10 May Prof. J.J.M. van Dongen, MD, PhD, Berend Houwen Award Lecture, Flow cytometric Immune Monitoring, ISLH-2018 Conference, Brussels, Belgium

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1 International Society for Laboratory Hematology 2018 Conference, Brussels, BE, 9-12 May 2018 Berend Houwen Lecture Flow cytometric immune monitoring in patient care Jacques J.M. van Dongen Founder of International Society of Laboratory Hematology Dept. Immunohematology & Blood Transfusion, Leiden University Medical Center, Leiden, NL J.J.M. van Dongen on behalf of and 1. Collaboration between academia, industry and diagnostic laboratories 2. Innovation & Standardization 3. Education & Training EuroFlow is an independent scientific consortium, which aims at innovation in flow cytometry for improvement of diagnostic patient care - Initiated in 2004 (FP6 STREP LSH-2004) - Formal project duration: April 2006-Oct 2009 (LSHB-CT ) - Sustained based on collective IP and patents and collective revenues European networks for laboratory diagnostics Since 1996 BMH4-CT CA FP4 BIOMED-2 program Since 2001 BMH-CMT CA FP2 BIOMED-1 program + I-BFM-SG and Pre-BMT-SG Since 2006 LSHB-CT FP6 STREP LSH-2004 March 2018 Chairmen: J.J.M. van Dongen & A. Orfao 22 laboratories in 11 countries Participants: Aims: Based on experience and participation in (inter)national clinical trails - Research & Innovation of diagnostic patient care - Standardization of laboratory diagnostics - Quality Assessment, including QA evaluation meetings - Continuous education: Seminars, Workshops, and Trainings EuroFlow-based Immune Monitoring 1. EuroFlow strategies and tools for immune monitoring Standardization of (pre-) analytical procedures (also sample processing, instrument setting, etc.) and data analysis Selection of appropriate antibodies and fluorochrome combinations 8-color immunostainings: multiple design-testing-redesign rounds New software for multidimensional data analysis using differentiationmaturation-activation pathway concepts Collective development of reference databases for diagnosis & classification and for automated (objective) gating & data analysis 2. Immune monitoring added to diagnosis/classification & MRD EuroFlow-based immune monitoring of immunotherapies: antibodies, CAR-T-cells, check point inhibitors. High throughput flow cytometric cellular immune monitoring of: innate cells (monocytes- DCs), CD4 T-cells, CD8 T-cells & NK-cells, B-cell subsets (including B-cell subsets and plasma cell subsets) Immunodeficiencies and vaccination as first models: clear baseline Standardization of pre-analytical and analytical procedures Standardization and EQA: Basis for ISO accreditation 1

2 PERtussIS COrrelates of Protection Europe Identification Monocyte and dendritic cells in PB Classical EuroFlow Subsetting CD1c+ CD1c+ cmo CD141+ cmo pdc Innate cells CD4 and CD8 T-cells and B-cells CD62LcMo imo ncmo 0 day1 day3 day5 day7 day10 day14 day21 3 mo 6 mo 12 mo The PERISCOPE project has received funding from the Innovative Medicines Initiative 2 Joint Undertakingunder grant agreementno This Joint Undertakingreceives support fromthe EuropeanUnion shorizon2020researchandinnovationprogrammeandefpiaandbmgf. 11 color- combination 17 (sub)populations Responsible scientists: Cristina Teodosio, Magda Berkowska and Daniela Damasceno Validation of monocyte-dc tube INTRA ASSAY VARIABILITY (n=7; 2 replicates/sample; 2 technicians) REPRODUCIBILITY (Multicentricstudy) (n=16; 3 centers; 4 different cytometers) T-cells Responsible scientists: Cristina Teodosio, Magda Berkowska and Daniela Damasceno 0 day 1 day 3 day 5 day 7 day 10 day 14 day 21 3 mo 6 mo 12 mo CD4+ T-cell maturation pathway in healthy adults Dissection of CD4+ T-cell compartment Tested in 6 peripheral blood samples from healthy adults in USAL (Spain), 4 in LUMC (Netherlands) and 3 in RIVM (Netherlands) total: 13 samples (merged) Central Terminal effector /17 Transitional Effector /17 /17 14-color combination: ~85 CD4+ T-cell subsets in blood 2

3 A total of ~85 different CD4+ T-cell subsets identified by the Periscope-EUROFLOW CD4+ T-cell tube Major CD4 T-cell subsets Maturation stage Functional T-cell subsets Classical CD4+ T cells (~ 40 cell subsets) Regulatory T cells (CD127lo/CD25hi) (~ 25 cell subsets) Follicular helper T cells (CXCR5+) (~ 20 cell subsets) Central Transitional Effector Central Transitional Effector Central Transitional Effector TH1 TH2 TH17 TH22 TH1/TH17 OtherTH CD4+ T cellsubsets* Treg-TH1-like Treg-TH2-like Treg-TH17-like Treg-TH22-like Treg-TH1/TH17-like Other Treg-TH CD4+ T cell subsets* T follicular regulatory cells (Tfr) TFH1 (or Tfh--like) TFH2 (or Tfh--like) TFH17 (or Tfh--like) Other TregH CD4+ T cell subsets* * Between 4 and 5 TH subsets other than the classical ones, identified by unique chemokine receptor profiles Color codes: general identification of T and NK cells,, functional markers,, maturation markers, activation marker SSC vs. CD45 CD8+ T cells weregated Additionally identification and subsetting of NK cells Dissection of CD8+ T-cell compartment CD27 vs. CD28 Central 13-color combination: ~45 CD8+ and NK cell subsets in blood Transitional Effector Earlyeffector Terminal effector Dissection of the blood B-cell compartment Plasma cells IgM IgA1 IgA2 IgG1 IgM IgG2 IgA1 IgG3 IgA2 IgG4 0 day1 day3 day5 day7 day10 day14 day21 3 mo 6 mo 12 mo Detection of ~25 B-cell subsets IgG1 IgG2 IgG3 IgG4 CD27- and CD27+ Identification of blood B-cell subsets in APS view IgM(D)+ Memory Non-Swt Responsible scientists: Mirjam van der Burg & Martin Perez 3

4 IGH class switch analysis in B-cells and plasma cells Memory Non-Switched 14 colour- combination ~115 populations B-cell subsets clasification (Number of subsets: N=115) Maturation compartments Phenotypic subsets Isotypes /Transitional IgM ++ IgD + (64 subsets) (48 subsets) CD20+CD21+CD24 + CD5+ IgM + IgD ++ CD27+CD62L- IgM ++ IgD + CD27- CD24++ CD27+ IgG1 + IgG2 + IgG3 + IgG4 + IgA1 + IgA2 + CD27- IgD + CD20+CD138- IgM + CD20-CD138- IgG1 + IgG2 + IgG3 + CD20-CD138-CD62L- IgG4 + CD20-CD138+ CD5- CD27+CD62L- CD27-CD62L- CD20++CD21- CD24- CD62L- CD20+CD138-CD62L- CD20-CD138+CD62L- IgA1 + IgA2 + IgD + Age-related patterns for plasma cells, B-cells, and Ig levels Infant(9m) Young adult(19y) Elderly(90y) IgM(D)+ Memory B- cells Cells/μl Cells/μl, JACI 2018 Soluble Ig levels y: years m: months Ig: immunoglobulin IGHC: immunoglobulin heavy chain constant region mg/dl Age E. Blanco et al., JACI 2018 IGH class switch analysis in B-cells and plasma cells Innate cells CD4 and CD8 T-cells and B-cells EuroFlow-based next generation flowcytometry in four 12-color tubes: - 11-color monocyte-macrophage and dendritic cells (DC): completed: ~15 subsets - 14-color CD4+ T-cell populations: completed: ~85 subsets - 13-color 0 day CD8+ 1 dayt-cell 3 day subsets 5 dayand 7 NK-cells: day 10 completed: day 14 day ~45 21subsets 3 mo 6 mo 12 mo - 14-color,, plasma cells, : ready: ~115 subsets Memory Non-Switched 14 colour- combination ~115 populations Extra analysis: - Labeled antigen - Labeled vaccine - Auto-antigen, e.g. CEP1 4

5 EuroFlow-based Immune Monitoring 1. EuroFlow strategies and tools for immune monitoring Standardization of (pre-) analytical procedures (also sample processing, instrument setting, etc.) and data analysis Selection of appropriate antibodies and fluorochrome combinations 8-color immunostainings: multiple design-testing-redesign rounds New software for multidimensional data analysis using differntiation-maturationactivation pathway concepts Collective development of reference databases for diagnosis & classification and for automated (objective) gating & data analysis 2. Immune monitoring added to diagnosis/classification & MRD EuroFlow-based monitoring of immunotherapies: antibodies, CAR-T-cells, check point inhibitors. High throughput flow cytometric cellular immune monitoring of: innate cells (monocytes- DCs), CD4 T-cells, CD8 T-cells & NK-cells, B-cell subsets (including B-cell subsets and plasma cell subsets) Immunodeficiencies and vaccination as first models: clear baseline European inter-disciplinary collaboration formed the basis for innovation and standardization in EuroFlow-based high-throughput diagnostic flow cytometry. Conclusions on cellular Immune Monitoring with standardized EuroFlow strategies and tools 1. EuroFlow strategies and tools aim at recognition of differentiationmaturation pathways with detailed subsetting to detect aberrancies from normal, using standardized methods and PCA for rapid data analysis. 2. Up to 5-10 million events can be analyzed, so that detailed studies of small subsets are possible. 3. Fast and easy methodology (within 2 to 3 hours; 3 to 4 hours in case of intracellular stainings), including 10 to 15 minutes for data analysis. 4. Dried tubes will contribute to: speed, easy performance, high quality, reliability (no mistakes), better inter-laboratory comparability. 5. Reference data bases are being build for each EuroFlow tube (tube set). 6. Automated gating (no subjectivity), based on cell population annotation according to reference data bases (with normal and aberrant samples). EuroFlow tools are designed for broad application in the fields of Hematology and Immunology, consistently using same methods Acknowledgements & Collaborations in Immune Monitoring Thank you IHB, Immune Monitoring Section Cristina Teodosio Magda Berkowska Pieter van der Pol Indu Khatri Wouter van den Bossche Kyra van der Pan Annieck Diks Gita Naber Alita van der Sluijs Sandra de Bruin Sandra Vloemans Rick Groenland Bas de Mooij Marieke Bitter Bart Lubbers Châtelaine Voets Jacques JM van Dongen CANCER RESEARCH CENTER Julia Almeida Martin Perez-Andres Daniela Damasceno Juan Flores Vitor Botafogo Quentin Lecrevisse Alberto Orfao WP5 Task 5.6 USAL, Salamanca, ES LUMC, Leiden, NL RIVM, Bilthoven, NL RUMC, Nijmegen, NL UTU, Turku, FI UOXF, Oxford, UK US, Southampton, UK MRC, Gambia, GM ULB, Brussels, BE EuroFlow is an independent scientific consortium, which aims at innovation in flow cytometry for improvement of diagnostic patient care 5