KILLING OF UNINFECTED (BYSTANDER) T CELLS BY HIV

The fact that only a small fraction of the CD4⁺ T cells in HIV-infected persons harbors the virus at any given time points to an important role for the death of uninfected bystander cells in the clinical evolution of AIDS.¹²⁹ However, as with direct killing, the underlying mechanism for bystander cell death remains poorly understood.

CD4⁺ T Cell Apoptosis Mediated by Cell-Cell Interactions:

Death Receptors

One mechanism implicated in the HIV-induced apoptosis of bystander cells involves the upregu­lation of FasL expression on infected cells. Subsequent interaction with Fas on neighboring cells triggers bystander cell apoptosis (Figure 13.3). In contrast to the Fas receptor, which is widely distributed throughout the body, FasL expression is principally restricted to lymphocytes and natural killer (NK) cells. FasL expression is activated by the transcription factors NF-κB, AP-1, nuclear factor of activated T cells (NF-AT), and Sp-1.¹³⁰ Additionally, FasL is stored in the cytoplasm in secretory vesicles and expressed as a 40 kDa membrane-bound protein that can be cleaved by matrix metalloproteinase to release a 28 kDa soluble ligand (sFasL).¹³¹

In HIV-infected persons, FasL is upregulated on peripheral blood mononuclear cells (PBMCs), and increased levels of sFasL can be detected in the plasma.¹³²,¹³³ This increase in FasL expression on PBMCs seems to correlate with disease progression, because it occurs at higher levels in patients with CD4⁺ T cell counts of fewer than 200 cells∕μL.

Other death receptors and ligands, such as the TNF-α receptor and TRAIL and its receptors, may also play a role in HIV-induced bystander killing (Figure 13.4). TNF-α is a multifunctional cytokine produced primarily by activated monocytes and macrophages. Although TNF-α can contribute to T cell apoptosis in the presence of HIV-infected macrophages,¹⁴²’¹⁴³ the importance of increased TNF-α expression in the death of directly infected vs. bystander cells remains to be established. T cells from HIV-infected subjects seem to be more sensitive to TRAIL compared with activated T cells from normal donors.¹⁴⁴ Anti-TRAIL antibodies also partially block activation- induced apoptosis of T cells from asymptomatic HIV-positive patients.¹⁴⁵ Again, as with TNF-α, firm evidence implicating TRAIL as a major mediator of bystander T cell apoptosis in HIV-infected patients is lacking.

The HIV Accessory Genes Tat, Vpr, and Nef as Mediators

of Bystander Cell Death

Several HIV-encoded gene products have been implicated as initiators of cell-death signaling involving uninfected bystander cells. We will focus on Tat, Vpr, and Nef in this discussion. As discussed above, Tat kills cells either by direct or indirect mechanisms, whereas Vpr acts directly through targeting of the MMP. Due to the protein-transducing properties of these proteins, it is possible that these same processes extend to bystander cells (Figure 13.4). Several lines of evidence also suggest a role for Nef as a mediator of bystander T cell apoptosis. Nef is present in significant quantities in the sera of HIV-infected patients.¹⁴⁶ Soluble Nef has been shown to cause the death of uninfected CD4⁺ T cells in vitro by a process termed apoptotic cytolysis.^147-149 A recent study highlighted a direct role for Nef in bystander killing, showing that soluble Nef protein targets CD4⁺ T cells for apoptosis through a direct interaction with the CXCR4 co-receptor (Figure 13.4).¹⁵⁰

Nef might also play a role in bystander killing independent of its presence in the extracellular media. Within infected cells, Nef interacts with and modulates the activity of a number of kinases that play a role in apoptosis: the Ser/Thr p21-activated kinase (PAK),¹⁵¹ the apoptosis signal­regulating kinases (ASK1),¹⁵² and a number of the Src-family kinases (Hck, Lck, and Lyn). Gele- ziunas et al. showed that the assembly with Nef inhibits the catalytic activity of ASK1, which, in turn, inhibits both Fas and TNF-α mediated apoptosis.¹⁵² The authors suggest a model by which Nef promotes the killing of bystander cells through the induction of FasL^153-155 and TNF-α production while simultaneously protecting the HIV-infected host from these same proapoptotic signals through its interference with ASK1 function.

Accelerated T Cell Turnover: The Effects of HIV on the Thymus

and Bone Marrow

T cell dynamics in HIV-infected patients have been studied extensively using in vivo methods of DNA labeling with both radioactive and nonradioactive labels. After administration, these labels are metabolized into nucleotides that are incorporated in a stable manner into dividing cells during DNA synthesis. During administration of the label, uptake can be employed as a marker of cell division, and, after administration, the rate at which the label disappears within specific cellular subsets provides information on the half-life of labeled cells. The fractional turnover rate of each cell population can then be delineated.

During HIV infection, 4 ? 10⁷ to 2 ? 10⁹ CD4⁺ T cells are replaced in the blood each day.¹⁵⁶ Initially, it was thought that this upper estimate exceeded the proliferative capacity of the CD4⁺ T cells. However, recent studies revealed that HIV-infected patients treated with potent drug regimens produce new CD4⁺ and CD8⁺ T cells at abnormally high rates.^157-160 In addition to its ability to promote high turnover of peripheral CD4⁺ T cells, HIV can infect thymic precursor cells, leading to greatly impaired production of new CD4⁺ T cells. This process, termed “regen­erative failure,” further contributes to profound loss of CD4⁺ T cells occurring in untreated HIV infection.^161-164

It is worth considering this phenomenon in greater detail. The primary supply route for naive T cells before they settle in the lymph node involves the bone marrow-thymus axis, where there are several precursor cells that are susceptible to HIV infection. Although the thymus involutes over time and intrathymic production of T cells diminishes progressively with increasing age, the residual functioning thymic tissue present in adults might be quickly eliminated by infection with HIV.^165,166 in particular, thymocytes are susceptible to infection with X4-tropic strains of HIV.^167-171 There is mounting evidence that viral infection is associated with thymic pathology in HIV-infected humans,^172-176 severe combined immunodeficient (SCID)-hu mice,^177-179 and simian immunodefi­ciency virus-infected nonhuman primates.^180,181 A number of different thymocyte populations are sensitive to HIV infection, including CD3^-CD4⁺CD8^- intrathymic progenitor cells, CD3⁺CD4⁺CD8⁺ cortical thymocytes, and CD3⁺CD4⁺CD8^- or CD3^-CD4^-CD8⁺ medullary thymocytes.¹⁶⁵,^177-179 Because the thymus is more active in children and young adults, these individuals may be less sensitive to the peripheral destruction of CD4⁺ T lymphocytes.

Another hypothesis that has received great interest of late is the idea that activation-induced cell death is an underlying mechanism of CD4⁺ T cell depletion. In this model, the loss of T cell homeostasis is due principally to an increased state of T cell activation leading to activation-induced apoptosis. The driving force behind the immune activation is likely the immune response to the virus and consequent release of cytokines. Thus, this loss of T cell homeostasis is a consequence of the immune response to the virus rather than infection of cells by the virus.¹⁶¹ This hypothesis clearly merits further investigation.

The Role of Chemokine Receptors in HIV-Mediated Bystander Killing

As discussed previously, Env plays a role in both direct cell killing and bystander cell killing (Figure 13.2A, Figure 13.3, and Figure 13.4). Again, this activity depends on the ability of Env to interact with both CD4 and the chemokine co-receptor. The mechanism by which engagement of the CD4 receptor by HIV Env leads to accelerated T cell death is poorly understood.

It seems evident then that Env-mediated bystander killing involves not only CD4 binding but also co-receptor engagement. Using an ex vivo human lymphoid culture system that preserves the natural lymphoid environment, Jekle and colleagues showed that bystander apoptosis occurs when cultures are infected with CXCR4-dependent strains of HIV.¹⁹⁵ Only minimal cell death is observed with CCR5-tropic viruses in this system, perhaps reflecting the relative paucity of CCR5-expressing cells in these unstimulated cultures. Of note, bystander apoptosis was not detected in CXCR4- expressing CD8⁺ or B cells, implicating a key role for CD4 in the response. However, interaction with CXCR4 is also required, as indicated by a blockade of the cytopathic response with the CXCR4 antagonist AMD3100 or with anti-CXCR4 monoclonal antibodies. A recent study showed that both CCR5 and CXCR4 activation by gp120 on the surface of cells can induce the death of uninfected CD4⁺ and CD8⁺ T cells in a different culture system (Figure 13.4).¹⁹6 Interestingly, engagement of CCR5 by HIV gp120 induces caspase-8-dependent cell death, whereas engagement of CXCR4 induces a caspase-independent form of cell death.

Finally, as discussed earlier, Env can mediate bystander killing due to syncytia formation (Figure 13.3). Using an experimental system based principally on transfected fibroblast cell lines, Kroemer and colleagues showed that in syncytia-induced cell death, cdk1 activity is necessary for p53 phosphorylation involving mTOR and that activated phospho-p53 subsequently upregulates expression of the Bax proapoptotic factor.^197-199 Intriguingly, activated forms of mTOR and p53 are found in lymph node biopsies from HIV-infected patients.¹⁹⁸