injected with Alexa488 labeled OVA via the intradermal (ID) or subcutaneous (SC) route and draining LNs were harvested 24 hrs later. (A) Representative FACS plots showing cells gated on Alexa488+/MHC-IIhigh cells from ID or SC injected mice. The percentages of CD301b+CD11c+ (CD301b+ dDC), EpCAM+CD11c+ (LC) and CD103+CD11c+ (CD103+ dDC) cells in each plot are shown. (B and C) Each circle represents the percentage of each DC subset among Alexa488+/MHC-IIhigh cells (B) and total cell number of each DC subset in draining LN (C) of individual mice, and horizontal bar shows the mean. The experiments were independently performed 10 times. *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 6.
Langerin positive cells are required for diminishment of IgE production.
B6/J and Langerin-DTR mice continuously received 3 times of DT and OVA by intradermal (ID) or subcutaneous (SC) injection with 2 μg of OVA in 2 μl saline. Blood samples were collected at 3 days before the first sensitization (day -3) and 18 days after the first sensitization (day 18). (A) Timeline for injection and blood sampling. (B–D) Serum concentrations of OVA-specific IgE (E), IgG1 (F) and IgG2a (G) were determined by ELISA. Each bar shows the mean ± SD of 8 mice per group. *P < 0.05.
Fig 7.
Repetitive desensitization via the intradermal route inhibits antigen-specific IgE elevation and enhances antigen-specific IgG production for the long-term.
(A) Timeline for sensitization, desensitization and blood sampling. BALB/c mice were used in this experiment. (B–D) Serum concentrations of OVA-specific IgE (B), IgG1 (C) and IgG2a (D) were determined by ELISA. Data are the mean ± SD of 15 mice per group.