Individual Motile CD4(+) T Cells Can Participate in Efficient Multikilling through Conjugation to Multiple Tumor Cells

Abstract

T cells genetically modified to express a CD19-specific chimeric antigen receptor (CAR) for the investigational treatment of B-cell malignancies comprise a heterogeneous population, and their ability to persist and participate in serial killing of tumor cells is a predictor of therapeutic success. We implemented Timelapse Imaging Microscopy in Nanowell Grids (TIMING) to provide direct evidence that CD4(+)CAR(+) T cells (CAR4 cells) can engage in multikilling via simultaneous conjugation to multiple tumor cells. Comparisons of the CAR4 cells and CD8(+)CAR(+) T cells (CAR8 cells) demonstrate that, although CAR4 cells can participate in killing and multikilling, they do so at slower rates, likely due to the lower granzyme B content. Significantly, in both sets of T cells, a minor subpopulation of individual T cells identified by their high motility demonstrated efficient killing of single tumor cells. A comparison of the multikiller and single-killer CAR(+) T cells revealed that the propensity and kinetics of T-cell apoptosis were modulated by the number of functional conjugations. T cells underwent rapid apoptosis, and at higher frequencies, when conjugated to single tumor cells in isolation, and this effect was more pronounced on CAR8 cells. Our results suggest that the ability of CAR(+) T cells to participate in multikilling should be evaluated in the context of their ability to resist activation-induced cell death. We anticipate that TIMING may be used to rapidly determine the potency of T-cell populations and may facilitate the design and manufacture of next-generation CAR(+) T cells with improved efficacy.

Figure 1. High-throughput single-cell analysis of CAR⁺ T-cell cytolytic functionality in nanowell grids

(A) Schematic of second-generation CD19-specific CAR (CD19RCD28) that signals through chimeric CD28/CD3-ζ. (B) Phenotypic characterization of the CAR⁺ T cells from two separate donors. The total CD3⁺CAR⁺ population was gated to reveal the frequencies of CD4⁺ and CD8⁺ CAR⁺ T-cell populations. (C) Representative composite micrographs illustrating the ability of single CAR⁺ T cells to kill, and to undergo apoptosis, when incubated with tumor cells confined within nanowells. Scale bar 50 µm. (D) Comparison of the cytolytic responses measured by the single-cell assay and population-level ⁵¹Cr release assay, at an E:T ratio of 1:1. The numbers in parentheses for the single-cell assay report the total number of events observed. (E) Donut plots summarizing the frequency of killing outcomes of the interaction between CAR⁺ T cells, derived from these two donors, and CD19⁺EL4 target cells. Representative micrographs illustrating each of these interactions are shown in Figure S5.

Figure 2. CAR8 cells can be classified into different subgroups based on their the motility and conjugation periods with NALM-6 tumor cell (E:T 1:1)

Schematic depicting the effector parameters used to describe their interaction with single NALM-6 tumor cells: (A) Red bar indicates periods of conjugation, blue arrow indicates timepoint at which conjugation was first observed, and green line indicates time to target death since first conjugation. (B) Aspect ratio of polarization describes the ratio of major and minor axis fitted to an ellipse. (C) d_Well represents the average displacement of the centroid of the effector cell between successive seven minute time points. The mean: (D) motility, (E) time to first conjugation, and (F) killing efficiency, of single CAR8 cells in each of three different subgroups. Each circle represents a single cell. P-values for multiple comparisons were computed using parametric one-way ANOVA.

Figure 3. Multi-killer CAR8 cells engage in simultaneous conjugations leading to multiplexed killing (E:T 1:2-5)

(A) Distribution of the number of simultaneous conjugations of individual CAR8 cells when incubated with increasing number of NALM-6 tumor cells. The mean: (B) motility, (C) time to first conjugation, and (D) killing efficiency, of individual multi-killer CAR8 cells. P-values for multiple comparisons were computed using parametric one-way ANOVA.

Figure 4. Subpopulation of CAR4 cells, identified based on their motility, can engage in efficient killing (E:T 1:1)

(A) Phenotypic characterization of the CAR⁺ T cells from two separate donors that comprise of predominantly CD4⁺CAR⁺ T cells. The mean: (B) motility, and (C) killing efficiency, of single CAR4 cells in each of three different subgroups. (D) Comparison of the means of the killing efficiencies between single CAR8 and CAR4 cells within the S1 subgroups. Each circle represents a single cell in panels B-D; CAR4 cells are represented using grey circles and CAR8 cells are represented using black circles. (E) Comparative Kaplan –Meier estimators depicting the differences in killing efficiencies of the entire population of CAR4 cells and CAR8 cells. P-values for multiple comparisons (B/C) were computed using a parametric one-way ANOVA, and dual comparisons (D/E) computed using unpaired two-tailed t-test.

Figure 5. Multi-killer CAR4 cells demonstrated delayed kinetics of killing in comparison to CAR8 cells (E:T 1:2-5)

Comparisons between the mean: (A) motility, and (B) killing efficiency, of single multi-killer CAR8 cells and CAR4 cells. Each circle represents a single cell; CAR4 cells are represented using grey circles and CAR8 cells are represented using black circles. (C) Box and whisker plots (extremities indicate 99% confidence intervals) displaying intracellular expression of Granzyme B identified by immunofluorescent staining and flow-cytometry. CAR4 cells (from donors PB5858 and PB333038) and CAR8 cells (from donors PB243566 and PB281848) were profiled using mAb against CD4/CD8/CAR and GzB. P-values were computed using parametric one-way ANOVA for multiple comparisons or t-tests for dual comparisons. (D) Flow cytometric killing assay (E:T = 5:1) of CAR4 cells incubated with three separate target cell lines (Daudi-β2m, NALM-6 and CD19⁺EL4) in the absence or presence of 5mM EGTA blockade.

Figure 6. Frequency and kinetics of killer-cell apoptosis are dependent on functional conjugations with multiple NALM-6 tumor cells

(A) Comparisons of the mean kinetics of effector apoptosis of individual single killer CAR⁺ T cells (E:T 1:1) with multi-killer CAR⁺ T cells (E:T 1:2-5). Each circle represents a single-cell; CAR4 cells are represented using grey circles and CAR8 cells are represented using black circles. (B) Frequency of killer-cell apoptosis as a function of tumor cell density.