The density of Tbet+ tumor-infiltrating T lymphocytes reflects an effective and druggable preexisting adaptive antitumor immune response in colorectal cancer, irrespective of the microsatellite status.

Purpose: The recent success of anti-PD1 antibody in metastatic colorectal cancer (CRC) patients with microsatellite instability (MSI), known to be associated with an upregulated Th1/Tc1 gene signature, provides new promising therapeutic strategies. However, the partial objective response highlights a crucial need for relevant, easily evaluable, predictive biomarkers. Here we explore whether in situ assessment of Tbet+ tumor infiltrating lymphocytes (TILs) reflects a pre-existing functional antitumor Th1/Tc1/IFNγ response, in relation with clinicopathological features, microsatellite status and expression of immunoregulatory molecules (PD1, PDL1, IDO-1). Methodology: In two independent cohorts of CRC (retrospective n = 80; prospective n = 27) we assessed TILs density (CD3, Tbet, PD1) and expression profile of PDL1 and IDO-1 by immunohistochemistry/image analysis. Furthermore, the prospective cohort allowed to perform ex vivo CRC explant cultures and measure by Elisa the IFNγ response, at baseline and upon anti-PD1 treatment. Results: The density of Tbet+ TILs was significantly higher in MSI CRC, especially in the medullary subtype but also in a subgroup of MSS (microsatellite stable), and positively correlated with PD1 and PDL1 expression, but not with IDO-1. Finally, a high number of Tbet+ TILs was associated with a favorable overall survival. These Tbet+ TILs were functional as their density positively correlated with basal IFNγ levels. In addition, the combined score of Tbet+ PD1+ TILs coupled with IDO-1 expression predicted the magnitude of the IFNγ response upon anti-PD1. Conclusion: Altogether, immunohistochemical quantification of Tbet+ TILs is a reliable and accurate tool to recapitulate a preexisting Th1/Tc1/IFNγ antitumor response that can be reinvigorated by anti-PD1 treatment.

The RFN riboswitch of Bacillus subtilis is a target for the antibiotic roseoflavin produced by Streptomyces davawensis.

The riboflavin (vitamin B(2)) biosynthetic genes in Bacillus subtilis are transcribed simultaneously from the riboflavin promoter (P(rib)). The 5'-end of the nascent rib-mRNA carries a flavin mononucleotide (FMN) binding riboswitch, which regulates gene expression. The antibiotic roseoflavin from Streptomyces davawensis is a naturally occurring riboflavin analog, its mechanism of action is largely unknown. A recombinant B. subtilis strain carrying a copy of P(rib)-RFN fused to a promoterless lacZ reporter gene in the chromosomal amyE locus was grown in a minimal medium. Upon addition of roseoflavin to the growth medium the apparent LacZ activity in this strain was not significantly reduced. Similar experiments carried out on recombinant B. subtilis strains oversynthesizing the flavin transporters RibU (B. subtilis) or RibM (S. davawensis) produced still other results. In these strains, roseoflavin (as well as riboflavin) repressed LacZ synthesis indicating that the RFN riboswitch is a target for roseoflavin (or roseoflavin mononucleotide), which may at least in part explain its antibiotic activity.