Immunophenotyping of Newly Diagnosed and Recurrent Glioblastoma Defines Distinct Immune Exhaustion Profiles in Peripheral and Tumor-infiltrating Lymphocytes

Abstract

<b>Purpose:</b> Immunotherapeutic treatment strategies for glioblastoma (GBM) are under investigation in clinical trials. However, our understanding of the immune phenotype of GBM-infiltrating T cells (tumor-infiltrating lymphocytes; TILs) and changes during disease progression is limited. Deeper insight is urgently needed to therapeutically overcome tumor-induced immune exhaustion.<b>Experimental Design:</b> We used flow cytometry and cytokine assays to profile TILs and peripheral blood lymphocytes (PBLs) from patients with GBM, comparing newly diagnosed or recurrent GBM to long-term survivors (LTS) and healthy donors. TCR sequencing was performed on paired samples of newly diagnosed and recurrent GBM.<b>Results:</b> We identified a clear immune signature of exhaustion and clonal restriction in the TILs of patients with GBM. Exhaustion of CD8⁺ TILs was defined by an increased prevalence of PD-1⁺, CD39⁺, Tim-3⁺, CD45RO⁺, HLA-DR⁺ marker expression, and exhibition of an effector-/transitional memory differentiation phenotype, whereas KLRG1 and CD57 were underrepresented. Immune signatures were similar in primary and recurrent tumors; however, restricted TCR repertoire clonality and a more activated memory phenotype were observed in TILs from recurrent tumors. Moreover, a reduced cytokine response to PHA stimulation in the blood compartment indicates a dysfunctional peripheral T-cell response in patients with GBM. LTS displayed a distinct profile, with abundant naïve and less exhausted CD8⁺ T cells.<b>Conclusions:</b> TILs and PBLs exhibit contrasting immune profiles, with a distinct exhaustion signature present in TILs. While the exhaustion profiles of primary and recurrent GBM are comparable, TCR sequencing demonstrated a contracted repertoire in recurrent GBM, concomitant with an increased frequency of activated memory T cells in recurrent tumors. <i>Clin Cancer Res; 24(17); 4187-200. ©2018 AACR</i><i>See related commentary by Jackson and Lim, p. 4059</i>.