TREM2-overexpressing transgenic (TREM2-TG) mice show a higher NK cell population than wild type (WT) mice
To investigate the effect of TREM2 on NK cell development, we analyzed NK populations in previously generated TREM2-overexpressing transgenic (TREM2-TG) and wild type (WT) mice using flow cytometry [33]. The expression of NK receptor repertoires, NK1.1 + population percentage, and their absolute numbers in the spleen, BM, and liver were higher in TREM2-TG mice than in WT mice (Fig. 1A and B). Furthermore, the absolute number of NK cells expressing the NKG2A/NKG2C/NKG2E receptor was higher in the spleen (Additional file 1, Fig. S1, left panel) and liver (right panel) of TREM2-TG mice than that in those of WT mice. A slight increase in the BM of TREM2-TG mice (middle panel) was also observed. Similarly, the percentage and absolute numbers of Ly49C/F/H/I+ and Ly49D+ NK cells in the spleen and BM of TREM2-TG mice were significantly higher than in those of WT mice (Fig. 1B and Additional file 1, Fig. S1).
To investigate whether NK cells express TREM2, we isolated BM cells from WT and TREM2-TG mice, stained them with NK-specific markers, and performed flow cytometry analysis. The t-SNE density plots of the BM cells of WT and TREM2-TG mice are shown in Fig. 1C–E left and middle panels, respectively. The cell population showed both NK1.1+ (Cluster 2, indigo) and TREM2 surface protein (red) expression (Fig. 1C, third panel). Furthermore, the NK1.1+TREM2+ (indigo and red) cell population was increased in the BM of TREM2-TG mice (Fig. 1C, red arrow) compared with that of WT mice. Likewise, Fig. 1D shows the CD122+ cells (Cluster 6, indigo) and TREM2 + cell population (red, third panel), demonstrating an increase in CD122+TREM2+ double-positive (indigo and red) cells in TREM2-TG mice (red circle). We further analyzed the TREM2 expression in the CD3−CD122+NK1.1+ pNK cells. As shown in Fig. 1E, TREM2-expressing pNK cells (CD3−CD122+NK1.1+) were increased in TREM2-TG mice compared with that in WT mice. In addition, the TREM2 + pNK cell population (TREM2+CD122+NK1.1+) was significantly increased in the BM of TG mice (4.68%) compared with that in the BM of WT mice (1.63%; Fig. 1F). These data showed that TREM2 was expressed in CD3−CD122+NK1.1+ pNK cells
Reverse transcription-polymerase chain reaction (RT-PCR) was performed to identify whether TREM2 regulates NK cell function-associated genes using splenic NK1.1+ cells from WT and TREM2-TG mice. The expression levels of IFN-γ (3.29 ± 1.158-fold increase), perforin (18.25 ± 5.3-fold increase), and granzyme B (57 ± 16.97-fold increase) were significantly higher in the spleens of TREM2-TG mice than in the spleens of WT mice (Additional file 1, Fig. S2A). Consequently, TREM2-TG splenic NK cells showed significantly higher NK cell-mediated cytotoxicity than those of WT mice (Additional file 1, Fig. S2B). Interestingly, no differences were observed in CD4+ T, CD8+ T, and B220+ cell populations and their absolute numbers in the spleen between WT and TREM2-TG mice (Additional file 1, Fig. S3).
Inhibition of TREM2 signaling reduces NK cell populations in vivo
To verify the effect of TREM2 on NK cell development in vivo, we inhibited TREM2 signaling in WT mice via intraperitoneal injection of a TREM2-Ig fusion protein or a humanized (hu)-Ig control. Three days after injection, the spleen, BM, and liver cells were isolated, and NK cell populations and the expression of NK-specific receptors were analyzed via flow cytometry. The NK1.1+ cell population was reduced in the spleens of TREM2-Ig-injected mice, with respect to both the percentage of the splenocyte population and absolute number of NK1.1+ cells, compared with that in the spleens of hu-Ig-injected control mice (3.525% ± 0.32% vs. 5% ± 0.5%; Fig. 2A and Additional file 1, Fig. S4). Similarly, the NK1.1+ population percentage and the absolute number of NK1.1+ cells were lower in the BM and liver of TREM2-Ig-injected mice compared with that in the BM and liver of hu-Ig-injected control mice (Fig. 2A). Furthermore, the NK1.1+/NKG2A/C/E+ cell population percentage was lower in the spleens of TREM2-Ig-injected mice (1.9%) than that in the spleens of hu-Ig-injected control mice (2.4%; Additional file 1, Fig. S4, left panel). Both the NK1.1+/NKG2A/C/E+ (0.6% vs. 1.1%) and NK1.1+/Ly49D+ (0.5% vs. 1%) populations were reduced in the BM of TREM2-Ig-injected mice than in the BM of control mice, and the absolute number of NK1.1+ cells was also decreased by TREM2 signal inhibition (TREM2-Ig treatment; Fig. 2B and Additional file 1, Fig. S4, middle panel). In addition, both the NK1.1+/NKG2A/C/E+ (4.9% vs. 6.3%) and NK1.1+/Ly49D+ (2% vs. 2.4%) populations, as well as the absolute number of NK1.1+ cells were decreased in the livers of TREM2-Ig-injected mice compared with those in the livers of control mice (Fig. 2B and Additional file 1, Fig. S4, right panel). However, the absolute numbers of NK1.1+/NKA/C/E+, Ly49C/F/H/I+, and Ly49D+ cells were significantly decreased in the spleen (Fig. 2B, left panel) and BM (Fig. 2B, middle panel), but not in the liver (Fig. 2B, right panel) of TREM-Ig-injected mice compared with those in hu-Ig-injected mice (Fig. 2B, right panel). Together, these data indicated that inhibition of TREM2 signaling by TREM2-Ig resulted in a decrease in the number of NK cells and the expression of their signature surface receptors in vivo.
TREM2 promotes NK cell differentiation in vitro
Our results showed that TREM2 signaling increased the number of NK cells in vivo. However, this is not sufficient to conclude that TREM2 enhances commitment to the NK cell fate and differentiation of the NK cell lineage. Therefore, to determine the effects of TREM2 on the differentiation of NK cells, we isolated c-kit+ Lin−HSCs from the BM of WT and TREM2-TG mice and differentiated them into pNK and mNK cells in vitro. During NK cell differentiation, pNK cells were treated with TREM2-Ig or hu-Ig to inhibit TREM2 signaling. As a result, the percentage and absolute numbers of NK1.1+/NKG2A/C/E+ cells were approximately 2-fold higher in mNK cells derived from hu-Ig-treated pNK cells of TREM2-TG mice (25%) than in their counterparts derived from hu-Ig-treated pNK cells of WT mice (14%; Fig. 3A). However, this NK1.1+ cell population dramatically decreased when both WT-pNK (46.8% ± 0.8–10.6% ± 2.7%) and TREM2-TG-pNK (53.8% ± 2.8–13.3% ± 0.88%) cells were treated with TREM2-Ig during differentiation (Fig. 3A and B, upper panel). The absolute number of NK1.1+ cells decreased by 3.97- (WT) and 4.77-fold (TREM2-TG) after treatment with TREM2-Ig (Fig. 3C). Additionally, the percentage and absolute number of NK cells expressing Ly49C/F/H/I or Ly49D were reduced in both NK cells derived from WT-pNK (6.7–1.8% or 3.9–2.2%, respectively) and TREM2-pNK (7.3–4.7% or 7.1–3.1%, respectively) after TREM2-Ig treatment during differentiation (Fig. 3A and C). In contrast, the total number of cells differentiated from TREM2-TG-pNK or WT-pNK cells did not differ significantly, regardless of TREM2-Ig treatment (Fig. 3B, lower panel). Subsequently, we performed RT-PCR analyses to identify the expression of NK cell-associated genes regulated by TREM2 (Fig. 3D and Additional file 1, Fig. S5). NK cells that differentiated from TREM2-TG-pNK cells treated with hu-Ig showed increased IFN-γ (3.97 ± 0.63-fold) and Fas ligand (4.5 ± 0.3-fold) expression compared with that in NK cells derived from WT-pNK cells treated with hu-Ig. We also observed increased expression levels of granzyme B (1.8 ± 0.36-fold), perforin (1.25 ± 0.05-fold), and TNF-related apoptosis-inducing ligand (TRAIL; 1.9 ± 0.22-fold) in NK cells derived from TREM2-TG-pNK cells treated with hu-Ig compared with those in NK cells derived from WT-pNK cells treated with hu-Ig. In contrast, the expression levels of these genes were reduced in NK cells derived from both WT-pNK and TG-pNK cells when TREM2 signaling was inhibited by TREM2-Ig (Fig. 3D). Moreover, the expression levels of E4bp4, Id2, CD122, and CD123 increased in TREM2-TG-pNK-derived NK cells compared with those in WT-pNK-derived NK cells treated with hu-Ig, whereas the expression levels of these genes in differentiated NK cells decreased significantly after TREM2-Ig treatment (Additional file 1, Fig. S5).
TREM2 signaling inhibits tumor progression
As described above, inhibition of the TREM2 signaling pathway by TREM2-Ig reduced NK cell receptor and NK cell-associated gene expression, along with the absolute number of NK cells in vitro (Fig. 3). To confirm whether TREM2 affects tumor progression in vivo, we injected TREM2-TG or WT mice with B16F10 melanoma cells after intraperitoneal injection of hu-Ig or TREM2-Ig. As shown in Fig. 4A, on day 25, the tumor volume in WT mice treated with TREM2-Ig (WT + TREM2-Ig) was significantly higher than that in WT mice treated with hu-Ig (WT + hu-Ig). Differences in tumor volume between the two groups became even more prominent after day 25. Additionally, on day 25, the tumor volume in TREM2-TG mice treated with TREM2-Ig (TG + TREM2-Ig; 1988.14 ± 426.2 mm3) was higher than that in TREM2-TG mice treated with hu-Ig (TG + hu-Ig; 970.3 ± 257.11 mm3). Furthermore, on day 31, the tumor volume was significantly lower in TG + hu-Ig mice (1700.82 ± 171.142 mm3) than in WT + hu-Ig mice (3088.09 ± 808.67 mm3). On average, the tumor volume in TREM2-TG mice (3718.48 ± 1095.74 mm3) was approximately 2-fold lower than that in WT mice (7915.32 ± 839.09 mm3). These data showed that tumor progression in TREM2-TG mice was significantly reduced compared with that in WT mice, and tumor progression in both TREM2-TG and WT mice increased upon TREM2-Ig treatment. In addition, differences in survival rate were observed upon inhibition of TREM2 signaling. The survival rate of tumor-bearing WT mice injected with TREM2-Ig was 14.2%, which was significantly lower than that of tumor-bearing WT mice injected with hu-Ig (42.8%), tumor-bearing TREM2-TG mice injected with TREM-Ig (71.4%), and tumor-bearing TREM2-TG mice injected with hu-Ig (100%) on day 38 (Fig. 4B). Furthermore, the number of metastatic melanomas in the lungs of TREM2-Ig-injected WT mice (24 ± 4, B16F10 cell spots) was higher than that in the lungs of hu-Ig-injected WT mice (3 ± 1, B16F12 cell spots; Fig. 4C). Surprisingly, B16F10 melanoma cells were rarely observed in the lungs of hu-Ig-injected TREM2-TG mice, whereas melanoma cells were apparent in the lungs of TREM2-Ig-injected TREM2-TG mice (19 ± 1, B16F10 cell spots).
Adoptive transfer of TREM2-TG BM cells promotes tumor regression
As mentioned above, TREM2-TG mice showed a significantly lower tumor volume and rare metastatic tumor spots compared with WT mice when they were injected with B16F10 melanoma cells. This may be related to the effects of TREM2-overexpressing monocytes/macrophages or DCs, which secrete cytokines and indirectly activate T cells and NK cells. To investigate whether TREM2-overexpressing NK cells cause tumor regression, we transplanted CD45.2 TG-BMs (TG to WT) or CD45.2 WT-BMs (WT to WT) into sub-lethally irradiated WT recipients (CD45.1). Four weeks after adoptive transplantation, we analyzed the NK cell population in various organs in each group using flow cytometry. Higher proportions of NK cells (NK1.1+/CD3−/CD45.2+) were detected in the spleen (3.51% vs. 0.73%), BM (1.0% vs. 0.36%), and lungs (10% vs. 7%) of recipient mice (CD45.1 WT) engrafted with CD45.2 TG-BMs than in those engrafted with CD45.2 WT-BMs (Fig. 5A). These data indicated that TREM2-TG mice had a larger NK cell population than WT mice.
We then used an in vivo tumor model to determine whether TREM2 signaling affects the antitumor effect of NK cells. To this end, we subcutaneously transplanted B16-F10 melanoma cells into WT mice (CD45.1) engrafted with BMs (CD45.2) from WT or TREM2-TG mice and measured the tumor volume every other day. The tumor volume measured 21 days post-inoculation in mice transplanted with TREM2-TG-BMs (TG to WT, 1079 ± 221.5 mm3) was lower than that in mice that received WT-BMs (WT to WT controls, 3122.7 ± 1269 mm3; Fig. 5B). Furthermore, 27 days post-inoculation, the survival rate (75%) of tumor-bearing mice transplanted with TREM2-TG-BMs was significantly higher than that of tumor-bearing mice that had received WT-BMs (0%; Fig. 5C). To observe lung metastatic melanoma, we sacrificed mice from each group on day 14. Several melanomas (large black spots, 14 ± 1) were observed in the lung tissues of WT BM recipients (open bars, Fig. 5D); however, few melanomas were detectable (2 ± 1) in the counterparts transplanted with TREM2-TG cells (solid bars, Fig. 5D).
TREM2 regulates NK cell differentiation via PI3K signaling
TREM2-DAP12 signaling, triggered by TREM2 ligand binding, may promote or inhibit proinflammatory responses, induce obesity [33], and mediate neurodegeneration [34, 38]. DAP12, an adaptor protein of TREM2, mediates downstream signaling via the cytoplasmic ITAM domain, which recruits SYK and activates PI3K, phospholipase C, and Vav signaling cascades [42]. To investigate how TREM2 signaling regulates NK cell differentiation, we treated pNK cells differentiated from WT-HSCs or TG-HSCs with the PI3K inhibitor Ly294002 during their differentiation into mNK cells. After 14 days, differentiated mNK cells were stained with NK-specific markers and analyzed via flow cytometry (Additional file 1, Fig. S6A). In the absence of the PI3K inhibitor, the population of NK1.1+/NKG2ACE+ cells differentiated from TREM2-TG-pNK cells (dimethyl sulfoxide (DMSO) control; Additional file 1, Fig. S6A, lower panel) was 2-fold higher than that of NK cells differentiated from WT-pNK cells (DMSO control; Fig. S6A, upper panel). Interestingly, NK1.1+/NKG2ACE+ cell populations derived from both WT- and TREM2-TG-pNK cells decreased (10-fold) after Ly294002 treatment during NK cell maturation.
We also analyzed the expression of NK cell-associated genes in mNK cells differentiating in the presence or absence of the PI3K inhibitor. The expression levels of IFN-γ, perforin, and granzyme B were increased by 4- to 5-fold in mNK cells differentiated from TREM2-TG-pNK cells than that in mNK cells differentiated from WT-HSCs. Similarly, the expression levels of Fas ligand, TRAIL, and IL-15Rα were higher in mNK cells differentiated from TREM2-TG-pNK cells than those in cells differentiated from WT-pNK cells. With the exception of E4bp4 and IL-15Rα, NK cell-related gene expression levels significantly decreased in mNK cells treated with Ly49294002 during NK cell differentiation (Additional file 1, Fig S6). In particular, the expression level of Id2 decreased by more than 2-fold in mNK cells after treatment with Ly294002.