Background
T cells have emerged as a promising candidate for cancer immunotherapy due to major histocompatibility complex (MHC) independence, epithelial tissue residency and cytotoxic activity.1 T cell receptors undergo V(D)J recombination similarly to αβT. Single cell studies show distinct functional and temporal properties based on variable gene usage; the most abundant subtypes are V1 and V2.2 3 Demonstrating the posited mechanism of anti-tumor T cells in formalin fixed paraffin embedded (FFPE) biopsies is difficult due to the sparsity of cells in the tumor microenvironment (TME) and lack of V gene subtyping reagents. We applied the exquisite selectivity of NanoString GeoMx to transcriptionally profile T cells in solid and hematological malignancies and developed a complementary in-situ RNA hybridization (ISH) assay for elucidating V gene usage.
Methods
T cells were identified with fluorescently tagged antibodies for T cell receptor (H-41, Santa Cruz Biotechnology) and CD3 (UMAB54, OriGene). Using the GeoMx instrument T cells (CD3+ TCR+) and adjacent αβ T cells (CD3+ TCR–) were profiled using a whole genome panel that included TCR V and J genes. Tumor microarrays (TMA) and biopsies of lymphoma, breast, head and neck, and prostate cancers were screened for T cells using an immunohistochemical assay; digital pathology was used to count cells. Custom ISH probes (BaseScope, ACD) were designed targeting V1, V2, and V9 and used in a chromogenic assay to subtype T cells.
Results
Fluorescence based cell segmentation on the GeoMx instrument successfully differentiated rare T from abundant αβ T cells in the TME (figure 1). Differential gene expression analysis showed expected T cell genes such as TRDC, TRDV1, TRVD2, GNLY, and KLRC2, higher in T cells, whereas TRBC1, CD8A and CD4 were higher in αβ T cells. In solid tumors V-gene usage estimates from GeoMx showed V1, V2 and V9 were abundant as expected;4 interestingly some patients had multiple V subunits expressed. Diffuse large B-cell lymphoma (DLBCL) samples contained both V1 and V2; a recent study shows tumor cell of origin may underly this distribution.5 ISH staining was used to confirm the subtype at the single cell level.
Conclusions
V-gene subtyping and transcriptomic profiling of rare T cells from FFPE tumor samples was achieved using a positive fluorescent selection strategy. This technique has the potential to uncover genes in T cells associated with anti-tumor effects, guiding therapeutic target discovery and patient stratification.
Abstract 1382 Figure 1
Example fluorescence strategy for T cell selection. T cells were selected for transcriptional profiling in DLBCL tissue using fluorescently labeled antibodies directed against TCR and CD3. The GeoMx platform enables specific, rare cell profiling from a complex tumor tissue
References
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