TAG HIV Basic Science, Vaccines, and Cure Project Blog: Innate immunity

Evidence of HIV-Induced Pyroptosis in Gut CD4 T Cells

Having recently criticized a widely publicized paper on HIV-induced pyroptosis for not providing much information relating to CCR5-tropic HIV infection, I’m duty bound to highlight a new study that includes some data addressing this topic. Pyroptosis is an inflammatory form of cellular suicide that has been shown by Gilad Doitsh from Warner Greene’s laboratory to result from abortive HIV infection of resting “bystander” CD4 T cells in lymphoid tissue, leading to a massive depletion of these cells. The effect is dramatic with CXCR4-tropic HIV isolates, because the majority of resting CD4 T cells express the CXCR4 receptor. In a paper published in Science earlier this year, Doitsh’s research group identified the human IFI16 protein as the sensor that detects HIV genetic material in these cells, leading to pyroptosis. Doitsh has also demonstrated that the phenomenon can be induced by CCR5-tropic HIV isolates in CCR5-expressing CD4 T cells, but because CCR5 is most commonly expressed on activated cells and is very rarely present on resting CD4 T cells, the number of bystander cells that might die as a result of pyroptosis in the setting of CCR5-tropic HIV infection is as yet unclear. The new study, published in Retrovirology by Amanda Steele and colleagues, reports evidence that pyroptosis occurs in gut CD4 T cells after exposure to CCR5-tropic HIV, despite the fact that most of these cells appear to be activated (and therefore potentially susceptible to productive, rather than just abortive, infection).

Unlike the work of Doitsh et al, which primarily used a lymphoid tissue culture system derived from tonsillar material (human lymphocyte aggregate culture or HLAC), Steele created a culture model from gut cells, naming it the Lamina Propria Aggregate Culture (LPAC). Infection of these cultures with a CCR5-tropic HIV isolate led to a statistically significant depletion of CD4 T cells after six days. Further analyses showed that the extent of the depletion correlated with the magnitude of productive HIV infection (as measured by p24 antigen expression), but that the majority of dying CD4 T cells were uninfected and undergoing necrosis associated with expression of caspase 1, an indication of pyroptosis (as opposed to apoptosis, the less inflammatory type of cellular suicide seen with productive infection). Approximately 63% of the dying CD4 T cells were negative for p24 and displayed a necrotic phenotype, while 13% were positive for p24 and apoptotic. Inhibition of caspase 1 did not affect productively infected cells but was able to reduce the extent of CD4 T cell depletion in the cultures from 36% to 18%, providing evidence that pyroptosis was contributing substantially to CD4 T cell death.

To try and model how HIV infection might interact with microbial translocation (the leakage of gut bacteria into the tissues and systemic circulation), the researchers conducted similar experiments with the addition of a commensal gut bacterial species, Escherichia coli. The presence of E. coli significantly enhanced CD4 T cell depletion, but also, surprisingly, seemed to cause a profound shift in the type of cell death, from pyroptosis (associated with expression of caspase 1) to apoptosis (associated with expression of caspase 3). This was established by testing the effects of caspase inhibitors on the extent of CD4 T cell death in the cultures; in the presence of E. coli, a caspase 1 inhibitor no longer had an effect, while a caspase 3 inhibitor significantly reduced CD4 T cell depletion. The authors suggest that perhaps the threshold for apoptosis is reduced in the presence of microbial products, and they propose a biphasic model of gut HIV pathogenesis in which pyroptosis dominates early on, contributing to the breakdown of gut wall integrity, followed by the onset of microbial translocation and increased HIV replication and CD4 T cell apoptosis.

One possible question pertaining to the interpretation of these studies is whether the involvement of caspase 1 is always synonymous with pyroptosis caused by abortive HIV infection. In one study cited by Steele and collagues, which measured caspase 1 expression in CD4 and CD8 T cells from HIV-positive individuals, a significant sixfold upregulation was documented in CD8 T cells, and a non-significant increase of threefold in CD4 T cells. Not having any expertise in caspase 1 biology (to say the least), I’m not clear how these differences might be explained or how they relate to the use of caspase 1 as a marker of pyroptosis (it seems unlikely that the caspase 1 upregulation documented in CD8 T cells was due to abortive HIV infection). Additional research is likely to clarify current uncertainties, as this has clearly become an important and expanding area of investigation.

In another development linked to the work of the Greene laboratory on pyroptosis, a separate paper published in Clinical & Experimental Immunology by Sara Nissen and colleagues reports that the expression of IFI16, the DNA-sensing protein, is significantly elevated in untreated HIV-positive people. Levels of IFI16 expression were found to correlate positively with viral load and the activation of central memory and effector memory CD4 T cells (measured by the CD38 marker). There was also an inverse correlation between IFI16 and CD4 T cell count. The researchers note these findings are consistent with the proposed role of IFI16 in HIV-induced CD4 T cell death, and indicate a relationship between IFI16 and immune activation (although further work is needed to discern if elevated IFI16 expression is a cause or consequence of immune activation—or both).

Retrovirology. 2014 Feb 4;11(1):14. doi: 10.1186/1742-4690-11-14.

Microbial exposure alters HIV-1-induced mucosal CD4+ T cell death pathways Ex vivo.

Steele AK, Lee EJ, Manuzak JA, Dillon SM, Beckham JD, McCarter MD, Santia ML, Wilson CC.

Abstract

BACKGROUND: Early HIV-1 infection causes massive CD4+ T cell death in the gut and translocation of bacteria into the circulation. However, the programmed cell death (PCD) pathways used by HIV-1 to kill CD4+ T cells in the gut, and the impact of microbial exposure on T cell loss, remain unclear. Understanding mucosal HIV-1 triggered PCD could be advanced by an ex vivo system involving lamina propria mononuclear cells (LPMCs). We therefore modeled the interactions of gut LPMCs, CCR5-tropic HIV-1 and a commensal gut bacterial species, Escherichia coli. In this Lamina Propria Aggregate Culture (LPAC) model, LPMCs were infected with HIV-1BaL by spinoculation and cultured in the presence or absence of heat killed E.coli. CD4+ T cell numbers derived from flow cytometry and viable cell counts were reported relative to mock infection. Viable cells were identified by viability dye exclusion (AqVi), and intracellular HIV-1 Gag p24 protein was used to identify infected cells. Annexin V and AqVi were used to identify apoptotic versus necrotic cells. Caspase-1 and Caspase-3 activities were blocked using specific inhibitors YVAD and DEVD, respectively.

RESULTS: CD4+ T cell depletion following HIV-1 infection was reproducibly observed by 6 days post infection (dpi). Depletion at 6 dpi strongly correlated with infection frequency at 4 dpi, was significantly blocked by Efavirenz treatment, and was primarily driven by p24-negative cells that were predominantly necrotic. HIV-1 infection significantly induced CD4+ T-cell intrinsic Caspase-1 activity, whereas Caspase-1 inhibition, but not Caspase-3 inhibition, significantly blocked CD4+ T cell depletion. Exposure to E.coli enhanced HIV-1 infection and CD4+ T depletion, and significantly increased the number of apoptotic p24+ cells. Notably, CD4+ T cell depletion in the presence of E.coli was partially blocked by Caspase-3, but not by Caspase-1 inhibition.

CONCLUSIONS: In the LPAC model, HIV-1 induced Caspase-1 mediated pyroptosis in bystander CD4+ T cells, but microbial exposure shifted the PCD mechanism toward apoptosis of productively infected T cells. These results suggest that mucosal CD4+ T cell death pathways may be altered in HIV-infected individuals after gut barrier function is compromised, with potential consequences for mucosal inflammation, viral dissemination and systemic immune activation.

Clin Exp Immunol. 2014 Mar 5. doi: 10.1111/cei.12317. [Epub ahead of print]

Innate DNA sensing is impaired in HIV patients and IFI16 expression correlates to chronic immune activation.

Nissen SK1, Højen JF, Andersen KL, Kofod-Olsen E, Berg RK, Paludan SR, Ostergaard L, Jakobsen MR, Tolstrup M, Mogensen TH.

Abstract

The innate immune system has been recognized to play a role in the pathogenesis of HIV infection, both by stimulating protective activities, and through contribution to chronic immune activation, the development of immunodeficiency, and progression to AIDS. A role for DNA sensors in HIV recognition has recently been suggested, and the aim of the present study was to describe the influence of HIV infection on expression and function of intracellular DNA sensing. Here we demonstrate impaired expression of interferon-stimulated genes in responses to DNA in PBMCs from HIV positive individuals, irrespective of whether patients receive antiretroviral treatment. Furthermore, we show that expression levels of the DNA sensors IFI16 and cGAS were increased in treatment-naïve patients, and for IFI16 expression was correlated with high viral load and low CD4 cell count. Finally, our data demonstrate a correlation between IFI16 and CD38 expression, a marker of immune activation, in CD4+ central and effector memory T cells, which may indicate that IFI16-mediated DNA sensing and signaling contributes to chronic immune activation. Altogether, the present study demonstrates abnormal expression and function of cytosolic DNA sensors in HIV patients, which may have implications for control of opportunistic infections, chronic immune activation, and T cell death.

March 21, 2014 in Immune activation, Inflammation, Innate immunity, Pathogenesis, Pyroptosis | Permalink | Comments (0)

Probiotic/Prebiotic Supplement Combination Shows Benefits in SIV-Infected Pigtailed Macaques

Over the years, a number of small studies have been published suggesting that there might be benefits associated with probiotic and/or prebiotic supplementation in people with HIV infection. Probiotics typically comprise live microbes that play a key role in maintaining gut health—commonly known as “good bacteria”—while prebiotics are food ingredients intended to stimulate the expansion and activity of these bacteria. The past decade has seen a renewed interest in gut health in HIV infection due to evidence that the virus severely depletes CD4 T cells in the GI tract, leading to diminished immune surveillance, compromised gut-wall integrity, and microbial translocation (the leakage of gut bacteria into the systemic circulation). Additionally, suppression of HIV replication by antiretroviral therapy (ART) does not necessarily restore gut CD4 T-cell numbers, and markers of microbial translocation have been associated with poor immune reconstitution despite ART.

To try to address the question of whether a combination of probiotics and prebiotics (referred to as synbiotic treatment) might serve as a useful adjunct to ART, Nichole Klatt and colleagues conducted a study in the animal model of SIV-infected pigtailed macaques. The results are published in the February issue of the Journal of Clinical Investigation. The researchers report that, compared to ART alone, adjunctive synbiotic treatment (the specific products used were VSL#3 and Culturelle) had positive effects on gut CD4 T cells and antigen-presenting cells, and also reduced fibrosis in local lymph tissue. The results appear encouraging, and were discussed in further detail at the recent CROI by senior author Jason Brenchley (the third talk in the symposium entitled Is Something Bugging You?).

Something that isn’t mentioned in the JCI paper, or addressed directly in Brenchley’s talk, is that previous work from this research group has shown that gut integrity in pigtailed macaques is unusually compromised even in uninfected animals; in a separate new paper published in the March 15 issue of the Journal of Immunology, they show that this correlates with the faster progression to AIDS that occurs in this monkey species. On the one hand, this may make pigtailed macaques a useful worst-case scenario in terms of modeling the contribution of microbial translocation to HIV pathogenesis and studying interventions like probiotics, but, on the other hand, the extent to which findings from this extreme situation can be extrapolated to humans is as yet unclear.

Available published data on synbiotic treatment in people with HIV appear limited, either involving individuals not on ART or on very short-term follow-up. As far as I can discern from a PubMed search, no trials have used the specific combination of VSL#3 and Culturelle. Currently, clinicaltrials.gov lists one open clinical trial of the probiotic Biola that is enrolling individuals both on and off ART; study sites are limited to Oslo, Norway, and Stockholm, Sweden. According to U.S.-based researchers, there are plans afoot to conduct a randomized, controlled clinical trial to assess whether the promising results from the Brenchley laboratory can be translated to humans with HIV infection.

Published in Volume 123, Issue 2 (February 1, 2013)

J Clin Invest. 2013;123(2):903–907. doi:10.1172/JCI66227.

Brief Report

Probiotic/prebiotic supplementation of antiretrovirals improves gastrointestinal immunity in SIV-infected macaques

Nichole R. Klatt1, Lauren A. Canary1, Xiaoyong Sun2, Carol L. Vinton1, Nicholas T. Funderburg3, David R. Morcock4, Mariam Quiñones1, Clayton B. Deming5, Molly Perkins1, Daria J. Hazuda6, Michael D. Miller7, Michael M. Lederman3, Julie A. Segre5, Jeffrey D. Lifson4, Elias K. Haddad2, Jacob D. Estes4 and Jason M. Brenchley1

1National Institute of Allergy and Infectious Diseases (NIAID), NIH, Bethesda, Maryland, USA.

2VGTI-Florida, Port St. Lucie, Florida, USA.

3Division of Infectious Diseases, Case Western Reserve University, Cleveland, Ohio, USA.

4SAIC Frederick Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland, USA.

5National Human Genome Research Institute (NHGRI), NIH, Bethesda, Maryland, USA.

6Merck Research Labs, West Point, Pennsylvania, USA.

7Gilead Sciences Inc., Foster City, California, USA.

HIV infection results in gastrointestinal (GI) tract damage, microbial translocation, and immune activation, which are not completely ameliorated with suppression of viremia by antiretroviral (ARV) therapy. Furthermore, increased morbidity and mortality of ARV-treated HIV-infected individuals is associated with these dysfunctions. Thus, to enhance GI tract physiology, we treated SIV-infected pigtail macaques with ARVs, probiotics, and prebiotics or with ARVs alone. This synbiotic treatment resulted in increased frequency and functionality of GI tract APCs, enhanced reconstitution and functionality of CD4+ T cells, and reduced fibrosis of lymphoid follicles in the colon. Thus, ARV synbiotic supplementation in HIV-infected individuals may improve GI tract immunity and thereby mitigate inflammatory sequelae, ultimately improving prognosis.

The Journal of Immunology, March 15, 2013, vol. 190 no. 6 2959-2965

Rate of AIDS Progression Is Associated with Gastrointestinal Dysfunction in Simian Immunodeficiency Virus–Infected Pigtail Macaques

Lauren A. Canary*, Carol L. Vinton*, David R. Morcock†, Jordan B. Pierce*, Jacob D. Estes†, Jason M. Brenchley* and Nichole R. Klatt*,‡

*Laboratory of Molecular Microbiology, Program in Barrier Immunity and Repair, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892;

†AIDS and Cancer and Virus Program, Science Applications International Corporation-Frederick, Frederick National Laboratory for Cancer Research, Frederick, MD 21702; and

‡Department of Pharmaceutics, Washington National Primate Research Center, University of Washington, Seattle, WA 98121

During HIV/SIV infection, mucosal immune system dysfunction and systemic immune activation are associated with progression to AIDS; however, it is unclear to what extent pre-existing gastrointestinal damage relates to disease progression postinfection. Pigtail macaques (PTM) are an excellent model in which to assess mucosal dysfunction in relation to HIV/SIV pathogenesis, as the majority of these animals have high levels of gastrointestinal damage, immune activation, and microbial translocation prior to infection, and rapidly progress to AIDS upon SIV infection. In this study, we characterized the mucosal immune environment prior to and throughout SIV infection in 13 uninfected PTM and 9 SIV-infected PTM, of which 3 were slow progressors. This small subset of slow progressors had limited innate immune activation in mucosal tissues in the periphery, which was associated with a more intact colonic epithelial barrier. Furthermore, we found that preinfection levels of microbial translocation, as measured by LPS-binding protein, in PTM correlated with the rate of progression to AIDS. These data suggest that pre-existing levels of microbial translocation and gastrointestinal tract dysfunction may influence the rate of HIV disease progression.

March 15, 2013 in Conferences & meetings, Immune activation, Innate immunity, Pathogenesis, Treatment | Permalink | Comments (0)

More on the Adaptive Recollections of Natural Killer Cells

As mentioned in a couple of recent posts, evidence is emerging that natural killer (NK) cells can sometimes specifically recognize pathogens and retain memory of prior encounters (a property once thought to be restricted to T cells and B cells). A new paper in Nature Immunology adds to this evidence, showing that NK cells can specifically respond to antigens from several different pathogens including influenza, vesicular stomatitis virus and HIV-1. Importantly, these NK cell responses were more robust upon second encounter, indicating the development of antigen-specific memory (also sometimes described as “recall”). The researchers report that the capacity for developing memory appears to be linked to NK cell expression of a chemokine receptor named CXCR6.

In discussing the implications of their results, the study authors echo prior papers on this subject by stating that NK cell memory should be explored in the context of vaccine research. They also suggest that the observed linkage between NK cell memory and CXCR6 may be relevant to a recently published study (abstract appended below) that identified an association between a genetic polymorphism in the CXCR6 gene and long-term non-progression of HIV infection; i.e. the impact of this gene variant on disease progression could be due to enhanced HIV-specific NK cell responses.

Nat Immunol. 2010 Oct 24. [Epub ahead of print]

Critical role for the chemokine receptor CXCR6 in NK cell-mediated antigen-specific memory of haptens and viruses.

Paust S, Gill HS, Wang BZ, Flynn MP, Moseman EA, Senman B, Szczepanik M, Telenti A, Askenase PW, Compans RW, von Andrian UH.

[1] Harvard Medical School, Department of Pathology, Boston, Massachusetts, USA. [2] The Ragon Institute of MIT, MGH and Harvard, Charlestown, Massachusetts, USA.

Abstract

Hepatic natural killer (NK) cells mediate antigen-specific contact hypersensitivity (CHS) in mice deficient in T cells and B cells. We report here that hepatic NK cells, but not splenic or naive NK cells, also developed specific memory of vaccines containing antigens from influenza, vesicular stomatitis virus (VSV) or human immunodeficiency virus type 1 (HIV-1). Adoptive transfer of virus-sensitized NK cells into naive recipient mice enhanced the survival of the mice after lethal challenge with the sensitizing virus but not after lethal challenge with a different virus. NK cell memory of haptens and viruses depended on CXCR6, a chemokine receptor on hepatic NK cells that was required for the persistence of memory NK cells but not for antigen recognition. Thus, hepatic NK cells can develop adaptive immunity to structurally diverse antigens, an activity that requires NK cell-expressed CXCR6.

J Infect Dis. 2010 Sep 15;202(6):908-15.

Multiple-cohort genetic association study reveals CXCR6 as a new chemokine receptor involved in long-term nonprogression to AIDS.

Limou S, Coulonges C, Herbeck JT, van Manen D, An P, Le Clerc S, Delaneau O, Diop G, Taing L, Montes M, van't Wout AB, Gottlieb GS, Therwath A, Rouzioux C, Delfraissy JF, Lelièvre JD, Lévy Y, Hercberg S, Dina C, Phair J, Donfield S, Goedert JJ, Buchbinder S, Estaquier J, Schächter F, Gut I, Froguel P, Mullins JI, Schuitemaker H, Winkler C, Zagury JF.

Chaire de Bioinformatique, Conservatoire National des Arts et Métiers.

Abstract

BACKGROUND: The compilation of previous genomewide association studies of AIDS shows a major polymorphism in the HCP5 gene associated with both control of the viral load and long-term nonprogression (LTNP) to AIDS.

METHODS: To look for genetic variants that affect LTNP without necessary control of the viral load, we reanalyzed the genomewide data of the unique LTNP Genomics of Resistance to Immunodeficiency Virus (GRIV) cohort by excluding "elite controller" patients, who were controlling the viral load at very low levels (<100 copies/mL).

RESULTS: The rs2234358 polymorphism in the CXCR6 gene was the strongest signal (P=2.5 x 10(-7); odds ratio, 1.85) obtained for the genomewide association study comparing the 186 GRIV LTNPs who were not elite controllers with 697 uninfected control subjects. This association was replicated in 3 additional independent European studies, reaching genomewide significance of P(combined)=9.7 x 10(-10). This association with LTNP is independent of the CCR2-CCR5 locus and the HCP5 polymorphisms.

CONCLUSIONS: The statistical significance, the replication, and the magnitude of the association demonstrate that CXCR6 is likely involved in the molecular etiology of AIDS and, in particular, in LTNP, emphasizing the power of extreme-phenotype cohorts. CXCR6 is a chemokine receptor that is known as a minor coreceptor in human immunodeficiency virus type 1 infection but could participate in disease progression through its role as a mediator of inflammation.

October 26, 2010 in HIV-specific T cell immunity, Innate immunity, Natural killer cells, Pathogenesis, Vaccines | Permalink | Comments (0)

Innate Immunity: Not Always Non-Specific

Components of the innate immune system generally respond to pathogens by either recognizing common features (known as pathogen-associated molecular patterns, or PAMPs) or via receptors that scan for signs of distress on infected cells (such as the absence of cell surface molecules called MHC class I receptors, which are essentially immunological alarm bells that some pathogens nefariously disable). Recently, it has been shown that innate immunity can sometimes be mobilized to mount a more specific response to individual pathogens than was once thought possible.

As covered in a previous post, Louise Kuhn’s research group has discovered evidence that natural killer (NK) cells recognize and retain memory of HIV peptides, and these NK responses may play a role in preventing mother-to-child HIV transmission. In a new paper in the Journal of Infectious Diseases, the same group of researchers reports that NK cell recognition of HIV peptides is also associated with lower HIV viral loads and higher CD4 counts in the a cohort of 79 HIV‐infected, postpartum women. In addition, the study found that the magnitude of the NK cell response was linked to the number of CD4 T cells targeting the HIV Gag protein, and the authors state that: “These findings can be interpreted in the following ways: (1) in vivo CD4 Gag responses may provide help for HIV‐specific NK cells, (2) HIV‐specific NK cells provide help to maintain and preserve CD4 T cell responses, or (3) NK‐CD4 T cell responses help each other in a bidirectional fashion. All these possibilities may in turn also lead to better quality CD8 T cell responses.”

Two recent basic immunology papers offer further evidence of linkages between specific, adaptive immune responses by CD4 T cells and innate immunity. In one case, memory CD4 T cells targeting rabies are shown to mobilize natural killer cells upon secondary exposure to the virus. The authors conclude that induction of NK cell responses may be an under-appreciated mechanism by which vaccine-induced memory CD4 T cells mediate protective immunity. These findings may even deserve consideration in the context of the Thai prime-boost HIV vaccine trial, as memory CD4 T cell responses were induced against HIV Gag p24 in around 50% of recipients in a small (n=71) immunogenicity analysis. The second paper, from Susan Swain’s research group at the Trudeau Institute, reports that memory CD4 T cells can induce production of multiple innate inflammatory cytokines and chemokines in the lung, and that this is associated with earlier control of influenza virus infection. Taken together, the results of these studies suggest that the ability of vaccine-induced memory T cell responses to recruit innate immunity should be specifically evaluated in future research.

The Journal of Infectious Diseases 2010;202:000–000

DOI: 10.1086/656535

MAJOR ARTICLE

Natural Killer Cells That Respond to Human Immunodeficiency Virus Type 1 (HIV‐1) Peptides Are Associated with Control of HIV‐1 Infection

Caroline T. Tiemessen,1 Sharon Shalekoff,1 Stephen Meddows‐Taylor,1 Diana B. Schramm,1 Maria A Papathanasopoulos,2 Glenda E. Gray,5 Gayle G. Sherman,2,4 Ashraf H. Coovadia,3 and Louise Kuhn6

1AIDS Virus Research Unit, National Institute for Communicable Diseases and Department of Virology, 2Department of Molecular Medicine and Haematology, and 3Empilweni Clinic, Coronation Women and Children Hospital, Enhancing Childhood HIV Outcomes, University of the Witwatersrand, and 4National Health Laboratory Services, Johannesburg, and 5Perinatal HIV Research Unit, Chris Hani Baragwanath Hospital, Soweto, South Africa; and 7Gertrude H. Sergievsky Centre, College of Physicians and Surgeons and Department of Epidemiology, Mailman School of Public Health, Columbia University, New York, New York

Human immunodeficiency virus (HIV)–specific natural killer (CD3− cells), CD4, and CD8 T cellular responses were determined in 79 HIV‐1–infected women in response to HIV‐1 peptide pools (Gag, Pol, Nef, Reg, and Env) with use of a whole‐blood intracellular cytokine staining assay that measures interferon–γ and/or interleukin–2. HIV‐specific CD3− cell responses to any region (Env and Reg predominantly targeted) were associated with lower viral load (P=.031) and higher CD4 T cell count (P=.015). Env‐specific CD3− cell responses were stronger in women who had both Gag CD4 and CD8 T cell responses and, in turn, was associated with lower viral load (P=.005). CD3− cell responders had significantly higher representation of CD4 T cell responses to Env and Reg (P=.012 and P=.015, respectively) and higher magnitudes of CD4 T cell responses (P=.017 and P=.037, respectively) than did nonresponders. Peptide‐specific natural killer cells are associated with markers of less severe disease progression among HIV‐1–infected women (lower viral load and higher CD4 T cell count) and with stronger HIV‐specific T cell responses.

The Journal of Immunology, 2010, 185, 2808 -2818

doi:10.4049/jimmunol.1000844

NK Cells as Effectors of Acquired Immune Responses: Effector CD4+ T Cell-Dependent Activation of NK Cells Following Vaccination

Amir Horowitz,* Ron H. Behrens,*, Lucy Okell,* Anthony R. Fooks, and Eleanor M. Riley*

*Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine; Department of Travel Medicine, Hospital for Tropical Diseases, London; and Rabies and Wildlife Zoonoses Group, World Health Organization Collaborating Centre of Rabies and Rabies-Related Viruses, Veterinary Laboratories Agency, Surrey, United Kingdom

We characterized vaccine-induced cellular responses to rabies virus in naive adult volunteers. Contrary to current paradigms, we observed potent and prolonged in vitro NK cell cytokine production and degranulation responses after restimulation of PBMCs with inactivated rabies virus in vaccinated, but not in unvaccinated, individuals. This "recall" NK cell response was absolutely dependent on Ag-specific IL-2 from CD45RO+ CD4+ T cells as well as IL-12 and IL-18 from accessory cells. Importantly, NK cells represented over 70% of all IFN-–secreting and degranulating cells in the first 12–18 h after virus rechallenge indicating they may be required for rapid control of infection after vaccination. Activation of NK cells may be a critical function of IL-2–secreting effector memory T cells. Although IL-2–dependent postvaccination NK cell activation has been reported previously, this is the first time the magnitude of this effect and its contribution to the overall vaccine-induced response has been appreciated and the mechanisms of NK activation postvaccination have been elucidated. Our data will allow standard protocols for evaluating vaccine-induced immunity to be adapted to assess NK cell effector responses.

Nat Med. 2010 May;16(5):558-64, 1p following 564. Epub 2010 May 2.

Memory CD4+ T cells induce innate responses independently of pathogen.

Strutt TM, McKinstry KK, Dibble JP, Winchell C, Kuang Y, Curtis JD, Huston G, Dutton RW, Swain SL.

Trudeau Institute, Saranac Lake, New York, USA.

Abstract

Inflammation induced by recognition of pathogen-associated molecular patterns markedly affects subsequent adaptive responses. We asked whether the adaptive immune system can also affect the character and magnitude of innate inflammatory responses. We found that the response of memory, but not naive, CD4(+) T cells enhances production of multiple innate inflammatory cytokines and chemokines (IICs) in the lung and that, during influenza infection, this leads to early control of virus. Memory CD4(+) T cell-induced IICs and viral control require cognate antigen recognition and are optimal when memory cells are either T helper type 1 (T(H)1) or T(H)17 polarized but are independent of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) production and do not require activation of conserved pathogen recognition pathways. This represents a previously undescribed mechanism by which memory CD4(+) T cells induce an early innate response that enhances immune protection against pathogens.

October 15, 2010 in HIV-specific T cell immunity, Innate immunity, Natural killer cells, Pathogenesis, Vaccines | Permalink | Comments (0) | TrackBack (0)

Natural Immunity to HIV Infection

The November 1, 2010 supplement to the Journal of Infectious Diseases features a suite of articles about individuals who remain HIV negative despite repeated exposure to the virus. The articles represent summaries of oral presentations given at the International Symposium on Natural Immunity to HIV that was held in Winnipeg, Manitoba, Canada, in November 2009. The potential importance of studying highly exposed but seronegative individuals was emphasized by Gregg Gonsalves in a report on HIV basic science that TAG issued in 1993; seventeen years later, a concerted effort appears to be underway to better understand the phenomenon and glean lessons for designing biomedical prevention approaches (particularly vaccines). There has already been a follow-up to the Winnipeg meeting: in July of this year, the National Institutes of Health sponsored a workshop in Rockville to further discuss the formation of an international research consortium (the workshop agenda is available online).

Unfortunately the JID supplement is not open access, the content will become freely available after 12 months (I emailed the journal to politely lobby for lifting the subscriber restriction, to no avail). However a report from the Winnipeg symposium that describes key presentations and outlines the consortium model was prepared by the International Center for Infectious Diseases and is posted to their website.

October 11, 2010 in Conferences & meetings, HIV-specific humoral immunity, HIV-specific T cell immunity, Innate immunity, Transmission, Vaccines | Permalink | Comments (0) | TrackBack (0)

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.

July 29, 2009 in Immune activation, Innate immunity, Pathogenesis | Permalink | Comments (0) | TrackBack (0)

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.

July 23, 2009 in Immune activation, Innate immunity, Pathogenesis | Permalink | Comments (0) | TrackBack (0)

Blurring the Line Between Innate and Adaptive Immunity: HIV-Specific NK Cell “Memory” Associated with Lack of Mother-to-Child Transmission

Traditionally it has been thought that the capacity for immunological memory resides only in cells classified as part of the adaptive immune system: T cells and B cells. But over the past few years there have been hints that perhaps other cell types have the capacity to develop a memory phenotype. Most recently, a paper from Lewis Lanier’s laboratory has strongly suggested that natural killer (NK) cells can develop memory against murine CMV; transfer of the putative memory cells into naïve animals was shown to be protective against an MCMV challenge.

Now, the “Cutting Edge” section of the new issue of the Journal of Immunology contains the first report of possible NK memory in humans. The paper comes from Louise Kuhn’s research group, which has specialized in studying the factors associated with prevention of mother-to-child transmission (MTCT) of HIV. Kuhn and colleagues were initially studying HIV-specific CD4 and CD8 T cell responses in a cohort of mother-infant pairs (these data have been recently published in AIDS) when they identified cytokine responses to HIV peptides coming from non-T cells. Further investigations demonstrated that the cells were NK cells, and that the responses were primarily to peptides from HIV Env and Regulatory proteins.

The findings prompted the researchers to analyze whether the presence of the novel NK responses had any association with the risk of MTCT. The analysis revealed that 28 of 49 (57.1%) non-transmitting mothers and 13 of 44 (29.5%) uninfected infants had detectable responses. In stark comparison, only one of 15 (6.7%) transmitting mothers and one out of 18 (5.6%) of the infected infants displayed responses (p = 0.001 for mothers and p = 0.049 for infants).

In discussing their results, the authors write: “The absence of these responses among uninfected controls suggests that the responses are not due to a direct process of peptide binding to cell surface receptors and triggering of these innate immune cells. Their detection in the context of HIV-1 exposure among uninfected infants indicates that detectable antigenic load is not required in vivo. Taken together, these results support a hypothesis of a "memory" type response. If this is indeed found to be the case, this would represent the first such report that indicates the possibility of NK cell ‘memory’ in humans.”

Although it was written prior to the publication of Lewis Lanier’s MCMV data, an excellent background review on the possibility of NK cell memory can be found in the July 2006 IAVI Report. The article is written by Galit Alter and Marcus Altfeld, who are also actively exploring the subject in the context of HIV infection. Given the struggles to develop an HIV vaccine, the possibility of inducing novel memory responses against the virus surely deserves careful and urgent evaluation.

The Journal of Immunology, 2009, 182: 5914-5918.

doi:10.4049/jimmunol.0900419

Cutting Edge: Unusual NK Cell Responses to HIV-1 Peptides Are Associated with Protection against Maternal-Infant Transmission of HIV-1

Caroline T. Tiemessen2,*, Sharon Shalekoff*, Stephen Meddows-Taylor*, Diana B. Schramm*, Maria A. Papathanasopoulos, Glenda E. Gray¶, Gayle G. Sherman, Ashraf H. Coovadia and Louise Kuhn||

* AIDS Virus Research Unit, National Institute for Communicable Diseases, Department of Molecular Medicine and Haematology, University of the Witwatersrand Medical School; National Health Laboratory Services, Empilweni Clinic, Coronation Women and Children Hospital, Enhancing Childhood HIV Outcomes, University of the Witwatersrand, Johannesburg, South Africa; ¶ Perinatal HIV Research Unit, Chris Hani Baragwanath Hospital, Soweto, South Africa; and || Gertrude H. Sergievsky Center, College of Physicians and Surgeons and Department of Epidemiology, Mailman School of Public Health, Columbia University, New York, NY 10032

Most infants exposed to HIV-1 in utero and at delivery do not acquire infection. We show that mothers and infants who have CD3-negative cells that respond to HIV-1 peptides are substantially less likely to transmit and acquire infection, respectively. The CD3-negative cells, shown to be NK cells, respond with remarkable specificity and high magnitude to HIV-1 peptides from Env (envelope) and Reg (regulatory) protein regions, as measured by a whole blood intracellular cytokine assay only in the context of HIV-1 infection or exposure. These findings identify an important new measure of protective immunity to HIV-1 that highlights the importance of innate immunity in preventing the establishment of HIV-1 infection.

May 05, 2009 in Innate immunity, Natural killer cells, Pathogenesis, Transmission, Vaccines | Permalink | Comments (0) | TrackBack (0)

TAG HIV Basic Science, Vaccines, and Cure Project Blog

By Richard Jefferys, Project Director at Treatment Action Group (TAG). Established thanks to a grant from the Michael J. Palm Foundation.

More on the Links Between the CD4/CD8 Ratio, Immunological Perturbations, and Risk of Illness and Death

Evidence of HIV-Induced Pyroptosis in Gut CD4 T Cells

Probiotic/Prebiotic Supplement Combination Shows Benefits in SIV-Infected Pigtailed Macaques

More on the Adaptive Recollections of Natural Killer Cells

Innate Immunity: Not Always Non-Specific

Natural Immunity to HIV Infection

Study Uncovers Sex Differences in Immune Activation

Lymphatic Tissue Gene Expression During HIV Infection

Blurring the Line Between Innate and Adaptive Immunity: HIV-Specific NK Cell “Memory” Associated with Lack of Mother-to-Child Transmission

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