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. 2015 Apr 9;125(15):2418-27.
doi: 10.1182/blood-2014-08-597070. Epub 2015 Feb 19.

Plant-based oral tolerance to hemophilia therapy employs a complex immune regulatory response including LAP+CD4+ T cells

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Plant-based oral tolerance to hemophilia therapy employs a complex immune regulatory response including LAP+CD4+ T cells

Xiaomei Wang et al. Blood. .

Abstract

Coagulation factor replacement therapy for the X-linked bleeding disorder hemophilia is severely complicated by antibody ("inhibitor") formation. We previously found that oral delivery to hemophilic mice of cholera toxin B subunit-coagulation factor fusion proteins expressed in chloroplasts of transgenic plants suppressed inhibitor formation directed against factors VIII and IX and anaphylaxis against factor IX (FIX). This observation and the relatively high concentration of antigen in the chloroplasts prompted us to evaluate the underlying tolerance mechanisms. The combination of oral delivery of bioencapsulated FIX and intravenous replacement therapy induced a complex, interleukin-10 (IL-10)-dependent, antigen-specific systemic immune suppression of pathogenic antibody formation (immunoglobulin [Ig] 1/inhibitors, IgE) in hemophilia B mice. Tolerance induction was also successful in preimmune mice but required prolonged oral delivery once replacement therapy was resumed. Orally delivered antigen, initially targeted to epithelial cells, was taken up by dendritic cells throughout the small intestine and additionally by F4/80(+) cells in the duodenum. Consistent with the immunomodulatory responses, frequencies of tolerogenic CD103(+) and plasmacytoid dendritic cells were increased. Ultimately, latency-associated peptide expressing CD4(+) regulatory T cells (CD4(+)CD25(-)LAP(+) cells with upregulated IL-10 and transforming growth factor-β (TGF-β) expression) as well as conventional CD4(+)CD25(+) regulatory T cells systemically suppressed anti-FIX responses.

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Figures

Figure 1
Figure 1
Modulation of antibody formation by oral administration of bioencapsulated FIX in hemophilia B mice. (A) Timeline and schedule for oral gavages (FIX or WT plant material, n = 8 per experimental group), IV FIX administration, and blood collection for antibody assays. (B) Inhibitor titers (in BU/mL plasma). (C) FIX-specific IgG titers (IgG1, IgG2a, and IgG2b (ng/mL). (D) FIX-specific IgE titers (ng/mL) and (E) FIX-specific IgA titers (in ng/mL for circulating IgA and in ng/100 μg total IgA for fecal titers). (F) At the end of the experiment, in vitro splenocyte cultures were established. Cells were stimulated with or without 10 μg/mL FIX antigen for 48 hours, followed by quantitative RT-PCR analysis. “Fold increase” is change in RNA transcripts of FIX vs mock stimulated. All gene expression was compared with that of glyceraldehyde-3-phosphate dehydrogenase. Fold-change was calculated using the 2ΔΔCt quantification method. The dotted horizontal line indicates 1-fold change. (B-F) Average ± standard error of the mean (SEM).
Figure 2
Figure 2
Increases of tolerogenic DCs in hemophilia B mice. (A) Flow cytometry analysis of CD11c and PDCA-1 staining in the spleen, MLNs, and PPs in hemophilia B mice following oral delivery of FIX plant material and IV challenge with FIX. Control groups were fed with WT plant material (wt fed) or with FIX material but not challenged with IV injections of FIX (FIX fed no injection). (B) CD103 and CD11c staining of MLN and PP cells of the same mice. Data points are for individual mice. Averages ± SEM are also indicated (n = 3-5 per experimental group). Shown are data from 2 independent experiments that showed similar results.
Figure 3
Figure 3
Changes in Treg frequencies in hemophilia B mice. Flow cytometric analyses of spleen, MLN, ILN, and PP cells of hemophilia B mice following oral delivery of FIX plant material and IV challenge with FIX. Control groups were fed with WT plant material (wt fed) or were fed with FIX material but not challenged with IV injections of FIX (FIX fed no injection). (A) CD4+CD25+FoxP3+ Tregs; (B,C) CD4+CD25LAP+ Tregs; and (D) Tr1 cells (CD49b+LAG-3+CD4+). (C) Independent experiment comparing the CD4+CD25LAP+ Treg frequencies of mice that were fed with FIX and intravenously challenged either with FIX or with and identical antigen dose of keyhole limpet hemocyanin (KLH). Data points are for individual mice. Averages ± SEM are also indicated (n = 3-6 per group). Representative flow cytometry plots are shown for spleen, MLNs, and PPs. (E,F) Quantitative RT-PCR analysis of IL-10 (E) and TGF-β (F) expression in different subsets of magnetically sorted cells from FIX-fed and control mice after a 48-hour lymphocyte culture established for individual animals (and comprising pooled MLN cells and splenocytes). Data indicate average cytokine expression levels (± SEM) for FIX-stimulated relative to unstimulated cultures (n = 6 mice/cell type, except for CD4+CD25+ cells, which was n = 3).
Figure 4
Figure 4
Increases of gut-homing receptor expressing CD4+ T cells in hemophilia B mice. (A) Representative examples of flow cytometric analysis of CCR9 and integrin α4β7 staining. Frequencies of CCR9 (B) and α4β7 (C) expressing cells in the spleen, MLNs, and PPs following oral delivery of FIX plant material and IV challenge with FIX. Control groups were fed with WT plant material (wt fed) or with FIX material but not challenged with IV injections of FIX (FIX fed no injection). Data points are for individual mice, with average ± SEM also indicated (n = 4-6 per group).
Figure 5
Figure 5
Plant-based oral tolerance induces CD4+CD25+ Tregs and LAP+CD25CD4+ Tregs. (A) Experimental outline of adoptive transfer. CD4, CD4+CD25, and CD4+CD25+ cells were purified via magnetic sorting from spleens and MLNs of FIX-fed/intravenously challenged hemophilia B mice and pooled. Doses of 106 cells per mouse were adoptively transferred into strain-matched mice via tail vein injection. Control cells were from naive mice. After 24 hours, all recipient mice (n = 5-8 per group) were challenged with 1 IU FIX in adjuvant via subcutaneous injection. IgG titers against FIX (B) were determined 3 weeks later. (C) Purified CD4+CD25 cells were further divided into LAP+ and LAP cells via magnetic sorting. The cells were adoptively transferred (n = 4-6 per group), followed by immunization with FIX. IgG titers against FIX (D) were determined 3 weeks later. Control mice had received cells from naive mice. Data are average ± SEM. Shown are data from 2 independent experiments that showed similar results.
Figure 6
Figure 6
Uptake of luminal FIX antigen by APCs in the small intestine. Immunohistochemistry of small intestine of hemophilia B mice. (A) Representative sections of villi from duodenum, jejunum, ileum, or (B) PP stained with antibody against FIX (red), F4/80 (green), and CD11c (blue) of mice fed with CTB-FIX expressing plant material. (C) Same stain of villi from ileum of a control mouse fed with WT material (NC, negative control). (D) Staining with antibody against FIX (red), CD103 (green), and CD11c (blue) of villi or (E) PPs from a mouse fed with CTB-FIX expressing or (F) WT plant material. Triangles in panel A point to yellow FIX+F4/80+ cells (yellow). Purple cells in panels A, B, and D are FIX+CD11c+ DCs. White/light blue cells in panels D and E are FIX+CD11c+CD103+ DCs (examples are indicated with triangles).
Figure 7
Figure 7
Reversal of inhibitor formation and anaphylaxis in hemophilia B mice. (A) Feeding and FIX administration schedule. Numbers in circle indicate time point of blood collection. (B) Inhibitor titers in BU/mL, (C) IgG1 titers against FIX at weeks 6, 15, and 21 of the experiment. Control mice were fed with WT plant material (control, black squares, n = 5). A second group received FIX transgenic plant material during weeks 6 to 14 followed by WT plant material (fed 1, gray triangles, n = 6), and a third group received FIX transgenic plant material for weeks 6 through 20 (fed 2, black triangles, n = 4). Data are average ± SEM. (D) Survival analysis in the same set of experimental mice. (E) IgE formation against FIX in plasma of the same mice.

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