Single Cell Immune Profiling Reveals Distinct T Cell Clones and Functional States in in-Vitro Expanded Cord Blood Derived Gamma Delta T Cells
Authors: Tan Kar Wai , Li Zhihui , Lai Joey , Guo Dianyan , Linn Yeh Ching , Hwang William YK , Li Shang , Eaves Connie J. , Goh Yeow Tee , Cheung Alice 
Published: February 2021

Gamma-delta (γδ) T cells represent a special class of unconventional T cells defined by their expression of the somatically rearranged T cell receptor (TCR) γ and δ chains. Unlike TCRαβ, it has been reported that different TCRγδ are also able to bind to their antigens in the context of non-classical MHC-like molecules or totally independent of the MHC complexes. Additionally, a variety of NK receptors are known to be expressed by γδ T cells, conferring their ability to sense alternative classes of cancer-associated antigens in a multimodal manner. Such a diverse mode of antigen recognition possibly endows γδ T cells with a wide spectrum of functional activation program.
Our team had previously explored the potential of expanding cord blood (CB) derived γδ 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 γδ 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 TCRγδ clonotypes, representing 18.6% of the starting 4,000 FACS purified γδ T cells seeded for expansion. The largest 10% of the clones was found to make up 60.9% of the total retrieved cells, demonstrating a significant extent of clonal focusing in our expansion cultures. Consistent to our FACS analysis, Vδ1 is the predominant TRD chain in the expanded cultures, accounting for 61.2% of all clones. Vγ4 is the most prevalent TRG chain making up to 24.9% of all clones regardless of the paired Vδ subtype. Notably, however, the largest γδ T cell clone did not utilize Vγ4, indicating that Vγ4 clones, although frequent, are not the most proliferative clone. These data are supportive of the adaptive characteristics of CB-gdTs, likely in a TCRγδ dependent manner.
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 γδ T cells: the Proliferative, Cytotoxic γδ T cells (P-CT), Differentiated Cytotoxic γδ T cells (D-CT) and Late Activated Cytotoxic γδ 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 γδ T cell differentiation and activation in culture, we found that in 85 (11.5%) of the γδ T cell clones bearing more than 10 cells each, all clones contain cells distributed across the 3 different γδ T cell subsets. Further analysis did not reveal any relationship between the relative proportion of the subsets within each clone with clone size nor any specific type of delta/gamma chain.
Taken together, our high-resolution transcriptome analysis suggests that as CB-gdT expand and differentiate in culture, they are likely to adopt dynamic memory and signal -specific functional programs. More importantly, our data highlights the rich clonal and cellular composition of in vitro expanded CB-gdT. These unique characteristics of our CB-gdT can overcome the challenges of tumor heterogeneity and cell persistence, with the potential of improving outcomes in cell immunotherapy.

Disclosures
Tan: Tessa Therapeutics Ltd: Current Employment.