Eye-mediated induction of specific immune tolerance to encephalitogenic antigens

Abstract

Aims: Administration of antigens into the anterior chamber (AC) of the eye induces a form of antigen-specific immune tolerance termed anterior chamber-associated immune deviation (ACAID). This immune tolerance effectively impairs host delayed-type hypersensitivity (DTH) responses. We hypothesized that ACAID could be generated in BALB/c mice following AC inoculation of the encephalitogenic antigens myelin oligodendrocyte glycoprotein (MOG) and myelin basic protein (MBP).

Methods: We used DTH assays and local adoptive transfer (LAT) assays to test whether MOG/MBP-induced ACAID following their administration into the AC, whether they elicited this immune tolerance via CD8(+) T cells, and whether their AC coadministration (MOG/MBP) induced specific immune tolerance to one or both antigens.

Results: We showed that MOG/MBP-induced AC-mediated specific immune tolerance, as evident from impaired DTH responses. This antigen-driven DTH suppression was solely mediated via splenic CD8(+) T cells as confirmed by LAT assays. Finally, a single AC injection with both antigens was sufficient to induce specific immune tolerance to these antigens, as evident from DTH and LAT assays.

Conclusion: ACAID T-cell regulation could be used as a therapeutic tool in the treatment of complicated autoimmune diseases that involve multiple antigens such as multiple sclerosis.

Figure 1

AC‐injected MOG or MBP inhibits DTH responses in Balb/c mice via induced peripheral tolerance. ACAID was induced in Balb/c mice via the AC injection of MOG or MBP (except for the control mice) followed by the s.c. immunization with MOG in CFA or MBP in CFA on day 7. On day 14, the mice were challenged with MOG or MBP (500 μg/20 μL) intradermally in the left ear pinna. 20‐μL PBS was injected into the right ear pinna as internal control. Positive control mice received the s.c. immunization with MOG/MBP in CFA on day 7 and were subsequently challenged intradermally with MOG or MBP on day 14. Negative control mice received only intradermal injections of MOG or MBP on day 14. Ear swelling responses were determined after 24 h (A) and 48 h (B) of the intradermal injection of MOG or MBP. P values < 0.05 were considered significant (*).

Figure 2

MOG/MBP coinjection via the AC of the eye in Balb/c mice inhibits MOG/MBP‐induced DTH responses. ACAID induction was performed by injecting Balb/c mice once with the MOG/MBP cocktail via the AC (except for the control mice) followed by the s.c. immunization with MOG/MBP in CFA on day 7. On day 14, the mice were challenged with MOG/MBP, MOG alone, or MBP alone (500 μg/20 μL) intradermally in the left ear pinna. 20‐μL PBS was injected into the right ear pinna as internal control. Positive control mice received the s.c. immunization with MOG/MBP in CFA on day 7 and were subsequently challenged intradermally with MOG/MBP, MOG, or MBP on day 14. Negative control mice received only intradermal injections of MOG/MBP, MOG, or MBP on day 14. Ear swelling responses were determined after 24 h (A) and 48 h (B) of the intradermal injection of MOG/MBP, MOG, or MBP. P values < 0.05 were considered significant (*).

Figure 3

In vivo‐generated putative regulatory spleen cells of mice that received a coinjection of MOG/MBP in the AC inhibit MOG/MBP‐induced DTH responses. On day 14, spleen cells were isolated from Balb/c mice that received a coinjection of MOG/MBP in the AC on day 0 and a s.c. immunization of MOG/MBP on day 7. These putative regulatory spleen cells were mixed with immune spleen cells (isolated from MOG/MBP subcutaneously immunized donors) and the MOG/MBP antigen cocktail. The suspension was injected into the left ear pinna of naïve mice to test for regulatory functions of the putative suppressor cells by monitoring the reduction in ear swelling responses after 24 (A) and 48 h (B). Two positive controls were included: The first included OVA‐specific regulatory spleen cells isolated from mice that received AC‐injected OVA mixed with immune spleen cells from MOG/MBP subcutaneously immunized mice and the MOG/MBP antigen cocktail. The second included naive spleen cells, immune spleen cells from MOG/MBP subcutaneously immunized mice, and the MOG/MBP antigen cocktail. Both controls were deficient in MOG/MBP‐specific regulatory splenocytes. A negative control group included naïve spleen cells and the MOG/MBP antigen cocktail injected into the ear pinna of naive mice. Another negative control involved the injection of the MOG/MBP antigen cocktail. P values < 0.05 were considered significant (*).

Figure 4

In vivo‐generated putative regulatory splenic T cells of mice that received a coinjection of MOG/MBP in the AC inhibit MOG/MBP‐induced DTH responses. On day 14, splenic T cells (CD90.2 microBeads) were isolated from Balb/c mice that received a coinjection of MOG/MBP in the AC on day 0 and a s.c. immunization of MOG/MBP on day 7. These putative regulatory splenic T cells were mixed with immune spleen cells (isolated from MOG/MBP subcutaneously immunized donors) and the MOG/MBP antigen cocktail. The suspension was injected into the left ear pinna of naïve mice to test for regulatory functions of the putative suppressor cells by monitoring the reduction in ear swelling responses after 24 (A) and 48 h (B). Two positive controls were included: The first included OVA‐specific regulatory spleen cells isolated from mice that received AC‐injected OVA mixed with immune spleen cells from MOG/MBP subcutaneously immunized mice and the MOG/MBP antigen cocktail. The second included naive spleen cells, immune spleen cells from MOG/MBP subcutaneously immunized mice, and the MOG/MBP antigen cocktail. A negative control group included naïve spleen cells and the MOG/MBP antigen cocktail injected into the ear pinna of naive mice. Another negative control involved the injection of the MOG/MBP antigen cocktail. P values < 0.05 were considered significant (*).

Figure 5

In vivo‐generated putative regulatory splenic CD8⁺ T cells of mice that received a coinjection of MOG/MBP in the AC inhibit MOG/MBP‐induced DTH responses. On day 14, splenic CD8⁺ T cells (CD8⁺ microBeads) were isolated from Balb/c mice that received a coinjection of MOG/MBP in the AC on day 0 and a s.c. immunization of MOG/MBP on day 7. These putative regulatory splenic CD8⁺ T cells were mixed with immune spleen cells (isolated from MOG/MBP subcutaneously immunized donors) and the MOG/MBP antigen cocktail. The suspension was injected into the left ear pinna of naïve mice to test for regulatory functions of the putative suppressor cells by monitoring the reduction in ear swelling responses after 24 (A) and 48 h (B). Two positive controls were included: The first included OVA‐specific regulatory spleen cells isolated from mice that received AC‐injected OVA mixed with immune spleen cells from MOG/MBP subcutaneously immunized mice and the MOG/MBP antigen cocktail. The second included naive spleen cells, immune spleen cells from MOG/MBP subcutaneously immunized mice, and the MOG/MBP antigen cocktail. A negative control group included naïve spleen cells and the MOG/MBP antigen cocktail injected into the ear pinna of naive mice. Another negative control involved the injection of the MOG/MBP antigen cocktail. P values < 0.05 were considered significant (*).