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. 2017 Jun:179:8-16.
doi: 10.1016/j.clim.2017.01.016. Epub 2017 Feb 20.

Therapeutic antibody targeting of indoleamine-2,3-dioxygenase (IDO2) inhibits autoimmune arthritis

Lauren M F Merlo  1 , Samantha Grabler  1 , James B DuHadaway  1 , Elizabeth Pigott  1 , Kaylend Manley  1 , George C Prendergast  2 , Lisa D Laury-Kleintop  1 , Laura Mandik-Nayak  3
Affiliations

Therapeutic antibody targeting of indoleamine-2,3-dioxygenase (IDO2) inhibits autoimmune arthritis

Lauren M F Merlo et al. Clin Immunol. 2017 Jun.

Abstract

Rheumatoid arthritis (RA) is a debilitating inflammatory autoimmune disease with no known cure. Recently, we identified the immunomodulatory enzyme indoleamine-2,3-dioxygenase 2 (IDO2) as an essential mediator of autoreactive B and T cell responses driving RA. However, therapeutically targeting IDO2 has been challenging given the lack of small molecules that specifically inhibit IDO2 without also affecting the closely related IDO1. In this study, we develop a novel monoclonal antibody (mAb)-based approach to therapeutically target IDO2. Treatment with IDO2-specific mAb alleviated arthritis in two independent preclinical arthritis models, reducing autoreactive T and B cell activation and recapitulating the strong anti-arthritic effect of genetic IDO2 deficiency. Mechanistic investigations identified FcγRIIb as necessary for mAb internalization, allowing targeting of an intracellular antigen traditionally considered inaccessible to mAb therapy. Taken together, our results offer preclinical proof of concept for antibody-mediated targeting of IDO2 as a new therapeutic strategy to treat RA and other autoantibody-mediated diseases.

Keywords: Antibody therapy; B cells; Experimental arthritis; IDO; Murine model.

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Figures

Figure 1
Figure 1. Monoclonal antibody specifically recognizes IDO2
Liver protein lysates (10μg) from IDO1 ko, IDO2 ko, or wt C57BL/6 mice were immunoblotted with monoclonal IDO2 Ig (clone 4–3.8) and detected with anti-mouse Igκ-HRP. Blots were then probed with anti-αtubulin, followed by anti-rabbit-HRP as a loading control. (A) Representative blot of 3 total. (B) Graph shows the mean ratio of IDO2 / αtubulin ± SEM for n=3 blots. P-values were calculated by one way-ANOVA followed by comparison of means with Tukey's post-hoc multiple comparison correction. * p < 0.05, n.s. = not significant
Figure 2
Figure 2. IDO2 Ig inhibits joint inflammation in KRN.g7 mice
(A,B) KRN.g7 mice were injected once with 0.5mg control mouse Ig or IDO2 Ig before (21 days of age) or after (28 days of age) the onset of arthritis. (C) IDO1 ko or (D) IDO2 ko KRN.g7 mice were injected once with 0.5 mg control mouse Ig or IDO2 Ig at 21 days of age. (A,C,D) Rear ankles were measured as an indication of arthritis and represented as mean ankle thickness ± SEM for n=9 KRN.g7, n=10 IDO1 ko KRN.g7, and n=8 IDO2 ko KRN.g7 mice per treatment group. (B) Metatarsal joint from KRN.g7 mice treated with control Ig, IDO2 Ig (pre-arthritis), or IDO2 Ig (post-arthritis). Representative sections from a total of n=8 mice per treatment group. Scale bar = 100μm. (A) P-values were calculated by one way-ANOVA followed by comparison of means with Tukey's post-hoc multiple comparison correction. (C,D) P-values were calculated using a 2-tailed unpaired t-test. *** p < 0.001, n.s. = not significant
Figure 3
Figure 3. IDO2 Ig inhibits joint inflammation in second model of arthritis, CIA
CIA was induced in DBA/1J mice by immunization with collagen + CFA (day 0) and collagen + IFA (day 21, arrows). Mice were treated with (A,B) 0.5mg IDO2 Ig or mouse Ig (weekly) or (C) carrier or 400mg/kg 1MT (b.i.d) starting on day -1. (A,C) Arthritis is indicated as a clinical score (0–4 per paw for a maximum of 16 per mouse). n=18 mouse Ig, n=18 IDO2 Ig, n=8 carrier, and n=8 1MT-treated mice. P-values were calculated using a 2-tailed unpaired t-test. (B) Representative front and rear paws from CIA mice treated with mouse Ig or IDO2 Ig on day 49 post-immunization. Mouse Ig-treated mice developed robust arthritis (left panel), whereas IDO2 Ig-treated mice developed only mild arthritis (right panel) or no arthritis at all (middle panel). n=9 mice per group. *** p < 0.001, n.s. = not significant
Figure 4
Figure 4. IDO2 Ig alters autoreactive T and B cell differentiation
(A,B) Frequency of CD4+ T helper cell subpopulations were measured by flow cytometry by intracellular staining for the transcription factors T-bet (Th1), GATA-3 (Th2), RORγt (Th17), Bcl-6 (Tfh), and FoxP3 (Treg). Percentages are shown for CD4+ cells expressing (A) a single transcription factor or (B) co-expressing FoxP3 with a second effector transcription factor. Graphs show mean % ± SEM of each subpopulation out of total CD4+ T cells for n=9 mouse Ig, n=5 IDO2 Ig (pre-arthritis), and n=9 IDO2 Ig (post arthritis) treated KRN.g7 mice, pooled from 3 independent experiments. (C) Cells from the joint dLNs were cultured for 4h in PMA (50ng/ml) + ionomycin (500ng/ml) + 3μg/ml Brefeldin A. Intracellular IL-21 was measured by flow cytometry. Graph shows % IL-21+ cells ± SEM out of total CD4+ T cell populations from n=12 mouse Ig, n=9 IDO2 Ig (pre-arthritis), and n=9 IDO2 Ig (post arthritis) treated KRN.g7 mice, pooled from 4 independent experiments. P-values were calculated by one way-ANOVA followed by comparison of means with Tukey's post-hoc multiple comparison correction. (D) Number of anti-GPI ASCs was measured by ELISpot. Graph shows mean number anti-GPI ASCs ± SEM for n=15 control Ig, n=11 IDO2 Ig (pre-arthritis), and n=9 IDO2 Ig (post-arthritis). P-values were calculated with Kruskal-Wallis non-parametric ANOVA followed by comparison of means with Dunn's multiple comparison correction. * p< 0.05, ** p < 0.01, *** p < 0.001, n.s. not significant.
Figure 5
Figure 5. IDO2 Ig is internalized, but does not signal via FcγRIIb
(A) Purified wild-type or FcγRIIb knock-out (ko) B cells were cultured in vitro for 24h with anti-CD40 (2μg/ml) + IL-21 (100ng/ml). IDO2-Ig-PE or isotype control-PE was incubated with the cells for the last 2h of culture. The cells were then analyzed by flow cytometry; graph shows internalization of IDO2-Ig (red) overlaid isotype control Ig (gray). Representative plots or images from 3 independent experiments. (B,C) Purified B cells from wt or FcγRIIb ko B6 mice were stimulated for 15 min. with 10μg/ml anti-FcγRIIb (AT130–5), whole IgM, or IDO2 Ig and analyzed for pFcγRIIb by Western blot with αtubulin as a loading control. (B) Representative blot and (C) ratio of pFCγRIIb / αtubulin from 3 independent experiments. P-values were calculated by one way-ANOVA followed by comparison of means with Tukey's post-hoc multiple comparison correction. ** p< 0.01, *** p < 0.001, n.s. = not significant
Figure 6
Figure 6. FcγRIIb expression on B cells is required for IDO2 Ig activity in vivo
(A) Schematic of adoptive transfer strategy. 3.5 × 105 IDO2 ko KRN CD4+ T cells and 1×106 wt or FcγRIIb ko I-Ag7/b B cells were adoptively transferred into IDO2 ko TCRα ko IDO2 ko C57BL/6 (I-Ag7/b) hosts. The day before cell transfer, host mice were injected with 0.5mg IDO2 Ig or control mouse Ig. (B) Rear ankles were measured as an indication of arthritis and represented as mean ankle thickness ± SEM for n=11 mice that received FcγRIIb ko B cells and n=13 mice that received wt B cells per treatment group, pooled from 3 independent experiments. P-values were calculated by one way-ANOVA followed by comparison of means with Tukey's post-hoc multiple comparison correction. ** p < 0.01, n.s. not significant.
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