Natural cytotoxicity receptor splice variants orchestrate the distinct functions of human natural killer cell subtypes

The natural cytotoxicity receptors NKp46/NCR1, NKp44/NCR2 and NKp30/NCR3 are critical for natural killer (NK) cell functions. Their genes are transcribed into several splice variants whose physiological relevance is not yet fully understood. Here we report that decidua basalis NK (dNK) cells of the pregnant uterine mucosa and peripheral blood NK (pNK) cells, two functionally distinct subsets of the physiological NK cell pool, display differential expression of NKp30/NCR3 and NKp44/NCR2 splice variants. The presence of cytokines that are enriched within the decidual microenvironment is sufficient to convert the splice variant profile of pNK cells into one similar to that of dNK cells. This switch is associated with decreased cytotoxic function and major adaptations to the secretome, hallmarks of the decidual phenotype. Thus, NKp30/NCR3 and NKp44/NCR2 splice variants delineate functionally distinct NK cell subsets. To our knowledge, this is the first conclusive evidence underlining the physiological importance of NCR splice variants.


Supplementary Figure 2.
Differential expression of NCR isoforms by dNK cells and pNK cells results in differential activation upon receptor cross-linking. (a) Freshly-isolated dNK and pNK cells cultured overnight with IL15 (10 ng ml -1 ) were stimulated for 4 hours with a single or combination of two specific mAbs. All the reactions were carried in the presence of fluorochrome-conjugated anti-CD107a mAb and monensin. CD107a expression was analysed by flow cytometry on CD3 neg CD56 pos cells. Representative FACS plots from three independent donors are shown. (b) Differential effect of specific receptor ligation on cell signalling. dNK or overnight IL15-cultured pNK cells were stimulated for 20min with anti-NKp30, -NKp44 and -NKp46 antibodies. Cleared cell lysates (10 µg of total cellular proteins) were separated by 4-15% denaturinggradient gel and electrotransferred. Proteins were successively immunoblotted with the indicated Ab or with anti-β-actin Ab to verify protein loading. Black arrow heads indicate the protein of interest.

Modulation of NK cell receptor expression by Cytokines enriched in the decidual environment. (a)
Flow cytometry analysis showing a representative CD3 neg CD56 pos NK cell expression of CD16, NKG2D or NKG2A after 6 days of culture with different cytokine combinations. Isotype matched control (grey), pNK cells cultured in complete media (continuous black) and pNK cells cultured in media supplemented with cytokine cocktail (dashed black). The percentage of positive cells is given for each condition. (b) Graphs represent Mean Fluorescence Intensity (MFI) of pNK cells after 6 days of culture with or without cytokines and freshly-isolated dNK cells. Data on graphs represent mean values ± s.e.m. from at least 4 independent donors. *p<0.05, **p<0.01, ***p<0.001, ns: not significant, one-way analysis of variance with Bonferroni post-test.

Supplementary Figure 4.
Cytokine treatment induces the acquisition of specific dNK cell markers with no impairment of pNK cell proliferation. The expression of dNK cell specific markers was analysed by flow cytometry in freshly isolated dNK cells and in pNK cells after 6 days of culture in media supplemented or not with different cytokine combinations. Data on graphs represent the percentage of positive cells for a dNK cell specific marker (CD9, CD49a, CD103) and two chemokine receptors that are differentially expressed in the two NK cell subsets (CXCR3 and CXR4). The percentage of positives cells is given for each marker. Data represent mean values ± s.e.m. from at least 4 independent donors. ***p<0.001, one-way analysis of variance with Bonferroni post-test.(b,c,d) The impact of cytokines on pNK cell proliferation and survival were monitored by counting cells on hemocytometer before the analysis of PI positive cells by flow cytometry. Cell number after 3 days of culture (b), 6 days (c), the percentage of cell survival after 6 days of culture (d). Each data point represents mean values ± s.e.m. from 4 independent donors performed in triplicates, one-way analysis of variance with Bonferroni post-test.

Decidual cytokines affect pNK cell degranualtion. (a) CD107a cell surface expression in pNK cells cultured for 6 days and freshly-isolated dNK cells after 4 hours of NCR ligation. NK cells stimulated with
anti-NKp30, -NKp44, -NKp46 antibodies or Isotype matched controls and chilled on ice. Cellular degranulation (CD107a expression) was then assessed by flow cytometry on CD3 neg CD56 pos cells. Representative graphs of 6 independent donors are presented. (b) Degranulation was evaluated after engagement of NKp46, NKp44 alone or co-engagement of both receptors on cytokine treated cells. Data represent mean values ± s.e.m. from at least 4 independent donors. **p<0.01, ***p<0.001, one-way analysis of variance with Bonferroni post-test.

RT-PCR
Total cellular RNA was isolated from dNK or pNK cells using an RNeasy kit (Qiagen, France). First-strand cDNA was synthesized from 1 μg total RNA using SuperScript III reverse transcriptase and random primers, according to the manufacturer's protocol (Life Technologies, France). PCR primers for NCR3 10 , NCR2 and the actin housekeeping transcripts were designed using an NCBI primer blast and their positions are indicated in Supplementary Fig. 1a and Table 1. PCR reactions were performed in a DNA thermal cycler (Perkin-Elmer) as follows: initial incubation at 55°C for 2 min, denaturation at 95°C for 10 min followed by 30 cycles (35 s at 95°C, 45 s at 56°C, 45 s at 72 °C). PCRs were normalized to that of β-actin housekeeping gene run for 25 cycles.

Cumulative CD107a staining
To assay cumulative degranulation, CD107a fluorochrome-conjugated antibody (BD-Pharmingen) was added at the beginning of stimulation along with 5 µg ml -1 monensin (Sigma Aldrich). After 4 h, cells were chilled at 4°C, washed in PBS and stained for CD3 and CD56 with specific antibodies. Flow Cytometry gates for positive CD107a expression were defined on CD3 neg CD56 pos unstimulated controls.