FULL-LENGTH ARTICLE Clinical Studies Common phenotypic dynamics of tumor-infiltrating lymphocytes across different histologies upon checkpoint inhibition: impact on clinical outcome Vinicius Araujo B. de Lima 1, *, Annie Borch 2 , Morten Hansen 3 , Arianna Draghi 3 , Iben Spanggaard 1 , Kristoffer Rohrberg 1 , Sine Reker Hadrup 2 , Ulrik Lassen 1 , Inge Marie Svane 3 1 Rigshospitalet, Department of Oncology, Phase 1 Unit, Copenhagen, Denmark 2 Department of Health Technology, Technical University of Denmark, Lyngby, Denmark 3 National Center for Cancer Immune Therapy, Department of Oncology, Herlev Hospital, Herlev, Denmark ARTICLE INFO ABSTRACT Background: Immune checkpoint inhibitors (ICIs) have revolutionized the cancer therapeutic landscape and our perception of interactions between the immune system and tumor cells. Despite remarkable progress, disease relapse and primary resistance are not uncommon. Understanding the biological processes that tumor-infiltrating lymphocytes (TILs) undergo during ICI, how this affects the tumor microenvironment (TME) and, ultimately, clinical outcome is, therefore, necessary to further improve treatment efficacy. Aim: In the current study, we sought to characterize TILs from patients with metastatic solid tumors undergo- ing ICI correlating flowcytometric findings with clinical outcome. Methods: In total, 20 patients with 10 different metastatic solid tumors treated with ICIs targeting programmed- cell death-1 (PD-1)/PD-L1 axis were included in this study. The phenotype of T cells deriving from biopsies obtained prior to treatment initiation and on-treatment was investigaded. Analyses were focused on T cells’ degree of differentiation and activity and how they correlate with transcriptomic changes in the TME. Results: Data indicate that patients benefitting from ICIs accumulate CD8+central memory T cells. TILs developed an effector-like phenotype over time, which was also associated with a cytolytic gene signature. In terms of mod- ulation of T-cell responses, we observed that high expression of checkpoint molecules pre-treatment (i.e., PD-1, lymphocyte activation gene-3 [LAG-3], B and T-lymphocyte attenuator [BTLA] and T-cell immunoglobulin and mucin domain containing-3 [TIM-3]) was associated with similar gene signature and correlated to treatment benefit. Increasing expression of LAG-3 and BTLA in the CD8 compartment and their co-expression with PD-1 during treatment were, however, a common feature for patients who failed to respond to ICIs. Conclusions: Besides identifying immune profiles suggestive of response to ICI, our results provide a more nuanced picture regarding expression of checkpoint molecules that goes beyond T-cell anergy. © 2020 International Society for Cell and Gene Therapy. Published by Elsevier Inc. All rights reserved. Key Words: immune checkpoint prediction tumor microenvironment tumor-infiltrating lymphocytes phenotype Background The presence of immune cells in the tumor microenvironment (TME) has been long known [1], but it is only over the past decades that targeting the immune system as treatment strategy against can- cer has gained popularity, especially due to remarkable results yielded by immune checkpoint inhibitors (ICIs) [2À4]. The rationale behind ICIs is to revert the state of T-cell dysfunc- tionality where continuous TCR stimulation by chronic antigen expo- sure in an immune suppressive milieu has led to impairment of effective T-cell activity and finally tumor immune scape. However, for a big fraction of patients, reverting T-cell exhaustion is still a chal- lenge and acquired resistance to the therapy is not unusual [5]. In this context, the quest for biomarkers has proven to be quite laborious because T-cell activity upon ICIs is reliant on several features. Most studies have focused on assessment of external factors spanning from genetic determinants (e.g., tumor mutational burden [6]) to type of somatic mutations and which tumor antigens they can give rise to * Correspondence: Vinicius A.B. de Lima, MD, Rigshospitalet, Department of Oncol- ogy, Phase 1 unit, Blegdamsvej 9, 2100 Copenhagen Ø, Denmark. E-mail address: vinicius.araujo.barbosa.de.lima@regionh.dk (V.A.B. de Lima). https://doi.org/10.1016/j.jcyt.2020.01.010 1465-3249/© 2020 International Society for Cell and Gene Therapy. Published by Elsevier Inc. All rights reserved. Cytotherapy 22 (2020) 204À213 Contents lists available at ScienceDirect CYTOTHERAPY journal homepage: www.isct-cytotherapy.org