Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase (G6PI)-Induced RA Mice

Summary

This protocol uses G6PI mixed peptides to construct rheumatoid arthritis models that are closer to that of human rheumatoid arthritis in CD4⁺ T cells and cytokines. High purity invariant natural killer T cells (mainly iNKT2) with specific phenotypes and functions were obtained by in vivo induction and in vitro purification for adoptive immunotherapy.

Abstract

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T (iNKT) cells. Patients with active RA present fewer iNKT cells, defective cell function, and excessive polarization of Th1. In this study, an RA animal model was established using a mixture of hGPI325-339 and hGPI469-483 peptides. The iNKT cells were obtained by in vivo induction and in vitro purification, followed by infusion into RA mice for adoptive immunotherapy. The in vivo imaging system (IVIS) tracking revealed that iNKT cells were mainly distributed in the spleen and liver. On day 12 after cell therapy, the disease progression slowed down significantly, the clinical symptoms were alleviated, the abundance of iNKT cells in the thymus increased, the proportion of iNKT1 in the thymus decreased, and the levels of TNF-α, IFN-γ, and IL-6 in the serum decreased. Adoptive immunotherapy of iNKT cells restored the balance of immune cells and corrected the excessive inflammation of the body.

Introduction

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic, progressive invasiveness with 0.5–1% incidence^1,2. The underlying pathogenesis is attributed to the abnormal proliferation of autoreactive CD4⁺ and CD8⁺ T cells, manifested by an increase in the proportion of CD4⁺IFN-γ⁺ and CD4⁺IL-17A⁺ T cells, and the reduced number of CD4⁺IL-4⁺ and CD4⁺CD25⁺FoxP3⁺ T cells. Therefore, the secretion of inflammatory cytokines is increased, and an excessive inflammatory rea....

Protocol

All experimental mice (150 in total) were healthy male DBA/1 mice, 6–8 weeks old (20.0 ± 1.5 g), reared in a specific pathogen-free (SPF) environment. There is no special treatment before modeling. The experiment was divided into a healthy control group (15 mice), a model control group (15 mice), and a cell therapy group (55 mice). This study was approved by the Animal Welfare and Ethical Committee of Hebei University.

1. Constructing the Disease Model

1. Duplicating the RA.......

Discussion

iNKT cells are special T cells that bridge innate and adaptive immunity and are mainly developed from CD4⁺⁺/CD8⁺ thymocytes. iNKT cells have diverse immunoregulatory functions and interact with other immune cells by direct contact and secretion of different cytokines²³, affecting dendritic cells (DCs), macrophages, neutrophils, B cells, T cells, and NK cell differentiation and development²⁴. α-GalCer.......

Materials

Name	Company	Catalog Number	Comments
Alexa Fluor 647 Mouse Anti-PLZF	BD	563490	America
Anti-PE MicroBeads	Miltenyi	130-048-801	Germany
Columns	Miltenyi	MS	Germany
Cryogenic Centrifuge	Beckman	Allegra® X-15R	America
DiR	Thermo Fisher Scientific	D12731	America
Embedding Center	Tianjin Aviation Electromechanical Co., Ltd.	BMJ-1	China
FITC Hamster Anti-Mouse TCR β Chain	BD	553170	America
Flow cytometer	BD	Accuri C6	America
Freund's complete adjuvant	Sigma	F5881	America
hGPI325-339 (IWYINCFGCETHAML)	Karebay Biochem	18062202	China
hGPI469-483 (EGNRPTNSIVFTKLT)	Karebay Biochem	18062203	China
In Vivo Imaging System	PerkinElmer	caliper IVIS lumina II	America
Ionomycin Calcium	Cayman	10004974	America
KRN7000	AdipoGen	AG-CN2-0013	America
Mouse CD1d Tetramer-PE	MBL	TS-MCD-1	Japan
Mouse percoll	Solarbio	P8620	China
Optical Microscope	Olympus	Olympus-II	Japan
PerCP-CyTM5.5 Mouse anti-ROR-ϒt	BD	562683	America
PerCP-CyTM5.5 Mouse anti-T-bet	BD	561316	America
Pertussis toxin	Sigma	P7208	America
phorbol esters	Cayman	10008014	America
Red Blood Cell Lysis Buffer	BD	555899	America
RPMI-1640	Biological Industries	01-100-1ACS	Israel
Th1/Th2/Th17 cytokines kit	BD	560485	America
Ultramicrotome	Leica	Leica EM UC6	Germany