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Phenotypic Changes and Impaired Function of Peripheral γδ T Cells in Patients With Sepsis

Liao, Xue-Lian*; Feng, Ting; Zhang, Jiang-Qian*; Cao, Xing*; Wu, Qi-Hong; Xie, Zhi-Chao*; Kang, Yan*; Li, Hong

Author Information
doi: 10.1097/SHK.0000000000000857

Abstract

INTRODUCTION

Sepsis results in life-threatening organ failure due to dysregulated host response to infection. It is the leading cause of mortality in most intensive care units (ICUs) and its global incidence is gradually increasing (1, 2). T lymphocytes play fundamental roles in the immunological response to sepsis. Previous studies have shown a marked loss of conventional αβ T cells (cluster of differentiation [CD]4+ and CD8+ T cells) in septic patients. Surviving αβ T cells show a phenotype shift from a pro-inflammatory Th-1 phenotype to an anti-inflammatory Th-2 phenotype, characterized by the decrease in pro-inflammatory cytokines and increase in inhibitory cytokine production as well as the change of surface molecules, such as programmed death receptor 1 (PD-1) and programmed death ligand 1 (PDL-1) (3). Therapies aiming to improve the function of these immune cells may represent an important and new direction for sepsis management.

Gamma delta T (γδ T) cell was identified after the αβ T cell and consists of a small population of T lymphocytes (approximately 2%–10% of circulating T cells). γδ T cell exhibits features of both innate and adaptive immunity and plays an indispensable role in host defense, immune surveillance, and homeostasis (4) In contrast to traditional αβ T cells, γδ T cells do express arranged TCR but they do not need the classic antigen presentation to be activated. In addition, they show unique tissue localization immunomodulatory functions (4, 5). Previous studies in septic mice yielded controversial results. While the most data demonstrated that γδ T cell-deficient septic mice had increased mortality, some studies showed that γδ T cells played an anti-infection role via up-regulating inflammation levels (6–8). Currently, the generally accepted role of γδ T cells is that they contribute to host defense but also protect host from organ dysfunction through regulating the magnitude of inflammation (9). The data obtained from mouse model provide important information to understand this unique subset of T cells. However, these results may not represent the functions of γδ T cells in human, since the majority of human peripheral γδ T subsets are Vγ9Vδ2 T cells, which are not present in mouse and non-human primates (10).

γδ T cells in septic patients have been previously reported (11–14), but more extensive studies are still necessary to understand their exact roles in this type of disease. Andreu-Ballester et al. (11) have demonstrated that there was a reduction in γδ T cells among all the lymphocyte subsets and such reduction was associated with high mortality. Thus far, the functional changes of γδ T cells in sepsis have not been well defined. Galley et al. (12), attempted to study the functional changes of γδ T cells after in vitro culture for 7 days in the presence of IL-2 and bisphosphonate zoledronic acid. They found that γδ T cells from septic patients could not proliferate as much as these from healthy controls, implying the impaired responses of γδ T cells in septic patients. Because the functions of γδ T cells are achieved by successful activation and appropriate cytokine expression, it is critical to investigate the activation status and their cytokine production upon stimulation. In the current study, we evaluated the phenotype and function of peripheral γδ T cells in patients with severe sepsis and septic shock (septic patients), and investigated their associations with clinical features and prognosis.

PATIENTS AND METHODS

Patient and control subjects

The patients and healthy controls were recruited during the period from October 2014 and June 2015. The patients were admitted in one of the three ICUs at the West China Hospital of Sichuan University. Written consent was obtained from each patient's legally authorized representative (since all enrolled patients were discerned to be too ill to provide valid consent) and all of the enrolled healthy volunteers. The study protocol (http://clinicaltrials.govnumberNCT02361970) was approved by the Ethics Committee of the West China Hospital and conformed to the Declaration of Helsinki.

Severe sepsis and septic shock was defined according to 2012 Surviving Sepsis Campaign guidelines (2). Patients were eligible for study inclusion if they: were ≥18 year old; had a known or suspected infection based on clinical data at the time of admission and ≥2 signs of systemic inflammation and sepsis-induced dysfunction of at least one organ or system. The precise methods of patient selection including threshold of each indicator were shown in supplementary File 1, https://links.lww.com/SHK/A569. Patients were excluded if they had one of the following conditions: autoimmune disease, history of transplantation, acute tuberculosis, chronic hepatitis B or C infection, HIV infection, active malignancy, and long-term use of cortical steroids. Healthy control subjects were adult volunteers from our hospital staff and their family members with no significant acute or chronic illnesses.

Study design and data collection

Patients admitted to the ICU were consecutively screened on the admission day, and if they were eligible for the study, their following clinical data on the first day of ICU admission were recorded: age, gender, primary diagnosis, comorbidities, origin, parameters for Acute Physiology and Chronic Health Evaluation II (APACHE II) (15), sepsis-related organ failure assessment (SOFA) (16) scores, time between the onset of acute organ failure and ICU admission, routine blood tests (such as white blood cell count, neutrophil count, lymphocyte count, procalcitonin, interleukin-6, lactate, etc.), radiological results, primary infection source, microbiological findings. Then, their peripheral blood samples were collected for the γδ T cell study, including in vivo percentage, surface markers, and intracellular cytokines upon harvest as well as CD69 and IFN-γ expression after ex-vivo stimulation. All patients enrolled in the study were followed up to day 28 after ICU admission to obtain the clinical outcome.

Collecting and processing sample

Strictly abiding by the principle of aseptic manipulation, 20 mL heparinized blood was collected from septic patients within 24 h after admission into ICU via peripheral venipuncture. Healthy control sample were collected following the same procedure. The blood samples were immediately transported and processed in the research laboratory. Briefly, peripheral blood mononuclear cells (PBMCs) were isolated by ficoll-hypaque density gradient using the standard protocol.

Activation of γδ T cells

As shown in Supplemental Fig. 1, https://links.lww.com/SHK/A569, the isolated PBMCs were divided into two sets, one set was used for flow cytometry analysis for surface markers and basal level of cytokine expression in γδ T cells; the second set of PBMCs were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum for 24 h (for CD69) or 6 h (for IFN-γ) with the corresponding stimulation. The stimulus for CD69 expression was pamidronate (PAM, 9 mg/mL) (17). The stimulation for IFN-γ measurement included phorbol-myristate acetate (PMA, 50 ng/mL) and ionomycin (1 μg/mL) for 6 h with the addition of brefeldin A during the last 4 h of culture (2 μg/mL) (18).

Flow cytometry

Cells were stained for surface markers with the following antibodies: anti-human CD3 (1.25 μL, BD Bioscience, Franklin Lakes, NJ), antihuman TCR-γδ (1.25 μL, BD Bioscience, Franklin Lakes, NJ), antihuman CD69 (1.25 μL, Biolegend, San Diego, Calif), antihuman NKG2D (1.25 μL, Biolegend, San Diego, Calif), and antihuman PD-1 (1.25 μL, Biolegend, San Diego, Calif). For intracellular staining, cells were fixed, permeabilized and stained with anti-human IFN-γ (1.25 μL, Biolegend, San Diego, Calif), antihuman IL-10 (1.25 μL, Biolegend, San Diego, Calif), antihuman IL-17 (1.25 μL, Biolegend, San Diego, Calif), and anti-human TGF-β (1.25 μL, Biolegend, San Diego, Calif). Corresponding isotype antibodies were used in parallel for staining controls. All samples were acquired by flow cytometry (BD Bioscience, Franklin Lakes, NJ) and analyzed using FlowJo software (Tree Star, Inc, Ashland, Oreg). TCR-γδ+ cells in CD3-positive cells of total PBMCs are the target cells for the determination of percentage, surface marker, and intracellular cytokine analysis. The flow plot and gate settings are shown in supplementary Figs. 2, 3, and 4, https://links.lww.com/SHK/A569.

Statistics

Continuous variables were described as mean ± standard deviation or median (interquartile range, IQR). Comparisons between independent groups were performed with unpaired t test and paired data with the paired t test or Wilcoxon test. Category variables were described as frequencies, and compared using Chi-square test or Fisher exact test. Association between two different variables was performed using Pearson correlation test. To evaluate the effects of surface markers and intracellular cytokines of γδT cells on mortality, we first performed univariate analysis between survivors and non-survivors, incorporating different factors that may relate to patient prognosis, such as age, septic shock, severity scores, chronic organ dysfunction, site of infection, pathogen, CD69, IFN-γ, etc. (19). Second, based on the results of univariate analyses and clinical relevance, variables with significant difference (P < 0.05) were incorporated into multiple regression models. Due to the sample size, we avoid further comparison for some comprehensive parameters such as APACHE II score, which contain multiple single factors, such as age, chronic organ dysfunction, and ingredients of SOFA score. For the purpose of affirming the significant effects of CD69 and IFN-γ of γδ T cells on mortality, multivariate regression analyses also included stepwise backward selection to reveal the most relevant associated parameters for mortality to validate the findings (20). Odds ratios (ORs) with 95% confidence intervals (CIs) and the corresponding P values were calculated for each risk factor. Quality of regression models was assessed with Hosmer–Lemeshow tests for model calibration. All tests were two-sided, and P < 0.05 was considered to be statistically significant. Statistical analyses were performed using SPSS for Windows (version 22.0, USA).

RESULTS

Clinical characteristics of study subjects

During the study period (from October 2014 to June 2015), we screened 1,328 patients. Based on the diagnostic criteria, 202 patients were admitted into our ICUs with a diagnosis of severe sepsis or septic shock. According to the inclusion and exclusion criteria, a total of 107 patients were included into the current study (Fig. 1). 61.7% of patients were male, the average age was 58.1 ± 15.6 years. The APACHE II and SOFA score was 19.6 ± 7.8 and 8.96 ± 4.0, respectively. Overall, septic shock patients accounted for 49.5% of the total patients admitted to ICUs. 29.9% patients had at least one chronic organ dysfunction. Abdomen and lung infection were the most common infection sites. A bacteriological confirmation was obtained in 61.7% of these septic patients. The time between acute organ dysfunction and ICU admission was 43 h (IQR, 20–120). As compared with the 45 healthy controls, patients have the similar gender distribution. No difference of gender was detected between two groups (P > 0.05). Because the healthy control and the patient groups showed difference in age, both groups were further divided into two sub-groups of age to facilitate the age-matched comparisons. Previous researches determined that greater difference of γδ T cell percentage exists between ≤65-year-old group and >65-year-old group (21). Therefore, we divided the patients and controls into two age groups ≤65 year old and >65 year old for comparisons. Clinical characteristics of the patients upon enrollment are presented in Table 1.

F1-8
Fig. 1:
A flow chart for subject recruitment, inclusion and exclusion process.
T1-8
Table 1:
Clinical characteristics of septic patients on ICU admission

Percentage and phenotypic changes of γδ T cells in septic patients

Septic patients showed a significant decrease in the percentage of γδ T cells (1.45 ± 1.24%) in CD3+ cells as compared with the healthy controls (5.03 ± 2.65%), P < 0.001. Such difference was observed in the overall septic patients as well as their two age subgroups (Fig. 2A). Similar to the healthy subjects, γδ T cells in septic patient also show decreasing trend with the increase of age (r = −0.207, P = 0.033) (22). Among numerous functional surface markers on γδ T cells, CD69, NKG2G, PD-1 expressed are frequently used to evaluate the early activation, costimulatory and inhibitory functions of γδ T cells, respectively (19). Our results showed that γδ T cells in septic patients had a higher percentage of CD69+ cells (Fig. 2B) and lower percentage of NKG2D+ cells (Fig. 2C) than healthy controls regardless of age. Of note, the γδ T cell did not show the difference in the expression of PD-1 between septic patients and healthy control (Fig. 2D).

F2-8
Fig. 2:
Percentage of γδ T cells and function-related surface markers on γδ T cells in septic patients and healthy controls.Antibody staining was performed for the freshly prepared PBMCs. CD3+ TCR-γδ+ cells were gated and the percentage of positive cells was analyzed by and flow cytometry analysis. The results were compared between septic patients and controls as the overall group or as age-matched subgroup. A, Percentage of γδ T cells in CD3+ cells was significantly lower in septic patients than healthy controls. B, Percentage of CD69+ γδ T cells was significantly higher, while (C) NKG2D+ γδ T cells were significantly lower in septic patients than in healthy controls. D, PD-1 expression on γδ T cells was similar between the patients and controls. All data were expressed as means ± SD. Unpaired t test or Wilcoxon test was used. * P < 0.05, ** P < 0.01, *** P < 0.001. γδ T indicates gamma delta T; CD, cluster of differentiation; NKG2D, natural-killer group 2 member D; NS, non-significant; PBMCs, peripheral blood mononuclear cells; PD-1, programmed death receptor 1.

Cytokine expression of γδ T cells

To determine the functions of γδ T cells in prompting the inflammation and regulating the immune responses, it is critical to examine the cytokines that they are producing. Accordingly, we analyzed two pro-inflammatory cytokines (IFN-γ and IL-17) and two anti-inflammatory cytokines (IL-10 and TGF-β) in septic patients and controls. As shown in Figure 3, both pro-inflammatory (Fig. 3, A and B) and anti-inflammatory cytokines (Fig. 3, C and D) showed higher levels in septic patients than in the healthy controls.

F3-8
Fig. 3:
Cytokine expressions by γδ T cells in septic patients and healthy controls.γδ T cells isolated from fresh blood were fixed, permeabilized, and stained for intracellular cytokines. CD3+ TCR-γδ+ cells were gated and cytokine expressions were analyzed using flow cytometry. The results were compared between septic patients and controls as the overall group or as age-matched subgroup. The positive cells of (A) IFN-γ, (B) IL-17, (C) IL-10, and (D) TGF-β were higher in septic patients as compared with healthy controls. Data were expressed as mean ± SD. Unpaired t test or Wilcoxon test was used. * P < 0.05, ** P < 0.01, *** P < 0.001. γδ T indicates gamma delta T; CD, cluster of differentiation; IFN-γ, interferon-γ; IL, interleukin; TCR, T cell receptor; TGF-β, transforming growth factor-β.

Responses of γδ T cells upon stimulation

After we found the significant difference of γδ T cells in frequency, phenotypic status, and the basal levels of cytokine expression between septic patients and healthy controls, we further determined whether the responses of these cells upon the antigen stimulation were also different in septic patients. Considering the future clinical feasibility and based on our previous study (17), we select two short-term stimulatory methods (Supplemental Fig. 1, https://links.lww.com/SHK/A569). Among all the markers and cytokines tested in the pilot study, only CD69, IFN-γ expressions were elevated significantly after the short-term stimulation in healthy controls. Therefore, we cultured these cells in the presence of appropriate stimuli and studied their CD69 and IFN-γ expressions. As shown in Figure 4, the activation marker, both CD69 (Fig. 4A) and IFN-γ expression (Fig. 4B) were significantly lower in septic patients than in healthy controls, despite their higher basal levels (before stimulation) in patients than controls. CD69 expressions increased to 48.30 ± 19.57% in healthy controls but only to 17.39 ± 8.69% in septic patients. IFN-γ expressions markedly elevated to 65.86 ± 16.39% in healthy controls but only to 23.66 ± 17.37% in septic patients. The results indicated that γδ T cells were activated in sepsis. Their responsiveness to new antigen stimulation was significantly lower as compared with γδ T cells from healthy controls.

F4-8
Fig. 4:
CD69 and IFN-γ expressions by γδ T cells before and after stimulation in different groups.γδ T cells were stimulated in the culture and subjected to surface staining for CD69 and intracellular staining for IFN-γ (unstimulated cells were analyzed in parallel as the before stimulation control). The percentages of (A) CD69+ and (B) IFN-γ+ γδ T cells were compared between septic patients and controls in in two age-matched subgroups, the age group ≤ 65 years old, as well as in age group >65 years old, respectively. The percentages of (C) CD69+ and (D) IFN-γ+ γδ T cells were compared between survival and non-survival patients. Data were expressed as mean ± SD. Unpaired t test or Wilcoxon test was used. * P < 0.05, ** P < 0.01, *** P < 0.001. γδ T indicates gamma delta T; CD, cluster of differentiation; IFN-γ, interferon-γ; NS, non-significant.

Prognostic assessment

Based on the above findings, we were interested in determining whether the levels of CD69 and IFN-γ expression after stimulation were associated with the prognosis. As shown in Figure 4C and D and Supplementary Fig. 3, https://links.lww.com/SHK/A569, survivors expressed a significant higher level of CD69 and IFN-γ than non-survivors (18.64 ± 9.54 vs. 14.60 ± 5.59 for CD69 and 28.72 ± 17.25 vs. 12.45 ± 11.52 for IFN-γ, respectively, P < 0.05). Then, multiple logistic regression models were performed incorporating clinical variables with significant difference (P < 0.05) in the univariate analysis (excluding some interactive factors) (Table 1), together with CD69 and IFN-γ expression after stimulation. The all-possible regression model showed SOFA score and IFN-γ (after stimulation) were the significant factors for mortality in this population, with odds ratios of 1.268 and 0.910, respectively. A stepwise backward-selection procedure reproduced this finding in a reduced model with six variables (age, SOFA score, chronic organ dysfunction, fungal infection, CD69, IFN-γ), also showing an OR for mortality of 0.908 (95% CI: 0.853–0.996) for IFN-γ (after stimulation) (Table 2).

T2-8
Table 2:
Multilogistic regression for risk factors of mortality in septic patients

DISCUSSION

In the current study, we found that there were changes in function-related markers of γδ T cells in the blood of septic patients. To our knowledge, this is the first study that evaluated relationship of function-related markers of γδ T cells and septic patient prognosis.

The phenotypic changes of γδ T cells in sepsis were interesting. The basal level of CD69+ cells in septic patients was higher in septic patients than in healthy controls. As an early activation marker of γδ T lymphocytes, its increase reflects the involvement of γδ T in defending sepsis. Our results were consistent with a previous study conducted by Matsushima et al. (18), who found that CD69+ γδ T cells were higher in 23 patients with sepsis than healthy controls. However, CD69 expression on γδ T cells upon stimulation has not been evaluated and whether CD69 changes after stimulation were associated with patient prognosis remains unknown. Therefore, we measured CD69 expression after stimulation and analyzed whether its expression has an implication on patient's prognosis. We found that septic patients showed impaired CD69 expression after stimulation, especially in patients with poor prognosis. However, this change was not significant in the multiple regression analysis.

Another function-related marker of γδ T cell is NKG2D, which is known to play an important role in γδ T cell-mediated anti-infection and antitumor cytotoxicity through PI3K-dependent and protein kinase cθ-dependent mechanisms (23, 24). However, NKG2D expression on γδ T cell in septic patients has not been studied previously. Our results showed that NKG2D expression was greatly decreased in septic patients. Many cytokines, such as IL-2, IL-7, IL-12, and IL-15, could increase the expression of NKG2D (25, 26). It was suggested that cytokine stimulation might be a potential strategy to modulate NKG2D expression and γδ T cell function. PD-1 is also a function-related marker of γδ T cell and plays an inhibitory role. However, no difference of PD-1 expression was detected between septic patients and controls. Our results support the notion that therapy targeting PD-1 receptor on γδ T cells may not be as effective as on αβ T cells (27).

Similar to αβ T cells, γδ T cells also produce cytokines. Basal level expression of both pro-inflammatory (IFN-γ, IL-17) and anti-inflammatory (IL-10, TGF-β) cytokines by γδ T cells in septic patient was detected and showed a higher level than that in healthy controls. These results suggested that both pro- and anti-inflammatory functions of γδ T cells have been initiated in sepsis, which are consistent with the dual roles of γδ T cells. The two mutually antagonizing functions of γδ T cells may be finely regulated to optimize immune response to maximally defending infection while minimizing the damage of self-tissues. Previous studies showed a large number of IL-17-secreting γδ T cells and γδ T cells were considered a main source of IL-17 (28, 29). Most studies concerning IL-17-producing γδT cells were from mice (30, 31). In our study, the percentage of IL-17+ γδ T cells in peripheral blood was less than 1%, which is much lower than those in the mucosa, lung, and peritoneal cavity (28, 29). γδ T cells may have functional difference depending on their localization in human (28, 32). Galley et al. (12) augured that γδ T cells retained their functional activity in sepsis according to IFN-γ level in PBMC culture supernatant after stimulation with IL-2 plus the bisphosphonate zoledronic. However, the level of IFN-γ in supernatant could be influenced by the accumulation effect as well as the cell phenotype change during a long-time culture (7 days). To minimize the impact from these two factors, we adopted the ex vivo stimulation protocol and performed the intracellular staining, which may represent better in vivo functional status of γδ T cells. Our results indicated that the expression of IFN-γ by γδ T cells in response to PMA stimulation in septic patients was significantly lower than that in healthy controls, it was suggested that the stimulus-induced IFN-γ production was impaired at the single cell level in septic patients. Bessoles et al. (24) demonstrated that reducing NKG2D expression by siRNA resulted in a significant decrease in cytokine production by γδ T cells. Since the basal level of IFN-γ is higher before stimulation and lower after stimulation in septic patients when compared with corresponding healthy controls, it remains unknown whether the IFN-γ expression is under the regulation of NKG2D expression.

Our study had some limitations. First, the included patients were not in the same sepsis stage, and we could not evaluate the dynamic changes of γδ T cells at the different time points of the disease course. However, it does not impede us learn useful knowledge of γδ T cells in sepsis from the current study. Take IFN-γ secretion as an example, no matter the patient was in “early” or “late” phase (divided by persistent organ failure ≤ or >48 h), the ex vivo IFN-γ secretion of γδ T cells after stimulation was decreased as compared with healthy controls. It was 29.98 ± 17.71% and 16.97 ± 14.37% for septic patients with persistent organ dysfunction < or ≥48 h, respectively, as compared with 65.86 ± 23.66% for healthy control. It seemed that ex vivo response of γδ T cells decreased more obviously in patients in “late” phase than “early” phase (P < 0.001). This result was similar to prior studies on αβ T cells (32). Second, the subjects were divided into two age groups for age-matched comparisons, the average age of the healthy controls was still relatively younger than septic patients. Third, in the stimulation test, we only evaluate CD69 and IFN-γ expression of γδ T cells, which increased most significantly among all the markers tested. In the future study, other stimuli should be tried to evaluate other markers of γδ T cells in sepsis. Lastly, we found some interesting phenotypic and functional changes of γδ T cells in sepsis, but the underlying mechanisms remain to be elucidated.

In conclusion, the current study demonstrated the phenotypic changes of γδ T cells in septic patients. The impaired IFN-γ expression by γδ T cells after the antigen stimulation is associated with mortality in septic patients. Our data provide some new insights in understanding the functions of γδ T cells in sepsis. The mechanisms underlying the phenotypic and cytokine-expressing alteration in septic patients need further investigation.

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Keywords:

CD69; gamma delta T cells; IFN-γ; NKG2D; septic patient; APACHE II; Acute Physiology and Chronic Health Evaluation II; CD; cluster of differentiation; CI; confidential interval; HIV; human immunodeficiency virus; ICU; intensive care unit; IFN-γ; interferon-γ; IL; interleukin; IQR; interquartile range; NKG2D; natural-killer group 2 member D; OR; odds ratio; PAM; pamidronate; PBMC; peripheral blood mononuclear cell; PBS; phosphate buffer saline; PCT; procalcitonin; PD-1; programmed death receptor 1; PDL-1; programmed death ligand 1; PMA; phorbol 12-myristate 13-acetate; RRT; renal replacement therapy; SD; standard deviation; SOFA; sepsis-related organ failure assessment score; TCR; T cell receptor; TGF-β; transforming growth factor-β; Th; T helper cell; WBC; white blood cell

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