October

October 2021

The following articles have been recommended for further reading in the field of cancer immunotherapy by Dr. Sandra Demaria, Co-Section Editor for the Reviews Section.

“Tumor-derived exosomes drive immunosuppressive macrophages in a pre-metastatic niche through glycolytic dominant metabolic reprogramming” by Samantha M Morrissey et al

Immunosuppressive macrophage infiltration is known to be an essential initial step to establish pre-metastatic niches, yet the upstream mechanisms driving the phenotypes of niche macrophages are not known. Samantha M Morrissey et al demonstrate that soluble tumor-derived exosomes (TDEs) induce an NF-κB-driven metabolic reprogramming resulting in increased PD-L1 expression and an immunosuppressive phenotype to set the stage for the pre-metastatic niche. TDEs isolated from a murine lung adenocarcinoma cell line (LLC) increased micro-metastases in LLC exosome-treated mice, with preferential increased PD-L1 mRNA and protein expression on lung interstitial macrophages (IMs). TLR-2 was required for the TDE-induced increase in PD-L1 expression in vivo and in vitro, and expression of the ligand decreased after exposure to TDEs in TLR2-deficient backgrounds. NF-κB inhibition also led to decreased TDE-induced macrophage PD-L1 expression and it was confirmed that exposure to TDEs resulted in increased phosphorylation of NF-κB in the macrophages. Upon stimulation with TDEs, M2 macrophages had increased glucose uptake and increased expression of GLUT-1, HIF1-α, and LDHA. Increased lactate secretion and mRNA expression of the gene encoding the lactate exporter MCT4 was also demonstrated in TDE-stimulated macrophages, leading to lactate-induced PD-L1 upregulation. Phosflow assay and confocal microscopy further confirmed lactate-induced phosphorylation and nuclear translocation of NF-κB. In vivo, depletion of RAB27a (necessary for exosome secretion) in a breast cancer line metastatic to lungs led to diminished lung-resident infiltrating macrophages with low PD-L1 expression. In vitro, TDEs from a human non-small cell lung cancer (NSCLC) cell line increased PD-L1 expression on CD14⁺ macrophages, which then inhibited CD4⁺ and CD8⁺ T cell proliferation and production of IFN-ƴ. Stimulation with recombinant human HMGB-1 and lactate increased PD-L1 expression in TDE-stimulated CD14⁺ macrophages. Lastly, draining LN (dLN) macrophages of NSCLC patients with N1-N3 disease were found to have higher expression of the exosome release genes YKT6 and TSG101 compared to Nx/N0 dLNs.

    

Why this matters: These findings uncover tumor-derived signals that set the stage for the pre-metastatic niche by causing TLR2/ NF-κB-dependent glycolytic metabolic reprogramming in macrophages that results in PD-L1 upregulation. The novel pathway responsible for skewing macrophages toward an immunosuppressive phenotype involving HMGB-1, HIF1-α, and GLUT-1 might offer future targets or biomarkers for development to predict or prevent metastatic disease.

 

“Commensal bacteria and fungi differentially regulate tumor responses to radiation therapy” by Stephen L Shiao et al

While the composition of bacterial commensals in the gut has been associated with response and resistance to chemotherapy and immunotherapy, the influence of the gut microbiota on radiation therapy (RT) outcomes is not known and the non-prokaryotic constituents have been understudied. Stephen L Shiao and colleagues demonstrate differential outcomes after radiotherapy in murine models depending on fungi and bacteria in the gut. In specific pathogen-free (SPF) mice with orthotopic mammary fat pad tumors, delay of tumor growth or prolonged survival after 16 Gy of RT were both decreased if radiation was preceded by antibiotics. Notably, antibiotic treatment depleted bacteria by >2,000-fold in the mice, but expanded fungi populations by >2,000-fold. Antifungal treatment enhanced RT efficacy in murine mammary tumors and melanoma models, primarily enhancing tumor cell death. Analysis of tumor tissue from antibiotic-treated mice one week following RT revealed increased CD11b⁺F4/80⁺ tumor-associated macrophages, while the number and percentage of CD4⁺ and CD8⁺ T cells was not affected. Conversely, tumor tissue from mice that received antifungals showed reduced CD4⁺ T cells, increased CD8⁺ T cells, and no change in the number of activated CD69⁺CD8⁺ and CD25⁺CD4⁺ T cells or activated CD11b⁺F4/80⁺MHCII⁺ macrophages. Furthermore, antifungal treatment increased the cytotoxic effector capacity after RT as evidenced by a reduction in PD-1⁺CD8⁺ and PD-1⁺CD4⁺ T cells and CD206⁺F4/80⁺ macrophages, and higher numbers of Graynzme B-expressing CD8⁺ T cells. The most striking effect of antibiotic therapy on the SPF mouse microbiome was the dramatic expansion of fungal populations, particularly Saccharomyces and Candida. Following tumor radiation of SPF mice that were robustly colonized by C. albicans, there was less delay in tumor regrowth, worse overall survival, higher numbers of exhausted T cells in the tumor, increased tumor cell division, and less tumor cell death. Finally, high mRNA expression of the Dectin-1 receptor (CLEC7A), which recognizes fungal β-glucans, in macrophages and dendritic cells from triple-negative breast tumors correlated with worse survival outcomes. Furthermore, dectin-1 deficient mice demonstrated an enhanced response to RT. 

 

Why this matters: This report emphasizes the role of fungal outgrowth that follows antibiotic treatment in dampening the efficacy of cancer radiotherapy, an effect largely due to decreased anti-tumor immunity. The deleterious effect of fungal overgrowth was mediated via the myeloid dectin-1 receptor for b-glucans found in fungal cell walls.  High expression of dectin-1 in human breast cancer was associated with worse survival, suggesting that targeting commensal fungi and/or targeting therapeutically  dectin-1 may increase the efficacy of radiotherapy and possibly other therapies.

“PD-1-stimulated T cell subsets are transcriptionally and functionally distinct” by Shalom Lerrer et al

The role that PD-1 plays in CD4⁺ T cell signaling is not well understood. Building on an observation that PD-1 signaling’s influence on proliferation and maturation may be uncoupled, Shalom Lerrer et al used transcriptomics to understand differential responses to PD-L1 and PD-L2 across T cell subsets. In CD4⁺ T cells, PD-L1 ligation altered the expression levels of more genes compared to PD-L2. Most genes initially regulated by the TCR were differentially expressed upon subsequent treatment with PD-L1 or PD-L2, particularly in naïve and central memory T cells. By ontogeny, PD-1-regulated genes in naïve and central memory T cells were primarily associated with metabolism, cell cycle regulation, T cell activation and differentiation, and production of IL-4, 7, 10, and 12. The PD-1-regulated genes in effector memory T cells, by contrast were primarily associated with IL-8 and MCP-1 production, adhesion, and migration. Among the PD-1-induced differentially expressed genes that were not coregulated by TCR signaling, little overlap was seen between T cell subsets. Naïve T cells showed a signature enriched for T cell migration and chemotaxis; central memory T cells were enriched for pathways involved in T helper responses and differentiation; effector memory T cells had a signature characterized by IL-4 responses, calcium signaling, and JAK-STAT pathway function. Ligation of the same PD-1 receptor with PD-L1 versus PD-L2 resulted in upregulation of non-identical catalogues of transcription factors, however, STAT1 and STAT2 being the most prevalent across all three T cell subsets. STAT1 and STAT2 mRNA expression increased with TCR ligation and then further with PD-L1 and PD-L2 ligation. Transcriptomic data from a real-world database of melanoma patients treated with pembrolizumab showed lower expression levels of both STAT1 and STAT2 in patients who responded to treatment. Similarly, an inverse relationship between 5-year overall survival and STAT1 expression was observed in data from human patients with lung adenocarcinoma.

 

Why this matters: PD-1 ligation by PD-L1 or PD-L2 results in a distinct transcriptional program that varies by T cell subtype. The pronounced STAT1 and STAT2 expression in T cell subsets upon PD-L1 and PD-L2 ligation may represent a future target for biomarker development or strategy to overcome resistance to immune checkpoint blockade.

 

“Low dose radiotherapy reverses tumor immune desertification and resistance to immunotherapy” by Fernanda G Herrera et al

While high-dose radiation therapy is known to synergize with immune checkpoint blockade, little is known about the effects of low-dose RT (LDRT). In pre-clinical models and a phase I trial that included eight patients with T cell-excluded solid tumors, Fernanda G Herrera and colleagues show that LDRT leads to a more favorable tumor microenvironment and synergizes with checkpoint blockade. In murine ID8 models of ovarian cancer, LDRT (1 Gy weekly) was associated with an influx of lymphocytes, NK cells, macrophages, and dendritic cells into the intraepithelial tumor compartment. Combination treatment with LDRT, cyclophosphamide, PD-1/CTLA-4 blockade, and an agnostic αCD40 Ab (RACIM) led to an 83.5% tumor response (15% cure) and was associated with an influx of tumor-infiltrating lymphocytes (TILs) enriched for activated effector T cells, particularly exhausted (Tex) and progenitor-exhausted PD1⁺TCF1^- clonotypes with IFNƴ expression. RACIM also induced repolarization of intratumoral macrophages from M2 to M1 and enhanced numbers of antigen-presenting cDC2/MoDC dendritic cell (DCs) with upregulated Rae1. This receptor, a stress-response ligand to the natural killer group 2D (NKG2D) costimulatory receptor, was also significantly upregulated in both CD4⁺ and CD8⁺ exhausted T cells from RACIM-treated tumors. However, NKG2D expression was not induced by the regimen upon omission of anti-CTLA-4 or anti-PD-1 blockade. In a phase I clinical trial, eight patients with pre-treated, immunotherapy-naïve solid tumors with <5 intraepithelial CD8⁺ cells per high-power field (“immune dessert”) were treated with combination LDRT (0.5 or 1 Gy per fraction every 2 weeks, total dose 6 or 13 Gy, respectively), cyclophosphamide, ipilimumab, nivolumab, and aspirin (RACIN). After treatment, 37.5% achieved reduced size of irradiated lesions, 87.5% achieved disease control, and 12.5% had disease progression (generally outside the field of LDRT), with an iRECIST ORR of 12.5%. The rate of grade ≥ 3 immune-related adverse events was 25%, including cases of colitis, hepatitis, and myocarditis, but no new safety signals emerged. Similar to mouse models, RACIN led to increases in Th1, CD8+, and T effector-memory signatures localized to tumor islets in responsive tumors, whereas non-responding tumors displayed M2 macrophage upregulation signatures mainly in tumor stroma.

Why this matters: Low dose radiation reprograms immune desert tumors by stimulating innate and adaptive immunity offering a potential strategy to overcome resistance to immunotherapy.