Suppression of R5-type of HIV-1 in CD4+ NKT cells by Vδ1+ T cells activated by flavonoid glycosides, hesperidin and linarin

We established transfectants expressing T cell receptors (TCRs) either for Vγ1 and Vδ1 (1C116) or for Vγ2 and Vδ2 (2C21) using the TCR-deficient Jurkat T cell line J.RT3-T3.5. The amount of IL-2 secreted from these γδ T cell clones accurately indicated TCR-dependent stimulation. Clone 2C21 was specifically stimulated by previously reported ligands for Vγ2Vδ2 (Vδ2)-TCR such as isopentenyl pyrophospate (IPP), ethylamine, or risedronate. In contrast, clone 1C116 was strongly stimulated through the Vγ1Vδ1 (Vδ1)-TCR by flavonoid glycosides such as hesperidin and linarin, having both rutinose at the A ring and methoxy (-OCH3) substitution at the B ring. Additionally, hesperidin and linarin showed stimulatory activity for peripheral blood mononuclear cell (PBMC)-derived T cells expressing Vδ1-TCR; these activated Vδ1+ T cells also secreted IL-5, IL-13, MIP-1α, MIP-1β and RANTES. Such PBMC-derived Vδ1+ T cells stimulated by hesperidin and linarin suppressed R5-HIV-1-NL(AD8) viral replication in CD4+ NKT cells in a dose-dependent manner. To the best of our knowledge, this is the first demonstration that flavonoid glycosides activate functional Vδ1+ T cells.

The development of anti-HIV-1 drugs, particularly the newer class of anti-retroviral drugs such as integrase inhibitors (IN), raltegravir, elvitegravir, and dolutegravir, now permits HIV-1 to be successfully eradicated from the circulating blood of HIV-1-infected individuals 1 . Nevertheless, in most cases, HIV-1 virions still re-emerge within a few months after interruption of anti-retroviral therapy (ART) 2,3 . These findings suggest that immunity to HIV-1 able to prevent the generation of circulating virus does not arise in most ART-treated infected individuals. Although free virions and HIV-1-p24 antigen (p24)-positive cells were not seen in the blood of patients receiving current effective ART, proviral DNA and HIV-1 p24 antigen could still be detected in the ileum of the same patient. Indeed, we have recently confirmed that Vα24 + natural killer T (NKT) cells 4 were the major p24-positive cells among the HIV-1-infected CD4 + cells in the ileum samples, suggesting that the innate NKT cells in the mucosal compartment are the most likely candidates for the origin of the HIV-1 that emerges after ART is interrupted 5 .
The majority of emergent HIV-1 after interrupting ART is macrophage-tropic and infects CD4 + , CCR5-expressing cells (R5-tropic) rather than CXCR4 expressing CD4 + T cells (X4-tropic) 5 . Additionally, we have verified that CD4 + NKT cells from peripheral blood mononuclear cells (PBMCs) predominantly express CCR5 rather than CXCR4 and are infected with R5-tropic HIV-1, such as the NL(AD8) isolate, which expands efficiently in the CD4 + NKT cells 5,6 , as has been reported 7 . In addition, we found that CD8αα + but not CD8αβ + T cells have the ability to inhibit R5-tropic HIV-1 replication in the CD4 + NKT cells and confirmed that replication of the NL(AD8) isolate in the CD4 + NKT cells was efficiently suppressed by CD8αα + γδ T cells, in particular Vγ1Vδ1(Vδ1)-T cell receptor (TCR)-expressing γδ T cells, mainly through chemokines such as macrophage inflammatory proteins 1alpha (MIP-1α) and MIP-1β and RANTES 6 . These results suggest that CD8αα + Vδ1 + T cells may contribute to control of R5-tropic HIV-1 replication in persistently infected CD4 + NKT cells.
However, the Vδ1 + T cells were obtained from freshly-isolated PBMC-derived T cells, which were variably activated by the procedure of their purification process with specific antibodies. Thus, the suppressing effect of the Vδ1 + T cells on HIV-1 replication in CD4 + NKT cells was uneven. Therefore, to examine more stable effects of (2019) 9:7506 | https://doi.org/10.1038/s41598-019-40587-6 www.nature.com/scientificreports www.nature.com/scientificreports/ Vδ1 + T cells, we used here resting Vδ1 + T cells purified according to a procedure by Shamshiev et al. 8 as responders to confirm the suppressing effects.
Human γδ T cells consist mainly of two distinct subsets, Vδ1-TCR-expressing Vδ1 + T cells and Vδ2-TCR-expressing Vδ2 + T cells. These lymphocytes play important roles in bridging innate and adaptive immunity, but their recognition mechanisms remain poorly understood. Approximately 70% of T lymphocytes express the Vδ2-TCR and can be expanded and activated by phospho-antigens such as the cholesterol biosynthesis-related substance, isopentenyl pyrophosphate (IPP) 9 , or synthetic bisphosphonates, such as pamidronate disodium and zoledronic acid 10 , as well as alkylamines such as ethylamine 11 . In contrast, the Vδ1 + T cell subset is predominantly present in the intestinal epithelia and responds to MICA and MICB (MHC class I chain-related, A and B; MIC) self-antigens, as well as CD1-molecule related lipid antigens 12,13 , mediating responses to tumorigenesis or viral infection 14 . In contrast to αβ TCRs, which require antigen processing and subsequent presentation antigenic peptides by MHC molecules, γδ TCRs are believed to recognize antigens directly 15-17 . In the present study, to identify antigenic ligands for the Vδ1-TCR, we first established two distinct clones from human γδT cell lines with stable proliferating ability and that express either Vγ1 and Vδ1 or Vγ2 and Vδ2 TCR. Full-length cDNAs encoding the TCR-γ1/TCR-δ1 chain or the TCR-γ2/TCR-δ2 chain were obtained from each T cell clone and transfected as pairs into a TCR-deficient Jurkat T cell line, J.RT3-T3.5 18 . We successfully established two distinct transfected clones expressing either Vγ1 and Vδ1 (1C116) or Vγ2 and Vδ2 (2C21). After confirming that clone 2C21 specifically responded to produce IL-2 upon stimulation by IPP, alkylamines such as ethylamine, and risedronate, we exposed the other clone 1C116 to various candidate antigenic molecules, including phytochemicals, such as alkaloids (compounds containing nitrogen), terpenoids (compounds derived from C5 isoprene units) and phenolics (compounds having aromaticity).
Among the flavonoids, we discovered two compounds, hesperidin and linarin, with very similar structures (C6-C3-C6 structure) containing a flavonoid glycoside that will specifically stimulate clone 1C116. Such flavonoid glycosides are well-known for their antioxidant, anti-inflammatory, anti-thrombogenic, anti-arteriosclerosis and anti-carcinogenic properties. Here, we demonstrate that both flavonoid glycosides suppressed the replication of R5-type HIV-1 in CD4 + NKT cells through the activation of Vδ1-TCR-bearing resting Vδ1 + T cells.

Establishment of two distinct transfectants expressing T cell receptors (TCRs) either for Vγ1
and Vδ1 (1C116) or for Vγ2 and Vδ2 (2C21) from TCR-deficient Jurkat T cell line J.RT3-T3.5. As described in the Methods section, we established single cell clones bearing either Vδ1-TCR or Vδ2-TCR with stable proliferation capacity. Full-length cDNAs encoding both TCR-γ1 and TCR-δ1 chains were isolated from a Vδ1 + T cell clone. Similarly, the full-length cDNAs encoding both TCR-γ2 and TCR-δ2 chains were obtained from a Vδ2 + T cell clone ( Supplementary Fig. S1). These plasmids were then doubly transfected into a TCRdeficient Jurkat T cell line, J.RT3-T3.5, generating two distinct transfected clones expressing either Vγ1 and Vδ1 (1C116) or Vγ2 and Vδ2 (2C21). These J.RT3-T3.5-derived transfectants were maintained in the presence of 1 mg/mL G418 sulfate and 0.5 mg/mL hygromycin B to maintain selection of γδ TCR-bearing cells.
As shown in Fig. 1A, clone 1 C116 was stained with monoclonal antibodies (mAbs) for pan-γδ and Vδ1 but not Vδ2, whereas clone 2C21 was stained with pan-γδ and Vδ2 but not Vδ1. Since both clones express CD3, when they are stimulated through the TCR, they can secrete IL-2 in the presence of PMA (Fig. 1B). Therefore, the amount of IL-2 secreted from these γδ T cell clones seems to be an excellent indicator for TCR-dependent stimulation.
Isopentenyl pyrophospate (IPP) is known to stimulate functional responses from Vδ2 + T cells 9,19 , although Vavassori et al. has been reported that Vδ2 + cells are not directly stimulated by IPP 20 . However, Bukowski, et al. reported 19 that an IPP-like phosphoantigen MEP directly stimulated Vγ2/Vδ2 + TCRs-tranfected Jurkat T cells such as clone 2C21. On the basis of these findings, we examined whether IPP stimulates clone 2C21 to secrete IL-2. As shown in Fig. 1C, we observed that clone 2C21 secreted IL-2 when stimulated with IPP, whereas clone 1C116 did not. Vδ2 + T cells can also be activated by other molecules, like various alkylamines such as ethylamine, propylamine or butylamine 11 , or amino-bisphosphonates used for the treatment of osteoporosis 21 . Similarly, clone 2C21 was stimulated to secrete IL-2 by ethylamine in a dose-dependent manner until 16.7 mM, but was not stimulated by methylamine while more than 33.5 mM of ethylamine were toxic and the secretion of IL-2 by clone 2C21 was abrogated (Fig. 1D). Moreover, clone 2C21 was functionally activated by amino-bisphosphonate such as risedronate (Fig. 1E). These findings indicate that clone 2C21 but not clone 1C116 is specifically stimulated by these various molecules acting through the Vδ2-TCR.
Screening of various phytochemicals for their stimulatory activity on clones 1C116 or 2C21 to secrete IL-2. It is well known that some plant metabolites have biological activities. Recently, such natural compounds have been called "phytochemicals" 22 . According to their metabolic pathways and their chemical structures, phytochemicals are mainly classified into alkaloids (compounds containing nitrogen), terpenoids (compounds derived from C5 isoprene units) and phenolics (compounds having aromaticity). Among phytochemicals, the phenolics make up a large group and include one of the most ubiquitous botanical products called flavonoids (compounds having C6-C3-C6 structure), which are well-known for their many beneficial properties, showing antioxidant, anti-inflammatory, anti-thrombogenic, anti-arteriosclerosis and anti-carcinogenic activity, and they are considered to be important resources for drug discovery 23 .
To examine whether flavonoids might act in part through immune cells, we screened several compounds for their capacity to activate clones 1C116 and 2C21. As shown in Fig. 2A and Table 1, some flavonoids stimulated clone 1C116 but not clone 2C21 to secrete large amounts of IL-2. In particular, flavonoids having a rutinose disaccharide such as 6-O-α-L-rhamnosyl-D-glucose on the A ring together with methoxy (-OCH3) substitution on www.nature.com/scientificreports www.nature.com/scientificreports/ the B ring, such as hesperidin and linarin, strongly induced clone 1C116 to secrete IL-2. The fact that such stimulatory potency was totally abrogated by the treatment with anti-γδ TCR-specific antibody strongly indicates that the recognition of the flavonoid glycoside was initiated through the Vδ1-TCR (Fig. 2B). Moreover, when treated www.nature.com/scientificreports www.nature.com/scientificreports/ with rutinose-deficient hesperidin (hesperetin) or rutinose-deficient linarin (acacetin), IL-2 secretion from clone 1C116 was not detected or weakly detected (about one-sixth of linarin), respectively. Additionally, when treated with flavonoids that have a rutinose disaccharide at the A ring that lack a methoxy (-OCH3) substitution at the B ring, such as isorhoifolin, clone 1C116 did not secrete IL-2 (Fig. 2C).
Taken together, these findings clearly show that clone 1C116 is specifically and strongly stimulated through its Vδ1-TCR by flavonoid glycosides having both rutinose at the A ring and methoxy (-OCH3) substitution at the B www.nature.com/scientificreports www.nature.com/scientificreports/ ring. Furthermore, hesperidin and linarin showed striking stimulatory activity on clone 1C116. Thus, we focused on hesperidin and linarin and examined whether these flavonoid glycosides could suppress the replication of R5-type HIV-1 in CD4 + NKT cells through the activation of Vδ1-TCR-expressing Vδ1 + T cells.
Expansion and activation of Vδ1 + T cells in the blood stimulated by flavonoid glycosides, hesperidin and linarin. We first examined whether human Vδ1 + T cells can be stimulated to proliferate and undergo functional activation by hesperidin and linarin. To perform this experiment, we first labeled PBMCs with CFSE. Then, the labeled cells were incubated with 100 μg/mL hesperetin, hesperidin, acacetin or linarin, or 0.01% DMSO for 14 days in 48-well plates containing 0.5 mL of CCM with 5% AB human serum and 100 U/mL recombinant IL-2. After culture, the cells were incubated with anti-Vδ1-APC antibody and anti-CD25-PE/Cy7 antibody at 4 °C for 30 min and were analyzed by flow-cytometry. When compared with unstimulated control or vehicle DMSO, hesperetin and acacetin did not significantly stimulate PBMC-derived Vδ1 + T cells, but hesperidin and linarin expanded the number of PBMC-derived Vδ1 + T cells (Fig. 3A, upper panel and Supplementary  Fig. S2). Additionally, hesperidin and linarin activated these cells to express CD25 (Fig. 3A, lower panel and Supplementary Fig. S2). The capacity of flavonoid glycosides to induce proliferation and activation of Vδ1 + T cells by was confirmed in repeat experiments (Fig. 3B). In contrast, neither Vδ2 + T cells and αβ + T cells responded to hesperidin and linarin (Fig. 3A,B).
Cytokine and chemokine profiles secreted from Vδ1 + T cells stimulated with flavonoid glycosides. These experiments revealed that flavonoid glycosides such as hesperidin and linarin can stimulate Vδ1 + T cells through their TCRs. We have reported previously that chemokines such as CCL3 (MIP-1α), CCL4 (MIP-1β) and CCL5 (RANTES) secreted by Vδ1 + T cells suppress R5-HIV-1-NL(AD8) viral replication in CD4 + NKT cells 6 . In addition, they activate Vδ1 + T cells through enhanced MICA/MICB expression on NL(AD8)-infected CD4 + NKT cells 6 . We, thus, asked whether the flavonoid glycoside-activated Vδ1 + T cells would secrete MIP-1α, MIP-1β and RANTES. To perform these experiments, we sorted and expanded Vδ1-TCR expressing T cells from PBMCs (Fig. 4A) and stimulated them with various flavonoid glycosides to examine the cytokine profiles. Surprisingly, we found that the predominant cytokines observed in the supernatant of flavonoid glycoside-activated Vδ1 + T cells were IL-5 and IL-13 ( Fig. 4B) but not IL-17 and IFN-γ, a profile very close to that of innate lymphocytes 2 (ILC-2) rather than that of γδ T cells 24 . Moreover, we examined the effect of three doses of hesperidin and linarin (10 µg/ mL, 30 µg/mL, 100 µg/mL) and measured the amounts of secreted cytokines and chemokines. In comparison with the effect of control DMSO, the secreted amounts of IL-5, IL-13, as well as MIP-1α, MIP-1β and RANTES were all enhanced (Fig. 4C). These findings strongly suggested that PBMCs stimulated by flavonoid glycosides such as hesperidin and linarin seem to suppress R5-HIV-1-NL(AD8) viral replication in CD4 + NKT cells.
Suppression in the replication of R5-type of HIV-1 through Vδ1 + T cells activated by flavonoid glycosides, hesperidin and linarin. CD4 + Vα24 + NKT cells were induced from human PBMCs (Fig. 5A, left panel) and infected with R5-tropic NL(AD8) HIV-1. The virus-infected NKT cells were cultured with or without Vδ1 + T cells stimulated with hesperidin, linarin, or DMSO. Addition of more than 100 μg/mL of hesperidin or linarin to the culture medium for interfered with NKT cell replication/survival, indicating that more than 100 μg/ mL was toxic (Fig. 5A, right panel). At lower concentrations that avoided this toxicity, flavonoid glucosides did not suppress R5-HIV-1-NL(AD8) production by infected CD4 + NKT cells in the absence of Vδ1 + T cells (Fig. 5B,  left panel), but a measurable amount of suppression of viral replication was observed in the presence of Vδ1 + T cells and the flavonoids (Fig. 5B, right panel and Fig. 5C). Conversely, resting Vδ1 + T cells alone did not suppress R5-HIV-1-NL(AD8) production by infected CD4 + NKT cells. Taken together, these findings suggest that resting Vδ1 + T cells may contribute to control R5 tropic HIV-1 replication in CD4 + NKT cells, when stimulated by flavonoid glycosides such as hesperidin and linarin. So far as we know, this is the first demonstration that flavonoid glycoside will activate Vδ1 + T cells and yield a functional outcome.

Discussion
In the present study, we have successfully established two distinct γδ TCR-transfected clones, 1C116 and 2C21, expressing either Vδ1-TCR or Vδ2-TCR, respectively. 2C21 is specifically stimulated to secrete IL-2 through the expressed Vδ2-TCR by any of several previously reported antigenic molecules such as IPP, ethylamine or by amino-bisphosphonates such as risedronate.  www.nature.com/scientificreports www.nature.com/scientificreports/ Using a similar technical approach, we found that another γδ TCR-transfected clone, 1C116, is specifically stimulated through its Vδ1-TCR by flavonoid glycosides such as hesperidin and linarin, which have both rutinose at the A ring and methoxy (-OCH3) substitution at the B ring. To our knowledge, this is the first demonstration www.nature.com/scientificreports www.nature.com/scientificreports/ that Vδ1 + cells specifically recognize flavonoids, especially flavonoid glycosides. Moreover, the identified flavonoid glycosides not only activated the engineered Jurkat clone expressing Vδ1, but also stimulated PBMC-derived Vδ1 + T cells to secrete both IL-5 and IL-13 cytokines as well as the chemokines MIP-1α, MIP-1β and RANTES. Consistent with our prior data, these mediators suppressed R5-HIV-1-NL(AD8) viral replication in CD4 + NKT cells. Therefore, the newly identified flavonoid glycosides that stimulate Vδ1 + T cells may constitute a new class of anti-HIV drugs able to act in the mucosal compartment to suppress the R5-type of HIV-1.
A number of studies have shown that many plants produce various flavonoids as defense factors against microbes and toxins, offering protection against pathogenic bacteria, fungi and viruses. Although the exact mechanisms behind their anti-microbial properties are not fully understood, two flavonoids, such as hesperidin and www.nature.com/scientificreports www.nature.com/scientificreports/ www.nature.com/scientificreports www.nature.com/scientificreports/ its aglycone, hesperetin, showed various biological properties, including anti-oxidant, anti-inflammatory and anti-cancer effects 25 . It should be noted that polyphenols have intensively been studied due to their beneficial effects in both cardiovascular diseases and cancer.
Among polyphenols, quercetin is one of the most studied compounds. It is found in apples, berries, oranges, grapes, onions and tea, and it is metabolized and then absorbed by the intestinal microbiota. High levels of unabsorbed flavonoid compounds in the gut play an important role in the intestine's health. The remaining flavonoids may suppress the growth of many intestinal pathogenic microbiota. Recently, it was demonstrated that one flavonoid glycoside called quercetin 3-β-O-D-glucoside (Q3G) has the ability to protect mice from Ebola, even when given only 30 min prior to infection 26 . Moreover, a number of reports on flavonoids, including on quercetin and its derivatives, demonstrated anti-viral activities for a variety of viruses, such as influenza virus 27,28 , hepatitis C virus 29 , Chikungunya virus 30 and Epstein-Barr virus 31 . Therefore, a number of flavonoids having quercetin-like structures will suppress the viral replication of various viruses, but most of these flavonoids will not activate both Vδ1 + T cells and Vδ2 + T cells. Only a limited repertoire of Vδ1 + T cells have a stronger ability to suppress viral replication than the effective flavonoids, and those Vδ1 + T cells can be mediated through flavonoid glycosides, such as hesperidin and linarin.
R5-type of HIV-1 replication can be observed in mucosal CD4 + NKT cells with the invariant Vα24 TCR, even in individuals given effective HAART-treatment 5 . Our current findings suggest that exposure of the Vδ1 + T cells in such patients to flavonoid glycosides such as hesperidin and linarin may contribute to limiting R5-type of HIV-1 replication. Finally, the findings also imply that the treatment of not only a variety of viral infections but also other virus-related diseases such as malignancies or several autoimmune-diseases might be enhanced by activation of host Vδ1 + T cells with flavonoid glycosides. (H. saimiri) and establishment of a γ1δ1 + T cell clone. PBMCs were obtained from a healthy individual by gradient centrifugation using Ficoll-Hypaque (GE Healthcare, Uppsala, Sweden). The isolated PBMCs were labeled with anti-pan γδ-TCR mAb (B6.1; BD Bioscience, San Diego, CA), and then γδ T cells were positively selected by FACSAriaII (BD Bioscience, Mountain View, CA). The purified γδ T cells were stimulated in complete culture medium (CCM) 32 composed of RPMI-1640 (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% heat-inactivated fetal calf serum (FCS) (HyClone, Logan, UT), 5 mM HEPES buffer (Thermo Fisher Scientific), 100 U/ml penicillin (Thermo Fisher Scientific), 100 μg/ml streptomycin (Thermo Fisher Scientific), 2 mM L-glutamine (Thermo Fisher Scientific), 2 mM sodium pyruvate (Thermo Fisher Scientific), 2 mM nonessential amino acids (Thermo Fisher Scientific), 2 mM-modified vitamins (Thermo Fisher Scientific), and 0.5 μM 2-mercaptoethanol (2-ME) (Thermo Fisher Scientific) for 3 days with 1 μg/mL phytohemagglutinin (PHA) (Sigma-Aldrich, St. Louis, MO). Then, the cells were infected with the 488 strain of H. saimiri subgroup C (a gift of Dr. M. Yasukawa, Ehime University, Japan) as described previously 33 , followed by further culture in the presence of 10 U/mL recombinant interleukin 2 (IL-2) (Shionogi, Osaka, Japan). Single cells were sorted FACSAriaII, resulting in the isolation of γ1δ1 + T cell clones showing stable proliferation over several months without mitogenic stimulation. The study was performed in accordance with the Declaration of Helsinki and under the approval of the Review Board of Nippon Medical School, and all human participants gave written informed consent.
These constructed plasmids were doubly transfected into the TCR-deficient Jurkat cells, J.RT3-T3.5 (ATCC, Manassas, VA), by electroporation at 240 to 270 V. Two or three days after the transfection, the transfected cells were cultured in the presence of 1 mg/mL G418 sulfate (Thermo Fisher Scientific) and 0.5 mg/mL hygromycin B (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) to select γδ TCR-bearing cells. Clones were isolated by limiting the dilution at which drug-resistant cells were plated at 0.5 or 1 cells/well in 96-well microtiter plates, and the cells were analyzed for γδ-TCR expression by flow cytometry. 1C116 and 2C21 clones that stably expressed Vδ1-and Vδ2-TCRs, respectively, were obtained and used in this study. www.nature.com/scientificreports www.nature.com/scientificreports/ by FACSCantoII (BD Biosciense) using FlowJo software (BD Biosciense). Additionally, to do the TCR-blocking assay, the established γδ-TCR transfected clones were incubated with 20 μg/mL anti-pan-γδ mAb (clone B1: BioLegend) at room temperature for an hour, and the following stimulation assay was performed.

Induction of resting Vδ1 + T cells from human PBMCs.
According to the procedure reported previously 8 , polyclonal Vδ1 + T cells freshly obtained from human PBMCs were incubated with 2 μg/mL PHA with 1 × 10 6 /mL irradiated PBMCs and were further cultured for an additional 21 days in 24-well culture plates containing CCM supplemented with 5% AB human serum and 100 U/mL rIL-2, and the medium was half-exchanged every 3-4 days. After the initial culture, the Vδ1 + cells were cultured by the same procedure for 14 days to rest.
Measurement of the amount of cytokine and chemokine production from Vδ1 + T cells stimulated by flavonoids. The Vδ1 + T cells (5 × 10 4 ) were cultured in 200 μl of CCM supplemented with 5% AB human serum and 100 U/mL rIL-2 in round-bottom 96-well plates. In the cell cultures, 25 ng/mL PMA and 1 μg/mL ionomycin (Sigma-Aldrich), 10, 30 or 100 μg/mL hesperidin or linarin or 0.01, 003 or 0.1% DMSO were added into the well. After 3 days, 150 μl of culture supernatant was collected from each well and stored at −80 °C until the measurement of cytokines and chemokines.

Infection of CD4 + NKT cells with NL(AD8) HIV
Then, 1 × 10 4 NL(AD8)-infected NKT cells were cultured with or without resting 1 × 10 4 Vδ1 + cells in 200 μl of CCM containing 20 U/mL IL-2 in round-bottom 96-well plates. In the cell cultures, 3, 10 or 30 μg/mL hesperidin or linarin or 0.003, 001 or 0.03% DMSO (vehicle control) was added into the well. On day 3, after the cultures were initiated, 100 μl of culture supernatant was collected from each well and stored at −80 °C until the measurement of p24 antigen. HIV-1 capsid protein p24 production in the culture supernatant was also measured using the ELISA kit (Sino Biological, Beijing. China).
Statistical analyses. The results were analyzed using an ANOVA with Dunnett's post hoc test, and were presented as the mean ± SEM. Differences at p < 0.05 were considered significant. The statistical analysis was performed using Statview 5.0 software (SAS Institute Inc., Cary, NC).

Study approval.
The study was conducted in accordance with the guidelines of the Declaration of Helsinki and principles of Good Clinical Practice and approved by the Review Board of Nippon Medical School, and all human participants gave written informed consent.