Single cell multiomics analysis of immune

cells in obese mice

BD, the BD Logo and Rhapsody are trademarks of Becton, Dickinson and Company or its affiliates. All other trademarks are the
property of their respective owners. © 2019 BD. All rights reserved.
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 Background
• Diets high in fat can lead to obesity, which is a major risk factor for the development
of various metabolic and inflammatory diseases.

• Here, we demonstrate the usage of the BD Rhapsody™ Single-Cell Analysis system

along with BD® AbSeq assays, the BD Rhapsody™ Immune Response Panel and the
BD® Mouse Immune Single-Cell Multiplexing Kit (SMK) to characterize obesity-caused
chronic inflammation through analyses of immune-cell composition and gene
expression profiles in high fat diet (HFD) mice.

• At six weeks of age, mice are placed on either a control diet (10% diet-induced
obesity (DIO) or a high fat diet (60% DIO) for 17 weeks before the start of the
experiment.

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property of their respective owners. © 2019 BD. All rights reserved.
 Experimental approach and workflow

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 Panel design
30-plex BD® AbSeq Panel 10-color Flow Cytometry Panel

CD1d CD25 CD184 Marker Fluorochrome

CD4 CD44 CD197 (CCR7) 7-AAD PerCP-Cy™5.5
CD5 CD45R (B220) CD223 (LAG-3) B220 BUV737
CD8β CD49a CD274 CD3ε APC
CD9 CD49b CD279 (PD-1) CD4 APC-H7
CD11b CD62L I-A/I-E CD8α BUV395
CD11c CD64 IgD
CD11b APC-R700
CD19 CD69 IgM
CD45 FITC
CD21/CD35 CD103 Ly-6C/Ly-6G
CD69 BUV605
CD23 CD182 (CXCR2) TCRβ
F4/80 PE
TCRγδ BV480

• The BD AbSeq panel contains 30 proteins for cell lineage, differentiation, fate and
function.
• A companion 10-color flow cytometry panel with selected, overlapping specificities
was designed to assess flow cytometry and BD AbSeq concordance.
• The BD Rhapsody Immune Response Panel includes 400 genes involved in immune
responses.
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property of their respective owners. © 2019 BD. All rights reserved.
 Frequency of hematopoietic cells in mouse
lymphoid and epididymal adipose tissues

Control Diet

High Fat Diet (HFD)

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 Characterization of immune cell populations
in adipose tissue

CD45+

A HFD causes a
decrease in the ratio of
CD4/CD8 in adipose
CD45+CD3+ tissue.

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property of their respective owners. © 2019 BD. All rights reserved.
 Sequencing metrics
Total Reads
2,000,000,000

1,500,000,000

1,000,000,000

500,000,000

0
mRNA AbSeq SMK Total

Sample Tag Metrics

Sample Name ST Cell # Rep1 Rep2 Total
Mean Reads/Cell Control Adipose Tissue ST 1 972 928 1900
Control Bone Marrow ST 2 1501 1527 3028
25000
Control Spleen ST3 1566 1337 2903
20000 Control Thymus ST4 1074 1006 2080
15000 HFD Adipose Tissue ST5 1196 1029 2225
HFD Bone Marrow ST6 1471 1308 2779
10000
HFD Spleen ST7 1389 1229 2618
5000 HFD Thymus ST8 1129 1039 2168
0 Multiplet 1048 818 1866
mRNA AbSeq SMK Undetermined 72 42 114
Total 11418 10263 21681

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property of their respective owners. © 2019 BD. All rights reserved.
 The combination of BD AbSeq assays and the BD
Rhapsody Immune Response Panel improves cell
clustering

t-SNE: Ab Alone t-SNE: mRNA Alone t-SNE: Abs + mRNA

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property of their respective owners. © 2019 BD. All rights reserved.
 Dimensionality reduction using t-SNE plots
All Tissues
(HFD and Control Diet)

Key:
Thymus
Spleen
Adipose Tissue
Bone Marrow

Cell Islands Created From

Concatenation of All Tissues

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property of their respective owners. © 2019 BD. All rights reserved.
 BD AbSeq assays identify major cell
populations in each tissue
The heat map shows the most highly expressed proteins in each tissue.

Thymus Spleen Adipose Tissue Bone Marrow

Myeloid Cell
Subsets

Myeloid Cell
Subsets

B Cells

T Cells

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property of their respective owners. © 2019 BD. All rights reserved.
 Robust identification of target cell populations
All Bone Adipose
Tissues Marrow Tissue Spleen Thymus

CD19
(B Cell Marker)

TCR beta
Key:
(T Cell Marker)

CD11b
(Myeloid Cell
Marker)

CD49b
(NK Cell
Marker)

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property of their respective owners. © 2019 BD. All rights reserved.
 Single cell protein and mRNA correlation
AbSeq (Protein) mRNA
CD1d Cd1d1 Protein-mRNA Correlation
CD4 (AbSeq) Cd4
CD5 (AbSeq) Cd5
CD8b (AbSeq) Cd8b1
CD9 (AbSeq) Cd9
CD11b Itgam
CD11c Itgax
CD19 Cd19
CD21_CD35 Cr2
CD23 Fcer2a

Correlation
Coefficient
CD25 Il2ra
CD44 Cd44
CD45R (AbSeq) Ptprc
CD49a (AbSeq) Itga1
CD49b (AbSeq) Itga2
CD62L (AbSeq) Sell
CD69 (AbSeq) Cd69
CD103 Itgae
CD182 (CXCR2) (AbSeq) Cxcr2
CD184 Cxcr4
CD197 Ccr7
CD223 Lag3
CD274 Cd274
CD279 Pdcd1
I-A_I-E (AbSeq) H2-Ab
IgD (AbSeq) Ighd
IgM (AbSeq) Ighm
Ly-6G_Ly-6C (AbSeq) Ly6g_Ly6c
TCR-beta (AbSeq) Tcr

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property of their respective owners. © 2019 BD. All rights reserved.
 Unbiased cell clustering identifies multiple
cell phenotypes across different samples

Key:

Key:

t-SNE t-SNE

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property of their respective owners. © 2019 BD. All rights reserved.
 Unbiased clustering reveals disease-associated
cell phenotypes

Key:

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property of their respective owners. © 2019 BD. All rights reserved.
 Subsets of clusters show differences in cell
number for adipose tissues of HFD mice
Bone Marrow Thymus
40 80
30 60

% Cells

% Cells
20 40
10 20
0 0
PG-1
PG-2
PG-3
PG-4
PG-5
PG-6
PG-7
PG-8
PG-9
PG-10
PG-11
PG-12

PG-1
PG-2
PG-3
PG-4
PG-5
PG-6
PG-7
PG-8
PG-9
PG-10
PG-11
PG-12
Clusters Clusters

Spleen Adipose Tissue

80
40
60
% Cells

30

% Cells
40
20
20 10
Key: 0 0
PG-1
PG-2
PG-3
PG-4
PG-5
PG-6
PG-7
PG-8
PG-9
PG-10
PG-11
PG-12
Control HFD

PG-10
PG-11
PG-12
PG-1
PG-2
PG-3
PG-4
PG-5
PG-6
PG-7
PG-8
PG-9
Clusters Clusters

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property of their respective owners. © 2019 BD. All rights reserved.
 Unbiased clustering reveals disease-associated
cell phenotypes

Key:

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property of their respective owners. © 2019 BD. All rights reserved.
 t-SNE plots of adipose tissue of control and
HFD mice

Key:
Control
HFD

Adipose Tissue t-SNE Plots

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property of their respective owners. © 2019 BD. All rights reserved.
 HFD causes drastic changes to immune cell
composition in adipose tissue
Key:

PG-12 PG-12
PG-7
PG-7

PG-5
PG-1 PG-5 PG-1

Fat t-SNE Fat t-SNE

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property of their respective owners. © 2019 BD. All rights reserved.
 HFD increases the frequency of B cells in
adipose tissue
Control HFD

1.6% 5.7%

PG-7 PG-7

CD19 (AbSeq)

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 HFD induces the B cells similar to B-2
regulatory cells
Adipose Tissue
9
8 Key:
7
6
5
4
3
2
1

B-2 regulatory cells (CD19hi,

CD1d+/hi, CD21+, CD23 +/-,
IgMhi, IgDlo/mid) regulate
inflammatory responses with
IL-10 production.

Shaikh et al., 2014

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property of their respective owners. © 2019 BD. All rights reserved.
 HFD causes drastic changes in immune cell
composition in adipose tissue

Key:

PG-5 PG-5
PG-1 PG-1

Fat t-SNE Fat t-SNE

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property of their respective owners. © 2019 BD. All rights reserved.
 Distinct populations of myeloid cells in the
adipose tissue of obese mouse
Accumulation of Adipose Tissue
Loss of CD11c+ Cell Subsets in HFD
Macrophages in HFD

Control HFD Control HFD

27.6% 35.9%
PG-5 PG-5
28.4% 12.2%
PG-1 PG-1

Adgre1 CD11c (AbSeq)

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 Adipose tissue macrophages in obese mouse
exhibit an inflammatory gene signature
Control HFD
PG-1 PG-1
Control HFD
Lgals3
Trem2
Top 10 Up Cd9
Cxcl1
in PG-1 HFD Il1b
q-value

Mmp12
Cd72
Spp1
Il1m Trem2
Qpct
I-A_I-E (Ab) (Promotes Diet-induced Obesity)
CD64 (Ab)
CD1d (Ab)
CD49b (Ab)
Fold-Change Fcna
HFD vs Control Cd163
F13a1
Top 10 Down Tfrc
Cd38
in PG-1 HFD Mmp9

Park M. et al, Diabetes, 2015 CD163

Junghwan Baek, Diabetes, 2018 (Controls Inflammation)
David A. et al, PNAS, 2018

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property of their respective owners. © 2019 BD. All rights reserved.
 HFD causes upregulation of genes associated with
inflammation in adipose tissue macrophages

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 CD11c+ cells express genes related to
immunoregulation, cell migration and adipogenesis

PG-5 PG-5
Control HFD Upregulated in HFD:
CD44 (AbSeq)

Top 10 Up Lgals1 Lgals1: Induces Adipogenesis

q-value

in PG-5 HFD Mmp12

CD63
Trem2
Clec10a Clec10a: Inflammatory Monocytes
CD9
Clec4e Clec4e: Induces Pro-inflammatory
Clec4d ytokines
Fold-change
Chil3
HFD vs Control Down CD5 (AbSeq)
in PG-5 HFD
CD49b (AbSeq)
Il6

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 HFD causes drastic changes in immune cell
composition in the epididymal adipose tissue

PG-12

Key:
Control

HFD
Fat
Fatt-SNE
t-SNE Fatt-SNE
Fat t-SNE

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 Unique population of cytotoxic cells in the adipose
tissue of an obese mouse exhibits signs of
exhaustion
PG-12 PG-12
Control HFD
CD279 (AbSeq)
Control HFD
Top 10 Up Cd8b1
Cd8a
4.4% PG-12 10.1% PG-12 in PG-12

q-value
Ctla4
HFD Gzmk
Pdcd1
Tigit
Eomes
Cxcr5
Lag3

CD64 (AbSeq)

Fold-change Jchain
CD9 (AbSeq)
HFD vs Control Klra7
Il1b
Itgax
CD8b (AbSeq)
Top 10 Down Klra21

in PG-12 HFD Hmox1

Klra5
Tnfsf13

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property of their respective owners. © 2019 BD. All rights reserved.
 A HFD causes upregulation of genes associated
with T-cell activation in cytotoxic cells

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 Cytotoxic cells infiltrate the adipose tissue of obese
mice and acquire a profile of exhausted cells

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 Cytotoxic cells from epididymal adipose tissue, but
not spleen tissue, exhibit signs of exhaustion in
obese mice HFD Adipose Tissue HFD Spleen
Up-regulated in HFD
adipose tissue compared
to HFD spleen

HFD Adipose Tissue Control Spleen

Up-regulated in HFD
adipose tissue compared
to control spleen

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 Analysis of cytotoxic cell states across
different tissues
A high fat diet induces the transition to an exhausted cell phenotype.

Tissue Diet Exhausted/Activated

PD-1+, ITGAE+
PD-1+, ITGAE-
PD-1-, ITGAE+
PD-1-, ITGAE-

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 Adipose tissue-infiltrating cytotoxic cells
from HFD mouse co-express PD-1 and Tigit
Monocle of Adipose Tissue CD8+ Cells
Key:
1
Key: 2
Control 3
HFD 4
5

CD103 (AbSeq) PD-1 (AbSeq) Pdcd1 Tigit CD49a (AbSeq) CD69 (AbSeq) Lag3

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 Working model
Low-Grade Chronic
Healthy Adipose Tissue Inflammation in Adipose Tissue
Myeloid Cells Myeloid Cells
CD11b F4/80 CD11b
CD11b F4/80 CD11b CD11c Trem2
CD163
CD11c Cell Migration
Clec10a CXCL1
Clec4e IL-1

Anti-Inflammatory Signals Inflammatory Signals

Adipogenesis Cell Activation

Galectin-1 CD1d
(Lgals1) PD-1
CD8 PD-1 CD8
CD1d

CD49a CD49a

Adipocytes Cytotoxic Cells B Cells Adipocytes Cytotoxic Cells B Cells

PD-1
Cell Exhaustion CD8 PD-1

Lag3
Tigit
Tigit

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 Summary
• The BD Single-Cell Multiplexing Kit allows assessment of immune cells from bone marrow,
spleen, thymus and epididymal adipose tissue from control and high fat diet mice
simultaneously.
• The BD Single-Cell Multiplexing Kit provides high-confidence information on relationships
between samples without batch effects.
• BD AbSeq assays allow effective cell clustering, cell type identification and obesity related
phenotyping of immune cells.
• B cells similar to B-2 regulatory cells in adipose tissue increased in mice given the high fat
diet.
• Inflammatory and adipogenic genes were increased in adipose tissue myeloid cells in mice
given the high fat diet.
• Cytotoxic cells were activated/exhausted in adipose tissue of mice given the high fat diet.

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property of their respective owners. © 2019 BD. All rights reserved.
 Thank you!

For Research Use Only. Not for use in diagnostic or therapeutic procedures.
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property of their respective owners. © 2019 BD. All rights reserved.