Additional file 1 of Multimeric immunotherapeutic complexes activating natural killer cells towards HIV-1 cure
Creators: name: Schober, Rafaëla
name: Brandus, Bianca
name: Laeremans, Thessa
name: Iserentant, Gilles
name: Rolin, Camille
name: Dessilly, Géraldine
name: Zimmer, Jacques
name: Moutschen, Michel
name: Aerts, Joeri L.
name: Dervillez, Xavier
name: Seguin-Devaux, Carole
Publisher: name: figshare
Year: 2024
Subjects: subject: Immunology
subject: FOS: Clinical medicine

description: Additional file 1: Fig. S1. NaMiX bind to their respective receptors on NK cells. A The molecules containing the anti-KIR scFv were incubated for 30 min with HEK293F cells expressing KIR2DL1 (left), KIR2DL2 (middle) and KIR2DL3 (right) and stained with anti-His and anti-IL-15 for flow cytometry analysis. B The molecules containing the anti-NKG2A scFv were incubated with the stable cell line NK-92MI expressing NKG2A and stained with anti-His and anti-IL-15 for flow cytometry analysis. C PBMCs from different donors were stained for extracellular markers including KIR2DL1/DS1, KIR2DL2/L3/DS2 and NKG2A to identify and phenotype CD3-CD56+CD16+ NK cells (left) and CD3+CD8+ T (right) cells using anti-CD3, CD8, CD14, CD16, CD19 and CD56 antibodies. D All NaMiX were incubated with human PBMCs and stained for NK cell markers, anti-His and anti-IL-15 for flow cytometry analysis. Data were expressed as the mean value ± SD. Fig. A and B represent three independent experiments with three different donors. Fig. C and D represent six and three independent experiments, respectively, with three and six different donors. NaMiX engrafted with IL-15Rα/IL-15 using C4bpα or C4bpβ and expressing the anti-NKG2A or the anti-KIR scFv are so called: α.anti-NKG2A.IL-15, β.anti-NKG2A.IL-15, α.anti-KIR.IL-15 and β.anti-KIR.IL-15, respectively while the control NaMiX without IL-15Rα/IL-15 expressing the anti-NKG2A scFv is termed β.anti-NKG2A and the control condition without any molecules as medium. Data were expressed as the mean value ± SD. Statistical analysis was performed using a one-way ANOVA and post-hoc Tukey test (*p &lt; 0.05, **p &lt; 0.005, ***p &lt; 0.001, ****p &lt; 0.0001). Fig. S2. NaMiX increased STAT5 phosphorylation in NK and CD8+ T cells. A PBMCs were incubated with the NaMiX molecules for 1, 10, 20 or 40 min, stained on ice to gate for live CD3-CD56+CD16+ NK (upper panel) and live CD3+CD8+ T cells (lower panel), permeabilized on ice and stained for intra-cellular pSTAT5 for flow cytometry analysis. The left panels represent time-dependent pSTAT5 phosphorylation of one representative donor. Right panel represents the data of three different healthy donors at 1 min incubation. NaMiX engrafted with IL-15Rα/IL-15 using C4bpα or C4bpβ and expressing the anti-NKG2A or the anti-KIR scFv are so called: α.anti-NKG2A.IL-15, β.anti-NKG2A.IL-15, α.anti-KIR.IL-15 and β.anti-KIR.IL-15, respectively while the control NaMiX without IL-15Rα/IL-15 expressing the anti-NKG2A scFv is termed β.anti-NKG2A, the recombinant human IL-15 as rhIL-15, and the control condition without any molecules as medium. Data were expressed as the mean value ± SD. Statistical analysis was performed using a one-way ANOVA and post-hoc Tukey test (***p &lt; 0.001, ****p &lt; 0.0001). B PBMCs were incubated with the indicated molecules for 1 minute, stained on ice to gate for CD3-CD56+CD16+ NK cells and CD3+CD8+ T cells using anti-CD3, CD8, CD14, CD16, CD19 and CD56 antibodies, permeabilized on ice and stained for intracellular pSTAT5 for imaging flow cytometry. Fig. S3. IL-15 stimulation and blocking of NKG2A-HLA-E interaction were required for NK cell activation by NKG2A NaMiX. A Human PBMCs were incubated for 5 h with the NKG2A NaMiX and HLA-E expressing K562 cells in the presence of anti-CD107a mAb. Natural Killer cells were further stained for extracellular markers to identify CD107a expression. B Cells were also permeabilized and further stained for IFN-γ. C Human PBMCs were incubated for 5 h with the NaMiX and HLA-E expressing K562 pre-stained with CellTrace Violet. Cells were further stained for Live/Dead and analyzed by flow cytometry. The Figure represents three independent experiments with 3 different donors. NaMiX engrafted with IL-15Rα/IL-15 using C4bpα or C4bpβ and expressing the anti-NKG2A scFv are so called α.anti-NKG2A.IL-15 and β.anti-NKG2A.IL-15, respectively while the control NaMiX without IL-15Rα/IL-15 expressing the anti-NKG2A scFv is termed β.anti-NKG2A and the control condition without any molecules as medium. Data were expressed as the mean value ± SD. Statistical analysis was performed using a one-way ANOVA and post-hoc Tukey test (*p &lt; 0.05, **p &lt; 0.005, ***p &lt; 0.001, ****p &lt; 0.0001). Fig. S4. NaMiX increased viral inhibition capacity of NK cells against HIV-1 CD4+ T cells. Autologous CD4+ T cells and NK cells were purified by microbeads positive and negative selection, respectively, from HIV-1 individuals under antiretroviral therapy. CD4+ T cells were superinfected by spinoculation with HIV III-B and incubated with stimulated NK cells for two or 5 days at an E:T of 1:1. A p24 intracellular staining of CD4+ T cells of one representative donor (left panel) and HIV-1 RNA in the supernatant measured by ddPCR of one representative donor (right panel). The experiment has been performed with three different patients showing the same trend but with high baseline variability among them. B PBMCs of three healthy donors were pre-incubated for 48 h with the different molecules and stimulated with ACH2 cells for 5 h. Cells were further stained for extracellular markers to identify CD25 and CD69 expression on CD3-CD56+CD16+ NK cells and CD3+CD4+ T cells using anti-CD3, CD4, CD14, CD16, CD19 and CD56 antibodies. The Figure represents three independent experiments with three different donors. Data were expressed as the mean value ± SD. Statistical analysis was performed using a one-way ANOVA and post-hoc Tukey test (*p &lt; 0.05, **p &lt; 0.005, ***p &lt; 0.001, ****p &lt; 0.0001). C Representative expression of CD25 and CD69 on CD4+ T and NK cells pre-activated by NaMiX for 48 h for one donor included in panel B. D HIV-1 mRNA release in the supernatant of ACH2 cells stimulated for 24 h by human PBMCs prestimulated by NaMiX for 48 h. Three independent experiments were performed with three different healthy donors. Data were expressed as the mean value ± SD. Statistical analysis was performed using a one-way ANOVA and post-hoc Tukey test. Fig. S5. Humanized NSG mice transgenic for human IL-15 develop functional NK cells compared to NSG mice. NSG mice (n=4) or NSG tg huIL-15 mice (n=4) were humanized with human CD34+ cells from umbilical cord blood. After 4 months, mice were sacrificed and organs harvested. Cells were stained to gate on CD3-CD56+CD16+ NK cells with anti-CD3, CD14, CD16, CD19 and CD56 antibodies. A Representation of the dot plot of NK cells from the spleen of NSG (left) or NSG tghuIL-15 (right). B Comparison of NK cells in blood, lung, bone marrow (BM) and spleen in NSG mice and NSG tg huIL-15 mice. C Comparison of NK subpopulations CD56dim (left) and CD56bright (right) in blood, lung, BM and spleen. D Cells from blood, lung, BM and spleen were stained for NK cell markers such as KIR2DL1/DS1 (left) KIR2DL2/3/DS2 (middle) and NKG2A (right). Data were expressed as the mean value ± SD. Statistical analysis was performed using unpaired Student t test. (**p&lt;0.005, ***p&lt;0.0005, ****p&lt;0.00005). E NK cells from humanized NSG mice transgenic for human IL-15 are cytotoxic as compared to NSG mice. Splenocytes were incubated with K562 cells for 5 h together with anti-CD107a (left graph), and were further permeabilized and stained with anti-perforin and anti-IFN-γ and anti-CD3, CD14, CD16, CD19 and CD56 antibodies to gate on CD3-CD56+CD16+ NK cells. K562 cells were pre-stained with CellTrace Violet and incubated with splenocytes for 5 h and all cells were stained for Live/Dead to determine cytotoxicity induced by NK cells. Data were expressed as the mean value ± SD. Statistical analysis was done using unpaired Student t test. (*p&lt;0.05, ***p&lt;0.0005).