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CAR/FoxP3-engineered T regulatory cells target the CNS and suppress EAE upon intranasal delivery.

Fransson M, Piras E, Burman J, Nilsson B, Essand M, Lu B, Harris RA, Magnusson PU, Brittebo E, Loskog AS - J Neuroinflammation (2012)

Bottom Line: CD4+ T cells were modified utilizing a lentiviral vector system to express a chimeric antigen receptor (CAR) targeting myelin oligodendrocyte glycoprotein (MOG) in trans with the murine FoxP3 gene that drives Treg differentiation.The engineered Tregs demonstrated suppressive capacity in vitro and could efficiently access various regions in the brain via i.n cell delivery.CNS-targeting Tregs delivered i.n. localized to the CNS and efficiently suppressed ongoing inflammation leading to diminished disease symptoms.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden.

ABSTRACT

Background: Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS). In the murine experimental autoimmune encephalomyelitis (EAE) model of MS, T regulatory (Treg) cell therapy has proved to be beneficial, but generation of stable CNS-targeting Tregs needs further development. Here, we propose gene engineering to achieve CNS-targeting Tregs from naïve CD4 cells and demonstrate their efficacy in the EAE model.

Methods: CD4+ T cells were modified utilizing a lentiviral vector system to express a chimeric antigen receptor (CAR) targeting myelin oligodendrocyte glycoprotein (MOG) in trans with the murine FoxP3 gene that drives Treg differentiation. The cells were evaluated in vitro for suppressive capacity and in C57BL/6 mice to treat EAE. Cells were administered by intranasal (i.n.) cell delivery.

Results: The engineered Tregs demonstrated suppressive capacity in vitro and could efficiently access various regions in the brain via i.n cell delivery. Clinical score 3 EAE mice were treated and the engineered Tregs suppressed ongoing encephalomyelitis as demonstrated by reduced disease symptoms as well as decreased IL-12 and IFNgamma mRNAs in brain tissue. Immunohistochemical markers for myelination (MBP) and reactive astrogliosis (GFAP) confirmed recovery in mice treated with engineered Tregs compared to controls. Symptom-free mice were rechallenged with a second EAE-inducing inoculum but remained healthy, demonstrating the sustained effect of engineered Tregs.

Conclusion: CNS-targeting Tregs delivered i.n. localized to the CNS and efficiently suppressed ongoing inflammation leading to diminished disease symptoms.

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Engineered Tregs localize to the CNS when administered intranasally in naive mice. Treg cells were administered intranasally in the right nostril and the distribution of green fluorescent protein (GFP) in horizontal cryosections of the brain of naïve mice was studied 24 hours after the delivery. The schematic drawing describes a selective immunofluorescence in various brain regions (green spots). GFP immunofluorescence is present in the granular and to a lower extent in the external plexiform layer of the olfactory bulb (B, C), lateral septal nucleus (E), central medial thalamic nucleus (F), ectorhinal cortex (H), medial genic nucleus (I) and cerebellum (K, L) of a CAR Treg-treated naïve mouse. Corresponding areas showing no GFP fluorescence in a PBS-treated naïve mouse are (A, D, G, J). Enlargements of areas in olfactory bulb and cerebellum (as indicated by boxes) are depicted in C and L. Detail of GFP immunofluorescence in the central medial thalamic nucleus and medial genic nucleus (F, I). Cell nuclei (blue) are stained with DAPI Original magnification 10× (A, B, D, E, G, H, J, K) and 40× (C, F, I, L).
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Figure 3: Engineered Tregs localize to the CNS when administered intranasally in naive mice. Treg cells were administered intranasally in the right nostril and the distribution of green fluorescent protein (GFP) in horizontal cryosections of the brain of naïve mice was studied 24 hours after the delivery. The schematic drawing describes a selective immunofluorescence in various brain regions (green spots). GFP immunofluorescence is present in the granular and to a lower extent in the external plexiform layer of the olfactory bulb (B, C), lateral septal nucleus (E), central medial thalamic nucleus (F), ectorhinal cortex (H), medial genic nucleus (I) and cerebellum (K, L) of a CAR Treg-treated naïve mouse. Corresponding areas showing no GFP fluorescence in a PBS-treated naïve mouse are (A, D, G, J). Enlargements of areas in olfactory bulb and cerebellum (as indicated by boxes) are depicted in C and L. Detail of GFP immunofluorescence in the central medial thalamic nucleus and medial genic nucleus (F, I). Cell nuclei (blue) are stained with DAPI Original magnification 10× (A, B, D, E, G, H, J, K) and 40× (C, F, I, L).

Mentions: CAR Tregs co-expressing GFP and CARαMOG-FoxP3 were used to evaluate in vivo targeting upon i.n. cell delivery in naïve mice. The overall localization of GFP immunofluorescence is illustrated in the schematic drawing in Figure 3. The green fluorescence was mainly localized in clusters of cells in the granular layer and the external plexiform layer of the olfactory bulb (Figure 3B, C), in the lateral septal nucleus (Figure 3E), in the central medial thalamic nucleus (Figure 3F), in the ectorhinal cortex (Figure 3H), in the medial genic nucleus (Figure 3I) and in the Purkinje cell layer and white matter of the cerebellum (Figure 3K, L). In addition, green immunofluorescence was observed in anterior olfactory nucleus and anterior orbital cortex (data not included). The green immunofluorescence was only observed in the soma and was preferentially present in the perinuclear part (Figure 3F, I). Although a unilateral dose of cells was given, the localization of immunofluorescence occurred both on the ipsilateral and contralateral sides of the brain. In the vehicle control animal no, or extremely weak, green background immunofluorescence could be detected (Figure 3A, D, G, J). The localization of immunofluorescence is summarized in Table 1.


CAR/FoxP3-engineered T regulatory cells target the CNS and suppress EAE upon intranasal delivery.

Fransson M, Piras E, Burman J, Nilsson B, Essand M, Lu B, Harris RA, Magnusson PU, Brittebo E, Loskog AS - J Neuroinflammation (2012)

Engineered Tregs localize to the CNS when administered intranasally in naive mice. Treg cells were administered intranasally in the right nostril and the distribution of green fluorescent protein (GFP) in horizontal cryosections of the brain of naïve mice was studied 24 hours after the delivery. The schematic drawing describes a selective immunofluorescence in various brain regions (green spots). GFP immunofluorescence is present in the granular and to a lower extent in the external plexiform layer of the olfactory bulb (B, C), lateral septal nucleus (E), central medial thalamic nucleus (F), ectorhinal cortex (H), medial genic nucleus (I) and cerebellum (K, L) of a CAR Treg-treated naïve mouse. Corresponding areas showing no GFP fluorescence in a PBS-treated naïve mouse are (A, D, G, J). Enlargements of areas in olfactory bulb and cerebellum (as indicated by boxes) are depicted in C and L. Detail of GFP immunofluorescence in the central medial thalamic nucleus and medial genic nucleus (F, I). Cell nuclei (blue) are stained with DAPI Original magnification 10× (A, B, D, E, G, H, J, K) and 40× (C, F, I, L).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 3: Engineered Tregs localize to the CNS when administered intranasally in naive mice. Treg cells were administered intranasally in the right nostril and the distribution of green fluorescent protein (GFP) in horizontal cryosections of the brain of naïve mice was studied 24 hours after the delivery. The schematic drawing describes a selective immunofluorescence in various brain regions (green spots). GFP immunofluorescence is present in the granular and to a lower extent in the external plexiform layer of the olfactory bulb (B, C), lateral septal nucleus (E), central medial thalamic nucleus (F), ectorhinal cortex (H), medial genic nucleus (I) and cerebellum (K, L) of a CAR Treg-treated naïve mouse. Corresponding areas showing no GFP fluorescence in a PBS-treated naïve mouse are (A, D, G, J). Enlargements of areas in olfactory bulb and cerebellum (as indicated by boxes) are depicted in C and L. Detail of GFP immunofluorescence in the central medial thalamic nucleus and medial genic nucleus (F, I). Cell nuclei (blue) are stained with DAPI Original magnification 10× (A, B, D, E, G, H, J, K) and 40× (C, F, I, L).
Mentions: CAR Tregs co-expressing GFP and CARαMOG-FoxP3 were used to evaluate in vivo targeting upon i.n. cell delivery in naïve mice. The overall localization of GFP immunofluorescence is illustrated in the schematic drawing in Figure 3. The green fluorescence was mainly localized in clusters of cells in the granular layer and the external plexiform layer of the olfactory bulb (Figure 3B, C), in the lateral septal nucleus (Figure 3E), in the central medial thalamic nucleus (Figure 3F), in the ectorhinal cortex (Figure 3H), in the medial genic nucleus (Figure 3I) and in the Purkinje cell layer and white matter of the cerebellum (Figure 3K, L). In addition, green immunofluorescence was observed in anterior olfactory nucleus and anterior orbital cortex (data not included). The green immunofluorescence was only observed in the soma and was preferentially present in the perinuclear part (Figure 3F, I). Although a unilateral dose of cells was given, the localization of immunofluorescence occurred both on the ipsilateral and contralateral sides of the brain. In the vehicle control animal no, or extremely weak, green background immunofluorescence could be detected (Figure 3A, D, G, J). The localization of immunofluorescence is summarized in Table 1.

Bottom Line: CD4+ T cells were modified utilizing a lentiviral vector system to express a chimeric antigen receptor (CAR) targeting myelin oligodendrocyte glycoprotein (MOG) in trans with the murine FoxP3 gene that drives Treg differentiation.The engineered Tregs demonstrated suppressive capacity in vitro and could efficiently access various regions in the brain via i.n cell delivery.CNS-targeting Tregs delivered i.n. localized to the CNS and efficiently suppressed ongoing inflammation leading to diminished disease symptoms.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden.

ABSTRACT

Background: Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS). In the murine experimental autoimmune encephalomyelitis (EAE) model of MS, T regulatory (Treg) cell therapy has proved to be beneficial, but generation of stable CNS-targeting Tregs needs further development. Here, we propose gene engineering to achieve CNS-targeting Tregs from naïve CD4 cells and demonstrate their efficacy in the EAE model.

Methods: CD4+ T cells were modified utilizing a lentiviral vector system to express a chimeric antigen receptor (CAR) targeting myelin oligodendrocyte glycoprotein (MOG) in trans with the murine FoxP3 gene that drives Treg differentiation. The cells were evaluated in vitro for suppressive capacity and in C57BL/6 mice to treat EAE. Cells were administered by intranasal (i.n.) cell delivery.

Results: The engineered Tregs demonstrated suppressive capacity in vitro and could efficiently access various regions in the brain via i.n cell delivery. Clinical score 3 EAE mice were treated and the engineered Tregs suppressed ongoing encephalomyelitis as demonstrated by reduced disease symptoms as well as decreased IL-12 and IFNgamma mRNAs in brain tissue. Immunohistochemical markers for myelination (MBP) and reactive astrogliosis (GFAP) confirmed recovery in mice treated with engineered Tregs compared to controls. Symptom-free mice were rechallenged with a second EAE-inducing inoculum but remained healthy, demonstrating the sustained effect of engineered Tregs.

Conclusion: CNS-targeting Tregs delivered i.n. localized to the CNS and efficiently suppressed ongoing inflammation leading to diminished disease symptoms.

Show MeSH
Related in: MedlinePlus