ILDR2-Fc Is a Novel Regulator of Immune Homeostasis and Inducer of Antigen-Specific Immune Tolerance

Abstract

ILDR2 is a member of the Ig superfamily, which is implicated in tricellular tight junctions, and has a putative role in pancreatic islet health and survival. We recently found a novel role for ILDR2 in delivering inhibitory signals to T cells. In this article, we show that short-term treatment with ILDR2-Fc results in long-term durable beneficial effects in the relapsing-remitting experimental autoimmune encephalomyelitis and NOD type 1 diabetes models. ILDR2-Fc also promotes transplant engraftment in a minor mismatch bone marrow transplantation model. ILDR2-Fc displays a unique mode of action, combining immunomodulation, regulation of immune homeostasis, and re-establishment of Ag-specific immune tolerance via regulatory T cell induction. These findings support the potential of ILDR-Fc to provide a promising therapeutic approach for the treatment of autoimmune diseases.

FIGURE 1.

Short-term treatment with ILDR2-mFc induces long-term beneficial effect in active and adoptive models of R-EAE and prevents development of T1D in NOD mice. (A) R-EAE was induced in SJL/J mice using PLP_139–151 in CFA. At the onset of remission (day 19), mice were treated i.p. with ILDR2-mFc (10, 30, or 100 μg per dose) or control Ig (mIgG2a; 100 μg per dose). Treatment was given three times a week for 2 wk. Mean clinical scores of 10 mice per group are presented. **p < 0.01 versus control Ig, *p < 0.05 versus ILDR2-mFc (30 μg). (B) Adoptive-transfer EAE was induced by i.v. injection of 5 × 10⁶ PLP_139–151 blast cells into recipient SJL/J mice. Starting on the day of cell transfer, mice were treated with ILDR2-mFc or control Ig (mIgG2a) at 100 mg per dose, three times a week for 2 wk. Mean clinical scores of 15 mice per group are presented. ***p < 0.0001 versus control Ig. (C) Adoptive-transfer EAE was induced by i.v. injection of 3 × 10⁶ PLP_139–151 blast cells into recipient SJL/J mice. At the onset of disease remission (day 17), mice were treated with ILDR2-mFc or control Ig (mIgG2a) at 100 mg per dose each, three times a week for 2 wk. Mean clinical scores of 10 mice per group are presented. **p < 0.01 versus control Ig. (D) NOD mice were treated with ILDR2-mFc or control Ig (mIgG2a) at 100 μg per dose, three times a week for 2 wk, starting from 10 wk of age. Blood glucose levels were monitored weekly from 8 to 30 wk of age. Presented are the percentage normal glycemic mice (n = 14 or 15 mice per group). One representative experiment of two or three independent experiments is presented for each experimental set. ***p < 0.001 versus control Ig.

FIGURE 2.

ILDR2-mFc treatment inhibits myelin epitope-specific epitope spreading in the R-EAE model. (A) On day 76 postdisease induction, five mice from each treatment group were analyzed for recall responses to spread epitopes, via injection of 10 μg of PLP_178–191 in one ear and MBP_84–104 into the opposite ear. The level of ear swelling was assayed 24 h postchallenge. The data are presented as the mean net ear swelling. (B) Recall responses were also carried on splenocytes. On day 76 postdisease induction, total splenocytes were collected from five representative mice from each treatment group and activated in the presence of OVA_323–339, PLP_139–151, PLP_178–191, or MBP_84–104 (20 μg/ml). Cells were pulsed with 1 mCi of tritiated thymidine at 24 h and harvested 72 h postculture set up. Cell proliferation in recall responses was also carried out using splenocytes from day-45 mice (C) or IFN-γ, IL-17, and IL-4 secretion was analyzed by LiquiChip following 72 h of culture (D–F). One representative experiment of two or three independent experiments is presented for each experimental set. *p < 0.05, **p < 0.01, ***p < 0.001 versus control Ig.

FIGURE 3.

ILDR2-mFc downregulates Th1 and Th17 differentiation and enhances Th2 and Treg differentiation in vitro. Naive CD4⁺ T cells isolated from DO11.10 mice were activated with OVA_323–339 peptide (20 μg/ml) in the presence of irradiated APCs under Th1-, Th17-, or Th2-driving conditions, as described in Materials and Methods. Soluble ILDR2-mFc or control Ig (mIgG2a) (5 mg/ml) was added to these cultures. (A–F) Supernatants were collected after 72 h and analyzed for cytokine production via LiquiChip. Lymph node cells from PLP_139–151/CFA primed mice were reactivated ex vivo with PLP_139–151 in the presence of ILDR2-mFc or control Ig. The level of cytokine secretion (G) was assessed in triplicate, and the phenotype of the resultant cells was assessed via flow cytometry (H). These ex vivo-reactivated blasts were transferred to naive SJL/J mice and evaluated for transfer R-EAE induction (I). (J) Freshly isolated CD4⁺CD25⁻ T cells were activated for 4 d with plate-bound anti-CD3 (2 μg/ml) and coimmobilized with 10 μg/ml ILDR2-mFc or control Ig (mIgG2a), in the presence of soluble anti-CD28 (1 μg/ml), with IL-2 (5 ng/ml) over the indicated range of TGF-β concentrations. The number of CD25⁺Foxp3⁺ cells was determined via FACS. Data represent mean ± SD of duplicate wells. One representative experiment of two or three independent experiments is presented for each experimental set. *p < 0.05, **p < 0.01, ***p < 0.001, ^#p < 0.0001 ILDR2-mFc versus control Ig.

FIGURE 4.

Abolishment of ILDR2-mFc-mediated tolerance by Treg blockade with anti-IL-10 or anti–TGF-β, and transient Treg neutralization with anti-CD25 Abs. SJL/J mice were primed with PLP_139–151/CFA. Starting from disease remission, mice were treated with ILDR2-mFc or control Ig (mIgG2a). These treatments were followed by a second injection of anti–IL-10 or control Ab (rat IgG1) (days 20–31; n = 5, experiment completed once) (A) or anti–TGF-β or control Ab (mouse IgG1) (B) (days 20–31; n = 15, experiment completed twice). All treatments were given i.p. at 100 μg per dose, three times a week for 2 wk. In the latter study, splenocytes were analyzed on day 44 for the presence of total live cells, CD45^hi immune cells, and total CD4⁺ T cells (C), and the cell number for different Foxp3⁺ Treg subsets was determined (D). A few mice died in these studies as a result of disease exacerbation and were not scored further: two mice died on day 28 in the anti–IL-10 + ILDR2-mFc treatment group in the study presented in (A). In the study presented in (B), one mouse died on day 31 in the anti–TGF-β + control Ig group, and one died on day 36 in the control Ab + ILDR2-mFc group; in the anti–TGF-β + ILDR2-mFc treatment group, one mouse died on day 35, and another died on day 36. (E) In another study, transient Treg neutralization was induced 2 wk after the last ILDR2-mFc or control Ig (mIgG2a) administration by two injections of anti-CD25 or control Ab (rat IgM), at 500 μg per dose, on days 46 and 48 (indicated by arrows). Statistical analysis of anti-CD25 effect was carried out starting from the day of anti-CD25 or control Ab administration until day 58, because of the transient effect of the anti-CD25 Ab. One representative experiment of two is presented. *p < 0.05 versus control Ig.

FIGURE 5.

ILDR2-mFc induces Ag-specific immune tolerance in the R-EAE model, which can be transferred to naive mice. SJL/J mice were primed with PLP_139–151/CFA. Starting from disease remission (day 20), mice (n = 10) were treated i.p. with ILDR2-mFc or control Ig (mIgG2a) at 100 μg per dose, three times a week for 2 wk. (A) Mice were followed for clinical disease. (B) On day 42, 10 d after the final treatment, spleens were harvested and pooled. A sample of pooled splenocytes was analyzed for CD25⁺Foxp3⁺ expression via FACS. T cells were sorted from these donor mice, and 5 × 10⁶ splenic CD4⁺ T cells were transferred to naive recipient SJL/J mice (n = 5) (C and D). Two days later, adoptive-transfer R-EAE was induced in the recipient mice by i.v. injection of 5 × 10⁶ PLP_139–151 (C) or PLP_178–191 (D) blast cells. Recipients were followed for clinical disease symptoms. One representative experiment of two is presented. *p < 0.05, **p < 0.01, ***p < 0.001 versus control Ig.

FIGURE 6.

ILDR2-hFc regulates immune human T cell responses by downregulating Th1 and Th17 and upregulating Th2 differentiation. Naive human CD4⁺ T cells were isolated from total PBMCs from four healthy human donors and cocultured with autologous irradiated PBMCs. T cells were then activated with anti-CD3 (0.5 μg/ml) and soluble anti-CD28 (0.5 μg/ml) in the presence of Th1 (A and B), Th17 (C), or Th2 (D–F) differentiation-promoting conditions, as detailed in Materials and Methods. ILDR2-hFc or control Ig (hIgG1) was added at 10 μg/ml. Supernatants were collected at 96 h, and cytokine levels were evaluated in duplicate. One representative experiment of two is presented. ***p < 0.001 versus control Ig.

FIGURE 7.

Immunomodulatory effect of ILDR2-hFc on MS patient PBMCs. Total PBMCs were isolated from the blood of MS patients (n = 6–10) and cultured in vitro with MBP_84–99 peptide (20 μg/ml) in the presence of ILDR2-hFc or control Ig (hIgG1) (10 μg/ml each), which were added at time of culture initiation. After 5 d in culture, proliferative responses were measured via [³H]TdR incorporation (A), and supernatants were collected for cytokine analysis (B–I). There was no detectible TGF-β for four of six MS patient samples. The data are presented as CPM or specific cytokine level for each MS patient sample tested using control Ig (0 μg/ml) versus ILDR2-hFc (10 μg/ml). *p < 0.05, ***p < 0.001.