cd56 cd4  (Miltenyi Biotec)

Journal: bioRxiv

Article Title: NK cell immunotherapy administered at the time of HIV recrudescence is associated with viral control

doi: 10.1101/2025.11.19.688897

Figure Lengend Snippet: A) Schematic figure showing the overview of NK cell production and the overview of the mouse study timeline. Red circles on the timeline indicate blood collection, black dots indicate scheduled necropsies. Schematic of transgene design: MND promoter, CAR (CD4-MBL-CD8TM-4-1BB-CD3z), CXCR5, IL-15, the BgH PolyA tail, with separation mediated by P2A and T2A sites, and inverted terminal repeats (ITRs). Schematic showing expression of all transgene components as well as base editor-mediated knock out of PD-1 on the NK cell surface. Figure made using Biorender.com. B) Expression of CAR (MBL) and CXCR5 molecules was confirmed in the cell product using flow cytometry. Cells were pre-gated sequentially on lymphocytes, singlets, live cells, CD56+, and CD3-. C) PD-1 knockout was detected at the RNA level by RT-PCR, CAR NK (blue) relative to control NK cells (black). D) IL-15 expression in CAR NK (blue) and control NK (black) cells via IL-15 ELISA of cell culture supernatant. E) CAR (blue) and control (black) cell proliferation over one week post-thaw. F) The specific lysis of HIV Envelope P815s by CAR (blue) or control (black) NK cells via DELFIA cytotoxicity assay. 5:1, 10:1, and 20:1 E:T ratios were tested. All assays were performed in duplicate or triplicate. Data are represented as mean + standard deviation (error bars).

Article Snippet: After a 2-day recovery, CAR NK cells were sorted on CD56+ CD4+ using the MACSQuant Tyto Cell Sorter (Miltenyi Biotec) and expanded for an additional 7 days following the procedure described above.

Techniques: Expressing, Knock-Out, Flow Cytometry, Reverse Transcription Polymerase Chain Reaction, Control, Enzyme-linked Immunosorbent Assay, Cell Culture, Lysis, Cytotoxicity Assay, Standard Deviation

Journal: bioRxiv

Article Title: NK cell immunotherapy administered at the time of HIV recrudescence is associated with viral control

doi: 10.1101/2025.11.19.688897

Figure Lengend Snippet: NK (CD56+) cells in CAR (blue), control (black), or PBS (red) treated groups from A) Peripheral Blood, B) Lymph node (at necropsies), and C) Spleen (at necropsies). CAR+CXCR5+ NK cells over time in D) Peripheral Blood, E) Lymph node (at necropsies), and F) Spleen (at necropsies). Flow was pre-gated on Lymphocytes, Singlets, Live Cells, human CD45+, mouse CD45-, CD3+, and then through CD56+ MBL+ CXCR5+ (see Fig. S4 for gating strategy). Median viral loads (green), NK cell levels (orange), post-treatment in G) controllers or H) non-controllers. I) Median human hematopoietic cell counts (CD45+)/ ul blood of controllers (gray) or non-controllers (pink).

Article Snippet: After a 2-day recovery, CAR NK cells were sorted on CD56+ CD4+ using the MACSQuant Tyto Cell Sorter (Miltenyi Biotec) and expanded for an additional 7 days following the procedure described above.

Techniques: Control

Journal: bioRxiv

Article Title: NK cell immunotherapy administered at the time of HIV recrudescence is associated with viral control

doi: 10.1101/2025.11.19.688897

Figure Lengend Snippet: CD4:CD8 ratios over time in A) CAR NK-treated animals (blue), B) control NK-treated animals (black), and C) PBS-treated animals (red). CD4:CD8 ratios in CAR (blue), control NK (black), and PBS (red) treated groups at D) -6 DPT, E) 6 DPT, F) 14 DPT, G) 28 DPT, H) 42 DPT, and I) 56 DPT. Lines represent median values. CD4:CD8 ratios were determined by flow cytometry and pre-gated on Lymphocytes, Singlets, Live Cells, Human CD45+, Mouse CD45-, CD3+ (see gating strategy in Fig. S4 ).

Article Snippet: After a 2-day recovery, CAR NK cells were sorted on CD56+ CD4+ using the MACSQuant Tyto Cell Sorter (Miltenyi Biotec) and expanded for an additional 7 days following the procedure described above.

Techniques: Control, Flow Cytometry

Journal: EMBO Molecular Medicine

Article Title: LILRB1-HLA-G axis defines a checkpoint driving natural killer cell exhaustion in tuberculosis

doi: 10.1038/s44321-024-00106-1

Figure Lengend Snippet: ( A ) Flow diagram summarizing the participant recruitment in this study. See “Methods” and Dataset for details of inclusion criteria and demographic information of participants included, respectively. ( B ) Representative results of fluorescence-activated cell sorting (FACS)-based analyses for frequencies of immune cell subsets in peripheral blood of individuals from HC, LTBI, and ATB groups. Each immune cell subsets were characterized using standard cell subset definitions. NK cell gating: FSC, SSC, CD3 – , CD19 – , CD56 bright/dim ; T cell gating: FSC, SSC, CD3 + , CD19 – , CD56 – ; B cell gating: FSC, SSC, CD3 – , CD19 + , CD56 – ; monocyte gating: FSC, SSC, CD3 – , CD19 – , CD14 + . ( C ) The number of immune cell subsets in peripheral blood of individuals ( n ) from HC, LTBI, and ATB groups. Statistical significance was determined using one-way ANOVA with Tukey’s post-hoc test ( C ). .

Article Snippet: Before co-culture, CD3 – CD56 + CD4 + CD14 + NK cells were prestained with 1 μg/mL Hoechst 33342 (Cell signaling Technology, Cat# 4082) at 37 °C for 10 min for visualization.

Techniques: Fluorescence, FACS

Journal: EMBO Molecular Medicine

Article Title: LILRB1-HLA-G axis defines a checkpoint driving natural killer cell exhaustion in tuberculosis

doi: 10.1038/s44321-024-00106-1

Figure Lengend Snippet: ( A ) Correlation analysis of expression between LILRB1 and other markers including GZMB, PRF1, IFN-γ, and TNF-α based on CyTOF single-cell data ( n = 35,000 cells). ( B – D ) Percentages of CD107a + ( B ), IFN-γ + ( C ), or TNF-α + ( D ) cells within total NK cells. ( E , F ) Supernatant levels of GZMB ( E ) and PRF1 ( F ) in MDM-NK cell co-cultures. ( G ) Percentages of apoptotic cells within total NK cells. ( H ) Mtb survival in MDM-NK cell co-cultures. For ( B – H ), HC donor-derived NK cells with overexpression of MSCV or MSCV-LILRB1 were co-cultured with or without Mtb-infected MDMs (Mtb-Mφ) for 24 h. ( I ) Histological and immunofluorescence analysis of human lung sections containing non-necrotic or necrotic TB granulomas or healthy control tissues adjacent to granulomatous lesions. Left, representative images (scale bars, 200 μm) for hematoxylin and eosin (H&E) staining. Right, representative images (scale bars, 10 μm) for cells stained with antibodies against LILRB1 (blue), CD56 (green), and CD69 or CD103 (red). Nuclei were stained with DAPI (gray). ( J , K ) Quantitation of LILRB1 expression in CD56 + CD69 + or CD56 + CD69 – NK cells ( J ) and CD56 + CD103 + or CD56 + CD103 – NK cells ( K ). For ( I – K ), lung sections from 5 ATB patients were examined. Data are mean ± SEM ( n = 5 donors per group) in ( B – H , J , K ). Statistical significance was determined using two-way ANOVA ( B – G , J , K ) or one-way ANOVA ( H ) with Tukey’s post-hoc test. Results are representative of three independent experiments. .

Article Snippet: Before co-culture, CD3 – CD56 + CD4 + CD14 + NK cells were prestained with 1 μg/mL Hoechst 33342 (Cell signaling Technology, Cat# 4082) at 37 °C for 10 min for visualization.

Techniques: Expressing, Derivative Assay, Over Expression, Cell Culture, Infection, Immunofluorescence, Control, Staining, Quantitation Assay

Journal: EMBO Molecular Medicine

Article Title: LILRB1-HLA-G axis defines a checkpoint driving natural killer cell exhaustion in tuberculosis

doi: 10.1038/s44321-024-00106-1

Figure Lengend Snippet: ( A ) Representative results of FACS-based analysis for percentages of CD3 – CD56 + CD4 + CD14 + cells within total CD3 – CD56 + NK cells from the peripheral blood of individuals in HC, LTBI, or ATB groups. ( B ) Percentages of CD3 – CD56 + CD4 + CD14 + cells within total CD3 – CD56 + NK cells in the peripheral blood of individuals ( n ) from HC, LTBI, or ATB groups. Box-whisker plot indicates the interquartile range (box), the median value (line within the box), and the maximum and minimum value (whiskers). ( C ) The number of CD3 – CD56 + CD4 + CD14 + cells in the peripheral blood of individuals ( n ) from HC, LTBI, or ATB groups. ( D , E ) Percentages of IFN-γ + ( D ) and TNF-α + ( E ) cells in total CD3 – CD56 + CD4 + CD14 + cells. CD3 – CD56 + CD4 + CD14 + cells were co-cultured with or without Mtb-Mφ at a ratio of 3:1 for 24 h in the presence of anti-LILRB1 (GHI/75) mAb or IgG control. Data are mean ± SEM ( n = 6 donors per group) in ( D , E ). Statistical significance was determined using one-way ANOVA with Tukey’s post-hoc test for ( B – E ). Results are representative of three independent experiments. .

Article Snippet: Before co-culture, CD3 – CD56 + CD4 + CD14 + NK cells were prestained with 1 μg/mL Hoechst 33342 (Cell signaling Technology, Cat# 4082) at 37 °C for 10 min for visualization.

Techniques: Whisker Assay, Cell Culture, Control

Journal: EMBO Molecular Medicine

Article Title: LILRB1-HLA-G axis defines a checkpoint driving natural killer cell exhaustion in tuberculosis

doi: 10.1038/s44321-024-00106-1

Figure Lengend Snippet: ( A , B ) Representative images (left) and quantitation (right) for immunofluorescence examining the granule polarization ( A ) and cytotoxic activity ( B ) of CD3 – CD56 + CD4 + CD14 + cells (effector cells) towards Mtb-infected MDMs (Mtb-Mφ). CD3 – CD56 + CD4 + CD14 + cells were co-cultured with Mtb-Mφ at a ratio of 3:1 for 24 h in the presence of anti-LILRB1 (GHI/75) mAb or IgG control. Bacteria were stained with Alexa Fluor 488 succinimidyl ester (green) before infection. CD3 – CD56 + CD4 + CD14 + cells were prestained with Hoechst (blue) for visualization, and were labeled with LysoTracker (red; white arrows) to monitor lytic granule polarization towards target cells, a characteristic of NK cells during degranulation. MDM lysis was determined by uptake of DRAQ7 (red; black arrows). Scale bars, 10 μm. Approximately 100 cells were examined. N.D., not detectable. ( C ) Time-lapse analysis (left) and quantitation (right) of calcein release from Mtb-Mφ. CD3 – CD56 + CD4 + CD14 + cells were co-cultured with Mtb-Mφ as in ( A , B ) for 12 h and were then monitored for 2 h. Bacteria were stained with Alexa Fluor 594 succinimidyl ester (red) before infection. MDMs were preloaded with calcein (green), an indicator of cell viability that was released from dead cells lysed by effector cells (arrows). Scale bars, 10 μm. See also Movies and . ( D – G ) Percentages of CD107a + ( D ), GZMB + ( E ), PRF1 + ( F ), and apoptotic ( G ) cells in total CD3 – CD56 + CD4 + CD14 + cells. CD3 – CD56 + CD4 + CD14 + cells were co-cultured with or without Mtb-Mφ at a ratio of 3:1 for 24 h in the presence of anti-LILRB1 (GHI/75) mAb or IgG control. ( H ) Mtb survival in MDM-effector cell co-cultures. MDMs were infected with Mtb, followed by co-culture with or without CD3 – CD56 + CD4 + CD14 + cells as in ( D – G ) for 24 h. Data are mean ± SEM [ n = 5 donors per group in ( A – C ) and n = 6 donors per group in ( D – H )]. Statistical significance was determined using two-way ANOVA ( A – C ) or one-way ANOVA ( D – H ) with Tukey’s post-hoc test. Results are representative of three independent experiments. .

Article Snippet: Before co-culture, CD3 – CD56 + CD4 + CD14 + NK cells were prestained with 1 μg/mL Hoechst 33342 (Cell signaling Technology, Cat# 4082) at 37 °C for 10 min for visualization.

Techniques: Quantitation Assay, Immunofluorescence, Activity Assay, Infection, Cell Culture, Control, Bacteria, Staining, Labeling, Lysis, Co-Culture Assay

Journal: EMBO Molecular Medicine

Article Title: LILRB1-HLA-G axis defines a checkpoint driving natural killer cell exhaustion in tuberculosis

doi: 10.1038/s44321-024-00106-1

Figure Lengend Snippet: ( A ) Schematic representation of the experimental design. PBMCs from individuals in HC, LTBI, or ATB group were transplanted to NCG mice. Two weeks later, mice were infected with Mtb by aerosol (~100 CFUs) for 4 weeks with treatment of PBS (as control), IgG control, or anti-LILRB1 blocking pAb every 3 days, together with treatment of corn oil (as control) or SCH772984 every 2 days. ( B ) Representative images (upper) and quantitation (lower) for H&E staining, acid-fast staining, and immunostaining of lung sections. Scale bars, 1 mm, 10 μm, and 10 μm, respectively. For immunostaining, NK cells were stained with anti-human CD56 antibody (red), apoptotic cells were stained with TUNEL (green), and nuclei were stained with DAPI (blue). Black arrows indicate granuloma-like inflammatory lesions. Black arrowheads indicate Mtb. White arrowheads indicate TUNEL-positive NK cells. ( C , D ) Enzyme-linked immunosorbent assay (ELISA) of GZMB ( C ) or PRF1 ( D ) in the lungs. ( E , F ) Bacterial CFUs in the lungs ( E ) or spleens ( F ). For ( B – F ), each group (HC, LTBI, or ATB) includes 5 donors, and each donor’s PBMCs were transplanted to four mice, which were subjected to four different treatments as indicated (PBS, IgG control, anti-LILRB1 blocking pAb, and anti-LILRB1 blocking pAb along with SCH772984, respectively). Data are mean ± SEM ( n = 5 mice per treatment group). Statistical significance was determined using two-way ANOVA with Tukey’s post-hoc test ( B – F ). Results are representative of two independent experiments. .

Article Snippet: Before co-culture, CD3 – CD56 + CD4 + CD14 + NK cells were prestained with 1 μg/mL Hoechst 33342 (Cell signaling Technology, Cat# 4082) at 37 °C for 10 min for visualization.

Techniques: Infection, Aerosol, Control, Blocking Assay, Quantitation Assay, Staining, Immunostaining, TUNEL Assay, Enzyme-linked Immunosorbent Assay

Journal: EMBO Molecular Medicine

Article Title: LILRB1-HLA-G axis defines a checkpoint driving natural killer cell exhaustion in tuberculosis

doi: 10.1038/s44321-024-00106-1

Figure Lengend Snippet: ( A ) Schematic representation of the experimental design. Control PBMCs or NK cell-depleted (ΔNK) PBMCs from ATB patients were transplanted to NCG mice. Two weeks later, mice were infected with Mtb by aerosol (~100 CFUs) for 4 weeks with treatment of IgG control or anti-LILRB1 blocking pAb every 3 days. ( B , C ) Representative images (left) and quantitation (right) for H&E staining ( B ) and immunostaining ( C ) of lung sections. Scale bars, 1 mm and 10 μm, respectively. For immunostaining, NK cells were stained with anti-human CD56 antibody (red), apoptotic cells were stained with TUNEL (green), and nuclei were stained with DAPI (blue). Black arrows indicate granuloma-like inflammatory lesions. White arrows indicate TUNEL-positive NK cells. ( D , E ) ELISA of GZMB ( D ) or PRF1 ( E ) in the lungs. ( F , G ) Bacterial CFUs in the lungs ( F ) or spleens ( G ). For ( B – G ), a total of 6 ATB donors were included for collecting PBMCs, and each donor’s PBMCs were divided into two treatment groups, of which one was subjected to NK cell deprivation (ΔNK PBMCs) and one was not (control PBMCs). Then, the control PBMCs or ΔNK PBMCs from each donor were transplanted to two mice, of which one was treated with anti-LILRB1 blocking pAb and one was treated with IgG control. Data are mean ± SEM ( n = 6 mice per treatment group). Statistical significance was determined using two-way ANOVA with Tukey’s post-hoc test ( B – G ). Results are representative of two independent experiments. .

Article Snippet: Before co-culture, CD3 – CD56 + CD4 + CD14 + NK cells were prestained with 1 μg/mL Hoechst 33342 (Cell signaling Technology, Cat# 4082) at 37 °C for 10 min for visualization.

Techniques: Control, Infection, Aerosol, Blocking Assay, Quantitation Assay, Staining, Immunostaining, TUNEL Assay, Enzyme-linked Immunosorbent Assay

Journal: EMBO Molecular Medicine

Article Title: LILRB1-HLA-G axis defines a checkpoint driving natural killer cell exhaustion in tuberculosis

doi: 10.1038/s44321-024-00106-1

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Before co-culture, CD3 – CD56 + CD4 + CD14 + NK cells were prestained with 1 μg/mL Hoechst 33342 (Cell signaling Technology, Cat# 4082) at 37 °C for 10 min for visualization.

Techniques: Recombinant, Western Blot, Sequencing, Modification, Cell Isolation, Enzyme-linked Immunosorbent Assay, Isolation, Antibody Labeling, Transfection, Lysis, Protease Inhibitor, Blocking Assay, Software, Flow Cytometry, Cytometry