Mechanisms of Natural Killer T cell effector differentiation – NKTdiff
NKTdiff
NKT cells represent a critical component of the mammalian immune system. Due to their wide functional repertoire, they are poised as interesting candidates for future cell-based immunotherapies. This is likely due to the presence of discrete functional subsets that emerge from a complex and ill-defined developmental program in the thymus. In this context, our proposal endeavours to better define the mechanisms associated with the acquiistion of these functions.
Mechanisms of Natural Killer T cell effector differentiation
NKT cells develop in the thymus, and branch away from the conventional aß T cells at the DP stage, upon positive selection by adjacent CD1d-expressing cortical DP. TCR signals are a hallmark of NKT cell positive selection, and likely drive their effector memory phenotype and innate effector program. Subsequent NKT cell effector differentiation relies on many cues such as transcription factors (TF), cytokine/chemokine receptor signalling and surface cell-cell interactions. <br />However, the precise steps of NKT cell differentiation in the thymus remain controversial. Two non-mutually exclusive models have been proposed: a linear maturation model and a lineage differentiation model. Historically, a linear maturation model has been reported in which newly-selected stage 0 (CD24+) NKT cells sequentially mature into stages 1 to 3 NKT cells through the loss of CD24 and acquisition of CD44 and NK1.1 markers. The lineage differentiation model suggests that NKT cells develop in discrete functional subsets based on the same “signature” TF used in CD4+ Thelper subsets. In this model, the three subsets (NKT1, NKT2 and NKT17) are believed to derive from a common progenitor. However, the factors and signaling cascades driving the development of these subsets remain poorly defined.<br />As NKT cell development and functions are intimately linked, a better understanding of the developmental trajectories and checkpoints that control NKT cell effector fate decision will provide important clues into the biology of this T cell lineage with potential outcomes on immune regulation and disease susceptibility.<br />Thus, by combining transcriptomic and biological approaches, the objectives of NKTdiff aim at better understanding the molecular and cellular events that control the functional differentiation of NKT cells as well as how an alteration in this process can influence NKT cell functions in diseases.
Specifically, our four complementary tasks are to:
1) Define the functional development trajectories of NKT cells based on transcriptomic, mathematical models and biological assays.
2) Evaluate the role of FHL2 on NKT cell development and homeostasis based on flow cytometry, biochemical approaches and in vivo experiments.
3) Dissect the molecular mechanisms underlying the control of FHL2 on NKT cell development based on flow cytometry, development of new gene-targeted mice and transcriptomic approaches.
4) Assess the influence of FHL2 deficiency on NKT cell functions during infection and cancer using in vivo models, flow cytometry and gene-targeted animals.
Task 1: To define the developmental model of NKT cells
From this task, we now suggest a new developmental path for NKT cells. This model differs from the previous models of development and suggest that functional differentiation of NKT cells is carried out in 2 steps: A first step of intense proliferation following positive selection in which NKT cells display an immature phenotype associated with a strong capacity to secrete Th2-like cytokines. Next, a second step a functional diversification in which NKT cells acquire either a Th1- (NKT1) or a Th17-like (NKT17) profile. In addition, our data suggest that NKT1 comprise additional functional subsets based on single cell transcriptomic profiling.
Task 2: To evaluate the role of the transcription cofactor FHL2 in NKT cell development and homeostasis.
Our data confirm the key role of FHL2 in NKT cell development in a cell-intrinsic manner. In addition, we have been able to demonstrate that FHL2 specifically controlled NKT1 differentiation. However, we did not observe any clear role for FHL2 in thymic egress of NKT cells. Thus, the lack of NKT cells in the periphery maps the one observed in the thymus. In line, NKT cells from FHL2-deficient mice failed to secrete IFN-g upon stimulation.
Task 3: To study the FHL2-dependent molecular events involved in NKT cell development.
Our preliminary data enabled us to define a list of putative “upstream” and “downstream” candidates in the regulation of FHL2 expression and functions that may be involved in NKT cell development. Among those, the activity of the cardinal transcription factor PLZF seems to be negatively regulated by FHL2. This is in line with the importance of PLZF downmodulation during acquisition of the NKT1 profile.
Regarding task 1, we pursue our work to confirm the new proposed model with in vivo approaches. To do so, methods to perform intrathymic injection have been implemented in the lab. Various labelled NKT cell subsets at various stages of their development will be injected and track after several days to monitor their outcome.
Regarding task 3, our in silico data suggest that various soluble environmental factors could control FHL2 expression/function in NKT cells. This work that was not recorded in the initial proposal is currently under investigation according to its original nature. In parallel, in order to explore the molecular partners of FHL2 in NKT cells, a gene-targeted mouse presenting a tag on the N-terminal part of Fhl2 gene is currently under development.
At this 18 month mark, our new model of NKT cell development has been published in Cell Reports (Baranek et al., PMID : 32905761). These data call into question the overall model of NKT cell development. In addition, our work suggest the existence of a higher functional heterogeneity in NKT cells than initially thought.
COVID-19 pandemic interrupted our experimental work for more than 6 months (March to August 2020). However, we have initiated during this period a clinical project on patients with severe presentation of COVID-19 and hospitalized in ICU department. Thus, we observed in biological fluids of these patients a strong alteration in the biology of unconventional T cells including NKT cells. In addition, these alterations were predictive of the outcome of the patients. This work has been published in the Journal of Experimental Medicine (Jouan et al., PMID : 32886755) and confirmed by subsequent studies.
Natural Killer T (NKT) cells represent a particular lineage of thymus-derived T cells, which occupy a special niche spanning the border of the innate/adaptive continuum. These cells possess a wide functional repertoire that allow them to play key roles in many immune responses poising them at interesting candidates for future cell-based immunotherapies. This is likely due to the presence of discrete functional subsets that emerge from a complex and ill-defined developmental program in the thymus. In this context, our proposal endeavours 1) to define the dynamics and trajectories of NKT cell effector differentiation and 2) to investigate the molecular factors that dictate the fate of NKT cell effector functions. Deciphering these mechanisms will undoubtedly provide important clues into the biology of this T cell lineage and therefore will bring new insights on immune education and disease susceptibility.
Project coordination
Christophe PAGET (Christophe PAGET)
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
Partner
CEPR Christophe PAGET
Help of the ANR 318,600 euros
Beginning and duration of the scientific project:
February 2020
- 36 Months
