Role of CD4 T Cell in Relapsing-Remitting Experimental Autoimmune Encephalomyelitis

Summary

An experimental autoimmune encephalomyelitis (EAE) model in mice was employed to investigate the role of CD4 T cells in the initial and relapse of EAE from the perspective of the activation phase and immune effect function.

Abstract

Multiple sclerosis (MS) is an autoimmune disease characterized by the infiltration of immune cells and demyelination in the central nervous system (CNS). Experimental autoimmune encephalomyelitis (EAE) serves as a prototypic animal model for studying MS. In this study, we aimed to investigate the role of CD4 T cells in the initiation and relapse of EAE, focusing on the activation phase and immune response. To create the EAE mice model, female mice were immunized with myelin oligodendrocyte glycoprotein (MOG)35-55 emulsified with complete Freund's adjuvant (CFA). Clinical scores were assessed daily, and results demonstrated that mice in the EAE group exhibited a classic relapsing-remitting pattern. Hematoxylin-eosin (H&E) and luxol fast blue (LFB) staining analysis revealed significant infiltration of inflammatory cells in the CNS and demyelination in EAE mice. Regarding the activation phase, both CD4⁺CD69⁺ effector T (Teff) cells and CD4⁺CD44⁺CD62L^- effector memory T (Tem) cells may contribute to the initiation of EAE, however, the relapse stage was probably dominated by CD4⁺CD44⁺CD62L^- Tem cells. Additionally, in terms of immune function, helper T (Th)1 cells are primarily involved in initiating the EAE. However, both Th1 and Th17 cells contribute to the relapse stage, and the immunosuppressive function of regulatory T (Treg) cells was inhibited during the EAE pathological process.

Introduction

Multiple sclerosis (MS) is an autoimmune disease that is characterized by the infiltration of the central nervous system (CNS) with immune cells and demyelination^1,2. A recent study has shown it to be the most common disabling disease affecting young people, with its incidence continuously increasing worldwide³. Experimental autoimmune encephalomyelitis (EAE) is a prototypic animal model for MS that simulates many aspects of the inflammatory phase of the human MS⁴. From the perspective of immune functions, the initial CD4 T cells that have not yet encountered ant....

Protocol

A total of 18 female C57BL/6J mice aged six to eight weeks were randomly divided into a control group (n = 6) and an EAE group (n = 12) for this study. Mice were purchased from the experiment animal center of Ningxia Medical University. Mice were housed in a temperature- and humidity-controlled room with a 12 h light/dark cycle, and free access to fresh water and food. Ethics approval was obtained from the Experimental Animal Ethics Committee of Ningxia Medical University before the study began.

Discussion

MS is the most prevalent autoimmune demyelinating disease of the CNS, which affects millions of people worldwide¹⁷. EAE is the typical animal model for simulating the clinical pathological features of MS¹⁸. Studies have demonstrated that individuals with MS experience cognitive impairment and other disabilities due to the degeneration of axons and neurons in the CNS^19,20,21. At the.......

Materials

Name	Company	Catalog Number	Comments
Anesthesia machine evaporator	Norvap	20-17368
Isoflurane	Sigma-Aldrich (Shanghai) Trading Co.Ltd.	792632
70 μm cell strainer	XIYAN Co.,Ltd.	15-1070
Alexa Fluor 700 anti-mouse CD45 Antibody	Biolegend	103128
APC anti-mouse CD4 Antibody	Biolegend	100616
Automatic cell counter	Jiangsu JIMBIO technology Co., LTD	JIMBIO iCyta S2
BD* Cytometric Bead Array (CBA) Mouse Th1/Th2/Th17 CBA Kit	BD Biosciences	560485
Biotin anti-mouse CD16/32 Antibody	Biolegend	101303
Brilliant Violet 510 anti-mouse CD69 Antibody	Biolegend	104532
BV786 Rat Anti-Mouse CD62L(MEL-14)	BD Pharmingen	563109
Column Tissue&Cell Protein Extraction Kit	Shanghai Epizyme Biomedical Technology Co., Ltd	PC201Plus
Complete Freund's Adjuvant	Sigma-Aldrich (Shanghai) Trading Co.Ltd.	SLCH4887
Dehydrator	DIAPATH	Donatello
Disposable sterilized syringe (1 mL)	Yikang Group	210820
Disposable sterilized syringe (2.5 mL)	Yikang Group	210820
Disposable sterilized syringe (5 mL)	Yikang Group	210820
Dyeing machine	DIAPATH	Giotto
Embedding machine	Wuhan Junjie Electronics Co., Ltd	JB-P5
Ethanol	SCRC	100092683
Fetal Bovine Serum	Procell Life Science&Technology Co.,Ltd.	FSP500
FITC Hamster Anti-Mouse CD3e(145-2C11)	BD Pharmingen	553062
Flow cytometer	Becton,Dickinson and Company	FACSCelesta
Flow cytometer	Becton,Dickinson and Company	Accuri C6
Flow Jo software	BD Biosciences	10.8.1
Frozen platform	Wuhan Junjie Electronics Co., Ltd	JB-L5
Glass slide	Servicebio	G6004
HE dye solution set	Servicebio	G1003
Hematoxylin-Eosin solution	Servicebio	G1002
High speed refrigerated centrifuge	Thermo Fisher Scientific	Mogafugo8R
Imaging system	Nikon	NIKON DS-U3
Luxol fast blue staining kit	Servicebio	G1030
Ms CD44 BV421 IM7	BD Pharmingen	564970
Mycobacterium tuberculosis H37RA	BD Pharmingen	231141
Myelin oligodendrocyte glycoprotein (MOG35-55)	AnaSpec	AS-60130-1
Neutral gum	SCRC	10004160
Organizer	KEDEE	KD-P
Oven	Labotery	GFL-230
Pathology slicer	Leica	RM2016
Pertussis Toxin from Bordetella pertussis	Sigma-Aldrich (Shanghai) Trading Co.Ltd.	P7208
Phosphate buffered saline	Servicebio	G4202
Pipette 0.5-10 μL	DLAB Scientific	7010101004
Pipette 100-1000 μL	DLAB Scientific	7010101014
Pipette 20-200 μL	DLAB Scientific	7010101009
RPMI-1640	Procell Life Science&Technology Co.,Ltd.	PM150110
Tee valve	Guangdong Kanghua Medical Co., LTD	A06
Tissue spreader	Zhejiang Kehua Instrument Co.,Ltd	KD-P
Upright optical microscope	Nikon	NIKON ECLIPSE E100
Xylene	SCRC	10023418