Scott G. Kitchen

The CCR5 and CXCR4 Coreceptors—Central to Understanding the Transmission and Pathogenesis of Human Immunodeficiency Virus Type 1 Infection

In this review, we will discuss what is known, what is suspected, and what still remains obscure about the central role played by coreceptor expression and usage in the transmission and pathogenic consequences of human immunodeficiency virus type 1 (HIV-1) infection. An emphasis will be on the HIV-1 phenotypic variants that are defined by their usage of the CCR5 or CXCR4 coreceptors, and how the different cellular tropism of these variants influences how and where HIV-1 replicates in vivo. We will also review what might happen when coreceptor antagonists are used clinically to treat HIV-1 infection.

Generation of HIV latency during thymopoiesis.

The use of combination antiretroviral therapy results in a substantial reduction in viremia, a rebound of CD4+ T cells and increased survival for HIV-infected individuals. However, this treatment does not result in the total eradication of HIV. Rather, the virus is thought to remain latent in a subset of cells, where it avoids elimination by the immune system. In this state the virus is capable of reactivation of productive infection following cessation of therapy. These latently infected cells are very few in number and it has thus been difficult to determine their origin and to study the molecular nature of the latent viral genome. HIV replication is linked to cellular gene transcription and requires target cell activation. Therefore, should an activated, infected cell become transcriptionally inactive prior to cytopathic effects, the viral genome might be maintained in a latent state. We used the SCID-hu (Thy/Liv) mouse model to establish that activation-inducible HIV can be generated at high frequency during thymopoiesis, a process where previously activated cells mature towards quiescence. Moreover, we showed that these cells can be exported into the periphery where the virus remains latent until T-cell receptor stimulation, indicating that the thymus might be a source of latent HIV in humans.

Upregulation of CD4 on CD8+ T cells: CD4dimCD8bright T cells constitute an activated phenotype of CD8+ T cells

Aside from an intermediate stage in thymic T‐cell development, the expression of CD4 and CD8 is generally thought to be mutually exclusive, associated with helper or cytotoxic T‐cell functions, respectively. Stimulation of CD8+ T cells, however, induces the de novo expression of CD4. We demonstrate that while superantigen (staphylococcal enterotoxin B, SEB) and anti‐CD3/CD28 costimulation of purified CD8+ T cells induced the expression of CD4 on CD8+ T cells by 30 and 17%, respectively, phytohaemagglutinin (PHA) stimulation did not induce CD4 expression on purified CD8+ T cells but significantly induced the expression of both CD4 on CD8 (CD4dimCD8bright) and CD8 on CD4 (CD4brightCD8dim) T cells in unfractionated peripheral blood mononuclear cells (PBMC). The level of the PHA‐mediated induction of CD4dimCD8bright and CD4brightCD8dim was at 27 and 17%, respectively. Depletion of CD4+ T cells from PBMC abrogated this PHA‐mediated effect. Autologous CD4+ and CD8+ T‐cell co‐cultures in the presence of PHA induced this CD4dimCD8bright T‐cell expression by 33%, demonstrating a role for CD4 cells in the PHA‐mediated induction of the double positive cells. The induction of CD4dimCD8bright was independent of a soluble factor(s). Phenotypic analysis of CD4dimCD8bright T cells indicated significantly higher levels of CD95, CD25, CD38, CD69, CD28, and CD45RO expression than their CD8+CD4− counterparts. CD4dimCD8bright T cells were also negative for CD1a expression and were predominately T‐cell receptor (TCR) αβ cells. Our data demonstrate that CD4dimCD8bright T cells are an activated phenotype of CD8+ T cells and suggest that CD4 upregulation on CD8+ T cells may function as an additional marker to identify activated CD8+ T cells.

In vivo suppression of HIV by antigen specific T cells derived from engineered hematopoietic stem cells.

The HIV-specific cytotoxic T lymphocyte (CTL) response is a critical component in controlling viral replication in vivo, but ultimately fails in its ability to eradicate the virus. Our intent in these studies is to develop ways to enhance and restore the HIV-specific CTL response to allow long-term viral suppression or viral clearance. In our approach, we sought to genetically manipulate human hematopoietic stem cells (HSCs) such that they differentiate into mature CTL that will kill HIV infected cells. To perform this, we molecularly cloned an HIV-specific T cell receptor (TCR) from CD8+ T cells that specifically targets an epitope of the HIV-1 Gag protein. This TCR was then used to genetically transduce HSCs. These HSCs were then introduced into a humanized mouse containing human fetal liver, fetal thymus, and hematopoietic progenitor cells, and were allowed to differentiate into mature human CD8+ CTL. We found human, HIV-specific CTL in multiple tissues in the mouse. Thus, genetic modification of human HSCs with a cloned TCR allows proper differentiation of the cells to occur in vivo, and these cells migrate to multiple anatomic sites, mimicking what is seen in humans. To determine if the presence of the transgenic, HIV-specific TCR has an effect on suppressing HIV replication, we infected with HIV-1 mice expressing the transgenic HIV-specific TCR and, separately, mice expressing a non-specific control TCR. We observed significant suppression of HIV replication in multiple organs in the mice expressing the HIV-specific TCR as compared to control, indicating that the presence of genetically modified HIV-specific CTL can form a functional antiviral response in vivo. 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.

Differential tropism of HIV-1 isolates for distinct thymocyte subsets in vitro.

ObjectiveUnderstanding the interaction between HIV and developing thymocytes is crucial in determining how HIV infection perturbs the immune system. We determined which thymocyte subsets can harbor and express HIV. DesignHIV expression in mature and immature thymocytes obtained from surgical specimens from non-infected children was determined after in vitro infection with the syncytium-inducing, cytopathic NL4–3 and the non-syncytium-inducing, relatively noncytopathic JR-CSF isolates. MethodsIntracellular staining for the HIV p24gag antigen was combined with cell surface phenotyping to determine thymocyte subsets expressing HIV. Infection was quantitated by polymerase chain reaction on sorted subsets. ResultsNL4–3 replicated faster and to higher titers and caused a more severe decrease of all CD4-bearing thymocytes than did JR-CSF. In addition, both immature CD1+ and mature CD1− thymocytes expressed NL4–3, whereas only mature CD1 -cells expressed JR-CSF. The tropism of NL4–3 for these immature cells suggests a mechanism for a more profound impact on T-cell maturation than that seen with JR-CSF. We also found that thymocytes lacking cell surface CD4 (CD4-CD8− and CD4-CD8+ subsets) expressed virus with either isolate late in infection, when viral levels were high. The CD4-CD8− cells expressing HIV were mature CD3bright T-cell receptor (TCR)α/βbright cells. ConclusionsThese results show that NL4–3 can be expressed by thymocytes at immature and mature stages of differentiation and cause severe loss of CD4+ cells. Thus, tropism of a virus for immature cells can affect the capability of the thymus to produce new T lymphocytes leading to a greater impact on development and functions of the immune system. It is proposed that this in vitro model can be used to study pathogenic mechanisms in the thymus.

Stem cell-based anti-HIV gene therapy.

Human stem cell-based therapeutic intervention strategies for treating HIV infection have recently undergone a renaissance as a major focus of investigation. Unlike most conventional antiviral therapies, genetically engineered hematopoietic stem cells possess the capacity for prolonged self-renewal that would continuously produce protected immune cells to fight against HIV. A successful strategy therefore has the potential to stably control and ultimately eradicate HIV from patients by a single or minimal treatment. Recent progress in the development of new technologies and clinical trials sets the stage for the current generation of gene therapy approaches to combat HIV infection. In this review, we will discuss two major approaches that are currently underway in the development of stem cell-based gene therapy to target HIV: one that focuses on the protection of cells from productive infection with HIV, and the other that focuses on targeting immune cells to directly combat HIV infection.

Initial Virological and Immunologic Response to Highly Active Antiretroviral Therapy Predicts Long-Term Clinical Outcome

Little is known about the long-term clinical outcomes for human immunodeficiency virus (HIV)-infected patients who have received highly active antiretroviral therapy (HAART). Determining factors associated with long-term clinical outcomes early in the course of treatment may allow modifications to be made for patients who are at a greater risk of treatment failure. To evaluate these factors, we studied 213 HIV-infected patients who had received HAART for at least 115 weeks. In the univariate analysis, virological response, which was measured as the change in virus load from baseline at month 3 of treatment, was the single best predictor of clinical outcome (relative hazard, 0.722; P=.001), independent of virological suppression. In the multivariate analysis, virological response and immunologic response, which was measured as an increase in CD4 cell count of >200 cells/mm(3), resulted in better prediction of clinical outcomes than did use of either variable alone (P=.02). Our results indicate that changes in virus load and immunologic response together are good predictors of clinical outcome and can be assessed after the initiation of HAART, which would allow clinicians to identify patients early in the course of therapy who are at greater risk of negative outcome.

Targeting type I interferon–mediated activation restores immune function in chronic HIV infection

Chronic immune activation, immunosuppression, and T cell exhaustion are hallmarks of HIV infection, yet the mechanisms driving these processes are unclear. Chronic activation can be a driving force in immune exhaustion, and type I interferons (IFN-I) are emerging as critical components underlying ongoing activation in HIV infection. Here, we have tested the effect of blocking IFN-I signaling on T cell responses and virus replication in a murine model of chronic HIV infection. Using HIV-infected humanized mice, we demonstrated that in vivo blockade of IFN-I signaling during chronic HIV infection diminished HIV-driven immune activation, decreased T cell exhaustion marker expression, restored HIV-specific CD8 T cell function, and led to decreased viral replication. Antiretroviral therapy (ART) in combination with IFN-I blockade accelerated viral suppression, further decreased viral loads, and reduced the persistently infected HIV reservoir compared with ART treatment alone. Our data suggest that blocking IFN-I signaling in conjunction with ART treatment can restore immune function and may reduce viral reservoirs during chronic HIV infection, providing validation for IFN-I blockade as a potential therapy for HIV infection.

HIV-specific Immunity Derived From Chimeric Antigen Receptor-engineered Stem Cells

The human immunodeficiency virus (HIV)-specific cytotoxic T lymphocyte (CTL) response is critical in controlling HIV infection. Since the immune response does not eliminate HIV, it would be beneficial to develop ways to enhance the HIV-specific CTL response to allow long-term viral suppression or clearance. Here, we report the use of a protective chimeric antigen receptor (CAR) in a hematopoietic stem/progenitor cell (HSPC)-based approach to engineer HIV immunity. We determined that CAR-modified HSPCs differentiate into functional T cells as well as natural killer (NK) cells in vivo in humanized mice and these cells are resistant to HIV infection and suppress HIV replication. These results strongly suggest that stem cell-based gene therapy with a CAR may be feasible and effective in treating chronic HIV infection and other morbidities.

Generation of T Lineage Cells from Human Embryonic Stem Cells in a Feeder Free System

Human embryonic stem cells (hESC) have the potential to revolutionize certain medical treatments, including T‐cell‐based therapies. However, optimal approaches to develop T cells from hESC are lacking. In this report, we show that T‐cell progenitors can be derived from hESC cultured as embryoid bodies (EBs). These EB‐derived T‐cell progenitors give rise to phenotypically and functionally normal cells of the T lineage when transferred into human thymic tissue implanted in immunocompromised mice, suggesting that introduction of these progenitors into patients may also yield functional T cells. Moreover, hematopoietic progenitors demonstrating T‐cell potential appeared to be CD45+/CD34+, resembling those found in normal bone marrow. In contrast to T cells developed from hESC cocultured on murine stromal cells, the EB‐derived T cells also expressed normal levels of CD45. Importantly, the EB system eliminates the previous need for murine cocultures, a key impediment to developing a protocol for T‐cell progenitor derivation suitable for clinical use. Furthermore, following lentiviral‐mediated introduction of a vector expressing enhanced green fluorescent protein into hESC, stable transgene expression was maintained throughout differentiation, suggesting a potential for gene therapy approaches aimed at the augmentation of T‐cell function or treatment of T‐cell disorders. STEM CELLS 2009;27:100–107