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. 2013:2013:345092.
doi: 10.1155/2013/345092. Epub 2013 Nov 26.

Antigen-specific gene therapy after immunisation reduces the severity of collagen-induced arthritis

Tove Eneljung  1 , Sara Tengvall  1 , Pernilla Jirholt  1 , Louise Henningsson  1 , Rikard Holmdahl  2 , Kenth Gustafsson  3 , Inger Gjertsson  1
Affiliations

Antigen-specific gene therapy after immunisation reduces the severity of collagen-induced arthritis

Tove Eneljung et al. Clin Dev Immunol. 2013.

Abstract

Reestablishment of tolerance induction in rheumatoid arthritis (RA) would be an optimal treatment with few, if any, side effects. However, to develop such a treatment further insights in the immunological mechanisms governing tolerance are needed. We have developed a model of antigen-specific tolerance in collagen type II (CII) induced arthritis (CIA) using lentivirus-based gene therapy. The immunodominant epitope of CII was inserted into a lentivirus vector to achieve expression on the MHC class II molecule and the lentiviral particles were subsequently intravenously injected at different time points during CIA. Injection of lentiviral particles in early phases of CIA, that is, at day 7 or day 26 after CII immunisation, partially prevented development of arthritis, decreased the serum levels of CII-specific IgG antibodies, and enhanced the suppressive function of CII-specific T regulatory cells. When lentiviral particles were injected during manifest arthritis, that is, at day 31 after CII immunisation, the severity of arthritis progression was ameliorated, the levels of CII-specific IgG antibodies decreased and the proportion of T regulatory cells increased. Thus, antigen-specific gene therapy is effective when administered throughout the inflammatory course of arthritis and offers a good model for investigation of the basic mechanisms during tolerance in CIA.

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Figures

Figure 1
Figure 1
Lentiviral gene constructs, design of experiments, integration, and histopathological scoring. (a) Lentiviral constructs: LNT-GFP and LNT-Ii-CII. LTR: long terminal repeat; cPPT: central polypurine tract; SFFV: spleen focus-forming virus promoter; GFP: green fluorescent peptide; WPRE: woodchuck posttranscriptional regulatory element; Ii: invariant chain; CII: collagen type II peptide amino acids 259–270. (b) Integration of lentiviral vectors analysed in splenocytes at termination of experiments after lentiviral treatment at day 7, 26, or 31 after immunisation. The number of lentiviral particles is expressed as integrated lentiviral particle per cell. Data were analysed by Mann-Whitney U test. Closed circles represent LNT-GFP, squares represents LNT-Ii-CLIP, and open circles represent LNT-Ii-CII treated mice. (c) Histopathological scoring in the knee joints at day 56 after CII immunization. Mice were injected with LNT-GFP at day 26. (I) Healthy joint; (II) synovitis and bone erosions grade 1; (III) synovitis grade 3 and bone and cartilage destruction grade 2; (IV) synovitis and bone and cartilage destruction grade 3. S = synovia, C = cartilage, B = bone, M = meniscus, and bone and cartilage destruction are marked with arrows. Scale bar, 100 μm.
Figure 2
Figure 2
Clinical and histopathological development of arthritis, serum levels of anti-CII IgG, and T regulatory cells after i.v. injection of lentiviral particles at day 7 after CII immunisation. (a) Design of experiments. Immunisation at day 0 with collagen type II (CII) in complete Freund's adjuvants (CFA), lentiviral injection with LNT-GFP or LNT-Ii-CII at day 7 after CII immunisation, booster at day 21 with CII in incomplete Freund's adjuvants (IFA), and termination of experiments at days 42–63. (b) The clinical frequency of arthritis. (c) The clinical severity of arthritis (LNT-GFP days 0–40 n = 24, days 42–52 n = 14, LNT-Ii-CII days 0–40 n = 20, and days 42–52 n = 10). (d) The severity of histopathological synovitis and bone-cartilage erosivity. (e) Serum levels of CII-specific IgG antibodies at days 42 and 52. (f) The frequency of CD4+CD25+Foxp3+  Tregs in spleen and lymph node at termination of experiment. Closed circles represent LNT-GFP and open circles represent LNT-Ii-CII treated mice. In Figure 2(b) the P value is calculated using logistic regression, in Figure 2(c) the P value is calculated using linear regression, and in Figures 2(d), 2(e), and 2(f) the P value is calculated using Mann Whitney U test. *P < 0.05 and **P < 0.01.
Figure 3
Figure 3
Clinical and histopathological development of arthritis, serum levels of anti-CII IgG, and T cell response after i.v. injection of lentiviral particles at day 26 after CII immunisation. (a) Design of experiments. Immunisation at day 0, lentiviral injection with LNT-GFP or LNT-Ii-CII at day 26 after CII immunisation, booster at day 21, and termination of experiments at days 54–63. (b) The clinical frequency of arthritis. (c) The clinical severity of arthritis (LNT-GFP days 0–43 n = 23, days 45–54 n = 22, days 56–63 n = 7, LNT-Ii-CII days 0–54 n = 22, and days 56–63 n = 7). (d) The severity of histopathological synovitis and bone-cartilage erosivity. (e) Histopathological findings in the knee joints at day 56 after CII immunization in a mouse injected with LNT-GFP (top panel) that shows synovitis and bone and cartilage destruction grade 2. The bottom panel shows the knee joint from an LNT-Ii-CII mouse with a nonarthritic knee joint at the same time point. S = synovia, C = cartilage, B = bone, M = meniscus, and bone and cartilage destruction are marked with arrows. Scale bar, 100 μm. (f) Serum levels of CII-specific IgG antibodies at days 45 and 56. (g) Suppressive capacity of T cells measured as levels of IFN-γ in supernatants from cocultures of T cell subsets and CD11c+ cells. The two first bars represent supernatants from cocultures containing CD4+CD25− and CD11c+ cells, the following two CD4+CD25+ cells and CD11c+ cells, and finally 4 bars represent a mix of CD4+CD25− and CD4+CD25+ in a ratio of 1 : 1 or 1 : 10 cocultured with CD11c+ cells. Closed circles represent LNT-GFP and open circles represent LNT-Ii-CII treated mice. In Figure 3(b) the P value is calculated using logistic regression, in Figure 3(c) the P value is calculated using linear regression, and in Figures 3(d) and 3(f) the P values are calculated using Mann Whitney U test. In Figure 3(g) black bars represent cells from LNT-GFP treated mice and white bars represent cells from LNT-Ii-CII treated mice. The P value is calculated using two-way ANOVA comparing the increasing IFN-γ levels from T cell cocultures from LNT-GFP mice in ratios from 1 : 1 to 1 : 10 with respect to IFN-γ levels from cells from LNT-Ii-CII mice in ratios from 1 : 1 to 1 : 10. *P < 0.05 and **P < 0.01.
Figure 4
Figure 4
Lentiviral treatment at day 31. (a) Design of experiment: Immunisation at day 0, booster at day 28, lentiviral injection with LNT-Ii-CLIP or LNT-Ii-CII at day 31 after CII immunization, and termination of experiments at day 54. LNT-Ii-CLIP construct, CLIP: class-II associated invariant chain peptide. (b) The clinical severity of arthritis (left y-axis) and levels of CII-specific antibodies (right x-axis). (c) The severity of histopathological synovitis and bone-cartilage erosivity (LNT-Ii-CLIP n = 5, LNT-Ii-CII days n = 6). (d) The gating strategy of CD4+CD25+ and subsequently Foxp3+ Helios+ cells. (e) The frequency of CD4+CD25+Foxp3+ Helios+  Tregs in blood at indicated time points after i.v. injection of LNT-Ii-CLIP or LNT-Ii-CII at day 31. The P values are calculated using Mann Whitney U test. *P < 0.05 and **P < 0.01.
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