*Telomerase reverse transcribes telomere DNA onto the ends of linear chromosomes and retards cellular aging. In contrast to most normal somatic cells, which show little or no telomerase activity, immune cells up-regulate telomerase in concert with activation. Nevertheless, during aging and chronic HIV-1 infection, there are high proportions of dysfunctional CD8؉ CTL with short telomeres, suggesting that telomerase is limiting. The present study shows that exposure of CD8 ؉ T lymphocytes from HIV-infected human donors to a small molecule telomerase activator (TAT2) modestly retards telomere shortening, increases proliferative potential, and, importantly, enhances cytokine/chemokine production and antiviral activity. The enhanced antiviral effects were abrogated in the presence of a potent and specific telomerase inhibitor, suggesting that TAT2 acts primarily through telomerase activation. Our study is the first to use a pharmacological telomerase-based approach to enhance immune function, thus directly addressing the telomere loss immunopathologic facet of chronic viral infection. The Journal of Immunology, 2008, 181: 7400 -7406.T elomeres, the TTAGGG tandem repeats at chromosome ends, become progressively shorter in dividing somatic cells (1). Numerous studies have shown associations between leukocyte telomere shortening and risk of disease, suggesting that these cells may serve as biomarkers for the cumulative burden of inflammation, chronic immune activation, or oxidative stress. Indeed, hypertension and increased insulin resistance are associated with shorter leukocyte telomere length in the participants of the Framingham heart study (2), and telomere shortening in peripheral blood leukocytes chronicles ischemic heart disease risk in older people (3). Leukocyte telomere length also correlates with bone mineral density, and shorter telomeres are seen in women with osteoporosis (4). Finally, in both aging and chronic HIV-1 infection, there are increased proportions of CD8 ϩ T lymphocytes with shortened telomeres, reduced proliferative capacity and altered effector function (5). Thus, strategies to retard or prevent telomere loss may lead to novel treatments for a variety of human pathologies.T and B lymphocytes transiently up-regulate telomerase, the cellular reverse transcriptase that adds telomeric DNA to the ends of chromosomes (6 -8). Such telomerase activity is believed to mitigate the losses of replicative capacity and function caused by chronic antigenic stimulation, oxidative stress, and cellular aging (9, 10). However, the ability of CD8 ϩ T lymphocytes to up-regulate telomerase is lost after repeated encounters with Ag, and continued chronic stimulation ultimately leads to critically short telomeres and other changes associated with replicative senescence (10, 11).We hypothesized that sustained telomerase activity in chronically activated CD8 ϩ T lymphocytes might prevent or delay immune dysfunction associated with aging and/or chronic disease. In previous studies, we showed that gene transduction of ...

Although the use of chimeric antigen receptors (CARs) based on single-chain antibodies for gene immunotherapy of cancers is increasing due to promising recent results, the earliest CAR therapeutic trials were done for HIV-1 infection in the late 1990s. This approach utilized a CAR based on human CD4 as a binding domain and was abandoned for a lack of efficacy. The growing number of HIV-1 broadly neutralizing antibodies (BNAbs) offers the opportunity to generate novel CARs that may be more active and revisit this modality for HIV-1 immunotherapy. We used sequences from seven well-defined BNAbs varying in binding sites and generated single-chain-antibody-based CARs. These CARs included 10E8, 3BNC117, PG9, PGT126, PGT128, VRC01, and X5. Each novel CAR exhibited conformationally relevant expression on the surface of transduced cells, mediated specific proliferation and killing in response to HIV-1-infected cells, and conferred potent antiviral activity (reduction of viral replication in log 10 units) to transduced CD8 ؉ T lymphocytes. The antiviral activity of these CARs was reproducible but varied according to the strain of virus. These findings indicated that BNAbs are excellent candidates for developing novel CARs to consider for the immunotherapeutic treatment of HIV-1. R ecent years have seen a surge in immunotherapeutic approaches for treating malignancy, including numerous promising human trials of chimeric antigen receptor (CAR) gene therapy to generate tumor-specific T cells, based on the importance of CD8 ϩ T lymphocytes (CTLs) in tumor surveillance and malignant cell clearance through cytotoxicity. The general approach has been to identify monoclonal antibodies that bind a tumor cell surface antigen and use a single-chain version of the antibody as an artificial T cell receptor by genetic fusion to the CD3 chain signaling domain. As opposed to native T cell receptors (TCRs), CARs have the advantage of being major histocompatibility complex (MHC) unrestricted and therefore broadly applicable across human individuals and are also unaffected by tumor cell immune evasion through MHC downregulation. IMPORTANCE While chimeric antigen receptors (CARsNotably, one of the earliest tested clinical applications of CARs was for the treatment of HIV-1 infection. In 1994, Roberts et al. designed two virus-specific CARs using CD4 or a single-chain antibody as the binding domain for recombinant gp120 on the surface of cells (1), and these CARs were shown subsequently to have the direct capacity to kill HIV-1-infected cells and suppress viral replication at levels similar to those of HIV-1-specific CTL clones isolated from infected persons (2). Based on these data, the CD4-based CAR, consisting of the CD4 extracellular and transmembrane domains fused to the CD3 intracellular signaling domain (CD4 Ϫ ), was advanced to clinical trials starting in the late 1990s, using retroviral transduction of autologous peripheral blood T lymphocytes and reinfusion. Unfortunately, this effort was abandoned after these trials showed...

Cytotoxic T lymphocytes (CTLs) are crucial for immune control of viral infections. "Functional avidity," defined by the sensitizing dose of exogenously added epitope yielding half-maximal CTL triggering against uninfected target cells (SD 50 ), has been utilized extensively as a measure of antiviral efficiency. However, CTLs recognize infected cells via endogenously produced epitopes, and the relationship of SD 50 to antiviral activity has never been directly revealed. We elucidate this relationship by comparing CTL killing of cells infected with panels of epitope-variant viruses to the corresponding SD 50 for the variant epitopes. This reveals a steeply sigmoid relationship between avidity and infected cell killing, with avidity thresholds (defined as the SD 50 required for CTL to achieve 50% efficiency of infected cell killing [KE 50 ]), below which infected cell killing rapidly drops to none and above which killing efficiency rapidly plateaus. Three CTL clones recognizing the same viral epitope show the same KE 50 despite differential recognition of individual epitope variants, while CTLs recognizing another epitope show a 10-fold-higher KE 50 , demonstrating epitope dependence of KE 50 . Finally, the ability of CTLs to suppress viral replication depends on the same threshold KE 50 . Thus, defining KE 50 values is required to interpret the significance of functional avidity measurements and predict CTL efficacy against virus-infected cells in pathogenesis and vaccine studies.

No abstract

The recent success in ribosome structure determination by cryoEM has opened the door to defining structural differences between ribosomes of pathogenic organisms and humans and to understand ribosome-targeting antibiotics. Here, by direct electron-counting cryoEM, we have determined the structures of the Leishmania donovani and human ribosomes at 2.9 Å and 3.6 Å, respectively. Our structure of the leishmanial ribosome elucidates the organization of the six fragments of its large subunit rRNA (as opposed to a single 28S rRNA in most eukaryotes, including humans) and reveals atomic details of a unique 20 amino acid extension of the uL13 protein that pins down the ends of three of the rRNA fragments. The structure also fashions many large rRNA expansion segments. Direct comparison of our human and leishmanial ribosome structures at the decoding A-site sheds light on how the bacterial ribosome-targeting drug paromomycin selectively inhibits the eukaryotic L. donovani, but not human, ribosome.