key: cord-0870564-3uem0e22
authors: Qin, Gang; Mao, Huawei; Zheng, Jian; Sia, Sin Fun; Liu, Yinping; Chan, Ping-Lung; Lam, Kwok-Tai; Peiris, J. S. Malik; Lau, Yu-Lung; Tu, Wenwei
title: Phosphoantigen-Expanded Human γδ T Cells Display Potent Cytotoxicity against Monocyte-Derived Macrophages Infected with Human and Avian Influenza Viruses
date: 2009-09-01
journal: J Infect Dis
DOI: 10.1086/605413
sha: c247fdaf4d9be6e73e744bc36112f563fdb3ebb9
doc_id: 870564
cord_uid: 3uem0e22

BackgroundInfluenza virus is a cause of substantial annual morbidity and mortality worldwide. The potential emergence of a new pandemic strain (eg, avian influenza virus) is a major concern. Currently available vaccines and anti-influenza drugs have limited effectiveness for influenza virus infections, especially for new pandemic strains. Therefore, there is an acute need to develop alternative strategies for influenza therapy. γδ T cells have potent antiviral activities against different viruses, but no data are available concerning their antiviral activity against influenza viruses MethodsIn this study, we used virus-infected primary human monocyte-derived macrophages (MDMs) to examine the antiviral activity of phosphoantigen isopentenyl pyrophosphate (IPP)–expanded human Vγ9Vδ2 T cells against influenza viruses ResultsVγ9Vδ2 T cells were selectively activated and expanded by IPP from peripheral blood mononuclear cells. IPP-expanded Vγ9Vδ2 T cells efficiently killed MDMs infected with human (H1N1) or avian (H9N2 or H5N1) influenza virus and significantly inhibited viral replication. The cytotoxicity of Vγ9Vδ2 T cells against influenza virus–infected MDMs was dependent on NKG2D activation and was mediated by Fas–Fas ligand and perforin–granzyme B pathways ConclusionOur findings suggest a potentially novel therapeutic approach to seasonal, zoonotic avian, and pandemic influenza—the use of phosphoantigens to activate γδ T cells against influenza virus infections

proportion (2%-10%) of T lymphocytes in the blood and peripheral organs of most adult animals and humans [6] [7] [8] . In humans, Vg9Vd2 T cells make up the majority of peripheral blood and lymphoid organ gd T cells [9] . Human gd T cells share characteristics of T cells, natural killer (NK) cells, and antigen-presenting cells [6] [7] [8] . Therefore, gd T cells are thought to represent one of the first lines of the host immune defense.

The antiviral activities of gd T cells have been demonstrated in different models [10] [11] [12] . In the mouse model, gd T cells were shown to contribute to recovery from influenza pneumonia [13, 14] , but no data are available on the contribution of gd T cells at early stages of influenza virus infections. Activated mouse gd T cells showed profound cytotoxicity against hemagglutinin (H1 or H3) expressing target cells in a non-major histocompatibility complex-restricted manner [15] . However, it is still unknown whether human gd T cells have antiviral activities against human or avian fluA viruses or what their underlying mechanisms are.

Vg9Vd2 T cells are specifically activated in an HLA-unrestricted manner by small nonpeptidic phosphoantigens, which are metabolites of isoprenoid biosynthesis pathways in all organisms [16] . The most potent phosphoantigen is hydroxydimethylallyl-pyrophosphate, produced through a nonmevalonate pathway in microorganisms such as mycobacteria. Natural isopentenyl pyrophosphate (IPP), an intermediate produced through the mevalonate pathway that also leads to cholesterol synthesis in mammalian cells, was found to selectively activate and expand human Vg9Vd2 T cells in vitro or in vivo [17, 18] . Vg9Vd2 T cells expanded by synthetic phosphoantigens were demonstrated to have antiviral potential against other viruses [19, 20] , but no data are available concerning their antiviral activities against human and avian fluA viruses.

One characteristic of the host immune response to fluA virus infection is the influx of both macrophages and T lymphocytes into the lungs [21] . We recently demonstrated that the macrophage is one of the major target cells for avian H5N1 virus in human lungs apart from alveolar epithelial cells [22] . Moreover, H1N1 and H5N1 viruses have been shown to replicate efficiently in both human primary lung epithelial cell cultures and macrophages in vitro [23] . Using the model of fluA virusinfected monocyte-derived macrophages (MDMs), we have found that macrophages infected with human and avian fluA viruses express differential proinflammatory cytokines and chemokines [23, 24] and exhibit differential apoptosis-inducing capability in T cells through the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) pathway [25] .

In the present study, we used the similar virus-infected MDMs model to examine the cytotoxicity of phosphoantigenexpanded Vg9Vd2 T cells against fluA viruses. Specifically, we demonstrated for the first time that IPP-expanded Vg9Vd2 T cells could efficiently kill MDMs infected with human (H1N1) and avian (H9N2 and H5N1) fluA viruses and inhibit virus replication. The cytotoxicity of IPP-expanded Vg9Vd2 T cells was dependent on NKG2D activation and mediated by perforin-granzyme B and Fas-Fas ligand (FasL) pathways. Our findings suggest that phosphoantigen could be used to activate Vg9Vd2 T cells against fluA infections, in particular for avian fluA virus infections.

Peripheral blood mononuclear cells (PBMCs) were isolated from buffycoat preparations of blood from healthy donors from the Hong Kong Red Cross by Ficoll-Paque (Pharmacia) gradient centrifugation, as described elsewhere [26] . The research protocol was approved by the Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster. PBMCs were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum. IPP (Sigma) was added on days 0 and 3, to a final concentration of 6 mg/mL. Recombinant human interleukin 2 (IL-2) (Invitrogen) was added every 3 days beginning on day 3, to a final concentration of 500 IU/mL. After being cultured for 14 days, the cells were purified by negative selection with a TCRg/d + T cell isolation kit (Miltenyi Biotec), in accordance with the manufacturer's instructions.

Culture of MDMs. Human MDMs were generated from mononuclear cells, as described elsewhere [25] . Briefly, adherent monocytes were seeded in 96-well flat-bottomed plates at cells/well or in 24-well plates at cells/well. They 5 5 1 ϫ 10 5 ϫ 10 were then re-fed by RPMI 1640 supplemented with 5% autologous serum and allowed to differentiate to macrophages for 14 days. The purity of monocytes, as determined by flow cytometry with anti-CD14 monoclonal antibody, was consistently 190%.

Virus preparation, titration, and infection. Human fluA H1N1 (A/Hong Kong/54/98) and avian fluA H9N2 (A/Quail/ Hong Kong/G1/97) and H5N1 (A/Hong Kong/483/97) viruses were used. All the viruses were cultured in Madin-Darby canine kidney cells (American Type Culture Collection), as described elsewhere [25] . The virus titer was determined by daily observation of cytopathic effect, and the median tissue culture infective dose was calculated according to the Reed-Muench formula. Differentiated MDMs on day 14 were infected by fluA viruses at a multiplicity of infection (MOI) of 2. After 1 h of viral adsorption, the cells were washed with phosphate-buffered saline to remove unadsorbed virus.

Cytotoxicity assay. Vg9Vd2 T cells (effector) were cocultured with H1N1-, H9N2-, or H5N1-infected MDMs (target) at different effector-to-target (E:T) ratios for 4-6 h. Afterward, nonadherent cells were harvested directly. Adherent cells were detached with 0.25% trypsin-ethylenediaminetetraacetic acid. All of the adherent and nonadherent cells were then stained with anti-CD3 to identify Vg9Vd2 T cells and ethidium homodimer 2 (EthD-2) to identify dead cells [27] . The cytotoxicity of Vg9Vd2 T cells against virus-infected MDMs was assessed by flow cytometry as the percentage of EthD-2 + cells in the CD3 Ϫ population.

To evaluate cell-cell contact requirement for Vg9Vd2 T cell cytotoxicity, a transwell system was used (24 wells; pore size, 0.4 mm; Millipore). MDMs (target) in the bottom well were infected with H1N1 or H9N2 virus at an MOI of 2. Vg9Vd2 T cells (effector) were added directly into the bottom wells or into transwell inserts at an E:T ratio of 10:1. In some inserts, the same amount of virus-infected MDMs was added to activate Vg9Vd2 T cells. After 6 h, the MDMs in the bottom wells were harvested and analyzed for cell death, as described above.

Blocking assay. Vg9Vd2 T cells (effector) were cocultured with H1N1 virus-infected or H9N2 virus-infected MDMs (target) at an E:T ratio of 10:1 for 6 h. The neutralization antibodies anti-NKG2D (10 mg/mL; 1D11, BD Biosciences), anti-FasL (10 mg/mL; 100419, R&D Systems), anti-TRAIL (10 mg/ mL; RIK-2, R&D Systems) and their respective isotype controls were added in the coculture for blocking NKG2D-, FasL-, and TRAIL-mediated pathways, respectively [28] . For blocking perforin and granzyme B, the perforin inhibitor concanamycin A (CMA) (1 mg/mL; Sigma) and the granzyme B inactivator Bcl-2 (1 mg/mL; R&D Systems) were used, as described elsewhere [29] . Cytotoxicity was analyzed by flow cytometry, as described below, and calculated as the percentage of inhibition relative to that in controls.

Flow cytometry. Cells were stained for surface markers with the following antibodies: anti-CD3 (HIT3a), anti-T cell receptor (TCR)-g9 (B3), anti-TCR-d2 (B6), anti-NKG2D (1D11), anti-TRAIL (RIK-2), anti-CD107a (H4A3) (BD Biosciences), anti-TCR-gd (5A6.E9), anti-CD14 (TUK4), and anti-FasL (Alf-2.1) (Invitrogen). For intracellular staining, cells were fixed, permeabilized, and then stained with anti-perforin (Pfp, dG9) and anti-granzyme B (GrB, GB11) antibodies (BD Biosciences) or their respective isotype controls. All samples were acquired using a BD FACSAria cell sorter (BD Biosciences) and were analyzed by means of FlowJo software (version 8.8.3; Tree Star).

) were infected 5 1 ϫ 10 with H1N1, H9N2, or H5N1 virus at an MOI of 2. One hour later, unadsorbed virus was washed away carefully and the MDMs were cultured alone or with Vg9Vd2 T cells for 6 1 ϫ 10 48 h. The cells and supernatant were then harvested, and total RNA was extracted by means of TRIzol LS reagent (Invitrogen), in accordance with the manufacturer's instructions. Complementary DNA was synthesized with oligo(dT) [12] [13] [14] [15] [16] [17] [18] primer and Superscript II reverse transcriptase (Invitrogen). Viral matrix gene copies were quantified on the basis of a SYBR green fluorescence signal after polymerase chain reaction (forward primer, 5 -CTTCTAACCGAGGTCGAAACG-3 ; reverse primer, 5 -GGCATTTTGGACAAAGCGTCTA-3 ) by means of the ABI PRISM 7700 sequence detection system (Applied Biosystems). Results were expressed as the number of target gene copies per MDMs. 5 1 ϫ 10 Statistical analysis. Data were expressed as means ‫ע‬ standard errors of the mean. Statistical significance was determined by the Student t test or nonparametric tests using GraphPad Prism software (version 5). Differences were considered significant at . P ! .05

Consistent with previous findings [18, 19] , we found that IPP and IL-2 could selectively expand Vg9Vd2 T cells. Freshly iso- 

To determine whether there was a decreased viral load in virus-infected MDMs after coculture with Vg9Vd2 T cells, H1N1-, H9N2-, or H5N1-infected MDMs (target) were cultured alone or with IPP-expanded Vg9Vd2 T cells (effector) at an E:T ratio of 10:1. After 2 days of coculture, H1N1, H9N2, and H5N1 fluA virus matrix gene copies from whole virusinfected MDMs and culture supernatants were significantly re- 

Vg9Vd2 T cells is dependent on cell-cell contact, we used a transwell culture system. As shown in figure 4A and 4B, Vg9Vd2 T cells lost their cytotoxicity during H1N1 or H9N2 virus infections when Vg9Vd2 T cells were physically separated from virus-infected MDMs. However, when virus-infected MDMs were put with Vg9Vd2 T cells, cytotoxicity toward targets in the bottom wells was also observed, although their cytolytic activities were much lower than those in the direct cell-cell contact coculture. These data suggest that the cytotoxicity of Vg9Vd2 T cells is dependent on cell-cell contact and requires activation of virus-infected MDMs; diffusible soluble factors, such as granules released from activated Vg9Vd2 T cells, may also be involved.

We then sought to confirm granule release during the killing of fluA virus-infected MDMs by Vg9Vd2 T cells. The expressions of CD107a (lysosome-associated membrane protein 1), a marker associated with the degranulation of NK cells and cytotoxic T lymphocytes (CTLs) [30] , were significantly upregulated in Vg9Vd2 T cells after coculture with H1N1-and H9N2-infected MDMs for 4 h, compared with mock-infected MDMs ( figure 4C ). These results suggest that virus-infected MDMs trigger more intensive granule release from Vg9Vd2 T cells.

Because IPP-expanded Vg9Vd2 T cells also expressed high or medium levels of NKG2D, Fas, and TRAIL, we further determined whether NKG2D, Fas-FasL, and TRAIL pathways were involved in their cytotoxicity. Using neutralizing antibodies for NKG2D and FasL, we found that blockades of NKG2D and FasL significantly inhibited the cytolytic activities of Vg9Vd2 T cells against H1N1-infected MDMs (64.08% ‫ע‬ inhibition by NKG2D blocking; by FasL 9.15% 41.34% ‫ע‬ 9.51% blocking). In contrast, there was no significant change in their cytolytic activity against H1N1-infected MDMs after treatment with TRAIL-blocking antibody (figure 5A). Similar results were also found during the killing of H9N2-infected MDMs by Vg9Vd2 T cells ( figure 5B ). These results demonstrate that both the NKG2D and the Fas-FasL pathway are also involved in the killing of MDMs infected with human and avian fluA viruses by Vg9Vd2 T cells.

To further confirm the involvement of cytolytic granule release in the killing of virus-infected MDMs by Vg9Vd2 T cells, the perforin-specific inhibitor CMA and granzyme B inactivator Bcl-2 were used. As shown in figure 5A , the cytolytic activities of Vg9Vd2 T cells against H1N1-infected MDMs were sig- Figure 5 . Dependency on NKG2D activation and mediation by Fas-Fas ligand (FasL) and perforin-granzyme B pathways for Vg9Vd2 T cell cytotoxicity. Vg9Vd2 T cells (effector) were cocultured with H1N1 virus-infected (A) or H9N2 virus-infected (B) human monocyte-derived macrophages (MDMs) (target) at an effector-to-target ratio of 10:1 for 6 h. The perforin inhibitor concanamycin A (CMA), the granzyme B inactivator Bcl-2, anti-NKG2D (aNKG2D), anti-tumor necrosis factor-related apoptosis-inducing ligand (aTRAIL), and anti-FasL (aFasL) blocking antibodies or their respective isotype controls were used as described in Methods. Cytotoxicity was analyzed by flow cytometry and calculated as the percentage of inhibition relative to that of controls. Data are means from 4 experiments; error bars represent standard errors of the mean. * for the comparison with isotype P ! .05 control. gIgG, goat immunoglobulin G; mIgG1, mouse immunoglobulin G1; mIgG2b, mouse immunoglobulin G2b.

nificantly abrogated after CMA treatment (57.25% ‫ע‬ 9.77% inhibition) or Bcl-2 treatment ( inhibition). 28.42% ‫ע‬ 4.41% Similar results were also observed during the killing of H9N2infected MDMs by Vg9Vd2 T cells (figure 5B). These results indicate that the perforin-granzyme B pathway is involved in the cytotoxicity of Vg9Vd2 T cells against MDMs infected with human and avian fluA viruses.

gd T cells have been reported to play an important role in the defense against pathogens and tumors, and they have broad antiviral activities against different viruses [10, 12, 31, 32] . In human in vitro systems, gd T cells have been shown to have potent cytolytic activities against virus-infected cells, suppressing the replication of human immunodeficiency virus, hepatitis B virus, herpes simplex virus, vaccinia virus, human cytomegalovirus, and severe acute respiratory syndrome coronavirus [20, [33] [34] [35] . In the present study, we focused on the cytotoxic properties of gd T cells in influenza virus infection, because cell-mediated cytotoxicity is the major mechanism to eliminate virus-infected cells and thus to eliminate potential sources of new virus. In particular, as a component of innate immunity, gd T cells may act as early responders in viral control and clearance, compared with specific CTL activity. We showed that IPP-expanded Vg9Vd2 T cells could kill ∼50% of human H1N1 virus-infected MDMs after 6 h of coculture. Most importantly, similar potent cytotoxic activities of these cells against MDMs infected with the newly emerged avian fluA H5N1 virus and its precursor H9N2 virus were also found after 4-6 h of coculture. Furthermore, we found that IPP-expanded Vg9Vd2 T cells significantly inhibited H1N1, H9N2, and H5N1 viral replication by eliminating virus-infected MDMs. To the best of our knowledge, ours is the first study to demonstrate that IPPexpanded gd T cells have rapid and potent antiviral activity against both human and avian fluA viruses.

Consistent with findings of other in vitro studies [17, 18] , we found that the phosphoantigen IPP could selectively activate and expand human Vg9Vd2 T cells from PBMCs in the presence of IL-2. During 2 weeks of stimulation by IPP and IL-2, Vg9Vd2 T cells were expanded by ∼36-fold. In fact, phosphoantigens have demonstrated the potential to facilitate largescale in vitro expansion of functional gd T cells for use in adoptive immunotherapy for tumors [36] and infectious diseases in different models [19, 20] . Although the lack of murine counterparts of Vg9Vd2 T cells has dramatically hampered efforts to understand the in vivo roles of phosphoantigens in Vg9Vd2 T cells, the selective activation and expansion of these cells by phosphoantigens were also confirmed in some in vivo models, such as cynomolgus monkeys and severe combined immunodeficient mice engrafted with human peripheral blood leukocytes (human PBL-SCID mice) [17, 37] . Therefore, phosphoantigens such as IPP could be an alternative for treating human and avian fluA infections via targeting Vg9Vd2 T cells. Indeed, synthetic phosphoantigens named therapeutic amino-bisphosphonates (pamidronate and zoledronate) have been commonly used clinically to treat osteoporosis through Vg9Vd2 T cell-mediated lysis of osteoclasts [38, 39] .

One concern regarding gd T cell-based immunotherapy as induced by phosphoantigens is whether these cells can traffic to infected sites, such as the lungs, during the fluA virus infection. Because gd T cells express chemokine (C-C motif) receptor 5 and are capable of diapedesis [40] , these cells should be able to migrate to inflammatory sites. A more recent study in a macaque model of Mycobacterium tuberculosis infection demonstrated that phosphoantigen-specific gd T cells could accumulate at all inflammatory sites in lymphoid and nonlymphoid tissues (including the lungs) [41] , suggesting that the strategy of targeting gd T cells by phosphoantigens may be feasible for the treatment of human and avian fluA virus infections. Use of nonhuman primate models or human PBL-SCID mice to further evaluate gd T cell-based immunotherapy for influenza virus infections in vivo may accelerate its future clinical application.

Although NKG2D was originally described as an activating receptor for NK cells, it has also been recognized as a potent costimulatory receptor of the cytotoxic functions of human Vg9Vd2 T cells [42, 43] . It has recently been demonstrated that NKG2D can directly activate Vg9Vd2 T cells and trigger their release of cytolytic granules through recognition of the NKG2D ligand [44] . In humans, it has been identified that stress-inducible major histocompatibility complex class I-related proteins A and B and members of the UL16-binding protein family (ULBP1-4 and RAET1G) are the ligands for NKG2D [45] . In different tumor models, NKG2D-mediated cytotoxicity was reported to be involved in the lysis of tumor cells by gd T cells [18, 46] . In the present study, we showed that most IPP-expanded Vg9Vd2 T cells expressed NKG2D and that the cytotoxicity of Vg9Vd2 T cells against H1N1-and H9N2-infected MDMs was significantly blocked by NKG2D neutralizing antibody, suggesting that the killing of influenza virus-infected cells by Vg9Vd2 T cells requires NKG2D activation and recognition. That only influenza virus-infected MDMs expressed up-regulated major histocompatibility complex class I-related protein B, an NKG2D ligand [47] , could also explain why IPPexpanded Vg9Vd2 T cells killed only H1N1-, H9N2-, and H5N1-infected MDMs but not mock-infected MDMs in the present study.

The Fas-FasL-mediated pathway was also shown to be involved in the killing of Listeria monocytogenes-infected macrophages by murine gd T cells in vivo [48] . We found that IPPexpanded Vg9Vd2 T cells expressed high levels of Fas and that blockade of the Fas-FasL pathway significantly inhibited the cytotoxicity of Vg9Vd2 T cells against H1N1-and H9N2-infected MDMs, indicating that the Fas-FasL-mediated pathway is also involved in the killing of influenza virus-infected cells by Vg9Vd2 T cells.

We recently demonstrated that TRAIL was significantly upregulated by both H9N2 and H5N1 avian fluA viruses in human MDMs, compared with H1N1, and that avian fluA virus-infected MDMs could induce T cell apoptosis through the TRAIL pathway [25] . The present study, however, did not show involvement of the TRAIL pathway during the killing of H1N1or H9N2-infected MDMs by Vg9Vd2 T cells; blockade of the TRAIL pathway did not reduce the cytotoxicity of Vg9Vd2 T cells against either H1N1-or H9N2-infected MDMs.

Using the transwell system, we demonstrated that the killing of fluA virus-infected MDMs by Vg9Vd2 T cells was dependent on cell-cell contact and required the activation of virus-infected cells. In addition, CD107a expression was increased only in Vg9Vd2 T cells cocultured with fluA virus-infected MDMs and not in those cocultured with mock-infected MDMs, supporting the hypothesis that granule release from Vg9Vd2 T cells requires virus-infected cell activation. Using the perforin inhibitor CMA and the granzyme B inactivator Bcl-2, we confirmed that perforin and granzyme B facilitated the cytolytic responses of Vg9Vd2 T cells to fluA virus-infected MDMs, which is consistent with the findings of other studies in tumor and other virus-infected cells [18, 20, [33] [34] [35] . Therefore, human Vg9Vd2 T cells closely resemble NK and CD8 + T cells in their cytotoxic function, using a predominantly granule-exocytosis mechanism to kill virus-infected cells.

In summary, we have demonstrated for the first time that Vg9Vd2 T cells can recognize and efficiently kill MDMs infected with human and avian fluA viruses and thus contribute to virus clearance. The cytotoxicity of Vg9Vd2 T cells against fluA virus-infected MDMs is dependent on NKG2D activation and is mediated by the Fas-FasL and perforin-granzyme B pathways. Our study suggests a novel approach of using phosphoantigens to activate Vg9Vd2 T cells against fluA virus infections-newly emerged avian fluA virus infections in particular.

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