Publications by authors named "Matthew F Krummel"

113 Publications

A "data sharing trust" model for rapid, collaborative science.

Cell 2021 Feb;184(3):566-570

ImmunoX Initiative, Department of Microbiology and Immunology, University of California, San Francisco, 513 Parnassus Ave, San Francisco, CA 94143-0511, USA; UCSF CoLabs, University of California, San Francisco, 513 Parnassus Ave, San Francisco, CA 94143-0511, USA; Division of Rheumatology, Department of Medicine, University of California, San Francisco, 513 Parnassus Ave, San Francisco, CA 94143-0511, USA. Electronic address:

Complex datasets provide opportunities for discoveries beyond their initial scope. Effective and rapid data sharing and management practices are crucial to realize this potential; however, they are harder to implement than post-publication access. Here, we introduce the concept of a "data sharing trust" to maximize the value of large datasets.
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http://dx.doi.org/10.1016/j.cell.2021.01.006DOI Listing
February 2021

Global absence and targeting of protective immune states in severe COVID-19.

Nature 2021 Jan 25. Epub 2021 Jan 25.

Department of Pathology, University of California San Francisco, San Francisco, CA, USA.

Although infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has pleiotropic and systemic effects in some individuals, many others experience milder symptoms. Here, to gain a more comprehensive understanding of the distinction between severe and mild phenotypes in the pathology of coronavirus disease 2019 (COVID-19) and its origins, we performed a whole-blood-preserving single-cell analysis protocol to integrate contributions from all major immune cell types of the blood-including neutrophils, monocytes, platelets, lymphocytes and the contents of the serum. Patients with mild COVID-19 exhibit a coordinated pattern of expression of interferon-stimulated genes (ISGs) across every cell population, whereas these ISG-expressing cells are systemically absent in patients with severe disease. Paradoxically, individuals with severe COVID-19 produce very high titres of anti-SARS-CoV-2 antibodies and have a lower viral load compared to individuals with mild disease. Examination of the serum from patients with severe COVID-19 shows that these patients uniquely produce antibodies that functionally block the production of the ISG-expressing cells associated with mild disease, by activating conserved signalling circuits that dampen cellular responses to interferons. Overzealous antibody responses pit the immune system against itself in many patients with COVID-19, and perhaps also in individuals with other viral infections. Our findings reveal potential targets for immunotherapies in patients with severe COVID-19 to re-engage viral defence.
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http://dx.doi.org/10.1038/s41586-021-03234-7DOI Listing
January 2021

SCENITH: A Flow Cytometry-Based Method to Functionally Profile Energy Metabolism with Single-Cell Resolution.

Cell Metab 2020 Dec;32(6):1063-1075.e7

Aix Marseille Univ, CNRS, INSERM, CIML, Centre d'Immunologie de Marseille-Luminy, Marseille, France; Institute for Research in Biomedicine (iBiMED) and Ilidio Pinho Foundation, Department of Medical Sciences, University of Aveiro, 3810-193 Aveiro, Portugal; International Associated Laboratory (LIA) CNRS "Mistra", 13288 Marseille Cedex 9, France.

Energetic metabolism reprogramming is critical for cancer and immune responses. Current methods to functionally profile the global metabolic capacities and dependencies of cells are performed in bulk. We designed a simple method for complex metabolic profiling called SCENITH, for single-cell energetic metabolism by profiling translation inhibition. SCENITH allows for the study of metabolic responses in multiple cell types in parallel by flow cytometry. SCENITH is designed to perform metabolic studies ex vivo, particularly for rare cells in whole blood samples, avoiding metabolic biases introduced by culture media. We analyzed myeloid cells in solid tumors from patients and identified variable metabolic profiles, in ways that are not linked to their lineage or their activation phenotype. SCENITH's ability to reveal global metabolic functions and determine complex and linked immune-phenotypes in rare cell subpopulations will contribute to the information needed for evaluating therapeutic responses or patient stratification.
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http://dx.doi.org/10.1016/j.cmet.2020.11.007DOI Listing
December 2020

Global Absence and Targeting of Protective Immune States in Severe COVID-19.

bioRxiv 2020 Oct 29. Epub 2020 Oct 29.

While SARS-CoV-2 infection has pleiotropic and systemic effects in some patients, many others experience milder symptoms. We sought a holistic understanding of the severe/mild distinction in COVID-19 pathology, and its origins. We performed a whole-blood preserving single-cell analysis protocol to integrate contributions from all major cell types including neutrophils, monocytes, platelets, lymphocytes and the contents of serum. Patients with mild COVID-19 disease display a coordinated pattern of interferon-stimulated gene (ISG) expression across every cell population and these cells are systemically absent in patients with severe disease. Severe COVID-19 patients also paradoxically produce very high anti-SARS-CoV-2 antibody titers and have lower viral load as compared to mild disease. Examination of the serum from severe patients demonstrates that they uniquely produce antibodies with multiple patterns of specificity against interferon-stimulated cells and that those antibodies functionally block the production of the mild disease-associated ISG-expressing cells. Overzealous and auto-directed antibody responses pit the immune system against itself in many COVID-19 patients and this defines targets for immunotherapies to allow immune systems to provide viral defense.

One Sentence Summary: In severe COVID-19 patients, the immune system fails to generate cells that define mild disease; antibodies in their serum actively prevents the successful production of those cells.
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http://dx.doi.org/10.1101/2020.10.28.359935DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7605559PMC
October 2020

Global Absence and Targeting of Protective Immune States in Severe COVID-19.

Res Sq 2020 Oct 28. Epub 2020 Oct 28.

Department of Pathology, San Francisco, 513 Parnassus Ave, HSW512, San Francisco, CA 94143-0511, USA.

While SARS-CoV-2 infection has pleiotropic and systemic effects in some patients, many others experience milder symptoms. We sought a holistic understanding of the severe/mild distinction in COVID-19 pathology, and its origins. We performed a wholeblood preserving single-cell analysis protocol to integrate contributions from all major cell types including neutrophils, monocytes, platelets, lymphocytes and the contents of serum. Patients with mild COVID-19 disease display a coordinated pattern of interferonstimulated gene (ISG) expression across every cell population and these cells are systemically absent in patients with severe disease. Severe COVID-19 patients also paradoxically produce very high anti-SARS-CoV-2 antibody titers and have lower viral load as compared to mild disease. Examination of the serum from severe patients demonstrates that they uniquely produce antibodies with multiple patterns of specificity against interferon-stimulated cells and that those antibodies functionally block the production of the mild disease-associated ISG-expressing cells. Overzealous and autodirected antibody responses pit the immune system against itself in many COVID-19 patients and this defines targets for immunotherapies to allow immune systems to provide viral defense.
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http://dx.doi.org/10.21203/rs.3.rs-97042/v1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7605560PMC
October 2020

Lessons of COVID-19: A roadmap for post-pandemic science.

J Exp Med 2020 09;217(9)

Department of Pathology, University of California, San Francisco, San Francisco, CA.

The response to the COVID-19 crisis across most research institutions mandated ceasing nonessential research activities in order to minimize the spread of the virus in our communities. With minimal notice, experiments were terminated, cell lines were frozen, mouse colonies were culled, and trainees were prevented from performing bench research. Still, despite the interruption of experimental productivity, the shutdown has proven for many PIs and trainees that doing and thinking science are not activities that are bound to the laboratory. Furthermore, the shutdowns have solidified important emerging trends and forced us to further innovate to get the most out of working remotely. We hope that some of these innovations, hard-gained in this difficult time, will persist and develop into new paradigms-lessons that will improve our science and our relationship to the climate and community beyond the current pandemic.
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http://dx.doi.org/10.1084/jem.20201276DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7392636PMC
September 2020

The NK cell-cancer cycle: advances and new challenges in NK cell-based immunotherapies.

Nat Immunol 2020 08 20;21(8):835-847. Epub 2020 Jul 20.

Translational Research Program, Public Health Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA.

Natural killer (NK) cells belong to the innate immune system and contribute to protecting the host through killing of infected, foreign, stressed or transformed cells. Additionally, via cellular cross-talk, NK cells orchestrate antitumor immune responses. Hence, significant efforts have been undertaken to exploit the therapeutic properties of NK cells in cancer. Current strategies in preclinical and clinical development include adoptive transfer therapies, direct stimulation, recruitment of NK cells into the tumor microenvironment (TME), blockade of inhibitory receptors that limit NK cell functions, and therapeutic modulation of the TME to enhance antitumor NK cell function. In this Review, we introduce the NK cell-cancer cycle to highlight recent advances in NK cell biology and to discuss the progress and problems of NK cell-based cancer immunotherapies.
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http://dx.doi.org/10.1038/s41590-020-0728-zDOI Listing
August 2020

ZipSeq: barcoding for real-time mapping of single cell transcriptomes.

Nat Methods 2020 08 6;17(8):833-843. Epub 2020 Jul 6.

Department of Pathology, University of California San Francisco, San Francisco, CA, USA.

Spatial transcriptomics seeks to integrate single cell transcriptomic data within the three-dimensional space of multicellular biology. Current methods to correlate a cell's position with its transcriptome in living tissues have various limitations. We developed an approach, called 'ZipSeq', that uses patterned illumination and photocaged oligonucleotides to serially print barcodes ('zipcodes') onto live cells in intact tissues, in real time and with an on-the-fly selection of patterns. Using ZipSeq, we mapped gene expression in three settings: in vitro wound healing, live lymph node sections and a live tumor microenvironment. In all cases, we discovered new gene expression patterns associated with histological structures. In the tumor microenvironment, this demonstrated a trajectory of myeloid and T cell differentiation from the periphery inward. A combinatorial variation of ZipSeq efficiently scales in the number of regions defined, providing a pathway for complete mapping of live tissues, subsequent to real-time imaging or perturbation.
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http://dx.doi.org/10.1038/s41592-020-0880-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7891292PMC
August 2020

Visualizing Synaptic Transfer of Tumor Antigens among Dendritic Cells.

Cancer Cell 2020 06;37(6):786-799.e5

Department of Pathology, University of California San Francisco, San Francisco, CA 94143, USA. Electronic address:

Generation of tumor-infiltrating lymphocytes begins when tumor antigens reach the lymph node (LN) to stimulate T cells, yet we know little of how tumor material is disseminated among the large variety of antigen-presenting dendritic cell (DC) subsets in the LN. Here, we demonstrate that tumor proteins are carried to the LN within discrete vesicles inside DCs and are then transferred among DC subsets. A synapse is formed between interacting DCs and vesicle transfer takes place in the absence of free exosomes. DCs -containing vesicles can uniquely activate T cells, whereas DCs lacking them do not. Understanding this restricted sharing of tumor identity provides substantial room for engineering better anti-tumor immunity.
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http://dx.doi.org/10.1016/j.ccell.2020.05.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7671443PMC
June 2020

Tumor Immune Profiling-Based Neoadjuvant Immunotherapy for Locally Advanced Melanoma.

Ann Surg Oncol 2020 Oct 2;27(11):4122-4130. Epub 2020 Jun 2.

Department of Medicine, Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA, USA.

Background: The frequency of "exhausted" or checkpoint-positive (PD-1CTLA-4) cytotoxic lymphocytes (Tex) in the tumor microenvironment is associated with response to anti-PD-1 therapy in metastatic melanoma. The current study determined whether pretreatment Tex cells in locally advanced melanoma predicted response to neoadjuvant anti-PD-1 blockade.

Methods: Pretreatment tumor samples from 17 patients with locally advanced melanoma underwent flow cytometric analysis of pretreatment Tex and regulatory T cell frequency. Patients who met the criteria for neoadjuvant checkpoint blockade were treated with either PD-1 monotherapy or PD-1/CTLA-4 combination therapy. Best overall response was evaluated by response evaluation criteria in solid tumors version 1.1, with recurrence-free survival (RFS) calculated by the Kaplan-Meier test. The incidence and severity of adverse events were tabulated by clinicians using the National Cancer Institute Common Terminology Criteria for Adverse Events version 4.

Results: Of the neoadjuvant treated patients, 10 received anti-PD-1 monotherapy and 7 received anti-CTLA-4/PD-1 combination therapy. Of these 17 patients, 12 achieved a complete response, 4 achieved partial responses, and 1 exhibited stable disease. Surgery was subsequently performed for 11 of the 17 patients, and 8 attained a complete pathologic response. Median RFS and overall survival (OS) were not reached. Immune-related adverse events comprised four grade 3 or 4 events, including pneumonitis, transaminitis, and anaphylaxis.

Conclusion: The results showed high rates of objective response, RFS, and OS for patients undergoing immune profile-directed neoadjuvant immunotherapy for locally advanced melanoma. Furthermore, the study showed that treatment stratification based upon Tex frequency can potentially limit the adverse events associated with combination immunotherapy. These data merit further investigation with a larger validation study.
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http://dx.doi.org/10.1245/s10434-020-08648-7DOI Listing
October 2020

Spacer-Mediated Control of Coumarin Uncaging for Photocaged Thymidine.

J Org Chem 2020 03 5;85(5):2945-2955. Epub 2020 Feb 5.

Michigan Nanotechnology Institute for Medicine and Biological Sciences, University of Michigan Medical School, Ann Arbor, Michigan 48109, United States.

Despite its importance in the design of photocaged molecules, less attention is focused on linker chemistry than the cage itself. Here, we describe unique uncaging properties displayed by two coumarin-caged thymidine compounds, each conjugated with () or without () an extended, self-immolative spacer. Photolysis of using long-wavelength UVA (365 nm) or visible (420, 455 nm) light led to the release of free thymidine along with the competitive generation of a thymidine-bearing recombination product. The occurrence of this undesired side reaction, which is previously unreported, was not present with the photolysis of , which released thymidine exclusively with higher quantum efficiency. We propose that the spatial separation between the cage and the substrate molecule conferred by the extended linker can play a critical role in circumventing this unproductive reaction. This report reinforces the importance of linker selection in the design of coumarin-caged oligonucleosides and other conjugates.
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http://dx.doi.org/10.1021/acs.joc.9b02617DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7293860PMC
March 2020

Regulatory T cells use arginase 2 to enhance their metabolic fitness in tissues.

JCI Insight 2019 12 19;4(24). Epub 2019 Dec 19.

Department of Dermatology.

Distinct subsets of Tregs reside in nonlymphoid tissues where they mediate unique functions. To interrogate the biology of tissue Tregs in human health and disease, we phenotypically and functionally compared healthy skin Tregs with those in peripheral blood, inflamed psoriatic skin, and metastatic melanoma. The mitochondrial enzyme, arginase 2 (ARG2), was preferentially expressed in Tregs in healthy skin, increased in Tregs in metastatic melanoma, and reduced in Tregs from psoriatic skin. ARG2 enhanced Treg suppressive capacity in vitro and conferred a selective advantage for accumulation in inflamed tissues in vivo. CRISPR-mediated deletion of this gene in primary human Tregs was sufficient to skew away from a tissue Treg transcriptional signature. Notably, the inhibition of ARG2 increased mTOR signaling, whereas the overexpression of this enzyme suppressed it. Taken together, our results suggest that Tregs express ARG2 in human tissues to both regulate inflammation and enhance their metabolic fitness.
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http://dx.doi.org/10.1172/jci.insight.129756DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6975275PMC
December 2019

Dendritic cells in cancer immunology and immunotherapy.

Nat Rev Immunol 2020 01 29;20(1):7-24. Epub 2019 Aug 29.

Immunobiology Laboratory, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain.

Dendritic cells (DCs) are a diverse group of specialized antigen-presenting cells with key roles in the initiation and regulation of innate and adaptive immune responses. As such, there is currently much interest in modulating DC function to improve cancer immunotherapy. Many strategies have been developed to target DCs in cancer, such as the administration of antigens with immunomodulators that mobilize and activate endogenous DCs, as well as the generation of DC-based vaccines. A better understanding of the diversity and functions of DC subsets and of how these are shaped by the tumour microenvironment could lead to improved therapies for cancer. Here we will outline how different DC subsets influence immunity and tolerance in cancer settings and discuss the implications for both established cancer treatments and novel immunotherapy strategies.
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http://dx.doi.org/10.1038/s41577-019-0210-zDOI Listing
January 2020

Tuning the Tumor Myeloid Microenvironment to Fight Cancer.

Front Immunol 2019 25;10:1611. Epub 2019 Jul 25.

Department of Pathology, University of California, San Francisco, San Francisco, CA, United States.

The tumor microenvironment (TME) of diverse cancer types is often characterized by high levels of infiltrating myeloid cells including monocytes, macrophages, dendritic cells, and granulocytes. These cells perform a variety of functions in the TME, varying from immune suppressive to immune stimulatory roles. In this review, we summarize the different myeloid cell populations in the TME and the intratumoral myeloid targeting approaches that are being clinically investigated, and discuss strategies that identify new myeloid subpopulations within the TME. The TME therapies include agents that modulate the functional activities of myeloid populations, that impact recruitment and survival of myeloid subpopulations, and that functionally reprogram or activate myeloid populations. We discuss the benefits, limitations and potential side effects of these therapeutic approaches.
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http://dx.doi.org/10.3389/fimmu.2019.01611DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6673698PMC
October 2020

Pulmonary environmental cues drive group 2 innate lymphoid cell dynamics in mice and humans.

Sci Immunol 2019 06;4(36)

Inflammation, Repair and Development, National Heart and Lung Institute, Imperial College London, London, UK.

Group 2 innate lymphoid cells (ILC2s) are enriched in mucosal tissues (e.g., lung) and respond to epithelial cell-derived cytokines initiating type 2 inflammation. During inflammation, ILC2 numbers are increased in the lung. However, the mechanisms controlling ILC2 trafficking and motility within inflamed lungs remain unclear and are crucial for understanding ILC2 function in pulmonary immunity. Using several approaches, including lung intravital microscopy, we demonstrate that pulmonary ILC2s are highly dynamic, exhibit amoeboid-like movement, and aggregate in the lung peribronchial and perivascular spaces. They express distinct chemokine receptors, including CCR8, and actively home to CCL8 deposits located around the airway epithelium. Within lung tissue, ILC2s were particularly motile in extracellular matrix-enriched regions. We show that collagen-I drives ILC2 to markedly change their morphology by remodeling their actin cytoskeleton to promote environmental exploration critical for regulating eosinophilic inflammation. Our study provides previously unappreciated insights into ILC2 migratory patterns during inflammation and highlights the importance of environmental guidance cues in the lung in controlling ILC2 dynamics.
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http://dx.doi.org/10.1126/sciimmunol.aav7638DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6744282PMC
June 2019

Unleashing Type-2 Dendritic Cells to Drive Protective Antitumor CD4 T Cell Immunity.

Cell 2019 04 4;177(3):556-571.e16. Epub 2019 Apr 4.

Department of Pathology, University of California, San Francisco, San Francisco, CA 94143, USA; UCSF Immunoprofiler Initiative, University of California, San Francisco, San Francisco, CA 94143, USA. Electronic address:

Differentiation of proinflammatory CD4 conventional T cells (T) is critical for productive antitumor responses yet their elicitation remains poorly understood. We comprehensively characterized myeloid cells in tumor draining lymph nodes (tdLN) of mice and identified two subsets of conventional type-2 dendritic cells (cDC2) that traffic from tumor to tdLN and present tumor-derived antigens to CD4 T, but then fail to support antitumor CD4 T differentiation. Regulatory T cell (T) depletion enhanced their capacity to elicit strong CD4 T responses and ensuing antitumor protection. Analogous cDC2 populations were identified in patients, and as in mice, their abundance relative to T predicts protective ICOS PD-1 CD4 T phenotypes and survival. Further, in melanoma patients with low T abundance, intratumoral cDC2 density alone correlates with abundant CD4 T and with responsiveness to anti-PD-1 therapy. Together, this highlights a pathway that restrains cDC2 and whose reversal enhances CD4 T abundance and controls tumor growth.
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http://dx.doi.org/10.1016/j.cell.2019.02.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6954108PMC
April 2019

Immunity as a continuum of archetypes.

Science 2019 04;364(6435):28-29

Department of Pathology, University of California, San Francisco, CA 94143-0511, USA.

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http://dx.doi.org/10.1126/science.aau8694DOI Listing
April 2019

Adventitial Stromal Cells Define Group 2 Innate Lymphoid Cell Tissue Niches.

Immunity 2019 03 26;50(3):707-722.e6. Epub 2019 Feb 26.

Department of Laboratory Medicine, University of California, San Francisco, San Francisco, CA 94143, USA; Diabetes Center, University of California, San Francisco, San Francisco, CA 94143, USA. Electronic address:

Type 2 lymphocytes promote both physiologic tissue remodeling and allergic pathology, yet their physical tissue niches are poorly described. Here, we used quantitative imaging to define the tissue niches of group 2 innate lymphoid cells (ILC2s), which are critical instigators of type 2 immunity. We identified a dominant adventitial niche around lung bronchi and larger vessels in multiple tissues, where ILC2s localized with subsets of dendritic and regulatory T cells. However, ILC2s were most intimately associated with adventitial stromal cells (ASCs), a mesenchymal fibroblast-like subset that expresses interleukin-33 (IL-33) and thymic stromal lymphopoietin (TSLP). In vitro, ASCs produced TSLP that supported ILC2 accumulation and activation. ILC2s and IL-13 drove reciprocal ASC expansion and IL-33 expression. During helminth infection, ASC depletion impaired lung ILC2 and Th2 cell accumulation and function, which are in part dependent on ASC-derived IL-33. These data indicate that adventitial niches are conserved sites where ASCs regulate type 2 lymphocyte expansion and function.
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http://dx.doi.org/10.1016/j.immuni.2019.02.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6553479PMC
March 2019

Paracrine costimulation of IFN-γ signaling by integrins modulates CD8 T cell differentiation.

Proc Natl Acad Sci U S A 2018 11 22;115(45):11585-11590. Epub 2018 Oct 22.

Department of Pathology, University of California, San Francisco, CA 94143;

The cytokine IFN-γ is a critical regulator of immune system development and function. Almost all leukocytes express the receptor for IFN-γ, yet each cell type elicits a different response to this cytokine. Cell type-specific effects of IFN-γ make it difficult to predict the outcomes of the systemic IFN-γ blockade and limit its clinical application, despite many years of research. To better understand the cell-cell interactions and cofactors that specify IFN-γ functions, we focused on the function of IFN-γ on CD8 T cell differentiation. We demonstrated that during bacterial infection, IFN-γ is a dominant paracrine trigger that skews CD8 T cell differentiation toward memory. This skewing is preferentially driven by contact-dependent T cell-T cell (T-T) interactions and the localized IFN-γ secretion among activated CD8 T cells in a unique splenic microenvironment, and is less sensitive to concurrent IFN-γ production by other immune cell populations such as natural killer (NK) cells. Modulation of CD8 T cell differentiation by IFN-γ relies on a nonconventional IFN-γ outcome that occurs specifically within 24 hours following infection. This is driven by IFN-γ costimulation by integrins at T-T synapses, and leads to synergistic phosphorylation of the proximal STAT1 molecule and accelerated IL-2 receptor down-regulation. This study provides evidence of the importance of context-dependent cytokine signaling and gives another example of how cell clusters and the microenvironment drive unique biology.
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http://dx.doi.org/10.1073/pnas.1804556115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6233119PMC
November 2018

Macrophages promote epithelial proliferation following infectious and non-infectious lung injury through a Trefoil factor 2-dependent mechanism.

Mucosal Immunol 2019 01 18;12(1):64-76. Epub 2018 Oct 18.

Department of Pathobiology, University of Pennsylvania School of Veterinary Medicine, Philadelphia, PA, 19104, USA.

Coordinated efforts between macrophages and epithelia are considered essential for wound healing, but the macrophage-derived molecules responsible for repair are poorly defined. This work demonstrates that lung macrophages rely upon Trefoil factor 2 to promote epithelial proliferation following damage caused by sterile wounding, Nippostrongylus brasiliensis or Bleomycin sulfate. Unexpectedly, the presence of T, B, or ILC populations was not essential for macrophage-driven repair. Instead, conditional deletion of TFF2 in myeloid-restricted CD11c TFF2 mice exacerbated lung pathology and reduced the proliferative expansion of CD45 EpCAM pro-SPC alveolar type 2 cells. TFF2 deficient macrophages had reduced expression of the Wnt genes Wnt4 and Wnt16 and reconstitution of hookworm-infected CD11c TFF2 mice with rWnt4 and rWnt16 restored the proliferative defect in lung epithelia post-injury. These data reveal a previously unrecognized mechanism wherein lung myeloid phagocytes utilize a TFF2/Wnt axis as a mechanism that drives epithelial proliferation following lung injury.
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http://dx.doi.org/10.1038/s41385-018-0096-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6301101PMC
January 2019

A natural killer-dendritic cell axis defines checkpoint therapy-responsive tumor microenvironments.

Nat Med 2018 08 25;24(8):1178-1191. Epub 2018 Jun 25.

Department of Pathology, University of California San Francisco, San Francisco, CA, USA.

Intratumoral stimulatory dendritic cells (SDCs) play an important role in stimulating cytotoxic T cells and driving immune responses against cancer. Understanding the mechanisms that regulate their abundance in the tumor microenvironment (TME) could unveil new therapeutic opportunities. We find that in human melanoma, SDC abundance is associated with intratumoral expression of the gene encoding the cytokine FLT3LG. FLT3LG is predominantly produced by lymphocytes, notably natural killer (NK) cells in mouse and human tumors. NK cells stably form conjugates with SDCs in the mouse TME, and genetic and cellular ablation of NK cells in mice demonstrates their importance in positively regulating SDC abundance in tumor through production of FLT3L. Although anti-PD-1 'checkpoint' immunotherapy for cancer largely targets T cells, we find that NK cell frequency correlates with protective SDCs in human cancers, with patient responsiveness to anti-PD-1 immunotherapy, and with increased overall survival. Our studies reveal that innate immune SDCs and NK cells cluster together as an excellent prognostic tool for T cell-directed immunotherapy and that these innate cells are necessary for enhanced T cell tumor responses, suggesting this axis as a target for new therapies.
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http://dx.doi.org/10.1038/s41591-018-0085-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6475503PMC
August 2018

Subcellular Localization of Antigen in Keratinocytes Dictates Delivery of CD4 T-cell Help for the CTL Response upon Therapeutic DNA Vaccination into the Skin.

Cancer Immunol Res 2018 07 15;6(7):835-847. Epub 2018 May 15.

Division of Tumor Biology and Immunology, The Netherlands Cancer Institute-Antoni van Leeuwenhoek, Amsterdam, the Netherlands.

In a mouse model of therapeutic DNA vaccination, we studied how the subcellular localization of vaccine protein impacts antigen delivery to professional antigen-presenting cells and efficiency of CTL priming. Cytosolic, membrane-bound, nuclear, and secretory versions of ZsGreen fluorescent protein, conjugated to MHC class I and II ovalbumin (OVA) epitopes, were expressed in keratinocytes by DNA vaccination into the skin. ZsGreen-OVA versions reached B cells in the skin-draining lymph node (dLN) that proved irrelevant for CTL priming. ZsGreen-OVA versions were also actively transported to the dLN by dendritic cells (DC). In the dLN, vaccine proteins localized to classical (c)DCs of the migratory XCR1 and XCR subtypes, and-to a lesser extent-to LN-resident cDCs. Secretory ZsGreen-OVA induced the best antitumor CTL response, even though its delivery to cDCs in the dLN was significantly less efficient than for other vaccine proteins. Secretory ZsGreen-OVA protein proved superior in CTL priming, because it led to engagement of antigen-loaded XCR1, but not XCR1, cDCs. Secretory ZsGreen-OVA also maximally solicited CD4 T-cell help. The suboptimal CTL response to the other ZsGreen-OVA versions was improved by engaging costimulatory receptor CD27, which mimics CD4 T-cell help. Thus, in therapeutic DNA vaccination into the skin, mere inclusion of helper epitopes does not ensure delivery of CD4 T-cell help for the CTL response. Targeting of the vaccine protein to the secretory route of keratinocytes is required to engage XCR1 cDC and CD4 T-cell help and thus to promote CTL priming. .
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http://dx.doi.org/10.1158/2326-6066.CIR-17-0408DOI Listing
July 2018

Understanding the tumor immune microenvironment (TIME) for effective therapy.

Nat Med 2018 05 23;24(5):541-550. Epub 2018 Apr 23.

Department of Pathology, University of California, San Francisco, San Francisco, CA, USA.

The clinical successes in immunotherapy have been both astounding and at the same time unsatisfactory. Countless patients with varied tumor types have seen pronounced clinical response with immunotherapeutic intervention; however, many more patients have experienced minimal or no clinical benefit when provided the same treatment. As technology has advanced, so has the understanding of the complexity and diversity of the immune context of the tumor microenvironment and its influence on response to therapy. It has been possible to identify different subclasses of immune environment that have an influence on tumor initiation and response and therapy; by parsing the unique classes and subclasses of tumor immune microenvironment (TIME) that exist within a patient's tumor, the ability to predict and guide immunotherapeutic responsiveness will improve, and new therapeutic targets will be revealed.
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http://dx.doi.org/10.1038/s41591-018-0014-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5998822PMC
May 2018

Trefoil Factor 2 Promotes Type 2 Immunity and Lung Repair through Intrinsic Roles in Hematopoietic and Nonhematopoietic Cells.

Am J Pathol 2018 05 16;188(5):1161-1170. Epub 2018 Feb 16.

Division of Experimental Medicine, University of California San Francisco, San Francisco, California. Electronic address:

Trefoil factors (TFFs) are small secreted proteins that regulate tissue integrity and repair at mucosal surfaces, particularly in the gastrointestinal tract. However, their relative contribution(s) to controlling baseline lung function or the extent of infection-induced lung injury are unknown issues. With the use of irradiation bone marrow chimeras, we found that TFF2 produced from both hematopoietic- and nonhematopoietic-derived cells is essential for host protection, proliferation of alveolar type 2 cells, and restoration of pulmonary gas exchange after infection with the hookworm parasite Nippostrongylus brasiliensis. In the absence of TFF2, lung epithelia were unable to proliferate and expressed reduced lung mRNA transcript levels for type 2 response-inducing IL-25 and IL-33 after infectious injury. Strikingly, even in the absence of infection or irradiation, TFF2 deficiency compromised lung structure and function, as characterized by distended alveoli and reduced blood oxygen levels relative to wild-type control mice. Taken together, we show a previously unappreciated role for TFF2, produced by either hematopoietic or nonhematopoietic sources, as a pro-proliferative factor for lung epithelial cells under steady-state and infectious injury conditions.
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http://dx.doi.org/10.1016/j.ajpath.2018.01.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5906739PMC
May 2018

TIM-3 Regulates CD103 Dendritic Cell Function and Response to Chemotherapy in Breast Cancer.

Cancer Cell 2018 01;33(1):60-74.e6

Department of Immunology, H. Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive SRB-2, Tampa, FL 33612, USA; Department of Breast Oncology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612, USA. Electronic address:

Intratumoral CD103 dendritic cells (DCs) are necessary for anti-tumor immunity. Here we evaluated the expression of immune regulators by CD103 DCs in a murine model of breast cancer and identified expression of TIM-3 as a target for therapy. Anti-TIM-3 antibody improved response to paclitaxel chemotherapy in models of triple-negative and luminal B disease, with no evidence of toxicity. Combined efficacy was CD8 T cell dependent and associated with increased granzyme B expression; however, TIM-3 expression was predominantly localized to myeloid cells in both human and murine tumors. Gene expression analysis identified upregulation of Cxcl9 within intratumoral DCs during combination therapy, and therapeutic efficacy was ablated by CXCR3 blockade, Batf3 deficiency, or Irf8 deficiency.
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http://dx.doi.org/10.1016/j.ccell.2017.11.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5764109PMC
January 2018

Partially exhausted tumor-infiltrating lymphocytes predict response to combination immunotherapy.

JCI Insight 2017 Jul 20;2(14). Epub 2017 Jul 20.

Department of Medicine and.

Background: Programmed death 1 (PD-1) inhibition activates partially exhausted cytotoxic T lymphocytes (peCTLs) and induces tumor regression. We previously showed that the peCTL fraction predicts response to anti-PD-1 monotherapy. Here, we sought to correlate peCTL and regulatory T lymphocyte (Treg) levels with response to combination immunotherapy, and with demographic/disease characteristics, in metastatic melanoma patients.

Methods: Pretreatment melanoma samples underwent multiparameter flow cytometric analysis. Patients were treated with anti-PD-1 monotherapy or combination therapy, and responses determined by Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST v1.1) criteria. peCTL and Treg levels across demographic/disease variables were compared. Low versus high peCTL (≤20% vs. >20%) were defined from a previous study.

Results: One hundred and two melanoma patients were identified. The peCTL fraction was higher in responders than nonresponders. Low peCTL correlated with female sex and liver metastasis, but not with lactate dehydrogenase (LDH), tumor stage, or age. While overall response rates (ORRs) to anti-PD-1 monotherapy and combination therapy were similar in high-peCTL patients, low-peCTL patients given combination therapy demonstrated higher ORRs than those who received monotherapy. Treg levels were not associated with these factors nor with response.

Conclusion: In melanoma, pretreatment peCTL fraction is reduced in women and in patients with liver metastasis. In low-peCTL patients, anti-PD-1 combination therapy is associated with significantly higher ORR than anti-PD-1 monotherapy. Fewer tumor-infiltrating peCTLs may be required to achieve response to combination immunotherapy.

Trial Registration: UCSF IRB Protocol 138510FUNDING. NIH DP2-AR068130, K08-AR062064, AR066821, and Burroughs Wellcome CAMS-1010934 (M.D.R.). Amoroso and Cook Fund, and the Parker Institute for Cancer Immunotherapy (A.I.D.).
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http://dx.doi.org/10.1172/jci.insight.93433DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5518562PMC
July 2017

The Lung is a Host Defense Niche for Immediate Neutrophil-Mediated Vascular Protection.

Sci Immunol 2017 Apr;2(10)

Department of Critical Care, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada.

Bloodstream infection is a hallmark of sepsis, a medically emergent condition requiring rapid treatment. However, upregulation of host defense proteins through toll-like receptors and NFκB requires hours after endotoxin detection. Using confocal pulmonary intravital microscopy, we identified that the lung provides a TLR4-Myd88-and abl tyrosine kinase-dependent niche for immediate CD11b-dependent neutrophil responses to endotoxin and Gram-negative bloodstream pathogens. In an model of bacteremia, neutrophils crawled to and rapidly phagocytosed sequestered to the lung endothelium. Therefore, the lung capillaries provide a vascular defensive niche whereby endothelium and neutrophils cooperate for immediate detection and capture of disseminating pathogens.
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http://dx.doi.org/10.1126/sciimmunol.aam8929DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5472445PMC
April 2017

Visualizing dynamic microvillar search and stabilization during ligand detection by T cells.

Science 2017 05;356(6338)

Department of Pathology, University of California, San Francisco, CA 94143, USA.

During immune surveillance, T cells survey the surface of antigen-presenting cells. In searching for peptide-loaded major histocompatibility complexes (pMHCs), they must solve a classic trade-off between speed and sensitivity. It has long been supposed that microvilli on T cells act as sensory organs to enable search, but their strategy has been unknown. We used lattice light-sheet and quantum dot-enabled synaptic contact mapping microscopy to show that anomalous diffusion and fractal organization of microvilli survey the majority of opposing surfaces within 1 minute. Individual dwell times were long enough to discriminate pMHC half-lives and T cell receptor (TCR) accumulation selectively stabilized microvilli. Stabilization was independent of tyrosine kinase signaling and the actin cytoskeleton, suggesting selection for avid TCR microclusters. This work defines the efficient cellular search process against which ligand detection takes place.
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http://dx.doi.org/10.1126/science.aal3118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6364556PMC
May 2017

Liver Metastasis and Treatment Outcome with Anti-PD-1 Monoclonal Antibody in Patients with Melanoma and NSCLC.

Cancer Immunol Res 2017 05 14;5(5):417-424. Epub 2017 Apr 14.

University of California, San Francisco, San Francisco, California.

We explored the association between liver metastases, tumor CD8 T-cell count, and response in patients with melanoma or lung cancer treated with the anti-PD-1 antibody, pembrolizumab. The melanoma discovery cohort was drawn from the phase I Keynote 001 trial, whereas the melanoma validation cohort was drawn from Keynote 002, 006, and EAP trials and the non-small cell lung cancer (NSCLC) cohort from Keynote 001. Liver metastasis was associated with reduced response and shortened progression-free survival [PFS; objective response rate (ORR), 30.6%; median PFS, 5.1 months] compared with patients without liver metastasis (ORR, 56.3%; median PFS, 20.1 months) ≤ 0.0001, and confirmed in the validation cohort ( = 0.0006). The presence of liver metastasis significantly increased the likelihood of progression (OR, 1.852; < 0.0001). In a subset of biopsied patients ( = 62), liver metastasis was associated with reduced CD8 T-cell density at the invasive tumor margin (liver metastasis group, = 547 ± 164.8; liver metastasis group, = 1,441 ± 250.7; < 0.016). A reduced response rate and shortened PFS was also observed in NSCLC patients with liver metastasis [median PFS, 1.8 months; 95% confidence interval (CI), 1.4-2.0], compared with those without liver metastasis ( = 119, median PFS, 4.0 months; 95% CI, 2.1-5.1), = 0.0094. Thus, liver metastatic patients with melanoma or NSCLC that had been treated with pembrolizumab were associated with reduced responses and PFS, and liver metastases were associated with reduced marginal CD8 T-cell infiltration, providing a potential mechanism for this outcome. .
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http://dx.doi.org/10.1158/2326-6066.CIR-16-0325DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5749922PMC
May 2017

The lung is a site of platelet biogenesis and a reservoir for haematopoietic progenitors.

Nature 2017 04 22;544(7648):105-109. Epub 2017 Mar 22.

Department of Medicine, University of California, San Francisco (UCSF), San Francisco, California 94143, USA.

Platelets are critical for haemostasis, thrombosis, and inflammatory responses, but the events that lead to mature platelet production remain incompletely understood. The bone marrow has been proposed to be a major site of platelet production, although there is indirect evidence that the lungs might also contribute to platelet biogenesis. Here, by directly imaging the lung microcirculation in mice, we show that a large number of megakaryocytes circulate through the lungs, where they dynamically release platelets. Megakaryocytes that release platelets in the lungs originate from extrapulmonary sites such as the bone marrow; we observed large megakaryocytes migrating out of the bone marrow space. The contribution of the lungs to platelet biogenesis is substantial, accounting for approximately 50% of total platelet production or 10 million platelets per hour. Furthermore, we identified populations of mature and immature megakaryocytes along with haematopoietic progenitors in the extravascular spaces of the lungs. Under conditions of thrombocytopenia and relative stem cell deficiency in the bone marrow, these progenitors can migrate out of the lungs, repopulate the bone marrow, completely reconstitute blood platelet counts, and contribute to multiple haematopoietic lineages. These results identify the lungs as a primary site of terminal platelet production and an organ with considerable haematopoietic potential.
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http://dx.doi.org/10.1038/nature21706DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5663284PMC
April 2017