Evaluation of T follicular regulatory cells as novel cellular targets of cancer immunotherapy - Project Summary Immune checkpoint blockade (ICB) targeting CTLA-4 or PD-1 rapidly assumed its role as a standard treatment for solid tumors and can lead to dramatic, long-lasting responses; nonetheless, fewer than 30% of patients respond to monotherapy with either agent. Combination therapy results in better long-term survival outcomes, but also causes more frequent and severe immune-related adverse events (irAEs). Several novel immunotherapies are currently being explored to evaluate their anti-tumor capacity. Crucially however, most of these targets suffer from on-target, off-cell effects, as other immune cell types can express high levels of these molecules. Hence, as low overall response rates, off-cell effects and widespread immune related toxicity severely limit both treatment efficacy and monotherapy and combination therapy options, there is urgent need to develop novel immunotherapy targets that exhibit a more restricted expression profile. We have recently demonstrated that PD-1+ follicular regulatory T (TFR) cells were prevalent in tumor tissues of several cancer types in humans and mice, and that they were critical cellular determinants of anti-PD-1 treatment efficacy. TFR cells were primarily located within tertiary lymphoid structures (TLS) and exhibited superior suppressive capacity and in vivo persistence when compared to regulatory T (TREG) cells, suggesting a key role for TFR cells in impairing patient survival and impeding immunotherapy treatment efficacy. While we have shown that intratumoral TFR cells derive from TREG precursor cells, the mechanisms and transcription factors (TFs) that are driving this differentiation step are largely unknown. In Aim1, we propose to employ single-cell RNA-seq, single-cell ATAC-seq and micro- scaled ChIP assays to fully characterize the transcriptomic signatures of tumor-infiltrating TREG cells, transitioning (4-1BB+) TREG cells and TFR cells. Elucidating the enhancer profiles in different developmental stages of TREG to TFR differentiation is likely to provide crucial insights into the TFs that instruct this differentiation step. These analyses will define genes and TFs that are pivotal for the heightened suppressive capacity of TFR cells, as well as for their maintenance or differentiation. Strategies to deplete TREG cells or to curb their functionality with the aim of enhancing anti-tumor immunity are being intensively investigated. Crucially however, most of these approaches are based on antibodies (i.e., ADCC-optimized for TREG cell depletion; i.e. anti-CTLA-4), which have an inherently long half-life in vivo, thus increasing the likelihood of causing irAEs. Conversely, our data imply that alternative dosing regimens of Phosphoinositide 3-kinase δ (PI3Kδ) inhibitors might offer a pathway to target TFR cells more specifically. We propose that PI3Kδ represents a novel and appealing immunotherapy target in solid tumors. In Aim 2 and Aim 3, we will assess whether alternative dosing regimens of PI3Kd inhibitors can be utilized to effectively and safely exploit the immunomodulatory impact of PI3Kδ inhibitors in solid cancers and whether a transient depletion or inhibition of TFR cells might suffice to restrict the immunosuppressive milieu in the tumor and thus drive anti-tumor immunity without causing toxicity.
