Developmental dynamics of post-selection thymic DN iNKT

Abstract

Background: Invariant natural killer T (iNKT) cells develop in the thymus and branch off from the maturation pathway of conventional T cell at the DP stage. While different stages of iNKT cellular development have been defined, the actual time that iNKT cell precursors spend at each stage is still unknown.

Methodology/principal finding: Here we report on maturation dynamics of post-selection DN iNKT cells by injecting wild-type DP(dim) thymocytes into the thymus of TCRα(-/-) mice and using the Vα14-Jα18 rearrangements as a molecular marker to follow the maturation dynamics of these cells.

Conclusion/significance: This study shows that the developmental dynamics of DN iNKT cells in DP(dim) are very rapid and that it takes less than 1 day to down-regulate CD4 and CD8 and become DN. These DN cells are precursors of peripheral DN iNKT cells and appear in the spleen in 1-2 days. Thymic DN iNKT residents are predominantly derived from cells that quickly return from the periphery. The expansion of a very small subset of DN iNKT precursors could also play a small role in this process. These data are an example of measuring T cell maturation in the thymus and show that the maturation dynamics of selected DN iNKT cells fall within the same general time frame as conventional αβ T cells.

Figure 1. Thymocytes with iNKT rearrangements are found in the DP^dim population.

A. FACS profile of thymocytes based on CD4 and CD8 expression and defined gates for DP^dim and DP^med thymocytes. B. FACS profile of thymocytes based on CD3 expression and defined gates for CD3^neg, CD3^med and CD3^hi. C. FACS profile of thymocytes based on CD4 and CD8 expression through CD3^neg and CD3^med populations. Numbers inside DP^dim gates represent the number of collected cells. D. Rearrangement analysis of Vα14-Jα18 in different sorted DP populations. The asterisk shows the population (CD3^med DP^dim) in which iNKT cells are observed, and lane “M” represents the iNKT rearrangement in the spleen of wild type C57BL/6 mouse and serves as size marker.

Figure 2. iNKT rearrangements in TCRα^−/− mice after intrathymic injection of DP^dim cells.

The X axis shows days post injection, and the Y axis shows the percent of mice analyzed that had iNKT rearrangements. A. DN thymocytes were analyzed. The arrows and asterisks indicate statistically significant changes. Fisher’s exact test was used to compute the P value from contingency table (day 1& 3, P = 0.04; day 2&3, P = 0.03). B. Total splenocytes were analyzed. The correlation between the two linear regression lines was calculated based on the Zar’s method (P = 0.008). The actual data for each time point are in Table 1.

Figure 3. DN iNKT cells can return to the thymus.

DP^dim thymocytes were injected in the thymi of four TCR Cα^−/− mice and 6 days post transfer, the thymi were pooled and DN thymocytes were sorted. A.iNKT rearrangement from DN thymocytes 6 days post transfer of DP^dim. The newly generated DN day 6 thymocytes were transferred to the periphery of TCR Cα^−/− mice. B. iNKT rearrangements in the thymus at different days post peripheral transfer of day 6 DN thymocytes.

Figure 4. DN iNKT cells that are generated from DP^dim injection express CD44.

A. FACS profile of DN thymocytes based on CD44 and CD25 expression for thymocytes collected at day 2 and day 6 post DP^dim injection. The sort gates for the four populations are shown. B. iNKT rearrangement in DN thymocytes from the four gated populations at day 2 and day 6, post DP^dim injection. “N.D.” not done due to the low number of CD44⁺ CD25⁺ cells on day 2. Lane “M” represents the iNKT rearrangement in the spleen of wild-type C57BL/6 mouse and serves as size marker. C. iNKT rearrangement analysis in the DN thymocyte subsets 18 hours after thymic injection of sorted CD44⁻ (Top row), and CD44⁺ (middle row) DP^dim cells. The bottom row shows iNKT rearrangement in DN thymocyte subsets from a C57BL/6 mouse representing the steady state pattern.

Figure 5. iNKT lineages generated from wild type DP^dim injection in TCRα^−/− mice can be long lived.

A. iNKT arrangement in DN thymocytes and in splenocytes at day 6, day 7, and day 14 post DP^dim injection. Top and bottom rows represent separate experiments. The middle column “M” shows iNKT rearrangement as a marker. The dates post injection of wild type DP^dim are shown at the top of each band. B. iNKT rearrangement in thymus and spleen 28 days post DP^dim injection. Thymocytes from three TCRα^−/− mice were pooled at day 28 post injection, and DN thymocytes were sorted based on the expression of CD44 and CD25 (first row). At this time newly generated iNKTs are predominantly CD44⁺CD25⁻ with a small population that is CD44⁻CD25⁻. The second row shows the iNKT rearrangement in the spleens of the same three mice.

Figure 6. A model for the developmental dynamics of DN iNKT cells.

The green box represents the thymus, and the red box the periphery. Filled ovals outlined in black identify cells transferred in the different experiments. Filled arrows and the timing next to them show the experimental findings. Open ovals represents specific compartments. Open arrows and the timing next to them are based on our interpretation. The gel insert shows the iNKT rearrangement in CD4⁺CD8^low and CD8⁺CD4^low cells.