CyTOF analyses in rheumatoid arthritis

Deepak Rao, MD PhD

Rheumatology, Immunology, Allergy, BWH

Agenda: CyTOF analyses

• Analysis of synovial tissue • Panel for analysis of synovial cells • Comparison of CyTOF and flow data • Traditional gating and viSNE visualization

• Analysis of synovial T cell function • Cells stimulated in vitro then analyzed for activation markers and cytokines

• Broad phenotyping of circulating T cells (PBMC) • RA, GCA, PMR

Synovial Biopsy

Arthroplasty

Single cell suspension

Single cell suspension

gentleMACS

Trituration

Synovial workflow – Tissue disaggregation

Kevin Wei, AMP

Synovial workflow

Assess cell yield and cell viability

Step 1. Determine analytical methods

1. < 100,000 cells

2. <500,000 cells

3. >500,000 cells

RNAseq only

RNAseq + Cytof (50%) (50%)

RNAseq + Cytof (limit CyTOF to 200,000 cells)

Single cell suspension

Kevin Wei, AMP

Step 2. Collect 1000 cells for low input RNAseq

Single cell suspension

Once 1000 cells collected, Exchange collection tube (step 3) and continue collection for each subset

Step 3. collect remaining cells into FACS buffer

Single cell suspension

Lysis buffer

Step 4. Sort single cells into 384 well plate

T B M F

T B M F

FACS buffer

T B M F

Collect cells until sample is depleted Collect up to 144 cells for

each subtype

Synovial workflow – Cell sorting

Kevin Wei, AMP

Synovial CyTOF: RA synovial biopsy CyTOF Panel CD45 VE-Cadherin

CD66b VCAM-1

CD14 Podoplanin

CD64 Cadherin-11

CD16 FAP

CD11c CD146

CCR2 CD90

CD3 CD34

CD4 CD19

CD8 CD20

CD69 CD38

CD45RO CD138

CXCR5 IgD

PD-1 IgM

ICOS IgA

HLA-DR FcRL4

TCRgd RANKL

FoxP3 Live/dead

t-SN

E1

t-SNE2 viSNE analysis

AMP

RA Synovial Tissue: Flow cytometry analysis

RA Synovial Tissue: CyTOF analysis

Parallel analysis of the same synovial sample by flow and mass cytometry

Synovial CyTOF: RA Synovial Biopsy

CD4 CD3 CD45

CD

8

CD

14

Pod

op

lan

in Monocytes/Macs

T cells

CD8+ T cells

CD4+ T cells Leukocytes

Fibroblasts

Biaxial gating of dot plots AMP

CD34

CD4 CD3 CD45

CD

8

CD

14

Pod

op

lan

in

CD

90

Monocytes/Macs

T cells

CD8+ T cells

CD4+ T cells Leukocytes

Fibroblasts

Synovial CyTOF: RA Synovial Biopsy

CD11c CD34

CD4 CD3 CD45

CD

8

CD

14

Pod

op

lan

in

CC

R2

CD

90

Monocytes/Macs

T cells

CD8+ T cells

CD4+ T cells Leukocytes

Fibroblasts

Synovial CyTOF: RA Synovial Biopsy

CD38 CD11c CD34

CD4 CD3 CD45

CD

8

CD

14

Pod

op

lan

in

CD

19

CC

R2

CD

90

Monocytes/Macs

T cells

CD8+ T cells

CD4+ T cells Leukocytes

Fibroblasts

Synovial CyTOF: RA Synovial Biopsy

ICOS CD38 CD11c CD34

CD4 CD3 CD45

CD

8

CD

14

Pod

op

lan

in

CD

19

CC

R2

CD

90

PD

-1

Monocytes/Macs

T cells

CD8+ T cells

CD4+ T cells Leukocytes

Fibroblasts

Synovial CyTOF: RA Synovial Biopsy

Synovial CyTOF: RA synovial biopsy CyTOF Panel CD45 VE-Cadherin

CD66b VCAM-1

CD14 Podoplanin

CD64 Cadherin-11

CD16 FAP

CD11c CD146

CCR2 CD90

CD3 CD34

CD4 CD19

CD8 CD20

CD69 CD38

CD45RO CD138

CXCR5 IgD

PD-1 IgM

ICOS IgA

HLA-DR FcRL4

TCRgd RANKL

FoxP3 Live/dead

t-SN

E1

t-SNE2 viSNE analysis

AMP

CyTOF: RA synovium

CD4+ T cells in a Seropositive RA synovial tissue

CyTOF: RA synovium

CD4+ T cells in a Seropositive RA synovial tissue

Pathologically expanded peripheral B cell-helper T cells in RA

Cytokine production after in vitro stimulation RNAseq transcriptomics

In vitro functional assays: B cell help Immunofluorescence microscopy

PD-1 CD20 CD3

Analysis of synovial T cells: 3-D culture

Lauren Henderson

In vitro stimulation

CyTOF

Analysis of synovial T cells: 3-D culture

Lauren Henderson

In vitro stimulation

Immunophenotyping of PBMCs by CyTOF

• Identify immune cell populations with altered frequencies in specific autoimmune diseases • Analytical methods in the Raychaudhuri lab – Chamith Fonseka

• Determine expression of many different proteins on specific immune cell populations of interest

• Data generated so far: • RA vs controls (n=7 each)

• RA vs controls w/ and w/o stimulation (Raychaudhuri lab, n=~30 each)

• RA, OA, SLE, controls (AMP, Lederer lab, in progress)

• GCA, PMR, seronegative RA, controls (n=4 each)

CyTOF: Memory CD4+ T cells from blood

CyTOF: Memory CD4+ T cells from blood

CyTOF for analysis of rheumatic disease samples

• Powerful, accessible discovery tool to measure 35+ markers on cell samples • Staining protocols have undergone substantial maturation and now regularly utilized

(Raychaudhuri, Lederer, Brenner labs)

• Panels developed for broad analysis of synovial cells

• Panels developed to phenotype T cells, B cells, myeloid cells in detail

• Simple data analysis strategies that we can teach you (e.g. viSNE)

• New, powerful analysis methods under development in the Raychaudhuri lab