Michael Palm Basic Science, Vaccines & Prevention Project Weblog

CD4 T Cells Can Wield a Chemokine Shield Against HIV

A new paper from Joseph Casazza and colleagues at the NIH’s Vaccine Research Center reports that some memory CD4 T cells can shield themselves against HIV infection by releasing beta chemokines. The researchers were following up on prior studies showing that CD4 T cells targeting HIV antigens are preferentially infected with the virus compared to CD4 T cells of other specificities (such as those targeting CMV). 

Looking at CMV-specific CD4 T cells in detail, they found that release of the chemokines MIP-1 alpha and MIP-1 beta is significantly correlated with resistance to HIV infection. These chemokines can inhibit HIV infection, both by blocking virus interactions with the CCR5 co-receptor and causing CCR5 downregulation from the cell surface. The researchers also report that the ability of CMV-specific CD4 T cells to make beta chemokines is linked to the maturational status of the cells; more mature CD4 T cells produce more chemokines. Maturation refers to the progressive acquisition of functions that occurs after a CD4 T cell is initially stimulated. This process is also referred to as differentiation and it typically progresses as CD4 T cells divide.

In contrast with CMV-specific cells, the study found that HIV-specific CD4 T cells rarely produce beta chemokines and are significantly less differentiated. This suggests that HIV infection inhibits the maturation of HIV-specific CD4 T cells, and is consistent with a study of acutely infected individuals which reported that the vast preponderance of HIV DNA is found in HIV-specific CD4 T cells that would normally be expected to mature into functional “memory” cells (these developing memory cells can be identified based on expression of the receptor for IL-7).

Casazza et al note that this research lays the groundwork for the development of strategies aiming to induce CD4 T cell responses against HIV that can self-protect against infection. The next step is to evaluate whether specific types of immunization can bias the virus-specific CD4 T cell response toward the production of the beta chemokines MIP-1 alpha and MIP-1 beta. If a successful approach can be discovered, it could conceivably be very useful for both preventive and therapeutic vaccine development.

PLoS Pathog 5(10): e1000646. doi:10.1371/journal.ppat.1000646

Autocrine Production of β-Chemokines Protects CMV-Specific CD4+ T Cells from HIV Infection

Joseph P. Casazza1*, Jason M. Brenchley2, Brenna J. Hill2, Ribka Ayana1, David Ambrozak1, Mario Roederer3, Daniel C. Douek4, Michael R. Betts5, Richard A. Koup1

1 Immunology Laboratory, Vaccine Research Center, NIAID, NIH, Bethesda, Maryland, United States of America, 2 Immunopathogenesis Unit, Lab of Molecular Microbiology, NIAID, NIH, Bethesda, Maryland, United States of America, 3 ImmunoTechnology Section, Vaccine Research Center, NIAID, NIH, Bethesda, Maryland, United States of America, 4 Human Immunology Section, Vaccine Research Center, NIAID, NIH, Bethesda, Maryland, United States of America, 5 Department of Microbiology, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America

Abstract Top

Induction of a functional subset of HIV-specific CD4+ T cells that is resistant to HIV infection could enhance immune protection and decrease the rate of HIV disease progression. CMV-specific CD4+ T cells, which are less frequently infected than HIV-specific CD4+ T cells, are a model for such an effect. To determine the mechanism of this protection, we compared the functional response of HIV gag-specific and CMV pp65-specific CD4+ T cells in individuals co-infected with CMV and HIV. We found that CMV-specific CD4+ T cells rapidly up-regulated production of MIP-1α and MIP-1β mRNA, resulting in a rapid increase in production of MIP-1α and MIP-1β after cognate antigen stimulation. Production of β-chemokines was associated with maturational phenotype and was rarely seen in HIV-specific CD4+ T cells. To test whether production of β-chemokines by CD4+ T cells lowers their susceptibility to HIV infection, we measured cell-associated Gag DNA to assess the in vivo infection history of CMV-specific CD4+ T cells. We found that CMV-specific CD4+ T cells which produced MIP-1β contained 10 times less Gag DNA than did those which failed to produce MIP-1β. These data suggest that CD4+ T cells which produce MIP-1α and MIP-1β bind these chemokines in an autocrine fashion which decreases the risk of in vivo HIV infection.

New Screening Tool for Drugs that Reverse HIV Latency

The major obstacle to maximizing HIV elimination from the body is the existence of latently infected cells (for an excellent introduction to the topic, see this free access minireview from J. Virology). These cells harbor HIV DNA that has integrated into the chomosomal DNA, and virus particles are typically not produced unless the cell is stimulated in some way. Latently infected cells are thus invisible to the immune system, and while antiretrovirals can prevent any virus they produce from infecting other cells, the drugs cannot excise the integrated HIV DNA. This has led researchers to pursue new approaches to awakening dormant HIV, but the pursuit has been slowed by a reliance on cell lines, due to the lack of a physiologically relevant system for generating latently infected primary CD4 T cells.

In a new paper in the Journal of Clinical Investigation, Hung-Chih Yang and colleagues from Bob Siliciano’s laboratory at Johns Hopkins report the development of a new screening system for identifying latency-reversing drugs. The researchers introduced the survival gene Bcl-2 into CD4 T cells in vitro and this facilitated the generation of large numbers of long-lived latently infected cells. As a proof of concept, one compound (named 5HN) that potently reactivated latent HIV without activating the CD4 T cells was identified from a screen of several thousand drugs. The researchers note, however, that 5HN is likely too toxic for human use. Screens of other compounds are ongoing and the researchers hope to eventually identify candidates suitable for clinical testing.

J. Clin. Invest. doi:10.1172/JCI39199. 

Technical Advance

Small-molecule screening using a human primary cell model of HIV latency identifies compounds that reverse latency without cellular activation (free access to full text)

Hung-Chih Yang1, Sifei Xing1,2, Liang Shan1,2, Karen O’Connell1, Jason Dinoso1,2, Anding Shen3, Yan Zhou1, Cynthia K. Shrum1, Yefei Han1, Jun O. Liu2, Hao Zhang4, Joseph B. Margolick4 and Robert F. Siliciano1,5

1 Department of Medicine and 2 Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. 3 Department of Biology, Calvin College, Grand Rapids, Michigan, USA. 4 Department of Molecular Microbiology and Immunology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland, USA. 5 Howard Hughes Medical Institute, Baltimore, Maryland, USA.

Published October 1, 2009

Received for publication March 16, 2009, and accepted in revised form July 29, 2009.

The development of highly active antiretroviral therapy (HAART) to treat individuals infected with HIV-1 has dramatically improved patient outcomes, but HAART still fails to cure the infection. The latent viral reservoir in resting CD4+ T cells is a major barrier to virus eradication. Elimination of this reservoir requires reactivation of the latent virus. However, strategies for reactivating HIV-1 through nonspecific T cell activation have clinically unacceptable toxicities. We describe here the development of what we believe to be a novel in vitro model of HIV-1 latency that we used to search for compounds that can reverse latency. Human primary CD4+ T cells were transduced with the prosurvival molecule Bcl-2, and the resulting cells were shown to recapitulate the quiescent state of resting CD4+ T cells in vivo. Using this model system, we screened small-molecule libraries and identified a compound that reactivated latent HIV-1 without inducing global T cell activation, 5-hydroxynaphthalene-1,4-dione (5HN). Unlike previously described latency-reversing agents, 5HN activated latent HIV-1 through ROS and NF-κB without affecting nuclear factor of activated T cells (NFAT) and PKC, demonstrating that TCR pathways can be dissected and utilized to purge latent virus. Our study expands the number of classes of latency-reversing therapeutics and demonstrates the utility of this in vitro model for finding strategies to eradicate HIV-1 infection.

Forced Retirement for Tired T Cells?

One of the signature immunological changes that accompanies aging is the accumulation of CD8 T cells lacking the CD28 co-stimulatory molecule (dubbed CD8+ CD28- T cells). Several studies have found that in elderly cohorts, increased numbers of CD8+CD28- T cells are associated with a significantly elevated risk of mortality. These studies have also reported that elderly people with CMV infection have higher numbers of CD8+CD28- T cells than comparable CMV seronegative individuals. These findings have parallels in HIV because CD8+CD28- T cells accumulate far more rapidly in people with HIV than in uninfected individuals, and a recent study reported that increased numbers of these cells are associated with more rapid progression to AIDS and death.

Current evidence suggests that CD8+ CD28- T cells are functionally impaired due to chronic stimulation and represent a “senescent” population. The evidence includes shortened telomeres (which shorten in cells as a result of proliferation), resistance to cell death (apoptosis), reduced response to normal immune signaling and production of high levels of interferon gamma. There are also studies indicating that this functional profile causes CD8+ CD28- T cells to contribute to detrimental inflammatory processes in the elderly (sometimes referred to as “inflammaging”).

Suspicions that CD8+ CD28- T cells are bad actors in a variety of disease processes, including HIV, has led some researchers to propose that they should simply be removed, although exactly how this might be accomplished has not been detailed (at least to the best of my knowledge). The goal would be to allow more functional T cells to expand and take up the “immunological space” being occupied by the senescent CD8+ CD28- T cells.

This therapeutic idea prompted Birgit Weinberger and colleagues to take a preliminary look at whether antigens being targeted by CD8+ CD28- T cells are also targeted by other CD8+ CD28+ T cells, based on the concern that removal of the CD28- population might inadvertently eliminate important immune responses. The study sampled T cells from six donors with a median age of 70. Responses to a CMV epitope from the pp65 protein were measured, and the researchers found that they were present among both CD8+ CD28- and CD8+ CD28+ T cells.

Although they stress the preliminary nature of the work, the authors interpret their findings as suggesting: “Elimination of CD28- T cells in order to create more immunological space and consequently to allow the regeneration and rejuvenation of the aged T cell system would…not lead to the loss of important specificities. Viral control would be intact due to the recruitment and propagation of the relevant clones from naïve or early memory T cell populations.” Due to the increased attention being paid to immunosenescence, both in HIV-infected and uninfected individuals, it seems likely that the potential for removing CD28- cells will be explored in animal models before too long.

Immunology Letters

Article in Press, Uncorrected Proof

doi:10.1016/j.imlet.2009.08.008   

CD28−CD8+ T cells do not contain unique clonotypes and are therefore dispensable

Birgit Weinberger a, Kathrin Welzl a, Dietmar Herndler-Brandstetter a, Walther Parson b and Beatrix Grubeck-Loebenstein a.

a Institute for Biomedical Aging Research, Austrian Academy of Sciences, Rennweg 10, 6020 Innsbruck, Austria

b Institute of Legal Medicine, Innsbruck Medical University, Müllerstr. 44, 6020 Innsbruck, Austria

Abstract

Highly differentiated CD28 effector T cells which accumulate in a variety of diseases and also with increasing age contribute to inflammatory processes, limit immunological space and diversity, and are associated with immunological dysfunction and reduced responses to vaccination. Elimination of CD28 T cells has been suggested as a measure for immunological rejuvenation but may lead to the loss of important T cell specificities. Using T cells specific for the immunodominant CMV-derived epitope NLVPMVATV as a model, we show that the same clonotypes are present in CD8+CD28+ naïve/early memory and CD8+CD28 effector T cells. Therefore, CD28 cells do not seem to contain clones which are not present in the residual population. The elimination of effector T cells would not lead to the loss of important specificities, as relevant clonotypes could be recruited and propagated from naïve or early memory T cell subsets in the case of exposure to pathogen. 

Trends in Immunology: Free Access Issue on Immune Senescence

The journal Trends in Immunology has published a special issue on immune senescence, and access to all articles is being offered free of charge (thanks to the sponsorship of the National Institutes of Health). The parallels between immune senescence in uninfected elderly individuals and people with HIV are increasingly well-recognized and include:

• Depletion of naive CD4 and CD8 T cells
• Involution of the thymus and decreased thymic output
• Skewing of the CD4:CD8 T cell ratio
• Accumulation of dysfunctional CD8 T cells lacking the CD28 co-stimulatory molecule
• Decreased responses to new immunizations
• Decreased recall (memory) T responses to common antigens

Research into immune senescence and its association with illness and mortality has gained considerably more attention recently, and may have the potential to offer important insights into the pathogenesis of HIV infection. Additional background on the topic, along with a link to a webcast presentation by immune senescence expert Rita Effros, can be found in a post from January of this year, "Accelerated Aging of the Immune System in HIV Infection."

Low Viral Loads in Elite Controllers

Several years ago, Bruce Walker and colleagues from Partners AIDS Research in Boston launched the largest ever effort to research immunological control of HIV infection, the international HIV controllers study. The project is now proceeding under the aegis of the new Ragon Institute of Massachusetts General Hospital, created recently thanks to the generous support of the Ragon family. The HIV controllers study is collecting blood samples from both elite controllers, defined based on having a viral load less than 50 copies in the absence of any treatment, and viremic controllers, defined based on a viral load of between 50 and 2000 copies in the absence of treatment. A number of papers have been published already describing results from the study, and the latest has just appeared online in the Journal of Infectious Diseases.

The purpose of this investigation was to quantify the level of viral load among elite controllers using an ultra-sensitive assay that can measure down to a single copy of HIV RNA per ml. Among 90 individuals studied, the median viral load level was just 2 copies/mL. A longitudinal analysis of a subset of 31 participants demonstrated that 2-5-fold fluctuations in viral load were common. In terms of the relationship with immune responses, the researchers document a significant correlation between the breadth and potency of neutralizing antibody responses against HIV and the level of viral load detected. The breadth and magnitude of HIV-specific CD8 T cell responses, as measured solely by interferon-gamma production, did not show a correlation with viral load at these low levels.  

An analysis of the relationship between viral load and the slope of CD4 decline was also conducted. The median time for which multiple CD4 measurements were available was 3.6 years (range 1 – 17.3), and the median number of measurements per person was seven. Although immune activation has previously been associated with CD4 T cell declines in elite controllers, in this analysis the median value of the slope per year was +11 cells/mm3.

Due the limited duration of follow up and number of measurements, the researchers note that in many cases the values were not significantly different from zero, indicating CD4 T cell counts were essentially stable over time. To try and get a better sense of which participants might be experiencing significant changes in CD4 counts, the researchers subsequently focused on individuals whose slopes were statistically different from zero. These analyses revealed that 5 out of 27 individuals (19%) with viral load levels less than 1 copy/mL experienced significant CD4 T cell count increases over time. The same was true for only 3 out of 50 individuals (3%) with HIV viral loads greater than 1 copy/mL. Conversely, 8 individuals experienced significant CD4 T cell declines, and all showed viral load levels over 1 copy/mL. An overall examination of the link between viral load level and CD4 T cell count slope showed a weak but significant correlation, with higher viral loads associated with CD4 T cell decline (r=-0.23; p=0.04).

Overall, the results suggest that the use of a viral load assay that is 250-fold more sensitive than those currently commercial available can reveal important information regarding elite control of HIV.

The Journal of Infectious Diseases 2009;200:000–000

DOI: 10.1086/605446

MAJOR ARTICLE

Persistent Low‐Level Viremia in HIV‐1 Elite Controllers and Relationship to Immunologic Parameters

Florencia Pereyra,1,2 Sarah Palmer,3,7 Toshiyuki Miura,1,6 Brian L. Block,1 Ann Wiegand,3 Alissa C. Rothchild,1 Brett Baker,1 Rachel Rosenberg,1 Emily Cutrell,1 Michael S. Seaman,4 John M. Coffin,5 and Bruce D. Walker1,6

1The Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology, and Harvard, 2Brigham and Women’s Hospital, Division of Infectious Diseases, 4Division of Viral Pathogenesis, Beth Israel Deaconess Medical Center, and 5Department of Molecular Biology and Microbiology, Tufts University, Boston Massachusetts; 3HIV Drug Resistance Program, National Cancer Institute, National Institutes of Health, Frederick, and 6Howard Hughes Medical Institute, Chevy Chase, Maryland; and 7Virology Department, Swedish Institute for Infectious Disease Control and Karolinska Institute, Solna, Sweden

Background. Human immunodeficiency virus type 1 (HIV‐1) elite controllers are able to control virus replication to levels below the limits of detection by commercial assays, but the actual level of viremia in these individuals is not well defined. Here, we quantify plasma HIV‐1 RNA in elite controllers and correlate this with specific immunologic parameters.

Methods. Plasma HIV‐1 RNA levels were quantified in 90 elite controllers with use of a real time reverse‐transcriptase polymerase chain reaction assay with a sensitivity of 0.2 copies/mL. HIV‐1–specific immune responses and longitudinal CD4+ T cell counts were examined.

Results. The median plasma HIV‐1 RNA level was 2 copies/mL (interquartile range, 0.2–14 copies/mL). A longitudinal analysis of 31 elite controllers demonstrated 2–5–fold fluctuations in viremia in the majority of individuals; 6 had persistent levels below 1 copy/mL. Viremia correlated directly with HIV‐1–specific neutralizing antibodies and Western blot reactivity but not with CD8+ T cell responses. Absolute CD4+ T cell decrease was more common among individuals with detectable viremia (p=.04).

Conclusions. Low‐level viremia is present in the majority of elite controllers and is associated with higher HIV‐1–specific antibody responses. Absolute CD4+ T cell loss is more common among viremic individuals, suggesting that even very low‐level viremia has negative consequences over time.

Received 29 January 2009; accepted 21 April 2009; electronically published 4 August 2009.

Reprints or correspondence: Florencia Pereyra, MD, Ragon Institute, Massachusetts General Hospital, 149 13th St, Charlestown, MA 02129

Potential conflicts of interest: none reported

Presented in part: 15th Conference on Retroviruses and Opportunistic Infections, Boston, Massachusetts, February 2008 (abstract 349).

Financial support: National Institutes of Health (AI28568 and AI30914 to B.D.W.), the Bill and Melinda Gates Foundation, the International AIDS Vaccine Initiative, and the Mark and Lisa Schwartz Foundation.

Study Uncovers Sex Differences in Immune Activation

There is a large body of evidence showing that innate and adaptive immune responses can differ in women and men, but the reasons for these differences are only partly understood (as reviewed recently in Nature Reviews Immunology). In HIV infection, it has been reported that average viral loads in early infection are lower in women than men, but women face a 1.6-fold higher risk of developing AIDS compared to men with the same viral load. Marc Hellerstein’s group has also presented data demonstrating that women have significantly higher T cell turnover compared to men with similar CD4 T cell counts. The mechanistic basis for these differences has been unclear.

A paper in Nature Medicine now offers a possible explanation for the observed sex differences in HIV pathogenesis. Angela Meier and colleagues from Marcus Altfeld’s group at the Ragon Institute of Massachusetts General Hospital have previously shown that HIV stimulates an innate immune receptor called toll-like receptor-7 (TLR7). In the new study, they report that a key immune cell called a plasmacytoid dendritic cell (pDC) responds differently to TLR7 stimulation depending on the sex of the donor. Specifically, significantly more pDC from women produced interferon-alpha compared to pDC from men. The study authors note that this finding has also been reported with a different TLR7 stimulus, a drug called imiquimod.

The researchers went on to evaluate the role of sex hormones, finding that the percentage of pDC producing interferon-alpha after TLR7 stimulation was significantly correlated with plasma progesterone concentrations. There was also a trend toward lower percentages of pDC producing interferon-alpha in postmenopausal women compared to premenopausual women.

Because interferon-alpha induces the expression of the immune activation marker CD38 on CD8 T cells in vitro, the researchers decided to explore whether women might show higher levels of immune activation than men despite comparable viral loads. In a comparison of 109 women and 514 men participating in the ACTG 384 trial (all treatment naive), women were found to have significantly higher levels of CD8 T cell activation than men after adjustment for viral load. On average, women had 4.6% more activated CD8 T cells. As immune activation levels are the strongest predictor of disease progression in HIV infection, the study results offer a compelling explanation for why women have a higher risk of developing AIDS compared to men with the same viral load.

The authors conclude: “Modulation of the TLR7 pathways in pDCs could…represent a unique approach to reduce HIV-1–associated pathogenesis and might have implications that go beyond HIV-1 infection, as differential susceptibility to several RNA viruses have been described for men and women, and autoimmune diseases that show sex differences in their incidence, such as systemic lupus erythematosus, have also been shown to involve the TLR7 and TLR8 pathway.”

Nature Medicine

Published online: 13 July 2009 | doi:10.1038/nm.2004

Letter

Sex differences in the Toll-like receptor–mediated response of plasmacytoid dendritic cells to HIV-1

Angela Meier1,8, J Judy Chang1,8, Ellen S Chan2, Richard B Pollard3, Harlyn K Sidhu1, Smita Kulkarni4, Tom Fang Wen1, Robert J Lindsay1, Liliana Orellana2, Donna Mildvan5, Suzane Bazner1,6, Hendrik Streeck1, Galit Alter1, Jeffrey D Lifson7, Mary Carrington4, Ronald J Bosch2, Gregory K Robbins6 & Marcus Altfeld1,6

Manifestations of viral infections can differ between women and men1, and marked sex differences have been described in the course of HIV-1 disease. HIV-1–infected women tend to have lower viral loads early in HIV-1 infection but progress faster to AIDS for a given viral load than men2, 3, 4, 5, 6, 7. Here we show substantial sex differences in the response of plasmacytoid dendritic cells (pDCs) to HIV-1. pDCs derived from women produce markedly more interferon- (IFN-) in response to HIV-1–encoded Toll-like receptor 7 (TLR7) ligands than pDCs derived from men, resulting in stronger secondary activation of CD8+ T cells. In line with these in vitro studies, treatment-naive women chronically infected with HIV-1 had considerably higher levels of CD8+ T cell activation than men after adjusting for viral load. These data show that sex differences in TLR-mediated activation of pDCs may account for higher immune activation in women compared to men at a given HIV-1 viral load and provide a mechanism by which the same level of viral replication might result in faster HIV-1 disease progression in women compared to men. Modulation of the TLR7 pathway in pDCs may therefore represent a new approach to reduce HIV-1–associated pathology.

1. Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard University, Boston, Massachusetts, USA.

2. Harvard School of Public Health, Boston, Massachusetts, USA.

3. University of California–Davis Medical Center, Sacramento, California, USA.

4. Cancer and Inflammation Program, Laboratory of Experimental Immunology and Science Applications International Corporation–Frederick, National Cancer Institute, Frederick, Maryland, USA.

5. Beth Israel Medical Center, New York, New York, USA.

6. Division of Infectious Diseases, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA.

7. AIDS and Cancer Virus Program, Science Applications International Corporation–Frederick, National Cancer Institute, Frederick, Maryland, USA.

8. These authors contributed equally to this work.

Lymphatic Tissue Gene Expression During HIV Infection

The new issue of the Journal of Immunology features a paper from Ashley Haase’s group looking at lymphoid tissue gene expression in HIV infection. The study, led by Qingsheng Li, uses microarray technology to investigate gene expression changes during the acute, asymptomatic and late (AIDS) stages of disease compared to healthy uninfected individuals. The analyses reveal that the expression of 46 genes is significantly altered (in most cases upregulated) at all stages of infection; half of these genes have known functions in immunity, particularly immune activation. Examples include CD38 (the well-known immune activation marker that predicts the pace of disease progression in HIV infection), CCR5, pro-apoptotic genes and nine different interferon-related genes.

Acute infection is associated with altered expression of another 358 unique genes, more than any other stage of infection. Again, in most cases (81%) these genes are upregulated and a large proportion are related to immune activation (e.g. proinflammatory cytokines, signal transduction molecules, activators of cell proliferation, mediators of cell cycle progression and the pro-apoptotic Fas-Fas pathway). However, there are also increases in the expression of genes associated with dampening the immune response such as IDO, an enzyme with powerful immune-suppressing properties. A suite of additional genes involved in innate immunity are also upregulated during acute infection, including the toll-like receptors TLR7 and TLR8 and numerous components of the interferon pathway.

The asymptomatic stage of infection is associated with a dramatic shift in gene expression, with levels returning to baseline in most cases. In addition to the 46 genes with altered expression at all stages of infection, only 18 genes are uniquely altered in asymptomatic individuals. The majority relate to immune activation and lymphoid tissue remodeling and repair.

In people with AIDS, 183 genes are uniquely altered and - in contrast to the other stages of infection - most of these genes (84%) are downregulated. Examples include genes involved in immune activation, apoptosis, and tissue remodeling and repair. A substantial proportion of the genes belong to the CD28 T cell co-stimulatory pathway, consistent with the accumulation of dysfunctional CD28-negative T cells that occurs over the course of HIV infection. The researchers also note that at this stage of disease “decreased expression could be due to loss of cells expressing these genes, decreased expression in a particular cell type, or both.”

In the discussion section of the paper, the authors conclude: “the major finding of this study is that there are stage-specific transcriptional signatures in lymphoid tissues during HIV-1 infection.” The results set the stage for additional investigations into the consequences of the documented changes in gene expression using a comprehensive systems biology approach. The ultimate aim is “not only to better understand viral pathogenesis during the various stages of HIV-1 disease but also to identify adjunctive approaches to improving treatment and immune reconstitution.”

FIGURE 2. In human LT of HIV-1-infected subjects, the general functional categories of genes with altered expression (1.7 times the level at baseline, p < 0.05). A, Common to all stages (C1); B, unique to the acute stage (S1); C, unique to the asymptomatic stage (S2); and D, unique to the AIDS stage (S3). Green and red letters indicate, respectively, decreased and increased expression. The size of each sector in a pie diagram is proportional to the number of genes in its category (in parentheses). All genes and their names derived from abbreviations can be found in supplemental Table I.

CREDIT: The Journal of Immunology, 2009, doi:10.4049/jimmunol.0803222

The Journal of Immunology, 2009, doi:10.4049/jimmunol.0803222

Published online July 13, 2009

Microarray Analysis of Lymphatic Tissue Reveals Stage-Specific, Gene Expression Signatures in HIV-1 Infection

Qingsheng Li2,*, Anthony J. Smith2,*, Timothy W. Schacker, John V. Carlis, Lijie Duan*, Cavan S. Reilly and Ashley T. Haase3,*

*Department of Microbiology  Division of Infectious Diseases, Department of Medicine, Medical School  Department of Computer Science and Engineering, Institute of Technology, and  Division of Biostatistics, School of Public Health, University of Minnesota, Minneapolis, MN 55455

Untreated HIV-1 infection progresses through acute and asymptomatic stages to AIDS. Although each of the three stages has well-known clinical, virologic, and immunologic characteristics, much less is known of the molecular mechanisms underlying each stage. In this study, we report lymphatic tissue microarray analyses, revealing for the first time stage-specific patterns of gene expression during HIV-1 infection. We show that although there is a common set of key genes with altered expression throughout all stages, each stage has a unique gene expression signature. The acute stage is most notably characterized by increased expression of hundreds of genes involved in immune activation, innate immune defenses (e.g., RIG-1, MDA-5, TLR7 and TLR8, PKR, APOBEC3B, 3F, 3G), adaptive immunity, and in the proapoptotic Fas-Fas ligand pathway. Yet, quite strikingly, the expression of nearly all acute stage genes return to baseline levels in the asymptomatic stage, accompanying partial control of infection. This transition from acute to asymptomatic stage is tied to increased expression of a diverse array of immunosuppressive genes (e.g., CLEC12B, ILT4, galectin-3, CD160, BCMA, FGL2, LAG3, GPNMB). In the AIDS stage, decreased expression of numerous genes involved in T cell signaling identifies genes contributing to T cell dysfunction. These common and stage-specific gene expression signatures identify potential molecular mechanisms underlying the host response and the slow, natural course of HIV-1 infection.

Tracing HIV Reservoirs

Two new papers offer differing perspectives on the reservoirs of HIV that persist despite effective antiretroviral therapy. Nicolas Chomont and colleagues demonstrate that when memory CD4 T cells containing integrated HIV proliferate (as most memory CD4 T cells do occasionally in a process known as homeostatic self-renewal), they copy the HIV provirus along with their own genomes. When CD4 T cell numbers decline, homeostatic proliferation occurs more frequently and Chomont’s paper shows that this is associated with an increase in the number of latently infected memory CD4 T cells. The researchers describe the cells that undergo more frequent proliferation in this setting as “transitional memory” T cells. At earlier stages of infection when the CD4 T cell pool is relatively intact, the reservoir of infected memory CD4 T cells is found to be far smaller and integrated virus is primarily located in “central memory” cells that divide less frequently.

Based on these findings, the study authors suggest that anticancer drugs that interfere with memory T cell proliferation should be studied for their potential to deplete the HIV reservoir. However, given the potential toxicities associated with inhibiting T cell proliferation, the risk/benefit of such trials would need to be carefully evaluated. A more ideal therapy would be one that only targeted dividing CD4 T cells containing HIV DNA, but it is currently unclear whether such an approach is within the realm of possibility. 

The second paper - by Timothy Brennan and colleagues from Bob Siliciano’s laboratory - uses genetic analyses of HIV sequences to show that there is a reservoir of virus that seems to be coming from a cell type other than memory CD4 T cells. The study finds that in most cases, the residual virus detectable in individuals on suppressive ART is genetically distinct from the virus found in memory CD4 T cells. The authors note in their conclusion: “Numerous laboratories are actively pursuing various eradication strategies, most of which involve some aspect of targeting and purging the latent reservoir in resting memory CD4+ T cells.  If much of the residual viremia of patients undergoing HAART comes from another reservoir or compartment as suggested here, then eradication strategies will have to include ways to target and purge this additional reservoir to be successful.”

Nature Medicine

Published online: 21 June 2009 | doi:10.1038/nm.1972

HIV reservoir size and persistence are driven by T cell survival and homeostatic proliferation

Nicolas Chomont1,2,3, Mohamed El-Far1,2,3, Petronela Ancuta3, Lydie Trautmann1,2,3, Francesco A Procopio1,2,3, Bader Yassine-Diab1,2,3, Geneviève Boucher1, Mohamed-Rachid Boulassel4, Georges Ghattas5, Jason M Brenchley6, Timothy W Schacker7, Brenna J Hill8, Daniel C Douek8, Jean-Pierre Routy4,9, Elias K Haddad1,2,3,9 & Rafick-Pierre Sékaly1,2,3,9,10,11

1. Laboratoire d'Immunologie, Centre de Recherche du Centre Hospitalier de l'Université de Montréal (CR-CHUM) Saint-Luc, Montréal, Québec, Canada. 2. Laboratoire d'Immunologie, Département de Microbiologie et d'Immunologie, Université de Montréal, Québec, Canada. 3. Institute National de la Santé et de la Recherche Médicale U743, CR-CHUM, Université de Montréal, Montréal, Québec, Canada. 4. Immunodeficiency Service and Division of Hematology, Royal Victoria Hospital, McGill University Health Centre (MUHC), McGill University, Montréal, Québec, Canada. 5. Department of Gastroenterology, MUHC, Montréal, Québec, Canada. 6. Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, US National Institutes of Health, Bethesda, Maryland, USA. 7. Department of Medicine, University of Minnesota, Minneapolis, Minnesota, USA. 8. Human Immunology Section, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, US National Institutes of Health, Bethesda, Maryland, USA. 9. Department of Microbiology and Immunology, McGill University, Montréal, Québec, Canada. 10. Department of Microbiology and Immunology, Oregon Health and Science University, Portland, Oregon, USA. 11. Vaccine and Gene Therapy Institute, Port-Ste Lucy, Florida, USA.

Abstract

HIV persists in a reservoir of latently infected CD4+ T cells in individuals treated with highly active antiretroviral therapy (HAART). Here we identify central memory (TCM) and transitional memory (TTM) CD4+ T cells as the major cellular reservoirs for HIV and find that viral persistence is ensured by two different mechanisms. HIV primarily persists in TCM cells in subjects showing reconstitution of the CD4+ compartment upon HAART. This reservoir is maintained through T cell survival and low-level antigen-driven proliferation and is slowly depleted with time. In contrast, proviral DNA is preferentially detected in TTM cells from aviremic individuals with low CD4+ counts and higher amounts of interleukin-7–mediated homeostatic proliferation, a mechanism that ensures the persistence of these cells. Our results suggest that viral eradication might be achieved through the combined use of strategic interventions targeting viral replication and, as in cancer, drugs that interfere with the self renewal and persistence of proliferating memory T cells.

JVI Accepts, published online ahead of print on 17 June 2009

J. Virol. doi:10.1128/JVI.02568-08

Analysis of HIV-1 Viremia and Provirus in Resting CD4+ T Cells Reveals a Novel Source of Residual Viremia in Patients on Antiretroviral Therapy

Timothy P. Brennan, John O. Woods, Ahmad R. Sedaghat, Janet D. Siliciano, Robert F. Siliciano, and Claus O. Wilke*

Department of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, Baltimore, MD 21205; Institute for Cell and Molecular Biology, The University of Texas at Austin, Austin, TX 78712; Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD 21205; Howard Hughes Medical Institute, Baltimore, MD 21205; Center for Computational Biology and Bioinformatics and Section of Integrative Biology, The University of Texas at Austin, Austin, TX 78712

Abstract

Highly active antiretroviral therapy (HAART) can reduce HIV-1 viremia to clinically undetectable levels. Despite this dramatic reduction, some virus is present in the blood. Additionally, a long-lived latent reservoir for HIV-1 exists in resting memory CD4+ T cells. This reservoir is believed to be a source of the residual viremia and is the focus of eradication efforts. Here, we employ two measures of population structure, analysis of molecular variance and the Slatkin-Maddison test, to demonstrate that the residual viremia is genetically distinct from proviruses in resting CD4+ T cells, but that proviruses in resting and activated CD4+ T cells belong to a single population. Residual viremia is genetically distinct from proviruses in activated CD4+ T cells, monocytes, and unfractionated peripheral blood mononuclear cells. The finding that some of the residual viremia in patients on HAART stems from an unidentified cellular source other than CD4+ T cells has implications for eradication efforts.

Illuminating Early Events in HIV Infection Using Single Genome Amplification

Two papers in the current Journal of Experimental Medicine offer unprecedented insight into the initial interactions between virus and host after HIV infection. An accompanying commentary by Zabrina Brumme and Bruce Walker eloquently articulates what these studies have achieved: “By identifying persons before seroconversion, pinpointing the transmitted virus, and assessing immune responses to that particular variant as it evolves, they provide a novel view of host and viral dynamics during the earliest stages of infection.”

In both studies the researchers use an optimized version of a technique called Single Genome Amplification (SGA), originally developed by Sarah Palmer and colleagues at the National Cancer Institute. While costly and labor-intensive, this technique allows sequencing of the HIV genome without many of the potential confounding errors that can occur with standard PCR. The researchers also used the sequences obtained by SGA to synthesize peptides for CD8 T cell response assays; this allowed detailed tracking of the impact of CD8 T cell responses on the virus genome.

The study results echo prior work from these groups suggesting that most HIV transmission events involve a single isolate; in 11 out of 12 cases SGA showed that all detected sequences were related to a single infecting virus. The remaining individual was infected with two viruses that could be unambiguously identified based on their sequences. In terms of viral evolution after infection, the researchers found that between transmission and peak viremia, diversification of HIV sequences was essentially random and showed no evidence of selection pressure from host immune responses. Subsequently, between 9-16 days later, the effects of selection became obvious, particularly effects attributable to HIV-specific CD8 T cell responses. By 32-45 days postinfection, almost the entire replicating virus population in each subject studied was replaced by viruses with mutations at two to five distinct loci in the genome, evincing selection pressure from both CD8 T cell and neutralizing antibody responses (and other unidentified sources also, perhaps innate and/or CD4 T cell immune responses).

The level of detail involved in the study also allowed the researchers to document virus escape from CD8 T cell responses earlier than has previously been reported. Mathematical modeling of the data indicated that HIV-specific CD8 T cells are more efficient at killing virus-infected cells during acute infection than prior estimates have suggested. Discussing the implications of their findings for T-cell-based vaccines, the authors state: “Modeling implied that a single T cell response was contributing as much as 15–35% of viral decline with multiple T cell responses. The implication of these observations is that vaccine-induced HIV-1–specific T cells will contribute to control of acute viremia if they are activated early in subsequent HIV-1 infection. However, because of the very rapid escape that occurs within the first few weeks of infection, T cell vaccines will need to stimulate a considerable breadth of T cell responses, clearly greater than the median of three epitopes induced by the Merck vaccine.”

Both papers are the work of researchers supported by the Center for HIV/AIDS Vaccine Immunology (CHAVI); without the substantial funding committed to this project by the National Institutes of Health, this work would not have been possible. 

Published online June 1, 2009

doi:10.1084/jem.20091094

The Journal of Experimental Medicine, Vol. 206, No. 6, 1215-1218

COMMENTARY

Tracking the culprit: HIV-1 evolution and immune selection revealed by single-genome amplification

Zabrina L. Brumme and Bruce D. Walker

Z.L. Brumme and B.D. Walker are at the Ragon Institute of MGH, MIT and Harvard, Charlestown MA 02129; Z.L. Brumme is at the Faculty of Health Sciences, Simon Fraser University, Burnaby BC V5A 1S6, Canada

ABSTRACT

Early control of HIV-1 infection is determined by a balance between the host immune response and the ability of the virus to escape this response. Studies using single-genome amplification now reveal new details about the kinetics and specificity of the CD8+ T cell response and the evolution of the virus during early HIV infection.

Published online June 1, 2009

doi:10.1084/jem.20090365

The Journal of Experimental Medicine, Vol. 206, No. 6, 1253-1272

ARTICLE

The first T cell response to transmitted/founder virus contributes to the control of acute viremia in HIV-1 infection

Nilu Goonetilleke1, Michael K.P. Liu1, Jesus F. Salazar-Gonzalez2, Guido Ferrari3, Elena Giorgi4, Vitaly V. Ganusov4, Brandon F. Keele2, Gerald H. Learn2, Emma L. Turnbull5, Maria G. Salazar2, Kent J. Weinhold3, Stephen Moore1, CHAVI Clinical Core B, Norman Letvin6, Barton F. Haynes3, Myron S. Cohen7, Peter Hraber4, Tanmoy Bhattacharya4,8, Persephone Borrow5, Alan S. Perelson4, Beatrice H. Hahn2, George M. Shaw2, Bette T. Korber4,8, and Andrew J. McMichael1

1 Medical Research Council Human Immunology Unit, Weatherall Institute of Molecular Medicine, Oxford University, Oxford OX3 9DS, England, UK

2 Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294

3 Duke University Medical Research, Duke University, Durham, NC 27710

4 Los Alamos National Laboratory, Theoretical Division, Los Alamos, NM 87545

5 The Jenner Institute, Oxford University, Compton RG20 7NN, England, UK

6 BIDMC, Harvard University, Boston, MA 02115

7 HIV Prevention Trials Unit, University of North Carolina, Chapel Hill, NC 27599

8 The Santa Fe Institute, Santa Fe, NM 87501

Identification of the transmitted/founder virus makes possible, for the first time, a genome-wide analysis of host immune responses against the infecting HIV-1 proteome. A complete dissection was made of the primary HIV-1–specific T cell response induced in three acutely infected patients. Cellular assays, together with new algorithms which identify sites of positive selection in the virus genome, showed that primary HIV-1–specific T cells rapidly select escape mutations concurrent with falling virus load in acute infection. Kinetic analysis and mathematical modeling of virus immune escape showed that the contribution of CD8 T cell–mediated killing of productively infected cells was earlier and much greater than previously recognized and that it contributed to the initial decline of plasma virus in acute infection. After virus escape, these first T cell responses often rapidly waned, leaving or being succeeded by T cell responses to epitopes which escaped more slowly or were invariant. These latter responses are likely to be important in maintaining the already established virus set point. In addition to mutations selected by T cells, there were other selected regions that accrued mutations more gradually but were not associated with a T cell response. These included clusters of mutations in envelope that were targeted by NAbs, a few isolated sites that reverted to the consensus sequence, and bystander mutations in linkage with T cell–driven escape.

Published online June 1, 2009

doi:10.1084/jem.20090378

The Journal of Experimental Medicine, Vol. 206, No. 6, 1273-1289

ARTICLE

Genetic identity, biological phenotype, and evolutionary pathways of transmitted/founder viruses in acute and early HIV-1 infection

Jesus F. Salazar-Gonzalez1, Maria G. Salazar1, Brandon F. Keele1, Gerald H. Learn1, Elena E. Giorgi2,3, Hui Li1, Julie M. Decker1, Shuyi Wang1, Joshua Baalwa1, Matthias H. Kraus1, Nicholas F. Parrish1, Katharina S. Shaw1, M. Brad Guffey1, Katharine J. Bar1, Katie L. Davis1, Christina Ochsenbauer-Jambor1, John C. Kappes1, Michael S. Saag1, Myron S. Cohen4, Joseph Mulenga5, Cynthia A. Derdeyn6, Susan Allen6, Eric Hunter6, Martin Markowitz7,8, Peter Hraber2, Alan S. Perelson2, Tanmoy Bhattacharya2,9, Barton F. Haynes10, Bette T. Korber2,9, Beatrice H. Hahn1, and George M. Shaw1

1 University of Alabama at Birmingham, Birmingham, AL 35294

2 Los Alamos National Laboratory, Los Alamos, NM 87545

3 University of Massachusetts, Amherst, MA 01002

4 The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599

5 Zambia-Emory HIV Research Project, Lusaka, Zambia

6 Emory University, Atlanta, GA 30329

7 Aaron Diamond AIDS Research Center, New York, NY 10016

8 The Rockefeller University, New York, NY 10065

9 Santa Fe Institute, Santa Fe, NM 87501

10 Duke University Medical Center, Durham, NC 27710

Identification of full-length transmitted HIV-1 genomes could be instrumental in HIV-1 pathogenesis, microbicide, and vaccine research by enabling the direct analysis of those viruses actually responsible for productive clinical infection. We show in 12 acutely infected subjects (9 clade B and 3 clade C) that complete HIV-1 genomes of transmitted/founder viruses can be inferred by single genome amplification and sequencing of plasma virion RNA. This allowed for the molecular cloning and biological analysis of transmitted/founder viruses and a comprehensive genome-wide assessment of the genetic imprint left on the evolving virus quasispecies by a composite of host selection pressures. Transmitted viruses encoded intact canonical genes (gag-pol-vif-vpr-tat-rev-vpu-env-nef) and replicated efficiently in primary human CD4+ T lymphocytes but much less so in monocyte-derived macrophages. Transmitted viruses were CD4 and CCR5 tropic and demonstrated concealment of coreceptor binding surfaces of the envelope bridging sheet and variable loop 3. 2 mo after infection, transmitted/founder viruses in three subjects were nearly completely replaced by viruses differing at two to five highly selected genomic loci; by 12–20 mo, viruses exhibited concentrated mutations at 17–34 discrete locations. These findings reveal viral properties associated with mucosal HIV-1 transmission and a limited set of rapidly evolving adaptive mutations driven primarily, but not exclusively, by early cytotoxic T cell responses.

Killer Helpers

CD4 T cells are typically portrayed as playing a supportive role in the generation and maintenance of immune responses, as their alternative name – helper cells – suggests. But over the past decade, studies have shown that there are a number of settings in which antigen-specific CD4 T cells exert direct cytotoxic activity (a capacity that was once believed to be an artifact of prolonged in vitro culture).

Two new papers describe the presence of cytotoxic virus-specific CD4 T cells in SIV and HIV infection, respectively. The first study, led by Jonah Sacha, obtained Gag- and Nef-specific CD4 T cells from a group of elite controller macaques infected with SIVmac239. The researchers had noticed that, after an experiment in which CD8 T cells were depleted, the animals experienced a rebound in viral load that was subsequently brought back down to undetectable levels. This regaining of control over viral replication was associated with an expansion of their Gag- and Nef-specific CD4 T cell responses, an observation that prompted the current study.

In Sacha’s in vitro analyses, cytotoxic Gag- and Nef-specific CD4 T cells were unable to recognize SIV-infected CD4 T cells, but efficiently recognized and killed infected macrophages. Recognition occurred very rapidly, within 1-2 hours, with activity peaking between 6 and 24 hours. The cytotoxic CD4 T cells consistently eliminated 30-40% of SIV-infected macrophages in culture. Sacha and colleagues conclude by stating “on the basis of the data presented here, we speculate that Gag- and Nef-specific CD4􏰀 T cells may play a more important role in the antiretroviral immune response than previously appreciated.”

The second paper, by Nan Zheng and colleagues from Kumamoto University, identifies cytotoxic Nef-specific CD4 T cells in 4/9 individuals analyzed. In this study, the Nef-specific CD4 T cells are reported to kill both HIV-infected macrophages and CD4 T cells, but the activity against macrophages is shown to be superior. 

Taken together, the findings suggest that it might be worth investigating whether cytotoxic virus-specific CD4 T cells can be induced by vaccination. Perhaps of note, one previous report from Ron Desrosiers laboratory has suggested that effector CD4 T cells with a cytotoxic phenotype (expressing perforin and the degranulation marker CD107) contribute to the protection obtained with live-attenuated SIV vaccines.

The PNAS paper is Open Access, click the title link for the full PDF. 

PNAS Published online before print May 28, 2009, doi: 10.1073/pnas.0813106106

Gag- and Nef-specific CD4+ T cells recognize and inhibit SIV replication in infected macrophages early after infection

Jonah B. Sacha a,1,2, Juan P. Giraldo-Vela a,1, Matthew B. Buechler a, Mauricio A. Martins a, Nicholas J. Maness a, Chungwon Chung a, Lyle T. Wallace a, Enrique J. León a, Thomas C. Friedrich b, Nancy A. Wilson a, Atsunobu Hiraoka  and David I. Watkins a

a Departments of aPathology and Laboratory Medicine andb  Pathobiological Sciences, University of Wisconsin, Madison, WI 53715; and c Osaka Dental University, Osaka 540-0008, Japan

1J.B.S. and J.P.G.-V. contributed equally to this work.

Abstract

The precise immunological role played by CD4+ T cells in retroviral infections is poorly defined. Here, we describe a new function of these cells, the elimination of retrovirus-infected macrophages. After experimental CD8+ cell depletion, elite controlling macaques with set-point viral loads ≤500 viral RNA copies/mL mounted robust Gag- and Nef-specific CD4+ T cell responses during reestablishment of control with ≥54% of all virus-specific CD4+ T cells targeting these 2 proteins. Ex vivo, these simian immunodeficiency virus (SIV)-specific CD4+ T cells neither recognized nor suppressed viral replication in SIV-infected CD4+ T cells. In contrast, they recognized SIV-infected macrophages as early as 2 h postinfection because of presentation of epitopes derived from virion-associated Gag and Nef proteins. Furthermore, virus-specific CD4+ T cells displayed direct effector function and eliminated SIV-infected macrophages. These results suggest that retrovirus-specific CD4+ T cells may contribute directly to elite control by inhibiting viral replication in macrophages.

JVI Accepts, published online ahead of print on 20 May 2009

J. Virol. doi:10.1128/JVI.00513-09

Strong ability of Nef-specific CD4+ cytotoxic T cells to suppress HIV-1 replication in HIV-1-infected CD4+ T cells and macrophages

Nan Zheng, Mamoru Fujiwara, Takamasa Ueno, Shinichi Oka, and Masafumi Takiguchi

Division of Viral Immunology and Division of Infectious Disease, Center for AIDS Research, Kumamoto University, 2-2-1 Honjo, Kumamoto 860-0811; and AIDS Clinical Center, International Medical Center of Japan, 1-21-1, Toyama, Shinjuku, Tokyo 162-8655, Japan

Abstract

A restricted number of studies have shown that HIV-1-specific cytotoxic CD4+ T cells are present in HIV-1-infected individuals. However, the roles of this type of CD4+ T cell in the immune responses against an HIV-1 infection remain unclear. In this study, we identified novel Nef epitope-specific HLA-DRB1*0803-restricted cytotoxic CD4+ T cells. The CD4+ T cell clones specific for Nef187-203 showed strong IFN-production after having been stimulated with autologous B-LCLs infected with Nef recombinant vaccinia virus or pulsed with heat-inactivated virus particles, indicating the presentation of the epitope antigen through both exogenous and endogenous MHC class II processing pathways. Nef187-203-specific CD4+ T cell clones exhibited strong cytotoxic activity against both HIV-1-infected macrophages and CD4+ T cells from an HLA-DRB1*0803+ donor. In addition, these Nef-specific cytotoxic CD4+ T cell clones exhibited strong ability to suppress HIV-1 replication in both macrophages and in CD4+ T cells in vitro. Nef187-203-specific cytotoxic CD4+ T cells were detected in cultures of peptide-stimulated PBMC and in ex vivo PBMC from 40% and 20% of DRB1*0803+ donors, respectively. These results suggest that HIV-1-specific CD4+ T cells may directly control HIV-1-infection in vivo by suppressing virus replication in HIV-1 natural host cells.