Soluble factors secreted by glioblastoma cell lines facilitate recruitment, survival, and expansion of regulatory T cells: implications for immunotherapy

Abstract

In patients with glioma, the tumor microenvironment can significantly impact pro-inflammatory immune cell functions. However, the mechanisms by which this occurs are poorly defined. Because immunosuppressive regulatory T cells (Treg) are over represented in the tumor microenvironment compared with peripheral blood, we hypothesized that the tumor may have an effect on Treg survival, migration, expansion, and/or induction of a regulatory phenotype from non-Treg conventional CD4+ T cells. We defined the impact of soluble factors produced by tumor cells on Treg from healthy patients in vitro to determine mechanisms by which gliomas influence T cell populations. We found that tumor-derived soluble factors allowed for preferential proliferation and increased chemotaxis of Treg, compared with conventional T cells, indicating that these mechanisms may contribute to the increased Treg in the tumor microenvironment. Conventional T cells also exhibited a significantly increased expression of pro-apoptotic transcripts in the presence of tumor-derived factors, indicating that survival of Treg in the tumor site is driven by exposure to soluble factors produced by the tumor. Together, these data suggest that tumor burden may induce increased Treg infiltration, proliferation, and survival, negating productive anti-tumor immune responses in patients treated with immunotherapies. Collectively, our data indicate that several mechanisms of Treg recruitment and retention in the tumor microenvironment exist and may need to be addressed to improve the specificity of immunotherapies seeking to eliminate Treg in patients with glioma.

Fig. 1.

Patients with GBM have an increased percentage of Treg in circulation relative to healthy controls, and Treg are overrepresented within the tumors. (A) Lymphocytes were isolated from blood and tissue collected during glioblastoma multiforme (GBM) surgical resections. T cells were defined as CD3 + CD4 +, and Tregs were defined as CD3 + CD4 + CD25 + FoxP3 + T cells. Representative FACS plots for gating Treg are depicted. (B) FoxP3 + expression in tumor infiltrating lymphocytes (TIL) and peripheral blood lymphocytes (PBL) from patients with GBM is depicted (n = 20).

Fig. 2.

Tumor-derived soluble factors preferentially attract Treg. (A) 1 × 10⁵ CFSE labeled Treg were cocultured with 1 × 10⁵ unlabeled conventional T cells in 0.5 µm transwell plates. Cells from the bottom chambers were analyzed using flow cytometry after culturing in complete T cell medium alone (left histogram), U87 TCM (center histogram), and SF767 TCM (right histogram). Representative histograms are shown. (B) Fold migration of both Treg and conventional T cells are shown over baseline migration for cells incubated in medium with U87 or SF767 TCM, CCL22 at 10 ng/mL or 50 ng/mL, and CCL5 at 30 ng/mL (n = 3).

Fig. 3.

Soluble factors produced by glioblastoma cells promote Treg proliferation while suppressing conventional T cell proliferation. (A) Isolated conventional T cells, labeled with PKH26, were cultured alone or with CFSE labeled Treg, in either complete T cell medium, U87 TCM, or SF767 TCM (not shown). Treg and conventional T cells were cultured in a 2:1 ratio. Proliferation was measured using flow cytometry. Representative FACS plots are shown for PKH26/CFSE dot plots. (B) Percent of proliferating cells Treg and conventional T cells from the same coculture are shown with the rate of conventional T cells cultured alone. Varying conditions include in complete T cell medium with or without stimulation and in the presence of U87 or SF767 TCM. (C) CFSE-labeled conventional T cells were plated with varying numbers of Treg or conventional T cells (n = 3). Proliferation of conventional T cells was measured using flow cytometry as described in the Materials and Methods section.

Fig. 4.

Conventional T cells transiently increase expression of Treg associated proteins FoxP3 and TGF-β in response to soluble tumor factors and are functionally suppressive. (A) Freshly isolated conventional CD4 + T cells were cultured in complete T cell medium or U87 cultured medium (TCM). Cells were harvested at Day 3, 5, 7, 10 poststimulation and stained for Treg markers, FoxP3 and TGF-β in addition to CD3, CD4, and CD25. Representative FACS plot are shown. (B) Time course of TGF-β (Top) and FoxP3 (Bottom) expression on CD4 + CD25 conventional T cells following stimulation in the presence of TCM (n = 3). (C) The ratio of expression of Treg associated markers, FoxP3 and TGF-β, induced by culturing in the presence of TCM: complete T cell media (n = 3). (D) T cells cultured in normal T cell media and those cultured in TCM (with tumor) were isolated at Day 7. CD25 + T cells from both conditions were then cultured with freshly isolated and CFSE-labeled, CD3/CD28 stimulated, autologous conventional T cells. Histograms depict the proliferation of CFSE labeled conventional T cells cultured with autologous CD25 + CD4 T cells.

Fig. 5.

Conventional CD4 + T cells increase pro-apoptotic mRNA expression in the presence of tumors. Magnetically purified Treg and conventional T cells were cultured in T cell medium (TCM). Following isolation of RNA, whole cell reverse transcription and Quantitative PCR was conducted to measure mRNA levels of known pro-apoptotic proteins BAK, BAX, BID, BAD, and BIM and standardized to HPRT gene expression. (A) Relative mRNA expression for Treg and conventional T cells for pro-apoptotic proteins without tumor conditioning (left) or following coculture with TCM (right). Bar graphs also show expression levels for unstimulated (NS) CD25+ Treg and CD25- conventional T cells and following 72 h CD3/CD28 stimulation (S) (n = 3). (B) Transcript expression of Treg and conventional T cells following stimulation in the presence of TCM were normalized to gene expression in unstimulated Treg or conventional T cell subsets (n = 3).

Fig. 6.

Treg populations in circulation correlate with tumor burden. Magnetic resonance imaging showing T1 contrast with FLARE indicate the tumor burden at the time of PBL analysis (Left). Percentage of Treg relative to total CD4 + T cells in PBL were measured at 3 time points for patients with high-grade gliomas: prior to tumor resection, following tumor resection, and prior to a subsequent surgery for tumor recurrence. CD25 + FoxP3 + cells were analyzed by flow cytometry and expressed as a percent of total CD4 + CD3 + T cells. (representative patient data) (B) Analysis of patients prior to surgical resection, following gross total resection, and at the time of surgery for tumor recurrence as defined by pathology (n = 7).