UCLA researchers in the Department of Microbiology, Immunology, and Molecular Genetics have developed a novel methodology that enables droplet-based, single-cell sequencing of RNA in parallel with quantification of intracellular phenotype. They performed intracellular staining with subsequent selection of T Cells expressing certain cytokine or transcription factors and cloned out antigen specific T Cell Receptors (TCRs). This is a dramatic advance in multi-omic single-cell analysis and may prove useful for the identification of therapeutic TCRs and evaluation of vaccine and other immune stimulants.
BACKGROUND:
Cancer therapy has followed three main categories historically: surgery, chemotherapy (small molecule therapeutics), and radiotherapy. While these therapies have been successful in a subsect of patient populations, many patients are non-responsive. The ineffectiveness of these treatments largely stems from the inability to selectively target cancer cells: leading to unintended side effects observed during therapy. Recently, much attention has been devoted to the idea of harnessing the immune system to more selectively target cancer cells. The immune system is capable of recognizing and eliminating tumors, relying on cytotoxic T cells that distinguish cancer cells through antigen binding.
Adoptive cell therapy (ACT) is a powerful type of immunotherapy, which involves generation of tumor-specific T cells that can be reinfused into patients to increase T cell responses to cancer. Tumor specific T cells are generated by engineering them to express an immune receptor. A critical bottleneck in the development of ACT is the identification of tumor specific immune receptors, such as a TCR. It can be difficult to generate a tumor specific TCR. Existing techniques focus on physical staining of the TCR or surface activation marker selection after T cell stimulation. These techniques proved to be quite laborious and/or identify non-antigen specific TCRs. Therefore, a current need exists to efficiently clone antigen specific TCRs for the effective harnessing of ACT for oncology.
INNOVATION:
Dr. Owen Witte and colleagues in the Department of Microbiology, Immunology, and Molecular Genetics have developed a novel methodology that enables single cell TCR sequencing in cells that have undergone intracellular staining. Antigen specific T cells are commonly quantified by their expression of TNF⍺ and IFNγ. Furthermore, the T cell phenotype is often defined by the expression of certain transcription factors. However, no protocol for high-throughput, single-cell sequencing post intracellular staining exists. The team defined conditions that allow for highly multiplexed T cell stimulation and subsequent highly specific selection of reactive cells for TCR alpha/beta cloning and reconstruction. This advance may prove useful for potential therapeutic agents and the identification of immune-epitopes. This technology, when coupled with traditional T cell stimulation and priming, promises to be an effective methodology in the development of more potent ACT therapies for both allogeneic tumor treatments as well as strategies for personalized TCR immunotherapy against neoantigens. Furthermore, inventors sequence T regulatory cell TCRs which confirm the utility of the technology in autoimmune diseases. Importantly, the technology has significant implications in other fields, like vaccine development. Development of vaccines for SARS-CoV-2 as well as other pathogens with significant medical need rely on measuring induction of neutralizing antibodies, however, T cell memory is also known to be required for protection against certain pathogens. The methodology reported here can confirm vaccine induction of effective CD4 and CD8 T Cell responses, which can be a validation step for vaccine design in diseases where CD4 and CD8 T cell memory is critical.
APPLICATIONS:
• Cancer Immunotherapy
• Vaccine Development
• Identification of novel reactivities defined by phenotype
• Adoptive cellular therapy
• Kits for intracellular staining that is compatible with downstream single cell gene expression
ADVANTAGES:
• Preserves TCR mRNA post intracellular staining, to allow for TCR reconstruction
• Allows quantification of intracellular phenotype in parallel with single-cell RNA sequencing, leading to more frequent identification of potent TCRs
• Allows for increased selection of antigen specific TCRs compared to current techniques (e.g. pMHC reagents, CD137, CD107)
• Validation of vaccine candidates by showing induction of T cell receptors with potent activity against infected targets
• Multiomic quantification of gene expression and intracellular proteome
DEVELOPMENT-TO-DATE:
The study has been validated in vitro: used to efficiently recover TCR alpha/beta pair mRNA via a droplet-based single-cell sequencing platform from cells that have been fixed and permeabilized. Virus specific full length TCRs have been sequenced based on intracellular TNF⍺ and IFNγ. Furthermore, T regulatory cell TCRs have been sequenced from human T cells identified by FOXP3 transcription factor.
RELATED MATERIALS:
Method to clone human t cell receptors from single lymphocytes based on functional profiling (UCLA reference: 2020-152)