Loss-of-Function in SMAD4 Might Not Be Critical for Human Natural Killer Cell Responsiveness to TGF-β

We characterized the NK cell phenotype and function in three family members with Hereditary Hemorrhagic Telangiectasia (HHT) due to heterozygous SMAD4 mutations. Loss-of-function mutation in this gene did not induce developmental effects to alter CD56bright or CD56dim NK cell subset proportions in peripheral blood; and did not result in major differences in either their IL-15-induced proliferation, or their cytokine secretion response to TGF-β1. These data suggest that SMAD4 plays a redundant role in downstream TGF-β signaling in NK cells.


INTRODUCTION
Natural killer (NK) cells are bone marrow-derived innate lymphocytes that are abundant in blood and lymphoid tissues and possess spontaneous anti-tumor activities (1). These activities are triggered by engagement of receptors to stress-induced ligands and lack of engagement of inhibitory self-ligands (2). The role of NK cell immunosurveillance in homeostasis is critical to identify early transformed cells, to prevent metastasis of circulating tumor cells to different body compartments (3,4). The physiology of the inhibitory NK cell checkpoints is currently under investigation and is being targeted for translational immunotherapeutic development (5). Both murine and human NK cells are dependent on gamma chain cytokine signaling (e.g., IL-2 or IL-15) for their development, differentiation, homeostasis, and priming (6). In contrast, TGF-β signaling was shown to override NK cell metabolism in both species to inhibit their effector functions (7,8). In addition to inhibiting effector functions, TGF-β signaling was also shown to upregulate tissue residency features in NK cells, suggesting its role in potentially differentiating conventional NK cells into more pro-angiogenic and less cytotoxic innate lymphoid cell (ILC)1-like cells (9,10).
The TGF-β superfamily is a well-conserved pathway and is highly homologous across different species. The canonical signal transduction from the TGF-βRI/II heterocomplex phosphorylates SMAD2 and 3 and activates a signal transduction mediated by a complex assembled by several potential hetero/homo-combinations of phosphorylated SMAD2 and SMAD3. Subsequent interactions with the molecule SMAD4 translocate the complex to the cell nucleus to induce transcriptional activity (11). Although it is not a receptor / substrate, SMAD4 is believed to be necessary to aid pSMAD2/3 -triggered TGF-β gene responses. However, the specific function that SMAD4 adds to the transcriptional complexes is still unclear (12).
In humans, Hereditary Hemorrhagic Telangiectasia (HHT) results from autosomal dominant loss-of-function mutations for specific components of the TGF-β superfamily: ENG, encoding endoglin (HHT1), ACVRL1 (HHT2), or more rarely SMAD4. Patients with HHT typically present with mucosal telangiectasia resulting in epistaxis, gastrointestinal bleeding, and anemia. Other clinical features include multi-organ arteriovenous malformations, and colonic polyps (commonly observed only in SMAD4-HHT patients). Under clinical management by specialized services, these patients can have a relatively normal lifespan (13). Studies phenotyping immune cell development in these patients are yet to be comprehensively performed. One recent report characterizing functions of IL-2-expanded NK cells from a patient with a SMAD4-loss-of-function mutation, demonstrated augmented response to TGF-β signaling and suggested an unexpected inhibitory role of SMAD4 in TGF-β signaling (14). However, observations from a single patient case report cannot be reliably generalized.
In this current report, we studied different aspects of NK cell phenotype and function from a mother, son and daughter with HHT classified as SMAD4-loss-of-function. Complementary to Cortez et al. we aimed to access a larger number of steady-state observations from different NK cell subsets, and to characterize responsiveness to TGF-β1 in vitro in a number of different assays, ranging from proliferation to cytotoxicity and cytokine production. We observed a number of parameters that suggest that SMAD4 plays a redundant role into responsiveness to TGF-β1 in human NK cells, with mutated cells displaying minimal differences in numbers, subset proportions, proliferation, cytotoxicity, and cytokine production along different maturation phenotypic stages.

CASE REPORTS
Patient HHT 1949F, a 69-years-old woman, had suffered from minor epistaxis and major bowel symptoms, mainly constipation, since her teenage years. At age 37 years she underwent partial colectomy for colonic cancer arising in a polyp. An episode of hematemesis from a bleeding gastric polyp necessitated partial gastrectomy, and she is now prone to recurrent hypoglycemic episodes. There was no history of frequent infective episodes, and she reported normal wound healing. Her father suffered from frequent and copious nosebleeds and died from a cerebrovascular event aged 56 years. A diagnosis of Juvenile Polyposis/Hereditary Hemorrhagic Telangiectasia (JP/HHT) was confirmed by identification of a frameshift mutation in SMAD4 (NP_005350.1:p.Ser232GInfs * 3), leading to a premature stop codon. Her son (Patient HHT 1965M,) and daughter (Patient HHT 1967F, described below have both inherited the SMAD4-mutation. Patient HHT 1965M, aged 53, is the son of the above, inherited the same SMAD4 mutation. He underwent Whipple's surgery in his early 20s, for upper GI bleeding from extensive polyps. At 46-years of age, he suffered large bowel intussusception from polyps. Recent identification of significant iron deficiency anemia led to extensive endoscopic procedures including antegrade push enteroscopy, colonoscopy, and Pill-cam surveillance. Multiple ulcerated jejunal polyps were removed endoscopically, though many remain. Three polyps were also removed from the descending colon, the rectum, and the anorectal verge. Other significant past history included five episodes of pneumonia, starting in childhood.
Patient HHT 1967F, aged 51, daughter of HHT 1949F, experience significant skeletal pain and deterioration of bones and teeth. She suffered from numerous co-morbidities since childhood, including abdominal pain and rectal bleeding. She has recurrent kidney stones and previous pyleonephritis. Ongoing blood loss requires frequent iron infusions, and she undergoes SMAD4 mutation-related active surveillance for bowel cancer.

Reagents
Commercial antibodies and reagents to detect human epitopes and stimulating cytokines used in this study are listed below:

Patients
Inclusion required a clinical diagnosis of HHT, and confirmation of the causative mutation.

NK Cell Preparations and Culture Conditions
Heparinized peripheral blood (∼30 mL) was obtained for each patient or healthy age-matched donor and processed by Ficoll-

Statistical Analysis
Statistical analyses (as shown in the Figure legends) were performed using GraphPad Prism 7 software.

Ethics
This study was carried out in accordance with approval of the Melbourne Health and Walter and Eliza Hall Institute of Medical Research's Human Research Ethics Committee (approval number: 2013.081). All subjects gave written informed consent for participation and publication.

RESULTS AND DISCUSSION
TGF-β signaling in NK cells is associated with: phosphorylation in SMAD2 and 3, inhibition of IL-15-induced metabolism/proliferation, simultaneous downregulation of CD44, CD49e, and Eomes, and upregulation of CD16 and CD49a expression (7,10). SMAD family member 4 (SMAD4) belongs to the SMAD family of transcription factor proteins which combine in heterocomplexes with SMAD2 and 3, and still have an unclear role during the signal transduction (11,12). In this case report, we obtained samples from patients carrying a loss-of-function mutation in the SMAD4 (p.Ser232GInfs * 3) expected to generate a protein without function and prone to be degraded upon expression. We accessed by western blot the SMAD4 protein expression, from fresh isolated NK cell by negative selection, two SMAD4-mutants and 2 health donors, and confirmed a lower expression of SMAD4 in both patient samples (data not shown). SMAD4-mutant and healthy donor NK cell phenotypes were assessed by gating the CD56 bright and CD56 dim subsets from the viable CD3 neg , CD14 neg , CD20 neg , CD45 + , CD66b neg , NKp46 + PBMC population. Although all donors were clinically classified as infection-free at time of donation, SMAD4-mutant CD56 bright and CD56 dim NK cells displayed decreased numbers in blood (Supplementary Figure 1). By overlaying the respective NK cell subsets (CD56 bright or dim ) from healthy donors (HD1, 2, and 3) and patients (HHT1, 2, and 3), multiparametric flow cytometry comparison of cell surface markers revealed no consistent/significant differences were observed in either of the SMAD4-deficient samples for CCR7, CD16, CD49a, CD49e, CD62L, and Eomes expression (Figures 1A-F, respectively, and Supplementary Figure 2). CD62L expression can subdivide CD56dim NK cells into a CD56 dim CD62L + population, which displays an intermediate maturation status between the CD56 bright NK cells (all CD62L + ) and CD56 dim CD62L neg subsets (17). SMAD4-deficient donor HHT2 displayed a striking upregulation of CD62L in CD56 dim cells, but this effect was not seen in HHT1 or 3 who showed levels similar to the healthy donors (Supplementary Figure 2).
Considering that TGF-β1 is a pleiotropic cytokine we also assessed if its levels were systemically increased at steady-state due to the clinical conditions associated with the SMAD4 mutation. However, we could not observe any major increase of TGF-β1 in the plasma of SMAD4-deficient samples (data not shown).
To ascertain the role of SMAD4 in the NK cell, CD56 subsets were sterilely sorted by negative selection to achieve high purity, and compared in in vitro functional assays in the presence of activating cytokines and presence or absence of TGF-β1 to examine their responsiveness to this cytokine. Combined with IL-15, IL-12, and IL-18 efficiently upregulate and sustain expression of cytokines such as GM-CSF and IFNγ in human NK cells (18,19), in mechanisms governed by Eomes (to drive the NK cell maturation program) and T-bet (to drive transcriptional regulation of cytokine genes such as IFN-γ) (20). IFN-γ and GM-CSF expression was decreased in SMAD4-deficient NK cells from all subjects by TGF-β1stimulation, similarly to health donors NK cells (Figures 2A,B;  Supplementary Figure 3). By contrast, T-bet expression was not lowered, while Eomes was further inhibited in the presence of TGF-β1 (Figures 2C,D; Supplementary Figure 3). Considering that those effector molecules, among others, are involved in NK cell immunosurveillance and control of target tumor cells, we next speculated that the cytotoxicity function of the SMAD4deficient NK cells might be differentially affected.
Cortez et al. assessed IFN-γ production as an activation outcome of SMAD4-deficient NK cells from one donor after co-culture with K562 targets, therefore the target cell killing was not measured (14). To address the role of SMAD4 in cytotoxicity, we next sorted highly purified CD56 dim NK cells, the more cytotoxic subset [(21) and Supplementary Figure 3], from healthy and SMAD4-deficient donors and primed them with IL-15 prior to adding K562 targets in a 4:1 ratio as in the study of Cortez et al. By using the Annexin V and PI method of K562 apoptosis quantification (22), we first observed a high viability in target cell cultures without effector cells (Figure 3A), which was dramatically decreased after the addition of IL-15-primed control or SMAD4-deficient NK cells for 4 h ( Figure 3B). However, live and death in K562 cells were not significantly changed in coculture with SMAD4-deficient or WT NK cells in presence of IL-15-treated NK cells This result suggested that cytotoxicity of SMAD4-deficient NK cells is not suggested to be affected by the mutation.
IL-15 signaling is a critical survival factor which also induces NK cell priming and proliferation. This is attracting great interest in manipulating this pathway to enhance NK cell function for immunotherapy development (5, 6). TGF-β signaling was previously shown by us and others to be an antagonistic pathway that efficiently represses IL-15-induced proliferation in NK cells FIGURE 4 | SMAD4-deficient NK cells display similar susceptibility to TGF-β-mediated inhibition of cellular proliferation as WT cells. CTV-labeled NK from three health donors (A) and three SMAD4-mutated donors (B), were cultured in presence of rIL-15 and with or without addition of rTGF-β1 as indicated. For each condition at 40, 84, and 132 h intervals, the total live cells were enumerated and mean division number was calculated. Circle dots represent health donors, and squares represent HHT donors. Color code was applied to represent the expression of each parameter in each donor alongside (black = donor #1, blue = donor #2 and red = donor #3). Results are representative from triplicate of each biological replicate. (7,23). We next examined whether SMAD4-deficient NK cells would display differential proliferation potential in response to IL-15 (Figures 4A,B). Conversely, TGF-β1 efficiently reduced the mean division number of NK cells from both healthy donors (Figure 4A), and SMAD4-defficient cells (Figure 4B). These results suggest that SMAD4 deficiency does not cooperate with SMAD2 and 3 to suppress IL-15-induced proliferation in human NK cells.

CONCLUDING REMARKS
A limitation inherent in reports based on a single or few donors, is the risk of over-interpretation from a limited sampling size. SMAD4 mutations are rare, and obtaining a large sample cohort is challenging. In the current study we described the NK cell phenotype of three family members carrying the same loss-of-function SMAD4 mutation. Although a previous study suggested that SMAD4 acts as an inhibitory molecule to the TGF-β signaling in human NK cells, our results suggests that this molecule plays a redundant role for human NK cell development and function. The clinical impact of SMAD4 mutations on immunity has not been well-characterized, due to the rarity of the condition, and the predominance of other clinical features. Future studies using a larger cohort may determine the redundancy of specific signaling members of the TGF-β superfamily for NK cell biology, and help us understand the complexity of their roles in the immune status of patients carrying mutations in these genes.

ETHICS STATEMENT
This study was carried out in accordance with approval of the Melbourne Health and Walter and Eliza Hall Institute of Medical Research's Human Research Ethics Committee (approval number: 2013.081). All subjects gave written informed consent in accordance with the Declaration of Helsinki, and donors provided informed consent for publication.

AUTHOR CONTRIBUTIONS
LH, GR and JR assisted with the performance of experiments. FS-F-G conducted the experiments and analyzed the data. NH, CS, IW, and FS-F-G interpreted the data and reviewed and revised the manuscript. LH, and IW genotyped, diagnosed, and recruited the patients for the study. LH, NH, IW, and FS-F-G cowrote the manuscript, reviewed the manuscript, and agreed with all aspects of work. FS-F-G and IW designed and supervised the study. All authors approved the manuscript and agreed with all aspects of the work.

FUNDING
This work is supported by project grants from the National Health and Medical Research Council (NHMRC) of Australia (#1124784, #1066770, #1057852, #1124907 to NH; and #1140406 to FS-F-G). FS-F-G. was supported by a NHMRC Early Career Fellowship (1088703)