Toll-like Receptor 7/8 Pathway Is a Therapeutic Target in Primary CNS Lymphoma

CNS lymphomas remain among the deadliest forms of Non-Hodgkin lymphoma. Factors contributing to therapeutic resistance include the tumor microenvironment, including tumor-associated macrophages and microglia, which may display an immunosuppressive M2 phenotype that promotes tumor growth. Tumor-associated macrophages (TAMs), however, are highly plastic and can acquire an anti-tumor, M1 phenotype, induced via stimulation by pattern recognition receptors, of which the most important class are the toll-like receptors (TLRs).

Applying both syngeneic and non-syngeneic CNS lymphoma models, including immortalized B-cell lymphoma cells and patient-derived xenografts of CNS lymphoma, we demonstrated that IFN-g deficiency, using IFN-g knockout mice, promotes CNS lymphoma progression. Transcriptional profiling of TAMs in these models via RNA-Seq provided evidence of TLR7/8 pathway activation (TLR7 is the active murine analog of human TLR8). Using immunofluorescence co-localization, we confirmed high expression of TLR7 by TAMs in these preclinical tumor models.

We next evaluated expression of TLRs on tumor-associated macrophages in diagnostic clinical specimens of PCNSL and determined that TLR8, but not TLR9, is selectively upregulated by tumor-associated macrophages and microglia in the CNS lymphoma microenvironment. Expression of TLR8 was not detected on lymphoma cells. Results were confirmed by immunohistochemistry and by in situ hybridization which localized expression of TLR8 transcripts to the largest CD68+ macrophages. Immunohistochemical analysis of TLR8 expression in 60 diagnostic specimens of PCNSL in patients treated with MTR chemotherapy demonstrated that low TLR8 correlated with short PFS and OS. Using transcriptional profiling of an independent multicenter set of 20 frozen diagnostic specimens of PCNSL, we demonstrated that low expression of TLR7 and TLR8 each significantly associated with shorter OS. Expression of TLR7 and TLR8 each correlated with increased CD4 and CD8 transcripts in PCNSL.

We demonstrated that the expression of TLR8 by human macrophages, differentiated in vitro from PBMCs, is induced with 24 hours treatment with IFN-g, yielding M1 macrophages, but not by IL-4. Taken together, these demonstrate that TLR8 is a macrophage marker with prognostic significance in PCNSL. These results suggest that pharmacologic agonists of the TLR7/8 pathway have significant potential to activate the anti-tumor potential of tumor-associated macrophages in CNS lymphomas.

Given these findings, in collaboration with Gilead, we performed preclinical therapeutic evaluations of a human TLR8 agonist, which activates mouse TLR7, via stereotactic injection into left hemisphere of non-tumor bearing brains in BALB/c mice, demonstrating evidence for pharmacodynamic potency and safety. Within one week, the stereotactic injection of TLR7 agonist, but not vehicle, induced a marked perivascular accumulation of Iba1+ activated macrophages, diffusely, several mm beyond the injection site, as demonstrated by immunohistochemistry. This observation was confirmed by dual-color immunofluorescence that showed co-localization of perivascular Iba1+/iNOS+ M1 macrophages diffusely along the vasculature of medium-sized, CD31+ blood vessels, one week after stereotactic injection of the murine TL7 agonist into brain.

We also detected an accumulation of CD8+ T cells adjacent to the injection site in brains exposed to the murine TLR7 agonist, but not vehicle. These results demonstrate that pharmacologic activation of the TLR7 pathway induces accumulation of Iba1+ M1 macrophages, and CD8+ T cells, and suggest that TLR7 activation may promote macrophage entry via the cerebral vasculature, independent of brain tumor pathogenesis. Notably, among biological replicates of mice that received 2 and 5 mg of TLR7 agonist, there was no evidence of overt toxicity after one week of observation.

In parallel, we determined that weekly systemic administration of the TLR7 agonist has single agent activity in the delay of intracranial tumor progression of the A20 CNS lymphoma model, in immunocompetent mice. These data suggest that pharmacologic activation of the TLR7/8 pathway, via systemic as well as intra-CNS delivery, has significant potential to program the myeloid microenvironment to an anti-tumor phenotype, with significant translational therapeutic potential in CNS lymphoma.