Innate-like Cytotoxic Function of Bystander-Activated CD8+ T Cells Is ...

Article

Innate-like Cytotoxic Function of BystanderActivated CD8+ T Cells Is Associated with Liver Injury in Acute Hepatitis A Graphical Abstract

Authors Jihye Kim, Dong-Yeop Chang, Hyun Woong Lee, ..., Su-Hyung Park, Hyung Joon Kim, Eui-Cheol Shin

Correspondence park3@kaist.ac.kr (S.-H.P.), mdjoon@cau.ac.kr (H.J.K.), ecshin@kaist.ac.kr (E.-C.S.)

In Brief

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During acute hepatitis A (AHA), non-HAV-specific memory CD8+ T cells are activated Non-HAV-specific CD8+ T cells are activated by IL-15 produced by HAV-infected cells CD8+ T cells of AHA patients exert TCR-independent, innatelike cytotoxicity Innate-like cytotoxicity of CD8+ T cells is associated with liver injury in AHA

Kim et al., 2018, Immunity 48, 161–173 January 16, 2018 ª 2017 Elsevier Inc. https://doi.org/10.1016/j.immuni.2017.11.025

During acute hepatitis A, hepatitis A virus (HAV)-infected cells produce IL-15 that induces TCR-independent bystander activation of non-HAV-specific memory CD8+ T cells. These CD8+ T cells exert innate-like cytotoxicity triggered by NKG2D and NKp30 without TCR engagement. The severity of liver injury is associated with activation and innate-like cytotoxicity of non-HAV-specific CD8+ T cells, but not the activation of HAVspecific T cells. Thus, IL-15-induced bystander-activated CD8+ T cells are implicated in host injury during acute viral infection.

Immunity

Article Innate-like Cytotoxic Function of Bystander-Activated CD8+ T Cells Is Associated with Liver Injury in Acute Hepatitis A Jihye Kim,1,8 Dong-Yeop Chang,2,8 Hyun Woong Lee,3,8 Hoyoung Lee,1,8 Jong Hoon Kim,2 Pil Soo Sung,2 Kyung Hwan Kim,2 Seon-Hui Hong,1 Wonseok Kang,2 Jino Lee,2 So Youn Shin,2 Hee Tae Yu,2 Sooseong You,2 Yoon Seok Choi,2 Insoo Oh,2 Dong Ho Lee,4 Dong Hyeon Lee,5 Min Kyung Jung,2 Kyung-Suk Suh,6 Shin Hwang,7 Won Kim,5 Su-Hyung Park,1,2,* Hyung Joon Kim,3,* and Eui-Cheol Shin1,2,9,* 1Biomedical

Science and Engineering Interdisciplinary Program, KAIST, Daejeon 34141, Republic of Korea School of Medical Science and Engineering, KAIST, Daejeon 34141, Republic of Korea 3Department of Internal Medicine, Chung-Ang University Hospital, Seoul 06973, Republic of Korea 4Department of Surgery, College of Medicine, The Catholic University of Korea, Daejeon St. Mary’s Hospital, Daejeon 34943, Republic of Korea 5Department of Internal Medicine, Seoul Metropolitan Government Seoul National University Boramae Medical Center, Seoul 07061, Republic of Korea 6Department of Surgery, Seoul National University College of Medicine, Seoul 03080, Republic of Korea 7Department of Surgery, Asan Medical Center, University of Ulsan College of Medicine, Seoul 05505, Republic of Korea 8These authors contributed equally 9Lead Contact *Correspondence: park3@kaist.ac.kr (S.-H.P.), mdjoon@cau.ac.kr (H.J.K.), ecshin@kaist.ac.kr (E.-C.S.) https://doi.org/10.1016/j.immuni.2017.11.025 2Graduate

SUMMARY

Acute hepatitis A (AHA) involves severe CD8+ T cellmediated liver injury. Here we showed during AHA, CD8+ T cells specific to unrelated viruses became activated. Hepatitis A virus (HAV)-infected cells produced IL-15 that induced T cell receptor (TCR)-independent activation of memory CD8+ T cells. TCR-independent activation of non-HAV-specific CD8+ T cells were detected in patients, as indicated by NKG2D upregulation, a marker of TCR-independent T cell activation by IL-15. CD8+ T cells derived from AHA patients exerted innate-like cytotoxicity triggered by activating receptors NKG2D and NKp30 without TCR engagement. We demonstrated that the severity of liver injury in AHA patients correlated with the activation of HAV-unrelated virus-specific CD8+ T cells and the innate-like cytolytic activity of CD8+ T cells, but not the activation of HAV-specific T cells. Thus, host injury in AHA is associated with innate-like cytotoxicity of bystander-activated CD8+ T cells, a result with implications for acute viral diseases.

INTRODUCTION During viral infection, tissue damage is caused not only by the direct cytopathic effects of the infecting virus, but also by host immune responses, known as immunopathology. Innate and adaptive immune responses contribute to the development of immunopathology through diverse mechanisms (Rouse and

Sehrawat, 2010). In particular, the destruction of infected cells by CD8+ T cells is known to be the primary cause of tissue damage in non-cytopathic viral infections, such as hepatitis A virus (HAV), hepatitis B virus (HBV), and hepatitis C virus (HCV) (Guidotti and Chisari, 2006; Rehermann and Nascimbeni, 2005; Shin et al., 2016; Siegl and Weitz, 1993). CD8+ T cell-mediated immunopathogenesis during viral infection has largely been investigated using an HBV transgenic mouse model in which adoptive transfer of HBV-specific CD8+ T cells into liver-specific HBV transgenic mice causes acute necroinflammatory liver disease (Ando et al., 1993). In this model, HBV-specific CD8+ T cells directly induce the apoptotic death of hepatocytes (Ando et al., 1993, 1994), and liver injury is further amplified by the recruitment of antigen-nonspecific lymphomononuclear cells into the liver (Ito et al., 2009; Kakimi et al., 2001; Sitia et al., 2004). A study of patients with chronic HBV infection also suggested that both HBV-specific CD8+ T cells and antigen-nonspecific CD8+ T cells are involved in liver injury during HBV infection (Maini et al., 2000). Activation and proliferation of antigen-independent bystander CD8+ T cells and the function of bystander CD8+ T cells during viral infection have remained controversial. Though some studies have demonstrated the proliferation of bystander CD8+ T cells during viral infection in mouse models (Haring et al., 2002; Tough et al., 1996; Tough and Sprent, 1996), other studies have suggested that bystander activation of CD8+ T cells is insignificant in terms of their size and functional role (Chen et al., 2005; Ehl et al., 1997; Murali-Krishna et al., 1998; Zarozinski and Welsh, 1997). In humans, the activation of CD8+ T cells not specific to the infecting virus has been observed during acute viral infections, including primary human immunodeficiency virus (HIV) infection, acute HBV infection, and hantavirus infection (Doisne et al., 2004; Sandalova et al., 2010; Tuuminen et al., 2007), but not after vaccination with live-attenuated viruses Immunity 48, 161–173, January 16, 2018 ª 2017 Elsevier Inc. 161

(Miller et al., 2008). To date, antigen-independent bystander activation of CD8+ T cells and their possible immunopathologic roles in viral infection have not been elucidated clearly. In the present study, we investigated the activation status and function of CD8+ T cells and the mechanisms by which CD8+ T cells mediate host injury in acute hepatitis A (AHA), which is caused by HAV infection and manifests as severe liver injury in adults (Walker et al., 2015). Though HAV-specific CD8+ T cells are thought to be responsible for liver injury in AHA (Siegl and Weitz, 1993), a recent study showed that HAV-specific CD8+ T cells do not exhibit effector function in HAV-infected chimpanzees (Zhou et al., 2012). In this context, we examined HAVspecific and non-HAV-specific CD8+ T cells in AHA patients and studied a mechanism of immune-mediated liver injury in AHA. Here, we demonstrate that CD8+ T cells specific to HAVunrelated viruses are activated and proliferate during AHA. HAV-infected cells produced IL-15, which induced TCR-independent activation of CD8+ T cells. CD8+ T cells isolated from AHA patients exerted innate-like cytolytic activity, which was triggered by ligation of NKG2D and NKp30 without TCR engagement. We also showed that hepatocytes from HAV-infected liver overexpressed NKG2D ligands, making them potential cytolytic targets of bystander-activated HAV-unrelated virus-specific CD8+ T cells. Finally, we observed that liver injury during AHA is associated with innate-like cytotoxic function of bystanderactivated CD8+ T cells. Taken together, our results show a human viral disease in which host injury is associated with innate-like cytolytic activity of bystander-activated CD8+ T cells. RESULTS CD8+ T Cell Activation and the Expression of Cytotoxic Molecules Correlate with the Severity of Liver Injury during AHA First, we examined the activation status of peripheral blood CD8+ T cells in AHA patients at the time of admission and found that the frequency of activated (CD38+HLA-DR+) cells was significantly increased compared to healthy donors (Figure 1A). Next, we determined whether there is a correlation between the activation of CD8+ T cells and severity of liver injury evaluated by serum alanine aminotransferase (ALT) levels. The percentage of activated CD38+HLA-DR+ CD8+ T cells in peripheral blood significantly correlated with serum ALT levels at the time of admission (Figure 1B). In addition, these activated CD8+ T cells expressed high levels of proliferation marker Ki-67, cytotoxic molecules (e.g., perforin, granzyme A, and granzyme B), and NK-activating receptors (e.g., NKG2D and NKp30) (Figure 1C). Further analysis revealed that the frequency of Ki-67+ cells among CD8+ T cells in the peripheral blood was significantly increased in AHA patients compared to healthy donors (Figure 1D) and significantly correlated with serum ALT levels at the time of admission (Figure 1E). The percentage of highly cytotoxic (perforin+granzyme B+) cells was also significantly increased in AHA patients compared to healthy donors (Figure 1F) and significantly correlated with serum ALT levels (Figure 1G). Taken together, these data substantiate CD8+ T cell-mediated immunopathogenesis during AHA (Siegl and Weitz, 1993). 162 Immunity 48, 161–173, January 16, 2018

HAV-Unrelated Virus-Specific CD8+ T Cells Are Activated during AHA To elucidate the antigen specificity of activated CD38+HLA-DR+ CD8+ T cells, we used HLA class I multimers. At the time of admission, HAV-specific CD8+ T cells were successfully detected in the peripheral blood and presented an activated phenotype of CD38+HLA-DR+ (Figure 2A). Next, we examined the activation status of CD8+ T cells specific for viruses other than HAV, including influenza A virus (IAV), Epstein-Barr virus (EBV), and cytomegalovirus (CMV). Unexpectedly, a considerable proportion of HAV-unrelated virus-specific CD8+ T cells in the peripheral blood of AHA patients presented an activated phenotype (CD38+HLA-DR+) at the time of admission regardless of virus specificity (Figure 2B), whereas IAV-specific, EBVspecific, and CMV-specific CD8+ T cells were minimally activated in the peripheral blood of healthy donors (Figure 2C). Statistical analyses revealed that the activation of IAV-specific, EBV-specific, and CMV-specific CD8+ T cells in AHA patients was significantly higher than that in healthy donors (Figure 2D). We also examined the activation status of peripheral blood CD8+ T cells specific for other HAV-unrelated viruses, including vaccinia virus (VV) and respiratory syncytial virus (RSV). VVspecific and RSV-specific CD8+ T cells were highly activated in AHA patients at the time of admission (Figure 2E). Moreover, both HAV-specific CD8+ T cells and CMV-specific CD8+ T cells were highly activated in HAV-infected liver from a patient with fulminant AHA (Figure 2F). Analysis of the peripheral blood from an AHA patient showed that activation of HAV-unrelated virus-specific CD8+ T cells and HAV-specific CD8+ T cells was apparent based not only on the CD38+HLA-DR+ phenotype, but also by high levels of granzyme B and Ki-67 expression (Figure 2G). Thus, both HAV-unrelated virus-specific CD8+ T cells and HAV-specific CD8+ T cells present an activated phenotype with proliferation and overexpression of cytotoxic molecules during AHA. HAV-Infected Cells Produce IL-15, Inducing AntigenIndependent Activation of CD8+ T Cells We hypothesized that the activation of HAV-unrelated virusspecific CD8+ T cells is driven by cytokines known to activate T cells independent of antigen. To this end, we sought to identify which cytokines are increased during AHA. Among cytokines measured, IL-2 was not detected in the sera of healthy donors and AHA patients, and though IL-7 was detected in serum, it was not increased in AHA patients (Figure 3A). However, serum IL-15 and IL-18 were significantly increased in AHA patients compared to healthy donors (Figure 3A). Next, we studied whether in vitro cytokine treatment activates the CD8+ T cells of healthy donors in the absence of antigen stimulation. IL-15 treatment maximally increased the frequency of activated CD38+HLA-DR+ CD8+ T cells in CD8+ T cells from healthy donors (Figure 3B). Similar results were observed with the frequency of Ki-67+CD8+ T cells after cytokine treatment (Figure 3B). IL-15induced activation was also observed in IAV-specific, EBVspecific, and CMV-specific CD8+ T cells from healthy donors. IL-15 treatment significantly increased the frequency of CD38+HLA-DR+CD8+ T cells (Figure 3C) and induced the overexpression of Ki-67, granzyme B, and perforin (Figure 3D). Taken together, these data indicate that IL-15 can activate

Figure 1. Activation and Proliferation of Peripheral Blood CD8+ T Cells in AHA Patients PBMCs acquired from 75 AHA patients at the time of admission and 10 healthy donors were analyzed by flow cytometry. (A and B) The percentage of CD38+HLA-DR+ cells in peripheral blood CD8+ T cells of AHA patients was compared with that of healthy donors (A) and analyzed for a correlation with serum ALT levels at the time of admission (B). (C) Expression of Ki-67, perforin, granzyme A, granzyme B, NKG2D, and NKp30 was analyzed in peripheral CD38+HLA-DR+CD8+ and CD38 HLA-DR CD8+ T cells. Representative data from a single AHA patient are presented. (D and E) The percentage of Ki-67+ cells in peripheral blood CD8+ T cells of AHA patients was compared with that of healthy donors (D) and analyzed for a correlation with serum ALT levels at the time of admission (E). (F and G) The percentage of perforin+granzyme B+ cells in peripheral blood CD8+ T cells of AHA patients was compared with that of healthy donors (F) and analyzed for a correlation with serum ALT levels at the time of admission (G). Error bars represent standard deviation (SD). ***p < 0.001.

CD8+ T cells from healthy donors, resulting in the CD38+HLADR+ and Ki-67+granzyme B+perforin+ phenotype observed in AHA patients. These findings suggest that IL-15 is the major cytokine driving the activation of non-HAV-specific CD8+ T cells during AHA. We found that in vitro HAV infection induced IL-15 production in HepG2 cells (Figure 3E). Immunofluorescent staining also revealed IL-15 expression in liver tissues from AHA patients with fulminant liver injury. IL-15 staining was evident in HAV antigen-positive cells (Figure 3F), but not in CD11c+HLA-DR+ dendritic cells and CD68+ macrophages (Figure S1). The majority of IL-15-stained cells were HAV antigen positive (Figure 3G), indicating that IL-15 was produced by HAV-infected cells. We confirmed overexpression of IL15 mRNA in liver tissues from AHA patients using quantitative real-time PCR (Figure 3H).

Because IL-15 needs to be trans-presented by IL-15Ra in order to be biologically active, we also examined IL-15Ra expression on CD14+ monocytes; IL-15Ra expression was upregulated on peripheral blood CD14+ monocytes from AHA patients compared to healthy donors (Figure 3I, left). Notably, IL-15Ra was expressed at higher levels on intrahepatic CD14+ cells compared to peripheral blood CD14+ monocytes during AHA (Figure 3I, right). Activation of HAV-Unrelated Virus-Specific CD8+ T Cells during AHA Is Bona Fide Bystander Activation without TCR Stimulation Activation of IAV-specific, VV-specific, and RSV-specific CD8+ T cells (Figures 2D and 2E) indicates that these cells are antigen independently activated in the absence of cognate antigen Immunity 48, 161–173, January 16, 2018 163

Figure 2. Activation of HAV-Specific CD8+ T Cells and CD8+ T Cells Specific to HAV-Unrelated Viruses in AHA Patients (A and B) HAV-specific CD8+ T cells (A) and CD8+ T cells specific to unrelated viruses, such as IAV, EBV, or CMV (B), were detected using HLA class I multimers and their activation status was evaluated by CD38 and HLA-DR staining of PBMCs from AHA patients at the time of admission. Representative data are presented (P39, P69, and P74 for HAV multimers, P5 for IAV multimer, P59 for EBV multimer, and P64 for CMV multimer). (C) IAV-specific, EBV-specific, or CMV-specific CD8+ T cells were detected using HLA class I multimers, and their activation status was evaluated by CD38 and HLA-DR staining of PBMCs from healthy donors. Representative data are presented. (D) The percentage of CD38+HLA-DR+ cells in virus-specific CD8+ T cells in peripheral blood was analyzed in AHA patients and healthy donors. Error bars represent SD. **p < 0.01 and ***p < 0.001. (E) VV-specific or RSV-specific CD8+ T cells were detected using HLA class I multimers, and their activation status was evaluated by CD38 and HLA-DR staining of PBMCs from AHA patients at the time of admission. Representative data are presented. (F) Intrahepatic lymphocytes were isolated from an explanted liver from a patient with fulminant AHA (P10) during liver transplantation. HAV-specific and CMV-specific CD8+ T cells were detected using HLA class I multimers, and their activation status was evaluated by CD38 and HLA-DR staining. (G) Expression of Ki-67 and granzyme B was analyzed in HAV-specific, EBV-specific, and CMV-specific CD8+ T cells and total CD8+ T cells of peripheral blood from an AHA patient at the time of admission.

164 Immunity 48, 161–173, January 16, 2018

Figure 3. IL-15 Expression during HAV Infection and Activation of CD8+ T Cells by IL-15 (A) Serum levels of IL-2, IL-7, IL-15, and IL-18 were determined by sandwich ELISA in AHA patients at the time of admission and healthy donors. (B) Peripheral blood CD8+ T cells from five healthy donors were treated with the indicated cytokine, and FACS analyses for CD38/HLA-DR (left) and Ki-67 (right) were performed. (C) Peripheral blood CD8+ T cells from three healthy donors were treated with IL-15, and the percentage of CD38+HLA-DR+ cells among IAV-specific, EBV-specific, or CMV-specific CD8+ T cells was determined. (D) Peripheral blood CD8+ T cells from healthy donors were treated with IL-15, and the expression of Ki-67/granzyme B and perforin/granzyme B was analyzed in the gate of IAV-specific, EBV-specific, or CMV-specific CD8+ T cells. Representative data from a single donor are presented. (E) HepG2 cells were infected with HAV and stained with anti-HAV and anti-IL-15 7 days after infection. (F) Immunofluorescent staining was performed with anti-HAV (green) and anti-IL-15 (red) in liver tissues without viral hepatitis and liver tissues obtained during liver transplantation from patients with fulminant AHA. Nuclei were stained with DAPI (blue). Merged images are also presented. Scale bar = 20 mm. Representative data from a single patient are presented. (G) The percentage of HAV antigen-positive cells among IL-15-positive cells was determined in liver tissues from five patients with fulminant AHA. (H) The expression of IL-15 was examined by TaqMan real-time quantitative PCR in liver tissues without viral hepatitis (n = 5) and liver tissues with AHA (n = 7). (I) IL-15Ra expression was examined in CD14+ monocytes. The data are compared between peripheral blood CD14+ monocytes from healthy donors and AHA patients (left), and between peripheral blood CD14+ monocytes and intrahepatic CD14+ cells of AHA patients (right). Representative data are presented. Error bars represent SD. *p < 0.05, **p < 0.01, and ***p < 0.001. See also Figure S1.

during AHA, but activation of EBV-specific and CMV-specific CD8+ T cells can be due to antigen-independent activation or reactivation of latent EBV or CMV. To clarify this issue, we measured serum DNA titers of EBV and CMV in serial samples from AHA patients taken during the course of AHA, from the acute phase to the convalescent phase. Among 14 patients, 3 (21.4%) and 7 (50.0%) presented evidence of EBV and CMV reactivation, respectively (representative cases in Figures S2A and S2C). Strikingly, however, activation of EBV-specific or CMV-specific CD8+ T cells was apparent during the acute phase of AHA, even in the absence of EBV or CMV reactivation (Figures S2B and S2D). We further clarified whether the activation of HAV-unrelated virus-specific CD8+ T cells during AHA is due to IL-15 or TCR stimulation. We focused on NKG2D expression, which has been shown to be increased on activated CD38+HLA-

DR+CD8+ T cells during AHA (Figure 1C). NKG2D expression was significantly increased on CD45RO+ memory CD8+ T cells after IL-15 treatment of healthy donors’ peripheral blood mononuclear cells (PBMCs) but not anti-CD3 stimulation (Figures 4A and 4B). Combined stimulation with anti-CD3 and IL-15 did not significantly increase NKG2D expression (Figures 4A and 4B). This finding was confirmed by stimulating CMV pp65495-specific CD8+ T cells with the cognate epitope peptide (NLVPMVATV), a control peptide, IL-15, or in combination. NKG2D expression was significantly increased on CMV pp65495-specific CD8+ T cells after IL-15 treatment in the absence of the cognate epitope peptide, whereas TCR stimulation with the cognate peptide did not increase NKG2D expression (Figure 4C). Thus, NKG2D upregulation on memory CD8+ T cells was an indicator of activation by IL-15 in the absence of TCR stimulation. TCRmediated interference in IL-15-induced NKG2D upregulation Immunity 48, 161–173, January 16, 2018 165

was significantly abrogated in part by Calpain inhibitor I (Figure 4D), indicating that TCR-stimulated calpain activity contributes to this phenomenon. In addition, decreased protein levels of Jak1 following anti-CD3 stimulation (Figure S3) might explain the additional mechanism of TCR-mediated interference in IL-15-induced NKG2D upregulation. Previous studies also demonstrated roles of calpain activity (Noguchi et al., 1997) and Jak1 downregulation (Katz et al., 2014) in the TCR-mediated inhibition of common cytokine receptor g chain signaling. Next, we examined the expression of NKG2D in HAV-specific or HAV-unrelated virus-specific CD8+ T cells in AHA patients and healthy donors. As expected, NKG2D expression on CMVspecific CD8+ T cells from healthy donors did not differ from the expression on CD45RO+CD8+ T cells from healthy donors (Figure 4E). However, CMV-specific or RSV-specific CD8+ T cells, but not HAV-specific CD8+ T cells, acquired at the time of admission from AHA patients expressed significantly higher levels of NKG2D than CD45RO+CD8+ T cells from healthy donors (Figure 4E). When we examined the expression level of NKG2D on CMV-specific CD8+ T cells from AHA patients with or without CMV reactivation, NKG2D expression on CMV-specific CD8+ T cells from AHA patients with CMV reactivation tended to be lower than those without CMV reactivation (Figure 4F). These data demonstrate that CD8+ T cells specific for HAV-unrelated viruses, such as CMV, were activated by IL-15 in the absence of TCR stimulation during AHA.

Figure 4. Increased Expression of NKG2D in IL-15-Treated CD8+ T Cells from Healthy Donors and CD8+ T Cells Specific to HAVUnrelated Viruses from AHA Patients (A and B) Sorted CCR7 CD8+ T cells from healthy donors (n = 10) were treated with anti-CD3, IL-15, or a combination for 48 hr. NKG2D expression was examined in the gate of CD45RO+ memory CD8+ T cells (A). Representative data from a single donor are presented (B). (C) PBMCs from healthy donors (n = 5) were treated with the CMV pp65495 epitope peptide (NLVPMVATV), a control peptide (SIINFEKL), IL-15, or a combination for 48 hr. NKG2D expression was examined in the gate of CMV pp65495 pentamer+ CD8+ T cells. (D) PBMCs from healthy donors (n = 4) were treated with anti-CD3, IL-15, or a combination for 48 hr in the presence or absence of Calpain inhibitor I. NKG2D expression was examined in the gate of CD45RO+ memory CD8+ T cells. (E) NKG2D expression levels were examined in the gate of CD45RO+ memory CD8+ T cells (n = 6) or CMV-specific CD8+ T cells (n = 5) in peripheral blood from healthy donors and in the gate of HAV-specific (n = 6), CMV-specific (n = 6), or RSV-specific (n = 3) CD8+ T cells in peripheral blood from AHA patients at the time of admission.


CD8+ T Cells from AHA Patients Exert NKG2D and NKp30-Dependent Innate-like Cytotoxic Activity Next, we assessed whether the cytolytic activity of CD8+ T cells from AHA patients is triggered in the absence of TCR engagement. Therefore, we determined whether CD8+ T cells from AHA patients can lyse K562 cells, which do not express HLA class I and are readily lysed by NK cells. We found that K562 cells were lysed not only by IL-15-activated CD8+ T cells isolated from healthy donors (Figure 5A) but also by intrahepatic CD8+ T cells isolated from the livers of AHA patients (Figure 5B), indicating that these CD8+ T cells exerted innate-like (or NK-like) cytolytic activity without TCR engagement. In addition, IL-15-activated CD8+ T cells isolated from healthy donors and intrahepatic CD8+ T cells isolated from the livers of AHA patients readily killed liver-derived Huh-7 cells (Figures 5C and 5D). To identify a potential mechanism for the innate-like cytolytic activity of CD8+ T cells from AHA patients, the expression of activating NK receptors was examined. First, we studied the expression pattern of activating NK receptors in IL-15-activated CD8+ T cells from healthy donors and found that the expression of both NKG2D and NKp30 was increased by IL-15 treatment (Figure 5E). In AHA patients, upregulation of NKG2D and NKp30 was observed on activated CD38+HLA-DR+CD8+ T cells in the intrahepatic compartment (Figure 5F), similar to the peripheral blood (Figure 1C). Of note, the expression of NKG2D and NKp30 on peripheral CD8+ T cells was not increased in an (F) NKG2D expression levels were examined in CMV-specific CD8+ T cells from peripheral blood of AHA patients with (n = 2) or without (n = 4) CMV reactivation at the time of admission. Error bars represent SD. *p < 0.05, **p < 0.01, and ***p < 0.001. See also Figures S2 and S3.

Figure 5. NKG2D-Dependent and NKp30-Dependent Innate-like Cytolytic Activity of CD8+ T Cells from AHA Patients (A–D) PKH26-labeled K562 cells (A and B) or Huh-7 cells (C and D) were co-cultured with IL-15-treated peripheral blood CD8+ T cells from healthy donors (A and C; n = 4) or intrahepatic CD8+ T cells isolated from the liver tissues of two patients with fulminant AHA (P10 and P48) or two non-AHA patients (non-AHA #1 and non-AHA #2) (B and D) at various effector:target (E:T) ratios. Cytotoxicity was evaluated by staining with TO-PRO-3-iodide and flow cytometric analysis. Error bars represent SD (A and C) or standard error of the mean (SEM) for triplicate experiments (B and D). (E) IL-15-treated peripheral blood CD8+ T cells from healthy donors were analyzed for the expression of activating NK receptors. Representative data are presented. (F) Expression of NKG2D and NKp30 was analyzed in intrahepatic CD38+HLA-DR+ and CD38 HLA-DR CD8+ T cells from AHA patients. Representative data are presented. (G–J) K562 cells (G and H) or P815 cells (I and J) were co-cultured with intrahepatic CD8+ T cells isolated from the liver tissues of patients with fulminant AHA (G and I; n = 2) or IL-15-treated peripheral blood CD8+ T cells from healthy donors (H and J) in the presence of the indicated antibodies. Experiments with IL-15treated CD8+ T cells were performed with cells from three healthy donors, and representative data from a single donor are presented. The E:T ratio in the K562 killing assay was 20:1 (G) or 10:1 (H), and the E:T ratio in P815 redirected killing assay was 40:1 (I and J). Error bars represent SD (G and I) or SEM for triplicate experiments (H and J). (K) CD8+ T cells isolated from the peripheral blood of healthy donors (n = 7) or AHA patients (n = 11) were co-cultured with PKH26-labeled K562 cells at a 10:1 ratio. Cytotoxicity was evaluated by staining with TO-PRO-3-iodide and presented as bar graphs. (legend continued on next page)

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Figure 6. Expression of NKG2D Ligands in Liver Tissues from AHA Patients (A) Immunofluorescent staining was performed with anti-HAV (green) and anti-MIC-A (red) in liver tissues without viral hepatitis and liver tissues obtained during liver transplantation from patients with fulminant AHA. Nuclei were stained with DAPI (blue). Merged images are presented. Scale bar = 20 mm. Representative data are presented. (B) The expression of MIC-A was examined by immunoblot analysis in liver tissues without viral hepatitis (n = 3) and liver tissues with AHA (n = 5). (C) The expression of MIC-A and MIC-B was examined by TaqMan real-time quantitative PCR in liver tissues without viral hepatitis (n = 5) and liver tissues with AHA (n = 7). Error bars represent SD.

asymptomatic HAV-infected patient with anti-HAV IgM-positive and normal serum ALT level (4,000 U/L) and exerted strong innate-like cytotoxicity, but those from healthy donors did not (Figure 5M). Livers of AHA Patients Overexpress NKG2D Ligands We also investigated whether the liver expresses ligands triggering the innate-like cytolytic activity of bystander-activated CD8+ T cells during AHA. As NKp30 ligands are not known, we investigated the expression of NKG2D ligands MIC-A and MIC-B in liver tissue from AHA patients. We found overexpression of MIC-A in HAV-infected liver tissue, but not in control liver tissue (Figure 6A). Interestingly, MIC-A expression was detected not only in HAV antigen-positive cells, but also in HAV antigennegative cells in HAV-infected liver tissue (Figure 6A). Thus, the NKG2D ligand is expressed on both HAV-infected and uninfected hepatocytes in HAV-infected liver tissue, and these cells may be innate-like cytolytic targets of bystander-activated CD8+ T cells in AHA patients. We confirmed increased expression of MIC-A protein in liver tissue from AHA patients by immunoblotting (Figure 6B), as well as increased MICA and MICB mRNA levels (Figure 6C). Activation of HAV-Unrelated Virus-Specific CD8+ T Cells and Innate-like Cytolytic Activity Correlates with Liver Injury during AHA To address the question of whether HAV-specific or HAVunrelated virus-specific CD8+ T cells contribute to liver injury in

(L) The percentage of CD38+HLA-DR+ cells in peripheral blood CD8+ T cells was analyzed for a correlation with the K562 cytotoxicity of CD8+ T cells isolated from peripheral blood at the time of admission in 11 AHA patients. (M) CMV pp65495-specific CD8+ T cells isolated from the peripheral blood of healthy donors (n = 4) or AHA patients (n = 4; P3, P35, P64, and P71) were co-cultured with PKH26-labeled K562 cells at a 10:1 ratio. Cytotoxicity was evaluated by staining with TO-PRO-3-iodide and compared between healthy donors and AHA patients. Error bars represent SD. *p < 0.05, **p < 0.01, and ***p < 0.001.


Figure 7. Correlation of Liver Injury with Activated CD8+ T Cells Specific to HAV-Unrelated Viruses and Innate-like Cytolytic Activity of CD8+ T Cells during AHA (A and B) Number of CD38+HLA-DR+ IAV MP58-specific (n = 14), EBV BMLF1259-specific (n = 12), CMV pp65495-specific (n = 26), and HAV 3D2026-specific (n = 18) CD8+ T cells per microliter of peripheral blood (A) and the percentage of them in the gate of each virus-specific peripheral CD8+ T cells (B) were analyzed for a correlation with serum ALT levels at the time of admission of AHA patients. (C and D) Direct ex vivo IFN-g ELISpot assay was performed with peptide mixes for HAV VP2 and 3C in PBMCs from 68 AHA patients (C) or with a mix of 27 HLAA2-restricted epitope peptides in PBMCs from 35 HLA-A2-positive AHA patients (D). The specific IFN-g spot number was analyzed for a correlation with serum ALT levels at the time of admission. (E) The VP2 and 3C-specific IFN-g spot number (left) and the percentage of CD38+HLA-DR+ cells in peripheral blood CD8+ T cells (right) were analyzed for a correlation with the serum HAV RNA titer at the time of admission of AHA patients. (legend continued on next page)


AHA, we analyzed their activation in correlation with serum ALT levels at the time of admission. Unexpectedly, the number of CD38+HLA-DR+CD8+ T cells specific to HAV-unrelated viruses (IAV, EBV, or CMV) but not the number of CD38+HLA-DR+ HAV-specific CD8+ T cells significantly correlated with serum ALT levels (Figure 7A). This was also the case with the percentage of CD38+HLA-DR+ cells among CD8+ T cells specific to HAV-unrelated viruses (IAV, EBV, or CMV) but not among HAVspecific CD8+ T cells (Figure 7B). These data indicate that HAV-unrelated virus-specific CD8+ T cells are associated with liver injury during AHA. Next, we examined HAV-specific T cell activity in PBMCs from AHA patients at the time of admission by direct ex vivo IFN-g ELISpot assays, which were performed with overlapping peptides (OLPs) spanning HAV VP2 and 3C proteins in order to comprehensively assess the HAV-specific IFN-g response regardless of the patient’s HLA allotype. The IFN-g response against VP2 and 3C inversely correlated with serum ALT levels (Figure 7C), indicating that HAV-specific T cells may not be major effectors in liver injury during AHA. In addition, direct ex vivo IFN-g ELISpot assays were performed with a mix of HLA-A2restricted HAV epitope peptides in HLA-A2+ patients. The IFN-g response against the mix of HLA-A2-restricted HAV epitopes inversely correlated with serum ALT levels (Figure 7D), corroborating the IFN-g response to VP2 and 3C. Interestingly, the IFN-g responses against the VP2 and 3C OLPs inversely correlated with serum HAV RNA titer, but the frequency of CD38+HLA-DR+ cells among total CD8+ T cells did not (Figure 7E), demonstrating an anti-viral role of HAV-specific T cells rather than a role in liver damage. Furthermore, the innate-like cytolytic activity of peripheral blood CD8+ T cells from AHA patients significantly correlated with serum ALT levels at the time of admission (Figure 7F). We examined changes in the phenotype of peripheral blood CD8+ T cells during the course of AHA, from the time of admission to the time of discharge (average 8 days after admission). The frequency of CD38+HLA-DR+ cells among total CD8+ T cells or HAV-unrelated virus (IAV, EBV, or CMV)-specific CD8+ T cells, but not among HAV-specific CD8+ T cells, significantly decreased with decreasing serum ALT levels (Figure 7G). In addition, the expression of NKG2D in CD8+ T cells specific to HAV-unrelated viruses significantly decreased (Figure 7H). Taken together, these findings suggest that liver injury during AHA is associated with activation of HAV-unrelated virusspecific CD8+ T cells and their innate-like cytotoxicity. DISCUSSION Activation of bystander CD8+ T cells during viral infection and the function of these cells during infection have been controversial in both mouse models (Chen et al., 2005; Ehl et al., 1997; Haring et al., 2002; Murali-Krishna et al., 1998; Tough et al., 1996; Tough

and Sprent, 1996; Zarozinski and Welsh, 1997) and human viral infections (Doisne et al., 2004; Miller et al., 2008; Sandalova et al., 2010; Tuuminen et al., 2007). Here, we investigated this issue during AHA, which is accompanied by severe liver injury in adults, by examining activation markers, proliferation markers, and cytotoxic molecules in CD8+ T cells specific for viruses other than HAV. We found that a considerable proportion of these CD8+ T cells are activated to proliferate and exhibit increased expression of perforin and granzymes. Furthermore, the activation of HAV-unrelated virus-specific CD8+ T cells correlates with the severity of liver injury, indicating a contribution to the immunopathology of AHA. Conflicting results in previous studies of bystander activation of CD8+ T cells during viral infection may be due to several factors. First, different viruses may have different abilities to induce bystander activation of CD8+ T cells. Vaccination with live-attenuated viruses was shown not to activate unrelated virus-specific CD8+ T cells (Miller et al., 2008), whereas viral infection activated unrelated virus-specific CD8+ T cells (Doisne et al., 2004; Sandalova et al., 2010; Tuuminen et al., 2007). Second, the size of the CD8+ T cell memory pool may determine the degree of bystander activation in each individual because memory cells are more susceptible to IL-15-induced activation and proliferation than naive cells (Weng et al., 2002). This may explain why HAV infection results in different clinical outcomes depending on the age of patients. In this regard, antigen-independent bystander activation would be more vigorous in adults, particularly the elderly, who have a large pool of memory CD8+ T cells (McCloskey et al., 1997), resulting in severe hepatitis, compared to children who present with subclinical infection. The naive or memory status of CD8+ T cells needs to be considered in studies of bystander activation, especially when TCR-transgenic CD8+ T cells are used to investigate bystander activation in mouse models of viral infection. When we observed activation and proliferation of CD8+ T cells specific for other viruses in AHA patients, we considered the possibility of heterologous immunity (Rehermann and Shin, 2005; Selin et al., 2004) in which these T cells could be activated by cross-reactive epitopes of HAV proteins rather than bona fide bystander activation. It was previously shown that cross-reactive CD8+ T cells primed by past viral infections can cause severe immunopathology during infection of heterologous viruses (Chen et al., 2001) or strains (Mongkolsapaya et al., 2003). This mechanism was also proposed to explain cases of severe liver injury in HCV infection (Urbani et al., 2005). However, in our study we excluded the possibility of heterologous immunity for the following reasons: it is unlikely that all six HLA class I multimers for IAV, EBV, CMV, VV, and RSV specificity used in the current study detect cross-reactive CD8+ T cells with HAV, and these epitope peptides do not exhibit significant amino acid sequence homology with HAV proteins. Another possible cause of the activation of CD8+ T cells specific for other viruses is the reactivation of latent infections,

(F) K562 cytotoxicity of CD8+ T cells isolated from peripheral blood was analyzed for a correlation with serum ALT levels at the time of admission. (G) Serum ALT levels (upper left), the percentage of CD38+HLA-DR+ cells among total CD8+ T cells (upper right), HAV-unrelated virus (IAV, EBV or CMV)-specific CD8+ T cells (lower left), and HAV-specific CD8+ T cells (lower right) were examined in the peripheral blood at the time of admission and discharge (average 8 days after admission). ***p < 0.001. (H) Expression level of NKG2D in HAV-unrelated virus (CMV, VV, or RSV)-specific CD8+ T cells were examined in peripheral blood at the time of admission and discharge (average 8 days after admission). ***p < 0.001.


such as EBV and CMV. Though 3 (21.4%) and 7 (50.0%) of 14 AHA patients presented evidence of EBV and CMV reactivation, respectively, activation of EBV-specific or CMV-specific CD8+ T cells was apparent in the acute phase of AHA, irrespective of EBV or CMV reactivation. These data indicate that EBV-specific or CMV-specific CD8+ T cells can be activated during AHA even in the absence of EBV or CMV reactivation. It was also demonstrated that EBV-specific and CMV-specific CD8+ T cells were activated without EBV and CMV reactivation in patients with acute viral infections, such as hepatitis B, dengue fever, influenza, and adenoviral infection (Sandalova et al., 2010). More importantly, activation of IAV-specific, VV-specific, and RSVspecific CD8+ T cells in our study strongly supports our conclusion that memory CD8+ T cells specific for HAV-unrelated viruses were antigen independently activated in the absence of cognate antigens during AHA. In the current study, we also clarified whether TCR stimulation is involved in the activation of HAV-unrelated virus-specific CD8+ T cells during AHA. As NKG2D is upregulated during the activation of memory CD8+ T cells by IL-15 in the absence of TCR engagement by cognate antigens, the level of NKG2D expression was measured to distinguish between TCR-induced activation and IL-15-induced bystander activation of memory CD8+ T cells. We found that NKG2D expression was significantly increased on HAV-unrelated virus-specific CD8+ T cells from AHA patients, but not HAV-specific CD8+ T cells, demonstrating that HAV-unrelated virus-specific CD8+ T cells were activated by IL-15 in the absence of TCR stimulation during AHA. In the present study, we found that IL-15 plays a main role in antigen-independent activation of CD8+ T cells. Expression of IL-15 and IL-15Ra was upregulated in AHA patients, and in vitro IL-15 treatment of PBMCs from healthy donors recapitulated the activated phenotype of CD8+ T cells from AHA patients. In addition, IL-15-treated CD8+ T cells overexpressed NKG2D and NKp30 and exerted innate-like cytotoxicity in an NKG2D and NKp30-dependent manner. IL-15 is a member of the common g-chain family of cytokines and is important in the prolonged maintenance of memory T cells (Ma et al., 2006; Waldmann, 2006). IL-15 is also known to be involved in bystander activation of CD8+ T cells, as demonstrated in mice (Judge et al., 2002) and humans (Sandalova et al., 2010). Furthermore, IL-15-treated CD8+ T cells overexpress NK receptors and exhibit TCR-independent cytotoxicity (Correia et al., 2011; Meresse et al., 2004; Tamang et al., 2006). These previous reports are consistent with our findings in AHA patients and support our conclusion that IL-15 is the major cytokine driving antigen-independent activation of CD8+ T cells and their innate-like cytolytic ability. In many acute viral infections, including HIV infection (Leeansyah et al., 2013), simian immunodeficiency virus infection (Eberly et al., 2009; Jacquelin et al., 2014), dengue virus infection (Azeredo et al., 2006), and RSV infection (Estripeaut et al., 2008), the concentration of IL-15 in the blood or tissues is increased. During viral infection, IL-15 is typically produced in the early acute phase prior to peak viremia, and its concentration in the blood decreases rapidly immediately after the early acute phase (Azeredo et al., 2006; Eberly et al., 2009; Estripeaut et al., 2008; Jacquelin et al., 2014; Leeansyah et al., 2013). This may explain why IL-15 was undetectable in some patients with AHA. In

further analysis, we found that serum IL-15 levels at the time of admission did not correlate with ALT levels or the frequency of activated CD8+ T cells among AHA patients (data not shown). This result is anticipated from the fact that the concentration of IL-15 in the blood decreases rapidly after the early acute phase of viral infection (Azeredo et al., 2006; Eberly et al., 2009; Estripeaut et al., 2008; Jacquelin et al., 2014; Leeansyah et al., 2013). Peak viremia in HAV infection is then followed by acute symptoms with serum ALT peaks, leading to hospitalization (Shin et al., 2016). Therefore, the concentration of IL-15 in the blood may already be decreased in our cohort at the time of admission. Antigen-independent activation of CD8+ T cells and their innate-like cytolytic activity should be explored further in other viral diseases. A recent study reported that bystander-activated CD8+ T cells exert NKG2D-dependent, innate-like cytolytic activity in mouse infection models of vesicular stomatitis virus and Listeria monocytogenes (Chu et al., 2013). In this model, bystander-activated CD8+ T cells contributed to the early control of Listeria infection. In contrast, bystander-activated CD8+ T cells were associated with tissue injury in the current study. In AHA, tissue injury-associated roles of bystander-activated CD8+ T cells may be related to HAV not being cytopathic to host cells. In human viral infection, acute hepatitis B and acute hepatitis C are of interest because they, like AHA, are known to be mediated by CD8+ T cell immunopathogenesis. Notably, EBV- or CMV-specific CD8+ T cells are activated in the absence of EBV or CMV reactivation in acute hepatitis B patients (Sandalova et al., 2010), though their function(s) in host injury are not clear. In IAV infection, a pathological role of IL-15 has been shown. When infected with lethal IAV, IL-15-deficient mice have reduced mortality with attenuated pneumonia (Nakamura et al., 2010). In the present study, we suggested that bystander-activated CD8+ T cells contribute to liver injury during AHA. Although antigen-independent activation of bystander CD8+ T cells is known (Doisne et al., 2004; Sandalova et al., 2010; Tuuminen et al., 2007), the immunopathological role of these cells was not previously reported in human viral diseases. Here, we report a human viral disease in which host injury is associated with innate-like cytolytic activity of antigen independently activated CD8+ T cells not specific for the infecting virus. Our current study raises the question of whether bystander CD8+ T cells contribute to host injury in other human viral diseases, and whether IL-15 or NKG2D can be a therapeutic target in the treatment of the immunopathology of viral diseases. STAR+METHODS Detailed methods are provided in the online version of this paper and include the following: d d d d

KEY RESOURCES TABLE CONTACT FOR REAGENT AND RESOURCE SHARING EXPERIMENTAL MODEL AND SUBJECT DETAILS B Patients and Specimens METHOD DETAILS B Flow Cytometry B Quantification of Serum Cytokines Immunity 48, 161–173, January 16, 2018 171

B

In Vitro Cytokine Stimulation In Vitro HAV Infection B Immunofluorescence Staining of Liver Tissues + B CD8 T Cell Separation B Cytotoxicity Assay B IFN-g ELISpot Assay B RNA Extraction, cDNA Synthesis, and Real-Time Quantitative PCR B Quantification of EBV and CMV DNA B Immunoblotting QUANTIFICATION AND STATISTICAL ANALYSIS B

d

SUPPLEMENTAL INFORMATION

Doisne, J.M., Urrutia, A., Lacabaratz-Porret, C., Goujard, C., Meyer, L., Chaix, M.L., Sinet, M., and Venet, A. (2004). CD8+ T cells specific for EBV, cytomegalovirus, and influenza virus are activated during primary HIV infection. J. Immunol. 173, 2410–2418. Eberly, M.D., Kader, M., Hassan, W., Rogers, K.A., Zhou, J., Mueller, Y.M., Mattapallil, M.J., Piatak, M., Jr., Lifson, J.D., Katsikis, P.D., et al. (2009). Increased IL-15 production is associated with higher susceptibility of memory CD4 T cells to simian immunodeficiency virus during acute infection. J. Immunol. 182, 1439–1448. Ehl, S., Hombach, J., Aichele, P., Hengartner, H., and Zinkernagel, R.M. (1997). Bystander activation of cytotoxic T cells: studies on the mechanism and evaluation of in vivo significance in a transgenic mouse model. J. Exp. Med. 185, 1241–1251.

Supplemental Information includes three figures and one table and can be found with this article online at https://doi.org/10.1016/j.immuni.2017.11.025.

Estripeaut, D., Torres, J.P., Somers, C.S., Tagliabue, C., Khokhar, S., Bhoj, V.G., Grube, S.M., Wozniakowski, A., Gomez, A.M., Ramilo, O., et al. (2008). Respiratory syncytial virus persistence in the lungs correlates with airway hyperreactivity in the mouse model. J. Infect. Dis. 198, 1435–1443.

ACKNOWLEDGMENTS

Guidotti, L.G., and Chisari, F.V. (2006). Immunobiology and pathogenesis of viral hepatitis. Annu. Rev. Pathol. 1, 23–61.

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Sitia, G., Isogawa, M., Iannacone, M., Campbell, I.L., Chisari, F.V., and Guidotti, L.G. (2004). MMPs are required for recruitment of antigen-nonspecific mononuclear cells into the liver by CTLs. J. Clin. Invest. 113, 1158–1167. Sung, P.S., Cheon, H., Cho, C.H., Hong, S.H., Park, D.Y., Seo, H.I., Park, S.H., Yoon, S.K., Stark, G.R., and Shin, E.C. (2015). Roles of unphosphorylated ISGF3 in HCV infection and interferon responsiveness. Proc. Natl. Acad. Sci. USA 112, 10443–10448. Tamang, D.L., Redelman, D., Alves, B.N., Vollger, L., Bethley, C., and Hudig, D. (2006). Induction of granzyme B and T cell cytotoxic capacity by IL-2 or IL-15 without antigens: multiclonal responses that are extremely lytic if triggered and short-lived after cytokine withdrawal. Cytokine 36, 148–159. Tough, D.F., and Sprent, J. (1996). Viruses and T cell turnover: evidence for bystander proliferation. Immunol. Rev. 150, 129–142. Tough, D.F., Borrow, P., and Sprent, J. (1996). Induction of bystander T cell proliferation by viruses and type I interferon in vivo. Science 272, 1947–1950. € la €inen, E., Ma €kela €, S., Ala-Houhala, I., Ennis, F.A., Hedman, Tuuminen, T., Keka K., Mustonen, J., Vaheri, A., and Arstila, T.P. (2007). Human CD8+ T cell memory generation in Puumala hantavirus infection occurs after the acute phase and is associated with boosting of EBV-specific CD8+ memory T cells. J. Immunol. 179, 1988–1995. Urbani, S., Amadei, B., Fisicaro, P., Pilli, M., Missale, G., Bertoletti, A., and Ferrari, C. (2005). Heterologous T cell immunity in severe hepatitis C virus infection. J. Exp. Med. 201, 675–680. Waldmann, T.A. (2006). The biology of interleukin-2 and interleukin-15: implications for cancer therapy and vaccine design. Nat. Rev. Immunol. 6, 595–601. Walker, C.M., Feng, Z., and Lemon, S.M. (2015). Reassessing immune control of hepatitis A virus. Curr. Opin. Virol. 11, 7–13. Weng, N.P., Liu, K., Catalfamo, M., Li, Y., and Henkart, P.A. (2002). IL-15 is a growth factor and an activator of CD8 memory T cells. Ann. N Y Acad. Sci. 975, 46–56. Zarozinski, C.C., and Welsh, R.M. (1997). Minimal bystander activation of CD8 T cells during the virus-induced polyclonal T cell response. J. Exp. Med. 185, 1629–1639. Zhou, Y., Callendret, B., Xu, D., Brasky, K.M., Feng, Z., Hensley, L.L., Guedj, J., Perelson, A.S., Lemon, S.M., Lanford, R.E., and Walker, C.M. (2012). Dominance of the CD4(+) T helper cell response during acute resolving hepatitis A virus infection. J. Exp. Med. 209, 1481–1492.


STAR+METHODS KEY RESOURCES TABLE

REAGENT or RESOURCE

SOURCE

IDENTIFIER

V500 Anti-human CD3 (clone UCHT1)

BD Biosciences

Cat# 561417; RRID: AB_10611584

Pacific Blue Anti-human CD3 (clone UCHT1)

BD Biosciences

Cat# 558117; RRID: AB_397038

AlexaFluor 700 Anti-human CD3 (clone UCHT1)

BD Biosciences

Cat# 557943; RRID: AB_396952

V450 Anti-human CD8 (clone RPA-T8)

BD Biosciences

Cat# 560348; RRID: AB_1645582

APC-H7 Anti-human CD8 (clone SK1)

BD Biosciences

Cat# 560273; RRID: AB_1645482

BV711 Anti-human CD8 (clone RPA-T8)

BD Biosciences

Cat# 563676

PerCP-Cy5.5 Anti-human CD14 (clone M5E2)

BD Biosciences

Cat# 561116; RRID: AB_2033939

APC Anti-human CD14 (clone M5E2)

BD Biosciences

Cat# 561708; RRID: AB_10896130

Antibodies

PerCP-Cy5.5 Anti-human CD19 (clone HIB19)

BD Biosciences

Cat# 561295; RRID: AB_10644017

Pe-Cy7 Anti-human CD38 (clone HIT2)

BD Biosciences

Cat# 560677; RRID: AB_1727473

PerCP-Cy5.5 Anti-human CD45RO (clone UCHL1)

BD Biosciences

Cat# 560607; RRID: AB_1727500

PE Anti-human CCR7 (clone 3D12)

BD Biosciences

Cat# 561008; RRID: AB_2033950

APC-H7 Anti-human HLA-DR (clone G46-4)

BD Biosciences

Cat# 561358; RRID: AB_10611876

FITC Anti-human granzyme A (clone CB9)

BD Biosciences

Cat# 558905; RRID: AB_297156

AlexaFluor 647 Anti-human granzyme B (clone GB11)

BD Biosciences

Cat# 561999; RRID: AB_10897997

FITC Anti-human granzyme B (clone GB11)

BD Biosciences

Cat# 561998; RRID: AB_10894005

FITC Anti-human perforin (clone d G9)

BD Biosciences

Cat# 556577; RRID: AB_396470

PE Anti-human perforin (clone d G9)

BD Biosciences

Cat# 556437; RRID: AB_396419

FITC Anti-Ki-67 (clone B56)

BD Biosciences

Cat# 556026; RRID: AB_396302

PE-Cy7 Anti-Ki-67 (clone B56)

BD Biosciences

Cat# 561283; RRID: AB_10716060

APC Anti-human NKG2D (clone 1D11)

BD Biosciences

Cat# 562064; RRID: AB_10926199

PE Anti-human NKG2D (clone 1D11)

BD Biosciences

Cat# 561815; RRID: AB_10896282

PE Anti-human NKp30 (clone p30-15)

BD Biosciences

Cat# 558407; RRID: AB_647240

Purified NA/LE Anti-human NKG2D (clone 1D11)

BD Biosciences

Cat# 562164; RRID: AB_10893343

Purified NA/LE Anti-human NKp46 (clone 9-E2/NKp46)

BD Biosciences

Cat# 557847; RRID: AB_2149297

Purified Anti-human IgG1 (clone G17-1)

BD Biosciences

Cat# 555868; RRID: AB_396186

Purified mouse IgG2a, k isotype control (clone G18-21)

BD Biosciences

Cat# 554126; RRID: AB_479661

APC Anti-human NKG2C/CD159c (clone 134591)

R&D Systems

Cat# FAB138A; RRID: AB_416838

Human NKp30/NRC3 antibody

R&D Systems

Cat# MAB18491; RRID: AB_2149445

APC Anti-human NKp44/NCR2 (clone 253415)

R&D Systems

Cat# FAB22491A; RRID: AB_2149422

Fluorescein-conjugated Anti-human NKp46/NRC1 (clone 195314)

R&D Systems

Cat# FAB1850F; RRID: AB_2235763

APC Anti-human IL-15 (clone 34559)

R&D Systems

Cat# IC2471A; RRID: AB_10714827

Anti-human IL-15 antibody

R&D Systems

Cat# AF315; RRID: AB_355296

PE Anti-human IL-15 R alpha (clone 151303)

R&D Systems

Cat# FAB1471P; RRID: AB_2124706

Fluorescein-conjugated Anti-human CCR7 (clone 150503)

R&D Systems

Cat# FAB197F; RRID: AB_2259847

Human MIC-A antibody

R&D Systems

Cat# AF1300; RRID: AB_2143622

PE-eFlour 610 Anti-human CD14 (clone 61D3)

Invitrogen

Cat# 61-0149-42; RRID: AB_2574534

PE-eFlour 610 Anti-human CD19 (clone HIB19)

Invitrogen

Cat# 61-0199-42; RRID: AB_2574538

CD3 monoclonal antibody (clone OKT3)

eBioscience

Cat# 16-0037-81; RRID: AB_468854

Goat pAb to Rb IgG (HRP)

Abcam

Cat# ab97051; RRID: AB_10679369

Anti-CD11c antibody (clone EP1347Y)

Abcam

Cat# ab52632; RRID: AB_2129793

Anti-HLA-DR antibody (cloneTAL1B5)

Abcam

Cat# ab20181; RRID: AB_445401 (Continued on next page)

e1 Immunity 48, 161–173.e1–e5, January 16, 2018

Continued REAGENT or RESOURCE

SOURCE

IDENTIFIER

Anti-CD68 antibody (clone KP1)

Abcam

Cat# ab74704; RRID: AB_1310061

AlexaFlour 647 donkey Anti-mouse IgG (polyclonal)

Abcam

Cat# ab150107

Donkey Anti-goat IgG (HRP)

Abcam

Cat# ab97110; RRID: AB_10679463

JAK (6G4) rabbit mAb

Cell Signaling

Cat# 3344; RRID: AB_2265054

GAPDH (14C10) rabbt mAb

Cell Signaling

Cat# 2118; RRID: AB_561053

AlexaFluor 488 donkey Anti-mouse IgG

Jackson ImmunoResearch Laboratories

Cat# 715-546-151; RRID: AB_2340846

AlexaFluor 488 donkey Anti-rabbit IgG


Cat# 711-546-152; RRID: AB_2313584

AlexaFlour 594 donkey Anti-goat IgG


Cat# 705-586-147; RRID: AB_2340432

FITC donkey Anti-mouse IgG


Cat# 715-006-151; RRID: AB_2340762

Cy3 donkey Anti-goat IgG


Cat# 705-166-147; RRID: AB_2340413

ATCC

Cat# VR-1402

R&D Systems

Cat# 202-IL-010

Bacterial and Virus Strains HAV (HM175/18f) Chemicals, Peptides, and Recombinant Proteins Recombinant human IL-2 protein Recombinant human IL-18/IL-1F4 protein

R&D Systems

Cat# 9124-IL

Recombinant human IL-7 protein

Peprotech

Cat# 200-07

Recombinant human IL-15 protein

Peprotech

Cat# 200-15

CMV peptide NLVPMVATV

Peptron

Customized

SIINFEKL peptide

Peptron

Customized

Ethidium monoazide bromide (EMA)

Invitrogen

Cat# E1374

To-Pro-3 Iodide

Invitrogen

Cat# T3605

HAV VP2 & 3C pentadecamer overlapping peptides

Mimotopes

Customized

26 HLA-A2-restricted epitope peptides

Mimotopes

Customized

Calpain Inhibitor I

Sigma-Aldrich

Cat# A6185

Ficoll-paque PREMIUM

GE Healthcare Life Science

Cat# 17-5442-02

Brefeldin A protein transport inhibitor

BD Biosciences

Cat# 555029

Monensin protein transport inhibitor

BD Biosciences

Cat# 554724

RIPA lysis and extraction buffer

Thermo Scientific

Cat# 89900

Critical Commercial Assays

Intracellular fixation & permeabilization buffer set

eBioscience

Cat# 88-8824-00

Human IL-2 DuoSet ELISA

R&D Systems

Cat# DY202


R&D Systems

Cat# DY207


R&D Systems

Cat# DY247


R&D Systems

Cat# DY318-05

LIVE/DEAD fixable aqua dead cell stain kit

Invitrogen

Cat# L34957

LIVE/DEAD fixable red dead cell stain kit

Invitrogen

Cat# L23102

DAPI (4’, 6-diamidino-2-phenylindole, dihydrochloride)

Invitrogen

Cat# D1306

RNeasy mini kit

QIAGEN

Cat# 74106

First strand cDNA synthesis kit

Marligen Biosciences

Cat# 11801-025

Taqman gene expression master mix

Applied Biosystems

Cat# 4369016

PKH26 red fluorescent cell linker kit for general cell membrane labeling

Sigma-Aldrich

Cat# PKH26GL-1KT

Human CD8 microbeads

Miltenyi Biotec

Cat# 130-045-201

Anti-PE microbeads

Miltenyi Biotec

Cat# 130-048-801

Human CD8+ T cell isolation kit

Miltenyi Biotec

Cat# 130-096-495 (Continued on next page)

Immunity 48, 161–173.e1–e5, January 16, 2018 e2

Continued REAGENT or RESOURCE

SOURCE

IDENTIFIER

HepG2

ATCC

Cat# HB-8065

K562

ATCC

Cat# CCL-243

Huh-7

KCLB

Cat# 60104

P815

ATCC

Cat# TIB-64

EBV UL132 TaqMan probe

Thermo Scientific

Vi03453400_s1

CMV IR2 TaqMan probe

Thermo Scientific

Customized

MIC-A TaqMan probe

Thermo Scientific

Hs00741286_m1

MIC-B TaqMan probe

Thermo Scientific

Hs00792952_m1

B-actin TaqMan probe

Thermo Scientific

Hs01060665_g1

FlowJo (version 10.7)

Tree Star

https://www.flowjo.com/solutions/flowjo/ downloads

Prism (version 6)

GraphPad

N/A

Experimental Models: Cell Lines

Oligonucleotides

Software and Algorithms

Other LLYNCCYHV (HAV 3D2026) HLA-A*0201 Pentamer

Proimmune

Customized

HPRLAQRIL (HAV 3D1876) HLA-B*0702 Pentamer

Proimmune

Customized

LLYNCCYHV (HAV 3D2026) HLA-A*0201 tetramer

Schulte et al. (2011)

N/A

GILGFVFTL (IAV MP58) HLA-A*0201 Pentamer

Proimmune

Cat# F007-2

GLCTLVAML (EBV BMFL1259) HLA-A*0201 Pentamer

Proimmune

Cat# F001-2

NLVPMVATV (CMV pp65495) HLA-A*0201 Pentamer

Proimmune

Cat# F008-2

TPRVTGGGAM (CMV pp65417) HLA-B*0702 Pentamer

Proimmune

Cat# F045-2

KVDDTFYUV (VV HRP274) HLA-A*0201 Pentamer

Proimmune

Cat# F703-2

KMLKEMGEV (RSV NP137) HLA-A*0201 Pentamer

Proimmune

Cat# F801-2

CONTACT FOR REAGENT AND RESOURCE SHARING All requests for reagents and resources can be directed to and will be fulfilled by the Lead Contact, Eui-Cheol Shin (ecshin@kaist. ac.kr). EXPERIMENTAL MODEL AND SUBJECT DETAILS Patients and Specimens The study population included 74 patients diagnosed with AHA and hospitalized in Chung-Ang University Hospital (Seoul, Republic of Korea). All patients were seropositive for both anti-HAV IgM and IgG and had clinical and laboratory features of acute hepatitis. Whole blood and serum were collected; PBMCs were isolated by Ficoll (GE Healthcare) density-gradient centrifugation and cryopreserved. In addition, fresh liver tissue was obtained from AHA patients with fulminant liver injury (n = 2) during liver transplantation at Seoul National University Hospital (Seoul, Republic of Korea). Information on the 76 AHA patients are listed in Table S1. As controls, liver tissue samples without viral hepatitis were obtained during operations, such as cholecystectomy, adrenalectomy, and partial liver resection for intrahepatic duct stones, at Daejeon St. Mary’s Hospital (Daejeon, Republic of Korea). Intrahepatic lymphocytes were isolated by mechanical homogenization and Ficoll density-gradient centrifugation. We recruited 7 additional AHA patients hospitalized at Seoul Metropolitan Government Seoul National University Boramae Medical Center (Seoul, Republic of Korea) to study VV-specific and RSV-specific T cells as well as HAV-specific and CMV-specific T cells. We also obtained frozen liver tissue from AHA patients with fulminant liver injury (n = 7) and liver tissue samples without viral hepatitis (n = 5) from Asan Medical Center (Seoul, Republic of Korea) and Pusan National University Hospital (Busan, Republic of Korea), respectively, for immunofluorescence, immunoblotting, and quantitative real-time PCR. This study was conducted according to the principles of the Declaration of Helsinki and approved by the institutional review boards of each institution from which samples were obtained (Chung-Ang University Hospital, Daejeon St. Mary’s Hospital, Seoul Metropolitan Government Seoul National University Boramae Medical Center, Asan Medical Center, and Pusan National University Hospital). Written informed consent was obtained from each patient or control prior to inclusion in the study. e3 Immunity 48, 161–173.e1–e5, January 16, 2018

METHOD DETAILS Flow Cytometry Cryopreserved PBMCs were thawed and stained with fluorochrome-conjugated antibodies for 30 min at 4 C. Following cell surface staining, cells were permeabilized using intracellular staining buffer (eBioscience) to stain intracellular markers, such as granzymes, perforin and Ki-67. For HLA multimer staining, cells were stained first with ethidium monoazide (EMA) to identify dead cells, and then with an HLA multimer for 15 min at room temperature. Multicolor flow cytometry was performed using an LSR II instrument (BD Biosciences) and data were analyzed using FlowJo software (Treestar). Quantification of Serum Cytokines IL-2, IL-7, IL-15 and IL-18 were quantified in serum samples using enzyme-linked immunoassays (R&D Systems) according to the manufacturer’s instructions. In Vitro Cytokine Stimulation PBMCs from healthy donors were cultured in the presence of IL-2 (10 ng/mL; R&D Systems), IL-7 (10 ng/mL; Peprotech), IL-15 (10 ng/mL; Peprotech), IL-18 (50 ng/mL; R&D Systems) or IFN-b (10 ng/mL; Peprotech) for 72 hr and examined for expression of CD38, HLA-DR, Ki-67 or granzyme B in the gate of total CD8+ T cells or HLA multimer+ CD8+ T cells. For the detection of NKG2D expression in memory CD8+ T cells, sorted CCR7 CD8+ T cells from healthy donors were cultured in the presence of coated antiCD3 (1 mg/mL; eBioscience), IL-15 (10 ng/mL; Peprotech) or a combination for 48 hr and examined for expression of NKG2D in the gate of CD45RO+ CD8+ T cells. For Calpain Inhibitor I (Sigma-Aldrich) treatment, PBMCs were pre-treated with Calpain inhibitor I (0.3 mM) for 1 hr prior to stimulation with anti-CD3 or IL-15. For the stimulation of CMV-specific CD8+ T cells with cognate epitope peptide or IL-15, PBMCs from healthy donors were treated with CMV pp65495 epitope peptide (NLVPMVATV; 0.5 mg/mL), a control peptide (SIINFEKL; 0.5 mg/mL), IL-15 (10 ng/mL) or in combination for 48 hr. NKG2D expression was examined in the gate of CMV pp65495 pentamer+ CD8+ T cells. In Vitro HAV Infection HepG2 cells were infected with HAV (HM175) at 5 multiplicity of infection. The cells were stained with anti-HAV (Abcam) and antiIL-15 (R&D Systems) 7 days post-infection. Immunofluorescence Staining of Liver Tissues Liver tissues were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) and sectioned (10 mm). The tissue sections were incubated with blocking solution (5% donkey serum in PBS-T [0.02% Triton X-100 in PBS]) for 1 hr at room temperature, and then for 3 hr with the following antibodies: anti-HAV (Abcam), anti-IL-15 (R&D Systems), or anti-MIC-A (R&D Systems). After several washes in PBS, the tissue sections were incubated for 2 hr with anti-mouse IgG-FITC and anti-goat IgG-Cy3 (both from Jackson ImmunoResearch Laboratories). In the case of staining for CD11c, HLA-DR and CD68, tissues were stained overnight with antiIL-15; rabbit anti-human CD11c (EP1347Y), mouse anti-human HLA-DR (TAL 1B5), mouse anti-human CD68 (KP1) (all from Abcam) as primary antibodies. AlexaFluor 488-conjugated donkey anti-rabbit IgG and anti-mouse IgG, AlexaFluor 594-conjugated donkey anti-goat IgG (all from Jackson ImmunoResearch Laboratories); AlexaFluor 647-conjugated donkey anti-mouse IgG (Abcam) were used as secondary antibodies. Finally, tissues were stained for 5 min with 4,6-diamidino-2-phenylindole (DAPI) (Invitrogen). For control experiments, the primary or secondary antibody was substituted with serum. Images were obtained using a Zeiss LSM 510 or LSM780 confocal microscope equipped with argon and helium-neon lasers (Carl Zeiss). CD8+ T Cell Separation CD8+ T cells were separated from PBMCs or intrahepatic lymphocytes using CD8 microbeads (Miltenyi Biotec). The purity was > 97%. In some patients, CMV-specific CD8+ T cells were separated from the CD8+ T cell population using PE-labeled CMV pentamer (Proimmune) and anti-PE microbeads (Miltenyi Biotec). Purified cells were used for cytotoxicity assays. Cytotoxicity Assay IL-15-treated healthy donors’ CD8+ T cells, CD8+ T cells isolated from PBMCs of AHA patients or CD8+ T cells isolated from intrahepatic lymphocytes were used as effector cells in cytotoxicity assays. Target cells (i.e., K562 or Huh-7 cells) were labeled with PKH26 (Sigma-Aldrich) and co-cultured with effector CD8+ T cells at various effector:target ratios. After 12 hr, TO-PRO-3-iodide (Invitrogen) was added to each culture at a final concentration of 0.5 mM and the cells were immediately analyzed by flow cytometry. The background or maximum TO-PRO-3-iodide staining was obtained by incubating target cells with medium or detergent, respectively. The percent specific cytotoxicity was calculated as: (% TO-PRO-3-iodide+PKH26+ cells in co-culture – % TO-PRO3-iodide+PKH26+ cells in background) / (% TO-PRO-3-iodide+PKH26+ cells in maximum – % TO-PRO-3-iodide+PKH26+ cells in background) 3 100. Anti-NKG2D (BD Biosciences), anti-NKp30 (R&D Systems) or anti-NKp46 (BD Biosciences) was used for blocking experiments. Mouse IgG1 (BD Biosciences) or IgG2a (R&D Systems) monoclonal antibodies were used as isotype controls. For FcgR-dependent redirected killing assays, P815 cells were used as target cells.

Immunity 48, 161–173.e1–e5, January 16, 2018 e4

IFN-g ELISpot Assay Duplicate cultures of 300,000 PBMCs/well were set up in ELISpot plates. PBMCs were stimulated with peptide mixes for HAV VP2 and 3C prepared from 42 pentadecamer peptides (Mimotopes) overlapping by 10 amino acids for each mix, respectively. The final concentration of each peptide was 1 mg/mL. After 30 hr in culture, IFN-g spots were developed and counted using an ELISpot reader (C.T.L.). The number of specific spots was calculated by subtracting the number of spots in negative control wells from the number in peptide-stimulated wells. In HLA-A2-positive patients, IFN-g ELISpot assays were also performed using a mix of 26 HLA-A2restricted epitope peptides. RNA Extraction, cDNA Synthesis, and Real-Time Quantitative PCR Total RNA isolation, cDNA synthesis, and TaqMan real-time quantitative PCR were performed as described previously (Sung et al., 2015). TaqMan Gene Expression Assays (Applied Biosystems) were used to determine the mRNA levels of target genes (MIC-A and MIC-B). The results were standardized to the mRNA level of b-actin, and the data are presented as mean ± standard deviation. Quantification of EBV and CMV DNA DNA was isolated from 200 mL of patient serum. EBV and CMV DNA were quantified by UL132- and IR2-detecting PCR primers and TaqMan probes (Applied Biosystems), respectively. Cases with DNA titers > 1,000 copies/mL during the acute phase of AHA and a decrease during the convalescent phase were considered viral reactivation. The lower detection limit of both assays was 100 copies/mL of serum. Immunoblotting To prepare total cell lysates, tissues or cells were lysed with RIPA buffer (Thermo Fisher Scientific). Ten micrograms of each cell lysate were loaded on SDS-PAGE gels. The antibodies used for immunoblotting were as follows: MIC-A (1:200, goat, R&D Systems), JAK1 (1:1000, Rabbit; Cell Signaling), GAPDH (1:1000, Rabbit; Cell Signaling), horseradish peroxidase (HRP)-conjugated goat IgG (1:5000; abcam) and HRP-conjugated rabbit IgG (1:5000, abcam). QUANTIFICATION AND STATISTICAL ANALYSIS Statistical analyses were performed using Prism software. To assess the significance of correlation, Pearson correlation tests were performed. Changes in the phenotype of peripheral blood CD8+ T cells during the course of AHA were analyzed by the paired t test, and other non-parametric comparisons were analyzed by the Mann-Whitney U-test. All tests of significance were 2-tailed and p % 0.05 considered significant.

e5 Immunity 48, 161–173.e1–e5, January 16, 2018