Edited by: Fabrizio Mattei, Istituto Superiore di Sanità, Italy
Reviewed by: Shahram Salek-Ardakani, Pfizer, United States; Wing-Kin Syn, Ralph H Johnson Veterans Affairs Medical Center, United States
Specialty section: This article was submitted to Molecular Innate Immunity, a section of the journal Frontiers in Immunology
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Natural killer T-cells are a subset of innate-like T-cells with the ability to bridge innate and adaptive immunity. There is great interest in harnessing these cells to improve tumor therapy; however, greater understanding of invariant NKT (iNKT) cell biology is needed. The first step is to learn more about NKT development within the thymus. Recent studies suggest lineage separation of murine iNKT cells into iNKT1, iNKT2, and iNKT17 cells instead of shared developmental stages. This review will focus on these new studies and will discuss the evidence for lineage separation in contrast to shared developmental stages. The author will also highlight the classifications of murine iNKT cells according to identified transcription factors and cytokine production, and will discuss transcriptional and posttranscriptional regulations, and the role of mammalian target of rapamycin. Finally, the importance of these findings for human cancer therapy will be briefly discussed.
Natural killer T-cells belong to the T lymphocyte family and are found in many different tissues within the body (
Natural killer T-cells are divided into two groups according to their TCR chains. Type I NKT cells, also called invariant NKT (iNKT) cells, use a distinct invariant TCR α-chain with limited TCR β-chain repertoires, while Type II NKT (NKT_II) cells express broad ranges of different TCR chain combinations (
Antigen recognition by NKT cells and their development within the murine thymus will be discussed. Recent publications suggest a classification of murine iNKT lineages according to their transcription factor (TF) expression and cytokine secretion. Therefore, the author will discuss transcriptional and posttranscriptional regulation of iNKT cell development and function, and the role of Mammalian Target of Rapamycin (mTOR) within iNKT cell subsets. This new lineage concept will be compared to the previous categorization into three developmental stages.
Unlike convT cells, iNKT cells bear a semi-invariant TCR, upon rearrangement of a single TCR α chain with a unique Jα segment, in combination with limited TCR β-chains usage. This results in a rearranged Vα14-Jα18/Vβ8, Vβ7, or Vβ2 TCR in mice and Vα24-Jα18/Vβ11 in humans (
Both NKT cell types share the recognition of various lipid antigens presented on CD1d molecules (
CD4−CD8− lymphoid precursors travel from bone marrow
Further differentiation and maturation of CD69+CD24+ iNKT precursor cells is initiated by parallel binding to the co-stimulatory signaling lymphocytic activation molecules (SLAMs), SLAMF1, and SLAMF6, which signal downstream
Three developmental iNKT stages based on cell surface molecule expression of CD44 and NK1.1 have been described (Figure
Schematic fluorescence-activated cell scanning plot depicting the three identified invariant NKT (iNKT) subsets within the described developmental stages, according to NK1.1 and CD44 expression. Red dots are iNKT2 cells, green dots are iNKT17 cells, and blue dots are iNKT1 cells. The beige dot represents a stage 0 iNKT cell, which expresses the transcription factors Erg2 and PLZF, and decreases CD24 and CD69 expression during the development into stage 1 NKT cells (
The new classification of iNKT cells alternative to the shared developmental stages favors clear lineage separation (
The categorization of iNKT subsets was done
Using this method, transcriptome analyses showed three distinct populations in principle component analyses (PCA) (
So far, the iNKT1 subset has been defined by the upregulation of
iNKT1, iNKT2, and iNKT17 displayed with their transcription factors (TF), cell surface molecules, and cytokine secretion. Diagram legends: – inhibiting, ↑ upregulated, → expressed TF (
In order to produce IFNγ,
The iNKT2 and iNKT17 cell subsets cannot be easily separated from one another. iNKT2 cells were defined by literature to upregulate either
By cell surface molecule classification, iNKT2 cells are thought to belong to developmental stage 1 and 2, sharing stage 2 with iNKT17 cells (
A recent publication highlights the possible importance of SAP for driving an iNKT2 fate. SAP-deficient mice showed decreased expression of
The serine protease SerpinB1 is associated with regulation of TH17 and IL-17-producing γδ T-cells (
Initially, it seems contradicting that only iNKT2 cells are affected by decreased PLZF expression, as iNKT17 and iNKT2 cells are thought to express the same developmental stage surface molecules and were both shown to express PLZF. High expression of PLZF might not be mandatory for iNKT17 differentiation, but may be needed for iNKT2 and iNKT17 to separate from an iNKT1 fate, as mature iNKT1 cells show low PLZF expression. In favor of this is the cross antagonism of TH1 and TH2 (
As an antagonism of
Interestingly, the deficiency of
Besides transcriptional regulation, the mTOR pathway has also been described to regulate iNKT cell fate. mTOR is a serine/threonine kinase, which regulates cell growth and metabolism. Two different mTOR complexes can be found: mTOR complex 1 containing Raptor, which is involved in “translation initiation, autophagy inhibition, lipid synthesis” (
In CD4creRaptorfl/fl mice, the authors reported an accumulation of iNKT cells within stage 0, two-third in stage 1, one-third in stage 2, and an absent stage 3 (
Published literature is controversial regarding, which of the described iNKT subsets is affected in CD4creRictorfl/fl mice. Two papers showed a cell intrinsic defect in iNKT cell development in the absence of Rictor (
Of note, autophagy has also been described to play an essential role in iNKT cell development (
Human and murine iNKT cells can both be divided into CD4+ and CD4−CD8− cells, while human iNKT cells can also express CD8 (
It is known that cell fates determine the overall direction of the immune response, for example, IFNγ production, seen in human NK, T-cells, and iNKT cells, is important for antitumor responses (
A recent Phase I clinical trial adoptively transferred iNKT cells into stage IIIB–IV melanoma patients after
Looking at these data within this review, one can find studies in favor of the developmental stage theory and studies against it. In favor of undergoing developmental stages is the distinct cut-off at stage 2 in
All in all, murine iNKT cell development still seems to be puzzling. Overall some differences in iNKT subset detection may be semantic and depends on individual mouse strain used. Furthermore, microbial effects in mice within different breeding facilities may influence different iNKT subset composition seen within different publications. Nevertheless, more insight will be gained by deeper transcriptional analyses parallel to phenotyping, as these analyses are currently limited to 20 fluorophores. Unbiased approaches such as Cytof or tSNE may further reveal iNKT cell differences and may account for the observed mouse strain specific differences. Furthermore, both approaches can reveal more insights into human iNKT cell development and highlight how these cells can be used more effectively in cancer therapy.
SB has designed, written, revised, and approved of the review herself. She is accountable for all aspects in this review.
The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The author acknowledges the MSc Integrated Immunology course at the University of Oxford. This review originated from its teaching, learning, and assessment activities, and the course defrayed the publication charges. The author also acknowledges Dr Mariolina Salio for critical reading the review.
CD, cluster of differentiation; CDR, complementarity-determining regions; convT-cell, conventional T-cells; DP, double-positive; FACS, fluorescence-activated cell scanning; GATA-3, GATA-binding protein 3; GM-CSF, granulocyte macrophage colony-stimulating factor; IFNγ, interferon gamma; IgE, immunoglobulin E; IL, interleukin; IL-XR, interleukin X receptor; ILCs, innate lymphoid cells; iNKT cells, invariant natural killer T-cells; MAIT, mucosal-associated invariant T-cells; MHC, major histocompatibility complex; MIR, modulator of immune recognition; miRNA, MicroRNA; mTOR, mammalian target of rapamycin; mTORC, mTOR complex; NK, natural killer; NKAP, NFKB activating protein; NKT cells, natural killer T-cells; NKT_II cells, type II NKT cell; PBMC, peripheral blood mononuclear cell; PCA, principle component analyses; pLN, peripheral lymph nodes; PLZF, promyelocytic leukemia zinc finger; RORγT, RAR-related orphan receptor gamma; SAP, SLAM-associated protein; SLAM, signaling lymphocytic activation molecule; TCR, T-cell receptor; TF, transcription factors; UTR, untranslated region; wt, wildtype.