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.

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.

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.

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.

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.

Influence of Self-MHC Class I Recognition on the Dynamics of NK Cell Responses to Cytomegalovirus Infection.

Although interactions between inhibitory Ly49 receptors and their self-MHC class I ligands in C57BL/6 mice are known to limit NK cell proliferation during mouse CMV (MCMV) infection, we created a 36-marker mass cytometry (CyTOF) panel to investigate how these inhibitory receptors impact the NK cell response to MCMV in other phenotypically measurable ways. More than two thirds of licensed NK cells (i.e., those expressing Ly49C, Ly49I, or both) in uninfected mice had already differentiated into NK cells with phenotypes indicative of Ag encounter (KLRG1+Ly6C-) or memory-like status (KLRG1+Ly6C+). These pre-existing KLRG1+Ly6C+ NK cells resembled known Ag-specific memory NK cell populations in being less responsive to IL-18 and IFN-α stimulation in vitro and by selecting for NK cell clones with elevated expression of a Ly49 receptor. During MCMV infection, the significant differences between licensed and unlicensed (Ly49C-Ly49I-) NK cells disappeared within both CMV-specific (Ly49H+) and nonspecific (Ly49H-) responses. This lack of heterogeneity carried into the memory phase, with only a difference in CD16 expression manifesting between licensed and unlicensed MCMV-specific memory NK cell populations. Our results suggest that restricting proliferation is the predominant effect licensing has on the NK cell population during MCMV infection, but the inhibitory Ly49-MHC interactions that take place ahead of infection contribute to their limited expansion by shrinking the pool of licensed NK cells capable of robustly responding to new challenges.

A conserved immune trajectory of recovery in hospitalized COVID-19 patients.

Many studies have provided insights into the immune response to COVID-19; however, little is known about the immunological changes and immune signaling occurring during COVID-19 resolution. Individual heterogeneity and variable disease resolution timelines obscure unifying immune characteristics. Here, we collected and profiled >200 longitudinal peripheral blood samples from patients hospitalized with COVID-19, with other respiratory infections, and healthy individuals, using mass cytometry to measure immune cells and signaling states at single cell resolution. COVID-19 patients showed a unique immune composition and an early, coordinated and elevated immune cell signaling profile, which correlated with early hospital discharge. Intra-patient time course analysis tied to clinically relevant events of recovery revealed a conserved set of immunological processes that accompany, and are unique to, disease resolution and discharge. This immunological process, together with additional changes in CD4 regulatory T cells and basophils, accompanies recovery from respiratory failure and is associated with better clinical outcomes at the time of admission. Our work elucidates the biological timeline of immune recovery from COVID-19 and provides insights into the fundamental processes of COVID-19 resolution in hospitalized patients.

Single-cell analysis by mass cytometry reveals metabolic states of early-activated CD8+ T cells during the primary immune response.

Memory T cells are thought to rely on oxidative phosphorylation and short-lived effector T cells on glycolysis. Here, we investigated how T cells arrive at these states during an immune response. To understand the metabolic state of rare, early-activated T cells, we adapted mass cytometry to quantify metabolic regulators at single-cell resolution in parallel with cell signaling, proliferation, and effector function. We interrogated CD8+ T cell activation in vitro and in response to Listeria monocytogenes infection in vivo. This approach revealed a distinct metabolic state in early-activated T cells characterized by maximal expression of glycolytic and oxidative metabolic proteins. Cells in this transient state were most abundant 5 days post-infection before rapidly decreasing metabolic protein expression. Analogous findings were observed in chimeric antigen receptor (CAR) T cells interrogated longitudinally in advanced lymphoma patients. Our study demonstrates the utility of single-cell metabolic analysis by mass cytometry to identify metabolic adaptations of immune cell populations in vivo and provides a resource for investigations of metabolic regulation of immune responses across a variety of applications.

Systemic dysfunction and plasticity of the immune macroenvironment in cancer models.

Understanding of the factors governing immune responses in cancer remains incomplete, limiting patient benefit. In this study, we used mass cytometry to define the systemic immune landscape in response to tumor development across five tissues in eight mouse tumor models. Systemic immunity was dramatically altered across models and time, with consistent findings in the peripheral blood of patients with breast cancer. Changes in peripheral tissues differed from those in the tumor microenvironment. Mice with tumor-experienced immune systems mounted dampened responses to orthogonal challenges, including reduced T cell activation during viral or bacterial infection. Antigen-presenting cells (APCs) mounted weaker responses in this context, whereas promoting APC activation rescued T cell activity. Systemic immune changes were reversed with surgical tumor resection, and many were prevented by interleukin-1 or granulocyte colony-stimulating factor blockade, revealing remarkable plasticity in the systemic immune state. These results demonstrate that tumor development dynamically reshapes the composition and function of the immune macroenvironment.

Elevated leptin disrupts epithelial polarity and promotes premalignant alterations in the mammary gland.

Obesity is a highly prevalent and modifiable breast cancer risk factor. While the role of obesity in fueling breast cancer progression is well established, the mechanisms linking obesity to breast cancer initiation are poorly understood. A hallmark of breast cancer initiation is the disruption of apical polarity in mammary glands. Here we show that mice with diet-induced obesity display mislocalization of Par3, a regulator of cellular junctional complexes defining mammary epithelial polarity. We found that epithelial polarity loss also occurs in a 3D coculture system that combines acini with human mammary adipose tissue, and establish that a paracrine effect of the tissue adipokine leptin causes loss of polarity by overactivation of the PI3K/Akt pathway. Leptin sensitizes non-neoplastic cells to proliferative stimuli, causes mitotic spindle misalignment, and expands the pool of cells with stem/progenitor characteristics, which are early steps for cancer initiation. We also found that normal breast tissue samples with high leptin/adiponectin transcript ratio characteristic of obesity have an altered distribution of apical polarity markers. This effect is associated with increased epithelial cell layers. Our results provide a molecular basis for early alterations in epithelial architecture during obesity-mediated cancer initiation.

Silver nanoparticles selectively treat triple-negative breast cancer cells without affecting non-malignant breast epithelial cells in vitro and in vivo.

Silver nanoparticles (AgNPs) show promise for treatment of aggressive cancers including triple-negative breast cancer (TNBC) in preclinical cancer models. For clinical development of AgNP-based therapeutics, it will be necessary to clearly define the specific physicochemical features of the nanoparticles that will be used, and to tie these properties to biological outcomes. To fill this knowledge gap, we performed thorough structure/function, mechanistic, safety, and efficacy studies to assess the potential for AgNPs to treat TNBC. We establish that AgNPs, regardless of size, shape, or stabilizing agent, are highly cytotoxic to TNBC cells at doses that are not cytotoxic to non-malignant breast epithelial cells. In contrast, TNBC cells and non-malignant breast epithelial cells are similarly sensitive to exposure to silver cation (Ag+), indicating that the nanoparticle formulation is essential for the TNBC-specific cytotoxicity. Mechanistically, AgNPs are internalized by both TNBC and non-malignant breast cells, but are rapidly degraded only in TNBC cells. Exposure to AgNPs depletes cellular antioxidants and causes endoplasmic reticulum stress in TNBC cells without causing similar damage in non-malignant breast epithelial cells. AgNPs also cause extensive DNA damage in 3D TNBC tumor nodules in vitro, but do not disrupt the normal architecture of breast acini in 3D cell culture, nor cause DNA damage or induce apoptosis in these structures. Lastly, we show that systemically administered AgNPs are effective at non-toxic doses for reducing the growth of TNBC tumor xenografts in mice. This work provides a rationale for development of AgNPs as a safe and specific TNBC treatment.

Autocrine TGFß Is a Survival Factor for Monocytes and Drives Immunosuppressive Lineage Commitment.

Transforming growth factor ß (TGFß) is an effector of immune suppression and contributes to a permissive tumor microenvironment that compromises effective immunotherapy. We identified a correlation between TGFB1 and genes expressed by myeloid cells, but not granulocytes, in The Cancer Genome Atlas lung adenocarcinoma data, in which high TGFB1 expression was associated with poor survival. To determine whether TGFß affected cell fate decisions and lineage commitment, we studied primary cultures of CD14+ monocytes isolated from peripheral blood of healthy donors. We discovered that TGFß was a survival factor for CD14+ monocytes, which rapidly executed an apoptotic program in its absence. Continued exposure to TGFß in combination with granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 6 (IL6) amplified HLA-DRlowCD14+CD11b+CD33+ myeloid-derived suppressor cells (MDSCs) at the expense of macrophage and dendritic cell (DC) differentiation. MDSCs generated in the presence of TGFß were more effective in suppressing T-cell proliferation and promoted the T regulatory cell phenotype. In contrast, inhibition of TGFß signaling using a small-molecule inhibitor of receptor kinase activity in CD14+ monocytes treated with GM-CSF and IL6 decreased MDSC differentiation and increased differentiation to proinflammatory macrophages and antigen-presenting DCs. The effect of autocrine and paracrine TGFß on myeloid cell survival and lineage commitment suggests that pharmacologic inhibition of TGFß-dependent signaling in cancer would favor antitumor immunity.