Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Sep 30;10(1):144.
doi: 10.1186/s40478-022-01447-z.

Ex vivo expanded human regulatory T cells modify neuroinflammation in a preclinical model of Alzheimer's disease

Alireza Faridar  1 , Matthew Vasquez  2 , Aaron D Thome  1 , Zheng Yin  2 , Hui Xuan  1 , Jing Hong Wang  1 , Shixiang Wen  1 , Xuping Li  3 , Jason R Thonhoff  1 , Weihua Zhao  1 , Hong Zhao  2 , David R Beers  1 , Stephen T C Wong  2 , Joseph C Masdeu  1 , Stanley H Appel  4
Affiliations

Ex vivo expanded human regulatory T cells modify neuroinflammation in a preclinical model of Alzheimer's disease

Alireza Faridar et al. Acta Neuropathol Commun. .

Abstract

Background: Regulatory T cells (Tregs) play a neuroprotective role by suppressing microglia and macrophage-mediated inflammation and modulating adaptive immune reactions. We previously documented that Treg immunomodulatory mechanisms are compromised in Alzheimer's disease (AD). Ex vivo expansion of Tregs restores and amplifies their immunosuppressive functions in vitro. A key question is whether adoptive transfer of ex vivo expanded human Tregs can suppress neuroinflammation and amyloid pathology in a preclinical mouse model.

Methods: An immunodeficient mouse model of AD was generated by backcrossing the 5xFAD onto Rag2 knockout mice (5xFAD-Rag2KO). Human Tregs were expanded ex vivo for 24 days and administered to 5xFAD-Rag2KO. Changes in amyloid burden, microglia characteristics and reactive astrocytes were evaluated using ELISA and confocal microscopy. NanoString Mouse AD multiplex gene expression analysis was applied to explore the impact of ex vivo expanded Tregs on the neuroinflammation transcriptome.

Results: Elimination of mature B and T lymphocytes and natural killer cells in 5xFAD-Rag2KO mice was associated with upregulation of 95 inflammation genes and amplified number of reactive microglia within the dentate gyrus. Administration of ex vivo expanded Tregs reduced amyloid burden and reactive glial cells in the dentate gyrus and frontal cortex of 5xFAD-Rag2KO mice. Interrogation of inflammation gene expression documented down-regulation of pro-inflammatory cytokines (IL1A&B, IL6), complement cascade (C1qa, C1qb, C1qc, C4a/b), toll-like receptors (Tlr3, Tlr4 and Tlr7) and microglial activations markers (CD14, Tyrobp,Trem2) following Treg administration.

Conclusions: Ex vivo expanded Tregs with amplified immunomodulatory function, suppressed neuroinflammation and alleviated AD pathology in vivo. Our results provide preclinical evidences for Treg cell therapy as a potential treatment strategy in AD.

Keywords: Adaptive immune system; Alzheimer’s disease; Amyloid pathology; Inflammation; Microglia; Regulatory T cells.

PubMed Disclaimer

Conflict of interest statement

The authors of this manuscript declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Treg treatment suppress Amyloid pathology. A ELISA quantification of mice brain homogenates shows a trend toward an increased levels of SDS-soluble and formic acid treated insoluble Aβ40 and Aβ42 in 5xFAD-Rag2KO, compared to 5xFAD-WT. Ex vivo expanded Treg administration decreased both soluble and insoluble Aβ40 and Aβ42 burden in 5xFAD-Rag2KO (10-month-old mice, 10 per group; sex-balanced). B, C Representative images of Aβ immunostaining (6E10) of the dentate gyrus (DG) and frontal cortex (FC) in 5xFAD-WT, 5xFAD-Rag2KO and Treg-treated 5xFAD-Rag2KO. D–I Quantification of the 6E10-positive amyloid aggregates in the DG and FC. The percentage of area covered by Aβ in the DG were increased in 5xFAD-Rag2KO compared to 5xFAD-WT. Number of plaques and their signal intensity were comparable between 5xFAD-Rag2KO and 5xFAD-WT in both DG and FC. Ex vivo expanded Treg administration reduced area covered by plaque, number of plaques and their signal intensity in both DG and FC of 5xFAD-Rag2KO mice (n = 6–7 per group). J Immunostaining of Tregs (CD3 in red, Foxp3 in green) and cell nuclei (DAPI in blue) in the DG of 10-month-old 5xFAD-Rag2KO mice treated with human Tregs or Phosphate-buffered saline (PBS). K, L Quantification of number of CD3+Foxp3+ Tregs in the DG and FC of 4 groups of mice including Treg treated 5xFAD-Rag2KO, Treg treated WT-Rag2KO, PBS treated 5xFAD-Rag2KO and also PBS treated WT-Rag2KO mice. Numbers shown as averages ± SEM with one-way ANOVA. P-values are *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Scale bar, 100 μm
Fig. 2
Fig. 2
Treg administration reduces number of activated microglia in the dentate gyrus. A Representative images of Iba1 positive-microglia (red) and 6E10-positive Aβ plaques (green) in the dentate gyrus (DG) of WT-WT, 5xFAD-WT, 5xFAD-Rag2KO and Treg-treated 5xFAD-Rag2KO (n = 6 per group, sex balanced). B Quantification of the number of Iba+ microglia in the DG; while number of Iba1+microglia was increased in 5xFAD-WT mice, compared to WT-WT, lack of adaptive immune system in 5xFAD-Rag2KO and subsequent Treg administration had no effect on the number of Iba1+ microglia. C Quantification of the number of Iba1+ microglia within 20 μm of the plaque surface; Treg administration reduced number of plaque- associated Iba1+ microglia. D Representative images of CD68-positive activated microglia (red) and 6E10-positive Aβ plaques (green) in the dentate gyrus. Quantification of CD68-positive activated microglia in the DG (E) and CD68-positive microglia within 20 μm of the plaque surface (F), revealed decreased number of total and plaque-associated CD68+ microglia in 5xFAD-Rag2KO following Treg administration. Numbers shown as averages ± SEM with one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. Scale bar, 100 μm
Fig. 3
Fig. 3
Treg administration reduces number of reactive astrocytes. Representative images of GFAP positive-reactive astrocytes (green) and Aβ 1–42 positive plaques (red) in the dentate gyrus (DG) (A) and frontal cortex (FC) (F) of WT-WT, 5xFAD-WT, 5xFAD-Rag2KO and Treg-treated 5xFAD-Rag2KO (n = 6 per group, sex balanced). B–E Quantification of the number of GFAP+ reactive astrocytes in the DG and FC; number of GFAP+ reactive astrocytes was increased in 5xFAD-WT mice, compared to WT-WT. Lack of adaptive immune system in 5xFAD-Rag2KO had no effect on the number of GFAP+ reactive astrocytes, compared to 5xFAD-WT. Decreased number of total and plaque-associated GFAP+ reactive astrocytes in 5xFAD-Rag2KO were noted following Treg administration. Numbers shown as averages ± SEM with one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. Scale bar, 100 μm
Fig. 4
Fig. 4
Modification of inflammation network following Treg administration. Volcano Plots showing fold changes vs. p-values of nCounter Mouse Neuroinflammation panel in A 5xFAD-WT vs. WT-WT; 73 immune related genes were upregulated (green dots) and 5 genes were down regulated (blue dots) in 5xFAD-WT (n = 4 in each groups). B Compared with WT-Rag2KO, 95 genes were upregulated (green dots) and 7 genes were down-regulated (blue dots) in 5xFAD-Rag2KO. The 10 most upregulated genes and all down-regulated genes are labeled in the figures. C Treg-treated 5xFAD-Rag2KO vs. 5xFAD-Rag2KO; the pathologic upregulation of inflammation related genes in 5xFAD-Rag2KO were modified following Treg administration. D The network representation and subcellular assignment of the enriched pathway in Treg-treated 5xFAD-Rag2KO vs. untreated 5xFAD-Rag2KO. The enriched network were centered in down-regulation of pro-inflammatory cytokines (IL1A&B, IL6, Tnfa, IFNγ), complement activation (C1qa, C1qb, C1qc, C4a/b), toll like receptors (Tlr3, Tlr4 and Tlr7), myeloid activation markers (CD14, Tyrobp,Trem2) and intra-nuclear binding motifs of interferon-regulatory factors (IRF3 and IRF7)
See this image and copyright information in PMC

References

    1. Schwartzentruber J, Cooper S, Liu JZ, Barrio-Hernandez I, Bello E, Kumasaka N, et al. Genome-wide meta-analysis, fine-mapping and integrative prioritization implicate new Alzheimer's disease risk genes. Nat Genet. 2021;53(3):392–402. doi: 10.1038/s41588-020-00776-w. - DOI - PMC - PubMed
    1. Jansen IE, Savage JE, Watanabe K, Bryois J, Williams DM, Steinberg S, et al. Genome-wide meta-analysis identifies new loci and functional pathways influencing Alzheimer's disease risk. Nat Genet. 2019;51(3):404–413. doi: 10.1038/s41588-018-0311-9. - DOI - PMC - PubMed
    1. McQuade A, Blurton-Jones M. Microglia in Alzheimer's Disease: Exploring How Genetics and Phenotype Influence Risk. J Mol Biol. 2019;431(9):1805–1817. doi: 10.1016/j.jmb.2019.01.045. - DOI - PMC - PubMed
    1. Welikovitch LA, Do Carmo S, Magloczky Z, Malcolm JC, Loke J, Klein WL, et al. Early intraneuronal amyloid triggers neuron-derived inflammatory signaling in APP transgenic rats and human brain. Proc Natl Acad Sci U S A. 2020;117(12):6844–6854. doi: 10.1073/pnas.1914593117. - DOI - PMC - PubMed
    1. Masliah E, Sisk A, Mallory M, Mucke L, Schenk D, Games D. Comparison of neurodegenerative pathology in transgenic mice overexpressing V717F beta-amyloid precursor protein and Alzheimer's disease. J Neurosci. 1996;16(18):5795–5811. doi: 10.1523/JNEUROSCI.16-18-05795.1996. - DOI - PMC - PubMed

Publication types

MeSH terms