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Overcoming challenges in cellular analysisMultiparameter analysis of rare cells

January 28, 2015

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January 28, 2015

Andrea Cossarizza, M.D., Ph.D.University in Modena and Reggio

Emilia School of MedicineModena, Italy

David Cousins, Ph.D.University of LeicesterLeicester, UK

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ANDREA COSSARIZZA

Multiparameteranalysis of rare cells

• Rare cell analysis: background and keypoints

OUTLINE OF THE TALK


• Main problems in the detection of such cells

OUTLINE OF THE TALK



• Possible solutions: from hardware to software

OUTLINE OF THE TALK



• Possible solutions: from hardware to software

• Rare cells in the immune system: the case of iNKT

OUTLINE OF THE TALK

• >30 years ago: enumeration of fetal red blood cells in the maternal circulation at a frequency of 1/10,000 to 1/100,000 by Cupp.

BACKGROUND


• Now: detection and quantitation of several rare cell populations in blood or bone marrow.

BACKGROUND


• Now: detection and quantitation of several rare cell populations in blood or bone marrow.

• Essential tool in the diagnosis and monitoring of hematological cancers and immunological disorders, as well as in the identification of Ag‐specific cells.

BACKGROUND

• Rare‐event analysis is the art of finding a needle in a haystack

WARNING

• Rare‐event analysis is the art of finding a needle in a haystack

• The frequency of the event of interest, and the signal‐to‐noise ratio of the method used to detect the event are the two most important factors.

WARNING

• ‘‘Rare‐event analysis’’: detection of events that occur at a frequency of 1 in 1,000 (0.1%) or less, although the record claimed in the literature has long stood at 1 cell in 10,000,000 (0.00001%) for tumor cells spiked into peripheral blood.

KEY POINTS

• ‘‘rare‐event analysis,’’: detection of events that occur at a frequency of 1 in 1,000 (0.1%) or less, although the record claimed in the literature has long stood at 1 cell in 10,000,000 (0.00001%) for tumor cells spiked into peripheral blood.

• Detecting an event at low frequency requires a high signal‐to‐noise ratio and the acquisition of a large number of events.

KEY POINTS

• Ag‐specific T cells• NKT and iNKT cells• Circulating endothelial cells and precursors• Stem cells (CD34+)• Particular lymphocytes subpopulations• Circulating tumor cells• Polyfunctional assays• ..........

IMMUNOLOGIST'S INTERESTS

Open pre‐analytical questions

• How much blood from patients?

• How much blood from patients• Lack of available standardized method


• How much blood from patients• Lack of available standardized method• Enriched or non enriched populations


• How much blood from patients• Lack of available standardized method• Enriched or non enriched populations• How many markers/colors


• How much blood from patients• Lack of available standardized method• Enriched or non enriched populations• How many markers/colours• How many cells


• How much blood from patients• Lack of available standardized method• Enriched or non enriched populations• How many markers/colours• How many cells • Exclusion of doublets, dead cells and debris:

use of a DUMP CHANNEL


Number of events to acquire

CV (%) 1 2.5 5 10 20Positive cells required

10,000 1,600 400 100 25

Frequency

EVENT NUMBER TO ACQUIRE %

l/n 10 10 100,000 16,000 4,000 1,000 250 1 100 1,000,000 160,000 40,000 10,000 2,500 0.1 1,000 10,000,000 1,600,000 400,000 100,000 25,0000.01 10,000 100,000,000 16,000,000 4,000,000 1,000,000 250,000 0.001 100,000 1,000,000,000 160,000,000 40,000,000 10,000,000 2,500,000

• Which instrument, and which performances

Open analytical questions

• Which instrument, and which performances• Flow cytometer rates of acquisition


• Which instrument, and which performances• Flow cytometer rates of acquisition• Maximize the signal‐to‐noise ratio of the

cells of interest from the background



cells of interest from the background• Data acquisition: instrument clean and the

background level of noise below the threshold




background level of noise below the threshold

• Spill over and carry over




background level of noise is below the threshold

• Spill over and carry over• Adequate software


Our previous experiencePolyfunctional analysis of Ag‐specific cells

2012

THE INTERBETWEENERS: INNATE‐LIKE LYMPHOCYTES

Types of lymphocyte that blur the traditional boundaries betweeninnate and adaptive immunity


Invariant Natural Killer T cells(iNKT)

Poised to robustly produce cytokines more rapidly than conventional naïve

T cells




Mucosal associated invariant T cells(MAIT)


T cells

Preferentiallylocalized in the mucosal tissues




Mucosal associated invariant T cells(MAIT)

T cells


T cells

Pre‐programmed to acquire their

effector functionsbefore egress from

thymus

Preferentiallylocalized in the mucosal tissues


iNKT cells MAIT cellsFrequency

(% among human PBMCs)

0.01‐1% 1‐10%

Receptors semi‐invariantVα24‐Jα18 TCR,NK receptors

semi‐invariantVα7.2‐Jα33 TCR, high

levels of CD161,IL‐18Rα.

Antigen recognized glycolipid antigenspresented by CD1d

microbial antigenspresented by MR1

Subsets CD4+, CD8+, and CD4‐CD8‐

CD4+, CD8+, and CD4‐CD8‐

Function Regulatory Effector‐memoryphenotype

DIFFERENT CHARACTERISTICS OF INKT AND MAIT CELLS

Gating strategy for iNKT cellsand their main subsets

SSC

CD3 DUMP CHANNEL(CD14,CD19)

Vα24Jα18Vβ11 TCR

SSC

SSC

CD8

CD4

CD161

CD161

SSC

SSC

Gating strategy for iNKT cellsand their main subsets

SSC

CD3 DUMP CHANNEL(CD14,CD19)

Vα24Jα18Vβ11 TCR

SSC

SSC

NKT cells and Multiple Sclerosis (MS)

Berzins SP., Nat Rev Immunol. 2011

Studies on a subset of NKT cells

Polyfunctionality of iNKT cells in patients affected by different forms of Multiple Sclerosis (Relapsing‐Remitting RR, Primary Progressive PR, Secondary Progressive SP), in the framework of a project sponsored by the Italian Foundation for Multiple Sclerosis ‐ FISM

• 3 RR patients (treated with Natalizumab)

• 2 PR patiens• 5 SP patients• 5 CTR (healthy subjects)

• PBMCs isolation from >30 mL of blood• Stimulation with PMA (100 ng/ml) plus ionomycin (1 g/ml) for 4 hrs• ICS with following markers:

Live Dead (Aqua)CD3 PE‐CY5 CD4 AF700CD8 APC‐CY7iTCR (V24‐J18) PEIFN‐gamma FITCIL‐4 APCIL‐17 BV421TNF‐alpha BV605

Methods

• PBMCs isolation from >30 mL of blood• Stimulation with PMA (100 ng/ml) plus ionomycin (1 g/ml) for 4 hrs• ICS with following markers:

Live Dead (Aqua)CD3 PE‐CY5 CD4 AF700CD8 APC‐CY7iTCR (V24‐J18) PEIFN‐gamma FITCIL‐4 APCIL‐17 BV421TNF‐alpha BV605

• 10‐20 millions events acquired on Attune NxT (Life Technologies/Thermo Fisher), at a speed up to 35,000 cells/second

• mAbs titrated on 20 millions PBMCs• Compensation matrix set using single stained samples and FMO (Fluorescence

Minus One) approach

Methods

Gating strategy

FSC‐H

FSC‐A

Live Dead

CD3

CD4

CD8

CD3

iTCR

CD4

CD8

Live Dead

CD3

iTCR

TNF‐

TNF‐

IL‐17A

23,053 iNKT cells

6,720,084 T cells

Junk removal

72.40.08

39.6

56.32.4

29.0 1.5

52.217.3

1.7

IL‐17A

IFN‐ IFN‐

TNF‐

IL‐4

TNF‐

TNF‐

IL‐17A

GATED ON CD8+ T CELLS

GATED ON CD4+ T CELLS

GATED ON CD4‐CD8‐ T CELLS

Analysis of CD3+ T cells

0

GATED ON CD8+ iNKT CELLS

GATED ON CD4+ iNKT CELLS

GATED ON CD4‐CD8‐ iNKT CELLS

IFN‐

IL‐17A

IFN‐

TNF‐

IL‐4

TNF‐

TNF‐

IL‐17A

Analysis of iNKT cells

Software: Attune NxTFlowJo using Boolean Gate function Pestle to transfer filesSPICE to plot data

First analysis of the polyfunctionality of: • CD4+ T cells• CD8+ T cells• CD4‐CD8‐ T cells

Then, evaluation of the polyfunctionality of the rare cells of interest, i.e.:

• CD4+ iNKT cells• CD8+ iNKT cells• CD4‐CD8‐ iNKT cells

Considering 4 intracellular cytokines, there are 24 (=16) different cell populations, well represented by using SPICE.

Data analysis

CTRRR(Nat)

PP

CD4+ T CELLSresponse

SP

CD8+ T CELLSresponse

CTRRR(Nat)

PP SP

CD4‐CD8‐ T CELLSresponse

CTRRR(Nat)

PP SP

CD4+ iNKT CELLSresponse

RR(Nat)

PP SP CTR

CD8+ iNKT CELLSresponse

RR(Nat)

PP SP CTR

CD4‐CD8‐ iNKT CELLSresponse

RR(Nat)

PP SP CTR

• CECs and EPCs are extremely rare events (0.1 – 0.0001% in buffy coat)

• Absence of standardized protocol

• Lack of unique markers

• The needle and the damage done (by the venipuncture)...

Circulating Endothelial Cells (CEC)Circulating Endothelial Cell Precursors (EPC)

FSC‐A

FSC‐H

CD45‐BV 510 LIVE DEAD AQUA

SSC

Syto16

SSC

CD34‐BV 605

SSC

TimeSSC

CD31‐APC‐CY7

CD13

3‐AP

C

CD276‐PE

SSC

CD309‐PECY7

GATED ON CD133‐,CD31+

GATED ON CD133+,CD31+

Gated on Syto16

Gating strategy for their identification

EPC

CEC

CONCLUSIONS

• Studying rare cells requires careful attention, optimal methodologies in all phases, including collection of biological samples, adequate software and hardware.

CONCLUSIONS


• I have shown you some examples (besides Ag‐specific cells) that could be of interest for immunologists.

CONCLUSIONS


• I have shown you some examples (besides Ag‐specific cells) that could be of interest for immunologists.

• "Next generation" instruments that work at a very high speed and sensitivity are now allowing an easy detection and analysis of such cells.

ACKNOWLEDGEMENTS

SARA DE BIASI, PhD

More acknowledgments

Neurology Clinic, NOCSAE, ModenaPatrizia Sola, MD PhD

Diana Ferraro, MD PhD

Francesca Vitetta, MD

Anna Maria Simone, MD

Chair of Pathology and Immunology, ModenaProf. Marcello Pinti, PhDMilena Nasi, PhDLara Gibellini, PhDDr. Regina BartolomeoDr. Elena Bianchini

Elisa Nemes, PhD (now at Univ. of Cape Town, South Africa)Enrico Lugli, PhD (now at Humanitas Institute, Milan, Italy)

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January 28, 2015




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Studying human innate lymphoid cells using multi‐parameter flow cytometry

Prof David CousinsDepartment of Infection, Immunity

and InflammationNIHR Leicester Respiratory BRU

University of Leicester

Allergic Mechanisms ‐ Th2 diseases

Akdis CA. Therapies for Allergic Inflammation. Nature Medicine 2012.

Innate Lymphoid Cells (ILCs)

• Lymphocyte‐like innate cells, lacking known lineage specific markers.

• Derived from an ID2+ common ILC precursor.

• Currently subdivided into 3 classes based on effector function (i.e. cytokine output).

• ILC1: IFN

• ILC2: IL‐5/IL‐13.

• ILC3: IL‐17/IL‐22.

• Early cytokine producers during immune response. Spits, H. et al. Nat Rev Immunol 13, 145–149

(2013)

Type 2 responses – 2013

Walker, Barlow & McKenzie. Innate lymphoid cells — how did we miss them?Nature Reviews Immunology 2013; 13, 75‐87.

Type 2 Innate Lymphoid Cells (ILC2s) ‐mice

• ILC2s (nuocytes) first identified in the small intestine using an IL‐13 eGFPreporter mouse (Neill et al. Nature 2010).

Type 2 Innate Lymphoid Cells (ILC2s) ‐mice

• ILC2 numbers have been found to increase in multiple mouse models of airways disease (Stockinger lab/Umetsu lab/Hendriks lab).

• ILC2s are responsible for early source of Type 2 cytokines (Halim et al. 2014).

(■)

(■)

(□)

Wild type + Papain

Rag1‐/‐ + Papain

Wild type untreated

Influenza infectionChang et al. Nat Immunol, 2011 vol. 12 (7) 631

Human ILC2s – Challenges

• Lack of Lineage specific markers

• Cannot use fluorescent reporters

• Cells are rare!

(Neill et al. 2010)

Type 2 Innate Lymphoid Cells (ILC2s) ‐ humans

• lung parenchyma and BAL (Monticelli et al. 2011).

a

c

TCR

,C

D11

c

CD11b

CD

56

CD19

CD

56

CD127

CD

3

CD3

Lung ILC

BAL

TCR

,C

D11

c

CD3 CD11b

CD

56

CD19

CD

56

CD127

CD

3

ILC

105

103

102

0

104

105

103

102

0

104

105

103

102

0

104

105

103

102

0

104

0102 103 104 105 0102 103 104 105 0 102 103 104 105 0 102 103 104 105

105

103

102

0

104

105

103

102

0

104

105

103

102

0

104

105

103

102

0

104

0102 103 104 105 0102 103 104 105 0 102 103 104 105 0 102 103 104 105

b HC

71105

104

103

102

102103104

CRTH2HC

*

CRS

4

1050

0

CD

117

CR

TH2+ c

ells

(% o

f CD

45+

popu

latio

n)2

225

CRS

33 11

3224

3210

• Fetal and adult lung and nasal polyps, CRTh2positive (Mjosberg et al. 2011).

Human ILC2s – Aims

Multi parameter flow cytometry:

• Determine whether an ILC2 population could be identified in human peripheral blood.

• Optimise a protocol for ILC2 isolation and culture.

• Phenotype human ILC2s based on surface marker expression and secreted inflammatory mediators.

• Examine a role in experimental rhinovirus infection.

Instrumentation

• Life Technologies Attune acoustic focussing flow cytometero High flow rate – 1ml/minute with low variationo Good for rare eventso New machine NxT capable of 14 colours

• BD FACS Aria II flow sortero 5 laser instrument – very flexibleo Can sort cells – enrichment needed prior to sorting

ILC2s were Identified in Human Peripheral Blood

PBMC magnetic depletion strategy developed using mouse anti‐human CD3/14/16/19 and pan mouse IgG Dynabeads for ILC2 enrichment.

CRTh2

Hematopoietic Lineage cocktail – FITC (ebio):CD2/3/14/16/19/56/235a + CD123.

ILC2s Have a Distinct Phenotype

• ILC2s: Lin-, : CD34-, CRTH2+ CD127+,CD45+CXCR3- CCR4+IL17BR+ with populations of CD117 and CCR6 positive cells.

ILC2s express IL‐13 ex vivo and after in vitro proliferation

ILC2s after 7 days of culturewith rIL2/7/25/33

PBMCs stimulated withPMA/Ionomycin 4hrs

ILC2s proliferate in response to IL25/IL33 (Day 7)

ILC2 activation ‐ time‐course

Rhinovirus infected BEC cultures Nasal mucosal fluid

IL‐25 is required for RV‐induced asthma exacerbation

• RV infection induces ILC2s in mouse asthma model• RV induced inflammation inhibited by anti IL17RB


David Jackson et al.Am J Respir Crit Care Med 190, 1373‐1382DOI: 10. 1164/rccm.201406

IL-33–Dependent Type 2 Inflammation during Rhinovirus-inducedAsthma Exacerbations In Vivo


• RV infection of asthmatics increases type‐2 cytokine release• IL‐33 correlates with cytokine release and symptoms

David Jackson et al.Am J Respir Crit Care Med 190, 1373‐1382DOI: 10. 1164/rccm.201406

IL-33–Dependent Type 2 Inflammation during Rhinovirus-inducedAsthma Exacerbations In Vivo


A, RV infection of Bronchial epithelial cells

B, Cytokine release from ILC2s stimulated with supernatant from BEC cultures

Conclusions

• Multi‐parameter flow cytometry is a useful tool in identifying ILCs

• Human ILC2s can be identified in peripheral blood (Lin‐CD45+CRTh2+CD127+CD25+)

• ILC2s can be cultured in vitro in an isolated system• ILC2s expand in response to both IL‐25 and IL‐33

• Experimental rhinovirus challenge induces both IL‐25 and IL‐33.• Both may contribute to ILC2 activation in asthma exacerbations.

Therapeutic opportunities?

Andreakos et al. IL‐25: The missing link between allergy, viral infection, and asthma? Sci. Transl. Med. 6, 256fs38 (2014).

IL‐33

AcknowledgementsBatika Rana (PhD Student)Celine Parmentier (PhD Student)Joanne McDonald (PhD Student)Cate Weston (post‐doc)Paul LavenderTak LeeHolly BowenGreg WoszczekRebecca BeavilMatthew Arno (KCL Genomics Centre)BRC Flow Cytometry CoreBRC Genomics Core Melissa Lennartz‐WalkerHolly FosterElisabeth Fuerst

Funded by:

Collaborators:Andrew McKenzie (MRC LMB)Sebastian Johnston (IC)Mike Edwards (IC)James Pease (IC)Ross Walton (IC)Nathan Bartlett (IC)Roberto Solari (IC/GSK)

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Instructions for Viewers - Science | AAAS › sites › default › files › All slides... · 2017-01-25 · Instructions for Viewers • To share webinar via social media: • To