Killing Of Virus-infected Cells Research Articles

Role of NK Cells in Skin Wound Healing of Mice.

NK cells are best known for their killing of virus-infected cells and tumor cells via release of cytotoxic factors. However, NK cells can also produce growth factors and cytokines, and thus have the potential to influence physiological processes such as wound healing. In this study, we test the hypothesis that NK cells play a physiological role in skin wound healing of C57BL/6J mice. Immunohistochemical and flow cytometry assays showed that NK cells accumulate in excisional skin wounds, peaking on day 5 postinjury. We also found that NK cells proliferate locally in wounds, and blocking IL-15 activity locally reduces NK cell proliferation and accumulation in wounds. Wound NK cells exhibit primarily a mature CD11b+CD27- and NKG2A+NKG2D- phenotype and express LY49I and proinflammatory cytokines such as IFN-γ, Tnf-a, and Il-1β. Systemic depletion of NK cells resulted in enhanced re-epithelization and collagen deposition, suggesting a negative role for these cells in skin wound healing. Depletion of NK cells did not influence accumulation of neutrophils or monocytes/macrophages in wounds but did reduce expression of IFN-γ, Tnf-a, and Il-1β, indicating that NK cells contribute to proinflammatory cytokine expression in wounds. In short, NK cells may impede physiological wound healing via production of proinflammatory cytokines.

Abstract 5514: Therapeutic potential of activated natural killer cells in nasopharyngeal carcinoma

Abstract One of the key features of nasopharyngeal carcinoma (NPC) is dense lymphocyte infiltration. Despite elevated lymphocyte populations in tumor microenvironment, these lymphocytes have modest killing activity. One of the possible speculations is constitutive STAT3 overexpression within NPC microenvironment which results in immunosuppression of cytolytic immune cells. Natural killer (NK) cells act as first line defense of innate immunity through cytolytic killing of virus-infected cells and/or cancer cells. Unlike T cells, NK killing does not require antigen presentation and therefore would be effective even for MHC defective cancers. This makes NK therapy attractive in form of autologous and allogeneic transfer. Here we hypothesized that activated NK cells would show enhanced anti-NPC activity. We also sought to investigate the potential of combined NK with STAT3 inhibitor in NPC treatment. In this study, NK cells from peripheral blood mononuclear cells (PBMC) of healthy donors (N=3) were isolated and expanded. Three Epstein Barr Virus (EBV) positive NPC cell lines (NPC43, C666-1, C17) were employed in this study. Naïve and activated NK cells were subjected to NPC coculture at various effector: target (E:T) cell ratios. In combination treatment with NK cells, a small molecule inhibitor Napabucasin was employed for STAT3 treatment. NK killing activity was assessed through flow cytometry analysis of NPC cell death. The current study demonstrated significant elevation of NK activation marker expressions after 4 weeks of ex vivo expansion (CD69 34.7±2.3 fold, p=0.002; NKG2D 4.2±0.8 fold, p=0.018). Concomitantly, ex vivo activated NK cells demonstrated augmented killing activity against NPC cells at a dose-dependent manner, compared to their naïve status (p<0.001). In NPC43, net cytotoxicity of expanded NK was 35.5±7.2% compared to naïve NK with only 2.5±4.3% (p=0.003). NPC43 was most sensitive towards NK killing, followed by C666-1, then C17. This corresponds to NPC43’s low PD-L1 protein expression, a known downstream STAT3 target and immune checkpoint molecule. In addition, activated NK could kill chemoresistant NPC cells indiscriminately compared to the paralleled parental cells (18.5±2.3% and 16.1±3.8%, p=0.589). Importantly, augmented NPC43 cell death was observed in combined treatment (47.1±2.1%) compared to single treatment (NK alone: 9.2±6.5%; Napabucasin alone: 25.0±4.5%). In summary, this work demonstrated encouraging therapeutic potential of both NK therapy and in combination with STAT3 inhibitor Napabucasin for NPC treatment. Citation Format: Shinyee Hui, Kwok Wai Lo, Mingjing Xu, Siu Tim Cheung. Therapeutic potential of activated natural killer cells in nasopharyngeal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5514.

The Role of the Inhibitory Ligand HVEM and Its Receptors CD160 and BTLA in the Regulation of Anti-retroviral T Cell Responses

Specific CD8+ T cells are crucial for the control of viruses. However, during many chronic viral infections these cells become dysfunctional. Immune checkpoint receptors, like PD-1 expressed on CD8+ T cells, contribute to this functional suppression during chronic infection. However, during the acute phase of infection virus-specific CD8+ T cells express high levels of PD-1 but are fully competent in killing virus-infected cells and there is increasing evidence that the biological activity of inhibitory receptors is strongly influenced by the availability of their respective ligands. We determined the expression of ligands for inhibitory receptors on infected myeloid cells during the acute phase of Friend retroviral (FV) infection. FV infection of granulocytes, monocytes, and macrophages strongly increased the cell surface expression of PD-L1 and the recently described ligand HVEM for inhibitory receptors BTLA and CD160. In addition, the infection of human myeloid cells in vitro with HIV also enhanced the expression of PD-L1 and HVEM. In infected mice, the upregulation of inhibitory ligands on infected cells was accompanied by enhanced frequencies of FV-specific CD8+ T cells that express PD-1, and the inhibitory receptors CD160 and BTLA. To define the functional effects of HVEM on activated CD8+ T cells, FV-infected mice were treated with blocking antibodies that prevented the interaction of HVEM with its two receptors, CD160 or BTLA, alone or in combination with anti-PD-L1 antibodies. Blocking the interaction of HVEM with CD160 and BTLA improved the production of cytotoxic molecules and the elimination of FV-infected cells. This effect was augmented when the therapy was combined with anti-PD-L1 antibodies, resulting in an additional expansion of cytotoxic CD8+ T cells. Thus, the ligand HVEM for the inhibitory receptors CD160 and BTLA downregulates the functionality of CD8+ T cells during retroviral infection and are potential targets for the immunomodulatory therapy of chronic viral infections.

Therapeutic Potential of IL-15 and N-803 in HIV/SIV Infection.

IL-15, a proinflammatory cytokine critical for the generation, maintenance, and homeostasis of T cell responses, is produced naturally in response to HIV/SIV infection, but has also demonstrated therapeutic potential. IL-15 can boost CD4+ and CD8+ T cell and NK cell proliferation, activation, and function. However, IL-15 treatment may cause aberrant immune activation and accelerated disease progression in certain circumstances. Moreover, the relationship between the timing of IL-15 administration and disease progression remains unclear. The IL-15 superagonist N-803 was developed to expand the therapeutic potential of IL-15 by maximizing its tissue distribution and half-life. N-803 has garnered enthusiasm recently as a way to enhance the innate and cellular immune responses to HIV/SIV by improving CD8+ T cell recognition and killing of virus-infected cells and directing immune cells to mucosal sites and lymph nodes, the primary sites of virus replication. N-803 has also been evaluated in “shock and kill” strategies due to its potential to reverse latency (shock) and enhance antiviral immunity (kill). This review examines the current literature about the effects of IL-15 and N-803 on innate and cellular immunity, viral burden, and latency reversal in the context of HIV/SIV, and their therapeutic potential both alone and combined with additional interventions such as antiretroviral therapy (ART) and vaccination.

HIV-1 Latency and Viral Reservoirs: Existing Reversal Approaches and Potential Technologies, Targets, and Pathways Involved in HIV Latency Studies.

Eradication of latent human immunodeficiency virus (HIV) infection is a global health challenge. Reactivation of HIV latency and killing of virus-infected cells, the so-called “kick and kill” or “shock and kill” approaches, are a popular strategy for HIV cure. While antiretroviral therapy (ART) halts HIV replication by targeting multiple steps in the HIV life cycle, including viral entry, integration, replication, and production, it cannot get rid of the occult provirus incorporated into the host-cell genome. These latent proviruses are replication-competent and can rebound in cases of ART interruption or cessation. In general, a very small population of cells harbor provirus, serve as reservoirs in ART-controlled HIV subjects, and are capable of expressing little to no HIV RNA or proteins. Beyond the canonical resting memory CD4+ T cells, HIV reservoirs also exist within tissue macrophages, myeloid cells, brain microglial cells, gut epithelial cells, and hematopoietic stem cells (HSCs). Despite a lack of active viral production, latently HIV-infected subjects continue to exhibit aberrant cellular signaling and metabolic dysfunction, leading to minor to major cellular and systemic complications or comorbidities. These include genomic DNA damage; telomere attrition; mitochondrial dysfunction; premature aging; and lymphocytic, cardiac, renal, hepatic, or pulmonary dysfunctions. Therefore, the arcane machineries involved in HIV latency and its reversal warrant further studies to identify the cryptic mechanisms of HIV reservoir formation and clearance. In this review, we discuss several molecules and signaling pathways, some of which have dual roles in maintaining or reversing HIV latency and reservoirs, and describe some evolving strategies and possible approaches to eliminate viral reservoirs and, ultimately, cure/eradicate HIV infection.

Sialic Acids and Their Influence on Human NK Cell Function.

Sialic acids are sugars with a nine-carbon backbone, present on the surface of all cells in humans, including immune cells and their target cells, with various functions. Natural Killer (NK) cells are cells of the innate immune system, capable of killing virus-infected and tumor cells. Sialic acids can influence the interaction of NK cells with potential targets in several ways. Different NK cell receptors can bind sialic acids, leading to NK cell inhibition or activation. Moreover, NK cells have sialic acids on their surface, which can regulate receptor abundance and activity. This review is focused on how sialic acids on NK cells and their target cells are involved in NK cell function.

Targeting Natural Killer Cells for Improved Immunity and Control of the Adaptive Immune Response.

Natural killer (NK) cells are critical for targeting and killing tumor, virus-infected and stressed cells as a member of the innate immune system. Recently, NK cells have also emerged as key regulators of adaptive immunity and have become a prominent therapeutic target for cancer immunotherapy and infection control. NK cells display a diverse array of phenotypes and function. Determining how NK cells develop and are regulated is critical for understanding their role in both innate and adaptive immunity. In this review we discuss current research approaches into NK cell adaptive immunity and how these cells are being harnessed for improving cancer and vaccination outcomes.

Understanding T cell signaling using membrane reconstitution.

T cells are central players of our immune system, as their functions range from killing tumorous and virus-infected cells to orchestrating the entire immune response. In order for T cells to divide and execute their functions, they must be activated by antigen-presenting cells (APCs) through a cell-cell junction. Extracellular interactions between receptors on T cells and their ligands on APCs trigger signaling cascades comprised of protein-protein interactions, enzymatic reactions, and spatial reorganization events, to either stimulate or repress T cell activation. Plasma membrane is the major platform for T cell signaling. Recruitment of cytosolic proteins to membrane-bound receptors is a common critical step in many signaling pathways. Membranes decrease the dimensionality of protein-protein interactions to enable weak yet biologically important interactions. Membrane resident proteins can phase separate into micro-islands that promote signaling by enriching or excluding signal regulators. Moreover, some membrane lipids can either mediate or regulate cell signaling by interacting with signaling proteins. While it is critical to investigate T cell signaling in a cellular environment, the large number of signaling pathways involved and potential crosstalk have made it difficult to obtain precise, quantitative information on T cell signaling. Reconstitution of purified proteins to modelmembranes provides a complementary avenue for T cell signaling research. Here, I review recent progress in studying T cell signaling using membrane reconstitution approaches.

Pigs that recover from porcine reproduction and respiratory syndrome virus infection develop cytotoxic CD4+CD8+ and CD4+CD8- T-cells that kill virus infected cells.

Porcine reproductive and respiratory syndrome virus (PRRSV) infection is difficult to control because the virus undergoes antigenic variation during infection and also modulates the protective host immune response. Although current vaccines do not provide full protection, they have provided insight into the mechanisms of protection. Live PRRSV vaccines induce partial protection before the appearance of neutralizing antibody, suggesting cell-mediated immunity or other mechanisms may be involved. Herein, we demonstrate recovery from infection is associated with development of cytotoxic T-lymphocytes (CTL) that can kill PRRSV-infected target cells. Initial experiments showed survival of PRRSV-infected monocyte derived macrophage (MDM) targets is reduced when overlaid with peripheral blood mononuclear cells (PBMC) from gilts that had recovered from PRRSV infection. Further studies with PBMC depleted of either CD4+ or CD8+ T-cells and positively selected subpopulations of CD4+ and CD8+ T-cells showed that both CD4+ and CD8+ T-cells were involved in killing. Examination of killing at different time points revealed killing was biphasic and mediated by CTL of different phenotypes. CD4+CD8+high were associated with killing target cells infected for 3–6 hours. CD4+CD8- CTL were associated with killing at 16–24 hours. Thus, all the anti-PRRSV CTL activity in pigs was attributed to two phenotypes of CD4+ cells which is different from the anti-viral CD4-CD8+ CTL phenotype found in most other animals. These findings will be useful for evaluating CTL responses induced by current and future vaccines, guiding to a novel direction for future vaccine development.

Differentiation of hematopoietic stem and progenitor cells to NK cells in a stroma-free, serum-free culture system

Abstract Natural Killer (NK) cells play an important role in innate immunity by secreting proinflammatory cytokines and killing tumor cells and virus-infected cells. Human NK cells can be generated by culturing CD34+ hematopoietic stem and progenitor cells (HSPCs) with stromal cells and cytokines. The use of stroma (and serum) is, however, not desirable in clinical applications. We developed a culture method for generating large numbers of NK cells from purified CD34+ cord blood (CB) HSPCs in the absence of serum and stromal cells. CD34+ CB cells were isolated by EasySep magnetic separation, seeded into culture plates coated with a Notch ligand and cultured for two weeks in StemSpan Serum-Free Expansion Medium (SFEM II) supplemented with SCF, TPO, Flt3L and IL-7. These conditions promote expansion of HSPCs and their differentiation into CD7+CD5+ lymphoid progenitors. Cells were then transferred to non-coated plates and cultured for two more weeks in StemSpan SFEM II supplemented with SCF, Flt3L, IL-7, IL-15 and a small molecule, UM729, to promote differentiation into NK cells. After culture, on average 76% (range: 54–90%, n=12) of cells expressed the NK cell marker CD56 and 58% (range: 25–80%) coexpressed CD56 and NKp46 (a NK cell activating receptor). A more mature CD56+CD16+ NK cell subset also emerged in these cultures (5%, range: 1–11%). The yield of CD56+ NK cells was ~13,000 (600–41,000) per initial CD34+ cell. The NK cells were functional and able to kill K562 target cells. These results show that HSPCs can expand and differentiate into NK cells under stroma- and serum-free culture conditions. This novel culture system will be useful for studies directed toward the development of cancer immunotherapies where large numbers of NK cells are needed.

A-106 Eradication of HIV; molecular therapeutic paths to a functional cure

The eradication of functional replicative HIV is within our collective grasp. Several emerging modes of targeted therapeutics, ranging from chimeric antigen receptor (CAR) T-cell targeted killing of virus infected cells to targeted genomic editing and/or excision of latent provirus, may prove promising with regards to eradicating the spread of functional HIV. We have recently developed 2 distinct molecular approaches that may facilitate the eradication of HIV. The first approach is the systemic activation of HIV followed by a polyvalent “swarm” of HIV-gp120 bi-specific CAR CD4+/CD8+ T-cells. Systemic activation of latent HIV can occur by removing anti-retroviral therapy (ART) and/or by targeted activation of provirus. To activate provirus we developed a biologically deliverable recombinant zinc finger protein activator (ZFP-362-VPR), that functionally activates HIV in an LTR directed and specific manner. Notably, ZFP-362-VPR can be used in combination with a swarm of differentially HIV-gp120 targeted bi-specific CAR CD4+/CD8+ T-cells, to specifically kill virus infected cells. A second approach to eradicate HIV is the targeted genomic editing and/or excision of integrated virus. The approach we describe here builds on biologically deliverable recombinant protein technologies to direct the excision and/or targeted mutations to loci in the provirus, ultimately rendering the virus nonreplicative. Collectively, these emerging molecular therapeutic strategies juxtaposed with current antiretroviral therapy may one day result in the eradication of HIV.

Time Intervals in Sequence Sampling, Not Data Modifications, Have a Major Impact on Estimates of HIV Escape Rates.

The ability of human immunodeficiency virus (HIV) to avoid recognition by humoral and cellular immunity (viral escape) is well-documented, but the strength of the immune response needed to cause such a viral escape remains poorly quantified. Several previous studies observed a more rapid escape of HIV from CD8 T cell responses in the acute phase of infection compared to chronic infection. The rate of HIV escape was estimated with the help of simple mathematical models, and results were interpreted to suggest that CD8 T cell responses causing escape in acute HIV infection may be more efficient at killing virus-infected cells than responses that cause escape in chronic infection, or alternatively, that early escapes occur in epitopes mutations in which there is minimal fitness cost to the virus. However, these conclusions were challenged on several grounds, including linkage and interference of multiple escape mutations due to a low population size and because of potential issues associated with modifying the data to estimate escape rates. Here we use a sampling method which does not require data modification to show that previous results on the decline of the viral escape rate with time since infection remain unchanged. However, using this method we also show that estimates of the escape rate are highly sensitive to the time interval between measurements, with longer intervals biasing estimates of the escape rate downwards. Our results thus suggest that data modifications for early and late escapes were not the primary reason for the observed decline in the escape rate with time since infection. However, longer sampling periods for escapes in chronic infection strongly influence estimates of the escape rate. More frequent sampling of viral sequences in chronic infection may improve our understanding of factors influencing the rate of HIV escape from CD8 T cell responses.

Complex antigen presentation pathway for an HLA-A*0201-restricted epitope from Chikungunya 6K protein.

BackgroundThe adaptive cytotoxic T lymphocyte (CTL)-mediated immune response is critical for clearance of many viral infections. These CTL recognize naturally processed short viral antigenic peptides bound to human leukocyte antigen (HLA) class I molecules on the surface of infected cells. This specific recognition allows the killing of virus-infected cells. The T cell immune T cell response to Chikungunya virus (CHIKV), a mosquito-borne Alphavirus of the Togaviridae family responsible for severe musculoskeletal disorders, has not been fully defined; nonetheless, the importance of HLA class I-restricted immune response in this virus has been hypothesized.Methodology/Principal findingsBy infection of HLA-A*0201-transgenic mice with a recombinant vaccinia virus that encodes the CHIKV structural polyprotein (rVACV-CHIKV), we identified the first human T cell epitopes from CHIKV. These three novel 6K transmembrane protein-derived epitopes are presented by the common HLA class I molecule, HLA-A*0201. One of these epitopes is processed and presented via a complex pathway that involves proteases from different subcellular locations. Specific chemical inhibitors blocked these events in rVACV-CHIKV-infected cells.Conclusions/SignificanceOur data have implications not only for the identification of novel Alphavirus and Togaviridae antiviral CTL responses, but also for analyzing presentation of antigen from viruses of different families and orders that use host proteinases to generate their mature envelope proteins.

Emerging Targets for Developing T Cell-Mediated Vaccines for Human Immunodeficiency Virus (HIV)-1.

Human immunodeficiency virus (HIV)-1 has infected >75 million individuals globally, and, according to the UN, is responsible for ~2.1 million new infections and 1.1 million deaths each year. Currently, there are ~37 million individuals with HIV infection and the epidemic has already resulted in 35 million deaths. Despite the advances of anti-retroviral therapy (ART), a cost-effective vaccine remains the best long-term solution to end the HIV-1 epidemic especially given that the vast majority of infected individuals live in poor socio-economic regions of the world such as Sub-Saharan Africa which limits their accessibility to ART. The modest efficacy of the RV144 Thai trial provides hope that a vaccine for HIV-1 is possible, but as markers for sterilizing immunity are unknown, the design of an effective vaccine is empirical, although broadly cross-reactive neutralizing antibodies (bNAb) that can neutralize various quasispecies of HIV-1 are considered crucial. Since HIV-1 transmission often occurs at the genito-rectal mucosa and is cell-associated, there is a need to develop vaccines that can elicit CD8+ T cell immunity with the capacity to kill virus infected cells at the genito-rectal mucosa and the gut. Here we discuss the recent progress made in developing T cell-mediated vaccines for HIV-1 and emphasize the need to elicit mucosal tissue-resident memory CD8+ T (CD8+ Trm) cells. CD8+ Trm cells will likely form a robust front-line defense against HIV-1 and eliminate transmitter/founder virus-infected cells which are responsible for propagating HIV-1 infections following transmission in vast majority of cases.

Distinct expression profile of HCMV encoded miRNAs in plasma from oral lichen planus patients

BackgroundOral lichen planus (OLP) is a T cell-mediated autoimmune disease. The aetiology and molecular mechanisms of OLP remain unclear. Human cytomegalovirus (HCMV) infection is a causal factor in the development of various diseases, but the clinical relevance of HCMV in OLP has not been thoroughly investigated.MethodsIn the present study, we firstly examined twenty-three HCMV-encoded microRNA (miRNA) expression profiles in plasma from training set that including 21 OLP patients and 18 healthy controls using RT-qPCR technology. Dysregulated miRNAs were subsequently confirmed in another larger cohort refereed as validation set consisting of 40 OLP patients and 33 healthy controls. HCMV DNA in peripheral blood leukocytes (PBLs) was also measured in an additional cohort of 13 OLP patients and 12 control subjects. Furthermore, bioinformatics analyses, luciferase reporter assay and western blotting were also performed to predict and verify the direct potential targets of HCMV-encoded miRNAs.ResultsThe RT-qPCR results showed that the plasma levels of five HCMV-encoded miRNAs including hcmv-miR-UL112-3p, hcmv-miR-UL22a-5p, hcmv-miR-UL148d, hcmv-miR-UL36-5p and hcmv-miR-UL59 were significantly increased in OLP patients in both training and validation sets. HCMV DNA in PBLs was also significantly higher in OLP patients than in control subjects. Additionally, by using a combination of luciferase reporter assay and western blotting, we demonstrated that cytomegalovirus UL16-binding protein 1, a molecule that mediates the killing of virus-infected cells by natural killer cells, is a direct target of hcmv-miR-UL59.ConclusionsOur results demonstrate a distinct expression pattern of HCMV-encoded miRNAs in OLP patients, which may provide insight into the relationship between HCMV infection and OLP, and warrants additional study in the diagnosis and aetiology of OLP.

Impact of Steroids on Natural Killer Cells Against Cytotoxicity and Hepatitis C Virus Replication

BackgroundNatural killer (NK) cells play important roles in killing tumor and virus-infected cells. Immunosuppression used after organ transplantation is thought to increase the risk of tumor recurrence and viral infections. However, the effect of immunosuppressive drugs on NK cells has not yet been clearly established. Therefore, we examined the effect of immunosuppression on NK cells. MethodsNK cells were cultured for 7 days in the presence of interleukin-2 (100 U/mL) with or without the following immunosuppressive drugs: tacrolimus, cyclosporine A, corticosteroid (methylprednisolone [MP]), mycophenolate mofetil, and rapamycin. The effect of the drugs on NK cell activation was tested on the basis of the following: NK cell phenotype, NK cell proliferation, cytotoxicity against K562 cells, cytokine production by NK cells, and anti-hepatitis C virus (HCV) activity with HCV genomic replicon cells. ResultsNK cells showed relatively robust functions in the presence of tacrolimus and cyclosporine A. Mycophenolate mofetil and rapamycin significantly prevented only NK cell proliferation (P < .05). In contrast, MP significantly inhibited the proliferation, cytotoxicity, and anti-HCV effect (10.9%, 18.5%, and 1.9%, respectively) of NK cells. Furthermore, MP specifically inhibited the expression of NK cell activation markers and the production of interferon-γ (P < .05). ConclusionsCorticosteroids have distinct effects on NK cells, which may have important implications for NK cell function in cytotoxicity and HCV effect after transplantation.

Dynamic acquisition of HTLV-1 tax protein by mononuclear phagocytes: Role in neurologic disease

Pathology of HTLV-1 associated myelopathy/Tropical spastic paraparesis (HAM/TSP) is believed to be the result of “bystander damage” involving effector CD8 (+) T lymphocytes (CTLs) killing of virus infected cells. But the specific cellular events leading up to tissue injury are still unclear. Here, we developed the Microscopy Imaging of Cytotoxic T lymphocyte assay with Fluorescence emission (MI-CaFé), an optimized visualization analysis to explore the interactions between CTLs and virus infected or viral antigen presenting target cells. Various cell-to-cell formations can be observed and our results demonstrate elevated frequencies of CTL-target cell conjugates in HAM/TSP patient PBMCs compared to control PBMCs. Furthermore, HTLV-1 Tax protein expression can be localized at the cell-cell junctions and also tracked moving from an infected cell to a CD14 (+) mononuclear phagocyte (MP). Activation of CD14 (+) MPs in HAM/TSP patient PBMCs and antigenic presentation of HTLV-1 Tax by MPs can be inferred by their spontaneous cytotoxicity after 18h of in vitro culture. Given that CD4 (+) T lymphocytes are the primary reservoirs of HTLV-1 and MPs are scavenger cells responsible for pathogen clearance, spontaneous cytotoxicity against MPs in HAM/TSP PBMCs suggests a mechanism of chronic inflammation, secondary to low level of persistent virus infection within the central nervous system.

Expanding specificity of class I restricted CD8+ T cells for viral epitopes following multiple inoculations of swine with a human adenovirus vectored foot-and-mouth disease virus (FMDV) vaccine

The immune response to the highly acute foot-and-mouth disease virus (FMDV) is routinely reported as a measure of serum antibody. However, a critical effector function of immune responses combating viral infection of mammals is the cytotoxic T lymphocyte (CTL) response mediated by virus specific CD8 expressing T cells. This immune mechanism arrests viral spread by killing virus infected cells before new, mature virus can develop. We have previously shown that infection of swine by FMDV results in a measurable CTL response and have correlated CTL killing of virus-infected cells with specific class I major histocompatibility complex (MHC) tetramer staining. We also showed that a modified replication defective human adenovirus 5 vector expressing the FMDV structural proteins (Ad5-FMDV-T vaccine) targets the induction of a CD8+ CTL response with a minimal humoral response. In this report, we show that the specificity of the CD8+ T cell response to Ad5-FMDV-T varies between cohorts of genetically identical animals. Further, we demonstrate epitope specificity of CD8+ T cells expands following multiple immunizations with this vaccine.

Balancing viral replication in spleen and liver determines the outcome of systemic virus infection

The innate immune system limits virus replication during systemic infection by producing type I interferons (IFN-I) but still has to allow viral replication to achieve maximal innate and adaptive immune activation. Some spleen and lymph node resident antigen presenting cells (APCs) show limited response to IFN-I due to expression of the endogenous inhibitor of IFN-I signaling, Usp18. Therefore, virus in this spleen niche replicates despite high levels of IFN-I. This enforced viral replication leads to an exorbitant propagation of viral antigens and viral RNA. Viral antigen leads to massive activation of the adaptive immune system, while viral RNA to activated innate immunity. In contrast to these APCs, liver resident Kupffer cells, take up most of the systemic virus and suppress its replication in response to IFN-I. In addition, virus specific CD8 + T cells which are primed in the spleen migrate to the liver and kill virus infectedcells. In this review we discuss the different mechanisms, which influence immune activation in spleen and antiviral mechanisms in the liver and how they determine the outcome of virus infection.

PD-L1 Expression on Retrovirus-Infected Cells Mediates Immune Escape from CD8+ T Cell Killing.

Cytotoxic CD8+ T Lymphocytes (CTL) efficiently control acute virus infections but can become exhausted when a chronic infection develops. Signaling of the inhibitory receptor PD-1 is an important mechanism for the development of virus-specific CD8+ T cell dysfunction. However, it has recently been shown that during the initial phase of infection virus-specific CD8+ T cells express high levels of PD-1, but are fully competent in producing cytokines and killing virus-infected target cells. To better understand the role of the PD-1 signaling pathway in CD8+ T cell cytotoxicity during acute viral infections we analyzed the expression of the ligand on retrovirus-infected cells targeted by CTLs. We observed increased levels of PD-L1 expression after infection of cells with the murine Friend retrovirus (FV) or with HIV. In FV infected mice, virus-specific CTLs efficiently eliminated infected target cells that expressed low levels of PD-L1 or that were deficient for PD-L1 but the population of PD-L1high cells escaped elimination and formed a reservoir for chronic FV replication. Infected cells with high PD-L1 expression mediated a negative feedback on CD8+ T cells and inhibited their expansion and cytotoxic functions. These findings provide evidence for a novel immune escape mechanism during acute retroviral infection based on PD-L1 expression levels on virus infected target cells.