klrg1 rabbit mab  (R&D Systems)

Journal: Cell Reports Medicine

Article Title: KLRG1 expression on natural killer cells is associated with HIV persistence, and its targeting promotes the reduction of the viral reservoir

doi: 10.1016/j.xcrm.2023.101202

Figure Lengend Snippet: Expression of KLRG1 in NK cells during HIV infection The expression of the KLRG1 receptor was measured by flow cytometry in different subjects. (A) Opt-SNE plots showing the distribution of KLRG1 + NK cells within the total pool of CD56 + NK cells in HIV-negative donors (HD) and people with HIV (HIV + ). The Volcano plot shows the difference in KLRG1 expression between both cohorts. (B) Frequency of KLRG1 + NK cells in a cohort of HD (n = 14) and PWH (n = 40). (C) Percentage of KLRG1 + NK in CD56 dim NK cells in HD and in HIV + with detectable viral loads (VIR) or on antiretroviral treatment with suppressed viremia (ART). (D) Percentage of KLRG1 + NK in CD56 bright NK cells in HD and HIV + in VIR or ART subjects. For (B), (C), and (D), median with range is represented. Statistical comparisons were performed using the Mann-Whitney test. ∗p < 0.05; ∗∗p < 0.01. (E–H) Spearman correlations between the percentage of KLRG1 + NK cells and (E) absolute CD4 + T cell count, (F) viral load during untreated infection (n = 16) (dot in parentheses was not included for the statistical analysis), (G) HIV RNA molecules per million CD4 + T cells during ART (n = 13), or (H) HIV DNA molecules per million CD4 + T cells during ART (n = 13) are represented. (I) Representative micrograph of a lymph node section from one HIV + VIR individual stained with anti-CD56 (purple), anti-KLRG1 (yellow), and anti-p24 (red) antibodies. White boxes indicate regions where NK cells show expression of both CD56 and KLRG1. The right panels correspond to zoomed views of CD56 + KLRG1 + cells. (J) Graph showing the number of KLRG1 + cells per mm 2 in the B and T zones from uninfected, ART-suppressed, and VIR lymph node samples. B and T cell zones were identified based on the intensity of the DAPI staining and the morphology of the cell nuclei. (K) Frequency of KLRG1 + cells within CD56 + NK cells in the lymph nodes from an HD, a VIR (#42), and an ART-suppressed (#41) PWH. Each dot corresponds to one B cell follicle. The percentage of NK cells expressing KLRG1 in the B cell zone or the intrafollicular region and in the T zone or the extrafollicular region was represented in orange and blue dots, respectively. Median and min-max rank is represented. Intra-sample comparisons between values inside or outside the B cell follicles from HD, VIR, and ART were performed using the Wilcoxon test, and the inter-sample comparisons between PWH and HD were performed using the Mann-Whitney test. See also Figures S1 and and Table S1 .

Article Snippet: Rabbit-polyclonal anti-human KLRG1 , Abcam , Cat#ab235951.

Techniques: Expressing, Infection, Flow Cytometry, MANN-WHITNEY, Cell Counting, Staining

Journal: Cell Reports Medicine

Article Title: KLRG1 expression on natural killer cells is associated with HIV persistence, and its targeting promotes the reduction of the viral reservoir

doi: 10.1016/j.xcrm.2023.101202

Figure Lengend Snippet: Characterization of NK cells expressing KLRG1 in HIV infection The expression of different NK markers was quantified by flow cytometry in different subjects: healthy donors (HD) n = 14, HIV + viremic (VIR) n = 17, and HIV + ART-treated individuals (ART) n = 23. (A) Volcano plot showing the statistically significant differences in the cluster composition between cohorts HIV + and HD. (B) Reduction of dimensionality analysis of the expression of different receptors in total (CD56 + ) NK cells. (C) Heatmap showing the differences in the mean fluorescence expression of the different NK cell receptors, in HD, VIR and ART cohorts, regarding the different cell clusters identified in Figure 2 B and represented in different colors. (D) Volcano plots showing the statistically significant differences in the cluster composition between cohorts. (E–L) Percentage of expression of different receptors in KLRG1 + and KLRG1 – NK cells. All graphs represent the median and ranges. Statistical comparisons intra-cohort were performed using the Wilcoxon matched-pairs signed-rank test, and comparisons inter-cohort used the Mann-Whitney test. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. See also Figures S1 , , and and Table S1 .

Article Snippet: Rabbit-polyclonal anti-human KLRG1 , Abcam , Cat#ab235951.

Techniques: Expressing, Infection, Flow Cytometry, Fluorescence, MANN-WHITNEY

Journal: Cell Reports Medicine

Article Title: KLRG1 expression on natural killer cells is associated with HIV persistence, and its targeting promotes the reduction of the viral reservoir

doi: 10.1016/j.xcrm.2023.101202

Figure Lengend Snippet: Functional profile of KLRG1 + NK cells in ART-suppressed PWH MHC-I-devoid K562 cells were subjected to NK-killing assays (n = 10). Functional markers were measured by flow cytometry under different conditions. (A) Reduction of dimensionality analysis for the expression of KLRG1, CD57, CD16, NKG2C, HLA-DR, CD69, IFN-γ, and CD107a in total (CD56 + ) NK cells. Three conditions are represented: NK alone, NK + K562, and NK (IL15) + K562. (B) Violin graphs showing the frequency of the different clusters of NK cells identified in (A). Statistical comparisons were performed using the Kruskal-Wallis test when required. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. (C) Expression of CD107a and IFN-γ on the different clusters identified in (A). (D) Expression of CD69 and HLA-DR on the different clusters identified in (A). (E) Expression of KLRG1 on the different clusters of NK cells identified in (A). (F) Heatmap showing the intensity of expression of the different markers on the NK clusters represented in different colors. (G–I) Percentage (G) of HLA-DR + cells in total CD56 + KLRG1 + (yellow) or KLRG1 – (blue) NK cells, after co-culturing with the K562 target cells and with the additional IL-15 stimulus. Similarly, the values of other parameters are represented in (H) frequency of CD69 + cells and (I) percentage of cells expressing the CD107a degranulation marker. (J–M) Mean fluorescence intensity (MFI intensity) signal (J) for CD107a expression, (K) frequency of cells producing IFN-γ, (L) MFI for IFN-γ, and (M) percentage of polyfunctional, double-positive CD107a + IFN-γ + cells. Statistical comparisons were performed using Wilcoxon matched-pairs signed-rank test. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. See also Figures S4 and and Table S1 .

Article Snippet: Rabbit-polyclonal anti-human KLRG1 , Abcam , Cat#ab235951.

Techniques: Functional Assay, Flow Cytometry, Expressing, Marker, Fluorescence

Journal: Cell Reports Medicine

Article Title: KLRG1 expression on natural killer cells is associated with HIV persistence, and its targeting promotes the reduction of the viral reservoir

doi: 10.1016/j.xcrm.2023.101202

Figure Lengend Snippet: Targeting KLRG1 increases the functional capabilities of NK cells NK cells were cultured with ex vivo infected CD4 + T cells after stimulation with the anti-KLRG1 antibody. Functional parameters on NK cells and direct cell killing of the HIV + targets were quantified by flow cytometry. (A–D) Frequency (A) of IFN-γ + cells in NK KLRG1 + cells, (B) frequency of IFN-γ + cells in NK KLRG1 – cells, (C) mean fluorescence intensity (MFI) of CD107a in KLRG1 + cells, and (D) MFI of CD107a in KLRG1 – cells. (E) Reduction in p24 + after co-culturing ex vivo HIV-infected CD4 + T cells with NK cells previously stimulated with the anti-KLRG1 antibody. Fc blocker is shown as a control. (F) Expression of the molecule E-cadherin on CD4 + T cells in infected (p24 + ) and uninfected (p24 – ) cells after ex vivo HIV infection. (G) Natural cytotoxicity assay of ex vivo HIV-infected CD4 + T cells after co-culturing with isolated NK expressing or not the KLRG1 and stimulated with the anti-KLRG1 antibody. (H) ADCC assay of ex vivo HIV-infected CD4 + T cells after co-culturing with isolated NK expressing or not the KLRG1 and stimulated with the anti-KLRG1 antibody. The condition with plasma from an HIV-positive person is shown as a control. In (G) and (H), the CD4 + T cells and NK cells were isolated by FACS. (I) P24 + cells in samples from ART-suppressed PWH at baseline and after viral reactivation with PMA/ionomycin (LRA). The limit of detection is set up at 53 copies/million cells calculated by the formula 3∗SD of the mean percentage of p24 + cells detected in healthy donor (HD) samples. (J) Functional NK assays after reactivation of the natural latent HIV reservoir of isolated CD4 + T cells from ART-treated PWH. P24 + cells are quantified by flow cytometry as a measure of viral reactivation. This panel shows an example of the p24 staining. (K) Functional NK assays after reactivation of the natural latent HIV reservoir of isolated CD4 + T cells from ART-treated PWH. Summary graph for p24 levels in CD4 + T cells after the NK-killing assays. Palivizumab was used as a control antibody. (L) Intact proviral DNA assay (IPDA) in samples from six ART-suppressed subjects after performing the NK-killing assay with primary ex vivo reactivated CD4 + T cells with PMA and ionomycin and isolated NK cells. NK cells were treated with the anti-KLRG1 antibody or the isotype control IgG2. Percentages of intact proviral reductions are shown for each subject. Statistical comparisons were performed using the Wilcoxon matched-pairs signed-rank test, Friedman test, or Kruskal-Wallis test when required. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. See also Figure S6 and Table S1 .

Article Snippet: Rabbit-polyclonal anti-human KLRG1 , Abcam , Cat#ab235951.

Techniques: Functional Assay, Cell Culture, Ex Vivo, Infection, Flow Cytometry, Fluorescence, Expressing, Cytotoxicity Assay, Isolation, ADCC Assay, Staining

Journal: Cell Reports Medicine

Article Title: KLRG1 expression on natural killer cells is associated with HIV persistence, and its targeting promotes the reduction of the viral reservoir

doi: 10.1016/j.xcrm.2023.101202

Figure Lengend Snippet: Impact of targeting KLRG1 in CD4 + T cells and in the viral reservoir (A) Frequency of KLRG1 + CD4 + T cells in a cohort of healthy donors (HD, n = 13), viremic (VIR, n = 17), and ART-suppressed (n = 23) PWH measured by flow cytometry. (B) Spearman correlation between the percentage of KLRG1 + CD4 + T cells and the intracellular levels of HIV RNA. (C) Intracellular HIV RNA levels measured by qPCR in CD4 + T cells from ART PWH after stimulation with the anti-KLRG1 antibody or the isotype control antibody (n = 10). (D) Quantification by qPCR of total HIV DNA in KLRG1 + and KLRG1 – CD4 + T cells isolated by FACS (n = 4). (E) Representative image of a lymph node section from a VIR HIV-infected PWH (#42). p24 HIV protein is shown in red and KLRG1 in yellow. (F and G) Percentage and mean fluorescence intensity expression of KLRG1 in cells infected ex vivo with the HIV BaL strain (n = 9) or (G) the HIV NL4.3 strain (n = 11). Statistical comparisons were performed using the Wilcoxon matched-pairs signed-rank test or Friedman test when required. ∗p < 0.05, ∗∗p < 0.01. See also Figures S6 and and Table S1 .

Article Snippet: Rabbit-polyclonal anti-human KLRG1 , Abcam , Cat#ab235951.

Techniques: Flow Cytometry, Isolation, Infection, Fluorescence, Expressing, Ex Vivo

Journal: Cell Reports Medicine

Article Title: KLRG1 expression on natural killer cells is associated with HIV persistence, and its targeting promotes the reduction of the viral reservoir

doi: 10.1016/j.xcrm.2023.101202

Figure Lengend Snippet:

Article Snippet: Rabbit-polyclonal anti-human KLRG1 , Abcam , Cat#ab235951.

Techniques: Purification, Blocking Assay, Virus, Plasmid Preparation, Recombinant, Isolation, Staining, Software