CD56^dim NK cells expressing NKG2A/CD94 but lacking KIR2DLs degranulate similarly to CD56^bright NK cells in response to autologous HIV-infected T-cells.

<p>(A) Degranulation of the CD56^dim NK cell subset from five different donors based on their expression of NKG2A/CD94 and/or KIR2DL in response to HIV-infected T-cells (five subjects A-E). The response of CD56^bright NK cells to the same target cells is also given. (B) Summary of the percent degranulation by CD56^bright NK cells and the various CD56^dim NK cell subpopulations in response to autologous HIV-infected T-cells from five different donors. Bars represent the mean of the CD107a responses of each NK cell subpopulation of the different donors. Gray error bars represent the standard deviation of the mean Statistical significance (p≤0.05) of the differences in responses was determined using the Wilcoxon-ranked sum test.</p

CD56^bright NK cells lyse autologous HIV-infected T-cells to a greater extent than CD56^dim NK cells.

<p><b>(A)</b> An example of an experiment (from data presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005421#ppat.1005421.g003" target="_blank">Fig 3B</a>) to determine if CD107a is expressed on NK cell subsets expressing relatively high (CD56^bright) or low (CD56^dim) levels of CD56 when exposed to K562 cells or HIV-infected T-cells. Bold numbers to the right of the figures are percent of CD56^bright (upper quadrants) and CD56^dim (lower quadrants) NK cells that express CD107a on their surface after 4-hour exposure to target cells. (<b>B)</b> Percent of CD107a positive CD56^dim (black bars) and CD56^bright (white bars) NK cells from five subjects after exposure to autologous HIV-infected cells. (<b>C)</b> Ability of unsorted NK cells, sorted CD56^dim NK cells and sorted CD56^bright NK cells from two different donors (subjects A and B) to degranulate in response to K562 cells or HIV-infected T-cells. The HLA genotype for subject A was A*0201/A*1101 B*0702 (HLA-Bw6) /B*5601 (HLA-Bw6) and Cw*0102 (C1)/Cw*0401(C2). The HLA genotype for subject B was A*2901/A*1101 B*4403 (HLA-Bw4)/B*5101 (HLA-Bw4) and Cw*1502 (C2)/Cw*1601 (C1). Numbers in each plot represent the percent of CD107a⁺ NK cells of unfractionated, sorted CD56^dim or sorted CD56^bright NK cells after exposure to target cells. (<b>D)</b> Percent specific lysis of HIV-1 infected T-cells by sorted CD56^dim (black bars) and sorted CD56^bright (white bars) NK cells from three donors cultured overnight without or with 200 U/ml of IL-2. Error bars represent standard deviations of percent specific lysis of three replicates. The HLA genotype for the first subject was A*0201/A*1101, B*0702 (HLA-Bw6) /B*5601 (HLA-Bw6) and Cw*0102 (C1) /Cw*0401 (C2). The HLA genotype for the second subject was A*0101/A*2901, B*0801 (HAL-Bw6)/B*4403 (HLA-Bw4) and Cw*0701 (C1)/Cw*1601(C1). The HLA genotype for the third subject was A*1101/A*3101, B*5101 (HLA-Bw4)/B*4001 (HLA-Bw6) and Cw*0304 (C1)/Cw*1402 (C1). Numbers over bars between columns indicate <i>p</i>-values based on Mann-Whitney U-tests. <i>p</i>≤0.05 was considered statistically significant.</p

HLA-C on HIV-infected T-cells prevents KIR2DL expressing NK cells from degranulating.

<p>(A) Expression of HLA-C on HIV-infected T-cells [HIV-1 p24 antigen positive] (blue line) and uninfected (red line) CD4⁺ T-cells, as determined by soluble KIR2DL-IgG fusion protein staining and staining control (secondary antibody alone–green line). This is an example of three separate experiments. (B) Degranulation of NK cells expressing or lacking KIR2DL1 and/or KIR2DL2/3 from six donors with different HLA-C genotypes when exposed to HIV-infected T-cells. In general, KIR2DL1 recognizes group 2 HLA-C alleles (C2), while KIR2DL2/3 recognize group 1 HLA-C (C1). Degranulation responses from donors lacking specific KIR2DL alleles are not shown. (C) Ability of NK cells lacking or expressing the various HLA-C specific inhibitory receptors to degranulate when exposed to HIV-infected T-cells. Each point represents a specific donor; bars represent the mean response of a given population of NK cells. Gray error bars represent the standard deviation of the mean. Degranulation in response to exposure to HIV-infected cells was significantly different (p<0.05; Wilcoxon ranked sum test) between NK cells lacking all KIR2DL inhibitory receptors and NK cells possessing the indicated KIR2DLs. (D) Contributions of KIR2DL1 and/or KIR2DL2/3 on the ability of NK cells to degranulate when exposed to HIV-infected cells were determined by pretreating NK cells with blocking antibodies against KIR2DL1 (anti-CD158a) and/or KIR2DL2/3 (anti-CD158b). The HLA-C allele and KIR2DLs expressed by each donor are given above each figure. KIR2DL1 recognizes HLA-C with a lysine at position 80 (C2) while KIR2DL2/3 predominantly recognizes HLA-C molecules with an asparagine at position 80 (C1) but also may recognize HLA-C2. Fab’ fragments of CD16 blocking antibodies (Fc block) were added to all samples. Each plot represents a different subject in which the frequency of 2 X 10⁴ NK cells expressing CD107a was determined after 4-hour incubation with purified autologous HIV-infected T-cells in the absence or presence of anti-KIR2DL blocking antibodies.</p

NK cells possessing NKG2A/CD94 degranulate in response to autologous HIV-infected T-cells despite HLA-E surface expression.

<p>(A) An example of the ability of NK cells possessing and lacking NKG2A/CD94 to degranulate following exposure to HIV-infected T-cells (from data presented in Fig 1B). As target cell controls, NK cells were exposed to K562 cells or uninfected CD4⁺ T-cells. NK cells ability to degranulate in the absence of target cells are also shown. Bold numbers are percent of NKG2A/CD94⁺ (upper quadrants) and NKG2A/CD94^- (lower quadrants) NK cells that express CD107a on their surface after 4-hour culture with or without target cells. (B) Ability of NK cells lacking (black dots) and expressing (white dots) NKG2A/CD94 from nine different donors to degranulate following exposure to HIV-infected cells. Statistical significance (p≤0.05) was determined using the Wilcoxon-ranked sum test. Black Bars represent the mean of the CD107a responses of each NK cell subpopulation of the different donors. Gray error bars represent the standard deviation of the mean. (C) NK cells, from seven different donors, which possess the HLA-E specific activation receptor NKG2C/CD94, degranulate to the same degree as NKG2C/CD94^- NK cells when exposed to autologous HIV-infected T-cells. Statistical significance (p≤0.05) was determined using the Wilcoxon-ranked sum test. Bars represent the mean of the CD107a responses of each NK cell subpopulation of the different donors. Gray error bars represent the standard deviation of the mean. (D) Expression of CD107a on NKG2A/CD94^- NK cells (black bars) and NKG2A/CD94⁺ NK cells (white bars) from five HLA-Bw4⁺ (subjects A-E), or four HLA-Bw6⁺/HLA-Bw6⁺ (subjects F-I) donors in response to HIV-infected T-cells. (E) Impact of anti-NKG2A blocking antibodies on degranulation of CD94+ NK cells when exposed to HIV-infected T-cells in the presence of Fab fragments of anti-CD16 blocking antibody. As a control, NK cells were treated with Fab fragments of anti-CD16 antibodies alone (absence of HIV-infected T-cells) and an antibody of similar isotype as anti-NKG2A (isotype control). Numbers above quadrants one and two designate the response (CD107a expression) of the CD94^- and CD94⁺ NK cells. (F) Effect of a point mutation in HIV-1 Gag on the ability of NKG2A- NK cells (black bars) and NKG2A⁺ (white bars) NK cells to degranulate when exposed to T-cells infected with HIV-1 with wild-type Gag (AISPRTLNA) or HIV-1 with an N to F mutation in position 153 of Gag (AISPRTLFA). This is an example of three experiments. (G) Purified NK cells stained with fluorochrome-conjugated HLA-E tetramer containing the leader peptide of HLA-G (VMAPRTLFL), wild type HIV-1 p24 capsid peptide (AISPRTLNA) or HIV-1 p24 capsid peptide with an asparagine to phenylalanine substitution (AISPRTLFA). As a control, the same purified NK cells were stained with antibody to NKG2A. Staining control consisted of NK cells stained with fluorochrome-conjugated IgG1. Dot Plots are examples of the data shown in the bottom panels. (H) Ability of HLA-E tetramer with peptide containing an N to F mutation in position 153 of Gag (AISPRTLFA) to interact with CD56^bright NK cells when blocking antibodies to NKG2A were present. As controls CD56^bright NK cells were stained with HLA-E tetramer with peptide containing the leader peptide of HLA-G (VMAPRTLFL) or wild type HIV-1 p24 capsid peptide (AISPRTLNA) in the presence and absence of blocking antibodies to NKG2A.</p

Patient Cohort from AIDS Clinical Trial Group Study 5192.

<p>Patient Cohort from AIDS Clinical Trial Group Study 5192.</p

CD4+ and CD8+ T cell counts.

<p>The CD4 Cell Count (A) and the CD8 Cell Count (B) are portrayed over the course of interferon-alpha treatment during the ACTG 5192 Study. Week 0 corresponds to the patients' baseline cell counts before beginning Interferon-Alpha Therapy. Weeks 3 and 12 correspond to 3 weeks and 12 weeks of interferon-alpha Therapy. Interferon-Alpha therapy was stopped at Week 12, and therefore Week 18 corresponds to 6 weeks off therapy.</p

Co-expression of CD38 and HLA-DR on CD4+ and CD8+ T cells.

<p>The percentage of CD4+ T cells (A) and CD8+ T cells (B) co-expressing CD38 and HLA-DR is shown over the course of interferon-alpha treatment during the ACTG 5192 Study. Week 0 corresponds to the patients' baseline cell counts before beginning Interferon-Alpha Therapy. Weeks 3 and 12 correspond to 3 weeks and 12 weeks of interferon-alpha Therapy. Interferon-Alpha therapy was stopped at Week 12, and therefore Week 18 corresponds to 6 weeks off therapy. The bar represents the median value.</p

Cell cycle status of CD4+ T cell maturation subsets.

<p>The cell cycle status of CD4+ T cell maturation subsets, Central Memory CD4 +T Cells (A), Naive CD4+ T cells (B), Transitional Memory CD4+ T cells (C), and Effector Memory CD4+ Tcells (D), is shown over the course of interferon-alpha treatment during the ACTG 5192 Study. Central memory cells were defined as CD 27+CD45RA− and CCR7+, whereas naïve cells are CD27+, CD45RA+, and CCR7+, transitional memory cells are CD27+, CD45RA−, and CCR7−, and effector memory cells are CD27−, CD45RA−, and CCR7−. Week 0 corresponds to the patients' baseline cell counts before beginning Interferon-Alpha Therapy. Weeks 3 and 12 correspond to 3 weeks and 12 weeks of interferon-alpha therapy. Interferon-Alpha therapy was stopped at Week 12, and therefore Week 18 corresponds to 6 weeks off therapy. The bar represents the median value.</p

Effect of the N1FR2011N-Vpu on CD4, CD1d and NTB-A.

<p>(<b>A, B</b>) The N1FR2011 Vpu does not interact with CD4 (<b>A</b>) or reduce its cells surface expression (<b>B</b>). 293T cells were transfected with plasmids expressing Vpu-N-Luc and CD4-C-Luc and interaction efficiencies were determined as described in the Experimental Procedures. (<b>C, D</b>) Vpu-dependent reduction of CD1d (<b>C</b>) and NTB-A (<b>D</b>) surface expression in 293T cells. Shown are the levels of receptor cell surface expression for group M (n = 3), N1FR2011, and Cameroonian N (n = 7) Vpus relative to those measured in cells transfected with the eGFP only control vector. Each allele was measured in triplicate and shown are averages ±SDs.</p

Functional characterization of Vpus from Cameroonian HIV-1 N strains.

<p>(<b>A</b>) FACS analysis of 293T cells cotransfected with tetherin, CD4, CD1d or NTB-A expression vectors and pCGCG plasmids expressing eGFP alone (lanes 2 and 3) or together with the indicated <i>vpu</i> allele (lanes 4 to 9). eGFP expression levels used to calculate receptor downmodulation and the mean fluorescence intensities (MFIs) are indicated. (<b>B</b>) Effect of various Vpus on infectious virus release. 293T cells were cotransfected with HIV-1 ΔVpu NL4-3 (2 µg), pCGCG vectors coexpressing eGFP and Vpu (500 ng), and a construct expressing HU tetherin (125 ng). Viral supernatants were obtained two days later and used to measure the quantity of infectious HIV-1 in the culture supernatants by infecting TZM-bl indicator cells. Shown is the infectious virion yield relative to that obtained in the absence of tetherin (100%). The results were confirmed in two independent experiments and each symbol indicates infectious virus yield in the presence of one of the 25 <i>vpu</i> alleles analyzed. (<b>C–F</b>) Vpu-dependent reduction of (<b>C</b>) tetherin, (<b>D</b>) CD4, (<b>E</b>) CD1d and (<b>F</b>) NTB-A surface expression in 293T cells. Shown is the reduction in the levels of receptor cell surface expression relative to those measured in cells transfected with the eGFP only control vector. Each symbol represents n-fold downmodulation of the indicated receptor molecule by one individual <i>vpu</i> allele examined. Shown are average values derived from three experiments. <i>Vpu</i> alleles derived from HIV-1 M are color coded red, N blue (except for the DJO0131 Vpu highlighted in green) and SIVcpz black; those shown in panel A are indicated by open symbols in panels B to F.</p