The Synergistic Local Immunosuppressive Effects of Neural Stem Cells Expressing Indoleamine 2,3-Dioxygenase (IDO) in an Experimental Autoimmune Encephalomyelitis (EAE) Animal Model

<div><p>Neurodegenerative diseases provoke robust immunological reactions in the central nervous system (CNS), which further deteriorate the neural tissue damage. We hypothesized that the expression levels of indoleamine 2,3-dioxygenase (IDO), an enzyme that has potent immune suppressive activities, in neural stem cells (NSCs) would have synergistic therapeutic effects against neurodegenerative diseases, since NSCs themselves have low IDO expression. In this study, the synergistic immune suppressive effects of rat fetal NSCs expressing IDO (rfNSCs-IDO) were validated by mixed leukocyte reaction (MLR) <i>in vitro</i> and an experimental autoimmune encephalomyelitis (EAE) animal model <i>in vivo</i>. rfNSCs-IDO showed significantly more suppressive effects on T cell proliferation in the MLR compared to control rfNSCs (rfNSCs-Cont). Importantly, IDO inhibition using 1-methyl-DL-tryptophan (1-MT), an IDO inhibitor, reversed the synergistic effects, confirming IDO-specific effects in rfNSCs-IDO. In the EAE animal model, systemic rfNSCs-IDO injections resulted in significant local immune suppression in the cervical lymph nodes and CNS, evidenced by a reduction in the number of activated T lymphocytes and an increase in regulatory T cell numbers, which induced significantly fewer clinical symptoms and faster recovery. In contrast, rfNSCs-Cont failed to reduce symptoms in the EAE animal models, although they showed local immune suppression, which was significantly less than that in rfNSCs-IDO. Taken together, IDO expression in NSCs synergistically potentiates the immune suppression activities of NSCs and could be applicable for the development of therapeutic modalities against various neurodegenerative diseases.</p></div

Characterization of rfNSCs and generation of IDO expressing NSCs.

<p>rfNSCs were cultured in two different methods <i>in vitro</i>. (A) rfNSCs initially formed neurospheres under suspension culture conditions and were then maintained adherently on PLO-coated plates. Scale bars, 50 μm. (B) The expression of NSC markers (Nestin, Sox2, and Musashi1) was confirmed by RT-PCR. As shown, there was no difference in the expression of NSC markers in both culture methods. (C) Undifferentiated rfNSCs were positive for Nestin. However, differentiated rfNSCs showed an increased expression of neural cell markers such as Olig2 (oligodendrocyte), GFAP (astrocyte), and NeuN (neuron). Scale bars, 50 μm. (D) Control and IDO expressing vector map scheme. IDO and EmGFP genes were inserted into the lentiviral vector, which were expressed by dual promoters, the CMV promoter and the PGK promoter, respectively. (E) After infection, the expression of EmGFP as a selection marker was observed under fluorescent microscope. rfNSCs-Cont (left) and rfNSCs-IDO (right) expressed EmGFP. Scale bars, 100 μm. The expression level of IDO was confirmed by (F) RT-PCR and (G) western blot.</p

Molecular consequences of <i>NTRK1</i>-fusion.

<p>(<b>A</b>) <i>NTRK1</i> expression in 170 TCGA GBM samples (from 162 patients) with RNA-Seq data. Samples bearing <i>NTRK1</i>-fusion genes are marked and labeled. (<b>B</b>) Relationship between <i>NTRK1</i> expression and NGF/TrkA-downstream pathway activity in 526 TCGA GBM samples (from 526 patients) with microarray gene expression data. Samples with <i>NTRK1</i>-fusion are marked with red circles. Two other samples with outlier <i>NTRK1</i> expression are marked with blue circles (TCGA-32-4209, TCGA-19-5947).</p

Reduced inflammatory lesion by transplantation of rfNSCs-IDO.

<p>The EAE model exhibited the abnormal infiltration of lymphocytes into the brain. (A) Migration of rfNSCs to the cerebral cortex was confirmed by immunofluorescent staining. SN12C-GFP was used as a positive control. Scale bars, 50 μm. (B) Abnormal infiltration of lymphocytes into the cerebral cortex was observed in the HBSS control group, whereas the rfNSCs-Cont and rfNSCs-IDO groups showed less or no lymphocyte infiltration. Blue arrowheads indicated the magnified region of each group. Scale bars, 100 μm. (C) The infiltration of CD25⁺ activated T lymphocyte in the cerebral cortex was visualized by immunofluorescent staining. Red arrowheads indicate the magnified region of each group. Scale bars, 100 μm. (D) The thickness of the lymphocyte layer was measured at the cerebral cortex among the experimental groups. (n = 6, * <i>p</i> < 0.05, ** <i>p</i> < 0.01).</p

<i>BCAN-NTRK1</i> fusion.

<p>(<b>A</b>) Per-nucleotide read coverage of genomic regions along <i>BCAN</i> and <i>NTRK1</i>. The dotted line marks approximate positions where the fusion has occurred. (<b>B</b>) A schematic of spliced transcripts of the fusion gene. Bottom sequences in black are the reads that map onto the chimeric exon-exon splicing junction.</p

<i>NTRK1</i> Fusion in Glioblastoma Multiforme

<div><p>Glioblastoma multiforme (GBM) is the most aggressive form of brain tumor, yet with no targeted therapy with substantial survival benefit. Recent studies on solid tumors showed that fusion genes often play driver roles and are promising targets for pharmaceutical intervention. To survey potential fusion genes in GBMs, we analysed RNA-Seq data from 162 GBM patients available through The Cancer Genome Atlas (TCGA), and found that 3′ exons of neurotrophic tyrosine kinase receptor type 1 (<i>NTRK1</i>, encoding TrkA) are fused to 5′ exons of the genes that are highly expressed in neuronal tissues, neurofascin (<i>NFASC</i>) and brevican (<i>BCAN</i>). The fusions preserved both the transmembrane and kinase domains of <i>NTRK1</i> in frame. <i>NTRK1</i> is a mediator of the pro-survival signaling of nerve growth factor (NGF) and is a known oncogene, found commonly altered in human cancer. While GBMs largely lacked <i>NTRK1</i> expression, the fusion-positive GBMs expressed fusion transcripts in high abundance, and showed elevated <i>NTRK1</i>-pathway activity. Lentiviral transduction of the <i>NFASC-NTRK1</i> fusion gene in NIH 3T3 cells increased proliferation <i>in vitro</i>, colony formation in soft agar, and tumor formation in mice, suggesting the possibility that the fusion contributed to the initiation or maintenance of the fusion-positive GBMs, and therefore may be a rational drug target.</p></div

Regulated proliferation of lymphocytes by rfNSCs-IDO <i>in vivo</i>.

<p>Cervical lymph node samples were collected from animals of each group at 12 dpi. The number of T cells, B cells, activated T cells, and regulatory T cells isolated from sacrificed animals was quantitated by FACS analysis. (A) The number of T cells and B cells showed no significant difference among experimental groups. (B) However, the number of activated T cells was reduced in the rfNSCs-IDO group. (C) Moreover, the number of regulatory T cells was significantly increased compared to the other groups (n = 4, *** p < 0.001).</p

Suppression of T cell proliferation by rfNSCs expressing IDO.

<p>(A) The result of mixed leukocyte reaction showed that T cell proliferation was significantly suppressed by both rfNSCs-Cont and rfNSCs-IDO groups. Moreover, the T cell number in the rfNSCs-IDO group was significantly reduced compared to that in the rfNSCs-Cont group. (B) With the treatment of IDO inhibitor, 1-MT (1-methyl-DL-tryptophan) at a 0.5 mM concentration, the T cell number in the rfNSCs-IDO group became similar to that in the rfNSCs-Cont group. (n = 3, *** p < 0.001).</p

Immunosuppressive effect of rfNSCs-IDO in EAE animal model.

<p>Immunization with MOG and CFA induced acute inflammation in 6 week-old female SD rats. (A) EAE-induced rats exhibited swollen feet with hind limb paralysis. (B) The intravenous injection of rfNSCs-Cont, rfNSCs-IDO, or HBSS was performed through the rat tail vein at 5 dpi after EAE induction. The total EAE experiment took 37 days. (C) The rfNSCs-IDO group maintained similar body weight to non-EAE control group, whereas the HBSS group showed weight loss throughout the experimental period. (D) According to EAE clinical scoring chart, it was observed that the recovery rate of rfNSCs-IDO group was significantly faster than that of HBSS control group (n = 7 for each group, * <i>p</i> < 0.05, ** <i>p</i> < 0.01).</p

<i>NFASC-NTRK1</i> fusion.

<p>(<b>A</b>) Per-nucleotide read coverage (expression) of genomic regions along <i>NFASC</i> and <i>NTRK1</i>. The dotted line marks the DNA-level break-points in the two genes, as instructed by the fusion-point mapping result in panel B. (<b>B</b>) A schematic of pre-mRNAs of the <i>NFASC-NTRK1</i> fusion gene. Top and bottom sequences in black are the reads that map onto the DNA-level fusion-point. The fusion-point is mapped with slight ambiguity due to 2-nt-long micro-homology between the two break-points in the involved genes. (<b>C</b>) A schematic of spliced transcripts of the fusion gene. Bottom sequences in black are the reads that map onto the chimeric exon-exon splicing junction.</p