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Granulomatous arteritis characterizes the pathology of giant cell arteritis, granulomatous aortitis, and intracerebral varicella zoster virus (VZV) vasculopathy. Because intracerebral VZV vasculopathy and giant cell arteritis are strongly associated with productive VZV infection in cerebral and temporal arteries, respectively, we evaluated human aortas for VZV antigen and VZV DNA. Using 3 different anti-VZV antibodies, we identified VZV antigen in 11 of 11 aortas with pathologically verified granulomatous arteritis, in 1 of 1 cases of nongranulomatous arteritis, and in 5 of 18 control aortas (28%) obtained at autopsy. The presence of VZV antigen in granulomatous aortitis was highly significant (P = .0001) as compared to control aortas, in which VZV antigen was never associated with pathology, indicating subclinical reactivation. VZV DNA was found in most aortas containing VZV antigen. The frequent clinical, radiological, and pathological aortic involvement in patients with giant cell arteritis correlates with the significant detection of VZV in granulomatous aortitis.

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. Based on 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.

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BackgroundAdoptive 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.MethodsTIL 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 β variable region of the T-cell receptor (TCR) was sequenced to assess the T-cell repertoire.ResultsTIL 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.ConclusionsWe 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.