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MHC-E restricted CD8 T cells show promise in vaccine settings, but their development and specificity remain poorly understood. Here we focus on a CD8 T cell population reactive to a self-peptide (FL9) bound to mouse MHC-E (Qa-1b ) that is presented in response to loss of the MHC I processing enzyme ERAAP, termed QFL T cells. We find that mature QFL thymocytes are predominantly CD8αβ+CD4-, show signs of agonist selection, and give rise to both CD8αα and CD8αβ intraepithelial lymphocytes (IEL), as well as memory phenotype CD8αβ T cells. QFL T cells require the MHC I subunit β-2 microglobulin (β2m), but do not require Qa1b or classical MHC I for positive selection. However, QFL thymocytes do require Qa1b for agonist selection and full functionality. Our data highlight the relaxed requirements for positive selection of an MHC-E restricted T cell population and suggest a CD8αβ+CD4- pathway for development of CD8αα IELs. [ABSTRACT FROM AUTHOR]

Recognition and elimination of tumor cells by the immune system is crucial for limiting tumor growth. Natural killer (NK) cells become activated when the receptor NKG2D is engaged by ligands that are frequently upregulated in primary tumors and on cancer cell lines. However, the molecular mechanisms driving NKG2D ligand expression on tumor cells are not well defined. Using a forward genetic screen in a tumor-derived human cell line, we identified several novel factors supporting expression of the NKG2D ligand ULBP1. Our results show stepwise contributions of independent pathways working at multiple stages of ULBP1 biogenesis. Deeper investigation of selected hits from the screen showed that the transcription factor ATF4 drives ULBP1 gene expression in cancer cell lines, while the RNA-binding protein RBM4 supports ULBP1 expression by suppressing a novel alternatively spliced isoform of ULBP1 mRNA. These findings offer insight into the stress pathways that alert the immune system to danger. [ABSTRACT FROM AUTHOR]

Lytic gammaherpesvirus infection restricts host gene expression by promoting widespread degradation of cytoplasmic mRNA through the activity of the viral endonuclease SOX. Though generally assumed to be selective for cellular transcripts, the extent to which SOX impacts viral mRNA stability has remained unknown. We addressed this issue using the model murine gammaherpesvirus MHV68 and, unexpectedly, found that all stages of viral gene expression are controlled through mRNA degradation. Using both comprehensive RNA expression profiling and half-life studies we reveal that the levels of the majority of viral mRNAs but not noncoding RNAs are tempered by MHV68 SOX (muSOX) activity. The targeting of viral mRNA by muSOX is functionally significant, as it impacts intracellular viral protein abundance and progeny virion composition. In the absence of muSOX-imposed gene expression control the viral particles display increased cell surface binding and entry as well as enhanced immediate early gene expression. These phenotypes culminate in a viral replication defect in multiple cell types as well as in vivo, highlighting the importance of maintaining the appropriate balance of viral RNA during gammaherpesviral infection. This is the first example of a virus that fails to broadly discriminate between cellular and viral transcripts during host shutoff and instead uses the targeting of viral messages to fine-tune overall gene expression. [ABSTRACT FROM AUTHOR]

Activation-induced cytidine deaminase (AID) is specifically induced in germinal center B cells to carry out somatic hypermutation and class-switch recombination, two processes responsible for antibody diversification. Because of its mutagenic potential, AID expression and activity are tightly regulated to minimize unwanted DNA damage. Surprisingly, AID expression has been observed ectopically during pathogenic infections. However, the function of AID outside of the germinal centers remains largely uncharacterized. In this study, we demonstrate that infection of human primary naïve B cells with Kaposi's sarcoma-associated herpesvirus (KSHV) rapidly induces AID expression in a cell intrinsic manner. We find that infected cells are marked for elimination by Natural Killer cells through upregulation of NKG2D ligands via the DNA damage pathway, a pathway triggered by AID. Moreover, without having a measurable effect on KSHV latency, AID impinges directly on the viral fitness by inhibiting lytic reactivation and reducing infectivity of KSHV virions. Importantly, we uncover two KSHV-encoded microRNAs that directly regulate AID abundance, further reinforcing the role for AID in the antiviral response. Together our findings reveal additional functions for AID in innate immune defense against KSHV with implications for a broader involvement in innate immunity to other pathogens. [ABSTRACT FROM AUTHOR]

The endoplasmic reticulum-associated degradation (ERAD) pathway is responsible for disposing misfolded proteins from the endoplasmic reticulum by inducing their ubiquitination and degradation. Ubiquitination is conventionally observed on lysine residues and has been demonstrated on cysteine residues and protein N-termini. Ubiquitination is fundamental to the ERAD process; however, a mutant T-cell receptor α (TCRα) lacking lysine residues is targeted for the degradation by the ERAD pathway. We have shown that ubiquitination of lysine-less TCRα occurs on internal, non-lysine residues and that the same E3 ligase conjugates ubiquitin to TCRα in the presence or absence of lysine residues. Mass-spectrometry indicates that WT-TCRα is ubiquitinated on multiple lysine residues. Recent publications have provided indirect evidence that serine and threonine residues may be modified by ubiquitin. Using a novel peptide-based stable isotope labeling in cell culture (SILAC) approach, we show that specific lysine-less TCRα peptides become modified. In this study, we demonstrate that it is possible to detect both ester and thioester based ubiquitination events, although the exact linkage on lysine-less TCRα remains elusive.These findings demonstrate that SILAC can be used as a tool to identify modified peptides, even those with novel modifications that may not be detected using conventional proteomic work flows or informatics algorithms. [ABSTRACT FROM AUTHOR]

Natural killer (NK) cells are lymphocytes that play a major role in the elimination of virally-infected cells and tumor cells. NK cells recognize and target abnormal cells through activation of stimulatory receptors such as NKG2D. NKG2D ligands are self-proteins, which are absent or expressed at low levels on healthy cells but are induced upon cellular stress, transformation, or viral infection. The exact molecular mechanisms driving expression of these ligands remain poorly understood. Here we show that murine cytomegalovirus (MCMV) infection activates the phosphatidylinositol-3-kinase (PI3K) pathway and that this activation is required for the induction of the RAE-1 family of mouse NKG2D ligands. Among the multiple PI3K catalytic subunits, inhibition of the p110α catalytic subunit blocks this induction. Similarly, inhibition of p110α PI3K reduces cell surface expression of RAE-1 on transformed cells. Many viruses manipulate the PI3K pathway, and tumors frequently mutate the p110α oncogene. Thus, our findings suggest that dysregulation of the PI3K pathway is an important signal to induce expression of RAE-1, and this may represent a commonality among various types of cellular stresses that result in the induction of NKG2D ligands. [ABSTRACT FROM AUTHOR]

During a lytic gammaherpesvirus infection, host gene expression is severely restricted by the global degradation and altered 3' end processing of mRNA. This host shutoff phenotype is orchestrated by the viral SOX protein, yet its functional significance to the viral lifecycle has not been elucidated, in part due to the multifunctional nature of SOX. Using an unbiased mutagenesis screen of the murine gammaherpesvirus 68 (MHV68) SOX homolog, we isolated a single amino acid point mutant that is selectively defective in host shutoff activity. Incorporation of this mutation into MHV68 yielded a virus with significantly reduced capacity for mRNA turnover. Unexpectedly, the MHV68 mutant showed little defect during the acute replication phase in the mouse lung. Instead, the virus exhibited attenuation at later stages of in vivo infections suggestive of defects in both trafficking and latency establishment. Specifically, mice intranasally infected with the host shutoff mutant accumulated to lower levels at 10 days post infection in the lymph nodes, failed to develop splenomegaly, and exhibited reduced viral DNA levels and a lower frequency of latently infected splenocytes. Decreased latency establishment was also observed upon infection via the intraperitoneal route. These results highlight for the first time the importance of global mRNA degradation during a gammaherpesvirus infection and link an exclusively lytic phenomenon with downstream latency establishment. [ABSTRACT FROM AUTHOR]

To efficiently establish a persistent infection, Kaposi's sarcoma-associated herpesvirus (KSHV; also known as HHV8) dedicates a large amount of its coding potential to produce proteins that antagonize the immune system of its host. These viral immunomodulators interfere with both the innate and adaptive immune responses and most of them are homologous to cellular proteins, suggesting that they have been pirated from the host during viral evolution. In this Review, I present recent advances in the understanding of immune evasion by KSHV, with a particular focus on the virally encoded modulators of immune responses that are unique to this virus. [ABSTRACT FROM AUTHOR]

Classifies the HTLV-I transgenic models according to tissue tropism controlled by an HTLV-I long terminal repeat (LTR), pathogenesis linked to HTLV-I gene expression and therapeutic approaches. Description of the cell types able to support the viral expression in vivo; Use of Tax-transgenic mice to demonstrate that the Tax protein is oncogenic and able to induce muscular atrophy and arthropathies.

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes two RING finger E3 ubiquitin ligases (MIR1 and MIR2) that mediate ubiquitination and degradation of cellular proteins important for the establishment of an efficient antiviral immune response. MIR1 and MIR2 share 30% sequence identity; however, their substrate preferences are varied. MIR1 has been shown to primarily downregulate major histocompatibility complex class I (MHC-I), whereas MIR2 can downregulate a wide range of cell surface proteins. Many of the MIR substrates are thought to be present in lipid raft microdomains, a subregion of the plasma membrane known to be important for a wide range of signal transduction events. Palmitoylation is a posttranslational modification that increases recruitment of transmembrane proteins to lipid rafts. In this study, we investigated the importance of palmitoylation for MIR function. We present evidence that MIR2-mediated downregulation of MHC-I and platelet endothelial cell adhesion molecule 1 (PECAM-1) but not other substrates is inhibited in the presence of the drug 2-bromohexadecanoic acid (2-Br), a chemical inhibitor of palmitoylation. Biochemical analysis indicates that MIR2 is directly palmitoylated on cysteine 146. Mutation of this cysteine to a phenylalanine prevents MIR2 palmitoylation and blocks the ability of MIR2 to downregulate MHC-I and PECAM-I but not B7.2 and intercellular adhesion molecule 1 (ICAM-I), consistent with the phenotype observed after 2-Br treatment. Unpalmitoylated MIR2 does not interact with MHC-I and is thus unable to ubiquitinate and downregulate MHC-I from the cell surface. Furthermore, we observed that MIR2 is palmitoylated in vivo during lytic infection. Palmitoylation may act to regulate MIR2 function and localization during viral infection by allowing MIR2 to properly interact with and downregulate multiple substrates known to play an important role in the host immune response. [ABSTRACT FROM AUTHOR]

Modulation of T-cell receptor expression and signaling is essential to the survival of many viruses. The U24 protein expressed by human herpesvirus 6A, a ubiquitous human pathogen, has been previously shown to downregulate the T-cell receptor. Here, we show that U24 also mediates cell surface downregulation of a canonical early endosomal recycling receptor, the transferrin receptor, indicating that this viral protein acts by blocking early endosomal recycling. We present evidence that U24 is a C-tail-anchored protein that is dependent for its function on TRC40/Asna-1, a component of a posttranslational membrane insertion pathway. Finally, we find that U24 proteins from other roseoloviruses have a similar genetic organization and a conserved function that is dependent on a proline-rich motif. Inhibition of a basic cellular process by U24 has interesting implications not only for the pathogenicity of roseoloviruses but also for our understanding of the biology of endosomal transport. [ABSTRACT FROM AUTHOR]

Kaposi's sarcoma-associated herpesvirus encodes two homologous E3 ligases, MIR1 and MIR2, that mediate the ubiquitination and subsequent downregulation of several cell surface proteins, and in particular major histocompatibility complex class I (MHC-I) molecules. We have previously shown that, in addition to lysine ubiquitination, MIR1 has the unique ability of transferring ubiquitin onto MHC-I molecules lacking available lysine residues, in a cysteine-dependent manner. Here we report that MIR1 activity is maximal when either a lysine or cysteine residue is placed approximately 15 amino acids away from the transmembrane domain, whereas MIR2 preferentially targets residues, including cysteines, that are closer to the transmembrane domain. Thus MIR1 and -2 can distinguish their substrates based on the position of the lysine or cysteine residues, suggesting that these proteins have evolved to target different sets of surface molecules. These results indicate that the position of target residues within a substrate is an essential determinant of E3 ubiquitin ligase specificity. [ABSTRACT FROM AUTHOR]

We have performed a screen aimed at identifying human herpesvirus 6 (HHV-6)-encoded proteins that modulate immune recognition. Here we show that the U24 protein encoded by HHV-6 variant A downregulates cell surface expression of the T-cell receptor (TCR)/CD3 complex, a complex essential to T-cell activation and the generation of an immune adaptive response. In the presence of U24, the TCR/CD3 complex is endocytosed but is not recycled back to the plasma membrane. Instead, it accumulates in early and late endosomes. Interestingly, whereas CD3 downregulation from the cell surface is normally associated with T-cell activation, U24 downregulates CD3 independently of T-cell activation. Moreover, we found that U24-expressing T cells are resistant to activation by antigen-presenting cells. HHV-6 has evolved a unique mechanism of inhibition of T-cell activation that may impair the establishment of an adaptive immune response. Furthermore, lymphocyte activation creates an environment favorable to the reactivation and replication of lymphotropic herpesviruses. Thus, by inhibiting T-cell activation, HHV-6 might limit its reactivation and thus minimize immune recognition. [ABSTRACT FROM AUTHOR]

Kaposi's sarcoma-associated herpesvirus (KSHV) and its murine homolog, murine gammaherpesvirus 68 (MHV68), are lymphotropic viruses that establish latent infection in their host. Surprisingly, while B cells are the main viral reservoir in vivo, B-cell lines are poorly permissive to infection by either MHV68 or KSHV. Here, we report that most B-cell lines express very little to no cell surface heparan sulfate (HS), a glycosaminoglycan that is essential for infection by these viruses. We found that Ext1, a key enzyme in the biosynthesis of HS, was expressed at a low level in these cells. Transfection of B-cell lines with Ext1 restored high HS expression at the cell surface. Overexpression of Ext1 in murine A20 and M12 B-cell lines increased MHV68 surface binding and enhanced the efficiency of infection. Finally, although it was not sufficient to allow efficient infection, the expression of HS on BJAB cells promoted KSHV binding at the cell surface. Thus, our results indicate that MHV68 and KSHV cycles are blocked in B-cell lines at the binding step due to a lack of surface HS. [ABSTRACT FROM AUTHOR]

Host receptor usage by KSHV has been best studied using primary microvascular endothelial and fibroblast cells, although the virus infects a wide variety of cell types in culture and in natural infections. In these two infection models, KSHV adheres to the cell though heparan sulfate (HS) binding, then interacts with a complex of EphA2, xct, and integrins α3β1, αVβ3, αVβ5 to catalyze viral entry. We dissected this receptor complex at the genetic level with CRISPR-Cas9 to precisely determine receptor usage in two epithelial cell lines. Surprisingly, we discovered an infection mechanism that requires HS and EphA2 but is independent of αV- and β1-family integrin expression. Furthermore, infection appears to be independent of the EphA2 intracellular domain. We also demonstrated that while two other endogenous Eph receptors were dispensable for KSHV infection, transduced EphA4 and EphA5 significantly enhanced infection of cells lacking EphA2. IMPORTANCE Our data reveals an integrin-independent route of KSHV infection and suggests that multiple Eph receptors besides EphA2 can promote and regulate infection. Since integrins and Eph receptors are large protein families with diverse expression patterns across cells and tissues, we propose that KSHV may engage with several proteins from both families in different combinations to negotiate successful entry into diverse cell types. [ABSTRACT FROM AUTHOR]

Cell surface heparan sulfate (HS) is an important coreceptor for many cytokines, chemokines, and growth factors. In this study, we report that splenic murine B cells express very little HS and that upon infection with either gammaherpesvirus (murine gammaherpesvirus 68) or betaherpesvirus (murine cytomegalovirus), HS is rapidly upregulated at the surface of B cells. HS upregulation was not observed in mice deficient for the type I IFN (IFN-I) receptor. Additionally, treatment of wild-type mice with the IFN-I inducer polyinosine polycytidylic acid triggered HS expression at the B cell surface. Similarly, incubation of purified splenic B cells with IFN-I, TLR ligands, or BCR stimulators ex vivo resulted in a drastic increase in HS surface expression..We found that IFN-I induced an increase in the surface expression of HS-modified syndecan 4 as well as that of an unidentified heparan sulfate proteoglycan. Finally, IFN-I treatment increased B cell responsiveness to APRIL, a cytokine involved in B cell survival and T cellindependent B cell responses. Enzymatic removal of HS from IFN-I-treated B cells inhibited APRIL. Altogether, our results indicate that upon herpesvirus infection in mice, HS is rapidly upregulated at the surface of B cells due to the action of IFN-I, potentially increasing B cell responsiveness to cytokines. Induction of HS expression at the B cell surface by stimulators of the innate immune response likely plays a key role in the development of a robust immune response. [ABSTRACT FROM AUTHOR]

Multiple receptors within the innate immune system have evolved to recognize nucleic acids as signatures of viral infection. It is believed that this specificity is essential for viral detection, as viruses often lack other invariant features that can serve as suitable targets for innate receptors. One such innate receptor, TLR9, has been implicated in the detection of many dsDNA viruses. In this study, we investigate the detection of murine gammaherpesvirus 68 (MHV68) by TLR9. We find that the genomic DNA of the murine CMV, a very potent inducer of innate responses. Genome-wide analysis of the number of stimulatory versus nonstimulatory CpG motifs present in the genome of each virus reveals that the MHV68 genome contains only a fraction of the number of immunostimulatory motifs present in murine CMV. Notably, MHV68 appears to have selectively suppressed the number of stimulatory motifs through cytosine to thymine conversion. These data suggest that certain viruses may have evolved and modified their genomic content to avoid recognition by nucleic acid-sensing receptors of the innate immune system. [ABSTRACT FROM AUTHOR]

MHC-E restricted CD8 T cells show promise in vaccine settings, but their development and specificity remain poorly understood. Here we focus on a CD8 T cell population reactive to a self-peptide (FL9) bound to mouse MHC-E (Qa-1 b ) that is presented in response to loss of the MHC I processing enzyme ERAAP, termed QFL T cells. We find that mature QFL thymocytes are predominantly CD8αβ+CD4-, show signs of agonist selection, and give rise to both CD8αα and CD8αβ intraepithelial lymphocytes (IEL), as well as memory phenotype CD8αβ T cells. QFL T cells require the MHC I subunit β-2 microglobulin (β2m), but do not require Qa1 b or classical MHC I for positive selection. However, QFL thymocytes do require Qa1 b for agonist selection and full functionality. Our data highlight the relaxed requirements for positive selection of an MHC-E restricted T cell population and suggest a CD8αβ+CD4- pathway for development of CD8αα IELs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Manoharan Valerio, Arana, Guan, Chan, Yang, Kurd, Lee, Shastri, Coscoy and Robey.)

Endoplasmic reticulum (ER) aminopeptidase associated with antigen processing (ERAAP) trims peptide precursors in the ER for presentation by major histocompatibility (MHC)-I molecules to surveying CD8 + T-cells. This function allows ERAAP to regulate the nature and quality of the peptide repertoire and, accordingly, the resulting immune responses. We recently showed that infection with murine cytomegalovirus leads to a dramatic loss of ERAAP levels in infected cells. In mice, this loss is associated with the activation of QFL T-cells, a subset of T-cells that monitor ERAAP integrity and eliminate cells experiencing ERAAP dysfunction. In this study, we aimed to identify host factors that regulate ERAAP expression level and determine whether these could be manipulated during viral infections. We performed a CRISPR knockout screen and identified ERp44 as a factor promoting ERAAP retention in the ER. ERp44's interaction with ERAAP is dependent on the pH gradient between the ER and Golgi. We hypothesized that viruses that disrupt the pH of the secretory pathway interfere with ERAAP retention. Here, we demonstrate that expression of the Envelope (E) protein from Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) leads to Golgi pH neutralization and consequently decrease of ERAAP intracellular levels. Furthermore, SARS-CoV-2-induced ERAAP loss correlates with its release into the extracellular environment. ERAAP's reliance on ERp44 and a functioning ER/Golgi pH gradient for proper localization and function led us to propose that ERAAP serves as a sensor of disturbances in the secretory pathway during infection and disease., Competing Interests: Declaration of interests The authors declare no competing interests.

The persistence of the COVID-19 pandemic demands a dramatic increase in testing efficiency. Testing pooled samples for SARS-CoV-2 could meet this need; however, the sensitivity of RT-qPCR, the gold standard, significantly decreases with an increasing number of samples pooled. Here, we introduce DIVER, a method that quantifies intact virus and is robust to sample dilution. DIVER first tags viral particles with exogeneous oligonucleotides, then captures the tagged particles on ACE2-functionalized beads, and finally quantifies the oligonucleotide tags using qPCR. Using spike-presenting liposomes and Spike-pseudotyped lentivirus as SARS-CoV-2 models, we show that DIVER can detect 1×10 5 liposomes and 100 pfu lentivirus and can successfully identify positive samples in pooling experiments. Overall, DIVER is well-positioned for efficient sample pooling and expanded community surveillance.

Necroptosis is a form of necrotic cell death that requires the activity of the death domain-containing kinase RIP1 and its family member RIP3. Necroptosis occurs when RIP1 is deubiquitinated to form a complex with RIP3 in cells deficient in the death receptor adapter molecule FADD or caspase-8. Necroptosis may play a role in host defense during viral infection as viruses like vaccinia can induce necroptosis while murine cytomegalovirus encodes a viral inhibitor of necroptosis. To see how general the interplay between viruses and necroptosis is, we surveyed seven different viruses. We found that two of the viruses tested, Sendai virus (SeV) and murine gammaherpesvirus-68 (MHV68), are capable of inducing dramatic necroptosis in the fibrosarcoma L929 cell line. We show that MHV68-induced cell death occurs through the cytosolic STING sensor pathway in a TNF-dependent manner. In contrast, SeV-induced death is mostly independent of TNF. Knockdown of the RNA sensing molecule RIG-I or the RIP1 deubiquitin protein, CYLD, but not STING, rescued cells from SeV-induced necroptosis. Accompanying necroptosis, we also find that wild type but not mutant SeV lacking the viral proteins Y1 and Y2 result in the non-ubiquitinated form of RIP1. Expression of Y1 or Y2 alone can suppress RIP1 ubiquitination but CYLD is dispensable for this process. Instead, we found that Y1 and Y2 can inhibit cIAP1-mediated RIP1 ubiquitination. Interestingly, we also found that SeV infection of B6 RIP3 -/- mice results in increased inflammation in the lung and elevated SeV-specific T cells. Collectively, these data identify viruses and pathways that can trigger necroptosis and highlight the dynamic interplay between pathogen-recognition receptors and cell death induction.

NKG2D is a stimulatory receptor expressed by NK cells and some T cell subsets. Expression of the self-encoded ligands for NKG2D is presumably tightly regulated to prevent autoimmune disorders while allowing detection of infected cells and developing tumors. The NKG2D ligand Mult1 is regulated at multiple levels, with a final layer of regulation controlling protein stability. In this article, we report that Mult1 cell-surface expression was prevented by two closely related E3 ubiquitin ligases membrane-associated RING-CH (MARCH)4 and MARCH9, members of an E3 family that regulates other immunologically active proteins. Lysines within the cytoplasmic domain of Mult1 were essential for this repression by MARCH4 or MARCH9. Downregulation of Mult1 by MARCH9 was reversed by heat-shock treatment, which resulted in the dissociation of the two proteins and increased the amount of Mult1 at the cell surface. These results identify Mult1 as a target for the MARCH family of E3 ligases and show that induction of Mult1 in response to heat shock is due to regulated association with its E3 ligases.

NKG2D is a major stimulatory receptor expressed by natural killer (NK) cells and some T cells. The receptor recognizes major histocompatibility complex class I-like cell surface ligands that are poorly expressed by normal tissues but are often induced in transformed and infected cells. The existence of several NKG2D ligands in each individual, some with strikingly divergent protein sequences, raises the possibility that different ligands are regulated by distinct disease-associated stresses. The transcripts for some ligands, including murine UL16-binding proteinlike transcript 1 (Mult1), are abundant in certain normal tissues where cell surface expression is absent, suggesting the existence of translational or posttranslational regulation. We report here that under normal conditions, Mult1 protein undergoes ubiquitination dependent on lysines in its cytoplasmic tail and lysosomal degradation. Mult1 degradation and ubiquitination is reduced in response to stress imparted by heat shock or ultraviolet irradiation, but not by other forms of genotoxicity, providing a novel mechanism for stress-mediated cellular control of NKG2D ligand expression.