Analysis of regulatory T cell recruitment and relevance in human and mouse models of glioma

Glioma is a primary brain tumor of the central nervous system. Of notability, glioblastoma multiforme (GBM) is a highly malignant form of glioma with a mean lifespan of 15 months after diagnosis. Our laboratory was one of the first to demonstrate that regulatory T cells (Tregs; CD4 + FoxP3 + ) are increased in human GBM and that Treg depletion significantly increases the lifespan in a mouse model of glioma. In this study, we hypothesized that recruited thymus‐born natural Tregs (nTregs), but not peripherally induced Tregs (iTregs), are the primary source of Tregs in mouse glioma. To study this hypothesis, we used the intracranially‐injected GL261 (glioma) cell‐ and the RasB8 transgenic‐mouse model of glioma, as well as the analysis of human GBM tissue. To investigate the origin of glioma‐resident Tregs in mouse models that recapitulate glioma, we used a combination of Treg depletion (via systemic CD25 mAb) with or without thymectomy. Using confocal immunofluorescence, we were able to localize and count the number of Tregs in mouse glioma to calculate the percentage of Tregs out of the total number of CD4 + T cells, which was verified by flow cytometry. In addition, H&E histology and magnetic resonance imaging (MRI) of mouse brain was performed to analyze tumor growth and invasion over time. Finally, the total number and frequency of Tregs that were Helios + (a transcription factor expressed by nTregs, but not iTregs) was analyzed by 4‐color confocal immunofluorescence and confirmed by flow cytometry in the brain, thymus, cervical (draining) lymph nodes and spleen of mice with glioma, as well as human GBM. To ascertain the functional importance of nTregs vs. iTregs, a survival analysis of mice with glioma ± Treg depletion ± thymectomy was performed. A model of how Tregs populate glioma and the relevance to survival is presented. Grant Funding Source : R01CA138587