Activation-Induced Cell Death of Mucosal-Associated Invariant T Cells Is Amplified by OX40 in Type 2 Diabetic Patients This information is current as of September 23, 2021. Mei Zhang, Siqi Ming, Sitang Gong, Siping Liang, Yuanmei Luo, Zibin Liang, Can Cao, Juanfeng Lao, Yuqi Shang, Xingyu Li, Manni Wang, Guoquan Zhong, Lingqing Xu, Minhao Wu and Yongjian Wu

J Immunol published online 2 October 2019 Downloaded from http://www.jimmunol.org/content/early/2019/10/01/jimmun ol.1900367 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2019/10/01/jimmunol.190036 Material 7.DCSupplemental

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published October 2, 2019, doi:10.4049/jimmunol.1900367 The Journal of Immunology

Activation-Induced Cell Death of Mucosal-Associated Invariant T Cells Is Amplified by OX40 in Type 2 Diabetic Patients

Mei Zhang,*,1 Siqi Ming,†,1 Sitang Gong,‡ Siping Liang,† Yuanmei Luo,* Zibin Liang,x Can Cao,† Juanfeng Lao,† Yuqi Shang,† Xingyu Li,† Manni Wang,† Guoquan Zhong,* Lingqing Xu,* Minhao Wu,† and Yongjian Wu*,†,‡

Mucosal-associated invariant T (MAIT) cells play a key role in local and systemic immune responses. Studies suggest that type 2 diabetes (T2D) is associated with alterations in the human MAIT cell response. However, the mechanisms that regulate the survival and homeostasis of human MAIT cells are poorly defined. In this study, we demonstrate that the costimulatory TNF superfamily OX40 was highly expressed in MAIT cells of patients with T2D. Compared with OX40-negative MAIT cells, OX40-positive Downloaded from MAIT cells showed a high activation and a memory phenotype. Surprisingly, OX40 expression was negatively correlated with the frequency of MAIT cells in the peripheral blood of T2D patients. Increased cleaved -3 levels were observed in OX40+- expressing MAIT cells in T2D patients. In vitro, activated OX40 signaling by recombinant OX40L promoted caspase-3 activation and of MAIT cells. Inhibition of caspase-3 restored apoptosis of MAIT cells induced by OX40 signaling. These results identify OX40 as an amplifier of activation-induced cell death of human blood MAIT cells and shed new on the regulation of MAIT cells in the phase of immune responses in T2D. The Journal of Immunology, 2019, 203: 000–000. http://www.jimmunol.org/

ype 2 diabetes (T2D) is widely characterized as a chronic, Studies have indicated that proinflammatory cells such as Th1, low-grade inflammatory disease caused by long-term Th17, and CTL, which increase in obesity and diabetes, also T immune system imbalance, metabolic syndrome, or nu- modulate insulin signaling (3, 6). Conversely, a number of anti- trient excess associated with obesity (1, 2). In addition, T2D- inflammatorycells,suchasTh2andregulatoryTcell(Treg), associated complications in the kidneys, arteries, and eyes also have been associated with the protection of insulin sensitivity implicate inflammatory processes (3, 4). Furthermore, immune (7). Innate-like immune cells called mucosal-associated invariant T dysfunction in obesity and T2D has been linked with an increased (MAIT) cells are considered to have a proinflammatory role in by guest on September 23, 2021 incidence of infectious diseases, including periodontal infections, T2D (8–10). Magalhaes et al. (10) reported that MAIT cells influenza, bacterial pneumonia, nosocomial and surgical site in- produced a high level of IL-17 in the adipose tissue from fections, and others (5). Therefore, inflammation is considered a obese and T2D patients, and IL-17 can promote adipose tissue major driving force in T2D and the associated complications. inflammation. In humans, obesity and T2D induce the ex- pansion of populations of proinflammatory T cells, such as CD4+ Th1, Th17, and CD8+, whereas populations of innate *Affiliated Hospital of Guangzhou Medical University, Qingyuan People’s Hospital, Qingyuan 511518, China; †Zhongshan School of Medicine, Sun Yat-sen University, T cells such as gdT, MAIT, and invariant NKT cells (iNKT) Guangzhou, Guangdong 510080, China; ‡Department of Gastroenterology, Guangz- x are decreased (9, 10). These alterations reflect a loss of total hou Women and Children’s Medical Center, Guangzhou 510120, China; and Depart- homeostasis that may contribute to systemic and tissue ment of Thoracic Oncology, The Cancer Center of The Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai 519020, China inflammation. 1M.Z. and S.M. contributed equally to this article. MAIT cells are ab T cells with a limited TCR repertoire ORCIDs: 0000-0002-6899-0616 (S.G.); 0000-0002-8711-868X (M. Wu); 0000- (11). MAIT cells are uniquely activated by small-molecule vi- 0003-1234-8760 (Y.W.). tamin B metabolites presented by the ubiquitously expressed Received for publication April 30, 2019. Accepted for publication September 10, and nonpolymorphic MHC class 1–like molecule, MR1 (12). 2019. MAIT cells are present in the blood (1–10% of total T cells), This work was supported by grants from the National Natural Science Foundation of lungs, adipose tissue, and abundantly in the liver. Upon TCR China (81801571, 81770552, 31970893), the Guangdong Natural Science Fund for activation, MAIT cells produce large amounts of , Distinguished Young Scholars (2016A030306004), the Guangzhou Pearl River New Star Program (201610010064), and the Medical Science and Technology Research including IFN-g, TNF, , perforin, IL-17, and IL-22 Fund of Guangdong (A2019326). (13, 14). A recent study indicated that MAIT cells exhibit Address correspondence and reprint requests to Prof. Minhao Wu and Dr. Yongjian Wu, several defects in T2D and obese patients. MAIT cell fre- Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan 2nd Road, quency was dramatically reduced in patients with T2D and Guangzhou, Guangdong 510080, China. E-mail addresses: [email protected] (M.W.) and [email protected] (Y.W.) particularly in obese patients (10). A lower frequency of blood The online version of this article contains supplemental material. MAIT cells in T1D patients could result from their sustained Abbreviations used in this article: AICD, activation-induced cell death; BD, Becton activation, leading to cell exhaustion as indicated by their Dickinson; FasL, Fas ; FPG, fasting plasma glucose; iNKT, invariant NKT cell; expression of Bcl-2, CD25, and PD-1 (15). Decreased circu- MAIT, mucosal-associated invariant T; OX40L, OX40 ligand; PI, propidium iodide; lating MAIT cell frequency has been described in different T2D, type 2 diabetes; Treg, . diseases: hepatitis C, HIV, tuberculosis, inflammatory bowel Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 diseases, and severe infections (10, 16–19). The mechanisms

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1900367 2 OX40 PROMOTES AICD OF MAIT CELLS IN T2D PATIENTS underlying MAIT cell defects in T2D and obesity remain to be Flow cytometric analysis elucidated. The cell staining procedure used in this study was previously described by OX40 (also called CD134) is a T cell costimulatory molecule our study (27). Intracellular cleaved caspase-3 were stained using an in- belonging to the TNFR superfamily. OX40 is an ∼50-kDa tracellular fixation/permeabilization buffer set (eBioscience). Flow cyto- glycoprotein with a 49 aa cytoplasmic tail and a 186 aa ex- metric analysis was performed on FACS Canto II (Becton Dickinson tracellular region. OX40 is a well-characterized member of the [BD]), and data were analyzed using FlowJo software (Tree Star). Abs were purchased from BD Biosciences, eBioscience, or BioLegend, except TNFR family and has been cloned in the rat, mouse, and human where otherwise indicated. Abs used were as follows: anti-human CD3 (20); it is expressed broadly in T cells, including Th1, Th2, (clone UCHT1; BD), CD161 (clone HP-3G10; BioLegend), OX40 (clone Th9, Th17, Treg, and CTL (21). In physiological immune re- Ber-ACT35; BioLegend), TCR Va7.2 (clone 3C10; BioLegend), CD45RO sponses, OX40 delivers a potent costimulatory signal that re- (clone UCHL1; BD), CD69 (clone FN50; eBioscience), Fas (clone DX2; BioLegend), and cleaved caspase-3 (clone, D3E9; Cell Signaling sults in T cell activation, effector differentiation, and memory Technology). generation (22). During OX40 activation, BCL2 expression is sustained, leading to a survival advantage for CD4+ T cells and Cell sorting the development of long-lived memory cells (23). In addition, Human PBMCs were used to isolate MAIT cells by positive selection activation of OX40 enhances the induction of Th9 cells and using the magnetic cell sorting system from BD Biosciences. airway inflammation through the activation of the noncanonical TCRVa7.2+ cells (MAIT cells) were purified by positive selection using NF-kB pathway (24). OX40 also has been linked to the path- anti-human TCRVa7.2 biotin Ab and streptavidin-conjugated magnetic particles (BD). The purity of sorted MAIT cells was analyzed by gating ogenesis of autoimmune colitis, arthritis, experimental auto- CD3+ and analyzing TCRVa7.2 and CD161-positive cells using flow immune encephalomyelitis, asthma, and tissue fibrosis (22, 25). cytometry. Downloaded from In contrast to its well-publicized role in T cell survival, OX40 Annexin V/propidium iodide staining is a potent death receptor for iNKT (26). In liver iNKT, OX40 stimulation activates caspase-1 via TNFR-associated factor MAIT cells were treated with the indicated ligands for 12 h and then 6–mediated recruitment of the paracaspase MALT1 (26). Ac- collected, resuspended in binding buffer, and stained with Annexin V–FITC and propidium iodide (PI) (KeyGen Biotech, Nanjing, China) according to tivation of caspase-1 in iNKT results in the cleavage of the the manufacturer’s instructions. The stained cells were analyzed with flow pyroptotic protein gasdermin D, which generates a mem- cytometry (EPICS XL/MCL, Beckman Coulter, Fullerton, CA). Data were http://www.jimmunol.org/ brane pore–forming fragment, leading to pyroptotic cell death analyzed with FlowJo Software (Tree Star, Ashland, OR). (26). Nevertheless, the expression and function of OX40 in Western blot MAIT cells remain undefined. In this study, we explored the expression and function of Western blot was performed as described previously (28). Cells were rinsed OX40 on MAIT cells in T2D. We demonstrated that a sub- three times with ice-cold PBS (pH 7.4) and treated with lysis buffer containing + 1% (v/v) inhibitor mixture, 1 mM phenylmethylsulphonyl fluoride, stantial proportion of OX40 MAIT cells were induced in and 1 mM DTT. Cell lysates with equivalent protein amounts (20 mg) were the peripheral blood of patients with T2D. Furthermore, the loaded, separated by SDS-PAGE, and then transferred to polyvinylidene OX40+ MAIT cell subset had a high expression of activation difluoride membrane. The membranes were blocked in PBS-Tween 20 and memory molecules as well as of the death receptor Fas. (pH 7.4, 0.5% Tween 20) with 5% BSA and then incubated overnight by guest on September 23, 2021 with the primary Abs at 4˚C. Then, the membranes were incubated with OX40 expression was significantly negatively correlated with the appropriate HRP-conjugated secondary Abs at room temperature for the frequency of blood MAIT cells in T2D patients. They 1 h and at last visualized with an ECL (KeyGen Biotech) according to showed high expression of cleaved caspase-3, and OX40 signal the manufacturer’s instructions. promoted caspase-3 activation and apoptosis of MAIT cells. Statistical analysis The inhibition of caspase-3 blocked apoptosis of MAIT cells. These results identify OX40 as an amplifier of activation- Data analyses were performed in GraphPad Prism 5.0 Software (San Diego, induced cell death (AICD) of human blood MAIT cells and CA). Statistical significance was determined with Kruskal–Wallis or Mann– Whitney nonparametric tests or with ANOVA or Student t tests. Data are shed new light on the regulation of MAIT cells in the phase of shown as mean 6 SD unless stated otherwise. A p value , 0.05 was immune responses in T2D. regarded as statistically significant.

Materials and Methods Results Ethics statement Upregulation of OX40 in peripheral blood MAIT cells of T2D The cohort study was approved by the following institutional review To determine whether OX40 is expressed in MAIT cells of T2D, we boards: Affiliated Hospital of Guangzhou Medical University, Qingyuan analyzed the expression level of OX40 in MAIT cells by flow People’s Hospital, Guangzhou Women and Children’s Medical Center, and cytometry in human PBMCs of healthy participants (n = 44; Table Sun Yat-sen University prior to commencement of the study. All partici- + pants provided written informed consent for their participation in the study. I) and T2D patients (n = 53; Table I). OX40 cells were gated on + + + Only adult participants were enrolled. live CD3 TCRVa7.2 CD161 cells. We found that OX40 was highly expressed in human peripheral blood Vd2T cells of T2D Blood sample collection patients when compared with the healthy controls (Fig. 1A). Next, Whole blood from registered T2D patients (n = 53) and age-, gender-, we analyzed the percentage of OX40+ MAIT cells. We found that and body mass index–matched healthy donors (n = 44) was collected circulating MAIT cells in T2D patients showed a high frequency of from Qingyuan People’s Hospital (Qingyuan, China). Anthropometric OX40+ MAIT cells (14.06 6 1.14%; n = 53). In contrast, OX40+ and metabolic data were collected by experienced physicians. Subjects ∼ 6 underwent a standardized 75 g oral glucose tolerance test to investigate MAIT cells represented 7% (7.42 0.79%; n = 44) of all circu- glucose status. Study participants were categorized according to World lating MAIT cells in healthy donors (Fig. 1B). Together, these results Health Organization criteria: fasting plasma glucose (FPG) , 6 1 mmol/l demonstrate that OX40 is expressedoncirculatingMAITcellsandis and2hplasmaglucose, 7 8 mmol/l as normal glucose tolerance and increased in T2D patients. FPG $ 6 1 mmol/l or 2 h plasma glucose $ 7 8 mmol/l as impaired To investigate the expression of OX40 in other T cell subsets, glucose regulation or prediabetes. Detailed clinical characteristics and + laboratory information are shown in Table I. All study participants in we analyzed OX40 expression in iNKT (CD3 TCRVa24- this cohort were recruited at the same time. Ja18+).WefoundthatOX40wasupregulatedinperipheral The Journal of Immunology 3

2 Table I. Characteristics of study population increased in OX40+ MAITcellswhencomparedwithOX40 MAIT cells. Together, these results indicate that OX40+ Participants HC (n = 44) T2D (n = 53) MAIT cells could be a subset of activated T cells in T2D pa- Characteristic tients and, if activated, can express significant amounts of death Age (y) 45.28 (62.4) 40.56 (61.7) receptor Fas. Gender (Female/ 30/10 18/26 Male) OX40 expression is associated with the frequency of BMI (kg/m2) 23.69 6 3.19 24.23 6 3.26 MAIT cells WC (cm) 82.22 6 10.41 85.37 6 7.98* WHR 0.86 6 0.05 0.87 6 0.04* A published study has shown that circulating MAIT cell fre- SBP (mm Hg) 116.08 6 15.05 128.47 6 16.36* quency was dramatically decreased in T2D patients compared DBP (mm Hg) 75.67 6 11.09 84.25 6 9.36* with healthy controls (10). In this study, MAIT cells represented TG (mmol/l) 1.14 (0.75–1.77) 2.01 (0.88–2.95)** ∼3.9% (3.85 6 0.314%; n = 44) of all circulating CD3+ Tcells 6 6 TC (mmol/l) 5.12 0.99 5.17 1.14 in healthy donors (Fig. 3A, 3B). We found that circulating LDL-C (mmol/l) 2.83 6 0.84 3.16 6 0.81 HDL-C (mmol/l) 1.35 (1.12–1.49) 1.11 (0.82–1.42)pp T cells in T2D patients showed a low frequency of MAIT cells HbA1c (%) NA 6.8 (5.6–8.4) (1.72 6 0.147%; n = 53) (Fig. 3A, 3B). To analyze how OX40 HbA1c (mmol/l) NA 50 (39–68) expression was related to the frequency of MAIT cells, we FPG (mmol/l) 5.24 (4.7–5.5) 7.17 (5.6–8.7)pp performed a correlation analysis between OX40+ and MAIT cells FCP (mmol/l) 0.44 (0.33–0.62) 0.63 (0.36–0.78)* in both healthy controls and T2D patients. Significant correlations n 6 + Data are the percentage ( ), mean SD, and median (25th–75th percentile). were found between the levels of OX40 and MAIT cells in both Downloaded from BMI, body mass index; DBP, diastolic blood pressure; FCP, fasting C-peptide; 2 , HbA1c, glycated hemoglobin; HC, healthy controls, normal glucose tolerance healthy controls (n = 44; linear regression, r = 0.53; p 0.01; subjects; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density Fig. 3C) and T2D patients (n = 53; linear regression, r = 20.61; lipoprotein cholesterol; NA, not appropriate; SBP, systolic blood pressure; p , 0.001; Fig. 3D). Moreover, slight correlations were found TC, total cholesterol; TG, triglycerides; WC, waist circumference; WHR, waist-hip + ratio. between the levels of OX40 and NKT cells in T2D patients *p , 0 05, **p , 0 01 compared with the healthy controls. (n = 53; linear regression, r = 20.61; p , 0.001; Supplemental

Fig. 1C). http://www.jimmunol.org/ blood iNKT of T2D patients when compared with the healthy Activated OX40 induce AICD of MAIT cells in T2D patients controls (Supplemental Fig. 1A, 1B). + To elucidate whether cell death was involved in the decreased Increased activation and memory status in OX40 MAIT cells frequency of circulating MAIT cells in T2D patients, we quantified of T2D patients the expression of cleaved caspase-3 in MAIT cells isolated from To investigate the memory phenotype of MAIT cells expressing peripheral blood by flow cytometry. MAIT cells from T2D patients OX40, we analyzed memory marker CD45RO (Fig. 2A). The had a significantly higher proportion of cleaved caspase-3+ (n = 20; percentage of CD45RO-positive cells was significantly in- 16.96 6 0.986%) than cells from healthy controls (n = 20; 6.82 6 + creased in the OX40 MAIT cell population compared with 0.606%; p , 0.001; Fig. 4A, 4B), suggesting that MAIT cells had by guest on September 23, 2021 OX40– MAIT cells (Fig. 2A, 2B). Because OX40 has been higher apoptotic proportions in T2D disease. To investigate shown to be expressed in activated T cells (20), we next explored whether OX40 was involved in cell death of MAIT cells in T2D whether OX40+ MAIT cells display any features of an activation disease, we analyzed the level of cleaved caspase-3+ in OX40+ phenotype. Upregulation of the surface molecule CD69 was ob- MAIT cells of T2D patients. MAIT cells expressing OX40 had served on OX40+ MAIT cells compared with OX402 MAIT cells substantially increased cleaved caspase-3 (27.8 6 1.41%) than (Fig. 2C). In addition, a significantly higher level of death receptor OX40-negative cells (13.6 6 0.771; p , 0.001; Fig. 4C, 4D). Fas was observed in OX40+ MAIT cells compared with OX40– AICD is the of lymphocytes after the MAIT cells (Fig. 2C). The mean fluorescence intensity of repeated stimulation of TCRs (29). This process is vital to host CD69 and Fas were significantly upregulated in OX40+ regulatory mechanisms as well as to maintaining peripheral im- 2 MAIT cells compared with OX40 MAIT cells (Fig. 2D, 2E). mune tolerance. To determine the direct role of OX40 in inducing To analyze the functional activity of OX40+ MAIT cells, AICD of MAIT cells, we continued with the examination of PBMCs from T2D patients (n = 24) were stimulated with anti- cleaved caspase-3 by sorting MAIT cells from T2D patients in CD3/CD28 (1 mg/ml) and brefeldin A (10 mg/ml) for 12 h. plates coated with anti-CD3 Ab in the presence of recombinant Flow cytometry data showed that upon stimulation, the pro- OX40 ligand (OX40L). Next, cultured cells were collected and the duction of IL-17a (Fig. 2F, 2G) and IFN-g (Fig. 2H, 2I) was expression of cleaved caspase-3 was detected by Western blot.

FIGURE 1. Analysis of OX40+ MAIT cells in the test cohort. (A) Qualitative analysis of OX40 expression gated in MAIT cells from healthy controls and T2D patients. Profiles are gated in TCRa7.2+ CD161+ cells, and representative flow cytometric plots are shown. (B) The percentage of OX40+ MAIT cells in healthy controls (n = 44) and T2D patients (n = 51). ***p , 0.001, Student t tests. 4 OX40 PROMOTES AICD OF MAIT CELLS IN T2D PATIENTS Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 2. Alterative phenotype of OX40+ MAIT cells in T2D patients. (A and B) Expression of CD45RO on OX40+ or OX402 MAIT cells was determined by flow cytometry (top). The percentage of CD45RO+ expressing OX40+ MAIT cells (n = 45, bottom). (C–E) Expression of CD69 and FasonMAITcellswasdeterminedbyflowcytometry(top). The percentage of CD69 and Fas expressing OX40+ MAIT cells (n = 45, bottom). Overlays show blank (filled histograms) and surface markers (open histograms). The mean fluorescence intensity (MFI) of CD69 and Fas on OX40+ and OX402 MAIT cells are also shown. (F–I) PBMCs from T2D patients (n = 24) were stimulated with anti-CD3/CD28 (1 mg/ml) and brefeldin A (10 mg/ml) for 12 h. The percentages of IL-17a+ (F and H)andIFN-g+ (G and I) cells were analyzed by flow cytometry. ***p , 0.001, Student t tests.

MAIT cells stimulated with OX40L increased cleavage of caspase-3 inhibitor Z-DEVD-FMK in the presence of anti-CD3 caspase-3 in a time- and dose-dependent manner (Fig. 4E, 4F). Ab and recombinant OX40L. Indeed, Z-DEVD-FMK efficiently The data suggest that OX40 directly induces AICD of MAIT cells, prevented apoptosis induced by anti-CD3 and OX40L, suggest- which may regulate cellular immunity during T2D disease. ing that caspase-3 is essential for intracellular processing of AICD in MAIT cells (Fig. 5B). These data suggest that the AICD Inhibition of caspase-3 blocked OX40 induction of AICD mechanism in OX40-expressing MAIT cells requires cleavage of of MAIT cells caspase-3. To confirm that TCR-induced AICD was amplified by OX40 signal, we stimulated purified MAIT cells with OX40L in the Discussion presence of anti-CD3 mAb for 18 h. Apoptosis of MAIT cells was As a member of the TNFR superfamily, OX40 displays a broad determined by annexin V–PI staining. Activated OX40 slightly range of expression and regulatory functions in T cells. Increasing increased the percentages of annexin V+PI+ MAIT cells (referred evidence shows that OX40 is induced by infection or inflammation as apoptosis cells) in the absence of anti-CD3 mAb (Fig. 5A, 5B). and functions as an immune modulator in both CD4+ (including Combined with TCR signal induced by anti-CD3 mAb, OX40L Th1, Th2, Th9, Th17, and Treg) and CD8+ T cells (20, 21). significantly enhanced apoptosis of MAIT cells (Fig. 5A, 5B). To However, whether OX40 participates in the immune modulation examine whether cleavage of caspase-3 was essential for AICD of MAIT cells remains unknown. In this study, we demonstrated induced by OX40 in MAIT cells, we treated MAIT cells with the for the first time, to our knowledge, that OX40 was expressed on The Journal of Immunology 5

FIGURE 3. OX40 was associated with the frequency of MAIT cells in T2D patients. (A and B) Qualitative analysis of TCRa7.2+ CD161+ MAIT cells gated in CD3+ cells from healthy control and T2D participants. (C and D) Correlation between frequency of MAIT cells and OX40+ was analyzed in healthy participants (n =44)and T2D patients (n = 51) by SPSS software. **p , 0.01, ***p , 0.001, Student t tests and Spearman correla- tion. r, correlation coefficient. Downloaded from

MAIT cells and induced apoptosis (in this study as AICD) of expressed higher levels of surface and intracellular MAIT cells through cleaved caspase-3. cleaved caspase-3 than OX40 cells in T2D patients. Activation of Previous studies have reported that OX40 is expressed in OX40 with OX40L recombination protein could induce apoptosis classical CD4+ and CD8+ T cells, as well as NK, gdT, and of MAIT cells, indicating that OX40-induced cell death contrib- http://www.jimmunol.org/ NKT cells, and can act directly in combination with TCR stimu- uted to a reduced frequency of MAIT cells in peripheral blood of lation to enhance the activation and production of cytokines T2D patients. (30). In this study, we showed that OX40 was widely expressed in Apoptosis of MAIT cells occurs after activation by TCR and human MAIT cells and upregulated in T2D patients. Interestingly, costimulatory signals; this is known as AICD (31). Previous studies OX40 expression was found to be negatively correlated with the found that AICD is initiated at cell surface death receptors, such as frequency of MAIT cells. We speculated that OX40 may play an Fas and TNFR2 (32). The Fas receptor (also known as CD95 and important role in the alteration of MAIT cell frequency in T2D APO-1) is a member of the TNF-a family of death receptors that patients. Decreased MAIT cell frequency has been observed in mediate T cell responses (33). The exposure of activated T cells to by guest on September 23, 2021 T2D patients (10). In this study, we found that OX40+ MAIT cells IL-2 results in transcriptional induction of the encoding Fas

FIGURE 4. OX40 signal promoted caspase-3 activation of MAIT cells in T2D patients. (A and B) Representative histograms of cleaved caspase-3 of MAIT cells in healthy control and T2D patients as determined by flow cytometry. (C and D) Repre- sentative histograms of cleaved caspase-3 of OX40+ or OX402 MAIT cells in T2D patients as determined by flow cytometry. Numerical values denote the percentages of cleaved caspase-3+ cells. (E) Sorted TCRa7.2+ MAIT cells were stimulated with a differ- ent concentration of OX40L recombinant protein in the presence of anti-CD3 Ab (10 mg/ml). (F) Sorted TCRa7.2+ MAIT cells were stimulated with OX40L recombinant protein (10 mg/ml) and anti-CD3 Ab (10 mg/ml) at the indicated time. Expression of cleaved caspase-3 was determined by Western blot. Sample size in healthy controls and T2D patients was n = 20. ***p , 0.001, Student t tests. 6 OX40 PROMOTES AICD OF MAIT CELLS IN T2D PATIENTS Downloaded from http://www.jimmunol.org/

FIGURE 5. Activated OX40 induces AICD of MAIT cells in a caspase-3–dependent manner. (A and B) Sorted MAIT cells were stimulated with OX40L recombinant protein (10 mg/ml) and anti-CD3 Ab (10 mg/ml) at 18 h. (C and D) Anti-CD3 and OX40L-induced MAIT cells were treated with DMSO or caspase-3 inhibitor (Z-DEVD-FMK, 50 mM). Apoptosis of MAIT cells was determined by Annexin V/PI staining. The numbers indicate the percentages of Annexin V+/PI+ cells. Data are representative of at least three independent experiments. Student t tests, *p , 0.05, **p , 0.01, ***p , 0.001. ligand (FasL) and its expression on the cell surface (34). Fas re- broader than is reflected in professional APCs. It is reported that ceptor directly binds to and activates TCR components and re- additional immune cell types such as NK cells (38) and mast cells quires a functional TCR complex (35). Fas receptor-bound FADD (39), as well as structural cells such as vascular endothelial cells by guest on September 23, 2021 forms a death-inducing signaling complex with caspase-8 (as well (40) and smooth muscle cells (41), can express OX40L induced as with caspase-10 in human cells). Reactivation of T cells via the by the inflammatory cytokines (mast cells, smooth muscle cells) TCR and subsequent Fas/FasL interactions directly activate ef- or by receptors such as NKG2D (NK cells). Furthermore, signals fector that eventually trigger AICD (35). In our study, the through CD40, membrane BCR, and several TLR (TLR2, 4, and activation phenotype of OX40+ MAIT cells was confirmed by a 9), as well as inflammatory cytokines such as TSLP, GM-CSF, high expression of CD69 and Fas as well as a high production of IL-1, TNF, and IL-18 (42, 43), can upregulate OX40L expres- IFN-g and IL-17a in T2D patients, indicating that FasL/Fas sig- sion. Although there have been no studies on OX40L expression naling may participate in apoptosis of OX40+ MAIT cells. in diabetes and obesity, OX40L expression level or OX40L+ cell OX40 was first identified as a cell death receptor for liver- number may increase in association with low-grade chronic in- resident iNKT (26). OX40 signaling activates caspase-1 in flammation. In this study, we used OX40L recombination protein iNKT through the recruitment of MALT1, which leads to IL-1b to stimulate sorted MAIT cells, which specifically activated OX40 processing, as well as activation of the pyroptotic protein signaling. gasdermin D to induce pyroptotic cell death (26). In this study, In conclusion, we have shown that OX40 is upregulated in we found that OX40 signals trigger an effector caspase-3 in MAIT cells of T2D patients and promotes caspase-3–dependent MAIT cells, which in turn executes apoptosis by cleaving targeted apoptosis. Our findings shed new light on the activation and reg- cellular . Caspase-3 is activated in apoptotic cells both by ulation of MAIT cells in the phase of T2D disease. These findings extrinsic (death ligand) and intrinsic (mitochondrial) pathways broaden our knowledge of OX40 and may be used as a potential (34). After TNFR-mediated adaptor protein FADD activation, therapeutic approach for inflammatory disease. initiator caspase-8 and caspase-9 are recruited and cleaved, followed by cleavage of caspase-3 (34). Caspase-3 is essential Acknowledgments for apoptosis, whereas caspase-1 is required for pyroptosis. We thank all members of the clinical laboratory department at Qingyuan In our study, we found that OX40 was increased in iNKT of T2D People’s Hospital, Sun Yat-sen University for technical support. patients, which was slightly associated with the frequency of iNKT. OX40 induces distinct types of cell death depending on cell subsets in different inflammatory or infectious microenvironments. Disclosures OX40L (also known as gp34, CD252, and TNFSF4) is a type II The authors have no financial conflicts of interest. glycoprotein expressed in professional APCs, including activated B cells, mature conventional dendritic cells, Langerhans cells, References plasmacytoid DCs, and macrophages (36, 37). The expression of 1. Sell, H., C. Habich, and J. Eckel. 2012. Adaptive immunity in obesity and insulin these molecules in inflamed and diseased tissue might be much resistance. Nat. Rev. Endocrinol. 8: 709–716. The Journal of Immunology 7

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Supplementary Figure . Analysis of OX40+ iNKT cells in the test cohort.

(A) Qualitative analysis of OX40 expression gated in iNKT cells from healthy control and T2D participants. Profiles are gated in CD3+ TCRVα24-Jα18+ cells, and representative flow cytometric plots were shown. (B) The percentage of OX40+ iNKT cells in healthy controls (n= 44) and in active TB (n=51). ***p < 0.001. (C)

Correlation between frequency of iNKT cells and OX40+ was analyzed in T2D patients (n=51) by SPSS Software. R, correlation coefficient; *p < 0.05, ***p < 0.001.