gms | German Medical Science

Objective: The tumor microenvironment (TME) of glioblastoma (GBM) contributes to tumor growth and therapy resistance. To further dissect the role of the TME in tumor immunity, tumor-associated macrophages (TAM) as well as tumor-infiltrating lymphocytes (TIL) from primary tumor tissue were analyzed phenotypically and functionally.

Methods: Patients with newly diagnosed (nd) GBM were subjected to fluorescence-guided surgery. CD45⁺ tumor-infiltrating leucocytes were enriched immunomagnetically and analyzed by flow cytometry to characterize TAMs. TILs were analyzed for activation markers and IFNg production. Further, the expression of immune checkpoint regulators was assessed on TAM and TIL and their role in T-cell proliferation was determined.

Results: In CD45⁺ tumor-infiltrating leukocytes, 57.5% (n=11) represented CD14+ myeloid cells, mainly macrophages of the CD163⁺ HLA-DR⁺ M2 subtype, indicating a prevalence of an alternatively-activated M2c-like subset. Conditioned media (CM) of GBM cells attracted in-vitro differentiated M2, but barely any M0 or M1 macrophages. Moreover, CM induced activation of M2 macrophages towards the M2c subset. The cells expressed immune checkpoint receptors and had low T-cell stimulatory, but potent suppressive activity, which could be reversed by blocking the tryptophan-degrading enzyme indoleamine-2,3-dioxygenase (IDO) and the immune checkpoint receptor PD-L1, respectively.In line with these results, TILs (9.5% of CD45⁺ leukocytes; n=11) showed low frequencies of IFNg-producing CD4⁺ (9.9%) and CD8⁺ (3.6%) effector memory T cells, indicating an accumulation of inactive T cells. Further, these T cells showed high levels of PD-1 (CD4⁺: 83.6%; CD8⁺: 77.5%) as well as Tim-3 (CD4⁺: 20.9%; CD8⁺: 35.0%) expression together with a T-bet^highEomes^high phenotype, indicating exhaustion and terminal differentiation.

Conclusion: In this study, we report the prevalence of M2c-like macrophages in primary GBM, preferentially attracted and activated by GBM cells. They have poor T-cell stimulatory, but potent immunosuppressive activity, mediated at least in part by IDO and the PD-L1/PD-1 axis. Accumulation of these cells correlates with infiltrating effector T cells showing low IFNg-production and markers of exhaustion. These findings might add valuable insights for the development of therapies targeting the TME in order to generate more potent effector T cells.