The tumor microenvironment of benign and malignant salivary gland tumors.

BACKGROUND: Treatment of salivary gland tumors (SGTs) remains challenging. Little is known about the immune landscape of SGTs. We aimed to characterize the tumor microenvironment in benign and malignant SGTs. METHODS: Eleven benign and nine malignant tumors were collected from patients undergoing curative intent surgery. Specimens were analyzed using mass cytometry by time-of-flight. Immune cell populations were manually gated, and T cells were clustered using the FlowSOM algorithm. Population frequencies were compared between high-grade and low-grade malignancies, corrected for multiple hypothesis testing. RESULTS: There were trends towards increased CD4+ and CD8+ T cells among malignant tumors. High-grade malignancies exhibited trends towards higher frequencies of CD8+ PD-1+ CD39+ CD103+ exhausted T cells, CD4+ FoxP3+ TCF-1+ CD127- Tregs, and CD69+ CD25- CD4+ T cells compared to low-grade malignancies. CONCLUSION: SGTs exhibit significant immunologic diversity. High-grade malignancies tended to have greater infiltration of exhausted CD8+ T cells and Tregs, which may guide future studies for immunotherapy strategies.

Disrupting CD38-driven T cell dysfunction restores sensitivity to cancer immunotherapy.

A central problem in cancer immunotherapy with immune checkpoint blockade (ICB) is the development of resistance, which affects 50% of patients with metastatic melanoma1,2. T cell exhaustion, resulting from chronic antigen exposure in the tumour microenvironment, is a major driver of ICB resistance3. Here, we show that CD38, an ecto-enzyme involved in nicotinamide adenine dinucleotide (NAD+) catabolism, is highly expressed in exhausted CD8+ T cells in melanoma and is associated with ICB resistance. Tumour-derived CD38hiCD8+ T cells are dysfunctional, characterised by impaired proliferative capacity, effector function, and dysregulated mitochondrial bioenergetics. Genetic and pharmacological blockade of CD38 in murine and patient-derived organotypic tumour models (MDOTS/PDOTS) enhanced tumour immunity and overcame ICB resistance. Mechanistically, disrupting CD38 activity in T cells restored cellular NAD+ pools, improved mitochondrial function, increased proliferation, augmented effector function, and restored ICB sensitivity. Taken together, these data demonstrate a role for the CD38-NAD+ axis in promoting T cell exhaustion and ICB resistance, and establish the efficacy of CD38 directed therapeutic strategies to overcome ICB resistance using clinically relevant, patient-derived 3D tumour models.

Enhancing CAR-T cell metabolism to overcome hypoxic conditions in the brain tumor microenvironment.

The efficacy of chimeric antigen receptor T cell (CAR-T) therapy has been limited against brain tumors to date. CAR-T cells infiltrating syngeneic intracerebral SB28 EGFRvIII gliomas revealed impaired mitochondrial ATP production and a markedly hypoxic status compared with ones migrating to subcutaneous tumors. Drug screenings to improve metabolic states of T cells under hypoxic conditions led us to evaluate the combination of the AMPK activator metformin and the mTOR inhibitor rapamycin (Met+Rap). Met+Rap-pretreated mouse CAR-T cells showed activated PPAR-γ coactivator 1α (PGC-1α) through mTOR inhibition and AMPK activation, and a higher level of mitochondrial spare respiratory capacity than those pretreated with individual drugs or without pretreatment. Moreover, Met+Rap-pretreated CAR-T cells demonstrated persistent and effective antiglioma cytotoxic activities in the hypoxic condition. Furthermore, a single intravenous infusion of Met+Rap-pretreated CAR-T cells significantly extended the survival of mice bearing intracerebral SB28 EGFRvIII gliomas. Mass cytometric analyses highlighted increased glioma-infiltrating CAR-T cells in the Met+Rap group, with fewer Ly6c+CD11b+ monocytic myeloid-derived suppressor cells in the tumors. Finally, human CAR-T cells pretreated with Met+Rap recapitulated the observations with murine CAR-T cells, demonstrating improved functions under in vitro hypoxic conditions. These findings advocate for translational and clinical exploration of Met+Rap-pretreated CAR-T cells in human trials.

Lower frequencies of circulating suppressive regulatory T cells and higher frequencies of CD4+ naïve T cells at baseline are associated with severe immune-related adverse events in immune checkpoint inhibitor-treated melanoma.

BACKGROUND: Immune-related adverse events (irAEs) are major barriers of clinical management and further development of immune checkpoint inhibitors (ICIs) for cancer therapy. Therefore, biomarkers associated with the onset of severe irAEs are needed. In this study, we aimed to identify immune features detectable in peripheral blood and associated with the development of severe irAEs that required clinical intervention. METHODS: We used a 43-marker mass cytometry panel to characterize peripheral blood mononuclear cells from 28 unique patients with melanoma across 29 lines of ICI therapy before treatment (baseline), before the onset of irAEs (pre-irAE) and at the peak of irAEs (irAE-max). In the 29 lines of ICI therapy, 18 resulted in severe irAEs and 11 did not. RESULTS: Unsupervised and gated population analysis showed that patients with severe irAEs had a higher frequency of CD4+ naïve T cells and lower frequency of CD16+ natural killer (NK) cells at all time points. Gated population analysis additionally showed that patients with severe irAEs had fewer T cell immunoreceptor with Ig and ITIM domain (TIGIT+) regulatory T cells at baseline and more activated CD38+ CD4+ central memory T cells (TCM) and CD39+ and Human Leukocyte Antigen-DR Isotype (HLA-DR)+ CD8+ TCM at peak of irAEs. The differentiating immune features at baseline were predominantly seen in patients with gastrointestinal and cutaneous irAEs and type 1 diabetes. Higher frequencies of CD4+ naïve T cells and lower frequencies of CD16+ NK cells were also associated with clinical benefit to ICI therapy. CONCLUSIONS: This study demonstrates that high-dimensional immune profiling can reveal novel blood-based immune signatures associated with risk and mechanism of severe irAEs. Development of severe irAEs in melanoma could be the result of reduced immune inhibitory capacity pre-ICI treatment, resulting in more activated TCM cells after treatment.

A mass cytometry approach to track the evolution of T cell responses during infection and immunotherapy by paired T cell receptor repertoire and T cell differentiation state analysis.

T cell receptor (TCR) recognition followed by clonal expansion is a fundamental feature of adaptive immune responses. Here, we developed a mass cytometric (CyTOF) approach combining antibodies specific for different TCR Vα- and Vß-chains with antibodies against T cell activation and differentiation proteins to identify antigen-specific expansions of T cell subsets and assess aspects of cellular function. This strategy allowed for the identification of expansions of specific Vß and Vα chain expressing CD8+ and CD4+ T cells with varying differentiation states in response to Listeria monocytogenes, tumors, and respiratory influenza infection. Expanded Vß chain expressing T cells could be directly linked to the recognition of specific antigens from Listeria, tumor cells, or influenza. In the setting of influenza infection, we showed that the common therapeutic approaches of intramuscular vaccination or convalescent serum transfer altered the clonal diversity and differentiation state of responding T cells. Thus, we present a new method to monitor broad changes in TCR specificity paired with T cell differentiation during adaptive immune responses.

Epigenetic reprogramming shapes the cellular landscape of schwannoma.

Mechanisms specifying cancer cell states and response to therapy are incompletely understood. Here we show epigenetic reprogramming shapes the cellular landscape of schwannomas, the most common tumors of the peripheral nervous system. We find schwannomas are comprised of 2 molecular groups that are distinguished by activation of neural crest or nerve injury pathways that specify tumor cell states and the architecture of the tumor immune microenvironment. Moreover, we find radiotherapy is sufficient for interconversion of neural crest schwannomas to immune-enriched schwannomas through epigenetic and metabolic reprogramming. To define mechanisms underlying schwannoma groups, we develop a technique for simultaneous interrogation of chromatin accessibility and gene expression coupled with genetic and therapeutic perturbations in single-nuclei. Our results elucidate a framework for understanding epigenetic drivers of tumor evolution and establish a paradigm of epigenetic and metabolic reprograming of cancer cells that shapes the immune microenvironment in response to radiotherapy.

The lncRNA Malat1 inhibits miR-15/16 to enhance cytotoxic T cell activation and memory cell formation.

Proper activation of cytotoxic T cells via the T cell receptor and the costimulatory receptor CD28 is essential for adaptive immunity against viruses, intracellular bacteria, and cancers. Through biochemical analysis of RNA:protein interactions, we uncovered a non-coding RNA circuit regulating activation and differentiation of cytotoxic T cells composed of the long non-coding RNA Malat1 (Metastasis Associated Lung Adenocarcinoma Transcript 1) and the microRNA family miR-15/16. miR-15/16 is a widely and highly expressed tumor suppressor miRNA family important for cell proliferation and survival. miR-15/16 play important roles in T cell responses to viral infection, including the regulation of antigen-specific T cell expansion and memory. Comparative Argonaute-2 high-throughput sequencing of crosslinking immunoprecipitation (AHC) combined with gene expression profiling in normal and miR-15/16-deficient mouse T cells revealed a large network of hundreds of direct miR-15/16 target mRNAs, many with functional relevance for T cell activation, survival and memory formation. Among these targets, Malat1 contained the largest absolute magnitude miR-15/16-dependent AHC peak. This binding site was among the strongest lncRNA:miRNA interactions detected in the T cell transcriptome. We used CRISPR targeting with homology directed repair to generate mice with a 5-nucleotide mutation in the miR-15/16-binding site in Malat1. This mutation interrupted Malat1:miR-15/16 interaction, and enhanced the repression of other miR-15/16 target genes, including CD28. Interrupting Malat1 interaction with miR-15/16 decreased cytotoxic T cell activation, including the expression of interleukin 2 (IL-2) and a broader CD28-responsive gene program. Accordingly, Malat1 mutation diminished memory cell persistence in mice following LCMV Armstrong and Listeria monocytogenes infection. This study marks a significant advance in the study of long non-coding RNAs in the immune system by ascribing cell-intrinsic, sequence-specific in vivo function to Malat1. These findings have implications for T cell-mediated autoimmune diseases, antiviral and anti-tumor immunity, as well as lung adenocarcinoma and other malignancies where Malat1 is overexpressed.

Enhancing CAR-T Cell Metabolism to Overcome Hypoxic Conditions in the Brain Tumor Microenvironment.

The efficacy of chimeric antigen receptor (CAR)-T therapy has been limited against brain tumors to date. CAR-T cells infiltrating syngeneic intracerebral SB28-EGFRvIII glioma revealed impaired mitochondrial ATP production and a markedly hypoxic status compared to ones migrating to subcutaneous tumors. Drug screenings to improve metabolic states of T cells under hypoxic conditions led us to evaluate the combination of AMPK activator Metformin and the mTOR inhibitor Rapamycin (Met+Rap). Met+Rap-pretreated mouse CAR-T cells showed activated PPAR-gamma coactivator 1α (PGC-1α) through mTOR inhibition and AMPK activation, and a higher level of mitochondrial spare respiratory capacity than those pretreated with individual drugs or without pretreatment. Moreover, Met+Rap-pretreated CAR-T cells demonstrated persistent and effective anti-glioma cytotoxic activities in the hypoxic condition. Furthermore, a single intravenous infusion of Met+Rap-pretreated CAR-T cells significantly extended the survival of mice bearing intracerebral SB28-EGFRvIII gliomas. Mass cytometric analyses highlighted increased glioma-infiltrating CAR-T cells in the Met+Rap group with fewer Ly6c+ CD11b+ monocytic myeloid-derived suppressor cells in the tumors. Finally, human CAR-T cells pretreated with Met+Rap recapitulated the observations with murine CAR-T cells, demonstrating improved functions in vitro hypoxic conditions. These findings advocate for translational and clinical exploration of Met+Rap-pretreated CAR-T cells in human trials.

Commensal myeloid crosstalk in neonatal skin regulates long-term cutaneous type 17 inflammation.

Early life microbe-immune interactions at barrier surfaces have lasting impacts on the trajectory towards health versus disease. Monocytes, macrophages and dendritic cells are primary sentinels in barrier tissues, yet the salient contributions of commensal-myeloid crosstalk during tissue development remain poorly understood. Here, we identify that commensal microbes facilitate accumulation of a population of monocytes in neonatal skin. Transient postnatal depletion of these monocytes resulted in heightened IL-17A production by skin T cells, which was particularly sustained among CD4+ T cells into adulthood and sufficient to exacerbate inflammatory skin pathologies. Neonatal skin monocytes were enriched in expression of negative regulators of the IL-1 pathway. Functional in vivo experiments confirmed a key role for excessive IL-1R1 signaling in T cells as contributing to the dysregulated type 17 response in neonatal monocyte-depleted mice. Thus, a commensal-driven wave of monocytes into neonatal skin critically facilitates long-term immune homeostasis in this prominent barrier tissue.

The lncRNA Malat1 Inhibits miR-15/16 to Enhance Cytotoxic T Cell Activation and Memory Cell Formation.

Proper activation of cytotoxic T cells via the T cell receptor and the costimulatory receptor CD28 is essential for adaptive immunity against viruses, many intracellular bacteria and cancers. Through biochemical analysis of RNA:protein interactions, we uncovered a non-coding RNA circuit regulating activation and differentiation of cytotoxic T cells composed of the long non-coding RNA Malat1 (Metastasis Associated Lung Adenocarcinoma Transcript 1) and the microRNA family miR-15/16. miR-15/16 is a widely and highly expressed tumor suppressor miRNA family important for cell proliferation and survival. miR-15/16 also play important roles in T cell responses to viral infection, including the regulation of antigen-specific T cell expansion and T cell memory. Comparative Argonaute-2 high throughput sequencing of crosslinking immunoprecipitation (Ago2 HITS-CLIP, or AHC) combined with gene expression profiling in normal and miR-15/16-deficient T cells revealed a large network of several hundred direct miR-15/16 target mRNAs, many with functional relevance for T cell activation, survival and memory formation. Among these targets, the long non-coding RNA Malat1 contained the largest absolute magnitude miR-15/16-dependent AHC peak in T cells. This binding site was also among the strongest lncRNA:miRNA interactions detected in the T cell transcriptome. We used CRISPR targeting with homology directed repair to generate mice with a 5-nucleotide mutation in the miR-15/16 binding site in Malat1. This mutation interrupted Malat1:miR-15/16 interaction, and enhanced the repression of other miR-15/16 target genes, including CD28. Interrupting Malat1 interaction with miR-15/16 decreased cytotoxic T cell activation, including the expression of IL-2 and a broader CD28-responsive gene program. Accordingly, Malat1 mutation diminished memory cell persistence following LCMV Armstrong and Listeria monocytogenes infection. This study marks a significant advance in the study of long noncoding RNAs in the immune system by ascribing cell-intrinsic, sequence-specific in vivo function to Malat1. These findings have implications for T cell-mediated autoimmune diseases, antiviral and anti-tumor immunity, as well as lung adenocarcinoma and other malignancies where Malat1 is overexpressed.

Computational Methods for Single-Cell Proteomics.

Advances in single-cell proteomics technologies have resulted in high-dimensional datasets comprising millions of cells that are capable of answering key questions about biology and disease. The advent of these technologies has prompted the development of computational tools to process and visualize the complex data. In this review, we outline the steps of single-cell and spatial proteomics analysis pipelines. In addition to describing available methods, we highlight benchmarking studies that have identified advantages and pitfalls of the currently available computational toolkits. As these technologies continue to advance, robust analysis tools should be developed in tandem to take full advantage of the potential biological insights provided by these data.

Dynamic CD8+ T cell responses to cancer immunotherapy in human regional lymph nodes are disrupted in metastatic lymph nodes.

CD8+ T cell responses are critical for anti-tumor immunity. While extensively profiled in the tumor microenvironment, recent studies in mice identified responses in lymph nodes (LNs) as essential; however, the role of LNs in human cancer patients remains unknown. We examined CD8+ T cells in human head and neck squamous cell carcinomas, regional LNs, and blood using mass cytometry, single-cell genomics, and multiplexed ion beam imaging. We identified progenitor exhausted CD8+ T cells (Tpex) that were abundant in uninvolved LN and clonally related to terminally exhausted cells in the tumor. After anti-PD-L1 immunotherapy, Tpex in uninvolved LNs reduced in frequency but localized near dendritic cells and proliferating intermediate-exhausted CD8+ T cells (Tex-int), consistent with activation and differentiation. LN responses coincided with increased circulating Tex-int. In metastatic LNs, these response hallmarks were impaired, with immunosuppressive cellular niches. Our results identify important roles for LNs in anti-tumor immune responses in humans.

Single cell proteomics characterization of bone marrow hematopoiesis with distinct Ras pathway lesions.

Normal hematopoiesis requires constant prolific production of different blood cell lineages by multipotent hematopoietic stem cells (HSC). Stem- and progenitor- cells need to balance dormancy with proliferation. How genetic alterations impact frequency, lineage potential, and metabolism of HSC is largely unknown. Here, we compared induced expression of KRAS G12D or RasGRP1 to normal hematopoiesis. At low-resolution, both Ras pathway lesions result in skewing towards myeloid lineages. Single-cell resolution CyTOF proteomics unmasked an expansion of HSC- and progenitor- compartments for RasGRP1, contrasted by a depletion for KRAS G12D . SCENITH™ quantitates protein synthesis with single-cell precision and corroborated that immature cells display low metabolic SCENITH™ rates. Both RasGRP1 and KRAS G12D elevated mean SCENITH™ signals in immature cells. However, RasGRP1-overexpressing stem cells retain a metabolically quiescent cell-fraction, whereas this fraction diminishes for KRAS G12D . Our temporal single cell proteomics and metabolomics datasets provide a resource of mechanistic insights into altered hematopoiesis at single cell resolution.

Synthetic cytokine circuits that drive T cells into immune-excluded tumors.

Chimeric antigen receptor (CAR) T cells are ineffective against solid tumors with immunosuppressive microenvironments. To overcome suppression, we engineered circuits in which tumor-specific synNotch receptors locally induce production of the cytokine IL-2. These circuits potently enhance CAR T cell infiltration and clearance of immune-excluded tumors, without systemic toxicity. The most effective IL-2 induction circuit acts in an autocrine and T cell receptor (TCR)- or CAR-independent manner, bypassing suppression mechanisms including consumption of IL-2 or inhibition of TCR signaling. These engineered cells establish a foothold in the target tumors, with synthetic Notch-induced IL-2 production enabling initiation of CAR-mediated T cell expansion and cell killing. Thus, it is possible to reconstitute synthetic T cell circuits that activate the outputs ultimately required for an antitumor response, but in a manner that evades key points of tumor suppression.

Innate type 2 immunity controls hair follicle commensalism by Demodex mites.

Demodex mites are commensal parasites of hair follicles (HFs). Normally asymptomatic, inflammatory outgrowth of mites can accompany malnutrition, immune dysfunction, and aging, but mechanisms restricting Demodex outgrowth are not defined. Here, we show that control of mite HF colonization in mice required group 2 innate lymphoid cells (ILC2s), interleukin-13 (IL-13), and its receptor, IL-4Ra-IL-13Ra1. HF-associated ILC2s elaborated IL-13 that attenuated HFs and epithelial proliferation at anagen onset; in their absence, Demodex colonization led to increased epithelial proliferation and replacement of gene programs for repair by aberrant inflammation, leading to the loss of barrier function and HF exhaustion. Humans with rhinophymatous acne rosacea, an inflammatory condition associated with Demodex, had increased HF inflammation with decreased type 2 cytokines, consistent with the inverse relationship seen in mice. Our studies uncover a key role for skin ILC2s and IL-13, which comprise an immune checkpoint that sustains cutaneous integrity and restricts pathologic infestation by colonizing HF mites.

Mass cytometry reveals a conserved immune trajectory of recovery in hospitalized COVID-19 patients.

While studies have elucidated many pathophysiological elements of COVID-19, little is known about immunological changes during COVID-19 resolution. We analyzed immune cells and phosphorylated signaling states at single-cell resolution from longitudinal blood samples of patients hospitalized with COVID-19, pneumonia and/or sepsis, and healthy individuals by mass cytometry. COVID-19 patients showed distinct immune compositions and an early, coordinated, and elevated immune cell signaling profile associated with early hospital discharge. Intra-patient longitudinal analysis revealed changes in myeloid and T cell frequencies and a reduction in immune cell signaling across cell types that accompanied disease resolution and discharge. These changes, together with increases in regulatory T cells and reduced signaling in basophils, also accompanied recovery from respiratory failure and were associated with better outcomes at time of admission. Therefore, although patients have heterogeneous immunological baselines and highly variable disease courses, a core immunological trajectory exists that defines recovery from severe SARS-CoV-2 infection.

Lymph node colonization induces tumor-immune tolerance to promote distant metastasis.

For many solid malignancies, lymph node (LN) involvement represents a harbinger of distant metastatic disease and, therefore, an important prognostic factor. Beyond its utility as a biomarker, whether and how LN metastasis plays an active role in shaping distant metastasis remains an open question. Here, we develop a syngeneic melanoma mouse model of LN metastasis to investigate how tumors spread to LNs and whether LN colonization influences metastasis to distant tissues. We show that an epigenetically instilled tumor-intrinsic interferon response program confers enhanced LN metastatic potential by enabling the evasion of NK cells and promoting LN colonization. LN metastases resist T cell-mediated cytotoxicity, induce antigen-specific regulatory T cells, and generate tumor-specific immune tolerance that subsequently facilitates distant tumor colonization. These effects extend to human cancers and other murine cancer models, implicating a conserved systemic mechanism by which malignancies spread to distant organs.

A cytotoxic-skewed immune set point predicts low neutralizing antibody levels after Zika virus infection.

Although generating high neutralizing antibody levels is a key component of protective immunity after acute viral infection or vaccination, little is known about why some individuals generate high versus low neutralizing antibody titers. Here, we leverage the high-dimensional single-cell profiling capacity of mass cytometry to characterize the longitudinal cellular immune response to Zika virus (ZIKV) infection in viremic blood donors in Puerto Rico. During acute ZIKV infection, we identify widely coordinated responses across innate and adaptive immune cell lineages. High frequencies of multiple activated cell types during acute infection are associated with high titers of ZIKV neutralizing antibodies 6 months post-infection, while stable immune features suggesting a cytotoxic-skewed immune set point are associated with low titers. Our study offers insight into the coordination of immune responses and identifies candidate cellular biomarkers that may offer predictive value in vaccine efficacy trials aimed at inducing high levels of antiviral neutralizing antibodies.

A Comprehensive Atlas of Immunological Differences Between Humans, Mice, and Non-Human Primates.

Animal models are an integral part of the drug development and evaluation process. However, they are unsurprisingly imperfect reflections of humans, and the extent and nature of many immunological differences are unknown. With the rise of targeted and biological therapeutics, it is increasingly important that we understand the molecular differences in the immunological behavior of humans and model organisms. However, very few antibodies are raised against non-human primate antigens, and databases of cross-reactivity between species are incomplete. Thus, we screened 332 antibodies in five immune cell populations in blood from humans and four non-human primate species generating a comprehensive cross-reactivity catalog that includes cell type-specificity. We used this catalog to create large mass cytometry universal cross-species phenotyping and signaling panels for humans, along with three of the model organisms most similar to humans: rhesus and cynomolgus macaques and African green monkeys; and one of the mammalian models most widely used in drug development: C57BL/6 mice. As a proof-of-principle, we measured immune cell signaling responses across all five species to an array of 15 stimuli using mass cytometry. We found numerous instances of different cellular phenotypes and immune signaling events occurring within and between species, and detailed three examples (double-positive T cell frequency and signaling; granulocyte response to Bacillus anthracis antigen; and B cell subsets). We also explore the correlation of herpes simian B virus serostatus on the immune profile. Antibody panels and the full dataset generated are available online as a resource to enable future studies comparing immune responses across species during the evaluation of therapeutics.