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1.  Natural Killer Cells: Tolerance to Self and Innate Immunity to Viral Infection and Malignancy 
Natural killer (NK) cells are lymphocytes whose ability to identify and kill virally infected and malignant cells while sparing normal cells was poorly understood until the late 1980’s and the introduction of the “missing self’ hypothesis. According to this hypothesis, downregulation of major histocompatibility complex (MHC) class I molecules during viral infection or malignant transformation triggers NK activation (1). Since this hypothesis was first proposed, much has been learned about NK cell surface receptors, their role in the molecular basis of missing-self recognition, and the mechanisms underlying NK cell tolerance. In this review, we will discuss these mechanisms, as well as their relevance to viral infection and tumor immunity and stem cell transplantation.
doi:10.1016/j.bbmt.2009.10.009
PMCID: PMC3900292  PMID: 19835969
2.  Temporal effect of HLA-B*57 on viral control during primary HIV-1 infection 
Retrovirology  2013;10:139.
Background
HLA-B alleles are associated with viral control in chronic HIV-1 infection, however, their role in primary HIV-1 disease is unclear. This study sought to determine the role of HLA-B alleles in viral control during the acute phase of HIV-1 infection and establishment of the early viral load set point (VLSP).
Findings
Individuals identified during primary HIV-1 infection were HLA class I typed and followed longitudinally. Associations between HLA-B alleles and HIV-1 viral replication during acute infection and VLSP were analyzed in untreated subjects. The results showed that neither HLA-B*57 nor HLA-B*27 were significantly associated with viral control during acute HIV-1 infection (Fiebig stage I-IV, n=171). HLA-B*57 was however significantly associated with a subsequent lower VLSP (p<0.001, n=135) with nearly 1 log10 less median viral load. Analysis of a known polymorphism at position 97 of HLA-B showed significant associations with both lower initial viral load (p<0.01) and lower VLSP (p<0.05). However, this association was dependent on different amino acids at this position for each endpoint.
Conclusions
The effect of HLA-B*57 on viral control is more pronounced during the later stages of primary HIV-1 infection, which suggests the underlying mechanism of control occurs at a critical period in the first several months after HIV-1 acquisition. The risk profile of polymorphisms at position 97 of HLA-B are more broadly associated with HIV-1 viral load during primary infection and may serve as a focal point in further studies of HLA-B function.
doi:10.1186/1742-4690-10-139
PMCID: PMC3874665  PMID: 24245727
HLA-B*57; HLA-B; Acute HIV-1 infection; Primary HIV-1 infection; Viral load set point; MHC class I
3.  Identification of HIV-1-specific regulatory T-cells using HLA class II tetramers 
AIDS (London, England)  2012;26(16):10.1097/QAD.0b013e328358cc75.
Regulatory T cells (Tregs) are potent immune modulators, but their precise role in HIV pathogenesis remains incompletely understood. Most studies to date have focused on frequencies or phenotypes of “bulk” Treg populations. However, although antigen-specific Tregs have been reported in other diseases, HIV-1-epitope specific Tregs have not been described to date. We here report the first identification of functional HIV-1-Gag-specific regulatory T cells using human leukocyte antigen class II tetramer staining in HIV-1-infected individuals.
doi:10.1097/QAD.0b013e328358cc75
PMCID: PMC3825094  PMID: 22874519
4.  Innate Immune Activation Enhances HIV Acquisition in Women, Diminishing the Effectiveness of Tenofovir Microbicide Gel 
The Journal of Infectious Diseases  2012;206(7):993-1001.
The antiretroviral agent, tenofovir, formulated as a vaginal microbicide gel, reduces human immunodeficiency virus (HIV) acquisition by 39% in women. This study assessed the role of preexisting immune activation in HIV acquisition in women from the CAPRISA 004 trial, to identify potential strategies to increase the effectiveness of tenofovir gel. Systemic cytokine and cellular immune mediators (platelets and natural killer [NK] cells) were assessed in women at high risk for HIV assigned to either tenofovir or placebo gel in the CAPRISA 004 trial. Notwithstanding tenofovir gel use, women who acquired HIV had significantly higher systemic innate immune activation prior to infection than women who remained uninfected. Activation of both soluble (cytokine) and cellular (NK cells) immune mediators were associated with HIV acquisition, individually or in combination. Hence, an innate immune activation suppressant could be added to tenofovir gel as a potential combination gel strategy in developing the next generation of higher efficacy antiretroviral microbicides.
doi:10.1093/infdis/jis465
PMCID: PMC3501691  PMID: 22829639
5.  Harnessing Innate and Adaptive Immunity for Viral Vaccine Design 
Expert review of vaccines  2012;11(7):775-777.
doi:10.1586/erv.12.48
PMCID: PMC3760238  PMID: 22913253
6.  Evasion from NK cell-mediated immune responses by HIV-1 
Human immunodeficiency virus type 1 (HIV-1) mostly owes its success to its ability to evade host immune responses. Understanding viral immune escape mechanisms is prerequisite to improve future HIV-1 vaccine design. This review focuses on the strategies that HIV-1 has evolved to evade recognition by natural killer (NK) cells.
doi:10.1016/j.micinf.2012.05.001
PMCID: PMC3432664  PMID: 22626930
HIV-1; NK cells; escape; innate immunity; vaccine
7.  Differential regulation of TLR pathways in acute and chronic HIV-1 infection 
AIDS (London, England)  2012;26(5):533-541.
Objective and design
The objective of this study was to determine changes in TLR responses of monocytes, myeloid dendritic cells and plasmacytoid dendritic cells during primary and chronic HIV-1 infection. Toll-like receptors (TLRs) serve as important innate receptors to sense pathogens, and have been implicated in mediating immune activation in HIV-1 infection. Studies assessing the consequences of HIV-1 infection on the ability of innate immune cells to respond to TLR stimulation have come to varying conclusions.
Methods
Using intracellular flow cytometry, cytokine production by cryopreserved PBMCs from healthy controls and HIV-1 infected individuals were examined after TLR stimulation.
Results
We observed that the effect of HIV-1 infection on TLR responses not only depended on the stage of HIV-1 infection, but was also dependent on the individual receptor and cell type examined. Monocyte and mDC responses to TLR8 stimulation were associated with HIV-1 viral load and CD4+ T cell count, while pDC responses to TLR7 stimulation were not. Responses to TLR2 stimulation were not affected by HIV-1 infection while responses to TLR9 stimulation were universally decreased in all HIV-1 infected individuals examined regardless of treatment or clinical parameters.
Conclusion
Responsiveness to TLR7/8 stimulation, which have been shown to recognize HIV-1 ssRNA, did not decrease in chronic infection, and may represent a contributing factor to ongoing T cell immune activation in the setting of chronic viremic HIV-1 infection.
doi:10.1097/QAD.0b013e32834f3167
PMCID: PMC3738004  PMID: 22210629
HIV-1; Toll-like receptor; Innate immunity; Pathogenesis; Dendritic Cells; Monocytes
9.  Reduced frequencies of NKp30+ NKp46+, CD161+, and NKG2D+ NK cells in acute HCV infection may predict viral clearance 
Journal of hepatology  2010;55(2):278-288.
Background & Aims
While the majority of HCV-infected patients progress to chronic hepatitis, a small fraction of individuals are able to clear the virus. Resolution of infection occurs within the first few weeks to months of infection, suggesting that innate immune functions may be critical for early control. Epidemiologic data support a role for particular NK cell receptor bearing populations in this control, yet the mechanism by which NK cells respond to HCV early in infection is unknown.
Methods
Changes in the phenotype and function of NK cells were investigated in a cohort of 43 individuals identified during various stages of HCV infection with different clinical outcomes.
Results
Acute, chronic, and resolved HCV infections were characterized by an expansion of CD56neg NK cells. Furthermore, increased levels of HLA-C-binding KIR+ NK cells were observed in HCV resolvers, while all stages of HCV infection were associated with reduced percentages of NKG2D+, NKp30+, and NKp46+ NK cells, and a slight increase in the ability of NK cells to respond to target cells bearing the ligands for these receptors. In contrast, NKG2A+ and CD94+ NK cells were elevated in acute and chronic HCV infection, but not in resolved infection. Most importantly, in acute infection, lower frequencies of NKp30+, NKp46+, CD161+, and NKG2D+ NK cells were observed in patients who were subsequently able to clear HCV infection than in those becoming chronically infected.
Conclusions
These data implicate particular populations of NK cells in the early control and clearance of HCV infection.
doi:10.1016/j.jhep.2010.11.030
PMCID: PMC3729214  PMID: 21168454
Hepatitis C virus; Natural killer cells; Innate immunity; CD161; NK-p30; NKp46; Resolution of HCV infection; Acute infection
10.  Dysregulated Tim-3 expression on natural killer cells is associated with increased Galectin-9 levels in HIV-1 infection 
Retrovirology  2013;10:74.
Background
Natural killer (NK) cells constitutively express high levels of Tim-3, an immunoregulatory molecule recently proposed to be a marker for mature and functional NK cells. Whether HIV-1 infection modulates the expression of Tim-3 on NK cells, or the levels of its ligand Galectin-9 (Gal-9), and how signaling through these molecules affects the NK cell response to HIV-1 remains inadequately understood.
Results
We analyzed Tim-3 and Gal-9 expression in a cohort of 85 individuals with early and chronic HIV-1 infection, and in 13 HIV-1 seronegative control subjects. HIV-1 infection was associated with reduced expression of Tim-3 on NK cells, which was normalized by HAART. Plasma concentrations of Gal-9 were higher in HIV-1-infected individuals than in healthy individuals. Interestingly, Gal-9 expression in immune cells was significantly elevated in early infection, with monocytes and dendritic cells displaying the highest expression levels, which correlated with HIV-1 viral loads. In vitro, Gal-9 triggered Tim-3 downregulation on NK cells as well as NK cell activation.
Conclusions
Our data suggest that high expression levels of Gal-9 during early HIV-1 infection can lead to enhanced NK cell activity, possibly allowing for improved early control of HIV-1. In contrast, persistent Gal-9 production might impair Tim-3 activity and contribute to NK cell dysfunction in chronic HIV-1 infection.
doi:10.1186/1742-4690-10-74
PMCID: PMC3750478  PMID: 23866914
Tim-3; Gal-9; HIV-1; Innate immunity; NK cells
11.  Rapid Evolution of HIV-1 to Functional CD8+ T-Cell Responses in Humanized BLT Mice 
Science translational medicine  2012;4(143):143ra98.
Important advancements in the development of novel mouse/human chimeras through the engraftment of human immune cells and tissues into immunodeficient mice, including the recently described humanized BLT mouse model, holds great promise to facilitate the in vivo study of human immune responses. However, little data exists regarding the extent to which cellular immune responses in humanized mice accurately reflect those seen in humans. As a model pathogen we infected humanized BLT mice with HIV-1 and characterized HIV-1-specific immune responses and viral evolution during the acute phase of infection. HIV-1-specific CD8+ T cell responses in these mice were found to closely resemble those in humans in terms of their specificity, kinetics and immunodominance. Viral sequence evolution also revealed rapid and highly reproducible escape from these responses, mirroring the adaptations to host immune pressures observed during natural HIV-1 infection. Moreover, mice expressing the protective HLA-B*57 allele exhibited enhanced control of viral replication, and restricted the same CD8+ T cell responses to conserved regions of HIV-1 Gag that are critical to its control of HIV-1 in humans. These data reveal that the humanized BLT mouse model appears to accurately recapitulate human pathogen-specific cellular immunity and the fundamental immunological mechanisms required to control a model human pathogen, aspects critical to the utility of a small animal model for human pathogens.
doi:10.1126/scitranslmed.3003984
PMCID: PMC3685142  PMID: 22814851
12.  Preservation HIV-1-specific IFNγ+ CD4+ T cell responses in breakthrough infections following exposure to Tenofovir Gel in the CAPRISA 004 microbicide trial 
The CAPRISA004 trial demonstrated reduction of sexual HIV-1 acquisition in women using a vaginal microbicide containing tenofovir. A better understanding of the consequences of antiretroviral-containing microbicides for immune responses in individuals with intercurrent HIV-1 infection is needed for future trials combining the use of microbicides with HIV-1 vaccines. Investigation of immune responses in women who acquired HIV-1 whilst using tenofovir gel showed significantly higher (p=0.01) Gag-specific IFNγ+ CD4+ T-cell responses. The use of tenofovir containing gel around the time of infection can modulate HIV-1 immunity, and these immunological changes need to be considered in future trials combining vaccines and microbicides.
doi:10.1097/QAI.0b013e31824f53a9
PMCID: PMC3381429  PMID: 22362152
HIV-1; vaginal microbicide; tenofovir; HIV-1-specific CD4+ T cell help
13.  Preserved Function of Regulatory T Cells in Chronic HIV-1 Infection Despite Decreased Numbers in Blood and Tissue 
The Journal of Infectious Diseases  2012;205(10):1495-1500.
Regulatory T cells (Tregs) are potent immune modulators, but their role in human immunodeficiency virus type 1 (HIV-1) pathogenesis remains poorly understood. We performed a detailed analysis of the frequency and function of Tregs in a large cohort of HIV-1–infected individuals and HIV-1 negative controls. While HIV “elite controllers” and uninfected individuals had similar Treg numbers and frequencies, the absolute numbers of Tregs declined in blood and gut-associated lymphoid tissue in patients with chronic progressive HIV-1 infection. Despite quantitative changes in Tregs, HIV-1 infection was not associated with an impairment of ex vivo suppressive function of flow-sorted Tregs in both HIV controllers and untreated chronic progressors.
doi:10.1093/infdis/jis236
PMCID: PMC3415814  PMID: 22427677
14.  Preferential CTL targeting of Gag is associated with relative viral control in long-term surviving HIV-1 infected former plasma donors from China 
Cell Research  2012;22(5):903-914.
It is generally believed that CD8+ cytotoxic T lymphocytes (CTLs) play a critical role in limiting the replication of human immunodeficiency virus type 1 (HIV-1) and in determining the outcome of the infection, and this effect may partly depend on which HIV product is preferentially targeted. To address the correlation between HIV-1-specific CTL responses and virus replication in a cohort of former plasma donors (FPDs), 143 antiretroviral therapy naive FPDs infected with HIV-1 clade B' strains were assessed for HIV-1-specific CTL responses with an IFN-γ Elispot assay at single peptide level by using overlapping peptides (OLPs) covering the whole consensus clade B proteome. By using a Spearman's rank correlation analysis, we found that the proportion of Gag-specific CTL responses among the total virus-specific CTL activity was inversely correlated with viral loads while being positively correlated to CD4 counts, as opposed to Pol- and Env-specific responses that were associated with increased viral loads and decreased CD4 counts. In addition, Vpr-specifc CTL responses showed a similar protective effect with Gag responses, but with a much lower frequency of recognition. Significantly, we also observed an association between HLA-A*30/B*13/Cw*06 haplotype and lower viral loads that was probably due to restricted Gag-specific CTL responses. Thus, our data demonstrate the prominent role of Gag-specific CTL responses in disease control. The advantage of HLA-A*30/B*13/Cw*06 haplotype in viral control may be associated with the contribution of Gag-specific CTL responses in the studied individuals.
doi:10.1038/cr.2012.19
PMCID: PMC3343654  PMID: 22290423
human immunodeficiency virus type 1; cytotoxic T lymphocytes; human leukocyte antigen class I; Gag
15.  Changes in Natural Killer Cell Activation and Function during Primary HIV-1 Infection 
PLoS ONE  2013;8(1):e53251.
Background
Recent reports suggest that Natural Killer (NK) cells may modulate pathogenesis of primary HIV-1 infection. However, HIV dysregulates NK-cell responses. We dissected this bi-directional relationship to understand how HIV impacts NK-cell responses during primary HIV-1 infection.
Methodology/Principal Findings
Paired samples from 41 high-risk, initially HIV-uninfected CAPRISA004 participants were analysed prior to HIV acquisition, and during viraemic primary HIV-1 infection. At the time of sampling post-infection five women were seronegative, 11 women were serodiscordant, and 25 women were seropositive by HIV-1 rapid immunoassay. Flow cytometry was used to measure NK and T-cell activation, NK-cell receptor expression, cytotoxic and cytokine-secretory functions, and trafficking marker expression (CCR7, α4β7). Non-parametric statistical tests were used. Both NK cells and T-cells were significantly activated following HIV acquisition (p = 0.03 and p<0.0001, respectively), but correlation between NK-cell and T-cell activation was uncoupled following infection (pre-infection r = 0.68;p<0.0001; post-infection, during primary infection r = 0.074;p = 0.09). Nonetheless, during primary infection NK-cell and T-cell activation correlated with HIV viral load (r = 0.32'p = 0.04 and r = 0.35;p = 0.02, respectively). The frequency of Killer Immunoglobulin-like Receptor-expressing (KIRpos) NK cells increased following HIV acquisition (p = 0.006), and KIRpos NK cells were less activated than KIRneg NK cells amongst individuals sampled while seronegative or serodiscordant (p = 0.001;p<0.0001 respectively). During HIV-1 infection, cytotoxic NK cell responses evaluated after IL-2 stimulation alone, or after co-culture with 721 cells, were impaired (p = 0.006 and p = 0.002, respectively). However, NK-cell IFN-y secretory function was not significantly altered. The frequency of CCR7+ NK cells was elevated during primary infection, particularly at early time-points (p<0.0001).
Conclusions/Significance
Analyses of immune cells before and after HIV infection revealed an increase in both NK-cell activation and KIR expression, but reduced cytotoxicity during acute infection. The increase in frequency of NK cells able to traffic to lymph nodes following HIV infection suggests that these cells may play a role in events in secondary lymphoid tissue.
doi:10.1371/journal.pone.0053251
PMCID: PMC3541400  PMID: 23326405
16.  Induction of Strong HIV-1-specific CD4+ T Cell Responses using an HIV-1 gp120/NefTat Vaccine adjuvanted with AS02A in ARV Treated HIV-1-Infected Individuals 
Background
Induction of HIV-1-specific CD4+ T cell responses by therapeutic vaccination represents an attractive intervention to potentially increase immune control of HIV-1.
Methods
We performed a double-blinded, randomized, placebo-controlled clinical trial to determine the safety and immunogenicity of GSK Biologicals' HIV-1 gp120/NefTat subunit protein vaccine formulated with the AS02A adjuvant in subjects with well controlled chronic HIV-1 infection on HAART. Ten individuals received the vaccine; while adjuvant alone or placebo was given to five subjects each. Immunogenicity was monitored by intracellular cytokine flow cytometry and CFSE-based proliferation assays.
Results
The vaccine was well tolerated with no related SAEs. Vaccine recipients had significantly stronger gp120-specific CD4+ T cell responses which persisted until week 48 and greater gp120-specific CD4+ T cell proliferation activity as compared to controls. In the vaccine group, the number of participants that demonstrated positive responses for both gp120-specific CD4+ T cell IL-2 production and gp120-specific CD8+ T cell proliferation was significantly higher at week 6.
Conclusions
The gp120/NefTat/AS02A vaccine induced strong gp120-specific CD4+ T cell responses, and a higher number of vaccinees developed both HIV-1-specific CD4+ T cell responses and CD8+ T cell proliferation. The induction of these responses may be important in enhancing immune-mediated viral control.
doi:10.1097/QAI.0b013e3182373b77
PMCID: PMC3241906  PMID: 21963936
HIV-1; Vaccination; Therapeutic Vaccination; HIV-1-specific CD4+ cells
17.  Frequent and Strong Antibody-Mediated Natural Killer Cell Activation in Response to HIV-1 Env in Individuals with Chronic HIV-1 Infection 
Journal of Virology  2012;86(12):6986-6993.
Natural killer (NK) cells play a critical role in the control of HIV-1 infection, and NK cells that respond to HIV-1 peptides have been recently described. However, the mechanisms by which NK cells recognize HIV-1 antigens are not fully understood. We investigated NK cell activation in response to HIV-1 peptides during early and chronic HIV-1 clade B infection using a whole-blood assay and multiparameter flow cytometry. Antibody-mediated NK cell activation in response to HIV-1 peptides was not detected in HIV-1-uninfected individuals. In contrast, 79% of individuals with chronic infection and 22% of individuals with early infection had detectable gamma interferon (IFN-γ) NK cell responses to HIV-1 antigens (P < 0.00001). IFN-γ- and tumor necrosis factor alpha (TNF-α)-producing NK cells most frequently targeted Env gp120 (median of 4% and range of 0 to 31% of all NK cells). NK cells rarely targeted other HIV-1 proteins such as Gag, Pol, and Nef. Antibody-mediated NK cell responses to peptides mapped predominantly to Env protein, required the presence of plasma or plasma IgG, and resulted in lower CD16 expression on NK cells, suggesting an antibody-mediated activation of NK cells. Further studies are needed to assess the consequences of these antibody-mediated NK cell responses for HIV-1 disease progression and vaccine-induced protection from infection.
doi:10.1128/JVI.00569-12
PMCID: PMC3393568  PMID: 22496218
18.  Limited Immunogenicity of HIV CD8+ T-Cell Epitopes in Acute Clade C Virus Infection 
The Journal of Infectious Diseases  2011;204(5):768-776.
Background. Human immunodeficiency virus type 1 (HIV-1)–specific CD8+ responses contribute to the decline in acute peak viremia following infection. However, data on the relative immunogenicity of CD8+ T-cell epitopes during and after acute viremia are lacking.
Methods. We characterized CD8+ T-cell responses in 20 acutely infected, antiretroviral-naive individuals with HIV-1 subtype C infection using the interferon-γ enzyme-linked immunosorbent spot assay. Eleven of these had not fully seroconverted at the time of analysis. Viruses from plasma were sequenced within defined cytotoxic T-lymphocyte (CTL) cell epitopes for selected subjects.
Results. At approximately 28 days after estimated initial infection, CD8+ T-cell responses were directed against an average of 3 of the 410 peptides tested (range, 0–6); 2 individuals had no detectable responses at this time. At 18 weeks, the average number of peptides targeted had increased to 5 (range 0–11). Of the 56 optimal Gag CTL epitopes sequenced, 31 were wild-type in the infecting viruses, but only 11 of 31 elicited measurable CD8+ T-cell responses.
Conclusions. These data demonstrate that the majority of CD8+ responses are not elicited during acute HIV infection despite the presence of the cognate epitope in the infecting strain. There is a need to define factors that influence lack of induction of effective immune responses and the parameters that dictate immunodominance in acute infection.
doi:10.1093/infdis/jir394
PMCID: PMC3156105  PMID: 21844303
19.  Short Communication: CD8+ T Cell Polyfunctionality Profiles in Progressive and Nonprogressive Pediatric HIV Type 1 Infection 
AIDS Research and Human Retroviruses  2011;27(9):1005-1012.
Abstract
Pediatric HIV-1 infection is characterized by rapid disease progression and without antiretroviral therapy (ART), more than 50% of infected children die by the age of 2 years. However, a small subset of infected children progresses slowly to disease in the absence of ART. This study aimed to identify functional characteristics of HIV-1-specific T cell responses that distinguish children with rapid and slow disease progression. Fifteen perinatally HIV-infected children (eight rapid and seven slow progressors) were longitudinally studied to monitor T cell polyfunctionality. HIV-1-specific interferon (IFN)-γ+ CD8+ T cell responses gradually increased over time but did not differ between slow and rapid progressors. However, polyfunctional HIV-1-specific CD8+ T cell responses, as assessed by the expression of four functions (IFN-γ, CD107a, TNF-α, MIP-1β), were higher in slow compared to rapid progressors (p=0.05) early in infection, and was associated with slower subsequent disease progression. These data suggest that the quality of the HIV-specific CD8+ T cell response is associated with the control of disease in children as has been shown in adult infection.
doi:10.1089/aid.2010.0227
PMCID: PMC3332389  PMID: 21288139
20.  HLA-Cw*0102-Restricted HIV-1 p24 Epitope Variants Can Modulate the Binding of the Inhibitory KIR2DL2 Receptor and Primary NK Cell Function 
PLoS Pathogens  2012;8(7):e1002805.
Accumulating evidence suggests an important role for Natural Killer (NK) cells in the control of HIV-1 infection. Recently, it was shown that NK cell-mediated immune pressure can result in the selection of HIV-1 escape mutations. A potential mechanism for this NK cell escape is the selection of HLA class I-presented HIV-1 epitopes that allow for the engagement of inhibitory killer cell immunoglobulin-like receptors (KIRs), notably KIR2DL2. We therefore investigated the consequences of sequence variations within HLA-Cw*0102-restricted epitopes on the interaction of HLA-Cw*0102 with KIR2DL2 using a large panel of overlapping HIV-1 p24 Gag peptides. 217 decameric peptides spanning the HIV-1 p24 Gag consensus sequence were screened for HLA-Cw*0102 stabilization by co-incubation with Cw*0102(+)/TAP-deficient T2 cells using a flow cytometry-based assay. KIR2DL2 binding was assessed using a KIR2DL2-IgG fusion construct. Function of KIR2DL2(+) NK cells was flow cytometrically analyzed by measuring degranulation of primary NK cells after co-incubation with peptide-pulsed T2 cells. We identified 11 peptides stabilizing HLA-Cw*0102 on the surface of T2 cells. However, only one peptide (p24 Gag209–218 AAEWDRLHPV) allowed for binding of KIR2DL2. Notably, functional analysis showed a significant inhibition of KIR2DL2(+) NK cells in the presence of p24 Gag209–218-pulsed T2 cells, while degranulation of KIR2DL2(−) NK cells was not affected. Moreover, we demonstrated that sequence variations in position 7 of this epitope observed frequently in naturally occurring HIV-1 sequences can modulate binding to KIR2DL2. Our results show that the majority of HIV-1 p24 Gag peptides stabilizing HLA-Cw*0102 do not allow for binding of KIR2DL2, but identified one HLA-Cw*0102-presented peptide (p24 Gag209–218) that was recognized by the inhibitory NK cell receptor KIR2DL2 leading to functional inhibition of KIR2DL2-expressing NK cells. Engagement of KIR2DL2 might protect virus-infected cells from NK cell-mediated lysis and selections of sequence polymorphisms that increase avidity to KIR2DL2 might provide a mechanism for HIV-1 to escape NK cell-mediated immune pressure.
Author Summary
Distinguishing between “self” and “non-self” is one of the fundamental principles of immune responses against viral infections. Upon viral infection the peptide repertoire presented by HLA class I molecules changes, potentially providing signals that result in recognition and elimination of the infected cell by the host immune system. Viruses, in particular HIV-1, developed multiple strategies to escape T cell and Natural Killer (NK) cell-mediated immune pressure, including sequence variations that lead to the engagement of inhibitory receptors expressed on T cells and NK cells. The systematic approach used in this study led to the identification of an HLA-presented HIV-1 peptide that allows engagement of the inhibitory NK cell receptor KIR2DL2 and inhibition of NK function. Our findings help to elucidate the complex interaction between KIR molecules, such as KIR2DL2, and HLA/peptide complexes and provide a foundation for further studies investigating the role of sequence variations within HIV-1 epitopes on HLA/KIR interactions, and the ability of viruses to evade NK cell-mediated recognition.
doi:10.1371/journal.ppat.1002805
PMCID: PMC3395618  PMID: 22807681
21.  Impairment of CD1d-Restricted Natural Killer T Cells in Chronic HIV Type 1 Clade C Infection 
Abstract
Recent studies suggest that natural killer T (NKT) cells play a role in early antiviral pathogenesis and are rapidly depleted in chronic human immunodeficiency virus type 1 (HIV-1) clade B infection. We aimed to characterize the phenotypic and functional characteristics of NKT cells in HIV-1 clade C-infected Africans at different stages of HIV-1 disease. NKT cell frequencies, subsets, and ex vivo effector functions were assessed using multiparametric flow cytometry in a cross-sectional analysis of cryopreserved peripheral blood mononuclear cells from a cohort of 53 HIV-1 clade C chronically infected South African adults with CD4 T cell counts ranging from 94 to 839 cells/μl. We observed a significant decline of NKT cell numbers in advanced HIV-1 disease as well as activation and functional impairment of NKT cells in individuals with low CD4 T cell counts. The loss of NKT cells was largely driven by a reduction in the CD4+ and CD4–CD8– NKT cell subsets in advanced disease. These findings demonstrate significant impairment of the NKT cell compartment in progressive HIV-1 clade C disease that might play an important role in the modulation of immune function in HIV-1 infection.
doi:10.1089/aid.2010.0237
PMCID: PMC3083726  PMID: 20942750
22.  Mutiny or Scrutiny: NK cell modulation of DC function in HIV-1 infection 
Trends in immunology  2011;32(5):219-224.
Accumulating data suggests that Natural Killer (NK) cells are not only involved in the innate [ET1]antiviral response following infection, but are also intimately involved in shaping the quality of the adaptive immune response by modulating the functional properties of myeloid Dendritic Cells (DC) during the acute immune response to infection. In this role, NK cells ensure that only fully maturated, immunogenic DCs gain access to inductive sites, where they might prime effective antiviral adaptive immune responses. However, increasing evidence now suggests that several aspects of this crosstalk between NK cells and DCs are compromised during HIV infection, potentially contributing to immune dysfunction.
doi:10.1016/j.it.2011.02.003
PMCID: PMC3090508  PMID: 21411368
24.  Whole Genome Deep Sequencing of HIV-1 Reveals the Impact of Early Minor Variants Upon Immune Recognition During Acute Infection 
PLoS Pathogens  2012;8(3):e1002529.
Deep sequencing technologies have the potential to transform the study of highly variable viral pathogens by providing a rapid and cost-effective approach to sensitively characterize rapidly evolving viral quasispecies. Here, we report on a high-throughput whole HIV-1 genome deep sequencing platform that combines 454 pyrosequencing with novel assembly and variant detection algorithms. In one subject we combined these genetic data with detailed immunological analyses to comprehensively evaluate viral evolution and immune escape during the acute phase of HIV-1 infection. The majority of early, low frequency mutations represented viral adaptation to host CD8+ T cell responses, evidence of strong immune selection pressure occurring during the early decline from peak viremia. CD8+ T cell responses capable of recognizing these low frequency escape variants coincided with the selection and evolution of more effective secondary HLA-anchor escape mutations. Frequent, and in some cases rapid, reversion of transmitted mutations was also observed across the viral genome. When located within restricted CD8 epitopes these low frequency reverting mutations were sufficient to prime de novo responses to these epitopes, again illustrating the capacity of the immune response to recognize and respond to low frequency variants. More importantly, rapid viral escape from the most immunodominant CD8+ T cell responses coincided with plateauing of the initial viral load decline in this subject, suggestive of a potential link between maintenance of effective, dominant CD8 responses and the degree of early viremia reduction. We conclude that the early control of HIV-1 replication by immunodominant CD8+ T cell responses may be substantially influenced by rapid, low frequency viral adaptations not detected by conventional sequencing approaches, which warrants further investigation. These data support the critical need for vaccine-induced CD8+ T cell responses to target more highly constrained regions of the virus in order to ensure the maintenance of immunodominant CD8 responses and the sustained decline of early viremia.
Author Summary
The ability of HIV-1 and other highly variable pathogens to rapidly mutate to escape vaccine-induced immune responses represents a major hurdle to the development of effective vaccines to these highly persistent pathogens. Application of next-generation or deep sequencing technologies to the study of host pathogens could significantly improve our understanding of the mechanisms by which these pathogens subvert host immunity, and aid in the development of novel vaccines and therapeutics. Here, we developed a 454 deep sequencing approach to enable the sensitive detection of low-frequency viral variants across the entire HIV-1 genome. When applied to the acute phase of HIV-1 infection we observed that the majority of early, low frequency mutations represented viral adaptations to host cellular immune responses, evidence of strong host immunity developing during the early decline of peak viral load. Rapid viral escape from the most dominant immune responses however correlated with loss of this initial viral control, suggestive of the importance of mounting immune responses against more conserved regions of the virus. These data provide a greater understanding of the early evolutionary events subverting the ability of host immune responses to control early HIV-1 replication, yielding important insight into the design of more effective vaccine strategies.
doi:10.1371/journal.ppat.1002529
PMCID: PMC3297584  PMID: 22412369
25.  Impact of blood processing variations on Natural Killer cell frequency, activation, chemokine receptor expression and function 
Journal of immunological methods  2011;366(1-2):28-35.
Understanding the role of natural killer (NK) cells in human disease pathogenesis is crucial and necessitates study of patient samples directly ex vivo. Manipulation of whole blood by density gradient centrifugation or delays in sample processing due to shipping, however, may lead to artifactual changes in immune response measures. Here, we assessed the impact of density gradient centrifugation and delayed processing of both whole blood and peripheral blood mononuclear cells (PBMC) at multiple timepoints (2–24 hrs) on flow cytometric measures of NK cell frequency, activation status, chemokine receptor expression, and effector functions. We found that density gradient centrifugation activated NK cells and modified chemokine receptor expression. Delays in processing beyond 8 hours activated NK cells in PBMC but not in whole blood. Likewise, processing delays decreased chemokine receptor (CCR4 and CCR7) expression in both PBMC and whole blood. Finally, delays in processing PBMC were associated with a decreased ability of NK cells to degranulate (as measured by CD107a expression) or secrete cytokines (IFN-γ and TNF-α). In summary, our findings suggest that density gradient centrifugation and delayed processing of PBMC can alter measures of clinically relevant NK cell characteristics including effector functions; and therefore should be taken into account in designing clinical research studies.
doi:10.1016/j.jim.2011.01.001
PMCID: PMC3051017  PMID: 21255578
Natural Killer cells; flow cytometry; activation; delayed processing; PBMC; whole blood; ficoll; chemokine receptor

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