Abstract
Herein, the migration distribution and safety of specific phenotypic and functionally identified spleen-derived invariant natural killer T2 (iNKT2) cells after adoptive infusion in mice were studied. The proliferation and differentiation of iNKT cells were induced by intraperitoneal injection of α-galactosylceramide (α-GalCer) in vivo. Mouse spleens were isolated in a sterile environment. iNKT cells were isolated by magnetic-activated cell sorting columns (MS columns). Cytometric bead array (CBA) assay was used to detect cytokine secretion in the supernatant stimulated by iNKT cells. The basic life status of the mice was observed, and systematic quantitative scoring was conducted after injecting spleen-derived iNKT cells through the tail vein. An in vivo imaging system was used to trace the migration and distribution of iNKT cells in DBA mice. The percentage of the iNKT2 subgroup was the highest in 3 days after intraperitoneal injection of α-GalCer, and iNKT2 subsets accounted for more than 92% after separation and purification by magnetic-activated cell sorting (MACS). Anti-inflammatory cytokine IL-4 was mainly found in the supernatant of cell cultures. The adoptive infusion of iNKT cells into healthy mice resulted in no significant change in the basic life status of mice compared with the noninjected group. iNKT cells were detected in the lung, spleen, and liver, but no fluorescence was detected in lymph nodes and thymus. After dissecting the mice, it was found that there were no significant abnormalities in the relevant immune organs, brain, heart, kidney, lung, and other organs. Intraperitoneal injection of α-GalCer results in a large number of iNKT2 cells, mainly secreting anti-inflammatory cytokine IL-4, from the spleen of mice. After adoptive infusion, the iNKT2 cells mainly settled in the liver and spleen of mice with a satisfactory safety profile.
Highlights
Invariant natural killer T cells are a group of unique immune cells that exhibit the characteristics of both NK and T cells. iNKT cells are restricted by antigen-presenting molecule CD1d, which is of the MHC-I type. α-GalCer is a prototype ligand of iNKT cells and a potent stimulator of glycolipid antigens that activates iNKT cells [1,2,3,4]
The frequency of iNKT cells in the spleen of normal DBA/1 mice is approximately 2%, of which invariant natural killer T2 (iNKT2) accounts for 5.2%, iNKT1 accounts for 15.1%, and iNKT17 accounts for 9.2%
The frequency of iNKT cells was approximately 6% after intraperitoneal injection of αGalCer for 3 days, of which the iNKT2 subgroup accounts for 82.0%, iNKT1 accounts for 1.5%, and iNKT17 accounts for 0.4%
Summary
Invariant natural killer T (iNKT) cells are a group of unique immune cells that exhibit the characteristics of both NK and T cells. iNKT cells are restricted by antigen-presenting molecule CD1d, which is of the MHC-I type. α-GalCer is a prototype ligand of iNKT cells and a potent stimulator of glycolipid antigens that activates iNKT cells [1,2,3,4]. Th2 cytokines (IL-2, IL-4, IL-6, IL-10, IL-17, IFN-γ, and TNF-α) and regulate the differentiation of immune cells and the type of immune response Because of their cytotoxic effect, they are active in diseases such as cancer and infection and in type I diabetes, autoimmune encephalomyelitis (EAE), multiple sclerosis (MS), systemic lupus erythematosus (SLE), and other autoimmune diseases, and they play a role in immunosuppression when organs are rejected after transplantation [5,6,7,8]. Our previous study found that the intraperitoneal injection of α-GalCer effectively activates the iNKT2 cell subsets of the spleen Adoptive infusion of such iNKT2 cells effectively alleviates the clinical symptoms of RA [19, 20]. We used an in vivo imaging system of small animals to trace the migration and distribution of iNKT cells in mice and to evaluate the safety of adoptive infusion, so as to provide basic research data for adoptive immunotherapy of iNKT cells
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