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.
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.
BackgroundWhile immune checkpoint blockade is regarded as standard of care for treatment of non-small cell lung cancer (NSCLC), up to 50% of patients with metastatic NSCLC do not achieve an optimal response.1–3 Previous work by our group and others in adoptive cell therapy (ACT) of metastatic melanoma (MM) has shown that infusion of a CD8+-rich TIL product significantly improved clinical outcomes, yet traditional IL-2 expansion methods have resulted in a predominantly CD4+ NSCLC TIL expansion product.7–12 This preclinical study explores the feasibility of producing a tumor-specific, CD8+-enriched NSCLC TIL product for ACT with an improved culture method.MethodsTIL from resected NSCLC tumors were cultured using 1) the traditional method using 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). Expanded TIL were phenotyped using flow cytometry for CD4 and CD8 subset assessment and the CDR3-beta variable region of the T-cell receptor (TCR) involved in antigen binding was sequenced to assess the T-cell repertoire.ResultsIn a shorter manufacturing time (median of 14 days vs 27.5 days), TIL 3.0 expanded on average 5.3-times more NSCLC TIL (95% CI= 4.3–6.2, p<0.0001) and achieved a higher expansion success rate than the traditional TIL 1.0 method (100% vs 62.5%, respectively, p<0.0001). Additionally, TIL 3.0 greatly enriched for CD3+CD8+ TIL (81.8% vs 36.9%, p=0.001) and expanded a larger breadth of clonotypes (p=0.039) which shared greater homology with the total clonotypes found in the repertoire of the resected tumor (p=0.0007), and contained a greater fraction of the clones found at high frequency in the tumor (p<0.00001). TIL 3.0 also retained a higher proportion of putative tumor-specific TCR when compared to TIL 1.0 (p=0.0039), defined based on exclusion of known viral-specific TCR and other TCR found in the paired uninvolved lung tissue.ConclusionsThis study reports the feasibility of using the TIL 3.0 methodology to robustly expand a CD8+ T-cell repertoire which maintains the respective clonal hierarchy in NSCLC tumors and enriches for putative tumor-specific TIL clones. The robustness and speed of the new process may facilitate testing and implementing effective TIL ACT in NSCLC.ReferencesGaron EB, Rizvi NA, Hui R, Leighl N, Balmanoukian AS, Eder JP, et al. Pembrolizumab for the treatment of non-small-cell lung cancer. N Engl J Med 2015;372(21):2018–28.Borghaei H, Paz-Ares L, Horn L, Spigel DR, Steins M, Ready NE, et al. Nivolumab versus Docetaxel in Advanced Nonsquamous Non-Small-Cell Lung Cancer. N Engl J Med 2015;373(17):1627–39.Gettinger S, Horn L, Jackman D, Spigel D, Antonia S, Hellmann M, et al. Five-Year Follow-Up of Nivolumab in Previously Treated Advanced Non-Small-Cell Lung Cancer: Results from the CA209–003 Study. J Clin Oncol 2018;36(17):1675–84.Melioli G, Ratto G, Guastella M, Meta M, Biassoni R, Semino C, et al. Isolation and in vitro expansion of lymphocytes infiltrating non-small cell lung carcinoma: functional and molecular characterisation for their use in adoptive immunotherapy. Eur J Cancer 1994;30A(1):97–102.McGranahan N, Furness AJ, Rosenthal R, Ramskov S, Lyngaa R, Saini SK, et al. Clonal neoantigens elicit T cell immunoreactivity and sensitivity to immune checkpoint blockade. Science 2016;351(6280):1463–9.Rosenberg SA, Yang JC, Sherry RM, Kammula US, Hughes MS, Phan GQ, et al. Durable complete responses in heavily pretreated patients with metastatic melanoma using T-cell transfer immunotherapy. Clin Cancer Res 2011;17(13):4550–7.Besser MJ, Shapira-Frommer R, Treves AJ, Zippel D, Itzhaki O, Hershkovitz L, et al. Clinical responses in a phase II study using adoptive transfer of short-term cultured tumor infiltration lymphocytes in metastatic melanoma patients. Clin Cancer Res 2010;16(9):2646–55.Pilon-Thomas S, Kuhn L, Ellwanger S, Janssen W, Royster E, Marzban S, et al. Efficacy of adoptive cell transfer of tumor-infiltrating lymphocytes after lymphopenia induction for metastatic melanoma. J Immunother 2012;35(8):615–20.Radvanyi LG, Bernatchez C, Zhang M, Fox PS, Miller P, Chacon J, et al. Specific Lymphocyte Subsets Predict Response to Adoptive Cell Therapy Using Expanded Autologous Tumor-Infiltrating Lymphocytes in Metastatic Melanoma Patients. Clinical Cancer Research 2012;18(24):6758–70.Forget MA, Haymaker C, Hess KR, Meng YJ, Creasy C, Karpinets T, et al. Prospective Analysis of Adoptive TIL Therapy in Patients with Metastatic Melanoma: Response, Impact of Anti-CTLA4, and Biomarkers to Predict Clinical Outcome. Clin Cancer Res 2018;24(18):4416–28.Ben-Avi R, Farhi R, Ben-Nun A, Gorodner M, Greenberg E, Markel G, et al. Establishment of adoptive cell therapy with tumor infiltrating lymphocytes for non-small cell lung cancer patients. Cancer Immunol Immunother 2018;67(8):1221–30.Ma Y, Ou J, Lin T, Chen L, Wang J, Qiao D, et al. Phenotypic analysis of tumor-infiltrating lymphocytes from non-small cell lung cancer and their potential application for adoptive cell therapy. Immunopharmacol Immunotoxicol 2020;42(4):319–29Ethics ApprovalThis study was performed on NSCLC tumor tissue resected from 16 patients enrolled, following informed consent, in the ImmunogenomiC prOfiling of early-stage NSCLC (ICON) project. This study was approved by the University of Texas MD Anderson Cancer Center‘s Institutional Review Board (protocol number PA15-1112_MODCR001).
Lung cancer is the leading cause of cancer-related mortality in the United States. Lung adenocarcinoma (LUAD) is the most common subtype and the most epidemiologically and genetically heterogeneous. Pathologists have routinely observed phenotypic heterogeneity among LUAD primary tumors as reflected by distinct patterns of tumor growth. However, despite prior implication on the association of immune-genomic environment and prognosis, this information is not utilized clinically. Herein, applying multiplatform immune-genomic analysis, we investigate two distinct classification systems and demonstrate that high-grade patterns of growth are associated with a distinct immunogenic tumor microenvironment that is predicted with a favorable response to immunotherapy, a finding with growing importance in the era of adjuvant and neoadjuvant immunotherapy.
Background: A better understanding of the T cells in lung cancer and their distribution across tumor-adjacent lungs and the peripheral blood is needed to improve efficacy and minimize toxicity from immunotherapy to lung cancer patients. Methods: Here, we performed CDR3β TCR sequencing of 143 samples from 21 patients with early-stage NSCLC including peripheral blood mononuclear cells, tumor, tumor edges (<1cm from tumor), as well as adjacent lungs 1cm, 2cm, 5cm, and 10cm 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 by immunohistochemical staining and genomic features were derived by targeted sequencing of 1,021 cancer related genes. Results: Our study reveals a decreasing gradient in TIL homology with the 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: The exclusion in T cells at play across the lungs of patients with NSCLC may be potentially the mechanism for lung cancer occurrence.
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