Natural Killer T Cells Are Numerically and Functionally Deficient in Patients with Trauma

Trauma refers to sudden physical injury caused by an external force. It is one of the leading causes of death worldwide. According to the Global Burden of Disease Study 2017, injuries account for 4.48 million deaths annually or an age-standardized death rate of 57.9 per 100,000 individuals [1]. The injury severity score (ISS) is regarded as the gold standard to assess trauma severity. It is correlated with mortality, morbidity, and hospitalization time after trauma [2]. According to the ISS, traumatic injuries are classified into mild (ISS <9), moderate (9≤ ISS ≤15), and severe or major trauma (ISS >15) [2]. In the past, most people with major trauma died from initial blood loss without medical care. However, after major trauma many people are now able to survive due to advances in treating hemorrhage and coagulopathy [3]. In most survivors, traumatic injury often evokes two opposing immune responses: (1) systemic inflammatory response syndrome (SIRS) characterized by increased levels of inflammatory cytokines and immune cell activation, and (2) compensatory anti-inflammatory response syndrome (CARS) characterized by elevated levels of anti-inflammatory cytokines and immune paresis [4]. A study by Hazeldine et al. [4] reported that traumatic injury resulted in immediate immune dysfunction, supported by evidence of concomitant immune activation and suppression detected within minutes of injury. Although the purpose of the inflammatory response was once protective against infection, paradoxically, these marked immune alterations (i.e., CARS immediately following SIRS) now reduce the survivors’ resistance to infection. These marked immune alterations involve both innate and adaptive immune systems. Furthermore, survivors still remain at risk for developing multiple organ dysfunction syndrome and sepsis [5].

Human natural killer T (NKT) cells are a distinct subset of T cells that express an invariant Vα24-Jα18 T cell receptor (TCR) chain paired with the Vβ11 TCR chain. TCR αβ pairs can recognize self or foreign glycolipids such as α-galactosylceramide (α-GalCer) presented by CD1d, a major histocompatibility complex class I-like molecule as cognate antigen [6]. NKT cells play a bridging role between innate and adaptive immune cells, including dendritic cells, monocytes, NK cells, T cells, and B cells by rapidly producing large amounts of Th1 and Th2 cytokines such as interferon-γ (IFN-γ) and interleukin (IL)-4 [7, 8]. Furthermore, NKT cells play either a protective or harmful role in a broad range of diseases, including autoimmunity, cancer, infection, sepsis, and ischemia/reperfusion-related tissue injury [8-10].

Several recent studies have shown that trauma-induced immune alterations, such as elevated proinflammatory cytokines [11, 12], impaired leukocyte function [13, 14], and altered monocyte phenotype [15], are associated with or predictive of mortality, multiple organ failure, and sepsis [4]. Thus, monitoring alterations of the immune system in trauma patients may play a potential role in assessing future poor outcomes. However, these studies have been limited to monocytes and neutrophils among innate immune cells. Our previous study has shown that circulating NKT cells are deficient in trauma patients and that their deficiency is associated with the severity of trauma [16]. These findings suggest that NKT cells may play an important role in the innate immune response to traumatic injury. However, little is known about immune alterations in NKT cells in response to trauma. Accordingly, the aims of this study were: (i) to examine numbers and proliferative responses of NKT cells, (ii) to examine Th1/Th2 cytokine levels produced by NKT cells, and (iii) to determine the mechanism responsible for NKT cell dysfunction in trauma patients.

The study cohort was composed of 32 patients with trauma (11 females and 21 males; mean age ± standard deviation [SD]: 60.1 ± 15.6 years) who visited Chonnam National University Hospital Regional Trauma Center and 32 non-injured healthy controls (HC; 16 females and 16 males; mean age ± SD: 50.0 ± 10.8 years). Blood samples were usually obtained from patients within 12–36 h after traumatic injury. The subjects were enrolled among residents living in Jeollanam-do province, South Korea from October 2016 to September 2018. Patients were included in this study if they visited the emergency room within 36 h of traumatic injury and if they were older than 18 years. Patients were excluded if they died, were discharged within 36 h of hospitalization, were under the age of 18 years, or were pregnant. No subject in the control group had a documented history of respiratory disorders such as chronic obstructive pulmonary disease and pulmonary embolism, autoimmune disease, pregnancy, infectious diseases, recent surgery, malignancies, chronic liver, renal, or endocrine diseases. HC did not experience fever during 72 h prior to enrollment either. The clinical and laboratory characteristics of patients with trauma are summarized in Table 1.

The following monoclonal antibodies (mAbs) and reagents were used in this study: fluorescein isothiocyanate (FITC)-conjugated anti-CD3, FITC-conjugated annexin V, phycoerythrin (PE)-conjugated anti-6B11, PE-conjugated anti-CD3, PE-conjugated anti-IFN-γ, PE-conjugated anti-IL-4, PE-conjugated anti-CD69, PerCP-conjugated anti-CD45, and PE-conjugated mouse IgG isotype control (all from BD Biosciences, San Diego, CA, USA); PE-conjugated anti-programmed death-1 (anti-PD-1; eBioscience, San Diego, CA, USA), and allophycocyanin (APC)-conjugated anti-6B11 mAbs (BioLegend, San Diego, CA, USA). Cells were stained with combinations of appropriate mAb for 20 min at 4°C. Stained cells were analyzed on a Navios flow cytometer (Beckman Coulter, Brea, CA, USA) using Kaluza software (version 1.5a; Beckman Coulter).

Peripheral venous blood samples were collected into heparin-containing tubes and peripheral blood mononuclear cells (PBMCs) were isolated by density-gradient centrifugation using Ficoll-Paque Plus solution (Amersham Biosciences, Uppsala, Sweden). NKT cells were identified phenotypically as CD3+6B11+ cells by flow cytometry as described previously [17].

Proliferative abilities of NKT cells were assayed by flow cytometry as described previously [17]. Briefly, freshly isolated PBMCs were suspended in complete media supplemented with 10% fetal bovine serum (FBS; Gibco BRL, Grand Island, NY, USA), seeded into a 24-well plate at a density of 1 × 10⁶/well, and then cultured at 37°C in a 5% CO₂ humidified incubator for 7 days in the presence of IL-2 (100 IU/mL; BD PharMingen, San Jose, CA, USA) and α-GalCer (100 ng/mL; Alexis Biochemicals, Lausen, Switzerland) or 0.1% DMSO as a control. Cells were harvested and stained with FITC-conjugated anti-CD3, PE-conjugated anti-6B11, and PerCP-conjugated anti-CD45 mAbs. Percentages of CD3+6B11+ NKT cells were determined by flow cytometry using a CD45/SSC gate. The proliferation index was defined as the percentage of NKT cells (100 ng/mL α-GalCer) minus the percentage of NKT cells (0 ng/mL α-GalCer) on day 7 divided by the percentage of NKT cells on day 0. It was expressed as the fold increase.

To determine the effect of a proinflammatory cytokine cocktail and its blocking antibody, freshly isolated PBMCs were stimulated with a cytokine cocktail consisting of IL-6 (50 ng/mL; PeproTech, London, UK), IL-8 (10 ng/mL; PeproTech), and TNF-α (5 ng/mL; PeproTech) for 3 days in the presence or absence of cytokine inhibitors (i.e., blocking antibodies) and then cultured for 7 days in the presence of IL-2 (100 IU/mL) and α-GalCer (100 ng/mL) or DMSO as a control. Blocking antibodies against cytokines included anti-IL-6 (5 µg/mL), anti-IL-8 (5 µg/mL), and anti-TNF-α (5 µg/mL; all from BD Biosciences).

IFN-γ and IL-4 expression levels in NKT cells were detected by intracellular cytokine flow cytometry as described previously [18]. Briefly, freshly isolated PBMCs (1 × 10⁶/well) were incubated at 37°C in 1 mL of complete media consisting of RPMI 1640, 2 mM L-glutamine, 100 units/mL of penicillin, and 100 μg/mL of streptomycin supplemented with 10% FBS for 2 h in the presence of α-GalCer (100 ng/mL) or 0.1% DMSO as a control. For intracellular cytokine staining, 1 μL of brefeldin A (GolgiPlug; BD Biosciences) for 1 mL of cell culture was added. After incubation at 37°C in a 5% CO₂ humidified incubator for an additional 4 h, cells were stained with FITC-conjugated anti-CD3 and APC-conjugated anti-6B11 mAbs for 20 min at 4°C, fixed in 4% paraformaldehyde for 15 min at room temperature, and permeabilized with Perm/Wash solution (BD Biosciences) for 10 min. The cells were then stained with PE-conjugated anti-IFN-γ and PE-conjugated anti-IL-4 mAbs for 30 min at 4°C and analyzed by flow cytometry.

Plasma levels of IL-6, IL-8, and TNF-α were measured using commercially available enzyme-linked immunosorbent assay (ELISA) kits (R&D systems Inc., Minneapolis, MN, USA) according to instructions of the manufacturer.

Numbers and proliferation indices of NKT cells and expression levels of IFN-γ, IL-4, CD69, PD-1, and annexin V in NKT cells were compared between HC and patients by analysis of covariance after adjusting for age and sex using the Bonferroni correction for multiple comparisons. The Mann-Whitney U test was used to compare plasma levels of cytokines between HC and patients. The Wilcoxon matched-pairs signed-rank test was used to compare changes in proliferation indices of NKT cells after cytokine stimulation. Relationships between proliferation indices of NKT cells and clinical or laboratory parameters were examined using Spearman’s correlation analysis. Statistical significance was considered for p < 0.05. Statistical analysis and graphic works were performed using SPSS version 18.0 software (SPSS, Chicago, IL, USA) and GraphPad Prism version 5.03 software (GraphPad Software, San Diego, CA, USA), respectively.

A total of 32 patients with trauma were included in this study (Table 1). The severity of injury was categorized as mild (<9), moderate (9–15), and severe (>15) according to the ISS scoring system, the gold standard for evaluating injury severity. Of these patients, 6 (18.8%) with mild injury, 10 (31.3%) with moderate injury, and 16 (50.0%) with severe injury were found. Two (6.25%) patients died, including 1 with mild injury and 1 with severe injury.

Frequencies of CD3+6B11+ NKT cells represent the percentages among peripheral blood lymphocytes. Percentages of circulating NKT cells were significantly lower in patients than in HC (median 0.04 vs. 0.12%, p < 0.01; Fig. 1a). Absolute NKT cell numbers were calculated by multiplying NKT cell fractions by total lymphocyte numbers (per microliter of peripheral blood). Patients with trauma had significantly lower absolute NKT cell numbers than HC (median 0.46 vs. 1.60, p < 0.0001; Fig. 1b). Representative NKT cell percentages as determined by flow cytometry are shown (Fig. 1c). In addition, linear regression analysis showed that log-transformed NKT cell numbers were correlated with the lymphocyte count (p < 0.0001) and C-reactive protein level (p =0.030; online suppl. Table 1; see www.karger.com/doi/10.1159/000504324 for all online suppl. material).

The representative percentage of NKT cells among peripheral blood lymphocytes from an HC subject was markedly increased from 0.37% on day 0 to 12.9% on day 7 in response to α-GalCer, resulting in a proliferation index of 34.0. In contrast, a representative patient with trauma displayed a weak proliferative response of NKT cells (Fig. 2a). Overall proliferation indices were significantly lower in patients with trauma than those in controls (median 1.1 vs. 20.8, p < 0.0001; Fig. 2b). Furthermore, CFSE dilution assay showed that NKT cells from HC profoundly expanded and extensively divided 7 days after stimulation with α-GalCer. However, few NKT cells from patients with trauma expanded and divided (online suppl. Fig. 1).

To evaluate the clinical relevance of NKT cell function in 32 patients with trauma, we investigated the correlation between NKT cell proliferation indices and clinical parameters by Spearman’s correlation analysis. NKT cell proliferation indices were positively correlated with lymphocyte count (p = 0.022) but negatively correlated with creatinine levels (p = 0.032). However, NKT cell proliferation indices showed no significant correlation with gender, age, mortality, SIRS, infectious complications, APACHE score, SAPS score, ISS score, leukocyte count, neutrophil count, hemoglobin level, platelet count, bilirubin level, blood urea nitrogen level, C-reactive protein level, PaO₂ level, lactate level, bicarbonate level, prothrombin time, mean arterial pressure level, heart rate, or body temperature (Table 2).

We next measured levels of representative Th1/Th2 cytokine secretion by NKT cells of trauma patients. Percentages of IFN-γ+ NKT cells were found to be significantly lower in trauma patients than those in HC (median 3.13 vs. 11.0%, p < 0.05). However, percentages of IL-4+ NKT cells were comparable between patients and HC. In addition, patients had a significantly lower ratio of IFN-γ/IL-4 than HC (median ratio 0.66 vs. 1.61, p < 0.05; Fig. 3).

Our previous study showed deficiencies of circulating NKT cell numbers in patients with trauma [16]. To determine whether circulating NKT cell deficiency was associated with activation-induced cell death, we investigated the activation and apoptosis indicated by CD69 upregulation and annexin V staining, respectively, in circulating NKT cells. CD69+ and annexin V+ NKT cells were examined by flow cytometry. Percentages of CD69+ NKT cells were found to be significantly higher in trauma patients than those in HC (median 10.0 vs. 5.98%, p < 0.05; Fig. 4a, b). However, no significant difference was observed in annexin V+ NKT cell levels between patients and HC (Fig. 4c, d). To determine whether dysfunction of NKT cells was related to anergy or exhaustion, we examined expression levels of PD-1, a representative inhibitory receptor. Percentages of PD-1+ NKT cells were comparable between patients and HC (Fig. 4e, f).

Both percentages of annexin V+ NKT cells on day 0 and day 7 without α-GalCer were similar between trauma patients and HC. However, after stimulation with α-GalCer for 7 days, NKT cell apoptosis was higher in trauma patients than in HC (mean ± SEM: 50.2 ± 5.4 vs. 23.0 ± 4.7%, p < 0.05; Fig. 5). These findings suggest that increased NKT cell apoptosis in culture is related to the impaired proliferative response of NKT cells observed in trauma patients.

A variety of proinflammatory cytokines can affect NKT cell proliferation [17]. Thus, we measured plasma levels of proinflammatory cytokines such as IL-6, IL-8, and TNF-α using ELISA. Patients with trauma showed significantly higher plasma levels of IL-6, IL-8, and TNF-α than HC (median: IL-6, 92.3 vs. 0.7 pg/mL, p < 0.05; IL-8, 24.0 vs. 1.5 pg/mL, p < 0.005; TNF-α, 2.1 vs. 2.1 pg/mL, p < 0.05; Fig. 6a).

We next determined whether these proinflammatory cytokines could affect the NKT proliferative capacity (Fig. 6b). Proliferation indices were found to be significantly lower in cytokine-treated cultures than those in untreated cultures (median 6.3 vs. 30.2, p < 0.01). They were then significantly increased after treatment with blocking antibodies compared to cytokine-treated cultures (median 6.3 vs. 11.0, p < 0.05; Fig. 6c). However, these blocking antibodies partially reversed the dysfunction of NKT cells. Taken together, these results suggest that the proliferative dysfunction of NKT cells may be partly due to proinflammatory cytokines in trauma patients.

The present study showed that proliferation and IFN-γ production by circulating NKT cells in response to α-GalCer were reduced in trauma patients, although their capacity for IL-4 production was preserved. Suppression of proliferation and IFN-γ production in NKT cells has also been observed in other inflammatory or infectious diseases, such as systemic lupus erythematosus, tuberculosis, human immunodeficiency virus type 1 infection, and scrub typhus [18-21]. Suppression of cell proliferation and IFN-γ production has also been reported in adaptive T cells following severe injury or trauma-hemorrhage [22, 23]. One possible explanation for adaptive T cell suppression is that a specific subset of neutrophils in acute systemic inflammation can inhibit T cell responses via Mac-1 and the reactive oxygen species signaling pathway [22]. Another possibility is that myeloid-derived suppressor cells are induced after inflammation and suppress adaptive T cell responses [24]. Likewise, these two mechanisms can explain defective innate T cell responses, including NKT cells in trauma patients. Further studies are needed to elucidate these mechanisms in suppressed innate T cell responses in trauma patients.

Our previous data have shown that a numerical deficiency of circulating NKT cells is associated with severity grades of traumatic injury (i.e., APACHE score, SAPS score, and ISS score) [16]. In the present study, however, no significant correlation was observed between NKT cell proliferative indices and severity grades. Nevertheless, our results revealed that NKT cell proliferation indices were negatively correlated with serum creatinine levels in trauma patients, suggesting that NKT cell dysfunction might reflect renal dysfunction. Similarly, our previous study showed that numerical deficiency of circulating NKT cells is accompanied by lupus nephritis [19]. Collectively, these data suggest that the circulating NKT cell level and function may be used as a biomarker for predicting disease severity or organ dysfunction in trauma. However, the correlation with serum creatinine levels was weak. Moreover, SIRS tended to be negatively associated with NKT cell proliferation indices, although the p value did not reach statistical significance. This may be due to small numbers of enrolled subjects. Therefore, further analysis using a large sample size is required to determine the clinical relevance of NKT cell function.

CD69 is known to be an early activation marker, whereas PD-1 is considered a relative later activation marker [25, 26]. Our data demonstrated that trauma patients displayed higher levels of CD69+ NKT cells without upregulation of PD-1, indicating that NKT cells were activated early following trauma. In addition, our previous study reported the same phenomenon (i.e., early activation) that occurs to mucosal-associated invariant T cells, another subset of innate T cells, after traumatic injury [27]. Collectively, these findings suggest that trauma may induce early activation of innate T cells irrespective of their cell types. Interestingly, our additional data showed that CD69 expression levels were much higher in NKT cells compared to those in conventional T cells, suggesting that NKT cells might be activated much earlier than conventional T cells after trauma (online suppl. Fig. 2). Furthermore, previous studies have demonstrated that NKT cells are activated much more rapidly than conventional T cells in response to CD1d-dependent stimulations, such as α-GalCer or a variety of infectious agents [28-31]. Taken together, these results indicate that NKT cells are activated much earlier than conventional T cells, irrespective of CD1d-dependent or CD1d-independent stimulations.

Our data showed that NKT cells were more susceptible to apoptosis when they were stimulated with α-GalCer in trauma patients. This observation has also been reported in other inflammatory or infectious conditions, such as adult-onset Still’s disease (AOSD) and tuberculosis [17, 20]. In particular, AOSD and trauma patients have high levels of IL-6, IL-8, and TNF-α in sera [32]. A previous study using in vitro experiments showed that TNF-α can induce NKT cell death by blocking downstream of NF-κB activation during thymic development [33]. In addition, Tang et al. [34] reported that proinflammatory cytokine-activated Kupffer cells by lipids can promote hepatic NKT cell deficiency through activation-induced cell death. A previous study using a mouse model reported that IL-6 treatment can inhibit NKT cells in CD4+ T cell- and STAT3-dependent manners [35]. Collectively, these findings lead us to speculate that proinflammatory cytokine-activated antigen-presenting cells may induce circulating NKT cell deficiency through activation-induced cell death. Proinflammatory cytokines such as IL-6, IL-8, and TNF-α were also found to be able to suppress the proliferative function of NKT cells in response to α-GalCer.

This is the first study to assess the function of NKT cells in trauma patients. The present study demonstrates that circulating NKT cells are early activated and functionally impaired in IFN-γ production and proliferation following trauma via increased proinflammatory cytokines. In addition, NKT cell dysfunction might reflect renal dysfunction. These findings provide important insight into the trauma-related early innate immune response.

This study was supported by the National Research Foundation of Korea funded by the Korean Government (Grants 2019R1A-2C1003238, 2017R1D1A1B03029239, 2018R1A6A3A11042850 and 2019R1I1A1A01040762), and the Chonnam National University Hospital Biomedical Research Institute (Grant CRI16036-22 and CRI18092-1).

The study protocol was approved by the Institutional Review Board of Chonnam National University Hospital. Written informed consent was obtained from all participants in accordance with the Declaration of Helsinki.

The authors declare that they have no competing interests.

Y.-G.J., H.-M.J., Y.-N.C., J.-C.K., S.-J.K., and Y.-W.P. designed this study, collected clinical information, analyzed raw data, performed statistical analysis, and contributed to writing of the paper. Y.-G.J., H.-M.J., and Y.-N.C. performed the experiments. All authors read and approved the final version of the manuscript.

Y.-G. J. and J.-C. K. contributed equally to this work.