Induction of Ovalbumin-Specific Tolerance by Oral Administration of Lactococcus lactis Secreting Ovalbumin

LL-OVA induces APC-mediated T-cell proliferation. L lactis-derived OVA is bioactive because it induces proliferation of DO11.10 CD4⁺ T cells after preincubation of BM-DC with LL-OVA. BM-derived DC were generated as described in the Materials and Methods section. Two × 10⁶ BM-DC were cultivated with 4 × 10⁵ CFU LL-OVA or LL-pT1NX, with 1 μg or 1 mg OVA per mL or with no additive (/). After 4 hours, bacteria were killed with gentamycin. Twenty hours later, BM-DC were harvested, and their proliferation was assayed using DO11.10 CD4⁺ T cells. Two × 10⁵ DO11.10 CD4⁺ T cells were incubated with 6.67 × 10⁴, 2.22 × 10⁴, 7.41 × 10³, and 2.47 × 10³ BM-DC, corresponding to 1/0.33, 1/0.11, and 1/0.01, respectively, CD4⁺ T cells/BM-DC. Given the considerable higher proliferation after stimulation with 1 mg/mL OVA, this proliferation is shown in a separate graph.

OVA is actively produced by LL-OVA in the intestine and is delivered to the small and large intestines. Balb/c mice (n = 4) received 10 serial inocula of 2 × 10⁹ CFU of LL-OVA in 100 μL suspension at intervals of 30 minutes. One hour after the final inoculation, live bacteria (Table 1) and OVA (Figure 2) were quantified in the entire small intestines, cecums, and colons, including their luminal contents (solid bar), and in the intestinal tissue (open bar), as described in the Materials and Methods section. Detection limit of the ELISA = 1.5 ng. Results are representative of 2 individual experiments.

Oral feeding of LL-OVA significantly reduces DTH responses. DO11.10 mice were sensitized by subcutaneous injection of 100 μg OVA in CFA on day 1. Mice were orally treated with 200 mg OVA on day 8, 1 mg OVA on days 1–5, 1 μg OVA at days 1–5 and 8–12, or with LL-OVA or LL-pT1NX on days 1–5 and 8–12. Control mice received BM9. On day 11, mice were challenged with 10 μg OVA in 10 μL saline in the auricle of 1 ear and with 10 μL saline in the other. DTH responses are expressed as the mean difference in ear swelling between the OVA-injected and the saline-injected ears. Results summarize data of 4 independent experiments including 6 mice per group per experiment.

OVA-specific increase in IL-10 production after restimulation of cervical lymph node, spleen, and GALT cells. IL-10 secretion of pooled cervical lymph node cells was determined in the supernatants 24, 48, and 72 hours after restimulation. Data are represented as means of IL-10 secretion in pg/mL of at least 2 separate experiments (A). IL-10 production by bulk spleen and GALT cells was measured after ex vivo restimulation. Mice were fed BM9, 1 μg OVA, LL-pT1NX, or LL-OVA as described in Figure 3, and DTH was determined. Thereafter, bulk cell populations were isolated, and 1 × 10⁵ cells were restimulated ex vivo with 0.5 mg/mL OVA (B). To determine antigen-specific IL-10 secretion, cells were restimulated with anti-CD3/anti-CD28 (C). Data summarize 3 independent experiments including 6 mice per experiment.

Feeding of LL-OVA or LL-pT1NX significantly reduces the OVA-specific proliferation and IFN-γ production of bulk splenocytes. DO11.10 mice were immunized by subcutaneous injection of 100 μg OVA in CFA on day 1. Mice were treated orally with BM9 (control), LL-OVA, or LL-pT1NX on days 1–5 and 8–12. On day 12, bulk splenocytes were isolated and tested for OVA-specific proliferation, which is expressed as the mean cpm at different OVA concentrations, and for IFN-γ production after 72-hour ex vivo stimulation with 33 μg/mL OVA (upper panel). Data represent at least 3 separate experiments, including 4 mice per group per experiment.

Feeding with LL-OVA reduces the OVA-specific proliferation of CD4⁺ splenic T cells. The reduction is mediated by CD4⁺CD25⁻ cells and TGF-β. DO11.10 mice were sensitized by subcutaneous injection of 100 μg OVA in CFA at day 1. They were treated orally with BM9, LL-OVA, or LL-pT1NX on days 1–5 and 8–12. On day 12, spleens were isolated and splenic CD4⁺ T, CD4⁺CD25^high, CD4⁺CD25^intermediate, and CD4⁺CD25⁻ T cells were isolated. OVA-specific proliferation of CD4⁺ and CD4⁺CD25⁻ T cells from all groups were assayed as described in the Materials and Methods section. Proliferative response of CD4⁺ and CD4⁺CD25⁻ T cells was studied while blocking TGF-β with anti-TGF-β neutralizing antibody (1 μg/mL). Proliferative responses are expressed as the proliferation relative to that of CD4⁺ T cells of the BM9 group (A). After 72 hours of culture, supernatants were collected from the proliferation assays and tested for IFN-γ production (B). Data represent at least 2 separate experiments. On day 12, isolated CD4⁺CD25⁻ T-cell subsets of the mice treated with BM9, 1 μg OVA, LL-pT1NX, or LL-OVA were intracellularly stained for Foxp3 and CTLA-4, and flow cytometry was performed (C).

Adoptive transfer of CD4⁺CD25⁻ T cells of LL-OVA-treated mice into sensitized Balb/c mice induces OVA-specific tolerance in vivo. Three CD4⁺CD25 subpopulations were sorted: CD4⁺CD25^high, CD4⁺CD25^intermediate, and CD4⁺CD25⁻. On day 7, 5 × 10⁴ cells of each subpopulation were adoptively transferred into Balb/c mice that had been sensitized by subcutaneous injection of 100 μg OVA in CFA on days 0 and 1. Five days later, DTH measurements of the ear were performed.