Intradermal Delivery of Antigens Enhances Specific IgG and Diminishes IgE Production: Potential Use for Vaccination and Allergy Immunotherapy

<div><p>Skin is protected by a tough but flexible multilayered barrier and is a front line for immune responses against invading particles. For many years now, skin has been a tissue where certain vaccines are injected for the prevention of infectious disease, however, the detailed mechanisms of the skin immune response are not yet well understood. Using thin and small injection needles, we carefully injected OVA into a restricted region of mouse skin, i.e., intradermal (ID), and examined the antibody response in comparison with subcutaneous (SC) injection or epicutaneous patch administration of OVA. Epicutaneous patches induced a high IgE response against OVA, but IgG production was low. High IgG production was induced by both ID and SC injection, moreover, ID injection induced higher IgG production without any adjutants. Furthermore, OVA-specific IgE production was diminished by ID injection. We found that ID injection could efficiently stimulate skin resident DCs, drive Th1-biased conditions and diminish IgE production. The ID injection response was regulated by Langerin⁺ dermal DCs, because OVA was taken up mainly by these cells and, after transiently deleting them, the IgE response was no longer diminished and IgG1 production was enhanced. We also tested whether ID injection might be an effective allergy treatment by attempting to inhibit ongoing IgE production in mice with experimentally induced high serum IgE levels. Multiple ID injections of OVA were shown to prevent elevation of serum OVA-specific IgE after repeated allergen challenge. In contrast, SC OVA injection could only transiently inhibit the OVA-specific IgE production. These findings indicated that ID injection results in higher induction of antigen-specific IgG, and thus may be useful for vaccine delivery with little or no adjuvant components. Moreover, the observed diminishment of IgE and induction of Th1-biased immune responses suggest that ID may be a useful injection route for allergy immunotherapy.</p></div

The dominant DC subset that migrates into the draining LN depends on the route of immunization.

<p>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-II^high cells in each plot are indicated. (B and C) The percentage (B) and absolute number (C) of Alexa488⁺/MHC-II^high cells in draining LN cells from ID or SC injected mice. Each bar shows the mean ± SD of 10 mice per group. **<i>P</i> < 0.01 and ***<i>P</i> < 0.001. (D) Representative FACS plots showing cells gated on Alexa488⁺/MHC-II^high 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-II^high 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. *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001.</p

Langerin⁺ cells that migrate into the draining LN are efficiently decreased by DT injection in Langerin-DTR mice.

<p>(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-II^high 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-II^high 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. *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001.</p

Distinct distribution of injected fluid via intradermal or subcutaneous route.

<p>(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.</p

Langerin positive cells are required for diminishment of IgE production.

<p>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. *<i>P</i> < 0.05.</p

Intradermal antigen injection preferentially diminishes IgE antibody formation.

<p>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. *<i>P</i> < 0.05, ***<i>P</i> < 0.001.</p

Intradermal injection efficiently induces antigen-specific IgG production.

<p>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. *<i>P</i> < 0.05, ***<i>P</i> < 0.001.</p

Repetitive desensitization via the intradermal route inhibits antigen-specific IgE elevation and enhances antigen-specific IgG production for the long-term.

<p>(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.</p

Function of <i>i</i>NKT cell subtypes in the spleen.

<p>(A) Global gene expression profiles in <i>i</i>NKT subtypes in the thymus and spleen. Tree view representation of clustering analysis among the four <i>i</i>NKT subtypes in thymus and spleen from B6 and BALB/c. The values represent coefficients between the indicated panels. <i>r</i>²>0.95 in red, 0.85<<i>r</i>²<0.95 in orange, and <i>r</i>²<0.85 in blue. One representative experiment of three is shown. (B) Plasticity and stability of <i>i</i>NKT subtypes. The four <i>i</i>NKT cell subtypes in the thymus were sorted and each subtype (5×10⁵) was i.v. transferred into independent <i>Jα18</i>^−/− mice (<i>n</i> = 3). 10 d after transfer, α-GalCer/CD1d dimer⁺ TCRβ⁺ cells in spleen were analyzed by FACS for the expression of IL-17RB and CD4. Representative data from three experiments are shown. (C–F) In vitro cytokine production by splenic <i>i</i>NKT cell subtypes (red, CD4⁻ IL-17RB⁺; orange, CD4⁺ IL-17RB⁺; blue, CD4⁻ IL-17RB⁻; green, CD4⁺ IL-17RB⁻). Sorted splenic <i>i</i>NKT subtypes (5×10⁴ cells/100 µL) were co-cultured with BM-DCs (5×10³/100 µL) for 48 h in the presence of α-GalCer (100 ng/µL) (C), IL-12 (10 ng/µL) (D), IL-23 (10 ng/µL) (E), and IL-25 (10 ng/µL) (F). Levels of IFN-γ, IL-4, IL-9, IL-10, IL-13, IL-17A, and IL-22 in the supernatants were analyzed by ELISA or CBA. Data are mean ± SD of triplicate wells. One representative experiment of three is shown.</p

Involvement of IL-17RB⁺<i>i</i>NKT cells in the development of RSV-induced AHR.

<p>(A) Schematic showing the protocol for RSV-induced AHR. Mice were i.n. administered with RSV (10⁶ pfu) or PBS alone as a control 4 times at 10-day intervals. Mice were i.p. immunized with rec Gs/alum (50 µg/2 mg) 4 d after first RSV infections. Three days after the last RSV administration, mice were exposed i.n. to rec Gs and were measured 1 d later. (B) Development of RSV-induced AHR in BALB/c, but not in <i>Jα18</i>^−/− or <i>Il17rb</i>^−/− mice. Changes in R_L are depicted. The RSV-infected, rec Gs immunized, BALB/c mice had a greatly increased AHR compared to the other three groups. Results are expressed as the mean ± SEM. * <i>p</i><0.05 and ** <i>p</i><0.01. (C, D) Total and differential cell counts (C) and cytokines (D) in BAL fluid. BAL fluid was collected 24 h after challenge of the mice depicted in (B) with intranasal rec Gs. Results are expressed as the mean ± SEM. * <i>p</i><0.05 and ** <i>p</i><0.01. (E) Histological examination of lung tissues by H&E and PAS staining. RSV infected, rec Gs immunized, BALB/c, <i>Jα18</i>^−/− or <i>IL17rb</i>^−/−, mice were compared with control BALB/c mice (rec Gs alone). Bars indicate 100 µm. (F) AHR development after cell transfer of spleen IL-17RB⁺<i>i</i>NKT cells into <i>Jα18</i>^−/− mice. Indicated cell numbers of sorted IL-17RB⁺, IL-17RB⁻<i>i</i>NKT cells or total <i>i</i>NKT cells from spleen, or PBS control, were i.v. transferred into rec-Gs/alum-sensitized <i>Jα18</i>^−/− mice 24 h before RSV treatment (on the day 9, 19, and 29), and then challenged with rec Gs (24 h) and measurement of lung resistance (48 h). Each group of IL-17RB⁺<i>i</i>NKT cell-transferred mice was compared to other three groups. * <i>p</i><0.05, ** <i>p</i><0.01 calculated by Kruskal Wallis test. The results represent one out of four experiments with five mice in each group.</p