Figure 4.
Conventional CAR interactions of high affinity or high antigen density result in hyper-stabilization of underlying microvillar protrusion, which can be reduced using monomeric CAR. (A) Experiments were performed comparing CARs with an HA scFv against HER2-based off trastuzumab (4D5, KD = 0.0523 nM, red) and a lower affinity scFv made by substitution of three amino acids (see Table S1, mut4D5, KD = 3.63 nM, blue). (B) Bilayers were loaded with fluorescent quantum dots (QD605) with a height of 16 nm. Locations where the cell makes close contact with the bilayer (<16 nm) are visualized as holes in QD605 signal due to their size-based exclusion. QD605 signal is shown for an LA CAR T cell interacting with a lipid bilayer loaded with 6.25 ng HER2. Outlined contact is shown in C. Scale bar = 3 μm. (C) QD605 signal across 5 time points are shown for the same field of view, each on low HER2 bilayers (6.25 ng/well). Top: Microvillus from LA CAR T cell moves out of view. Bottom: Microvillus from HA CAR T cell remains across time points. Scale bars = 1 μm. (D) CAR-occupied close contact persistence times (blue, red) and CAR-negative close contact persistence times (gray) are shown for varying antigen densities and affinities. All CAR:HER2 interactions tested result in CAR-occupied MV contact stabilization above background CAR-negative contacts (gray). Persistence time is further increased in interactions of HA CAR (red), even at lowest HER2 densities on the bilayer. For LA CAR (blue), only high levels of HER2 yield similar persistence times to high-affinity CAR. Data is shown for at least 11 cells per condition across four experiments (n = 13, 13, 11, 15, 17, 15 cells per group from left to right, respectively). (E) LA CAR was retrovirally expressed in primary mouse OT-I T cells. Receptor-occupied MV persistence times are shown for OT-I:SL8 (green), LA CAR:Low HER2 (light blue), and LA CAR:High HER2 (dark blue) interactions. All cognate interactions are stabilized above background receptor-negative contacts (gray). CAR:High HER2 persistence is hyper-stable relative to TCR:pMHC stabilization. Data is shown for at least 10 cells per condition across three experiments (n = 16, 10, 10 cells per group from left to right, respectively). (F) Dimers (filled dots) and monomers (open dots) are compared on high HER2 bilayers (625 ng/well). Only monomeric LA CAR regains natural persistence time of TCR:pMHC contacts (green dashed line). All receptor-occupied contacts are stabilized above non-cognate antigen interactions (gray). Data is shown for at least 5 cells per condition across three experiments (n = 7, 7, 5, 13 cells per group from left to right, respectively). (D–F) Error bars represent SD and analyses shown are Šídák’s multiple comparisons tests.

Conventional CAR interactions of high affinity or high antigen density result in hyper-stabilization of underlying microvillar protrusion, which can be reduced using monomeric CAR. (A) Experiments were performed comparing CARs with an HA scFv against HER2-based off trastuzumab (4D5, KD = 0.0523 nM, red) and a lower affinity scFv made by substitution of three amino acids (see Table S1, mut4D5, KD = 3.63 nM, blue). (B) Bilayers were loaded with fluorescent quantum dots (QD605) with a height of 16 nm. Locations where the cell makes close contact with the bilayer (<16 nm) are visualized as holes in QD605 signal due to their size-based exclusion. QD605 signal is shown for an LA CAR T cell interacting with a lipid bilayer loaded with 6.25 ng HER2. Outlined contact is shown in C. Scale bar = 3 μm. (C) QD605 signal across 5 time points are shown for the same field of view, each on low HER2 bilayers (6.25 ng/well). Top: Microvillus from LA CAR T cell moves out of view. Bottom: Microvillus from HA CAR T cell remains across time points. Scale bars = 1 μm. (D) CAR-occupied close contact persistence times (blue, red) and CAR-negative close contact persistence times (gray) are shown for varying antigen densities and affinities. All CAR:HER2 interactions tested result in CAR-occupied MV contact stabilization above background CAR-negative contacts (gray). Persistence time is further increased in interactions of HA CAR (red), even at lowest HER2 densities on the bilayer. For LA CAR (blue), only high levels of HER2 yield similar persistence times to high-affinity CAR. Data is shown for at least 11 cells per condition across four experiments (n = 13, 13, 11, 15, 17, 15 cells per group from left to right, respectively). (E) LA CAR was retrovirally expressed in primary mouse OT-I T cells. Receptor-occupied MV persistence times are shown for OT-I:SL8 (green), LA CAR:Low HER2 (light blue), and LA CAR:High HER2 (dark blue) interactions. All cognate interactions are stabilized above background receptor-negative contacts (gray). CAR:High HER2 persistence is hyper-stable relative to TCR:pMHC stabilization. Data is shown for at least 10 cells per condition across three experiments (n = 16, 10, 10 cells per group from left to right, respectively). (F) Dimers (filled dots) and monomers (open dots) are compared on high HER2 bilayers (625 ng/well). Only monomeric LA CAR regains natural persistence time of TCR:pMHC contacts (green dashed line). All receptor-occupied contacts are stabilized above non-cognate antigen interactions (gray). Data is shown for at least 5 cells per condition across three experiments (n = 7, 7, 5, 13 cells per group from left to right, respectively). (D–F) Error bars represent SD and analyses shown are Šídák’s multiple comparisons tests.

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