A GMCSF-neuroantigen fusion protein is a potent tolerogen in experimental autoimmune encephalomyelitis (EAE) that is associated with efficient targeting of neuroantigen to APC

Abstract

Cytokine-NAg fusion proteins represent an emerging platform for specific targeting of self-antigen to particular APC subsets as a means to achieve antigen-specific immunological tolerance. This study focused on cytokine-NAg fusion proteins that targeted NAg to myeloid APC. Fusion proteins contained GM-CSF or the soluble extracellular domain of M-CSF as the N-terminal domain and the encephalitogenic 69-87 peptide of MBP as the C-terminal domain. GMCSF-NAg and MCSF-NAg fusion proteins were approximately 1000-fold and 32-fold more potent than NAg in stimulating antigenic proliferation of MBP-specific T cells, respectively. The potentiated antigenic responses required cytokine-NAg covalent linkage and receptor-mediated uptake. That is, the respective cytokines did not potentiate antigenic responses when cytokine and NAg were added as separate molecules, and the potentiated responses were inhibited specifically by the respective free cytokine. Cytokine-dependent targeting of NAg was specific for particular subsets of APC. GMCSF-NAg and MCSF-NAg targeted NAg to DC and macrophages; conversely, IL4-NAg and IL2-NAg fusion proteins, respectively, induced an 1000-fold enhancement in NAg reactivity in the presence of B cell and T cell APC. GMCSF-NAg significantly attenuated severity of EAE when treatment was completed before encephalitogenic challenge or alternatively, when treatment was initiated after onset of EAE. MCSF-NAg also had significant tolerogenic activity, but GMCSF-NAg was substantially more efficacious as a tolerogen. Covalent GMCSF-NAg linkage was required for prevention and treatment of EAE. In conclusion, GMCSF-NAg was highly effective for targeting NAg to myeloid APC and was a potent, antigen-specific tolerogen in EAE.

Figure 1.

The cytokine domains of GMCSF-NAg and MCSF-NAg had full biological activity and supported differentiation of distinct APC subsets. (A) Lewis rat bone marrow cells (10⁵/well) were cultured with designated concentrations of purified fusion proteins. Cultures were pulsed with [³H]thymidine during the last day of a 3-day culture. These data are representative of three experiments. (B) Bone marrow cells were cultured with 50 nM GMCSF-NAg (left panels) or 50 nM MCSF-NAg (right panels) for 10 days. Surface markers of adherent APC were analyzed by flow cytometry. These data are representative of three experiments.

Figure 2.

The cytokine domains of GMCSF-NAg and MCSF-NAg strongly promoted reactivity to the covalently linked NAg by a mechanism that was blocked competitively and specifically by the respective cytokine. (A and B) Irradiated splenic APC and MBP-specific RsL.11 T cells were cultured with designated concentrations of GMCSF-NAg, MCSF-NAg, or NAg (x-axis) in the presence or absence of 100 nM GM-CSF or 100 nM M-CSF. (C–E) Irradiated, splenic APC and RsL.11 T cells were cultured with 100 nM GM-CSF, 100 nM M-CSF, or no cytokine for 4 h before addition of designated concentrations (x-axis) of GMCSF-NAg (C), MCSF-NAg (D), or NAg (E). (C and D) See the legend for E. Cultures were pulsed with [³H]thymidine during the last day of a 3-day culture. These data are representative of three experiments. (A) GMCSF-NAg versus NAg or versus NAg + GM-CSF (P<0.001 for 1 pM–10 nM). (B) MCSF-NAg versus NAg or versus NAg + M-CSF (P<0.001 for 100 pM–1 nM). (C) GM-CSF versus M-CSF or versus “no addition” (P<0.001 for 10 pM–10 nM). (D) M-CSF versus GM-CSF or versus “no addition” (P≤0.001 for 100 pM–1 nM). (E) No significant difference was noted among the three groups.

Figure 3.

The cytokine domains of GMCSF-NAg and MCSF-NAg efficiently targeted NAg to myeloid APC subsets. Bone marrow cells were cultured with M-CSF (A) or GM-CSF (B) baculovirus supernatants (0.1% v/v) for 7 days and were used as APC. Bone marrow-derived APC (15×10³/well) were cultured with RsL.11 T cells and 2.5 mM aminoguanidine in the presence of designated concentrations of purified fusion protein (x-axis). (C and D) Splenic OX33⁺ B cells (C) and OX33⁺Ig⁺ B cells (D) were sorted by FACS and were cultured (2.5×10⁴/well) with RsL.11 T cells and designated concentrations of fusion protein or NAg (x-axis). (E and F) R1T cells were cultured for 24 h in complete RPMI without IL-2 to allow clearance of IL-2 from cell surface receptors. R1T cells were then cultured with irradiated RsL.11 T responders in the presence of designated concentrations of fusion protein or NAg (x-axis) in the presence or absence of the anti-RT1B mAb (OX6; anti-I-A mAb). After 4 h, IL-2 (0.4% v/v IL-2 baculovirus supernatant) was added to all wells. This assay measured IL-2-dependent proliferation of R1T cells unless these T cell APC were killed upon antigen presentation to irradiated RsL.11 responders. Cultures were pulsed with [³H]thymidine during the last day of a 3-day culture. Not all fusion proteins were used in each experiment. These data are representative of three experiments.

Figure 4.

GMCSF-NAg was a potent tolerogen that prevented subsequent induction of EAE by a mechanism that required covalent linkage of GM-CSF and NAg. Rats were pretreated with 4 nmoles GMCSF-NAg, MCSF-NAg, NAg, a mixture of GM-CSF and NAg (GM-CSF+NAg; 4 nmoles each), and a mixture of M-CSF and NAg (M-CSF+NAg; 4 nmoles each) on Days –21, –14, and –7 before challenge, with 50 μg DHFR-NAg in CFA on Day 0. Rats were scored twice/day at approximate 12-h intervals, and weights were recorded daily. Shown are the time courses of clinical signs and weight loss for Experiment 1 of Table 1 (A and B, respectively) as well as the compiled time courses of clinical signs and weight loss for Experiments 2 and 3 (C and D, respectively) of Table 1. Analysis was performed with one-way or two-way ANOVA and Bonferroni post-hoc tests. (A and B) GMCSF-NAg versus NAg (EAE severity): P ≤ 0.037 for Days 12–14; (weight loss): P ≤ 0.035 for Days 11–28; MCSF-NAg versus NAg (EAE severity): P ≤ 0.017 for Days 10.5 and 12–13; (weight loss): P ≤ 0.042 for Days 15–22; GMCSF-NAg versus MCSF-NAg (weight loss): P ≤ 0.029 for Days 16–23. GMCSF-NAg versus MCSF-NAg (EAE severity): P = 0.029 for Day 14. (C and D) GMCSF-NAg versus GM-CSF + NAg (EAE severity): P ≤ 0.002 for Days 9–12; (weight loss): P ≤ 0.029 for Days 10–19; GMCSF-NAg versus MCSF-NAg (EAE severity): P ≤ 0.02 for Days 13.5 and 14.5; (weight loss): P ≤ 0.012 for Days 14–20; GMCSF-NAg versus M-CSF + NAg (EAE severity): P ≤ 0.003 for Days 9.5 and 11–13.5; (weight loss): P ≤ 0.012 for Days 12–21.

Figure 5.

GMCSF-NAg blocked the progression of EAE when treatment was initiated after disease onset. Rats were challenged with 50 μg DHFR-NAg in CFA on Day 0 (Experiments 1–3) and also with pertussis toxin (400 ng i.p.) on Days 0 and 1 (Experiment 3). On the day of initial treatment, rats were matched into treatment groups based on clinical signs of EAE as described in Table 2. Rats were treated with 1 nmole of the designated proteins on Days 9, 10, 12, and 14 (Experiment 1) and Days 10, 11, and 13 (Experiment 2) or 4 nmoles on Day 8 and 1 nmole on Day 11 (Experiment 3). Shown are the compiled time-course data for clinical EAE (A) and weight loss (B) for Experiments 1, 2, and 3 of Table 2. Analysis was performed with two-way ANOVA and Bonferroni post-hoc tests. GMCSF-NAg versus NAg (EAE severity): P ≤ 0.015 for Days 10–12.5; (weight loss): P ≤ 0.007 for Days 12, 13, and 15–18; GMCSF-NAg versus GMCSF + NAg (EAE severity): P ≤ 0.022 for Days 11–13.5; (weight loss): P ≤ 0.022 for Days 12, 13, and 15–18.