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

Abstract

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. 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. 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. 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. .

Expression profiling of single cells and patient cohorts identifies multiple immunosuppressive pathways and an altered NK cell phenotype in glioblastoma

“…However, we favour TGF‐β as a modulator of the NK cell phenotype in GBM, as we show reduced expression of activation receptors coupled with increased expression of CD9, a tetraspanin induced by TGF‐β in NK cells . Mohme et al showed that infiltrating T cells expressed PD‐1, TIM‐3 and CD39 , characteristic of T cell exhaustion . Our analysis extended these findings by identifying CTLA‐4 and LAG‐3 on GBM‐infiltrating T cells.…”

“…a) and 5'‐nucleotidase ecto (NT5E) (encoding CD39) by the immune fraction (Fig. b), with Mohme et al demonstrating CD39 expression by GBM‐infiltrating T cells .…”

“…3a) and 5'-nucleotidase ecto (NT5E) (encoding CD39) by the immune fraction (Fig. 3b), with Mohme et al demonstrating CD39 expression by GBM-infiltrating T cells [13]. Furthermore, to assess whether this immunosuppressive network was induced in response to tumour, we analysed gene expression data derived from normal brain tissue [33] (Supporting information, Fig.…”

Expression profiling of single cells and patient cohorts identifies multiple immunosuppressive pathways and an altered NK cell phenotype in glioblastoma

“…This scRNA-seq data along with the GSC surface antigen screen shows that both tumour and immune infiltrating cells express receptors and ligands that together constitute a complex network of immunosuppression. For example, the combined action of CD73 and CD39 generate immunosuppressive adenosine (29); our data shows expression of CD73 by the tumour cells ( Figure 3A) and NT5E (encoding CD39) by the immune fraction ( Figure 3B), with Mohme et al demonstrating CD39 expression by GBM infiltrating T cells (13).…”

The role of CMV in glioblastoma and implications for immunotherapeutic strategies

“…3 CMV can infect many cell types including and, like all herpes viruses, can result in life long persistence after the initial acute infection. 4 Therefore, the immunosuppression associated with GBM 5,6 would be the perfect environment for CMV re-activation in tumor cells. CMV DNA and protein products have been discovered in multiple tumor types 7-9 including low grade glioma and higher grade glioma (e.g.…”