Figure 5.
Impaired movement of HA dimer CAR microclusters in synapses and reduced effector function. (A and B) TIRF imaging of CAR-mEmerald is shown for synapses with LA or HA CAR interacting with low or high HER2-loaded bilayers (A). Time point shown is 93.6 s following the initiation of imaging, which began as synapses were starting to form. Yellow lines were drawn to span CAR microclusters in synapse, indicating diameter quantified in B. Three lines were averaged and line assignment was blinded to account for manual drawing. For dimeric CARs, only LA:low HER2 synapses result in accumulation of CAR microclusters at the center, indicated by lower diameter. Monomerization of CARs improves centralization on high HER2. Data is shown for at least 7 cells pooled from a minimum two independent experiments per condition (n = 13, 13, 20, 20, 13, 7 cells per group from left to right, respectively). Error bars represent SD. Analyses shown are Šídák’s multiple comparisons tests. Scale bars = 2 μm. (C and D) Spots with tracking were assigned to CAR microclusters in Imaris. Examples of mobile (left, LA dimer on low HER2) and immobile (right, LA dimer on high HER2) microclusters are shown as a flower plot for 10 random tracks (C). Quantification is shown for the average displacement (left) and speed (right) of all CAR microclusters for a given cell (D, n = 3 cells per group, 30–294 tracks per cell). Limited mobility is apparent for dimeric CARs on high-HER2 loaded bilayers. On low-HER2 bilayers, LA CAR microclusters show increased mobility. Error bars represent SD. Analyses shown are Šídák’s multiple comparisons tests. (E) Low HER2-expressing MCF7 (open circle) and high HER2-expressing SKBR3 (solid circle) cell lines were used to assess differences between HER2 levels in cell-cell interactions in vitro. (F) Proliferation is induced across all CAR+ conditions with low and high HER2, as seen by VPD dilution at 96 h following co-incubation. Line marks VPD dilution indicating at least 3 replication cycles (quantified in G). (G) Percentage of cells that have undergone at least 3 replication cycles. Replicates from two independent experiments of different donors are pooled (n = 6). Dashed lines indicate individual donor trends. Error bars represent SD. Analysis shown is Šídák’s multiple comparisons test. (H) Intracellular staining with anti-IFN-γ-APC at 18 h following co-incubation. LA CAR induces greater IFN-γ than HA. Monomeric LA CAR has overall lower magnitude of response, but improves the dose response. Box and whiskers plot error bars showing minimum and maximum values (n = 12 samples per group pooled from four independent experiments and two donors). Analyses shown are Šídák’s multiple comparisons tests. (I) LA (blue) and HA (red) CARs were retrovirally expressed in primary mouse CD8+ T cells and co-cultured with MC38 cells expressing low- or high-HER2 for 24 h in normoxia (20% oxygen) before separation into normoxic or hypoxic (1.5% oxygen) cultures for an additional 6 d. Hypoxic co-cultures of HA CAR T cells with MC38-HER2-high cells have significantly more TOX high/PD1+ cells than LA CAR T cells, indicative of development towards an exhausted state. Box and whiskers plot error bars showing minimum and maximum values (n = 9 samples pooled from three independent experiments). Analysis shown is unpaired t test.

Impaired movement of HA dimer CAR microclusters in synapses and reduced effector function. (A and B) TIRF imaging of CAR-mEmerald is shown for synapses with LA or HA CAR interacting with low or high HER2-loaded bilayers (A). Time point shown is 93.6 s following the initiation of imaging, which began as synapses were starting to form. Yellow lines were drawn to span CAR microclusters in synapse, indicating diameter quantified in B. Three lines were averaged and line assignment was blinded to account for manual drawing. For dimeric CARs, only LA:low HER2 synapses result in accumulation of CAR microclusters at the center, indicated by lower diameter. Monomerization of CARs improves centralization on high HER2. Data is shown for at least 7 cells pooled from a minimum two independent experiments per condition (n = 13, 13, 20, 20, 13, 7 cells per group from left to right, respectively). Error bars represent SD. Analyses shown are Šídák’s multiple comparisons tests. Scale bars = 2 μm. (C and D) Spots with tracking were assigned to CAR microclusters in Imaris. Examples of mobile (left, LA dimer on low HER2) and immobile (right, LA dimer on high HER2) microclusters are shown as a flower plot for 10 random tracks (C). Quantification is shown for the average displacement (left) and speed (right) of all CAR microclusters for a given cell (D, n = 3 cells per group, 30–294 tracks per cell). Limited mobility is apparent for dimeric CARs on high-HER2 loaded bilayers. On low-HER2 bilayers, LA CAR microclusters show increased mobility. Error bars represent SD. Analyses shown are Šídák’s multiple comparisons tests. (E) Low HER2-expressing MCF7 (open circle) and high HER2-expressing SKBR3 (solid circle) cell lines were used to assess differences between HER2 levels in cell-cell interactions in vitro. (F) Proliferation is induced across all CAR+ conditions with low and high HER2, as seen by VPD dilution at 96 h following co-incubation. Line marks VPD dilution indicating at least 3 replication cycles (quantified in G). (G) Percentage of cells that have undergone at least 3 replication cycles. Replicates from two independent experiments of different donors are pooled (n = 6). Dashed lines indicate individual donor trends. Error bars represent SD. Analysis shown is Šídák’s multiple comparisons test. (H) Intracellular staining with anti-IFN-γ-APC at 18 h following co-incubation. LA CAR induces greater IFN-γ than HA. Monomeric LA CAR has overall lower magnitude of response, but improves the dose response. Box and whiskers plot error bars showing minimum and maximum values (n = 12 samples per group pooled from four independent experiments and two donors). Analyses shown are Šídák’s multiple comparisons tests. (I) LA (blue) and HA (red) CARs were retrovirally expressed in primary mouse CD8+ T cells and co-cultured with MC38 cells expressing low- or high-HER2 for 24 h in normoxia (20% oxygen) before separation into normoxic or hypoxic (1.5% oxygen) cultures for an additional 6 d. Hypoxic co-cultures of HA CAR T cells with MC38-HER2-high cells have significantly more TOX high/PD1+ cells than LA CAR T cells, indicative of development towards an exhausted state. Box and whiskers plot error bars showing minimum and maximum values (n = 9 samples pooled from three independent experiments). Analysis shown is unpaired t test.

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