Ganglioside-Functionalized Nanoparticles for Chimeric Antigen Receptor T-Cell Activation at the Immunological Synapse

Abstract

Chimeric Antigen Receptor (CAR) T cell therapy has proven to be an effective strategy against hematological malignancies but persistence and activity against solid tumors must be further improved. One emerging strategy for enhancing efficacy is based on directing CAR T cells to antigen presenting cells (APCs). Activation of CAR T cells at the immunological synapse (IS) formed between APC and T cell is thought to promote strong, persistent antigen-specific T cell-mediated immune responses but requires integration of CAR ligands into the APC/T-cell interface. Here, we demonstrate that CAR ligand functionalized, lipid-coated, biodegradable polymer nanoparticles (NPs) that contain the ganglioside GM3 (GM3-NPs) bind to CD169 (Siglec-1)-expressing APCs and localize to the cell contact site between APCs and CAR T cells upon initiation of cell conjugates. The CD169⁺ APC/CAR T-cell interface is characterized by a strong optical colocalization of GM3-NPs and CARs, enrichment of F-actin, and recruitment of ZAP-70, indicative of integration of GM3-NPs into a functional IS. Ligands associated with GM3-NPs localized to the APC/T-cell contact site remain accessible to CARs and result in robust T-cell activation. Overall, this work identifies GM3-NPs as a potential antigen delivery platform for active targeting of CD169 expressing APCs and enhancement of CAR T-cell activation at the NP-containing IS.

Keywords: CAR T cell activation; Siglec-1/CD169; antigen presenting cell; biomimetic nanomaterials; ganglioside GM3; lipid-coated polymeric nanoparticle.

Figure 1.. Design and characterization of GM3-FITC-NPs for αFITC Jurkat CAR T cell activation.

a) Scheme of αFITC CAR construct. The extracellular domain contains scFv and the intracellular domain comprises CD28, 4–1BB, and CD3ζ signaling domains. CAR is tagged with mCherry in the intracellular domain. b) Representative flow cytometry histograms (mCherry fluorescence) from transfected αFITC Jurkat CAR T cell (red) and wild-type Jurkat T cell (gray). c) Z-stack confocal sections of two αFITC Jurkat CAR T cells from bottom to top. Step size = 1 μm. Scale bar = 5 μm. d) Scheme of GM3-FITC-NPs and molecular structures of the lipid components (DPPC, Chol, GM3, Galcer). e) Hydrodynamic size and zeta potential of GM3-FITC-NPs, BLK-FITC-NPs (no gangliosides) and Galcer-FITC-NPs. f) Hydrodynamic diameter of GM3-FITC-NPs in cell culture media at 37 °C measured over 2 weeks. Data in e) and f) represent average ± standard deviation (n = 3).

Figure 2.. GM3-CD169-mediated targeting of GM3-FITC-NPs to Raji B/CD169 cell.

a-b) Representative flow cytometry histograms (FITC fluorescence) after incubation of specified NPs with Raji B/CD169 cells for 10 min at 37 °C. Conditions include cells without NPs treatment, and cells treated with GM3-FITC-NPs, BLK-FITC-NPs, and Galcer-FITC-NPs. c) Binding curve for GM3-FITC-NPs binding with Raji B/CD169 cells. Fitted with one-site binding model. Specific binding was obtained by total binding from Raji B/CD169 cells subtracted by non-specific binding from Raji B cells (n = 2). Data presented as average ± standard deviation. d-e) Confocal sections of GM3-FITC-NPs associated with Raji B/CD169 cells. The cells were incubated with the NPs for 10 min and subsequently maintained at 37 °C for 4 h. The cells were stained for CD169 (d) and LAMP-1 (e). Pearson correlation coefficients (R) for NP signal and CD169 or LAMP-1 signal of 50 randomly selected cells are included in the box plots. Scale bar = 5 μm.

Figure 3.. αFITC CAR T cell activation by GM3-FITC-NPs associated with Raji B/CD169 (APC-mediated activation).

a) CD69 expression by αFITC CAR T cells after direct activation through GM3-FITC-NPs with different FITC concentrations incorporated in the membrane. (n = 2) b-d) Characterization of αFITC CAR T cell activation through GM3-FITC-NPs associated with Raji B/CD169 cell (APC-mediated activation) by different markers. (b) Total FITC intensity of αFITC CAR T cell and Raji B/CD169 cell, (c) CD69 upregulation in αFITC CAR T cells and (d) IL-2 secretion for specified input volumes of GM3-FITC-NPs (2 mol% FITC). (n = 4) e-g) Ca²⁺ response of αFITC CAR T cell through GM3-FITC-NPs associated with Raji B/CD169 (APC-mediated activation). (e) Bright-field view of GM3-FITC-NPs presenting Raji B/CD169 cell before and after CAR-T cell addition. (f) Cellular Ca²⁺ activity in a CAR-T cell during the interaction with GM3-FITC-NPs presenting Raji B/CD169 cell monitored using the ratiometric Fura-2 AM dye. The ratio of the fluorescent intensities at 340 nm and 380 nm is plotted as function of time. (g) Representative time-lapse fluorescent images show Ca²⁺activity of the CAR T cell. Scale bar = 5 μm. Data presented as average ± standard deviation. p<0.05, p<0.01, p<0.001 by student t-test.

Figure 4.. GM3-FITC-NP-induced synapse formation between αFITC CAR-T cell and Raji B/CD169 cell.

a) Confocal section of cell conjugate formed by αFITC CAR T cell and Raji B/CD169 cell. GM3-FITC-NPs (green), CAR (red), nucleus (blue) and F-actin (gray) were labelled. b) Box plot of Pearson correlation coefficients (R) for CARs and NPs in individual cells. The average R is 0.62 ± 0.10. c, d) Box plots of CAR localization at the cell-cell interface (c) and GM3-FITC-NPs localization at the cell-cell interface (d). The average CAR and NP localization at the IS is 92% ± 6% and 76% ± 20%, respectively. (n = 22 for b)-d)). e) Confocal section of αFITC CAR T cell - Raji B/CD169 cell conjugate with CAR (red), GM3-NP (green), ZAP-70 (cyan), and F-actin (gray) labeling. Scale bar = 5 μm.

Figure 5.. Intracellular fate of GM3-NPs in mature DCs.

a-b) Confocal maps of two phenotypes of GM3-FITC-PLA NPs in DCs after 15 min incubation with NPs, and a subsequent chase of 1 h and 4 h and staining for CD169 and LAMP-1. (a) Random distribution from 1 h incubation (b) Cluster distribution from 4 h incubation. c) Dot plot of cluster phenotype occurrence over total cell number after 0 h, 1 h and 4 h incubation. 20 confocal images with a total 1008 cells (0 h), 860 cells (1 h) and 955 cells (4 h) were collected and analyzed. Each dot represents the occurrence of cluster phenotype of total cells in one confocal image. Scale bar = 5 μm. Data presented as average ± standard deviation. **** p<0.0001 by student t-test.

Figure 6.. DC-mediated CAR T cell activation and synapse formation between GM3-FITC-NP-loaded DCs and CAR T cells.

a) CD69 expression and b) IL-2 secretion for αFITC CAR T cells after incubation with DCs loaded with GM3-FITC-NPs or without. (n = 4) c) CD69 expression in CAR T cells as function of FITC signal intensity for direct activation by free GM3-NPs at different input concentrations (blue, n = 3) and APC-mediated activation (red, n = 4). d) IL-2 secretion by CAR T cells for direct activation by free GM3-NPs vs. for APC-mediated activation for the conditions marked by a dashed rectangle in (c). e) CD69 expression in CAR T cells as function of the FITC signal intensity for GM3-FITC-NPs, amph-FITC, and DC and CAR T cell mixture (without any FITC) as control. (n = 4) f) Intracellular distribution of amph-FITC in DC after 1 h incubation. Lysosome were stained and labelled in red. g) Cell conjugate formed by αFITC CAR T cell and DC after treatment with amph-FITC (green). h) Cell conjugate formed by αFITC CAR T cell and GM3-FITC-NP-loaded DC. GM3-NPs (green), CARs (red), nucleus (blue) and F-actin (gray) were labelled. i) Conjugate of αFITC CAR T cell and GM3-FITC-NP-loaded DC with CAR (red), GM3-NP (green), ZAP-70 (cyan), and F-actin (gray) labeling. Scale bar = 5 μm. Data presented as average ± standard deviation. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 by student t-test.

Figure 7.. DC-mediated activation of primary CAR T cells.

a) Representative flow cytometry histogram (mCherry fluorescence) of transfected αFITC CD8⁺ CAR T cell (red) and wild-type T cell (gray). b) CD69 upregulation and increased IFN-γ secretion αFITC CD8⁺ CAR T cell by GM3-FITC-NP-loaded DCs (n = 2). Data presented as average ± standard deviation. * p<0.05, ** p<0.01 by student t-test. c) CD69 expression in CD3⁺ CAR T cells as function of FITC signal intensity for direct activation by free GM3-NPs at different input concentrations (blue) and APC-mediated activation (red). Each plot represents results from the same DC and primary T cell donor. d) Fold increase of inhibitory receptors in CD3+ CAR T cells after 24 h of stimulation. Data represents average ± standard deviation from two independent experiments with DCs and primary T cells from two donors. The input concentrations of GM3-FITC-NPs used for the exhaustion experiments are marked with green arrows in Figure 7c.