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Apr 13, 2012

Engineered HSCs Differentiate into HIV-Specific CTLs that Hold Back HIV Infection In Vivo

  • Researchers have demonstrated that hematopoietic stem cells (HSCs) can be engineered to differentiate into HIV-specific cytotoxic CD8+ T cells capable of mounting a potent antiviral immune response that blocks HIV replication in a mouse model.  The team, at the University of California, Los Angeles (UCLA) David Geffen School of Medicine, built on previous work demonstrating that  human HSCs genetically modified with genes encoding a human HIV-specific T-cell receptor (TCR) can produce mature, fully functional T cells in human thymus implants in severe combined immunodeficient (SCID) mice.

    However, while this prior research showed that the resulting CD8+ T cells were capable of killing HIV antigen-expressing cells ex vivo, the SCID-human mouse model demonstrates poor peripheral reconstitution and function of human immune cells, and so it wasn’t possible to evaluate the ability of the HIV-specific HSC-derived cytotoxic T lymphocytes (CTLs) to suppress HIV replication in vivo.

    In their latest work the researchers took the technology a step further and introduced the genetically modified HSCs into a modified version of a humanized mouse model known as the nonobese diabetic (NOD)-SCID, common gamma chain knockout (γc−/−), humanized bone marrow, fetal liver, and thymus (the NSG-BLT) mouse model. These modified NSG-BLT animals can generate of peripheral human immune responses and represent an effective model for HIV infection and pathogenesis.

    The results showed that HSCs carrying the HIV-specific TCR were capable of differentiating into CTLs that suppressed HIV replication in vivo and prevented or at least slowed HIV-related damage to the animals’ human tissues. Jerome A. Zack, M.D., and colleagues report their approach and experimental results in PLoS Pathogens in a paper titled “In Vivo Suppression of HIV by Antigen Specific T Cells Derived from Engineered Hematopoietic Stem Cells.”

    The generation of CTLs that recognize viral antigens and kill virus-infected cells represents a critical component of the body’s natural antiviral responses. However, in the case of HIV infection, the CTL response fails to clear HIV from the body, and even when effective antiretroviral therapy (ART) is administered, the virus isn’t completely cleared. In fact levels of HIV-specific CTLs decline, probably because lower antigen levels fail to stimulate CTL persistence, the David Geffen researchers explain.

    Their earlier studies demonstrated that TCR-modified human HSCs can be directed to develop into mature CTLs in mice engrafted with human thymus, in the context of the proper HLA type. Expanding on these findings, examined the ability of genetically modified T cells derived from HSC transduced with a single HIV-specific TCR to suppress viral replication in vivo.

    NSG mice were implanted with human fetal liver-derived CD34+ HSCs that had been modified with a lentiviral vector carying the genes for a TCR targeting the HIV Gag SL9 epitope. Control HSCs were modified using a lentiviral vector containing a non-HIV-specific TCR with unknown specificity. The mice in addition received implants of human fetal thymus and liver under the kidney capsule. Initial analyses in this NSG-CTL model found that 53% of the CD45+ cells in the animals’ peripheral blood were of human origin, and there was a significant population of human CD34+ HSCs in the bone marrow, most of which co-expressed CD45, indicating they had lymphopoietic potential. The animals also generated significant populations of CD3-expressing T cells and CD19-expressing B cells in the bone marrow, indicating both that they were capable of multilineage human hematopoiesis and that other components of the human immune system were present in addition to T cells.

    The NSG-CTL and control mice were then infected with an HIV-1 variant engineered with a tag that enables the detection of HIV-infected cells using flow cytometry. Within two weeks levels of infected cells were lower, and there was less initial CD4 depletion n animals implanted with the HIV TCR-expressing HSCs than the control animals. And although by six weeks both the HSC-CTL mice and control mice demonstrated overall increases in virus-expressing cells,  HIV-specific TCR animals still demonstrated much lower levels of productively infected cells than the control mice, “indicating suppression of viral replication over time,” the team writes.

    Moreover, at the six week post-infection time point mice containing cells expressing the HIV-specific TCR exhibited significantly greater preservation of CD4+ T cells and higher CD4 to CD8 T cell ratios than mice expressing the control TCR. “Thus, genetic modification of HSCs with a single HIV-specific TCR produces peripheral T cells capable of suppressing cellular HIV expression and CD4 depletion in vivo,” the investigators claim.

    They separately assessed virus levels in the infected animals’ peripheral blood plasma using a novel quantitative PCR-based technique. This similarly confirmed that viral load at two weeks and six weeks post-infection was much lower in mice receiving the HIV-specific TCR HSCs than the control mice, which “suggested systemic suppression of HIV replication,” they add. Encouragingly, viral RNA analysis indicated that there had been no mutation of the specific SL9 viral epitope recognized by the TCR as a result of viral suppression.

    In fact T cells expressing transgenic HIV-specific TCRs were found in multiple organs in mice receiving genetically modified HSCs, and when compared with control mice, these animals exhibited much lower HIV levels in the spleen, bone marrow, and human thymus implant. Evaluation of peripheral blood CTLs expressing the HIV-specific transgenic TCRs confirmed that the cells possessed an effector phenotype, including loss of CD45RA and CCR7 expression that was indicative of antigen-specific induction of cellular differentiation. Further assessment of HIV-specific CTL expansion indicated that the greater the initial reconstitution of transgenic HIV-specific cells, the more effective the control of viral replication at the six week time point.

    “These studies provide a foundation and a model system that would allow the closer examination of human antiviral T-cell responses and the development of therapeutic strategies that target chronic viral infection,” the authors conclude. However, they point out, one apparent drawback with the humanized mouse model is that it demonstrates relatively limited immune responses to HIV. “The incomplete and varied immune reconstitution in the current humanized mouse systems results in differences in immune responses and kinetics of viral pathogenesis compared to natural HIV infection in humans,” they admit.

    And while HIV replication rates and viral loads persisted in the mice, they also didn’t reach levels observed in natural infection in humans. This lower level of viral replication may be one reason why viral escape mutants to the SL9-specific TCR didn’t occur in the reported studies.

    Nevertheless, they state, while natural antiviral T-cell immune responses are limited in current humanized mouse models, our studies suggest that the genetic programming of HSCs to produce T cells specific for HIV can overcome this limitation in this system and produce measurable T-cell responses that have a significant antiviral effect in vivo.

    “We found it startling that the use of a single HIV-specific TCR can result in significant HIV suppression while natural suppressive antiviral CTL responses are polyclonal … These results strongly suggest that stem cell-based gene therapy may be a feasible approach in the treatment of chronic viral infections and provide a foundation towards the development of this type of strategy.”


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