Virus-infected Target Cells Research Articles

Differential Immune-Modulating Activities of Cell Walls and Secreted Metabolites from Probiotic Bacillus coagulans JBI-YZ6.3 under Normal versus Inflamed Culture Conditions.

Spore-forming probiotic bacteria, including Bacillus coagulans, are resilient and produce a variety of beneficial metabolites. We evaluated the immune-modulating effects of the novel probiotic strain Bacillus coagulans JBI-YZ6.3, where the germinated spores, metabolite fraction, and cell wall fraction were tested in parallel using human peripheral blood mononuclear cell cultures under both normal and lipopolysaccharide-induced inflamed culture conditions. The expression of CD25 and CD69 activation markers was evaluated via flow cytometry. Supernatants were tested for cytokines, interferons, chemokines, and growth factors using Luminex arrays. The germinated spores were highly immunogenic; both the cell wall and metabolite fractions contributed significantly. Under normal culture conditions, increased levels of immune activation were observed as increased expressions of CD25 and CD69 relative to natural killer cells, suggesting an increased ability to attack virus-infected target cells. On monocytes, a complex effect was observed, where the expression of CD25 increased under normal conditions but decreased under inflamed conditions. This, in combination with increased interleukin-10 (IL-10) and decreased monocyte chemoattractant protein-1 (MCP-1) production under inflamed conditions, points to anti-inflammatory effects. The production of the stem cell-related growth factor granulocyte colony-stimulating Factor (G-CSF) was enhanced. Further research is warranted to characterize the composition of the postbiotic metabolite fraction and document the characteristics of immunomodulating agents secreted by this probiotic strain.

Changes in the natural killer cell repertoire and function induced by the cancer immune adjuvant candidate IMMUNEPOTENT-CRP

Natural killer (NK) cells are CD3–, CD16+, CD56+ that play a crucial role in immune response by recognizing and eliminating a variety of virus-infected, malignant, and antibody-coated target cells. We examined activation; repertoire changes and effector functions of human NK cells normal donors treated with IMMUNEPOTENT-CRP (I-CRP), a bovine dialyzable leukocyte extract (DLE) containing a mixture of low molecular weight molecules. I-CRP induces human NK cells activation and increase CD56Dim CD16– subset, without inducing proliferation. Human NK cells showed an increase on NKp30, NKp44, NKp46, NKG2D, NKG2C and KIR receptors, whereas no significant differences on CD160, CD85j and CD226 where observed. I-CRP-treated human NK cells exhibited an increased degranulation activity against K562 target cells, as shown by CD107a assay, and this correlates with cytotoxicity against K562 cells observed in calcein release assay. These results indicate that I-CRP can modify human NK cells receptor repertoire leading to an increased cytotoxic activity, supporting evidence for its use to stimulate NK cells.

Role of V-ATPase a3-Subunit in Mouse CTL Function.

CTLs release cytotoxic proteins such as granzymes and perforin through fusion of cytotoxic granules (CG) at the target cell interface, the immune synapse, to kill virus-infected and tumorigenic target cells. A characteristic feature of these granules is their acidic pH inside the granule lumen, which is required to process precursors of granzymes and perforin to their mature form. However, the role of acidic pH in CG maturation, transport, and fusion is not understood. We demonstrate in primary murine CTLs that the a3-subunit of the vacuolar-type (H+)-adenosine triphosphatase is required for establishing a luminal pH of 6.1 inside CG using ClopHensorN(Q69M), a newly generated CG-specific pH indicator. Knockdown of the a3-subunit resulted in a significantly reduced killing of target cells and a >50% reduction in CG fusion in total internal reflection fluorescence microscopy, which was caused by a reduced number of CG at the immune synapse. Superresolution microscopy revealed a reduced interaction of CG with the microtubule network upon a3-subunit knockdown. Finally, we find by electron and structured illumination microscopy that knockdown of the a3-subunit altered the diameter and density of individual CG, whereas the number of CG per CTL was unaffected. We conclude that the a3-subunit of the vacuolar adenosine triphosphatase is not only responsible for the acidification of CG, but also contributes to the maturation and efficient transport of the CG to the immune synapse.

Selective Engagement of FcγRIV by a M2e-Specific Single Domain Antibody Construct Protects Against Influenza A Virus Infection.

Lower respiratory tract infections, such as infections caused by influenza A viruses, are a constant threat for public health. Antivirals are indispensable to control disease caused by epidemic as well as pandemic influenza A. We developed a novel anti-influenza A virus approach based on an engineered single-domain antibody (VHH) construct that can selectively recruit innate immune cells to the sites of virus replication. This protective construct comprises two VHHs. One VHH binds with nanomolar affinity to the conserved influenza A matrix protein 2 (M2) ectodomain (M2e). Co-crystal structure analysis revealed that the complementarity determining regions 2 and 3 of this VHH embrace M2e. The second selected VHH specifically binds to the mouse Fcγ Receptor IV (FcγRIV) and was genetically fused to the M2e-specific VHH, which resulted in a bi-specific VHH-based construct that could be efficiently expressed in Pichia pastoris. In the presence of M2 expressing or influenza A virus-infected target cells, this single domain antibody construct selectively activated the mouse FcγRIV. Moreover, intranasal delivery of this bispecific FcγRIV-engaging VHH construct protected wild type but not FcγRIV−/− mice against challenge with an H3N2 influenza virus. These results provide proof of concept that VHHs directed against a surface exposed viral antigen can be readily armed with effector functions that trigger protective antiviral activity beyond direct virus neutralization.

Identification of a Porcine Liver EomeshighT-betlow NK Cell Subset That Resembles Human Liver Resident NK Cells.

Natural killer (NK) cells are cells of the innate immunity and play an important role in the defense against viral infections and cancer, but also contribute to shaping adaptive immune responses. Long-lived tissue-resident NK cells have been described in man and mouse, particularly in the liver, contributing to the idea that the functional palette of NK cells may be broader than originally thought, and may include memory-like responses and maintaining tissue homeostasis. Remarkably, liver resident (lr)NK cells in man and mouse show substantial species-specific differences, in particular reverse expression patterns of the T-box transcription factors Eomesodermin (Eomes) and T-bet (EomeshighT-betlow in man and vice versa in mouse). In pig, compared to blood NK cells which are CD3−CD8αhigh cells, the porcine liver contains an abundant additional CD3−CD8αdim NK cell subpopulation. In the current study, we show that this porcine CD3−CD8αdim liver NK population is highly similar to its human lrNK counterpart and therefore different from mouse lrNK cells. Like human lrNK cells, this porcine NK cell population shows an EomeshighT-betlow expression pattern. In addition, like its human counterpart, the porcine liver NK population is CD49e− and CXCR6+. Furthermore, the porcine EomeshighT-betlow liver NK cell population is able to produce IFN-γ upon IL-2/12/18 stimulation but lacks the ability to kill K562 or pseudorabies virus-infected target cells, although limited degranulation could be observed upon incubation with K562 cells or upon CD16 crosslinking. All together, these results show that porcine EomeshighT-betlow NK cells in the liver strongly resemble human lrNK cells, and therefore indicate that the pig may represent a unique model to study the function of these lrNK cells in health and disease.

A Subset of CXCR5+CD8+ T Cells in the Germinal Centers From Human Tonsils and Lymph Nodes Help B Cells Produce Immunoglobulins.

Recent studies indicated that CXCR5+CD8+ T cells in lymph nodes could eradicate virus-infected target cells. However, in the current study we found that a subset of CXCR5+CD8+ T cells in the germinal centers from human tonsils or lymph nodes are predominately memory cells that express CD45RO and CD27. The involvement of CXCR5+CD8+ T cells in humoral immune responses is suggested by their localization in B cell follicles and by the concomitant expression of costimulatory molecules, including CD40L and ICOS after activation. In addition, CXCR5+CD8+ memory T cells produced significantly higher levels of IL-21, IFN-γ, and IL-4 at mRNA and protein levels compared to CXCR5−CD8+ memory T cells, but IL-21-expressing CXCR5+CD8+ T cells did not express Granzyme B and perforin. When cocultured with sorted B cells, sorted CXCR5+CD8+ T cells promoted the production of antibodies compared to sorted CXCR5−CD8+ T cells. However, fixed CD8+ T cells failed to help B cells and the neutralyzing antibodies against IL-21 or CD40L inhibited the promoting effects of sorted CXCR5+CD8+ T cells on B cells for the production of antibodies. Finally, we found that in the germinal centers of lymph nodes from HIV-infected patients contained more CXCR5+CD8+ T cells compared to normal lymph nodes. Due to their versatile functional capacities, CXCR5+CD8+ T cells are promising candidate cells for immune therapies, particularly when CD4+ T cell help are limited.

Viral infection sensitizes hepatocytes towards CD8 T cell mediated antiviral immunity

Virus-specific cytotoxic T Lymphocytes (CTLs) are crucial for the elimination of viral infections. So far, CTL-mediated killing of virus infected target cells is believed to depend on cell-cell contact depended recognition of antigen presented on MHC class I molecules. Therefore, the mechanisms determining the efficiency of target cell killing have been mainly investigated in an immune-centric fashion. Previous work on the non-canonical CTL effector function in our group shows selective killing of virus-infected hepatocytes by secreted TNF independent of direct target/effector cell contact. Therefore, we aimed to investigate the role of the target cells in antiviral immunity during experimental adenovirus infection as well as in HBV infection.

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.

A Conserved HIV-1-Derived Peptide Presented by HLA-E Renders Infected T-cells Highly Susceptible to Attack by NKG2A/CD94-Bearing Natural Killer Cells.

Major histocompatibility class I (MHC-I)-specific inhibitory receptors on natural killer (NK) cells (iNKRs) tolerize mature NK cell responses toward normal cells. NK cells generate cytolytic responses to virus-infected or malignant target cells with altered or decreased MHC-I surface expression due to the loss of tolerizing ligands. The NKG2A/CD94 iNKR suppresses NK cell responses through recognition of the non-classical MHC-I, HLA-E. We used HIV-infected primary T-cells as targets in an in vitro cytolytic assay with autologous NK cells from healthy donors. In these experiments, primary NKG2A/CD94+ NK cells surprisingly generated the most efficient responses toward HIV-infected T-cells, despite high HLA-E expression on the infected targets. Since certain MHC-I-presented peptides can alter recognition by iNKRs, we hypothesized that HIV-1-derived peptides presented by HLA-E on infected cells may block engagement with NKG2A/CD94, thereby engendering susceptibility to NKG2A/CD94+ NK cells. We demonstrate that HLA-E is capable of presenting a highly conserved peptide from HIV-1 capsid (AISPRTLNA) that is not recognized by NKG2A/CD94. We further confirmed that HLA-C expressed on HIV-infected cells restricts attack by KIR2DL+ CD56dim NK cells, in contrast to the efficient responses by CD56bright NK cells, which express predominantly NKG2A/CD94 and lack KIR2DLs. These findings are important since the use of NK cells was recently proposed to treat latently HIV-1-infected patients in combination with latency reversing agents. Our results provide a mechanistic basis to guide these future clinical studies, suggesting that ex vivo-expanded NKG2A/CD94+ KIR2DL- NK cells may be uniquely beneficial.

Pseudorabies Virus US3 Protein Kinase Protects Infected Cells from NK Cell-Mediated Lysis via Increased Binding of the Inhibitory NK Cell Receptor CD300a.

Several reports have indicated that natural killer (NK) cells are of particular importance in the innate response against herpesvirus infections. As a consequence, herpesviruses have developed diverse mechanisms for evading NK cells, although few such mechanisms have been identified for the largest herpesvirus subfamily, the alphaherpesviruses. The antiviral activity of NK cells is regulated by a complex array of interactions between activating/inhibitory receptors on the NK cell surface and the corresponding ligands on the surfaces of virus-infected cells. Here we report that the US3 protein kinase of the alphaherpesvirus pseudorabies virus (PRV) displays previously uncharacterized immune evasion properties: it triggers the binding of the inhibitory NK cell receptor CD300a to the surface of the infected cell, thereby providing increased CD300a-mediated protection of infected cells against NK cell-mediated lysis. US3-mediated CD300a binding was found to depend on aminophospholipid ligands of CD300a and on group I p21-activated kinases. These data identify a novel alphaherpesvirus strategy for evading NK cells and demonstrate, for the first time, a role for CD300a in regulating NK cell activity upon contact with virus-infected target cells. Herpesviruses have developed fascinating mechanisms to evade elimination by key elements of the host immune system, contributing to their ability to cause lifelong infections with recurrent reactivation events. Natural killer (NK) cells are central in the innate antiviral response. Here we report that the US3 protein kinase of the alphaherpesvirus pseudorabies virus displays a previously uncharacterized capacity for evasion of NK cells. Expression of US3 protects infected cells from NK cell-mediated lysis via increased binding of the inhibitory NK cell receptor CD300a. We show that this US3-mediated increase in CD300a binding depends on aminophospholipids and on cellular p21-activated kinases (PAKs). The identification of this novel NK cell evasion strategy may contribute to the design of improved herpesvirus vaccines and may also have significance for other PAK- and CD300a-modulating viruses and cancer cells.

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.

A robust and scalable TCR-based reporter cell assay to measure HIV-1 Nef-mediated T cell immune evasion

HIV-1 evades cytotoxic T cell responses through Nef-mediated downregulation of HLA class I molecules from the infected cell surface. Methods to quantify the impact of Nef on T cell recognition typically employ patient-derived T cell clones; however, these assays are limited by the cost and effort required to isolate and maintain primary cell lines. The variable activity of different T cell clones and the limited number of cells generated by re-stimulation can also hinder assay reproducibility and scalability. Here, we describe a heterologous T cell receptor reporter assay and use it to study immune evasion by Nef. Induction of NFAT-driven luciferase following co-culture with peptide-pulsed or virus-infected target cells serves as a rapid, quantitative and antigen-specific measure of T cell recognition of its cognate peptide/HLA complex. We demonstrate that Nef-mediated downregulation of HLA on target cells correlates inversely with T cell receptor-dependent luminescent signal generated by effector cells. This method provides a robust, flexible and scalable platform that is suitable for studies to measure Nef function in the context of different viral peptide/HLA antigens, to assess the function of patient-derived Nef alleles, or to screen small molecule libraries to identify novel Nef inhibitors.

Endocytosis of cytotoxic granules in CD8 T lymphocytes is required for multiple killing of target cells (IRM14P.448)

Abstract Cytotoxic (CD8+) T lymphocytes (CTLs) eliminate tumorigenic and virus-infected target cells by releasing the contents of cytotoxic granules (CGs) at the immunological synapse. Even though CTLs have only a limited number of pre-existing fusogenic CGs, they can efficiently kill multiple target cells upon activation. We therefore hypothesized that, following CG fusion, the remaining components are recycled and subsequently refilled with cytotoxic substances to become available for the next round of fusion, thus enabling efficient multiple killing. We visualized endogenous CGs and their recycling in real-time in CTLs from synaptobrevin2-mRFP knock-in mice by fluorescently-labeled anti-RFP antibodies. We found that endocytosis (recycling) of CGs was blocked by Dynasore, an inhibitor of dynamin. Additionally, clathrin was colocalized with endocytosing CGs at the plasma membrane following dynasore pretreatment. Thus, endocytosis of CGs is clathrin- and dynamin-dependent. Importantly, although CTLs pre-treated with Dynasore showed no immediate effect on CG exocytosis, they exhibited a 50% deficiency in killing target cells after 2 hours of incubation. Our data strongly suggest that recycling of CGs plays an important role in CTLs for executing multiple killing of target cells.

Fas/FasL pathway participates in regulation of antiviral and inflammatory response during mousepox infection of lungs.

Fas receptor-Fas ligand (FasL) signalling is involved in apoptosis of immune cells as well as of the virus infected target cells but increasing evidence accumulates on Fas as a mediator of apoptosis-independent processes such as induction of activating and proinflammatory signals. In this study, we examined the role of Fas/FasL pathway in inflammatory and antiviral response in lungs using a mousepox model applied to C57BL6/J, B6. MRL-Faslpr/J, and B6Smn.C3-Faslgld/J mice. Ectromelia virus (ECTV) infection of Fas- and FasL-deficient mice led to increased virus titers in lungs and decreased migration of IFN-γ expressing NK cells, CD4+ T cells, CD8+ T cells, and decreased IL-15 expression. The lungs of ECTV-infected Fas- and FasL-deficient mice showed significant inflammation during later phases of infection accompanied by decreased expression of anti-inflammatory IL-10 and TGF-β1 cytokines and disturbances in CXCL1 and CXCL9 expression. Experiments in vitro demonstrated that ECTV-infected cultures of epithelial cells, but not macrophages, upregulate Fas and FasL and are susceptible to Fas-induced apoptosis. Our study demonstrates that Fas/FasL pathway during ECTV infection of the lungs plays an important role in controlling local inflammatory response and mounting of antiviral response.

Prevention of hepatitis C virus infection by adoptive allogeneic immunotherapy using suicide gene-modified lymphocytes: an in vitro proof-of-concept.

Hepatitis C virus (HCV)-induced, end-stage liver disease is a major indication for liver transplantation, but systematic graft reinfection accelerates liver disease recurrence. Transplantation recipients may be ineligible for direct-acting antivirals, owing to toxicity, resistance or advanced liver disease. Adoptive immunotherapy with liver graft-derived, ex vivo-activated lymphocytes was previously shown to prevent HCV-induced graft reinfections. Alternatively, the applicability and therapeutic efficacy of adoptive immunotherapy may be enhanced by 'ready for use' suicide gene-modified lymphocytes from healthy blood donors; moreover, conditional, prodrug-induced cell suicide may prevent potential side effects. Here, we demonstrate that allogeneic suicide gene-modified lymphocytes (SGMLs) could potently, dose- and time-dependently, inhibit viral replication. The effect occurs at effector:target cell ratios that exhibits no concomitant cytotoxicity toward virus-infected target cells. The effect, mediated mostly by CD56+ lymphocytes, is interleukin-2-dependent, IFN-γ-mediated and, importantly, resistant to calcineurin inhibitors. Thus, post-transplant immunosuppression may not interfere with this adoptive cell immunotherapy approach. Furthermore, these cells are indeed amenable to conditional cell suicide; in particular, the inducible caspase 9 suicide gene is superior to the herpes simplex virus thymidine kinase suicide gene. Our data provide in vitro proof-of-concept that allogeneic, third-party, SGMLs may prevent HCV-induced liver graft reinfection.

Reply to “CD8 + T Cells Are Essential for Controlling Acute Friend Virus Infection in C57BL/6 Mice”

Reply to “CD8 ⁺ T Cells Are Essential for Controlling Acute Friend Virus Infection in C57BL/6 Mice”

Organ-specific phenotypic and functional features of NK cells in humans

Natural killer (NK) cells kill virus-infected and tumor target cells without prior sensitization. Each NK cell expresses a multitude of activating and inhibitory receptors, and the interplay of signals determines the outcome of NK cell activity. NK cell-mediated cytolysis of target cell involves polarized degranulation at effector-target interface. Peripheral blood NK cell constitutes about 10% of lymphocytes, and approximately 90% of peripheral blood NK cells are CD56(dim)CD16(+); however, there is a distinct subset of NK cells, CD56(bright)CD16(-), expressed by certain lymphoid organs which are able to produce large amounts of cytokines including interferon-γ, tumor necrosis factor, and granulocyte-macrophage colony-stimulating factor, but the cytotoxicity is attained only on their prolonged activation. In this review, we discuss the accumulated data on distinct phenotypes of NK cells in human uterus, liver, intestine, skin, and lung and also attempt to correlate their phenotype with corresponding activity and functions, with significant stress on the role of NK cells in pathology in the specific organs. Our detailed understanding of altered NK cell activity in different organs and their inherent cytotoxic activity against tumor target cells will help us design better immunotherapeutic strategies in NK cell-mediated cancer therapies.

HLA-A2, HLA-B44 and HLA-DR15 are associated with lower risk of BK viremia

Human leucocyte antigens (HLAs) modulate immunity to polyomavirus BK (BKV). Identification of HLAs that alter the course of infection will facilitate risk stratification, and customization of pre-emptive intervention strategies. We performed a retrospective cohort study with 998 kidney transplant patients with BKV infection status confirmed by polymerase chain reaction (PCR). Clinical parameters and donor-recipient matching for specific HLAs were examined in relation to occurrence of viremia. An emphasis was placed on donor-recipient matching rather than the actual frequency of specific HLA-alleles, since a successful immune response requires sharing of HLAs between a virus-infected target cell and the anti-viral effector cell. Using multivariate statistics, low risk of BK viremia was associated with matching of HLA-A2 [hazard ratio (HR) 0.51, 95% confidence interval (CI) 0.28-0.85], HLA-B44 (HR 0.31, 95% CI 0.076-0.85) and HLA-DR15 (HR 0.35, 95% CI 0.084-0.93) (P < 0.05), whereas high risk of viremia was associated with male gender (HR 2.38, 95% CI 1.46-4.09, P < 0.001). HLAs that associated with a lower predisposition to the development of BK viremia have been identified. Evaluation of donor-recipient mismatching for these HLAs could potentially be used to (i) fine tune virus screening strategies for BKV in individual patients and (ii) facilitate discovery of major histocompatibility complex (MHC) class I and II binding peptides that can elicit clinically meaningful BKV-specific immunity.

CD8 down-regulation and functional impairment of SIV-specific cytotoxic T lymphocytes in lymphoid and mucosal tissues during SIV infection

Functional impairment of virus-specific T cells is a hallmark of HIV/SIV infection, but the underlying mechanisms of this dysfunction are not well understood. To address this, we simultaneously analyzed the expression and intensity of CD8 and inhibitory PD-1 on CTL in blood and lymphoid tissues in SIV-infected rhesus macaques. The intensity (mean channel fluorescence) of CD8 expression was transiently down-regulated in early SIV infection (10-14 dpi), despite an increase in CD8(+) T cell proliferation. In chronic infection, CD8 expression was maintained at low levels on CD8(+) T cells in all tissues. Interestingly, Gag-specific CTLs were clearly divided into CD8high- and CD8low-expressing populations in SIV-infected macaques, and CD8low Gag-specific cells increased with disease progression, especially in lymphoid tissues when compared with peripheral blood or in Gag-vaccinated controls. Moreover, the CD8low CTL population secreted lower levels of cytokines upon SIV antigen stimulation and exhibited lower proliferative capacity during infection compared with the CD8high CTL population. Meanwhile, intensity of PD-1 expression on Gag-specific CTL in chronic infection was significantly higher than in acute SIV infection, although the frequencies of PD-1+ Gag-specific cells were similar in acute and chronic stages. In summary, down-regulation of CD8 expression and higher expression of PD-1 on SIV-specific CTLs could coordinately attenuate SIV-specific CTL responses and their ability to recognize virus-infected target cells, especially in lymphoid tissues, resulting in failure to contain viremia, and continued persistence and replication of HIV in lymphoid tissue reservoirs.

Synaptobrevin2 is the v-SNARE required for cytotoxic T-lymphocyte lytic granule fusion

Cytotoxic T lymphocytes kill virus-infected and tumorigenic target cells through the release of perforin and granzymes via fusion of lytic granules at the contact site, the immunological synapse. It has been postulated that this fusion process is mediated by non-neuronal members of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex protein family. Here, using a synaptobrevin2-monomeric red fluorescence protein knock-in mouse we demonstrate that, surprisingly, the major neuronal v-SNARE synaptobrevin2 is expressed in cytotoxic T lymphocytes and exclusively localized on granzyme B-containing lytic granules. Cleavage of synaptobrevin2 by tetanus toxin or ablation of the synaptobrevin2 gene leads to a complete block of lytic granule exocytosis while leaving upstream events unaffected, identifying synaptobrevin2 as the v-SNARE responsible for the fusion of lytic granules at the immunological synapse.