We demonstrate that antigen-specific gut-homing CD4+α4β7+ T cells that develop in response to oral immunization can be reprogrammed in LNs following cutaneous antigen exposure to migrate to the skin and elicit allergic skin inflammation.
BALB/c and C57BL/6 mice orally immunized with OVA and CT as adjuvant developed Th2-dominated allergic skin inflammation in response to cutaneous challenge with antigen, with increased epidermal thickening, dermal infiltration of CD4+
T cells and eosinophils, and increased expression of Th2 cytokines (Figures and ), all features of acute AD skin lesions. In contrast to C57BL/6 mice, BALB/c mice had no detectable upregulation of Ifng
mRNA expression in EC-challenged skin, despite a mixed antigen-specific Th1 and Th2 systemic response. This is consistent with our previous observation that Ifng
mRNA expression is upregulated in EC-sensitized skin of C57BL/6, but not BALB/c, mice despite a vigorous systemic mixed Th2 and Th1 response in both (19
). The factors that promote IFN-γ secretion by skin-infiltrating T cells in C57BL/6, but not in BALB/c, are not known. The less robust infiltration by eosinophils and CD4+
cells at sites of cutaneous challenge in orally immunized C57BL/6 controls compared with BALB/c mice also mirrors our previous findings in EC-sensitized mice of these 2 strains (21
Allergic skin inflammation did not develop at sites of cutaneous antigen challenge in orally immunized Ccr4–/–
mice (Figure ). This essential role for CCR4 is consistent with the notion that CCR4 is a nonredundant, necessary component of skin-specific lymphocyte trafficking, as demonstrated by the observation that in adoptive transfer experiments. only antigen-specific CCR4+
T cells, but not antigen-specific Ccr4–/–
T cells, home to the skin (17
Purified antigen-activated CD4+α4β7+ MLN T cells from BALB/c and C57BL/6 mice orally immunized with OVA, which expressed no detectable E-lig or CCR4 on their surface and no detectable Ccr4 mRNA, transferred the allergic inflammatory skin response to EC challenge (Figures and ). It is unlikely that contaminating CD4+CCR4+ cells would have accounted for the ability of CD4+α4β7+ cells to transfer allergic inflammation to WT recipients. The percentage of cells detected in the CCR4 gate in purified CD4+α4β7+ MLN T cells from WT mice was comparable to that detected in CD4+α4β7+ MLN T cells from Ccr4–/– mice, suggesting that the signal could be nonspecific and that very few, if any, CD4+CCR4+ cells contaminated the purified CD4+α4β7+ T cell populations. EC challenge with OVA of mice that had received no donor cells, or of mice that received CD4+α4β7+ T cells from donors orally immunized with saline, failed to elicit allergic skin inflammation. This makes it unlikely that recipient T cells were responsible for the observed allergic skin inflammation. Transfer of allergic skin inflammation by CD4+α4β7+ T cells was strictly dependent on their ability to express the skin-homing receptor CCR4 (Figure ), as CD4+α4β7+ T cells purified from MLNs of orally immunized Ccr4–/– mice failed to transfer allergic skin inflammation.
Purified α4β7–CCR4–E-lig– T cells directly isolated from orally immunized mice transferred allergic skin inflammation, albeit less vigorously than α4β7+ donor cells (Supplemental Figure 6). This suggests that both T effector memory and T central memory cells might have participated in driving allergic skin inflammation following cutaneous antigen challenge in orally sensitized mice.
We directly demonstrated that antigen-activated purified CD4+
T cells from OVA-TCR transgenic DO11.10 mice orally immunized with OVA acquire E-lig and CCR4 expression and migrate to sites of cutaneous antigen challenge in the recipients (Figure ). Donor T cells that expressed skin-homing receptors were highly enriched in antigen-challenged skin compared with DLNs. Since antigen-specific T cells that infiltrate cutaneous sites of antigen introduction do not proliferate locally (22
), their selective enrichment in antigen-challenged skin suggests that donor cells that may have acquired skin-homing receptors in DLNs selectively migrated to sites of cutaneous antigen challenge. It is possible that reprogramming of gut-homing T cells by skin-derived DCs may also occur in secondary lymphoid organs other than skin DLNs, because following skin painting with OVA-FITC, CD11c+
DCs were detected in MLNs and, to a lesser extent, in the spleen (Supplemental Figure 10). Reprogramming of gut-homing T cells by skin-derived DCs would be consistent with the observation that the ability of DCs to program T cell homing depends on their milieu (23
). The 2-fold increase in the accumulation of recipient-derived CD4+
cells that expressed skin-homing receptors in OVA-challenged skin is consistent with the previously demonstrated influx of non–antigen-specific T cells into inflamed skin (22
). Use of DC-depleted mice is needed to define the role of DCs in the reprogramming of T cells to express skin-homing receptor. While our adoptive transfer experiments clearly show that CD4+
MLN T cells can be reprogrammed to home to the skin following cutaneous antigen challenge, it is possible that in addition, antigen can be carried following oral immunization to peripheral LNs where it can induce antigen-specific T cells to express skin-homing receptors. This may contribute to the development of allergic skin inflammation in orally sensitized mice cutaneously challenged with antigen.
Our results establish a critical role of LNs in reprogramming gut-homing CD4+
T cells that developed in response to oral immunization to migrate to the skin. CD4+
OT-II cells failed to migrate to OVA-challenged skin of Lta–/–
recipients, which lack LNs (Figure ). The critical role of LNs in reprogramming homing of T cells is consistent with previous observations that a subset of antigen-specific T cells disseminate throughout lymphoid tissues and acquire additional tissue-homing phenotype according to their new lymphoid tissue microenvironment (25
). It is likely that skin DLNs were involved in redirecting CD4+
T cells to home to the skin, since DCs in these LNs present cutaneously introduced antigen to recirculating T cells and antigen-presenting DCs from PLNs upregulate E-lig and P-Lig expression on CD8+
T cells in vitro (12
). The weaker migration of CD4+
OT-II cells compared with CD4+
DO11.10 cells to OVA-challenged skin is likely due to genetic differences between C57BL/6 and BALB/c mice.
Vitamin D3 (vit-D3) has been described to program homing to the skin (27
). A role for vit-D3 in reprogramming α4
cells to home to the skin was supported by the observation that E-lig+
donor T cells isolated from DLNs of OVA-sensitized skin upregulated mRNA expression of the vit-D3–metabolizing enzymes 25-hydroxylase Cyp27a1
and 1-hydroxylase Cyp27b1
compared with E-lig–
cells isolated from the same DLNs. There was no detectable signal for Cyp2r1
in either E-lig+
cells (Supplemental Figure 11A). In addition, CD11c+
DCs isolated from skin DLNs of unsensitized mice expressed significantly higher levels of all 3 enzymes compared with CD11c+
DCs isolated from MLNs from the same mice (Supplemental Figure 11B). This is consistent with a role for DC-derived vit-D3 in reprogramming T cells to express skin-homing receptors. Furthermore, tape stripping significantly upregulated Cyp27a1
mRNA expression in CD11c+
DCs isolated from the skin 6 hours after mechanical injury, compared with CD11c+
DCs isolated from unmanipulated skin (Supplemental Figure 11C). This suggests that upregulation of vit-D3–metabolizing enzymes following mechanical injury, such as inflicted by scratching in AD patients, may contribute to the ability of DCs that carry cutaneously introduced antigens to program T cells to express skin-homing receptors.
The resetting of the migratory program of gut-homing antigen-specific T cells following cutaneous challenge with antigen demonstrated in this study may explain the flare-up of skin lesions following cutaneous contact with food allergens in orally sensitized patients with AD.