TAG HIV Basic Science, Vaccines, and Cure Project Blog

Modeling CD4 T cell Depletion

A new paper in PLoS Medicine by Andrew Yates and colleagues makes a valiant attempt to mathematically model the impact of HIV infection on CD4 T cell homeostasis in humans. The authors carefully review recent published data on the factors that are involved in the maintenance of CD4 T cells, including variables relating to the major CD4 subsets (naïve and memory) and potential states (activated versus resting) in order to try and ascertain how HIV infection and HIV-related immune activation might lead to the gradual decline in peripheral blood memory CD4 T cell counts that occurs during chronic HIV infection. The resultant data shows that, based on the authors’ assumptions, HIV infection should lead to a much more rapid decline of memory CD4 T cell numbers than in fact occurs.

To look more closely at the assumptions that underlie the mathematical model, the authors posit that memory CD4 T cells turn over (in a process known as homeostatic self renewal) about once every 60 days, and that this turnover occurs more often (once ever 30 days) under conditions of memory CD4 T cell depletion. These assumptions are in broad agreement with the published literature. More speculatively - and controversially - the authors assume that this turnover renders the cells susceptible to HIV infection, despite the fact central memory CD4 T cells are not thought to express CCR5 during this process. The estimated lifespan of HIV infected CD4 T cells in the model is 0.5 days, and this is derived from a David Ho paper published in Science in 1996. The authors also construct a more complex model that takes into account antigen driven CD4 T cell activation as well as homeostatic turnover, which predicts that memory CD4 T cell depletion should occur even faster than predicted by the simpler model.

There are some other potentially controversial aspects of the paper: not least, the authors dispense with recent data describing the rapid depletion of mucosal CD4 T cells by focusing on the subsequent chronic phase of infection. The paper states that the goal was to keep the model as simple as possible and the results are based on changes to total body memory CD4 T cell numbers and do not appear to take into account redistribution of memory CD4 T cells from the blood into the lymphoid tissue (this phenomenon is known to account for a significant amount of memory CD4 T cell loss from the blood in HIV infection). Also, as far back as 1994 the late Janis Giorgi and colleagues reported that people with AIDS are not preferentially depleted of phenotypically defined memory CD4 T cells as the peripheral blood CD4 T cell count declines, a finding echoed by a comprehensive analysis of total body CD4 T cell counts in SIV-infected macaques which found that total CD4 T cell numbers were slightly increased during the asymptomatic phase of the infection and did not decline significantly until the late stage of disease. However, it may be that these phenomenon - like the slow decline of peripheral blood memory CD4 T cells - would, based on the model, also not be explained solely by elevated memory CD4 T cell activation and turnover. The authors also list some additional caveats and considerations in the discussion section of the paper, and suggest that viral adaptation to the immune system environment may play a role in memory CD4 T cell depletion.

In an interesting supplemental analysis, the authors adapt the model to include the possibility that every time a CD4 T cell is activated, there is a small probability that the CD4 T cell will become exhausted and dysfunctional. The analysis demonstrates that, under these circumstances, the model would in fact predict the slow accumulation of dysfunctional memory CD4 T cells and the progressive lowering of CD4 T cell counts over a period of years that occurs in people with HIV infection. Given that there is abundant evidence that dysfunctional memory CD4 T cells do accumulate in people with HIV, this analysis may offer some important insights into the role of T cell exhaustion in HIV pathogenesis, although the authors note that additional data is needed to fully explore this possibility.

One final odd note about this study is that the Imperial College Press Office produced an accompanying press release which has the potential to be extremely misleading. The release is entitled “HIV's effect on white blood cells questioned by new research” which some media outlets have changed to “Study Refutes Theory on HIV's Effect on White Blood Cells” because of the press release’s subtitle which says “Scientists have refuted a longstanding theory of how HIV slowly depletes the body's capacity to fight infection, in new research published today.” The only problem – and it’s quite a large problem – is that it is entirely unclear which theory (if any) is being refuted. According to the press release, “one popular theory has been the ‘runaway’ hypothesis” - but there is no such hypothesis! The phrase “runaway” is coined by the study authors in the PLoS Medicine paper, without citation, and the phrase appears nowhere in the literature on HIV pathogenesis. I emailed the Imperial College press office just in case I’d missed a citation somewhere, but they have not yet responded.

UPDATE: I received an email from Jaroslav Stark acknowledging that there is indeed no "runaway" hypothesis.

"The term "runaway" was indeed our own since we needed a simple term with which to refer to such a class of  models within the manuscript. As I am sure you are aware, a number of authors, including Grossman, Hellerstein, Hazenberg, Feinberg & Silvestri have proposed such explanations. I agree that these have
never been clearly unified into a single theory in the literature. I do not think that we suggest anywhere that this is the case, but rather we identify the common denominator in all of these various theories and then show that this always leads to a much quicker decline in CD4+ numbers than is observed in practice."

UPDATE 2: The BBC website seems to have developed an odd habit of publishing stories many weeks or months after the fact; on Saturday June 23 they unfortunately decided to add to the misinformation surrounding this modeling exercise with a story headlined "HIV infection theory challenged." Stark is quoted as saying "our new interdisciplinary research has thrown serious doubt on one popular theory of how HIV affects these cells, and means that further studies are required to understand the mechanism behind HIV's distinctive slow process of cellular destruction." Researchers focusing on the role of immune activation in HIV pathogenesis will doubtless be surprised to learn that not only have they all agreed on one theory, it has apparently also become popular!

UPDATE 3: In a surfeit of annoyance at the inaccuracy of the media coverage of this paper, I now realize - with the help of a thoughtful and informative email from Dr. Stark - that the first few iterations of this post were overly (and inaccurately) critical of the paper itself. Although the lifespan of infected memory CD4 T cells assumed in the model is taken from a David Ho paper that posits cytopathicity as the driving force causing cell death, altering the lifespan of infected cells such that it is equivalent to uninfected cells may not necessarily change the model significantly (although this analysis has not yet been performed). It is perhaps asking a lot to expect researchers engaged in the complex and arcane art of modeling CD4 T cell homeostasis to be savvy about the way a media story can be abused (as regrettably, this story has been by the cult-like group known as AIDS denialists).

UPDATE 4: The global circulation of the extremely misleading press release associated with this study continues: Popular HIV Theory "Wrong," We've Got it Wrong About HIV, Theory of How HIV Attacks is Wrong. After trying to be generous toward the authors of the paper, it is hard to escape the conclusion that - whether it was naivete or something worse - they have made a huge and unwelcome contribution to the public misunderstanding of HIV science.

PLoS Med 4(5): e177

Understanding the Slow Depletion of Memory CD4+ T Cells in HIV Infection (free access)

Andrew Yates1,2*, Jaroslav Stark3,4, Nigel Klein5, Rustom Antia1, Robin Callard2,5

1 Department of Biology, Emory University, Atlanta, Georgia, United States of America, 2 Centre for Mathematics and Physics in the Life Sciences and Experimental Biology, University College London, London, United Kingdom, 3 Department of Mathematics, Imperial College, London, United Kingdom, 4 Centre for Integrative Systems Biology at Imperial College, Imperial College London, United Kingdom, 5 Immunobiology Unit, Institute of Child Health, London, United Kingdom

Background

The asymptomatic phase of HIV infection is characterised by a slow decline of peripheral blood CD4+ T cells. Why this decline is slow is not understood. One potential explanation is that the low average rate of homeostatic proliferation or immune activation dictates the pace of a “runaway” decline of memory CD4+ T cells, in which activation drives infection, higher viral loads, more recruitment of cells into an activated state, and further infection events. We explore this hypothesis using mathematical models.

Methods and Findings

Using simple mathematical models of the dynamics of T cell homeostasis and proliferation, we find that this mechanism fails to explain the time scale of CD4+ memory T cell loss. Instead it predicts the rapid attainment of a stable set point, so other mechanisms must be invoked to explain the slow decline in CD4+ cells.

Conclusions

A runaway cycle in which elevated CD4+ T cell activation and proliferation drive HIV production and vice versa cannot explain the pace of depletion during chronic HIV infection. We summarize some alternative mechanisms by which the CD4+ memory T cell homeostatic set point might slowly diminish. While none are mutually exclusive, the phenomenon of viral rebound, in which interruption of antiretroviral therapy causes a rapid return to pretreatment viral load and T cell counts, supports the model of virus adaptation as a major force driving depletion.

PLoS Med 4(5): e193

PERSPECTIVES

Time Scales of CD4+ T Cell Depletion in HIV Infection (free access)

Rob J. De Boer

Unraveling the Causes of Immune Activation

Picking up on the theme of the last blog posting, a new study in the “in press” section of J. Virology investigates the potential causes of immune activation in HIV infection. The researchers - led by Angela Meier from Marcus Altfeld’s laboratory at Partners AIDS Research Center - carefully document the close parallel between declining viral load levels in response to treatment and declining levels of immune activation markers (CD38 and HLA-DR) on CD8 T cells, concluding that “the dramatic decline of immune activation following the suppression of HIV-1 viremia by HAART strongly suggests a direct causal effect of HIV-1 itself, or components of the virus, on immune activation.”

The study authors explore this question further by examining the ability of HIV to encode single-stranded RNA sequences that can directly activate the immune system via toll-like receptors (TLRs). The authors focus on uridine-rich sequences (uridine is one of the nucleoside components of RNA) because these types of sequences have previously been shown to interact with TLRs. They synthesized nine different uridine-rich sequences from the laboratory HIV strain HXB2 and found that all of them could induce significant amounts of cytokine production by plasmacytoid dendritic cells and monocytes in vitro. The researchers also demonstrate that this activity depends on interactions between the RNA and the TLR7 and TLR8 by using inhibitors of these TLRs and also by using cells from mice lacking MyD88 (an adaptor protein that is essential for signaling through TLR7 and TLR8).

The question of whether these HIV-derived RNA sequences can also activate CD4 and CD8 T cells via their effects on dendritic cells and monocytes is also partially addressed; the researchers report that they can induce upregulation of the activation marker CD69 on CD4 and CD8 T cells but these T cells do not produce cytokines. Data on the activation markers CD38 and HLA-DR is not reported. Notably, other researchers attempting to induce T cell activation using TLR ligands have also reported CD69 upregulation and elevated expression of CD38 after overnight culture, but with little effect on HLA-DR expression, perhaps calling into question the relevance of these data to the immune activation documented in HIV infection (which is associated with elevated CD38 and HLA-DR expression). In order to try and address this issue definitively, several research groups are now planning in vivo studies of TLR inhibitors in SIV and HIV infection.

JVI Accepts, published online ahead of print on 16 May 2007

J. Virol. doi:10.1128/JVI.00421-07

MyD88-dependent Immune Activation mediated by HIV-1-encoded TLR Ligands

Angela Meier, Galit Alter, Nicole Frahm, Harlyn Sidhu, Bin Li, Aranya Bagchi, Nickolas Teigen, Hendrik Streeck, Hans-Juergen Stellbrink, Judith Hellman, Jan van Lunzen, and Marcus Altfeld*

Partners AIDS Research Center, Massachusetts General Hospital and Division of AIDS, Harvard Medical School, Boston, MA, USA; Department of Anesthesia and Critical Care, Massachusetts General Hospital, Boston, MA, USA; IPM Study Center, Hamburg,Germany; University Medical Center Hamburg-Eppendorf, Infectious Disease Unit, Hamburg, Germany

Immune activation is a major characteristic of HIV-1 infection and a strong prognostic factor for HIV-1 disease progression. The underlying mechanisms leading to immune activation in viremic HIV-1 infection however are not fully understood. Here we show that following initiation of HAART, the immediate decline of immune activation is closely associated with the reduction of HIV-1 viremia, which suggests a direct contribution of HIV-1 itself to immune activation. To propose a mechanism, we demonstrate that the ssRNA of HIV-1 encodes for multiple uridine-rich TLR7/8 ligands that induce strong MyD88-dependent pDC and monocyte activation, as well as accessory cell-dependent T cell activation. HIV-1-encoded TLR ligands may therefore directly contribute to the immune activation observed during viremic HIV-1 infection. These data provide initial rationale for inhibiting the TLR pathway to directly reduce the chronic immune activation induced by HIV-1 and the associated immune pathogenesis.

HIV's Impact on the Lymph Nodes

In the new issue of the journal Blood, Angélique Biancotto and colleagues from Mike Lederman’s lab at Case Western Reserve University provide a detailed description of the immunological perturbations of the lymph node caused by HIV infection. The researchers found significantly elevated levels of activated CD4 and CD8 T cells in lymph node samples from HIV-infected individuals compared to uninfected controls, consistent with the increasingly well-described role of immune activation in HIV pathogenesis. Activation was assessed by expression of the marker CD38 and 73.3% of the T cells from infected nodes were CD38+ compared to 13.7% in uninfected nodes. A more detailed analysis of CD38 expression on different subsets of CD4 and CD8 T cells showed that levels were elevated on all subsets (naïve, effector memory and central memory CD4 and CD8 T cells). Looking at these subsets also revealed that infected lymph nodes had consistently higher proportions of both CD4 and CD8 effector memory T cells (effector memory T cells are short-lived cells that are generated when T cells are activated; their normal role is to traffic out of the lymph nodes and into the tissues). Consistent with this result, more T cells in infected nodes expressed CD95, a molecule associated with apoptosis (cell death).

Other findings were that the proportions of CD4 T cells was reduced (37.1% vs. 81.7%) in infected lymph nodes while the fraction of CD8 T cells was increased (63.2% vs. 22.6%); the numbers of antigen-presenting cells (called plasmacytoid and myeloid dendritic cells) were also reduced (0.73% vs. 27.1% and 0.46% vs. 8.9%, respectively). Lastly, an analysis of cytokines and chemokines found that IL-1beta, IL-2, IL-10, IL-12 and IL-15 levels were all significantly elevated in infected nodes while concentrations of MIP-1beta, SDF-1beta and IP-10 were significantly diminished.

In discussing the results, the authors suggest that the activation they documented is not the result of specific engagement of the T cell receptor by antigen but rather a non-specific “bystander” effect. However, in an accompanying commentary Zvi Grossman notes that the data do not necessarily entirely rule out a role for direct T cell activation by HIV and/or other microbial antigens. This distinction may seem arcane, but because T cell activation correlates with disease progression it is critically important to understand the exact cause (or causes) of this phenomenon. Grossman also offers a schematic drawing representing the potential impact of HIV infection on the lymph node environment.

Grossman concludes by saying: “Are the changes in the cellular and cytokine profiles in secondary lymphoid organs truly ‘abnormal,’ or are they essentially reversible manifestations of immune activation and its physiological controls? To what extent do they reflect irreversible tissue destruction and loss of function? A better understanding of these issues may reveal, in the authors' words, ‘new targets for immune-based interventions to slow HIV-1 disease progression.’”

Blood, 15 May 2007, Vol. 109, No. 10, pp. 4116-4117  (full text available free)

Looking at HIV-infected lymph nodes
Zvi Grossman, Tel Aviv University

Blood, 15 May 2007, Vol. 109, No. 10, pp. 4272-4279

Abnormal activation and cytokine spectra in lymph nodes of people chronically infected with HIV-1

Angélique Biancotto1, Jean-Charles Grivel1, Sarah J. Iglehart1, Christophe Vanpouille1, Andrea Lisco1, Scott F. Sieg2, Robert Debernardo2, Kristen Garate2, Benigno Rodriguez2, Leonid B. Margolis1, and Michael M. Lederman2

1 Laboratory of Molecular and Cellular Biophysics, National Institute of Child Health and Human Development, Bethesda, MD; 2 Center for AIDS Research, Case Western Reserve University/University Case Medical Center, Cleveland, OH

There is growing recognition that HIV-1 infection leads to an activation of the immune system that includes perturbations of cytokine expression, redistribution of lymphocyte subpopulations, cell dysfunction, and cell death. Here, we explored the relationships between HIV-1 infection and immune activation in chronically HIV-1–infected human lymph nodes. In addition to CD4 T-cell depletion, we found increased effector T-cell frequencies associated with profound up-regulation of an activation marker CD38 in naive, central memory, and effector CD4+ and CD8+ T cells. Likewise, Fas death receptor (CD95) was more frequently detectable on T cells from HIV-1 nodes. Dendritic cell (DC) depletion was dramatic, with plasmacytoid DCs (PDCs) 40-fold and myeloid DCs (MDCs) 20-fold less frequent in HIV+ nodes than in control nodes. Cytokine dysregulation was evident, with IL-2 and IL-15 as much as 2 or 3 logs greater in infected nodes than in control nodes. Thus, activated effector cells are inappropriately attracted and/or retained in lymphoid tissue in chronic HIV-1 infection. High-level cytokine expression in turn activates and retains more cells at these sites, leading to lymphadenopathy and massive bystander activation that characterizes HIV-1 infection. Strategies targeting these activation pathways may lead to new therapies.

Reservoirs Revisited

When highly active antiretroviral therapy (HAART) first became available, some scientists expressed the opinion that long term suppression of HIV replication might lead to the virus being eradicated from the body. But because HIV can integrate into cellular DNA and persist in a latent (inactive) state, the majority of the scientific community were skeptical of these claims. This skepticism was borne out by subsequent studies demonstrating the long term persistence of latent HIV in resting memory CD4 T cells. A new study from Anthony Fauci's laboratory at the National Institute of Allergy and Infectious Diseases (NIAID) now once again attempts to resurrect the possibility of eradication by claiming that, in people treated early in infection, infected resting memory CD4 T cell numbers decline with a half-life of 4.6 months. The authors extrapolate from their data that HIV could theoretically be eradicated by 7.7 years of HAART.  Given the many potential sanctuaries for latent HIV in the human body (which include not just CD4 T cells but also macrophages and potentially other cell types), this claim is severely lacking in biological plausibility.

In an accompanying commentary, David Margolis and Nancie Archin politely highlight the study's limitations:

"...although Chun et al.'s study finds that the half-life of latently infected resting CD4+ T cells in this cohort is the shortest ever reported (4.6 months), a close examination of the data suggests that, at least in 4 of the patients studied, a second, slower phase of decay appears to exist. If so, the frequency of infection may not diminish to much less than 1 cell per billion after years of therapy. In this case, given the 110 billion resting CD4+ T cells carried by the average human, the few infected cells remaining may be enough to reignite infection. And, of course, the possibility exists that HIV may rarely persist in cells other than resting CD4+ T cells or that circulating resting cells somehow underrepresent infection in resting cells in the tissue."

The Journal of Infectious Diseases    2007;195:000

EDITORIAL COMMENTARY

Eliminating Persistent HIV Infection: Getting to the End of the Rainbow

David M. Margolis1,2,3 and Nancie M. Archin1

Departments of 1Medicine, 2Microbiology and Immunology, and 3Epidemiology, University of North Carolina at Chapel Hill, Chapel Hill

The Journal of Infectious Diseases    2007;195:000

Brief Report

Decay of the HIV Reservoir in Patients Receiving Antiretroviral Therapy for Extended Periods: Implications for Eradication of Virus

Tae-Wook Chun,1 J. Shawn Justement,1 Susan Moir,1 Claire W. Hallahan,2 Janine Maenza,3 James I. Mullins,3,4,5 Ann C. Collier,3 Lawrence Corey,3,5 and Anthony S. Fauci1

1Laboratory of Immunoregulation and 2Biostatistics Research Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland; Departments of 3Medicine, 4Microbiology, and 5Laboratory Medicine, University of Washington School of Medicine, Seattle

(See the editorial commentary by Margolis and Archin, on pages XXX–XX.)

The persistence of latently infected resting CD4+ T cells has been clearly demonstrated in human immunodeficiency virus (HIV)–infected individuals receiving effective antiviral therapy. However, estimates of the half-life of this viral reservoir have been quite divergent. We demonstrate clear evidence for decay of this HIV reservoir in patients who initiated antiviral therapy early in infection. The half-life of this latent viral reservoir was estimated to be 4.6 months. It is projected that it will take up to 7.7 years of continuous therapy to completely eliminate latently infected resting CD4+ T cells in infected individuals who initiate antiviral therapy early in HIV infection.