αDEC205/PLP ameliorates EAE induced by adoptive transfer of pathogenic PLP139–151-specific T cells. PLP139–151-specific T-cell lines were generated as described in Materials and Methodsand 5 × 10⁶ cells were adoptively transferred into naïve SJL/J mice i.v. into the tail veins. One day later, mice were immunized i.p. with either 1 μg of αDEC205/PLP mAb (n = 5) or GL117/PLP mAb (n = 5), and then PT (200 ng) was injected i.v. on day 3. Mice were monitored for 30 d. αDEC205/PLP-treated mice were protected, whereas the GL117/PLP-treated mice developed severe disease (P < 0.02 at 30 d).

Effect of preadministration of αDEC205/PLP on disease course. (A and B) EAE was induced in SJL/J mice by immunizing with PLP 139–151 with or without preadministration of 1 μg of fusion mAbs (αDEC205/PLP mAb, αDEC205/HA mAb, αDEC205 mAb, or GL117/PLP mAb) in sterile PBS on either (A) day minus 10 or (B) day minus 15. Mice were then immunized with 75 μg of PLP139–151 in CFA s.c. on day 0 followed by PT (200 ng) i.v. on day 1. Appearance of clinical signs of EAE was monitored daily, and disease severity was scored as described in Materials and Methods. Mean EAE scores for 5–10 mice in each group are shown. The majority of mice in groups immunized with PLP139–151 that had received αDEC205 mAb, αDEC205/HA mAb, or GL117/PLP mAb were dead by day 12 whereas disease was ameliorated in those that received αDEC205/PLP. (A) On day 15, αDEC205/PLP mAb (n = 5) vs. GL117/PLP mAb (n = 5) (P < 0.01). (B) On day 15, αDEC205/PLP mAb (n = 13) vs. GL117/PLP mAb (n = 9) (P < 0.001). All scoring was performed double blind. The data shown are representative of three to six separate experiments. (C) Effect of preimmunization with fusion mAbs together with MPLA. MPLA (10 μg) was administered together with either αDEC205/PLP mAb (n = 8) or GL117/PLP (n = 5) i.p. 10 d before induction of EAE with PLP139–151 in CFA s.c. and PT (200 ng) i.v. as above. Mice that received MPLA + αDEC205/PLP mAb were not significantly different from controls (P > 0.05). A representative of two independent experiments is shown. All scoring was performed double blind.

Effect of αDEC205/PLP on splenocyte proliferation and number of IL-17–producing cells. (A) SJL/J mice were preimmunized with 1 μg of different fusion antibodies. After 10 d, mice were immunized with PLP139–151 followed by i.v. PT as described in Fig. 1. Seventeen days after disease induction, splenocytes were removed and challenged with a titration of PLP139–151. On day 4 of the proliferation assay, cells were pulsed with ³[H]-thymidine; 16 h later, proliferative response was measured as cpm. (B) IL-17 ELISPOT analysis of mouse splenocytes isolated on day 17. Splenocytes were plated onto precoated plates as described in protocols from eBioscience’s IL-17 ELISPOT kit and stimulated with 10 μg/mL PLP139–151. Unstimulated wells were used as controls. A representative of two independent experiments is shown. (C) Quantification of IL-17 ELISPOT. Statistics: none vs. αDEC205/PLP mAb (P < 0.02); αDEC205/PLP mAb vs. GL117/PLP mAb (P < 0.006). Spots per million were calculated by multiplying the average of triplicate wells (2 × 10⁵ cells) by 5-fold.

Adoptive transfer (ATx) of CD4⁺ T cells from anti-DEC205/PLP139–151 mAb preimmunized mice ameliorates induction of PLP139–151-induced EAE. Two independent experiments are presented (A and B). (A) The 5 × 10⁶ CD4 + T cells enriched from splenocytes from SJL mice preimmunized on day 10 i.p. with a 1 μg fusion mAbs (αDEC205 /PLP mAb or GL117/PLP mAb) were adoptively transferred i.v. into the tail veins, followed by immunization on day 1 with 75 μg of PLP139–151 in CFA and PT i.v. the following day. Controls received PBS injections. Mean disease scores of five mice/group are shown. At days 20–21, αDEC205 /PLP mAb ATx vs. GL117/PLP mAb ATx were compared (P < 0.02). (B) An additional identical experiment is shown in which SJL mice were preimmunized at day 10 i.p. with 1 μg of αDEC205/PLP mAb only. Mice were monitored for clinical signs of EAE for 30 d. All scoring was performed double blind.

Effect of αDEC205/PLP on adoptively transferred CD4⁺ Vβ6⁺ TCR 5B6 transgenic (tg) T cells. Splenocytes were isolated from B10.S mice that carry a transgenic TCR 5B6 recognizing PLP139–151 presented on I-A^s. Splenocytes were enriched for Vβ6⁺ CD4⁺ tg T cells using Miltenyi CD4-positive selection kits (purity ∼89%). (A and B) The 10 × 10⁶ T cells were injected i.v. into naïve B10.S rag^−/− mice along with 1 μg i.p. of fusion antibodies (αDEC205/PLP mAb or GL117/PLP mAb). Splenocytes (A) and axillary lymph nodes (B) were removed 10 d later. Single cell suspensions were stimulated with PLP139–151 for 4 d, and ³H-thymidine incorporation was measured. The αDEC205/PLP mAb-treated Vβ6⁺ CD4⁺ tg T cells did not proliferate in response to PLP139–151 peptide, whereas Vβ6⁺ CD4⁺ tg T cells treated with GL117/PLP mAb proliferated (P < 0.03). (C) Vβ6⁺ TCR 5B6 tg CD4⁺ T cells were stimulated by cross-linking using plate-bound CD3 and CD28 mAb coated overnight to detect cytokine production. Supernatants from the proliferation assay were removed 3 d after stimulation, and cytokines were measured by Luminex assay as described in Materials and Methods. IL-17 was significantly reduced upon administration of 1 μg of αDEC205/PLP mAb compared with a control group treated with 1 μg of GL117/PLP mAb (P < 0.005). (D) Splenocytes used were obtained in A. FACS analysis of gated CD4⁺ cells stained for intracellular Foxp3 was carried out using CD4-FITC and Foxp3-PE.