SUMMARY:
UCLA researchers from the Department of Microbiology, Immunology, and Molecular Genetics have developed a novel method to clone human T cell receptors from single lymphocytes.
BACKGROUND:
Immunotherapy is a growing therapy type for the treatment of disease, especially cancer, by activating or repressing the immune system. One major type of immunotherapy is Adoptive Cell Transfer (ACT). In ACT, peripheral blood monocyte cells (PBMCs) are harvested from a patient, modified to increase tumor cell recognition, and then re-introduced into the patient. The critical step of harvesting PBMCs from the patient is the lower incidence of treatment rejection: where several alternative immunotherapy options have had issues with successful adoption in the clinic. Currently, a critical bottleneck in the ACT treatment process is the isolation of T cells from the patient with a high affinity to the disease antigen. In this step, isolation of the mRNA sequence of the T cell receptor (TCR) that encodes the high affinity for targeted cells of the therapy must be done in such a way as not to degrade the sequence. While many potential methods in this process have been proposed, many are low throughput and not clinically translatable. Once the mRNA encoding the TCR is isolated, this sequence can be used to modify other T cells to selectively target cells: enriching the number of T cells that can effectively target and ablate cancer cells. Therefore, a desperate need exists to isolate cell-specific TCR sequences for the effective clinical translation of ACT.
INNOVATION:
UCLA researchers have developed a novel method to perform intracellular staining of T cells while preserving human TCR mRNA quality at the single-cell level. This technique allows for single-cell FACS sorting of human cytokine-producing cells, followed with TCR mRNA paired alpha and beta sequencing. UCLA researchers are able to generate cDNA sequences covering the variable regions of the TCR to allow reconstruction of the full-length TCRs and evaluate the function of individual clones in healthy donor PBMCs.
POTENTIAL APPLICATIONS:
• Cancer immunotherapy
• Discovery of novel reactivities defined by phenotype
ADVANTAGES:
• Preserves TCR mRNA
• Allows for analysis of up to 18 transcription factors and cytokines, depending on the FACS
machine
DEVELOPMENT-TO-DATE:
This approach has been confirmed with multiple peptide antigens, including well-described viral epitopes from cytomegalovirus and Epstein-Barr virus. UCLA researches have shown they can capture human, intracellularly stained T cells and single-cell sequence TCR alpha and beta pair cDNA. Identified clones have been cloned into retrovirus vectors and tested for reactivity to the cognate peptide in donor PBMCs. TCR clones have been tested for the ability to kill target cell lines expressing full-length CMB protein. Derived clones successfully recognize endogenous processed peptide and kill target cell lines at an efficiency comparable to clinical-grade TCRs used in adoptive cell therapies.
Related Papers (from the inventors only)
Thomsen, E. R. et al. Fixed single-cell transcriptomic characterization of human radial glial diversity. Nat. Methods 13, 87–93 (2016)