Transcriptional analysis of natural killer T cell development

Type 1 NKT cells are an innate-like population of T cells that rapidly respond to both cytokine and TCR stimulation by the production of pro-inflammatory and immunoregulatory cytokines. Different from conventional αβT cells, which express a diverse repertoire of TCR sequences that are generated by random rearrangement and are positively selected by thymic epithelium expressing MHC Class I and Class II products, type 1 NKT cells express a highly restricted TCR (Vα14-Jα18 in mice, and the homologous Vα24-Jα18 chain in humans, paired with a restricted range of β chains) and are positively selected by ligating β2 Microglobulin (β2M)/CD1d and SLAM family members expressed on CD4⁺CD8⁺ (double positive – DP) cortical thymocytes. In the periphery of mice, type 1 NKT cells express a 'memory' or 'activated' like surface phenotype (CD62L⁻CD69⁺CD44hi) and the great majority is either CD4⁺CD8⁻ (single positive; SP) or CD4⁻CD8⁻ (double negative; DN). Studies on NKT cell development indicated that they are originated from the same progenitor as conventional T cells, and branched off from the mainstream lineage at DP stage. NKT cells' development is thymus dependent, they develop in fetal thymic organ culture; neonatal thymectomy on the third day of life selectively depletes them. NKT cells are not found in the peripheral tissues of mice until 1-2 weeks after birth. Many efforts have been made to understand the molecular mechanisms that govern their commitment and homeostasis. However, the very low numbers of developing NKT cells, especially DP NKT cells at Stage 0, makes it difficult to analyse transcriptional programs controlling NKT cell development. To further dissect the events surrounding NKT cell lineage commitment and to examine transcriptional factors controlling early NKT cell ontogeny, a mouse model with increased numbers of immature type 1 NKT cells was generated on the SLAM-deficient, NOD background. These mice were found to have greatly increased numbers of CD24⁺NK1.1⁻ DP NKT cells with the characteristics of pre-selection and Stage 0 NKT cells. This provides an opportunity to compare the transcriptional profiles of these very immature CD24⁺NK1.1⁻ DP NKT cells with those of conventional T cells (CD24⁺NK1.1⁻ DP T cells), and those of more mature NKT cell subsets, including CD24⁺NK1.1⁻ CD4⁺ NKT cells and CD24⁺NK1.1⁻ DN NKT cells. Expression levels of a total of 35,556 transcripts of each biological samples of above four different cell populations were obtained by microarray analysis. Principal component analysis indicated that the four populations clearly separated, in order, across principal component 1 from CD24⁺NK1.1⁻ DP T cells, to CD24⁺NK1.1⁻ DP NKT cells, to CD24⁺NK1.1⁻ CD4⁺ NKT cells, and finally to CD24⁺NK1.1⁻ DN NKT cells. Comparison of gene expression between these populations has provided an overall transcriptional profile during TCR validation, positive selection and lineage commitment of NKT cells. These findings have further confirmed phenotypic changes during NKT cell development observed by previous studies and suggest that immature DP NKT cells are pre-selection progenitors of NKT cells. Our transcriptional regulatory network approach mapped TCR validation to the transition from DP T to DP NKT cells, while positive selection and lineage commitment were associated with the transition from DP NKT to CD4 NKT cells. This is the first time that the effects of positive and negative selection have been examined on their actual population – the immature DP NKT cells. We confirm by in vivo experimentation that both positive and negative selection occur at the latter transition, separating for the first time in any T cell population the events associated with TCR validation from those associated with positive selection. NOD. Vα14Tg mice provide a model to study the earliest identifiable stages of NKT cell commitment and differentiation, and to help dissect factors controlling the numbers and function of this important immunoregulatory population.