Because of the well-established role of Treg in suppressing productive immune responses, we hypothesized that the accumulation of Treg in tumor tissue from patients with GBM may be achieved through multiple mechanisms, including preferential Treg chemoattraction.12,16
We first confirmed the findings of previous studies that demonstrate an overrepresentation of Treg in the tumor microenvironment, compared with Treg in circulation.15
Similar to previous reports, we found an increased percentage of Treg in the tumor tissue, compared with that in circulation. Patients with GBM had a mean of 38.5% Tregs, as defined by flow cytometric analysis of CD3, CD4, CD25, and FoxP3 expression at the tumor site (n
= 20) (Fig. A). This population was significantly more than11.9% in the peripheral blood of the same patients (Fig. B). Percentages in the peripheral blood were still higher than those observed in healthy individuals or patients with benign meningioma undergoing intracranial surgery (5%–10%; data not shown), suggesting that GBM tumor burden has both a local and systemic effect on CD4 T cell populations.
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 (more ...)
Tumors secrete various soluble factors, including chemoattractant molecules that have the potential to attract Tregs from the periphery to the tumor site. We performed a transwell assay to determine whether CFSE-labeled Treg preferentially migrated in response to tumor cell–derived soluble factors, compared with unlabeled conventional T cells (Fig. A). Gliomas have been shown to secrete CCL22,16
and we found that this chemokine preferentially attracted Treg. We also tested a panel of other tumor-derived chemokines, including CCL5, CCL2, CCL10, CXCL10, CCL22, CCL17, CXCL13, and CCL20, and found that, as previously published,19
only CCL22 had a statistically significant effect on Treg migration. SF767 Tumor TCM, containing a variety of chemokines and cytokines, had 2.5-fold preference for Treg migration over that of conventional T cells, compared with a 1.5-fold preference for the highest dose of CCL22 alone (Fig. B). Furthermore, antibody blocking of the CCL22 receptor CCR4 did not reduce Treg migration to the level observed for conventional T cells cultured with TCM (data not shown). Together, these data suggest that soluble factors produced by glioma cells, including CCL22, can induce preferential migration of Treg.
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 (more ...)
Because all human T cells will increase expression of the canonical Treg markers CD25 and FoxP3 after stimulation, we first labeled Treg with CFSE and conventional T cells with PKH26—2 fluorescent dyes that are distinguishable by FACS. Once labeled, we subjected the mixed culture to CD3/CD28 stimulation and coculture with TCM. We found that the 2 cell populations were easily detectable, and the dyes diluted as expected after T cell stimulation (Fig. A). To demonstrate that the Treg isolated from healthy individuals were functional, we cocultured stimulated conventional T cells and added increasing numbers of Treg. The addition of as few as 2 × 104 Treg significantly reduced conventional T cell proliferation (Fig. B). For subsequent coculture experiments, we therefore modified the ratio of Treg to conventional T cells to 1:1.8. In doing so, we sought to minimize Treg-mediated suppression in coculture and to establish a coculture with physiologically relevant ratios based on tumor-infiltrating T cell data from patients with GBM (Fig. . We also analyzed the proliferation of conventional T cells in the absence of any Treg after stimulation and found that conventional T cells cultured in TCM in the absence of Treg proliferate after stimulation (mean = 33.5%) (Fig. C) , suggesting that soluble tumor-derived factors inhibit conventional T cell proliferation independently. In contrast, when cultured with TCM, Treg proliferation is significantly greater than that observed for conventional T cells (mean = 45.6%). In the tumor microenvironment, where both Treg and conventional T cells are present in ratios as great as 5 Treg to 1 conventional T cell, it is likely that there is an even greater reduction in the ability of conventional T cells to proliferate in response to stimulation (mean = 25.4%).
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 (more ...)
Many recent studies have focused on T cell plasticity and polarizing conditions, which can conditionally induce Treg phenotypes in conventional T cells. If this mechanism is exploited by GBM, immunotherapies that are predicated on initiating T cell responses and recruitment to the tumor site may instead promote tumor evasion through converted Treg-mediated immune suppression. We therefore examined the effects of malignant gliomas on T cell plasticity to determine whether conventional T cells may be converted to a suppressive Treg after recruitment to the tumor microenvironment. We cultured a population of conventional T cells in the presence of tumor-conditioned medium and observed Treg-associated markers FoxP3 and TGFβ over the incubation (Fig. A). Initially, there was a significant increase in the percentage of cells expressing both FoxP3 and TGFβ (Fig. A and B). The percentages of conventional T cells expressing TGFβ and FoxP3 after stimulation in the presence of TCM were 3.3 and 2.4 times greater, respectively, than those of conventional T cells stimulated without TCM (Fig. C). By day 10, however, these percentages were equivalent, suggesting that conversion to a suppressive Treg phenotype is transient and may be apparent only during peak stimulation (Fig. C).
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 (more ...)
Conventional T cells that were transiently induced to increase expression of FoxP3 and TGFβ after TCM culture (converted Treg) were phenotypically similar to Treg (4B and data not shown), but it was unclear whether they were functionally suppressive. To address this, we cultured with autologous CFSE-labeled conventional T cells with converted Treg and found that, similar to thymically derived Treg, converted Treg suppressed conventional T cell proliferation (Fig. D). Our data suggest that soluble factors produced by gliomas can upregulate Treg-associated markers in conventional T cells after stimulation and that these cells are functionally suppressive, even if only transiently. Collectively, our data support the notion that patients with a minimal tumor burden would be preferred candidates for T cell–based immunotherapies to prevent even temporary increases in functional Treg during immune responses.
In cases of some immune cells in the tumor microenvironment, such as myelomonocytic cells, tumor cells may actively promote the survival of immune cells that support tumor immune escape and prevent anti-tumor immune responses.26,27
We therefore sought to determine whether tumor-derived soluble factors influenced the survival of Treg, compared with conventional T cells, as determined by changes in the expression of pro- and anti apoptotic genes. We found that expression of anti-apoptotic genes Bcl2, Bcl-XL, IAP-1, and IAP-2 did not vary significantly after stimulation or in the presence of tumor TCM (data not shown). After stimulation in the absence of TCM, both Treg and conventional T cells decreased transcription of pro-apoptotic genes Bak, Bax, and Bim. The most striking reduction was seen in Bax transcription in both Treg (8.2-fold) and conventional T cells (10.6-fold) (Fig. A). After culture with TCM, Treg significantly reduced pro-apoptotic genes Bax, Bak, and Bim, even in the absence of stimulation (Fig. A). In contrast, conventional T cells increased expression of Bax, Bak, and Bim, suggesting a possible mechanism of preferential Treg survival in the tumor microenvironment. For example, Treg reduced Bax expression from 31.4 to 12.2 (61.2% reduction) relative to HPRT without stimulation, compared with conventional T cells, which increased Bax expression after TCM culture from 26.9 to 43.1, relative to HPRT. Indeed, tumor TCM coculture increased transcript levels for conventional T cells of several pro-apoptotic genes, with a mean 1.98-fold increase after stimulation in the presence of TCM, whereas Treg expression of these genes significantly decreased, compared with unstimulated Treg (Fig. B).
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 (more ...)
Having demonstrated that preferential migration, proliferation, and survival may contribute to the suppressive tumor microenvironment, we sought to determine whether the effects of tumor-mediated increases in Treg are restricted to the tumor microenvironment or whether the tumor may also exert a systemic effect. Increased survival, trafficking, and proliferation of Treg may in part explain the increase in Treg percentages in the circulation of patients harboring a GBM tumor. Therefore, we sought to determine whether tumor burden is associated with circulating Treg percentages in patients with GBM. To test this, we compared MRIs used to determine tumor burden and analyzed percentages of Treg by flow cytometry in patients within 48 h of the image acquisition. We analyzed a small cohort of patients prior to tumor resection, a mean of 34.8 days after the procedure, and again prior to surgery for tumor recurrence. We observed a significant decrease in circulating Treg populations after a gross total tumor resection (more than 0%) from 16.1% to 5.97%, which rebounded to 14.5% by the time of recurrence (Fig. A). We then examined the trend in a subset of patients opting for repeat surgery at the time of recurrence. Of the 7 patients analyzed, we found that, although not always statistically significant, the percentage of circulating regulatory T cells decreases after gross total resection and increases at the time of surgery for tumor recurrence (Fig. B). Together, these data suggest an association between tumor burden in patients with glioma and percentages of circulating Treg. Current experiments seek to evaluate a larger and prospectively analyzed cohort of patients to determine whether the observed trend is reproducible. These data suggest that circulating Treg may be useful as an indicator of tumor burden in patients with GBM.
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 (more ...)