Mechanisms by Which NK T Cells Become the Predominant T Cell Subset in Mice after Irradiation.

Previously, we found that the percentage of NK T cells among all T cells in the spleen of mice treated with fractionated irradiation to the lymphoid tissues (Total lymphoid irradiation; TLI) with a total dose of 4,080 cGy increased markedly due to greater reduction in the absolute number of non-NK T cells as compared to NK T cells. The underlying mechanisms of the change in the T cell subsets after irradiation remained to be established. In the current study, C57BL/6 mice were given escalating single doses of 240, 1,000, 2,000 and 3,000 cGy total body irradiation (TBI). Splenocytes were harvested at 4 or 24 hours after irradiation, and the percentage and absolute number of NK T and non-NK T cells was determined. At the same time, the intracellular level of the anti-apoptotic protein, Bcl-2 was assayed by flow cytometry. In some studies, the turnover rate of NK T cells and non-NK T cells was examined by injection of BrdU and intracellular staining. At 4 hours after all doses of irradiation, neither the NK T nor non-NK T cell subset had a significant change in percentage or absolute number as compared to untreated controls. However, at 24 hours the percentage of NK T cells among all T cells had progressively increased with increased doses of TBI from 3% in the untreated controls to 65% in mice given 3,000 cGy. Whereas the absolute number of non-NK T cells decreased at least 1000 fold, the absolute number of NK T cells decreased approximately 50 fold after 3,000 cGy. The BrdU incorporation of NK T cells from irradiated mice was markedly reduced as compared to untreated mice, and was similar to that of non NK T cells in these irradiated mice. 8–12% of NK T cells and non NK T cells in untreated mice expressed a high level Bcl-2. As the dose of TBI increased progressively, the percentage of Bcl-2^hi cells increased progressively to 89% amongst NK T cells and 70% amongst non-NK T cells. At each irradiation dose, the percentage of Bcl-2^hi cells amongst NK T cells was higher than amongst non-NK T cells. There were 40×10³ Bcl-2^hi NK T cell and 10×10³ Bcl-2^hi non-NK T cells surviving per spleen at 24 hours after 3000 cGy TBI. The absolute number of Bcl-2^hi NK T cells decreased by about two fold while the absolute number of Bcl-2^hi non-NK T cells decreased by about 100 fold. These results indicate that the increased percentage of NK T cells amongst all T cells after irradiation is due to greater radioresistance rather than to more rapid replenishment of NK T cells as compared to non-NK T cells. We are investigating whether Bcl-2 plays a critical role in the extraordinary radioresistance of the NK T cells.