Interleukin 15 throughout murine natural killer cell biology

Natural killer (NK) cells are innate immune cells that mount responses against virally infected, transformed and allogeneic transplanted cells. Equipped with cytolytic molecules and death receptors, NK cells mediate cytotoxicity against cells expressing low levels of MHC class I (MHC-I) or high levels of stress-induced molecules. NK cells are also immune-modulatory, releasing various cytokines and chemokines as well as kill immature immune cells. A tight regulation is available to endow NK cells with capacity to distinguish self and non-self, while making sure that they are well functional. Numerous extrinsic and intrinsic factors become parts of the regulatory network to control NK cell development, maturation and functional acquisition.

Interleukin 15 (IL-15) is an indispensable cytokine throughout NK cell biology. In this thesis, a few novel aspects of IL-15 in NK cell biology under non-inflammatory conditions were reported. In paper I, the importance of IL-15 expressed by dendritic cells (DCs) for NK cell homeostasis, maturation, and functions at steady-state were investigated. In paper II, the coordination of IL-15 with activating signaling DNAX-1-associated receptor (DNAM-1) in controlling the expression of linker for activation of T cells (LAT), an activating signaling molecule, was studied. In paper III, we studied the functional impact of brief contact with IL-15 (“priming”) and its underlying molecular mechanism. Finally, in paper IV, the roles of forkhead box transcription factors of the O class (FOXO) transcription factors in NK cell development in relation to IL-15 receptor (IL-15R) expression and other transcription factors were explored (Figure 1).

In summary, we have found that under non-inflammatory conditions, the presence of DCs is required to maintain NK cell homeostasis in regards to apoptosis and proliferation. NK cell maturation and receptor expression were also perturbed upon DC depletion. Importantly, DC derived IL-15 was required for “missing self” reactivity of NK cells in the absence of infection (paper I). At steadystate, the brief contacts with IL-15 are functionally relevant as five-minute treatment with IL-15 augmented degranulation, cytokine production, and calcium flux triggered by activating receptor stimulation, as well as cytotoxicity against YAC1 cells (paper III). Short-time IL-15 stimulation induced phosphorylation of activating signaling molecules, which is Janus Kinase 3 (JAK3) dependent. The functional impact of short time IL-15 stimulation remained up to three hours after IL15 removal. In paper II, IL-15 stimulation was found to induce expression of LAT especially in DNAM-1+ NK cells. The absence of DNAM-1 or its ligand, CD155, reduced LAT expression. The heightened level of LAT expression in DNAM-1+ NK cells endows them with better responsiveness to NK1.1 stimulation, as measured by calcium flux, cytokine production and degranulation.

In the last paper, paper IV, mouse models with specific deletion of FOXO1 and FOXO3 in hematopoietic cells (Vav1+iCre FOXO1,3flox/flox) were employed to study the roles of these transcription factors in NK cell development. Upon depletion of FOXO1,3 in Vav1+ cells, NK cell development was blocked at the transition from pre-NK cell progenitors (preNKP) (CD122- or IL15Rβ-) to refined NK cell progenitors (rNKP) (CD122+), resulting in very few committed NK cells in both bone marrow (BM) and spleen. The few NK cells developing in the absence of FOXO1,3 were less mature and display perturbed inhibitory and activating receptor expression. The transplantation experiment demonstrated that the effects of FOXO1,3 on NK cell development, maturation and receptor expression were NK-cell-progenitor intrinsic. The experiment employing RNA sequencing (RNA-seq) and assay for transposase-accessible chromatin using sequencing (ATAC-seq) revealed a possible mechanism underlying the roles of FOXO1,3 in controlling NK cell development. The expression and/or DNA binding of ETS proto-oncogene 1 (ETS1), transcription factor T cell factor 7 (TCF7), and CD122 were affected in different stages of NK cell development in the absence of FOXO1,3. With the reduction in CD122 (IL-15Rβ), mature NK cells from both BM and spleen displayed an increase rate in undergoing apoptosis.

Taken together, FOXO1,3 controlled IL-15R expression on NK progenitors and committed NK cells, which contributes to maintain NK cell population in mice. In summary, we have found that, under non-inflammatory condition, IL-15 regulated NK cell homeostasis and functions, brief contacts with IL-15 were functionally relevant, and the cellular effect was coordinated with DNAM-1 signaling via controlling LAT expression. Lastly, FOXO1,3 were identified as novel transcription factors which control IL-15R expression and NK cell development.

List of scientific papers

I. Luu, T.T., Ganesan, S., Wagner, A.K., Sarhan, D., Meinke, S., Garbi, N., Hammerling, G., Alici, E., Karre, K., Chambers, B.J., Hoglund*, P. & Kadri*, N. Independent control of natural killer cell responsiveness and homeostasis at steady-state by CD11c+ dendritic cells. Sci Rep. 2016;6:37996. *Shared last authors.
https://doi.org/10.1038/srep37996

II. Luu, T.T., Wagner, A.K., Schmied, L., Meinke, S., Freund, J.E., Kambayashi, T., Ravens, I., Achour, A., Bernhardt, G., Chambers, B., Hoglund, P. & Kadri, N. IL-15 and CD155 expression regulate LAT expression in murine DNAM1(+) NK cells, enhancing their effectors functions. Eu J Immunol. 2020;50(4):494-504.
https://doi.org/10.1002/eji.201948233

III. Luu, T.T., Schmied, L., Nguyen, N.-A., Wiel, C., Mohammad, D., Meinke, S., Bergö, M., Alici, E., Kadri, N., Ganesan, S. & Höglund, P. Short-term IL-15 priming leaves a long-lasting signalling imprint in mouse NK cells independent of a metabolic switch. [Manuscript]

IV. Luu, T.T., Søndergaard, J.N.#, Peña-Pérez, L.#, Kharazi, S., Meinke, S., Schmied, L., Nicolai F., Heshmati, Y., Kierczak, M., Bouderlique, T., Wagner, A.K., Chambers, B.J., Achour, A., Kutter, C., Höglund, P., Månsson*, R. & Kadri*, N. Foxo1 and Foxo3 cooperatively control NK cell development. #Shared second authors, *Shared last authors. [Manuscript]