Multi-resolution deep learning characterizes tertiary lymphoid structures and their prognostic relevance in solid tumors

BACKGROUND Tertiary lymphoid structures (TLSs) are dense accumulations of lymphocytes in inflamed peripheral tissues, including cancer, and are associated with improved survival and response to immunotherapy in various solid tumors. Histological TLS quantification has been proposed as a novel predictive and prognostic biomarker, but lack of standardized methods of TLS characterization hampers assessment of TLS densities across different patients, diseases, and clinical centers. METHODS We introduce an approach based on HookNet-TLS, a multi-resolution deep learning model, for automated and unbiased TLS quantification and identification of germinal centers in routine hematoxylin and eosin stained digital pathology slides. We developed HookNet-TLS using n = 1019 manually annotated TCGA slides from clear cell renal cell carcinoma, muscle-invasive bladder cancer, and lung squamous cell carcinoma. RESULTS Here we show that HookNet-TLS automates TLS quantification across multiple cancer types achieving human-level performance and demonstrates prognostic associations similar to visual assessment. CONCLUSIONS HookNet-TLS has the potential to be used as a tool for objective quantification of TLS in routine H&E digital pathology slides. We make HookNet-TLS publicly available to promote its use in research

Modified tumour antigen-encoding mRNA facilitates the analysis of naturally occurring and vaccine-induced CD4 and CD8 T cells in cancer patients

The development of effective anti-cancer vaccines requires precise assessment of vaccine-induced immunity. This is often hampered by low ex vivo frequencies of antigen-specific T cells and limited defined epitopes. This study investigates the applicability of modified, in vitro-transcribed mRNA encoding a therapeutically relevant tumour antigen to analyse T cell responses in cancer patients. In this study transfection of antigen presenting cells, by mRNA encoding the tumour antigen NY-ESO-1, was optimised and applied to address spontaneous and vaccine-induced T cell responses in cancer patients. Memory CD8+ T cells from lung cancer patients having detectable humoral immune responses directed towards NY-ESO-1 could be efficiently detected in peripheral blood. Specific T cells utilised a range of different T cell receptors, indicating a polyclonal response. Specific killing of a panel of NY-ESO-1 expressing tumour cell lines indicates recognition restricted to several HLA allelic variants, including a novel HLA-B49 epitope. Using a modified mRNA construct targeting the translated antigen to the secretory pathway, detection of NY-ESO-1-specific CD4+ T cells in patients could be enhanced, which allowed the in-depth characterisation of established T cell clones. Moreover, broad CD8+ and CD4+ T cell responses covering multiple epitopes were detected following mRNA stimulation of patients treated with a recombinant vaccinia/fowlpox NY-ESO-1 vaccine. This approach allows for a precise monitoring of responses to tumour antigens in a setting that addresses the breadth and magnitude of antigen-specific T cell responses, and that is not limited to a particular combination of known epitopes and HLA-restriction

Efficient Generation of Multipotent Mesenchymal Stem Cells from Umbilical Cord Blood in Stroma-Free Liquid Culture

BACKGROUND: Haematopoiesis is sustained by haematopoietic (HSC) and mesenchymal stem cells (MSC). HSC are the precursors for blood cells, whereas marrow, stroma, bone, cartilage, muscle and connective tissues derive from MSC. The generation of MSC from umbilical cord blood (UCB) is possible, but with low and unpredictable success. Here we describe a novel, robust stroma-free dual cell culture system for long-term expansion of primitive UCB-derived MSC. METHODS AND FINDINGS: UCB-derived mononuclear cells (MNC) or selected CD34(+) cells were grown in liquid culture in the presence of serum and cytokines. Out of 32 different culture conditions that have been tested for the efficient expansion of HSC, we identified one condition (DMEM, pooled human AB serum, Flt-3 ligand, SCF, MGDF and IL-6; further denoted as D7) which, besides supporting HSC expansion, successfully enabled long-term expansion of stromal/MSC from 8 out of 8 UCB units (5 MNC-derived and 3 CD34(+) selected cells). Expanded MSC displayed a fibroblast-like morphology, expressed several stromal/MSC-related antigens (CD105, CD73, CD29, CD44, CD133 and Nestin) but were negative for haematopoietic cell markers (CD45, CD34 and CD14). MSC stemness phenotype and their differentiation capacity in vitro before and after high dilution were preserved throughout long-term culture. Even at passage 24 cells remained Nestin(+), CD133(+) and >95% were positive for CD105, CD73, CD29 and CD44 with the capacity to differentiate into mesodermal lineages. Similarly we show that UCB derived MSC express pluripotency stem cell markers despite differences in cell confluency and culture passages. Further, we generated MSC from peripheral blood (PB) MNC of 8 healthy volunteers. In all cases, the resulting MSC expressed MSC-related antigens and showed the capacity to form CFU-F colonies. CONCLUSIONS: This novel stroma-free liquid culture overcomes the existing limitation in obtaining MSC from UCB and PB enabling so far unmet therapeutic applications, which might substantially affect clinical practice

Acquired resistance to anti-PD1 therapy in patients with NSCLC associates with immunosuppressive T cell phenotype

Immune checkpoint inhibitor treatment has the potential to prolong survival in non-small cell lung cancer (NSCLC), however, some of the patients develop resistance following initial response. Here, we analyze the immune phenotype of matching tumor samples from a cohort of NSCLC patients showing good initial response to immune checkpoint inhibitors, followed by acquired resistance at later time points. By using imaging mass cytometry and whole exome and RNA sequencing, we detect two patterns of resistance¨: One group of patients is characterized by reduced numbers of tumor-infiltrating CD8$^{+}$ T cells and reduced expression of PD-L1 after development of resistance, whereas the other group shows high CD8$^{+}$ T cell infiltration and high expression of PD-L1 in addition to markedly elevated expression of other immune-inhibitory molecules. In two cases, we detect downregulation of type I and II IFN pathways following progression to resistance, which could lead to an impaired anti-tumor immune response. This study thus captures the development of immune checkpoint inhibitor resistance as it progresses and deepens our mechanistic understanding of immunotherapy response in NSCLC

The Tumor Immune Landscape and Architecture of Tertiary Lymphoid Structures in Urothelial Cancer

Candidate immune biomarkers have been proposed for predicting response to immunotherapy in urothelial cancer (UC). Yet, these biomarkers are imperfect and lack predictive power. A comprehensive overview of the tumor immune contexture, including Tertiary Lymphoid structures (TLS), is needed to better understand the immunotherapy response in UC. We analyzed tumor sections by quantitative multiplex immunofluorescence to characterize immune cell subsets in various tumor compartments in tumors without pretreatment and tumors exposed to preoperative anti-PD1/CTLA-4 checkpoint inhibitors (NABUCCO trial). Pronounced immune cell presence was found in UC invasive margins compared to tumor and stroma regions. CD8+PD1+ T-cells were present in UC, particularly following immunotherapy. The cellular composition of TLS was assessed by multiplex immunofluorescence (CD3, CD8, FoxP3, CD68, CD20, PanCK, DAPI) to explore specific TLS clusters based on varying immune subset densities. Using a k-means clustering algorithm, we found five distinct cellular composition clusters. Tumors unresponsive to anti-PD-1/CTLA-4 immunotherapy showed enrichment of a FoxP3+ T-cell-low TLS cluster after treatment. Additionally, cluster 5 (macrophage low) TLS were significantly higher after pre-operative immunotherapy, compared to untreated tumors. We also compared the immune cell composition and maturation stages between superficial (submucosal) and deeper TLS, revealing that superficial TLS had more pronounced T-helper cells and enrichment of early TLS than TLS located in deeper tissue. Furthermore, superficial TLS displayed a lower fraction of secondary follicle like TLS than deeper TLS. Taken together, our results provide a detailed quantitative overview of the tumor immune landscape in UC, which can provide a basis for further studies

Combinations of Toll-like receptor 8 agonist TL8-506 activate human tumor-derived dendritic cells

BackgroundDendritic cells (DCs) are professional antigen presenting cells that initiate immune defense to pathogens and tumor cells. Human tumors contain only few DCs that mostly display a non-activated phenotype. Hence, activation of tumor-associated DCs may improve efficacy of cancer immunotherapies. Toll-like receptor (TLR) agonists and interferons are known to promote DC maturation. However, it is unclear if DCs in human tumors respond to activation signals and which stimuli induce the optimal activation of human tumor DCs.MethodsWe first screened combinations of TLR agonists, a STING agonist and interferons (IFNs) for their ability to activate human conventional DCs (cDCs). Two combinations: TL8-506 (a TLR8 agonist)+IFN-γ and TL8-506+Poly(I:C) (a TLR3 agonist) were studied in more detail. cDC1s and cDC2s derived from cord blood stem cells, blood or patient tumor samples were stimulated with either TL8-506+IFN-γ or TL8-506+Poly(I:C). Different activation markers were analyzed by ELISA, flow cytometry, NanoString nCounter Technology or single-cell RNA-sequencing. T cell activation and migration assays were performed to assess functional consequences of cDC activation.ResultsWe show that TL8-506 synergized with IFN-γ or Poly(I:C) to induce high expression of different chemokines and cytokines including interleukin (IL)-12p70 in human cord blood and blood cDC subsets in a combination-specific manner. Importantly, both combinations induced the activation of cDC subsets in patient tumor samples ex vivo. The expression of immunostimulatory genes important for anticancer responses including CD40, IFNB1, IFNL1, IL12A and IL12B were upregulated on stimulation. Furthermore, chemokines associated with CD8$^{+}$ T cell recruitment were induced in tumor-derived cDCs in response to TL8-506 combinations. In vitro activation and migration assays confirmed that stimulated cDCs induce T cell activation and migration.ConclusionsOur data suggest that cord blood-derived and blood-derived cDCs are a good surrogate to study treatment responses in human tumor cDCs. While most cDCs in human tumors display a non-activated phenotype, TL8-506 combinations drive human tumor cDCs towards an immunostimulatory phenotype associated with Th1 responses on stimulation. Hence, TL8-506-based combinations may be promising candidates to initiate or boost antitumor responses in patients with cancer

Eosinophils suppress Th1 responses and restrict bacterially induced gastrointestinal inflammation.

Eosinophils are predominantly known for their contribution to allergy. Here, we have examined the function and regulation of gastrointestinal eosinophils in the steady-state and during infection with or We find that eosinophils are recruited to sites of infection, directly encounter live bacteria, and activate a signature transcriptional program; this applies also to human gastrointestinal eosinophils in humanized mice. The genetic or anti-IL-5-mediated depletion of eosinophils results in improved control of the infection, increased inflammation, and more pronounced Th1 responses. Eosinophils control Th1 responses via the IFN-γ-dependent up-regulation of PD-L1. Furthermore, we find that the conditional loss of IFN-γR in eosinophils phenocopies the effects of eosinophil depletion. Eosinophils further possess bactericidal properties that require their degranulation and the deployment of extracellular traps. Our results highlight two novel functions of this elusive cell type and link it to gastrointestinal homeostasis and anti-bacterial defense

Graft-versus-host disease, but not graft-versus-leukemia immunity, is mediated by GM-CSF–licensed myeloid cells

Allogeneic hematopoietic cell transplantation (allo-HCT) not only is an effective treatment for several hematologic malignancies but can also result in potentially life-threatening graft-versus-host disease (GvHD). GvHD is caused by T cells within the allograft attacking nonmalignant host tissues; however, these same T cells mediate the therapeutic graft-versus-leukemia (GvL) response. Thus, there is an urgent need to understand how to mechanistically uncouple GvL from GvHD. Using preclinical models of full and partial MHC-mismatched HCT, we here show that the granulocyte-macrophage colony-stimulating factor (GM-CSF) produced by allogeneic T cells distinguishes between the two processes. GM-CSF drives GvHD pathology by licensing donor-derived phagocytes to produce inflammatory mediators such as interleukin-1β and reactive oxygen species. In contrast, GM-CSF did not affect allogeneic T cells or their capacity to eliminate leukemic cells, retaining undiminished GvL responses. Last, tissue biopsies and peripheral blood mononuclear cells from patients with grade IV GvHD showed an elevation of GM-CSF–producing T cells, suggesting that GM-CSF neutralization has translational potential in allo-HCT