Single Cell Immune Profiling of in vitro expanded cord blood derived gamma-delta T cells (CB-gdT)

Our team had previously explored the potential of expanding cord blood (CB) derived ?d T cells (CB-gdT) as well as their corresponding ability to target primary acute myeloid leukemia (AML) cells. Using a feeder cell line-based in vitro expansion protocol, we achieved a clinically relevant scale expansion of ?d T cells over a period of 14 days. These cells exhibit variable degree of potency against a range of human AML cell lines and primary patient samples. In order to dissect the cellular and molecular programs governing the activation, differentiation and functional states of our in vitro expanded CB-gdT, we performed multiplex single cell sequencing analysis using the 10X Genomics Chromium System. After initial quality check and filtering, data from a total of 4,276 cells were retrieved. Among which, we identified 742 unique TCR?d clonotypes, representing 18.6% of the starting 4,000 FACS purified ?d T cells seeded for expansion. Consistent to our FACS analysis, Vd1 is the predominant TRD chain in the expanded cultures, accounting for 61.2% of all clones. Based on uniform manifold approximation and projection for dimension reduction (UMAP), all cells were clustered into 11 subsets. Key cytotoxic genes including GZMB, GZMA and NKG7 were all highly expressed across all clusters, indicating that the expanded cells were indeed functionally cytotoxic. Comparing against multiple curated gene sets, we have identified 3 main subsets of ?d T cells: the Proliferative, Cytotoxic ?d T cells (P-CT), Differentiated Cytotoxic ?d T cells (D-CT) and Late Activated Cytotoxic ?d T cells (LA-CT). P-CT (~46% of all cells) shows an expression profile positively associated with cell proliferation as well as increased cell surface expression of memory T cell markers CD27, CCR7 and CD62L. Similar to cytotoxic genes, genes associated with TCR signaling and interferon response were found to be expressed across all cell clusters, yet with elevated levels in D-CT and LA-CT. Furthermore, cell surface expression of different NK receptors including NKG2D, DNAM1 and NKp30 are more enriched in LA-CT compared to the other 2 subsets, suggesting the acquisition of additional NK receptor related functions in this group of cells. Consistent with the concept of progressive ?d T cell differentiation and activation in culture, we found that in 85 (11.5%) of the ?d T cell clones bearing more than 10 cells each, all clones contain cells distributed across the 3 different ?d T cell subsets. This is the first report on single cell immune profiling of in vitro expanded gdT cells derived from human CB.