T-Cell Receptor Repertoire Sequencing in the Era of Cancer Immunotherapy.

T cells are integral components of the adaptive immune system, and their responses are mediated by unique T-cell receptors (TCR) that recognize specific antigens from a variety of biological contexts. As a result, analyzing the T-cell repertoire offers a better understanding of immune responses and of diseases like cancer. Next-generation sequencing technologies have greatly enabled the high-throughput analysis of the TCR repertoire. On the basis of our extensive experience in the field from the past decade, we provide an overview of TCR sequencing, from the initial library preparation steps to sequencing and analysis methods and finally to functional validation techniques. With regards to data analysis, we detail important TCR repertoire metrics and present several computational tools for predicting antigen specificity. Finally, we highlight important applications of TCR sequencing and repertoire analysis to understanding tumor biology and developing cancer immunotherapies.

Spatial heterogeneity of T cell repertoire across NSCLC tumors, tumor edges, adjacent and distant lung tissues.

Background: A better understanding of T cells in lung cancer and their distribution across tumor-adjacent lungs and peripheral blood is needed to improve efficacy and minimize toxicity from immunotherapy to lung cancer patients. Methods: Here, we performed CDR3ß TCR sequencing of 136 samples from 20 patients with early-stage NSCLC including peripheral blood mononuclear cells, tumors, tumor edges (<1 cm from tumor), as well as adjacent lungs 1 cm, 2 cm, 5 cm, and 10 cm away from the tumor to gain insight into the spatial heterogeneity of T cells across the lungs in patients with NSCLC. PD-L1, CD4, and CD8 expression was assessed using immunohistochemical staining, and genomic features were derived by targeted sequencing of 1,021 cancer-related genes. Multiplex immunohistochemistry against PD-1, CTLA4, LAG3, and TIM3 was performed on four patients to assess T cell exhaustion. Results: Our study reveals a decreasing gradient in TIL Tumor Infiltrating Lymphocytes homology with tumor edge, adjacent lungs, and peripheral blood but no discernible distance-associated patterns of T cell trafficking within the adjacent lung itself. Furthermore, we show a decrease in pathogen-specific TCRs in regions with high T cell clonality and PD-L1 expression. Conclusions: Exclusion in T exhaustion cells at play across the lungs of patients with NSCLC may potentially be the mechanism for lung cancer occurrence.

Combined IL-2, agonistic CD3 and 4-1BB stimulation preserve clonotype hierarchy in propagated non-small cell lung cancer tumor-infiltrating lymphocytes.

BACKGROUND: Adoptive cell transfer (ACT) of tumor-infiltrating lymphocytes (TIL) yielded clinical benefit in patients with checkpoint blockade immunotherapy-refractory non-small cell lung cancer (NSCLC) prompting a renewed interest in TIL-ACT. This preclinical study explores the feasibility of producing a NSCLC TIL product with sufficient numbers and enhanced attributes using an improved culture method. METHODS: TIL from resected NSCLC tumors were initially cultured using (1) the traditional method using interleukin (IL)-2 alone in 24-well plates (TIL 1.0) or (2) IL-2 in combination with agonistic antibodies against CD3 and 4-1BB (Urelumab) in a G-Rex flask (TIL 3.0). TIL subsequently underwent a rapid expansion protocol (REP) with anti-CD3. Before and after the REP, expanded TIL were phenotyped and the complementarity-determining region 3 ß variable region of the T-cell receptor (TCR) was sequenced to assess the T-cell repertoire. RESULTS: TIL 3.0 robustly expanded NSCLC TIL while enriching for CD8+ TIL in a shorter manufacturing time when compared with the traditional TIL 1.0 method, achieving a higher success rate and producing 5.3-fold more TIL per successful expansion. The higher proliferative capacity and CD8 content of TIL 3.0 was also observed after the REP. Both steps of expansion did not terminally differentiate/exhaust the TIL but a lesser differentiated population was observed after the first step. TIL initially expanded with the 3.0 method exhibited higher breadth of clonotypes than TIL 1.0 corresponding to a higher repertoire homology with the original tumor, including a higher proportion of the top 10 most prevalent clones from the tumor. TIL 3.0 also retained a higher proportion of putative tumor-specific TCR when compared with TIL 1.0. Numerical expansion of TIL in a REP was found to perturb the clonal hierarchy and lessen the proportion of putative tumor-specific TIL from the TIL 3.0 process. CONCLUSIONS: We report the feasibility of robustly expanding a T-cell repertoire recapitulating the clonal hierarchy of the T cells in the NSCLC tumor, including a large number of putative tumor-specific TIL clones, using the TIL 3.0 methodology. If scaled up and employed as a sole expansion platform, the robustness and speed of TIL 3.0 may facilitate the testing of TIL-ACT approaches in NSCLC.