Shannon Turley's research while affiliated with Centre d'Immunologie de Marseille-Luminy and other places

Background TGFβ signaling is associated with non-response to immune checkpoint blockade in patients with advanced metastatic cancers, particularly in patients with the immune excluded phenotype. While previous work demonstrates that converting tumors from excluded to inflamed phenotypes requires attenuation of PD-L1 and TGFβ signaling, the underlying cellular mechanisms remain largely unclear. Methods We performed single-cell RNA-seq (n=5 animals per treatment) on myeloid, stromal, tumor, and T cells, as well as TCR-seq (n=7 animals per treatment) on T cells from anti-PD-L1 and anti-TGFβ antibody-treated mice bearing subcutaneous EMT6 tumors on day 7 after initiation of treatment. We performed flow cytometry to characterize and quantify T cell subtypes from treated tumors (n>=10 animals per treatment). Furthermore, we measured T cell motility (n=8 animals per treatment) in a dorsal skinfold chamber from IFNγ-YFP reporter mice bearing EMT6-mApple tumor cells with anti-gp120 as control or anti-PD-L1 and anti-TFGβ combination treatment. To determine the impact of IFNγ response on the effect of combination treatment, we tracked tumor growth in mice with anti-IFNγ treated EMT6 tumors (n=10 per treatment) or with IFNGR1 KO EMT6 tumors (n=10 per group). Results We show that TGFβ and PD-L1 restrain intratumoral stem cell-like CD8 T cell (TSCL) expansion and maintain progenitor-exhausted and dysfunctional CD8 T cells. Upon combined TGFβ/PD-L1 blockade, TSCL expand and can differentiate into IFNγhi CD8 T effector cells that show enhanced motility and accumulate in the tumor microenvironment. Ensuing IFNγ signaling transforms myeloid, stromal, and tumor niches to yield a broadly immune-supportive ecosystem. Blocking IFNγ either through knockout of IFNGR1 in tumor cells or blocking IFNγ abolishes the effect of anti-PD-L1/TGFβ combination therapy. Conclusions Our data suggest that TGFβ works with PD-L1 to prevent TSCL expansion and replacement of exhausted CD8 T cells, thereby maintaining the T cell compartment in a dysfunctional state. Ethics Approval All animal activities in the research studies here presented were conducted under protocols approved by the Genentech Institutional Animal Care and Use Committee (IACUC).

Background Single cell transcriptomics has led to the generation of large-scale atlases to understand cellular heterogeneity across both healthy and diseased tissues at the highest resolution. Such atlases have allowed for in silico inferences of fibroblast ontogeny and function, but without in vivo substantiation are limited in their utility to inspire novel fibroblast-directed therapies to improve disease outcome. In cancer, single cell studies identified the emergence of a myofibroblast population, in both mice and humans, uniquely marked by a highly restricted leucine rich repeat containing protein, LRRC15. This cancer-associated myofibroblast population expresses a multitude of extracellular matrix (ECM) associated and immunosuppressive genes. Clinically, high expression of an LRRC15⁺ CAF gene signature in bulk RNAseq data from cancer patients correlated with lack of response to anti-programmed death ligand-1 (aPDL1) checkpoint blockade therapy. It remains unclear if LRRC15⁺ CAFs are the cause of this lack of response, or if they represent a read-out of tumor-intrinsic features driving the association. Also missing are in vivo substantiation of the cellular and molecular signals driving LRRC15⁺ myofibroblast development and the direct impact LRRC15⁺ CAFs have on anti-tumor immunity. Methods To address these gaps, we paired newly developed preclinical genetic tools in murine tumor models of pancreatic cancer with a large clinical dataset of human stromal cell-sorted expression data from 159 cancer patients across 13 tumor types to understand LRRC15⁺ CAF development and function. Results In mouse models of pancreatic cancer, we provide in vivo genetic evidence that TGFβR2 signaling in healthy Dermatopontin (DPT)⁺ universal fibroblasts is essential for development of tumor-associated LRRC15⁺ myofibroblasts. Analysis of tumors from 159 patients across 13 indications revealed a conserved axis from universal fibroblasts to LRRC15⁺ myofibroblasts. This axis is the predominant driver of fibroblast lineage diversity in human cancers. Using newly developed Lrrc15-Diphtheria toxin receptor knock-in mice to selectively deplete LRRC15⁺ CAFs, we show loss of this population markedly reduced total tumor fibroblast content and recalibrated the CAF composition towards universal fibroblasts. This, in turn, relieved direct suppression of tumor-infiltrating CD8⁺ T cells to enhance their effector function and significantly augmented tumor regression in response to anti-PDL1 immune checkpoint blockade. Conclusions Collectively, these findings demonstrate that TGFβ-dependent LRRC15⁺ CAFs dictate the tumor-fibroblast setpoint to promote tumor growth, directly suppress CD8⁺ T cell functionality, and limit responsiveness to checkpoint blockade.¹ Development of treatments that restore the homeostatic fibroblast setpoint by diminishing pro-disease LRRC15⁺ myofibroblasts may improve patient survival and response to immunotherapy. Reference • Krishnamurty AT, Shyer JA, Thai M, Gandham V, Buechler MB, Yang YA, Pradhan RN, Wang AW, Sanchez PL, Qu Y, Breart B, Chalouni C, Dunlap D, Ziai J, Elstrott J, Zacharias N, Mao W, Rowntree RK, Sadowsky J, Lewis GD, Pillow TH, Nabet BY, Banchereau R, Tam L, Caothien R, Bacarro N, Roose-Girma M, Modrusan Z, Mariathasan S, Muller S*, Turley SJ*. LRRC15⁺ myofibroblasts dictate the stromal setpoint to suppress tumor immunity. Nature, 2022; 611 :148–154

Sensory neurons of the vagal ganglia (VG) innervate lungs and play a critical role in maintaining airway homeostasis. However, the specific VG neurons that innervate lungs, and the mechanisms by which these neurons sense and respond to airway insults, are not well understood. Here, we identify a subpopulation of lung-innervating VG neurons defined by their expression of Tmc3 . Single cell transcriptomics illuminated several subpopulations of Tmc3+ sensory neurons, revealing distinct Piezo2 - and Trpv1 -expressing subclusters. Furthermore, Tmc3 deficiency in VG neurons leads to global and subcluster specific transcriptional changes related to metabolic and ion channel function. Importantly, we show that broncho-constriction and dilation can be modulated through inhibition or activation of Tmc3+ VG neurons resulting in a decrease or increase of end-expiratory lung volume, respectively. Together, our data show that Tmc3 is a marker of lung-innervating neurons and may play a pivotal role in maintaining fundamental inspiratory and expiratory processes. Significance Harnessing the neuronal mechanisms that regulate lung function offers potential alternatives to existing corticosteroid treatment regimens for respiratory illness associated with acute bronchoconstriction including asthma, COPD, and emphysema. Our findings define Transmembrane channel-like 3 , Tmc3 , as a marker of lung-innervating sensory neurons, identify distinct subpopulations of Tmc3 + neurons with unique transcriptional profiles, and show that activation or inhibition of these neurons has a significant impact on airway function. Our work highlights potential avenues of novel targeted intervention in respiratory conditions driven by dysfunctional neuronal reflexes.

Background Single cell transcriptomics has led to the generation of large-scale atlases to understand cellular heterogeneity across both healthy and diseased tissues at the highest resolution. Such atlases have allowed for in silico inferences of fibroblast ontogeny and function, but without in vivo substantiation are limited in their utility to inspire novel fibroblast-directed therapies to improve disease outcome. In cancer, single cell studies identified the emergence of a myofibroblast population, in both mice and humans, uniquely marked by a highly restricted leucine rich repeat containing protein, LRRC15. This cancer-associated myofibroblast population expresses a multitude of extracellular matrix (ECM) associated and immunosuppressive genes. Clinically, high expression of an LRRC15⁺ CAF gene signature in bulk RNAseq data from cancer patients correlated with lack of response to anti-programmed death ligand-1 (aPDL1) checkpoint blockade therapy. It remains unclear if LRRC15⁺ CAFs are the cause of this lack of response, or if they represent a read-out of tumor-intrinsic features driving the association. Also missing are in vivo substantiation of the cellular and molecular signals driving LRRC15⁺ myofibroblast development and the direct impact LRRC15⁺ CAFs have on anti-tumor immunity. Methods To address these gaps, we paired newly developed preclinical genetic tools in murine tumor models of pancreatic cancer with a large clinical dataset of human stromal cell-sorted expression data from 159 cancer patients across 13 tumor types to understand LRRC15⁺ CAF development and function. Results In mouse models of pancreatic cancer, we provide in vivo genetic evidence that TGFβR2 signaling in healthy Dermatopontin (DPT)⁺ universal fibroblasts is essential for development of tumor-associated LRRC15⁺ myofibroblasts. Analysis of tumors from 159 patients across 13 indications revealed a conserved axis from universal fibroblasts to LRRC15⁺ myofibroblasts. This axis is the predominant driver of fibroblast lineage diversity in human cancers. Using newly developed Lrrc15-Diphtheria toxin receptor knock-in mice to selectively deplete LRRC15⁺ CAFs, we show loss of this population markedly reduced total tumor fibroblast content and recalibrated the CAF composition towards universal fibroblasts. This, in turn, relieved direct suppression of tumor-infiltrating CD8⁺ T cells to enhance their effector function and significantly augmented tumor regression in response to anti-PDL1 immune checkpoint blockade. Conclusions Collectively, these findings demonstrate that TGFβ-dependent LRRC15⁺ CAFs dictate the tumor-fibroblast setpoint to promote tumor growth, directly suppress CD8⁺ T cell functionality, and limit responsiveness to checkpoint blockade. Development of treatments that restore the homeostatic fibroblast setpoint by diminishing pro-disease LRRC15⁺ myofibroblasts may improve patient survival and response to immunotherapy.

To better define the control of immune system regulation, we generated an atlas of microRNA (miRNA) expression from 63 mouse immune cell populations and connected these signatures with assay for transposase-accessible chromatin using sequencing (ATAC–seq), chromatin immunoprecipitation followed by sequencing (ChIP–seq) and nascent RNA profiles to establish a map of miRNA promoter and enhancer usage in immune cells. miRNA complexity was relatively low, with >90% of the miRNA compartment of each population comprising <75 miRNAs; however, each cell type had a unique miRNA signature. Integration of miRNA expression with chromatin accessibility revealed putative regulatory elements for differentially expressed miRNAs, including miR-21a, miR-146a and miR-223. The integrated maps suggest that many miRNAs utilize multiple promoters to reach high abundance and identified dominant and divergent miRNA regulatory elements between lineages and during development that may be used by clustered miRNAs, such as miR-99a/let-7c/miR-125b, to achieve distinct expression. These studies, with web-accessible data, help delineate the cis-regulatory elements controlling miRNA signatures of the immune system. Brown and colleagues generated an atlas of miRNA expression profiles from primary mouse immune cell populations and connected these signatures with ATAC–seq, ChIP–seq and nascent RNA profiles to establish a map of miRNA promoter and enhancer usage in immune cells.

Nature Immunology’s 20th anniversary is a good opportunity to reminisce about the ImmGen collective endeavor — its goals, successes and horror stories — and the group’s exploration of various modes of scientific publishing.

CD4 effector lymphocytes (T ) are traditionally classified by the cytokines they produce. To determine the states that T cells actually adopt in frontline tissues in vivo, we applied single-cell transcriptome and chromatin analyses to colonic T cells in germ-free or conventional mice or in mice after challenge with a range of phenotypically biasing microbes. Unexpected subsets were marked by the expression of the interferon (IFN) signature or myeloid-specific transcripts, but transcriptome or chromatin structure could not resolve discrete clusters fitting classic helper T cell (T ) subsets. At baseline or at different times of infection, transcripts encoding cytokines or proteins commonly used as T markers were distributed in a polarized continuum, which was functionally validated. Clones derived from single progenitors gave rise to both IFN-γ- and interleukin (IL)-17-producing cells. Most of the transcriptional variance was tied to the infecting agent, independent of the cytokines produced, and chromatin variance primarily reflected activities of activator protein (AP)-1 and IFN-regulatory factor (IRF) transcription factor (TF) families, not the canonical subset master regulators T-bet, GATA3 or RORγ. + eff eff eff H H

Sepsis is an aggressive inflammatory syndrome, killing 7.3 million people per year. In mouse and human systems, we have identified highly differentiated, immune-specialized myofibroblasts called Fibroblastic Reticular Cells (FRCs) as a new cell therapy candidate for sepsis. One injection of ex-vivo expanded allogeneic FRCs significantly reduced mortality when administered at therapeutic timepoints. When administered 4 hrs after cecal ligation and puncture (CLP) in mice, FRCs prevented peritonitis progressing to blood sepsis, reducing deaths by 50%, while mesenchymal stromal cells (MSCs) were ineffective. FRCs also reduced lung pathology and lowered systemic concentrations of pathogenic cytokines. Rarely for putative sepsis therapies, we found that when FRCs were administered very late, as mice approached euthanasia endpoints (16 hrs), survival was still extremely robust: 44% of FRC-treated mice survived compared to 9% of controls. Production of the prominent vasodilator and immunosuppressant NOS2 was a primary molecular mechanism. Human FRCs showed strong similarities to mouse FRCs and exhibited desirable traits for a cell therapy candidate, including low variance between unrelated donors. Importantly, human FRCs constitutively secrete high levels of soluble cytokine decoy receptors against major mediators of sepsis. Together these data describe a novel anti-inflammatory cell type and provide robust pre-clinical evidence for therapeutic efficacy in multiple models of sepsis.

We assessed gene expression in tissue macrophages from various mouse organs. The diversity in gene expression among different populations of macrophages was considerable. Only a few hundred mRNA transcripts were selectively expressed by macrophages rather than dendritic cells, and many of these were not present in all macrophages. Nonetheless, well-characterized surface markers, including MerTK and FcγR1 (CD64), along with a cluster of previously unidentified transcripts, were distinctly and universally associated with mature tissue macrophages. TCEF3, C/EBP-α, Bach1 and CREG-1 were among the transcriptional regulators predicted to regulate these core macrophage-associated genes. The mRNA encoding other transcription factors, such as Gata6, was associated with single macrophage populations. We further identified how these transcripts and the proteins they encode facilitated distinguishing macrophages from dendritic cells.