A multi-omics approach toinvestigate the heterogeneityof memory T cells
Immunology applications for theBD LSRFortessa™ X-20 cell analyzerFeatures• Design complex, high-resolution panels of up to 18 colors
for a deeper understanding of the biology of immune cells• Maximize panel resolution and minimize spillover by spreading
fluorochromes across laser lines• Standardize the performance of flow cytometry panels acrossBD platforms, analyzers and sorters• Correlate gene expression with protein expression at the 		single-cell level• Expand multicolor flow cytometry panels based on biologicalinformation obtained from genomic analysis

As researchers extend the boundaries of cellular knowledge anddiscovery, they need tools for deeper and detailed phenotypicand molecular analyses. For example, the post-thymic activationand development of T cells involves a heterogeneous populationof cells at different stages of differentiation, including naïve (TN),stem cell memory (TSCM), central memory (TCM), effector memory(TEM) and terminal effector T cells (TEff). Studies have shown thatthese different T-cell subsets are themselves highly heterogeneous, and their identification and characterization requires
not one or a few but, rather, a combination of several markers.
This heterogeneity has heightened interest within the scientificcommunity to find new markers that might provide insights
into the T-cell differentiation and development pathway andpotentially identify new cell populations. Such exploratory
studies require development of multi-omics approaches thatcombine high-dimensional flow cytometry and single-cell RNA
sequencing (scRNA-Seq) for phenotypic and genomic analyses
at the single-cell level.

Figure 1
9–color panel design

Transfer to sorter

TCM

Single–cell sort

CD45RO PE
PE-Cy7Cy7TEMBD Precise plate

Naive-like

CD197 AF647
Deeper understandingof biology
Amplify target
genes and create NGS library

16–color panel design...AGTCCAATC

Self renewal
survival

Effector function
differentiation
senescence

Figure 1. Multi-omics workflow to identify markers for deeper characterization of immune cells
A 9-color panel was optimized on a BD LSRFortessa X-20 cell analyzer for detection of CD8⁺ TN, TSCM, TCM and TEM subsets, and then transferred to a BD FACSAria Fusioncell sorter for single-cell isolation of these subsets. Cells were deposited individually into a BDTM Precise plate, followed by target gene amplification, library preparation andscRNA-Seq. Differential gene-expression analysis was performed to identify genes encoding surface markers that might better identify the CD8⁺ memory cell subsets.
Finally, a 16-color panel was designed based on the RNA-Seq analysis, including potential markers to discriminate different CD8⁺ subsets.

With up to 5 lasers and 18 fluorescence parameters, the BD
LSRFortessa™ X-20 cell analyzer enables the development ofintegrated workflows to investigate these phenotype-genotype
relationships. In this data sheet, we describe a complete
workflow solution (Figure 1) in which a BD cell analyzer, afluorescence-activated cell sorter and a sequencing-ready library
preparation platform are used to identify, sort and characterizedifferent CD8⁺ T-cell subsets at the single-cell level.
We began by designing a 9-color panel that was optimized on theBD LSRFortessa X-20 analyzer for clearly resolving the CD8⁺ T-cellsubsets. This optimized panel was transferred to a BD FACSAria™
Fusion cell sorter to isolate single cells into BD™ Precise 96-wellplates for downstream genomic analysis. We used scRNA-Seq
to analyze gene expression of 200 T-cell specific genes, to identify
markers that might further differentiate T cell subsets of interest.
Taking advantage of the BD LSRFortessa X-20 system’s multiple
detectors, we designed an expanded 16-color flow cytometry
panel to validate those markers’ usefulness for further phenotypicanalysis.
Table 1 shows the 9-color panel designed to identify CD8⁺
TN, TSCM, TCM and TEM cell subsets on the BD LSRFortessa X-20.

Fluorochromes were chosen and assigned to each marker considering antigen co-expression and density as well as fluorochromebrightness and spillover. By distributing fluorochromes across thefive lasers and accounting for cross-laser excitation and emission
spectra, it is possible to design multicolor panels with minimal
spectral overlap and maximum resolution.
Table 1. Nine-color T-cell subset panel for cell analyzer and sorter
BD LSRFortessa X-20 5-laser cell analyzer configuration: B-2, R-3, YG-3, V-6, UV-4
BD FACSAria Fusion 5-laser cell sorter configuration: B-2, R-3, YG-4, V-6, UV-3

MarkerClone

Laser

Fluorochrome/Dye

CD8

RPA-T8Ultraviolet (355 nm)

BUV395

CD27

M-T271

Ultraviolet (355 nm)

BUV737CD183

1C6/CXCR3

Violet (405 nm)

BV421

CD3

UCHT1

Violet (405 nm)

BV510

CD45RA

HI100

Blue (488 nm)

BB515

CD19/CD14/CD4

HIB19/MφP/SK3

Blue (488 nm)

PerCP-Cy™5.5

Live/Dead—Blue (488 nm)

7-AAD

CD95

DX2

Yellow-Green (561 nm)

PE

CD45RO

UCHL1Yellow-Green (561 nm)

PE-Cy™7

CD197 (CCR7)150503

Red (640 nm)

Alexa Fluor® 647

Figure 2 shows the gating strategy used to identify CD8⁺ T-cell subsets. After excluding lineage and dead cells, CD3⁺CD8⁺ cells weregated for analysis of CD45RO and CD197 expression. TEM and TCM cells were defined as CD45RO⁺CD197⁻ and CD45RO⁺CD197⁺ respectively.
The CD45RO⁻CD197⁺ naïve-like population contains TN cells and a population of long-lived memory T cells endowed with enhanced selfrenewal and multipotency. These cells, called stem cell memory T cells (TSCM), can be distinguished from the TN cells based on high expressionof CD183 (CXCR3) and CD95.¹ CD27 and CD45RA were also included in the panel to further illuminate the heterogeneity of CD8⁺ cells asshown by the presence of noncytotoxic CD45RA⁻CD27⁺ memory cells and cytotoxic CD45RA⁺CD27⁻ effector cells.2

CD8 BUV395
CD3 BV510

CD3 BV510

NN

SCM

SCM

CMCMEMEM

CD45RA BB515

TCM

TSCM

CD45RA BB515

TEM

CD8⁺CD95 PE

CD3⁺CD45RO PE-Cy7

CD14/CD19/CD4
PerCP-Cy5.5 + 7-AAD

Figure 2ATNNaive-like

CD197 AF647

CD183 BV421

CD27 BUV737

CD27 BUV737

CD3 BV510

CD3 BV510

NN

SCM

SCMCMCMEMEM
CD45RA BB515

TEM

TCM

TSCM

CD45RA BB515

CD8 BUV395

CD8⁺

CD95 PE

CD3⁺

CD45RO PE-Cy7

CD14/CD19/CD4
PerCP-Cy5.5 + 7-AAD

Figure 2B

TN

Naive-like

CD197 AF647

CD183 BV421

CD27 BUV737

CD27 BUV737

Figure 2. Nine-color CD8⁺ T-cell panel on the BD LSRFortessa X-20 and BD FACSAria Fusion systems
Method: Peripheral blood mononuclear cells (PBMCs) were isolated from a healthy donor. Cells were pre-stained with CD197 (CCR7) at 37°C. After 15 minutes, cells
were stained with a premade cocktail of antibodies (Table 1) and BD Horizon™ Brilliant Stain Buffer at RT for 30 minutes. BD Pharmingen™ 7-AAD was added before
acquisition to exclude dead cells from analysis. Instruments: Samples from the same staining were acquired on both (A) the BD LSRFortessa X-20 cell analyzer and (B)the BD FACSAria Fusion cell sorter with standardized mean fluorescence intensity (MFI) target values to maintain consistency between instruments. Data was analyzedusing both BD FACSDiva™ and FlowJo® software. Gating strategy: Red arrows denote gating hierarchy. CD8⁺ T cells were identified by excluding doublet cells and definedby lineage/7-AAD⁻ populations and CD3⁺ T cells. From the CD8⁺ population, naïve-like (CD45RO⁻CCR7⁺), central memory (TCM, CD45RO⁺CCR7⁺) and effector memory
T cells (TEM, CD45RO⁺CCR7⁻) were identified. Stem cell memory (TSCM, CD183highCD95⁺) and naïve T cells (TN, CD183⁺/⁻CD95⁻) were gated from the naïve-like T cells.
Biexponental scaling for each instrument was set for optimal visualization. Overall, the target populations were similarly resolved upon cross-platform standardization.

With the subset identification panel optimized on the BD
LSRFortessa X-20 cell analyzer, we transferred it to the BD
FACSAria Fusion cell sorter to enable single-cell isolation of thecells for downstream genomic analysis. We standardized acrossplatforms by staining human whole blood with the BD Horizon™
CD4 Fluorochrome Evaluation Kit and adjusting voltages on
the BD FACSAria Fusion to match the MFI recorded on the BD
LSRFortessa X-20. This approach resulted in overall similar resolution
and clear detection of the target T-cell subsets without designingor optimizing a panel specifically for cell sorting (Figure 2B).
The cells were then sorted individually into a BD Precise 96-wellplate for downstream genomic analysis. BD Precise kits contain acomplete set of reagents for preparation of a sequencing-readylibrary. BD Precise kits also incorporate molecular indexing technology that enables highly accurate mRNA molecule quantificationand eliminates noise that would otherwise result from PCR amplification bias. Finally, the technology allows samples from each well
to be pooled into a single tube while maintaining the identity ofeach sample.
Following targeted scRNA-Seq using a panel of 200 T-cell-specificgenes, we performed a comparative analysis of the gene expression
profiles from the sorted CD8⁺ cell subsets. We specifically focusedon the analysis of genes coding for surface markers that could
be prospectively used for flow cytometry analysis and sorting,upon validating the correlation between mRNA and protein
expression. We identified four candidate genes that weredifferentially expressed in TN, TSCM, TCM and TEM CD8⁺ subsets,
which coded for the surface markers CD161, CD314, CD127 andCD184. For example, CD161 mRNA expression was exclusive toTCM, and TEM cells, while CD184 and CD314 mRNA expression was
higher in TSCM than in other subsets (data not shown).
Taking advantage of the many fluorescent parameters available
on the BD LSRFortessa X-20, we expanded the original 9-color panel
to a new 16-color panel (Table 2) to validate the differential protein
expression of the gene markers identified from the scRNA-Seqstudy. The panel included two additional markers, CD58 andCD122, previously reported to improve the identification ofTSCM,1,3 as well as CD4, to comprehensively and simultaneously
characterize memory subsets of both CD4⁺ and CD8⁺ T cells.
This approach significantly increases the amount of biologicalinformation that can be obtained from a single tube.

Table 2. Sixteen-color T-cell subset panel with expanded marker set
BD LSRFortessa X-20 5-laser cell analyzer configuration: B-2, R-3, YG-3, V-6, UV-4
MarkerClone

Laser

Fluorochrome/DyeCD8

RPA-T8Ultraviolet (355 nm)

BUV395

CD127

HIL-7R-M21

Ultraviolet (355 nm)

BUV737CD183

1C6/CXCR3

Violet (405 nm)

BV421

CD161

DX12Violet (405 nm)

BV510

CD184

12G5Violet (405 nm)

BV605

CD45RO

UCHL1

Violet (405 nm)

BV650

CD58

1C3Violet (405 nm)

BV711

CD4

SK3

Violet (405 nm)

BV786

CD122

Mik-β3

Blue (488 nm)

BB515

CD19/CD14

HIB19/MφP

Blue (488 nm)

PerCP-Cy5.5

Live/Dead—Blue (488 nm)

7-AAD

CD95

DX2

Yellow-Green (561 nm)

PE

CD197 (CCR7)150503

Yellow-Green (561 nm)

PE-CF594

CD314

1D11

Yellow-Green (561 nm)

PE-Cy7

CD27

M-T271

Red (640 nm)

APC

CD45RA

HI100

Red (640 nm)

APC-H7CD3

UCHT1

Red (640 nm)

Alexa Fluor® 700

The flow cytometry data (Figure 3) partially recapitulated andextended the results of the genomic analysis. For example, consistent with the scRNA-Seq data, CD161 was exclusively expressedin TCM and TEM cell subsets. These findings agree with previous
characterizations of CD161⁺ T cells, validating the multi-omics
approach for identification of subset-specific markers that aredifferentially expressed at the molecular and protein level. We
observed similar expression patterns for CD4⁺ and CD8⁺ subsets
for all markers tested except CD314, which was expressed by all
CD8⁺ subsets but not by CD4⁺ subsets.
The experiment demonstrates the usefulness of integrating
flow-cytometry-based cell analysis and sorting with downstream
genomic analysis to discover potential novel markers that can better
identify and characterize cell populations of interest. scRNA-seq
allows for the screening of a large number of genes. The list ofprospective markers for phenotypic discrimination and cell sorting
can be narrowed down after differential gene expression analysis,
selection of genes coding for surface markers and validation byflow cytometry. The BD LSRFortessa X-20 lets you analyze moremarkers in a single tube by spreading key protein markers acrossmultiple laser lines and detectors, allowing you to delve deeply into
the biology of individual lineages and discover interrelationships
between markers that would be lost across multiple tubes.

Figure 3A
CD8⁺

CD3 AF700

CD95 PE

CD45RO BV650

CD3⁺ T cells

CD4⁺
T cellsCD8 BUV395

CD14/CD19
PerCP-Cy5.5 + 7-AAD

CD8⁺ T cells

CD4 BV786

CD197 PE-CF594

CD183 BV421

CD95 PE

CD45RO BV650

CD4⁺CD197 PE-CF594

CD183 BV421

Figure 3B
Gated on CD8⁺ subsets
NSCM
CMEM
CD27 APC

CD45RA APC-H7

CD161 BV510

CD127 BUV737

CD184 BV605

CD314 PE-Cy7

CD122 BB515

CD58 BV711

CD45RA APC-H7

CD161 BV510

CD127 BUV737

CD184 BV605

CD314 PE-Cy7

CD122 BB515

CD58 BV711

Gated on CD4⁺ subsets

NSCM
CMEM
CD27 APC

Figure 3. Sixteen-color CD8⁺ and CD4⁺ T-cell panel on the BD LSRFortessa X-20 cell analyzer
The 9-color CD8⁺ T-cell panel (Figure 1) was expanded to 16 colors on the BD LSRFortessa X-20 to analyze the expression level of markers reported in the literatureand identified from the scRNA-Seq analysis. The staining protocol and gating strategy (A) were similar to Figure 2, adding additional markers and gates for CD4⁺ as wellas CD8⁺ subsets. Results (B): Histogram overlays show expression levels of potentially differentiating markers within CD8⁺ and CD4⁺ subsets. The data provides a comprehensive
phenotypic analysis of multiple T-cell subsets. For example, CD161 was exclusively expressed in TCM and TEM cells, in agreement with the gene expression analysis andprevious publications. CD58 expression gradually increased as cells differentiated toward the TEM phenotype. CD314 was uniformly expressed by all CD8⁺ subsets but by
no CD4⁺ subsets.

References
1. Gattinoni L, Lugli E, Ji Y, et al. A human memory T-cell subset with stem cell-like properties. Nat Med. 2011;17:1290–1297.
2. Hamann D, Baars PA, Rep MG, et al. Phenotypic and functional separation of memory and effector human CD8⁺ T cells.
J Exp Med. 1997;186:1407-18.
3. Lugli E, Gattinoni L, Roberto A, et al. Identification, isolation and in vitro expansion of human and nonhuman primate
T stem cell memory cells. Nat Protoc. 2013;8:33-42.

Systems and software
Description
BD LSRFortessa™ X-20 Cell Analyzer – Special Order

Ordering information: Reagents
Description

Cat. No.

BD Pharmingen™ Alexa Fluor® 700 Mouse Anti-Human CD3557943

BD Horizon™ BV510 Mouse Anti-Human CD3563109

BD Pharmingen™ PerCP-Cy™5.5 Mouse Anti-Human CD4566316

BD Horizon™ BV786 Mouse Anti-Human CD4563877

BD Horizon™ BUV395 Mouse Anti-Human CD8563795

BD Pharmingen™ PerCP-Cy™5.5 Mouse Anti-Human CD14562692

BD Pharmingen™ PerCP-Cy™5.5 Mouse Anti-Human CD19561295

BD Horizon™ BUV737 Mouse Anti-Human CD27

564301

BD Pharmingen™ APC Mouse Anti-Human CD27

561786

BD Pharmingen™ APC-H7 Mouse Anti-Human CD45RA

560674

BD Horizon™ BB515 Mouse Anti-Human CD45RA

564552

BD Pharmingen™ PE-Cy™7 Mouse Anti-Human CD45RO

560608

BD Horizon™ BV650 Mouse Anti-Human CD45RO

563750

BD OptiBuild™ BV711 Mouse Anti-Human CD58742729

BD Pharmingen™ PE Mouse Anti-Human CD95

555674

BD Horizon™ BB515 Mouse Anti-Human CD122

564688

BD Horizon™ BUV737 Mouse Anti-Human CD127

564300

BD Horizon™ BV510 Mouse Anti-Human CD161

563212

BD Horizon™ BV421 Mouse Anti-Human CD183 (CXCR3)562558

BD OptiBuild™ BV605 Mouse Anti-Human CD184 (CXCR4)740418

BD Horizon™ PE-CF594 Mouse Anti-Human CD197 (CCR7)

562381

BD Pharmingen™ Alexa Fluor® 647 Mouse Anti-Human CD197 (CCR7)

560816

BD Pharmingen™ PE-Cy™7 Mouse Anti-Human CD314 (NKG2D)562365

BD Pharmingen™ 7-AAD

559925

BD Horizon™ Human CD4 Fluorochrome Evaluation Kit

566352

BD Horizon™ Brilliant Stain Buffer

563794

Class 1 Laser Product.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Cy™ is a trademark of GE Healthcare. Cy™ dyes are subject to proprietary rights of GE Healthcare and Carnegie Mellon University, andare made and sold under license from GE Healthcare only for research and in vitro diagnostic use. Any other use requires a commercial
sublicense from GE Healthcare, 800 Centennial Avenue, Piscataway, NJ 08855-1327, USA.
Alexa Fluor® is a registered trademark of Life Technologies Corporation.
FlowJo® is a registered trademark of FlowJo, LLC, a wholly owned subsidiary of BD.
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