Mature tertiary lymphoid structures are key niches of tumour-specific immune responses in pancreatic ductal adenocarcinomas.

OBJECTIVE: To better understand the immune microenvironment of pancreatic ductal adenocarcinomas (PDACs), here we explored the relevance of T and B cell compartmentalisation into tertiary lymphoid structures (TLSs) for the generation of local antitumour immunity. DESIGN: We characterised the functional states and spatial organisation of PDAC-infiltrating T and B cells using single-cell RNA sequencing (scRNA-seq), flow cytometry, multicolour immunofluorescence, gene expression profiling of microdissected TLSs, as well as in vitro assays. In addition, we performed a pan-cancer analysis of tumour-infiltrating T cells using scRNA-seq and sc T cell receptor sequencing datasets from eight cancer types. To evaluate the clinical relevance of our findings, we used PDAC bulk RNA-seq data from The Cancer Genome Atlas and the PRINCE chemoimmunotherapy trial. RESULTS: We found that a subset of PDACs harbours fully developed TLSs where B cells proliferate and differentiate into plasma cells. These mature TLSs also support T cell activity and are enriched with tumour-reactive T cells. Importantly, we showed that chronically activated, tumour-reactive T cells exposed to fibroblast-derived TGF-ß may act as TLS organisers by producing the B cell chemoattractant CXCL13. Identification of highly similar subsets of clonally expanded CXCL13 + tumour-infiltrating T cells across multiple cancer types further indicated a conserved link between tumour-antigen recognition and the allocation of B cells within sheltered hubs in the tumour microenvironment. Finally, we showed that the expression of a gene signature reflecting mature TLSs was enriched in pretreatment biopsies from PDAC patients with longer survival after receiving different chemoimmunotherapy regimens. CONCLUSION: We provided a framework for understanding the biological role of PDAC-associated TLSs and revealed their potential to guide the selection of patients for future immunotherapy trials.

The STING ligand cGAMP potentiates the efficacy of vaccine-induced CD8+ T cells.

Pathogen recognition receptor (PRR) agonists are currently being developed and tested as adjuvants in various formulations to optimize the immunogenicity and efficacy of vaccines. Using an original in vitro approach to prime naive precursors from unfractionated human peripheral blood mononuclear cells, we assessed the influence of cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), a ligand for the stimulator of interferon genes (STING), on the induction of antigen-specific CD8+ T cells. We found that 2'3'-cGAMP and 3'3'-cGAMP were especially potent adjuvants in this system, driving the expansion and maturation of functionally replete antigen-specific CD8+ T cells via the induction of type I IFNs. The biological relevance of these findings was confirmed in vivo using two mouse models, in which 2'3'-cGAMP-adjuvanted vaccination elicited protective antitumor or antiviral CD8+ T cell responses. These results identify particular isoforms of cGAMP as effective adjuvants that may find utility in the development of novel immunotherapies and vaccines.

Naïve CD8+ T-Cells Engage a Versatile Metabolic Program Upon Activation in Humans and Differ Energetically From Memory CD8+ T-Cells.

Background: Characterization of the intracellular biochemical processes that regulate the generation and maintenance of effector and memory CD8+ T-cells from naïve precursors is essential for our understanding of adaptive immune responses and the development of immunotherapies. However, the metabolic determinants of antigen-driven activation and differentiation remain poorly defined, especially in humans. Methods: We used a variety of different approaches, including gene expression profiling and measurements of nutrient flux, to characterize the basal and activation-induced energetic requirements of naïve and phenotypically-defined subsets of human memory CD8+ T-cells. Findings: Profound metabolic differences were apparent as a function of differentiation status, both at rest and in response to stimulation via the T cell receptor (TCR). Of particular note, resting naïve CD8+ T cells were largely quiescent, but rapidly upregulated diverse energetic pathways after ligation of surface-expressed TCRs. Moreover, autophagy and the mechanistic target of rapamycin (mTOR)-dependent glycolytic pathway were identified as critical mediators of antigen-driven priming in the naïve CD8+ T cell pool, the efficiency of which was dampened by the presence of neutral lipids and fatty acids. Interpretation: These observations provide a metabolic roadmap of the CD8+ T-cell compartment in humans and reveal potentially selective targets for novel immunotherapies.

Epigenetic Control of Macrophage Shape Transition towards an Atypical Elongated Phenotype by Histone Deacetylase Activity.

Inflammatory chronic pathologies are complex processes characterized by an imbalance between the resolution of the inflammatory phase and the establishment of tissue repair. The main players in these inflammatory pathologies are bone marrow derived monocytes (BMDMs). However, how monocyte differentiation is modulated to give rise to specific macrophage subpopulations (M1 or M2) that may either maintain the chronic inflammatory process or lead to wound healing is still unclear. Considering that inhibitors of Histone Deacetylase (HDAC) have an anti-inflammatory activity, we asked whether this enzyme would play a role on monocyte differentiation into M1 or M2 phenotype and in the cell shape transition that follows. We then induced murine bone marrow progenitors into monocyte/macrophage differentiation pathway using media containing GM-CSF and the HDAC blocker, Trichostatin A (TSA). We found that the pharmacological inhibition of HDAC activity led to a shape transition from the typical macrophage pancake-like shape into an elongated morphology, which was correlated to a mixed M1/M2 profile of cytokine and chemokine secretion. Our results present, for the first time, that HDAC activity acts as a regulator of macrophage differentiation in the absence of lymphocyte stimuli. We propose that HDAC activity down regulates macrophage plasticity favoring the pro-inflammatory phenotype.