Characterization of the early stages of thymic NKT cell development

Abstract

Upon reaching the mature heat stable antigen (HSA)low thymic developmental stage, CD1d-restricted Vα14-Jα18 thymocytes undergo a well-characterized sequence of expansion and differentiation steps that lead to the peripheral interleukin-4/interferon-γ–producing NKT phenotype. However, their more immature HSAhigh precursors have remained elusive, and it has been difficult to determine unambiguously whether NKT cells originate from a CD4+CD8+ double-positive (DP) stage, and when the CD4+ and CD4−CD8− double-negative (DN) NKT subsets are formed. Here, we have used a CD1d tetramer-based enrichment strategy to physically identify HSAhigh precursors in thymuses of newborn mice, including an elusive DPlow stage and a CD4+ stage, which were present at a frequency of ∼10−6. These HSAhigh DP and CD4+ stages appeared to be nondividing, and already exhibited the same Vβ8 bias that characterizes mature NKT cells. This implied that the massive expansion of NKT cells is separated temporally from positive selection, but faithfully amplifies the selected TCR repertoire. Furthermore, we found that, unlike the DN γδ T cells, the DN NKT cells did not originate from a pTα-independent pathway bypassing the DP stage, but instead were produced during a short window of time from the conversion of a fraction of HSAlow NK1.1neg CD4 cells. These findings identify the HSAhigh CD4+ stage as a potential branchpoint between NKT and conventional T lineages and between the CD4 and DN NKT sublineages.

CD1d-restricted iNKT cells, the ‘Swiss-Army knife’ of the immune system

“…This explains the relatively late appearance of NKT cells in the thymus [4] and is consistent with random generation of the canonical Vα14-Jα18 rearrangement within a common T cell progenitor pool. Furthermore, the frequency of the earliest identified NKT cell precursor was estimated to be 1 cell per 10 6 thymocytes [7]. Together, these data support the notion that Vα14-Jα18 rearrangement occurs randomly at very low frequency.…”

The Tec Kinases Itk and Rlk Regulate NKT Cell Maturation, Cytokine Production, and Survival

“…3A). Recently, an earlier stage of NKT cell development has been described in which cells are either HSA high DP low or HSA high CD4 ϩ/high (50). Because this stage precedes early developmental expansion, we considered the possibility that the substantial decrease in NKT cell numbers seen in Itk Ϫ/Ϫ and Itk/Rlk Ϫ/Ϫ mice was due to a block at this stage.…”

Dicer-Dependent MicroRNA Pathway Controls Invariant NKT Cell Development

“…CD4CD8 double positive (DP) precursors that have randomly rearranged the canonical invariant TCR are positively selected into the iNKT cell lineage upon recognition of endogenous agonist lipids, presented by CD1d on DP thymocytes (1,2). Postselection iNKT cells undergo an ordered phenotypic maturation characterized by four stages: the first, detectable immediately after positive selection, is the HSA high CD69 ϩ stage 0, which becomes HSA low CD44 low NK1.1 Ϫ stage 1, followed by CD44 high NK1.1 Ϫ stage 2, and finally by the mature CD44 high NK1.1 ϩ stage 3, which occurs both in the thymus and periphery (3)(4)(5). Maturation is accompanied by a massive cellular expansion between stage 1 and 2 (4).…”

“…Invariant NK T (iNKT) 3 cells recognize both self and exogenous lipids presented by the MHC class I-like molecule CD1d and play a role in infection, autoimmunity, allergy, and cancer (1).…”