Michael Palm Basic Science, Vaccines & Prevention Project Weblog

Homing in on the Cause of Disrupted Hematopoiesis in HIV Infection

A new study in the free access Journal of Clinical Investigation makes a compelling case that the HIV protein Nef is responsible for the hematopoietic abnormalities (anemia, granulocytopenia, and thrombocytopenia) that have been reported in people with HIV infection. Researchers led by Stéphane Prost report that Nef interacts with a nuclear receptor called PPARγ, causing suppression of two signaling proteins, STAT5A and STAT5B, known to be important for the development of hematopoietic stem cells (HSCs). The study used multiple different approaches to verify that a specific region of the nef gene which is conserved among different HIV-1 and SIV strains triggered HSC dysfunction. The authors conclude by suggesting that the study “provides new clues for the development of novel drugs targeting PPARγ activity to cure hematopoietic disorders, including those affecting seropositive/AIDS patients.”

In an accompanying commentary, Frank Kirchhoff and Guido Silvestri praise the work but also caution that “much work will be required to define the mechanism by which extracellular Nef induces PPARγ and to determine whether the levels of Nef in blood and bone marrow are sufficient to impair HSC function during HIV infection.” They also note that “as STAT5 signaling plays a critical role in T cell development and function, an additional question is whether Nef also affects T cell homeostasis by suppressing STAT5A/B function in other, more mature T cell subsets.”

J. Clin. Invest. - (2008). doi:10.1172/JCI35487.

Commentary

Is Nef the elusive cause of HIV-associated hematopoietic dysfunction?

Frank Kirchhoff1 and Guido Silvestri2

1Institute of Virology, University of Ulm, Ulm, Germany.
2Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

HIV-associated hematological abnormalities involve all lineages of blood cells, thus implying that the virus impairs the function of early HSCs. However, the underlying mechanisms of this defect are unknown, particularly since HSCs are largely resistant to HIV-1 infection. In this issue of the JCI, Prost and colleagues show that the viral accessory protein Negative factor (Nef) plays a potentially critical role in the pathogenesis of HIV/SIV-associated hematopoietic dysfunction by affecting the clonogenic potential of HSCs. Soluble Nef induces PPARγ in uninfected HSCs, thereby suppressing the expression of STAT5A and STAT5B, two factors necessary for proper HSC function. The identification of this novel activity of extracellular Nef defines a new mechanism of HIV/SIV pathogenesis and suggests that approaches aimed at increasing STAT5A and STAT5B expression may be considered in HIV-infected individuals with prominent hematological abnormalities. The results also raise the question of whether dysregulation of hematopoiesis by extracellular Nef plays a role in the development of T cell immunodeficiency and the high levels of chronic immune activation associated with AIDS.

J. Clin. Invest. doi:10.1172/JCI33037.

Research Article

Human and simian immunodeficiency viruses deregulate early hematopoiesis through a Nef/PPARγ/STAT5 signaling pathway in macaques

Stéphane Prost1,2,3, Mikael Le Dantec1, Sylvie Augé4,5, Roger Le Grand1, Sonia Derdouch1, Gwenaelle Auregan1,6, Nicole Déglon6, Francis Relouzat2,3, Anne-Marie Aubertin7, Bernard Maillere8, Isabelle Dusanter-Fourt4,5 and Marek Kirszenbaum1

1Immunovirology Division and
2Innovative Therapy Division, Institute of Emerging Diseases and Innovative Therapies, CEA, Fontenay-aux-Roses, France.
3INSERM-CEA-Paris XI, UMR U733, CEA, Fontenay-aux-Roses, France.
4Institut Cochin, Université Paris Descartes, CNRS (UMR8104), Paris, France.
5INSERM U567, Paris, France.
6CEA, I2BM et programme MIRCen, Orsay, France.
7Laboratoire de Virologie, Université Louis Pasteur, Strasbourg, France.
8CEA, IBITEC-S, Service d’Ingénierie Moleculaire des Protéines, Saclay, France.

Infection of primates by HIV-1 and SIV induces multiple hematological abnormalities of central hematopoietic origin. Although these defects greatly contribute to the pathophysiology of HIV-1 infection, the molecular basis for altered BM function remains unknown. Here we show that when cynomolgus macaques were infected with SIV, the multipotent potential of their hematopoietic progenitor cells was lost, and this correlated with downregulation of STAT5A and STAT5B expression. However, forced expression of STAT5B entirely rescued the multipotent potential of the hematopoietic progenitor cells. In addition, an accessory viral protein required for efficient SIV and HIV replication and pathogenicity, “Negative factor” (Nef), was essential for SIV-mediated impairment of the multipotent potential of hematopoietic progenitors ex vivo and in vivo. This newly uncovered property of Nef was both conserved between HIV-1 and SIV strains and entirely dependent upon the presence of PPARγ in targeted cells. Further, PPARγ agonists mimicked Nef activity by inhibiting STAT5A and STAT5B expression and hampering the functionality of hematopoietic progenitors both ex vivo and in vivo. These findings have extended the role of Nef in the pathogenicity of HIV-1 and SIV and reveal a pivotal role for the PPARγ/STAT5 pathway in the regulation of early hematopoiesis. This study may provide a basis for investigating the potential therapeutic benefits of PPARγ antagonists in both patients with AIDS and individuals with hematopoietic disorders.

Transmission of CD8 T Cell Escape Mutants is Associated with Lower Viral Loads in Newly Infected Individuals

A new study by Paul Goepfert and colleagues offers compelling evidence that CD8 T cell responses can pressure HIV into mutating in ways that compromise viral fitness. CD8 T cells target tiny slices of viral proteins called epitopes, which are displayed by infected cells as a sort of alarm signal; CD8 T cells that recognize a particular epitope (via a docking bay-type structure called a T cell receptor or TCR) can mediate destruction of an infected cell by releasing cell-destroying substances such as perforin and granzyme B. HIV mutations that impact the structure of an epitope can abrogate CD8 T cell recognition and this phenomenon is called immune escape (it is loosely analogous to the way mutations can allow the virus to resist the effects of antiretroviral drugs). If a particular epitope-specific CD8 T cell response is effective, viruses with escape mutations in that epitope are at a selective advantage because they can persist despite the presence of the immune response. However, certain parts of HIV can tolerate mutations more easily than others, and studies have shown that the Gag protein is so vital to replication that mutations affecting epitopes in Gag can impair the ability of the virus to replicate in a lab dish (in vitro).

To assess whether these observations are relevant in people, Goepfert et al analyzed data from 114 epidemiologically linked transmission pairs in Zambia (the individuals had been participants in a larger cohort of “discordant” couples in which one partner was HIV-infected; despite counseling and increased condom use in the cohort, transmission still occurred at a rate of approximately 8% per year). The researchers looked for evidence of CD8 T cell escape mutations in Gag and Nef and then analyzed whether the transmission of HIV containing mutations impacted viral load in the newly infected individual. The timepoint for the analysis was 6 months after infection, because 579 of the 610 mutations documented in the transmitting partners were still present in recipients at this time point. The results showed that higher numbers of CD8 T cell escape mutations in the Gag protein of transmitted viruses were significantly associated with lower viral loads in the newly infected individuals (no such effect was seen for Nef). Further analyses revealed that the effect was most consistent for Gag epitopes targeted by HLA-B-restricted CD8 T cells (HLA genes manufacture the CD8 T cell TCR and thus govern the epitope structures that a CD8 T cell can recognize). The researchers hypothesized that the impact on HIV replication of mutations in epitopes targeted by HLA-B-restricted CD8 T cells would be most prominent in individuals lacking the same HLA-B genes themselves, and indeed this turned out to be the case: when the analysis was restricted to only these study participants the associations between more mutations in Gag and lower viral load became much stronger (p=0.0003).

In discussing their results, the study authors note that there was a ~10-fold difference in viral loads when individuals infected with viruses containing less than two escape mutations in Gag were compared to those with more than six such mutations, suggesting that infection with these multiple escape mutants may slow disease progression (although longer term follow up of a subset of participants indicates that viral load is, as is typical, increasing over time). They also state that: “these data imply that for CTL-based HIV vaccines to effectively control viral load, they must simultaneously target multiple Gag epitopes, thereby ensuring that fitness constraints prevent the virus from easily mutating.” In an accompanying news brief, JEM editor Hema Bashyam speculates that the study “might explain why a T cell vaccine that induces immune responses against two Gag epitopes failed in a recent trial.” In fact, recipients of the Merck HIV vaccine developed responses to an average of just one Gag epitope.

The Journal of Experimental Medicine
Published online 21 April 2008
doi:10.1084/jem.20072457

BRIEF DEFINITIVE REPORT

Transmission of HIV-1 Gag immune escape mutations is associated with reduced viral load in linked recipients

Paul A. Goepfert1,2, Wendy Lumm4, Paul Farmer4, Philippa Matthews5, Andrew Prendergast5, Jonathan M. Carlson6,7, Cynthia A. Derdeyn4,8, Jianming Tang1,2, Richard A. Kaslow3, Anju Bansal1, Karina Yusim10, David Heckerman6, Joseph Mulenga11, Susan Allen9, Philip J.R. Goulder5,12,13, and Eric Hunter4,8

1 Department of Medicine, 2 Department of Microbiology, and 3 Department of Epidemiology, University of Alabama at Birmingham, Birmingham, AL 35294
4 Emory Vaccine Center at Yerkes National Primate Research Center, Atlanta, GA 30322
5 Department of Pediatrics, The Peter Medawar Building for Pathogen Research, University of Oxford, Oxford OX1 3SY, England, UK
6 Microsoft Research, Redmond, WA 98052
7 Department of Computer Science and Engineering, University of Washington, Seattle, WA 98195
8 Department of Pathology and Laboratory Medicine and 9 Department of Global Health, Emory University, Atlanta, GA 30322
10 Los Alamos National Laboratory, Los Alamos, NM 87545
11 Zambia-Emory HIV Research Group, Lusaka, Zambia
12 HIV Pathogenesis Program, Doris Duke Medical Research Institute, University of KwaZulu-Natal, Durban 4013, South Africa
13 Partners AIDS Research Center, Massachusetts General Hospital, Charlestown, MA 02129

CORRESPONDENCE Paul A. Goepfert: paulg@uab.edu

In a study of 114 epidemiologically linked Zambian transmission pairs, we evaluated the impact of human leukocyte antigen class I (HLA-I)–associated amino acid polymorphisms, presumed to reflect cytotoxic T lymphocyte (CTL) escape in Gag and Nef of the virus transmitted from the chronically infected donor, on the plasma viral load (VL) in matched recipients 6 mo after infection. CTL escape mutations in Gag and Nef were seen in the donors, which were subsequently transmitted to recipients, largely unchanged soon after infection. We observed a significant correlation between the number of Gag escape mutations targeted by specific HLA-B allele–restricted CTLs and reduced VLs in the recipients. This negative correlation was most evident in newly infected individuals, whose HLA alleles were unable to effectively target Gag and select for CTL escape mutations in this gene. Nef mutations in the donor had no impact on VL in the recipient. Thus, broad Gag-specific CTL responses capable of driving virus escape in the donor may be of clinical benefit to both the donor and recipient. In addition to their direct implications for HIV-1 vaccine design, these data suggest that CTL-induced viral polymorphisms and their associated in vivo viral fitness costs could have a significant impact on HIV-1 pathogenesis.

HLA Class II Associations with Resistance & Susceptibility to HIV Infection

Following a similar theme to a recently posted study indicating that certain class II HLA genes – which impact antigen presentation to CD4 T cells – are associated with elite control of HIV replication in infected individuals, a new paper just published in the journal AIDS adds to the literature showing significant associations between class II HLA genes and resistance/susceptibility to HIV infection. The data comes from a large cohort study of sex workers in the Pumwani district of Nairobi, approximately 10% of whom persistently resist HIV infection despite an estimated ~60 unprotected exposures per year.

AIDS. 22(7):807-816, April 23, 2008.

BASIC SCIENCE

Human leukocyte antigen-DQ alleles and haplotypes and their associations with resistance and susceptibility to HIV-1 infection.

Hardie, Rae-Anne; Luo, Ma; Bruneau, Brigitte; Knight, Erin; Nagelkerke, Nico JD; Kimani, Joshua; Wachihi, Charles; Ngugi, Elizabeth N; Plummer, Francis A

Abstract:

Objectives: To determine the association of DQ antigens with resistance and susceptibility to HIV-1.

Design: Despite repeated exposure to HIV-1, a subset of women in the Pumwani Sex Worker cohort established in Nairobi, Kenya in 1985 have remained HIV-1 negative for at least 3 years and are classified as resistant. Differential susceptibility to HIV-1 infection is associated with HIV-1 specific CD4+ and CD8+ T cell responses. As human leukocyte antigen-DQ antigens present viral peptides to CD4+ cells, we genotyped human leukocyte antigen -DQ alleles for 978 women enrolled in the cohort and performed cross-sectional and longitudinal analyses to identify associations of human leukocyte antigen -DQ with resistance/susceptibility to HIV-1.

Methods: DQA1 and DQB1 were genotyped using taxonomy-based sequence analysis. SPSS 13.0 was used to determine associations of DQ alleles/haplotypes with HIV-1 resistance, susceptibility, and seroconversion rates.

Results: Several DQB1 alleles and DQ haplotypes were associated with resistance to HIV-1 infection. These included DQB1*050301 (P = 0.055, Odds Ratio = 12.77, 95% Confidence Interval = 1.44-112), DQB1*0603 and DQB1*0609 (P = 0.037, Odds Ratio = 3.25, 95% Confidence Interval = 1.12-9.47), and DQA1*010201-DQB1*0603 (P = 0.044, Odds Ratio = 17.33, 95% Confidence Interval = 1.79-168). Conversely, DQB1*0602 (P = 0.048, Odds Ratio = 0.68, 95% Confidence Interval = 0.44-1.05) and DQA1*010201-DQB1*0602 (P = 0.039, Odds Ratio = 0.64, 95% Confidence Interval = 0.41-1.03) were overrepresented in the HIV-1 infected population. DQA1*0504-DQB1*0201, DQA1*010201-DQB1*0201, DQA1*0402-DQB1*0402 and DQA1*0402-DQB1*030101 genotypes were only found in HIV-1 positive subjects (Odds Ratio = 0.30-0.31, 95% Confidence Interval = 0.03-3.70), and these women seroconverted rapidly. The associations of these DQ alleles and haplotypes with resistance and susceptibility to HIV-1 were independent of the previously reported human leukocyte antigen-DRB*01, human leukocyte antigen A2/6802, and human leukocyte antigen-A*2301.

Conclusion: The associations of DQ alleles and haplotypes with resistance and susceptibility to HIV-1 emphasize the importance of human leukocyte antigen-DQ and CD4 in anti-HIV-1 immunity.

CTLA-4 Blockade Can Increase SIV Replication

The expression of the molecule CTLA-4 by T cells is typically associated with suppression of T cell activation. Prior studies have shown that CTLA-4 is elevated in individuals with progressing HIV infection compared to healthy controls. Furthermore, levels of CTLA-4 on HIV-specific CD4 T cells are significantly higher in individuals with progressive disease compared to "elite controllers," suggesting that expression of the molecule may contribute to the failure of the immune response to control HIV replication. These data have led to the suggestion that blockade of CTLA-4 may deserve evaluation as a therapeutic approach to treating HIV. One prior study in macaques with longstanding (>96 weeks) SIV infection reported that two doses of an anti-CTLA-4 antibody (MDX-010, manufactured by a company called Medarex) slightly reduced SIV RNA in lymphoid tissue compared to antiretroviral therapy (ART) alone.

However, a new paper by the same research group now reports that MDX-010 increases immune activation and consequently increase SIV replication in gut-associated lymphoid tissue of macaques (see abstract below). In this study, MDX-010 was administered immediately prior to SIV infection and, in a second group of animals, during early SIV infection (four doses given at weeks 22, 27, 32 & 37 after infection). The authors do not offer much in the way of explanation for the differing study outcomes, beyond noting that mucosal sites were not evaluated in the prior work. Notably, there was no evidence of enhancement of SIV-specific T cell responses in either this or the previous study. Because regulatory T cells (Tregs) preferentially express CTLA-4, the authors argue that their findings suggest "a limited contribution of Treg to suppression of (SIV-specific) immune responses in vivo." However, the relative specificity of MDX-010 for Tregs versus other T cells that may transiently express CTLA-4 (such as effector T cells) remains controversial and uncertain. The findings imply that MDX-010 is unlikely to be a useful HIV treatment; one human phase I safety study has been conducted by Medarex but the only reference to the results to be found online is in an IAVI Report article on Tregs, in which the clinician responsible for the trial states that the treatment was "safe and well tolerated." The company does not appear to be conducting further trials in HIV infection.

The Journal of Immunology, 2008, 180: 5439-5447.

Immune Activation Driven by CTLA-4 Blockade Augments Viral Replication at Mucosal Sites in Simian Immunodeficiency Virus Infection

Valentina Cecchinato2,*, Elzbieta Tryniszewska2,*,, Zhong Min Ma¶, Monica Vaccari*, Adriano Boasso, Wen-Po Tsai*, Constantinos Petrovas||, Dietmar Fuchs#, Jean-Michel Heraud3,*, David Venzon, Gene M. Shearer, Richard A. Koup||, Israel Lowy**, Christopher J. Miller¶ and Genoveffa Franchini4,*

* Animal Models and Retroviral Vaccines Section, Experimental Immunology Branch, and Biostatistics and Data Management Section, National Cancer Institute, Bethesda, MD 20892; Department of Microbiology Diagnostics, Medical University of Bialystok, Bialystok, Poland; ¶ California National Primate Research Center, University of California, Davis, CA 95616; || Immunology Laboratory, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892; # Division of Biological Chemistry Biocentre, Innsbruck Medical University, Innsbruck, Austria; and ** Medarex, Bloomsbury, NJ 08804

The importance of chronic immune activation in progression to AIDS has been inferred by correlative studies in HIV-infected individuals and in nonhuman primate models of SIV infection. Using the SIVmac251 macaque model, we directly address the impact of immune activation by inhibiting CTLA-4, an immunoregulatory molecule expressed on activated T cells and a subset of regulatory T cells. We found that CTLA-4 blockade significantly increased T cell activation and viral replication in primary SIVmac251 infection, particularly at mucosal sites, and increased IDO expression and activity. Accordingly, protracted treatment with anti-CTLA-4 Ab of macaques chronically infected with SIVmac251 decreased responsiveness to antiretroviral therapy and abrogated the ability of therapeutic T cell vaccines to decrease viral set point. These data provide the first direct evidence that immune activation drives viral replication, and suggest caution in the use of therapeutic approaches for HIV infection in vivo that increase CD4+ T cell proliferation.

HLA Class II Associations with Viral Load Control

Last week’s NIAID vaccine summit featured much handwringing pessimism regarding the potential for adaptive immunity to control HIV replication, leading Guiseppe Pantaleo to remind attendees about the strong and consistent association between the class I HLA allele B*57 and elite control/long term non progression. Class II HLA alleles, which influence CD4 as opposed to CD8 T cell responses, have been less well studied, although some associations with slowed disease progression and resistance to HIV infection have been reported. At the recent Keystone HIV pathogenesis conference in Banff, Rachel Owen from UCSF presented new data indicating that the class II HLA allele DRB1*13 is significantly overrepresented among individuals who maintain viral loads in the absence of therapy, suggesting that CD4 T cell responses contribute to the phenomenon. The findings echo a recently published report on class II alleles and elite control in SIV-infected macaques (see second abstract, below).

As a side note, the Keystone HIV pathogenesis conference occurs in parallel with an HIV vaccine meeting and has done so for many years. However, next year the titles of the two meetings will be changed to “Prevention of HIV/AIDS” and “HIV Immunobiology: From Infection to Immune Control.”

Keystone Symposia: HIV Pathogenesis (X8), March 27 - April 1, 2008

HLA Class II associations in HIV infection: Controllers versus Non-controllers

Rachel E. Owen1, 2, Elizabeth Sinclair3, C. Lorrie Epling3, Jeffrey N. Martin3, Steven G. Deeks3, Philip J. Norris1, 2,3.

1Blood Systems Research Institute, 270 Masonic Avenue, San Francisco, CA 94118, USA, 2Department of Laboratory Medicine and 3Department of Medicine, University of California San Francisco, San Francisco, CA 94143, USA.

Strong CD4+ and CD8+ T cell responses are detected in individuals who control viraemia, suggesting but not proving that these cells may be causally related to virus control. The consistent association between certain class I alleles (e.g., B5701) and virus control provides strong evidence that CD8 T cells are able to effectively exert control. However, the role of CD4+ T cells in controlling HIV infection is not fully understood, and few HLA class II gene associations have been made.

We investigated HLA class II gene associations in chronically infected HIV+ individuals from the UCSF SCOPE cohort and categorized individuals as: (1) “controllers”: defined as plasma HIV RNA levels <10,000 copies/ml (in absence of therapy, n= 38); and (2) “non-controllers”: defined as plasma HIV RNA levels >10,000 copies/ml (n=290).

We found an increased frequency of the HLA-DRB1*13 allele in the controllers compared to the non-controllers (42.1% vs. 24.1%, p= 0.029), consistent with previous studies. Some DRB1*13 haplotypes were more common in controllers than non-controllers (p= <0.05 for each pair-wise comparison): DRB1*13 plus DQB1*03 (24% vs. 10%), DQB1*04 (8% vs. 0.7%), DQB1*06 (37% vs. 18%), DRB3*03 (13% vs. 2%) and DRB4*01 (21% vs. 8%). Controllers had higher CD4+ T cell IFN and IL-2 responses than non-controllers, measured by intracellular cytokine staining, following gag stimulation (mean IFN response 0.7% vs. 0.3%; mean IL-2 response 0.3% vs. 0.1%). No difference in the CD4+ IFN or IL-2 response following env or pol stimulation was measured. A small number of DRB1*13 expressing controllers had a trend towards higher CD4+ IFN and IL-2 responses following gag stimulation, when compared to controllers not expressing the DRB1*13 allele or non-controllers.

The epidemiological link between class II alleles and virus control is consistent with observations made with class I alleles, and argues that antigen-specific CD4+ T cells are playing a role in control of viraemia. These responses would be important to study in the pathogenesis of acute HIV infection and in HIV vaccine trials.

This work was supported by the National Institutes of Health to the UCSF-GIVI Center for AIDS Research, 5P30AI027763, and to the UCSF Clinical and Translational Sciences Institute, U54RR023566.

J Virol. 2008 Jan;82(2):859-70. Epub 2007 Nov 7.

The major histocompatibility complex class II alleles Mamu-DRB1*1003 and -DRB1*0306 are enriched in a cohort of simian immunodeficiency virus-infected rhesus macaque elite controllers.

Giraldo-Vela JP, Rudersdorf R, Chung C, Qi Y, Wallace LT, Bimber B, Borchardt GJ, Fisk DL, Glidden CE, Loffredo JT, Piaskowski SM, Furlott JR, Morales-Martinez JP, Wilson NA, Rehrauer WM, Lifson JD, Carrington M, Watkins DI.

Department of Pathology and Laboratory Medicine, University of Wisconsin-Madison, 555 Science Dr., Madison, WI 53711, USA.

The role of CD4(+) T cells in the control of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) replication is not well understood. Even though strong HIV- and SIV-specific CD4(+) T-cell responses have been detected in individuals that control viral replication, major histocompatibility complex class II (MHC-II) molecules have not been definitively linked with slow disease progression. In a cohort of 196 SIVmac239-infected Indian rhesus macaques, a group of macaques controlled viral replication to less than 1,000 viral RNA copies/ml. These elite controllers (ECs) mounted a broad SIV-specific CD4(+) T-cell response. Here, we describe five macaque MHC-II alleles (Mamu-DRB*w606, -DRB*w2104, -DRB1*0306, -DRB1*1003, and -DPB1*06) that restricted six SIV-specific CD4(+) T-cell epitopes in ECs and report the first association between specific MHC-II alleles and elite control. Interestingly, the macaque MHC-II alleles, Mamu-DRB1*1003 and -DRB1*0306, were enriched in this EC group (P values of 0.02 and 0.05, respectively). Additionally, Mamu-B*17-positive SIV-infected rhesus macaques that also expressed these two MHC-II alleles had significantly lower viral loads than Mamu-B*17-positive animals that did not express Mamu-DRB1*1003 and -DRB1*0306 (P value of <0.0001). The study of MHC-II alleles in macaques that control viral replication could improve our understanding of the role of CD4(+) T cells in suppressing HIV/SIV replication and further our understanding of HIV vaccine design.

Understanding Control of HIV Replication in the Absence of Therapy

Individuals who maintain control of HIV replication in the absence of any therapy may offer important clues to aid the development of both vaccines and novel immune-based therapies. Many research groups are studying such individuals, with the largest and most ambitious research project being the elite controller study helmed by Bruce Walker from Partners AIDS Research Center in Boston. In a recent issue of the Journal of Infectious Diseases, lead investigator Florence Pereyra and colleagues report preliminary data from 126 individuals who have enrolled into the study. Participants are divided into two categories: elite controllers, defined as those with viral load consistently below 50 copies/mL (the limit of detection for the viral load test) and viremic controllers, whose viral loads are consistently below 2000 copies/mL. The JID paper contains data on 66 individuals in the former category and 60 in the latter; 30 individuals with chronic, progressive infection recruited over the same time period are also included for comparative purposes.

The major finding is that controllers (both elite and viremic) are a heterogeneous group with regard to many of the factors that previous studies have associated with viral load control. In terms of host genetics, HLA B*57 alleles were significantly overrepresented among controllers, but the researchers emphasize that the frequency of HLA B*57 was considerably lower than reported previously in smaller cohorts. When the analysis was expanded to any HLA allele that has been associated with a favorable prognosis in HIV infection, 68% of elite controllers and 60% of viremic controllers were found to carry at least one such allele, compared with 37% of the chronic progressors, “leaving almost one-third of HIV controllers without any known relatively protective HLA alleles.” CCR5 and CCR2 polymorphisms that have been associated with slowed disease progression were not overrepresented among controllers. Absolute CD4 counts were different between elite and viremic controllers (884 vs. 602 cells), in line with a recent paper showing that CD4 declines can still occur – albeit very slowly - despite low or even undetectable viral loads.

Randomly selected subsets from each group of study participants were further evaluated for HIV-specific immune responses. In terms of CD8 T cell responses (as measured by interferon gamma ELISpot), elite controllers displayed the lowest breadth and magnitude of HIV-specific CD8 T cells. Median breadth was 15, 19 and 27 epitopes among elite controllers, viremic controllers and chronic progressors respectively while average magnitude was 5428, 6253 and 8300 spot-forming cells (SFC) per million PBMC (peripheral blood mononuclear cells). The simplistic interpretation of these data is that the CD8 T cell response cannot be important, but the broader, larger responses associated with higher viral loads are to be expected due to the persistent recruitment from the naïve CD8 T cell pool that can occur in the setting of chronic infection, and because interferon gamma production is the last function to be lost by exhausted cells (so a large, broad response does not necessarily equate to an effective response). When the researchers looked at IL-2 and interferon gamma production together, responses were significantly higher in elite controllers than viremic controllers or progressors (both for HIV-specific CD4 T cells and CD8 T cells), but there was still heterogeneity in that these responses were not detectable in some individuals. Interestingly, elite controllers had the highest ratio of IL-2+interferon-gamma-producing HIV-specific CD4 T cells to HIV-specific CD8 T cells while chronic progressors had the lowest, suggesting – as have many prior studies - that CD4 T cell help plays an important, perhaps undervalued, role in the immune response to HIV. Adding to the heterogeneity, elite controllers also displayed the widest range of CD8 T cell interferon gamma ELISpot responses in terms of both breadth and magnitude, with responses ranging from 2-101 epitopes in breadth and from less than 500 SFC to over 25,000 SFC in magnitude. Despite the range of responses, the vast majority of both elite and viremic controllers displayed responses to many more CD8 T cell epitopes than have been reported to date in recipients of T cell-based HIV vaccine candidates (the average response in the recent Merck trial was to three epitopes).

Consistent with previously published studies, the HIV Gag protein was preferentially targeted among controllers. In progressors, Gag, Pol and Nef were targeted equally and more targeting of Env was observed than in controllers. The researchers also looked at neutralizing antibody responses. Plasma samples from both viremic controllers and progressors showed neutralizing activity against viruses sampled from the same groups as well as against laboratory HIV strains. There were no differences in neutralizing antibody titers between these two groups. Elite controllers had significantly lower neutralizing antibody responses overall but, again, heterogeneity was seen: some individuals had broad neutralizing antibody responses whereas others had very low or undetectable responses.

In concluding, the authors state that: “although elite and viremic controllers share some immunologic features and are distinct from persons with chronic progressive infection, the elite controllers are a distinct subgroup. However, even within these groups there is substantial heterogeneity in all of the parameters studied, which suggests that there are as-yet-undefined viral or host factors or combinations of factors that contribute to this remarkable phenotype.” They also cite the elite controller study linked to above and their hope that this large collaborative effort will better define the pathways that lead to durable control of HIV replication.

Two other recent studies offer additional perspectives on the same topic. In the first, Shiv Ghandi and colleagues show that the antiretroviral activity of APOBEC proteins does not appear to explain elite control of HIV replication. In the second, recently presented at CROI, researchers from Barbara Shacklett’s lab at UC Davis demonstrate that controllers have significantly more polyfunctional HIV-specific CD4 and CD8 T cells in their rectal mucosa, a novel finding indicating that analyses of additional compartments beyond the blood may add to the understanding of the elite controller phenomenon.

The Journal of Infectious Diseases 2008;197:563–571
DOI: 10.1086/526786
MAJOR ARTICLE

Genetic and Immunologic Heterogeneity among Persons Who Control HIV Infection in the Absence of Therapy

Florencia Pereyra,1,2 Marylyn M. Addo,1 Daniel E. Kaufmann,1 Yang Liu,5 Toshiyuki Miura,1 Almas Rathod,1 Brett Baker,1 Alicja Trocha,1,4 Rachel Rosenberg,1 Elizabeth Mackey,1 Peggy Ueda,1 Zhigang Lu,1 Daniel Cohen,3 Terri Wrin,5 Christos J. Petropoulos,5 Eric S. Rosenberg,1 and Bruce D. Walker1,4

1Partners AIDS Research Center, Massachusetts General Hospital and Division of AIDS, Harvard Medical School, 2Brigham and Women’s Hospital, Division of Infectious Diseases, and 3Fenway Community Health Care Center, Boston, Massachusetts; 4Howard Hughes Medical Institute, Chevy Chase, Maryland; and 5Monogram Biosciences, South San Francisco, California

Background: Spontaneous control of human immunodeficiency virus (HIV) infection has been documented in a minority of HIV-infected individuals. The mechanisms behind this outcome remain largely unknown, and a better understanding of them will likely influence future vaccine strategies. Methods: HIV-specific T cell and antibody responses as well as host genetics were examined in untreated HIV-infected patients who maintain comparatively low plasma HIV RNA levels (hereafter, controllers), including those with levels of <50 RNA copies/mL (elite controllers, n=64), those with levels of 50–2000 copies/mL (viremic controllers, n=60); we also examined HIV-specific T cell and antibody responses as well as host genetics for patients with levels of >10,000 copies/mL (chronic progressors, n=30). Results: CD8+ T cells from both controller groups preferentially target Gag over other proteins in the context of diverse HLA class I alleles, whereas responses are more broadly distributed in persons with progressive infection. Elite controllers represent a distinct group of individuals who have significantly more CD4 and CD8 T cells that secrete interferon-γ and interleukin-2 and lower levels of HIV-neutralizing antibodies. Individual responses were quite heterogeneous, and none of the parameters evaluated was uniquely associated with the ability to control viremia. Conclusions: Elite controllers are a distinct group, even when compared to persons with low level viremia, but they exhibit marked genetic and immunologic heterogeneity. Even low-level viremia among HIV controllers was associated with measurable T cell dysfunction, which has implications for current prophylactic vaccine strategies.

Journal of Virology, March 2008, p. 3125-3130, Vol. 82, No. 6
doi:10.1128/JVI.01533-07

Role of APOBEC3G/F-Mediated Hypermutation in the Control of Human Immunodeficiency Virus Type 1 in Elite Suppressors

Shiv K. Gandhi,1 Janet D. Siliciano,1 Justin R. Bailey,1 Robert F. Siliciano,1,2 and Joel N. Blankson1

Department of Medicine, Johns Hopkins University School of Medicine,1 Howard Hughes Medical Institute, Baltimore, Maryland 212052

While many studies show that the APOBEC3 family of cytidine deaminases can inhibit human immunodeficiency virus type 1 (HIV-1) replication, the clinical significance of this host defense mechanism is unclear. Elite suppressors are HIV-1-infected individuals who maintain viral loads below 50 copies/ml without antiretroviral therapy. To determine the role of APOBEC3G/F proteins in the control of viremia in these patients, we used a novel assay to measure the frequency of hypermutated proviral genomes. In most elite suppressors, the frequency was not significantly different than that observed in patients on highly active antiretroviral therapy. Thus, enhanced APOBEC3 activity alone cannot explain the ability of elite suppressors to control viremia.

15th Conference on Retroviruses & Opportunistic Infections
Abstract #355

Polyfunctional HIV-specific T Cells in Rectal Mucosa of HIV Controllers

April Ferre*1, P Hunt2, D Young1, J Garcia1, H Yee2, R Pollard1, S Deeks2, and B Shacklett1

1Univ of California, Davis, US and 2Univ of California, San Francisco, US

Background: Among the HIV-infected population exists a unique group of individuals who achieve control over HIV replication in the absence of ART. The study of such individuals is crucial to understanding how the immune system may effectively control viral replication and limit progression to AIDS. Methods: In this study we examined cell-mediated immune responses in 26 HIV controllers (viral load <2000 copies/mL), 14 non-controllers (viral load >10,000 copies/mL), and 10 HAART-suppressed individuals (viral load <50 copies/mL) in peripheral blood mononuclear cells (PBMC) and rectal mucosa. Of the controllers, 14 were classified as elite controllers, individuals who maintain plasma viral load <75 copies/mL in the absence of ART. This group is believed to encompass <1% of all HIV-infected individuals. In intracellular cytokine assays, we measured the production of 3 cytokines (interferon-γ, interleukin-2, tumor necrosis factor-α), 1 chemokine (MIP-1β), and the cytolytic granule marker CD107 in response to stimulation by HIV-1 Gag peptides. We hypothesized that “polyfunctional” T cells, capable of producing multiple antiviral factors, are critical in limiting viral replication and disease progression. Results: Mucosal CD8 T cell responses in controllers were significantly stronger and more complex than those in HAART-suppressed individuals (p = 0.0006). Differences between controllers and non-controllers were more subtle, but included a higher frequency of 4-function HIV-specific CD8 T cells in rectal mucosa of controllers than in non-controllers (p = 0.002). CD4 T cell responses were less complex and of lower magnitude than CD8 responses, but several controllers had unusually strong, polyfunctional mucosal CD4 responses. Conclusions: These findings demonstrate that many controllers mount strong and complex HIV-specific T cell responses in mucosal tissues. These polyfunctional cells may play an important role in immune surveillance of gut mucosa, as suggested by their relative enrichment among individuals who appear to be controlling HIV replication in absence of therapy.

Evanescing Th17 CD4 T Cells & HIV Pathogenesis

A slew of new studies presented at CROI revealed that a recently discovered subset of CD4 T cells – dubbed T-helper type 17 or Th17 cells for short – appear preferentially depleted in HIV and pathogenic SIV infections. Th17 cells were identified as a separate lineage of CD4 T cells in 2005; they are characterized by the production of the cytokines IL-17 and IL-22 and have been shown to be involved in both pro-inflammatory autoimmune diseases, and in protection against certain pathogens such as Klebsiella pneumonia and Salmonella typherium. The cytokines IL-17 and IL-22 also play a role in protecting mucosal sites from pathogens and maintaining the integrity of the mucosal barrier. Prior studies conducted outside of the HIV field indicate that Th17 cells make up less than 1% of circulating CD4 T cells in healthy people with slightly higher levels (~1-2%) found in the gut. The cytokine IL-23 appears involved in the development of Th17 CD4 T cells, which arise – like other Th subsets – from the activation of naïve CD4 T cells. The development of Th17 cells is also suppressed under certain conditions, particularly by the presence of type I & II interferons (e.g. alpha interferon and interferon gamma).

At CROI, Mirka Paiardini presented data showing that Th17 cell levels were similar or slightly increased in the blood of HIV-infected individuals compared to uninfected controls. But in the gut, the percentage of CD4 T cells producing IL-17 after in vitro stimulation (with the pan-T cell stimulating agents PMA and ionomycin or anti-CD3) was significantly lower in people with HIV and antiretroviral therapy did not fully restore the proportion of Th17 cells to those seen in uninfected individuals. Paiardini also showed that there was a significant inverse correlation between immune activation levels in both gut and peripheral blood (as assessed by the proportion of CD4 T cells expressing the proliferation marker Ki67) and the proportion of Th17 cells in the gut.

It has recently been reported that non-pathogenic SIV infection of sooty mangabeys is associated with gut CD4 T cell loss, so Paiardini measured Th17 levels in the gut of mangabeys to ascertain if they were also depleted, as they appear to be in HIV-infected humans. He reported that the proportion of gut Th17 cells remained stable in sooty mangabeys despite the overall reduction in the percentage of gut CD4 T cells. Paiardini hypothesized that preservation of gut Th17 cells in mangabeys preserves the integrity of the mucosal barrier and thus prevents translocation of microbial products and systemic immune activation, supporting this hypothesis with slides demonstrating the health of the epithelial barrier in SIV-infected animals. However it is perhaps worth noting that – as is so often the case with research results – cause and effect are not necessarily easy to tease apart. Immune activation in HIV infection is associated with elevated levels of type I interferons, so if Th17 cells are generated dynamically in the gut, immune activation would be expected to inhibit their differentiation and thus reduce the proportion of Th17 cells. In sooty mangabeys, where SIV infection does not result in persistent immune activation, Mark Feinberg has shown that type I interferons are not upregulated, suggesting that preservation of Th17 cells could be a consequence rather than a cause of the observed lack of activation.

Valentina Cecchinato from Genoveffa Franchini’s lab at the National Cancer Institute sounded a similar theme to Paiardini, showing that in a study of SIV-infected rhesus macaques, the percentage of Th17 cells was reduced in the gut while the proportion of Th1 (interferon gamma producing) CD4 T cells was unaffected. In contrast, both populations were depleted from the peripheral blood. Further analyses revealed an inverse correlation between SIV viral load and percentages of Th17 cells in the colon and rectum. But when Cecchinato looked for evidence of microbial translocation by measuring blood levels of lipopolysaccharides (LPS), there was no correlation between LPS levels and the proportion of Th17 cells in the gut (or anywhere else). This finding may be explained by the technical challenges associated with LPS measurement, but it could also be an indication that Th17 loss is an effect rather than a cause of virus-driven immune activation in these animals.

In the last of three talks on the topic, David Favre from UCSF showed that the proportion of Th17 cells also declines at mucosal sites in SIV-infected pigtailed macaques. Favre focused on the balance between T regulatory (Treg) cells (another CD4 T cell subset with the ability to suppress immune responses) and Th17 cells, noting that the balance is maintained in African green monkeys, a species that – like sooty mangabeys – does not develop immunodeficiency as a result of SIV infection. In contrast, the loss of Th17 cells dysregulated the Th17/Treg balance in pigtailed macaques.

At the close of CROI, conference chair Mario Stevenson highlighted these presentations as representing an important advance in the understanding of HIV pathogenesis. However, until the cause and effect relationship between immune activation and Th17 cell decreases is better understood, the data should probably be interpreted with caution.

Webcasts for the presentations are available on the CROI website (the fourth oral abstract session on Wednesday).

Study abstracts:

15th Conference on Retroviruses & Opportunistic Infections
Session 36 Oral Abstracts
New Insights into Mechanisms of Viral Pathogenecity

Abstract #115

Preferential Loss of Th17 CD4 T Cells in the Gastrointestinal Tract of HIV-infected Individuals but Not SIV-infected Sooty Mangabeys

Barbara Cervasi*1, J Brenchley2, M Paiardini1, S Gordon1, A Asher2, I Frank1, J Else3, D Douek2, and G Silvestri1
1Univ of Pennsylvania, Philadelphia, US; 2Vaccine Res Ctr, NIAID, NIH, Bethesda, MD, US; and 3Yerkes Natl Primate Res Ctr, Emory Univ, Atlanta, GA, US

Background: A new subset of CD4 T helper cells, designated Th17, has been recently identified, leading to a reappraisal of the classically proposed dichotomy in which memory CD4 T cells belong exclusively to either a Th1 or Th2 lineage. While Th17 cells may be involved in the pathogenesis of autoimmune diseases, emerging data suggest that they are important in mucosal immunity to extra-cellular bacteria infection. Here we examined the frequency, antigen specificity, functionality, phenotype, and infection frequency of Th17 cells in the peripheral blood and gastrointestinal tract of HIV-infected and uninfected humans and simian immunodeficiency virus (SIV) -infected and uninfected sooty mangabeys, a natural, non-progressing host species of SIV infection.

Methods: Peripheral blood- and gastrointestinal tract-derived cells were obtained from 30 HIV-infected individuals and 15 healthy controls; peripheral blood, gastrointestinal, and lymph node cells were also obtained from 12 naturally SIV-infected and 10 uninfected sooty mangabeys. Phenotypical and functional characterization of Th17 cells was performed by 10-color flow cytometry.

Results: Interleukin (IL) -17+, interferon (IFN) -g–, IL-4–, IL-2+, IL23R+ memory CD4+ Th17 cells can be identified in all the examined tissues of humans and sooty mangabeys, with the majority of Th17 cells being CCR5– in peripheral blood, but CCR5+ in the gastrointestinal tract. The main results of this study were: There is significant depletion of Th17 cells in the gastrointestinal tract of HIV-infected individuals than in uninfected individuals; SIV-infected sooty mangabeys maintain healthy frequencies of Th17 cells in the the gastrointestinal tract; Th17 cells are infected by HIV in vivo, but not preferentially so compared to Th1 cells; and human Th17 cells are specific for bacterial and fungal antigens, but not common viral antigens.

Conclusions: The preferential loss of Th17 in the the gastrointestinal tract of HIV-infected individuals represents a new mechanism of mucosal immune dysfunction during HIV infection. These data thus further elucidate the immunodeficiency of HIV disease and may provide a mechanistic basis for the enteropathy and associated microbial translocation that characterize HIV infection. Conversely, maintenance of gastrointestinal Th17 cells may account for the non-progressive nature of non-pathogenic SIV infection in sooty mangabeys.

Abstract #116

Preferential Loss of Th17 T Cells at Mucosal Sites Predicts AIDS Progression in Simian Immunodeficiency Virus-infected Macaques

Valentina Cecchinato*1, C Trindade1, J M Heraud1, A Laurence2, J Brenchley3, E Tryniszewska1,4, D Venzon1, D Douek3, J O'Shea2, and G Franchini1
1NCI, NIH, Bethesda, MD, US; 2Natl Inst of Arthritis and Musculoskeletal and Skin Diseases, NIH, Bethesda, MD, US; 3NIAID, NIH, Bethesda, MD, US; and 4Med Univ of Bialystok, Poland

Background: Depletion of CD4+ T cells in the gut is necessary, but not sufficient to cause AIDS in animal models, raising the possibility that differential depletion of CD4+ T cell subtypes may be important. Th17 is a recently identified lineage of effector CD4+ T-helper interleukin-17 (IL-17) -secreting cells, important in mucosal immunity to extracellular bacteria. Here we investigated whether the frequency of Th17 lymphocytes is modified during simian immunodeficiency virus (SIV) infection, particularly at mucosal sites.

Methods: We enrolled in the study 31 rhesus macaques, either naive or SIV-infected. Blood and lymphoid tissues were collected 2 weeks after infection or at the time of necropsy. The frequency of CD4+IL-17+ cells in tissues was determined by intracellular cytokines staining following stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin. Flow cytometric analysis was performed to determine Th17 phenotype. Quantification of SIV Gag DNA in sorted memory CD4 T cells was performed by quantitative polymerase chain reaction (PCR). Human CD4+ T cells were infected in vitro with SIVmac251 and analyzed for IL-17 and p27 expression. Differences between groups were assessed by t-test. The Spearman rank correlation method was used to determine the correlation between the frequency of Th17 in tissues and viral load in plasma.

Results: Our data demonstrate that Th17 cells express CCR5 and can be infected in vitro and in vivo. We found that healthy macaques have a significantly higher number of Th17 T cells in the gut than in systemic tissues. Importantly, in the SIVmac251 model, Th17 cells are preferentially depleted within the first weeks of infection and their frequency is not restored to normal levels over time. Furthermore, we found that SIVmac251-infected macaques that could effectively control viral replication maintain normal levels of Th17 T cells in all tissues, and that there is a negative correlation between Th17 cell frequency at mucosal sites and plasma virus level, suggesting their importance in HIV/SIV pathogenesis.

Conclusions: Because Th17 cells play a central role in innate and adaptive immune response to extracellular bacteria, these data provide a rational explanation for the chronic enteropathy in HIV infection. Thus, therapeutic approaches to reconstitute Th17 numbers and/or function may be of benefit in HIV infection.

Abstract #117LB

Primary SIV Infection Causes Rapid Loss of the Balance between TH17 and T Regulatory Cell Populations in Pathogenic Infection of Non-human Primates

David Favre*1, S Lederer2, B Kanwar1, Z M Ma3, S Proll2, Z Kasakow1, C Miller3, M Katze2, and J McCune1
1Univ of California, San Francisco, US; 2Univ of Washington, Seattle, US; and 3Univ of California, Davis, US

Background: Progression to AIDS is observed after simian immunodeficiency virus (SIV) infection of some but not all non-human primates. To better define the basis for this dichotomy, we performed a comparative analysis of non-pathogenic infection of the African green monkey and pathogenic infection of the Asian pigtailed macaque. We hypothesized that a critical distinction between pathogenic and nonpathogenic SIV infections may lie in a shift in the equilibrium between pro- and anti-inflammatory host immune responses during acute infection.

Methods: To address this hypothesis, we investigated early virologic, immunologic, and gene expression events that occurred in the peripheral blood and in lymphoid organs, including the colonic mucosa, after acute infection of pigtailed macaques and African green monkeys with the same SIVagm.sab92018 isolate. In particular, we explored the absolute number and relative frequency of TH17 cells, which produce the pro-inflammatory cytokine interleukin (IL) -17, and FoxP3+ T regulatory cells (Treg), the function of which is instead immunosuppressive.

Results: After infection with SIVagm.sab92018, both African green monkeys and pigtailed macaques developed high peak and set point viral loads, but only pigtailed macaque showed generalized CD4+ T cell depletion and sustained immune activation and inflammation. In addition, the following novel features were observed. First, signs of chronic T cell activation and inflammation were detected in the colonic mucosa of both species, while systemic immune activation (e.g., in the blood and lymph node) was only sustained in pigtailed macaques. Second, an increase in the frequency of FoxP3+ Treg was detected early in the blood and in lymph nodes after infection of African green monkeys, but only at later time points in the colon of both African green monkeys and pigtailed macaques. Finally, and unexpectedly, the TH17 population was maintained in infected African green monkeys, but rapidly lost during the course of progressive SIV disease in pigtailed macaques.

Conclusions: These results suggest that balanced representation of both Treg and TH17 cells plays a unique and possibly determinative role in the setting of acute lentiviral infection. Because IL-17 acts to preserve the integrity of the mucosal barrier, thus enhancing host defenses against microbial agents, maintenance of robust TH17 function in mucosal tissue during SIV infection may decrease systemic inflammation and disease progression by preventing the immune activation that would otherwise occur after microbial translocation and spread.

Functional Impairment of MTB-Specific Memory Cells in HIV Infection

Following on from yesterday’s posting about central memory T cells, a new paper in the Journal of Infectious Diseases offers what may be a clearer view of the impact of the loss of this cell population in HIV infection. Cheryl Day and collaborators (from the University of KwaZulu Natal in Durban and Partners AIDS Research in Boston) investigated memory CD4 and CD8 T cell responses to Mycobacterium tuberculosis (MTB) in a cohort of individuals with HIV infection and latent (asymptomatic) TB, finding that cells producing IL-2 – a signature cytokine of central memory T cells - are depleted compared to cells producing TNF-alpha and interferon gamma or interferon gamma alone. Furthermore, they report an inverse correlation between HIV viral load and the proportion of IL-2-producing MTB-specific CD4 T cells, stating that the data “suggests that MTB-specific CD4 T cells in HIV-1-positive subjects may gradually lose IL-2 secretion capacity as HIV-1 disease progresses.” The authors note that further studies are required to identify the mechanisms contributing to the impairment of MTB-specific T cell responses in people with HIV, and the data also suggest that more studies of the impact of antiretroviral therapy on the functional profile of MTB-specific T cell responses are needed.

The Journal of Infectious Diseases 2008;197:000–000
DOI: 10.1086/529048

MAJOR ARTICLE

Detection of Polyfunctional Mycobacterium tuberculosis–Specific T Cells and Association with Viral Load in HIV-1–Infected Persons

Cheryl L. Day,1,3,4,a Nompumelelo Mkhwanazi,1 Sharon Reddy,1 Zenele Mncube,1 Mary van der Stok,1 Paul Klenerman,2 and Bruce D. Walker1,3,4,5

1HIV Pathogenesis Programme, Doris Duke Medical Research Institute, University of KwaZulu Natal, Durban, South Africa; 2Nuffield Department of Medicine, The Peter Medawar Building for Pathogen Research, Oxford University, Oxford, United Kingdom; 3Partners AIDS Research Center, Massachusetts General Hospital, and 4Division of AIDS, Harvard Medical School, Boston, Massachusetts; and 5Howard Hughes Medical Institute, Chevy Chase, Maryland

Background. The human immunodeficiency virus type 1 (HIV-1) epidemic is associated with a significant increase in the incidence of tuberculosis (TB); however, little is known about the quality of Mycobacterium tuberculosis (MTB)–specific cellular immune responses in coinfected individuals.

Methods. A total of 137 HIV-1–positive individuals in Durban, South Africa, were screened with the use of overlapping peptides spanning Ag85A, culture filtrate protein 10 (CFP-10), early secretory antigen target 6 (ESAT-6), and TB10.4, in an interferon (IFN)–γ enzyme-linked immunospot (ELISPOT) assay. Intracellular cytokine staining for MTB-specific production of IFN-γ, tumor necrosis factor (TNF)–α, and interleukin (IL)–2 was performed, as was ex vivo phenotyping of memory markers on MTB-specific T cells.

Results. A total of 41% of subjects responded to ESAT-6 and/or CFP-10, indicating the presence of latent MTB infection. The proportion of MTB-specific IFN-γ+/TNF-α+ CD4+ cells was significantly higher than the proportion of IFN-γ+/IL-2+ CD4+ cells (p=.0220), and the proportion of MTB-specific IL-2–secreting CD4 cells was inversely correlated with the HIV-1 load (p=.0098). MTB-specific CD8 T cells were predominately IFN-γ+/TNF-α+/IL-2−. Ex vivo memory phenotyping of MTB-specific CD4 and CD8 T cells indicated an early to intermediate differentiated phenotype for the population of effector memory cells.

Conclusions. Polyfunctional MTB-specific CD4 and CD8 T cell responses are maintained in the peripheral blood of HIV-1–positive individuals, in the absence of active disease, and the functional capacity of these responses is affected by HIV-1 disease status.

The Journal of Infectious Diseases 2008;197:000–000
DOI: 10.1086/529049

EDITORIAL COMMENTARY

T Cells and Tuberculosis: Beyond Interferon-γ

Ajit Lalvani and Kerry A. Millington

Tuberculosis Immunology Group, Department of Respiratory Medicine, National Heart and Lung Institute, Imperial College London, London, United Kingdom
Received 18 December 2007; accepted 19 December 2007; electronically published 4 March 2008.

FOXO3a and the Survival of Central Memory CD4 T cells in HIV Infection

Recent research on the pathogenesis of HIV and SIV infections has identified the depletion of “central memory” (TCM) CD4 T cells as being a crucial – perhaps the crucial – factor precipitating the development of clinical immunodeficiency and AIDS. As covered previously on the blog, TCM are a subset of memory T cells endowed with the capacity for self-renewal and capable of prodigious proliferation if they encounter a pathogen that they recognize. The research group of Rafik-Pierre Sekaly at the University of Montreal recently identified the transcription factor forkhead box O3a (FOXO3a) as a key mediator of TCM survival. FOXO3a controls several genes involved in the cell cycle (the process of cell division) and apoptosis (cell death) such as bim, but when FOXO3a is phosphorylated, it is excluded from the cell nucleus and thus cannot promote expression of these genes. In their paper published in the Journal of Experimental Medicine in late 2006 (see abstract, below), Catherine Riou and colleagues showed that TCM expressed higher levels of the transcriptionally inactive phosphorylated forms of FOXO3a and, consequently, lower levels of the cell-death promoting protein Bim. They also reported that FOXO3a phosphorylation can be promoted synergistically by signaling through the T cell receptor (TCR) and cytokines known to enhance memory T cell survival such as IL-7.

Now, in paper just published online by Nature Medicine, the same group demonstrate that HIV-infected elite controllers (individuals controlling viral load to less than 50 copies in the absence of any treatment) have higher levels of phosphorylated FOXO3a in both TCM and effector memory CD4 T cells (TEM) compared to individuals on successful antiretroviral therapy (ART). Conversely, levels of the pro-apoptotic protein Bim were higher in TCM and TEM from people on ART compared to elite controllers. Consistent with these findings, TCM and TEM from elite controllers survived repeated rounds of in vitro stimulation far better than those from individuals on ART. The researchers also used various techniques to inactivate FOXO3a in TCM and TEM cells from ART-treated study subjects and found that these interventions made their in vitro lifespan comparable with TCM and TEM from the elite controllers.

In discussing the results, the authors note that it is possible signaling pathways are perturbed in the cells of individuals on ART such that the integration of TCR and cytokine signals does not cause FOXO3a phosphorylation in the normal way. They also suggest that specific genetic polymorphisms affecting cell signaling pathways could be present in elite controllers; ongoing whole-genome analyses (in a study led by Partners AIDS Research Center in Boston) will be able to address this possibility. The authors close by stating that their work confirms the importance of maintaining TCM integrity in preventing HIV disease progression, and that approaches with the potential to enhance TCM survival may deserve study as therapies for HIV infection.

Nat Med. 2008 Mar 2 [Epub ahead of print]

Transcription factor FOXO3a controls the persistence of memory CD4(+) T cells during HIV infection.

van Grevenynghe J, Procopio FA, He Z, Chomont N, Riou C, Zhang Y, Gimmig S, Boucher G, Wilkinson P, Shi Y, Yassine-Diab B, Said EA, Trautmann L, Far ME, Balderas RS, Boulassel MR, Routy JP, Haddad EK, Sekaly RP.

[1] Laboratoire d'Immunologie, Centre de Recherche, Hôpital Saint-Luc, Centre Hospitalier de l'Université de Montréal, 264 Boulevard Rene-Levesque Est, Montréal, Québec H2X 1P1, Canada. [2] Laboratoire d'Immunologie, Département de Microbiologie et d'Immunologie, Université de Montréal, 264 Boulevard Rene-Levesque Est, Montréal, Québec H3T 1J4, Canada. [3] Institut national de la Santé et de la Recherche médicale U743, Centre de Recherche, Centre Hospitalier de l'Universite de Montréal, 264 Boulevard Rene-Levesque Est, Montreal, Québec H2X 1P1, Canada.

The persistence of central memory CD4(+) T cells (T(CM) cells) is a major correlate of immunological protection in HIV/AIDS, as the rate of T(CM) cell decline predicts HIV disease progression. In this study, we show that T(CM) cells and effector memory CD4(+) T cells (T(EM) cells) from HIV(+) elite controller (EC) subjects are less susceptible to Fas-mediated apoptosis and persist longer after multiple rounds of T cell receptor triggering when compared to T(CM) and T(EM) cells from aviremic successfully treated (ST) subjects or from HIV(-) donors. We show that persistence of T(CM) cells from EC subjects is a direct consequence of inactivation of the FOXO3a pathway. Silencing the transcriptionally active form of FOXO3a by small interfering RNA or by introducing a FOXO3a dominant-negative form (FOXO3a Nt) extended the long-term survival of T(CM) cells from ST subjects to a length of time similar to that of T(CM) cells from EC subjects. The crucial role of FOXO3a in the survival of memory cells will help shed light on the underlying immunological mechanisms that control viral replication in EC subjects.

J Exp Med. 2007 Jan 22;204(1):79-91. Epub 2006 Dec 26.

Convergence of TCR and cytokine signaling leads to FOXO3a phosphorylation and drives the survival of CD4+ central memory T cells.

Riou C, Yassine-Diab B, Van grevenynghe J, Somogyi R, Greller LD, Gagnon D, Gimmig S, Wilkinson P, Shi Y, Cameron MJ, Campos-Gonzalez R, Balderas RS, Kelvin D, Sekaly RP, Haddad EK.

Laboratoire d'Immunologie, Centre de Recherche, Hôpital Saint-Luc du Centre Hospitalier de l'Université de Montréal (CHUM), Montréal, Québec H2X 1P1, Canada.

The molecular events involved in the establishment and maintenance of CD4+ central memory and effector memory T cells (TCM and TEM, respectively) are poorly understood. In this study, we demonstrate that ex vivo isolated TCM are more resistant to both spontaneous and Fas-induced apoptosis than TEM and have an increased capacity to proliferate and persist in vitro. Using global gene expression profiling, single cell proteomics, and functional assays, we show that the survival of CD4+ TCM depends, at least in part, on the activation and phosphorylation of signal transducer and activator of transcription 5a (STAT5a) and forkhead box O3a (FOXO3a). TCM showed a significant increase in the levels of phosphorylation of STAT5a compared with TEM in response to both IL-2 (P<0.04) and IL-7 (P<0.002); the latter is well known for its capacity to enhance T cell survival. Moreover, ex vivo TCM express higher levels of the transcriptionally inactive phosphorylated forms of FOXO3a and concomitantly lower levels of the proapoptotic FOXO3a target, Bim. Experiments aimed at blocking FOXO3a phosphorylation confirmed the role of this phosphoprotein in protecting TCM from apoptosis. Our results provide, for the first time in humans, an insight into molecular mechanisms that could be responsible for the longevity and persistence of CD4+ TCM.

Genetic Associations with Control of HIV Replication

At the recent CROI meeting, David Goldstein from Duke University gave a plenary presentation on genetic determinants of viral load set point in HIV-infected individuals. Goldstein’s talk was an update on work published recently with CHAVI (Center for AIDS Vaccine Immunology) collaborator Amalio Telenti from the University of Lausanne. Their approach involved analyzing a staggering 500,000 different single nucleotide polymorphisms (SNPs) present in the human genome to see if they were associated with lower viral load set points (although Goldstein noted that this initial work is restricted to SNPs documented in >1% of the population and thus excludes rare polymorphisms). As reported in the Science paper, three sets of significant associations emerged. The first was a SNP in a gene called HCP5. This is linked to an immune response gene called HLA B*5701 which is well known to be over-represented among HIV-infected long-term non-progressors (HLA B*5701 makes a receptor on CD8 T cells which appears particularly good at recognizing HIV epitopes). However, HCP5 is also an endogenous retroviral element (a part of the human genome derived from an ancient retroviral infection which gained access the human germ line by infecting an egg or sperm cell) and so Goldstein initially speculated that the SNP in HCP5 might somehow have a direct anti-HIV effect. At CROI, he reported that studies in which the SNP-containing version of HCP5 was overexpressed in cells showed no inhibition of HIV replication, suggesting that the SNP is mediating its effect via other means (exactly how is under investigation).

The second association uncovered by Goldstein’s work was with a SNP in the gene for HLA-C. HLA-C molecules are part of a family called class I HLA molecules that are involved in the recognition of pathogens by CD8 T cells. Thousands of HLA molecules are displayed on the surface of every cell in the body (except red blood cells) and their job is to constantly lift cellular debris from inside the cell and display it to passing CD8 T cells. If an HLA molecule displays a protein fragment (epitope) from a pathogen that has infected the cell, this can trigger the CD8 T cell to release toxic enzymes, which cause the cell to die. The majority of HLA molecules on cells belong to classes HLA-A and HLA-B, while HLA-C molecules are less frequent and, as a result, less studied. But because HIV’s Nef protein is known to cause a reduction in HLA-B molecules on infected cells (as a means of escaping the immune response), Goldstein’s hypothesis is that the SNP he has identified causes more HLA-C molecules to be expressed, thereby making it easier for CD8 T cells to identify HIV-infected cells. He is currently collaborating with Andrew McMichael at Oxford University to measure the effect of the SNP on HLA-C expression.

The third association described by Goldstein involves a set of seven SNPs in or near two genes, ZNRD1 and RNF39. In the Science paper, this association was only seen with disease progression (as assessed by time to a CD4 T cell count of less than 350) but at CROI Goldstein reported that in an expanded analysis involving 1,000 more individuals (in addition to the 446 studied initially), an association with viral load set point was also documented. ZNRD1 encodes a protein involved in RNA transcription and, interestingly, was also identified in the recent, widely publicized study of host proteins needed by HIV to replicate. The function of RNF39 remains to be determined. Goldstein stressed that the discovery of all these associations is a first step, and efforts are underway to uncover the mechanisms by which the identified SNPs mediate their effects.

To give a sense of how a combination of genetic factors can have a profound impact on HIV disease progression, Goldstein showed an analysis that included the SNPs in the HCP5, HLA-C, and ZNRD1/RNF39 genes and two other known favorable genetic polymorphisms in CCR5 and CCR2 genes (CCR5Δ32 and CCR2 V64I). HIV-infected individuals with no favorable mutations in any of these genes showed an average time of less than two years from infection to a CD4 T cell count of less than 350. In contrast, people with one or two favorable mutations in at least four of these genes did not experience a CD4 T cell decline to this level for an average period of more than eight years.

Goldstein’s group is now embarking on an effort to uncover genetic associations with the magnitude of antibody responses generated against a vaccine, using data from the North American efficacy trial of AIDSVAX. This is a potentially important area of study because the ability of an individual to generate a high titer antibody response was correlated with reduced susceptibility to HIV infection in the trial cohort. Some researchers have suggested an analogy with the association between the magnitude of anti-Ad5 antibody responses and susceptibility to HIV infection seen in the placebo group of the recent Merck vaccine trial, so Goldstein’s work may have the potential shed light on that mystery also. Another important area of ongoing study mentioned by Goldstein is an analysis of genetic associations with viral load set point restricted to African American individuals.

The webcast of David Goldstein’s talk is available on the CROI website, it is the second presentation on Monday.

Shortly after CROI, a study of untreated HIV-infected individuals with viral loads under 50 copies – a group now dubbed “elite controllers” – highlighted the complexities and limitations of genetic associations. In a research letter published in the journal AIDS, Yefei Han and colleagues from Bob Siliciano’s laboratory at Johns Hopkins report that, of 16 elite controllers analyzed, only four possessed the SNP in HLA-C identified in the CHAVI work and none had the SNP in HCP5 (although two had HLA B*5702 and HLA B*5703 genes, which are closely related to HLA B*5701). The researchers did find the HCP5 SNP in two other infected individuals with HLA B*5701, one of whom had a low but detectable viral load and the other with progressive disease. The authors also note that the frequency of the HLA-C SNP in the elite controllers was not significantly different from what would be expected in a larger population of people from the same ethnic background. Citing these data and an example of an individual in their cohort who developed an increasing viral load after developing a CD8 T cell escape mutation, they argue that adaptive immune responses are likely an important contributor to the control of viral replication in elite controllers.

AIDS. 22(4):541-544, February 19, 2008.

The role of protective HCP5 and HLA-C associated polymorphisms in the control of HIV-1 replication in a subset of elite suppressors.

Research Letters

Han, Yefei; Lai, Jun; Barditch-Crovo, Patricia; Gallant, Joel E; Williams, Thomas M; Siliciano, Robert F; Blankson, Joel N

Abstract:

Elite suppressors (ES) are untreated HIV-1-infected patients who maintain undetectable viral loads. A recent whole-genome analysis identified two independent polymorphisms associated with low viral loads in untreated HIV-1 infection. We screened 16 ES; none were positive for the protective HLA complex 5 gene polymorphism, and only four were positive for the protective polymorphism associated with the HLA-C gene. These results suggest that some ES control viremia by mechanisms independent of the newly-identified genetic factors.