Designed improvement to T-cell immunotherapy by multidimensional single cell profiling

Abstract

BackgroundAdoptive cell therapy based on the infusion of chimeric antigen receptor (CAR) T cells has shown remarkable efficacy for the treatment of hematologic malignancies. The primary mechanism of action of these infused T cells is the direct killing of tumor cells expressing the cognate antigen. However, understanding why only some T cells are capable of killing, and identifying mechanisms that can improve killing has remained elusive.MethodsTo identify molecular and cellular mechanisms that can improve T-cell killing, we utilized integrated high-throughput single-cell functional profiling by microscopy, followed by robotic retrieval and transcriptional profiling.ResultsWith the aid of mathematical modeling we demonstrate that non-killer CAR T cells comprise a heterogeneous population that arise from failure in each of the discrete steps leading to the killing. Differential transcriptional single-cell profiling of killers and non-killers identified CD137 as an inducible costimulatory molecule upregulated on killer T cells. Our single-cell profiling results directly demonstrate that inducible CD137 is feature of killer (and serial killer) T cells and this marks a different subset compared with the CD107apos (degranulating) subset of CAR T cells. Ligation of the induced CD137 with CD137 ligand (CD137L) leads to younger CD19 CAR T cells with sustained killing and lower exhaustion. We genetically modified CAR T cells to co-express CD137L, in trans, and this lead to a profound improvement in anti-tumor efficacy in leukemia and refractory ovarian cancer models in mice.ConclusionsBroadly, our results illustrate that while non-killer T cells are reflective of population heterogeneity, integrated single-cell profiling can enable identification of mechanisms that can enhance the function/proliferation of killer T cells leading to direct anti-tumor benefit.

Multidimensional single-cell analysis identifies a role of CD2-CD58 interactions for clinical antitumor T cell responses

“…Unlike the small numbers of infusion product T cells available for profiling, 19-28z CAR T cells manufactured from healthy donors can be assayed in larger numbers and we next profiled two preclinical 19-28z CAR T cells products (>95% CAR + CD8 + ), specifically focusing on serial killing (17). We identified 1,178 nanowells of interest populated with a single live T cell, 2 to 5 NALM-6 tumor cells, and one or more beads.…”

“…We set up a TIMING assay with CAR T cells, without the confounding influence of tumor cells to record basal migration. Single migratory or non-migratory T cells were retrieved ( Figure 2a, Supplementary Video 2 & 3 ), and we performed direct quantitative PCR (qPCR)-based amplification since it has higher sensitivity than single-cell RNA-sequencing (17). Seven genes were differentially expressed between the two groups: CXCR3 and IL18R1 (chemokine and cytokine receptors); LAG3, CD244, CD58, and CD2 (inhibition/activation receptors) were upregulated in migratory T cells; whereas CD69 (activation marker) was down-regulated in migratory T cells ( Figure 2b ).…”

“…We generated CAR T cells as previously described (17). Briefly, we activated PBMCs using OKT3 and anti CD28 antibodies for 48 hours.…”

Cytotoxic T Lymphocytes Targeting a Conserved SARS-CoV-2 Spike Epitope are Efficient Serial Killers

“…More broadly, TIMING is an integrated platform that leverages advances in microfabrication, microscopy and AI to enable the profiling of thousands of time-lapse microscopy experiments in parallel. As we have shown previously, the ability to integrate the dynamic profile with downstream clonal expansion or linked transcriptional profiling makes TIMING the only platform capable of generating dynamic multiomic datasets [ 18 , 19 ].…”

Cytotoxic T lymphocytes targeting a conserved SARS-CoV-2 spike epitope are efficient serial killers

“…More broadly, TIMING is an integrated platform that leverages advances in microfabrication, microscopy and AI to enable the profiling of thousands of timelapse microscopy experiments in parallel. As we have shown previously, the ability to integrate the dynamic profile with downstream clonal expansion or linked transcriptional profiling makes TIMING the only platform capable of generating dynamic multi-omic datasets 16,17 .…”