Stromal cell diversity associated with immune evasion in human triple-negative breast cancer.

The tumour stroma regulates nearly all stages of carcinogenesis. Stromal heterogeneity in human triple-negative breast cancers (TNBCs) remains poorly understood, limiting the development of stromal-targeted therapies. Single-cell RNA sequencing of five TNBCs revealed two cancer-associated fibroblast (CAF) and two perivascular-like (PVL) subpopulations. CAFs clustered into two states: the first with features of myofibroblasts and the second characterised by high expression of growth factors and immunomodulatory molecules. PVL cells clustered into two states consistent with a differentiated and immature phenotype. We showed that these stromal states have distinct morphologies, spatial relationships and functional properties in regulating the extracellular matrix. Using cell signalling predictions, we provide evidence that stromal-immune crosstalk acts via a diverse array of immunoregulatory molecules. Importantly, the investigation of gene signatures from inflammatory-CAFs and differentiated-PVL cells in independent TNBC patient cohorts revealed strong associations with cytotoxic T-cell dysfunction and exclusion, respectively. Such insights present promising candidates to further investigate for new therapeutic strategies in the treatment of TNBCs.

Monogenic mutations differentially affect the quantity and quality of T follicular helper cells in patients with human primary immunodeficiencies.

BACKGROUND: Follicular helper T (TFH) cells underpin T cell-dependent humoral immunity and the success of most vaccines. TFH cells also contribute to human immune disorders, such as autoimmunity, immunodeficiency, and malignancy. Understanding the molecular requirements for the generation and function of TFH cells will provide strategies for targeting these cells to modulate their behavior in the setting of these immunologic abnormalities. OBJECTIVE: We sought to determine the signaling pathways and cellular interactions required for the development and function of TFH cells in human subjects. METHODS: Human primary immunodeficiencies (PIDs) resulting from monogenic mutations provide a unique opportunity to assess the requirement for particular molecules in regulating human lymphocyte function. Circulating follicular helper T (cTFH) cell subsets, memory B cells, and serum immunoglobulin levels were quantified and functionally assessed in healthy control subjects, as well as in patients with PIDs resulting from mutations in STAT3, STAT1, TYK2, IL21, IL21R, IL10R, IFNGR1/2, IL12RB1, CD40LG, NEMO, ICOS, or BTK. RESULTS: Loss-of-function (LOF) mutations in STAT3, IL10R, CD40LG, NEMO, ICOS, or BTK reduced cTFH cell frequencies. STAT3 and IL21/R LOF and STAT1 gain-of-function mutations skewed cTFH cell differentiation toward a phenotype characterized by overexpression of IFN-γ and programmed death 1. IFN-γ inhibited cTFH cell function in vitro and in vivo, as corroborated by hypergammaglobulinemia in patients with IFNGR1/2, STAT1, and IL12RB1 LOF mutations. CONCLUSION: Specific mutations affect the quantity and quality of cTFH cells, highlighting the need to assess TFH cells in patients by using multiple criteria, including phenotype and function. Furthermore, IFN-γ functions in vivo to restrain TFH cell-induced B-cell differentiation. These findings shed new light on TFH cell biology and the integrated signaling pathways required for their generation, maintenance, and effector function and explain the compromised humoral immunity seen in patients with some PIDs.

A quantitative risk assessment for the incursion of lumpy skin disease virus into Australia via long-distance windborne dispersal of arthropod vectors.

Lumpy skin disease (LSD) is an infectious disease of cattle and water buffalo caused by lumpy skin disease virus (LSDV). It is primarily transmitted mechanically by biting insects. LSDV has spread from Africa to the Middle-East, the Balkans, Caucasus, Russia, Kazakhstan, China, Asia and India, suggesting that a wide variety of arthropod vectors are capable of mechanical transmission. In 2022, LSD was detected in Indonesia, heightening awareness for Australia's livestock industries. To better understand the risk of LSDV incursion to Australia we undertook a quantitative risk assessment (QRA) looking at windborne dispersal of arthropod vectors, assuming a hypothetical situation where LSD is endemic in south-east Asia and Papua New Guinea. We estimated the risk of LSDV incursion to be low, with a median incursion rate of one incursion every 403 years, based on a model where several infectious insects (i.e. a 'small batch' of 3-5) must bite a single bovine to transmit infection. The incursion risk increases substantially to one incursion every 7-8 years if a bite from a single insect is sufficient for transmission. The risk becomes negligible (one incursion every 20,706 years) if bites from many insects (i.e. a 'large batch' of 30-50 insects) are necessary. Critically, several of our parameter estimates were highly uncertain during sensitivity analyses. Thus, a key outcome of this QRA was to better prioritise surveillance activities and to understand the key research gaps associated with LSDV in the Australasian context. The current literature shows that multiple vectors are required for successful bovine-to-vector transmission of LSDV, suggesting that our estimate of one outbreak every 403 years more accurately represents the risk to Australia; however, the role of single insects in transmission has not yet been evaluated. Similarly, attempts to transmit LSDV between bovines by Culicoides have not been successful, although midges were the highest risk vector category in our model due to the high vector-to-host ratio for midges compared to other vector categories. Our findings provide further insight into the risk of LSD to Australian cattle industries and identify the Tiwi Islands and areas east of Darwin as priority regions for LSDV surveillance, especially between December and March.

Quantitative ctDNA Detection in Hepatoblastoma: Implications for Precision Medicine.

Hepatoblastoma is characterized by driver mutations in CTNNB1, making it an attractive biomarker for a liquid biopsy approach utilizing circulating tumor DNA (ctDNA). This prospective observational study sought to ascertain the feasibility of ctDNA detection in patients with hepatoblastoma and explore its associations with established clinical indicators and biomarkers, including serum Alpha-fetoprotein (AFP). We obtained 38 plasma samples and 17 tumor samples from 20 patients with hepatoblastoma. These samples were collected at various stages: 10 at initial diagnosis, 17 during neoadjuvant chemotherapy, 6 post-operatively, and 5 at disease recurrence. Utilizing a bespoke sequencing assay we developed called QUENCH, we identified single nucleotide variants and deletions in CTNNB1 ctDNA. Our study demonstrated the capability to quantitate ctDNA down to a variant allele frequency of 0.3%, achieving a sensitivity of 90% for patients at initial diagnosis, and a specificity of 100% at the patient level. Notably, ctDNA positivity correlated with tumor burden, and ctDNA levels exhibited associations with macroscopic residual disease and treatment response. Our findings provide evidence for the utility of quantitative ctDNA detection in hepatoblastoma management. Given the distinct detection targets, ctDNA and AFP-based stratification and monitoring approaches could synergize to enhance clinical decision-making. Further research is needed to elucidate the interplay between ctDNA and AFP and determine the optimal clinical applications for both methods in risk stratification and residual disease detection.

Antigen-driven EGR2 expression is required for exhausted CD8+ T cell stability and maintenance.

Chronic stimulation of CD8+ T cells triggers exhaustion, a distinct differentiation state with diminished effector function. Exhausted cells exist in multiple differentiation states, from stem-like progenitors that are the key mediators of the response to checkpoint blockade, through to terminally exhausted cells. Due to its clinical relevance, there is substantial interest in defining the pathways that control differentiation and maintenance of these subsets. Here, we show that chronic antigen induces the anergy-associated transcription factor EGR2 selectively within progenitor exhausted cells in both chronic LCMV and tumours. EGR2 enables terminal exhaustion and stabilizes the exhausted transcriptional state by both direct EGR2-dependent control of key exhaustion-associated genes, and indirect maintenance of the exhausted epigenetic state. We show that EGR2 is a regulator of exhaustion that epigenetically and transcriptionally maintains the differentiation competency of progenitor exhausted cells.

A single-cell and spatially resolved atlas of human breast cancers.

Breast cancers are complex cellular ecosystems where heterotypic interactions play central roles in disease progression and response to therapy. However, our knowledge of their cellular composition and organization is limited. Here we present a single-cell and spatially resolved transcriptomics analysis of human breast cancers. We developed a single-cell method of intrinsic subtype classification (SCSubtype) to reveal recurrent neoplastic cell heterogeneity. Immunophenotyping using cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) provides high-resolution immune profiles, including new PD-L1/PD-L2+ macrophage populations associated with clinical outcome. Mesenchymal cells displayed diverse functions and cell-surface protein expression through differentiation within three major lineages. Stromal-immune niches were spatially organized in tumors, offering insights into antitumor immune regulation. Using single-cell signatures, we deconvoluted large breast cancer cohorts to stratify them into nine clusters, termed 'ecotypes', with unique cellular compositions and clinical outcomes. This study provides a comprehensive transcriptional atlas of the cellular architecture of breast cancer.

Unique and shared signaling pathways cooperate to regulate the differentiation of human CD4+ T cells into distinct effector subsets.

Naive CD4(+) T cells differentiate into specific effector subsets-Th1, Th2, Th17, and T follicular helper (Tfh)-that provide immunity against pathogen infection. The signaling pathways involved in generating these effector cells are partially known. However, the effects of mutations underlying human primary immunodeficiencies on these processes, and how they compromise specific immune responses, remain unresolved. By studying individuals with mutations in key signaling pathways, we identified nonredundant pathways regulating human CD4(+) T cell differentiation in vitro. IL12Rß1/TYK2 and IFN-γR/STAT1 function in a feed-forward loop to induce Th1 cells, whereas IL-21/IL-21R/STAT3 signaling is required for Th17, Tfh, and IL-10-secreting cells. IL12Rß1/TYK2 and NEMO are also required for Th17 induction. Strikingly, gain-of-function STAT1 mutations recapitulated the impact of dominant-negative STAT3 mutations on Tfh and Th17 cells, revealing a putative inhibitory effect of hypermorphic STAT1 over STAT3. These findings provide mechanistic insight into the requirements for human T cell effector function, and explain clinical manifestations of these immunodeficient conditions. Furthermore, they identify molecules that could be targeted to modulate CD4(+) T cell effector function in the settings of infection, vaccination, or immune dysregulation.