Matthew H. Spitzer

Mass Cytometry: Single Cells, Many Features

Technology development in biological research often aims to either increase the number of cellular features that can be surveyed simultaneously or enhance the resolution at which such observations are possible. For decades, flow cytometry has balanced these goals to fill a critical need by enabling the measurement of multiple features in single cells, commonly to examine complex or hierarchical cellular systems. Recently, a format for flow cytometry has been developed that leverages the precision of mass spectrometry. This fusion of the two technologies, termed mass cytometry, provides measurement of over 40 simultaneous cellular parameters at single-cell resolution, significantly augmenting the ability of cytometry to evaluate complex cellular systems and processes. In this Primer, we review the current state of mass cytometry, providing an overview of the instrumentation, its present capabilities, and methods of data analysis, as well as thoughts on future developments and applications.

Palladium-based mass tag cell barcoding with a doublet-filtering scheme and single-cell deconvolution algorithm

Mass-tag cell barcoding (MCB) labels individual cell samples with unique combinatorial barcodes, after which they are pooled for processing and measurement as a single multiplexed sample. The MCB method eliminates variability between samples in antibody staining and instrument sensitivity, reduces antibody consumption and shortens instrument measurement time. Here we present an optimized MCB protocol. The use of palladium-based labeling reagents expands the number of measurement channels available for mass cytometry and reduces interference with lanthanide-based antibody measurement. An error-detecting combinatorial barcoding scheme allows cell doublets to be identified and removed from the analysis. A debarcoding algorithm that is single cell–based rather than population-based improves the accuracy and efficiency of sample deconvolution. This debarcoding algorithm has been packaged into software that allows rapid and unbiased sample deconvolution. The MCB procedure takes 3–4 h, not including sample acquisition time of ∼1 h per million cells.

Risks and benefits of testosterone therapy in older men

In young men (defined as age<50 years) with classic hypogonadism caused by known diseases of the hypothalamus, pituitary or testes, testosterone replacement therapy induces a number of beneficial effects, for example, the development of secondary sex characteristics, improvement and maintenance of sexual function, and increases in skeletal muscle mass and BMD. Moreover, testosterone treatment in this patient population is associated with a low frequency of adverse events. Circulating testosterone levels decline progressively with age, starting in the second and third decade of life, owing to defects at all levels of the hypothalamic-pituitary-testicular axis. In cohort studies, testosterone levels are associated weakly but consistently with muscle mass, strength, physical function, anaemia, BMD and bone quality, visceral adiposity, and with the risk of diabetes mellitus, coronary artery disease, falls, fractures and mortality. However, the clinical benefits and long-term risks of testosterone therapy--especially prostate-related and cardiovascular-related adverse events--have not been adequately assessed in large, randomized clinical trials involving older men (defined as age>65 years) with androgen deficiency. Therefore, a general policy of testosterone replacement in all older men with age-related decline in testosterone levels is not justified.In young men (defined as age <50 years) with classic hypogonadism caused by known diseases of the hypothalamus, pituitary or testes, testosterone replacement therapy induces a number of beneficial effects, for example, the development of secondary sex characteristics, improvement and maintenance of sexual function, and increases in skeletal muscle mass and BMD. Moreover, testosterone treatment in this patient population is associated with a low frequency of adverse events. Circulating testosterone levels decline progressively with age, starting in the second and third decade of life, owing to defects at all levels of the hypothalamic–pituitary–testicular axis. In cohort studies, testosterone levels are associated weakly but consistently with muscle mass, strength, physical function, anaemia, BMD and bone quality, visceral adiposity, and with the risk of diabetes mellitus, coronary artery disease, falls, fractures and mortality. However, the clinical benefits and long-term risks of testosterone therapy—especially prostate-related and cardiovascular-related adverse events—have not been adequately assessed in large, randomized clinical trials involving older men (defined as age >65 years) with androgen deficiency. Therefore, a general policy of testosterone replacement in all older men with age-related decline in testosterone levels is not justified.

Conditional density-based analysis of T cell signaling in single-cell data

Introduction Cellular circuits sense the environment, process signals, and compute decisions using networks of interacting proteins. Emerging high-dimensional single-cell technologies such as mass cytometry can measure dozens of protein epitopes simultaneously in millions of individual cells. With thousands of individual cells, each providing a point of data on co-occurring protein states, it is possible to infer and quantify the functional forms of the relationships between proteins. However, in practice these underlying relationships are typically obscured by statistical limitations of the data, hence rendering the analysis and interpretation of single-cell data challenging. We developed computational methods, tailored to single-cell data, to more completely define the function and strength of signaling relationships. Quantitative characterization of T cell signaling. (A) The pCD3ζ-pSLP76 signaling interaction shown as (I) a scatterplot, (II) a kernel density estimate, and (III) by using a conditional DREVI method. (IV) Shape features are extracted and quantified. (B) DREVI plots of a signaling cascade downstream of TCR show the time-varying nature of edge shapes and strengths

An interactive reference framework for modeling a dynamic immune system

Single-cell measurements map immunity Multiple characteristics of individual cells define cell types and their physiological states. Spitzer et al. quantitated the abundance of 39 different cell surface proteins or transcription factors on individual cells of the mouse immune system. They used these measurements to create a map that clustered similar individual cells into groups corresponding to cell type and function. Their extensible experimental platform will allow the inclusion of other data types and data from independent laboratories. Science, this issue 10.1126/science.1259425 Cytometry meets mass spectrometry to create a functional map of the immune system. INTRODUCTION Immune cells constitute an interacting hierarchy that coordinates its activities according to genetic and environmental contexts. This systemically mobile network of cells results in emergent properties that are derived from dynamic cellular interactions. Unlike many solid tissues, where cells of given functions are localized into substructures that can be readily defined, the distribution of phenotypically similar immune cells into various organs complicates discerning any modest differences between them. Over decades of investigation into immune functions during health and disease, research has necessarily focused on understanding the individual cell types within the immune system, and, more recently, toward identifying interacting cells and the messengers they use to communicate. RATIONALE Methods of single-cell analysis, such as flow cytometry, have led the effort to enumerate and quantitatively characterize immune cell populations. As research has accelerated, our understanding of immune organization has surpassed the technical limitations of fluorescence-based flow cytometry. With the advent of mass cytometry, which enables measuring significantly more features of individual cells, most known immune cell types can now be identified from within a single experiment. Leveraging this capability, we set out to initiate an immune system reference framework to provide a working definition of immune organization and enable the integration of new data sets. RESULTS To build a reference framework from mass cytometry data, we developed a novel algorithm to transform the single-cell data into intuitive maps. These Scaffold maps provide a data-driven interpretation of immune organization while also integrating conventional immune cell populations as landmarks to orient the user. By applying Scaffold maps to data from the bone marrow of wild-type C57BL/6 mice, the method reconstructed the organization within this complex developmental organ. Using this sample as a reference point, the unique organization of immune cells within various organs across the body was revealed. The maps recapitulated canonical cellular phenotypes while revealing reproducible, tissue-specific deviations. The approach revealed influences of genetic variation and circadian rhythms on immune structure, permitted direct comparisons of murine and human blood cell phenotypes, and even enabled archival fluorescence-based flow cytometry data to be mapped onto the reference framework. CONCLUSION This foundational reference map provides a working definition of systemic immune organization to which new data can be integrated to reveal deviations driven by genetics, environment, or pathology. Beyond providing an analytical framework to understand immune organization from the unified data set generated here, the approaches we describe can serve as a data repository for collating experimental data from the research community, including gene expression and mutational analysis. Efforts that characterize cellular behavior in this open-source approach will continue to improve upon the initiating reference presented here to reveal the inherent structure in biological networks of immunity for clinical benefit. Building a dynamic immune system reference framework. By combining mass cytometry with the Scaffold maps algorithm, the cellular organization of any complex sample can be transformed into an intuitive and interactive map for further analysis. By first choosing one foundational sample as a reference (i.e., the bone marrow of wild-type mice), the effects of any perturbation can be readily identified in this framework. Immune cells function in an interacting hierarchy that coordinates the activities of various cell types according to genetic and environmental contexts. We developed graphical approaches to construct an extensible immune reference map from mass cytometry data of cells from different organs, incorporating landmark cell populations as flags on the map to compare cells from distinct samples. The maps recapitulated canonical cellular phenotypes and revealed reproducible, tissue-specific deviations. The approach revealed influences of genetic variation and circadian rhythms on immune system structure, enabled direct comparisons of murine and human blood cell phenotypes, and even enabled archival fluorescence-based flow cytometry data to be mapped onto the reference framework. This foundational reference map provides a working definition of systemic immune organization to which new data can be integrated to reveal deviations driven by genetics, environment, or pathology.

Allogeneic IgG combined with dendritic cell stimuli induce antitumour T-cell immunity

Whereas cancers grow within host tissues and evade host immunity through immune-editing and immunosuppression, tumours are rarely transmissible between individuals. Much like transplanted allogeneic organs, allogeneic tumours are reliably rejected by host T cells, even when the tumour and host share the same major histocompatibility complex alleles, the most potent determinants of transplant rejection. How such tumour-eradicating immunity is initiated remains unknown, although elucidating this process could provide the basis for inducing similar responses against naturally arising tumours. Here we find that allogeneic tumour rejection is initiated in mice by naturally occurring tumour-binding IgG antibodies, which enable dendritic cells (DCs) to internalize tumour antigens and subsequently activate tumour-reactive T cells. We exploited this mechanism to treat autologous and autochthonous tumours successfully. Either systemic administration of DCs loaded with allogeneic-IgG-coated tumour cells or intratumoral injection of allogeneic IgG in combination with DC stimuli induced potent T-cell-mediated antitumour immune responses, resulting in tumour eradication in mouse models of melanoma, pancreas, lung and breast cancer. Moreover, this strategy led to eradication of distant tumours and metastases, as well as the injected primary tumours. To assess the clinical relevance of these findings, we studied antibodies and cells from patients with lung cancer. T cells from these patients responded vigorously to autologous tumour antigens after culture with allogeneic-IgG-loaded DCs, recapitulating our findings in mice. These results reveal that tumour-binding allogeneic IgG can induce powerful antitumour immunity that can be exploited for cancer immunotherapy.

Expression of specific inflammasome gene modules stratifies older individuals into two extreme clinical and immunological states

Low-grade, chronic inflammation has been associated with many diseases of aging, but the mechanisms responsible for producing this inflammation remain unclear. Inflammasomes can drive chronic inflammation in the context of an infectious disease or cellular stress, and they trigger the maturation of interleukin-1β (IL-1β). Here we find that the expression of specific inflammasome gene modules stratifies older individuals into two extremes: those with constitutive expression of IL-1β, nucleotide metabolism dysfunction, elevated oxidative stress, high rates of hypertension and arterial stiffness; and those without constitutive expression of IL-1β, who lack these characteristics. Adenine and N4-acetylcytidine, nucleotide-derived metabolites that are detectable in the blood of the former group, prime and activate the NLRC4 inflammasome, induce the production of IL-1β, activate platelets and neutrophils and elevate blood pressure in mice. In individuals over 85 years of age, the elevated expression of inflammasome gene modules was associated with all-cause mortality. Thus, targeting inflammasome components may ameliorate chronic inflammation and various other age-associated conditions.

A gut bacterial pathway metabolizes aromatic amino acids into nine circulating metabolites

The human gut microbiota produces dozens of metabolites that accumulate in the bloodstream, where they can have systemic effects on the host. Although these small molecules commonly reach concentrations similar to those achieved by pharmaceutical agents, remarkably little is known about the microbial metabolic pathways that produce them. Here we use a combination of genetics and metabolic profiling to characterize a pathway from the gut symbiont Clostridium sporogenes that generates aromatic amino acid metabolites. Our results reveal that this pathway produces twelve compounds, nine of which are known to accumulate in host serum. All three aromatic amino acids (tryptophan, phenylalanine and tyrosine) serve as substrates for the pathway, and it involves branching and alternative reductases for specific intermediates. By genetically manipulating C. sporogenes, we modulate serum levels of these metabolites in gnotobiotic mice, and show that in turn this affects intestinal permeability and systemic immunity. This work has the potential to provide the basis of a systematic effort to engineer the molecular output of the gut bacterial community.

Sildenafil increases serum testosterone levels by a direct action on the testes.

Phosphodiesterase‐5‐inhibitors, such as sildenafil, increase intracavernosal cyclic guanosine monophosphate levels, which results in corporal smooth muscle relaxation and penile erection. Here, we determined the effects of sildenafil administration on the hypothalamic‐pituitary‐gonadal axis in men with erectile dysfunction and low testosterone levels. The Testosterone and Erectile Dysfunction trial (ClinicalTrials.gov # NCT00512707) initially administered an optimized dose of sildenafil to 140 men, aged 40–70 years with erectile dysfunction, low serum total testosterone (<11.4 nmol/L; 330 ng/dL) and/or free testosterone (<173 pmol/L; 50 pg/mL) over 3–7 weeks. Sex steroids and gonadotropins were measured at baseline and after sildenafil optimization in a longitudinal study without a separate control group. Serum testosterone, dihydrotestosterone (DHT) and oestrogens were measured using liquid chromatography‐tandem mass spectrometry. Administration of an optimized dose of sildenafil was associated with mean increases of 3.6 nmol/L (103 ng/dL; p < 0.001) and 110 pmol/L (31.7 pg/mL; p < 0.001) in total and free testosterone levels respectively. This was accompanied by parallel increases in serum DHT (0.17 nmol/L; 4.9 ng/dL; p < 0.001) and oestradiol (14 pmol/L; 3.7 pg/mL; p < 0.001) and significant suppression of luteinizing hormone (change −1.3 units/L; p = 0.003) levels, suggesting a direct effect at the testicular level. Androstenedione and oestrone increased by 1.3 nmol/L (38 ng/dL; p = 0.011) and 10.7 pmol/L (2.9 pg/mL; p = 0.012), respectively, supporting a possible effect of sildenafil on adrenal steroidogenesis. In conclusion, sildenafil administration was associated with increased testosterone levels likely ascribable to a direct effect on the testis.

ApoE Receptor 2 Regulates Synapse and Dendritic Spine Formation

Background Apolipoprotein E receptor 2 (ApoEr2) is a postsynaptic protein involved in long-term potentiation (LTP), learning, and memory through unknown mechanisms. We examined the biological effects of ApoEr2 on synapse and dendritic spine formation—processes critical for learning and memory. Methodology/Principal Findings In a heterologous co-culture synapse assay, overexpression of ApoEr2 in COS7 cells significantly increased colocalization with synaptophysin in primary hippocampal neurons, suggesting that ApoEr2 promotes interaction with presynaptic structures. In primary neuronal cultures, overexpression of ApoEr2 increased dendritic spine density. Consistent with our in vitro findings, ApoEr2 knockout mice had decreased dendritic spine density in cortical layers II/III at 1 month of age. We also tested whether the interaction between ApoEr2 and its cytoplasmic adaptor proteins, specifically X11α and PSD-95, affected synapse and dendritic spine formation. X11α decreased cell surface levels of ApoEr2 along with synapse and dendritic spine density. In contrast, PSD-95 increased cell surface levels of ApoEr2 as well as synapse and dendritic spine density. Conclusions/Significance These results suggest that ApoEr2 plays important roles in structure and function of CNS synapses and dendritic spines, and that these roles are modulated by cytoplasmic adaptor proteins X11α and PSD-95.