Liver Restores Immune Homeostasis after Local Inflammation despite the Presence of Autoreactive T Cells

The liver must keep equilibrium between immune tolerance and immunity in order to protect itself from pathogens while maintaining tolerance to food antigens. An imbalance between these two states could result in an inflammatory liver disease. The aims of this study were to identify factors responsible for a break of tolerance and characterize the subsequent restoration of liver immune homeostasis. A pro-inflammatory environment was created in the liver by the co-administration of TLR ligands CpG and Poly(I:C) in presence or absence of activated liver-specific autoreactive CD8⁺ T cells. Regardless of autoreactive CD8⁺ T cells, mice injected with CpG and Poly(I:C) showed elevated serum ALT levels and a transient liver inflammation. Both CpG/Poly(I:C) and autoreactive CD8⁺T cells induced expression of TLR9 and INF-γ by the liver, and an up-regulation of homing and adhesion molecules CXCL9, CXCL10, CXCL16, ICAM-1 and VCAM-1. Transferred CFSE-labeled autoreactive CD8⁺ T cells, in presence of TLR3 and 9 ligands, were recruited by the liver and spleen and proliferated. This population then contracted by apoptosis through intrinsic and extrinsic pathways. Up-regulation of FasL and PD-L1 in the liver was observed. In conclusion, TLR-mediated activation of the innate immune system results in a pro-inflammatory environment that promotes the recruitment of lymphocytes resulting in bystander hepatitis. Despite this pro-inflammatory environment, the presence of autoreactive CD8⁺ T cells is not sufficient to sustain an autoimmune response against the liver and immune homeostasis is rapidly restored through the apoptosis of T cells.     

Parasite Fate and Involvement of Infected Cells in the Induction of CD4+ and CD8+ T Cell Responses to Toxoplasma gondii

During infection with the intracellular parasite Toxoplasma gondii, the presentation of parasite-derived antigens to CD4⁺ and CD8⁺ T cells is essential for long-term resistance to this pathogen. Fundamental questions remain regarding the roles of phagocytosis and active invasion in the events that lead to the processing and presentation of parasite antigens. To understand the most proximal events in this process, an attenuated non-replicating strain of T. gondii (the cpsII strain) was combined with a cytometry-based approach to distinguish active invasion from phagocytic uptake. In vivo studies revealed that T. gondii disproportionately infected dendritic cells and macrophages, and that infected dendritic cells and macrophages displayed an activated phenotype characterized by enhanced levels of CD86 compared to cells that had phagocytosed the parasite, thus suggesting a role for these cells in priming naïve T cells. Indeed, dendritic cells were required for optimal CD4⁺ and CD8⁺ T cell responses, and the phagocytosis of heat-killed or invasion-blocked parasites was not sufficient to induce T cell responses. Rather, the selective transfer of cpsII-infected dendritic cells or macrophages (but not those that had phagocytosed the parasite) to naïve mice potently induced CD4⁺ and CD8⁺ T cell responses, and conferred protection against challenge with virulent T. gondii. Collectively, these results point toward a critical role for actively infected host cells in initiating T. gondii-specific CD4⁺ and CD8⁺ T cell responses.     

Control of Simian Immunodeficiency Virus Replication by Vaccine-Induced Gag- and Vif-Specific CD8+ T Cells

For development of an effective T cell-based AIDS vaccine, it is critical to define the antigens that elicit the most potent responses. Recent studies have suggested that Gag-specific and possibly Vif/Nef-specific CD8⁺ T cells can be important in control of the AIDS virus. Here, we tested whether induction of these CD8⁺ T cells by prophylactic vaccination can result in control of simian immunodeficiency virus (SIV) replication in Burmese rhesus macaques sharing the major histocompatibility complex class I (MHC-I) haplotype 90-010-Ie associated with dominant Nef-specific CD8⁺ T-cell responses. In the first group vaccinated with Gag-expressing vectors (n = 5 animals), three animals that showed efficient Gag-specific CD8⁺ T-cell responses in the acute phase postchallenge controlled SIV replication. In the second group vaccinated with Vif- and Nef-expressing vectors (n = 6 animals), three animals that elicited Vif-specific CD8⁺ T-cell responses in the acute phase showed SIV control, whereas the remaining three with Nef-specific but not Vif-specific CD8⁺ T-cell responses failed to control SIV replication. Analysis of 18 animals, consisting of seven unvaccinated noncontrollers and the 11 vaccinees described above, revealed that the sum of Gag- and Vif-specific CD8⁺ T-cell frequencies in the acute phase was inversely correlated with plasma viral loads in the chronic phase. Our results suggest that replication of the AIDS virus can be controlled by vaccine-induced subdominant Gag/Vif epitope-specific CD8⁺ T cells, providing a rationale for the induction of Gag- and/or Vif-specific CD8⁺ T-cell responses by prophylactic AIDS vaccines.     

Broadening of Virus-Specific CD8+ T-Cell Responses Is Indicative of Residual Viral Replication in Aviremic SIV Controllers

Control of HIV replication is a rare immunological event, providing clues to understand the viral control mechanism. CD8⁺ T-cell responses are crucial for virus control, but it is unclear whether lasting HIV containment can be achieved after establishment of infection. Here, we describe lasting SIV containment in a macaque AIDS model. Analysis of ten rhesus macaques that controlled viremia for 2 years post-infection found accumulation of proviral gag and nef CD8⁺ T-cell escape mutations in four of them. These four controllers mounted CD8⁺ T cells targeting Gag, Nef, and other viral proteins at 4 months, suggesting that broadening of CD8⁺ T-cell targets can be an indicator of the beginning of viral control failure. The remaining six aviremic SIV controllers, however, harbored proviruses without mutations and showed no or little broadening of their CD8⁺ T-cell responses in the chronic phase. Indeed, three of the latter six exhibiting no change in CD8⁺ T-cell targets showed gradual decreases in SIV-specific CD8⁺ T-cell frequencies, implying a concomitant reduction in viral replication. Thus, stability of the breadth of virus-specific CD8⁺ T-cell responses may represent a status of lasting HIV containment by CD8⁺ T cells.     

Anti‐inflammatory and immune‐modulatory impacts of berberine on activation of autoreactive T cells in autoimmune inflammation

Autoreactive inflammatory CD4⁺ T cells, such as T helper (Th)1 and Th17 subtypes, have been found to associate with the pathogenesis of autoimmune disorders. On the other hand, CD4⁺ Foxp3⁺ T regulatory (Treg) cells are crucial for the immune tolerance and have a critical role in the suppression of the excessive immune and inflammatory response promoted by these Th cells. In contrast, dendritic cells (DCs) and macrophages are immune cells that through their inflammatory functions promote autoreactive T‐cell responses in autoimmune conditions. In recent years, there has been increasing attention to exploring effective immunomodulatory or anti‐inflammatory agents from the herbal collection of traditional medicine. Berberine, an isoquinoline alkaloid, is one of the main active ingredients extracted from medicinal herbs and has been shown to exert various biological and pharmacological effects that are suggested to be mainly attributed to its anti‐inflammatory and immunomodulatory properties. Several lines of experimental study have recently investigated the therapeutic potential of berberine for treating autoimmune conditions in animal models of human autoimmune diseases. Here, we aimed to seek mechanisms underlying immunomodulatory and anti‐inflammatory effects of berberine on autoreactive inflammatory responses in autoimmune conditions. Reported data reveal that berberine can directly suppress functions and differentiation of pro‐inflammatory Th1 and Th17 cells, and indirectly decrease Th cell‐mediated inflammation through modulating or suppressing other cells assisting autoreactive inflammation, such as Tregs, DCs and macrophages.     

Hepatitis B Virus-Infected HepG2hNTCP Cells Serve as a Novel Immunological Tool To Analyze the Antiviral Efficacy of CD8+ T Cells In Vitro

CD8⁺ T cells are the main effector lymphocytes in the control of hepatitis B virus (HBV) infection. However, limitations of model systems, such as low infection rates, restrict mechanistic studies of HBV-specific CD8⁺ T cells. Here, we established a novel immunological cell culture model based on HBV-infected HepG2^hNTCP cells that endogenously processed viral antigens and presented them to HBV-specific CD8⁺ T cells. This induced cytolytic and noncytolytic CD8⁺ T-cell effector functions and reduction of viral loads.     

Noncytolytic CD8+ Cell Mediated Antiviral Response Represents a Strong Element in the Immune Response of Simian Immunodeficiency Virus-Infected Long-Term Non-Progressing Rhesus Macaques

The ability of long term non progressors to maintain very low levels of HIV/SIV and a healthy state, involves various host genetic and immunological factors. CD8+ non-cytolytic antiviral response (CNAR) most likely plays an important role in this regard. In order to gain a deeper insight into this unique phenomenon, the ability of CD8+ T cells to suppress viral replication in vitro was investigated in 16 uninfected, longitudinally in 23 SIV-infected long-term non-progressing (LTNPs), and 10 SIV-infected rhesus macaques with progressing disease. An acute infection assay utilizing CD4⁺ cells from MHC-mismatched monkeys to avoid cytolytic responses was employed. The study has identified CNAR as a long-term stable activity that inversely correlated with plasma viral load. The activity was also detected in CD8⁺ cells of uninfected macaques, which indicates that CNAR is not necessarily a virus specific response but increases after SIV-infection. Physical contact between CD4⁺ and CD8⁺ cells was mainly involved in mediating viral inhibition. Loss of this activity appeared to be due to a loss of CNAR-expressing CD8+ cells as well as a reduction of CNAR-responsive CD4+ cells. In contrast, in vitro viral replication did not differ in CD4+ cells from un-infected macaques, CNAR(+) and CNAR(-) LTNPs. A role for transitional memory cells in supporting CNAR in the macaque model of AIDS was questionable. CNAR appears to represent an important part of the immune response displayed by CD8+ T cells which might be underestimated up to now.     

HIV Replication Is Not Controlled by CD8+ T Cells during the Acute Phase of the Infection in Humanized Mice

HIV replication follows a well-defined pattern during the acute phase of the infection in humans. After reaching a peak during the first few weeks after infection, viral replication resolves to a set-point thereafter. There are still uncertainties regarding the contribution of CD8⁺ T cells in establishing this set-point. An alternative explanation, supported by in silico modeling, would imply that viral replication is limited by the number of available targets for infection, i.e. CD4⁺CCR5⁺ T cells. Here, we used NOD.SCID.gc^-/- mice bearing human CD4⁺CCR5⁺ and CD8⁺ T cells derived from CD34⁺ progenitors to investigate the relative contribution of both in viral control after the peak. Using low dose of a CCR5-tropic HIV virus, we observed an increase in viral replication followed by “spontaneous” resolution of the peak, similar to humans. To rule out any possible role for CD8⁺ T cells in viral control, we infected mice in which CD8⁺ T cells had been removed by a depleting antibody. Globally, viral replication was not affected by the absence of CD8⁺ T cells. Strikingly, resolution of the viral peak was equally observed in mice with or without CD8⁺ T cells, showing that CD8⁺ T cells were not involved in viral control in the early phase of the infection. In contrast, a marked and specific loss of CCR5-expressing CD4⁺ T cells was observed in the spleen and in the bone marrow, but not in the blood, of infected animals. Our results strongly suggest that viral replication during the acute phase of the infection in humanized mice is mainly constrained by the number of available targets in lymphoid tissues rather than by CD8⁺ T cells.     

CD40 Activation Rescues Antiviral CD8+ T Cells from PD-1-Mediated Exhaustion

The intrahepatic immune environment is normally biased towards tolerance. Nonetheless, effective antiviral immune responses can be induced against hepatotropic pathogens. To examine the immunological basis of this paradox we studied the ability of hepatocellularly expressed hepatitis B virus (HBV) to activate immunologically naïve HBV-specific CD8⁺ T cell receptor (TCR) transgenic T cells after adoptive transfer to HBV transgenic mice. Intrahepatic priming triggered vigorous in situ T cell proliferation but failed to induce interferon gamma production or cytolytic effector function. In contrast, the same T cells differentiated into cytolytic effector T cells in HBV transgenic mice if Programmed Death 1 (PD-1) expression was genetically ablated, suggesting that intrahepatic antigen presentation per se triggers negative regulatory signals that prevent the functional differentiation of naïve CD8⁺ T cells. Surprisingly, coadministration of an agonistic anti-CD40 antibody (αCD40) inhibited PD-1 induction and restored T cell effector function, thereby inhibiting viral gene expression and causing a necroinflammatory liver disease. Importantly, the depletion of myeloid dendritic cells (mDCs) strongly diminished the αCD40 mediated functional differentiation of HBV-specific CD8⁺ T cells, suggesting that activation of mDCs was responsible for the functional differentiation of HBV-specific CD8⁺ T cells in αCD40 treated animals. These results demonstrate that antigen-specific, PD-1-mediated CD8⁺ T cell exhaustion can be rescued by CD40-mediated mDC-activation.     

Identification of SIV Nef CD8 T cell epitopes restricted by a MHC class I haplotype associated with lower viral loads in a macaque AIDS model

Virus-specific CD8⁺ T-cell responses are crucial for the control of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) replication. Multiple studies on HIV-infected individuals and SIV-infected macaques have indicated association of several major histocompatibility complex class I (MHC-I) genotypes with lower viral loads and delayed AIDS progression. Understanding of the viral control mechanism associated with these MHC-I genotypes would contribute to the development of intervention strategy for HIV control. We have previously reported a rhesus MHC-I haplotype, 90-120-Ia, associated with lower viral loads after SIVmac239 infection. Gag_206–216 and Gag_241–249 epitope-specific CD8⁺ T-cell responses have been shown to play a central role in the reduction of viral loads, whereas the effect of Nef-specific CD8⁺ T-cell responses induced in all the 90-120-Ia⁺ macaques on SIV replication remains unknown. Here, we identified three CD8⁺ T-cell epitopes, Nef_9–19, Nef_89–97, and Nef_193–203, associated with 90-120-Ia. Nef_9–19 and Nef_193–203 epitope-specific CD8⁺ T-cell responses frequently selected for mutations resulting in viral escape from recognition by these CD8⁺ T cells, indicating that these CD8⁺ T cells exert strong suppressive pressure on SIV replication. Results would be useful for elucidation of the viral control mechanism associated with 90-120-Ia.     

Systemic LPS Translocation Activates Cross-Presenting Dendritic Cells but Is Dispensable for the Breakdown of CD8+ T Cell Peripheral Tolerance in Irradiated Mice

Lymphodepletion is currently used to enhance the efficacy of cytotoxic T lymphocyte adoptive transfer immunotherapy against cancer. This beneficial effect of conditioning regimens is due, at least in part, to promoting the breakdown of peripheral CD8⁺ T cell tolerance. Lymphodepletion by total body irradiation induces systemic translocation of commensal bacteria LPS from the gastrointestinal tract. Since LPS is a potent activator of the innate immune system, including antigen presenting dendritic cells, we hypothesized that LPS translocation could be required for the breakdown of peripheral tolerance observed in irradiated mice. To address this issue, we have treated irradiated mice with antibiotics in order to prevent LPS translocation and utilized them in T cell adoptive transfer experiments. Surprisingly, we found that despite of completely blocking LPS translocation into the bloodstream, antibiotic treatment did not prevent the breakdown of peripheral tolerance. Although irradiation induced the activation of cross-presenting CD8⁺ dendritic cells in the lymphoid tissue, LPS could not solely account for this effect. Activation of dendritic cells by mechanisms other than LPS translocation is sufficient to promote the differentiation of potentially autoreactive CD8⁺ T cells into effectors in irradiated mice. Our data indicate that LPS translocation is dispensable for the breakdown of CD8⁺ T cell tolerance in irradiated mice.     

Distinct APC Subtypes Drive Spatially Segregated CD4+ and CD8+ T-Cell Effector Activity during Skin Infection with HSV-1

Efficient infection control requires potent T-cell responses at sites of pathogen replication. However, the regulation of T-cell effector function in situ remains poorly understood. Here, we show key differences in the regulation of effector activity between CD4⁺ and CD8⁺ T-cells during skin infection with HSV-1. IFN-γ-producing CD4⁺ T cells disseminated widely throughout the skin and draining lymph nodes (LN), clearly exceeding the epithelial distribution of infectious virus. By contrast, IFN-γ-producing CD8⁺ T cells were only found within the infected epidermal layer of the skin and associated hair follicles. Mechanistically, while various subsets of lymphoid- and skin-derived dendritic cells (DC) elicited IFN-γ production by CD4⁺ T cells, CD8⁺ T cells responded exclusively to infected epidermal cells directly presenting viral antigen. Notably, uninfected cross-presenting DCs from both skin and LNs failed to trigger IFN-γ production by CD8⁺ T-cells. Thus, we describe a previously unappreciated complexity in the regulation of CD4⁺ and CD8⁺ T-cell effector activity that is subset-specific, microanatomically distinct and involves largely non-overlapping types of antigen-presenting cells (APC).     

A Correlate of HIV-1 Control Consisting of Both Innate and Adaptive Immune Parameters Best Predicts Viral Load by Multivariable Analysis in HIV-1 Infected Viremic Controllers and Chronically-Infected Non-Controllers

HIV-1 infected viremic controllers maintain durable viral suppression below 2000 copies viral RNA/ml without anti-retroviral therapy (ART), and the immunological factor(s) associated with host control in presence of low but detectable viral replication are of considerable interest. Here, we utilized a multivariable analysis to identify which innate and adaptive immune parameters best correlated with viral control utilizing a cohort of viremic controllers (median 704 viral RNA/ml) and non-controllers (median 21,932 viral RNA/ml) that were matched for similar CD4⁺ T cell counts in the absence of ART. We observed that HIV-1 Gag-specific CD8⁺ T cell responses were preferentially targeted over Pol-specific responses in viremic controllers (p = 0.0137), while Pol-specific responses were positively associated with viral load (rho = 0.7753, p = 0.0001, n = 23). Viremic controllers exhibited significantly higher NK and plasmacytoid dendritic cells (pDC) frequency as well as retained expression of the NK CD16 receptor and strong target cell-induced NK cell IFN-gamma production compared to non-controllers (p<0.05). Despite differences in innate and adaptive immune function however, both viremic controllers (p<0.05) and non-controller subjects (p<0.001) exhibited significantly increased CD8⁺ T cell activation and spontaneous NK cell degranulation compared to uninfected donors. Overall, we identified that a combination of innate (pDC frequency) and adaptive (Pol-specific CD8⁺ T cell responses) immune parameters best predicted viral load (R² = 0.5864, p = 0.0021, n = 17) by a multivariable analysis. Together, this data indicates that preferential Gag-specific over Pol-specific CD8⁺ T cell responses along with a retention of functional innate subsets best predict host control over viral replication in HIV-1 infected viremic controllers compared to chronically-infected non-controllers.     

ADAR1 Facilitates HIV-1 Replication in Primary CD4+ T Cells

Unlike resting CD4⁺ T cells, activated CD4⁺T cells are highly susceptible to infection of human immunodeficiency virus 1 (HIV-1). HIV-1 infects T cells and macrophages without activating the nucleic acid sensors and the anti-viral type I interferon response. Adenosine deaminase acting on RNA 1 (ADAR1) is an RNA editing enzyme that displays antiviral activity against several RNA viruses. Mutations in ADAR1 cause the autoimmune disorder Aicardi-Goutieères syndrome (AGS). This disease is characterized by an inappropriate activation of the interferon-stimulated gene response. Here we show that HIV-1 replication, in ADAR1-deficient CD4⁺T lymphocytes from AGS patients, is blocked at the level of protein translation. Furthermore, viral protein synthesis block is accompanied by an activation of interferon-stimulated genes. RNA silencing of ADAR1 in Jurkat cells also inhibited HIV-1 protein synthesis. Our data support that HIV-1 requires ADAR1 for efficient replication in human CD4⁺T cells.     

Human Cytomegalovirus Latency-Associated Proteins Elicit Immune-Suppressive IL-10 Producing CD4+ T Cells

Human cytomegalovirus (HCMV) is a widely prevalent human herpesvirus, which, after primary infection, persists in the host for life. In healthy individuals, the virus is well controlled by the HCMV-specific T cell response. A key feature of this persistence, in the face of a normally robust host immune response, is the establishment of viral latency. In contrast to lytic infection, which is characterised by extensive viral gene expression and virus production, long-term latency in cells of the myeloid lineage is characterised by highly restricted expression of viral genes, including UL138 and LUNA. Here we report that both UL138 and LUNA-specific T cells were detectable directly ex vivo in healthy HCMV seropositive subjects and that this response is principally CD4⁺ T cell mediated. These UL138-specific CD4⁺ T cells are able to mediate MHC class II restricted cytotoxicity and, importantly, show IFNγ effector function in the context of both lytic and latent infection. Furthermore, in contrast to CD4⁺ T cells specific to antigens expressed solely during lytic infection, both the UL138 and LUNA-specific CD4⁺ T cell responses included CD4⁺ T cells that secreted the immunosuppressive cytokine cIL-10. We also show that cIL-10 expressing CD4⁺ T-cells are directed against latently expressed US28 and UL111A. Taken together, our data show that latency-associated gene products of HCMV generate CD4⁺ T cell responses in vivo, which are able to elicit effector function in response to both lytic and latently infected cells. Importantly and in contrast to CD4⁺ T cell populations, which recognise antigens solely expressed during lytic infection, include a subset of cells that secrete the immunosuppressive cytokine cIL-10. This suggests that HCMV skews the T cell responses to latency-associated antigens to one that is overall suppressive in order to sustain latent carriage in vivo.     

CD3+/TCR-αβ− Cells Are Important in Protecting Spinal Cord Tissues against Theiler's Virus Strain GD VII Infection

Intravenous infection with Theiler's virus strain GD VII causes acute encephalomyelitis in mice. Endogenous IFN-γ produced in the spinal cord is important to protect the tissue in mice infected with this virus. Neither CD4⁺ cells nor CD8⁺ cells infiltrated the spinal cords of infected mice until Day 9 postinfection. However, the number of CD3⁺/TCR-γδ⁺ cells increased in the spinal cords of mice infected with the virus. These cells resided in the spinal cords of normal mice, and produced IFN-γ as a result of stimulation by immobilized anti-CD3 mAb. Elimination of CD3⁺ cells by the administration of a specific mAb augmented viral replication and suppressed production of endogenous IFN-γ. Depletion of TCR-αβ⁺ cells and ASGM1⁺ cells did not affect the viral replication, and did not alter the production of IFN-γ. Therefore, CD3⁺/TCR-αβ^– cells producing IFN-γ play an important role in the protection of the spinal cord against Theiler's virus infection. These results suggest that CD3⁺/TCR-αβ^– cells might be identical to TCR-γδ⁺ cells.     

Oseltamivir Prophylaxis Reduces Inflammation and Facilitates Establishment of Cross-Strain Protective T Cell Memory to Influenza Viruses

CD8⁺ T cells directed against conserved viral regions elicit broad immunity against distinct influenza viruses, promote rapid virus elimination and enhanced host recovery. The influenza neuraminidase inhibitor, oseltamivir, is prescribed for therapy and prophylaxis, although it remains unclear how the drug impacts disease severity and establishment of effector and memory CD8⁺ T cell immunity. We dissected the effects of oseltamivir on viral replication, inflammation, acute CD8⁺ T cell responses and the establishment of immunological CD8⁺ T cell memory. In mice, ferrets and humans, the effect of osteltamivir on viral titre was relatively modest. However, prophylactic oseltamivir treatment in mice markedly reduced morbidity, innate responses, inflammation and, ultimately, the magnitude of effector CD8⁺ T cell responses. Importantly, functional memory CD8⁺ T cells established during the drug-reduced effector phase were capable of mounting robust recall responses. Moreover, influenza-specific memory CD4⁺ T cells could be also recalled after the secondary challenge, while the antibody levels were unaffected. This provides evidence that long-term memory T cells can be generated during an oseltamivir-interrupted infection. The anti-inflammatory effect of oseltamivir was verified in H1N1-infected patients. Thus, in the case of an unpredicted influenza pandemic, while prophylactic oseltamivir treatment can reduce disease severity, the capacity to generate memory CD8⁺ T cells specific for the newly emerged virus is uncompromised. This could prove especially important for any new influenza pandemic which often occurs in separate waves.     

Us3 Kinase Encoded by Herpes Simplex Virus 1 Mediates Downregulation of Cell Surface Major Histocompatibility Complex Class I and Evasion of CD8+ T Cells

Detection and elimination of virus-infected cells by CD8⁺ cytotoxic T lymphocytes (CTLs) depends on recognition of virus-derived peptides presented by major histocompatibility complex class I (MHC-I) molecules on the surface of infected cells. In the present study, we showed that inactivation of the activity of viral kinase Us3 encoded by herpes simplex virus 1 (HSV-1), the etiologic agent of several human diseases and a member of the alphaherpesvirinae, significantly increased cell surface expression of MHC-I, thereby augmenting CTL recognition of infected cells in vitro. Overexpression of Us3 by itself had no effect on cell surface expression of MHC-I and Us3 was not able to phosphorylate MHC-I in vitro, suggesting that Us3 indirectly downregulated cell surface expression of MHC-I in infected cells. We also showed that inactivation of Us3 kinase activity induced significantly more HSV-1-specific CD8⁺ T cells in mice. Interestingly, depletion of CD8⁺ T cells in mice significantly increased replication of a recombinant virus encoding a kinase-dead mutant of Us3, but had no effect on replication of a recombinant virus in which the kinase-dead mutation was repaired. These results indicated that Us3 kinase activity is required for efficient downregulation of cell surface expression of MHC-I and mediates evasion of HSV-1-specific CD8⁺ T cells. Our results also raised the possibility that evasion of HSV-1-specific CD8⁺ T cells by HSV-1 Us3-mediated inhibition of MHC-I antigen presentation might in part contribute to viral replication in vivo.