HIV-1 Tat affects the programming and functionality of human CD8+ T cells by modulating the expression of T-box transcription factors
Sforza, Fabioa,*; Nicoli, Francescoa,*; Gallerani, Eleonoraa; Finessi, Valentinaa; Reali, Evab; Cafaro, Aurelioc; Caputo, Antonellad; Ensoli, Barbarac; Gavioli, Riccardoa
aDepartment of Life Sciences and Biotechnology, University of Ferrara, Ferrara
bLaboratory of Translational Immunology, I.R.C.C.S. Istituto Ortopedico Galeazzi, Milan
cNational AIDS Center, Istituto Superiore di Sanità, Rome
dDepartment of Molecular Medicine, University of Padova, Padova, Italy.
*Dr Fabio Sforza and Dr Francesco Nicoli contributed equally to the writing of this article.
Correspondence to Professor Riccardo Gavioli, PhD, Department of Life Sciences and Biotechnology, University of Ferrara, via Fossato di Mortara 74, 44121 Ferrara, Italy. E-mail: firstname.lastname@example.org
Received 29 January, 2014
Revised 20 April, 2014
Accepted 22 April, 2014
Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's Website (http://www.AIDSonline.com).
Objective: HIV infection is characterized by several immune dysfunctions of both CD8+ and CD4+ T cells as hyperactivation, impairment of functionality and expansion of memory T cells. CD8+ T-cell dysfunctions have been associated with increased expression of T-bet, Eomesdermin and pro-inflammatory cytokines, and with down-regulation of CD127. The HIV-1 trans-activator of transcription (Tat) protein, which is released by infected cells and detected in tissues of HIV-positive individuals, is known to contribute to the dysregulation of CD4+ T cells; however, its effects on CD8+ T cells have not been investigated. Thus, in this study, we sought to address whether Tat may affect CD8+ T-cell functionality and programming.
Methods: CD8+ T cells were activated by T-cell receptor engagement in the presence or absence of Tat. Cytokine production, killing capacity, surface phenotype and expression of transcription factors important for T-cell programming were evaluated.
Results: Tat favors the secretion of interleukin-2, interferon-γ and granzyme B in CD8+ T cells. Behind this functional modulation we observed that Tat increases the expression of T-bet, Eomesdermin, Blimp-1, Bcl-6 and Bcl-2 in activated but not in unstimulated CD8+ T lymphocytes. This effect is associated with the down-regulation of CD127 and the up-regulation of CD27.
Conclusion: Tat deeply alters the programming and functionality of CD8+ T lymphocytes.
HIV is one of the major plagues in the world for the number of people infected and deaths per year . The most devastating damages caused by HIV infection are observed at the level of cellular immunity, and include the depletion of CD4+ T cells and important dysfunctions of both CD8+ and CD4+ T cells as impairment of functionality [2,3], exhaustion , increased T-cell proliferation , susceptibility to apoptosis [6,7] and expansion of memory T cells [8–10]. This status of chronic immune activation and immune senescence involves the whole T-cell compartment, including uninfected and non-HIV-specific T cells , is also present during antiretroviral therapy (ART) and contributes to the appearance of AIDS-defining and nondefining diseases . Different mechanisms contribute to these phenomena, including CD4+ T-cell loss, viral replication and effects of HIV proteins such as glycoprotein 120, negative regulatory factor and trans-activator of transcription (Tat) [5,11].
Several studies have reported that the HIV-1 Tat protein activates CD4+ T cells and increases pro-inflammatory cytokine production in both HIV-infected and uninfected cells [13–15]. In fact, Tat, in addition to be required for viral replication and infectivity [16,17], is released extracellularly , even during ART , and enters neighboring cells, affecting their functionality [20–24]. Moreover, it has been demonstrated that anti-Tat immunity is important for disease control and restoration of immune functions [25,26], suggesting that Tat may contribute to immune activation. In accordance with this hypothesis, we have recently shown in murine models that Tat favors the activation and the expansion of antigen-specific cytotoxic T lymphocytes (CTLs) and modulates antiviral responses causing dysfunctions similar to those observed in HIV-infected individuals [22,27].
Dysfunctionality of T lymphocytes in HIV-positive patients has been linked to the deep modification of their transcriptional profile [28,29]. In particular, it has been shown that CD8+ T cells from HIV-positive patients display an effector phenotype and a simultaneous increased expression of two T-box transcription factors, T-bet and Eomesdermin (Eomes), which correlate with viral load and decrease after ART . T-bet and Eomes are the master regulators of effector and memory functions in CD8+ T cells . Indeed, it has been shown that, although both transcription factors are important for the generation of different memory T-cell subsets, higher T-bet expression favors a short-lived effector phenotype, whereas higher Eomes expression is important for development of central memory T cells [31,32]. Moreover, T-bet and Eomes expression is modulated by B-cell lymphoma 6 protein (Bcl-6) and B lymphocyte-induced maturation protein 1 (Blimp-1) . Bcl-6 plays a central role in survival and proliferation of T cells, whereas Blimp-1 is required for the development of CTLs and terminal effector cells [33–35]. These transcription factors are regulated by or regulate interferon (IFN)-γ and interleukin (IL)-2 signaling [34–36]. As we have shown in murine models that Tat enhances T-cell receptor stimulation favoring IFN-γ production , and several works describe that Tat increases IL-2 release in CD4+ T cells [13,15], we sought to determine whether Tat may favor the activation of human CD8+ T cells affecting the expression of these transcription factors. We demonstrate here for the first time that CD8+ T cells activated in the presence of Tat enhance their effector functions and display an increased expression of those transcription factors important for T-cell programming.
Thus, this study provides evidence that Tat modulates the functionality and the fate of human CD8+ T lymphocytes, suggesting that this viral protein contributes to T-cell dysfunctions during HIV infection.
Materials and methods
Human cells and culture conditions
Buffy coats from healthy volunteers who provided consent were obtained from the University Hospital of Ferrara. Peripheral blood lymphocytes (PBLs) were separated by use of Ficoll–Hypaque (Lonza, Basel, Switzerland) density gradient centrifugation followed by 90 min of adhesion on a plastic support at 37°C to remove monocytes.
Peripheral blood lymphocytes (3 × 106) were cultured in 2 ml of Roswell Park Memorial Institute medium (RPMI) (Gibco, Life Technologies, Carlsbad, California, USA) containing 10% fetal calf serum (complete medium) in the absence or presence of the Tat protein in 24-well flat bottom polystyrene plates coated overnight at 4°C with PBS or anti-CD3 monoclonal antibody (mAb) (0.5 μg/ml; R&D Systems, Minneapolis, Minnesota, USA). Soluble anti-CD28 mAb (0.1 μg/ml; R&D Systems), Tat and anti-Tat immune sera were added, when indicated, after cell seeding. In the blocking experiments with anti-integrin antibodies, cells were preincubated with 10 μg/ml of anti-α5β1 and anti-αvβ3 antibodies (Merck Millipore, Billerica, Massachusetts, USA) on rotation in RPMI + 0.05% bovine serum albumin for 1 h at room temperature.
CD8+ T cells were sorted by MACS magnetic negative selection (Miltenyi Biotec, Bergish Gladbach, Germany) according to manufacturer's instructions (purity >95% assessed by fluorescence activated cell sorting).
HIV-1 Tat from the human T-lymphotropic virus type IIIB isolate (BH10 clone) was expressed in Escherichia coli and purified by heparin-affinity chromatography and HPLC, as described previously . The lyophilized Tat protein was stored at −80°C to prevent oxidation, reconstituted in degassed buffer before use, and handled, as described . Endotoxin concentration was undetectable (detection threshold: 0.05 EU/μg).
Generation of CTL cultures
Epstein-Barr virus (EBV)-specific and survivin-specific CTL cultures were obtained by stimulation of PBLs with peptide-pulsed T2 cells , in the absence or presence of Tat (see supplementary materials and methods, http://links.lww.com/QAD/A525).
Cytotoxicity and Elispot assays
The cytotoxic activity of CTL cultures was assayed against peptide-pulsed target cells in standard 5-h 51Cr-release assays . For Elispot assays, CTLs were seeded on 96-well Elispot plates precoated with an anti-IFN-γ or antigranzyme B mAb and stimulated with EBV-derived CD8+ peptides (see supplementary materials and methods, http://links.lww.com/QAD/A525).
Reverse transcription and quantitative real-time PCR
DNase-treated total RNA was isolated from cells and cDNA was PCR-amplified. For each RNA, the relative levels were calculated by the 2−ΔΔCT method using human 18S as housekeeping gene (see supplementary materials and methods, http://links.lww.com/QAD/A525).
Tat enhances interleukin-2 and interferon-γ production in CD8+ T cells
Tat is released by infected cells and is detected in the tissues and in the sera of HIV-infected individuals at concentrations within the nanomolar range [18,39–41]. As some reports showed that Tat enhances the release of IL-2 and pro-inflammatory cytokines from activated PBLs [13,42], we first sought to determine whether the amounts of secreted Tat usually found in vivo may account for this effect. To this aim, PBLs from healthy donors were activated with anti-CD3/CD28 in the absence or presence of different doses of Tat (from 0.001 to 1 μg/ml), and IL-2 mRNA levels were measured after 4 h by quantitative PCR (qPCR). As shown in Fig. 1a, a 75-fold increase of IL-2 mRNA was observed in PBLs activated in the absence of Tat compared to untreated PBLs, whereas the presence of Tat induced a 150–200-fold increase of IL-2 mRNA expression. This effect was observed at similar levels for all Tat doses except at 0.001 μg/ml, and it was abolished after incubation with anti-Tat-positive sera (Fig. S1, http://links.lww.com/QAD/A525). Similar results were obtained at 24 h after activation (Fig. 1b), demonstrating that this effect is long-lasting. As the highest fold increase was observed at 0.1 μg/ml of Tat, this dose was chosen to perform the subsequent experiments.
It is already known that Tat favors the release of IL-2 from CD4+ T cells ; however, the effect of Tat on CD8+ T lymphocytes has never been investigated. Thus, we evaluated whether Tat affects the expression of IL-2 mRNA in CD8+ T cells purified from unstimulated or activated PBLs cultured in the absence or presence of Tat. Interestingly, Tat significantly increased the expression of IL-2 mRNA in CD8+ T cells purified from activated PBLs (Fig. 1c), and this effect was further confirmed by intracellular cytokine staining (Fig. S2, http://links.lww.com/QAD/A525).
We next examined whether the presence of Tat could also modulate IFN-γ production. Consistent to what was observed for IL-2 production, Tat dramatically enhanced IFN-γ mRNA expression in CD8+ T cells purified from activated PBLs (Fig. 1d). Of note, Tat did not induce IL-2 or IFN-γ production in CD8+ T cells purified from unstimulated PBLs (Fig. 1c and d).
These results demonstrate that physiological concentrations of Tat enhance the production of IL-2 and IFN-γ in CD8+ T cells activated with anti-CD3/CD28.
Tat affects the expression of T-bet, Eomesdermin and other key transcription factors in activated CD8+ T cells
T-bet and Eomes are transcription factors that are up-regulated during HIV infection  and that control IFN-γ production [30,43]. Since we have shown here that Tat enhances IFN-γ production in human CD8+ T cells stimulated by T-cell receptor engagement, we next characterized the expression of T-bet and Eomes in CD8+ T cells purified from unstimulated or activated PBLs cultured in the absence or presence of Tat. Moreover, the expression of other transcription factors important for T-cell functionality, survival and programming, such as Blimp-1, Bcl-6 and Bcl-2, was analyzed.
As shown in Fig. 2, mRNA levels of all five transcription factors measured were significantly increased in CD8+ T cells purified from PBLs activated in the presence of Tat. Notably, Tat up-regulated not only genes required for effector functions (as T-bet, Eomes and Blimp-1), but also transcription factors important for memory development (Bcl-6 and Eomes) and T-cell survival (Bcl-2). Tat did not significantly increase transcription factor expression in CD8+ T cells purified from unstimulated PBLs, although the results obtained show a tendency of a Tat-mediated enhancement of the two memory-related transcription factors Eomes and Bcl-6.
To assess whether the increased mRNA levels resulted in increased protein expression, T-bet and Eomes proteins were evaluated by western blotting in CD8+ T cells at 24 and 48 h after activation. As shown in Fig. 3a, CD8+ T cells purified from PBLs activated in the presence of Tat exhibited an increase of T-bet and Eomes expression 48 h after the activation.
Extracellular Tat is known to activate CD4+ T cells by binding with its Arg-Gly-Asp (RGD) region the αvβ3 and α5β1 integrins . To understand whether the enhancement of transcription factor expression induced by Tat was integrin-mediated, PBLs were preincubated with Abs directed against αvβ3 and α5β1 and subsequently activated with anti-CD3/CD28 in the absence or presence of Tat. As shown in Fig. 3b, transcription factor expression was not up-regulated by Tat in CD8+ T cells purified from PBLs activated in the presence of anti-integrin Abs, suggesting that the binding of Tat with αvβ3 and α5β1 may be required for the enhancement of transcription factor expression. However, we have to point out that preincubation with anti-integrin antibodies affected CD8+ T-cell activation induced by anti-CD3/CD28. In particular, integrin blocking prevented the mRNA level increase of Bcl-6 and Bcl-2 and down-modulated Eomes (compare Fig. 2 and Fig. 3b), but did not affect the increase of T-bet and Blimp-1 in activated CD8+ T cells. Thus, our results indicate that the Tat-mediated increase of T-bet and Blimp-1 is abolished by integrins blocking, whereas the role of the binding of Tat to integrins on the expression of Eomes, Bcl-6 and Bcl-2 remains to be elucidated.
Taken together, these data demonstrate that Tat favors the activation of CD8+ T cells affecting the expression of transcription factors crucial for T-cell programming and functionality.
Tat down-regulates CD127 expression and modulates T-cell fate
It has been recently shown that the expression of T-bet and Eomes in memory CD8+ T cells from HIV-infected individuals is associated with decreased expression of the IL-7 receptor CD127, and increased IFN-γ and granzyme B levels . As Tat up-regulates T-bet and Eomes (Figs. 2 and 3), as well as IFN-γ (Fig. 1), we then assessed whether Tat could affect CD127 expression in activated CD8+ T cells. Moreover, we also measured the expression of CD25, the alpha chain of the receptor for IL-2, whose production is modulated by Tat. As shown in Fig. 4a, activation of PBLs with anti-CD3/CD28 increased the expression of CD25 and decreased the expression of CD127 on CD8+ T cells. The presence of Tat did not affect the percentage of CD8+ T cells expressing CD25, whereas it decreased the fraction of CD8+ T lymphocytes expressing CD127. Interestingly, this effect was mediated by Tat in both unstimulated and activated CD8+ T cells.
As T-bet, Eomes, Bcl-6, Blimp-1 and Bcl-2 regulate at different extent the T-cell programming and memory development, we next sought to determine the fate of CD8+ T cells exposed for a longer time to Tat. To this aim, we evaluated the expression of the memory markers CD45RO and CD27 and of the exhaustion marker programmed cell death 1 (PD-1) in unstimulated or activated PBLs cultured for up to 8 days in the absence or presence of Tat. In long-term cell cultures, the presence of Tat did not modulate the expression of CD45RO (Fig. 4b) and PD-1 (Fig. S3, http://links.lww.com/QAD/A525), whereas it increased the expression of CD27 in activated but not in unstimulated CD8+ T cells (Fig. 4b). Interestingly, CD27 expression was not affected by Tat after 24 or 48 h of culture (not shown). These results suggest that the Tat-mediated modulation of T-bet, Eomes and the other transcription factors may be associated with the CD127 down-regulation and the accumulation of CD27+CD8+ T cells.
Tat favors the activation of antigen-specific naive and memory CD8+ T cells
We next assessed whether the presence of Tat in long-term cell cultures could also affect activation and functionality of antigen-specific memory and naive CD8+ T cells. To this aim, PBLs obtained from healthy human leukocyte antigen (HLA) class I-typed EBV-seropositive donors were stimulated ex vivo with cells pulsed with EBV-derived CTL peptide epitopes in the absence or presence of Tat. Specifically, PBLs were stimulated with the subdominant HLA-A2-restricted Cys-Leu-Gly-Gly-Leu-Leu-Thr-Met-Val (CLG) or Tyr-Leu-Gln-Gln-Asn-Trp-Trp-Thr-Leu (YLQ) epitope [45,46], or with the immunodominant HLA-A11-restricted Ile-Val-Thr-Asp-Phe-Ser-Val-Ile-Lys (IVT) epitope . The cytotoxic activity of each CTL culture generated in the absence or presence of Tat was tested against autologous phytohaemagglutinin (PHA) blasts, pulsed or not with the relevant synthetic peptide, in a standard 51Cr-release assay. As shown in Fig. 5a–c, all the three CTL cultures generated in the presence of Tat exhibited higher percentages of specific lysis compared to those generated in the absence of Tat.
To determine whether the Tat protein also favors the activation of naive T cells, PBLs from HLA-A2 healthy donors were stimulated with the synthetic Glu-Leu-Thr-Leu-Gly-Glu-Phe-Leu-Lys-Leu (ELT) peptide in the absence or presence of the Tat protein. The ELT peptide is a CTL epitope, presented by HLA-A2 [47,48], belonging to the antiapoptotic protein Survivin, which is overexpressed in tumor cells . No T-cell reactivity against this epitope is normally detected in healthy individuals . The specificity of CTL cultures was tested against PHA blasts, pulsed or not with the ELT peptide, by 51Cr-release assays (Fig. 5d). HLA-A2-positive PHA blasts pulsed with the ELT peptide were efficiently lyzed only by CTL cultures generated in the presence of Tat, demonstrating that Tat favors the priming of naive CD8+ T cells.
These observations suggest that Tat favors the activation of CD8+ T cells, but do not clarify whether the increased cytotoxic activity observed in CTL cultures generated in the presence of Tat depends on a higher number or a higher functionality of epitope-specific CD8+ T cells. To address this issue, CTL cultures specific for the HLA-A2-restricted CLG epitope were generated in the absence of Tat and were then left untreated or preincubated with the Tat protein 24/48 h before the cytotoxic activity. As shown in Fig. 5e, CTL cultures lyzed target cells at similar levels, suggesting that Tat does not enhance effector functions, but rather must be present at the time of the priming, thus favoring CTL expansion. To confirm this hypothesis, CLG and YLQ-specific CTLs generated in the absence or presence of Tat were assayed in IFN-γ and granzyme B Elispot assays to evaluate differences in the number of antigen-specific T cells. As shown in Fig. 5f, CTL cultures generated in the presence of Tat exhibited higher numbers of both IFN-γ and granzyme B CLG and YLQ-specific CTLs, suggesting that Tat favors the expansion of epitope-specific and actively secreting CD8+ T cells.
We demonstrate here that the HIV-1 Tat protein, which is released by infected cells and found extracellularly in HIV-positive individuals [18,39,40], favors the activation and effector functions of CD8+ T cells (Figs. 1 and 5). Interestingly, behind this functional modulation we observed that Tat increases the expression of T-bet, Eomes, Blimp-1, Bcl-6 and Bcl-2 in activated but not in unstimulated CD8+ T lymphocytes (Fig. 2), leading to the down-regulation of CD127 and the up-regulation of CD27 (Fig. 4). The Tat-mediated increase of T-bet and Blimp-1 require the binding of Tat to integrins (Fig. 3). Thus, these results are indicating that the programming and functionality of CD8+ T cells are deeply altered by the amount of Tat close to the concentrations measured in HIV-infected individuals. Indeed, it has been demonstrated that the plasma of HIV-positive patients may contain up to 40 ng/ml of soluble Tat [39,41], value which probably reaches higher concentrations in tissues where Tat is sequestered by glycosaminoglycans and heparan sulfate proteoglycans of the extracellular matrix [18,41]. Moreover, it has been proposed that Tat continues to be secreted even during HAART , as confirmed by the immune restoration observed after the induction of anti-Tat immunity in HIV-infected HAART-treated individuals .
It is known that Tat favors IL-2 secretion in CD4+ T cells . Here we show for the first time that CD8+ T cells activated in the presence of Tat also exhibited increased production of IL-2 (Fig. 1). Several mechanisms may account for this effect, as it has been reported that Tat favors the activation of transcription factors required for IL-2 transcription, like nuclear factor kappa-light-chain-enhancer of activated B cells [13,14], nuclear factor of activated T-cells  and activator protein 1 . Moreover, Tat superinduces factors binding to the CD28-responsive element (CD28RE), which mediates IL-2 gene activation by CD28 costimulation [13,14].
The results also demonstrate that naive and memory CD8+ T cells activated in vitro in the presence of Tat exhibit an increased IFN-γ production and cytotoxic activity (Figs. 1 and 5). The effect was abolished when Tat was added after the stimulation, suggesting that Tat favors the expansion and the functionality of effector cells only if present at the beginning of the stimulation. It is likely that Tat potentiates the production of cytokines and cytolytic molecules through the induction of T-bet, Eomes and Blimp-1 (Fig. 2), which control at different levels the transcription of IFN-γ, perforins and granzymes [30,43,52]. The interaction of the RGD domain of Tat with αvβ3 and α5β1 integrins seems to be necessary, as we observed that the Tat-mediated up-regulation of the T-bet and Blimp-1 was abolished by integrin blocking. It is known that Tat mediates the activation of the extracellular signal-regulated kinases (ERK)/mitogen-activated protein kinase and phosphoinositide 3-kinase/RAC-alpha serine/threonine-protein kinase (Akt) pathways through its RGD domain [53,54], and both ERK and Akt are involved in T-bet induction [55,56]. Moreover, the ERK pathway also favors Eomes and Blimp-1 up-regulation [57,58].
Of note, an increased production of effector molecules like IFN-γ and granzymes, as well as an enhancement of T-bet and Eomes in CD8+ T cells, is observed in HIV-infected individuals [29,59–62]. Thus, our in-vitro observations suggest that Tat may be responsible for, or contribute to, all these effects in vivo.
The role of Tat on T-cell survival is highly debated [63–66]. We report that Tat enhances the expression of the antiapoptotic marker Bcl-2 in activated CD8+ T cells. However, the up-regulation of Bcl-2 does not appear to be due to a direct effect of Tat on Bcl-2 expression, as instead demonstrated in CD4+ T cells , since it was observed after activation with anti-CD3/CD28 and Tat further increased it. Thus, our results indicate that Tat may differently affect Bcl-2 expression in CD4+ and CD8+ T cells. Interestingly, the presence of Tat did not modulate PD-1 expression (Fig. S3, http://links.lww.com/QAD/A525), a marker of exhaustion up-regulated in HIV-specific CD8+ T cells which poorly control the infection [67,68].
We found that CD8+ T cells activated in the presence of Tat exhibited increased levels of Blimp-1, which favors the development of effector memory T cells [34,52]. Intriguingly, we also observed the up-regulation of Bcl-6, which promotes the development of a central memory phenotype and is repressed by Blimp-1 , suggesting a Tat-mediated mechanism that deserves further investigations. Moreover, the presence of Tat favors the expression of CD27, a hallmark of incomplete differentiation to effector cells [10,69,70], and causes CD127 down-regulation not only in unstimulated CD8+ T cells, as previously demonstrated [71,72], but also in activated CD8+ T lymphocytes. Interestingly, CD127 down-regulation is observed in HIV-infected individuals in association with immune activation [73,74], higher levels of T-bet and Eomes, and increased granzyme B and IFN-γ release . In conclusion, our results indicate that Tat modulates programming and secretory capacity of CD8+ T cells, suggesting that it may be involved in the development of CD8+ T lymphocytes with an effector profile as observed during HIV infection [2,29]. We propose a model by which HIV, through the release of Tat, may affect T-bet and Eomes expression, thus contributing to immune activation and to a profound and long-lasting modulation of CD8+ T-cell responses. Thus, our observations provide new hints on the role that Tat may play in CD8+ T-cell dysfunctionalities during HIV infection, suggesting that the induction anti-Tat immune responses may be a valuable tool to protect HIV-infected individuals from immune dysfunctions.
F.S., F.N., and R.G. conceived and designed the experiments and analyzed the data. F.S., F.N., E.G., V.F., and E.R. performed the experiments. F.S., F.N., A.C., A.C., B.E., and R.G. wrote the manuscript.
We would like to thank Mirko Pinotti, Silvia Cellini, Matteo Rosa and Nicola Cavallari for technical assistance.
Funding: This work was supported by grants from the University of Ferrara and by the Gilead Fellowship Program.
Conflicts of interest
F.S. was partially supported by the ‘Carlo Fornasini’ Foundation. F.N. was partially supported by the Italian Center of Biotechnology (CIB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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CD8+; Eomes; HIV; immune activation; T-cell programming; Tat; T-bet
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