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DOI: 10.1002/eji.201545480

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TIGIT expression levels on human NK cells correlate with functional heterogeneity among healthy individuals Feng Wang, Hongyan Hou, Shiji Wu, Qing Tang, Weiyong Liu, Min Huang, Botao Yin, Jing Huang, Lie Mao, Yanfang Lu and Ziyong Sun Department of Clinical Laboratory, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Human NK cells display extensive phenotypic and functional heterogeneity among healthy individuals, but the mechanism responsible for this variation is still largely unknown. Here, we show that a novel immune receptor, T-cell immunoglobulin and ITIM domain (TIGIT), is expressed preferentially on human NK cells but shows wide variation in its expression levels among healthy individuals. We found that the TIGIT expression level is related to the phenotypic and functional heterogeneity of NK cells, and that NK cells from healthy individuals can be divided into three categories according to TIGIT expression. NK cells with low levels of TIGIT expression show higher cytokine secretion capability, degranulation activity, and cytotoxic potential than NK cells with high levels of TIGIT expression. Blockade of the TIGIT pathway significantly increased NK-cell function, particularly in NK cells with high levels of TIGIT expression. We further observed that the TIGIT expression level was inversely correlated with the IFN-γ secretion capability of NK cells in patients with cancers and autoimmune diseases. Importantly, we propose a novel mechanism that links TIGIT expression with NK-cell functional heterogeneity, and this mechanism might partially explain why individuals have different susceptibilities to infection, autoimmune disease, and cancer.

Keywords: Cytokine secretion r Cytotoxity r Human



r

NK cells

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TIGIT

Additional supporting information may be found in the online version of this article at the publisher’s web-site

Introduction NK cells are key effectors in innate immunity and play critical roles in the early control of infections and malignancies [1]. In recent years, there has been a growing concern that human NK cells can display extensive phenotypic and functional heterogeneity among individuals [2–7]. For example, the level of CD56 surface expression on NK cells varies significantly among individuals [8]. Addi-

Correspondence: Dr. Ziyong Sun e-mail: [email protected]  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

tionally, the release of IFN-γ and TNF-α by NK cells in response to mycobacteria varies 1000-fold among individuals [5]. Furthermore, the activation potential of human NK cells in response to Plasmodium falciparum-infected erythrocytes is distinctly variable among different donors [2, 9, 10]. These findings indicate that the functional heterogeneity of NK cells among individuals may be associated with the susceptibility to infection in human population. To date, only a few studies have been devoted to dissecting the basic functional heterogeneity in individual NK-cell behavior. There is growing evidence that heterogeneous killercell immunoglobulin-like receptor (KIR) genotypes among NKcell clones is related to the differing abilities of NK cells during www.eji-journal.eu

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disease states [5, 11, 12]. Given the importance of NK-cell heterogeneity and our incomplete understanding of this mechanism, further studies are needed. NK cells depend on a vast combinatorial array of receptors to initiate effector functions. Both activating and inhibitory receptors expressed on NK cells regulate their activity when interacting with tumors, virus-infected cells, and bacteria, as well as with healthy cells [1]. The best-known inhibitory receptors of NK cells are KIRs and CD94-NKG2A, whose physical ligands are MHC class 1 (MHC-1) molecules that are expressed on healthy cells to protect them from NK-cell lysis [13–15]. Other NK inhibitory receptors that do not interact with MHC-1 molecules also exist [16], such as IRp60, CEACAM1, and CD300a [17–19]. It was recently shown that a new inhibitory receptor, named T-cell immunoglobulin and ITIM domain (TIGIT), is expressed mainly on T cells and NK cells [20–22]. Poliovirus receptor (PVR, also known as CD155) is identified as the physical ligand of TIGIT, and TIGIT/PVR engagement suppresses T-cell activation through regulation of IL-10 secretion by dendritic cells [20]. TIGIT also has an intrinsic inhibitory function to T cells independent of antigenpresenting cells [23, 24]. Additionally, activation of TIGIT signaling can downregulate IFN-γ secretion and cytotoxicity in both human and mouse NK cells [22, 25, 26]. Very recently, it was reported that TIGIT alleviates liver injury through negatively regulating NK-cell activation in murine acute viral hepatitis and also that TIGIT is a safeguard molecule to improve liver regeneration through negatively regulating NK–hepatocyte crosstalk [27, 28]. Furthermore, regulatory T cells, also through expression of the coinhibitory molecule TIGIT, selectively inhibit proinflammatory Th1 and Th17 cell responses [29]. Although previous studies have documented the role of the TIGIT pathway in regulating NK-cell function, whether this new inhibitory receptor is associated with the functional heterogeneity of NK cells in different individuals is unknown. Here, we made the surprising observation that the TIGIT expression level on human NK cells shows wide variation among healthy individuals. We further found that NK cells with low levels of TIGIT expression had a higher cytokine secretion capability, degranulation activity, and cytotoxic potential than NK cells with high levels of TIGIT expression. Overall, our work identifies a novel mechanism by which TIGIT regulates NK-cell functional heterogeneity, and this might partially explain why individuals have different susceptibilities to infection, autoimmune disease, and cancer.

Results

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of TIGIT+ cells in peripheral blood cells ranged in a descending order from: CD56dim NK cells > NK cells > CD4+ CD25high regulatory T cells > CD8+ CD45RO+ memory T cells > CD4+ CD45RO+ memory T cells > CD8+ T cells > CD4+ T cells > CD4+ CD25low T cells > CD3+ CD56+ NKT cells. TIGIT was detected at only very low levels on B cells, monocytes, DC, and neutrophils (Fig. 1B and C; Supporting Information Fig. 1). We further observed that the expression of TIGIT on NK cells was unresponsive to stimulation with IL-12 or lipopolysaccharide (Fig. 1D). Interestingly, although TIGIT expression on NK cells seemed to be stable in vitro, it showed wide variation among different individuals. By analyzing the levels of TIGIT expression on NK cells in 199 healthy individuals, we observed that the percentages of TIGIT+ NK cells ranged from 20 to 90% (mean, 62.57%; median, 64.4%; Fig. 1E). The frequency distribution analysis showed that most of the tested individuals had TIGIT expression levels between 30 and 90% (Fig. 1F). We also examined the levels of CD69 expression on these NK cells and found that CD69 expression on NK cells was stable and showed no difference between the high or low TIGIT expression individuals (Supporting Information Fig. 1E). These data suggest that TIGIT expression levels on human NK cells display extensive variation among different individuals. Additionally, we measured the CD226 expression levels on NK cells because TIGIT exerts its function by competing with CD226 for the same ligand, PVR [30]. We observed that CD226 expression levels on NK cells also showed broad variation among different individuals. Furthermore, the expression levels of CD226 and TIGIT on NK cells were significantly correlated with one another (Fig. 1G).

Variation in TIGIT expression levels is related to NK-cell phenotype Next, we tried to determine whether the TIGIT expression level is related to the phenotype of NK cells. We found that TIGIT expression levels on NK cells had no correlation with the expression levels of activation markers CD25 and CD69, and the percentages of CD25+ and CD69+ cells were also not significantly different between TIGIT− and TIGIT+ NK cells (Fig. 2A and B). However, we observed that the TIGIT expression level was significantly inversely correlated with the CD107a and perforin expression levels in NK cells and that the expression of background CD107a on TIGIT− NK cells was significantly higher than that on TIGIT+ NK cells (Fig. 2C, D and E). Our results suggest that the TIGIT expression level is related to the degranulation potential of NK cells.

TIGIT expression levels on human NK cells show wide variation among healthy individuals To determine the range of TIGIT expression in healthy individuals, we used flow cytometry to assess the expression of TIGIT on a variety of cell types. We observed that TIGIT was expressed preferentially on NK cells, CD4+ CD25high regulatory T cells, and CD8+ CD45RO+ memory T cells (Fig. 1A). The mean percentages  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

TIGIT expression is inversely correlated with cytokine secretion and degranulation of NK cells We further investigated the relationship between the TIGIT expression level and NK-cell function. After IL-12 stimulation, the production of IFN-γ in TIGIT− NK cells was significantly higher www.eji-journal.eu

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Figure 1. TIGIT expression on human NK cells shows wide variation among health individuals. (A) Representative FACS histograms showing the expression of TIGIT on peripheral blood CD4+ T cells, CD8+ T cells, CD8+ CD45RO+ T cells, CD4+ CD25high T cells, and NK cells. The MFI of TIGIT is also shown as mean + SEM (n = 10 subjects per group, right). Data are from a single experiment representative of three. ** p < 0.01, *** p < 0.001 (Mann–Whitney U test), relative to the MFI of TIGIT on CD4+ T cells. (B) Representative FACS plots showing the expression of TIGIT on peripheral blood CD19+ B cells, CD14+ monocytes, CD11c+ DC, and CD13+ neutrophils. (C) Representative FACS plots showing the expression of TIGIT on peripheral blood CD4+ T cells, CD8+ T cells, and NK cells. The percentages of TIGIT+ cells are shown as the mean + SEM (n = 10–14 subjects per group). Data are from a single experiment representative of three. (D) PBMCs isolated from healthy individuals were stimulated with IL-12 or LPS for 24 h. The percentages of TIGIT+ NK cells are shown as mean + SEM (n = 16–23 subjects per group). Data are from a single experiment representative of three. (E) Scatter plots showing TIGIT expression on NK cells in 199 healthy individuals. Horizontal bar indicates the median. (F) Frequency distribution analysis of the percentages of TIGIT+ NK cells in 199 healthy individuals. (G) FACS plots showing the expression of CD226 on NK cells from two representative healthy individuals. Correlation between TIGIT and CD226 expression on NK cells is also shown (Spearman’s rank correlation test). Each symbol represents an individual donor.

than that in TIGIT+ NK cells (Fig. 3A). This result indicates that TIGIT functions as a negative regulator of human NK cells. Furthermore, the TIGIT expression level on NK cells was significantly inversely correlated with their IFN-γ-producing capacity among different individuals (Fig. 3B). Based on the levels of TIGIT expression among healthy individuals, we divided NK cells into three categories as follows: low-level TIGIT expression NK cells (TIGIT%:30–50%), middlelevel TIGIT expression NK cells (TIGIT%:50–70%), and high-level TIGIT expression NK cells (TIGIT%:70–90%). We found that the low-level TIGIT expression NK cells had a significantly higher IFN-γ-producing capacity after IL-12 stimulation than the high-level TIGIT expression NK cells (Fig. 3C). We similarly observed that after LPS stimulation, the CD107a expression level was also significantly inversely correlated with

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the TIGIT expression level on NK cells among healthy individuals (Fig. 3D and E). Moreover, after LPS stimulation, the percentage of CD107a+ NK cells in the low-level TIGIT expression category was significantly higher than that in the high-level TIGIT expression group (Fig. 3F). To further confirm the relationship between TIGIT expression levels and NK-cell degranulation, NK cells in PBMCs were cocultured with K562 cells. Under the same stimulation conditions, we found a similar relationship between TIGIT and CD107a expression levels on NK cells (Fig. 3G and H). These data suggest that the TIGIT expression level is inversely correlated with cytokine secretion and degranulation of NK cells among healthy individuals. However, the expression levels of CD25 and CD69 did not have any significant differences between the low- and highlevel TIGIT expression NK cells after LPS stimulation (Supporting Information Fig. 2).

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Innate immunity

TIGIT expression variation is inversely correlated with the cytotoxic potential of NK cells We next evaluated the proliferation, apoptosis, and cytotoxicity of NK cells with varied TIGIT expression levels. Following IL-2 stimulation, there was no difference in the amount of cell proliferation between the low- and high-level TIGIT expression NK cells (Fig. 4A). Although LPS stimulation induced slightly higher levels of apoptosis than the spontaneous apoptosis levels observed in unstimulated NK cells, the apoptosis levels in unstimulated and LPS-stimulated cells were not significantly different between the low- and high-level TIGIT expression categories (Fig. 4B). Furthermore, our results show that although the cytotoxicities of unstimulated low- and high-level TIGIT expression NK cells were similar, after stimulation with IL-12 the NK cells with low levels of TIGIT expression showed a significantly higher cytotoxicity than the NK cells with high levels of TIGIT expression (Fig. 4C and D). These data suggest that the TIGIT expression level is inversely correlated with the cytotoxic potential of NK cells.

Blockade of the TIGIT pathway increases NK-cell function, especially in high TIGIT-expressing cells

Figure 2. The relationship between TIGIT expression level and NKcell phenotype. (A, B) PBMCs isolated from healthy individuals were analyzed by flow cytometry. Correlation (A) between TIGIT and CD25 expression or (B) between TIGIT and CD69 expression on NK cells is shown (Spearman’s rank correlation test). The expressions of (A) CD25 and (B) CD69 on TIGIT− or TIGIT+ NK cells are expressed as the mean + SEM (n = 8–12 subjects per group) and are from a single experiment representative of three. (C) Correlation between TIGIT and CD107a expression on NK cells (Spearman’s rank correlation test). (D) FACS plot showing the expressions of TIGIT and CD107a on NK cells from a representative healthy individual. CD107a expression on TIGIT− or TIGIT+ NK cells is shown as the mean + SEM (n = 9 subjects per group). Data are from a single experiment representative of three. **p < 0.01 (Student’s t-test). (E) FACS histograms showing the expression of perforin in NK cells from two representative healthy individuals. Correlation between TIGIT and perforin expression in NK cells (Spearman’s rank correlation test). (A–C, E) Each symbol represents an individual donor.

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We next investigated whether a blockade of the TIGIT pathway would affect NK-cell function. Our results showed that addition of a TIGIT Fc chimera protein significantly increased the IL-12stimulated IFN-γ production by NK cells with high levels of TIGIT expression but not by total NK cells (Fig. 5A and B). Furthermore, the blockade of the TIGIT pathway by a TIGIT Fc protein significantly increased LPS-stimulated CD107a expression levels on both total and high-level TIGIT expression NK cells (Fig. 5C and D). A similar change was observed in the expression levels of IL-12-stimulated perforin; like the others, its expression levels were also significantly increased by blocking the TIGIT pathway, especially in NK cells with high levels of TIGIT expression (Fig. 5E and F). Additionally, we examined the effect of another functional anti-TIGIT monoclonal antibody on cytokine secretion by NK cells purified from healthy individuals. We observed that this antibody also increased the IL-12-stimulated IFN-γ production by high-level TIGIT expression NK cells (Fig. 5G). However, we did not observe a statistically significant difference in the proliferation, activation, or apoptosis of NK cells before or after blocking the TIGIT pathway (Supporting Information Fig. 3). We found that the TIGIT receptor PVR is expressed on human peripheral blood CD14+ monocytes (Fig. 5H). To further investigate the effect of blocking TIGIT signaling on NK cells, CD14+ monocytes were depleted from PBMCs by magnetic beads and the IFN-γ production of the remaining cells was assessed. Our results show that IFN-γ production after IL-12 stimulation was significantly increased in NK cells from monocyte-depleted PBMCs (Fig. 5I). These data confirm that the TIGIT pathway negatively regulates NK-cell function, especially in the high-level TIGIT expression category. www.eji-journal.eu

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Figure 3. TIGIT expression levels correlate with differential cytokine secretion and degranulation of NK cells. (A) PBMCs isolated from healthy individuals were stimulated with IL-12 for 24 h. The percentages of IL-12-stimulated IFN-γ in TIGIT− or TIGIT+ NK cells were measured by flow cytometry. Data are shown as the mean + SEM (n = 21 subjects per group) and are from a single experiment representative of three. ***p < 0.001 (Student’s t-test). (B) FACS plots showing the expressions of TIGIT and IL-12-stimulated IFN-γ in NK cells from two representative healthy individuals. Correlation between TIGIT expression and percentages of IL-12-stimulated IFN-γ in NK cells is also shown (Spearman’s rank correlation test). (C) The percentages of IFN-γ+ NK cells before and after stimulation with IL-12 in different TIGIT expression groups were evaluated by flow cytometry. Data are expressed as the mean + SEM (n = 8–15 subjects per group) and are from a single experiment representative of three. *p < 0.05, ***p < 0.001 (Mann–Whitney U test). (D–H) PBMCs were stimulated with LPS in the (D–F) absence or presence (G, H) of K562 cells for 24 h. (D) FACS plots showing the expressions of TIGIT and CD107a on NK cells from two representative healthy individuals. (E) Correlation between TIGIT and LPS-stimulated CD107a expression on NK cells (Spearman’s rank correlation test). (F, H) The percentages of CD107a+ NK cells in the (F) absence or (H) presence of K562 in different TIGIT expression groups were evaluated by flow cytometry. Data are shown as the mean + SEM (n = 5–8 subjects per group) and are from a single experiment representative of three. **p < 0.01 (Mann–Whitney U test). Individual 1: representing low-level TIGIT expression donor; Individual 2: representing high-level TIGIT expression donor. (B, E) Each symbol represents an individual donor.

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Figure 4. The relationship between TIGIT expression level and the proliferation, apoptosis, and cytotoxicity of NK cells. (A) Purified NK cells labeled with CFSE were stimulated with IL-2 for 5 days. FACS histograms showing the proliferation of NK cells from two representative individuals (M1: representing the percentage of NK-cell proliferation). The percentages of NK-cell proliferation in different TIGIT expression groups were evaluated by CFSE dilution by flow cytometry. Data are shown as the mean + SD (n = 5 subjects per group) and are from a single experiment representative of three. (B) PBMCs isolated from healthy individuals were stimulated with or without LPS for 24, 48, and 72 h. FACS plots showing the apoptosis of NK cells from two representative individuals. The apoptosis of NK cells in different TIGIT expression groups was analyzed at different time points and shown as the mean ± SD (n = 4 subjects per group) and from a single experiment representative of three experiments performed. *p < 0.05 (Mann–Whitney U test). (C) Purified NK cells stimulated with or without IL-12 for 24 h were collected as effector cells. K562 cells labeled with CFSE were used as target cells. Representative FACS plots showing the purity of effector cells and CFSE-labeled target cells. (D) Effector cells and target cells were coincubated at different (effector-to-target) E:T ratios for 6 h. CFSEhigh target cells were gated for analysis of PI intensities. FACS histograms showing the cytotoxicity of NK cells from two representative individuals (M1: representing the percentage of dead target cells). The cytotoxicity of NK cells in different TIGIT expression groups was analyzed and shown as the mean ± SD (n = 5 subjects per group) and from a single experiment representative of three experiments performed. **p < 0.01 (Mann– Whitney U test).

TIGIT expression variation on NK cells is related to cancer and autoimmune disease susceptibility Our above results demonstrated that variation in TIGIT expression levels among healthy individuals is associated with NK-cell functional heterogeneity. Thus, we hypothesized that the TIGIT expression level on NK cells might be related to disease susceptibility. As expected, we found that the TIGIT expression on NK cells from patients with gastrointestinal cancer was significantly higher than that on NK cells from healthy individuals, whereas  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

the opposite phenomenon occurred with TIGIT expression levels on NK cells from patients with autoimmune diseases (Fig. 6A). Although the mean values of TIGIT expression levels on NK cells from both latent tuberculosis infection (LTBI) patients and inactive Hepatitis B virus (HBV) carriers tend to be higher than that on NK cells from healthy individuals, these values are not significantly different from one another (Fig. 6A). We further investigated the relationship between TIGIT expression levels and NK-cell function in these disease states. We observed that the percentages of IFN-γ-producing NK cells after

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IL-12 stimulation were significantly increased in rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) patients but notably decreased in gastrointestinal cancer patients, when compared with that in healthy individuals (Fig. 6B and C). Furthermore, our results showed that TIGIT expression levels were also significantly inversely correlated with the IFN-γ-producing capability of NK cells in healthy individuals and in patients with these diseases (Fig. 6D). These data imply that variation in TIGIT expression levels among individuals might be associated with susceptibility to cancer and autoimmune disease.

Discussion Studies have increasingly demonstrated that NK cells are genetically, phenotypically, and functionally diverse, both at the human population level and within individuals [31–33]. This phenotypical and functional heterogeneity of NK cells might be associated with the varying susceptibilities to diseases, such as infection, autoimmune disease, and cancer, among individuals. There is growing evidence that NK-cell responses are finely tuned by a process involving an interaction between KIR on NK cells and host-specific MHC-1 molecules [34]. Other studies have shown that variations among individuals in the repertoires of KIR alleles expressed by NK cells can be associated with differences in NK-cell responses to pathogen-associated signals and with resistance or susceptibility to different diseases [11, 12]. Despite these findings, the mechanism that regulates NK-cell functional variation among different individuals is still largely unknown. The data presented here put forward a new mechanism for NK-cell functional heterogeneity. We found that a novel immune receptor TIGIT shows wide variation among healthy individuals, and this phenotypic difference is significantly inversely correlated with several NK-cell functions. TIGIT, as a surface protein containing an extracellular IgV-like domain, a transmembrane domain, and an intracellular domain which includes one ITIM motif, was recently identified as an inhibitory receptor that is expressed mainly on regulatory T cells, activated CD4+ and CD8+ T cells, and NK cells [20]. There are some discrepancies in the reported expression levels of TIGIT on different immune cells between publications [20–22], which is probably owing to the different mAbs used by the various groups [35]. Thus, to confirm the expression of TIGIT on human peripheral blood immune cells, we used a commercial APC-labeled antiTIGIT mAb (MBSA43, 17–9500) to detect TIGIT expression. We

Innate immunity

observed that TIGIT was expressed preferentially on CD56dim NK cells, CD4+ CD25high regulatory T cells, and CD8+ CD45RO+ memory T cells. The characteristic features of TIGIT expression that we observed can be summarized as: (1) CD56dim NK cells have a higher TIGIT expression level than CD56bright NK cells, (2) activated T cells have a higher TIGIT expression level than nonactivated T cells (CD25high CD4 > CD25dim CD4 > CD25low CD4), (3) memory T cells have a higher TIGIT expression level than naive T cells (CD45RO+ CD 8/CD4 > CD45RO− CD8/CD4), and (4) antigen-presenting cells have minimal levels of TIGIT expression. Furthermore, we also used a second commercially available PElabeled anti-TIGIT antibody (12-9500) to detect TIGIT expression and similar trends in the TIGIT expression levels were observed (Supporting information Fig. 1F). The relative TIGIT expression levels on peripheral immune cells suggest that this molecule might play crucial roles in the effector functions of innate and adaptive immunity. Regarding the regulation of TIGIT expression on NK cells, results from one study have shown that a variable proportion of human NK cells express TIGIT upon stimulation with phytohemagglutinin and IL-2 [21]. Furthermore, in an animal model, the expression levels of TIGIT on both splenic and hepatic NK cells are relatively low in healthy mice but are significantly upregulated during acute viral hepatitis [27]. However, we observed in our study that the TIGIT expression on human NK cells did not change significantly following stimulation with IL-12 or LPS in vitro, which is also consistent with a previous study [36]. Therefore, we speculate that the expression level of TIGIT on human NK cells might be naturally high, which is different from that on mouse NK cells. Moreover, by evaluating the TIGIT expression on NK cells from 199 healthy individuals, we observed that TIGIT expression levels on human NK cells showed wide variation among healthy individuals. We also found that low-level TIGIT expression NK cells had a higher degranulation activity, cytokine secretion capability, and cytotoxic potential than high-level TIGIT expression NK cells. These data suggest that the variation in TIGIT expression levels is inversely correlated with NK-cell function, which indicates that this variation among healthy individuals is associated with NK-cell functional heterogeneity. To further investigate the functional difference between highand low-level TIGIT expression NK cells, a recombinant TIGIT Fc chimera protein and another functional anti-TIGIT antibody were used in this study. The properties of agonistic versus nonagonistic antibodies are currently undefined, and this makes prediction of the behavior of a given antibody difficult. At least one



Figure 5. Blockade of the TIGIT pathway affects NK-cell function. (A–F) PBMCs isolated from healthy individuals were stimulated with IL-12 or LPS in the presence of TIGIT Fc protein or IgG control for 24 h. The expressions of (A, B) IFN-γ, (C, D) CD107a, and (E, F) perforin in NK cells were evaluated by flow cytometry. (A, C, and E) FACS plots and histograms showing the expressions of (A) IFN-γ, (C) CD107a, and (E) perforin in NK cells from two representative individuals. (B, D, and F) The percentages of (B) IFN-γ+ cells, (D) CD107a+ cells, and the MFI of (F) perforin in total NK cells or in NK cells with different TIGIT expression levels are shown. (G) Purified NK cells were stimulated with IL-12 in the presence of anti-TIGIT antibody or IgG control for 24 h. The percentages of IFN-γ+ cells in total NK cells or in NK cells with different TIGIT expression levels are shown. (H) PBMCs isolated from healthy individuals were analyzed by flow cytometry. Representative FACS histograms showing the expression of PVR on CD14+ monocytes. (I) PBMCs or monocyte-depleted PBMCs were stimulated with IL-12 for 24 h. Representative FACS dot plots showing IFN-γ expression in NK cells from PBMCs or monocyte-depleted PBMCs. The percentages of IFN-γ+ NK cells in different groups were shown. (B–I) There are 5–13 subjects in each different TIGIT expression group. Data are from a single experiment representative of three experiments performed. *p < 0.05, **p < 0.01 (Student’s t-test). Each pairwise comparison represents an individual donor.  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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Figure 6. Variation in TIGIT expression levels on NK cells from patients with cancer and autoimmune disease. (A) Patients with LTBI (n = 22), inactive HBV carriers (n = 23), and patients with SLE and RA (n = 14), and gastric and colon cancer (n = 22) were recruited for analysis of TIGIT expression by flow cytometry. The expression of TIGIT on NK cells from patients with above diseases was compared with TIGIT expression on NK cells from healthy individuals (n = 199). Each symbol represents an individual donor, and horizontal bars indicate the median. (B) FACS plots showing the expression of IFN-γ in NK cells from a representative healthy individual, a representative RA/SLE patient, and a representative gastric/colon cancer patient after IL-12 stimulation. (C) The percentages of IFN-γ+ NK cells before and after IL-12 stimulation in healthy individuals, RA/SLE patients, and gastric/colon cancer patients are shown. The percentages of IFN-γ+ NK cells after IL-12 stimulation are shown as the mean + SEM (n = 14–20 subjects per group) and are from a single experiment representative of three experiments performed. (D) Correlation between TIGIT expression and IL-12-stimulated IFN-γ production in NK cells from healthy individuals, RA/SLE patients, and gastric/colon cancer patients (Spearman’s rank correlation test). (A–C) **p < 0.01, ***p < 0.001 (Mann–Whitney U test).

study has shown that agonistic anti-TIGIT antibodies can inhibit T-cell responses [37]. However, we observed that the functional anti-TIGIT antibody used in our study increased IFN-γ secretion by NK cells, so we speculate that this functional antibody is a nonagonistic antibody that can be used to block the TIGIT pathway. Our findings agree with those of another group, who also reported that this antibody increased cytokine secretion [24]. Both the TIGIT Fc chimera protein and the anti-TIGIT antibody

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significantly increased IFN-γ production by NK cells with high levels of TIGIT expression, which indirectly suggests that high- and low-level TIGIT expression NK cells have different functions and that the functions of high-level TIGIT expression NK cells might be more inhibited than those of low-level TIGIT expression NK cells. We also observed that the TIGIT ligand PVR was expressed on CD14+ monocytes. After removing monocytes from PBMCs, the IFN-γ production secreted by the NK cells in the remaining

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mixture of cells was significantly increased following IL-12 stimulation. Together, these data suggest that blockade of TIGIT signaling increases NK-cell activity. Furthermore, CD56bright NK cells release high levels of cytokines, whereas CD56dim NK cells are potent cytotoxicity [8]. Our results show that TIGIT is expressed on both CD56bright and CD56dim NK cells. Thus, blockade of TIGIT signaling influences both the cytokine secretion and the cytotoxicity of NK cells. Impaired NK-cell function is found in cancer patients [38], which emphasizes the importance of NK-cell immune system surveillance in the prevention of malignancy. Conversely, NK-cell involvement in the direct killing of tissue cells and in IFN-γ secretion accelerates the progression of autoimmune diseases [39]. Thus, the human body needs to maintain an appropriate level of NK-cell function to stay healthy. Individuals with NK-cell function that is too low or too high may have an increased susceptibility to cancer or autoimmune disease, respectively. As expected, we observed that NK cells from patients with gastrointestinal cancer had a high-level of TIGIT expression but a lower IFN-γ secretion capability. In contrast, NK cells from patients with SLE or RA had a low-level of TIGIT expression but a higher IFN-γ secretion capability. Given that the expression level of TIGIT on NK cells is stable in vitro under different stimulation conditions, we speculate that the TIGIT expression level on NK cells might be inherent and is not increased or diminished in these disease states. These findings suggest a new mechanism for NK-cell functional heterogeneity and might shed light on one of the reasons why individuals have different susceptibilities to autoimmune disease and cancer. Taken together, our findings have revealed a new mechanism to explain NK-cell functional heterogeneity among healthy individuals. We found that the levels of TIGIT expression on NK cells show wide variation and that low TIGIT expression NK cells perform relatively high levels of cell functions compared to high TIGIT expression NK cells. Intriguingly, the TIGIT expression level on NK cells is also inversely correlated with NK-cell function in cancer and autoimmune disease. This study proposes a novel potential mechanism by which TIGIT regulates NK-cell functional heterogeneity, which might partially explain why individuals have different susceptibilities to infection, autoimmune disease, and cancer.

Materials and methods Study subjects This study was approved by the ethical committee of Tongji hospital, Tongji Medical College, Huazhong University of Science and Technology. Participants were recruited from Tongji Hospital, the largest hospital in central region of China. All participants were older than 18 years. After giving written informed consent, each participant was asked to complete a questionnaire about his or her previous medical conditions and treatments. Healthy volunteers were defined as those without any clinical symptoms or signs of  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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disease. Exclusion criteria were pregnancy, tuberculosis, HIV or HBV infection, diabetes mellitus or renal failure, and any drug intake. We also recruited patients with the following diseases: (1) inactive HBV carrier state, individuals with HBsAg+ over 6 months, HBeAg− and anti-HBe+ , and persistently healthy aminotransferases and undetectable serum HBV DNA levels [40]; (2) LTBI, individuals with positive T-SPOT.TB (Oxford Immunotec, Oxford, England) results but without clinical or radiographic evidence of active TB; (3) autoimmune disease, patients with autoimmune diseases such as SLE and RA were diagnosed by the revised criteria for SLE and RA proposed by the American College of Rheumatology [41, 42]; and (4) gastrointestinal cancer, patients with gastrointestinal cancers such as gastric and colon cancer were diagnosed by pathological examination.

Cell preparation and activation PBMCs were isolated from heparinized blood of healthy individuals and patients by using Ficoll–Hypaque density gradients (Sigma–Aldrich). Purified NK cells were obtained by negative selection from PBMCs using the NK cell Isolation Kit II (Miltenyi Biotec) according to the manufacturer’s protocol. CD14+ monocytes were depleted from PBMCs by using magnetic beads coupled to anti-CD14 mAb (Miltenyi Biotec). For cell stimulation studies, PBMCs, monocyte-depleted PBMCs, or purified NK cells were stimulated with IL-2 (1000 U/mL, R&D Systems), IL-12 (100 U/mL, BioLegend), or LPS (10 μg/mL, Sigma) for 24 h. In some experiments, recombinant human TIGIT Fc Chimera protein (5 μg/mL, R&D Systems), functional antihuman TIGIT antibody (5 μg/mL, eBioscience), or IgG control (R&D Systems) were added to the culture medium for blocking TIGIT pathway.

Flow cytometric analysis Cell surface staining was performed on PBMCs or whole blood cells (after lysis of erythrocytes). Monoclonal antibodies against the following antigens were added to the cell suspensions: CD3, CD56, CD69, CD107a, and TIGIT (eBioscience); CD4, CD8, CD25, CD45RO, CD19, CD11c, CD14, and CD13 (BD Pharmingen). Isotype controls with irrelevant specificities were included as negative controls. All of these cell suspensions were incubated for 30 min on ice. For intracellular staining, the cells were fixed and permeabilized, and stained with anti-IFN-γ and antiperforin mAbs (eBioscience). After washing, the pellets were resuspended in 500 μL cold staining buffer, followed by analysis with FACSCalibur cytometer (Becton Dickinson). Data analysis was performed using FlowJo version 7.6.1 software (TreeStar).

Cytotoxicity assay NK-cell cytotoxicity assay was performed as described previously [43]. In brief, NK cells purified from PBMCs of healthy individuals www.eji-journal.eu

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were stimulated with or without IL-12 (100 U/mL) for 24 h. After culture, the NK cells were collected as effector cells. K562 cells were labeled with CFSE (Sigma–Aldrich) and were used as target cells. Effector cells and target cells were coincubated at effectorto-target (E:T) ratios of 1:1, 5:1, and 10:1 in duplicate wells for 6 h at 37°C with 5% CO2 . Control tubes including only target cells were assayed to determine spontaneous cell death. After two washings, the cells were suspended in 200 μL, 1 × binding buffer (eBioscience). Five microliter of propidium iodide (eBioscience) was added to each tube and the cells were incubated for 5 min at room temperature in the dark. The cells were then quantified immediately by flow cytometry and the dead target cells were indicated as CFSEhigh PI+ cells.

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Conflict of interest: The authors declare no financial or commercial conflict of interest.

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Acknowledgments: This work was supported by the Infectious Diseases Control Project from Ministry of Health of China (2012zx10004-207) and the National Natural Science Foundation of China (81401639).  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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Abbreviations: HBV: Hepatitis B virus · KIR: killer-cell immunoglobulin-

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TIGIT: T-cell immunoglobulin and ITIM domain

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Full correspondence: Dr. Ziyong Sun, Department of Clinical Laboratory, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Fax: +86-02783663639 e-mail: [email protected]

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 C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Received: 16/1/2015 Revised: 3/6/2015 Accepted: 8/7/2015 Accepted article online: 14/7/2015

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