Probes for INS

Abstract

PURPOSE:

Blocking the immunosuppressive PD-1/PD-L1 pathway has antitumor activity in multiple cancer types, and PD-L1 expression on tumor cells and infiltrating myeloid cells correlates with the likelihood of response. We previously found that IFNG (interferon-gamma) was overexpressed by tumor-infiltrating lymphocytes in PD-L1(+) versus PD-L1(-) melanomas, creating adaptive immune resistance by promoting PD-L1 display. This study was undertaken to identify additional factors in the PD-L1(+) melanoma microenvironment coordinately contributing to immunosuppression.

EXPERIMENTAL DESIGN:

Archived, formalin-fixed paraffin-embedded melanoma specimens were assessed for PD-L1 protein expression at the tumor cell surface with IHC. Whole-genome expression analysis, quantitative (q)RT-PCR, IHC, and functional in vitro validation studies were used to assess factors differentially expressed in PD-L1(+) versus PD-L1(-) melanomas.

RESULTS:

Functional annotation clustering based on whole-genome expression profiling revealed pathways upregulated in PD-L1(+) melanomas, involving immune cell activation, inflammation, and antigen processing and presentation. Analysis by qRT-PCR demonstrated overexpression of functionally related genes in PD-L1(+) melanomas, involved in CD8(+) T-cell activation (CD8A, IFNG, PRF1, and CCL5), antigen presentation (CD163, TLR3, CXCL1, and LYZ), and immunosuppression [PDCD1 (PD-1), CD274 (PD-L1), and LAG3, IL10]. Functional studies demonstrated that some factors, including IL10 and IL32-gamma, induced PD-L1 expression on monocytes but not tumor cells.

CONCLUSIONS:

These studies elucidate the complexity of immune checkpoint regulation in the tumor microenvironment, identifying multiple factors likely contributing to coordinated immunosuppression. These factors may provide tumor escape mechanisms from anti-PD-1/PD-L1 therapy, and should be considered for cotargeting in combinatorial immunomodulation treatment strategies.

Abstract

BACKGROUND:

Alzheimer's disease (AD) is a chronic neurodegenerative disease with pathological hallmarks including the formation of extracellular aggregates of amyloid-beta (Aβ) known as plaques and intracellular tau tangles. Coincident with the formation of Aβ plaques is recruitment and activation of glial cells to the plaque forming a plaque niche. In addition to histological data showing the formation of the niche, AD genetic studies have added to the growing appreciation of how dysfunctional glia pathways drive neuropathology, with emphasis on microglia pathways. Genomic approaches enable comparisons of human disease profiles between different mouse models informing on their utility to evaluate secondary changes to triggers such as Aβ deposition.

METHODS:

In this study, we utilized two animal models of AD to examine and characterize the AD-associated pathology: the Tg2576 Swedish APP (KM670/671NL) and TgCRND8 Swedish plus Indiana APP (KM670/671NL + V717F) lines. We used laser capture microscopy (LCM) to isolate samples surrounding Thio-S positive plaques from distal non-plaque tissue. These samples were then analyzed using RNAsequencing.

RESULTS:

We determined age-associated transcriptomic differences between two similar yet distinct APP transgenic mouse models, known to differ in proportional amyloidogenic species and plaque deposition rates. In Tg2576, human AD gene signatures were not observed despite profiling mice out to 15 months of age. TgCRND8 mice however showed progressive and robust induction of lysomal, neuroimmune, and ITIM/ITAM-associated gene signatures overlapping with prior human AD brain transcriptomic studies. Notably, RNAseq analyses highlighted the vast majority of transcriptional changes observed in aging TgCRND8 cortical brain homogenates were in fact specifically enriched within the plaque niche samples. Data uncovered plaque-associated enrichment of microglia-related genes such as ITIM/ITAM-associated genes and pathway markers of phagocytosis.

CONCLUSION:

This work may help guide improved translational value of APP mouse models of AD, particularly for strategies aimed at targeting neuroimmune and neurodegenerative pathways, by demonstrating that TgCRND8 more closely recapitulates specific human AD-associated transcriptional responses.