TAG HIV Basic Science, Vaccines, and Cure Project Blog

Countervailing Selective Forces Shape HIV

It’s well documented that T-cell immune responses targeting HIV can cause selection of viral variants that evade recognition (immune escape). But some studies have reported that, surprisingly, HIV variability in regions targeted by T cells is lower compared to other locations in the genome, a phenomenon not seen with other RNA viruses. In a paper published last week in PLoS Biology, Rafael Sanjuán and colleagues offer a potential explanation. Based on mathematical modeling studies, they suggest that selection may favor the conservation of parts of HIV that efficiently activate CD4 T cells, because this provides the virus with target cells in which to replicate. This suggestion is consistent with evidence that HIV itself is often a major driver of immune activation (e.g. activation precipitously declines when HIV replication is suppressed by therapy), and with the finding that HIV preferentially infects HIV-specific CD4 T cells. The theory also fits with recent data showing that SIV can cause immune activation and progression to simian AIDS in the absence of any contribution from microbial translocation. 

The overall message is that while effective CD8 T-cell responses can drive the development of immune escape mutations and thus promote viral variation, HIV’s need to activate CD4 T-cell targets may act as a countervailing force selecting for conservation. The strength of the selective pressure favoring conservation, Sanjuán and colleagues note, will depend on the extent to which other stimuli—such as co-infections and/or microbial translocation—are contributing to immune activation (the greater their contribution, the less HIV needs to activate CD4 T cells itself).

One implication of the study, the researchers argue, is that vaccines that induce HIV-specific CD4 T-cell responses might create conditions favorable to viral replication. They suggest that in order to provide the CD4 T-cell help required to generate functional CD8 T-cell responses, non-HIV antigens that activate CD4 T cells should be included in vaccines. However, the model does not distinguish between the different types of CD4 T cell that may respond to HIV antigens (e.g. naïve or the many possible types of memory cells) and an alternative solution to this conundrum may be to try to design vaccines that create HIV-specific memory CD4 T cells that are relatively resistant to infection. Evidence that CMV-specific specific memory CD4 T cells are highly resistant to HIV suggests that this might be possible.

PLoS Biol 11(4): e1001523. doi:10.1371/journal.pbio.1001523

Immune Activation Promotes Evolutionary Conservation of T-Cell Epitopes in HIV-1

Rafael Sanjuán, Miguel R. Nebot, Joan B. Peris, José Alcamí

Abstract

The immune system should constitute a strong selective pressure promoting viral genetic diversity and evolution. However, HIV shows lower sequence variability at T-cell epitopes than elsewhere in the genome, in contrast with other human RNA viruses. Here, we propose that epitope conservation is a consequence of the particular interactions established between HIV and the immune system. On one hand, epitope recognition triggers an anti-HIV response mediated by cytotoxic T-lymphocytes (CTLs), but on the other hand, activation of CD4+ helper T lymphocytes (TH cells) promotes HIV replication. Mathematical modeling of these opposite selective forces revealed that selection at the intrapatient level can promote either T-cell epitope conservation or escape. We predict greater conservation for epitopes contributing significantly to total immune activation levels (immunodominance), and when THcell infection is concomitant to epitope recognition (trans-infection). We suggest that HIV-driven immune activation in the lymph nodes during the chronic stage of the disease may offer a favorable scenario for epitope conservation. Our results also support the view that some pathogens draw benefits from the immune response and suggest that vaccination strategies based on conserved TH epitopes may be counterproductive.

PLoS Biol 11(4): e1001521. doi:10.1371/journal.pbio.1001521

HIV Plays (and Wins) a Game of T Cell Brinkmanship

Roland G. Roberts

Viruses are locked into an adaptive arms race with the host immune system: the immune system adapts to recognize the virus, the virus adapts to evade the immune system, the cycle repeats. But in the case of HIV, it seems, sometimes it's good to be recognized. 

Selected Research Highlights from CROI 2013

Coverage of some subjectively selected presentations from the recent CROI meeting in Atlanta, in addition to the prior post on the case report of a potentially functionally cured infant.

Gene Modification of Virus-Specific CD4 T Cells 

Patrick Younan from the Fred Hutchinson Cancer Research Center delivered an interesting talk about transplantation of gene-modified stem cells in pigtailed macaques (abstract, webcast—third in session). The experiment used lentiviral vectors to deliver the gene for a virus entry inhibitor, C46 (also known as M87o, it has a similar mechanism of action to the approved antiretroviral Fuzeon), into stem cells along with a green fluorescent protein (GFP) marker to make the modified cells identifiable. Four macaques underwent stem cell transplantation, with two given C46-modified cells and two controls given cells with only the GFP marker. All were subsequently challenged with the dual-tropic SIV/HIV hybrid virus SHIV89.6P. The two controls displayed typical rapid CD4 T-cell loss, and one was euthanized at week 32 due to the onset of simian AIDS. In contrast, recipients of the modified cells recovered CD4 T-cell counts after an initial dip and showed significantly lower viral loads. Levels of gene-modified CD4 T cells peaked at around 90% during acute infection, but subsequently declined to pre-challenge levels (around 20% in one animal, 55% in the other) during follow-up. Despite the decline in modified CD4 T cells, levels of unmodified cells improved over time, suggesting a protective effect of the intervention on the overall CD4 T-cell pool.

Further studies revealed that superior SHIV-specific CD4 T-cell and antibody responses were associated with the salutary outcome. SHIV-specific CD4 T cells were not detectable in either of the controls. Younan found that a striking 85% of the SHIV-specific CD4 T cells in treated animals were gene-modified, suggesting that protection of these cells had allowed them to better perform their role of providing help to B cells and CD8 T cells. One potentially encouraging implication of this work is that gene therapy approaches might not have to protect all susceptible cells from HIV infection in order to offer benefit; if sufficient numbers of HIV-specific CD4 T cells can be protected, it is possible that these cells will do a better job of coordinating the immune response against HIV, leading to improved control of viral replication. This possibility is being investigated in ongoing trials of Sangamo BioSciences SB-728-T gene therapy, which aims to protect CD4 T cells by abrogating expression of the CCR5 coreceptor. 

Engineering SIV to Spare the CD4 T-Cell Help

Adrienne Swanstrom from the University of Pennsylvania described a novel approach to defining the role of infection of CD4 T cells in SIV pathogenesis, using an engineered version of the highly pathogenic SIVmac239—named iMac-ΔD385—that does not bind the CD4 molecule (abstract, webcast—seventh in session). In a preliminary experiment involving just two macaques, Swanstrom found that the modified virus replicated to levels comparable to the wild-type SIVmac239 during acute infection and targeted a variety of non-CD4 cell types, but was then robustly controlled by the immune response. Strong neutralizing antibody responses were detected, which is unusual in pathogenic SIV infection, and ongoing work is now looking at CD8 T-cell responses. The findings, at least so far, suggest that sparing CD4 T-cell help led to more effective CD4 T cell–dependent immune responses and superior control of SIV replication. 

CMV Vector Vaccination Leads to Apparent Clearance of SIV Infection

Louis Picker from the Vaccine and Gene Therapy Institute at Oregon Health & Science University gave an update on results obtained in macaques with a CMV-based vaccine against SIV (abstract, webcast—fourth in session). As Picker has shown in published work, the vaccine consistently facilitates strict control of a pathogenic SIVmac239 challenge in around 50% of immunized macaques. The remarkable news shared by Picker at CROI is that, over time, these protected animals appear to clear SIV infection. This claim is based on multiple criteria, including loss of detectable virus, waning of CD8 T-cell responses to viral antigens not included in the vaccine, and the failure to transmit infection to uninfected macaques despite transfer of over 50 million blood and/or tissue white blood cells (in contrast, similar transfers from elite controller animals or those on suppressive ART reliably transmitted infection). Picker noted that the CD8 T-cell responses in the vaccine recipients appear to “violate all the rules” in that they target very large numbers of different SIV epitopes and, in many cases, their ability to recognize the virus involves MHC class II molecules, which normally facilitate antigen recognition by CD4 T cells rather than CD8 T cells. Picker is now working to shed further light on these findings, as well as collaborating with the Vaccine and Gene Therapy Institute of Florida to try and develop a CMV vaccine vector that can safely be studied in humans.

Results from Multidose Vorinostat Trial

Sharon Lewin from Monash University in Melbourne debuted data from the first multidose trial of vorinostat (also known as SAHA) as a potential anti-HIV latency drug (abstract, webcast—ninth in session). Consistent with results published by David Margolis from a single-dose trial, expression of HIV RNA significantly increased among the twenty participants after 14 days of vorinostat. However, there was no evidence for reduction in the size of the latent HIV reservoir, suggesting that additional approaches will be needed to facilitate the elimination of the infected cells. As expected, vorinostat side effects were more prevalent as a result of the multiple dosing, particularly fatigue and lethargy.

Curing HIV Removes the Scars of the Past

One small but encouraging piece of news about the lone adult considered cured of HIV, Timothy Brown, was to be found in a presentation by Joyce Sanchez from the University of Minnesota (abstract, webcast—second in session). Sanchez’s study focused on lymphoid structure abnormalities in people with HIV, particularly fibrotic (scarring) damage to lymph tissue resulting from persistent HIV replication and associated immune activation. The extent of lymph tissue fibrosis can be quantified by measuring collagen deposition using imaging techniques. Sanchez showed that in gut lymph tissue, even HIV controllers (individuals with low viral loads in the absence of ART) have levels of collagen deposition that are higher than those of uninfected individuals (15.9% compared with 7%). However, samples from Timothy Brown showed levels of collagen deposition comparable to the uninfected study participants (6.8%), consistent with studies showing no HIV activity in his body (despite the occasional detection of viral genetic material that was reported last year).  

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.

CD4 T Cells Specific for Different Pathogens Vary in Susceptibility to HIV Infection

When it comes to susceptibility to HIV infection, not all CD4 T cells are created equal. There are a variety of factors that have been shown to influence how readily HIV gains entry and replicates, with the best-described distinction being between activated CD4 T cells, which are highly susceptible, and resting CD4 T cells, which are relatively resistant. The particular types of cytokines and chemokines that a CD4 T cell makes have also been shown to influence the efficiency of HIV infection. Less well understood is the influence of antigen specificity—the pathogen being targeted by the CD4 T cell. Evidence has been published showing that TB-specific CD4 T cells are highly susceptible, whereas those responding to the viral infection CMV are relatively resistant (with this resistance being associated with the release of beta-chemokines capable of binding the CCR5 receptor and blocking HIV entry). A study by Haitao Hu and colleagues from the U.S. Military HIV Research Program has now explored this issue further by looking at the susceptibility of various pathogen-specific CD4 T cells and the relationship with the genes that the different cells express.

The results confirm that CMV-specific CD4 T cells are highly resistant to HIV, but not just due to the release of beta-chemokines: gene expression analyses revealed that the cells also upregulate several innate antiviral factors such as IFIT1 (a protein that recognizes a particular form of viral RNA). Because of these factors, CMV-specific CD4 T cells displayed reduced susceptibility to HIV infection even when the researchers used antibodies to neutralize any effect of beta-chemokines. In contrast, CD4 T cells specific for tetanus toxoid (TT) and Candida exhibited a pro-inflammatory Th17-type profile and were highly permissive to HIV infection. The researchers suggest that these differences in susceptibility may contribute to the well-documented relationship between differing levels of immune deficiency and risk of specific opportunistic infections; i.e., candidiasis is typically an early sign of immune deficiency whereas active CMV disease occurs almost exclusively at extremely low CD4 T-cell levels.

Another important implication of the study is that HIV vaccines should aim to induce HIV-specific CD4 T cells with a profile that resembles CMV-specific responses. The researchers speculate that there may be a connection between their observations and the fact that the best results obtained to date in the stringent SIV/macaque model of vaccination have involved a CMV-based vaccine vector. Several research groups—including those of Louis Picker at the Oregon Health & Science University and Rafick-Pierre Sékaly at the Vaccine & Gene Therapy Institute of Florida—are now working to develop CMV-based HIV vaccine vectors with the aim of conducting human studies in both the therapeutic and preventive contexts.

Blood. Published online before print, December 20, 2012. doi: 10.1182/blood-2012-07-446278

Distinct gene expression profiles associated with the susceptibility of pathogen-specific CD4 T cells to HIV-1 infection

Haitao Hu1, Martin Nau1, Phil Ehrenberg1, Agnes-Laurence Chenine1, Camila Macedo1, Yu Zhou2, Z. John Daye3, Zhi Wei4, Maryanne Vahey1, Nelson L Michael1, Jerome H Kim1, Mary Marovich1, and Silvia Ratto-Kim1

1 US Military HIV Research Program, Walter Reed Army Institute of Research, Silver Spring, MD, United States;

2 Department of Pathology and Laboratory Medicine, Temple University, Philadelphia, PA, United States;

3 Division of Epidemiology and Biostatistics, University of Arizona Mel and Enid Zuckerman College of Public Health, Tucson, AZ, United States;

4 Department of Computer Science, New Jersey Institute of Technology, Newark, NJ, United States

ABSTRACT

In HIV infection, the CD4 responses to opportunistic pathogens like Candida albicans are lost early, but cytomegalovirus (CMV)-specific CD4 response persists. Little is currently known about HIV infection of CD4 T cells of different pathogen/antigen specificity. CFSE-labeled PBMC were stimulated with CMV, Tetanus Toxoid (TT) and Candida antigens and subsequently exposed to HIV. HIV infection was monitored by intracellular p24 in CFSE-low population. We showed that while TT- and Candida-specific CD4 T cells were permissive, CMV-specific CD4 T cells were highly resistant to both R5 and X4 HIV. Quantification of HIV DNA in CFSE-low cells showed a reduction of strong-stop and full-length DNA in CMV-specific cells compared to TT- and Candida-specific cells. β-chemokine neutralization enhanced HIV entry and infection in TT- and Candida-specific cells, whereas HIV infection in CMV-specific cells remained low despite increased HIV entry by β-chemokine neutralization, suggesting post-entry restriction of HIV replication by CMV-specific cells. Microarray analysis (GEO Accession Number: GSE42853) revealed distinct transcriptional profiles that involved selective upregulation of comprehensive innate antiviral genes in CMV-specific cells, whereas TT- and Candida-specific cells mainly upregulated Th17 inflammatory response. Our data suggest a mechanism for persistence of CMV-specific CD4 response and earlier loss of mucosal Th17-associated TT- and Candida-specific CD4 response in AIDS.

Lymph Node Fibrosis, CD4 T Cells, and Immune Reconstitution

One of the less well-publicized consequences of the persistent immune activation caused by HIV infection is a type of scarring damage to lymph tissues described as fibrosis. Some early studies of lymph nodes from HIV-infected individuals reported evidence of this problem, but it wasn’t until the publication of a study by the research group of Ashley Haase in 2002 that a connection was made between the extent of fibrosis (as measured by deposition of collagen) and maintenance of CD4 T-cell numbers. Haase and colleagues showed that there was an inverse correlation between lymph node fibrosis and the number of CD4 T cells measurable in the same node. Importantly, they also found that the degree of fibrosis was significantly associated with the magnitude of CD4 T-cell increases after initiation of antiretroviral therapy, with greater fibrosis linked to poorer CD4 T-cell recovery.

In the subsequent years, Haase’s group has delved further into the mechanisms underlying these findings. In 2011, they reported that fibrosis disrupts the fibroblastic reticular cell (FRC) network, which forms pathways along which T cells travel on their journey through lymph tissue. The FRC network provides fuel for maintaining T-cell health in the form of the cytokine IL-7, and fibrotic damage to FRCs was found to inhibit the ability of T cells to access IL-7, leading to cell death. The study also identified the cytokine lymphotoxin-β as critical for maintaining FRCs, and suggested that loss of CD4 T cells was linked to a decline in lymphotoxin-β production, further exacerbating the problem created by the fibrosis.

In an important paper published last summer that I neglected to write about at the time, the researchers confirm that CD4 T cells are the major source of lymphotoxin-β, thus demonstrating that fibrosis creates a vicious cycle by depleting factors needed for CD4 T-cell survival, leading to CD4 T-cell loss, which in turn removes a critical source of factors needed to maintain the FRC network that provides sustenance to CD4 T cells. The study offers evidence that this problem is relevant to not just HIV infection but also CD4 depletion after chemotherapy and irradiation in individuals with cancer. 

In an accompanying editorial, Steve Deeks from UCSF notes that the research suggests possible interventions that could be evaluated in the context of HIV-induced persistent immune activation and CD4 T-cell depletion: “These experimental interventions include drugs that remove pro-inflammatory pathogens and microbial products (eg, valganciclovir for CMV, rifaximin for gut microbes, sevelamer for lipopolysaccharide), drugs that directly prevent fibrosis (eg, angiotensin II receptor antagonists* and ACE inhibitors), and drugs that have more broad effects in reducing inflammation (eg, statins, nonsteroidal antinflammatory drugs, methotrexate, and mesalamine).” Deeks also points out that the best way of avoiding fibrotic damage to the lymph nodes is to suppress HIV replication as soon as possible after infection.

A more recent study, published in the Journal of Infectious Diseases this past October, offers further support for the conclusions of Haase et al. Researchers led by Brian Tabb from the laboratory of Jacob Estes at NCI–Frederick report that blocking the inflammatory cytokine TNF alpha in early SIV infection reduced lymphoid-tissue fibrosis and was associated with better preservation of CD4 T-cell numbers in macaques (without affecting SIV viral load). The authors conclude: “This initial study highlights the importance of early inflammatory responses to lentiviral infections and underscores the need for additional studies to ascertain the potential clinical benefits of adjunctive therapies to attenuate these responses and to improve patient outcomes.”

*TAG’s new HIV project director, Tim Horn, has recently led an effort to support AIDS Clinical Trial Group investigators seeking to obtain the angiotensin II receptor antagonist telmisartan (trade name: Micardis) from the manufacturer, Boehringer Ingelheim, for a study in people with HIV. Unfortunately, the company remains unwilling to provide drug for the planned trial, citing regulatory concerns. This situation highlights issues that will likely arise again in the future as investigators attempt to conduct exploratory studies of these types of potential adjunctive treatments in HIV infection: many of the candidate interventions are already indicated for other uses and are off-patent or toward the end of their patent life; additionally, the research is at such an early stage that it is probing questions of disease pathogenesis rather than carving a clear path toward an FDA-approved HIV indication. Activists are continuing to discuss possible approaches to addressing these issues in order to ensure that needed research can proceed.

Blood August 30, 2012 vol. 120 no. 9 1753-1754

HIV infection, lymphoid fibrosis, and disease

Steven G. Deeks

In this issue of Blood, Zeng and colleagues demonstrate in SIV-infected macaques and HIV-infected humans that CD4+ T cells actively maintain the fibroblastic reticular cell network in lymphoid tissues and that this network in turn is needed to maintain normal T-cell homeostasis and function.

Blood August 30, 2012 vol. 120 no. 9 1856–1867

Critical role of CD4 T cells in maintaining lymphoid tissue structure for immune cell homeostasis and reconstitution

Ming Zeng1, Mirko Paiardini2, Jessica C. Engram3, Greg J. Beilman4, Jeffrey G. Chipman4, Timothy W. Schacker5, Guido Silvestri2, and Ashley T. Haase1 

1Department of Microbiology, Medical School, University of Minnesota, Minneapolis, MN;

2Yerkes National Primate Research Center and Emory University, Atlanta, GA;

3University of Pennsylvania School of Medicine, Philadelphia, PA;

4Department of Surgery, University of Minnesota, Minneapolis, MN; and

5Department of Medicine, Medical School, University of Minnesota, Minneapolis, MN 

ABSTRACT

Loss of the fibroblastic reticular cell (FRC) network in lymphoid tissues during HIV-1 infection has been shown to impair the survival of naive T cells and limit immune reconstitution after antiretroviral therapy. What causes this FRC loss is unknown. Because FRC loss correlates with loss of both naive CD4 and CD8 T-cell subsets and decreased lymphotoxin-β, a key factor for maintenance of FRC network, we hypothesized that loss of naive T cells is responsible for loss of the FRC network. To test this hypothesis, we assessed the consequences of antibody-mediated depletion of CD4 and CD8 T cells in rhesus macaques and sooty mangabeys. We found that only CD4 T-cell depletion resulted in FRC loss in both species and that this loss was caused by decreased lymphotoxin-β mainly produced by the CD4 T cells. We further found the same dependence of the FRC network on CD4 T cells in HIV-1–infected patients before and after antiretroviral therapy and in other immunodeficiency conditions, such as CD4 depletion in cancer patients induced by chemotherapy and irradiation. CD4 T cells thus play a central role in the maintenance of lymphoid tissue structure necessary for their own homeostasis and reconstitution. 

J Infect Dis. (2012)

doi: 10.1093/infdis/jis643

First published online: October 19, 2012

Reduced Inflammation and Lymphoid Tissue Immunopathology in Rhesus Macaques Receiving Anti-TNF Treatment During Primary SIV Infection

Brian Tabb1,7, David R. Morcock1, Charles M. Trubey1, Octavio A. Quiñones2, Xing Pei Hao3, Jeremy Smedley4, Rhonda Macallister4, Michael Piatak Jr1, Levelle D. Harris5, Mirko Paiardini6, Guido Silvestri6, Jason M. Brenchley5, W. Gregory Alvord2, Jeffrey D. Lifson1 and Jacob D. Estes1 

1AIDS and Cancer Virus Program, SAIC-Frederick, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD

2Statistical Consulting, Data Management Services, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD

3Pathology and Histotechnology Laboratory, SAIC-Frederick, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD

4Laboratory Animal Science Program, SAIC-Frederick, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD

5Immunopathogenesis Unit, Lab of Molecular Microbiology, NIAID, NIH, Bethesda, MD

6Yerkes National Primate Research Center, Emory University, Atlanta, Georgia

7Present address: XYZ, Memphis, TN 

Abstract 

Background. HIV/SIV infections induce robust, generalized inflammatory responses that begin during acute infection and lead to pathological systemic immune activation, fibrotic damage of lymphoid tissues (LTs) and CD4+ T cell loss, pathogenic processes that contribute to disease progression.

Methods. To better understand the contribution of TNF, a key regulator of acute inflammation, to lentiviral pathogenesis, rhesus macaques (RMs) newly infected with SIVmac239 were treated for 12 weeks in a pilot study with adalimumab (Humira), a human anti-TNF mAb.

Results. Adalimumab did not affect plasma SIV RNA levels or measures of T-cell immune activation (CD38 or Ki67) in peripheral blood or lymph node T cells. However, compared to untreated RMs, adalimumab-treated RMs showed attenuated expression of pro-inflammatory genes, decreased infiltration of polymorphonuclear cells into the T cell zone (TZ) of LTs and weaker anti-inflammatory regulatory responses to SIV infection (i.e. fewer presumed alternatively activated (CD163+) macrophages, IL-10+ and TGFβ+ cells), along with reduced LT fibrosis and better preservation of CD4+ T cells.

Conclusions. While HIV/SIV replication drives pathogenesis, these data emphasize the contribution of the inflammatory response to lentiviral infection to overall pathogenesis, and suggest that early modulation of the inflammatory response may help attenuate disease progression.

J Infect Dis. first published online October 24, 2012

doi:10.1093/infdis/jis648

EDITORIAL:

Cooling off the Host Immune Response to Acute SIV Infection—Is Less More?

Nelson L. Michael

New Research on Gut CD4 T-Cell Depletion and HIV Pathogenesis

In both HIV and SIV infections, it has been shown that the most rapid and extensive loss of CD4 T cells occurs in the gut. As a consequence, a theory has emerged positing that gut CD4 T-cell depletion plays a central causative role in driving HIV pathogenesis; the proposed mechanism is that gut wall integrity becomes compromised, leading to the leakage of normally friendly bacteria from the digestive tract and into systemic circulation, which in turn contributes to persistent immune activation and, ultimately, progression to AIDS. However, some scientists have remained skeptical of this theory, suggesting instead that gut CD4 T-cell depletion is an effect of HIV infection, but not necessarily the primary cause of disease progression. The skeptics have gained some support from studies showing that severe gut CD4 T-cell depletion occurs during acute SIV infection in monkey species that experience no apparent ill effects from the virus (sooty mangabeys and African green monkeys). A paper published recently in the Journal of Virology now shows that the opposite phenomenon is also possible: a modified SIV that does not cause loss of gut CD4 T cells nevertheless causes persistent immune activation and progression to simian AIDS in rhesus macaques.

Published ahead of print, 14 November 2012, doi: 10.1128/JVI.01928-12

Loss of a Tyrosine-Dependent Trafficking Motif in the SIV Envelope Cytoplasmic Tail Spares Mucosal CD4 Cells but Does Not Prevent Disease Progression

Matthew W. Breed1, Andrea P.O. Jordan2, Pyone P. Aye1, Cornelis F. Lichtveld1, Cecily Midkiff1, Faith Schiro1, Beth S. Haggarty2, Chie Sugimoto1, Xavier Alvarez1, Netanya G. Sandler3, Daniel C. Douek3, Marcelo J. Kuroda1, Bapi Pahar1, Michael Piatak Jr4, Jeffrey D. Lifson4, Brandon F. Keele4, James A. Hoxie2 and Andrew A. Lackner1 1 Tulane National Primate Research Center, Covington, LA, USA 2 University of Pennsylvania, Philadelphia, PA, USA 3 Human Immunology Section, Vaccine Research Center, National Institutes of Allergy and Infectious Diseases, NIH, Bethesda, Maryland, USA 4 SAIC-Frederick Inc., Frederick National Laboratory for Cancer Research, Frederick, MD, USA

ABSTRACT

A hallmark of pathogenic SIV and HIV infection is the rapid and near complete depletion of mucosal CD4+ T-lymphocytes from the gastrointestinal tract. Loss of these cells and disruption of epithelial barrier function are associated with microbial translocation, which has been proposed to drive chronic systemic immune activation and disease progression. Here we evaluate in rhesus macaques, a novel attenuated variant of pathogenic SIVmac239, termed ΔGY, which contains a deletion of a Tyr and a proximal Gly from a highly conserved YxxØ trafficking motif in the envelope cytoplasmic tail. Compared to SIVmac239, ΔGY established a comparable acute peak of viremia, but only transiently infected lamina propria and caused little or no acute depletion of mucosal CD4+ T-cells and no detectable microbial translocation. Nonetheless, these animals developed T-cell activation, declining peripheral blood CD4+ T-cells and ultimately progressed with clinical or pathological features of AIDS. ΔGY-infected animals also showed no infection of macrophages or CNS tissues even in late stage disease. Although the ΔGY mutation persisted, novel mutations evolved including the formation of new YxxØ motifs in two of four animals. These findings indicate that disruption of this trafficking motif by the ΔGY mutation leads to a striking alteration in anatomic distribution of virus with sparing of lamina propria and a lack of microbial translocation. Because these animals exhibited wildtype levels of acute viremia and immune activation, our findings indicate that these pathological events are dissociable, and that immune activation unrelated to gut damage can be sufficient for the development of AIDS.

HIV Replication and Persistence in T Follicular Helper Cells

Following up on the recent post on T follicular helper cells (TFH), a study published on December 17 in the Journal of Experimental Medicine reports that TFH represent a major site of HIV replication and persistence. The finding is not necessariy surprising, because lymph node studies conducted in the late 1980s and early 1990s showed that CD4 T cells in areas named germinal centers bore a great burden of HIV infection, and it is now known that this is where TFH locate during an immune response. But it was not previously understood that these CD4 T cells comprise a discrete subset, and TFH are now becoming increasingly well characterized in terms of both identifying features (such as surface markers and cytokine secretion) and their function in providing help to B cells.

Matthieu Perreau and colleagues from the University of Lausanne investigated TFH in three different groups of HIV-positive individuals:

• 23 with untreated chronic infection and viral load >5,000 copies/mL
• 14 on antiretroviral therapy (ART) with viral load <20 copies/mL
• 3 long-term nonprogressors (LTNP) with low viral load (<1,000 copies/mL)

A control group of 13 HIV-negative individuals was also included. The study confirmed the prior finding that TFH are significantly expanded in the lymph nodes of people with HIV compared to controls and that this expansion correlates with alterations in B-cell subsets (a decline in long-lived resting naive and memory B cells and an increase in short-lived activated B cells). The percentage of TFH correlated significantly with peripheral blood HIV viral load (R = 0.6035, P = 0.002). Suppression of HIV replication by ART reduced TFH levels and shifted B-cell subsets back toward the distribution observed in controls. Echoing and extending the previous research by Madelene Lindqvist, a substantial proportion of the TFH were found to be specific for HIV, with responses to Gag and Pol proteins more commonly detected than those against Env (based on a test evaluating cytokine production after stimulation with Gag, Pol, or Env peptides).

An analysis of the different CD4 T-cell populations present in lymph nodes revealed that TFH contained the greatest amount of HIV DNA: assuming one HIV DNA copy per cell, approximately five percent of the TFH population was infected. After 72 weeks of ART there was a 1.5–2 log drop in the number of TFH containing HIV DNA, but a reservoir of infected cells remained detectable. Cell culture experiments showed that TFH were highly susceptible to HIV infection and replication compared to other CD4 T-cell subsets, with the difference being most notable among the LTNP. The expression of the proliferation marker Ki67 by TFH was found to correlate with HIV DNA levels and ability to support HIV replication in vitro. Consistent with the elevation in immune activation that occurs in HIV, the proportion of TFH expressing Ki67 was 50 percent higher in the untreated HIV-positive individuals than in HIV-negative controls.

The discussion section of the paper highlights several implications of the results, including:

• HIV itself—as opposed to other potential factors, like microbial translocation—appears to be the main driving force behind TFH activation and expansion, and the associated dysregulation of B cells.
• The preferential infection of TFH likely contributes to viral persistence, because germinal centers have been shown to offer limited access to CD8 T cells (which might otherwise kill HIV-infected cells). Follicular dendritic cells in lymph nodes have also long been known to be sites of extensive trapping of HIV virions, providing an ongoing source of viruses to infect TFH.
• The involvement of TFH in responses to new pathogens and vaccines offers an explanation for the reported association between intercurrent infections and vaccinations and transient HIV viral-load increases (activation of TFH by these stimuli provides additional targets for HIV replication).
• The expression of certain cell-surface markers (such as PD-1) by TFH may offer a means of targeting this population with interventions designed to reduce HIV persistence. A human trial of an antibody targeting PD-1 is currently in the works at the AIDS Clinical Trials Group (for more background, see this recent short review "Novel Approaches to Curing HIV" by the principal investigator for the trial, Hiroyu Hatano).

Journal of Experimental Medicine

Published December 17, 2012

The Rockefeller University Press, doi: 10.1084/jem.20121932

Article

Follicular helper T cells serve as the major CD4 T cell compartment for HIV-1 infection, replication, and production

Matthieu Perreau1, Anne-Laure Savoye1, Elisa De Crignis1, Jean-Marc Corpataux2, Rafael Cubas5, Elias K. Haddad5, Laurence De Leval3, Cecilia Graziosi1, and Giuseppe Pantaleo1,4

1Divisions of Immunology and Allergy and 2Thoracic Surgery, 3Institute of Pathology, and 4Swiss Vaccine Research Institute, Lausanne University Hospital, University of Lausanne, CH-1011 Lausanne, Switzerland

5Vaccine and Gene Therapy Institute of Florida, Port St. Lucie, Florida

M. Perreau and A.-L. Savoye contributed equally to this paper.

ABSTRACT

In the present study, we have investigated the distribution of HIV-specific and HIV-infected CD4 T cells within different populations of memory CD4 T cells isolated from lymph nodes of viremic HIV-infected subjects. Four memory CD4 T cell populations were identified on the basis of the expression of CXCR5, PD-1, and Bcl-6: CXCR5−PD-1−Bcl-6−, CXCR5+PD-1−Bcl-6−, CXCR5−PD-1+Bcl-6−, and CXCR5+PD-1+Bcl-6+. On the basis of Bcl-6 expression and functional properties (IL-21 production and B cell help), the CXCR5+PD-1+Bcl-6+ cell population was considered to correspond to the T follicular helper (Tfh) cell population. We show that Tfh and CXCR5−PD-1+ cell populations are enriched in HIV-specific CD4 T cells, and these populations are significantly increased in viremic HIV-infected subjects as compared with healthy subjects. The Tfh cell population contained the highest percentage of CD4 T cells harboring HIV DNA and was the most efficient in supporting productive infection in vitro. Replication competent HIV was also readily isolated from Tfh cells in subjects with nonprogressive infection and low viremia (<1,000 HIV RNA copies). However, only the percentage of Tfh cells correlated with the levels of plasma viremia. These results demonstrate that Tfh cells serve as the major CD4 T cell compartment for HIV infection, replication, and production.

Attack of the Killer Helpers (Part Two)

Among the many tasks of the immune system, the responsibility for recognizing and killing virus-infected cells falls largely to the subset of CD8 T cells designated cytotoxic T-lymphocytes (CTL). Whether CD4 T cells (traditionally called just “helper” cells) can exert cytotoxic functions has historically been controversial, but over the past decade studies have convincingly documented the existence of cytotoxic CD4 T-cell responses in a variety of different settings, including HIV and SIV infection (as previously covered on this blog). A new paper in the open-access journal Retrovirology from Jonah Sacha’s research group at Oregon Health and Science University now reports that not only are cytotoxic CD4 T-cell responses detectable in macaques controlling a pathogenic SIV isolate, but they can drive the selection of immune escape mutations. As the authors note, this represents compelling evidence that CD4 T cells can directly suppress viral replication.

Although I neglected to highlight it on the blog at the time of publication, a human study from the laboratory of Hendrick Streeck also argues for a key role of cytotoxic CD4 T-cell responses in controlling HIV. Published in Science Translational Medicine back in February of this year, the study showed that HIV-specific cytotoxic CD4 T-cell activity predicted superior control of viral load and slower disease progression (as assessed by time to CD4 T-cell count <350, time to ART initiation, or time to viral load >100,000 copies/mm³) in a cohort of acutely HIV-infected individuals.

Taken together, these results further underscore the importance of considering virus-specific CD4 T-cell responses, both in studies of natural control of HIV replication and in attempts to design effective immune-based therapies.

Retrovirology. 2012 Nov 6;9:91. doi: 10.1186/1742-4690-9-91.

CD8+ and CD4+ cytotoxic T cell escape mutations precede breakthrough SIVmac239 viremia in an elite controller.

Burwitz BJ, Giraldo-Vela JP, Reed J, Newman LP, Bean AT, Nimityongskul FA, Castrovinci PA, Maness NJ, Leon EJ, Rudersdorf R, Sacha JB.

Vaccine and Gene Therapy Institute, Oregon Health and Science University, 505 NW 185th, Beaverton, OR, 97006, USA.

Abstract

BACKGROUND: Virus-specific T cells are critical components in the containment of immunodeficiency virus infections. While the protective role of CD8+ T cells is well established by studies of CD8+ T cell-mediated viral escape, it remains unknown if CD4+ T cells can also impose sufficient selective pressure on replicating virus to drive the emergence of high-frequency escape variants. Identifying a high frequency CD4+ T cell driven escape mutation would provide compelling evidence of direct immunological pressure mediated by these cells. RESULTS: Here, we studied a SIVmac239-infected elite controller rhesus macaque with a 1,000-fold spontaneous increase in plasma viral load that preceded disease progression and death from AIDS-related complications. We sequenced the viral genome pre- and post-breakthrough and demonstrate that CD8+ T cells drove the majority of the amino acid substitutions outside of Env. However, within a region of Gag p27CA targeted only by CD4+ T cells, we identified a unique post-breakthrough mutation, Gag D205E, which abrogated CD4+ T cell recognition. Further, we demonstrate that the Gag p27CA-specific CD4+ T cells exhibited cytolytic activity and that SIV bearing the Gag D205E mutation escapes this CD4+ T cell effector function ex vivo. CONCLUSIONS: Cumulatively, these results confirm the importance of virus specific CD8+ T cells and demonstrate that CD4+ T cells can also exert significant selective pressure on immunodeficiency viruses in vivo during low-level viral replication. These results also suggest that further studies of CD4+ T cell escape should focus on cases of elite control with spontaneous viral breakthrough.

Sci Transl Med. 2012 Feb 29;4(123):123ra25. doi: 10.1126/scitranslmed.3003165.

HIV-specific cytolytic CD4 T cell responses during acute HIV infection predict disease outcome.

Soghoian DZ, Jessen H, Flanders M, Sierra-Davidson K, Cutler S, Pertel T, Ranasinghe S, Lindqvist M, Davis I, Lane K, Rychert J, Rosenberg ES, Piechocka-Trocha A, Brass AL, Brenchley JM, Walker BD, Streeck H.

Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard Medical School, Charlestown, MA 02129, USA.

Abstract

Early immunological events during acute HIV infection are thought to fundamentally influence long-term disease outcome. Whereas the contribution of HIV-specific CD8 T cell responses to early viral control is well established, the role of HIV-specific CD4 T cell responses in the control of viral replication after acute infection is unknown. A growing body of evidence suggests that CD4 T cells-besides their helper function-have the capacity to directly recognize and kill virally infected cells. In a longitudinal study of a cohort of individuals acutely infected with HIV, we observed that subjects able to spontaneously control HIV replication in the absence of antiretroviral therapy showed a significant expansion of HIV-specific CD4 T cell responses-but not CD8 T cell responses-compared to subjects who progressed to a high viral set point (P = 0.038). Markedly, this expansion occurred before differences in viral load or CD4 T cell count and was characterized by robust cytolytic activity and expression of a distinct profile of perforin and granzymes at the earliest time point. Kaplan-Meier analysis revealed that the emergence of granzyme A(+) HIV-specific CD4 T cell responses at baseline was highly predictive of slower disease progression and clinical outcome (average days to CD4 T cell count <350/μl was 575 versus 306, P = 0.001). These data demonstrate that HIV-specific CD4 T cell responses can be used during the earliest phase of HIV infection as an immunological predictor of subsequent viral set point and disease outcome. Moreover, these data suggest that expansion of granzyme A(+) HIV-specific cytolytic CD4 T cell responses early during acute HIV infection contributes substantially to the control of viral replication.

T Follicular Helper Cells and HIV

T follicular helper cells (TFH) are a subset of CD4 T cells that reside almost exclusively in the lymph nodes, where they specialize in helping B cells generate antibodies. In a testament to the complexity and difficulty of studying the immune system, TFH were only identified as a discrete subset of CD4 T cells at the turn of the millennium. Since that time, several features that define TFH have been described, including high levels of the chemokine receptor CXCR5, expression of the PD-1 molecule, secretion of the cytokine IL-21, and upregulation of the Bcl-6 gene. Studies have also found TFH to be important for driving the process of affinity maturation (wherein B cells producing antibodies with increased affinity for their antigen target are progressively generated and selected), and for supporting the development of long-lived memory B cells.

Because TFH are relatively new on the scene, very little is known about how they are affected by HIV infection. A few months ago in the Journal of Clinical Investigation, two research groups published studies shedding light on this question. Madelene Lindqvist and colleagues looked at TFH in people with HIV and found they were significantly expanded in lymph nodes, with a major contribution from HIV-specific TFH (predominantly targeting the Gag protein). TFH levels were approximately tenfold higher in HIV-positive individuals compared to HIV-negative controls. Although high viral load was associated with around double the levels of TFH compared to undetectable viremia on ART, there was no direct correlation between viral load levels and TFH numbers. Notably, the extent of TFH expansion correlated with hypersecretion of antibodies by B cells, strongly suggesting that dysregulation of TFH contributes to B-cell dysfunction and hypergammaglobulinemia in people with HIV; these features of the infection were first reported in the early 1980s, but the underlying mechanisms have been unclear. Constantinos Petrovas and colleagues report broadly similar findings from the SIV/macaque model, additionally providing evidence that TFH are susceptible to infection.

In an accompanying commentary, TFH expert Carola Vinuesa highlights a number of important questions pertaining to this CD4 T-cell subset in HIV infection. Firstly, it has become apparent that several of the rare antibodies capable of broad neutralizing activity against multiple HIV isolates have undergone an unusual degree of affinity maturation, suggesting that interactions between TFH and B cells were essential in supporting their development; a better understanding of how this occurs could prove essential to designing an efficacious vaccine. The other side of this equation is that most individuals with HIV fail to generate effective neutralizing antibodies, and it will also be important to learn how TFH dysregulation contributes to this failure. From the standpoint of cure research, Vinuesa notes that these studies imply that TFH could contribute significantly to the persistent reservoir of HIV-infected cells and may therefore require specific targeting by reservoir-eliminating strategies.

Several webcasts of scientific talks relating to TFH and HIV are available free online. At CROI in March 2012, Carola Vinuesa provided an introductory overview and Madelene Lindqvist and Constantinos Petrovas presented some of the results described in these papers. In September at AIDS Vaccine 2012, Hendrick Streeck discussed the latest findings from his laboratory and Rick Koup offered a perspective on TFH and vaccines.

CROI 2012 

Symposium: Ontogeny of a Protective Immune Response to HIV (3rd presentation in session)

Follicular Helper and Regulatory T Cells Control the Quality of Antibody Responses

Carola Vinuesa, John Curtin Sch of Med Res, Australian Natl Univ, Canberra, Australia

Oral Abstract: Immune, Cellular, and Viral Factors Influencing HIV–Host Interplay (5th and 6th presentations in session) 

SIV Infection Affects the Function, but not Survival, of Follicular T Helper Cells in Rhesus Macaques

Constantinos Petrovas, Vaccine Res Ctr, NIAID, NIH, Bethesda, MD, US

HIV-specific T Follicular Helper Cell Responses in Chronically Infected Individuals

Madelene Lindqvist, Ragon Inst of MGH, MIT, and Harvard, Boston, MA, US

AIDS Vaccine 2012

T Follicular Helper Cells in HIV Infection

Hendrik Streeck, Ragon Institute of MGH, MIT, and Harvard, Boston, MA, US

Follicular T helper cells in vaccination and lentivirus pathogenesis

Richard A. Koup, Vaccine Research Center, Bethesda, MD, US

 

Journal of Clinical Investigation abstracts: 

Published in Volume 122, Issue 9 (September 4, 2012)

J Clin Invest. 2012;122(9):3271–3280. doi:10.1172/JCI64314.

Research Article

Expansion of HIV-specific T follicular helper cells in chronic HIV infection

Madelene Lindqvist1, Jan van Lunzen2, Damien Z. Soghoian1, Bjorn D. Kuhl1, Srinika Ranasinghe1, Gregory Kranias1, Michael D. Flanders1, Samuel Cutler1, Naomi Yudanin3, Matthias I. Muller1, Isaiah Davis1, Donna Farber3, Philip Hartjen2, Friedrich Haag4, Galit Alter1, Julian Schulze zur Wiesch2 and Hendrik Streeck1

1Ragon Institute of MGH, MIT, and Harvard Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA.

2Infectious Diseases Unit, University Medical Center Eppendorf, Hamburg, Germany.

3Columbia Center for Translational Immunology, Columbia University, New York, New York, USA.

4Department of Immunology, University Medical Center Eppendorf, Hamburg, Germany. 

Authorship note: Madelene Lindqvist, Jan van Lunzen, and Hendrik Streeck contributed equally to this work.

HIV targets CD4 T cells, which are required for the induction of high-affinity antibody responses and the formation of long-lived B cell memory. The depletion of antigen-specific CD4 T cells during HIV infection is therefore believed to impede the development of protective B cell immunity. Although several different HIV-related B cell dysfunctions have been described, the role of CD4 T follicular helper (TFH) cells in HIV infection remains unknown. Here, we assessed HIV-specific TFH responses in the lymph nodes of treatment-naive and antiretroviral-treated HIV-infected individuals. Strikingly, both the bulk TFH and HIV-specific TFH cell populations were significantly expanded in chronic HIV infection and were highly associated with viremia. In particular, GAG-specific TFH cells were detected at significantly higher levels in the lymph nodes compared with those of GP120-specific TFH cells and showed preferential secretion of the helper cytokine IL-21. In addition, TFH cell expansion was associated with an increase of germinal center B cells and plasma cells as well as IgG1 hypersecretion. Thus, our study suggests that high levels of HIV viremia drive the expansion of TFH cells, which in turn leads to perturbations of B cell differentiation, resulting in dysregulated antibody production.

Published in Volume 122, Issue 9 (September 4, 2012)

J Clin Invest. 2012;122(9):3281–3294. doi:10.1172/JCI63039.

Research Article

CD4 T follicular helper cell dynamics during SIV infection

Constantinos Petrovas1, Takuya Yamamoto1, Michael Y. Gerner2, Kristin L. Boswell1, Kaska Wloka1, Emily C. Smith1, David R. Ambrozak1, Netanya G. Sandler3, Katherina J. Timmer3, Xiaoyong Sun4, Li Pan4, Amanda Poholek2, Srinivas S. Rao5, Jason M. Brenchley6, S. Munir Alam7, Georgia D. Tomaras7, Mario Roederer8, Daniel C. Douek3, Robert A. Seder9, Ronald N. Germain2, Elias K. Haddad4 and Richard A. Koup1

1Immunology Laboratory, Vaccine Research Center,

2Laboratory of Systems Biology, Lymphocyte Biology Section, and

3Human Immunology Section, Vaccine Research Center, National Institute of Allergy and Infectious Diseases (NIAID), NIH, Bethesda, Maryland, USA.

4VGTI Florida, Port St. Lucie, Florida, USA.

5Laboratory Animal Medicine, Vaccine Research Center, and

6Laboratory of Molecular Microbiology, NIAID, NIH, Bethesda, Maryland, USA.

7Duke Human Vaccine Institute, Duke University Medical School, Durham, North Carolina, USA.

8ImmunoTechnology Section and

9Cellular Immunology Section, Vaccine Research Center, NIAID, NIH, Bethesda, Maryland, USA.

CD4 T follicular helper (TFH) cells interact with and stimulate the generation of antigen-specific B cells. TFH cell interaction with B cells correlates with production of SIV-specific immunoglobulins. However, the fate of TFH cells and their participation in SIV-induced antibody production is not well understood. We investigated the phenotype, function, location, and molecular signature of TFH cells in rhesus macaques. Similar to their human counterparts, TFH cells in rhesus macaques represented a heterogeneous population with respect to cytokine function. In a highly differentiated subpopulation of TFH cells, characterized by CD150lo expression, production of Th1 cytokines was compromised while IL-4 production was augmented, and cells exhibited decreased survival, cycling, and trafficking capacity. TFH cells exhibited a distinct gene profile that was markedly altered by SIV infection. TFH cells were infected by SIV; yet, in some animals, these cells actually accumulated during chronic SIV infection. Generalized immune activation and increased IL-6 production helped drive TFH differentiation during SIV infection. Accumulation of TFH cells was associated with increased frequency of activated germinal center B cells and SIV-specific antibodies. Therefore, chronic SIV does not disturb the ability of TFH cells to help B cell maturation and production of SIV-specific immunoglobulins.

Published in Volume 122, Issue 9 (September 4, 2012)

J Clin Invest. 2012;122(9):3059–3062. doi:10.1172/JCI65175.

Commentary

HIV and T follicular helper cells: a dangerous relationship

Carola G. Vinuesa

Department of Pathogens and Immunity, John Curtin School of Medical Research, Australian National University, Canberra, Australia.

HIV infection leads to progressive destruction of infected CD4 T cells, hypergammaglobulinemia, and loss of memory B cells. Germinal centers, which are key to memory B cell formation and protective antibody responses, are major HIV reservoirs in which the virus replicates within T follicular helper (TFH) cells. In this issue of the JCI, the Koup and Streeck groups report that chronic SIV/HIV infection promotes TFH cell accumulation, which may drive B cell dysregulation. Their discoveries suggest that HIV harnesses TFH cells to evade the antibody response.

Antiviral Activity of Virus-Specific CD8 T Cells Linked to Rate of CD4 T Cell Decline in HIV Infection

The extent to which virus-specific CD8 T cell responses contribute to control of viral load and prevention of disease progression in HIV infection has long been a subject of conflicting data and controversy. Early tests of the ability of CD8 T cells to kill target cells expressing HIV antigens were misleading because they relied on extended culture with IL-2, a process that turned out to have restorative properties on otherwise dysfunctional cells. The advent of class I MHC tetramers, which facilitate the counting of CD8 T cells specific for individual HIV epitopes, was an advance but also initially caused confusion: an early paper using tetramers reported an inverse correlation between CD8 T cells specific for epitopes from HIV Gag and Pol and viral load, but a subsequent more comprehensive analysis of CD8 T cell responses to all viral proteins found the opposite: a positive correlation with viral load.

To some extent, these disparate findings have been reconciled by data indicating that CD8 T cell targeting of certain epitopes, especially those in Gag, may be particularly important in controlling HIV. But it has also become clear that cell function is critical, and a number of research groups have developed assays designed to directly measure the ability of CD8 T cells to kill HIV-infected CD4 T cells in vitro. For example, the laboratory of Mark Connors at NIAID has pioneered this approach and demonstrated that elite controllers display functionally superior HIV-specific CD8 T cells compared to individuals with progressive infection.

In new a study published in the current issue of the Journal of Infectious Diseases, Lucy Dorrell and colleagues use an assay developed in their lab to investigate the relationship between the killing function of HIV-specific CD8 T cells and the rate of decline in peripheral blood CD4 T cell counts. The researchers first established that the CD8 T cell antiviral activity they were measuring was specific: at a CD8:CD4 ratio of 2:1 in the in vitro assay, the median inhibition was 78% in HIV-positive individuals compared to 0% in HIV-negative controls. The activity was only seen when the CD4 T cell targets shared a class I HLA allele with the study participant from whom the CD8 T cells were obtained.

In an initial retrospective analysis, the slopes of CD4 T cell decline were determined in a group of 30 chronically infected individuals using a median of nine counts obtained over the course of 4.5 years. For individuals with a CD8 T cell inhibition value of 90%, the mean rate of CD4 T cell loss was 20 cells/mL per year (assuming a baseline count of 500 cells), whereas for an inhibition value of 50% the mean rate of loss was 85 cells/mL per year. The relationship between CD8 T cell antiviral activity and CD4 T cell decline was highly statistically significant (p < 0.0001). Notably, there was no correlation between the inhibitory capacity of the CD8 T cells and the magnitude of interferon gamma-producing HIV-specific T cell responses targeting Gag, other proteins, or the entire virus proteome.

The researchers also studied a cohort of 20 individuals recruited during acute HIV infection in China (in the Beijing PRIMO study) followed prospectively. CD4 T cell slopes were calculated from a median of 13.5 measurements over an average of around 2.5 years of follow-up. The results showed that the antiviral activity of CD8 T cells in early infection was predictive of the rate of CD4 T cell loss over time. The relationship was strongest when reduced ratios of CD8:CD4 T cells were used (either 1:10 or 1:100), leading the authors to state that: “titration of CD8 T cells to sub-optimal levels improved the capacity of the assay to distinguish fast and slow progressors.” The percentage of CD8 T cell inhibition was calculated to explain 73% of the variance in CD4 slopes at a CD8:CD4 ratio of 1:10. The researchers found that adding HIV viral load set point into a model together with percentage of CD8 T cell inhibition improved their ability to predict CD4 T decline: at a CD8:CD4 ratio of 1:10, the combined measures explained a remarkable 91% of the variance in the slope of CD4 T cell loss.

These findings make an important contribution to the understanding of differences in individual rates of CD4 T cell loss and disease progression among HIV-positive individuals. The researchers note, however, that their study has some limitations: their model assumes constant CD8 T cell antiviral activity, when in reality it is likely that this function becomes impaired over the course of infection. The retrospective nature of the analysis of the chronic infection cohort also makes discerning cause and effect difficult, and the prospective cohort only had limited samples from early infection available. To address these limitations, they recommend the conduct of a longitudinal study assessing CD8 T cell antiviral activity repeatedly from the earliest stages of infection onwards.

J Infect Dis. 2012 Jul 3. [Epub ahead of print]

Antiviral Inhibitory Capacity of CD8+ T cells Predicts the Rate of CD4+ T-Cell Decline in HIV-1 Infection.

Yang H, Wu H, Hancock G, Clutton G, Sande N, Xu X, Yan H, Huang X, Angus B, Kuldanek K, Fidler S, Denny TN, Birks J, McMichael A, Dorrell L.

Source

Oxford NIHR Biomedical Research Centre.

Abstract

Background. Rare human immunodeficiency virus type 1 (HIV-1)-infected individuals who maintain control of viremia without therapy show potent CD8+ T-cell-mediated suppression of viral replication in vitro. Whether this is a determinant of the rate of disease progression in viremic individuals is unknown.

Methods. We measured CD8+ T-cell-mediated inhibition of a heterologous HIV-1 isolate in 50 HIV-1-seropositive adults with diverse progression rates. Linear mixed models were used to determine whether CD8+ T-cell function could explain variation in the rate of CD4+ T-cell decline.

Results. There was a significant interaction between CD8+ T-cell antiviral activity in vitro and the rate of CD4+ T-cell decline in chronically infected individuals (P < .0001). In a second prospective analysis of recently infected subjects followed for up to 3 years, CD8+ T-cell antiviral activity strongly predicted subsequent CD4+ T-cell decline (P < .0001) and explained up to 73% of the interindividual variation in the CD4+ T-cell slope. In addition, it was inversely associated with viral load set point (r = -0.68 and P = .002).

Conclusions. The antiviral inhibitory capacity of CD8+ T cells is highly predictive of CD4+ T-cell loss in early HIV-1 infection. It has potential as a benchmark of effective immunity in vaccine evaluation.