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Therapies that target the programmed death-1 (PD-1) receptor have shown unprecedented rates of durable clinical responses in patients with various cancer types (1-5). One mechanism by which cancer tissues limit the host immune response is via upregulation of PD-1 ligand (PD-L1) and its ligation to PD-1 on antigen-specific [CD8.sup.+] T cells (termed adaptive immune resistance) (6,7). Here we show that pre-existing [CD8.sup.+] T cells distinctly located at the invasive tumour margin are associated with expression of the PD-1/PD-L1 immune inhibitory axis and may predict response to therapy. We analysed samples from 46 patients with metastatic melanoma obtained before and during anti-PD-1 therapy (pembrolizumab) using quantitative immunohistochemistry, quantitative multiplex immunofluorescence, and nextgeneration sequencing for T-cell antigen receptors (TCRs). In serially sampled tumours, patients responding to treatment showed proliferation of intratumoral [CD8.sup.+] T cells that directly correlated with radiographic reduction in tumour size. Pre-treatment samples obtained from responding patients showed higher numbers of CD8-, PD-1and PD-L1-expressing cells at the invasive tumour margin and inside tumours, with close proximity between PD-1 and PD-L1, and a more clonal TCR repertoire. Using multivariate analysis, we established a predictive model based on CD8 expression at the invasive margin and validated the model in an independent cohort of 15 patients. Our findings indicate that tumour regression after therapeutic PD-1 blockade requires pre-existing [CD8.sup.+] T cells that are negatively regulated by PD-1/PD-L1-mediated adaptive immune resistance., Recently, we reported sustained tumour regression in 38% of patients in a multi-institutional, international, phase 1 expansion study evaluating the safety and clinical activity of pembrolizumab (formerly known as MK-3475 [...]

The expression of the bzd genes involved in the anaerobic degradation of benzoate in Azoarcus sp. CIB is controlled by the specific BzdR transcriptional repressor at the [P.sub.N] promoter. This catabolic promoter is also subject to catabolite repression by some organic acids. In vivo and in vitro experiments have shown that BzdR behaves as a repressor of the PR promoter by overlapping the transcription initiation site as well as the -35 and -10 boxes, benzoyl-CoA being the inducer molecule. In addition, by using a [P.sub.N]::lacZ fusion both in Azoarcus sp. CIB and in an isogenic strain lacking the bzdR gene, we have shown that the succinate-dependent catabolite repression requires participation of the BzdR repressor.

The role of oxygen in the transcriptional regulation of the [P.sub.N] promoter that controls the bzd operon involved in the anaerobic catabolism of benzoate in the denitrifying Azoarcus sp. strain CIB has been investigated. In vivo experiments using [P.sub.N]::lacZ translational fusions, in both Azoarcus sp. strain CIB and Escherichia coli cells, have shown an oxygen-dependent repression effect on the transcription of the bzd catabolic genes. E. coli Fnr was required for the anaerobic induction of the [P.sub.N] promoter, and the oxygen-dependent repression of the bzd genes could be bypassed by the expression of a constitutively active Fnr* protein. In vitro experiments revealed that Fnr binds to the PN promoter at a consensus sequence centered at position -41.5 from the transcription start site overlapping the -35 box, suggesting that [P.sub.N] belongs to the class II Fnr-dependent promoters. Fnr interacts with RNA polymerase (RNAP) and is strictly required for transcription initiation after formation of the RNAP-[P.sub.N] complex. An fnr ortholog, the acpR gene, was identified in the genome of Azoarcus sp. strain CIB. The Azoarcus sp. strain CIB acpR mutant was unable to grow anaerobically on aromatic compounds and it did not drive the expression of the [P.sub.N]::lacZ fusion, suggesting that AcpR is the cognate transcriptional activator of the [P.sub.N] promoter. Since the lack of AcpR in Azoarcus sp. strain CIB did not affect growth on nonaromatic carbon sources, AcpR can be considered a transcriptional regulator of the Fnr/Crp superfamily that has evolved to specifically control the central pathway for the anaerobic catabolism of aromatic compounds in Azoarcus.

The sequences surrounding the -12/-24 motif of the m-xylene-responsive [[sigma].sup.54] promoter Pu of the Pseudomonas putida TOL plasmid pWW0 were replaced by various DNA segments of the same size recruited from PnifH [[sigma].sup.54] promoter variants known to have various degrees of efficacy and affinity for [[sigma].sup.54]-RNA polymerase (RNAP). In order to have an accurate comparison of the output in vivo of each of the hybrids, the resulting promoters were recombined at the same location of the chromosome of P. putida KT2442 with a tailored vector system. The promoters included the upstream activation sequence (UAS) for the cognate regulator of the TOL system (XyIR) fused to the -12/-24 region of the wild-type PnifH and its higher [[sigma].sup.54]-RNAP affinity variants PnifH049 and PnifH319. As a control, the downstream region of the glnAp2 promoter (lacking integration host factor) was fused to the XyIR UAS as well. When the induction patterns of the corresponding lacZ fusion strains were compared in vivo, we observed that promoters bearing the RNAP binding site of PnifH049 and PnifH319 were not silenced during exponential growth, as is distinctly the case for the wild-type Pu promoter or for the Pu-PnifH variant. Taken together, our results indicate that the promoter sequence(s) spanning the -12/-24 region of Pu dictates the coupling of promoter output to growth conditions.

We report here that the bzd genes for anaerobic benzoate degradation in Azoarcus sp. strain CIB are organized as two transcriptional units, i.e., a benzoate-inducible catabolic operon, bzdNOPQMSTUVWXYZA, and a gene, bzdR, encoding a putative transcriptional regulator. The last gene of the catabolic operon, bzdA, has been expressed in Escherichia coli and encodes the benzoate-coenzyme A (CoA) ligase that catalyzes the first step in the benzoate degradation pathway. The BzdA enzyme is able to activate a wider range of aromatic compounds than that reported for other previously characterized benzoate-CoA ligases. The reduction of benzoyl-CoA to a nonaromatic cyclic intermediate is carried out by a benzoyl-CoA reductase (bzdNOPQ gene products) detected in Azoarcus sp. strain CIB extracts. The bzdW, bzdX, and bzdY gene products show significant similarity to the hydratase, dehydrogenase, and ring-cleavage hydrolase that act sequentially on the product of the benzoyl-CoA reductase in the benzoate catabolic pathway of Thauera aromatica. Benzoate-CoA ligase assays and transcriptional analyses based on lacZ-reporter fusions revealed that benzoate degradation in Azoarcus sp. strain CIB is subject to carbon catabolite repression by some organic acids, indicating the existence of a physiological control that connects the expression of the bzd genes to the metabolic status of the cell.

Results show that the sigma(sup)54-dependent Pu promoter activity of the TOL plasmid is stimulated by the alarmone ppGpp in Pseudomonas putida. Data also indicate that the stimulatory effect is moderate because of mild transcription activity of the alarmone.

We have examined the effects on transcription initiation of promoter and enhancer strength and of the curvature of the DNA separating these entities on wild-type and mutated enhancer-promoter regions at the Escherichia coli [[Sigma].sup.54]-dependent promoters glnAp2 and glnHp2 on supercoiled and linear DNA. Our results, together with previously reported observations by other investigators, show that the initiation of transcription on linear DNA requires a single intrinsic or induced bend in the DNA, as well as a promoter with high affinity for [[Sigma].sup.54]-RNA polymerase, but on supercoiled DNA requires either such a bend or a high affinity promoter but not both. The examination of the DNA sequence of all nif gene activator- or nitrogen regulator I-[[Sigma].sup.54] promoters reveals that those lacking a binding site for the integration host factor have an intrinsic single bend in the DNA separating enhancer from promoter.

The regulation of aromatic degradation in obligate anaerobes is analyzed in the Fe(III)-respiring model organism Geobacter metallireducens GS-15. A two-component system and a [[sigma].sup.54]-dependent promoter are identified that are both involved in the regulation of the gene coding for benzoate-coenzyme A ligase, which has catalyzed the initial step of benzoate degradation.