In-depth immunophenotyping of patients with glioblastoma multiforme: Impact of steroid treatment

Abstract

Despite aggressive treatment regimens based on surgery and radiochemotherapy, the prognosis of patients with grade IV glioblastoma multiforme (GBM) remains extremely poor, calling for alternative options such as immunotherapy. Immunological mechanisms including the Natural Killer Group 2 member D (NKG2D) receptor-ligand system play an important role in tumor immune surveillance and targeting the NKG2D system might be beneficial. However, before considering any kind of immunotherapy, a precise characterization of the immune system is important, particularly in GBM patients where conventional therapies with impact on the immune system are frequently co-administered. Here we performed an in-depth immunophenotyping of GBM patients and age-matched healthy controls and analyzed NKG2D ligand expression on primary GBM cells ex vivo. We report that GBM patients have a compromised innate immune system irrespective of steroid (dexamethasone) medication. However, dexamethasone drastically reduced the number of immune cells in the blood of GBM patients. Moreover, higher counts of immune cells influenced by dexamethasone like CD45⁺ lymphocytes and non-Vδ2 γδ T cells were associated with better overall survival. Higher levels of NKG2D ligands on primary GBM tumor cells were observed in patients who received radiochemotherapy, pointing towards increased immunogenic potential of GBM cells following standard radiochemotherapy. This study sheds light on how steroids and radiochemotherapy affect immune cell parameters of GBM patients, a pre-requisite for the development of new therapeutic strategies targeting the immune system in these patients.

Keywords: Dexamethasone; NKG2D; gamma/delta T cells; glioblastoma multiforme.

Figure 1.

Immune cell subsets in the peripheral blood of GBM patients. Immune cell populations (cells/uL) were determined by FCM in GBM patients, treated with steroids (GBM-Dex, n = 22) or not (GBM, n = 13), and in age-matched HCs groups, all HC (n = 22) and HC-I (n = 14), respectively. Data between groups GBM/GBM-Dex, GBM/HC-I and GBM-Dex/HC were compared for immune cell subsets as indicated: (A) Lymphocytes (CD45⁺ cells), B cells (CD19⁺ cells), αβ T cells, CD3⁺CD8⁺ T cells, CD3⁺CD4⁺ T cells, and Tregs, defined as CD127⁻CD25^high CD3⁺CD4⁺ T cells. (B) Neutrophils (CD66b⁺ cells), monocytes (CD14⁺ cells). (C) The data obtained from GBM-Dex patients before surgery (BT, before therapy) was compared with the data from a later time point (after 2–5 months; AT, after therapy). Results shown for CD3⁺CD8⁺ cells (left panel), and neutrophils (right panel). The median values in (A) and (B) are compared by 2-tailed Wilcoxon's rank-sum test. Statistical significance is displayed as *** for p < 0.001, ** for p < 0.01 and * for p < 0.05.

Figure 2.

γδ T cells and NK cells in the peripheral blood of GBM patients. (A) Total γδ T cells (left panel) and γδ T cell subsets (Vδ2, middle panel; non-Vδ2, right panel) and (B) total NK cells (left panel) and NK cell-subsets (CD56^high, middle panel; CD56^dim, right panel) were analyzed in GBM patients treated with steroids (GBM-Dex, n = 22) or not (GBM, n = 13) and in age matched HCs groups, all HCs (n = 22) and HC-I (n = 14). Median values of numbers were compared between the GBM and GBM-Dex groups, GBM and HC-I groups and GBM-Dex and HC groups. (C) Serum levels of SDF-1α in GBM patients and HCs. Significance was analyzed by 2-tailed Wilcoxon's rank-sum test. Statistical significance is displayed as *** for p < 0.001, ** for p < 0.01 and * for p < 0.05.

Figure 3.

Analysis of NKG2D/NKG2DLs in GBM patients and HCs. (A) Absolute numbers of NKG2D⁺ lymphocytes (upper left panel). mRNA levels of full-length (NKG2D-FL) and truncated NKG2D (NKG2D-TR) (upper middle and right panel), and MICA (lower right panel) were measured by qRT-PCR. The ratio between NKG2D-FL and NKG2D-TR (lower left panel) was calculated by dividing the NKG2D-FL value by the NKG2D-TR value and compared between patients groups and HCs. Serum concentrations of sMICA were measured by Luminex assay in the sera of GBM patients and HCs (lower middle panel). (B) Spearman correlation between sMICA and αβ T-cells in HCs (left part). Spearman correlation between sMICA and γδ T cells in steroid untreated GBM patient group (right part). Spearman´s Rho (r) correlation coefficient is depicted in the respective graphs with the corresponding p-value. Statistical significance was analyzed by 2-tailed Wilcoxon's rank-sum test. Statistical significance is displayed as *** for p < 0.001, ** for p < 0.01 and * for p < 0.05.

Figure 4.

NKG2DLs are expressed and released from short-term GBM tumor cell cultures. Short term GBM primary tumor cells were analyzed for NKG2DLs expression by FCM and qRT-PCR. (A) Representative FCM analysis of NKG2DLs surface expression after staining with mAb MICA-PE, ULBP2/5/6-APC, ULBP3-PE, or ULBP2 followed by goat-anti-mouse (Gam) Ig-PE as indicated (gray histograms). Open histograms represent appropriate isotype controls. (B) Summary of data obtained from 12 short-term GBM primary cell cultures. mRNA levels (left panel) and FCM results (MFI normalized to isotype controls; right panel) of all analyzed NKG2DLs are depicted. (C) NKG2DLs levels in cell lysates (left panel) and cell culture supernatants (right panel) of GBM short-term cell cultures were analyzed with Luminex assay. Culture media of GBM short-term cell cultures were collected at 3 time points (day 1,2,3) and cells were lysed at the end of the experiment (on day 3). The median values of the amounts of sNKG2DLs (pg/mL) of 9 different GBM short-term cell cultures are depicted. (D) Soluble and cellular MICA (left panel) and ULBP2/5/6 (right panel) were analyzed separately in short-term GBM cell cultures from patients with primary GBM (Primary GBM, n = 2), patients with primary GBM treated with steroids (Primary GBM-Dex, n = 7), and patients with recurrent GBM (Recurrent GBM-Dex, n = 3).

Figure 5.

Kaplan-Meier survival analysis of GBM patients. (A) CD45⁺ cell and the percentages of CD3⁺ cells (% of CD45⁺ cells) (upper panel), non-Vδ2 γδ and Vδ2 γδ T cell numbers per µL blood (middle panel) and CD8⁺ NK cells (% of NK cells) (lower panel), and (B) the cellular content of ULBP2/5/6 (pg/mL) in short-term primary GBM cells was dichotomized (median as a cut off) and used to generate Kaplan-Meier curves in the GBM patient cohort (n = 32, censored 6). The levels of variables above (high) and below (low) median were compared by log-rank test (depicted p-values).

Figure 6.

Interplay between immunological parameters associated with survival of GBM patients. Interplay between the parameters associated with survival, CD3⁺CD8⁺ T cells/µL, steroid medication, recurrence of the disease, RCT and age of GBM patients (n = 26; GBM-Dex/GBM 17/9). Positive and negative influences are indicated with arrows and lines with flat ending, respectively. Negative and positive correlations between parameters are shown as double-headed arrows. For statistical analysis Spearman´s Rank correlation was used. Spearman´s Rho (r) correlation coefficient is depicted with statistical significance displayed as ** for p < 0.01 and * for p < 0.05.