Impact of Radiochemotherapy on Immune Cell Subtypes in High-Grade Glioma Patients

Abstract

Glioblastoma is a dreadful disease with very poor prognosis, median overall survival being <2 years despite standard-of-care treatment. This has led to the development of alternative strategies, among which immunotherapy is being actively tested. In particular, many clinical trials of therapeutic vaccination using peptides or tumor cells are ongoing. A major issue in implementing therapeutic vaccines in patients with high-grade glioma is that immune responses have to be elicited in the context of immunosuppressive treatments. Indeed, radiotherapy, chemotherapy, and steroids, which are part of the standard of care for patients with glioblastoma, are known to deplete leukocytes. Whether lymphopenia is beneficial or detrimental to elicitation of efficient immune responses is still debated. Here, in order to determine the impact of standard radiochemotherapy on immune cell subsets, we analyzed the phenotype and function of immune populations in 25 patients with high-grade glioma along concomitant radiochemotherapy and adjuvant chemotherapy with temozolomide. Thirteen healthy individuals were studied along the same period. We show that absolute T and B cell counts are reduced upon concomitant radiochemotherapy. Importantly, T cell counts were not restored long-term after discontinuation of treatment. In addition, the percentage of T regulatory cells among CD4 T cells was increased during the same period and was not decreased upon treatment discontinuation. Finally, we show that the ability of T cells to proliferate is transiently reduced after concomitant radiochemotherapy but is restored at the time of adjuvant TMZ cycles. Although not experimentally validated, transient reduction in proliferation associated with strong lymphopenia during radiochemotherapy may suggest that vaccine-induced T cell stimulation would be suboptimal in that period and that therapeutic vaccination should be performed outside radiochemotherapy administration. In addition, strategies aiming at depleting Treg cells should be implemented in future trials.

Keywords: cancer vaccines; glioma; immune subsets; immunotherapy; lymphopenia; radiotherapy; temozolomide.

Figure 1

Study scheme. Patients underwent surgery (day −30) followed by concomitant radiotherapy (60 Gy) and TMZ (75 mg/m²) (day 0–42). After a 4-week break (day 42–72), patients were given adjuvant TMZ (150–200 mg/m²) at day 1–5 of each month. TMZ was discontinued in case of progression or intolerability. Time points for peripheral blood analysis are indicated in boxes (pre TT: pre RX/TMZ therapy, post-RX/TMZ: post-RX/TMZ therapy, C1-4, at day 1 of TMZ cycle 1–4).

Figure 2

(A) The absolute lymphocyte counts (left panel) and neutrophil counts (right panel) are shown for patients (n = 25) before and upon treatment. (B) Absolute CD3, CD4, and CD8 T cell counts and CD4/CD8 ratio are shown for patients (n = 25) before and upon treatment. (C) Absolute B cell, monocyte, and CD16⁺ or CD56^high NK cell counts are shown for patients (n = 25) before and upon treatment. Wilcoxon signed rank test was used to test variation in absolute counts over time.

Figure 3

(A) The percentage of naïve (T_N), central memory (T_CM), effector memory (T_EM), and effector cells (T_E) in CD4 (upper panels) and CD8 (lower panels) are shown for patients (n = 25) before and upon treatment. (B) The percentages of CD4 (upper panel) and CD8 (lower panel) T cells expressing PD1 are shown for patients (n = 6) before and upon treatment. Data were analyzed using box and whiskers plots with outliers. Wilcoxon signed rank test was used to test variation in absolute counts over time.

Figure 4

(A) The percentage of granzyme B (GZB)⁺ cells among CD8 T cells is shown for patients (n = 25) before and upon treatment. (B) Proliferation of CD4 (upper panels) and CD8 (lower panels) T cells in response to SEB (panels to the left) or CD3/CD28 antibodies (panels to the right) is shown for patients (n = 18) before and upon treatment. Data were analyzed using box and whiskers plots with outliers. Wilcoxon signed rank test was used to test variation in absolute counts over time.

Figure 5

(A) The percentage of Treg (CD3⁺CD4⁺CD25⁺FoxP3⁺) cells among CD4 T cells is shown for patients (n = 25) before and upon treatment. (B) The percentage of Ki67⁺ (left) and of HLA-DR⁺ (right) Treg cells is shown for patients (n = 12) before and upon treatment. (C) The percentage of PD1⁺, LAG3⁺, and ICOS⁺ Treg cells is shown for patients (n = 6) over time. Data were analyzed using box and whiskers plots with outliers. Wilcoxon signed rank test was used to test variation in absolute counts over time.

Figure 6

Overall survival (OS, left) and progression-free survival (PFS, right) of the patients (n = 25).