Standardization of T cell immunomonitoring - correct protocol choices and additional sources of variation

CMI standardization techniques in evaluation of vaccine

response

15 17 September 2008 15–17 September 2008 Annecy, France

Standardization of T cell immunomonitoring - correct protocol choices and additional sources of variation

If you accompany clinical drug development with T cell-based monitoring assays you probably want to base important decisions on their results. In such a case you might find yourself back in one of the following situationsy g y g

A:An assay has recently

been established

B:An experienced operator

has left your lab

C:Established assays are

running smoothly in hand been established. Experience in the

technology is still low.

has left your lab. Experience of new staff

member is still low.

running smoothly in hand of experienced staff

members but you are a control freak.

What do you do now ?y

o neld

ew

External Validation in interlaboratoy testing projects

Open Network with large number of Members

1) E i

Panel Leader(s)

1) Experience2) Patronage3) Financing

Statistician( )

1) Panel design1) Help

2) Instructions3) Questionnaires4) Report forms5) Communication

Group of Participants

Panel Leader(s)

Adminis-tration

C t l

p

1) Testing 1) Analyse data

1) Help

Central Lab

) g2) Reporting3) Experience

2) Write reports3) Disseminate data4) Conclusions Database

1) Cell Samples2) Pre-testing3) Distribution

1) Help

CIMT IMMUNOGUIDING PROGRAM

“ ”“CIP”(www.c-imt.org)

Monitoring Panels

Workshops +Scientific Sessions

Co-operation with other

groups

Dissemination of Data

Tools and Services

HLA-peptide multimer ELISPOT

Intracellular Cytokine Stainingg

S.A. / T.K. / C.G.

A.M. / C.O. / C.M.B.

M.W. / A.L. / S.H.vdB

Panel members(24 centers from 8 European countries

“We can do more than just external validation”

Provide immediate feed-back about performance relative to the

group - „External Validation“.

Use the collected data sets to systematically investigate the

f f b d d d t l d tiperformance of subgroups and deduce protocol recommendations.

- „Protocol Optimization“.

Use the collected data sets to systematically identify sources of

variation and quantify their impact on total test variationvariation and quantify their impact on total test variation -

„Protocol Optimization“.

Use our data and experience to define guidelines -

„Harmonization and Standardization“.„

The first two Monitoring Panels

3 central labs organized the first 2 panels

C. Gouttefangeas (Tübingen) S.H. van der Burg (Leiden) C.M. Britten (Mainz - Leiden)

Design of the panels for ELISPOT and HLA-peptide Tetramer staining

12-13 labs from 6 European countries participated in the first two phasesp p p p Pre-tested PBMC from healthy donors were prepared centrally 2 model antigens were used Synthetic peptides and HLA-A2 tetramers were prepared centrally Recommendations were deduced from the results of phase I Experiments were repeated (Labs had to follow recommendations) Experiments were repeated (Labs had to follow recommendations) The influence of introduced protocol changes was verified in phase II

Two-step approach(Britten et al. CII 2008)

High variation of protocols despite first harmonization

Minimum requirements deduced from Phases I/2005 and II/2006: Minimum requirements deduced from Phases I/2005 and II/2006:

were mandatory in phase IV/2007

Use Triplicates !

Do not use allo-APC …were mandatory in phase IV/2007 Do not use allo APC

Use a resting time

Use ≥ 400.000 PBMC per well

1022subg. 1 (Thawing

Plain mediumMedium + SerumM di + DNAn

g

133

Subg. 6 (pref. protocol) with serumserum free

ents

11s bg 2 (medi m

37 °C

211

g ( gmedium) Medium + DNAse

Medium + Serum + DNAsePBS

g &

Cou

nti

n

1222BD

millipore hydrophilicmillipore hydrophobic

Subgr. 7 (plates)

ol &

Rea

ge

124

Subg. 3 (counting)Trypan

M hi

41

subg. 2 (medium temperature) RT

4°C

Thaw

ing

1231

Mabtech BD

PharmingenSubgr. 8 (Abs)P

roto

co

4Machine

8814h-20h

Subg. 4 (resting time)

2h-6h

tin

g

652Biosys (3000/5000)

AID (ERL2/2/2/3/4)CTL (2/2/3/4/4/5)

Subgr. 9 (Reader)ader

s

124

falcon tubesplates

Subg. 5 (how rest)Res

t

21

y ( )g ( )

A EL VIS (V2)Zeiss (4.1/4.4)R

ea

High variation of results obtained by different labs

R lt f ELISPOT l IV/2007 l d b A M d C Ott i d C B ittResults from ELISPOT panel IV/2007 led by A. Mander, C. Ottensmeier and C. Britten:

2 from 78571P2454PPPBGID02DetectedD5 / FluD4/ FluD3/CMVD3/ FluD2/CMVD2/ FluD1/CMV

0 f 7P + BGP + BGP + BGP + BGBGP + BGBGID055 from 75825PP + BG641757143196412766ID045 from 724000545451320BGBG1061923529ID032 from 78571P2454PPPBGID02

wer

at

e

5 from 6n.d.46875157212821P862116484ID131 from 7BGP + BG871BGP + BGBGP + BGID080 from 7P + BGP + BGP + BGP + BGBGP + BGBGID05

wit

h lo

ctio

n r

a

4 from 710714BG1594BG1515BGP + BGID235 from 734615P + BG18249187P + BG737713500ID214 from 72062P + BG5442963P + BG2637BGID16

Lab

s w

det

ec

3 from 6n.d.60911121BGP + BG10256P + BGID24

6 from 72459BG8594396774239608889ID076 from 74138210538644724P + BG421116216ID01

ers

7 from 71290346154921983692308533326087ID156 from 73419P1350681852174379713333ID116 from 7188783337792603BG26045639ID096 from 72459BG8594396774239608889ID07

Hig

h

erfo

rme

7 from 7279115190652326126667251010435ID197 from 71290346154921983692308533326087ID15

pe

Quantitative comparability of results ?

Th i hi h f lit ti l (!) t d th !

D1 / CMV D2 / Flu D2 / CMV D3 / Flu D3 / CMV D4 / Flu D5 / Flu

The six high performers qualitatively (!) reported the same responses !

ID01 16216 4211 P + BG 4724 864 21053 4138 6 from 7

ID07 8889 3960 7742 4396 859 BG 2459 6 from 7

ID09 5639 2604 BG 2603 779 8333 1887 6 from 7igh

or

mer

s

ID11 13333 3797 52174 6818 1350 P 3419 6 from 7

ID15 26087 5333 92308 9836 921 46154 12903 7 from 7

ID19 10435 2510 26667 3261 652 15190 2791 7 from 7

Hp

erfo

460022683904527344723373613433mean

12903461541350983692308533326087max

188783336522603774225105639min.

90,0472,6926,2650,5081,7828,3653,50CV

4141,6116488,50237,472662,9136576,241059,667186,95sdv

Same quality Different quantity

Frequencies indicated as 1 per x PBMCs

Many ways lead to Rome !

ID prior panels ReaderAbsMedium used PlatesRestingThawing

CTL IBDBD ELISPOT X-Vivo falcon L3

Guava PCA 9637

CTL wash di +ID01

BioSys 3000

Phar-mingen

millipore MAHA S45

10% human AB serum PANIscove

falcon tubes

lShort2Trypan

blueRTIscove + 10% AB ID07

Immuno-spot S3

BDplateno serum15tubesLong3PCA-96 System

37medium + benzonase

ID01

ers

BioSys 5000Mabtechmillipore

MSHA S4510%human

AB serum PAAIMDMgreiner tubes 50ml

Long2Trypan blue4IMDMID09

3000mingenMAHA S45AB serum PAN50mlblueserum

erfo

rme

AID ELR02Mabtechmillipore

MAIP S45

10% human AB serum LONZA

RPMIfalcon tubes 15ml

Short2Trypan blueRTRPMIID11

Hig

h p

e

10% FBS

Zeiss ELISPOT

Reader V4.1

MabtechMilliporeMSIP S45

5% human poolserumRPMI

falcon tubes 15ml

Short1CASY

Model TT Inovatis

37X vivo 15ID15

H

AID ELR04Mabtechmillipore

MSIP S4W

10% FBS Gibco

Invitrogen (US origin)

RPMI 1640?? tubesShort0Guava

ViaCount37AIM-VID19

We should harmonize results but not necessarily the reagents that come to use !

Effects of changing Acceptance Criteria

BG vs. detected responses(threshold >2‐fold BG) 

BG vs. detected responses(threshold >3‐fold BG) 

80

90

100

ected

80

90

100

tected

50

60

70

spon

ses de

te

50

60

70

spon

ses de

t

30

40

50

entage of res

20

30

40

50

entage of re

0

10

20

Perce

0

10

20

Perce

1000 10000 100000 1000000

Background in 1 per x PBMC

1000 10000 100000 1000000

Background in 1 per x PBMC

serum no serumAverage detection rate drops from 76% to 62%

T-cell immunomonitoring - 5 Challenges

High variation between protocols

P fi i P l No comparability of results from different labs

Proficiency Panels can help to solve these problems.

Lack of Validation

led toData

Lack of acceptance by regulatory authorities and investigators

CIMT / CIP can help to

communicate

Difficulty to show correlation with clinical events

Why standardize biomarkers that do not show a correlation with clinical events ?

Lack of correlation between results from T cell assaysand clinical events might be due to the fact that mostgtests have not been performed under highlystandardized conditions?standardized conditions?This is probably (only) a part of the explanation.

No correlation has been found for assays that monitori l f ti f th ll i i l tione single function of the cell in one single tissue

compartment.IFN-producing or HLA-peptide multimer binding cells fromthe peripheral blood in the great majority of cases.

Correlation with clinical events ?

“Clever” use of “mono-parametric” tests:

T cells that are found in the DTH (migration)de Vries IJ et al (2005) J Clin Oncol Immunomonitoring tumor-specific T cells in delayed-type de Vries IJ et al. (2005) J Clin Oncol. „Immunomonitoring tumor specific T cells in delayed type hypersensitivity skin biopsies after dendritic cell vaccination correlates with clinical outcome.“

T cells that are found in the BAL (migration)Janossy G et al. (2008) Cytometry B Clin Cytom. “The role of flow cytometry in the interferon-gamma-based diagnosis of active tuberculosis and its coinfection with HIV-1-A technically oriented review.”

Combining two or more “mono-parametric” assaysVan der Burg S.H. (unpublished)

Expand basic tests to measure multiple functions simultaneously:p p y

T cells that are able to produce several cytokines (“multi-functional”)Darrah PA et al. (2007) Nat Med. “Multifunctional TH1 cells define a correlate of vaccine-mediatedprotection against Leishmania major.”

Basic tests such as ELISPOT, staining with HLA-peptidemultimers and ICS will still have to be standardized as they will probably

protection against Leishmania major.

multimers and ICS will still have to be standardized as they will probablybe part of surrogate tests applied in the future.

What could be achieved so far ?

A larger number of labs now realize the importance of validation and standardization efforts.

Many participants want to improve the quality of Many participants want to improve the quality of their immunomontoring assays. (“passive effects”)

More and more labs want to join these panels More and more labs want to join these panels. (“waiting lists”)

Harmonization indeed results in improvement Harmonization indeed results in improvement.(“Britten et al. CII 2008 - free access”)

Fi t H i ti id li bli h dFirst Harmonization guidelines were published.(www.c-imt.org – Immunoguiding Program)

... but harmonization is a laborious process !

Harmonization will have to include several aspects such as…

Harmonized Q.C.

of cell material

Harmonized Assay

Protocol

Harmonized Data

Acquisition

Harmonized Acceptance

Criteria

… and the regular and specific education of all staff membersinvolved in handling, thawing, measuring, evaluating andg, g, g, greporting data from immunomonitoring as well as an externalvalidation of their performance on a regular base.

The Vision – T cell monitoring as surrogate marker to substitute for clinical endpoint testing

timeTreatment

Immune response Tumor size Patient survival

early late

Immune response Tumor size Patient survival

Clinical endpointsImaging techniquesT cell immunomonitoring

CIMT Monitoring Panel in 2008

• 24 participating labs24 participating labs• 8 European countries• Leiden, Tübingen (main organizers)• Southampton, Copenhagen, Berlin ( co-organizers)

Next meeting of the group will be at CIMT 2008Phase III/2007 (Tetramer)

- Resting phase of cells- Standardization of the analysis

CIMT 2008 in Mainz, Germany

Phase IV/2007 (ELISPOT)- Serum vs. serum-free media

Diff t R d

in Mainz, Germany(June 3rd – 5th 2009)

- Different Readers

Phase V/2008 (ICS)Comparison of individual protocolsSave the date !- Comparison of individual protocols

- Two-step approachSave the date !

Acknowledgements

CIMT Panel Organizers and Co-organizers:

C Gouttefangeas S H Van der Burg M Welters S Attig C C. Gouttefangeas, S.H. Van der Burg, M. Welters, S. Attig, C.

Ottensmeier, A. Mander, T Kollgaard, A. Letsch

CIMT Executive Board:

C. Huber, G. Schuler, H.G. Rammensee, U. Kalinke, P. Walden, C.J.M.

Melief, P.W. Johnson

CIMT supporters:

(Finances C.M.B. with ald d h l )Dr. Mildred-Scheel grant)