Name: single-cell analysis of immunophenotype and cytokine production in peripheral whole blood via mass cytometry
Uploaded: 2018-06-26T13:30:00
Description: Watch this Scientific Journal Video about Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry at JoVE.com

We would like to thank Aimee Pugh-Bernard for her intellectual input and helpful comments. This work was supported by the Boettcher Foundation Webb-Waring Biomedical Research Award and award number K23-1K23AR070897 from the NIH to Elena W.Y. Hsieh. She was also supported by award number K12-HD000850 from the Eunice Kennedy Shriver National Institute of Child Health and Human Development and the Lucile Packard Foundation for Children&#39;s Health, Stanford CTSA UL1 TR001085, and Child Health Research Institute of Stanford University.

All methods described here have been approved by the Colorado Multiple Institutional Review Board (COMIRB) of the University of Colorado. All described procedures below should be performed in a sterile tissue culture hood unless stated otherwise, with filtered pipette tips, and all reagents filtered.
1. Preparation of Reagents for Peripheral Whole Blood Processing
<ol>
	<li>Prepare ruxolitinib stock aliquots (10&#8211;15 &#956;L/vial for single use) at 10 mM by diluting the lyophilized reage...

<ol>
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Here we describe a novel, single-cell, proteomic approach to simultaneously assess multiple immune cell types and detect their various cytokine perturbations in the milieu of patient specific &#34;pathogenic&#34; peripheral blood plasma circulating factors. This method employs peripheral whole blood as the analytical vehicle, and mass cytometry as the tool for the evaluation of immune cellular phenotypic and functional abnormalities. The method is readily applicable to human and mice studies21, an...

Autoimmune diseases are a major cause of morbidity and mortality affecting 3-8% of the population. In the United States, autoimmune disorders are among the leading causes of death among young and middle-aged women (ages &#60;65 years)1,2. Autoimmune disorders are characterized by heterogeneous clinical presentation and diverse underlying immunological processes. The spectrum of heterogeneity is well represented across different disorders, such as joint involvement in rheumatoid arthritis (RA) and neurological disease in multiple sclerosis (MS). However, this level of heterogeneity is also exemplified within a single disorder, such as systemic lupus erythematosus (SLE): some patients may present with renal pathology, while others suffer from hematologic or joint involvement3.
The underlying immunopathogenesis in autoimmune disorders mirrors the clinical heterogeneity, involving auto- and hyper-activation of multiple innate and adaptive immune cell subsets, and concomitant dysregulated cytokine production. While cytokines play a pivotal role in the pathogenesis of autoimmune disease, understanding their specific role in the mechanism of disease has proven to be challenging. Cytokines are characterized by pleiotropy (one cytokine can have multiple effects on different cell types), redundancy (multiple cytokines can have the same effect), duality (one cytokine can have pro- or anti-inflammatory effects under different conditions), and plasticity (cytokines can be molded into a role different from its &#34;original&#34; one, depending on the environment)4,5,6. Consequently, population-level methods cannot distinguish heterogeneous cellular responses to the same &#34;cytokine milieu&#34;. Similarly, study designs that focus on one specific aspect of the immune system (a single cell type or cytokine), do not offer a global assessment of all the elements involved in complex autoimmune disease. While patient sera-based studies have afforded important insights into autoimmunity, they do not provide the specific cellular source of the dysregulated cytokines detected.
Recently, we developed a single-cell proteomic approach to simultaneously assess multiple immune cell types, and detect their various cytokine perturbations in the milieu of patient specific &#34;pathogenic&#34; peripheral blood plasma circulating factors. The workflow described here is characterized by the use of intact peripheral whole blood samples, as opposed to isolated peripheral blood mononuclear cells (PBMCs). Peripheral whole blood represents the most physiologically relevant vehicle to study systemic immune-mediated disease, including 1) non-mononuclear blood cells often involved in autoimmune disease (i.e., neutrophils, platelets), and 2) plasma circulating factors, such as nucleic acids, immune complexes, and cytokines, which have immune activating roles. To capture the &#34;intrinsic pathogenic&#34; dysregulated cytokine production, peripheral blood samples are processed immediately after the blood draw (T0, Time zero), and after 6 h of incubation at 37 &#176;C (physiological body temperature) with a protein transport inhibitor in the absence of any exogenous stimulating condition (T6, Time 6 h), to detect cytokine production (accumulation, T6-T0) that would reflect the &#34;intrinsic&#34; disease state (Figure 1). To study dysregulated processes that reflect over or under-activation of signaling pathways involved in immune responses relevant to the disease, peripheral blood samples are treated (6 h incubation at 37 &#176;C with a protein transport inhibitor) with an exogenous stimulating condition that reflects disease pathogenesis, such as Toll-Like-Receptor (TLR) agonists in the case of SLE (T6 + R848, Time 6 h with 1 &#181;g/mL R848), to detect cytokine production that would reflect a response to nucleic acids (comparing T0 vs. T6 vs. T6 + R848, Figure 1). To study immunomodulatory effects of available therapeutics ex vivo, as they pertain to the precise immune dysregulated processes for specific patients, peripheral blood samples are treated with a JAK inhibitor at the relevant therapeutic concentration (here, 5 uM ruxolitinib; T6 + 5R, Time 6 h with 5 uM ruxolitinib), to detect changes in &#34;intrinsic&#34; disease state in response to the drug (T0 vs. T6 vs. T6 + 5R, Figure 1). A JAK inhibitor was chosen for this study because JAK inhibitors have been shown to be successful in the treatment of autoimmune disorders such as RA.
To simultaneously evaluate the dysregulated processes described above in multiple immune cell subsets, peripheral blood samples from SLE patients and healthy controls were processed as described above and analyzed by mass cytometry. Mass cytometry, also known as Cytometry-Time-Of-Flight, offers single-cell analysis of over 40 parameters without issues of spectral overlap7,8,9. This technique utilizes rare earth metal isotopes in the form of soluble metal ions as tags bound to antibodies, instead of fluorophores10. Additional details regarding the mass cytometry technological platform (i.e., tuning and calibration, sample acquisition) can be found in Leipold et al. and McCarthy et al.11,12 The high-dimensionality of mass cytometry enables simultaneous measurement of multiple cytokines throughout innate and adaptive immune cell subsets with single-cell granularity (Table of Materials).
Current conventional clinical and laboratory parameters are often not sensitive or specific enough for detecting ongoing disease activity or the response to specific immunomodulators13, reflecting the need to better delineate the underlying immune changes that drive flare-ups. Given the pervasiveness of cytokine dysregulation in autoimmune disease, a plethora of treatment approaches that use antibodies or small molecular inhibitors targeting cytokines or signaling proteins involved in the regulation of cytokine production have recently emerged. In its basic format, the peripheral blood analytical approach described here provides a platform to identify patient-specific dysregulated cell subsets and their abnormal cytokine production in autoimmune disease with systemic manifestations. This methodology allows for the personalization of therapeutic choices as specific dysregulated cytokines can be identified, and specific treatment options can be tested ex vivo to assess their ability to immunomodulate the patient specific disease process.

<table>
	<tbody>
		<tr>
			<td>Ruxolitinib</td>
			<td>Santa Cruz</td>
			<td>SC-364729A</td>
			<td>Stock Conc: 10 mM; Final Conc: 5 &#956;M</td>
		</tr>
		<tr>
			<td>R848</td>
			<td>Invivogen</td>
			<td>tlrl-r848-5</td>
			<td>Stock Conc: 1 &#956;g/&#956;L; Final Conc: 1 &#956;g/mL</td>
		</tr>
		<tr>
			<td>LPS-EK</td>
			<td>Invivogen</td>
			<td>tlrl-eklps</td>
			<td>Stock Conc: 1 &#956;g/&#956;L; Final Conc: 0.1 &#956;g/mL</td>
		</tr>
		<tr>
			<td>Sterile PBS</td>
			<td>Lonza</td>
			<td>17-516F</td>
			<td></td>
		</tr>
		<tr>
			<td>Lyse/Fix Buffer</td>
			<td>BD biosciences</td>
			<td>558049</td>
			<td>Stock Conc: 5X; Final Conc: 1X (dilute in ddH2O)</td>
		</tr>
		<tr>
			<td>&#160;BD Phosflow perm/wash buffer I</td>
			<td>BD biosciences</td>
			<td>557885</td>
			<td>Stock Conc: 10X; Final Conc: 1:10 (dilute in ddH2O)</td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Gibco</td>
			<td>21870-076</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Azide (NaN3)</td>
			<td>Sigma-Aldrich</td>
			<td>S-8032</td>
			<td>Stock Conc: &#62;99.9%; Final Conc: 0.0002</td>
		</tr>
		<tr>
			<td>Protein Transport Inhibitor (PTI)</td>
			<td>eBiosciences</td>
			<td>00-4980-93</td>
			<td>Stock Conc: 500X; Final Conc: 1X</td>
		</tr>
		<tr>
			<td>DNA Intercalator</td>
			<td>Fluidigm</td>
			<td>201192B</td>
			<td>Stock Conc: 500 &#956;M; Final Conc: 0.1 &#956;M</td>
		</tr>
		<tr>
			<td>Cell Staining Media (CSM)</td>
			<td></td>
			<td></td>
			<td>PBS + 0.5% BSA, 0.02% NaN3</td>
		</tr>
		<tr>
			<td>MaxPar Barcode Perm Buffer</td>
			<td>Fluidigm</td>
			<td>201057</td>
			<td>Stock Conc: 10X; Final Conc: 1X</td>
		</tr>
		<tr>
			<td>20-plex Pd Barcode Set</td>
			<td>Fluidigm</td>
			<td>S0014</td>
			<td>Stock Conc: n/a; Final Conc: n/a</td>
		</tr>
		<tr>
			<td>EQ TM Four Element Calibration Beads</td>
			<td>Fluidigm</td>
			<td>201078</td>
			<td>Stock Conc: 10X; Final Conc: 1X</td>
		</tr>
		<tr>
			<td>16% MeOH-free Formaldehyde Solution</td>
			<td>Thermo</td>
			<td>28908</td>
			<td>Stock Conc: 16% (w/v); Final Conc: 1.6% (w/v)</td>
		</tr>
		<tr>
			<td>Sterile round bottom polystyrene tubes</td>
			<td>VWR</td>
			<td>60818-496</td>
			<td>Stock Conc: n/a; Final Conc: n/a</td>
		</tr>
		<tr>
			<td>Polypropylene cluster tubes</td>
			<td>Light Labs</td>
			<td>A-9001</td>
			<td>Stock Conc: n/a; Final Conc: n/a</td>
		</tr>
		<tr>
			<td>Helios CyTOF instrument</td>
			<td>Fluidigm</td>
			<td>Helios</td>
			<td>All solutions to be used in CyTOF analysis need to be free of metal contamination. ddH2O is used in the preparation of any solutions should have a resistivity of at least 18.0 M&#937;.cm. ddH2O and any self-prepared solutions should be stored in new plastic or glass bottles that have never been autoclaved.</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Antibodies used for Mass Cytometry</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surface markers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CD1c</td>
			<td>Biolegend</td>
			<td>L161</td>
			<td>Mass: 161</td>
		</tr>
		<tr>
			<td>CD3</td>
			<td>BD</td>
			<td>UCHT1</td>
			<td>Mass: 144</td>
		</tr>
		<tr>
			<td>CD4</td>
			<td>Biolegend</td>
			<td>SK3</td>
			<td>Mass: 174</td>
		</tr>
		<tr>
			<td>CD7</td>
			<td>BD</td>
			<td>M-T701</td>
			<td>Mass: 149</td>
		</tr>
		<tr>
			<td>CD8</td>
			<td>Biolegend</td>
			<td>SK1</td>
			<td>Mass: 142</td>
		</tr>
		<tr>
			<td>CD11b</td>
			<td>Fluidigm</td>
			<td>ICRF44</td>
			<td>Mass: 209</td>
		</tr>
		<tr>
			<td>CD11c</td>
			<td>BD</td>
			<td>B-ly6</td>
			<td>Mass: 152</td>
		</tr>
		<tr>
			<td>CD15</td>
			<td>BD</td>
			<td>HI98</td>
			<td>Mass: 115</td>
		</tr>
		<tr>
			<td>CD14</td>
			<td>Biolegend</td>
			<td>M5E2</td>
			<td>Mass: 154</td>
		</tr>
		<tr>
			<td>CD16</td>
			<td>eBioscience/Thermo</td>
			<td>B73.1</td>
			<td>Mass: 165</td>
		</tr>
		<tr>
			<td>CD19</td>
			<td>Santa Cruz</td>
			<td>SJ25C1</td>
			<td>Mass: 163</td>
		</tr>
		<tr>
			<td>CD21</td>
			<td>Biolegend</td>
			<td>Bu32</td>
			<td>Mass: 141</td>
		</tr>
		<tr>
			<td>CD27</td>
			<td>BD</td>
			<td>L128</td>
			<td>Mass: 155</td>
		</tr>
		<tr>
			<td>CD38</td>
			<td>Fluidigm</td>
			<td>HIT2</td>
			<td>Mass: 172</td>
		</tr>
		<tr>
			<td>CD45 total</td>
			<td>Biolegend</td>
			<td>HI30</td>
			<td>Mass: 89</td>
		</tr>
		<tr>
			<td>CD45RA</td>
			<td>Biolegend</td>
			<td>HI100</td>
			<td>Mass: 153</td>
		</tr>
		<tr>
			<td>CD56</td>
			<td>Miltenyi</td>
			<td>REA196</td>
			<td>Mass: 168</td>
		</tr>
		<tr>
			<td>CD66</td>
			<td>BD</td>
			<td>B1.1/CD66</td>
			<td>Mass: 113</td>
		</tr>
		<tr>
			<td>CD86</td>
			<td>Fluidigm</td>
			<td>IT2.2</td>
			<td>Mass: 150</td>
		</tr>
		<tr>
			<td>CD123</td>
			<td>Fluidigm</td>
			<td>6H6</td>
			<td>Mass: 143</td>
		</tr>
		<tr>
			<td>CD278/ICOS</td>
			<td>Biolegend</td>
			<td>C398.4A</td>
			<td>Mass: 156</td>
		</tr>
		<tr>
			<td>CD179/PD1</td>
			<td>Biolegend</td>
			<td>EH12.2H7</td>
			<td>Mass: 162</td>
		</tr>
		<tr>
			<td>IgD</td>
			<td>Biolegend</td>
			<td>IA6-2</td>
			<td>Mass: 146</td>
		</tr>
		<tr>
			<td>IgM</td>
			<td>Biolegend</td>
			<td>MHM-88</td>
			<td>Mass: 151</td>
		</tr>
		<tr>
			<td>CXCR5</td>
			<td>BD</td>
			<td>RF8B2</td>
			<td>Mass: 173</td>
		</tr>
		<tr>
			<td>HLADR</td>
			<td>Biolegend</td>
			<td>L243</td>
			<td>Mass: 167</td>
		</tr>
		<tr>
			<td>Cytokines</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IL-1&#945;</td>
			<td>Biolegend</td>
			<td>364-3B3-14</td>
			<td>Mass: 147</td>
		</tr>
		<tr>
			<td>IL-1&#946;</td>
			<td>Biolegend&#160;</td>
			<td>H1b-98</td>
			<td>Mass: 169</td>
		</tr>
		<tr>
			<td>IL-1RA</td>
			<td>Santa Cruz&#160;</td>
			<td>AS17</td>
			<td>Mass: 157</td>
		</tr>
		<tr>
			<td>IL-6</td>
			<td>Biolegend</td>
			<td>MQ2-13A5</td>
			<td>Mass: 164</td>
		</tr>
		<tr>
			<td>IL-8</td>
			<td>BD</td>
			<td>E8N1</td>
			<td>Mass: 160</td>
		</tr>
		<tr>
			<td>IL-12/IL-23p40</td>
			<td>Biolegend&#160;</td>
			<td>C8.6</td>
			<td>Mass: 171</td>
		</tr>
		<tr>
			<td>IL-17A</td>
			<td>Biolegend&#160;</td>
			<td>BL168</td>
			<td>Mass: 148</td>
		</tr>
		<tr>
			<td>IL23p19</td>
			<td>eBioscience/Thermo</td>
			<td>23dcdp</td>
			<td>Mass: 176</td>
		</tr>
		<tr>
			<td>MIP1&#946;</td>
			<td>BD</td>
			<td>D21-1351</td>
			<td>Mass: 158</td>
		</tr>
		<tr>
			<td>MCP1</td>
			<td>BD</td>
			<td>5D3-F7</td>
			<td>Mass: 170</td>
		</tr>
		<tr>
			<td>IFN&#945;</td>
			<td>Miltenyi&#160;</td>
			<td>LT27:295</td>
			<td>Mass: 175</td>
		</tr>
		<tr>
			<td>IFN&#947;</td>
			<td>Biolegend</td>
			<td>4S.B3</td>
			<td>Mass: 165</td>
		</tr>
		<tr>
			<td>PTEN</td>
			<td>BD</td>
			<td>A2B1</td>
			<td>Mass: 159</td>
		</tr>
		<tr>
			<td>TNF&#945;</td>
			<td>Biolegend</td>
			<td>Mab11</td>
			<td>Mass: 166</td>
		</tr>
		<tr>
			<td>Note: If the manufacturer is stated as Fluidigm, this antibody was purchased from Fluidigm with metal pre-conjugated. If the manufacturer is stated as other than Fluidigm, this antibody was self-conjugated using the MaxPar Multi-Metal Labeling&#160; Kit (Fluidigm Cat: 201300) according to manufacturer protocol.&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

single-cell analysis of immunophenotype and cytokine production in peripheral whole blood via mass cytometry

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies in an attempt to understand the precise mechanisms that lead to the breakdown of self-tolerance and subsequent autoimmunity. Approaches thus far have been based on the study of one specific aspect of the immune system (a single or few cell types or cytokines), and do not offer a global assessment of complex autoimmune disease. While patient sera-based studies have afforded important insights into autoimmunity, they do not provide the specific cellular source of the dysregulated cytokines detected. A comprehensive single-cell approach to evaluate cytokine production in multiple immune cell subsets, within the context of &#34;intrinsic&#34; patient-specific plasma circulating factors, is described here. This approach enables monitoring of the patient-specific immune phenotype (surface markers) and function (cytokines), either in its native &#34;intrinsic pathogenic&#34; disease state, or in the presence of therapeutic agents (in vivo or ex vivo).

Figure 1 demonstrates the workflow for the stimulation and processing of the peripheral blood samples, including allocation of blood sample aliquots, timing of the addition of stimulation agents, protein transport inhibitor cocktail, and incubation times until the red blood cell (RBC) lysis and fixation. The choice of stimulating agents will depend on the signaling and cytokine pathways that are targeted for assessment. For example, in the protocol described ...

Watch this Scientific Journal Video about Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry at JoVE.com

Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry

Here we describe a single-cell proteomic approach to evaluate immune phenotypic and functional (intracellular cytokine induction) alterations in peripheral whole blood samples, analyzed via mass cytometry.

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies ...

Research

JoVE Journal

Immunology and Infection

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Generation of Human Alloantigen-specific T Cells from Peripheral Blood

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A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood

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Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

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Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...