High-Throughput Single-Cell Functional and Molecular Profiling of Immune Cells in Cancer Immunotherapy

Immunotherapy has revolutionized the treatment of cancer and newer approaches including the adoptive transfer of genetically modified T cells reprogrammed to target tumor antigens have shown remarkable responses. Despite their promise, the efficacy of adoptive immunotherapy remains unpredictable due to the heterogeneous nature of the infusion products, patients’ characteristics, treatment regimens, and tumor burdens. Specifically with regards to the T-cell infusion product, there is a need to develop methodologies that allow for definition of potencies to understand the phenotypic, molecular, and functional contribution of infusion products at single-cell level. In the first part of this dissertation, we implemented Timelapse Imaging Microscopy in Nanowell Grids (TIMING) to demonstrate that while CD4+CAR+ (CAR4) cells killed at slower rate, most likely due to lower granzyme B content, they benefited from apoptosis resistance compared to CD8+CAR+ (CAR8) cells. These findings suggest that overall potency of multi-killing should be evaluated together in their context to resist apoptosis. In the second part of this dissertation, we developed single-cell multiplexed platforms comprising beads biosensors for detecting protein secretion, TIMING to monitor motility and cell-cell interactions, and microfluidic qPCR for transcriptional profiling. Analysis of thousands of single-cell interactions for over 5 hours revealed that the integrated behavior of polyfunctional T cells that kill and secrete IFN-γ was similar to those without IFN-γ secretion, suggesting cytolysis to be the dominant determinant of the interaction behavior and that killing enables faster synapse termination. In addition, tracking the speed of these cells by TIMING indicated that serial killer T cells may be identified based on their high out-of-contact basal motility. Transcriptional profiling of these single-cells confirmed that the motile cells expressed increased amounts of perforin and displayed an activated phenotype. In summary, these results highlight the heterogeneity of immune cells and thus, the need for definition of potency prior to infusion. We propose that single-cell platforms as demonstrated here are suitable to uncover the diversity and to help identify optimal functional and molecular biomarkers for applications in the clinic.

Name thesis:degree_name

Doctor of Philosophy

Level thesis:degree_level

Doctoral

Discipline thesis:degree_discipline

Chemical Engineering

Grantor

University of Houston

Year dc:date.issued

2015

Author dc:creator

• Liadi, Ivan

Advisor dc:contributor.advisor

• Varadarajan, Navin

Committee members dc:contributor.committeemember

• Willson, Richard C.
• Donnelly, Vincent M.
• Roysam, Badrinath
• Zhang, Xiaoliu Shaun

• Single cell
• TIMING
• High-throughput
• CAR T cells
• Cytokine
• Single-cell gene expression
• Beads

Statement dc:rights

• The author of this work is the copyright owner. UH Libraries and the Texas Digital Library have their permission to store and provide access to this work. UH Libraries has secured permission to reproduce any and all previously published materials contained in the work. Further transmission, reproduction, or presentation of this work is prohibited except with permission of the author(s).

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/10657/1929

OAI identifier oai:identifier

oai:uh-ir.tdl.org:10657/1929