NK cells are the first line of defense against viruses and malignant cells, and their natural functionality makes these cells a promising candidate for cancer cell therapy. The genetic modifications of NK cells, allowing them to overcome some of their inherent limitations, such as low proliferative potential, can enable their use as a therapeutic product. We demonstrate that hTERT-engineered NK cell cultures maintain a high percentage of cells in the S/G2 phase for an extended time after transduction, while the life span of NK cells is measurably extended. Bulk and clonal NK cell cultures pre-activated in vitro with IL-2 and K562-mbIL21 feeder cells can be transduced with hTERT more efficiently compared with the cells activated with IL-2 alone. Overexpressed hTERT was functionally active in transduced NK cells, which displayed upregulated expression of the activation marker HLA-DR, and decreased expression of the maturation marker CD57 and activating receptor NKp46. Larger numbers of KIR2DL2/3+ cells in hTERT-engineered populations may indicate that NK cells with this phenotype are more susceptible to transduction. The hTERT-modified NK cells demonstrated a high natural cytotoxic response towards K562 cells and stably expressed Ki67, a proliferation marker. Overall, our data show that ectopic hTERT expression in NK cells enhances their activation and proliferation, extends in vitro life span, and can be a useful tool in developing NK-based cancer cell therapies.

Adoptive cell transfer (ACT) is a powerful experimental approach to directly study T-cell-mediated immunity in vivo In the rhesus macaque AIDS virus model, infusing simian immunodeficiency virus (SIV)-infected animals with CD8 T cells engineered to express anti-SIV T-cell receptor specificities enables direct experimentation to better understand antiviral T-cell immunity in vivo Limiting factors in ACT experiments include suboptimal trafficking to, and poor persistence in, the secondary lymphoid tissues targeted by AIDS viruses. Previously, we redirected CD8 T cells to B-cell follicles by ectopic expression of the CXCR5 homing protein. Here, we modify peripheral blood mononuclear cell (PBMC)-derived CD8 T cells to express the CCR9 chemokine receptor, which induces preferential homing of the engineered cells to the small intestine, a site of intense early AIDS virus replication and pathology in rhesus macaques. Additionally, we increase in vivo persistence and overall systemic distribution of infused CD8 T cells, especially in secondary lymphoid tissues, by minimizing ex vivo culture/manipulation, thereby avoiding the loss of CD28+/CD95+ central memory T cells by differentiation in culture. These proof-of-principle results establish the feasibility of preferentially localizing PBMC-derived CD8 T cells to the small intestine and enables the direct experimental ACT-based assessment of the potential role of the quality and timing of effective antiviral CD8 T-cell responses to inhibit viral infection and subsequent replication in small intestine CD4 T cells. More broadly, these results support the engineered expression of homing proteins to direct CD8 T cells to target tissues as a means for both experimental and potential therapeutic advances in T-cell immunotherapies, including cancer.IMPORTANCEAdoptive cell transfer (ACT) of T cells engineered with antigen-specific effector properties can deliver targeted immune responses against malignancies and infectious diseases. Current T-cell-based therapeutic ACT relies on circulatory distribution to deliver engineered T cells to their targets, an approach which has proven effective for some leukemias but provided only limited efficacy against solid tumors. Here, engineered expression of the CCR9 homing receptor redirected CD8 T cells to the small intestine in rhesus macaque ACT experiments. Targeted homing of engineered T-cell immunotherapies holds promise to increase the effectiveness of adoptively transferred cells in both experimental and clinical settings.

ABSTRACT AIDS virus infections are rarely controlled by cell-mediated immunity, in part due to viral immune evasion and immunodeficiency resulting from CD4 + T-cell infection. One likely aspect of this failure is that antiviral cellular immune responses are either absent or present at low levels during the initial establishment of infection. To test whether an extensive, timely, and effective response could reduce the establishment of infection from a high-dose inoculum, we adoptively transferred large numbers of T cells that were molecularly engineered with anti-simian immunodeficiency virus (anti-SIV) activity into rhesus macaques 3 days following an intrarectal SIV inoculation. To measure in vivo antiviral activity, we assessed the number of viruses transmitted using SIVmac239X, a molecularly tagged viral stock containing 10 genotypic variants, at a dose calculated to transmit 12 founder viruses. Single-genome sequencing of plasma virus revealed that the two animals receiving T cells expressing SIV-specific T-cell receptors (TCRs) had significantly fewer viral genotypes than the two control animals receiving non-SIV-specific T cells (means of 4.0 versus 7.5 transmitted viral genotypes; P = 0.044). Accounting for the likelihood of transmission of multiple viruses of a particular genotype, the calculated means of the total number of founder viruses transmitted were 4.5 and 14.5 in the experimental and control groups, respectively ( P = 0.021). Thus, a large antiviral T-cell response timed with virus exposure can limit viral transmission. The presence of strong, preexisting T-cell responses, including those induced by vaccines, might help prevent the establishment of infection at the lower-exposure doses in humans that typically transmit only a single virus. IMPORTANCE The establishment of AIDS virus infection in an individual is essentially a race between the spreading virus and host immune defenses. Cell-mediated immune responses induced by infection or vaccination are important contributors in limiting viral replication. However, in human immunodeficiency virus (HIV)/SIV infection, the virus usually wins the race, irreversibly crippling the immune system before an effective cellular immune response is developed and active. We found that providing an accelerated response by adoptively transferring large numbers of antiviral T cells shortly after a high-dose mucosal inoculation, while not preventing infection altogether, limited the number of individual viruses transmitted. Thus, the presence of strong, preexisting T-cell responses, including those induced by vaccines, might prevent infection in humans, where the virus exposure is considerably lower.

Infection of CD4 T cells by HIV/SIV inhibits the formation of robust antiviral responses. Furthermore, HIV-specific CD4 T cells are preferentially infected and eliminated in vivo. This highlights the importance of protecting virus-specific CD4 T cells to effectively study their antiviral mechanisms. In this study, we have shown that African green monkey (Agm) TRIM5α restricts SIVmac239 replication in primary rhesus macaque CD4 T cells and synergizes with an intrinsic resistance present in some CD4 T-cell clones. Importantly, AgmTRIM5α-transduced effector CD4 T cells exhibit an increased ability to suppress viral replication in infected target cells. This increase in antiviral effector function was strongly linked to decreased infection and prolonged persistence of the AgmTRIM5α-expressing effectors. Hence, AgmTRIM5α restriction in rhesus CD4 T cells offers an approach to evaluate their anti-SIV effector functions. Currently, we are pursuing studies to determine the effectiveness of SIV-specific CD8 and CD4 T cells in controlling infection in an adoptive transfer model, where the genetically modified CD4 T-cell clones express AgmTRIM5α to prolong their persistence in vivo.

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes 12 pre-microRNAs that can produce 25 KSHV mature microRNAs. We previously reported single-nucleotide polymorphisms (SNPs) in KSHV-encoded pre-microRNA and mature microRNA sequences from clinical samples (V. Marshall et al., J. Infect. Dis., 195:645-659, 2007). To determine whether microRNA SNPs affect pre-microRNA processing and, ultimately, mature microRNA expression levels, we performed a detailed comparative analysis of (i) mature microRNA expression levels, (ii) in vitro Drosha/Dicer processing, and (iii) RNA-induced silencing complex-dependent targeting of wild-type (wt) and variant microRNA genes. Expression of pairs of wt and variant pre-microRNAs from retroviral vectors and measurement of KSHV mature microRNA expression by real-time reverse transcription-PCR (RT-PCR) revealed differential expression levels that correlated with the presence of specific sequence polymorphisms. Measurement of KSHV mature microRNA expression in a panel of primary effusion lymphoma cell lines by real-time RT-PCR recapitulated some observed expression differences but suggested a more complex relationship between sequence differences and expression of mature microRNA. Furthermore, in vitro maturation assays demonstrated significant SNP-associated changes in Drosha/DGCR8 and/or Dicer processing. These data demonstrate that SNPs within KSHV-encoded pre-microRNAs are associated with differential microRNA expression levels. Given the multiple reports on the involvement of microRNAs in cancer, the biological significance of these phenotypic and genotypic variants merits further studies in patients with KSHV-associated malignancies.

Published Ahead of Print 4 September 2013. 2013, 87(22):12237. DOI: 10.1128/JVI.01202-13. J. Virol. David Ott, Rolf Renne and Denise Whitby Quiñones, Alex Ray, Nazzarena Labo, Matthew Trivett, Soo-Jin Han, Vickie Marshall, Eugene Barsov, Octavio Level of Expression, and Silencing Activity Samples Can Affect MicroRNA Processing, Polymorphisms Identified in Clinical MicroRNA Single-Nucleotide Kaposi's Sarcoma-Associated Herpesvirus

MiRNAs are a class of non-coding RNA molecules between 19-25 nucleotides in length that have been shown to be involved in many biological processes by post-transcriptionally regulating gene expression. Aberrant miRNA expression has recently been associated with disease including many human cancers. Kaposi’s sarcoma associated herpesvirus (KSHV) encodes 12 miRNAs located within the latency associated region. We previously reported single nucleotide polymorphisms (SNP) in the sequence of KSHV encoded mature and pre-miRNAs from clinical samples [1]. An earlier report showed that a SNP in mir-K12-5 resulted in increased expression of the mature miRNA [2]. In the current study, we have analyzed three different classes of miRNA polymorphisms to determine if any affect mature miRNA processing and expression. The identified SNPs include single and multiple polymorphisms within the pre-miRNA transcript, single mutations within the terminal loop, and single sequence changes within the mature miRNA. We used four complimentary approaches to detect differences in miRNA processing and expression resulting from sequence polymorphisms. Analysis of KSHV miRNA expression levels in PEL cell lines using custom ABI real time qPCR assays showed differential expression that correlates with sequence variation. Lentiviral vectors constructed to express wild type and variant pre-miRNAs were transduced into 293T cells to make stably expressing cell lines. miRNA expression was assessed using custom ABI real time qPCR assays. Luciferase reporter assays were performed following transient transfections of each miRNA. In addition, in vitro maturation assays were performed to assess differences in Drosha/DGCR8 and Dicer cleavage between wild type and variant pre-miRNAs. Our results show that polymorphisms within the pre-miRNA sequence can cause subtle expression differences as in the case of KSHV miR-K12-6 or more profound changes as observed in miR-K12-5. This is also the case with SNPs located within the terminal loop as some miRNAs exhibited no discernable change, miR-K12-7 and miR-K12-10, while others can cause a reduction in mature miRNA transcripts as noted in miR-K12-4. Mutations within the mature transcript appear to have the most potential to effect mature transcript processing and expression. The polymorphism within miR-K12-2 results in reduction of mature miRNA levels while the multiple changes in miR-K12-9 leads to complete loss of the mature transcript. Our data clearly show that SNPs can affect pre-miRNA processing resulting in changes in mature miRNA expression levels. The biological significance of these phenotypic and genotypic variants merits further study.

Kaposi sarcoma-associated herpesvirus (KSHV) encodes 12 pre-microRNAs which potentially yield 25 mature microRNAs and have been shown to play prominent roles in the viral lifecycle including maintaining viral latency, evading the host immune response, and controlling lytic replication. We previously reported phylogenetic analysis of the microRNA-coding region of KSHV from Kaposi sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman disease (MCD) patients. We showed a high level of conservation for most sequences, but also a divergent cluster of 5 KSHV sequences including 2 from MCD patients [1]. We additionally observed single nucleotide polymorphisms (SNP) in the sequence of KSHV encoded mature and pre-miRNAs from clinical samples including a SNP in mir-K12-5 reported to result in increased expression of the mature miRNA [2]. To determine whether SNPs in other KSHV encoded miRNAs resulted in differences in miRNA processing and expression we used four complimentary approaches. Analysis of KSHV miRNA expression levels in PEL cell lines using custom ABI real time qPCR assays showed differential expression that correlates with sequence. Lentiviral vectors constructed to express wild type and variant pre-miRNAs were transduced into 293T cells to make stably expressing cell lines. miRNA expression was assessed using custom ABI real time qPCR assays. Luciferase reporter assays were performed following transient transfections of each miRNA. In addition, in vitro maturation assays were performed to assess differences in Drosha/DGCR8 and Dicer cleavage between wild type and variant pre-miRNAs. Our results indicate that polymorphisms within the pre-miRNA sequence can cause subtle expression differences as in the case of KSHV miR-K12-6 or more profound changes as observed in miR-K12-5. Our data clearly shows that SNPs can affect pre-miRNA processing resulting in changes in mature miRNA expression levels. To extend our studies on miRNA variation in MCD patients, KSHV miRNA sequences from 23 MCD patients and 7 patients with a newly described KSHV-associated inflammatory cytokine syndrome (KICS) were examined by amplification, cloning, and sequencing of a 646-bp fragment of K12/T0.7 encoding miRNA-K12-10 and miRNA-K12-12 and a 2.8-kbp fragment containing the remaining 10 pre-microRNAs. Phylogenetic analysis showed a distinct variant cluster consisting exclusively of MCD and KICS patients in all trees. Pearson’s chi-squared analysis revealed 40 single nucleotide polymorphisms (SNPs) at various loci were significantly associated with MCD and KICS risk. Additionally, cluster analysis of these SNPs generated several combinations of three SNPs as putative indicators of MCD and KICS risk. Taken together, these findings show that MCD and KICS patients frequently have unusual KSHV microRNA sequences and suggest association between the observed sequence variation and risk of MCD and KICS.

[This corrects the article DOI: 10.1371/journal.pone.0023703.].

Human and rhesus macaque primary antigen‐specific T cells derived from infected or immunized individuals or animals are a valuable material with which to study cellular immune responses against pathogens and tumors. Antigen‐specific T cells can be expanded in vitro but have a finite proliferative life span. After a limited period in culture, primary T cells undergo replicative senescence and stop dividing. This restricts their applicability to short‐term experiments and complicates their use in adoptive immunotherapy. The proliferative life span of primary human and rhesus macaque T cells can be considerably extended by ectopically expressed human telomerase reverse transcriptase (TERT). Antigen‐specific T cells transduced with TERT‐expressing retroviral vectors can proliferate and expand in culture for long periods of time while maintaining their primary T cell characteristics, including antigen‐specific responses. Thus, TERT‐immortalized T cells are an important and valuable resource for studying T cell–mediated immune responses and, potentially, for adoptive immunotherapy. Curr. Protoc. Immunol . 95:7.21B.1‐7.21B.20. © 2011 by John Wiley & Sons, Inc.

Studies using transformed human cell lines suggest that most SIV strains use CCR5 as co-receptor. Our analysis of primary rhesus macaque CD4(+) T-cell clones revealed marked differences in susceptibility to SIV(mac)239 infection. We investigated whether different levels of CCR5 expression account for clonal differences in SIV(mac)239 susceptibility. Macaque CD4(+) T-cells showed significant CCR5 downregulation 1-2days following CD3 mAb stimulation, which gradually recovered at resting state, 7-10days after activation. Exposure of clones to SIV(mac)239 during their CCR5(low) or CCR5(high) expression states revealed differences in SIV susceptibility independent of surface CCR5 levels. Furthermore, a CCR5 antagonist similarly reduced SIV(mac)239 infection of clones during their CCR5(low) or CCR5(high) expression states. Our data suggest a model where i) very low levels of CCR5 are sufficient for efficient SIV infection, ii) CCR5 levels above this threshold do not enhance infection, and iii) low level infection can occur in the absence of CCR5.

Telomeres are specialized repeats, present at the end of chromosomes, whose loss during cell division is followed by growth arrest, a central mechanism of replicative senescence in human cells. Telomere length in stem cells is maintained by telomerase, a specialized reverse transcriptase, whose function is to restore shortening telomeres. Unlike most somatic cell types, human T lymphocytes are capable of briefly reactivating telomerase expression at the time of stimulation. Telomerase expression in T lymphocytes is modulated by a variety of external stimuli and by viral infections. However, telomerase reactivation in stimulated, proliferating human T lymphocytes is limited and cannot prevent the ultimate onset of senescence. Ectopic telomerase expression can rescue human and macaque antigen-specific T cells from senescence. Primary T cells have been engineered with telomerase to have substantially extended replicative lifespans without the loss of primary cell functions or malignant transformation. ‘Immortal’ antigen-specific T-cell lines and clones overexpressing telomerase are an invaluable source of well-characterized quasi-primary T cells for research of T-cell biology and are potentially useful for immunotherapy of cancer and AIDS.

AIMS While numerous studies have reported on nanoparticle uptake by phagocytic cells, the mechanisms of this uptake are poorly understood. A metastudy of research focusing on biological particulate matter has postulated that nanoparticles cannot be phagocytosed and therefore must enter cells via pinocytosis. The purpose of this study was to identify the route(s) of uptake of gold nanoparticles in vitro and to determine if these route(s) depend on particle size. MATERIALS & METHODS The parent RAW264.7 cell line and its derivatives, transduced with a virus carrying siRNA to macrophage scavenger receptor A, were used as model phagocytes. Citrate-stabilized gold colloids were used as model nanoparticles. We used chemical inhibitors known to interfere with specific routes of particulate uptake. We developed multifocal light microscopy methods including multifocal stack analysis with NIH ImageJ software to analyze cell uptake. RESULTS Irrespective of size, gold nanoparticles are internalized by macrophages via multiple routes, including both phagocytosis and pinocytosis. If either route was blocked, the particles entered cells via the other route. CONCLUSION Gold nanoparticles with hydrodynamic sizes below 100 nm can be phagocytosed. Phagocytosis of anionic gold colloids by RAW264.7 cells is mediated by macrophage scavenger receptor A.