A Lysosome-Targeted Tetrazine for Organelle-Specific Click-to-Release Chemistry in Antigen Presenting Cells

Abstract

Bioorthogonal deprotections are readily used to control biological function in a cell-specific manner. To further improve the spatial resolution of these reactions, we here present a lysosome-targeted tetrazine for an organelle-specific deprotection reaction. We show that trans-cyclooctene deprotection with this reagent can be used to control the biological activity of ligands for invariant natural killer T cells in the lysosome to shed light on the processing pathway in antigen presenting cells. We then use the lysosome-targeted tetrazine to show that long peptide antigens used for CD8⁺ T cell activation do not pass through this organelle, suggesting a role for the earlier endosomal compartments for their processing.

Figure 1

Bioorthogonal chemistry to study the subcellular routing before antigen presentation of CD1d-glycolipid (top) and MHC-I-peptide (bottom) processing by DCs. With the use of a lysosome-specific tetrazine, the TCO-protecting group will only be removed when the antigens enter the lysosome before presentation at the cell surface. While the glycolipid antigens TCO-α-GalCer (1) and TCO-α-GalPhyto (2) pass the lysosome and will therefore activate the iNKT cells, the mbTCO-OVA18 (6) peptide does not enter the lysosome, resulting in no CD8⁺ T cell activation.

Figure 2

Evaluation of LysoTz (3). (A) Structure of DABCYL-TCO-BODIPY (9). (B) Turn-on rate of DABCYL-TCO-BODIPY (9, 1 μM) ± LysoTz 3 (10 μM) in H₂O measured by increased fluorescent signal (spectral properties can be found in Figure S2). (C) Confocal microscopy images of bone marrow derived DCs (BMDCs) stained with Hoechst 33342 (DNA), CellMask Orange Actin tracking stain and LysoTracker Deep Red as reference. Legend: 1. LysoTz (3) + DABCYL-TCO-BODIPY (9); 2. Negative control for 3; 3. Negative control for 9. All scale bars represent 20 μm.

Figure 3

Synthesis of TCO-protected α-GalCer (1) and α-GalPhyto (2). Reagents/conditions: (a) NIS, TMS-OTf, DCM, −40 °C, 67%; (b) PtO₂, H₂ (g), THF, rt; (c) TCO-NHS, DIPEA, DMAP, DMF, rt, 89% over two steps; (d) LiOH, THF, H₂O, rt, quant.; (e) Et₃N·3HF, THF, rt, 84%; (f) hexacosonoic acid, EDC·HCl, DIPEA, DMAP, DCM, rt, 31–34%; (g) Et₃N·3HF, THF, rt, 23%.

Figure 4

Characterization of iNKT cell activation with TCOα-galactosylceramide (α-GalCer, 1) and TCOα-galactosyl phytosphingosine (α-GalPhyto, 2) uncaged with tetrazines; the y-axis shows the IL-2 levels measured with ELISA as readout for DN32.D3 iNKT cell activation. (A) Structures of the six different tetrazines used for characterization. (B) iNKT cell activation measured by IL-2 levels upon addition of TCO α-GalCer (1, 0.3 μM) and TCO α-GalPhyto (2, 15 μM) at different concentrations. α-GalCer (4, 10 nM) and α-GalPhyto (5, 10 μM) were used as positive control. (C) iNKT cell activation measured upon uncaging of TCO with different tetrazines. (D) Concentration optimization of Tz-concentration for both 1 (left) and 2 (right)-based reagents; (E) IL-2 production with increasing Tz-incubation time showing near maximal uncaging after a 20-minutes Tz incubation. All experiments were performed in triplicate and with BMDCs from three different mice.

Figure 5

TCOα-galactosylceramide (α-GalCer) (1) and TCOα-galactosyl phytosphingosine (α-GalPhyto) (2) are both present in the lysosome before presentation by BMDCs; the y axis shows the IL-2 levels measured with ELISA as readout for DN32.D3 iNKT cell activation. (A) Experimental set-up for glycolipid uncaging with LysoTz (3) in BMDCs; (B) iNKT cell activation measured by IL-2 levels upon addition of TCOα-GalCer (1) and TCOα-GalPhyto (2) with thereafter addition of LysoTz (3); (C) experimental set-up for lysosomal-specific uncaging in BMDCs; (D) iNKT cell activation measured by IL-2 levels after preincubation with LysoTz (3) and after a wash addition of TCOα-GalCer (1) or TCOα-GalPhyto (2). All experiments were performed in triplicate and with BMDCs from three different mice.

Figure 6

SIINFEKL peptides do not enter the lysosome before presentation by DCs. B3Z T cell activation assay was used to assess the amount of “uncaged” antigen presented by D1 cells; the y axis shows the normalized absorbance at 570 nm. (A) T cell activation upon addition of 100 nM mbTCO-SIINFEKL (8) ± LysoTz (3). As control, LysoTz (3) is also added after peptide presentation and the samples are normalized between the negative cells only and the positive 100 nM SIINFEKL control. (B) T cell activation upon addition of 50 μM mbTCO-OVA18 (6) ± LysoTz (3). LysoTz (3) is also added as control after peptide presentation and the samples are normalized between the negative cells only and the positive 10 nM SIINFEKL (7) controls. All experiments were performed three times and in triplicate.