A kinetic investigation of interacting, stimulated T cells identifies conditions for rapid functional enhancement, minimal phenotype differentiation, and improved adoptive cell transfer tumor eradication

<div><p>For adoptive cell transfer (ACT) immunotherapy of tumor-reactive T cells, an effective therapeutic outcome depends upon cell dose, cell expansion <i>in vivo</i> through a minimally differentiated phenotype, long term persistence, and strong cytolytic effector function. An incomplete understanding of the biological coupling between T cell expansion, differentiation, and response to stimulation hinders the co-optimization of these factors. We report on a biophysical investigation of how the short-term kinetics of T cell functional activation, through molecular stimulation and cell-cell interactions, competes with phenotype differentiation. T cells receive molecular stimulation for a few minutes to a few hours in bulk culture. Following this priming period, the cells are then analyzed at the transcriptional level, or isolated as single cells, with continuing molecular stimulation, within microchambers for analysis via 11-plex secreted protein assays. We resolve a rapid feedback mechanism, promoted by T cell—T cell contact interactions, which strongly amplifies T cell functional performance while yielding only minimal phenotype differentiation. When tested in mouse models of ACT, optimally primed T cells lead to complete tumor eradication. A similar kinetic process is identified in CD8⁺ and CD4⁺ T cells collected from a patient with metastatic melanoma.</p></div

<i>In vivo</i> antitumor efficacy as a function of T₁ and molecular stimulation in an ACT immunotherapy model.

<p><b>A</b>. The mouse model ACT regimen that uses OT1 tumor antigen specific CD8⁺ T cells. <b>B</b>. Tumor growth curve (mean with error bars as s.e.m.) with the influence of the T₁ priming period. Five conditions are shown: a buffer solution control (PBS), T cells with no stimulation, and T cells with molecular stimulation (OT1 tetramer + anti-CD28) for varying T₁ periods. The figure represents one independent experiment with 5 mice per condition. The data are representative of two to three independent experiments (9–13 mice total per condition) with similar results. The inset chart provides statistical measures of the responses for the different conditions (n.s. = not significant, ** P < 0.005, * P < 0.05) <b>C</b>. Comparison between weak (anti-CD28 + anti-CD3) and strong (OT1 tetramer + anti-CD28), with a constant T₁ (= 16 hours) prior to ACT. PBS is the negative control. Representative luciferase images of tumor at day 3 following ACT, at day 12 for the negative control and at day 12 following ACT for the strong stimulation condition. Similar images, for strong stimulation, and showing the absence of tumor, we collected at day 25. (Enlarged versions of these images are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191634#pone.0191634.s018" target="_blank">S13 Fig</a>). The inset chart provides statistical measures of the responses for the different treatment conditions, as assessed by a Kruskal-Wallis non-parametric comparison (n.s. = not significant, ** P < 0.005, * P < 0.05). This figure represents one independent experiment with 4 mice per condition, and is representative of two to three independent experiments (8–13 mice total per condition) with similar results. (<b>D-E</b>) Representative images of fluorescent Tunel tissue staining (<b>D</b>) and CD137 staining (<b>E</b>) assay 4 days after ACT. (top) ACT with OT1 T cells with a 16-hour T₁ plus OT1 tetramer + anti-CD28 stimulation; (middle) with OT1 T cells with no stimulation; and (bottom) negative control (no ACT). The white line in (<b>D</b>) delineates the periphery of the tumor. Scale bar is 100 μm for Tunel staining and 50 μm for CD137 staining. <b>F</b>. Quantitation of staining and flow cytometry assays. (top) An intensity threshold is used to quantify Tunel staining of EG7 tumor cells (green bars), which is plotted with the average number (red bars) and size (middle graph) of CD137⁺ cells. Each histogram was based on 4 fields per section (<i>n</i> = 4–6 histological sections per animal; 4–5 animals per group). (bottom) Flow cytometry analysis of OT1 T cells <i>in vivo</i>. The percentage of CD8⁺ OT1 T cells among T cells in PBMC (blue) and in TILs (orange) are displayed. The x-axis labels apply to all plots. Values plotted are mean ± s.e.m (* <i>P</i> < 0.05, *** <i>P</i> < 0.001).</p

The effect of T₁ priming on T cells extracted from the peripheral blood of a melanoma cancer patient.

<p>Strong stimulation (PMA + ionomycin + anti-CD23 + anti-CD28) over a variable T₁ period increases the functionality and polyfunctionality of (<b>A</b>) CD8⁺ and (<b>B</b>) CD4+ T cells. The total time T = T₁+T₂ is held constant at 13 hours. <b>C</b>. The percentage of different phenotypes (described in detail in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191634#sec009" target="_blank">Materials and methods</a>) as a function of T₁ incubation period analyzed from multi-color flow cytometry of CD8⁺ T cells shows loss of the naïve phenotype, but no evidence of exhaustion or terminal differentiation. <b>D</b>. The expression level of naïve-associated and effector-associated genes as a function of T₁ for CD8+ T cells.</p

The short-time kinetics of OT-1 TCR-specific CD8+ T cell functional activation and phenotype differentiation.

<p><b>A</b>. Experimental design. T cells are stimulated in bulk culture for a time period T₁, during which those cells interact with each other. The supernatant is then flushed, and the cells are analyzed for phenotype using multi-color flow cytometry, transcriptome analysis, or they are resuspended and isolated, in the presence of molecular stimulant, within the microchambers of an SCBC, for a time T₂. During T₂, secreted proteins are captured by antibody arrays patterned within each microchamber. The total molecular stimulation time (T = T₁ + T₂) is constant. <b>B</b>. Protein secretion levels from single OT1 antigen-specific CD8+ T cells as T₁ is increased from 10 minutes (0.2 hrs) to 16 hours. The y-axis is the pSI, calculated for cells secreting 1, 2, 3, or more proteins, out of 11 measured. <b>C</b>. Flow cytometry measures of the expression of CD62L vs. CD44 or KLRG1 as T₁ increases from non-stimulated (n.s.) to 4 hrs to 16 hrs. <b>D</b>. The expression levels of naïve-associated and effector-associated genes as a function of increasing T₁.</p

Correlations of T cell aggregation and T cell functionality increase during the T₁ conditioning period, and a proposed associated mechanism.

<p><b>A</b>. Micrographs showing the kinetics of the aggregation of OT-1 T cells following tetramer stimulation, over the course of T₁, for cell densities of 2×10⁵ and 5×10⁵ cells/cm³. For the higher density culture, small, 2D cell aggregates are observed after T₁ = 5 hours (arrow), while 3D aggregates are observed by T₁ = 16 hours (arrow). At the lower cell density, 2D aggregates are observed by T₁ = 16 hours (arrow). Scale bar = 200 μm. <b>B</b>. Dynamics of the production of the CCL3 and IL2 cytokines following tetramer stimulation of OT-1 T cells, as cell density is varied. In both cases, CCL3 production precedes IL2 production, and the production of both proteins ramps up more quickly at higher cell density. <b>C</b>. Fluorescent micrographs showing the staining of CD25 (the IL2 receptor, in red) for non-stimulated (n.s.) (left column) and tetramer (and CD28) stimulated OT-1 T cells (right column), after T₁ = 16 hours. The cells were co-stained with the DAPI nuclear stain. Scale bar = 20 μm. <b>D</b>. Quantitation of the CD25 staining assays, measured in fluorescence intensity per cell (mean values ± s.e.m), using a threshold (*** <i>P</i> < 0.001, * <i>P</i> < 0.05). Statistics are based on 70–150 cells per condition. <b>E</b>. Drawing illustrating the dynamics of functional activation. The initial molecular stimulation promotes cell motility, via prompting CCL3 and CCL4 secretion. This, in turn, promotes increased contact interactions between T cells. Those interactions amplify the stimulation effects, leading to enhanced cell motility and additional contacts, in a fashion similar to a positive feedback loop. The feedback loop is established within the first one or two hours following molecular stimulation.</p