Epigenetic regulation of iNKT2 cell adoptive therapy on the imbalance of iNKT cell subsets in thymus of RA mice.

Epigenetic regulation affects the development and differentiation of iNKT cells. Our previous study found that the number of iNKT cells in thymus of RA mice was reduced and the ratio of subsets was unbalanced, but the related mechanism remains unclear. We adopted an adoptive infusion of iNKT2 cells with specific phenotypes and functions to RA mice and used the α-Galcer treatment group as control. The findings revealed that: 1. Adoptive treatment of iNKT cells decreased the proportion of iNKT1 and iNKT17 subsets in the thymus of RA mice, and increased the proportion of iNKT2 subsets. 2. Following treatment with iNKT cells, the expression of PLZF in thymus DP T cells was increased whereas the expression of T-bet in thymus iNKT cells was decreased in RA mice. 3. Adoptive therapy reduced the modification levels of H3Kb7me3 and H3K4me3 in the promoter regions of Zbtb16 (encoding PLZF) and Tbx21 (encoding T-bet) gene in thymus DP T cells and iNKT cells, and the reduction of H3K4me3 was particularly significant in the cell treatment group. Furthermore, adoptive therapy also upregulated the expression of UTX (histone demethylase) in thymus lymphocytes of RA mice. As a result, it is hypothesized that adoptive therapy of iNKT2 cells may affect the level of histone methylation in the promoter region of important transcription factor genes for iNKT development and differentiation, thereby directly or indirectly correcting the imbalance of iNKT subsets in the thymus of RA mice. These findings offer a fresh rationale and concept for the management of RA that targets.

Migration, Distribution, and Safety Evaluation of Specific Phenotypic and Functional Mouse Spleen-Derived Invariant Natural Killer T2 Cells after Adoptive Infusion.

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.