The molecular mechanisms responsible for the high immunosuppressive capacity of CD4⁺ regulatory T cells (Tregs) in tumors are poorly known. High-dimensional single cell profiling of T cells from chemotherapy-naïve individuals with non-small cell lung cancer identified the transcription factor IRF4 as specifically expressed by a subset of intratumoral CD4⁺ effector Tregs with superior suppressive activity. In contrast to the IRF4– counterparts, IRF4+ Tregs expressed a vast array of suppressive molecules, and their presence correlated with multiple exhausted subpopulations of T cells. Integration of transcriptomic and epigenomic data revealed that IRF4, either alone or in combination with its partner BATF, directly controlled a molecular program responsible for immunosuppression in tumors. Accordingly, deletion of Irf4 exclusively in Tregs resulted in delayed tumor growth in mice while the abundance of IRF4⁺ Tregs correlated with poor prognosis in patients with multiple human cancers. Thus, a common mechanism underlies immunosuppression in the tumor microenvironment irrespectively of the tumor type.

Metastasis is the spread of cancer cells from primary tumours to distant organs and is the cause of 90% of cancer deaths globally^1,2. Metastasizing cancer cells are uniquely vulnerable to immune attack, as they are initially deprived of the immunosuppressive microenvironment found within established tumours³. There is interest in therapeutically exploiting this immune vulnerability to prevent recurrence in patients with early cancer at risk of metastasis. Here we show that inhibitors of cyclooxygenase 1 (COX-1), including aspirin, enhance immunity to cancer metastasis by releasing T cells from suppression by platelet-derived thromboxane A₂ (TXA₂). TXA₂ acts on T cells to trigger an immunosuppressive pathway that is dependent on the guanine exchange factor ARHGEF1, suppressing T cell receptor-driven kinase signalling, proliferation and effector functions. T cell-specific conditional deletion of Arhgef1 in mice increases T cell activation at the metastatic site, provoking immune-mediated rejection of lung and liver metastases. Consequently, restricting the availability of TXA₂ using aspirin, selective COX-1 inhibitors or platelet-specific deletion of COX-1 reduces the rate of metastasis in a manner that is dependent on T cell-intrinsic expression of ARHGEF1 and signalling by TXA₂ in vivo. These findings reveal a novel immunosuppressive pathway that limits T cell immunity to cancer metastasis, providing mechanistic insights into the anti-metastatic activity of aspirin and paving the way for more effective anti-metastatic immunotherapies.

Adoptive T cell transfer (ACT) immunotherapy benefits from early differentiated stem cell memory T (Tscm) cells capable of persisting in the long term and generating potent antitumor effectors. Due to their paucity ex vivo, Tscm cells can be derived from naive precursors, but the molecular signals at the basis of Tscm cell generation are ill-defined. We found that less differentiated human circulating CD8 ⁺ T cells display substantial antioxidant capacity ex vivo compared with more differentiated central and effector memory T cells. Limiting ROS metabolism with antioxidants during naive T cell activation hindered terminal differentiation, while allowing expansion and generation of Tscm cells. N-acetylcysteine (NAC), the most effective molecule in this regard, induced transcriptional and metabolic programs characteristic of self-renewing memory T cells. Upon ACT, NAC-generated Tscm cells established long-term memory in vivo and exerted more potent antitumor immunity in a xenogeneic model when redirected with CD19-specific CAR, highlighting the translational relevance of NAC as a simple and inexpensive method to improve ACT.

Immune checkpoint inhibitors are used for treating patients with metastatic melanoma. Since the response to treatment is variable, biomarkers are urgently needed to identify patients who may benefit from such therapy. Here, we combine single-cell RNA-sequencing and multiparameter flow cytometry to assess changes in circulating CD8⁺ T cells in 28 patients with metastatic melanoma starting anti-PD-1 therapy, followed for 6 months: 17 responded to therapy, whilst 11 did not. Proportions of activated and proliferating CD8⁺ T cells and of mucosal-associated invariant T (MAIT) cells are significantly higher in responders, prior to and throughout therapy duration. MAIT cells from responders express higher level of CXCR4 and produce more granzyme B. In silico analysis support MAIT presence in the tumor microenvironment. Finally, patients with >1.7% of MAIT among peripheral CD8⁺ population show a better response to treatment. Our results thus suggest that MAIT cells may be considered a biomarker for patients responding to anti-PD-1 therapy.

In bacterial genomes, there are two mechanisms to terminate the DNA transcription: the “intrinsic” or Rho-independent termination and the Rho-dependent termination. Intrinsic terminators are characterized by a RNA hairpin followed by a run of 6–8 U residues relatively easy to identify using one of the numerous available prediction programs. In contrast, Rho-dependent termination is mediated by the Rho protein factor that, firstly, binds to ribosome-free mRNA in a site characterized by a C > G content and then reaches the RNA polymerase to induce its release. Conversely on intrinsic terminators, the computational prediction of Rho-dependent terminators in prokaryotes is a very difficult problem because the sequence features required for the function of Rho are complex and poorly defined. This is the reason why it still does not exist an exhaustive Rho-dependent terminators prediction program. In this study we introduce RhoTermPredict, the first published algorithm for an exhaustive Rho-dependent terminators prediction in bacterial genomes. RhoTermPredict identifies these elements based on a previously proposed consensus motif common to all Rho-dependent transcription terminators. It essentially searches for a 78 nt long RUT site characterized by a C > G content and with regularly spaced C residues, followed by a putative pause site for the RNA polymerase. We tested RhoTermPredict performances by using available genomic and transcriptomic data of the microorganism Escherichia coli K-12, both in limited-length sequences and in the whole-genome, and available genomic sequences from Bacillus subtilis 168 and Salmonella enterica LT2 genomes. We also estimated the overlap between the predictions of RhoTermPredict and those obtained by the predictor of intrinsic terminators ARNold webtool. Our results demonstrated that RhoTermPredict is a very performing algorithm both for limited-length sequences (F1-score obtained about 0.7) and for a genome-wide analysis. Furthermore the degree of overlap with ARNold predictions was very low. Our analysis shows that RhoTermPredict is a powerful tool for Rho-dependent terminators search in the three analyzed genomes and could fill this gap in computational genomics. We conclude that RhoTermPredict could be used in combination with an intrinsic terminators predictor in order to predict all the transcription terminators in bacterial genomes.

Staphylococcus aureus (Staph. aureus) is one of the major pathogens causing mastitis in dairy ruminants worldwide. The chronic nature of Staph. aureus infection enhances the contagiousness risk and diffusion in herds. In order to identify the factors involved in intra-mammary infection (IMI) and diffusion in dairy cows, we investigated the molecular characteristics of two groups of Staph. aureus strains belonging to ST8 and ST398, differing in clinical properties, through comparison of whole genome and whole transcriptome sequencing. The two groups of strains, one originated from high IMI prevalence herds and the other from low IMI prevalence herds, present a peculiar set of genes and polymorphisms related to phenotypic features, such as bacterial invasion of mammary epithelial cells and host adaptation. Transcriptomic analysis supports the high propensity of ST8 strain to chronicity of infection and to a higher potential cytotoxicity. Our data are consistent with the invasiveness and host adaptation feature for the strains GTB/ST8 associated to high within-herd prevalence of mastitis. Variation in genes coding for surface exposed proteins and those associated to virulence and defence could constitute good targets for further research.